Document EOdjvjaYBgmdRaQeK6MD8xxV

R&S 111617 BlO-MSDICAL RESEARCH DTO. CUME*NT DESCRI*PTION *FORM 63 68 69 76 Duplidate in all cards:--> irnmm year as-1961- File number [Right justify [Numeric only] Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded -by. one blank space JZ~ O -J &S.4 4 DG. __________________ -- fU_. 40 41 ....................... 77 78 Sub-Index Code 60 61 62 11 12 13 Title of Report: end with space-hyphen-hyphen-space. Follow with Index Terms, separated from each other with -comma-space. Avoid other punctuation; do not abbreviate. 12 * . 61 62 21 </ -t ' A- .J i- ` /.- / 22 V " ; . i ^ T. 1/ 1 1' h 23 s * 24 irce (Journal, Vol., Number, Pages, Date) 12 i" -*s j /' Imh * <_( 'i / i - j ' j j 61 62. 31 32 / - * Brief Su-^aT-y 12 10 SUMMARY: * 61 62 61 62 63 64 TOXICOLOGY AND APPLIED PHARMACOLOGY 44, 571-579 (1978) 000032$ Hepatic Macromolecular Binding Following Exposure to Vinyl Chloride1 P. G. Watanabe,2 J. A. Zempel, D. G. Pegg, and P. J. Gehring Toxicology Research Laboratory, Health and Environmental Research, 1803 Building, The Dow Chemical Company, Midland, Michigan 48640 ReceivedJuly 28,1977; accepted November 1,1977 Hepatic Macromolecular Binding Following Exposure to Vinyl Chloride. Watanabe, P. G., Zempel, J. A,, Pegg, D. G., and Gehring, P. J. (1978). Toxicol. Appl. Pharmacol., 44, 571-- 579. Covalent binding of radioactivity to hepatic macromolecules in rats exposed to ,4C-labeled vinyl chloride (VC) was studied to determine if VC-induced carcinogenesis may be related to electrophilic alkylation of macromolecules in vivo. Male Sprague-Dawley rats were exposed to 1, 10, 25, 50, 100, 250, 500, 1000, or 5000 ppm of [14C]VC for 6 hr. Following exposure) radioactivity covalently bound to hepatic macromolecules and purified nucleic acids (RNA, DNA) was determined. The total amount of [14C1VC metabolized and hepatic glutathione (GSH) content were also determined. The total amount of radioactivity bound to macro molecules 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 14C bound to macromolecules in the liver correlates with induction 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; Maltoni and Lefemine, 1975). The concept of a reactive metabolite of VC being responsible for the carcinogenic activity (Hefner et 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 (Bartsh et al., 1975; Malavielle et al., 1975; Rannug et aU 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 vinyl chloride projects being administered by the Manufacturing Chemists Association, Washington, D.C. 2 Author to whom all correspondence should be sent, manuscript No, B 600-173-77. 57 1 004I-008X/78/OU3-0571S02.00/0 Copyright 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed ui Great Britain R& s 111619 572 WATANABE ET AL. by reaction with 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 attributed to the reaction of electrophilic metabolites with intracellular macromolecules (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 al., 1975), protein sulfhydryl groups, RNA (Bolt et al., 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 al., 1975; Green and Hathway, 1975; 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 concentration 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 adds following exposure to various concentrations of uC-labeled VC. METHODS Materials. uC-labeled VC was synthesized from [ 1,2-14C ] 1,2-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, 1975). Each new batch of [l,2-14C]dichloroethane was analyzed and yielded [,4C]VC with a radiochemical purity of 95%. The synthesized [,4C]VC has been shown repeatedly in our laboratory to be 95 to 96% radiochemically pure (Wagner et al., 1975). Thus, while each synthesis of [14C]VC 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. Nonlabeied VC (Matheson Gas Products) of 99.9% minimum purity was mixed with the [i4C]material to obtain the desired specific activity. Typically 40 ml of the [14C]VC-helium gas mixture was injected into a 5 to 10-liter Saran bag (Anspec, Inc.) containing the desired quantity of nonlabeied VC. Animals. Male Sprague-Dawley rats (Spartan Research Laboratory) weighing 220 to 250 g were used throughout the study. All 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 [14C]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 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. 14Clabeled VC was metered into the chamber air flow (~6 liters/min) with a dual syringe VINYL CHLORIDE 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 /on. In addition, samples (1 ml) of the chamber atmosphere were analyzed at approximately hourly intervals during the exposure by gas chromatography (Watanabe et al., 1976c). At corresponding times, the UC activity was determined by bubbling 1-ml aliquots of the chamber atmosphere into a scintillation solution containing a mixture of Concifluor (Mallinckrodt Chemical), 2-methoxyethanol, toluene (6:11:83). The radioactivity was determined by counting in a Mark 11 or Mark HI 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 0.3 (SD), 9.3 0.2, 24.7 1.4, 51 2, 109 23,250 3, 511 11, 1020 13, and 4600 311. The respective specific activities were 132,000, 4801, 3170, 2528, 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 [14C]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 [MCjVC (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 --20C until analyzed. The carcass was analyzed for total radioactivity as described previously (Watanabe et aL, 1976a). Previous studies have shown that only a small percentage of radioactivity (<12%) is excreted as metabolites other than [14C]VC during 72 hr following a 6-hr inhalation exposure (Watanabe et al., 1976c). The large proportion of the 12% is comprised of UC02 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 14C-labeled VC to hepatic tissue (protein, nucleic adds, and lipid) was determined by the method of Jollow et al. (1973). The trichloroacetic acidpredpitable 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 bdow). 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 of frozen tissue was thawed slowly and homogenized in 50 ml of 0.15 M sodium chloride-0.04 M ethylenediaminetetraacetic 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 R&S 111620 R&S 111621 574 WATANABE ET AL. precipitate was washed with ethanol and dissolved in 20 ml of 0.015 m sodium chloride0.0015 m sodium citrate buffer (pH 7.0). RNA was precipitated by adding 1 vol of icecold 6 m potassium acetate (pH 7.5) and removed by centrifugation. DNA was precipitated from the supernatant by adding 2 vol of 95% ethanol. The resulting crude DNA pellet was dissolved in 20 ml of 0.015 m sodium chloride-0.0015 m sodium citrate buffer (pH 7.0) and the solution was centrifuged at 100,000 g 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 ml) and 2 vol of 2-ethoxyethanol. The precipitate was redissoved in 10 ml of 0.015 M sodium chloride-0.0015 m sodium citrate at 2C. The final DNA precipitation was accomplished using 5 ml of ice-cold isopropanol. RNA and DNA pellets were dried at 2C under reduced pressure and weighed. RNA and DNA were quantified by the orcinol and diphenylamine reactions, respectively (Keleti and Lederer, 1974). RESULTS Hepatic macromolecular binding, hepatic nonprotein sullhydryl content (primarily glutathione, GSH), 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. Hepadc 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 (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 1000 ppm is not understood fully, but it may be due to differences in respiratory parameters causing differences in the uptake of VC. Hepatic nonprotein 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 Metabolism of VC was not increased in rats exposed to 100 ppm of VC after pretreatment with phenobarbital. Macro molecular 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 1, 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 VINYL CHLORIDE MACROMOLECULAR BINDING TABLE 1 Total Metabolism, Hepatic Macromolecular Binding, and Hepatic Glutathione (GSH) Concentrations Following Inhalation Exposure {6 hr) to Vinyl Chloride (VC)" Nominal concentration A (pg of VC equivalents metabolized) B (pg or VC equivalents bound per g of protein) B/A x 100 Hepatic GSH (% control) Percentage of total l4C activity in liver bound to macromolecules 1 10 25 50 100 250 500 1000 5000 29.8 + 3.2 242 26 557 42 1181 93 2406 173 3826 345 6263 355 4257 765 9255 + 1,467 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 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 0.13 104 89 93 94 81* 70* 60* 51* 39* 20 + 3 21 2 21 2 20 + 2 25 + 2 22 + 2 25 + 3 22 2 22 + 3 Prelreatment with phenobarbetal* 100 2160 166 80.0 + 23.9 3.70 0.82 39 + 3 * Means SD. * Slatislically different from controls. Student's I lesl (p < 0.05). ' Rais were injected ip with sodium phenobarbital, 80 mg/ltg for 3 days prior to exposure. zzsin ssa L-aH  _,,tJ L/VV. V*%t - r 576 WATANABE ET AL. each exposure level because the specific activity of the [UC]VC varied with exposure and the different amounts of DNA and RNA analyzed. The sensitivity limits for the 1-, 100-, 250-, and 1000-ppm exposures were 0.007, 0.051, 0.108, and 0.30 fi% of VC equivalents/mg of DNA and 0.0001,0.009,0.018, and 0.05 fig of VC equivalents/mg of RNA. 30 (/) Fig. 1. Hepatic macromolecular binding and glutathione (GSH) depression expressed as a function of Ccroo> the logarithm of the exposure concentration to vinyl chloride (VC). Macromolecular binding () is expressed as microgram equivalents of VC bound per gram of protein (mean SD) GSH (A) is expressed as percentage ofcontrol. DISCUSSION The results indicated that the total amount of radioactivity bound to macromolecules in the liver 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 (Maltoni, 1975) indicate that the percentage induction of hepatic angiosarcoma in rats is linear between 50 and 500 ppm 'iV;;--.v.-!".: - i-i' V39VVV 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 believed to be reaction of electrophilic metabolites with intracellular macromolecules (Miller and Miller, 1971). The covalent binding to hepatic macromoiecules 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 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 a/., 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 >4C 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, initial studies by Bolt et al. (1976a) reported covalent binding of radioactivity to DNA and RNA following a 5-hr static exposure to 145 ppm of [I4CJVC. The specific activity used by Bolt et al. (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 [,4C]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 al. (1976a) confirm the conclusion that VC does not preferentially react with hepatic nucleic acids. It is important to emphasize that the methodologies employed only detect covalent 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 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 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 33 (/) R&S 111625 578 WATANABE ET AL. binding may reflect a nonspecific interaction merely due to an increase in the available protein binding sites. 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 evidence 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 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 adds. This is important because phenobarbital does increase the hepatic macromolecular binding of ,4C activity to macromolecules 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 adds as the mechanism for VC-induced cardnogenesis, is the need to determine the absence of such activity in target tissue rather than hepatocytes. Essentially all studies of metabolic and clinical parameters to date 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 toxidty. Toxidty may be induced in tissues with smaller capadty 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-ethylguanine in the nervous system (Goth and Rajewsky, 1974). Although other metabolizing organs such as the liver produce O-6-ethylguanine, the turnover rate of DNA mediated by repair mechanisms is suffident 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 adds or critical proteins. REFERENCES Barbin, A., Bresil, H., Croisy, A., Jacqijignon, P,, Malavielle, C,, Montesano, R., and Bartsch, H. (1975). Liver microsome mediated formation of alkylating agents from vinyl bromide and vinyl chloride. Biochem, Biopkys. Res. Commun. 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 1425. Bolt, H. M., Kappus, H., Buchter. A., and Bolt, W. (1976b). Disposition of l,2-14C-vinyI chloride in the rat. Arch. Toxicol. 35, 153-162. Bolt, H. M,, Kappus, H,, Kaufmann, R., Appel, K. E., Buchter, A., and Bolt, W. (1976a). Metabolism of ,4C-vinyl chloride in vitro and in vivo. INSERM 52, 151-164. Creech, J. L., and Johnson, M. N. (1974). Angiosarcoma of liver in the manufacture of polyvinyl chloride./, Occup. Med. 16, 150-151. VINYL CHLORIDE MACROMOLECULAR BINDING 519- Goth, R,, and Rajewsky, M. F. (1974). Persistence of 0-6-ethylguanine in rat brain DNA: Correlation with nervous system-specific carcinogenesis by ethylnitrosurea. Proc. Nat. Acad. Sci. USA 71, 639-643. Green. T., and Hathway, D. E. (1975). The biological fate in rats of vinyl chloride in relation to its carcinogenicity. Chem. Biol. Interae. 11,545-562. 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. Irving, C. C., and Veazey, R. A. (1968). Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from rat liver. Biochim. Biophys. Acta. 166, 246-248. Jollow, D. J., Mitchell, J. R., Potter, W. Z., Davis, D. C., Gillette, J. R., and Brodie, B. B. (1973). Acetaminophen-induced hepatic necrosis. II. J. Pharmacol. Exp, Ther. 187, 195- 202. Kappus, H., Bolt, H. M., Buchter, A., and Bolt, W. (1975). Rat liver microsomes catalyze covalent binding of 14C-vinyl chloride to macromolecules. Nature (London) 257,134-135, Keleh, G., and Lederer, W. H. (1974). Handbook of Methods for the Biological Sciences. Van Nostrand--Reinhold, New York. Laib, R. J., and Bolt, H. M. (1977). Alkylation of RNA by vinyl chloride metabolites in vitro and in vivo. Formation of l-N`-etheno adenosine. Arch. Toxicol., in press. Malavielle, C., Bartsch, H., Barbin, A_ Camus, A. M., and Montesano, R. (1975). Mutagenicity of vinyl chloride, chlorocthyleneoxide, chloroacetaldehyde and chloroethanol. Biochem. Biophys. Res. Commun. 63,363--370. Maltonl C. (1975). The value of predictive experimental environmental carcinogenesis. Ambio 4, 18-23. Maltonl C,, and Lefemine, G. (1975). Carcinogenicity assays of vinyl chloride: Current results. Ann. N. Y.Acad, Sci. 246,195-224. Miller, J. A,, and Miller, E. C. (1971). Chemical carcinogenesis: Mechanisms and approaches to its control. J. Nat. Cancer Inst. 47,5-14. Okuuara, E. (1970). Preparation of mammalian deoxyribonucleic acid by SDS-phenol treatment. Anal. Biochem. 37, 175-178. Rannug, U,, Johansson, A., Ramel, C~, and Wachtmeister, C. A. (1974). The mutagenicity of vinyl chloride after metabolic activation. Ambio 3, 194--197. Sedlak, J,, and Lindsay, R. M. (1968). Estimation of total protein-bound, and nonprotein sulfhydryl groups in tissue with EUman's Reagent, Anal. Biochem, 25,192-205. Van Duuren, B. L. (1975). On the possible mechanism of carcinogenic action of vinyl chloride. Ann.N. Y.Acad. Sci. 246,258-267. Wagner, E. R., and Muelder, W. W. (1975). A procedure for preparing ,4C-labeled vinyl chloride. Ann. N. Y. Acad. Set 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 chromatographic method for the preparation of l4C-labeled vinyl chloride. J. Labeled Compounds 11,535-542. Watanabe, P. G,, Hefner, R. E., Jr., and Gehring, P. J. (1976b). Vinyl chloride induced depression of hepatic nonprotein sulfhydryl content and effects on bromosulphthalein (BSP) clearance in rats. Toxicology 6,1-8. Watanabe, P. G., McGowan, G. R,, and Gehring. P. J. (1976a). Fate of 14C-vinyl chloride after single oral administration in rats. Toxicol. Appl. Pharmacol, 36,339-352. Watanabe, P. G., McGowan, G. R., Madrid, E. O., and Gehring, P. J. (1976c). Fate of l*C-vinyl chloride following inhalation exposure in rats. Toxicol. Appl. Pharmacol. 37,49-59. R&S 111626 DEPARTMENT OF HEALTH, EDUCATION. AND WELFARE PUBLIC HEALTH Sr HVICE NATIONAL INSTITUTES OF HEALTH BCTHESDA. MARYLAND 20014 July 29, 1975 NATIONAL CANCER INSTITUTE Dr. Albert C. Clark Vice President, Technical Director Manufacturing Chemists Association 1825 Connecticut Avenue, N. W. Washington, D. C. 20009 \ Dear Dr. Clark: Thank you very much for your letter of July 18 and the enclosed reports on vinyl chloride. These are very interesting reports which we have looked forward to reading. You have indicated that you will compare workers from vinyl monomer production, VC production and PVC fabrication plants. This report on comparisons would be most interesting since one may wonder what potential hazard prevails from PVC exposure where there is potential escape of the monomer. This, of course, would take us beyond the occupational setting and be an environmental problem (non-occupational). I would appreciate your thoughts on the matter. The report from Dow bv Gehrina. et al is a classical study and_one which I have followed since some preliminary data was given last year at the New York Academy of Sciences meeting in New York. This is real good work from the mechanistic standpoint. We certainly need more of such research to provide us with a better understanding of the action of a chemical on target organs and their metabolic fate. Thank you again for these excellent reports. I hope you will send us others as they become available. Sincerely H. F. Kraybill, Ph. D. Scientific Coordinator for Environmental Cancer 7/31/75 py to Mr. Freifeld DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE PUBLIC HEALTH SERVICE CENTER FOR DISEASE CONTROL JUL 311975 - 'V i NATIONAL INSTITUTE FOR OCCUPATIONAL' SAFETY AND HEALTH 5600 FISHERS LANE ROCKVILLE, MARYLAND 20852 O) l\5 00 July 29, 1975 I I Mr. Albert C. Clark Vice President, Technical Director Manufacturing Chemists Association 1825 Connecticut Avenue, N.W. Washington, D. C. 20009 Dear Mr. Clark: I appreciate receiving an updated report of the TabershawCooper vinyl chloride study and the progress report of the Dow Metabolism studies of vinyl chloride. X hope that we may continue to be kept up to date with any additional data in this regard. Sincerely yours, John'F. rinklea, M.D. Defector 7/31/75 ''Copy to Mr. Freifeld I > ( I (