MSU LIBRARIES ._:—. RETURNING MATERIALS: P1ace in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped be1ow. QUANTITATIVE CYTOTOXICITY AND MUTAGENICITY STUDIES EMPLOYING MAMMALIAN CELLS IN CULTURE By David James Doolittle A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1982 ABSTRACT QUANTITATIVE CYTOTOXICITY AND MUTAGENICITY STUDIES EMPLOYING MAMMALIAN CELLS IN CULTURE By David James Doolittle This thesis describes the characterization of an in_zi£gg_mamma- lian assay for the detection and study of toxic and mutagenic chemi— cals. The postmitochondrial supernatant (S-lS fraction) prepared from rat liver homogenates is used as enzyme source and V79 Chinese hamster cells as targets for chemically induced damage. The assay advances the field of genetic toxicology because in addition to deter- mining in a qualitative manner whether or not a chemical can be meta- bolically activated to a mutagen it may be used to elucidate quanti- tatively the relative importance of specific enzymes (ethoxyresorufin- 0-deethylase, ethylmorphine—N-demethylase, epoxide hydrase) in the metabolic toxification/detoxification of that chemical. To accomplish this objective the enzyme pattern contained in the 5-15 fraction is modulated by pretreating experimental animals with enzyme inducers (phenobarbital, 3-methylcholanthrene, B-naphtho- flavone or Aroclor 1254) and/or by adding enzyme inhibitors (a-naph- thoflavone, metyrapone, cyclohexene oxide) i§_git£g, The enzymic activity in a given S-lS fraction is determined, and the ability of David James Doolittle the preparation to produce mutagenicity and/or cytotoxicity in the presence of the test chemical is assessed. Correlations are made between the activity of a particular enzyme or enzyme ratio in the 3—15 fraction and the biological response produced by the test chemi— cal. Two correlation coefficients are determined, one relating enzyme activity to cytotoxicity and the other relating enzyme activity to mutagenicity. Benzo(a)pyrene (BP) was used to validate the assay. The results indicate that the rate of epoxide formation divided by the rate of epoxide destruction is an important determinant of the degree of BP- induced cytotoxicity and mutagenicity when it is metabolized ig_zi££g, As this ratio increases BP becomes more cytotoxic and more mutagenic towards V79 cells. The relative importance of selected enzymes in metabolically activating dimethylnitrosamine was also determined using the assay. The genetic toxicology of the potent bacterial mutagen 2,4-dinitrofluorobenzene (DNFB) was assessed in the assay. The results of this study indicate that neither DNFB, nor its microsomal metabolites, are mutagenic. These observations emphasize the need for caution when using data derived from bacterial systems for use in human risk assessment and indicate that mammalian assays are needed to determine accurately the mutagenic potential of suspected carcino- gens. to Esther, Dorothy and Joan, with love ii ACKNOWLEDGEMENTS I sincerely thank Drs. T.M. Brody, C.C. Chang, J.I. Goodman, R.A. Roth, J.E. Trosko and F. Welsch, who as members of my guidance committee have provided invaluable assistance throughout the course of this work. I am especially indebted to Dr. J.I. Goodman for his expert advice and continued support. I thank Ms. Michelle Paquin for excellent technical assistance and Ms. Diane Hummel for out- standing secretarial work. Finally, I would like to thank my friends Ed Schwartz, Elaine Faustman and Bonnie Baranyi for providing a pleasurable laboratory environment in which to conduct these studies. iii TABLE OF CONTENTS Page DEDICATI N ii ACKNOWLEDGEMENTS -— iii LIST OF TABLES vi LIST OF FIGURES viii LIST OF ABBREVIATIONS--- xi INTRODUCTION- 1 l. The need for short-term tests and their applications--- 1 2. Test system selection 5 3. Benzo(a)pyrene (BP) 10 4. Dimethylnitrosamine 15 5. 2,4-Dinitrofluorobenzene l7 6. Overall Objective 17 MATERIALS AND METHODS 19 1. Materials 19 2. Cell line and culture conditions — l9 3. Preparation of hepatic postmitochondrial supernatant—-— 20 4. Cytotoxicity and mutagenicity assays 21 5. Enzyme assays 26 A. Aryl hydrocarbon hydroxylase— 26 B. Ethoxyresorufin—O-deethylase 26 C. Ethylmorphine-N-demethylase 27 D. Epoxide hydrase 28 6. Protein assay 29 RESULTS-- 30 1. Enzyme activity in the 8—15 fraction 30 A. Aryl hydrocarbon hydroxylase 30 B. Ethoxyresorufin-O-deethylase 30 C. Ethylmorphine-N-demethylase 38 D. Epoxide hydrase 46 iv TABLE OF CONTENTS (continued) Page 2. The effects of a-naphthoflavone, metyrapone, and cyclo- hexene oxide on enzyme activity in the 3-15 fraction-—- 46 A. Ethoxyresorufin-O—deethylase activity 46 B. Ethylmorphine-N—demethylase Activity 52 C. Epoxide hydrase 52 3. Characterization of the V79 ouabain resistance mutagen- icity assay 52 4. Benzo(a)pyrene 60 5. Dimethylnitrosamine 92 6. 2,4-Dinitrofluorobenzene 101 A. Cytotoxicity studies 101 B. Mutagenicity studies 110 DISCUSSION 115 1. Preliminary characterization of the V79/ouabain resis- tance assay 115 2. Benzo(a)pyrene 118 3. Dimethylnitrosamine 128 4. 2,4—Dinitrof1uorobenzene 130 5. Significance 132 BIBLIOGRAPHY 137 Table 10 ll 12 13 LIST OF TABLES Aryl hydrocarbon hydroxylase activity in the 8-15 fraction Ethoxyresorufin—O-deethylase activity in the 8-15 fraction Ethylmorphine-N-demethylase activity in the 8-15 fraction Epoxide hydrase activity in the 8—15 fraction The effect of a-naphthoflavone on ethoxyresorufin-O— deethylase activity in the 8-15 fraction The effect of cyclohexene oxide or metyrapone on ethoxyresorufin-O-deethylase activity in the 8-15 fraction The effect of metyrapone on ethylmorphine-N-demethylase activity in the 3—15 fraction The effect of cyclohexene oxide or a-naphthoflavone on ethylmorphine-N-demethylase activity in the 3-15 fracO tion The effect of cyclohexene oxide on epoxide hydrase activity in the 8—15 fraction — - The effect of metyrapone or a-naphthoflavone on epoxide hydrase activity in the 8-15 fraction The influence of ouabain concentration on mutation frequency Cytotoxicity of the 8-15 fraction and NADPH generating system The cytotoxicity of benzo(a)pyrene in the absence of the 8—15 fraction vi Pace 35 41 47 50 51 53 54 55 56 57 63 64 65 LIST OF TABLES (continued) Table 14 15 16 17 18 19 20 21 22 23 24 Influence of the 8-15 concentration on benzo(a)pyrene- induced cytotoxicity Effect of the 8-15 concentration on benzo(a)pyrene- induced cytotoxicity and mutation frequency - The cytotoxicity of metyrapone, cyclohexene oxide and a-naphthoflavone The cytotoxicity and mutagenicity of benzo(a)pyrene in the presence of various concentrations of d-naphtho- flavone The cytotoxicity and mutagenicity of benzo(a)pyrene in the presence of various concentrations of cyclohexene oxide The cytotoxicity and mutagenicity of benzo(a)pyrene in the presence of various concentrations of metyrapone-~— The cytotoxicity of dimethylnitrosamine in the absence of the 8—15 fraction The cytotoxicity and mutagenicity of dimethylnitros- amine in the presence of various concentrations of metyrapone The cytotoxicity and mutagenicity of dimethylnitros- amine in the presence or various concentrations of a- naphthoflavone Effect of pretreatment with enzyme modulators on the Page 69 7O 74 75 77 78 93 99 100 detoxification of 2,4-DNFB by the 5-15 fraction -------- lll Mutagenicity of 2,4—DNFB in the absence of the 8-15 fraction vii 112 LIST OF FIGURES Figures Page 1 Structures of benzo(a)pyrene, dimethylnitrosamine and 2,4-dinitrofluorobenzene ll 2 Protocol for the determination of cytotoxicity and mutation frequency in V79 Chinese hamster cells -------- 22 3 Standard curve for the aryl hydrocarbon hydroxylase assay 31 4 Time course of the aryl hydrocarbon hydroxylase assay-- 33 5 Standard curve for the ethoxyresorufin-O-deethylase assay 36 6 Time course of the ethoxyresorufin-O-deethylase assay-- 39 7 Standard curve for the ethylmorphine-N-demethylase assay 42 8 Time course of the ethylmorphine-N—demethylase assay-—- 44 9 Time course of the epoxide hydrase assay 48 10 Growth curves for V79 Chinese hamster cells 58 ll Cytotoxicity of N-methyl-N—nitroso-Nl-nitroguanidine (MNNG) in the absence of the 8-15 fraction 61 12 Cytotoxicity of 20 pH benzo(a)pyrene in the presence of the 3-15 fraction derived from control or 3-methyl- cholanthrene (MC) pretreated animals 66 13 The relationship between cytotoxicity and mutagenicity when benzo(a)pyrene is incubated in the presence of the 8-15 fractions derived from rats pretreated with 3- methylcholanthrene (MC) or B-naphthoflavone (BNF) ------ 71 14 The relationship between ethoxyresorufin-O-deethylase activity in the 8-15 fraction and the biological re- sponse of V79 cells to benzo(a)pyrene— 80 viii LIST OF FIGURES (continued) Figure 15 16 17 18 19 20 21 22 23 24 Page The relationship between ethylmorphine-N-demethylase activity in the 8-15 fraction and the biological response of V79 cells to benzo(a)pyrene --- 82 The relationship between the ratio of ethoxyresorufin— O~deethylase to ethylmorphine—N-demethylase activity in the 8-15 fraction and the biological response of V79 cells to benzo(a)pyrene 84 The relationship between epoxide hydrase activity in the 8—15 fraction and the biological response of V79 cells to benzo(a)pyrene 86 The relationship between the ratio of ethylmorphine-N- demethylase to epoxide hydrase activity in the 8—15 fraction and the biological response of V79 cells to benzo(a)pyrene 88 The relationship between the ratio of ethoxyresorufin- O-deethylase to epoxide hydrase activity in the 8-15 fraction and the biological response of V79 cells to benzo(a)pyrene 90 Cytotoxicity of 100 mM dimethylnitrosamine in the pre- sence of the 8-15 fraction 94 The relationship between cytotoxicity and mutagenicity when dimethylnitrosamine is incubated in the presence of the 8-15 fraction 96 The relationship between ethoxyresorufin-O-deethylase activity in the 8—15 fraction and the biological re— sponse of V79 cells to dimethylnitrosamine 102 The relationship between ethylmorphine—N-demethylase activity in the 8—15 fraction and the biological re- sponse of V79 cells to dimethylnitrosamine 104 The relationship between the ratio of ethoxyresorufin- O-deethylase to ethylmorphine-N-demethylase activity in the 8-15 fraction and the biological response of V79 cells to dimethylnitrosamine — 106 LIST OF FIGURES (continued) Figure Page 25 The cytotoxicity of 2,4—dinitrofluorobenzene (2,4-DNFB) 108 26 The mutagenicity of 2,4-dinitrofluorobenzene (2,4-DNFB) and benzo(a)pyrene (B(a)P)- 113 AHH BP BPDE BPE BNF CCHO DMN DNFB EH EMND EROD HGPRT MC MNNG PB PBS PCB S-lS TCPO LIST OF ABBREVIATIONS aryl hydrocarbon hydroxylase a-naphthoflavone benzo(a)pyrene benzo(a)pyrene 7,8—diol, 9,10-epoxide benzo(a)pyrene 4,5-epoxide B-naphthoflavone cyclohexene oxide dimethylnitrosamine 2,4—dinitrofluorobenzene epoxide hydrase ethylmorphine-N-demethylase ethoxyresorufin-O-deethylase hypoxanthine-guanine phosphoribosyltransferase 3—methy1cholanthrene metyrapone N-methyl-N-nitroso—N'-nitro-guanidine phenobarbital phosphate buffered saline polychlorinated biphenyl 15,000 x g supernatant of a rat liver homogenate l,2-epoxy-3,3,3-trichloropropane xi INTRODUCTION 1. The Need for Short-Term Tests and Their Applications Methodologies must be developed which will facilitate the detec- tion and study of chemicals in our environment which are carcinogens. At the present time the primary method for identifying human carcino- gens is the rodent lifetime exposure bioassay. Unfortunately, the expense, in terms of both time and money of this assay limits its utility as a screening assay for the multitude of chemicals to which humans are exposed (for review see Ames, 1979). Therefore, many initial studies will be conducted utilizing so called "short-term tests." As their name implies, short-term tests deliver results on the biological activity of chemicals within only a few days or weeks. These assays have the additional advantage of being relatively inex- pensive. However, while short-term tests may give a fast and cost— efficient estimate of the carcinogenic potential of a given chemical there are serious problems in the extrapolation of results to yield a human risk assessment. Short-term tests do not cover all aspects of tumor formation in animals and humans and therefore these tests will never completely supplant tumor induction tests in mammals. Neverthe- less, short-term tests have been, and will continue to be, useful in determining if a given chemical is potentially carcinogenic to humans. 2 The current theoretical framework for the study of chemical carcinogenesis, first put forward thirty-four years ago (Berenblum and Shubik, 1947), divides the complex process into two major stages, initiation and promotion. The theory is supported by retrospective epidemiological studies which indicate that human carcinogenesis is a multistage process (Dorvlo 35 31., 1980). Initiation requires only a single exposure to a carcinogen, is heritable, and irreversible. Promotion is a multistage process (MUfson g£_§1,, 1979; Slaga e£_§l,, 1980a,b) where, in the presence of agents termed tumor promoters, an initiated cell is allowed to express itself phenotypically and form a tumor. If short-term tests are to be effectively utilized in the field of chemical carcinogenesis, it seems desirable that methods be developed which are capable of discriminating between complete car- cinogens and tumor promoters. Based on present scientific insights this has proved feasible because tumor promoters do not appear to damage DNA, whereas complete carcinogens, with few exceptions, cause DNA damage. Recently a short-termlin_yi£rg_method has been developed to detect and study tumor promoters (Yotti 25 31., 1979) which is based on the observation that tumor promoters inhibit metabolic cooperation in V79 Chinese hamster cells. The development of short-term tests aimed at the identification and study of complete carcinogens has received a great deal of attention in recent years. A variety of cell types, including bacteria, fungi, insects, plants and cultured mammalian cells have been employed. The available test methods can be divided into 3 three broad categories: those that detect mutations, those that detect gross chromosomal effects and those that measure DNA repair. This dissertation deals with the characterization of a short-term test based on the observation that carcinogens are capable of acting as mutagens (McCann g£_§l,, 1975; McCann and Ames, 1976). In some cases the degree of mutagenic response ig_zi££g_has been shown to correlate with the carcinogenic potency of the chemical in 3112 (Huberman and Sachs, 1976), while in other cases the mutagenic and carcinogenic potencies do not appear to correlate (Schut and Thor- geirsson, 1978; Bartsch st 31., 1980; Wislocki gt_§l,, 1980; Brambilla g£_§1,, 1981). There are several probable reasons which could explain why in_zit£g_responses do not always accurately predict in zizg_re— sponses. The two broad classes of inaccurate results in mutagenicity assays are false negative and false positive responses. A false nega— tive test outcome could be due to: a) the carcinogenic mechanism may be unsuitable for detection in the short-term test being employed, e.g., the chemical in question is a tumor promoter, b) the test chemi- cal may require metabolism into another form before it is active, and the conversion does not occur in the particular short-term test employed. This problem may be partially overcome by utilizing a battery of short-term tests. A false positive result in a short-term test may result from: a) inadequate carcinogenicity studies, i.e., the study was performed in the wrong species, at the wrong dose or via an inappropriate route of administration, b) pharmacologic factors are not taken into account in short-term tests. The absorption and 4 distribution pattern in_yigg may profoundly affect the biological response to a particular chemical. In the past mutagenicity assays have been used primarily as a qualitative prescreen to establish priorities for long—term carcino- genicity experiments. However, assays of the sort described in this dissertation are capable of testing a chemical's cytotoxic as well as mutagenic potential. This means that there are several other poten— tial applications of this test in toxicological studies besides car- cinogen screening. These include: 1) Quickly assessing the toxico- logical activity of a large number of compounds (e.g., structure- activity studies). This would be particularly useful when the test chemical is available in limited quantities because in_y}££g_assays require much less chemical than do in zi!g_studies. 2) Metabolism Studies. This assay may be utilized to efficiently study the follow- ing questions: A) Is a chemical a direct acting toxicant or does it require metabolic activation prior to exerting toxic effects? B) If a chemical requires metabolic activation, then what is the active metabolite? C) Why do many toxic chemicals exhibit species and/or organ specificity? The ability of a particular organ to metabolize a potential toxicant to an active form may contribute to organ speci— ficity. The metabolic potential of various organs could be analyzed quickly and inexpensively in a short-term test. The results from this type of analysis must be interpreted with caution because corre- lations between ianitrg_and in_!iyg_drug metabolism are not always perfect (Pelkonen g£_§1,, 1980). 3) Epidemiological Studies. One may analyze fecal, urine or blood samples for the presence of toxicants 5 utilizing this test. Such an analysis would be very useful in cases of suspected occupational or environmental exposure to toxic chemi— cals. 2. Test System Selection The assay has been designed to complement other previously existing short-term mutagenicity tests and is intended to be useful in the construction of a battery of short—term tests so that chemicals which may pose a risk to man can be more fully evaluated. Mbst chemical carcinogens must be metabolically converted into an electrophilic form in order to be active (for reviews see Miller, 1978; Miller and Miller, 1979). Cells in culture usually possess, at most, a limited capacity to metabolize foreign compounds (Huberman and Sachs, 1974, Meijer g£_§l,, 1980). Therefore, in_yi££g_assays designed to study putative carcinogens/mutagens often utilize a mamma- lian enzyme source in order to incorporate metabolism into the test system. There are presently two widely used mammalian activation systems employed in short—term tests. These are either metabolically competent feeder cells or tissue subcellular fractions, which are pri- marily derived from liver. Several assays which utilize mammalian feeder cells have been described. For example, assay systems developed by Huberman and collaborators (Huberman and Sachs, 1974, 1976; Huber- man gt_§l}, 1976, 1979), Langenbach g£_§l, (1978), Newbold g£_§l, (1977) and Gould (1980) utilize lethally irradiated hamster embryo cells, rat liver cells, BHK21 cells and rat mammary gland cells, respectively, as a source of xenobiotic metabolizing enzymes. These 6 systems have the advantage of producing metabolic profiles which approximate in yiyg_profiles (Selkirk, 1977; Glatt £5 31., 1981) and thus may prove particularly useful in the study of chemically induced, organ-specific carcinogenesis. For example, rat mammary gland cells appear unable to metabolically activate aflatoxin 31’ a potent liver carcinogen, whereas they can activate dimethylbenzanthracene, a mammary carcinogen (Gould, 1980). However, the cell in which the procarcinogen is initially metabolized may not necessarily be the cell which ulti- mately will suffer detrimental biological effects from that compound. That is, a procarcinogen could be metabolized to a proximate carcino- gen in one organ and subsequently be transported to a second organ where tumor initiation may result. In a situation like this the use of feeder cells derived from the organ in which the tumor appears as a source of metabolic potential may yield a misleading result. An additional problem with cell mediated activation systems is the fact that the parent compound must be transported into the feeder cell and the activated metabolite(s) must be transported out to the target cell. The serum concentration in the culture medium may influence the amount of parent compound transported into the feeder cells (Coulomb gt 31., 1981), and the high degree of chemical reactivity of the active metabolites indicates that much of what is produced will bind to nucleophilic sites in the feeder cells (Newbold 35 21,, 1977) thus eliminating their opportunity to produce biological effects in the target cells. Cell—mediated systems are also less useful than subcellular fractions for studies on the importance of alterations 7 in enzyme pattern and/or concentration in regard to the generation of mutagenic and/or cytotoxic metabolites from xenobiotics. For example, subcellular fractions have an obvious advantage over feeder cells in the evaluation of the relative importance of cytosolic, microsomal and nuclear enzymes in the process of metabolic activa- tion. Therefore, I chose to utilize the hepatic postmitochondrial supernatant as an enzyme source in these studies. Short—term tests utilizing mammalian subcellular fractions for activation and bacteria as target cells for chemically induced damage have been developed (Ames g£_§1,, 1973, 1975). However, there is a need for mutagenesis assays utilizing mammalian target cells. Some compounds may be metabolically activated to mutagens by enzymes found in bacterial, but not mammalian cells (Blumer §E_§l,, 1980, Speck_g£ a1}, 1981). The mutagenicity of some compounds is dependent upon bacterial repair processes (Ivanovic and Weinstein, 1980; McCoy gt 31,, 1981), which may differ from those found in mammalian cells. In addition, reports in the literature utilizing bacteria as target cells do not routinely quantify the toxicity of a given xenobiotic, precluding one's ability to express mutation frequency on a per- survivor basis. This information could be useful in comparing the potency of various mutagens (Peterson gt_§l,, 1979). The simultaneous measurement of cytotoxicity and mutagenicity may also aid in differ- entiating lesions which lead to mutagenesis from those responsible for cytotoxicity (Bradley £5 31,, 1980). I chose to utilize V79 Chinese hamster cells as targets for chemically—induced damage and to measure cytotoxicity and mutation frequency in parallel. 8 Two loci which are commonly monitored for the induction of muta- tions in mammalian cells are the Na+,K+-ATPase locus (Baker gt 31,, 1974; Chang g£_§13, 1978) and the hypoxanthine-guanine phosphoribo— syltransferase (HGPRT) locus (Chu and Malling, 1968; Krahn and Heidel- berger, 1977; O'Neill-gtual., l977a,b; Kuroki gt 31., 1977; Fox and Hodgkiss, 1981). 'Mutagenicity at the HGPRT locus is assessed by measuring resistance to purine analogs, 6—thioguanine or 8-azaguanine. Resistance to these compounds requires either a mutation which results in an abnormally functioning or non—functional HGPRT enzyme, or ab- sence of HGPRT, possibly due to an epigenetic alteration of gene expression. Mutagenicity at the Na+,K+—ATPase locus is monitored by measuring resistance to ouabain, an inhibitor of the enzyme. In view of the fact that a functional Na+,K+—ATPase is essential for cell survival ouabain resistance cannot be caused by events which result in the enzyme being absent or non-functional. Therefore, ouabain resis- tance is probably due to a mutation involving a base pair substitution in the portion of the Na+,K+-ATPase gene that codes for the ouabain binding site. There are several potential problems associated with the assessment of mutagenicity at the HGPRT locus. These include the phenomenon of metabolic cooperation which makes the assay very depen— dent upon cell density (Burk gt_§l,, 1968; Van Zeeland g£_§1,, 1972; Fox, 1975), the presence in serum of hypoxanthine (Peterson gt 31., 1976), as well as enzymes capable of degrading purine analogs (Van Zeeland and Simons, 1975), a long expression period which may necessi- tate subculturing prior to mutant selection, high background (Arlett 9 §E_al,, 1975), and finally the possibility that some mammalian cells which are resistant to the toxic effects of 8—azaguanine may arise as a result of non—mutational events, e.g., a stable shift in phenotypic expression (Harris, 1971; Sharp g£_§1,, 1973). For these reasons I chose to monitor the Na+,Kf—ATPase locus. The advantage of this assay system seems to be the short expression time, the absence of metabolic cooperation and high inducibility by mutagens. I recognize that the assessment of mutagenicity using ouabain resistance will fail to detect certain classes of mutagenic agents such as X-rays (Chang g£_ 31,, 1978) and gamma-rays (Arlett_g£flal., 1975) which can be effi- ciently detected by monitoring the HGPRT locus. In summary, I have chosen to utilize an in zi££g_mammalian assay system which employs the postmitochondrial supernatant prepared from a rat liver homogenate as an enzyme source and V79 Chinese hamster cells as the target for chemically-induced damage. Cytotoxicity is determined by measuring colony forming ability and mutagenicity is assessed at the locus coding for the membrane bound Na+,K+-ATPase by measuring development of resistance to ouabain. I have used this assay system to study quantitatively the meta- bolic activation of three chemical carcinogens which belong to three different classes of carcinogenic agents. The chemicals I chose to study are: a) Benzo(a)pyrene, a polycyclic aromatic hydrocarbon, b) dimethylnitrosamine, a nitrosamine, c) 2,4-dinitrofluorobenzene, an aromatic amine. 10 3. Benzo(a)pyrene (BP) BP (Figure l) is a common environmental pollutant (approximately 1320 tons emitted into the United States atmosphere in 1972, National Academy of Sciences, 1972), which requires metabolic activation and produces hepatocellular carcinomas in male Sprague-Dawley rats (Kita— gawa g£_31,, 1980). The different forms of cytochrome P-450 exhibit positional and stereospecificity in the metabolism of BP (Deutsch 33 31,, 1978; Wang, 1981). Therefore, the pattern of metabolites produced during the microsomal metabolism of BP may be altered by pretreating the experimental animals with either PB or MC (Holder 3; 31., 1974; Yang 3£_31,, 1975). Pretreatment with MC increases the production of the 4,5-diol, the 7,8-diol and the 9,10-diol, whereas PB pretreatment increases the production of only the 4,5-diol (Rasmussen and Wang, 1974). Two theories have been prOposed to relate the carcinogenic action of BP to the manner in which it is metabolized. The BP molecule con— tains two regions, the bay and the K, which are readily metabolized by mixed function oxidases. The theories relate the carcinogenic action of BP to metabolism in these regions; the K region theory pre- dicting that the critical reactions leading to the ultimate reactive species occur in the K region, while the bay region theory predicts that these reactions will occur in the bay region. The K region theory was proposed (Pullman and Pullman, 1955) based upon the electronic characteristics of the BP molecule. According to this theory the 4,5-epoxide of BP (BPE) is considered to be the most likely ultimate carcinogen. However, the DNA adducts formed in cultured mammalian 11 Figure 1. Structures of benzo(a)pyrene, dimethylnitrosamine, and 2,4-dinitrofluorobenzene. 12 12 1 11 2 10 3 90 O 8 4 7 6 5 BENZO (a) PYRENE DIMETHYLNITROSAMINE N02 : N02 F! 2, 4 - DINITROFLUOROBENZENE Figure l 13 cells exposed to BP were found to be different from those formed when BPE is reacted with DNA 13_y1££3_(Sims 33 31., 1974; Baird 33 31,, 1975). This work suggested that bay region diol-epoxides (BP 7,8-diol-9,10—epoxide) might be the ultimate carcinogenic species of BP (Sims E£“§£': 1974). According to the bay region theory, in order for BP to become mutagenic and carcinogenic it must be ini— tially acted upon at the 7,8 position, presumably by cytochrome P-448, to form the 7,8-oxide, followed by hydration catalyzed by epoxide hydrase to form the 7,8-dihydrodiol (Wood 33 31., 1976a; Neidle 33 31,, 1981). In the absence of epoxide hydrase the 7,8-oxide may non— enzymatically rearrange to a relatively non-reactive phenol. This scheme is supported by the observation that the addition of epoxide hydrase to a purified microsomal system decreases the production of phenols and increases the production of dihydrodiols (Holder 3£_31,, 1974), indicating that dihydrodiols and phenols share arene oxides as a common precursor. The 7,8-dihydrodiol may be further epoxi- dated, again presumably by cytochrome P-448 (King 33 31., 1976) to the highly reactive diol epoxides, 7,8-dihydrodiol-9,10-epoxide (BPDE). There is recent evidence to suggest that prostaglandin synthetase may also catalyze this reaction (Sivarajah 35 31., 1981). The BPDE's exist as a pair of diastereomers due to the fact that the 7-hydroxyl group may be either cis or trans to the 9,10-epoxide. BPDE I is the diastereomer in which the 7—hydroxyl group is trans to the 9,10 epoxide, and BPDE II is the diastereomer in which the 7-hydroxyl group is cis to the 9,10 epoxide. BPDE I appears to be the more biologically important diastereomer due to the following considerations: l4 1) When BP is metabolized 13_y1££3_by rat liver microsomes a single enantiomer, (-) r-7, t-8—dihydroxy-7,8-dihydrobenzo(a)pyrene, is produced. This enantiomer is further metabolized predominantly to the dial-epoxide, r-7, t-8-dihydroxy-t-9, 10-oxy-7, 8, 9, 10- tetrahydrobenzo(a)pyrene (BPDE I) (Yang 35 31., 1976). 2) BPDE I is more mutagenic to mammalian cells than is BPDE II (Huberman 3£_31,, 1976; WOod 3£_31,, 1977). 3) BPDE I is more active than BPDE II in transforming hamster embryo cells 13_y1££3_(Mager 3£_31,, 1977). 4) BPDE I is more carcinogenic than BPDE II (Slaga 33 31., 1979). 5) When human or bovine bronchial explants are exposed to BP the major DNA adduct results from BPDE I (Jeffrey 3£_31,, 1977). 6) When 10T% mouse embryo fibroblasts metabolize BP the major DNA adduct results from BPDE I (Brown 33 31., 1979). When BPDE I interacts with mammalian DNA several nucleic acid adducts are produced, some of which may cause distortion of the double helix (Hogan 33_31,, 1981). It is presently not known which of these nucleic acid adducts is(are) most critical with respect to the muta- genic and/or carcinogenic process. BPDE I produces multiple guanine, adenine and cytidine adducts but does not appear to bind covalently to thymidine (Jennette 3£_31,, 1977). The major adduct results from the covalent linkage of the N-2 of guanine to the lO-position of BPDE I (Jeffrey 33 31., 1976; Feldman 3£_31,, 1980; Osborne 3£_31,, 1981). BPDE I also interacts with guanine at the 0—6 and N-7 positions to a minor extent (Osborne 33 31., 1981). The deoxyadenosine adducts result 15 from the addition of the N—6 amino group of adenine to the 10—position of BPDE I (Jeffrey 3£_31,, 1979), and the cytidine adducts result from the covalent interaction of the N-4 cytidine with the lO—position of the BPDE (Jennette 35_31,, 1977). The BPDEs have been reported to be relatively poor substrates for epoxide hydrase (Wood 33 31., 1976b) but if acted upon prior to their interaction with critical cellular nucleophilic sites they can be detoxified to the non-reactive metabolite, 7,8,9,lO-tetrahydro- tetrol. Therefore, it is clear that the role of epoxide hydrase in the metabolism of BP can be either toxification or detoxification depending on the metabolite which it acts upon. Furthermore, it appears probable that the ratio of P—448 activity to epoxide hydrase activity could determine the concentration of the reactive diol epoxide at any given time. 4. Dimethylnitrosamine (DMN) DMN (Figure l) is a hepatocarcinogenic nitrosamine (Craddock, 1971; Uchida and Hirono, 1979) which requires metabolic activation in order to acquire mutagenic properties (Malling, 1971; Umeda and Saito, 1975; Kuroki 33_31,, 1977; Chin and Bosmann, 1980), although at high concentrations DMN is cytotoxic in the absence of metabolic activation due to its protein denaturing ability, which may result in cell mem— brane destruction (Argus and Arcos, 1978). The metabolic activation of DMN is catalyzed by mixed function oxidases collectively termed DMN demethylase (Czygan_3£_31., 1973). 16 DMN is dealkylated by DMN demethylase to a monoalkyl derivative which is spontaneously converted to the corresponding monoalkyl diazonium ion. This compound decomposes to yield a carbonium ion, which alky- lates nucleophilic sites in DNA and RNA (Lijinsky 3£_31,, 1968). The DNA alkylation products are primarily 06amethylguanine, N7—methyl- guanine and N3-methyladenine (Abanobi 33 31., 1980). The DMN demethylase activity in an 13_X1££3_metabolic activation system correlates, in some cases, with DMN-induced mutagenicity (Frantz and Malling, 1975; Hutton 3£_31,, 1979b; Yoshikawa 33 31., 1980), while in other instances DMN demethylase activity does not correlate with DMN—induced mutagenesis (Hutton 33_31,, 1979a). There is a correlation between the ability of a particular tissue to meta- bolize DMN 13“!1££3_and that tissue's susceptibility to DMN-induced carcinogenesis (Bartsch 3£_31,, 1975). Isolated microsomes from mouse livers exhibit only about half the DMN demethylase activity present in the crude homogenate (Lake 3£_31,, 1974; Lake 3£_31,, 1976; Anderson and Angel, 1980). The enhancing effect of the soluble frac- tion may be due to the presence of additional cofactors for the microsomal enzymes because the hepatic soluble fraction is unable to metabolize DMN in the absence of microsomes (Lake 3£_31,, 1975, 1976). Hepatic DMN demethylase activity (Czygan 3£_31,, 1973; Ander— son and Angel, 1980) as well as DMN's enzymic activation to a mutagen (Czygan 33,31,, 1973) are inducible by Aroclor 1254. Recent evidence suggests that DMN demethylase activity may involve multiple enzymes (Lake 33 31., 1974; Kroeger-Koepke and Michejda, 1979; l7 Haag and Sipes, 980), e.g. both P—450 and P—448 (Guttenplan 3£_31,, 1976). The role that these mixed function oxidases and other enzymes play in the metabolic activation of DMN is obscure and requires fur— ther study. 5. 2,4-Dinitrofluorobenzene (DNFB) DNFB (Figure 1), an aromatic amine used in the identification of N—terminal amino acids of polypeptides (Sanger, 1945) has been re— ported to be a potent, direct acting mutagen in bacteria (Hepe, 1979; Jagannath 3£H31,, 1980; Summer and Goggelmann, 1980) and yeast (Fahrig, 1979). However, 13,3133_studies employing mice (Bock 33 31., 1969) have indicated that DNFB lacks initiating properties. To investigate this apparent discrepancy I chose to determine if DNFB is a direct acting mutagen in mammalian cells or if it can be metabolically acti- vated to a form mutagenic towards mammalian cells. 6. Overall Objective The objective of this research was to establish and characterize a mammalian short-term test which will be useful in conducting meaning- ful mechanistic studies regarding the metabolic toxification/detoxi— fication of xenobiotics, particularly carcinogens. It has become apparent in recent years that most carcinogens are electrophilic in their ultimate form(s). The majority of carcinogens exist as procarcinogens which must be metabolically converted into an electrophilic form. The target cells commonly used in short—term tests possess, at most, a limited capacity to metabolize foreign 18 compounds. Therefore, 13;!1££3_assays designed to study carcinogens must utilize a mammalian enzyme source in order to incorporate meta- bolism into the test. The concentration and pattern of enzymes responsible for the metabolic toxification/detoxification of xeno- biotics in short-term tests is usually modulated by pretreating the mammals with selected compounds. To interpret properly the results obtained from these tests it is crucial that the effects of alterations in both the absolute and relative amounts of enzymes capable of meta- bolizing the putative toxicant on the outcome of the test be thoroughly examined and understood. MATERIALS AND METHODS 1. Materials Calf serum, antibiotics and culture medium were purchased from Gibco (Grand Island, NY). Ouabain, NADPH, NADP, NADH, glucose-6— phosphate, g1ucose—6-phosphate dehydrogenase, 3-methy1cholanthrene, benzo(a)pyrene, trypsin, 2,4-dinitrof1uorobenzene, dimethylnitros- amine, metyrapone, a-naphthoflavone, quinine sulfate, bovine serum albumin and semicarbazide-HCl were obtained from Sigma Chemical Co. (St. Louis, MO). Cyclohexene oxide and B-naphthoflavone were from Aldrich Chemical Co. (Milwaukee, WI). Phenobarbital was from Mallinc- krodt (St. Louis, MO), ethylmorphine (Dionin) from McKesson and Rob- bins (Lansing, MI), 7—ethoxyresorufin from Pierce Chemical Co. (Rock- ford, IL), resorufin from Eastman Organic Chemicals (Rochester, NY) and Giemsa stain (improved R66 solution) from Bio/medical Specialties (Santa Monica, CA). Aroclor 1254 brand of PCBs (Monsanto Chemical Co., St. Louis, MO) and [3H]styrene oxide (Amersham, Arlington Heights, IL) were gifts from Dr. J.B. Hook, Michigan State University, and 3-OH benzo(a)pyrene (IIT Research Institute, Chicago, IL) was a gift from Dr. R.A. Roth, Michigan State University. 2. Cell Line and Culture Conditions The cells used in these experiments are V79 Chinese hamster cells which grow in monolayer (obtained as a gift from Drs. J.B. Trosko and 19 20 C.C. Chang, Michigan State University). This cell line was derived from the lung of a normal, male Chinese hamster (Ford and Yerganian, 1958). The cells are routinely cultured at 37°C in humid air contain- ing 5% CO in modified Eagle's minimal essential medium (Gibco formula 2» 78-5470, with Earle's salts, 1.5 X essential amino acids, 1 X gluta- mine, 2 X non-essential amino acids, 1.5 X vitamins; without glucose, sodium bicarbonate, and phenol red) supplemented with 20% (v/v) calf serum, penicillin (100 units/ml), streptomycin (100 pg/ml), fungizone (0.25 ug/ml), sodium pyruvate (110 ug/ml), glucose (1 mg/ml), sodium chloride (0.83 mg/ml) and sodium bicarbonate (1 mg/ml). 3. Preparation of Hepatic Postmitochondrial Supernatant (S-15 Fraction) Male, Sprague-Dawley rats (Spartan Research Farms, Haslett, MI) weighing 180-240 g were maintained on a 12 hr light cycle (7 p.m.-7 a.m.), allowed free access to food (Wayne Lab-Blox, Allied Mills, Chicago, IL) and water, and they were acclimated to our animal quarters for at least 3 days prior to use. Groups of animals were pretreated with i.p. injections of 3MC (80 mg/kg body weight in peanut oil, 24 hr prior to sacrifice), BNF (80 mg/kg body weight in peanut oil, 24 hr prior to sacrifice), Aroclor 1254 brand of PCBs (500 mg/kg body weight in peanut oil, 96 hr prior to sacrifice) or phenobarbital (80 mg/kg body weight in isotonic NaCl solution, 96, 72, 48 and 24 hr prior to sacrifice). Controls received vehicle only. The hepatic postmitochondrial supernatant is prepared (Kuroki 35 31,, 1977) as aseptically as possible, at 0-4°C, just prior to use. 21 Animals are killed by a sharp blow to the back of the head followed by cervical dislocation. The liver is removed, washed with 0.9% saline, and homogenized with a Potter—Elvehjem type homogenizer in 3 volumes of sucrose—HEPES buffer (0.25 M sucrose containing 2 mM MgCl2 and 20 mM N-Z-hydroxyethylpiperazine-N'-2 ethanesulfonic acid, pH 7.4). The homogenate then undergoes two successive centrifugations (Sorvall RC 2—B refrigerated centrifuge); 9000 x g for 10 minutes followed by 15,000 x g for 20 minutes. The postmitochondrial supernatant obtained is referred to as the S-15 fraction. 4. Cytotoxicity and Mutagenicity Assays The assays are performed in the following manner (Figure 2): Stock cultures of V79 cells in the mid-logarithmic phase of growth are trypsinized (0.01% crystalline trypsin dissolved in phosphate buffered saline, pH 7.8, 37°C; 10 minutes) and the cell number deter- mined using a hemocytometer. The cells are diluted to the desired con- centration and seeded into 10 ml of warm (25°C) growth medium in 100 mm tissue culture dishes. The number of cells seeded per dish is 200- 2000 for cytotoxicity assessment and 1x105 to 5x105 for the determina— tion of mutation frequency. The actual number is dependent upon the anticipated toxicity of a particular treatment. The same stock popu- lation of V79 cells is used for parallel determinations of cytotoxicity and mutation frequency. The cells are allowed 6 hours to attach to the culture dishes. The growth medium is then removed and the treatment medium is added. The treatment medium consists of the following components, added in 22 .mahuso onu mo Houfinfiaca am .awmnmoo ou oocmu Imfimmu wo uaoanoao>oo onu wcflusmmoa kn Am.H.o.m .u.mv mmmmHuHHHom wcfiauom zcoaoo wcwunmmma he woafiaumuop ma hufioHNOuouho .mHHou Houmams ommcfinu ms> aw kudosvoum coaumuna pom muwowxououhu mo moaumdwahmuov use you Houououm .N madman 23 N ouowam mezm_meaz Imizmmmmm ozq mmmmu oosmm o» zo_»_oo< muei< m>m ozieammzmo Iasaz a: N Am guiizm mmmu: z_ mH-m 3: N Am mz_4Uzmsammi _ uz=iamz azuzquaefi zoieaezz wm_ZOEou ezzoa msaa z=_amz >e_u_xoe mzisam Hz=iamz alums 1137 1:55 flaming EEBEA :QEEAIIII m-a -oa>u a: N a: m comm z_.:m2mhz_ 203mg; IO TIME (MINUTES) Figure 4 35 TABLE 1 Aryl Hydrocarbon Hydroxylase Activity in the S-15 Fraction Pretreatment Enzyme Activity8 Controlb 132:45C 3-Methy1cholanthrened 420:41 B-Naphthoflavonee 426123 Aroclor 1254f 457:18 Phenobarbitalg l69il9 apmoles 3-OH benzo(a)pyrene/minute/mg S-15 pro- tein. bPeanut oil or 0.9% saline. CMean i S.E. of the values obtained from four animals. d8O mg/kg, i.p., 24 hr prior to sacrifice. 880 mg/kg, i.p., 24 hr prior to sacrifice. f500 mg/kg, i.p., 96 hr prior to sacrifice. g80 mg/kg, i.p., 96, 72, 48 and 24 hr prior to sacrifice. 36 Figure 5. Standard curve for the ethoxyresorufin-O-deethylase assay. Each point represents the mean of three replicates. 37 150- >100- *— :7) Z III-I *— E Z 9 U5 ‘..’..’ .5. 50- o OL- C I l I l l J o 100 zoo 300 400 500 RESORUFIN (pmoles) Figure 5 38 carrying known amounts of resorufin through the assay. In accordance with previous observations (Pohl and Fouts, 1980), the addition of 1.6 mg of bovine serum albumin per ml of reaction mixture increases apparent enzyme activity (2.5x) in this assay. Therefore, all EROD assays reported in this thesis contain bovine serum albumin (1.6 mg/ ml). The time course of the reaction is illustrated in Figure 6, and indicates that the reaction velocity is linear for at least 6 minutes at appropriate concentrations of S-15 protein. Therefore, when the ethoxyresorufin—O—deethylase activity in the S-lS fraction is assessed the reaction is terminated after 5 minutes, and the following amounts of S-15 protein are employed: Aroclor 1254 pretreated, 0.035 mg; MC, BNF, 0.070 mg; PB, non-treated, 0.150 mg. The results are expressed as pmoles resorufin/minute/mg S-15 protein (Table 2). C. Ethylmorphine-N-Demethylase (EMND) This assay is based on the enzymatic N-demethylation of ethylmorphine, a reaction believed to be catalyzed by cytochrome P-450. The product is formaldehyde, and the reaction velocity is quantified by comparison to a standard curve constructed by carrying known amounts of formaldehyde through the assay (Figure 7). The time course of the reaction is illustrated in Figure 8, and indicates that the reaction velocity is linear for at least fifteen minutes at appropriate concen— trations of S-15 protein. When ethy1morphine-N-demethy1ase activity in the S-15 fraction is assessed, the reaction is terminated after 10 minutes and the following amounts of S-15 protein are used: Aroclor 1254, PB, pretreated, 10 mg; MC, BNF, or non-treated, 20 mg. The results 39 Figure 6. Time course of the ethoxyresorufin—O-deethylase assay. H, 0.12 mg 3-methy1cholanthrene pretreated S-15 protein;H , 0.06 mg 3-methylcholanthrene pretreated S-15 protein;I-I , 0.24 mg non—induced S-15 protein; O-O , 0.12 mg non-induced S-15 protein. Each point represents the mean of three replicates. (pmoles ) RESORUFIN 1600 I400 I200 I000 800 600 400 200 4O . C 3"... —- ..:' ’_.—-—".—— J l J 2 4 6 TIME (minutes) Figure 6 41 TABLE 2 Ethoxyresorufin-O-Deethylase Activity in the S-15 Fraction Pretreatment Enzyme Activity3 Controlb 124: 28c 3-‘Methy1cholanthrened 2089:405 B-Naphthoflavonee 2166:336 Aroclor 1254f 4611:338 Phenobarbitalg 462il44 apmoles resorufin/minute/mg S-15 protein. bPeanut oil or 0.9% saline. CMean i S.E. of the values obtained from three animals. d80 mg/kg, i.p., 24 hr prior to sacrifice. e80 mg/kg, i.p., 24 hr prior to sacrifice. f500 mg/kg, i.p., 96 hr prior to sacrifice. g80 mg/kg, i.p., 96, 72, 48 and 24 hr prior to sacrifice. 42 Figure 7. Standard curve for the ethylmorphine-N-demethylase assay. Each point represents the mean of three replicates. ABSORBANCE 2.0—- I.5- mi- 0.5;- l _l l 1 l l l 0.8 L2 1.6 2.0 2.4 CD-- .0 .5 FORMALDEHYDE (AL moles) Figure 7 44 Figure 8. Time course of the ethylmorphine-N—demethylase assay. H, 10.7 mg Aroclor 1254 pretreated S-15 protein;H , 4.4 mg Aroclor 1254 pretreated S-15 protein; 0-0, 8.6 mg non-induced S-15 protein; 0". 3.6 mg non-induced S-15 protein. Each point represents the mean i S.E. of three replicates. ABSORBANCE I.5 I.0 0.5 I 45 TIME (MINUTES) Figure 8 46 are expressed as nmoles formaldehyde produced per minute per mg S-15 protein (Table 3). D. Epoxide Hydrase (EH) The assay is based on the conversion of [3H]styrene oxide to [3H]styrene dihydrodiol by epoxide hydrase. Differential extraction of the incubation mixture is employed to separate substrate from pro- duct and the reaction velocity is determined by assaying the product ([3H]styrene dihydrodiol) in a scintillation counter. The data in Figure 9 illustrate the time course of the reaction. At appropriate concentrations of S-15 protein the reaction velocity is linear for at least 9 minutes. Therefore, when assessing the epoxide hydrase activity in the S—15 fraction the reaction is terminated after 6 minutes and the following amounts of S-15 protein are employed: PB, Aroclor 1254 pre- treated, 0.25 mg; MC, BNF or non—treated, 0.50 mg. The results are expressed as nmoles styrene dihydrodiol produced per minute per mg S-15 protein (Table 4). 2. The Effects of a-Naphthoflavone (ANF), Metyrapone (MET), and Cyclohexene Oxide (CCHO) on Enzyme Activity in the S-15 Fraction A. Ethoxyresorufin—O-Deethylase (EROD) Activity ANF, an inhibitor of cytochrome P-448, inhibits EROD activity in a concentration-dependent fashion when added to S-15 fraction de- rived from animals pretreated with MC or Aroclor 1254 (Table 5). Con- versely, ANF does not inhibit EROD activity in the S-15 fraction derived from a non-induced animal (Table 5). 47 TABLE 3 Ethylmorphine-N-Demethylase Activity in the S-15 Fraction Pretreatment Enzyme Activitya Controlb 1.28:0.12C 3-Methy1cholanthrened 0.59:0.07 B—Naphthoflavonee O.56i0.03 Aroclor 1254f 3.29:0.27 Phenobarbitalg 4.15:0.04 anmoles formaldehyde/minute/mg S-15 protein. bPeanut oil or 0.9% saline. CMean i S.E. of the values obtained from three animals. d80 mg/kg, i.p., 24 hr prior to sacrifice. e80 mg/kg, i.p., 24 hr prior to sacrifice. f500 mg/kg, i.p., 96 hr prior to sacrifice. 880 mg/kg, i.p., 96, 72, 48 and 24 hr prior to sacrifice. 48 Figure 9. Time course of the epoxide hydrase assay. H , 0.4 mg Aroclor 1254 pretreated S-15 protein;t}——CI, 0.2 mg Aroclor 1254 pre— treated S-15 protein; I—I , 0.4 mg non—induced S-15 protein; O-O , 0.2 mg non-induced S—15 protein. Each point represents the mean of three replicates. 49 (D J 3 +5 9 ms (mmurss) Figure 9 50 TABLE 4 Epoxide Hydrase Activity in the S-15 Fraction Pretreatment Enzyme Activitya Controlb 0.93:0.10C 3-Methylcholanthrened 0.72:0.04 B-Naphthoflavonee 0.85:0.09 Aroclor 1254f 2.12:0.41 Phenobarbitalg 1.85:0.12 anmoles styrene dihydrodiol/minute/mg S-15 pro- tein. bPeanut oil or 0.9% saline. CMean i S.E. of the values obtained from three animals. d80 mg/kg, i.p., 24 hr prior to sacrifice. e80 mg/kg, i.p., 24 hr prior to sacrifice. f500 mg/kg, i.p., 96 hr prior to sacrifice. 880 mg/kg, i.p., 96, 72, 48 and 24 hr prior to sacrifice. 51 .mouMUfiHaou mounu mo .m.m H cmo2w .musuxaa cowuumou ozu :H coaumuuaooaoom .uaoaumouuoum oz.“U .mowmauomm ou uoana us ca ..Q.H .wx\wa oomo .mowwfiuomm ou scape u: «N ..Q.H .mx\ma own .N mHnMH mom .Houuaoo uam>aom mo mwmudouumm m mm mommoumxmm m.NHHn.mw n.0Hq.N m.~ Ho.m Smloa H.n Hm.mm m.owq.ma m.o Ha.m zoloa o.m “a.moa N.MHn.nm o.q Ho.qm anoa o.m Ho.ooa m.NHo.ooa wa.mawo.ooa mo pmeSUGHIcoz comma uoauou< nocmuzucmaonuahfiquIM mao>mawonu£am216 ucmaumouumum mhuw>wuo< mazuam aowuomum malm onu ca zuH>Huo< ommaxsuooQIOIGHmauomouxxonum no oco>mawozunmm216 mo uommmm 659 m m4maom mo mwmucooumm m mm wommmuaxmm w.o HB.MH m.H Ho.m n.~ Hm.oa SMIOH q.m Ho.Ho m.H Hm.na m.w Hm.aq ZqIOH o.HHHN.Nn N.m Hm.mq n.q Hw.no Emloa m.NHHo.ooa o.HHHo.OOH wo.HHHo.OOH mo woosvcchoz quH Hoaoou< HManumnoaosm v o n mcommuzuoz ucoaumouuoum mmuw>wuu< mazucm mandamus manm on“ as sua>auo< ommamnuoaoQIZIocwnmuoaaznum so ocoamumumz mo uuomwm one u m4m<8 55 TABLE 8 The Effect of Cyclohexene Oxide or a-Naphthoflavone on Ethylmorphine-N—Demethylase Activity in the a S-15 Fraction Compound Enzyme Activityb Cyclohexene Oxide 0C 100.0: 6.7d 10‘4M 97.3: 3.5 10'3M 91.1: 6.9 5x10-3M 95.6: 3.9 a-Naphthoflavone 0 100.0: 6.7 10‘7M 98.3:10.3 10'6M 85.3: 7.2 10'5M 103.0: 5.4 aS-15 fraction derived from animals pretreated with Aroclor 1254, 500 mg/kg, i.p., 96 hr prior to sacrifice. bExpressed as a percentage of solvent control, see Table 3. CConcentration in the reaction mixture. dMean : S.E. of three replicates. 56 TABLE 9 The Effect of Cyclohexene Oxide on Epoxide Hydrase Activity in the S-15 Fraction Enzyme Activitya Pretreatment Cyclohexene Oxide b c Aroclor 1254 3-Methy1cholanthrene d e 0 100.0:3.2 100.0:11.9 10'4M 73.4:5.0 84.9: 4.0 10'3M 29.9:2.5 52.0: 3.6 5x10'3M 20.7:2.4 22.9: 0.6 8Expressed as a percentage of solvent control, see Table 4. b500 mg/kg, i.p., 96 hr prior to sacrifice. C8O mg/kg, i.p., 24 hr prior to sacrifice. dConcentration in the reaction mixture. eMean i S.E. of three replicates. 57 TABLE 10 The Effect of Metyrapone or a-Naphthoflavone on Epoxide Hydrase Activity in the S—15 Fraction Compound Enzyme Activityb Metyrapone 0C 100.0: 2.1d 10'5M 81.0: 6.8 10'4M 111.0: 3.4 10'3M 142.0:11.8 a—Naphthoflavone 0 100.0: 1.2 10'7M 100.0: 2.5 10'6M 101.2: 2.3 10‘5M 102.7: 5.8 aS-15 fraction derived from animals pretreated with Aroclor 1254, 500 mg/kg, i.p., 96 hr prior to sacrifice. bExpressed as a percentage of solvent control, see Table 4. CConcentration in the reaction mixture. dMean i S.E. of three replicates. 58 Figure 10. Growth curves for V79 Chinese hamster cells. 1x105 cells were seeded into 75 cm2 flasks containing medium supplemented with the indicated concentration of calf serum (CS) or fetal calf serum (FCS). After various periods of time the cell number was determined. Each point represents the mean : S.E. of 8 determinations. 59 NUMBER OF CELLS (x 10'5) 1000 596ch 209608 . 159668 100 - 10%CS 1O '- 5%CS 1 I l l I I 1 0 20 40 60 80 100 120 140 HOURS OF GROWTH Figure 10 60 agents in a dose—dependent fashion. MNNG is a direct acting alkylating agent which is toxic to mammalian cells (Peterson 3£_31,, 1979), and, MNNG produced cytotoxicity in our V79 cells in a concentration-dependent manner in the absence of the S—15 fraction (Figure 11). MNNG is also a direct acting mutagen in mammalian cells and it induces ouabain resistant mutants in our V79 cells (Table 11). The spontaneous as well as the induced mutation frequency is markedly influenced by the ouabain concentration employed in the selective medium (Table 11). The S-15 fraction from.a MC-pretreated animal along with the NADPH generating system is non-toxic to V79 cells (Table 12). The S-15 fractions prepared from control animals or from animals pretreated with PB, BNF or Aroclor 1254 are similarly non—toxic (data not shown). 4. Benzo(a)pyrene (BP) Data on the cytotoxicity of BP in the absence of the S-15 frac- tion are presented in Table 13. The data in Figure 12 illustrate the cytotoxicity produced when a non-toxic concentration of BP (20 pM, Table 13) is titrated with non-toxic concentrations of the S—15 fraction (Table 12). When the S-15 is prepared from an animal pre- treated with MC cytotoxicity occurs, but when the S-15 fraction is pre- pared from an untreated animal there is no cytotoxicity. The cytotoxi- city curve is biphasic when MC pretreated S-15 is incorporated. When a portion of the S—15 fraction derived from a MC pretreated animal is inactivated by boiling (10 minutes) and various amounts are added to the reaction mixture along with 1.4 mg/ml active MC pretreated S-15 it can be seen that the BP is detoxified. 61 Figure 11. Cytotoxicity of N-methyl-N—nitroso-Nl—nitro-guanidine (MNNG) in the absence of the S-15 fraction. Cloning efficiency is expressed as a percentage of control. Values are the mean i S.E. of 3 replicates. CLONING EFFICIENCY 100 10 0.1 62 MNNG (11M) Figure 11 L 2 3 63 TABLE 11 The Influence of Ouabain Concentration on Mutation Frequency Cloning b Treatment Efficiencya Mutation Frequency Ouabain Concentration 0.5 mM 1.0 mM Control 100c 155d 1.3 (26) (2) 3 uM MNNG 9 399 152 (139) (53) 8Expressed as a percentage of solvent control. bMutants/106 viable cells. cMean of 3 replicates. dNumber of mutants recovered. 64 TABLE 12 Cytotoxicity of the S-15 Fraction and NADPH Generating System mg S-15 Proteina b Cloning Efficiency ml Reaction Mixture 0 100.0:2.4C 2.0 106.9:7.4 2.4 107.5:3.4 3.2 99.2:4.4 4.0 106.0:0.8 6.2 98.7:1.7 3Derived from animals pretreated with 3-methyl- cholanthrene, 80 mg/kg, i.p., 24 hr prior to sacrifice. b Expressed as a percentage of control. CMean i S.E. of 3 replicates. 65 TABLE 13 The Cytotoxicity of Benzo(a)pyrene in the Absence of the S-15 Fraction Benzo(a)pyrene (uM) Cloning Efficiencya 0 100:2.7b 5 101:5.1 20 99.1:6.2 50 95.6il.7 100 83.6:3.l a Expressed as a percentage of control. bMean i S.E. of 3 replicates. 66 .mmumofiaamp m mo .m.m H mama mnu ucmmoummu mosam> .Honuaoo mo mwmuaoouom m an oommoumxo m3.” xocofiuwmmo wsflaoao .ACIOV dowuomum malm woumouumum mamunuamaonuflEuoanm 623%.. Ha\wa q.a cows wcoam ousuxwa coauommu onu ou omoom mums mucooam mnoaum> mam .mm: on “cane mouoafla cos you mafiaaon he woum>fiuumcfi mmz cowuomnm malm voumouumue meoHSDcmHonoamzume1m mnu mo coauuom < .mHmaacm moumouuoum AIV mcmunuamaonoabtmalm no AIV omummuulao: 80.5 oo>fiumw cowuumuw malm ozu mo oucmmoum mzu :H oomummnmvouamn 2: on no mufioexououmu .NH musmwm 67 NH muswhe 95.5.: 20.55". _X_m5md m. m as hum” N _. o n 11 E A d AONEIOIddE ONINO'IO 68 It is apparent from Figure 12 that for S-15 derived from a MC pretreated animal the observed cytotoxicity is dependent upon the con- centration of the S-15 fraction in the reaction mixture. To determine whether this dependence on S-15 concentration would be apparent when the animals were pretreated with other types of enzyme inducers I chose to test the P—450 type inducer PB (Conney, 1967), a P-448 type inducer other than MC (BNF) (Boobis 3£_31,, 1977) and a mixed type inducer (Aroclor 1254) (Alvares 33 31., 1973; Parkinson 33 31., 1980). The results from this study are depicted in Table 14 and indicate that in all three cases, cytotoxicity is dependent upon the concentra- tion of S-15 protein in the reaction mixture. The results presented in Table 15 illustrate that the mutation frequency as well as the cyto- toxicity, is dependent upon the concentration of S-lS protein in the reaction mixture when the S-15 is derived from a MC pretreated animal. MC and BNF are P—448 type inducers (Boobis 33 31., 1977) and at the dose that I routinely employ both increase AHH activity to a similar degree (300% of control, Table l). The ability of S-15 fractions prepared from animals pretreated with these compounds was compared regarding their ability to produce mutagenic and/or cytotoxic metabo- lites from BP. The results are illustrated in Figure 13 and indicate that at equivalent levels of cytotoxicity these S-15 fractions produced differing degrees of mutagenicity. BNF pretreatment produced a greater degree of mutagenicity per degree of cytotoxicity than did MC pretreat— ment. 69 TABLE 14 Influence of the S-15 Concentration on Benzo(a)pyrene- Induced Cytotoxicity Cloning Efficiencya S-15 Source 10 uM Benzo(a)pyrene 20 pH Benzo(a)pyrene Phenobarbitalb 1.1c 83.3:6.5d <1 2.4 91.2:4.8 34.0:3.4 B—Naphthoflavonee 0.9 <1 <1 2.3 68.7:5.4 7.5i0.5 Aroclor 1254f 1.1 53.1:3.7 <1 2.4 101.8iS.7 19.3:3.0 8Expressed as a percentage of control. b80 mg/kg, i.p., 96, 72, 48 and 24 hr prior to sacrifice. cmg S-15 protein/ml reaction mixture. dMean i S.E. of 3 replicates. 680 mg/kg, i.p., 24 hr prior to sacrifice. f500 mg/kg, i.p., 96 hr prior to sacrifice. .vmum>ouou mufiMusa mo Honaszo .afimnmso 28 H :a coauooamm “maaoo manmfi> moa\muamu:zo .moumowaaou m mo .m.m H Goose .Houuaoo mo mwmuaoouoa m mm ommmoumxmn .ooam Ifiuomm ou Howum u: «N ..Q.H .wx\wa ow .mamunuamaonoamnumalm :ufiB moummuuoum mamafiam Scum om>aummm 70 AHmV Aaav Ame H.N m.NhH.ms 8.0 o.~hH.Ha m.o N.HHN.NQH .wa ~.~ :3 :3 8E a.mm N.OHN.m 8.~ “.mho.om 6N.o 6m.mhm.am ma 3.3 hoaoavoum huaowofimmm zoomsvoum hocoaofimmm mucosvoum moaoflofimmm coaumuaz wcficoau coaumunz waacoau coeumusz n wawcoao ousuxflz coauomom He 2: m.m 21 o.m z: m.~ camuoum malm ma m coaumuucmoaoo mamuzaAmvoucom kudosvmum coaumusz mam huHonouou%U wousocHlmamuhmAmvoucmm so cowumnuamoaoo malm mnu mo uoowmm ma mqmH>hsm oa\muamu=a mm wommoumxm n m .Amm oav Houuaou mo owmuaoouma m mm wommmumxmo .ooHMHuomm ou uowum up om ..e.a .mx\ma oomn .ooamwuomm ou Howua u: «N ..m.H .mx\wa owm nm.o w.HHm.oo wH.o H.MH~.NHH Emloa :m.o m.mHo.on mm.a a.mwo.mm zoned nm.o H.Hae.ow mo.~ a.mwo.wm anoa an.o o aha mm mm m mm H+w Ha o mucosvoum xucofiowmmm koamavoum chucofiowmmm Goaumuuaouaoo coaumusz wsfisoao ozowumDsz mafiaoao oao>mamonunmm215 Lemma uoaoou¢ oaounuamaonuamnquTm m mounom malm maopmamonunmmZIv mo mGOHumuuamuaoo waowum> mo oocmmmum onu ca ocou%mAmvoucom mo %uHoHamwmu=z mam >uHUonuou>o use NH mqm<8 76 derived from a MC pretreated animal, but does not affect either the cytotoxicity or mutagenicity of BP in the presence of the Aroclor 1254 induced S—15 fraction. CCHO increases the cytotoxicity and mutageni- city of BP in the presence of MO or Aroclor 1254 pretreated S-15 frac- tions (Table 18). At a concentration of lO-BM MET increases the cyto— toxicity and mutagenicity of BP in the presence of the S-15 fraction derived from an Aroclor 1254 pretreated animal but does not affect BP toxicity or mutagenicity when present at lower concentrations (Table 19). The effect of specific enzymes and/or specific enzyme patterns on the metabolic activation of BP was elucidated in the following manner: A) the activity of EH, EROD and EMND in S-15 fractions derived from animals pretreated with selected enzyme modulators is known (Tables 2, 3, 4). B) the inhibitory efficacy of various concentrations of MET, CCHO and ANF on these enzyme activities is known (Tables 5 to 10). C) knowing (A) and (B) the activity of a given enzyme in a given S-15 fraction in the presence of a given inhibitor concentration may be calculated and expressed as amount of product/minute/mg S—lS protein. D) the cytotoxicity and mutagenicity of BP in the presence of a particular S-15 fraction combined with a particular inhi- bitor is known (Tables 17, 18, 19). E) graphs relating the cytotoxicity 3£_mutagenicity of BP to the activity of a particular enzyme (or the ratio of two 77 .mocwauoumo no: .pohmmmm mHHou oaxmaw : m .vommmmm mHHoo moaxwaw .mmumowaaou m mo .m.m H amozm .muo>w>usm moa\muamuaa mm wowmounxmo .Amm ocv Houucoo mo ammuamouom o no vommoumxmo .oowmwuomm ou HOHHQ an mm ..m.« .mx\wa oomn .muamauumm ou uowum as am ..Q.H .wx\wa owm wo.m o.HHq.H .Q.z Hv Enloaxm mm.N «.8Hm.m e.g.z Hv EMIOH wm.a m.NHH.om mm.HH m.~am.w quoa mm.o m.me.w¢ mm.~ m~.NHn.om o hoamsvoum hoaofiofimmm %uaoovoum ohoaofiofiwmm coaumuuamuaoo coaumumz wcaaoau aowumusz mafiaoau mpfixo mamxonoaomo nsm~a uoaoou< monouzuamaonoahnuozlm mouaom malm mofixo ocmxosoaoho mo maowumuuaooaoo mDOHHm> mo oucmmmum osu cw ocouhmAmvoncmm mo muflowcmMMuaz mam huHonououxo 0:9 ma mqm<9 78 TABLE 19 The Cytotoxicity and Mutagenicity of Benzo(a)pyrene i2 the Presence of Various Concentrations of Metyrapone Metyrapone Cloning Mutation Concentration Efficiency Frequency 0 53.9:6.2d 0.5e 10'5M 52.1:4.9 0.8e 10'4M 58.1:8.3 0.3e 10'3M 26.7:2.8 1.9e aS-15 fraction derived from rats which had been pre- treated with Aroclor 1254, 500 mg/kg, i.p., 96 hr prior to sacrifice. bExpressed as a percentage of control (no BP). CExpressed as mutants/105 survivors. dMean i S.E. of 3 replicates. e15x105 cells assayed. 79 enzyme activities) were constructed by plotting the biolo- gical response to BP on the abscissa and enzyme activity in the S—15 fraction on the ordinate. The results of this analysis are illustrated in Figures 14 to 19. EROD aCtivity (Figure 14), EMND activity (Figure 15) or the ratio of EROD activity to EMND activity (Figure 16) in the S—lS fraction does not correlate with the induction of cytotoxicity or mutagenicity in the presence of BP. Conversely, EH activity (Figure 17) as well as the ratio between EMND activity and EH activity (Figure 18) do correlate with the induction of cytotoxicity and mutagenicity in the presence of BP. As the EH activity in the S-15 fraction increases, BP becomes less cytotoxic and less mutagenic and as the EMND/EH ratio increases, BP becomes more cytotoxic and more mutagenic. The correlations are sta— tistically significant (p<0.01) (EH—cytotoxicity, 59% of total varia— bility due to regression; EH-mutagenicity, 42% of total variability due to regression; EMND/EH-cytotoxicity, 62% of total variability due to regression; EMND/EH-mutagenicity, 46% of total variability due to re- gression). The ratio of EROD activity to EH activity in the S—15 fraction (Figure 19) correlates best with the production of toxic and mutagenic metabolites from BP (p<0.01, cytotoxicity - 79% of total variability due to regression; mutagenicity — 61% of total variability due to regression). As the ratio of EROD to EH activity in a given S—lS fraction increases, the cytotoxicity and mutagenicity of BP also increases indicating that the P-448/EH ratio in the S-lS fraction is important in determining the quantity of mutagenic and/or cytotoxic metabolites produced from BP. 8O .awmnmso SE o.H ca mmcwahouom mm3 mam mHHoo manmfi> oa\muamuss mm commmumxm ma Aan xocoav Imam coaumusz .mmumofiaeou omens mo some oau “comma on monam> .Houuaoo wo owMuamuumm m mm oommoumxo ma ADV kocmwofimwm mafiaoao .aaououm mHIm wa\ouscfie\5mauommu mmaoea mm. wmmmmuexo we >ua>fluum comm .ocoummAmvouamn ou mHHoo mm> mo mmaoemmu HmofiwOHOHn osu mam coauumum malm osu ca >ufl>auom Anommv mmmahzumooIOIawmanommu%xosuo comsuon masmaowumamu 6:9 .qH oudwfim 81 8H magmas >UZmUEn—m 02.20au m o. 0m 00— OD Do U o D o D 0 O D O HO HO 0 D >UZmDOw~E 203.332 000— Doom coon 000v 000m 0000 NOIIDVIH Sl-S 3H1 NI All/\IIDV 0083 82 .cfimomno 28 o.H :H mmaaahouow mms mam mHHmo oanma> moa\mucmu:a mm pommoumxm ma Anuv zoomsvouw coaumuoz .mmumuaaeou moucu mo coma mzu uaomoumou mooam> .Houuaoo mo ommuamoumm m mm wommouaxm ma ADV .Aocofiowwmm wcwcoao .awmuoum 31m wfi\munafia\ou%nmo.nmfiuom mo mmHoEa mm oommoum Ixm ma zufi>wuum 922m .maoummAmvouaoa ou maaoo mn> mo uncommon HmonOHOfln on“ new moauomuw malm msu CH muw>fiuom Amzzmv mmmamsumaopIZIoaasauoaaanuo Gooaumn manmcowumamu one .mH muomfim 83 3 0.33m >UZm_U_uEm OZ_ZO._U O— In >UZmDOw~E ZO:.<.—D<< On 00— 0.. O.N od 0.? All/“13V CINW3 NOIIDVEH SI'S 3H1 NI 84 .cwmnmso 28 o.H ca mocwaumumv mos mam mHHmu maamfi> OH \mucmusa mm commoumxm me Aan mucoswmum cowumusz .mmumuwamou woman mo cmoa mnu uammmu on mooam> .Houucoo mo mwmuamoumm m mm oommmumxm m.“ ADV hocmwofimmo wdwaoao .cfimuona malm we\ou=cfia\moznowamauom moaoad mm vommmuexm mm3 huH>Huom azzm mam unwououa malm me\mu::«a \afimnuomou moaoam mm mmmwmuaxm mm3 muw>fiuum comm .oamuxmAmvoucon ou mHHoo mn> mo mmaommmu HmonOHOHo was new soauumum mHIm onu as zuH>Huum Aazzmv ommHanumaouIZIocwsauoBH%£uo cu Anommv mmmH>SuoovIOIcamonomouhxosum mo oaumu on» cooSuon awamaoaumaou 05H .oH ouswwm 85 8H dogmas >UZm_U_“Ew OZ.ZO._U — m o— Om oo— 7 3 . _ mfiflu O D O. DU. 0 D O D E D O U. C D p n F —L o m V m N p o >UZmDOu~E ZO_._.<._.D<< I 1 000— ... Doom 1 000m 1 08¢ 1 000m L 0000 NOIIDVZH Sl-S 3H1 NI All/\IIDV ONWB/GOHS 86 .aamnmno :8 o.H CH wmsfiEMouov mos mam mHHoo manmfl> moa\muamuaa mm oommoumxm ma Amo.OI n H .nuIAuV hocmsv loom coaumuoz .moumofiaaou woman mo some msu uzommumou mmDHm> .Houuaoo mo ommucmuuoa m mm wommmuaxm ma :50 u H Iv moaofiowmmo wawcoao .awmuoua malm wa\muafiua\aowwou?¢fim 9553.6. mmaoad mm wommoumxo ma >ufi>fiuom mm .mamuheflmvoucmn ou maaoo-mm> mo mmaommou Hmuwwoaoan osu cam coauumum malm mau as zufi>fiuom Nmmv ommuv%£ mofixomo cmwBuon mfinmcowumamu 658 .NH ouswflm 87 S 8:me 09 88 .camnmso 25 o.H cw vocaauouoo mus mam mHHoo manmfir modmucmuaa mm wommmumxm ma 30.0 n u .UIUV 13:056on coaumusz .mmumofiamou mounu we come on... ucmmouaou moi}; .Houucoo mo owmuamouom m mm vommouexo m.“ 3561 n H .Iv mocofiofimmm wcacoao .aHououa maum wa\ouaaHE\H0Hpouw%£Hm ocmumum moaoaa mm commouexo mm3 huabfiuom mm mam mafimuoum mHIm me\muscfia\ov%nomamahom moaoad mm wommoumxo mm3 mufi>fiuom 922m .ocouka IAmvouaon ou mHHoo mm> mo mmcommou HmonOHOHn onu mam doeuomum malm onu aw >uH>fiuom AmmV ommuous moaxoao ou AQZZMV mmmaznumammIZImawnauoaaznuo mo owumu man cmmBumn magmaOfiumamu one .wa madman 89 ma 3&3 >UZwUEmm OZ_ZO._U m 9 v m N >UZmDOw~E ZOZ.<.:.:2 O NOIlDVlIzl Sl-S 3H1 NI All/\IIDV H3/0NW3 9O .neonoao 28 o.c ca vocfiahouoo mos woo maaoo oanofi> non \mucouaa mo mooooucxo ma Amm.o n H .nulnuv monoavoum coauouaz .oouooancou oounu mo cooa onu uaomoucou mosno> .Houuaoo mo owmuaoouoc o no oommoucxo we Ammo: n H . ’IOV euaowoammo wcfinono .cfiououc malm ma\oudafia\aowoouoenfio odoueuo moHoac mo mooooucxo mo3 eufi>wuoo cm moo mcfiououc malm wa\ousaaa\nfimsuomou moHoBc mo oommoucxo mos hufi>fiuoo comm .oaouhcaovounon ou mHHou mn> mo omnocmou HoofiwOHOfin onu new sowuooum malm onu an eufi>wuuo Ammv omouoen oowxoco ou Acommv omocenuoowIOIcHmonomouexonuo mo oauou onu coosuon canoaowuoaou one .mH ouowfic 91 oH mudwfim >UZw_U_u_u_m OZ_ZO._U >UZmDOw~E 20.63.22 Doom 000.... 0000 000m 0000— 0009 NOIlDVlH Sl-S 3H1 NI AllAIlDV H3/002I] 92 5. Dimethylnitrosamine (DMN) The cytotoxicity of DMN in the absence of the S-15 fraction is illustrated in Table 20. The cytotoxicity produced when 100 mM DMN is titrated with various concentrations of S—15 protein is depicted in Figure 20. It is apparent that the observed cytotoxicity is biphasic in the presence of all five S—lS fractions. To determine if DMN-induced cytotoxicity and mutagenicity were related it is necessary to use a variety of treatments such that various numbers of cells survive each treatment and to measure the mutation frequency of each treatment in parallel with the cytotoxicity assessment. Toward that end, I chose to assay the cytotoxicity and mutagenicity of three different concentrations of DMN (10, 50, and 100 mM) in the presence of two different concentrations of S-15 protein (1.3 mg/ml and 3.5 mg/ml). This protocol was performed using S—15 fractions prepared from untreated rats as well as from rats pretreated with four different enzyme inducers (PB, Aroclor 1254, BNF, MC). After combining all of these data and plotting them on one graph (Figure 21), I conclude that the log of cloning efficiency is linearly related to mutation frequency (p<0.01) when DMN is metabolically activated by the S-15 fraction. DMN is metabolically activated by (a) mixed function oxidase(s) termed DMN demethylase. I wished to determine: A) whether DMN deme— thylase activity was correlated with EROD activity (i.e., P-448), EMND activity (i.e., P—450), or the ratio of EROD to EMND activity and B) whether DMN demethylase activity could be inhibited by MET and/or ANF. 93 TABLE 20 The Cytotoxicity of Dimethylnitrosamine in the Absence of the S-15 Fraction Dimethylnitrosamine (mM) Cloning Efficiencya 0 100:2.9b 10 96.8:6.3 50 101.4:2.4 100 87.3:3.l 3Expressed as a percentage of control. bMean i S.E. of 12 replicates. 94 Figure 20. Cytotoxicity of 100 mM dimethylnitrosamine in the presence of the S—15 fraction. S-lS fractions were derived from animals pre- treated with phenobarbital (C>-()), Aroclor 1254 (tr—t3), B-naphthofla- vone (H), 3-methy1cholanthrene (H) or from animals which had not been pretreated (**). Cloning efficiency is expressed as a percentage of control. Values represent the mean of three replicates. CLONING EFFICIENCY (%) I00 50 5 U! 95 A I I O O O 1 1 1 1 1 2 4 6 8 I0 mg S-IS PROTEIV . ml REACTION MIX. Figure 20 96 Figure 21. The relationship between cytotoxicity and mutagenicity when dimethylnitrosamine is incubated in the presence of the S—15 fraction. Cloning efficiency is expressed as a percentage of control. Values represent the mean of three replicates. Mutation frequency was deter— mined in 1.0 mM ouabain (r = 0.96). .- N 8 8 '8' MUIANT%06 SURVIVORS a C3 97 L I00 I I 50 IO CLONING EFFICIENCY (X) Figure 21 b 98 The experimental approach for this series of experiments was iden— tical with that previously described for studies on the metabolic acti- vation of BP (see BP results section). Since the concentration of S—15 protein in the reaction mixture markedly influences the cytotoxic response to DMN (Figure 20), all samples in this series of experiments contained equal amounts of S-15 protein (2.4:0.1 mg/ml reaction mix— ture), while the DMN concentration was always 100 mM. MET (Table 21) does not appear to affect DMN induced cytotoxicity or mutagenicity in a consistent manner in the presence of the S—lS fractions derived from PB or non-treated animals. However, when the S—15 fraction is derived from an Aroclor 1254 pretreated animal, MET decreases DMN induced cytotoxicity and mutagenicity in a dose-dependent fashion. In the presence of an Aroclor 1254 pretreated S—lS fraction ANF decreases DMN induced cytotoxicity, but only at 10_5M, the highest concentration tested (Table 22). Due to the extremely low cloning 6M ANF (Table 22) the mutation fre- efficiency in the presence of 10- quency determination at this concentration of ANF is probably not very accurate. In view of the fact that the activities of EROD and EMND vary sub— stantially in S-15 fractions derived from animals pretreated in various manners (Tables 2 and 3) and that MET and ANF affect these enzymes in a rather complex fashion (Tables 5, 6, 7, 8) it is difficult to draw definitive conclusions from Tables 21 and 22. The analysis which is required involves calculating the activity of a given enzyme in a given S-15 preparation including, in most cases, an inhibitor and then plotting 99 .ooeommo mHHoo moaxmcn .oomomoo mHHou moaxqmfi .ooeoooo mHHoo moaxon .ooeommo manoo moaxomw .oouoowacou m mo .m.m H coo2m .mpo>H>unm moH\oucouaa mo oomooucxco .Acc oav conunoo mo owounoonoc o mo vooooucxcw .ooamwuuom ou uoeuc Mn om ..c.H .wn\wa oomo .oouoouuouc no: ouo3 muom .oofimwuuom ou uowuc un «N mam mo .Nn .om ..c.w .wn\wa owo n .Amo.ovcv oowuoo CH muonuo Ham Scum ucouowwwo enuaooemeawwmke .Amo.ovcv uonuo nomo aouw uaoquMfio enucoowmwawamo no «2H.Haw.am nu «m.~aq.oa hon «m.oow.o Scion mm kkm.owm.on nm q.¢Hm.NN no m.oHH.N Zoloa HHH m.onm.m nm «o.cno.qq em «m.onw.m ZmIOH wqm N.on.H no q.mwo.- me mm.o+m.n o moaoowouc monoeofimmm eonoawoum hocoaowmmm oeononvouc eocofiuewmm coauouuaoonoo nowuousz wchoao coeuousz maenoao coauounz o waHaoao onocoueuoz comma Hoaooud nooosocelaoz odouanuonononc oousom mnlm ococoueuoz mo onoauouuaoocou wooeum> mo oocooouc onu CH oceaomouuficahnuoaac mo huHUHnowmunz woo euwowxououeo one am mcm moa\muaousa mo oommoucxo ow HHUV honoavoum cowuousz .mouoowccou oounu mo noofi onu uaoooucou mosno> .Houunoo mo owoucoouoc o mo oommouc Ixo ma ADV mocowuwwco wnwcoao .neououc 31m ws\ou=aaa\swmaomou ooHoEc mo commoucxo we muH>Huoo comm .onfiaomouuwcamnuoaeo ou mcaoo.mn> mo omnocmou Hooawoaown onu woo coauooum mHIm one aw muw>fiuuo Acommv omonmnuoocloucfimouooouexonuo noo3uon cfinmnoauoaou one .NN ouswfic 103 mm moswam >UZm_UIuw 02.20.; m o— On Go— . _ . _ mu.:o cu n. mm 0 D O D a. am m_ a. 1m —u+., _ _ _ _ _ Tub 3” 18 o. a. m e o >UZmDOw~I 20:55.2 1 9 COO— OOON 000m Doom coon NOIIDVEH SI-S 3H1 NI AlIAllDV 0083 104 .neonmno 28 o.c CH moafieuouoo moB woo manoo oHnoH> moc\oueou:a mo oommoucxo me Aan eoaoavoum cocoons: .mouooaacou oounu we come one uaomoucou mo=Ho> .Houunoo mo owounoouoc o oo womoouc Ixo m.“ AOV moaofioflcwo waenoao .ceououc malm wa\ou=afia\oo%nooaoauom ooaofid mo woomoucxo ma mufi>auoo czzm .ocfiaomouufiaamnuoaao ou maaoo mn> mo omcocmou HoUHwOHOHn onu new coauoouw mnlm one ea muw>fluoo Aczzmv omocmnuoaooIZIonfincuoaamnuo nooBuon canonowuoaou one .mN ouawwc 105 .I b ca mm muswam >u2w_u_&u 02205 n o. . _ e _ _ . h o. a. a >UZmDOm~E 20:<.5<‘ DC] U 0.— ON o.m 06. Qm NOIIDVEH SI'S 3H1 NI All/“13V CINW3 106 .caonoso Ea o.c cw moafiauouoo oo3 new macoo oHnoa> moH\mucou:E mo oommoucxo on Anuv mucosvoum coauouaz .mouooaacou oounu co coma one udomoucou mosao> .Houucoo we owmunoouoc o no oommoucxo m.“ AOV mocoHUmeo mafiaoau .cwououc mHIm wa\ousfina \oomnomnoehom moaoan mo commoucxo moB euH>Huoo czzm mam mefiououc mHIm wa\ousnfia\afimnu Iomou moaoac mo oommoucxo mos mufi>fiuoo comm .oafiaomouuficnenuoaeo ou mHHoo om> mo omnocm ion HoUfiwOHOHn onu mam nowuooum mHIm one nfi mufi>fiuoo Aczzmv omoaenuoaooIZIocanHoaahnuo Ou Acommv ommnmnuoooIOInHmonomoumxonuo we onuou onu noo3uon cfinmnowuoaou one .cm ousmwc 107 am .4634: >UZm_U_uEm 02.2040 m is. .um azu— d n A _ D U.‘ D D B 0 D O U 0 D P... I. ... .T _ _ _ em a om o. a. a v o >02m30mmm 20.52.22 l 000— Doom Doom 1 000.. 1 000m NOLDVEH Sl-S 3H1 NI AIIAIIDV ONW3 /C|02IS 108 Figure 25. The cytotoxicity of 2,4-dinitrofluorobenzene (2,4—DNFB). (0), treatment in complete medium; (0), treatment in phosphate- buffered saline; (I), treatment in phosphate-buffered saline in the presence of the S-15 fraction from a non—induced animals. Each point represents the mean i S.E. of three replicates. PERCENT SURVIVAL 109 1N —— I; I 10" 0.5 '- o.‘ I I I 0.01 0.1 0.5 1.0 2.4-DNFB In”) 110 To determine whether or not the enzymes responsible for the detoxi- fication could be induced I pretreated animals with PB or MC and com— pared the cytotoxicity of DNFB in the resulting S-15 fractions with that derived from untreated animals. As illustrated in Table 23 these pretreatments did not appear to increase the ability of the S—15 fraction to detoxify DNFB, suggesting that the enzymes which are in- duced by these pretreatments are either not involved in the metabolic detoxification of DNFB or are not rate limiting in this instance. I was unable to test DNFB concentrations greater than 500 uM due to problems in the solubility of the compound. B. Mutagenicity Studies The experiments designed to determine whether DNFB is a direct acting mutagen in V79 cells were conducted in growth medium. The results are shown in Table 24. At doses resulting in 11.5 and 35.5 percent survival DNFB did not significantly change the mutation fre- quency at the ouabain locus, whereas UV irradiation used as a positive control did elevate the mutation frequency. In order to determine whether DNFB requires metabolic activa- tion in order to become mutagenic experiments were performed utilizing MC induced, PB induced and non-induced S—lS fractions. The results indicate that DNFB was not metabolically activated to a mutagen by these S-lS fractions (Figure 26). Note that the positive control BP in the presence of S—lS prepared from MC pretreated rats did increase the mutation frequency in a dose-dependent fashion. 111 TABLE 23 Effect of Pretreatment with Enzyme Modulators on the Detoxification of 2,4-DNFB by the S-15 Fraction Enzyme Sourcea Cloning Efficiencyb c Untreated 50.0 uMd 101.6:12.6e 500.0 uM 63.6: 4.1 3-Methylcholanthrenef 50.0 pM 97.1:10.4 500.0 uM 68.1: 1.0 Phenobarbitalg 50.0 uM 101.3: 9.1 500.0 uM 56.9: 5.8 aS—15 fraction prepared as described in Materials and Methods. bExpressed as a percentage of control (no 2,4-DNFB). cAnimals were not pretreated. dConcentration of 2,4-DNFB in the reaction mixture. eMean i S.E. of 3 replicates. f80 mg/kg, i.p., 24 hr prior to sacrifice. 380 mg/kg, i.p., 96, 72, 48 and 24 hr prior to sacrifice. 112 TABLE 24 Mutagenicity of 2,4-DNFB in the Absence of the S—15 Fraction Cloning Mutatio Treatment Efficiencya Frequency Control 100.0C 0.9 (1)d 1.0 uM 2,4-DNFB 35.5 1.4 (l) 2.5 uM 2,4-DNFB 11.5 2.3 (1) UV light (183/112) 9.0 184.3 (68) 8Expressed as % of control (no 2,4-DNFB). bMutants per 106 viable cells. CMean of three replicates. d Number of mutants recovered. 113 Figure 26. The mutagenicity of 2,4—dinitrof1uorobenzene (2,4—DNFB) and benzo(a)pyrene (B(a)P). (O), 2,4—DNFB with 3-methylcholanthrene induced S-15; (II), 2,4-DNFB with phenobarbital induced S-15; (CD), 2,4-DNFB with non-induced S-lS; (Cl), benzo(a)pyrene with 3—methyl- cholanthrene induced S-15 as a positive control. Each point repre- sents the mean i S.E. of three replicates. mums PER 100 VIABLE CELLS 114 8(0)? (MM) 0 10.0 20.0 T I 10- 14.- 12- 10— + 1 I I 0 0.5 5.0 2.4-ours (um Figure 26 DISCUSSION 1. Preliminary Characterization of the V79/Ouabain Resistance Assay In order to develop this assay system it was necessary to verify that the V79 cells in my possession were capable of detecting cyto- toxic agents in a sensitive, dose-dependent fashion. MNNG is a direct acting alkylating agent which induces dose-dependent cytotoxicity in my V79 cells at uM concentrations (Figure 11). The inclusion of the hepatic S-15 fraction allows a quantitation of cytotoxicity and mutation frequency for many of those compounds which become active only after being metabolized to reactive species. The S-15 fraction is always prepared just prior to use because mixed function oxidase activity may deteriorate when subcellular fractions are frozen (-80°C) and stored (Yoshikawa_3£ 31., 1980; Dent 3£_31,, 1981). At the time the S-15 fraction is being added to the reaction mixture an aliquot is taken for the determination of AHH activity (Table 1) and protein content. AHH activity is assessed to be certain that the animals have been pretreated prOperly and that the microsomal mixed function oxidase activity has not deteriorated during preparation of the S-15 fraction. 1 have chosen to assess the cytotoxic and mutagenic action of the test compounds following a two hour incubation period. It is probable 115 116 that at earlier time points (e.g., 5 minutes) the results would be different from those obtained after a two hour incubation due to pharma- cokinetic differences between various enzymes. Since the vast majority of xenobiotics must be metabolized 13_y1yg_prior to excretion it is appropriate to allow all of the xenobiotic introduced into the test system to be metabolized prior to stopping the reaction. When employing a two hour incubation it appears as if all of the xenobiotic is metabo- lized because if one increases the concentration of test compound a greater degree of cytotoxicity and mutagenicity is observed. Exposure of cells to xenobiotics is conducted in PBS to eliminate interaction of the test compound or its metabolites with constituents of the growth medium (e.g. serum proteins). Damage to DNA results in an inhibition of replication. The degree of inhibition is usually positively correlated with the amount of damage initially produced (Painter, 1978). In the assessment of mutagenicity at the Na+,K+-ATPase locus by measuring resistance to ouabain, it is desirable to add the ouabain-containing medium to the cells after allowing them to undergo an equal number of DNA replications regardless of treatment toxicity. This is important because the cells must replicate their DNA in order to convert a DNA adduct into a mutation. To accomplish this I monitor the cells with an inverted microscope and add ouabain—containing medium after the cells have divided four times (16 cell stage). The sponta- neous mutation frequency is <0.05 ouabain-resistant mutants per 105 viable cells (29.3x105 viable cells assayed, zero mutants recovered). I have investigated the ability of hepatic S-15 fractions to generate mutagenic and/or cytotoxic metabolites from BP, DMN and DNFB 117 13,21353, In an attempt to identify enzyme patterns which are optimal in this regard the enzyme pattern contained in the S-15 fraction is modulated by pretreating the animals with enzyme inducers. Some of the chemicals employed to influence hepatic enzyme activity such as PB (Peraino 33 31,, 1971), MC (Miller 33 31., 1958) and PCBs (Makiura 33 31,, 1974) have a protective effect on chemically—induced hepatocar— cinogenesis. There are several probable reasons for this: A) fewer reactive metabolites may be produced from a precarcinogen due to quali- tative changes in the manner in which it is metabolized (Miller 33 31., 1958). B) the reactive metabolites may be conjugated and excreted at a faster rate (Degen and Neumann, 1981; Chipman 3£_31,, 1981). C) the reactive metabolites may be detoxified at a faster rate (Leonard 33 31,, 1981). The protective effect might not be reflected in the results obtained from 13331££3_mutagenicity assays using hepatic sub- cellular fractions to activate chemical carcinogens (Stout and Becker, 1979). It must be recognized that we are testing for the potential to metabolize a putative mutagen/carcinogen to a reactive species which may or may not be identical with the net effect in an intact organ. That is, in an 13ly1££3_assay we are dealing with only portions of the pharmacodynamic picture present in an intact, functioning organ, and this fact must be kept in mind when interpreting results from 13_y13£9_ assays. However, by characterizing the metabolic parameters which are operational in 13:33££3_assays and understanding how they may influence the results obtained it should be possible to conduct meaningful mechanistic studies on the potential for metabolic toxification/detoxi- fication of xenobiotics. These studies represent a step in this direction. 118 2. Benzo(a)pyrene (BP) Concentrations of BP which are non—toxic in the absence of metabo- lism have been defined (Table 13). A non-toxic concentration of BP (20 uM) was selected, various concentrations of S—15 protein were added, and the production of toxic metabolites was assessed by measuring cyto— toxicity in V79 cells (Figure 12). The shape of the toxicity curve is dependent upon the source of the S-lS fraction. When the S—15 fraction is prepared from control animals I do not observe toxicity in the V79 cells. This could be due either to the inability of this preparation to generate toxic metabolites from BP, or alternatively it could be that this S-15 fraction has the ability to detoxify those toxic metabo- lites which are generated prior to their interaction with the V79 target cells. When the S—15 fraction is prepared from animals which have been pretreated with MC, a biphasic toxicity curve is produced. At low concetrations of S-15 protein (<0.8 mg S-15 protein/ml reaction mixture) an increase in S-15 protein concentration produces an increase in cytotoxicity, whereas at higher concentrations of S-lS protein (>0.8 mg S—15 protein/m1 reaction mixture) an increase in S-15 protein con- centration results in a decrease in observed cytotoxicity. There are two probable explanations for this biphasic effect: 1) the excess pro- tein may be acting as a nucleophilic trap for the activated metabolites of BP, preventing them from interacting with V79 cells (Malaveille 33 31,, 1979; Guenthner 3£_31,, 1979; Bartsch 35 31., 1980), or 2) the pattern of enzymatic activity present in the reaction mixture may shift from a pattern favoring the production of toxic metabolites from BP to a pattern of enzyme activity which is less favorable for the production 119 of toxic metabolites as the S—lS protein concentration is raised (Kuroki 35 31., 1979; Machanoff 3£_31,, 1981). In an attempt to differentiate between these two alternatives I have taken a fixed concentration of active S—15 protein and added to it various concentrations of inactive S-lS protein (Figure 12). The S—15 fraction was inactivated by heating to 100°C for 10 minutes. The results indicate that protein binding is an important detoxification mechanism, but at high concentrations of postmitochondrial supernatant it appears as though a shift in the pattern of metabolites produced occurs. This conclusion is based on the observation that the active S-15 does not produce detectable toxicity when present in high concen- trations, whereas the inactivated S—15 is unable to completely abolish the cytotoxic response to activated BP. To further investigate the interrelationship of S-lS protein concentration and cytotoxicity, S—15 fractions have been prepared from rats which have been pretreated with compounds known to modulate hepatic enzyme activity. The ability of such preparations to influence BP-induced cytotoxicity at several protein concentrations was assessed. I prepared the S-15 fraction from animals pretreated with a P-450 type inducer (PB) (Conney, 1967), a P—448 type inducer other than MC (BNF) (Boobis 3£_31,, 1977) and a mixed type inducer (Aroclor 1254) (Alvares_3£ 31., 1973; Parkinson 33 31,, 1980). In all three instances I observed BP concentration depen- dent cytotoxicity which was markedly influenced by the concentration of S—lS protein employed (Table 14). In accordance with previous results (Kuroki 33 31,, 1979; Malaveille 33 31., 1979) I have observed that the mutagenicity of BP is dependent upon the concentration of 120 postmitochondrial supernatant protein when the homogenate is derived from an animal which has been pretreated with MC (Table 15). The degree of mutagenicity and cytotoxicity generated from BP by metabolism depends on the relative levels of individual BP metabo- lites. This is due to the fact that the degree of cytotoxicity and mutagenicity induced in V79 cells by individual BP metabolites varies substantially (Huberman 3£_31,, 1976). The levels of the individual metabolites which are generated from BP depends on the pattern of enzy- matic activity present in the metabolic activation system. Therefore, I wished to investigate the role of specific enzymes and enzyme ratios in regard to the metabolic activation of BP to cytotoxic and mutagenic entities. High inducibility of AHH in humans has been associated with the development of lung cancer (Kellermann.3£_31,, 1973; Emery 33 31., 1978), which is believed to be initiated primarily by polycyclic aroma- tic hydrocarbons present in cigarette smoke. AHH activity in the post- mitochondrial supernatant prepared from rat liver has been reported to correlate with the ability of this fraction to metabolically acti- vate BP to compounds which bind to calf thymus DNA 13,y1££3_(Raineri 35_31,, 1981). However, AHH activity did not correlate with the amount of BP bound to DNA in an intact cell system (Gozukara 3£_31,, 1981). In order to study the effect of AHH induction on the metabolism of BP, I have induced hepatic AHH activity to equivalent levels (approxi— mately 300% of control, Table 1) using two different inducing agents (MC and BNF) and assessed the effect of this induction on cytotoxicity and mutation frequency in the presence of BP (Figure 13). Pretreatment 121 with MC or BNF leads to the appearance of a new hepatic microsomal protein band (presumably P-448) at the same molecular weight (55,000) in AHH responsive mice (Wang, 1981). I found that at any given level of cytotoxicity these two pretreatments produced differing mutation frequencies. These results indicate that the cytotoxic metabolites of BP may not be identical with the mutagenic metabolites. This conclu- sion is supported by the observation that when hamster or rat embryo cells were used as feeder cells to metabolically activate BP to com— pounds which are mutagenic and/or cytotoxic to V79 Chinese hamster cells, these two biological responses were not a constant ratio when the two cell types were compared (Baird 33 31., 1981). At equal toxi— city levels hamster embryo cells produced 3 times as many mutations in V79 cells as did rat embryo cells. The comparison between MC and BNF (Figure 13) also indicates that AHH activity is not the sole determinant for the generation of mutagenic metabolites from BP, confirming previous results using hepatic sub— cellular fractions from mice (Hutton 3£_31,, 1979a), hamsters (Hutton 33 31., 1979b), and guinea pigs (Baker 33 31., 1980). These data may be explained by the observation (Gurtoo 35_31,, 1980) that AHH activity as measured by the standard fluorometric assay (Nebert and Gelboin, 1968) does not correlate with the production of benzo(a)pyrene-7,8- dihydrodiol, the proposed proximate mutagenic form of BP (Huberman 33 31,, 1976). I have investigated the effect of AHH activity on the generation of toxic metabolites from BP by comparing the ability of the S—15 122 fractions prepared from control and MC pretreated animals to metabolize BP to toxic metabolites (Figure 12). Since the AHH activity/mg S—15 protein in MC pretreated S—15 is approximately 3 times that of control S—15 (Table 1) one may make the AHH activity/m1 reaction mixture equal for these two preparations by incorporating 3 times as much non-treated S—15 protein into the reaction mixture as compared to MC S-15 protein. The extra protein in the non-treated S-lS preparation would be expected to act as a nucleophilic trap for the activated, electrophilic BP metabolites responsible for cytotoxicity (Figure 12). Nevertheless, S-15 preparations derived from animals pretreated with MC, BNF, Aroclor 1254 or PB induce a significant degree of cytotoxicity at this concen- tration of S—15 protein (Figure 12, Table 14). However, the S-15 fraction prepared from control animals is still ineffective in metabo- lizing BP to compounds cytotoxic to V79 cells, indicating that the AHH activity contained in the reaction mixture is not the sole determinant for the induction of V79 cytotoxicity in the presence of BP. There are two probable reasons for this: A) the AHH assay does not detect the metabolism of BP to non-phenolic products (Yang 33 31., 1975) such as diolepoxides, which are capable of killing V79 cells (Huberman 33 31,, 1976). B) AHH activity in rat hepatic microsomes appears to be composed of two or more enzymes (Wiebel 33 31., 1971). The relative amounts of these enzymes in hepatic subcellular fractions vary depending on the type of inducer used (Wiebel and Gelboin, 1975). Therefore, even though "AHH activity" is equalized in two subcellular fractions, the enzymes which comprise "AHH" are probably not identical in the two preparations. 123 To examine the role of other enzymes in the metabolic activation of BP I chose to employ ANF, an inhibitor of cytochrome P-448, MET, an inhibitor of cytochrome P-450, and CCHO, an inhibitor of epoxide hydrase. The rationale for this approach was that, by inhibiting spe- cific enzymes in the S-15 fraction to various degrees and concurrently measuring the ability of these fractions to metabolically activate BP, it might be possible to elucidate the relative importance of specific enzymes and/or enzyme patterns. ANF is a synthetic flavonoid which acts as an 13:31353 inhibitor of cytochrome P-448 (Wiebel and Gelboin, 1975). When employed 13,31333 ANF inhibits primary (Selkirk 3£_31,, 1974) as well as secondary (Capdevila 3£_31,, 1975; King 3£_31,, 1976) oxidations of BP by micro- somal enzymes. In the presence of MC induced mouse liver microsomes and DNA ANF decreases the concentration of all DNA-BP adducts with the exception of the adduct formed from BP 4,5-oxide (Boobis 3£_31,, 1979). This indicates that the metabolism of BP to the 4,5-oxide is mediated by an ANF insensitive form of cytochrome P-450. The data in Table 5 illustrate that ANF inhibits EROD activity in a concentration dependent manner in S-15 fractions derived from animals pretreated with MO or Aroclor 1254, but does not significantly affect EROD activity in non-induced S-lS fractions. This result confirms and extends the observation that MC inducible AHH activity is inhibited by ANF, whereas AHH activity in a control animal is not inhibited by ANF (Wiebel 35 31., 1971). A possible reason for this specificity is that ANF may require metabolic activation (Nesnow and Bergman, 1981), probably at the number six position, before it is capable of inhibiting 124 AHH activity. This possibility is supported by the observation that hepatic microsomes from MC pretreated Sprague-Dawley rats metabolize ANF to the 5,6—epoxide, which inhibits AHH activity (Coombs 3£_31,, 1981). Uninduced microsomes may be unable to perform this oxidation. Further support for this hypothesis comes from the observation that, in order for ANF to have an inhibitory effect oanC induced microsomal drug oxidations, the number six position on the ANF molecule must be either unsubstituted or be substituted with an oxidizable moiety (Nesnow, 1979). ANF inhibits BP-induced cytotoxicity and mutagenicity in the pre- sence of a MC pretreated S-15 fraction, but does not alter BP-induced cytotoxicity or mutagenicity in the presence of the Aroclor 1254 pre- treated S-15 fraction (Table 17). This result is possibly due to the fact that EROD activity in an Aroclor 1254 pretreated animal is sub- stantially higher than in a MC pretreated animal (Table 2). Therefore, the EROD activity in the Aroclor preparation, even though significantly inhibited by ANF, may not have become rate limiting. Another possi- bility for the differential effect of ANF on BP activation is the fact that EH activity in an Aroclor 1254 pretreated animal is substan- tially higher than in a‘MC pretreated animal (Table 4). MET is an inhibitor of cytochrome P-450 which has been reported to have little effect on the BP metabolite-nucleoside profile produced by MC induced mouse liver microsomes (Boobis 33 31., 1979). MET inhi— bits EMND activity (Table 7) and EROD activity (Table 6) in a concen— tration dependent manner and, in accordance with previous results 125 (Vaz 33 31., 1981), stimulates EH activity (Table 10). .MET has no effect on the cytotoxicity and mutagenicity of BP when present at low concentrations, but at the highest concentration used (10-3M) MET increased the toxicity and mutagenicity of BP (Table 19). EH is a ubiquitous cellular enzyme found on nuclear as well as microsomal membranes, in mitochondria and in the cytosol (Gill and Hammock, 1981). I chose to examine the role of EH in the metabolic activation of BP because the majority of BP metabolites either are arene oxides or have arene oxides as intermediates. Selective inhibi- tion of EH by TCPO increases the mutagenicity of BP (Oesch, 1976; Rasmussen and Wang, 1974). However, these results must be interpreted with caution because TCPO itself is a direct acting mutagen (Guest and Dent, 1980). Strong evidence for a role of EH in the metabolic toxi— fication/detoxification of BP comes from the observation that the addition of purified EH decreases the mutagenicity of BP in the Ames assay (Oesch, 1976) and reduces the binding of 3H(-)t—7,8-diol metabo— lites to DNA in either purified reconstituted mixed function oxidase systems or intact cells (Gozukara 33 31., 1981). CCHO is an inhibitor of EH and has been reported to inhibit the formation of BP dihydrodiols in the presence of rat liver microsomes (Fahl 33 31., 1977). CCHO markedly potentiates the observed cytotoxicity and mutagenicity of BP in the presence of either a'MC or Aroclor 1254 pretreated S—15 fraction (Table 18). This result provides circumstantial evidence that the toxic and mutagenic metabolites of BP are epoxides. 126 In view of the fact that the activities of EROD, EH and EMND in the S-15 fraction vary substantially depending on the type of inducer used (Tables 2, 3, 4) and that the activities of these enzymes are altered in a rather complex fashion by inhibitors (Tables 5, 6, 7, 8, 9, 10), it is difficult to draw definitive conclusions about the meta— bolic activation of BP from the results of experiments in which a single S-15 fraction is combined with various concentrations of a single inhibitor. Metabolism of BP involves several enzymes acting both sequentially and in concert. The interrelationships between toxifica- tion and detoxification pathways is crucial in determining the levels of reactive intermediates available to interact with cellular macro— molecules. Therefore, in the study of BP metabolism patterns of enzyme activity should be considered in addition to studies on single enzymes. The required analysis involves pooling the data from multiple experiments using several different S—15 sources and several different inhibitors. One may then look for correlations between a particular enzyme or enzyme ratio in the S-15 fraction and the ability of the fraction to metabolically activate BP. This type of analysis indicates that EH is the only single enzyme to correlate significantly (p<0.0l) with BP induced cytotoxicity and mutagenicity (Figure 17). As the EH activity in the S-15 fraction increases the degree of cytotoxicity and mutagenicity which results when BP is metabolically activated by the S—15 fraction decreases. BPDE I is the most toxic and mutagenic metabolite of BP but BPE is also relatively cytotoxic and mutagenic (Huberman 3£_31,, 1976). Although the BPDEs are relatively poor sub— strates for EH (Wood_3£_a1., 1976b), they are detoxified to BP 127 7,8,9,10-tetrols by EH at some low rate. This is supported by the observation that in a purified, reconstituted mixed function oxidase system the addition of purified EH reduces the binding of 3H(—)t-7,8- diol metabolites to DNA (Gozukara 33 31., 1981). Presumably, as EH activity increases the rate of BPDE detoxification also increases, resulting in less V79 toxicity and mutagenicity. In addition, BPE may be detoxified by EH (Woood 33 31,, 1976a), presumably to the 4,5—diol. Therefore, although the relative roles of the BPDEs and BPE in inducing cytotoxicity and mutagenicity in this assay has not been determined, both of these reactive intermediates would be expected to be detoxified by EH. When I look at enzyme ratios I find two significant correlations (p<0.01), EMND/EH (Figure 18) and EROD/EH (Figure 19). This suggests that an important determinant of BP induced toxicity and mutagenicity is the rate of epoxide formation divided by the rate of epoxide destruc- tion. As the rate of epoxide formation increases and/or the rate of epoxide destruction decreases BP becomes more cytotoxic and more muta- genic. These data support the role of epoxides in mediating the cyto- toxic and mutagenic actions of BP. EMND/EH does not correlate as strongly with BP induced cytotoxicity and mutagenicity as does EROD/EH Since EMND activity measures 3 PB inducible form of P-450 (Table 3) and EROD activity measures a MC inducible form of P-450 (Table 2) this result confirms and extends previous findings (W00d e£_a1., 1976a) that MC inducible mixed function oxidases are better able to activate BP to a mutagen than are PB inducible mixed function oxidases. 128 3. Dimethylnitrosamine (DMN) The cytotoxicity curve which results when 100 mM DMN is titrated with various amounts of S-lS protein has a biphasic shape, regardless of whether the S-lS fraction is derived from an untreated rat or from one which has been pretreated with MC, BNF, Aroclor 1254 or PB (Figure 20). Maximal toxicity is seen between 2.1 and 3.5 mg S-15 protein/ml reaction mixture, depending on the pretreatment used. The S—lS fraction which has the highest EMND activity (PB, Table 3) pro— duces the greatest degree of cytotoxicity from DMN at all concentra- tions of S-15 protein. When the concentration of S-15 protein is 3.5 mg/ml or greater, it is apparent that an increase in the amount of S-15 protein produces a decrease in observed cytotoxicity. In view of the currently accepted hypothesis that the metabolic activation of DMN to a cytotoxic entity is due to a single mixed function oxidase catalyzed reaction, one would anticipate that the greater the amount of mixed function oxidase (i.e. S—lS fraction) in the reaction mixture, the greater the rate of DMN activation and the greater the amount of DMN induced cytotoxicity. Since DMN is less cytotoxic in the presence of large amounts of S-15 protein when compared to intermediate amounts, the excess protein is presumably acting as a nucleophilic trap for the DMN metabolite responsible for cytotoxicity. The data depicted in Figure 21 illustrate that cloning efficiency is linearly related to mutation frequency when DMN is metabolically activated by the S-15 fraction. This indicates either A) the cytotoxic metabolite of DMN is identical with the mutagenic metabolite or B) the cytotoxic and mutagenic metabolites of DMN are not identical, but are 129 produced from DMN by microsomal enzymes in a constant ratio. A high pressure liquid chromatography analysis of the metabolite profile in the reaction mixture would be useful in distinguishing between these two possibilities. The metabolic activation of DMN is currently believed to be cata— lyzed by mixed function oxidase(s) termed DMN demethylase (Czygan 35 31,, 1973). It is unclear whether DMN demethylase is a P—450 type enzyme, a P—448 type enzyme or is comprised of several mixed function oxidases, including both P-450 and P-448. Therefore, I wished to determine if DMN demethylase activity could be correlated with EROD activity, a reaction catalyzed by P-448, with EMND activity, a reaction catalyzed by P—450, or with the ratio of EROD activity to EMND acti— vity, which reflects the P-448/P—450 ratio. This study was facili— tated through the use of the 13_y1££3_enzyme inhibitors MET and ANF. ANF inhibits primarily P—448 (Tables 5, 8), whereas MET inhibits primarily P—450 but also has a significant inhibitory effect on P-448 (Tables 6, 7). The results of this study (Figures 22, 23, 24) indi- cate that the cytotoxicity and mutagenicity of DMN neither correlate with EROD activity or with EMND activity nor with the ratio of EROD to EMND activity. There are several probable explanations for these results: A) The mixed function oxidases whose activities are measured by the EROD and EMND assays and/or inhibited by ANF and MET are not the enzymes which metabolically activate DMN to toxic and mutagenic forms. For example, pretreatment of rats with isopropanol, acetone or ethanol increases the hepatic microsomal demethylation of DMN, but 130 does not significantly increase EMND activity (Maling 3£_31,, 1975). B) The mixed function oxidases which are inhibited by MET or ANF are responsible for the metabolic activation of DMN, but these enzymes, even when substantially inhibited by MET or ANF, are present in such high concentrations that they do not become rate limiting. C) The metabolic activation of DMN may be catalyzed by any one of several mixed function oxidases. When one or more of these enzymes are inhibited by MET or ANF, other forms of cytochrome P-450 are able to compensate with little or no change in the rate of DMN acti— vation. 4. 3,4-Dinitrofluorobenzene (DNFB) DNFB has been shown to be a potent and presumably direct acting mutagen in several species of bacteria (Hope, 1979; Summer and Goggel- mann, 1980; Jagannath 3£_31,, 1980; warren 33 31., 1981). However, 13hy1yg_studies performed on mouse skin have indicated that DNFB is not a complete carcinogen, but rather acts as a tumor promoter (Bock 3£_31,, 1969). To investigate this apparent discrepancy I chose to determine if DNFB is mutagenic in V79 cells. The results indicate that DNFB is extremely cytotoxic (Figure 25), but not detectably mutagenic (Table 24) in V79 Chinese hamster cells in the absence of the S-15 fraction. The addition of the S-lS fraction markedly reduces the cytotoxicity produced by DNFB (Figure 25). DNFB is not metabolically activated to a mutagen by the S—15 fraction as assessed by measuring ouabain resistance (Figure 26). 131 In additional studies employing V79 Chinese hamster cells, DNFB either in the presence or absence of the S-15 fraction was not muta- genic at the HGPRT locus as assessed by resistance to 6-thioguanine, did not cause unscheduled DNA synthesis, a measure of DNA excision repair, nor did it increase the frequency of sister chromatid ex- changes, which is another indicator of DNA damage (Warren 33 31., 1981). Taken together these results suggest that neither 2,4-DNFB or its microsomal metabolites damage DNA in V79 cells. Therefore, there are qualitative differences between bacteria and V79 cells in terms of the mutagenic response to DNFB. Two probable explanations may be proposed for the discordance between bacterial and mammalian cells. Since DNFB binds covalently to amino acids and proteins (Sanger, 1945) thus presumably leading to cytotoxicity, the relatively greater number of intracellular barriers to the DNA in mammalian cells (i.e., nuclear membrane, histones, etc.) as compared to bacteria may not allow DNA binding while maintaining viability. The survival data of DNFB treated cells indicate that proteins do provide a protective effect from cytotoxicity (Figure 25). Second and perhaps more likely is the possible existence of bacterial enzymes, which are lacking in mammals, that are capable of metabolizing DNFB to a mutagenic form. This possibility is supported by the existence of bacterial nitroreductases (Blumer EE“§£°’ 1980) essential for niridazole mutagenesis which are not found in mammalian liver homogenates. If this possibility were true, then one could speculate that DNFB may be a complete carcinogen in mammals when ingested orally and subsequently metabolized by intestinal bacteria, a situation 132 previously described for other compounds (Batzinger EE”§1°: 1978). These data emphasize the need for caution when using data derived from bacterial systems for use in human risk assessment. 5. Significance Most reports in the genetic toxicology literature dealing with short-term mutagenicity assays have addressed the qualitative question: Can a particular chemical be metabolically activated to a mutagen? The 13_y1££3_mammalian assay system developed and characterized in this thesis has advanced the field of genetic toxicology because in addition to answering the qualitative question posed above it may be used to elucidate the relative importance of specific enzymes and/or enzyme ratios in the metabolic toxification/detoxification of a chemi- cal. To accomplish these objectives the enzyme pattern contained in the hepatic S-15 fraction is modulated in two manners: 1) the experimental animals are pretreated with compounds known to alter hepatic enzyme activities and 2) selected enzymes in the S-15 fractions prepared from these animals are inhibited to various degrees 13_y1££3, The enzymic activity and pattern in a given S-lS fraction is determined, and the ability of the preparation to produce mutagenicity and/or cyto— toxicity in the presence of a particular xenobiotic is assessed. Prior to conducting the mutagenicity and cytotoxicity assays aimed at elucidating the importance of specific enzymes in the meta- bolic toxification/detoxification of a chemical three initial proce- dures must be performed: 1) the cytotoxicity of various concentrations 133 of the test chemical in the absence of metabolism must be determined, 2) the cytotoxic response to fixed concentration of the test chemical in the presence of various concentrations of S—15 fraction must be determined. The concentration of test chemicals employed in this procedure should be the highest concentration which is non—toxic in the absence of metabolism. The procedure should be performed under four conditions: A) with the S-15 fraction derived from animals pre— treated with a P-450 type inducer (e.g., PB); B) with the S-15 fraction derived from animals pretreated with a P-448 type inducer (e.g., MC); C) with the S—15 fraction derived from animals pretreated with a mixed type inducer (e.g., Aroclor 1254); and D) with the S-15 fraction from animals which have not been pretreated. This initial experiment is very important because the degree of observed cytotoxicity is biphasic and dependent on the type of pretreatment employed. At lower concentrations of S-15 protein an increase in the amount of S-15 fraction increases the degree of cytotoxic response, while at higher concentrations of S-15 protein an increase in the amount of S-15 fraction decreases the degree of cytotoxic response. The concentration of S-15 fraction which induces maximum cytotoxicity should be selected for use in the metabolic activation studies. The third of the orien- ting experiments is performed after a concentration of S-15 fraction has been selected and is aimed at assessing the effect of lowering the concentration of test chemical on cytotoxicity. The S-15 fraction which is most active in metabolizing the test chemical to a cytotoxic form should be employed in this procedure. The concentration of test chemical chosen for use in the metabolic activation studies should be 134 the concentration which produces a cloning efficiency of approximately 5%. This will allow an accurate assessment of cytotoxicity and muta- tion frequency. Once selected the concentration of test chemical and the amount of S-15 fraction in the reaction mixture must be maintained at a constant level in all cytotoxicity and mutagenicity assays. Correla- tions are made relating the activity of a particular enzyme or enzyme ratio in the S-lS fraction to the cytotoxicity or mutagenicity of the test chemical in the presence of the S-15 fraction. This is done graphically by plotting enzyme activity on the ordinate and biological response on the abscissa. Two correlation coefficients (r) are deter— mined, one relating enzyme activity to cytotoxicity and the other relating enzyme activity to mutagenicity. The significance of r is estimated from the expression: t = £§é§EZl-(N is the number of samples) The null hypothesis tested in this way is that r estimated from the sample represents a true correlation coefficient of zero. If the apparent correlation is real (p<0.05), the best fitting linear re- gression line is calculated and the strength of the correlation (r2, fraction of total variability due to regression) is determined. When BP is utilized as the test compound the degree of cytotoxi- city and mutagenicity observed in V79 cells was neither correlated with EROD or EMND activity, nor with the ratio of EROD activity to EMND activity in the S—15 fraction. On the other hand, there were significant correlations between the degree of BP activation, as 135 assessed by V79 cytotoxicity and mutagenicity, and EH activity, the ratio between EMND activity and EH activity, as well as the ratio of EROD activity to EH activity. The latter two findings indicate that the rate of epoxide formation divided by the rate of epoxide destruc- tion is an important determinant of the degree of BP induced cytotoxi- city and mutagenicity when it is metabolized 13_y1grg, As these ratios increase BP becomes more cytotoxic and more mutagenic towards V79 cells. In light of the fact that EH activity in the S-15 fraction correlates with the degree of cytotoxicity and mutagenicity produced by RP whereas EROD activity and EMND activity do not, the rate of epoxide destruction is more important than the rate of epoxide forma- tion in determining the biological response to BP in this 13_y1££p_ system. As EH activity in the S-15 fraction increases the degree of cytotoxicity and mutagenicity observed as a result of metabolism de— creases. These results verify that the experimental approach described in this thesis will allow one to elucidate the relative importance of various enzymes and/or enzyme patterns in the metabolic toxifica- tion/detoxification of a potentially genotoxic chemical. The results reported in this thesis also shed light on a discrep- ancy in the genetic toxicology literature regarding the carcinogenic potential of DNFB. Although DNFB is a potent bacterial mutagen, it does not initiate tumors when applied to mouse skin but rather acts as a tumor promoter at this site. According to the widely accepted two stage theory of chemical carcinogenesis initiators would be expected to be mutagenic whereas tumor promoters would not be expected to be mutagens. 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