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,> * TOMCOl OGY A\D APVLIf D PHARMACOLOGY 44. 57 1-579 ( | ^78) Hepatic Macromolecular Binding Following Exposure to Vinyl Chloride1 P. G. Watanabe.: J. A. Ze.mpel D. G. Pegg. and P. J. Gehring ToxiCoiogv Research Laboratory, Health ana Environmental Research. 1303 Building. The Don ' Chemical Company, \haland. Michigan 486*0 Received Jul\ 23. 1977. accepted \ovemner 1. 1977 Hcbauc Macromoiecular Binding Following Exposure to V nyl Chioride- Watanabe. P G. Zevipel. J a. Pegg, D. G.. and Gehring. P. J. i ! 978). To x:cot. Appt Pharmacol.. 44. 571 -- 579 Covalent binding of radioactivity to hepatic macromolecu.es in rats exposed to '*C labeled unvl chloride (VC) was studied to determine if VC induced carcinogenesis mas be related to electrophilic alkylation of macromoiecules in vivo. Male Sprague-Dawitv rats xverc exposed to 1. 10. 25. 50, 100, 250. 500. 1000. or 5000 ppm of i'*C!VC for 6 hr. Following exposure, radioactivity covalently bound to hepatic macromoiecules ana punned nucleic acids (RNA. DNA) was determined The total amount of ;'*CiVC metapoiizea and hepatic glutathione <GSH) content were also determined. The total amount cf radioactivus bound to macro moiecules in the liver did not increase proportionately to tne increase in the exposure concentration of V'C. A disproportionate decrease in macromo.ecular binding was observed as the concentration of VC increased- The covaient binding to hepatic macromoiecules was related to tne amount of VC metaooltged. At exposures greater than 50 ppm. tne amount of `*C bound to macromoiecules in the liver correlates with induction of hepatic angiosarcoma. There was no detectable binding of radioactivus to either DNA or RNa c. the user. Hepatic e'utathione content was significantly depressed only at exposure concentrations greater man 100 ppm Considerable etTort has been devoted to research on vinyl chloride (VC) since it was demonstrated to be carcinogenic ;n man and animals (Creech and Johnson, 1974; Maltom and Lefemine, 1975). The concept of a reactive metabolite of VC being responsible for the carcinogenic activity (Hefner et 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 a!.. 1975; Malavielle et al., 1975; Rannug et al.. 1974). Metabolites of VC have been identified in the urine of rats as conjugates of cysteine (Green and Hathway. 1975; Watanabe et al., 1976a). suggesting that VC is biotransformed to electrophilic metabolites and that the primary 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 al.. 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 study was funded by the companies supporting the vmvl chloride projects being administered by the \1 jnuiaeturm* Chemists Association. Washington, D C. Author to whom ail correspondence snould he sent, manuscript No 8 600* 1 73-77. n m | iiU I m\. 7X1144* ll<` IS/)' I"1 0 C\>p\riiril C fcv Academic Pfr**. 1 All rt^M* 4m' m an% foero reused Primed m Great 8n(jm 1100233 R&S160514 572 a \ r \n \m 11 bv reaction with glutathione. However. as the exposure is increased, detoxification will be impaired by the reduction of hepatic gluiainionc. Tr.is wi;| lead to an increase in the level of reactive metanolites and result in an increased reaction of these metabolites witn uitraceiluiar macrvimolecules. Chemical carcinogenesis has oeen auriouted to me reaction of electrophilic metabolites vuui intracellular macromolecules (Miller ar.c Miller. 1971). Recent reports have demonstrated that liver microsomal enzymes m intro :or~. reactive metabolites from VC wmch bind to the microsomes (Kappus ei ai. ;e`;i. protein sulfhydryl groups. R N A (Bolt et al.. 1975). anc adenosine of DN A i Bar bin et al.. 1975). In addition to reduced hepatic glutathione leading to an increase in the binding of reactive metabolites of VC with macromolecuies. suefi an effect may also be associated with other dose-dependent alterations in tne fate of \C in me boay. The dose dependence of the fate of VC has been elucidated kmetically ana attributed to saturable metaoolic pathways (Hefner et al.. 1975; Green and Hathway. 1975; Watanabe et al., 19763.0. In Liuo studies are needed to determine wnether cose-depencent disproportionate increases in the macromoiecular binamg of reactive m.etapobtes of VC with increasing exposure concentration may be associated wnh toxicity and carcinogenicity. Thus, the objective of this study was to characterize the binding of VC to neoatic macromolecuies and nucleic acids following exposure to various concentrations of ,JC-labe!ed VC. METHODS Materials. IJC-labeled V'C w-as synthesized from , i.2-:iC 1 I.I-dichloroethane (New England Nuclear. Lot Nos. 819-221 and 819-292. 5.0 and 4.8 mCi/mmol, respectively) immediately prior to use (Wagner and Muelder. !9~:j. Each new batch of ! 1.2-MC Idichloroethane was analyzed and yielded ||JC!VC with a radiochemical purity of 95"n. The synthesized IIJC1 VC has Deen snow n repeated;y in our laboratory to be 95 to 96t> radiochemicallv pure (Wagner et al.. 1975). Thus, wmie each synthesis of 1|4C|VC prior to each experiment was not analyzed, it was assumed to be of the same purity. Furthermore, no indication during a syntnesis which utilizes a gas chromatographic separation suggested that any deviation from previous syntheses had occurred. Nonlabelea VC (Maiheson Gas Products) of 99.9% minimum purity was mixed with the 1MC Imatenal to obtain the desired specific activity. Typically 40 ml of the 1IJCiVC-helium gas mixture was injected into a 5 to 10-liter Saran bag (Anspec. Inc.) containing the desired quantity of noniabeled VC. Animals. Male Sprague-Dawlev rats (Spartan Research Laboratory) weighing 220 to 250 g were used throughout the study. All animals were housec 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 o; rats (three to six animals per group) were exposed to [MCIVC (treated) or room air (controls) lor 6 hr between 9;00 am and 4:00 pm (EST). An additional group of five rats cretreatea wnn phenooarbitai (80 mg/kg/day, ip, 3 days prior to exposure) were exposed at the 100-ppm level. Exposure. Exposures were conducted in 30-liter glass inhalation chambers. 1JClabeled VC was metered into the chamber air flow (-6 liters/mm) with a dual syringe 01 00 234 R&5160515 VINYL CHLORIDE MACROMOLECL'LAR BINDING 573 pump. The analytical concentration of VC was monitored continuously by recirculating a fraction of the chamber atmosphere through an mfrareo soectrophotometer (Wilks) at a wavelength 10.6 um. In addition, samples (1 ml) of the cnamber atmosphere were anah zed at approximately hourly intervals during the exposure o> gas chromatograpny (Watanaoe et ai.. 1976c). At corresponding times, tne MC activity uas determined by bubbling 1-mi aliquots of the cnamber atmosphere into a scintillation solution containing a mixture of Concifluor (Maliincxroat Chemical), 2-methoxyethanol. toluene i6: 11:33). The radioactivity was determined by counting in a Mark II or Mark 111 liquid scintillation spectrometer (Searle Analytic. Inc.). The target 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 r 0.3 (SD). 9.3 r 0.2. 24.7 1.4. 5 1 r 2. 109 t 23, 250 3, 5 11 11. 1020 : 13. and 4600 311. The respecuve specific activities were 132.000. 4801. 3170. 2528. 1 '50. 837. 217, 301. and 50 dpm/ug of VC. The inhalation chamber was coeratec in a laboratory fume hood to prevent contamination of the working environment. After transit through the mhaiation chamoer tne 1UC]VC was adsorbed on activated charcoal. The charcoal traps were disposed of as radioactive waste accorcmg to standard regulations. Proceaure. Following the 6-hr exoosure to various concentrations of lIJC1VC (I5000 ppm), the rats were killed immediately bv a blow to the head. An aliquot of liver was sampled and used for determining hepatic nonprotein sulfhydry! content by a modification of the method of Sedlak and Lindsay (1968). Tne remaining liver was frozen immediately on dry ice and stored at --20C until analyzed. The carcass was analyzed for total radioactivity as descrioed previously (Watanabe ei ai. 1976a). Previous studies have shown that only a small percentage of radioactivity (<12%) is excreted as metabolites other than (1JC|YC during 72 hr followang a 6-hr inhalation exposure (W'atanabe ei ai, 1976c). The large proportion of the 12% is comprised of l4CO, excretion 72 hr after exposure. Furthermore, very little urine is excreted during the exposure period. Thus, the nonvolatile radioactivity determined in the tissue immediately after exposure is a good estimate of the total amount of metabolized VC. Macromolecular binding of l4C-Iabeled VC to hepatic tissue (protein, nucleic acids, and lipid) was determined by the method of follow et ai. (1973). The trichloroacetic acidprecipitable material following exhaustive solvent extraction was digested in 1 N KOH and the radioactivity was determined by liquid scintillation spectrometry. Hepatic nucleic acids (DNA 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 scintillation spectrometry (see below). Isolation of nucleic acids. RNA and DNA were isolated from rat liver by modification of the techniques described by Okuiiara (1970) and Irving and Veazey (1968). Approximately 10 g ot frozen tissue was thawed slowly and homogenized in 50 ml of 0.15 m sodium chtonde-0.04 m ethvlenediaminetetraacetic acid (EDTA) buffer (pH 8 .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 final concentration of 2%. Nucleic acids were precipitated using 1 vol of 95% ethanol and spooled onto a glass rod. The $ i Cu_j5 R&S160516 * 574 AATANABfc LT 4L precipitate was wasned with ethanol and dissolved in 20 mi ot'0.015 m sodium chloride0.0015 M sodium citrate Duller (pH 7.0). RNA was precimtatea by adding 1 vol or icecold o m potassium acetate (pH 7.5) ana remoxea by centrifugation. DNA was precipitated from tne suDernatant by adding 2 xoi of v5"u etr.anoi. The resulting crude DNA peilet was dissolved m 20 ml of 0.015 m soaium chlorice-0.0015 m sodium citrate buffer (pH 7.0'; and the solution was centrifuged at 100,000 ? for 60 min at 2C 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 precipitated by adding sodium acetate (4 g/100 mil and 2 voi of 2-ethoxvethanol. The precipitate was reaissoved in 10 ml of 0.015 m sodium chloride--0.0015 M sodium citrate at 23C. The final DNA precipitation was accomplished using 5 ml of ice-cold isoproDanol. RNA and DNA pellets were cried at 23C uncer reduced pressure and weigned. RNA ana DNA were quantified by tne orcmoi anc diohenylamme reactions, respectively (Keleti and Leaerer. 1974). RESULTS Hepatic macromolecuiar binding, hepatic nonprotem suifr.ycrvi content (primarily glutathione. GSH). and the total amount of VC metacoiizea following various exposure concentrations are summarized in Table i ana presentee grapnicallv in Figure 1. Covalent binding to hepatic macromolecules piotiea as a function of the log of the 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.point appeared to lie between 25 and 50 ppm. The metaoolism of VC and binding approacn a piateau at concentrations exceeding 250 ppm. Hepatic macromolecuiar binding correlated weii 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 (B/A x 100, Table I). This point is further substantiated by the constant fraction of bound versus total radioactivity m tne liver with increasing exposures. Although the value for total metabolism appears low for the 1000 ppm exposure, a corresponding reduction in macromoiecuiar binding 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 nonprqiein sulfhydryl content (primarily GSH) was not 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. Metaoolism of VC was not increased in rats exposed to 100 ppm of VC after pretreatment with pnenobarbital. Macromolecular binding, however, was increased markedly when compared to nonpretreated ammais. Isolation of RNA and DNA by a nondigestive procedure from the liver of animals exposed to 1, 100. 250, and 1000 ppm of VC failed to reveal any detectable radioactivity. The sensiuvity for detecting the radioactivity in DNA and RNA varied at U1C0236 R&S160517 VINYL CHLORIDE MACROMOLECLLAR BINDINC I A 111 I I I oi a i. Ml i Mini ism. Ml i* a i it M aiiujmcu i * m, ah Diniiini., ,\nij U i ta h< (ii ui \ miioni ((iSII) Coni inmimiow I ui i nvusn Kii \i u ms E'AI'OMJHt { fi Hit) in V IN t I (til OH II M (V('V' Nojniii.il oincciilriilioii 1 10 25 50 100 250 500 1000 5000 A {/ig or VC cumviiluiils mcOituiliml) 29 8 * 3.2 242 * 26 557 i 42 1181 l 91 2406 *173 .1826 * .14 5 6261 t 355 4257 * 765 9255 * 1.467 /( 0<g of VC ci|iiiviik'ms houiul per g of prutem} 0.5 1 0.08 3.1 t 0.2 12 2* 4 0 213 t 3.4 4 7.6 i 4.8 89 6 i 12.3 98 8 l 5 0 106 8 l 22 2 111.5 l 10 4 /;/.-f v 100 1.78 L 0.4 8 1.38 t 0.16 2 15 l 0 60 1.99 * 0.16 1 99 1 0.26 2.35 i 0.28 1.58 i 0 21* 2 55 i 0.58 1 12 * 0.13 1 IcjMtiL C iSI 1 Llllllrol) 104 84 41 91 HI* 70* 60* 5 1* .14* Percentol lulal 1'( ' ;ilIh ill ill In i'l l>omn1 to m.iciixiioIcluks 20 i 1 21 * 2 21 * 2 20 i 2 25 i 2 > > i "> 25 t 1 22 * 2 22 * 1 l'ri.'l teat men t mih pliL'mibiif hil.il' 100 2161) 1 160 800 t 23 9 1 1(1 i 082 " Means * SI > h Sl.ilisliciill) c. 1111V r ci 11 hum v-i mt i i nK . St mini Is / tol (ft > tl (IM 1 Hals w ere mjvUCiil IJ> Wilh soilmni phcnobarlntul HO Inr 5 da vs prior to exposure il *ji G9 o cz, ro oj "i etsogis'sy 57 fi WATANABE et al. each exposure level because the specific activity of the |I4C)VC varied with exposure and the different amounts of DNA and RN'A analyzed. The sensitivity limits for ihe 1-. I00-. 250-, and 1000-ppm exposures were 0.007. 0.051. 0.108, and 0.30 ,ug of VC equivaients/mg of DNA and 0.0001. 0.009. 0,018. and 0.05 u% of VC equivalents/mg of RN'A. R&S160519 E<oouj^ rz*' Fig I. Hepatic macromolecular binding and glutathione (GSH1 depression expressed as a function of the logantnm of the exposure concentration to vinyl chloride (VC). Macromolecular binding () is expressed a> microgram equivalents of VC bound per gram of protein fmean ~ SD) GSH {) is expressed as percentage of control. DISCUSSION The results indicated that the total amount of radioactivity bound to macromolecules in the liter did not increase proportionately to the increase in the exposure concentration of VC. Macromolecular binding was related directly 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 inflection points below 50 and above 250 ppm. respectively. Above 500 ppm binding appeared to plateau. Below 100 ppm binding was approximately proportional to the increase in exposure. It is particularly significant that available data (Maltom. 1975) indicate that the percentage induction of hepatic angiosarcoma in rats is linear between 50 and 500 ppm VINYL CHLORIDE MACROMOLECULAR BINDING 577 when expressed as the log of the exposure concentration. Above 2500 ppm tumor incidence is constant. This correlates with the plateau effect observed in total metabolism and hepatic macromolecular binding above 500 ppm in the present study. One mechanism of chemical carcinogenesis is beiieved to be reaction of electrophilic metabolites with intracellular macromolecules (Miller and Miller. 1971). The covalent binding to hepatic macromolecuies reflects the activation of VC to reactive metabolites. Therefore, both total metabolism of VC and covalent binding appear to correlate with 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 below 50 ppm is not clear. 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 bioassavs currently being concuctea at 25. 10, and 1 ppm become available. The reactive metabolites of VC are detoxified presumably by reaction with GSH (Watanabe et at.. 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 snowed that a single 50 ppm exposure to VC was in the threshold zone for depression of hepatic GSH (Watanabe et at.. 1976b). This suggests that carcinogenicity of VC is related to the decreased ability to detoxify the reactive metabolites of VC. Covalent binding of radioactivity to isolated nucleic acids (both RNA and DNA) was not detectable in any of the exposure groups tested. The detection limit in all groups was sufficient to detect the -iC activity if it were equailv distributed by weight throughout the components of the liver. Therefore, it was concluded that VC does not preferentially react with intracellular nucleic acids. In contrast to this, initial studies by Bolt et at. (1976a) reported covalent binding of radioactivity to DNA and RNA following a 5-hr static exposure to 145 ppm of [IJC]VC. The specific activity used by Bolt et at. (1976a) was higher than that used in our study and this may account for the difference in the two observations. However, more recently it has been reported by this group (Laib and Bolt. 1977) that greater than 95% of the radioactivity associated with RNA following inhalation exposure in rats to 1300 ppm of [ UC]VC for 5 hr is due to one carbon fragment incorporation of MC into the native nucleotides. Both our results and those of Bolt et at. (1976a) confirm the conclusion that VC does not preferentially react with hepatic nucleic acids. It is important to emphasize that the methodologies employed oniv detect covateni binding and exclude any other more subtle interactions. Therefore, 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. Pretreatment with phenobarbital did not increase the total metabolism of VC. Bolt et at. (1976b) have reported similar data showing that phenobarbital pretreatment failed to stimulate the uptake of V'C following a static inhalation exposure. Although total metabolism was not affected by phenobarbital. the macromolecular binding was increased markedly. The true significance of the increased binding is difficult to interpret since phenobarbital increases total protein in the liver, and the increase in 11 00239 R&St 60520 I J 578 waTanabe et al. binding may reflect a nonspecific interaction merely due to an increase in the available protein binding sues. 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 metabolites of VC to hepatic macromolecules as the exposure concentration is increased. Even more significantly, there was no eviaence for any preferential binding of electrophilic metabolites to nucleic acids of hepatocytes. This suggests that the carcino genic activity of VC may not be associated directly with this commonly accepted mechanism for carcinogenesis. Before excluding tms mecnamsm entirely, additional experiments are needed to show 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 ennance binding to nucleic acids. This is important because phenoDarbital does increase the nepatic macromolecular binding of MC activity to macromolecules in toto in rats exposed to 100 ppm even thougn it did not increase the total amount of VC metaoolized. These aspects are being explored. Even more important, pefore excluding aikylation 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 ail studies of metabolic and clinical parameters to date base been either conducted on or related to the hepatocvte. The hepatocytes may constitute primarily a means for detoxification since they are not particularly susceptible to VC induced toxicity. Toxicity may be induced in tissues with smaller capacity to detoxify the reactive metabolites of VC. The induction of tumors of the nervous system by ethylnitrosourea has been correlated with the persistence of 0-6-ethyiguanme in the nervous system (Goth and Rajewsky, 1974). Although other metabolizing organs such as the liver produce O-6-ethylguanme, the turnover rate of DNA mediated by repair mechanisms is sufficient to prevent induction of cancer. Likewise, the mechanism of carcinogenesis of VC may be due to an inability of the target tissue, in this case endothelium, to repair lesions whether the reaction be with nucleic acids or critical proteins. REFERENCES Barbin. A . Bresil. H.. Croisy. A.. Jacqltgnon. P., Malavielle. C., Montesano. R.. and Bartsch, H. (ly75). Liver microsome mediated formation of alkylating agents from vinyl bromide and vinyl chloride. Biochem. Btophvs. Res. Conunun. 67. 596-603- Bartsch. H.. Malavielle. C.. and Montesano. R. ( 1975). Human, rat. and mouse liver mediated mutagenicity of vinyl chloride in Salmonella typhtmunum strains. Ini.J. Cancer 15. 429-437. Bolt. H. M.. Kappls, H.. Blchter. A., and Bolt, W. ( 1975). Meiaoolism of vinyl chloride. Lancet 1425. 3olt. H. M.. Kappls. H.. Blchter. A., and Bolt, \v, (1976b). Disposition of l,2-'JC-vinyl chloride in the rat Arch. Tosicol. 35. 153-162. 3oi r. H. M . K appl s, H.. K al'fman.n. R,, appfl. K. ., Blchter. A., and Bolt. W. (1976a). Metabolism of IJC vinyl chloride in atro and in nco. INSERM 52. 15 1-164. Creech. J. L., and Johnson. M. N. (1974). Angiosarcoma of liver in the manufacture of poly vmv i chloride. J. Occup. Med. 16. 150-151. SI 00240 R&S160521 VINYL CHLORIDE MACROMOLECULAR BINDING 579 Goth. R.. and Rajewskv. M. F. (1974). Persistence of 0-6-ethylguanine in rat brain DNA: Correlation itn nervous system-specific carcinogenesis by ethyimtrosurea. Proc. Sat. Acad. Set. USA 71. 639-643. Green. T.. and Hathwaa. D, E. (1975). The biological fate in rats of vinyl chloride in relation to its carcinogenicity. Chem. Biol. Interac. 1 1. 545-567. Hefner, R. E.. Jr.. Watanabe. P. G.. and Gehring. P. J. (1975). Preliminary studies of the Tate of inhaled \ :nyl chloride monomer ( VCM) in rats. A nn. V. )' A cad. Sct. 246. 135-148. Irving. C. C,. and Veazey. R. a. (1968). Isolation of deoxyribonucleic acid and nbosomal ribonucleic acid from rat liver Bioehtm. Biophys. Acta. 166, 246-248. Jollow. D, J.. Mitchell. J. R.. Potter. W. 2.. Davis. D. C.. Gillette. J. R., and Brodie. B. B. (1973). Acetaminopnen-ir.cuced hepatic necrosis. II. J. Pharmacol. Exp. Ther. 187, 195- 202. Kappls. H.. Bolt. H. M.. Buchter. A., and Bolt. W. (1975). Rat liver mcrosomes catalyze covalent binding of l4C-vinyl chloride to macromolecules. Sature <London) 257, 134-135, Keleti. G.. and Lederer. W H. (1974). Handbook of Methods for the Biological Sciences. Van Nostrana-Reinhold. New York. Laib, R. J.. and Bolt. H. M. (1977). Alkylation of RNA b> vinyl chloride metabolites m enro and in rico. Formation of 1- V`-etheno adenosine. Arch. Toxicol.. :n press. Malavtelle. 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Biocnem. 25. 192-205. Van Duuren, B. L. (1975). On tne possible mechanism of carcinogenic action of vinv I chloride. Ann. X. Y. Acad. Sa. 246, 258-267. Wagner. E. R,. and Ml'ELDER. W, W, (1975). A procedure for preparing iJC labeled vinvl chloride. ,4nn V Y. Acad. Sc:. 246. 152- 153. Wagner. E. R.. Muelder. W, W.. Watanabe. P. G.. Hefner. R, E.. Jr.. Braun. W. H.. and Gehring, P. J. (1975). Gas cnromatographic method for tne preparation of IJC-labeled vinyl chloride. J. Labeled Compounds 1 1. 535-542. Watanabe, P. G.. Hefner. R. E.. Jr., and Gehring. P. J. (I976bi. Vinyl chloride induced depression of hepatic nonprotein sulfhydryl content and effects on Dromosulphthalein (BSP) clearance in rats. Toxicology 6, 1-8. W \tan vrf. P. G.. McGowan. G. R.. and Gehring. P. J (I9~6ai, Fate of IJC-vinyl chloride after single oral administration in rats. Toxicol. Appl. Pharmacol. 36. 339-352. W atanabe, P. G.. McGowan. G. R., Madrid, E. 0., and Gehring. P. J. (1976c). Fate of ,JC-v inyl chloride following inhalation exposure in rats. Toxicol. App.. Pharmacol. 37, 49-59 R&S160522