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R&S 112283 BIO-MEDICAL. RESEARCH DOCUMENT DESCRIPTION FORM 63 Duplicate in all cards: --^ 68 69 76 | UU0U3SS year as-1961- File number [Right justify [Numeric only] 3 77 78 Sub-Index Code Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded by one blank space 1 bu . JT~ 20 21 rt'szrr's/ / 40 4160 1 ~7~/3 , C4' 1 61 62 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 L -' ___________ 61 62 21 si*Jkr~ ---------- / 22 23 24 Source (Journal, Vol., Number, Pages, Date ) 12 7 61 62 31 . 32 Brief Summary 12 10 SUMMARY: 61 62 61 62 63 64 R&S 112284 < 0000365 THREE SPECIFIC ENZYMATIC ALTERATIONS IN LIVER OF RATS EXPOSED TO VINYL CHLORIDE1 Julie T, Du, Ruth Shelton and Carlo H. Tamburro Cancer Center and Digestive Diseases' and Nutrition Section Department of Medicine University of Louisville School of Medicine Louisville, Kentucky 40201^ 1This work was supported by a grant from B. F. Goodrich Company, Akron, Ohio, and the Manufacturing Chemists Association, Washington, D.C, ^Send reprint requests to Julie T. Du, Room 539 MDR Building, Digestive Diseases Section, 511 South Floyd Street, Louisville, Kentucky, 40201. ^Portions of this study have been presented in Fed. Proc., 35:329 (1976) Abstract. 1 <i sTM"ary Decreases in the activity of key gluconeogenic enzymes and increases in glucose-6-phosphate dehydrogenase and glutathione reductase have been shown in hepatoma. We exposed Sprague-Dawley rats to 10,000-20,000 ppm of vinyl chloride (VC), 4-8 hrs/day, 5 days/week for 1-4 weeks (14-137 hrs. of exposure) to Induce potential liver injury and angiosarcoma formation. Glucose-6-phosphatase, a key gluconeogenic enzyme in the liver micro somal fraction, decreased its activity 252 over control (p<0.05). Glu-- cose-6-phosphate dehydrogenase, an enzyme in the pentose pathway, increased its activity two-fold after more than 100 hrs, of exposure. Non-protein sulfhydryl levels (glutathione and/or cysteine) remained constant but the activity of the enzyme to regenerate the reduced glutathione, glutathione reductase Increased 50-602 during exposure to vinyl chloride (p<0.05) as a means of compensation thus holding the level of reduced glutathione constant* Other microsomal proteins and enzymes related to vinyl chloride metabolism, l.e. P-450, NADPH-cytochrome c_ reductase and mixed function Oxidase were unchanged in the same microsomal fraction. Further, there were no changes in the liver function tests such as serum aminotransferases, nor in liver cytochrome oxidase, a mitochondrial marker for normal cell respiration. The decrease in glucose-6-phosphatase, and increase in glucose-6-phosphate dehydrogenase and glutathione reductase are similar to the enzymatic altera tions in hepatomas. This may reflect an increase in rlbose-5-phosphate production associated with de novo nucleic acid biosynthesis prior to the development of angiosarcoma. 2 R&S 112285  iIntroduction Vinyl chloride has been shown to induce tumors including angiosar coma in laboratory animals (23,31) and angiosarcoma in man (4). At higher concentrations it is believed that vinyl chloride is metabolized by micro somal mixed function oxidase system (MFO) of the liver to toxic metabolites (2,13) and that these metabolites are detoxified by way of glutathione (13,18,33). When animals were pretreated with phenobarbital, an inducer of MFO, and exposed to 5Z vinyl chloride. Increased activities of serum alanine aminotransferase (SG7T), an enzyme used as a conventional liver function test, and serum sorbitol dehydrogenase, a liver specific enzyme, were observed (17)* There have been, to date no serial enzymatic studies performed to determine the progressive metabolic alterations that occur with vinyl chloride injury before and during the development of angiosar coma, a cancer of adjacent mesenchymal cells* In this study, we examined the enzymatic profiles in relation to cellular function of the liver prior to tumor formation by exposure of rats 4 to vinyl chloride. The approach Includes: 1) conventional clinical liver function tests In serum, including aspartic aminotransferase (SGOT), alanine aminotransferase (SGFT), lactic dehydrogenase (LDH), alkaline phosphatase (AP), bilirubin, albumin, cholesterol and triglyceride; 2) microsomal proteins and enzymes related to the metabolism of vinyl chloride (F-450, NADPH cyto chrome c_reductase and mixed function oxidase) and glutathione content; the mitochondrial marker, cytochrome oxidase; the microsomal marker, glucose-6-phosphatase (G-6-P ase) and a pentose phosphate shunt enzyme, glucose-6-phosphate dehydrogenase (G-6-PD) in liver. 3 R&S 112286 1 Materials and Methods Materials * NADPH, cytochrome and oxidized glutathione were purchased from Sigma Chemical Co., St. Louis, Missouri. Aniline and other chemical reagents were ACS grade. Double distilled water was used throughout. Animals and Experimental Design A series of four experiments was conducted in which Sprague-Davley male rats (300-500 g) were randomly placed Into either a group exposed to vinyl chloride gas (10,000-20,000 ppm) or a non-exposed control group. The randomization was performed prior to each of the four experiments. The animals were exposed In a closed chamber in which the atmosphere was constantly circulated. The chamber was a converted vinyl chloride poly merization vat, used for the manufacture of polyvinyl chloride in the B.F. Goodrich Plant, Louisville, Kentucky with a volume of 1,100 gallons; thus, the. respiration of the rats had a negligible effect on the gaseous composi tion of the enclosed atmosphere. The exposure schedule was 4 or 8 hours per day, 5 days per week, for 1-4 weeks. In the first experiment, three animals in each group were sacrificed after the animals had been exposed to vinyl chloride for total accumulated time periods of 14, 28 and 42 hours, a total of 18 animals including equal number of control. The second experi ment was a repeat of the first. For the third experiment, there were 5 animals per group sacrificed after 71 hours of exposure and 6 animals per group sacrificed after 103 hours, a total of 22 animals. In the fourth experiment 3 animals per group were sacrificed after exposures of 84 and 137 hours, a total of 12 animals. The control animals were kept at the 4 R&S 112287 .University animal care facility In order to guarantee the total absence of vinyl chloride exposure. Two to three hours after exposure, the ani mals were anesthetized with ether, blood was drawn by cardiac puncture and the animals were sacrificed Immediately. They were sacrificed at a fixed time of day to avoid diurnal effects. Sample Preparation The liver was excised rapidly and Immediately rinsed in ice cold 0.15 H KC1 with 0.02 M Trls buffer. A portion of the tissue was homogenized with 9 volumes of the cold KCl-Tris buffer In a Potter Elvehjem homogenizer. Each sample was prepared from a single organ and kept at 0 C during pre paration. Remaining liver was frozen rapidly and kept at -20 C. Subcellular Fractionation The homogenate was centrifuged at 600 , and 8,000 , and 100,000 to obtain nuclear, mitochondrial, microsomal and cytosol fraction, respectively, by the differential technique of Schneider and Hogeboom (27) . Centrifuga tion procedures were carried out at 0-4 C In a Sorvall supercentrifuge with a 9RA rotor and a Beckman Model L-5 ultracentrifuge with a swinging bucket rotor. Enzyme activities were determined in the Isolated subcellular frac tions. P-450, NADPH cytochrome c_ reductase and cytochrome oxidase were de termined In fresh samples and glucose-6-phosphatase, mixed function oxidase, glucose-6-phosphate dehydrogenase and glutathione reductase were determined In freshly Isolated fractions from frozen tissue. Glucose-6-phosphatase was estimated (12) by measuring Inorganic phos phate released. P-450 was estimated by the maximum absorption difference between the dlthionite-reduced cytochrome and its carbon monoxide complex 5 R&S 112288 (25)'. P-450 concentration is calculated by using 91,000 M*'^cm''^ as the ex tinction coefficient for the increase in peak height between 490 and 450 tnn. Cytochrome oxidase was measured as described by Wharton and Tzagoloff (39). NADPH cytochrome c_ reductase was determined by the method of Degroot and Dunn (5). Mixed function oxidase was determined by the method of Holtzman (15). Non-protein sulfhydryl-containing compound was estimated by the method of Sedlak and Lindsay (28). The rate of oxidation of NADPH by oxi dized glutathione was used as a standard measure of enzymatic activity of glutathione reductase (3). Glucose-6-phosphate dehydrogenase was measured spectrophotometrieally as the rate of NADPH formation (20). All enzyme assays were validated. The reaction rate was proportional to time and sample size. The protein content of the fractions was determined by the method of Lowry et al. (21). Conventional clinical biochemical studies of serum including aspartate aminotransferase (SGOT), alanine aminotransferase (SGPT), lactic dehydrogenase (LDH), alkaline phosphatase (AP), bilirubin, albumin, cholesterol and triglyceride were determined by Technicon Sequential Multiple Analyzer Computer (SMAC) system. Results Significant differences between the exposed and control groups were obtained in glucose-6-phosphatase, glutathione reductase and glucose-6phosphate dehydrogenase. A. Glucose-6-phosphatase. Chart 2A shows ninety-five percent con fidence intervals for D, the mean difference between the exposed group and the control groups in each experiment. (The confidence intervals for groups 6 R&S 112289 R&S 112290 mean differences on the enzyme discussed are based on the error mean square i from a two-factor analysis of variance with Interaction, the two main effects being time In hours and exposure-nonexposure. Such an analysis was performed for each of the four experimental occasions). There Is no significant difference between the two groups over the first two experiments since D 0 is included within both of the confidence intervals. For the third and fourth experiments, however, the level of glucose-6-phosphatase In the exposed animals was significantly less than that of the control animals.- Note that for each of the experiments we show only a single confidence Interval despite the fact that there are two or three time periods of exposure In each experiment. This results from the nonsignificance of the Interaction term in the analysis of variance, meaning that the differences between the exposed and control groups are uniform over the time periods In each experiment. This uniformity is also evident from the parallel profiles In Chart 2B which Is the composite curve of the group means of the specific activity with respect to time of exposure. From these experiments we observed that the mean activity of glucose-6-phosphatase de creased 25Z after approximately 71 hours of exposure to vinyl chloride gas. B. Glutathione reductase. Chart 3A shows the 95Z confidence inter vals for the difference between the exposed group and the control groups, and Chart 3B shows the composite curve of the group means of the specific activity with respect to exposure time. As shown by the 95Z confidence in tervals in Chart 3A, there Is a significant difference between the exposed and control groups in all four experiments except at 71 hours of exposure, which has a p value of 0.06 In the third experiment. This experiment is shown with two confidence intervals because on this occasion the interaction 7 R&S 112291 system to form chlorooxirane (2,13,19) and rearranges to chloroacetaldehyde (11). These two compounds are considered as the ultimate carcinogens (1, 17,30). They have been shown to be mutagenic in bacterial systems (8,10, 22,24), and to serve as an alkylating agent by reacting with adenosine (1) and to bind with protein (19). Under normal circumstances, the main detoxification route for vinyl chloride metabolism Is thought to be via glutathione. This view is supported by the early work of Johnson (18) and the recent discovery of sulfur-contain ing urinary metabolites of radioactive labeled vinyl chloride in rats (9, 33). The toxic metabolites can be further oxidized to chloroacetlc acid (13). It Is also possible that chlorooxlrane can be detoxified by microsomal epoxide hydrase. This idea is supported by the work of Kappus' group who demonstrated an Increase of Irreversible vinyl chloride protein binding capacity after Inhibiting epoxide hydrase by trichloropropene oxide (19). Based on these findings,, we postulate the metabolic fate of vinyl chloride as shown In Chart 1. Our study did not show a demonstrable change in the activity of mixed function oxidase, MDPH-cytochrcme reductase and the concentration of P-450 which are Involved with the oxidation of vinyl chloride (2,13). Drew's group (5) has studied the effect of vinyl chloride exposure on the activity of benzphetamine demethylatlon and the concentration of cytochrome P-450 in rat li ver. The result differed depending on the time Interval between cessation of exposure and sacrifice of animals. When they sacrificed animals 18 hours af ter exposure, there was no difference in demethylatron nor in cytochrome P-450. However, when they sacrificed 4 days later, there was a decrease in both demethylation capacity and cytochrome P-450. From Drew's and our results, one can probably conclude that the enzyme system for vinyl chloride oxidation is 9 R&S 112292 bf Che time and exposure factors was significant; i.e., the difference be tween the exposed and control groups after 103 hours was significantly greater than the difference after 71 hours. This can also be seen by the differences f the mean values at 71 and 103 hours as shown in Chart 3B. Hence, the level of glutathione reductase in exposed animals was about 25Z greater than that in nonexposed animals up to 71 hours, and the observed difference was further Increased to approximately 50Z after 84 hours of exposure up to 137 hours. C. Glucose-6-phosphate dehydrogenase. From the 95! confidence in tervals of Chart 4A one can see that there is no significant difference between the groups over the first two experiments from 14 to 42 hours. In the third and fourth experiments, however, the difference at 71 and 84 hours was not significant whereas that at 103 and 137 was. The difference can also be seen in Chart 4B which is a composite plot of the group means of the specific activity with respect to exposure time. After about 84 hours of exposure, the mean level of glueose-6-phosphate dehydrogenase in the exposed animals was about twice that of the nonexposed animals. The above differences occurred while other liver function tests, serum aminotransferases, LDH, alkaline phosphatase, etc., showed no signi ficant changes. Nor could we detect any change in mitochondrial marker, cytochrome oxidase in liver. Liver microsomal enzymes related to the metabolism of vinyl chloride, mixed function oxidase, P-450 and NADPH cyto chrome c. reductase also remained unchanged. Discussion Our present understanding of vinyl chloride carcinogenesis implies that the compound at high levels is oxidized by liver mixed function oxidase 8 R&S 112293 sot Inducible by its substrate, vinyl chloride, Jaeger, et_ al__(17) using serum, found elevated activities of both alanine aminotransferase and sor bitol dehydrogenase In rats pretreated with phenobarbltal and exposed to vinyl chloride. However, they could not detect any elevation in these serum enzymes in rats pretreated with, phenobarbltal and exposed consecutively to vinyl chloride for 5 days and they suggested that the initial exposure to vinyl chloride seemed to protect against acute injury on re-erposure. Under our experimental conditions, we found that the non-protein sulfhydryl content of liver (glutathione and/or cysteine) did not change in rats exposed to vinyl chloride. This finding is unexpected because Hefner and Watanabe (13,33) have found a depletion of non-protein sulf hydryl content in rats exposed to VC and Johnson (18) found a depletion of liver glutathione when chloroethanol was fed to rats. In a preliminary experiment,. we found that the. non-protein sulfhydryl content decreased sharply one hour after injection of chloroethanol, but the level went back to normal about 5 hours after injection and stayed normal afterwards (Du, J.T. and Tamburro, C.H.^; 40). Since we did not sacrifice our animals immediately af ter exposure; rather, we sacrificed 3 hours later, we might have missed the decrease seen by others. However, we did see an increase in the level glutathione reductase, which converts the oxidized glutathione to reduced glutathione In all four experiments. This would serve two purposes: to maintain the level of reduced glutathione for detoxification as a compensa tion by enzyme'and to supply NADP+ as a co-factor for various synthetic pathways. Including nucleic.acid synthesis. ^Unpublished data. 10 R&S 112294 In hepatoma the activity of key enzymes for gluconeo genes is have been shown to decrease in parallel with an Increased rate of tumor growth (34,36,37). In contrast, two enzymes for the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (29,38) and transaldolase (14) were found to be increased In all hepatomas regardless of growth rate. In addition, the activity of glutathione reductase was Increased In hepatomous liver induced by a chemical, diethyInitrosamine (26), Further, when carcinogens such as nitrosamlne and dimethyl aminoazobenzene were fed to rats, the activity of glucose-6-phosphatase decreased prior to and with the development of hepatomas (16,35).. Our finding of a decreased activity In glucose-6-phosphatase and an increased activity in glucose-6-phosphate dehydrogenase and in gluta thione reductase in rat liver after exposure to vinyl chloride may be an early biochemical alteration indicative of early liver injury associated with subsequent development of angiosarcoma. These differences were ob served before any sign of abnormalities were detectable by conventional liver function tests, serum: aminotransferases or by mitochondrial marker, cytochrome oxidase activity in liver. In hepatocytes, chlorooxirane may rearrange to chloroacetaldehyde, and then be detoxified via glutathione, and subsequently metabolized and excreted in the urine (Chart 1). In situations when glutathione is insuf ficient for complete detoxification of these metabolites or the activity of the detoxification enzymes is too low to detoxify them; chloroacetalde hyde may be reduced to chloroethanol, and then be transferred to mesenchy mal cells anti there be reoxidized to the aldehyde form, but may not be oxidized further to the acid or detoxified by glutathione due to metabolic limits of these mesenchymal cells. Alternatively, the mesenchymal cell 11 R&S 112295 *' pay act metabollcally similar to the hepatocyte in the oxidation of vinyl chloride, but may have a decreased or limited capability of detoxi fication leading to increased amounts of chloroacetaldehyde in contrast to the hepatocyte thereby allowing for Increased possibilities of DNA injury. There is an obvious difference in the cellular etiology of hepa toma (primary cell carcinoma) and angiosarcoma. The former is a cancer of hepatocytes and the latter, a cancer of mesenchymal cells. The concept that the hepatocyte's ability to detoxify the active metabolite of vinyl chloride, thereby protecting itself from DNA injury is given some further support by the observation of Maltoni etr al. (personal communications, 1976) who have produced primary hepatocellular carcinoma in new bom rats exposed to vinyl chloride and shown only the development of angiosarcoma in the' adult rats. The newborn rat liver may have hepatocytes which are more vulnerable to the toxic metabolites. Studies have already begun to con firm Maltoni's observation and the enzymatic capabilities of hepatocytes from newborn rats. Whether these metabolic changes reflect a precancerous alteration In the hepatocytes regardless of the cell origin of the cancer, as seen in primary liver cell cancer, must yet be determined. Studies are underway to follow these enzymatic changes until induction of these two cell types of liver cancer. Acknowledgement We should like to express our sincere thanks to the people in the B. F, Goodrich Plant in Louisville for their cooperation in exposure studies and Dr. R. A. Greenberg and Mr. J. F. Sandoz for their statistical help. 12 ` References 1- Barbln, A., Bresil, H., Crolsy, A., Jacquignon, P. Malaveille, C., Montesano, R., and Bartsch, H. Liver-Microscme-Mediated Formation of Alkylating Agents from Vinyl Bromide and Vinyl Chloride. Blochem. Biophys. Res. Comm. 67:596-603, 1975. 2. Bolt, H.M., Kappus, H., Buchter^ A., and Bolt, W. Metabolism of Vinyl Chloride. Lancet 1:1425, 1975. 3. Carlberg, I. and Mannervik, B, Purification and Characterization of the Flavoenzyme Glutothione Reductase from Rat Liver. J. Biol. Chem. 250: 5475-5480, 1975. 4. Creech, J.L., and Johnson, M.N, Angiosarcoma of Liver in the Manufacture of Polyvinyl Chloride. J. Occup. Med. 16:150-151, 1974. 5. Degroot, L.J. and Dunn, A.D. Electron-transport Enzymes of Calf Thyroid. Blochem. Biophys. Acta 92:205-222, 1964. 6. Drew,. R. T*, Harper, C., Gupta, B.N. and Talley,. F.A. Effects of Vinyl Chloride Exposures to Rats Pretreated with Phenobarbital, Envlr. Health Persp. 11:235-242, 1975. 7. Du, J.T. and Tamburro, C.H. Decreased Glucose-6-Phosphatase Activity In Liver In Vinyl Chloride Exposed Rats, Fed. Proc. 35:329, 1976. 8. Elmore, J.D., Wong, J.L., Laumbach, A.D., and Strelps, U.N. Vinyl Chloride Mutagenicity via the Metabolites Chlorooxlrane and Chloroacetaldehyde Mono mer Hydrate. Blochem. Biophys. Acta 442:405-419, 1976. 9. Green, T. and Hathway, D.E. The Biological Fate in Rats of Vinyl Chloride in Relation to its Oncogenicity, Chem.-Biol. Interactions, 11:545-562, 1975 13 R&S 112296 10. Greim, H., Bonse, G., Radwan, Z., Reichert, 0. and Henschler, D., Mutageni city In Vitro and Potential Carcinogenicity of chlorinated Ethylenes as a Function of Metabolic Oxirane Formation, 24:2013-2017, 1975. 11. Gross, H. and Freiberg, J. Alpha-Halo Ethers. 41. Existence of Chloroethylene Oxide. J. Prakt. Chem. 311:506-510, 1969. 12. Harper, A.E. Glucose-6-Phosphatase. In: H.U. Bergmeyer (ed*). Methods of Enzymatic Analysis, pp. 788-792. New York: Academic Press, Inc., 1965. 13. Hefner, R.E., Watanabe, P.G., and Gehrlng, P.J. Preliminary Studies of the Fate of Inhaled Vinyl Chloride Monomer in Rats. Ann. N. Y. Acad. Sci. 246:135-148, 1975. 14. Heinrich, P.C., Morris, H.P., and Weber, G. Increased Phosphoribosylpyrophosphate Synthestase Activity in Rapidly Growing Hepatomas. FEBS Letters 42:145-148, 1974. 15. Holtzman, J.L., Gram, T.E., Glgon, P.L., and Gillette, J.R. The Distri bution of the Components of Mixed-Function Oxidase between the Rough and the Smooth Endoplasmic Reticulum of Liver Cells. Biochem J. 110:407-412, ` 1968. 16. Isok, M.E., and Teras, L.E. Glucose-6-phosphatase Activity in Liver Carcinogenesis and in Transplantable Hepatoma in Mice. Voproxy Med. Khim. 19:568-570, 1973. 17. Jaeger, R.J., Reynolds, E.5., Connolly, R.B., Moslen, M.T., Szabo, A., and Murphy, S.M. Acute Hepatic Injury by Vinyl Chloride in Rats Pretreated with Phenobarbital. Nature 252:724-726, 1974. 18. Johnson, M.K. Metabolism of Chloroethanol in the Rat. Biochem. Pham. 16:185-199, 1967. 14 R&S 112297 *'19. Kappus, H., Bolt, H.M., Buchter, A.,-and Bolt, W. Liver Microsomal Uptake l of ^C Vinyl Chloride and Transformation to Protein Alkylating Metabolit in Vitro, Toxicol. Appl. Pharmacol. 37:461-471, 1976. 20. Lohr, G.W. and Waller, H.D. Glucose-6-phosphate Dehydrogenase. In: H.U. Bergneyer (ed.), Methods of Enzymatic Analysis, pp. 744-751. New York: Academic Press, Inc., 1965. 21. Lowry, O.E., Rosebrough, N.J., Farr, A.L., and Randall, R.J. Protein Measuremert with the Folin Phenol Reagent. J. Biol. Chem. 193:265275, 1951. 22. Malaveille, C., Bartsch, H., Barbin, A., Camus, A.M., nad Montesano, R. Mutagenicity of Vinyl Chloride, Chloroethylene Oxide, Chloroacetaldehyde and Chloroethanol. Blochem. Biophys^ Res. Comm. 63:363-370, 1975. 23. Maltoni, C. and Lefemlne, G. Carcinogenicity Bioassays of Vinyl Chloride: Current Results. Ann. N. Y. Acad. Scl. 246:195-218, 1975. 24. McCann, J., Simmon, V., Streitwieser, D., and Ames , B.N. Mutagenicity of Chloroacetaldehyde, A Possible Metabolic Product of 1,2-Dlchloroethane, Chloroethanol, Vinyl Chloride and Cyclophosphamide. Proc. Nat. Acad. Sci. 72:3190-3193, 1975. 25. Omura, T. and Sato, R. The Carbon Monoxide-binding Pigment of Liver Microsomes. I. Evidence for its Hemoproteln Nature. J. Biol. Chem. 239: 2370-2378, 1964. 26. Pinto, R. E. and Bartley, W. Glutathione Reductase and Glutathione Peroxi dase Activities in Hepatomous Livers of Rats Treated with Diethyl Nitrosamlne, FEBS Letters, 32:307-309, 1973. 27. Schenider, W.C. and Hogeboom, G.H. Intracellular Distribution of Enzymes V. Further Studies on the Distribution of Cytochrome _c in Rat Liver Homogenates. J. Biol. Chem. 183:123-128, 1950. 15 R&S 112298 R&S 112299 28. Sedlak, J. and Lindsay, R.H. Estimation of Total, Protein Bound and Non-Protein Sulfhydryl Groups in Tissue with Ellman's Reagent. Anal. Biochem. 25:192-205, 1968. 29. Selmeci, L.E. and Weber, G., Increased Glucose-6-Phosphate Dehydrogenase Concentration in Hepatoma 3924A: Enzymic and Immunological Evidence, FEBS Letters, 61:63-67, 1976. 30* Van Duuren, B.L. Possible Mechanism of Carcinogenic Action of Vinyl Chloride. Ann. N. Y. Acad. Sci. 246:258-267, 1975. 31. Viola, P.L., Blgotti, A., and Caputo, A. Oncogenic Response of Rat Skin, Lungs, and Bones to Vinyl Chloride. Cancer Res. 31:516-522, 1971. 32. Wataxxabe, P.G., Hefner, R.E., Jr., and Gehring, P.J. Vinyl ChlorideInduced Depression of Hepatic Non-protein Sulfhydryl Content and Effects on Bromosulphalein. (BSP) Clearance in Rats. Toxicol., 6:1-8, 1976. 33. Watanabe, P.G., McGowan, G.R., and Gehring, P'.J. Fate of (Vinyl-C-14) Chloride After Single Oral Administration In Rats. Toxicol. Appl. Pharmacol. 36:339-352, 1976. 34. Weber, G. The Molecular Correlation Concept. In: H. Busch (ed.) The Molecular Biology of Cancer pp. 487-521. New York: Academic Press, Inc., 1974. 35. Weber, G. and Cantero, A. Glucose-6-phosphatase Activity in Normal, Precancerous, and Neoplastic Tissues. Cancer Res. 15:105-108, 1955. 36. Weber, G. and Convery, H.J.H. Insulin: Inducer of Glucose-6-phosphate Dehydrogenase. Life Sci. 5:1139-1146, 1966. 37. Weber, G. and Lea, M.A. The Molecular Correlation Concept. In: H. Busch (ed.). Methods in Cancer Research, Vol. 2, pp. 523-578. New York: Academic Press, Inc., 1967. 16 R&S 112300 38. Weber, G. and Morris, H.P. Comparative Biochemistry of Hepatomas. III. Carbohydrate Enzymes In Liver Tumors of Different Growth Rates. Cancer Res. 23:987-994, 1963. 39. Wharton, D.C. and Tzagoloff, A. Cytochrome Oxidase from Beef Heart Mitochondria. In: R.W. Estabrook and M.E. Pullman (eds.). Methods in Enzymology, Vol. X, pp. 245-250. New York: Academic Press, Inc., 1967. 40. White, I.N.H., The Role of Liver Glutathione in the Acute Toxicity of Retrorslne to Rats, Chem-Blol. Interactions, 13:333-342, 1976. 17 Chart 1. The proposed metabolic fate f vinyl chloride. GSH, gluta thione; MFO, mimed function oxidase; VC, vinyl chloride. The detailed steps involved in the formation and detoxification of chloroethanol have not been demonstrated firmly in a biological system. Chlorooxirane can be reduced by sulfhydryl reagents such as cysteine and/or gluta thione to chloroethanol In vitro without adding an enzyme.^ It is likely that chloroethanol Is first oxidized to chloroacetaldehyde, conjugated with glutathione to form GSC52CH0, and then reduced to GSCHjCHjOH in vivo (18). g CO 10ESU 4Personal communication with Dr. John Wong, Professor, Department of Chemistry, University of Louisville, Louisville, Kentucky. zozzvv s'sa Cl CH a CHj (v c) ^ LIVER MFO Cl CH-CH, 'V & (chlorooxirane) * V ' . \ * DETOXIFICATION WITH GLUTATHIONE \ Cl CHnCH90H (chloroethanol) \ +GSH iGS CH9CHo0H | Cl CH2CHO GS CHjCHO (chloroacetaldehyde) 1 Cl C^COOH (chloroacetic acid) N-Ac-S-(2-HYDROXY ethyl) cysteine GS CHjCOOH 1 THIODIGLYCOLIC ACID  Chart 2. Part 2A sh vs the 95Z confidence Intervals for difference b tween vinyl chloride exposed and control groups on glucose-6-phosphatase. The ordinate denotes D, the difference between the exposed and control group* and the abscissa denotes the exposure time in hours. Part 2B shows the composite curve of the group means of the specific activity of glucose-6-phosphatase with respect to time of exposure. Experimental rats* shown with 0* were exposed to 10*000-20,000 ppm of vinyl chloride for various periods of time; and control rats* shown with were unexposed. Detailed exposure schedule* methods and statistical analysis of results were described in the text. Each point represents the mean of 6 Individual animals excepting periods of 71 hrv. In which only 5 animals were used and 84 and 137 hr. In which only 3 animals were involved. R&S 112303 Exposure 14.28.42 (hrs) 14.28.42 71.103 84.137 EXPOSURE TIME (hours) R&S 112305 j Chart 3. Part 3A shows the 95Z confidence Intervals for difference between vinyl chloride exposed and control groups on glutathione reductase. Part 3B shows the composite curve of the group means of the specific activity of glutathione reductase with respect to time of exposure. Experimental rats* shown with 0, were exposed to 10,000-20,000 ppm of vinyl chloride for various periods of time and control rats, shown withA, were unexposed. Same animals were used as in Chart 2. See text and footnote to Chart 2 for other experimental and statistical details. The mean value of the exposed is significantly more than that of the control groups throughout the entire experiment (p<0.05) except at 71 hours of exposure which has a p value of 0.06. tki t Chart 4. Fart 4A shows the 95Z confidence intervals for D, the mean dif ference between the exposed and control group. Fart 4B shows the composite curve of the group means of the specific activity of glucose-6-phosphate dehydrogenase with respect to time of exposure. See footnote to Chart 2 about the experimental and statistical details. There la no significant difference between the experimental and control groups up to 84 hours, since D 0 is Included in the confidence Interval, but the mean level of exposed group is significantly more than that of the control after 103 hours of exposure. R&S 112307 Exposure 14.2ft42 (hrs) 14.28.42 71.103 84.137 DO flo CO croo 00