Document 8VZBM2YZ2pagpvjvZkDoan7kZ

2 BIOCHEMICAL ALTERATIONS IN LIVERS OF RATS EXPOSED TO VINYL CHLORIDE julle T. Du, John P. Sandoz, Michael T. Tseng, Carlo H. Tamburro Division of Digestive Diseases and Nutrition, Department of Medicine, Department of Anatomy, and Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky Sprague-Dawhy rats were exposed to vinyl chloride to determine tht earliest sequential biochemical changes occurring with liver injury before angiosarcoma development. Activity of g/ucose-6-phosphatase, a key gluconeogenic eneyme in the liver microtome/ fraction, decrcated 25% with respect to controls after 70 h of exposure. Glucose-&phosphate dehydrogenase activity increased twofold after more than 100 h of exposure. Nonprottin sullhydryl levels (glutathione and/or cysteine) showed a slight but progressive elevation, whereas glutathione reductase activity increased 50-50% during exposure to vinyl chloride, NAQPH-cytochrome c reductase and mixed function oxidase were unchanged in the same microsomal fraction. There were no changes in seven conventional clinical biochemical liver tests or in four other markers of liver mitochondrial, cytosol, and microsomal function. No significant histological changes were found on light microscopic examination during this exposure period. However, with electron microscopy, dilation of rough endoplasmic reticulum urn seen in the animals exposed for more than 137 h. These enrymatic changes are considered to reflect early hepatocellular adaptation to vinyl chloride exposure with vary mild or limited hepatocellular Injury In Its earliest stage. INTRODUCTION Vinyl chloride has been shown to induce tumors, including angio* sarcoma, in laboratory animals (Maltoni and Lefemine, 1975; Viola et al., 1971) and angiosarcoma in humans (Creech and Johnson, 1974). At high concentrations, vinyl chloride is believed to be metabolized by the microsomal mixed function oxidase (MFO) system of the liver to toxic metabolites (Bolt et al., 1975; Hefner et al., 1975; Johnson, 1967; Watanabe et al., 1976b, 1976c). Animals pretreated with phenobarbital, an inducer of Wc wish to express our sincere thanks to the people in the B. F. Goodrich Plant in LouisviUe_Lfor their cooperation in the exposure studies. Or, R. ^-Greenberg for help with statistics, and'Ms. Ruth Shelton for technical assistance. This work was supported by a grant from the Manufacturing Chemists Association, Washing ton, D.C, Requests for reprints should be sent to Carlo M. Tamburro, Division of Digestive Diseases and Nutrition, Health Sciences Center, MOR 535, University of Louisville, Louisville, Kentucky 40232. 1119 Jownal of Toxicology and Environmental Health, 5:1119-1132, 1979 Copyright C 1979 by Hemisphere Publishing Corporation 009S-4108/79/051119-14S2.25 CMA 052805 1120 j.T. DUETAL. MFO, and exposed to 5% vinyl chloride (50,000 ppm) had increased serum alanine aminotransferase, a conventional biochemical indicator of liver injury, and increased serum sorbitol dehydrogenase, a liver specific enzyme (jaeger et al., 1974). However, there have been no sequential enzymatic studies to determine what progressive metabolic alterations of the hepatocyte occur during vinyl chloride exposure preceding the development of angiosarcoma. Although the hepatocyte is the major cell for oxidation and. probably detoxification of vinyi chloride and its metabolites, angiosarcoma develops not in the hepatocyte but in adjacent mesenchymal cells. During this time, the hepatocytes undergo changes (focal nodular hyperplasia) suggesting focal regeneration, probably from limited hepatocellular injury (Tamburro et al., 1979). The purpose of our study was to characterize the earliest sequential enzymatic changes that occurred in vinyl chloride exposure to elucidate the hepatocytes* role in vinyl chloride-induced injury and angiosarcoma develop* ment. Portions of this study have been presented elsewhere (Du and Tamburro, 1976). METHODS Animals and Experimental Design Sprague-Dawley male rats (300-500 g) were randomly assigned before each experiment to cither a group to be exposed to vinyl chloride gas (15,000 ppm) or a nonexposed control group kept outside the chamber in the animal room. Animals were fetf standard laboratory chow pellets ad libitum. Four sequential experiments were done. In experiments A and S, rats were exposed 2-4 h/d, 4 d/wk, for 1-2 wk with total accumulated exposure times of 14, 2S, and 42 h. In experiment C, rats were exposed 4-8 h/d, 5 d/wk, for 2-3 wk with total accumulated exposure periods of 71 and 103 h. In experiment D, rats were exposed 6-8 h/d, 5 d/wk, for 3-4 wk with' total accumulated exposure periods of 84 and 137 h. The animals were exposed in a modified chamber consisting of an airtight vat that had been used for manufacturing polyvinyl chloride. An aliquot of vinyl chloride (185 g) was added to the vat per 4-d period to make an average concentration of 15,000 4000 ppm. The air was constantly circulated by a stirrer. The chamber volume was 4400 I (1100 gal), so the' respiration of the rats had a negligible effect on the composition of the chamber's atmosphere. Experiments with an added control group exposed only to air in an identical modified vat showed no differences from the controls kept in the animal*room (Table 1). All animals were anesthetized with ether, had blood drawn by cardiac puncture, and were sacrificed at the same time of day (within 3 h after final exposure) to avoid diurnal effects. CMA 052806 EFFECTS QM ?.a7 \`iR 7'21 TA3LE 1. Compwton of Htpjtie njym Aeiivit:*; in Animal Control Croup* 8aMd in Animal Quartan and In tht Exposure Chamber* Entyme Air expowd Animal room Chamber* ClucawS-pnoiphau dehydfogtnxM Giuutitian* raducun 12.82 i 1.32c 4.77 t 0.27* ^Valuta art means t SEM (p 6), *Kpt in chamber 7 h/d, J d/wk, for 6 wk; total, 310 h. ^Not significant. *Not significant. 11.OS x 1.78* S.28 i 0.40* Sample Preparation Liver was excised rapidly and was immediately rinsed in ice-cold 0.15 M KQ with 0.02 M Tris buffer, pH 7.4. A portion of the tissue was homogenized with 9 volumes of the cold KCl-Tris buffer in a PotterElvehjem homogenizer. Each sample was prepared from a single organ and kept at 4C during preparation. Remaining liver was frozen rapidly and stored at --20C. For assays with frozen tissue, livers from control and experimental rats were frozen in an identical manner for the same length of time. Subceliular Fractionation and Biochemical Determination Homogenate was centrifuged at 600 and 8000 X g for 10 min and at 100,000 Ag for 60 min to obtain the nuclear, mitochondrial, microsomal, and cytosol fractions, respectively, by the differential technique of Schneider and Hogcboom (1950). Centrifugation was at 4C in a Sorvail refrigerated RC5 supercentrifugc with a fixed angle rotor and a Beckman model L-5 ultracentrifuge with a swinging bucket rotor. Enzyme activities and cytochrome P-450 were determined in the isolated subceliular frac tions: cytochrome oxidase in the mitochondrial fraction; NADPHcytochome c reductase, mixed function oxidase, cytochrome P-450, and glucose-6-phosphatase in the microsomal fraction; and glutathione reductase and giucose-6-phosphate dehydrogenase in the 100,000 X g supernatant fraction. Cytochrome P-450, NADPH-cytochrome c reductase, and cytochrome oxidase were determined in fresh samples; ail other determinations were in freshly isolated fractions from frozen tissue.~Glucose-6-phosphatase was estimated by measuring release of inorganic phosphate (Harper, 1965). Cytochrome P-450 was estimated by the maximum absorption difference between the dithionite-reduced cytochrome and its CO complex (Omura and Sato, 1964). The P-450 concentration was calculated by using 91,000 CMA 052S07 1122 J.T. DU ET AL. M~x cm-1 as the extinction coefficient for the increase in peak height between 490 and 450 nm. Cytochrome oxidase was measured as described by Wharton and Tzagoloff (1967), and NADPH-cytochrome c reductase according to Degroot and Dunn (1964). Mixed function oxidase was determined according to Holtzman et al. (1968) by measuring the hydroxylation of analine. Nonprotein sulfhydryl was estimated by the method of Sedlak and Lindsay (1968). Rate of oxidation of NADPH by oxidized glutathione was used as a measure of enzymatic activity of glutathione reductase (Carlberg and Mannervik, 1975). Glucose-6phosphate dehydrogenase was measured spcctrophotometrically, as the rate of NADPH formation (Lohr and Waller, 1965). Assays were conducted under conditions of linearity with respect to both time and protein. Protein content was determined by the method of Lowry et at. (1951). Conventional clinical analyses of serum included aspartate aminotransferase (AST, SGOT), alanine aminotransferase (ALT, SGPT), alkaline phosphatase, bilirubin,albumin, cholesterol, and triglyceride, determined by the technicon sequential multiple analyzer computer (SMAC) system. I Materials The NADPH, cytochrome c, and oxidized glutathione were obtained from Sigma Chemical Co., St. Louis, Mo. Analine and other chemicals were reagent grade. Double-distilled water was used throughout. Light and Electron Microscopy Small strips of liver were removed under ether anesthesia and immersed immediately in icc-cold 3% glutaraldchyde (pH 7.4). Tissues were sliced into small cubes and fixed for 2 h at 4C Subsequently, samples were washed overnight in phosphate buffer and postfixed in 1% osmium tetroxide for 1 h before being dehydrated in ascending alcohol and embedded in Epon. Tissue blocks were polymerized at 60C for 2 d. Thin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate before examination on a Philips 300 electron microscope. For ultrastructural analysis, control rats and rats exposed for 42 and 137 h to vinyl chloride were reviewed. For light microscopy, a block of tissue was fixed in buffered formalin and processed routinely for paraffin embedding. Sections 6 pm thick, were stained with hematoxylin and eosin before examination. RESULTS The protein contents (milligrams per gram of liver) of the subccllular fractions in control and vinyl chloride-exposed groups were the same throughout the exposure; therefore, the enzymatic results were expressed as micromoles converted per minute per milligram of subccllular protein. The statistical analysis (sec below) showed certain significant N CMA 052808 > [ 4. r'cCTS CN RAT LIVER enzymatic differences between the exposed and control groups in glucose6-pnosphatasc, glutathione reductase, and glucose-6-phosphatc dehydro genase. Glucose-6-phosphatase. No significant differences in g!ucose*6-phosphatase activity were detected between ihe two groups up to 42 h; however, after 71 h, the glucose*6-phosphatase activity was significantly less in the exposed than in the control animals. Figure 1(7 shows the 25% decrease in mean activity of glucose-6-phosphatasc after 71 h; this activity remained significantly lower up to 137 h. Figure 1 b shows the 95% confidence intervals for D, the mean difference between the exposed group and the control groups, in each experiment. <> FIGURE 1. (a) Composite curve of (roup nuns of specific activity of (lucosc-6-phosphatasc with respect to exposure time; (6) 95% confidence intervals for D, the mein difference between the exposed end control (roups. There Is no significant difference between the exposed and control (roups until after 42 h of exposure. After 71 h the mean level of the exposed (roup Is significantly less than that of the control (roup. CMA 052809 1124 J. T. DO ET AL. Glutathione Reductase. Figure 2, a and b, illustrates the significant differences {p<0.05) between the exposed and control groups in all 4 experiments except at 71 h of exposure (p<0.06). The glutathione reductase level was 25% greater in exposed than in nonexposed animals up to 71 h and approximately 50% greater after 84 and 137 h. Glucose-6-phosphate Dehydrogenase. There was no significant differ ence between exposed and control groups from 14 to 42 h and at 71 and 84 h. However, after 103 and 137 h of exposure there was a statistically significant difference as shown in Fig. 3, a and b. After about 100 h of exposure, the mean glucose-6-phosphate dehydrogenase level in exposed animals was about twice that in nonexposed animals. While these differences were occurring, none of the conventional biochemical liver function tests (e.g., serum aminotransferases, alkaline phosphatase) showed any significant change, nor were there any changes in cytochrome oxidase, mixed function oxidase, P-450, or NADPHcytochrome c reductase. FIGURE 2. (a) Composite curve of (roup meant of specific activity of glutathione reductase with respect to exposure time; (b) 05% confidence intervals tor D, the mean difference between the exposed and control (roup. The mean value for the exposed (roup is significantly (raster than that for the controls throu(hout the entire experiment (/* < 0.05) except at 71 h of exposure. CMA 052810 CHLOXiae F;C7S ZN '.aT Li 7 Lit mi () FIGURE 3. (a) Composite curve of {roup meins of specific activity of (lucaM-S-phosphlte dehydrogenise with respect to exposure time; (b) 95% confident* Intervals for O, the moan dlfforcnc* botwoon tho exposed and control (roups. Tho mean value for the exposed (roup is not siiniflcantly different from that for the control (roup until after 14 h of exposure. The concentration of reduced glutathione in the livers of rats decreased to 52% of the control value after a single 2-h exposure to 15,000 ppm vinyl chloride (Table 2). However, the reduced glutathione concentration appeared to be the same or slightly elevated when the rats underwent multiple exposures. This elevation was not significant at the 5% level. The vinyl chloride-exposed rats suffered a weight loss of 4-13% and the control group gained 2-10% (Table 3). All rats survived to the termination of the experiment without noticeable ill effects. ***-'r-" Statistical Analysis of Results The 95% confidence interval for group mean differences in each enzyme study was based on the error mean square from a two-factor analysis of variance with interaction,, the two main effects being exposure time in hours and cxposurc-noncxposure. This analysis was performed for CMA 052811 1126 J.T. DU ET AL. TABLE % Effect of Vinyl Chloride Exposure on Concentration of Liver Nonprotein Sulfhydryl Compound in Ran Exposure time (h) 14e 28 42 71 84 103 137 Concentration ratio (exposed/controi] 0.52 (3)* 1.02 (6) 0.96 (6) 0.82 (6) 1.21 (5) 1.07 (3) 1.49 (6) 1.29 (3) 'Single exposure, *Number of animals in control or experimental group is shown In parentheses. `Multiple exposures (4-6 h/d, 4-5 d/wk) for 14-137 h. TABLE 3. Body Wei|hts of Rats before and after Vinyl Chloride Exposure* Accumulated exposure M 14 28 42 71 84 103 137 Time Before After Before After Before After Before After Before After Before Affter Before After Control 442 6.2* (3)* 454 9.5 (3) 436 * 9.2 (3) 445 1 12.1 (3) 433 * 5.2 (3) 455 * 6.4 (3) 437.6* 12.2(5) 447* 19.4 (5) 412.7 * 7.9 (3) 455.0-1 6.8 (3) 453 12.9 (6) 465 * 16.9 (6) 404.7 a 1.2 (3) 42S.0 a 5.2 (3) Change (K> 3 2 +5 . +2 10 , +3 5 Vinyl chlorideexposed 453* 1.5(3) 421 * 4.1 (3) 448 * 6.0 (3) 408 * 3.0 (3) 450* 4.3 (3) 410* 2.8 (3) 422.7 * 9.2 (5) 385.0 * 4.2 (5) 425 * 17.4 (3) 399 * 72 (3) 441.5 * 6.8 (6) 397.0 * 5.7 (6) 415.0 15.7(3) 398 * 28 (3) Change <*) -7 -9 -9 -13 -6 -10 --4 'Multiple.exposure', to 1.5% vinyl chloride for 14-137 h, 4-6 h/d,4-5 d/wk. 6Results are expressed as mean * 5EM, `Number of animals h given in parentheses. CMA. 052812 VINYL CHLORIDE EFFECTS ON RAT LIVER VT17 each of the four experiments* Overall significant [p < 0.05) differences between the exposed and control groups are. found when the 95% confidence intervals (D values) do not contain zero. in Fig. Izr, there is no significant difference (p>0.05) between the 2 groups in experiments A and B because a D value of zero is within these two confidence intervals. However, for experiments C and 0, the con fidence intervals do not contain a D value of zero, indicating a significant difference (p<0.05) between the exposed and control groups. When the D value is less than zero (as in Fig. 16), the experimental group is significantly lower than the control throughout the experiment; when the D value is greater than zero (as in Figs. 2 and 3), the experimental group is significantly higher than the control. Further, in experiments C and D of Fig. 16, a single confidence interval is shown even though there are two time periods of exposure each. This results from the nonsignificance of the interaction term in the analysis of variance, meaning that the observed difference at 71 h of exposure is not statistically different from the difference at 103 h. Similar conclusions hold for experiment D in Fig. 16. Figure 26 shows 2 confidence intervals for the set of experiments C at 71 and 103 h because the interaction of the time and exposure factors was significant; that is, the difference between the exposed and control groups after 103 h of exposure was significantly greater than the difference after 71 h. This is also reflected by the differences of the mean values at 71 and 103 h as shown in Fig. 2a. For the same reasons, there are also two confidence intervals for the C (71 and 103 h) and D (S4 and 137 h) sets of experiments in Fig. 36. Morphological Studies Morphological light microscopic studies were performed in a blind (coded) and randomized fashion and did not show any evidence of hepatocellular injury or changes usually seen in the latter stages of vinyl chloride exposure. Besides some variations in glycogen content, no difference in the fine structure of the hepatocytes was observed between controls (Fig. 4) and those exposed to vinyl chloride for 42 h. Dilation of rough endoplasmic reticulum was observed in a small number of hepatocytes after 137 h of vinyl chloride exposure (Fig. 5). Although no noticeable change in the amount of smooth endoplasmic reticulum was associated with this change, a concomitant increase in cytoplasmic density was evident in these ceils. Another type of lesion found in other hepatocytes was characterized by the presence of small patches of dear spaces, which tended to aggregate near the cell periphery (Fig. 6). Such lesions usually affect the adjacent cell equally. Mo other cell type appeared to be affected by vinyl chloride exposure in this study. ms J.T. DUET AL. FIGURE 4. Portion of a hepatocyte from i control rat. A food complement of mitochondria and rough endoplasmic reticulum is shown (X 12,000). FIGURE 5. Dilation of RER (arrows) shown in hepatocyte 137 h after exposure to 13,000 ppm vinyl chloride during 2-3 wk (X 2,5,000). FIGURES. Subplasmalcmmal lesions (*) in hepatocyte* 137 h after exposure to 15,000 ppm vinyl Chloride during 2-3 wk. The' lesions are present in two adjacent ceils and the dear spaces seem to coalesce (x 12,000), ' DISCUSSION Decreased glucose-6-phosphatase activity and increased glucose6-phosphate dehydrogenase and glutathione reductase activity in rat liver after exposure to vinyl chloride (as in our studies) may be the biochemical alterations indicative of early liver injury, adaptation to increased, detoxi fication activity, or preparation for increased nucleic acid synthesis. These differences were observed before any abnormalities were detectable from conventional liver function tests, such as serum aminotransferases, or from changes in other subccllular organelle markers, such as mitochondrial cytochrome oxidase activity. In primary hepatocellular cancer (hepatomas), the activity of key enzymes for gluconcogenesis (glucosc-6-phosphatasc, etc.) decreased with increased rate of tumor growth (Weber, 1974; Weber and Convery, 1966; Weber and Lea, 1967). In contrast, two enzymes for the pentose phosphate pathway, glucosc-6-phosphate dehydrogenase (Sclmcci and CMA 052814 VINYL CJ.L0KI3E EFFECTS ON RaT lIV Ek Weber, 1976; Weber and Morris, 1953) and transaldolasc (Heinrich ct al., 1974), increased in all hepatomas. Further, the activity of gludosc-6phosphatase decreased before and during the development of hepatomas when carcinogens such as nitrosamine and dimcthylaminoazobenzene were fed to rats (Isok and Teras, 1973; Weber and Cantero, 1955). Whether our similar findings in this study are indicators of eventual cancer development (angiosarcoma) is not yet known. We used a shorter exposure period in order to identify the biochemical changes that would best reflect the morphological and cellular changes anticipated on the basis of previous human and animal studies. No attempt was made to determine what effects these shorter exposure periods would induce with long-term observation. This is now in progress. However, Maltoni and Lefemins (1975) showed that of 69 Sprague-Cawley rats exposed to 10,000 ppm vinyl chloride, 16 (26%) developed Zymbal gland carcinomas after 50 wk, 5 (8%) developed nephroblastomas after 59 wk, and 9 (15%) developed angiosarcomas after 64 wk. The vinyl chloride-exposed groups lost weight (4-13%) whereas the control group gained weight (2-10%) during the entire experimental period. However, the differences in the three enzymes cannot be accounted for by lack of dietary food intake in the experimental group, because fasting increases glucose-6-phosphatase (Ashmore et al., 1954) and decreases glucose-6-phosphate dehydrogenase (Winberry and Hoi ten, 1977), and we found decreased glutathione reductase and reduced glutathione content in fasted animals (unpublished data). Rats exposed to a high dose (5%) of vinyl chloride (Reynolds et al., 1975) had decreased mixed function oxidase activity. An in vitro study (Ivanetich et al., 1977) showed that the metabolites of vinyl chloride from the microsomal enzyme system decreased the levels of cytochrome P-450. In our studies; the cytochrome P-450 content and the activity of mixed function oxidase and NAOPH-cytochrome c reductase in rats repeatedly exposed to 1-2% vinyl chloride did not show any such changes. Sub sequent experiments in which rats were exposed to 28,000 ppm vinyl chloride for 70-210 h in 2-6 wk showed a significant decrease of cytochrome P-450 concentration (in preparation). Differences in dose level or experimental design may account.for these variations. Drew et al. (1975) found that the activity of mixed function oxidase depended on the time interval between cessation of exposure to vinyl chloride and sacrifice of animals. The main detoxification route for vinyl chloride metabolism is thought to be conjugation with glutathione (Johnson, 1967); sulfur-containing urinary metabolites of radioactiveiy labeled vinyl chloride have been found in rats' urine (Green and Hath way, 1975, 1977; Watanabe et al., 1976b, 1976c). We found that under the present experimental conditions- the nonprotein sulfhydryl content of liver (glutathione and/or cysteine) tended to CMA 052815 mo J. T. DU ET AL. increase in rats repeatedly exposed to vinyl chloride (Table 2). Although the difference was not significant in this experiment, subsequent exposure of rats to 28,000 ppm for 70, 140, and 210 h in 2, 4, and 6 wk led to a significant elevation of the nonprotcin sulfhydryl content in liver (Du and Tamburro, 1978). Watanabe et ai. (1976a) and Johnson (1967) found a depletion of nonprotein sulfhydryl content in rats after only a single exposure to vinyl chloride or chloroethanol. Hefner et al. (1975) found that a single vinyl chloride exposure reduced the glutathione content by one-half, but the decrease became smaller and even unnoticeable after repeated exposure to vinyl chloride. Fiala et al. (1976) showed that the reduced glutathione content increased after prolonged exposure of rats to various chemical carcinogens. These results are thought to reflect the exposed animals' attempt to make more glutathione to meet the unusually strong demand for detoxification. Glutathione reductase was elevated in our system after rats were exposed to vinyl chloride for 42 h. This enzyme generates reduced glutathione from its oxidized form as a compensatory mechanism to maintain the level of glutathione, and the simultaneous elevation of glucose-6-phosphate dehydrogenase regenerates NADPH, which can be used as a cofactor for various synthetic pathways including nucleic acid synthesis. Elevated glutathione reductase activity was found in rats with primary hepatocellular cancer induced by diethylnitrosamine (Pinto and Bartley, 1973). This consistent increase in glutathione reductase activity after exposure to vinyl chloride suggests that it may be one of the earliest biochemical manifestations of exposure and injury. These metabolic changes could also play a role in the early changes observed by light and electron microscopy. Hepatic lesions such as dilation of smooth endoplasmic reticulum and loss of microvilli were reported in mice as early as 1 mo after vinyl chloride exposure (Schaffner et al., 1976). In contrast, we observed dilation of rough endoplasmic reticulum and patchy, lesions near the piasmalemma. The dilated rough endoplasmic reticulum could be related to the increased enzyme synthesis (i.e, glucose* 6-phosphate dehydrogenase and glutathione reductase) induced by vinyl chloride. The absence of smooth endoplasmic reticulum proliferation in our animals suggests a comparatively milder effect in our short-term study. In a subsequent study where long-term effects of vinyl chloride exposure was assessed, dilation of smooth endoplasmic reticulum in hepatocytcs was observed (in preparation). The nature of the patchy lesions found near the piasmalemma remains to be established. The location of the lesion, however, suggests that some toxic agents may be entering or exiting the hepatocytcs in these sites. We believe these early enzymatic changes reflect adaptation of the liver cell to early mild injury. The alterations in gfuconeogencsis and in the pentose phosphate shunt appear to indicate the liver's adaptation while undergoing repetitive and prolonged exposure to the mildly toxic chemical CMA 052816 VINYL CHLORIDE EFFECTS ON RAT L1"ER . Ilii vinyl chloride, which, when inadequately metabolized, produces inter mediates that lead to the formation of cancer. REFERENCES Ashmore, )., Hastings, A. 8., and Nesbett, F. 8. 1954. The effect of diabetes and fasting on liver giucost-6-phosphatue. Prot. Natl. Acad. Sci. U.S.A. 40:673-678. Bolt, H. M., ICappus, H., Buehter, A., and Bolt, V/. 1975. Metabolism of vinyl chloride. Lancet 1:1425. Carlbsr-, I. and MtnneryiV, 3. 1975. Parificirien tr.i ch;."c;:r:ia:isn of the Ravctntyme glutathiont reductase from rat liver. /. 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