Document ExZN3x8Qq1EVvkyxy4G5e6a6b

R&S 022559 R & D REPORT (J(. ft:..?. / / f' DOW CHEMICAL U.S.A, RESTRICTED for uv* Within Ihr* t)).*. Ch:mi< ,i` Romp-in/ r.nly ut.v * ti ruM i Toxicology Research Laboratory 7 IVlFBCITOPl lAIIOIfOUHr HUMrjffT f ti(/r. IUJT K-00171J - {2-5) nATI I'. ViL. w Anril 7, 1977 l'lfOL<LI.M fiO 0, Oj 0( 7 1,5 5 Hepatic Macromoleculnr Binding Following E:-:: osurc to Vinyl 23 Chloride !u f nr ! P. G. Watonabe, J. A. Compel, D. G. Pegg, and p. J, Gehring PAGL3 IN t-ULt REPORT ____L Cm NUMBER itncfor also to career related reports and publications.) PATENT STATUS. disclosure sub'nitrpd iDESCmPTlVE SUMMARY WITH CONCLUSIONS case filed ;____ no patent action required Covalent oinding of radioactivity to hepatic macrcmolecv exposed to *^C-labeled vinyl chloride (VC) was studied to determine if VC induced carcinogenesis may be related to electrophilic alkyla tion of maeromcleculus in vjyo. Male Snraouo-Dawiov rats were exposed to 1, 10, 25, 50, 100, 230, 500, 1000, or 5000 pom -^c-VC for 6 hours. Following e; radioactivity covalently bound to hepatic macromolocules and pur led nucleic acids (RMA, DMA) were determined. The total amount of i`iC-VC metabolized and hepatic glutathione (GSi!) content was also determined. The total amount of radioactivity bound to macromolecules in the liver did not increase proportionately with the increase in the exposure concentration of VC. A disproportionate decrease in macro-molecular 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 bound to macro- molecules in the liver correlates with induction of hepatic angio sarcoma. There was no preferential binding of radioactivity to either DMA or RNA in the liver. Hepatic glutathione content was significantly depressed only at exposure concentrations greater than 100 ppm. TOXICOLOGY RESEARCH LABORATORY HEALTH AND ENVIRONMENTAL RESEARCH DOW CHEMICAL U.S.A. LOCATER SHEET FILE NO. HET K^17T-t-(24) DEAD STORAGE NO. 1477 TITLE OF REPORT: Hepatic macromolecular binding following exposure to vinyl chloride DATE REPORT ISSUED: 3D fio to roo M O) oo AUTHOR(s): p. g. Watanabe, J. A. Zcmpol, D. G. Pegg and P. J. Oehring Notebook(s) - (Number and Page(s): 1). NBK 13-5 (1-47) 43 2). 53 33 63 SPECIMENS: PATHOLOGY NUMBERS: 1)___________ 2)_________ 33___________ 43___________ ADDITIONAL DOCUMENTS: 13__________________________ LOCATION: Dead Storage 2 3. 3 3. 43. RESTRICTED FOR USE WITHIN THE DOW CHEMICAL COMPANY ONLY. THIS PAGE SHOULD FOLLOW THE DOW SUMMARY PAGE AND BE ATTACHED TO COPIES MAINTAINED ONLY IN THE TOXICOLOGY RESEARCH ARCHIVES AND QUALITY ASSURANCE UNIT FILES. P. G. Watanabe, J. A. Xor.pcjl, D. G. Pccjq and P. J. Gehrir.'j 3) 0) 0rroo 01 o xmBi .t hepatic MACP.o.MOi.ECur.AH BiumuG foi.j.ov.m;;r, CHLORIDE jkuh", to vi:;y:. Bv: P. C. Watanubo, J. A. Zc.t.;;ci , D. O. Hogg, and P. J. Gohrm ABSTRACT Covalent binding of radioactivity to hepatic macromolecuios in rats exposed to ^C-labelcd vinyl chloride (VC) was studied to determine if VC induced carcinogenesis may be related to electrophilic alkylation of macrcmolecules in vivo. Male Spraguc-P ic-y rats were exposed to 1, 10 , 25, 50, 100 , 250, 500, 1000 or 5000 ppm 1>,C-VC for 6 hours. Following exposure radicacti"ity covalently bound to hepatic macromolecuios and purified nucleic acids (RNA, DMA) were determined. The total amount of `*C-VC metabolized and hepatic glutathione (GSH) content was also determined. The total amount of raoioactivitbound to macromoleculcs in the liver did not increase propor tionately with 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 to macromolecuios in the liver correlates with induction of hepatic angiosarcoma. There was no preferential binding of radioactivity to either DMA or REA in the liver. Hepatic glutathione content was significantly depressed only at exposure concentrations greater than 100 ppm TflKESSSSSBr . 7.V -ayKE* tr- **->' INTRODUCTION Considerable effort has boon devoted to research on vinyl chloride (VC) since it was demonstrated to be carcinogenic in man and animals (Creech and Johnson, 10/4; Maltoni. and Lefcmine; 1975) . The concept of a reactive metabolite of VC being responsible for the carcinogenic activity (Hefner, ot al_. , 1975; Van Duuren, 1975) is supported by evidence of enhanced, mutagenic activity of VC to bacteria in the presence of microsomal enzyme activating systems (Bartsn, et al., 1975; Malnvielle, et a_I. , 1975; Rar.nug, et al > 1974). Metabolites of VC have been identified in the urine of rats as conjugates of cysteine (Green and Hathvay, 1975; Watanabe, ot al., 1976a), suggesting that VC is biotransformed to electrophilic metabolites and that tne primary detoxifica tion mechanism for these metabolites is corrugation with heoatic glutathione prior to excretion . Comp]imentary to these data was elucidation of a doso-r elated reduction of hepatic glutathione in rats exposed to 50-2000 pom VC for 7 hours (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 by reaction with glutathione. However, as the exposure is increased, detoxification will be impaired bv 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 metabolite;: with intracol lular mneremolecu 1 os. 2- - Chemical carcinogenesis has beer, attributed to the reaction of electrophiles with intracellular macromolecules (Miller and Miller, 1971). Recent reports have demonstrated that liver microsomal enzymes, iri vitro, form reactive metabolites from VC which bind to the microsomes (Kappus, et al_. , 1975) protein sulfhydryl groups, RNA (Bolt, et al., 1975) and adenosine of DNA (Barbin, et a_l. , 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 dependency of the fate of VC has been elucidated kinetically and attributed to saturable metabolic pathways (Hefner, et al^, 1975; Green and Hathway, 1975; and Watanabe, et al_. , 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 concentra tion may be associated with toxicity and carcinogenicity. Thus, the objective of this study was to characterize the binding of VC to hepatic macromolecules and nucleic acids following exposure to various concentrations of 14 C-labeled VC. R&S 022564 mi -3- METHODS Material. *4c-labeled VC was synthesized from (1,2-***C) 1,2-dichloroethane (Now England Nuclear, Lot #819-221 and 819-292, 5.0 and 4.8 mCi/mmole, respectively) immediately prior to use (Wagner and Muelder, 1975). The synthesized 14 ' C-VC has been reported to be 95-96% radiochemicallv pure (Wagner,, et al., 1975). Non-labeled VC (Matheson Gas Products) of 99.9% minimum purity was mixed with the 14 Cmaterial to obtain the desired specific activity. Typically, 14 40 ml of the C-VC, helium gas mixture was injected into a 5-10 liter Saran bag (Anspec, Inc.) containing the desired quantity of non-labeled VC. Animals. Male Sprague-Dawley rats (Spartan Research Laboratory) weighing 220-250 g were used throughout the study. All animals were housed in rooms in which a constant humidity, temperature and a 12 hour light-dark cycle (7 AM 7 PM, EST) were maintained. Food and water were provided ad libitum except during exposure. Exposures were conducted between 9:00 AM and 4:00 PM. Groups of rats (3-6) were exposed to 1, 10, 25, 50, 100, 250, 500, 1000, or 5000 ppm 14 C-VC for 6 hours. Control animals used for hepatic nonprotein sulfhydryl determinations (5/group) were exposed concomitantly to room air. An additional group of 5 rats pretreated with phenobarbitai (SO mg/kg/day, ip, 3 days prior to exposure) were exposed at the 100 ppm level. R&S 022565 R&S 022566 -4- Exposure. The rats were exposed under dynamic conditions to varying concentrations of ^C-VC (treated) or room air (control) in 30 % glass inhalation chambers. ^4C-labeled VC was metered into the chamber air flow (-6 i/min) with a dual syringe pump. The nominal concentration of VC was the ratio of the rate at which the VC gas was dispersed to the total chamber air flow. 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 u. In addition, samples (1 ml) of the chamber atmosphere were analyzed at approximate hour intervals during the exposure by gas chromatography (Watanabe, et a^., 1976c). At corres ponding times, the ^C-activitv was determined by bubbling 1 ml aliquots of the chamber atmosphere into a scintillation solution containing Concifluor (Mallinckrodt Chemical), 2methoxvethanol, toluene (6:11:83). The radioactivity was determined by counting in a Mark II or Mark III liquid scintillation spectrometer (Searlc Analytic, Inc.). The nominal concentrations of VC were 1, 10, 25, 50, 100, 250, 5G0, 1000, and 5000 ppm. The respective mean analytical concentrations measured by gas chromatography were 1.4+0.3 (SD), 9.30.2, 24.7+1.4, 51+2, 109+23, 250+3, 511+11, 1020+13 and 4600+311. The respective specific activities wore 132,000, 4801, 3170, 2528, 1750, 837, 237, 301, and 50 DPM/'.tg VC. \ T*jjsLi_5&7_* - sk -. T' ---- 1-- I- *----r . T*.-, ; *V,- .-i-C./.' ,, on activated charcoal. The charcoal traps were disposed of as radioactive waste according to standard regulations. Oro cn O) >1 i Procedure. Following the 6 hour exposure to varying conccntrations of 14 C-VC (1-5000 ppm) the rats were killed immedlately by a blow to the head. An aliquot of liver was sampled and used for determining hepatic nonprotcir. sulf'nydryl content by a modification of the method of Sedlak and Lindsay (19G3), The remaining liver was frozen immediately on dry ice and stored at -20C until analyzed. The carcass was analyzed for total radioactivity as described previously (VJatanabe, e_t a_l. , 1970a). The radioactivity determined in the tissue and carcass was non-volatile, therefore this radioactivity represented the total amount of metabolized VC. Protein binding of 14 C-labelcd VC to hepatic tissue was determined by the method of Jollow, et_ a_l. , (1973). Hepatic nucleic acid:: (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 scintillation spectrometry (sec below). .1 Isolation of Nucleic Acids. RNA and DMA were isolated from rat liver by modification of the techniques described by R&S 022568 6- - Okuiiara (1970) and Irviny and Vcazcy (1963). Aoproxirately 10 g frozen tissue was thawed slowly and homcqeni .ed in 50 mi 0.15M sodium chloride - 0.04M ethylenod famine to tr'i :v:; r: acid (EDTA) buffer (pH 8.0). Sodium dodocyl sulphate vl g) was added, followed by 1 volume 90" phenol solution. The mixture was mechanically stirred for 30 minutes at room temperature and the phenol and water phases separated by centrif ugation. The water p;;aso was collected and sodium acetate added to a final concentration of 21. Nucleic acids were precipitated using 1 volume 951 ethanol and spooled on a glass rod. The precipitate was washed with ethanol and dissolved in 20 ml 0.015M sodium chloride - 0.0015M sodium citrate buffer (pH 7.0). RNA was precipitated by addition of 1 volue ice cold 6M potassium acetate (pH 7.5) and rdmoved by centrifugation. DNA was prccioitated from t'nc supernatant by addition of 2 volumes 952- ethanol. The resulting crude DNA pellet was dissolved in 20 ml 0.015M sodium chloride-0.0015M sodium citrate buffer (pH 7.0) and the solution centrifuged at 100,000 x g for 60 minutes at 2C to remove glycogen. RNAasc (3 mg/50 ml) was added to the supernatant and the solution incubated at 37C for 30 minutes. One volume of phenol saturated with 0.15M sodium chloride-0.015M sodium citrate (pH 7.0) was added and' the mixture stirred for 30 minutes at room temperature. The aqueous phase was extracted with ether and DMA was -x*- *^-:r" precipitated by addition of sodium acetate C4 e/100 ml) and 2 volumes 2-etho:<ycthanol. The precipitate was rcdissolvcd in 10 ml 0.015M sodium chloride-0.001sodium citrate at 2C. The final DMA precipitation was accomplished usinc 5 ml ice cold isopropanol. RMA and DMA pellets were dryed at 2C under reduced pressure and weighed. RMA and DMA were quantified by the orcinol and dipher.ylamino reactions respectively (Keleti and Lodcrer, 197-!) . 3D CO O IO cNn) o> <o 1 i` R&S 022570 -8- RES LILTS Hepatic macromolecular binding, hepatic non-protein sulfhydryl content (primarily glutathione, HSU), and the total amount of VC metabolized following various exposure concentrations are summarized in Table 1 and presented graphically in Figure 1. Covalent binding to hepatic macromolecules plotted 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 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 tote] metabolized VC (B/A 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 total metabolism appears low for the 1000 ppm exposure, a corresponding reduction in macromolecular binding was not observed. The apparent discrepancy between total metabolism of VC at 500 and 1LO0 ppm is not understood fully, but it may be due to differences in respiratory parameters causing differences in the uptake of VC. ~v i -` -9Hepntic non-protein sulfhydrvl content (primarily GSM was not depressed significantly at 1, 10, 25 or 50 ppm. Only at concen trations 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 VC after protreatment with nhenobarbita1. Macromolecular binding, however, was increased markedly when compared to non-pretreated animals. Isolation of RNA and DMA by a non-digestive procedure from the liver of animals exposed to 1, 100, 250, and 1000 ppm VC failed to reveal any detectable radioactivity. The sensitivity was such that if the hepatic radioactivity was distributed uniformly by weight throughout the tissue, moasureable radio activity would have been detected in the iso]a ted nucleic acids. - n *- * R&S 022571 -10dtscuss 10:: The results indicated that the total nr,;ount of radioactivity bound to macromolecules in the liver did net increase proportionately with the increase in the exposure concentra tion of VC. Instead, covalent binding of metabolites of VC to hepatic macromolecules decreased disproportionately with the increase in exposure. Macromclecular binding was related directly to the total amount of VC which was metabolised over exposure concentrations ranging from 1 to 5000 ppm VC. Macro- molecular binding plotted as a function of the log of the exposure concentration gave a sigmoid-shaped curve. The s linear portion of the curve is bounded by lov; and high inflec tion 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 (Maltoni, 1975) indicate that the percent induction of hepatic angiosarvoma in rats is linear between 50 and 500 ppm 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 macrcmolecular binding above 500 ppm in the present study. One mechanism of chemical carcinogenesis is believed to be reaction, of o 1 ectronhi 1 ic metabolites with intracellular to Cl R&S 022573 -11- macromolcculcs (Miller and Miller, 1971). The covalent i^-i-riding to hepatic macrcmol ecu 1 c is reflects the activation of VC to reactive motnbol i ter.. Therefore, both total metabolism of VC and covalent binclinq appear to correlate with the induction of hepatic angiosarcoma in rats exposed to concen trations 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 higner levels indicates that the carcinogenic resnor.se o? the population may bo chanqeu at lower level exposures. However, twis hypo tries is can not be val ixahec. until the results carcinogenesis bioassays currently being conducted at 25, 10, and 1 ppm become available. The reactive metabolites of VC are detoxified presumable reaction with GDI! (Ivatanabe, et a] . , 197Gb) . Therefore, it is important that significant depression of hepatic GSH was dose--related only at levels of 100 nnm and greater. This is consistent with previous studies which showed that a single 50 ppm exposure to VC was in the threshold cone for depression of hepatic GGH (Katanabc, ct al., 197Gb). This suggests that carcinogenicity of VC is related to the degreased ability to detoxify the reactive metabolites of VC. Covalent binding of radioactivity to isolated nucleic acids (both R:;.* and DMA) was not detectable in any of the exposure groups tested. The detection limit in all groups was sufficient to detect the ^C-activity if it were equally 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 observation Bolt, e_t al_. , (1 976a) reported covalent binding of radioactivity to DMA and RMA following a 5 hour static exposure to 145 ppm 14 C-VC. The specific activity used by Bolt, e_t a_l. , (1976a) was higher than used in our study and this may account for the difference in the two observations. However, the amount of binding to nucleic acids reported by Bolt, et_ a_l. , (1976a) was net greater than that which could be predicted to be due to one carbon fragment incorporated into native nucleotides. The authors present data supporting the concept that the radioactivity associated with nucleic acids following exposure to ^C-VC is not due to incorporation into native nucleotides. However, due to the very small degree of binding observed, until the bound species can be identified in vivo this will remain a critical question. R&S 022574 ) 1 ? I j R&S 022575 1 3- Both our results ant! that of Bolt, e_t a_l., (1976a) confirm the conclusion that VC does not preferentially react with hepatic nucleic acids. It is important to emphasize that the methodo logies employed only detect covalent bindinn n-d exclude any other more subtle interactions. Therefore, a very small degree of covalent binding to nucleic acids docs not exclude the possibility of other interactions which result in the loss of the ability to control cellular replication. Pretreatment with phenobarbital did nor increase trie total metabolism of VC. Bolt, o_t a_l. , (19/6o) nas reported similar data showing that phenobarbital pretreatmont failed to stimulate the uptake of VC following a static inhalation exposure. Although total metabolism was not affected by phenobarbital, the protein binding was increases markedly. The true significance of the increased protein oindir.g is difficult to interpret since phenobarbital increases total protein in the liver, and the increase in binding may reflect a`nonspecific interaction merely due to an increase in the available protein binding sites. i In summary, the results of the studies reported herein do not associate the carcinogenic effect of VC with a dispropor tionate increase in binding of electrophilic metabolites of `''Trr'X'. -14- 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 nucleic acids of hepatocytcs. 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 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 enhance binding to nucleic acids. This is important because phenobarbital does increase the hepatic nacromolecular binding of 14 C activity to macromolecuj..es i.n to.to m rats exposed to 100 ppm even though it did not incrcas the total amount of VC metabolized. These aspects arc- being n.plored. Even more important before exclusion of 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 hepatocytcs. To date essentially all studios of metabolic and clinical parameters have been either conducted on or related to the hepatocytc. T'-T R&S 022576 -15- Thc hepatocyte may constitute primarily a means for detoxifi cation sinct they are not particularly susceptible to VC induced toxicity. Toxicity may be induced in tissues with smaller capacity to detoxify the reacti'"- metabolites of VC. r The induction of tumors of the nervous system by ethylnitrosourea has been correlated with the persistence of 0-6ethylguanine in the nervous system (Goth and Rajewsky, 1974). Although other metabolizing organs such as the liver produce 0-6-ethylguanine the turnover rate of DMA 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. R&S 022577 -16- REFERENCES Barbi.n, A., Brcsil, H., Croisy,' A., Jncquignon, P., Malaviclle, C., Montesano, R. and Bartsch, H. (1975). Liver microsonc mediated formation of alkylating naer.ts from vinyl bromide and vinyl chloride. Biochem. Biophys. P.es. Comm., 67, 596-603. Bartsch, II., 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, 1425. Bolt, H. M., Kappus, II., Kaufmann, R. , Appel, K. E., Buchter, A. and Bolt, W*. (1976a). Metabolism of 1''C-vinyl chloride in vitro and in vivo. IM5ERM, 52^ 151-164. Bolt, H. M. , Kappus, II., Buchter, A., and Bolt, W. (1976b). Disposition of 1,2-1 !`C-vinyl chloride in the rat, Arch. Toxicol., 35, 153-162. Creech, J. L. and Johnson, M. N. (1974). Angiosarcoma of liver in the manufacture of polvvinyl chloride, J. Occun. Med., 16, 150-151. Goth, R. and Rajewskv, M. F. (1974). Persistar.ee of 0-6ethylguanir.c in rat brain DMA: correlation with nervous system - specific carcinogenesis by ethyl.oi trosurea. Proc Mat. Acad. Sci. , 71_ (3), 639-643. Green, T. and Hathway, D. E. (1975). The biological fate in rats of vinyl chloride in relation to its carcinogenicity. Chen. Biol. Interac., 11, 545-562. Hefner, R. E. , Jr., Watanabe, P. G., and Ce'nring, P. J. (1975). Preliminary studies of the fate of inhaled vinyl chloride monomer (VCM) in rats, Ann. M.Y. Acad. Sci., 246, 135-148. Irving, C. C. and Veazey, R. A. (1968). Isolation of deoxy ribonucleic acid and ribosomal ribonucleic acid from rat liver, Biochem. Biophys. Acta., 166, 246-248. Jollow, D. J., Thorgcirsson, S. S., Potter, W. Z., Hashiinoto, M. and Mitchell, J. R. (1974). Acetaminophen-indeced hepatic necrosis VI., Pharmaco .1 otiy . 12 . 251-271. O R&S R&S 022579 H t .! e ..: i l, ! f >, L' A id k -17- Kappus, H., Bolt, H. M., Buchter, A. and Bolt, W. (3975). Rat liver microsomes catalyze covalent binding of 14c-vinyl chloride to macromolecules, Na tore, 257 , 13-1 -- 135 . Keleti, G. and Lederer, w. H. (197*3) . Handbook of Methods for the Biological Sciences, Van Hostrand Rcinhold Co., New York, N.Y. Malavielle, C., Bartsch, H., Barbin, A., Camus, A. M. and Montcsano, R. (1975 . Mutagenicity of vinyl chloride, chloroethyleneoxide, chloroacctnldehydc and chloroethancl. Diochem. Biophvs. Res. Comm., 63, 363-370. Maltoni, C. and Lefemine, G. (1975) . Carcinogenicity assays of vinyl chloride: Current Results. Ann. N.Y. Acad.* Sci., 246, 195-224. ;" Maltoni, C. (1975) . The value of predictive experimental environmental carcinogenesis. Anbio, 4, 13-23. Miller, J. A. and Miller, E. C. (1971) . Chemical carcinogenosis: mechanisms and aooroachos tc its control. J. Mat. Cancer Inst. , 47_, 5-14.'* Okuiiara, E. (1970). Preparation of mammalian deoxyribo nucleic acid by SDS-nhenol treatment. /Anal. 3iochen. , 37, 175-173. Rannug, U., Johansson, A., Ramcl, C. and Wachtmeistor, C. A. (1974). The mutagenicity of vinyl chloride after metabolic activation, Ambio, 3^ 194-197. Sedlak, J. and Lindsay, R. M. (1963). Estimation of total protein-bound, and nonprotein sulfhydryi groups in tissue with Ellman's Reagent, Analyt. Diochem., 25, 192-205 . Van Duuren, B. L. (1975). On ..be possible mechanism of carcinogenic action of vinvl chloride. Ann. N.Y. Acad. Sci. , 24 6 , 250-267. Wagner, E. R. and Muelder, W. VI. (1975) . A procedure for prcDnring l4C-labelcd vinvl chloride. Ann. N.Y. Acad. Sci., 24_6, 152-153. * Wagner, E. R. , Muelder, W. W., Watanabe, P. G., Hefner, R. E., Braun, W. H., and Gearing, P. J. (1975). Gas chromato graphic method for the preparation of :4C-labeled vinyl chloride, J. Labeled Compounds, 11, 535-542. Jr., f adware :.<r tjSSSL'.'.- 13- Watanabe, P. G., McGowan, C R., anc c<..-hrir.o, P. J. (10 7 6a). I-'ata of 1``C-vinyl chi'ratio afte'* single oral administra tion in rats, Toxicol. Appl. Pharraco 1 , 36, 339-352 . Katar.abo, P. G., Hefner, R. E. Jr., and Gehring, P. J. (1976b) Vinyl chloride induced depression of hepatic nor.protein sulfhydryl content and effects on bromosulphtnaloin (BSP) clearance in rats. Toxicology, 6, 1-3. Watanabc, P. G., McGowan, G. R., Madrid, E. 0., and Gehring, P. J. (1976c). Fate of 1``C-vinyl chloride following inhalation excosure in rats. Tox i.col . Paxil . Pharmacol . , 37, 49-59. 33 </) o rMo cn oo o ifi. i!i f; l, ? f' J i. , ! ' i; -V r* / Tut a L Metabolism, Hepatic Macrone i iru 1 a r Hindi up. and Hepatic C.luLatliione ^CSH) Levels i\> l lou* i m; Inhalation exposure (6 Hours) To Vinyl Chloride (VC) Conian 1 Cone . 1 it) 50 100 250 500 1000 5000 A i:p. VC Pcpiivalenls Meta he!ized 29.Si3.3 2'. 212(i 457i42 1 ,181 i93 2, 4 00 j 173 J,82613-0 (), 2(<31355 ',,2571765 lJ.25511,467 __________ P. ____ VC l'<|u i va 1 en t s l.-uind Per e. Protein 0.3.10.08 3. 1 '.0.2 12.214.0 2 3.313.4 47.614.8 89.6H2.3 93.815.0 106.8122.2 113.5110.4 jl/A x 100 1 .7810.48 1.38 *0. in 2. 1410.60 1 .9910.36 1.9910.26 3.3310.28 1 . 53.Ml. 20 2.3510.53 1.L2i0.13 Hepatic OSH (;' Control) i 04 89 93 94 8 i1 70h 60b 51b 39b .,Pereeu t of lot a 1 l -tC....-..A....e....t.i.v.. ilv m ,Li. ver Pound to Maerono1e,n 1es 20t3 21 *2 2 14 2 20i2 2512 22l2 2513 +'i 'y n 22i3 pretreuneni will] phcnobarbital1 100 2,1601166 80.0123.9 3.7010.82 ** Means 1 standard deviation Statistically different from controls. Student t-tost (p < 0.05) Rats were injected ip with sodium plienoharbi Lai , 80 mp./lti; for 3 days prior to exposure 390 L ,; t '[ i f i j .| 14 1 4 if H 1 j|> ' jj t' Figure 1 Hepatic protein binding and glutathione (GSIi) depression e:- pressed as a function of the logarithm of the exposure concen tration to vinyl chloride (VC). Protein binding (O ) '-V equivalents VC bound per g protein (mean + S . D . ) GSH (A) percent of control. R&S 022582 {, Ff ` l y\ *^ Ml *