Document a1yqG1GBnYJ6DXGReqRe1Qmn9

TOXICOLOGY ANO APPLIED PHARMACOLOGY +4, 57 1-579 (1978) Hepatic Macromolecular Binding Following Exposure to Vinyl Chloride1 P. G. Watanabe,- J. A. Zempel, D. G. Pegg, and P. J. Geh(ung Toxicology Research Laboratory, Health and Environmental Research, 1303 Building, The Dave Chemical Company, Midland, Michigan S3640 Received July 23,1977; accepted November l, 1977 Hepatic Sixcronclecuiar Binding Following Exposure to Vinyl CMoinie. W xta.va3E. P. G.. Zempel, J. A., Pegg. D G,, and Gearing. P. J. (1978). Toxicol, Appl, Pharmacol, 44. 571579, Covalent binding of radioactivity to hepatic macromolecules in rats exposed to ,4C-labeled vinyl chlordi (VC) was studied to determine if VC-induced carcinogenesis may be related to electrophilic aikylut.on of nacromolecuies in vivo. Male Sprague-Dawley rats were exposed to 1, 10. 25. 50. 100. 250, 500. 1000, or 5000 ppm of ("CiVC for 6 hr. Following exposure, radioacttv'ty cova entlu bound to itepa'ic macromolecules and purified nucleic acids (RNA. DNAi was determined. The total amount of (l4C|VC metabolized and hepatic glutathione (GSH) content were also determined. The total amount of radioactivity bound to macromolecules in the liver did not increase proportionately to the increase in the exposure concentration of VC. A disproportionate decrease in macromolecular binding was observed as the concentration of VC increased. The covalent binding to hepatic macromolecules was related to the amount of VC metabolized. At exposures greater than 50 ppm, the amount of '*C bound ^ tojr.acratr.oiecules in the liver correlates withjnductiori of hepatic angiosarcoma. There was no ' detectable binding of radioactivity to either DNA or RNA in the liver. Hepatic glutathione content was significantly depressed only at exposure concentrations greater than 100 ppm. Considerable effort has been devoted to research on vinyl chloride (VC) since it was demonstrated to be carcinogenic in man and animals (Creech and Johnson. 1974; Maitoni and Lefemine, 1975). The concept of a reactive metabolite of VC being responsible for the carcinogenic activity (Hefner er. at., 1975; Van Duuren. 1975) is supported by evidence of enhanced mutagenic activity of VC to bacteria in the presence of microsomal enzyme-activating systems (Bartsh et al,, 1975; Maiavielle es at., 1975; Rannufidf a!., 1974). Metabolites of VC have been identified in the urine of rats as conjugates of cysteine (Green and Hathway, 1975; Watanabe er at., 1976a), suggesting that VC is biotransformed to electrophilic metabolites and that the pCmary detoxification mechanism for these metabolites is conjugation with hepatic glutathione prior to excretion. Complementary to these data was elucidation of a dose-related reduction of hepatic glutathione in rats exposed to 50 to 2000 ppm of VC for 7 hr (Watanabe et at., 1976b). It was therefore hypothesized that reactive metabolites formed during exposure to low levels of VC (less than 50 ppm) will be readily detoxified ' This iiubv was funded by the companies supporting the vinyl chloride protects being administered by trie Manji'icturing Chemists Association. Washington, D C. : Aut.icr-o w hom ail correspondence mould be sent, manuscript No. 8 bOO i"3- 77. 1 f * t 'd4f 572 WATANABE et al. by reaction cv ith glutathione. However, as the exposure is increased, detoxification will be impaired by the reduction of hepatic glutathione. This will lead to an increase in the level of reactive metabolites and result in an increased reaction of these metabolites with intracellular macromolecules. Chemical carcinogenesis has been attnwuted to the reaction of electrophilic metabolites with intracellular macromolecuies (Miller and Miller, 1971). Recent reports have demonstrated that liver microsomal enzymes in vitro form reactive metabolites from VC which bind to the microsomes (Kappus et a!., 1975), protein sulfhydryl groups. RNA (Bolt etal., 1975), and adenosine of DNA (Barbin et al,, 1975). In addition to reduced hepatic glutathione leading to an increase in the binding of reactive metabolites of VC with macromolecules, such an effect may also be associated with other dose dependent alterations in the fate of VC in the body. The dose dependence of the fate of VC has been elucidated kinetically and attributed to saturable metabolic pathways (Hefner et ai, 1975; Green and Hathway, 1975; Watanabe et at., 1976a,c). In vivo studies are needed to determine whether dose-dependent disproportionate increases in the macromolecular binding of reactive metabolites of VC with increasing exposure concentration may be associated with toxicity and carcinogenicity. Thus, the objective of this study was to characterize the binding of VC to hepatic macromolecuies and nucleic adds following exposure to various concentrations of uC-labeiej).3Kl ***&' "C METHODS -~- Materials. uC-labeled VC was synthesized from [ 1,2-,4C) 1,2-dichloroethane (New England Nuclear, Lot Nos. 819-221 and 819-292, 5.0 and 4.8 mCi/mmoI, respectively) immediately prior to use (Wagner and Muelder, 1975). Each new batch of [I,2-uC|dichloroethane was analyzed and yielded [UC|VC with a radiochemical purity of 95%. The synthesized [14C|VC has been shown repeatedly in our laboratory to be 95 to 96% radiochemically pure (Wagner et ai, 1975). Thus, while each synthesis of [l4ClVC prior to each experiment was not analyzed, it was assumed to be of the same purity. Furthermore, no indication during a synthesis which utilizes a gas chromatographic separation suggested that any deviation from previous syntheses had occurred. Nonlabeled VC (Matheson Gas Products) of 99.9% minimum purity was mixed with the [MC [material to obtain the desired specific activity. Typically 40 ml of the !uC)VC-he!ium gas mixture was injected into a 5 to 10-liter Saran bag (Anspec, Inc.) containing the desired quantity of nonlabeled VC. Animals. Male Sprague-Dawlcy rats (Spartan Research Laboratory) weighing 220 to 250 g were used throughout the study. Al! animals were housed in rooms in which a constant humidity, temperature, and a 12-hr light-dark cycle (7 am-7 pm, EST) were maintained. Food and water were provided ad libitum except during exposure. Groups of rats (three to six animals per group) were exposed to (,4C)VC (treated) or room air (controls) for 6 hr between 9:00 am and 4:00 pm (EST). An additional group of five rats pretreated with phenobarbital (80 m^kg/day, ip, 3 days prior to exposure) were exposed at the 100-opm ievei. Exposure. Exposures were conducted in 30-liter glass inhalation chambers. ,JClabeled VC was metered into the chamber air flow (-6 liters/min) with a dual syringe . s. - _ r SL 097427 VINYt CWEOTUDE MACROMOLECULAR BINDING 573 pump. The analytical concentration of VC was monitored continuously by recirculating a fraction of the chamber atmosphere through an infrared spectrophotometer (Wilks) at a wavelength 10.6 ,um. In addition, samples (1 ml) of the chamber atmosphere w-ere analyzed at approximately hourly intervals during the exposure by gas chromatography (Watanabe e: ai, 1976c). At Corresponding times, the UC activity was determined by bubbling I-ml aliquots of the chamber atmosphere into a scintillation solution containing a mixture of Concifluor (Mailinckrodt Chemical), 2-methoxyethanol, toluene (6:11: S3). The radioactivity was determined by counting in a Mark II or Mark III liquid scintillation spectrometer (Searle Analytic, Inc.). The targe: concentrations of VC were 1, 10, 25, 50, 100, 250, 500, 1000, and 5000 ppm. The respective mean analytical concentrations measured by gas chromatography were 1.4 0.3 (SD), 9.3 0.2, 24.7 1.4, 51 2, 109 23, 250 r 3, 511 11, 1020 13, and 4600 311. The respective specific activities were 132,000, 4801, 3170, 2523, 1750, 837, 217, 301, and 50 dpm/#g of VC. The inhalation chamber was operated in a laboratory fume hood to prevent contamination of the working environment. After transit through the inhalation chamber the (MC|VC was adsorbed on activated charcoal. The charcoal traps were disposed of as radioactive waste according to standard regulations. Procedure. Following the 6-hr exposure to various concentrations of l,4C]VC (15000 ppm), the rats were killed immediately by a blow to the head. An aliquot of liver was sampled and used for determining hepatic nonprotein sulfhydryl content by a modification of the method of Sedlak and Lindsay (1968). The remaining liver was frozen immediately on dry ice and stored at -20JC until analyzed. The carcass was analyzed for total radioactivity as described previously (Wacanabe el ai, 1976a). Previous studies have shown that only a small percentage of radioactivity (<12%) is excreted as metabolites other than (`'ClVC during 72 hr following a 6-hr inhalation exposure (W'atanabe el ai, 1976c). The large proportion of the 12% is comprised of 'CO. excretion 72 hr after exposure. Furthermore, very little urine is excreted during the exposure period. Thus, the nonvolatile radioactivity determined in the tissue immediately arier exposure is a good estimate of the total amount of metabolized VC. Macrortoiecular binding of t4C-labeIed VC to hepatic tissue (protein, nucleic acids, and lipidi was iere-mirted by the method of Jollow et al. (1973). The trichloroacetic acidprectpitah'e -m-ai following exhaustive solvent extraction was digested in 1 n KOH and the racoactivity was determined by liquid scintillation spectrometry. Hepatic nucleic acids (DMA and RNA) were isolated from the 1-, 100-, 250-, and 1000-ppm exposure groups and radioactivity was determined by direct counting of the aqueous fractions by liquid sdntfttteon spectrometry (see below). [solution of nucleic acids. RNA and DNA were isolated from rat liver by modification of the techniques described by Okuiiara (1970) and Irving and Veazey (1963). Approximately 10 g of frozen tissue was thawed slowly and homogenized in 50 mi of 0.15 m sodium chioride-0.04 m ethylenediamineteiraacetic acid (EDTA) butTer (pH 3.0). Sodium dodecyl sulphate (1 g) was added, followed by 1 vol of 90% phenol solution. The mixture was mechanically stirred for 30 min at room temperature and the phenol and water phases were separated by centrifugation. The water phase was collected and sodium acetate was added to a rir.nl concentration cf 2b, Nucleic acids were precipitated using 1 vol of 95% ethanol arc spooled onto a giias red. The ,. - `L s tI 1 1 'p1 r* + t* s i*3 p* T*<r - precipitate was washed with ethanol and dissolved in 20 ml of0.015 m sodium chloride-- 0.0015 m sodium citrate buffer (pH 7.0). RNA was precipitated by adding 1 vol oficerCOld 6 m potassium acetate (pH 7.5) and removed by centrifugation. DMA was precipitated from the supernatant by adding 2 vol of 95% ethanol. The resulting crude DNA pellet was dissolved in 20 m! ofO.015 vt sodium chloride-0.0015 MSOdium citrate buffer (pH 7.0) and the solution was centrifuged at 100,000 g for 60 min at 2**C to remove glycogen. RNAase (3 mg/50 ml) was added to the Supernatant and the solution was incubated at 37'C for 30 min. One volume of phenol saturated with 0.15 M sodium chloride--0.015 m sodium citrate (pH 7.0) was added and the mixture was stirred for 30 min at room temperature. The aqueous phase was extracted with ether and DNA was precipita'ed by adding so 51 jc; restate (4 g/100 ml) and 2 vo! of 2-ethoxyethanol. The precipitate was redis-o'-ed in 10 m! of 0.015 M sodium chloride-0.0015 M sodium citra'e at 2JC. The finai DN'A precipitation was accomplished using 5 ml of ice-cold isopropanol. RNA and DNA pe'Vs were dried at 2C under reduced pressure arid weighed RNA and DNA were quantified by the orcinol and diphenylamine reactions, respectively (Keisti and Lederer, 1974). i 5W RESULTS Hepatic macromolecular binding, hepatic nonprocein sutfhydryl content (prima^lyLV; glutathione, GSH), and the total amount of VC metabolized following various exposit^ ' concentrations are summarized in Table t and presented graphically in Figure-11. Covalent binding tc hepatic macromolecules plotted as a function of the log of die exposure concentration was triphasic and best represented by a sigmoid curve. The linear portion of the binding curve extended from exposure concentrations of approximately 50 to 250 ppm. The lower inflection poinc appeared to lie between 25 and 50 ppm. The metabolism of VC and binding approach a plateau at concentrations exceeding 250 ppm. Hepatic macromolecular binding correlated well with the total amount of VC metabolized as evidenced by a lack of any obvious trend in the ratio of bound VC versus total metabolized VC (S/.4 x 100, Table 1). This point is further substantiated by the constant fraction of bound versus total radioactivity in the liver with increasing exposures. Although the value for totai metabolism appears low for the 1000 ppm exposure, a corresponding reduction in macromolecular bidding was not observed. The apparent discrepancy between total metabolism of VC at 500 and 1000 ppm is not understood fully, but it may be due to differences in respiratory parameters causing differences in the uptake of VC. Hepatic nonprocein suifhyJry! concent (primarily GSH) was noc depressed significantly at 1, 10, 25. or 50 ppm. Only at concentrations of 100 ppm or greater was a dose-related depression of hepatic GSH evident Metabolism of VC was not increased in rats exposed to 100 ppm of VC after pretreatment with phenobarbital. Macromolecular binding, however, was increased markedly when compared to nonpretreated animals. Isolation of RNA and DNA by a nondigestive procedure from the liver of animals exposed to I, 100, 250, and 1000 ppm of VC failed to reveal any detectable radioactivity. The sensitivity for detecting the radioactivity in DNA and RNA varied at f. SL 097429 *- >r V VINYL CHLORIDE MACROMOLECULAR BINDING TADLt I TOTAL MHTABOUSM, HUATiC MaCHUMUI ECU1-AH UlNDINO, AND Uhl'AHC Gl.UT AIHIDNI-- <GSlt) CoNt.I.NI RATIONS FOLLOWING INHALATION l-xi'osimi: (6 nit) to Vinyl Chloride (VC)'` Nominal con central ion 1 10 25 50 100 250 500 1000 5000 Pretreatment with phcnot>arbiiutr 100 'iV* af\C equivalents metabolized) 29.8 + 3.2 242 26 557 42 1181 ' 93 2-lHli-i 173 3826 + 345 6263 +- 365 4267 i 7o5 9255 < 1,467 2160 ! 166 B Gig of VC equivalents bound per g of protein) 0.5 0.08 3.3 0.2 12,2 4.0 23.3 3.4 47.6 4.8 89.6+12.3 98.8 5.0 106.8 + 22.2 113.5 10.4 BfA x 100 1,78 0.48 1.38 0.36 2.15 0.60 1.99 0.36 . 1.99 0.26 ' 2.35 + 0.28 ,1.58 0.20 2.55 0.58 1.12 0.13 l 80.0 23.9 3.70 0.82 Hepatic GSM (% control) 104 89 93 94 81* 70* 60* 51* 39* Percentage of ini.d "C activity m liver bound to macromolcculcs 20 3 21+2 21+2 20 + 2 25 t 2 . 22 + 2 25 3 22+2 22 3 39 3 * Moms t SO. * Si.msiivtiliy diftcrew from cutihoh, Sindeiil'i / jest {;> < 0.05). ` Rats were injected ip with sudnmi pliemibatlmiit, Ho mg/kg for 3 days prior lo exposure. SL 097430 576 W-\T.vSA3E Et al. an,, 't UXtttriihllM ilftiid* the logarithm of the exposure concentration to vuiy! chloride (VC). Macromolecular binding () is expressed as microgram equivalents of VC bound per gram of protein (mean SD) GSH (A) is expressed as percentage of control. DISCUSSION The results indicated that the total amount of radioactivity bound to macrcmolecules in the liver did not increase proportionately to the increase in the exposure concentration of V'C. Macromolecular binding -sas related directiv to the total amount Of VC which was metabolized over exposure concentrations ranging from 1 to 5000 ppm of VC. Macromolecular binding plotted as a function of the log of the exposure concentration gave a sigmoid-shaped curve. The linear portion of the curve is bounded by low and high irtr'eation pomts M'ow 50 and above 250 ppm, respectively. Above 500 ppm binding appeared to plateau. Below 100 pprn binding was approximately proportional to the .increase In exposure. (t is particularly significant that available data (Maltoni, 1975) indicate that the percentage induction of hepatic angiosarcoma in rats is linear between 50 and 500 ppm ' SL 097431 t iJw.'-jflidwa'iilir- VINYL CHLORIDE MACROMOLECL'LAR BINDING 577 when expressed as the log of the exposure concentration. Above 25riO ppm tumor incidence is constant. This correlates with the plateau effect observed in total metabolism and hepauc macromolecular binding above 500 ppm in the present study. One mechanism of chemical carcinogenesis is believed to be reaction of electrophilic 7 metabolites with intracellular macrorr.olecules (Miller and Miller, 1971). Jhe covalentbinding.to hepatic macromolecules refiects.the activation of VC to reactive metabolites. Therefore, both total metabolism of VC and covalent binding appear to correlate with To the induction of hepatic angiosarcoma in rats exposed to concentrations greater than 50 ppm. ' '' ' The toxicologic significance of the metabolism and covalent binding of VC at levels p. below 50 ppm is not dear. Deviation in these parameters from the log-linear relationship at higher levels indicates that the carcinogenic response of the population may be changed at lower level exposures. However, this hypothesis cannot be validated until the results of carcinogenesis bioassays currently being conducted at 25, 10, and 1 ppm become available. The reactive metabolites of VC are detoxified presumably by reaction with GSH (Watanabe et al., 1976b). Therefore, it is important that significant depression of hepatic GSH was dose related only at levels of 100 ppm and greater. This is consistent with previous studies which showed that a single 50 ppm exposure to VC was in the threshold zone for depression of hepatic GSH (Watanabe et al., 1976b). This suggests that carcinogenicity of VC is related _to_the decreased ability to detoxify the reactive jnetabolites of VC. A Covalent binding of radioactivity to isolated nucleic acids (both RNA and DNA) > was not detectablein any of the exposure groups tested. The detection limit in all groups f wai'liuxnaent to detect the l4C activity if it were equally distributed by weight I throughout the components of the liver. Therefore, it was concluded that VC does not j preferentially react with intracellular nucleic acids. In contrast to this, initial studies by 1 Bolt et al. (1976a) reported covalent binding of radioactivity to DN'A and RNA ' following a 5-hr static exposure to 145 ppm of (l'CiVC. The specific activity used by / Bolt et al. (1976a) was higher than that used in our study and this may account for the j difference in the tw a observations. However, more recently it has been reported by this j group (Lain and Bolt. 1977) that greater than 95o of the radioactivity associated with i RN A'following inhalation exposure ;n rats to 1300 ppm. of [^Ci'VC for 5 hr is due to . or.e carbon fraament incorporation of IJC ir.tothe n rtr-e n odd Both our results and those of Boit et al. (1976a^ confirm the conclusion that VC does not preferenrisfly react with hepatic nucleic acids. Itis i mportarajgj: mprusize that the methodotagies employed only detect covalent binding andexclude any other more subtle interactions. Vheretore, a very small degree of covalent binding to nucleic acids does not""exclude the possibility of other interactions which result in the loss of the^ ability to control cellular replication. Presentment with phenobarfcital did not increase the total metabolism of VC. Bolt et al. (1976b) have reported similar data showing that phenobarbital pretreatment failed to stimulate the uptake of VC following a static inhalation exposure. Although total metabolism was not affected by phenobarbita!. the mecrorr.clerular binding was ire:eased mu--,; if- The :me sigr.ficsr.ce of the :r.;rmsec be'e-g is diircult to interpret since phenobarcca! mceascs total protein in the liver, ard the increase in SL 097432 1 "V --S-Jt- t - -k-r vJS^HB8^<w,"T*X^i^j8UjfiT5- ^ t-'VvL*'3* -rfy&i - ,sfj5 ' . *,Sb'-"TASR52 -*'- 'r ''C. f*t?f:,- .a-. . -' wataia< mtal. - . 3^?'. . binding may reflect a nonspecific interaction merely due to an increase in the available protein binding sites. c In summary, the results of the studies reported herein do not associate the carcinogenic effect of VC with a disproportionate increase in binding of electrophilic fllSfaBolites of VC to hepatic macromolecules as the exposure concentration is increased. Even more significantly, there was no evidence for any preferential.binding of ( electrophilic metabolites to nucieic acids of hepacocytes. This suggests that the carcino genic activity of VC may not be associated directly with this commonly accepted , mechanism for carcinogenesis. Before excluding this mechanism entirely, additional experiments are needed to snow whether binding to nucleic acids may occur after repeated exposure since repeated exposure may induce preferentially alternate metabolic pathways. Another aspect relating to this is whether the administration of phenobarbital may enhance binding to nucleic acids. This is important because j phenobarbitai does increase the hepatic macromolecular binding of MC activity to I macromotecules in toto in rats exposed to 100 ppm even though it did not increase the total amount of VC metabolized. These aspects are being explored. Even more important, before excluding alkylation of nucleic acids as the mechanism . for VC-induced carcinogenesis, is the need to determine the absence of such activity in target tissue rather than hepatocytes. Essentially all studies of metabolic and dinkal parameters to dace have been either conducted on or related to the hepatocyte. The hepatocytes may constitute primarily a means for detoxification since they are not particularly susceptible to VC induced toxicity. Toxicity may be induced tn tissues f with smaller capacty to detoxify the reactive metabolites of VC. The induction of j tumors of the nervous system by ethylnitrosourea has been correlated with the persistence of O-6-ethylguanir.e in the nervous system (Goth and Rajewsky, 1974), Although other metabolizing organs such as the liver produce 0-6-ethylguanine, the turnover rate of DNA mediated by repair mechanisms is sufficient to prevent induction^ ^of cancer. Ljkwjse,jhe, mecranism ofcarcinogenesis of VC may be due to an inability 7 ] of the target tissue, in this case endothelium, to repair"lesions whether the reaction bc~7 with nucieic acids or critical proteins. - -- ~~ J REFERENCES Barslv, A., Bresil, H., Croisy, A., Jacquignon, P,, Mal.wielle, C., Montesano, R., and Bartsch, H. (1975). Liver microsome mediated formation of alkylating agents from vinyl bromide and vinyl chloride. Bioc/teu. Btopkys. Res. Common. 67, 596-603. Bartsch, H,, Mal.avielle. C., and Montesano. R. (1975). Human, rat, and mouse liver mediated mutagenicity of vinyl chloride in Salmonella ;yphi.murium strains. Ir.t. J. Cancer I5t 4:9-437. Bolt,' H. M.,Kappls. H.. 3uchter. A., and Bolt, W. (1975). Metabolism of vinyl chloride. Lancti 1425. BflftT, H- M-. Kappus, H., Blchter. A., and Bolt. W. (1976b). Disposition of IdZ-'X-vinyl chloride in the rat. .4 rch. Toxicol. 35, 153-162. Bolt, H. M., (Carpus. H,, (Caltmann, R,, Appel, K. E., Blchter, A,, and Bolt, W. (1976a). 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Ambio 4, 18-23. Maltonl C., and Lefe.mine, G. (1975). Carcinogenicity assays of vinyl chloride: Current % results. Ann. .V. Y.Acad. Sci. 246, 195-224. i Miller, J. A., and Miller, E. C. (1971). Chemical carcinogenesis: Mechanisms and approaches to its control./. Hat. Cancer fnst. 47, 5-14. Okliiara, E. (1970). Preparation of mammal'an deoxyribonucleic acid by SDS-phenol treatment. Anal. Biochem. 37, 175-178. Rannlg, U., Johansson, A., Ramel. C., and Wachtmeister. C. A, (1974). The mutagenicity of vinyl chloride after metabolic activation. Amid j, 194--197. Seolak, J., and Lindsay, R. M. (1968). Estimation of total protein-bound, and nonprotein suifhydryl groups in tissue with EUman's Reagent. Anal. Biochem. 25, 192-205. Van Dllren. 3. L. (1975). On the possible mechanism of carcinogenic action of vinyl chloride. Ann. .V. Y.Acad. Sci. 246, 253-267. Wagner. E. R., and Mdeldfr, W. W, (1975). A procedure for preparing `*C-labeled vinyl chloride. Ann. S. Y. Acad. Sci. 246, 152-153. Wagner, E. R-, Mlelder, W. W . Watanabe. P. G . Kef^er. R E.. Jr.. Braun, W. H., and Gehri.no, P. J. (1975). Gas chromatographic .r.e-hod 'or the prep citicr of "C-laceied vinyl chloride. J. Labeled Compounds 1 1, 535-542. Watanabe, PtO, Hefner, R. E., Jr., and Gfhr..ng. P. J. (19'pcl V'm.%! chloride induced depression of hepatic nonprotein sulfhydryl content and eifects on bromosuiphthuieln (3SP) clearance in rats. Toxicology 6. 1-8. Watanabe, P, G.. McGcwav, G R.. and ?. 3. (19"6al. Fate of MC-vinv! chhiriJe alter single oral id.T.i.'2i"otior. In riLi Toe.'.-d -. pci. P-.a.-macol. 56, 339-552. Watanabe. P. G.. MgGdwan G. R.. Map ... . E 0.. and Ge-..-.nt. P. J. (1976c). Fate of 1'C-vinyl chlor.de folio- :.r a -r.ha^t'on exp, - ,rc rats. Toxicol. Apr!. Pharmacol. 37, 49-59. SL 097434