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Volume 62, Number 1 January 1982 K- 17/1- Cloo) '*** ov ISSN 0041-008X R&S 134149 Applied Pharmacology Founding Editors Frederick Coulston Harry W. Hays Arnold J. Lehman Editors Emeritus C. Boyd Shaffer Gabriel L Plaa Robert A. Neal Associate Editors Marion W. Anders James E. Gibson Frederick Guengerich Curtis D. Klaassen Tom S. Miya Donald J. Reed F. Robens I^Kpeter Witschi Official Journal of the Society of Toxicology Editor: n A. WALLACE HAYES ,X.CsMEMilC:PRESS'^^^'^^| New Ynrk J.nnrirtn Tnrnntn /vwVrt ,^Snn Ernnmm toxicology and applied pharmacology 62, 1-10 (1982) K- 1711- Cteo) II8 Z. R&S 134150 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, 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~nitrophenoxy)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 /M50, the major protein in volved with vinyl chloride metabolism, was reduced after vinyl chloride exposure, confirming reports of others that vinyl chloride metabolites destroy /'-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 at., 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 at., 1975; Hefner et at., 1975; Kappus et al, 1976) and chloroacetaldehyde 1 Thia work was supported by a grant from the Man ufacturing Chemists Association, Washington. D.C. Portions of this study have been presented (Fed. Proc. n, 1545, 1978). 'Present address: Clement Associates, Inc., 1010 Wisconsin Avenue, N.W., Suite 660, Washington, D.C. ^^0007 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 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 at., 1975) and cytidine (Laib and Bolt, 1978), and to bind with protein (Bolt et al., 1976; Kappus et at., 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). Chloroacetaldehyde can be further oxidized to chloro- 0041-008X/8 2/010001 -10502.00/0 Copyright 19S2 by Aodemtc Prt**, Iftc. All righti of reproduction la toy form re*crvd. 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 ( -- 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-ex posed 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/wcck 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 f3ts' respirations had negligible effect on the composition of the chamber's atmosphere becat^M of the chamber's large volume. Animals were fed 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 cii2ch2oh (chloroethanql) DETOXIFICATION WITH GLUTATHIONE (chlorooxirane) Cl CH-CH, Y' 4 GS CH2CH20H <* _ GAST (CHLOROACETALOEHYDE) GS CH2CH0 4 N-Ac-S-(2-hydroxy ethyl) cysteine GS CHjCOOH 4 THIOOIGLYCOLIC ACID Cl ch2cooh (CMLORQACETIC acid) Fig. 1. The proposed metabolic fate of vinyl chloride. (GSH, glutathione; MFO. mixed-function oxidase; VC, vinyl chloride; GEST, glutathione S-cpoxide transferase; and GAST. glutathione S-aldehyde transferase). t r - - * , ' u .*1 -- j--i' M K * al VINYL CHLORIDE EFFECT ON ENZYMES killed approximately 20 hr after exposure at 1:00 pm each day. NADPH. glutathione, and glutathione disulfide were obtained from Sigma Chemical Company, St. Louis, Missouri; l,2-epoxy-3-(p-nitrophcnoxy)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 al, 1979). Each sample was prepared from a single organ and kept at 4"C 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 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), durathione-5-transferase activity was determined using the 100.000 X g supernatant fraction. 1.2-EpoxyJ-(/s-nitrophcnoxy)propane and p-nitrobenzyl chloride were the substrates for glutathione-5-epoxide transfer ase and glutathione-5-aralkyl transferase (CAST), 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-J(/>nilrophenoxy)propane and p-nilrobenzyl 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 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 pm 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-cpoxide transferase (GEST, Table 1) and glutathione-S-aralkyl transferase (GAST, Table 1) act ivities were signifi cantly higher than controls after 6 weeks of exposure, 37 and 45%, respectively. The cy tochromes /M50 content, on the other hand, TABLE 1 Sequential Changes (n Hepatic Nonprotein Sulfiiydryl, Cytochromes P-450 Content and Activities of Glutathione Reductase ano Glutathione-S-Transferases (Epoxide and Aralkyl) in Rats Exposed to Vinyl Chloride' Time (weeks) Treatment 0 2 4 6 Nonprotein sulfhydryt (pmol/g liver) Glutathione reductase (100 X pmol/min/mg protein) GESTO00 X pmol/ min/mg protein) GAST (10 X pmol / min/mg protein) Cytochrome P-450 (nmol/g liver) Norma! 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 U 15.5 1.3 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 6.9 + 0.4* 11.4 0.6*-' 7.9 0.3' 4.8 + 0.3* 8.9 0.7*' J 5.3 0.4' 7.7 1.0* 11.0 1.3*' 8.4 0.9' 2.4 0.3* 3.2 0.1*' 2.1 0.2' 15.5 1.4* 10.6 1.0*' 14.3 1.8' c H m -t 6 > a ->i 2 c 73 o ' 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 Ihe SEM from a group of six rals. * Normal vs vinyl chloride-exposed, p < 0.05. ' 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. Ctot IV.'- 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-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. 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* 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 gain 8* -2** 5* 13*-" 3* 6* 21*" <l*" \yJ * 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.) * Normal control vs vinyl chloride exposed, p < 0.05. ' Air coatrol vs vinyl chloride exposed, p < 0.05. ' Air control vs normal control, p < 0.05. 30 in to Ol 4* R&S 134156 VINYL CHLORIDE EFFECT ON ENZYMES 7 were characterized by an increased accu dized to monochloroacetic acid (Johnson, mulation of lysosomal-like substances in 1967). This would be consistent with the some of the sinusoidal lining cells as well as later increases in the aralkyl-transferases a greater tendency to accumulate lipids in and the finding by Hefner et al. (1975) that the interstitial cells (Fig. 2d). monochloroacetic acid is found only in the urine of rats exposed for an extended time DISCUSSION to higher levels (5000 ppm) of vinyl chloride. The increased use of alternate pathways, for chlorooxirane and chloroacetaldehyde de Glutathione conjugation is an important toxification, may reflect increased concen pathway for the metabolism of potentially tration of these active metabolites allowing harmful electrophilic metabolites of xeno- greater opportunity for DNA injury. biotics. Studies by Watanabeer al. (1976b,c) A single exposure to vinyl chloride de indicate this to be the major route for in creased hepatic nonprotein sulfhydryl com activation of the vinyl chloride metabolites. pounds in rats (Watanabe, 1976a); similar Glutathione-S'-transferases are a group of decreases of glutathione concentrations were cytosol enzymes catalyzing the reaction of produced in rats by other xenobiotics such glutathione and electrophilic compounds to as I,I-dichloroethylene (Jaeger et al., 1974a; form less toxic and more water-soluble con Reichert et al., 1978) and acetaminophen jugates. Their activity during chronic ex (Mitchell et al., 1973). In the present study, posure to xenobiotics, like vinyl chloride, repeated exposure to vinyl chloride caused could be a key determinate in the ultimate a significant increase of nonprotein sulfhy outcome of such exposures as illustrated by dryl concentrations (Table 1) analogous to the longer arrow in Fig. 1. The increase in the elevation of glutathione concentrations hepatic GEST activity at 4 weeks and the seen after the administration of carcinogens later increase in GAST activity at 6 weeks to rats (Fiala et al., 1976). In addition, the suggested that in the earlier stages of ex results showed that repeated exposure to vi posures most of the chlorooxirane interme nyl chloride also caused an increase in he diate is being adequately detoxified. During patic glutathione-5'-transferasc activity (Ta the later stages of chronic exposure, more ble 1) similar to that seen after the chlorooxirane may become rearranged to administration of phenobarbital and 3-meth- yield more chloroacetaldehyde and, in turn, ylcholanthrene to rats (Mukhtar and Bres- react with other available glutathione trans nick, 1976). These data suggest a mecha ferases or become further metabolized to nism for compensatory synthesis of hepatic chloroacctic acid. Alternatively, the exces glutathione and glutathione-iT-transferases sive chlorooxirane could rearrange sponta after repeated exposure to vinyl chloride. neously to form chloroethanol and be further The decreased concentration of cyto oxidized to chloroacetaldehyde, which in chromes P-450 found in rats after repeated turn may react with glutathione, or be oxi exposure to vinyl chloride (Table 1) is con- Fic. 2. (a) Portion of a hepatocyte from control. Stacks of rough endoplasmic reticulum (RER) arc 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 la a vinyl chloride-treated animal. Unlike lipid stored in hepatocytes. the shape of lipids (L) appeared irregular in these cells. 8000X, 8 DU, TSENG, AND TAMBURRO sistcnt with work by Reynolds et al. (1975). ATPase or cytochrome oxidase levels: The This decrease in cytochromes P-450 content only enzyme activity measured, glucose-6- was also shown in vitro (Guengerich and phosphatase, was reduced. Strickland, 1977; Ivanetich et a!., 1977) sug The authors believed that the altered glu gesting that a metabolite of vinyl chloride tathione metabolism, as reflected by the in destroys the cytochrome. Mixed-function creased nonprotein sulfhydryl content, and oxidase activity, with benzphetamine as sub the increased activities of glutathione-S- strate was unaltered. transferases and glutathione reductase, in With regard to the structural alterations rat liver after repeated exposure to high produced by vinyl chloride, the present find doses of vinyl chloride represent an early ings confirmed previous observations on the hepatocellular adaptation to vinyl chloride selective effect of this carcinogen in the en exposure. doplasmic reticulum (Du et al., 1979). A gradual increase in the number of hepatocytes affected and the involvement of both ACKNOWLEDGMENTS 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 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. Golgi hypertropy is noteworthy since this John Kreislc and Ms. Debra S. Eades for technical as organelle serves as the site of glycosylation sistance. and packaging of many exportable proteins. It may be inferred that the vinyl chloride effect is mainly an intracellular phenome REFERENCES non. The relatively late involvement of SER could reflect a further attempt at detoxifi cation by the hepatocyte. The tendency for Barbin, A., Bresil, H., Croisy, A., Jacqlugnon, P., Malaveille, C., Montesano, R., and Bartsch, H. (1975). Liver-microsome-mediatcd formation of 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. 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