Document VJQjzN4b4g2vkmyaXoGg0gbRZ

THE DOW CHEMICAL COMPANY February 24, 1976 BENNETT BUILDING 2030 DOW CENTER midland, Michigan wmo mar i 1976' Mr. Hilton Freifeld Manufacturing Chemists1 Assoc. 1825 Connecticut Ave., K.W. Washington, D.C, 20009 MEDICAL SYCS, Dear Mr. Freifeld: The attached protocol dated February 5, 1S76 has been prepared as an extension of the metabolic studies coordinated by the Panel. You may recall that at the time the previous metabolic studies were reported, the Dow group thought it inappropriate to recommend further studies since there was no obvious next step to be taken. The Dow Toxicology Research Group now feels they have a reasonable approach to studying metabolism. Therefore I request that a letter ballot be sent to The Technical Panel to determine their interest in re-establishing support for these proposed studies. The committee is aware, I*m sure, how valuable the metabolism data has been in discussing thresnolds with tns government agencies. The metabolic laboratory would like a rather prompt response so that they cah start "these studies or direct there interest to other problems. The members should recall that the Panel is also considering research at the University of Louisville. It is also possible that we may wish to consider various types of human epidemilogical studies since there appear to be rather important. Sincerely yours, T. R. TorkelscnChairman, MCA Tech. Panel on Vinyl Chloride Research dbg cc: MCA Technical Panel on Vinyl Chloride Research Attachment ASl 000l5252 PROTOCOL FOR CONTINUED STUDIES ON THE METABOLISM OF VINYL CHLORIDE Prepared for the companies sponsoring The Technical Task Group on Vinyl Chloride Research February 5, 1976 Toxicology Research Laboratory Health and Environmental Research Dow Chemical U.S.A. Midland, Michigan 48640 AS I 0000152 I. Introductory Summary This document was prepared in response for the need to con duct additional studies on the metabolism of vinyl chloride (VC) in order to assess more definitely the hazard of low level exposure to VC. Numerous studies on the pharmaco kinetics and metabolism of VC both in experimental animals and in vitro have indicated that the carcinogenesis of VC may be mediated by the metabolic formation of reactive metabolites. It appears that several metabolic pathways, dependent on the dose or exposure concentration, function in the biotransformation of VC in the body. Integration of the available information on the metabolism of VC has led to the theory that the carcinogenicity of VC is mediated by the reaction of metabolites of VC with intracellular macro molecules (DNA, RNA and protein). Although the fate of VC has been extensively studied, little information is avail able on the actual carcinogenic lesion and its relationship to metabolism. This proposal describes a series of studies designed to characterize the potential in vivo macromolecular binding of 14 C-labeled VC in the liver of rats exposed by inhalation to 000015254 AS I -2varying concentrations of VC. Parallel studies inves tigating in vitro metabolism and binding properties will also be conducted. If these initial studies are successful in elucidating 1) an effect consistent with induction of carcinogenesis by VC and 2) the primary metabolic pathways of VC biotransformation, then through additional studies it may be possible to relate the dose dependent metabolism of VC to carcinogenesis. The proposed project on the metabolism of VC will draw on the broad spectrum of resources of The Dow Chemical Company. Primary responsibility for the project will lie with the personnel of the Toxicology Laboratory, located in Midland, Michigan. The research will be under the direction of Dr. Perry Gehring, Director of the Toxicology Research Lab oratory; and Dr. Philip Watanabe, Senior Research Toxi cologist. The Toxicology Research Laboratory has had extensive experience with VC (Hefner, et al^, 1975, 1976; Wagner et al., 1975; Watanabe et al., 1976 a,b,c,d. The present studies are an extension of the previous work. AS I 000015255 -3- II Technical Proposal 1. Introduction Considerable effort has been devoted to research on VC since Creech and Johnson (1974) first associated the induction of liver disease and neoplasias in industrial workers exposed to VC. Studies in this laboratory have demonstrated that the fate of VC in the body following both ingestion and inhalation is dependent on the administered dose or exposure concentration (Watanabe et al., 1976a,b). These results are consistent with earlier studies (Hefner et al., 1975) which suggested that at least two metabolic pathways may be involved in transformation of VC in the body. Consider ation of these results in toto led to the hypothesis that at low doses VC may be detoxified innocuously and as the dose increases the primary detoxicating metabolic pathway becomes saturated. When this occurs another pathway(s) become operant and ultimately form reactive metabolites which react with macromolecules leading to the development of cancer. This hypothesis is consistent with studies showing an en hanced mutagenic response in certain studies of Salmonella typhimurium and E. coli if fortified liver homogenates or microsomal enzymes are present (Bartsch et^ al.., 1975; Malaveille et al., 1975; Rannug et al., 1975; Greim et al., 1975). ooooi^ 2&0 -4- Many alkylating chemicals are detoxified in the body by conjugation with nonprotein sulfhydryl groups (primarily glutathione, GSH, Mitchell et a^, 1973; Gillette, 1974) . Urinary metabolites of VC appear to be primarily conjugates of cysteine. This indicates that covalent binding of reactive VC metabolites with hepatic GSH is a major detoxi fication mechanism. Furthermore, significant dose-related reduction of hepatic glutathione content occurred in rats exposed to 50-2000 ppm VC for 7 hours. In rats exposed for 7 hours to 10 ppm, no depression of hepatic GSH content was observed (Watanabe et al., 1976). It is reasonable to expect that as the GSH content of liver is decreased a larger fraction of the reactive metabolites are binding to intracellular macromolecules (protein, DNA and RNA) rather than to GSH. If it can be demonstrated that metabolites of VC interact in vivo with protein and nucleic acids which are responsible for cellular proliferation, and this interaction is a function of the dose dependent metabolism, then such results would be consistent with predicting a dispropor tionate increase in toxicity including cancer as the ex posure level is increased. It is necessary to emphasize at this juncture that phar macokinetic and metabolism studies alone are insufficient AS! OOOO15257 -5- to assess toxicity. They are utilized to gain insight into the time related disposition of chemicals in the body which can explain in certain instances why toxic effects are produced at high doses and not at low doses. It is note worthy that both of the studies conducted thus far, (indi cating an altered fate of VC as the dose increases, and the dose related depression of hepatic GSH by VC) appear to correlate with the available data on the induction of carcinogenesis (Maltoni, et al., 1975a,b). Therefore this suggests that a threshold may exist for the production of cancer. Recent reports have demonstrated that microsomal enzyme preparations form reactive metabolites of VC which bind to rat liver microsomes (Kappus et al., 1975) protein sulfhydryl groups, RNA (Bolt et^ al., 1975) and adenosine of DNA (Barbin et. a_l., 1975). Reaction of VC metabolites with albumin has also been demonstrated to be mediated by a xanthine oxidase enzyme system (Bolt et al., 1975) in vitro. To determine whether the reaction of metabolites of VC with macromolecules may be pertinent to assessing the hazard of variuos magnitudes of exposure to VC, it is necessary to conduct studies in vivo. ASI 000015258 -6- Therefore the thrust of this research proposal is to charac terize the macromolecular binding of VC to hepatic protein and nucleic acids following exposure to various concentrations of 14 C-labeled VC. Since the metabolism of VC appears to be intimately associated with its binding pro perties, additional in vitro studies will be conducted to elucidate the primary metabolic pathways involved in the biotransformation of VC. 2. Objectives A. To determine the potential of reactive VC meta bolites to bind with hepatic macromolecules (protein, DNA or RNA) in vivo following exposure in rats to various concentrations of 14 C-labeled VC. B. To define the various metabolic pathways res ponsible for the biotransformation of VC. It is anticipated that in vitro techniques will be used to study both soluble (xanthine oxidase, glutathione transferases) as well as particulate (microsomal) enzyme systems. If the studies outlined above are successful in elucidating 1) a VC induced effect which can be related to carcinogenesis ASI 000015259 -7- and/or 2) the primary metabolic pathways responsible for VC biotransformation, then additional studies to integrate the relationship between dose dependent metabolism and carcino genesis may be warranted. Due to the complex nature of the task at hand the proposed studies are outlined as a general approach. Individual tests will be screened and either pursued or terminated after an appropriate evaluation. 3. Test Material 14 Labeled 1,2- C-VC will be used in the majority of the . 14 proposed studies. The methodology for synthesis of C-VC14 directly from 1,2- 14 C dichloroethane (New England Nuclear) has been established previously (Wagner et al., 1975). The 14 C-VC will be synthesized directly prior to use and main tained in the gaseous state to prevent polymerization and degradation. The chemical purity of the labeled VC will be compared to authentic vinyl chloride (Matheson Gas Products, minimum purity 99.9%). The radiochemical purity will be established by liquid scintillation counting of trapped effluent fractions from the gas chromatograph. 000015260 A.S I -8- 4. Animals Male Sprague-Dawley (Spartan substrain) rats will be used. Since alterations in hepatic glutathione levels may change the metabolic pattern of VC (Hefner, et al_. , 1974; Jaeger, et al., 1974) experiments will be initiated at a fixed time of day and food and water will be provided ad libitum. 5. Housing All animals will be kept in rooms in which a constant temperature, humidity, and a 12 hour light-dark cycle are maintained. Noise and activity in the animal rooms are minimal and constant from day to day. The animal facility is approved by the American Association for Laboratory Animal Science and the American Association for Accreditation of Laboratory Animal Care. 6. Binding of 14C-VC to Hepatic Macromolecules a. Exposure Techniques Rats will be exposed under dynamic conditions to 14 C-VC14 in a 30 1 glass inhalation chamber. 1,4 C-labeled VC will be metered into the chamber air flow (~6 1/min) with a precision dual syringe pump. The nominal concentration of VC will be determined from the ratio of the rate at which the VC gas is dispensed and the total chamber air flow. The ASI 000015261 -9- analytical concentration of VC will be continuously monitored by recirculating a fraction of the chamber atmosphere through an infrared spectrophotometer (Wilks) set at 10.6 nm. Samples (1 ml) of the chamber atmosphere will also be subjected to gas chromatography periodically throughout the exposure. At corresponding intervals, the 14 C-activity will be determined by bubbling 1 ml aliquots of the chamber atmosphere into a scintillation solution and the radio activity will be counted in a liquid scintillation spectro meter . The inhalation chamber will be operated in a laboratory fume hood to prevent contamination of the working environ- 14 ment. After transit through the inhalation chamber the CVC will be absorbed on activated charcoal or trapped in an appropriate liquid solvent. These traps will be disposed of as radioactive waste according to standard regulations. Air samples from all experimental rooms will be sampled period ically by the Industrial Hygiene Department of The Dow Chemical Company. b. Sample Preparation Following a single 6 hour exposure to varying concentrations AsI 0000 *5262 -10' of VC (10-1500 ppm) the rats will be killed immediately. An aliquot of liver will be sampled and used for determining hepatic nonprotein sulfhydryl content by a modification of the method of Sedlak and Lindsay (1968) . The remaining liver will be frozen immediately on dry ice and stored at -20C until analyzed. The remaining carcass will be analyzed for total radioactivity as described previously (Watanabe, et al., 1976a). Protein binding of 14 C-labeled VC to hepatic tissue will be determined by the method of Jollow et 14 al. (1973). Binding of C-VC to nucleic acids will be screened. Nucleic acids (DNA and RNA) will be isolated from rat liver following selected exposure levels (Kirby, 1957, 1962) and the 14 C-activity determined by liquid scintil lation counting. If a significant interaction is observed additional exposures may be necessary to characterize the binding properties. 7. In. Vitro Metabolism Previous work in this laboratory (Hefner et aT., 1975) in dicated that the metabolic uptake of VC in rats exposed to less than 100 ppm was inhibited dramatically by pretreat ment with ethanol. When exposed to greater than 220 ppm AST 000015263 -li ve, SKF 525-A, an inhibitor- of the mixed function oxidase (MFO) enzymes, caused a slight inhibition of metabolic uptake. More recently Bolt et al.. (1976), confirmed the effects of SKF 525-A, but also showed that 3-bromophenyl- 4 (5)-imidazole and 6-nitro-l,2,3-benzothiodiazole, potent inhibitors of cytochrome P-450 dependent MFO enzymes, completely blocked the metabolic uptake of VC in rats. Subsequent studies showed that VC binding to sulfhydryl containing proteins in vitro was facilitated by NADPH fortified MFO enzymes and the soluble enzyme xanthine oxidase. Therefore it appears that the metabolism of VC in vivo and binding properties in vitro are a function of both soluble and particulate enzymes. Considerable effort has been expended in studying the MFO mediated metabolism of VC while very little attention has been given to other meta bolic enzyme systems. The present study proposes to in vestigate both soluble (xanthine oxidase, GSH transferases) as well as microsomal metabolism in vitro to gain further information on the potential metabolic systems involved in the biotransformation of VC. It is anticipated that various chemical trapping agents such as 3,4-dichlorobenzenethiol (Gothe et^ al., 1974), 4-(4-nitrobenzyl) pyridine (Barbin et al., 1975) or GSH will be used to derivatize reactive intermediates AS I 000015264 -12- which can then be examined for identification. Both radio gas chromatography, liquid chromatography and gas chroma tography-mass spectrometry are expected to be utilized extensively in metabolite identification. Following eluci dation of in vitro VC metabolizing systems it may be appropriate to use selected inducers and inhibitors of the potential metabolic system to verify its function iri vivo. 8. Conclusion The proposed studies are designed to gain insight into the potential in vivo binding of VC to intracellular macro molecules (proteins and nucleic acids) which may be ul timately related to its mechanism of carcinogenicity. The secondary objective is to further characterize the bio transformation of VC by various enzyme systems in vitro in order to isolate reactive metabolites which may be related to the dose dependent metabolism and carcinogenicity of VC. The studies as outlined will be pursued only if preliminary results appear encouraging. This "experiment by experiment" approach is necessary to allow the flexibility required to optimize the return on investment to solve the complex problem of assessing the hazard of low level exposure to VC. 000015265 AS I -13It is anticipated that an equivalent of 2 man years plus necessary support personnel will be required for the studies. This amounts to $115,000 over about a one year time period. Interim reports will be issued as the data warrants. ASI 000015266 REFERENCES Barbin, A., Bresil, H., Croisy, A., Jacquignon, P., Malaveille, C., Montesano, R. and Bartsch, H. (1975). Liver microsome mediated formation of alkylating agents from vinyl bromide and vinyl chloride. Biochem. Biophys. Res. Comm., 67, 596-603. Bartsch, H., Malavielle, C., and Montesano, R, (1975). Human rat, and mouse liver mediated mutagenicity of vinyl chloride in Salmonella typhimurium strains. Int. J. Cancer, 15, 429-437. Bolt, H. M., Kappus, H., Buchter, A., and Bolt, W. (1975). Metabolism of vinyl chloride. Lancet, June 28, 1425. Bolt, H. M., Kappus, H., Buchter, A. and Bolt, W. (1976). Disposition of 1,2- 14 C-vinyl chloride in the rat, in manuscript. Creech, J. L. and Johnson, M. N. (1974). Angiosarcoma of liver in the manufacture of polyvinyl chloride, J. Occup. Med., 16, 150-151. Gillette, J. R. (1974a). A perspective on the role of chemically reactive metabolites of foreign compounds in toxicity - X. Biochem. Pharmacol., 23, 2785-2794. Gothe, R., Calleman, C. J., Ehrenberg, L. and Wachtmeister, C, A. (1974). Trapping with 3,4-dichlorobenzenethiol of reactive metabolites formed in vitro from the carcinogen vinyl chloride, Ambio, 3, 224-226. AS I 000015267 Greim, H., Bonse, G., Radwan, Z., Reichert, D., and Henschler, D. (1975) . Mutagenicity In vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation, in manuscript. Hefner, R. E. Jr., Watanabe, P. G., and Gehring, P. J. (1975). Preliminary studies of the fate of inhaled vinyl chloride monomer (VCM) in rats, Ann. N.Y. Acad. Sci., 246, 135-148. Hefner, R. E. Jr., Watanabe, P. G. and Gehring, P. J. (1976). Short Communication: Percutaneous absorption of vinyl chloride, Toxicol. Appl. Pharmacol., in press. Jollow, D. J., Thorgeirsson, S. S., Potter, W. Z., Hashimoto, M. and Mitchell, J. R. (1974). Acetaminophen-induced hepatic necrosis VI., Pharmacology, 12, 251-271. Kappus, H., Bolt, H. M., Buchter, A. and Bolt, W. (1975). Rat liver microsomes catalyze covalent binding of 14 C-rvmyl chloride to macromolecules, Nature, 257, 134-135. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R. and Brodie, B. B. (1973). Acetaminopheninduced hepatic necrosis IV. J. Pharmacol. Exp. Therap., 187, 211-217. Rannug, U., Johansson, A., Ramel, C. and Wachtmeister, C. A. (1974). The mutagenicity of vinyl chloride after metabolic activation, Ambio, 3, 194-197. 000015268 AS I Sedlak, J. and Lindsay, R. m. (1968). Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's Reagent, Analyt. Biochem., 25, 192-205. Wagner, E. R., Muelder, W. W., Watanabe, P. G., Hefner, R. E. Jr., Braun, W. H., and Gehring, P. J. (1975). Gas chromatographic method for the preparation of 14 C-labeled vinyl chloride, J. Labeled Compounds, in press. Watanabe, P. G., McGowan, G. R. and Gehring, P. J. (1976a). Fate of 14 C-vinyl chloride after single oral adminis tration in rats, Toxicol. Appl. Pharmacol, accepted for publication. Watanabe, P. G., McGowan, G. R., Madrid, E. 0. and Gehring, P. J. (1976b) . Fate of "^C-vinyl chloride following inhalation exposure in rats, in manuscript. Watanabe, P. G., Hefner, R. E. Jr., and Gehring, P. J. (1976c). Vinyl chloride induced depression of hepatic nonprotein sulfhydryl content and effects on bromosulphthalein (BSP) clearance in rats, Toxicology, in press. Watanabe, P. G., Hefner, R. E. Jr., McGowan, G. R. and Gehring, P. J. (1976d). Metabolism of vinyl chloride, Ann. Internal Med., in press. ASI 000015269