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TOXICOLOGY AND APPLIED PHARMACOLOGY 62, 1-10 (1982) RECEIVED APR 8 1982 The Effect of Repeated Vinyl Chloride Exposure on Rat Hepatic Metabolizing Enzymes1 Julie T. Du,- Michael T. Tseng, and Carlo H. Tamburro3 Liver Research Center, Division of Digestive Diseases and Nutrition, Departments of Medicine and Anatomy, and Regional Cancer Center, University of Louisville School of Medicine, Louisville. Kentucky 40292 Received July 17. 1980; accepted September 12, 1981 The Effect of Repeated Vinyl Chloride Exposure on Rat Hepatic Metabolizing Enzymes. Du, J. T , Tseng. M. T . and Tamburro, C. H. (1982). Toxicol. Appl. Pharmacol. 62, 110. Sprague-Dawley rats were exposed to 2.8% vinyl chloride for 2 (70 hr), 4 (140 hr), and 6 (210 hr) weeks to determine the sequential biochemical changes related to the oxidation and detoxification ability of hepatic tissue. Glutathione-S-transferase(s) activity using 1,2epoxy-(p-mtrophenoxy)propane and p-nitrobenzyl chloride as substrates was elevated 17 to 24, 28, and 35 to 42% after 2, 4, and 6 weeks of exposure, respectively, suggesting enzyme(s) induction. Reduced glutathione, the major substrate required to conjugate the toxic metabolites of vinyl chloride, was also consistently elevated. Similarly, the activity of glutathione reductase, the enzyme necessary for the regeneration of reduced glutathione from its oxidized form, was also increased following vinyl chloride exposure. Cytochromes f-450, the major protein in volved with vinyl chloride metabolism, was reduced after vinyl chloride exposure, confirming reports of others that vinyl chloride metabolites destroy P-450. No abnormalities of standard clinical biochemical blood tests of liver function were found during 6 weeks of vinyl chloride exposure. The only consistent ultrastructural modification was the dilation of endoplasmic reticulum. The biochemical and ultrastructural alterations could reflect early hepatocellular adaptation to vinyl chloride exposure. Vinyl chloride, at high concentrations, has been shown to be carcinogenic in both lab oratory animals (Maltoni and Lefemine, 1975; Viola et al., 1971) and man (Creech and Johnson, 1974). Present data support the metabolism of vinyl chloride by hepatic microsomal mixed-function oxidase system into toxic intermediates, chloroethylene ox ide (Bolt et al., 1975; Hefner et al., 1975; Kappus et al., 1976) and chioroacetaldehyde 1 This work was supported by a grant from the Man ufacturing Chemists Association, Washington. D.C. Portions of this study have been presented (Fed. Proc. 37, 1545, 1978). : Present address; Clement Associates, Inc., 1010 Wisconsin Avenue, N.W., Suite 660, Washington, D.C. 20007. ! Address requests for reprints to: Carlo H. Tam burro, University of Louisville. (Gross and Freiberg, 1969). These two in termediates are considered to be the ultimate carcinogens (Barbin et al., 1975; Jaeger et al., 1974b; Van Duuren, 1975), to be mu tagenic in bacterial systems (Elmore et al., 1976; Greim et al., 1975; Malaveille et al., 1975; McCann et al., 1975), to act as an alkylating agent by reacting with adenosine (Barbin et al., 1975) and cytidine (Laib and Bolt, 1978), and to bind with protein (Bolt et al., 1976( Kappus et al., 1976; Watanabe et al., 1978). Detoxification of these metab olites occurs mainly by conjugation with glu tathione and is catalyzed by hepatic gluta thione transferases; the conjugates are excreted in the urine as substituted cysteine derivatives (Watanabe et al., 1976b,c; Green and Hathway, 1975, 1977). Chloroacetaidehyde can be further oxidized to chloro- 1 0041-008X/82/010001-10S02.0O/0 Copyright 1982 by Academic Press, Inc. AU rights of reproduction in any form reserved. CMA 005730 2 DU, TSENG, AND TAMBURRO acetic acid (Hefner et al., 1975). These data are compiled in a metabolic scheme in Fig. 1 as an updated hypothesized metabolic fate of vinyl chloride in the adult rat. The me tabolism of chloroethylene oxide via epoxide hydratase is not listed in the scheme because its product has not been identified. There have been a few in vivo studies con cerning the effects of vinyl chloride exposure on hepatic concentrations of glutathione (Hefner et al., 1975; Watanabe et al., 1976c; Du and Tamburro, 1978), cytochromes P450 (Reynolds et al., 1975) and on mixedfunction oxidase activity (Drew et al., 1975; Reynolds et al., 1975). However, informa tion about the sequential alterations in he patic oxidation and detoxification of vinyl chloride, during prolonged exposure, simu lating the occurrence in workers, is still lack ing. It was reported previously that the en zymatic changes in rat liver following prolonged exposure to vinyl chloride were similar to those found in rat hepatoma (Du and Tamburro, 1976; Du et al., 1979). The sequential biochemical changes related to the hepatic oxidation and detoxification of vinyl chloride following prolonged exposure are reported here. METHODS Animats and experimental design. Eight- to tenweek-old Sprague-Dawley male rats ( -- 300g), sup plied by Laboratory Supply of Indianapolis, Indiana, were randomized prior to the experiment into three groups: a vinyl chloride-exposed group and the air-exposed group housed in identical chambers and a second control group housed in the University's Central Animal Care Center. The exposure level was 28,000 ppm vinyl chloride, 7 hr/day, 5 days/week for 2, 4, and 6 weeks. The exposure chambers were 4400-liter airtight vats. Vinyl chloride ( -- 300 to 340 g) was added to the vat to give a time-weighted average concentration of 28,000 1000 ppm. The chamber air was changed daily and the vinyl chloride concentration was determined by gas chromatography. The air was constantly circulated by a stirrer. The rats' respirations had negligible effect on the composition of the chamber's atmosphere because of the chamber's large volume. Animals were fed on standard laboratory chow pellets ad libitum. All animals were anesthetized with ether, blood was drawn from the inferior vena cava, and the animals were (v c) Cl CH - CH2 % ** Cl CH2CH20H (chloroethanol) DETOXIFICATION WITH GLUTATHIONE (chlqrooxirane) Cl CH-CH, \/2 v4 4 *' GS CH2CH20H A k* Cl CH2CH0 (t-GSHj , GAST (chloroacetaldehvde) GS CH2CH0 4 Cl ch2cooh N-Ac-S-(2-hydroxy ethyl) cysteine GS CH2C00H | T TH10DIGLYC0LIC ACID (CHL0R0ACETIC ACID) Fig. 1. The proposed metabolic fate of vinyl chloride. (GSH, glutathione; MFO, mixed-function oxidase; VC. vinyl chloride; GEST, glutathione 5-epoxide transferase; and GAST, glutathione 5-al dehyde transferase). CMA 005731 VINYL CHLORIDE EFFECT ON ENZYMES 3 killed approximately 20 hr after exposure at 1 00 pm eaeh day NADPH. glutathione, and glutathione disulfide were obtained from Sigma Chemical Company, St. Louis, Missouri: 1,2-epoxy-3-(p-mtrophenoxy)propane was purchased from Eastman Kodak Company, Rochester, New York, p-nitrobenzyl chloride was obtained from Matheson, Coleman and Bell, East Rutherford, New Jersey; benzphetamme was donated by the Upjohn Company, Kalamazoo, Michigan. Double-distilled water was used throughout. Sample preparation and biochemical determination. Homogenates and subcellutar fractions were prepared as described previously (Du et al., 1979). Each sample was prepared from a single organ and kept at 4C during preparation. The remaining liver was frozen rapidly in liquid nitrogen and stored at -70C. Cytochromes P450 concentrations were determined in the frozen mi crosomal fractions the day following sacrifice. The glu tathione (GSH) concentration, as well as glutathioneJ-transferase, glutathione reductase, and mixed-func tion oxidase activities were determined in the freshly fractionated frozen liver. For the assays using frozen tissue, the livers from control and experimental rats were frozen in an identical manner for the same length of time. Cytochromes P-450 concentration (Omura and Sato, 1964), nonprotein sulfhydryl content (Sedlak and Lindsay, 1968), and glutathione reductase activity (Carlberg and Mannervik, 1975) were determined in the microsomal or cytosol fractions by methods de scribed previously (Du et al., 1979). Glutathione-5-transferase activity was determined using the 100,000 X g supernatant fraction. 1,2-EpoxyJ-(p-nitrophenoxy)propane and p-nitrobenzyl chloride were the substrates for glutathione-S-epoxide transfer ase and glutathione-5-aralkyl transferase (GAST), re spectively. Enzyme activity was determined as described by others (Habig et al., 1974; Kaplowitz et al., 1975). All assays were linear functions of protein concentration and timed for at least 2 min. Solutions of 1,2-epoxy-3(/>-nitrophenoxy)propane and p-nitrobenzyl chloride were prepared in absolute ethanol; the final ethanol con centration in the incubation mixture was 0.5%. Mixedfunction oxidase activity was estimated in the micro somal fraction by measuring NADPH disappearance in the NADPH-depcndent demethylation reaction of benzpbetamine (Lu et al., 1972). The protein content was determined by the method of Lowry et al. (1951). The serum clinical liver tests including aspartate ami notransferase, alanine aminotransferase, alkaline phos phatase, bilirubin, cholesterol, and triglyceride were determined by Technicon sequential multiple analyzer computer (SMAC) system. Light and electron microscopy. Small strips of liver were removed under ether anesthesia, sliced into small cubes, placed immediately in ice-cold 1% osmium tetroxide (pH 7.4), and fixed for 2 hr at 4C. Subse quently, samples were washed overnight in phosphate buffer, dehydrated in ascending alcohol, and embedded in Epon. Tissue blocks were polymerized at 60C for 2 days. Thin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate before ex amination on a Philips 300 electron microscope. For ultrastructural analysis, three rats randomly selected from controls and groups exposed for 2, 4, and 6 weeks to vinyl chloride were studied. For light microscopy, a block of tissue was fixed in buffered formalin and processed routinely for paraffin embedding. Sections 6 m thick were stained with he matoxylin and eosin. Statistical analysis. Analysis of variance was per formed for the various groups at the different time pe riods and multiple comparisons were performed based on the results of the analysis of variance. RESULTS The protein content (mg protein/g liver) in the subcellular fractions in both control and vinyl chloride-exposed groups was the same throughout the exposure (data not shown); the enzymatic results, therefore, are expressed as micromoles of substrate con verted per minute per milligram of protein. There were no statistical differences be tween the normal and air-exposed groups in the glutathione and cytochromes P-450 con tents or in any of the enzyme activities. The nonprotein sulfhydryl content (Table 1) was significantly elevated from 26 to 54% at 2, 4, and 6 weeks in the vinyl chloride-exposed group compared to both control groups. Al though the nonprotein sulfhydryl content in creased with exposure, the increases were not statistically significant. Glutathione re ductase activity (Table 1) in the exposed group was increased by 53 to 77% at all three time periods. The increase in glutathione reductase was the same at 2 and 4 weeks of exposure but showed a further significant increase after 6 weeks of exposure. Glutathione-S-epoxide transferase (GEST, Table 1) and glutathione-5-aralkyl transferase (GAST, Table 1) activities were signifi cantly higher than controls after 6 weeks of exposure, 37 and 45%, respectively. The cy tochromes P-450 content, on the other hand, CMA 005732 DU, TSENG, AND TAMBURRO CMA 0 0 5 7 3 3 TABLE [ Sequential Changes in Hepatic Nonprotein Sulehyoryl, Cytochromes H-450 Content anu Activitiesoe Glut at hioni Ri ihk r asi and GLUTATH ION E-S-TR A NSE ERASES (EPOXIDE AND ARALKYL) IN RaTS EXPOSED TO VlNYL ChLORIOE"' Time (weeks) Nonprotein suirhydryl Gnnol/g liver) Glutathione reductase (100 x rtmol/min/mg protein) GEST (100 X pmol/ min/mg protein) GAST (10 X pmol/ min/mg protein) Cytochrome R-450 (nmol/g liver) Treatment Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Norma] control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control 0 7.9 0.3 -- -- 5.0 0.2 --__ 9.1 1.6 -- 2.4 0.4 -- -- 17.0 3.7 -- -- 2 7.8 0 4** 9.4 0 2*' 7.1 0.3- 4.3 0.4* 6.7 0.6*' 4.5 0.2` 7.8 0.4 9.1 + 1.7 8.1 + 1.2 2.1 + 0.3 2.6 + 0.4 2.2 + 0.2 17 1 1.7 13 2 + 1.1 15.5 + 1.3 4 7.1 0.4* 10 2 0 6*' 6.9 + 0 4' 4.2 + 0 4* 6.3 + 0.5*' 3.7 0.3' 7.5 1.0 9.7 0.7' 6.3 0.7' 1.9 + 0.2 2.4 0.3 1.9 0.2 19.7 1.3* 15.3 1.3* 19.6 2.7 6 6 9 0.4* 1 1 4 ! 0 6*' 79 ! 0 V 4 8 t l> V' 8.9 1 0 7*' 5.3 10 4' 7 7 1 1.0* 1 1 0 1.3*' 84 0 9' 2 4 i 0 3* 3.2 0 l*' 2.1 0 2' 15 5 + 1 4* 10.6 10*' 14.3 1.8' " Rats were exposed to 28,000 ppm of vinyl chloride; normal controls and the air controls were exposed to air only. Each number represents the mean and the SEM from a group of six rats. * Normal vs vinyl chloride-exposed, p < 0.05. 1 Air control vs vinyl chloride-exposed, p < 0.05. * Vinyl chloride (6 weeks) exposed vs vinyl chloride (2 and 4 weeks) exposed, p < 0.05. I VINYL CHLORIDE EFFECT ON ENZYMES was significantly lower than controls after 6 weeks of exposure to vinyl chloride (Table 1). No differences were found in the hepatic mixed-function oxidase activity or in the serum clinical liver tests. After 2 weeks of exposure, the two control groups had gained weight but the vinyl chloride-exposed group did not (Table 2). After 4 weeks of exposure, the normal control group housed at the an imal care center had gained significantly more weight than either the air-control or vinyl chloride-exposed group. After 6 weeks of exposure, however, the vinyl chloride-exposed group failed to gain weight; the nor mal control group gained more than the aircontrol group (Table 2). Morphological examination revealed poly hedral hepatocytes arranged in irregular plates interposed by vascular sinusoids in the livers of the control rats. This general cytoarchitecture was maintained after vinyl chloride exposure. Hepatocytes in control rats contained a prominent spherical nu cleus, numerous ovoid mitochondria, stacks of rough endoplasmic reticulum (RER), some aggregates of smooth endoplasmic re ticulum (SER), and varying amounts of ly- sosomes and glycogen particles (Fig. 2a).Few interstitial cells were scattered among the hepatocytes. These cells contained few cy toplasmic organelles and could be readily discerned at the light microscopic level by their hyperchromatic nuclei. The sinusoids were linked by fenestrated endothelium and some of the lining cells displayed phagocytic activity. After 2 to 6 weeks of vinyl chloride exposure, the principal organelle affected appeared to be the endoplasmic reticulum. Cistemae of the RER became dilated in a relatively small population of the hepato cytes in the 2-week treatment group. Four weeks after exposure to vinyl chloride, patches of dilated endoplasmic reticulum were prominently displayed in some hepa tocytes (Fig. 2b). At this stage the SER was relatively unaffected. In the 6-week treat ment group, vesiculation of SER and dis tention of RER were easily discernible in a large number of hepatocytes (Fig. 2c). How ever, other cell organelles showed no de monstrable change. These changes, though, are still beyond the resolving limit of the light microscope. The nonhepatocyte com ponents showed minimum changes which TABLE 2 Body Weights of Rats before and after Vinyl Chloride Exposure" Duration (week) 2 4 6 Treatment Normal control VC-exposed Air control Normal control VC-exposed Air control Normal control VC-exposed Air control Initial weight (g) 400 15 405 12 396 17 398 8 410 16 395 10 402 14 396 9 398 14 Final weight (g) 433 16 396 14 414 18 450 15 421 15 419 5 486 14 398 10 449 4 Percentage gam 8* -2" 5' 13" 3* 64 21" <1" 13" " Analysis of body weight was by regression analysis followed by an analysis of variance on the residuals from the regression equation. (Residual = observed final weight - predicted final weight from regression equation.) 4 Normal control vs vinyl chloride exposed, p < 0.05. c Air control vs vinyl chloride exposed, p < 0.05. 4 Air control vs normal control, p < 0.05. 6 DU. TSENG. AND TAMBURRO CMA 005735 VINYL CHLORIDE EFFECT ON ENZYMES 7 were characterized by an increased accu mulation of lysosomal-like substances in some of the sinusoidal lining cells as well as a greater tendency to accumulate lipids in the interstitial cells (Fig. 2d). DISCUSSION Glutathione conjugation is an important pathway for the metabolism of potentially harmful electrophilic metabolites of xenobiotics. Studies by Watanabe et al. (1976b,c) indicate this to be the major route for in activation of the vinyl chloride metabolites. Glutathione-S-transferases are a group of cytosol enzymes catalyzing the reaction of glutathione and electrophilic compounds to form less toxic and more water-soluble con jugates. Their activity during chronic ex posure to xenobiotics, like vinyl chloride, could be a key determinate in the ultimate outcome of such exposures as illustrated by the longer arrow in Fig. 1. The increase in hepatic GEST activity at 4 weeks and the later increase in GAST activity at 6 weeks suggested that in the earlier stages of ex posures most of the chlorooxirane interme diate is being adequately detoxified. During the later stages of chronic exposure, more chlorooxirane may become rearranged to yield more chloroacetaldehyde and, in turn, react with other available glutathione trans ferases or become further metabolized to chloroacetic acid. Alternatively, the exces sive chlorooxirane could rearrange sponta neously to form chloroethanol and be further oxidized to chloroacetaldehyde, which in turn may react with glutathione, or be oxi dized to monochloroacetic acid (Johnson, 1967). This would be consistent with the later increases in the aralkyl-transferases and the finding by Hefner et al. (1975) that monochloroacetic acid is found only in the urine of rats exposed for an extended time to higher levels (5000 ppm) of vinyl chloride. The increased use of alternate pathways, for chlorooxirane and chloroacetaldehyde de toxification, may reflect increased concen tration of these active metabolites allowing greater opportunity for DNA injury. A single exposure to vinyl chloride de creased hepatic nonprotein sulfhydryl com pounds in rats (Watanabe, 1976a); similar decreases of glutathione concentrations were produced in rats by other xenobiotics such as 1,1-dichloroethylene (Jaeger et al., 1974a; Reichert et al., 1978) and acetaminophen (Mitchell et al., 1973). In the present study, repeated exposure to vinyl chloride caused a significant increase of nonprotein sulfhydryl concentrations (Table 1) analogous to the elevation of glutathione concentrations seen after the administration of carcinogens to rats (Fiala et al., 1976). In addition, the results showed that repeated exposure to vi nyl chloride also caused an increase in he patic glutathione-5-transferase activity (Ta ble 1) similar to that seen after the administration of phenobarbital and 3-methylcholanthrenc to rats (Mukhtar and Bresnick, 1976). These data suggest a mecha nism for compensatory synthesis of hepatic glutathione and glutathione-5-transferases after repeated exposure to vinyl chloride. The decreased concentration of cyto chromes P~450 found in rats after repeated exposure to vinyl chloride (Table 1) is con- -- Fig. 2. (a) Portion of a hepatocyie from control. Stacks of rough endoplasmic reticulum (RER) are separated by many ovoid mitochondria (M). Chromatin is finely dispersed in the nucleus (N). 9300X. (b) Hepatocyte after 2 weeks of vinyl chloride exposure. Dilation of RER appeared widespread in these two cells. Bile (B) canaliculus appeared unaltered in these rats. 5300X. (c) Four weeks after vinyl chloride exposure. Golgi complex (G) appeared unaffected while cisternal dilation continued. Distinction between SER and RER is complicated by the detachment of ribosomes. Lipid droplets (L) and glycogen (GL) often accumulated. 9500X. (d) A fat-storing interstitial cell is surrounded by several hepatocytes in a vinyl chloride-treated animal. Unlike lipid stored in hepatocytes, the shape of lipids (L) appeared irregular in these cells. 8000X. CMA 005736 DU, TSENG, AND TAMBURRO sistent with work by Reynolds et al. (1975). This decrease in cytochromes F-ISO content was also shown in vitro (Guengerich and Strickland, 1977: Ivanetich et al., 1977) sug gesting that a metabolite of vinyl chloride destroys the cytochrome. Mixed-function oxidase activity, with benzphetamine as sub strate was unaltered. With regard to the structural alterations produced by vinyl chloride, the present find ings confirmed previous observations on the selective effect of this carcinogen in the en doplasmic reticulum (Du et al., 1979). A gradual increase in the number of hepatocytes affected and the involvement of both smooth and rough ER were shown in this study. The endoplasmic reticulum is the pri mary site of protein synthesis and its dilation suggested the presence of a vinyl chloriderelated effect. The lack of a concomitant Golgi hypertropy is noteworthy since this organelle serves as the site of glycosylation and packaging of many exportable proteins. It may be inferred that the vinyl chloride effect is mainly an intracellular phenome non. The relatively late involvement of SER could reflect a further attempt at detoxifi cation by the hepatocyte. The tendency for the interstitial cells to accumulate lipid and the heightened phagocytic activity correlate with increases in collagen formation and may be evidence of low-grade cellular injury. This may prove to be the initial histological response to vinyl chloride exposure. Ultrastructural responses to chronic in halation of vinyl chloride were reported by Feron et al. (1979). Unlike our findings, the principal effects observed were swollen mi tochondria and some proliferation of SER. The mitochondrial swelling presumably re flects the extensive vacuolization observed by light microscopy. The discrepancy prob ably resulted from differences in the dose and duration in vinyl chloride exposure since Feron et al. (1979) exposed rats to 5000 ppm vinyl chloride for 52 weeks. Such an exten sive mitochondrial lesion could result in bio chemical modifications such as a change in ATPase or cytochrome oxidase levels. The only enzyme activity measured, glucose-6phosphatase, was reduced. The authors believed that the altered glu tathione metabolism, as reflected by the in creased nonprotein sulfhydryl content, and the increased activities of glutathione-Stransferases and glutathione reductase, in rat liver after repeated exposure to high doses of vinyl chloride represent an early hepatocellular adaptation to vinyl chloride exposure. ACKNOWLEDGMENTS The authors wish to express their gratitude to the staff of the B. F. Goodrich Plant in Louisville, Kentucky, for their cooperation in the exposure studies, to Dr. Richard A. Greenberg for his review, to Mr. John P. Sandoz for his help with the statistical analysis, and Mr. John Kreisle and Ms. Debra S. Eades for technical as sistance. REFERENCES Barbin, A., Bresil, H., Croisy, A., Jacquignon, P., Malaveille, C., Montesano, R,, and Bartsch, H. (1975). Liver-microsome-mediated formation of alkylating agents from vinyl bromide and vinyl chlo ride. Biochem. Biophys. Res. Commun. 67, 596-603. Bolt, H. 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C H, (1978). Elevated gluUthione content, glutalhione-S-transferase and iiuuthione reductase in liver of rats exposed to vinyl chloride Fed Proc. 37. 1545. Dl. J T . Sxsdoz, J P . Tseng. M. T . and Tambi rro. C H. I I979) Biochemical alterations in liv ers of rats exposed to vinyl chloride J Toxicol nwran Health 5, 1119-1132 Elmore, J D , Wong, J L , Laumbach. a. D , and Streips, U N. (1976). Vinyl chloride mutagenicity via the metabolites chlorooxirane and chloroacetaldehyde monomer hydrate. Btochtm Btophys. Acta 442, 405-419, Feron. v j.. Spit. B. J,, Immel, H. R.. and Kroes, R (1979). One-year time sequence inhalation toxicity study of vinyl chloride in rats. III. Morphological changes in the liver Toxicology 13, 143-154. Fiala, S., Mohindru, A., Kettering, W. G., Fiala, A E., and Morris, H. P. ( 1976), Glutathione and gamma glutamyl transpeptidase in rat liver during chemical carcinogenesis. / Hat Cancer Inst. 57, 591598. Green, T , and Hathway, D. E. (1975). 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Hefner. R. E., Watanabe, P. G., and Gehrino, P. J. (1975). Preliminary studies of the fate of inhaled vinyl chloride monomer in rats. Ann. N. Y. Acad. Sci. 246, 135-148. Ivanetich, K. M., Aronson, I., and Katz, I. D. ( 1977). The interaction of vinyl chloride with rat he patic microsomal cytochrome P-450 in vitro. Biochem. Biophys. Res. Commun 74, 1411-1418. Jaeger, R. J., Connolly. R. B., and Murphy, S. D, (1974a). Effect of 18 hr. fast and glutathione deple tion on 1,1-dichloroethylene-induced hepatotoxicity and lethality in rats. Exp. Mol. Pathol. 20, 187-198. Jaeger. R. J. Reynolds, E. S. Connolly. R B, Moslen. M T,. Szabo, S., and Murphy, S. D. ( 1974b), Acute hepatic injury by vinyl chloride in rats pretreated with phenobarbttal Mature /London/ 252, 724-726. Johnson, M. K, (1967) Metabolism of chloroeihanol in the rat. Biochem Pharmacol. 16, 185-199 Kaplowitz, N , Kuhlenkamp. I., and Clifton, G ( 1975) Drug induction of hepatic glutathione-Stransferases in male and female rats. 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Maltoni, C, and Lefemine, C. (1975). Carcinoge nicity bioassays of vinyl chloride: Current results. Ann N Y Acad. Sci. 246, 195-218. McCann, J., Simmon, V.. Streitwieser, D,, and Ames, B. N. (1975). mutagenicity of chloroacetaldehyde, a possible metabolic product of 1,2-dichloroethane, chloroethanol, vinyl chloride and cyclo phosphamide. Proc. Nat. Acad. Sci. USA 72, 31903193. Mitchell, J, R., Jollow, D. J., Potter, W. A., Gillete, J. R., and Brodie, B. B. (1973). Acetamin ophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187, 211217. Mukhtar, H., and Bresnick, E. (1976). Effects of phenobarbital and 3-methylcholanthrene administra tion on glutathionc-5-epoxide transferase activity in rat liver. Biochem. Pharmacol. 25, 1081-1084. OmuRa, T., and Sato, R. (1964). The carbon monoxide-binding pigment of liver microsomes. I. Evi dence for its hemoprotein nature. J Biol. Chem. 239, 2370-2378. Reichert, D., Werner. H. W,, and Henschler, D. CMA 005738 10 DU, TSENG, AND TAMBURRO ( 1978) Role of liver glutathione in 1,1 -dichloroethv lene metabolism and hepatotoxicity in intact rats and isolated perfused rat liver. Arch. Toxicol. 41, 169178. Reynolds. E, S., Moslen, M T, Szabo. S, and JaEGER, R, J, (1975). Vinyl chloride-induced deac tivation of cytochrome P-450 and other components of the liver mixed function oxidase system: An in vivo study. Res. Commute Chem Pathol Pharmacol, 12, 685-694. Sedlak, J., and Lindsay, R. H. (1968). Estimation of total protein bound and non-protein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem. IS, 192-205. VanDuuren, B. L. (1975). Possible mechanism of car cinogenic action of vinyl chloride. Ann. ,S.Y Acad. Sci. 246, 258-267. Viola, P L,, Bigotti, A., and Caputo, A. (1971). Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res. 31, 516-522. Watanabe, P, G,, Hefner, R E,, Jr,, and Gehring, P. J. (1976a). Vinyl chloride-induced depression of hepatic non-protein sulfhydryl content and effects of bromosulphalein (BSP) clearance in rats. Toxicology 6, 1-8. Watanabe, P. G., McGowan, G. R., and Gehring, P, J. (1976b). Fate of '`C-vinyl chloride after single oral administration in rats. Toxicol. Appl. Pharma col. 36, 339-352. Watanabe, P. G., McGowan, G. R., Madrid, E. O., and Gehring, P. J. (1976c). Fate of 14C-vinyl chlo ride following inhalation exposure in rats, Toxicol. Appl. Pharmacol. 37, 49-50. Watanabe, P. G., ZeMPEL, J. A., Pegg, D. G., and Gehring, P. J. (1978). Hepatic macromolecular binding following exposure to vinyl chloride. Toxicol. Appl. Pharmacol. 44, 571-579. CMA 005739 TOXICOLOGY AND APPLIED PHARMACOLOGY 62. 1 - 10 ( 1982) The Effect of Repeated Vinyl Chloride Exposure on Rat Hepatic Metabolizing Enzymes1 Julie T. Du,2 Michael T. Tseng, and Carlo H. Tamburro3 Liver Research Center, Division of Digestive Diseases and Nutrition, Departments of Medicine and Anatomy, and Regional Cancer Center. University of Louisville School of Medicine, Louisville, Kentucky 40292 Received July 17, 1980; accepted September 12, 1981 The Effect of Repeated Vinyl Chloride Exposure on Rat Hepatic Metabolizing Enzymes. Du, J. T., Tseng, M. T, and Tamburro, C. H. (1982). Toxicol. Appl. Pharmacol 62, l10. Sprague-Dawley rats were exposed to 2.8% vinyl chloride for 2 (70 hr), 4 (140 hr), and 6 (210 hr) weeks to determine the sequential biochemical changes related to the oxidation and detoxification ability of hepatic tissue. Glutathione-S-transferase(s) activity using 1,2epoxy-(y>nitrophenoxy (propane and />-nitrobenzyl chloride as substrates was elevated 17 to 24, 28, and 35 to 42% after 2. 4, and 6 weeks of exposure, respectively, suggesting enzyme(s) induction. Reduced glutathione, the major substrate required to conjugate the toxic metabolites of vinyl chloride, was also consistently elevated. Similarly, the activity of glutathione reductase, the enzyme necessary for the regeneration of reduced glutathione from its oxidized form, was also increased following vinyl chloride exposure. Cytochromes P-450, the major protein in volved with vinyl chloride metabolism, was reduced after vinyl chloride exposure, confirming reports of others that vinyl chloride metabolites destroy P~450. No abnormalities of standard clinical biochemical blood tests of liver function were found during 6 weeks of vinyl chloride exposure. The only consistent ultrastructural modification was the dilation of endoplasmic reticulum. The biochemical and ultrastructural alterations could reflect early hepatocellular adaptation to vinyl chloride exposure. Vinyl chloride, at high concentrations, has been shown to be carcinogenic in both lab oratory animals (Maltoni and Lefemine, 1975; Viola et al., 1971) and man (Creech and Johnson, 1974). Present data support the metabolism of vinyl chloride by hepatic microsomal mixed-function oxidase system into toxic intermediates, chloroethylene ox ide (Bolt et al., 1975; Hefner et al., 1975; Kappus et al., 1976) and chloroacetaldehyde ' This work was supported by a grant from the Man ufacturing Chemists Association, Washington, D.C. Portions of this study have been presented (Fed. Proc. 37, 1545, 1978). ' Present address: Clement Associates, Inc., 1010 Wisconsin Avenue, N.W., Suite 660, Washington, D.C. 20007. 1 Address requests for reprints to: Carlo H. Tam burro, University of Louisville. (Gross and Freiberg, 1969). These two in termediates are considered to be the ultimate carcinogens (Barbin et al., 1975; Jaeger et al., 1974b; Van Duurcn, 1975), to be mu tagenic in bacterial systems (Elmore et al., 1976; Greim et al., 1975; Malaveille et al., 1975; McCann et al., 1975), to act as an alkylating agent by reacting with adenosine (Barbin et al., 1975) and cytidine (Laib and Bolt, 1978), and to bind with protein (Bolt et al., 1976; Kappus et al., 1976; Watanabe et al., 1978). Detoxification of these metab olites occurs mainly by conjugation with glu tathione and is catalyzed by hepatic gluta thione transferases; the conjugates are excreted in the urine as substituted cysteine derivatives (Watanabe et al., 1976b,c; Green and Hathway, 1975, 1977). Chloroacetal dehyde can be further oxidized to chloro- 1 0041-008X/82/010001-10S02.00/0 Copyright 1982 by Academic Preu, Inc All rights of reproduction in any form reserved. CMA 005740 2 DU. TSENG, AND TAMBURRO acetic acid (Hefner et al., 1975). These data are complied in a metabolic scheme in Fig. 1 as an updated hypothesized metabolic fate of vinyl chloride in the adult rat. The me tabolism of chloroethylene oxide via epoxide hydratase is not listed in the scheme because its product has not been identified. There have been a few in vivo studies con cerning the effects of vinyl chloride exposure on hepatic concentrations of glutathione (Hefneretal., 1975; Watanabeetai, 1976c; Du and Tamburro, 1978), cytochromes P450 (Reynolds et al., 1975) and on mixedfunction oxidase activity (Drew et al., 1975; Reynolds et al., 1975). However, informa tion about the sequential alterations in he patic oxidation and detoxification of vinyl chloride, during prolonged exposure, simu lating the occurrence in workers, is still lack ing. It was reported previously that the en zymatic changes in rat liver following prolonged exposure to vinyl chloride were similar to those found in rat hepatoma (Du and Tamburro, 1976; Du et al., 1979). The sequential biochemical changes related to the hepatic oxidation and detoxification of vinyl chloride following prolonged exposure are reported here. METHODS Animals and experimental design. Eight- to tenweek-old Sprague-Dawley male rats ( -- 300 g), sup plied by Laboratory Supply of Indianapolis. Indiana, were randomized prior to the experiment into three groups, a vinyl chloride-exposed group and the air-exposed group housed in identical chambers and a second control group housed in the University's Central Animal Care Center. The exposure level was 28,000 ppm vinyl chloride, 7 hr/day, 5 days/week for 2, 4, and 6 weeks. The exposure chambers were 4400-liter airtight vats. Vinyl chloride ( -- 300 to 340 g) was added to the vat to give a time-weighted average concentration of 28,000 1000 ppm. The chamber air was changed daily and the vinyl chloride concentration was determined by gas chromatography. The air was constantly circulated by a stirrer. The rats' respirations had negligible effect on the composition of the chamber's atmosphere because of the chamber's large volume. Animals were fed on standard laboratory chow pellets ad libitum. All animals were anesthetized with ether, blood was drawn from the inferior vena cava, and the animals were Fic. 1. The proposed metabolic fate of vinyl chloride. (GSH, glutathione; MFO, mixed-function oxidase; VC, vinyl chloride; GEST, glutathione 5-epoxide transferase; and GAST, glutathione S-aldehyde transferase). CMA 005741 VINYL CHLORIDE EFFECT ON ENZYMES 3 killed approximately 20 hr after exposure at 1 00 pm each day N ADPH, glutathione, and glutathione disulfide were obtained from Sigma Chemical Company, St Louis, Missouri: l,2-epox>-3-(p-nitrophenoxy)propane was purchased from Eastman Kodak. Company, Rochester, New York, p-nitrobenzyl chloride was obtained from Matheson, Coleman and Bell, East Rutherford, New Jersey, benzphetamine was donated by the L'pJohn Company, Kalamazoo, Michigan. Double-distilled water was used throughout. Sample preparation and biochemical determination. Homogenates and subcellular fractions were prepared as described previously (Du et al., 1979). Each sample was prepared from a single organ and kept at 4C during preparation. The remaining liver was frozen rapidly in liquid nitrogen and stored at -70C. Cytochromes P450 concentrations were determined in the frozen mi crosomal fractions the day following sacrifice. The glu tathione (GSH) concentration, as well as glutathioneY-transferase, glutathione reductase, and mixed-func tion oxidase activities were determined in the freshly fractionated frozen liver. For the assays using frozen tissue, the livers from control and experimental rats were frozen in an identical manner for the same length of time. Cytochromes P-450 concentration (Omura and Sato, 1964), nonprotein sulfhydryl content (Sedlak and Lindsay, 1968), and glutathione reductase activity (Carlberg and Mannervik, 1975) were determined in the microsomal or cytosol fractions by methods de scribed previously (Du et al., 1979). Glutathione-S-transferase activity was determined using the 100,000 X g supernatant fraction. 1,2-Epoxy?-(p-nitrophenoxy)propane and p-nitrobenzyl chloride were the substrates for glutathione-S-epoxide transfer ase and glutathione-S-aralkyl transferase (GAST), re spectively. Enzyme activity was determined as described by others (Habig et al., 1974; Kaplowitz et al., 1975). All assays were linear functions of protein concentration and timed for at least 2 min. Solutions of 1,2-epoxy-3(p-nitrophenoxy)propane and p-nitrobenzyl chloride were prepared in absolute ethanol; the final ethanol con centration in the incubation mixture was 0.5%. Mixedfunction oxidase activity was estimated in the micro somal fraction by measuring NADPH disappearance in the NADPH-dependent demethylation reaction of benzphetamine (Lu er al., 1972). The protein content was determined by the method of Lowry et al. (1951). The scrum clinical liver tests including aspartate ami notransferase, alanine aminotransferase, alkaline phos phatase, bilirubin, cholesterol, and triglyceride were determined by Technicon sequential multiple analyzer computer (SMAC) system. Light and electron microscopy. Small strips of liver were removed under ether anesthesia, sliced into small cubes, placed immediately in ice-cold 1% osmium tetroxide (pH 7.4), and fixed for 2 hr at 4C. Subse quently, samples were washed overnight in phosphate buffer, dehydrated in ascending alcohol, and embedded in Epon. Tissue blocks were polymerized at 60C for 2 days. Thin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate before ex amination on a Philips 300 electron microscope. For ultrastructural analysis, three rats randomly selected from controls and groups exposed for 2, 4, and 6 weeks to vinyl chloride were studied. For light microscopy, a block of tissue was fixed in buffered formalin and processed routinely for paraffin embedding. Sections 6 m thick were stained with he matoxylin and eosin. Statistical analysis. Analysis of variance was per formed for the various groups at the different time pe riods and multiple comparisons were performed based on the results of the analysis of variance. RESULTS The protein content (mg protein/g liver) in the subcellular fractions in both control and vinyl chloride-exposed groups was the same throughout the exposure (data not shown); the enzymatic results, therefore, are expressed as micromoles of substrate con verted per minute per milligram of protein. There were no statistical differences be tween the normal and air-exposed groups in the glutathione and cytochromes />-450 con tents or in any of the enzyme activities. The nonprotein sulfhydryl content (Table l) was significantly elevated from 26 to 54% at 2, 4, and 6 weeks in the vinyl chloride-exposed group compared to both control groups. Al though the nonprotein sulfhydryl content in creased with exposure, the increases were not statistically significant. Glutathione re ductase activity (Table 1) in the exposed group was increased by 53 to 77% at all three time periods. The increase in glutathione reductase was the same at 2 and 4 weeks of exposure but showed a further significant increase after 6 weeks of exposure. Glutathione-S-epoxide transferase (GEST, Table 1) and glutathione-S-aralkyl transferase (GAST, Table 1) activities were signifi cantly higher than controls after 6 weeks of exposure, 37 and 45%, respectively. The cy tochromes P-450 content, on the other hand. Cii* 005742 DU, TSENG, AND TAMBURRO CMA 0 0 5 7 4 3 TABLE i Sequential Changes in Hepatic Nonprotein Sul.ihyhryi, Cytochromes P-450 Content and Activities oe Gluiatiiioni Rujuitasi and Glutathione-.S'-Tr ANSI erases (Epoxide and Aralkyl) in Rats Exposed to Vinyl Cm oride" Time (weeks) Treatment 0 2 4 *1 Nonprolein sulfhydryt (pmol/g liver) Glutathione reductase (100 X nmol/min/mg protein) GEST (100 x nmol/ min/mg protein) GAST (10 X nmol/ min/mg protein) Cytochrome P-450 (nmol/g liver) Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control 7.9 0.3 -- -- 5.0 0.2 -- 9.1 1.6 -- " 2.4 0.4 -- -- 17.0 3.7 --__ 7.8 0.4* 9.4 0 2*` 7.1 0.3` 4.3 0 4* 6 7 0.6*' 4 5 0 2* 7.8 0.4 9.1 + 1.7 8.1 1.2 2.1 0.3 2.6 0.4 2.2 0.2 17.1 + 1.7 13 2 + l.l 15.5 13 7 1 0.4* 10 2 0 6*' 6.9 0 4' 4.2 0 4* 6.3 0.5*' 3.7 0.3' 7 5 1.0 9 7 0.76.3 0 7' J .9 0.2 2.4 + 0.3 19 0.2 19.7 1 3* 15.3 l 3* 19.6 2.7 6 9 0 4* 11.4 0 6* 7 9 0.3- 4 8 0 3* 8 V .1 0 7* 5 3 i 0 4- 7.7 1 0* il 0 i 1 3* 8.4 x 0.9' 2.4 x 0 3* 3.2 3 0 i`2.1 + 0 2- 15.5 1.4* 106 + l 0* 14.3 t 1.8 " Rats were exposed to 28,000 ppm of vinyl chloride; normal controls and the air controls were exposed to air only. Each number represents the mean and the SEM from a group of six rats. 1 Normal vs vinyl chloride-exposed, p < 0.05. ' Air control vs vinyl chloride-exposed, p < 0.05. J Vinyl chloride (6 weeks) exposed vs vinyl chloride (2 and 4 weeks) exposed, p < 0.05. 1 VINYL CHLORIDE EFFECT ON ENZYMES 5 was significantly lower than controls after 6 weeks of exposure to vinyl chloride (Table 1). No differences were found in the hepatic mixed-function oxidase activity or in the serum clinical liver tests. After 2 weeks of exposure, the two control groups had gained weight but the vinyl chloride-exposed group did not (Table 2). After 4 weeks of exposure, the normal control group housed at the an imal care center had gained significantly more weight than either the air-control or vinyl chloride-exposed group. After 6 weeks of exposure, however, the vinyl chloride-ex posed group failed to gain weight; the nor mal control group gained more than the aircontrol group (Table 2). Morphological examination revealed poly hedral hepatocytes arranged in irregular plates interposed by vascular sinusoids in the livers of the control rats. This general cytoarchitecture was maintained after vinyl chloride exposure. Hepatocytes in control rats contained a prominent spherical nu cleus, numerous ovoid mitochondria, stacks of rough endoplasmic reticulum (RER), some aggregates of smooth endoplasmic re ticulum (SER), and varying amounts of ly- sosomes and glycogen particles (Fig. 2a).Few interstitial cells were scattered among the hepatocytes. These cells contained few cy toplasmic organelles and could be readily discerned at the light microscopic level by their hyperchromatic nuclei. The sinusoids were linked by fenestrated endothelium and some of the lining cells displayed phagocytic activity. After 2 to 6 weeks of vinyl chloride exposure, the principal organelle affected appeared to be the endoplasmic reticulum. Cisternae of the RER became dilated in a relatively small population of the hepato cytes in the 2-week treatment group. Four weeks after exposure to vinyl chloride, patches of dilated endoplasmic reticulum were prominently displayed in some hepa tocytes (Fig. 2b). At this stage the SER was relatively unaffected. In the 6-week treat ment group, vesiculation of SER and dis tention of RER were easily discernible in a large number of hepatocytes (Fig. 2c). How ever, other ceil organelles showed no de monstrable change. These changes, though, are still beyond the resolving limit of the light microscope. The nonhepatocyte com ponents showed minimum changes which TABLE 2 Body Weiohts of Rats before and after Vinyl Chloride Exposure0 Duration (week) 2 4 6 Treatment Normal control VC-exposed Air control Normal control VC-exposed Air control Normal control VC-exposed Air control Initial weight (g) 400 15 405 12 396 17 398 8 410 16 395 10 402 14 396 9 398 14 Final weight (g) 433 16 396 14 414 18 450 15 421 15 419 5 486 14 398 10 449 4 Percentage gam 8* -2ie y 13" 3* 6" 21" <1" 13" ' Analysis of body weight was by regression analysis followed by an analysis of variance on the residuals from the regression equation. (Residual = observed final weight - predicted final weight from regression equation.) 4 Normal control vs vinyl chloride exposed, p < 0.05. r Air control vs vinyl chloride exposed, p < 0.05. 0 Air control vs normal control, p < 0.05. CH* 005744 CMA 005745 VINYL CHLORIDE EFFECT ON ENZYMES 7 were characterized by an increased accu mulation of lysosomal-like substances in some of the sinusoidal lining cells as well as a greater tendency to accumulate lipids in the interstitial cells (Fig. 2d). DISCUSSION Glutathione conjugation is an important pathway for the metabolism of potentially harmful electrophilic metabolites of xenobiotics. Studies by Watanabe et al. (1976b,c) indicate this to be the major route for in activation of the vinyl chloride metabolites. Glutathione-5-transferases are a group of cytosol enzymes catalyzing the reaction of glutathione and electrophilic compounds to form less toxic and more water-soluble con jugates. Their activity during chronic ex posure to xenobiotics, like vinyl chloride, could be a key determinate in the ultimate outcome of such exposures as illustrated by the longer arrow in Fig. 1, The increase in hepatic GEST activity at 4 weeks and the later increase in GAST activity at 6 weeks suggested that in the earlier stages of ex posures most of the chlorooxirane interme diate is being adequately detoxified. During the later stages of chronic exposure, more chlorooxirane may become rearranged to yield more chloroacetaldehyde and, in turn, react with other available glutathione trans ferases or become further metabolized to chloroacetic acid. Alternatively, the exces sive chlorooxirane could rearrange sponta neously to form chloroethanol and be further oxidized to chloroacetaldehyde, which in turn may react with glutathione, or be oxi dized to monochloroacetic acid (Johnson, 1967). This would be consistent with the later increases in the aralkyl-transferases and the finding by Hefner et al. (1975) that monochloroacetic acid is found only in the urine of rats exposed for an extended time to higher levels (5000 ppm) of vinyl chloride. The increased use of alternate pathways, for chlorooxirane and chloroacetaldehyde de toxification, may reflect increased concen tration of these active metabolites allowing greater opportunity for DNA injury. A single exposure to vinyl chloride de creased hepatic nonprotein sulfhydryl com pounds in rats (Watanabe, 1976a); similar decreases of glutathione concentrations were produced in rats by other xenobiotics such as 1,1-dichloroethylene (Jaeger et al., 1974a; Reichert et al., 1978) and acetaminophen (Mitchell et al., 1973). In the present study, repeated exposure to vinyl chloride caused a significant increase of nonprotein sulfhy dryl concentrations (Table 1) analogous to the elevation of glutathione concentrations seen after the administration of carcinogens to rats (Fiala et al., 1976). In addition, the results showed that repeated exposure to vi nyl chloride also caused an increase in he patic glutathione-5-transferase activity (Ta ble 1) similar to that seen after the administration of phenobarbitai and 3-methylcholanthrene to rats (Mukhtar and Bresnick, 1976), These data suggest a mecha nism for compensatory synthesis of hepatic glutathione and glutathione-5-transferases after repeated exposure to vinyl chloride. The decreased concentration of cyto chromes P-450 found in rats after repeated exposure to vinyl chloride (Table 1) is con- FIG. 2. (a) Portion of a hepatocyte from control. Stacks of rough endoplasmic reticulum (RER) are separated by many ovoid mitochondria (M). Chromatin is finely dispersed in the nucleus (N). 9300X. (b) Hepatocyte after 2 weeks of vinyl chloride exposure. Dilation of RER appeared widespread in these two cells. Bile (B) canaliculus appeared unaltered in these rats. 5300X. (c) Four weeks after vinyl chloride exposure. Golgi complex (G) appeared unaffected while cisternal dilation continued. Distinction between SER and RER is complicated by the detachment of ribosomes. Lipid droplets (L) and glycogen (GL) often accumulated. 9500X. (d) A fat-storing interstitial cell is surrounded by several hepatocytes in a vinyl chloride-treated animal. Unlike lipid stored in hepatocytes, the shape of lipids (L) appeared irregular in these cells. 8000X. C005746 8 DU, TSENG, AND TAMBURRO sistent with work by Reynolds et al. ( 1975), This decrease in cytochromes R-450 content was also shown in vitro (Guengerich and Strickland, 1977; [vanetich et al., 1977) sug gesting that a metabolite of vinyl chloride destroys the cytochrome, Mixed-function oxidase activity, with benzphetamine as sub strate was unaltered. With regard to the structural alterations produced by vinyl chloride, the present find ings confirmed previous observations on the selective effect of this carcinogen in the en doplasmic reticulum (Du et al., 1979). A gradual increase in the number of hepatocytes affected and the involvement of both smooth and rough ER were shown in this study, The endoplasmic reticulum is the pri mary site of protein synthesis and its dilation suggested the presence of a vinyl chloriderelated effect. The lack of a concomitant Golgi hypertropy is noteworthy since this organelle serves as the site of glycosylation and packaging of many exportable proteins. It may be inferred that the vinyl chloride effect is mainly an intracellular phenome non. The relatively late involvement of SER could reflect a further attempt at detoxifi cation by the hepatocyte. The tendency for the interstitial cells to accumulate lipid and the heightened phagocytic activity correlate with increases in collagen formation and may be evidence of low-grade cellular injury. This may prove to be the initial histological response to vinyl chloride exposure. Ultrastructural responses to chronic in halation of vinyl chloride were reported by Feron et al. (1979). Unlike our findings, the principal effects observed were swollen mi tochondria and some proliferation of SER. The mitochondrial swelling presumably re flects the extensive vacuolization observed by light microscopy. The discrepancy prob ably resulted from differences in the dose and duration in vinyl chloride exposure since Feron et al. (1979) exposed rats to 5000 ppm vinyl chloride for 52 weeks. Such an exten sive mitochondrial lesion could result in bio chemical modifications such as a change in ATPase or cytochrome oxidase levels. The only enzyme activity measured, glucose-6phosphatase, was reduced. The authors believed that the altered glu tathione metabolism, as reflected by the in creased nonprotein sulfhydryl content, and the increased activities of glutathione-Stransferases and glutathione reductase, in rat liver after repeated exposure to high doses of vinyl chloride represent an early hepatocellular adaptation to vinyl chloride exposure. acknowledgments The authors wish to express their gratitude to the staff of the B. F Goodrich Plant in Louisville, Kentucky, for their cooperation in the exposure studies, to Dr. Richard A, Greenberg for his review, to Mr. John P. Sandoz for his help with the statistical analysis, and Mr. John Kreisle and Ms. Debra S. Eades for technical as sistance. 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Prakt. Chem. 311, 506-510. Glengerich, F P., and Strickland, T. W. (1977). Metabolism of vinyl chloride: Destruction of the heme of highly purified liver microsomal cytochrome P-450 by a metabolite. Mol. Pharmacol. 13, 993-1004. Habig, W. H., Pabst, M. J.. and Jakoby, W. B. (1974). Glutathione-S-transferases. The first enzy matic step in mercapturic acid formation. J. Biol. Chem. 249, 7130-7139. Hefner, R. E., Watanabe, P. G., and Gehring, P, J. (1975). Preliminary studies of the fate of inhaled vinyl chloride monomer in rats. Ann. S. Y. Acad. Sci. 246, 135-148. [vanetich, K. M., Aronson, I., and Katz, 1. D, (1977). The interaction of vinyl chloride with rat he patic microsomal cytochrome P-450 in vitro. Biochem Biophys. Res. Commun 74, 1411-1418. Jaeger, R. J., Connolly, R. B., and Morphy, S. D. (1974a). Effect of 18 hr. fast and glutathione deple tion on 1,1-dichloroethylene-induced hepatotoxicity and lethality in rats. Exp. Mol. Pathol. 20, 187-198. Jaeger, R. J. Reynolds, E. S., Connolly, R. B, Moslen, M. T, Szabo. S.. and Murphy, S. D. (1974b) Acute hepatic injury by vinyl chloride in rats pretreated with phenobarbital, Sature lLondon) 252, 724-726. Johnson. M. K. (1967). Metabolism of chloroeihanol in the rat. Biochem. Pharmacol. 16, 185-199 Kaplowitz, N., Kuhlenkamp. J.. and Clifton, G, (1975). Drug induction of hepatic glutathione-Stransferases in male and female rats. Biochem. J 146, 351-356. Kappus, H,, Bolt, H M., Buchter. A., and Bolt, W. (1976). Liver microsomal uptake of ,'lC-vinyl chloride and transformation lo protein alkylating metabolites in vitro. Toxicol. Appi. Pharmacol 37, 461-471. Laib, R. J., and Bolt, H. M. (1978). Formation of 3, N4-ethenocytidine moieties in RNA by vinyl chloride metabolites in vitro and in vivo. Arch. Toxicol. 39, 235-240. Lowry, O. H., Rosebrough, N. J., Farr, A. L,, and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. / Biol. Chem. 193, 265275. Lu, A. Y. H., Kunztzman, R., West, S., Jacobson, M., AND Conney, A. H. (1972). Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and exogenous substrates. J Biol. Chem. 247, 1727-1734. Malaveille, C., Bartsch, H., Barbin. A., Camus, A. M., and Montesano, R. (1975). Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacetaldehyde and chloroethanol. Biochem Biophys. Res. Commun. 63, 363-370. Maltoni, C., and Lefemine, C. (1975). Carcinoge nicity bioassays of vinyl chloride: Current results. Ann N.Y. Acad. Sci. 246, 195-218. McCann, J,, Simmon, v,, Streitwieser, D., and Ames, B. N. (1975). mutagenicity of chloroacetaldehyde, a possible metabolic product of 1,2-dichloroethane, chloroethanol, vinyl chloride and cyclo phosphamide. Proc. Nat. Acad. Sci. USA 72, 3190-- 3193. Mitchell, J, R., Jollow, D. J,, Potter, W. a,, Gillete, J. R,, and Brodie, B, B. (1973). Acetamin ophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187, 211 -- 217. Mukhtar, H., and Bresnick, E. (1976). Effects of phenobarbital and 3-methylcholanthrene administra tion on glutathione-5-cpoxide transferase activity in rat liver. Biochem Pharmacol. 25, 1081-1084. Omura, T., and Sato, R. (1964). The carbon mon oxide-binding pigment of liver microsomcs. 1. Evi dence for its hemoprotcin nature. J. Biol. Chem. 239, 2370-2378. Reichert, D., Werner, H. W., and Henschler, D. 005748 10 DU, TSENG. AND TAMBURRO t 1978). Role of liver glutathione in 1,1-dichloroeth\ lene metabolism and hepatotoxicity in intact rats and isolated perfused rat liver Arch Toxicol 41, 169-- 178, Refolds, E. S., Moslen, M. T, Szabo, S., and Jaeger, R J (1975). Vinyl chlonde-mduced deac tivation of cytochrome P-450 and other components of the liver mixed function oxidase system: An in vivo study. Res, Commun. Chem. Pathol. Pharmacol 12, 685-694 Sedlak, J, and Lindsay, R. H, (1968) Estimation of total protein bound and non-protein sulfhydryl groups in tissue with Ellman's reagent. Anal, Biochem. 25, 192-205. VanDuuren, B. L. (1975). Possible mechanism of car cinogenic action of vinyl chloride. Ann. .V. Y Acad. Sci. 246, 258-267 Viola, P. L., Bigotti, A., and Caputo, A. (1971). Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res 31, 516-522. Watanabe, P. G., Hefner, R. E., Jr., and Gehring, P. J. (1976a). Vinyl chloride-induced depression of hepatic non-protein sulfhydryl content and effects of bromosulphalein (BSP) clearance in rats. Toxicology 6. 1-8. Watanabe, P. G., McGowan, G. R., and Gehring, P. J. (1976b). Fate of '"C-vinyl chloride after single oral administration in rats. Toxicol. Appl, Pharma col. 36, 339-352. Watanabe, P. G., McGowan. G. R.. Madrid, E. O , and Gehring, P. J, (1976c). Fate of "C-vinyl chlo ride following inhalation exposure in rats. Toxicol. Appl. Pharmacol. 37, 49-50. Watanabe, P G., Zempel, J. A., Pegg, D. G., and Gehring, P. J. (1978). Hepatic macromolecular binding following exposure to vinyl chloride. Toxicol. Appl. Pharmacol. 44, 571-579. 0057^9 CM* TOXICOLOGY AND APPLIED PHARMACOLOGY 62, 1-10 ( 1982) RECEIVED APR 3 1982 The Effect of Repeated Vinyl Chloride Exposure on Rat Hepatic Metabolizing Enzymes1 Julie T. Du,: Michael T. Tseng, and Carlo H. Tamburro3 Liver Research Center, Division of Digestive Diseases and Nutrition, Departments of Medicine and Anatomy, and Regional Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky 40292 Received July 17. 1980; accepted September 12, 1981 The Effect of Repeated Vinyl Chloride Exposure on Rat Hepatic Metabolizing Enzymes. Du, J. T., Tseng, M. T, and Tamblrro, C. H. (1982). Toxicol. Appl. Pharmacol. 62, 110. Sprague-Dawley rats were exposed to 2.8% vinyl chloride for 2 (70 hr), 4 (140 hr), and 6 (210 hr) weeks to determine the sequential biochemical changes related to the oxidation and detoxification ability of hepatic tissue. Glutathione-S-transferase(s) activity using 1,2epoxy-(p-mtrophenoxy)propane and p-mtrobenzyl chloride as substrates was elevated 17 to 24, 28, and 35 to 42% after 2, 4, and 6 weeks of exposure, respectively, suggesting enzyme(s) induction. Reduced glutathione, the major substrate required to conjugate the toxic metabolites of vinyl chloride, was also consistently elevated. Similarly, the activity of glutathione reductase, the enzyme necessary for the regeneration of reduced glutathione from its oxidized form, was also increased following vinyl chloride exposure. Cytochromes P-450, the major protein in volved with vinyl chloride metabolism, was reduced after vinyl chloride exposure, confirming reports of others that vinyl chloride metabolites destroy P-450. No abnormalities of standard clinical biochemical blood tests of liver function were found during 6 weeks of vinyl chloride exposure. The only consistent ultrastructural modification was the dilation of endoplasmic reticulum. The biochemical and ultrastructural alterations could reflect early hepatocellular adaptation to vinyl chloride exposure. Vinyl chloride, at high concentrations, has been shown to be carcinogenic in both lab oratory animals (Maltoni and Lefemine, 1975; Viola et al., 1971) and man (Creech and Johnson, 1974). Present data support the metabolism of vinyl chloride by hepatic microsomal mixed-function oxidase system into toxic intermediates, chloroethylene ox ide (Bolt et al., 1975; Hefner et al., 1975; Kappus et al., 1976) and chloroacetaldehyde 1 This work was supported by a grant from the Man ufacturing Chemists Association, Washington. D.C. Portions of this study have been presented (Fed. Proc. 37, 1545, 1978). ' Present address: Clement Associates, Inc., 1010 Wisconsin Avenue, N.W., Suite 660, Washington, D.C. 20007. ' Address requests for reprints to: Carlo H. Tamburro. University of Louisville. (Gross and Freiberg, 1969). These two in termediates are considered to be the ultimate carcinogens (Barbin et al., 1975; Jaeger et al., 1974b; Van Duuren, 1975), to be mu tagenic in bacterial systems (Elmore et al., 1976; Greim et al., 1975; Malaveille et al., 1975; McCann et al., 1975), to act as an alkylating agent by reacting with adenosine (Barbin et al., 1975) and cytidine (Laib and Bolt, 1978), and to bind with protein (Bolt et al., 1976; Kappus et al., 1976; Watanabe et al., 1978). Detoxification of these metab olites occurs mainly by conjugation with glu tathione and is catalyzed by hepatic gluta thione transferases; the conjugates are excreted in the urine as substituted cysteine derivatives (Watanabe et al., 1976b,c; Green and Hathway, 1975, 1977). Chloroacetal dehyde can be further oxidized to chloro- 1 0041-008X/82/010001-10S02.00/0 Copyright 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. CMA QQ 5750 2 DU, TSENG, AND TAMBURRO acetic acid (Hefner et al.. 1975). These data are compiled in a metabolic scheme in Fig, 1 as an updated hypothesized metabolic fate of vinyl chloride in the adult rat. The me tabolism of chloroethylene oxide via epoxide hydratase is not listed in the scheme because its product has not been identified. There have been a few in vivo studies con cerning the effects of vinyl chloride exposure on hepatic concentrations of glutathione (Hefner et al., 1975; Watanabe et al., 1976c; Du and Tamburro, 1978), cytochromes P450 (Reynolds et al., 1975) and on mixedfunction oxidase activity (Drew et al., 1975; Reynolds et al., 1975). However, informa tion about the sequential alterations in he patic oxidation and detoxification of vinyl chloride, during prolonged exposure, simu lating the occurrence in workers, is still lack ing. It was reported previously that the en zymatic changes in rat liver following prolonged exposure to vinyl chloride were similar to those found in rat hepatoma (Du and Tamburro, 1976; Du et al., 1979). The sequential biochemical changes related to the hepatic oxidation and detoxification of vinyl chloride following prolonged exposure are reported here. METHODS Animals and experimental design. Eight- to tenweek-old Sprague-Dawley male rats ( --300g), sup plied by Laboratory Supply of Indianapolis, Indiana, were randomized prior to the experiment into three groups: a vinyl chloride-exposed group and the air-exposed group housed in identical chambers and a second control group housed in the University's Central Animal Care Center. The exposure level was 28,000 ppm vinyl chloride, 7 hr/day, 5 days/week for 2, 4, and 6 weeks. The exposure chambers were 4400-liter airtight vats. Vinyl chloride ( -- 300 to 340 g) was added to the vat to give a time-weighted average concentration of 28,000 1000 ppm. The chamber air was changed daily and the vinyl chloride concentration was determined by gas chromatography. The air was constantly circulated by a stirrer. The rats' respirations had negligible effect on the composition of the chamber's atmosphere because of the chamber's large volume. Animals were fed on standard laboratory chow pellets ad libitum. All animals were anesthetized with ether, blood was drawn from the inferior vena cava, and the animals were (V c) Cl CH - CH2 Cl ch2ch2oh / (chloroethanol) detoxificati ON WITH glutathione (chlorooxirane) Cl ch-ch2 V GS CH,CH,0H , *A 4 Cl CH2CH0 (GSH) . GS CH2CH0 GAST (chloroacetaldehyde) 1 Cl ch2cooh N-Ac-S-(2-hydroxv ethyl) cysteine GS CHjCOOH THIODIGLYCOLIC ACID (CHLOROACETIC ACID) Fig. 1. The proposed metabolic fate of vinyl chloride. (GSH, glutathione; MFO, mixed-function oxidase; VC, vinyl chloride; GEST. glutathione 5-epoxide transferase; and GAST, glutathione 5-al dehyde transferase). CMA 005751 VINYL CHLORIDE EFFECT ON ENZYMES 3 killed approximately 20 hr after exposure at 1 00 pm each day N ADPH. glutathione, and glutathione disulfide were obtained from Sigma Chemical Company, St. Louis, Missouri. 1,2-epoxy-3-(|f-nitrophenoxy)propane was purchased from Eastman Kodak Company, Rochester. New York, p-nitrobenzyl chloride was obtained from Matheson, Coleman and Bell, East Rutherford, New Jersey; benzphetamine was donated by the Upjohn Company, Kalamazoo, Michigan. Double-distilled water was used throughout. Sample preparation and biochemical determination. Homogenates and subcellular fractions were prepared as described previously (Du et ai., 1979). Each sample was prepared from a single organ and kept at 4C during preparation. The remaining liver was frozen rapidly in liquid nitrogen and stored at -70C, Cytochromes P450 concentrations were determined in the frozen mi crosomal fractions the day following sacrifice. The glu tathione (GSH) concentration, as well as glutathione5,-transferase, glutathione reductase, and mixed-func tion oxidase activities were determined in the freshly fractionated frozen liver. For the assays using frozen tissue, the livers from control and experimental rats were frozen in an identical manner for the same length of time. Cytochromes R-450 concentration (Omura and Sato, 1964). nonprotein sulfhydryl content (Sedlak and Lindsay, 1968), and glutathione reductase activity (Carlberg and Mannervik, 1975) were determined in the microsomal or cytosol fractions by methods de scribed previously (Du et ai, 1979). Glutathione-5-transferase activity was determined using the 100,000 X g supernatant fraction. 1,2-EpoxyMp-nitrophenoxy)propane and p-nitrobenzyl chloride were the substrates for glutathione-S-epoxide transfer ase and glutathione-5-aralkyl transferase (GAST), re spectively. Enzyme activity was determined as described by others (Habig et ai, 1974; Kaplowitz et ai, 1975). All assays were linear functions of protein concentration and timed for at least 2 min. Solutions of 1,2-epoxy-3(p-nitrophenoxy)propane and p-nitrobenzyl chloride were prepared in absolute ethanol; the final ethanol con centration in the incubation mixture was 0.5%. Mixedfunction oxidase activity was estimated in the micro somal fraction by measuring NADPH disappearance in the NADPH-dependent demethylation reaction of benzphetamine (Lu et al,, 1972). The protein content was determined by the method of Lowry et ai (1951). The serum clinical liver tests including aspartate ami notransferase, alanine aminotransferase, alkaline phos phatase, bilirubin, cholesterol, and triglyceride were determined by Technicon sequential multiple analyzer computer (SMAC) system. Light and electron microscopy. Small strips of liver were removed under ether anesthesia, sliced into small cubes, placed immediately in ice-cold 1% osmium tetroxide (pH 7.4), and fixed for 2 hr at 4C. Subse quently, samples were washed overnight in phosphate buffer, dehydrated in ascending alcohol, and embedded in Epon. Tissue blocks were polymerized at 60C for 2 days. Thin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate before ex amination on a Philips 300 electron microscope. For ultrastructural analysis, three rats randomly selected from controls and groups exposed for 2, 4, and 6 weeks to vinyl chloride were studied. For light microscopy, a block of tissue was fixed in buffered formalin and processed routinely for paraffin embedding. Sections 6 am thick were stained with he matoxylin and eosin. Statistical analysis. Analysis of variance was per formed for the various groups at the different time pe riods and multiple comparisons were performed based on the results of the analysis of variance. RESULTS The protein content (mg protein/g liver) in the subcellular fractions in both control and vinyl chloride-exposed groups was the same throughout the exposure (data not shown); the enzymatic results, therefore, are expressed as micromoles of substrate con verted per minute per milligram of protein. There were no statistical differences be tween the normal and air-exposed groups in the glutathione and cytochromes />-450 con tents or in any of the enzyme activities. The nonprotein sulfhydryl content (Table 1) was significantly elevated from 26 to 54% at 2, 4, and 6 weeks in the vinyl chloride-exposed group compared to both control groups. Al though the nonprotein sulfhydryl content in creased with exposure, the increases were not statistically significant. Glutathione re ductase activity (Table 1) in the exposed group was increased by 53 to 77% at all three time periods. The increase in glutathione reductase was the same at 2 and 4 weeks of exposure but showed a further significant increase after 6 weeks of exposure. Glutathione-S-epoxide transferase (GEST, Table 1) and glutathione-5-aralkyl transferase (GAST, Table 1) activities were signifi cantly higher than controls after 6 weeks of exposure, 37 and 45%, respectively. The cy tochromes R-450 content, on the other hand. CH* 005752 TABLE I SEQUENTIAL CHANGES IN HEPATIC NONPROTEIN SULt HYIJRYL, CYTOCHROMES P-450 CONTENT AND AC11VI IIES Ol Gl UTAIHIONJ Kl 1)1 Jt I AM AND GLUTATH1ONE-S-TRANSEERASES (EPOXIDE AND ARALKYL) IN RaTS EXPOSED TO VlNYE CllEORIDI:" Time (weeks) Treatment 0 2 4 b DU. TSENG, AND TAMBURRO Nonprotein sulfhydryl (pmol/g liver) Glutathione reductase (100 X pmol/min/mg protein) GEST (100 X pmol/ min/mg protein) GAST (10 X *<mol/ min/mg protein) Cytochrome P-450 (nmoi/g liver) Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control 7.9 + 0.3 --_ 5.0 + 0.2 -- -- 9.1 1.6 -- -- 2.4 0.4 -- -- 17.0 3.7 -- -- 7.8 + 0 4** 9.4 0 2*J 7.1 0 3' 4.3 0.4* 67 0 6*" 4.5 0.2` 7.8 04 9.1 1.7 8.1 1.2 2.1 0.3 2.6 0.4 2.2 0.2 17.1 1.7 13.2 l.l 15.5 1.3 7 1 0 4* 10 2 0 6*' 6.9 i 0 4' 4.2 0 4* 6.3 0 5s' 3.7 0.3' 7.5 1.0 9.7 0.7' 6.3 0.7' 1 9 0.2 24 0.3 1.9 0.2 19.7 1.3* 15.3 + 1.3* 19.6 2.7 6V 0 41' 1 I 4 i 0 6*' 7 9 t 0 3' 4 8 t 0 3" 8,9 t 0 T' 5.3 0.4' 7.7 1.0* 1 1.0 1 3*' 8 4 0 9' 2.4 0 3* 3 2 0.1*' 2 l 0 2' 15 5 1.4* 10 6 1 0*' 14 3 1 8' " Rats were exposed to 28,000 ppm o( vinyl chloride; normal controls and the air controls were exposed to air only Each number represents the mean and the SEM from a group of six rats. * Normal vs vinyl chloride-exposed, p < 0.05. c Air control vs vinyl chloride-exposed, p < 0.05. * Vinyl chloride (6 weeks) exposed vs vinyl chloride (2 and 4 weeks) exposed, p < 0.05. VINYL CHLORIDE EFFECT ON ENZYMES 3 was significantly lower than controls after 6 weeks of exposure to vinyl chloride (Table 1). No differences were found in the hepatic mixed-function oxidase activity or in the serum clinical liver tests. After 2 weeks of exposure, the two control groups had gained weight but the vinyl chloride-exposed group did not (Table 2). After 4 weeks of exposure, the normal control group housed at the an imal care center had gained significantly more weight than either the air-control or vinyl chloride-exposed group. After 6 weeks of exposure, however, the vinyl chloride-ex posed group failed to gain weight; the nor mal control group gained more than the aircontrol group (Table 2). Morphological examination revealed poly hedral hepatocytes arranged in irregular plates interposed by vascular sinusoids in the livers of the control rats. This general cytoarchitecture was maintained after vinyl chloride exposure. Hepatocytes in control rats contained a prominent spherical nu cleus, numerous ovoid mitochondria, stacks of rough endoplasmic reticulum (RER), some aggregates of smooth endoplasmic re ticulum (SER), and varying amounts of ly- sosomes and glycogen particles (Fig. 2a).Few interstitial cells were scattered among the hepatocytes. These cells contained few cy toplasmic organelles and could be readily discerned at the light microscopic level by their hyperchromatic nuclei. The sinusoids were linked by fenestrated endothelium and some of the lining cells displayed phagocytic activity. After 2 to 6 weeks of vinyl chloride exposure, the principal organelle affected appeared to be the endoplasmic reticulum. Cisternae of the RER became dilated in a relatively small population of the hepato cytes in the 2-week treatment group. Four weeks after exposure to vinyl chloride, patches of dilated endoplasmic reticulum were prominently displayed in some hepa tocytes (Fig. 2b). At this stage the SER was relatively unaffected. In the 6-week treat ment group, vesiculation of SER and dis tention of RER were easily discernible in a large number of hepatocytes (Fig. 2c). How ever, other cell organelles showed no de monstrable change. These changes, though, are still beyond the resolving limit of the light microscope. The nonhepatocyte com ponents showed minimum changes which TABLE 2 Body Weights of Rats before and after Vinyl Chloride Exposure'1 Duration (week) 2 4 6 Treatment Normal control VC-exposed Air control Normal control VC-exposed Air control Normal control VC-exposed Air control Initial weight () 400 +15 405 12 396 17 398 8 410 16 395 10 402 14 396 9 398 14 Final weight (g) 433 16 396 14 414 18 450 15 421 15 419 5 486 14 398 10 449 4 Percentage gain 8* -2" 13" 3* 6" 21" <1" 13" * Analysis of body weight was by regression analysis followed by an analysis of variance on the residuals from the regression equation. (Residual = observed final weight - predicted final weight from regression equation.) b Normal control vs vinyl chloride exposed, p < 0.05. ' Air control vs vinyl chloride exposed, p < 0.05. d Air control vs normal control, p < 0.05. CMA 005754 6 DU, TSENG, AND TAMBURRO CMA 005 755 VINYL CHLORIDE EFFECT ON ENZYMES 7 were characterized by an increased accu mulation of lysosomal-like substances in some of the sinusoidal lining cells as well as a greater tendency to accumulate lipids in the interstitial cells (Fig. 2d). DISCUSSION Glutathione conjugation is an important pathway for the metabolism of potentially harmful electrophilic metabolites of xenobiotics. Studies by Watanabe et al. (1976b,c) indicate this to be the major route for in activation of the vinyl chloride metabolites. Glutathione-S-transferases are a group of cytosol enzymes catalyzing the reaction of glutathione and electrophilic compounds to form less toxic and more water-soluble con jugates. Their activity during chronic ex posure to xenobiotics, like vinyl chloride, could be a key determinate in the ultimate outcome of such exposures as illustrated by the longer arrow in Fig. 1. The increase in hepatic GEST activity at 4 weeks and the later increase in GAST activity at 6 weeks suggested that in the earlier stages of ex posures most of the chlorooxirane interme diate is being adequately detoxified. During the later stages of chronic exposure, more chlorooxirane may become rearranged to yield more chloroacetaldehyde and, in turn, react with other available glutathione trans ferases or become further metabolized to chloroacetic acid. Alternatively, the exces sive chlorooxirane could rearrange sponta neously to form chloroethanol and be further oxidized to chloroacetaldehyde, which in turn may react with glutathione, or be oxi dized to monochioroacetic acid (Johnson, 1967). This would be consistent with the later increases in the aralkyl-transferases and the finding by Flefner et al. (1975) that monochioroacetic acid is found only in the urine of rats exposed for an extended time to higher levels (5000 ppm) of vinyl chloride. The increased use of alternate pathways, for chlorooxirane and chloroacetaldehyde de toxification, may reflect increased concen tration of these active metabolites allowing greater opportunity for DNA injury. A single exposure to vinyl chloride de creased hepatic nonprotein sulfhydryl com pounds in rats (Watanabe, 1976a); similar decreases of glutathione concentrations were produced in rats by other xenobiotics such as 1,1-dichloroethylene (Jaeger et al., 1974a; Reichert et al., 1978) and acetaminophen (Mitchell et al., 1973). In the present study, repeated exposure to vinyl chloride caused -- a significant increase of nonprotein sulfhy dryl concentrations (Table 1) analogous to the elevation of glutathione concentrations seen after the administration of carcinogens to rats (Fiala et al., 1976). In addition, the results showed that repeated exposure to vi nyl chloride also caused an increase in he patic glutathione-5-transferase activity (Ta ble 1) similar to that seen after the administration of phcnobarbital and 3-methylcholanthrene to rats (Mukhtar and Bresnick, 1976). These data suggest a mecha nism for compensatory synthesis of hepatic glutathione and glutathione-5-transferases after repeated exposure to vinyl chloride. The decreased concentration of cyto chromes P-450 found in rats after repeated exposure to vinyl chloride (Table 1) is con- Fig. 2. (a) Portion of a hepatocyte from control. Stacks of rough endoplasmic reticulum (RER) are separated by many ovoid mitochondria (M). Chromatin is finely dispersed in the nucleus (N). 9300x. (b) Hepatocyte after 2 weeks of vinyl chloride exposure. Dilation of RER appeared widespread in these two cells. Bile (B) canaliculus appeared unaltered in these rats. 5300x. (c) Four weeks after vinyl chloride exposure. Golgi complex (G) appeared unaffected while cisternal dilation continued. Distinction between SER and RER is complicated by the detachment of ribosomes. Lipid droplets (L) and glycogen (GL) often accumulated. 9500X. (d) A fat-storing interstitial cell is surrounded by several hepatocytes in a vinyl chloride-treated animal. Unlike lipid stored in hepatocytes, the shape of lipids (L) appeared irregular in these cells. 8000X. CMA 005756 DU, TSENG, AND TAMBURRO sistent with work by Reynolds et al. (1975). This decrease in cytochromes P-450 content was also shown in vitro (Guengerich and Strickland, 1977; [vanetich et al., 1977) sug gesting that a metabolite of vinyl chloride destroys the cytochrome. Mixed-function oxidase activity, with benzphetamine as sub strate was unaltered. With regard to the structural alterations produced by vinyl chloride, the present find ings confirmed previous observations on the selective effect of this carcinogen in the en doplasmic reticulum (Du et al., 1979). A gradual increase in the number of hepatocytes affected and the involvement of both smooth and rough ER were shown in this study. The endoplasmic reticulum is the pri mary site of protein synthesis and its dilation suggested the presence of a vinyl chloriderelated effect. The lack of a concomitant Golgi hypcrtropy is noteworthy since this organelle serves as the site of glycosylation and packaging of many exportable proteins. It may be inferred that the vinyl chloride effect is mainly an intracellular phenome non. The relatively late involvement of SER could reflect a further attempt at detoxifi cation by the hepatocyte. The tendency for the interstitial cells to accumulate lipid and the heightened phagocytic activity correlate with increases in collagen formation and may be evidence of low-grade cellular injury. This may prove to be the initial histological response to vinyl chloride exposure. Ultrastructural responses to chronic in halation of vinyl chloride were reported by Feron et al. (1979). Unlike our findings, the principal effects observed were swollen mi tochondria and some proliferation of SER. The mitochondrial swelling presumably re flects the extensive vacuolization observed by light microscopy. The discrepancy prob ably resulted from differences in the dose and duration in vinyl chloride exposure since Feron et al. (1979) exposed rats to 5000 ppm vinyl chloride for 52 weeks. Such an exten sive mitochondrial lesion could result in bio chemical modifications such as a change in ATPase or cytochrome oxidase levels. The only enzyme activity measured, glucose-6phosphatase, was reduced. The authors believed that the altered glu tathione metabolism, as reflected by the in creased nonprotein sulfhydryl content, and the increased activities of glutathione-Stransferases and glutathione reductase, in rat liver after repeated exposure to high doses of vinyl chloride represent an early hepatocellular adaptation to vinyl chloride exposure. ACKNOWLEDGMENTS The authors wish to express their gratitude to the staff of the B. F. Goodrich Plant in Louisville, Kentucky, for thetr cooperation in the exposure studies, to Dr. Richard A. Greenberg for his review, to Mr. John P. Sandoz for his help with the statistical analysis, and Mr. John Kreisle and Ms. Debra S. Eades for technical as sistance. REFERENCES Barbin, A., Bresil, H., Croisy, A., Jacqoionon, P., Malaveille, C., Montesano, R., and Bartsch, H. (1975). Liver-microsome-mediated formation of alkylating agents from vinyl bromide and vinyl chlo ride. Biochem, Biophys. Res. Common. 67, 596-603. Bolt, H. 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