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%% 'F^Z'-., -Sfi- _t*l .l.St.**'A '*v\w <.: i*-- >-t VV :'r.'^<L- -v /-- ->-: < : ` *i' f. Jl 'ii5' r,'/t---i^ rn/. (Wjf\l i / ISSN 0041-008X 77zzs Number Completes Volume 50 Volume 50, Number 3 , ..Sl% * i V 3 "W Wt' , ^ September 30, 1979 R&S 133488 Applied Pharmaeology j Founding Editors Frederick Collision Harry W. Hays Arnold J. Lehman Associate Editors John L. Emmerson Seymour L. Friess Perry J. Gehring Tom S. Miya Jane F. Robens Hanspeter Witschi Official Journal of the Society of Toxicology Editor GABRIEL L. PLAA APAcademic Press New York and London isceral altera?> 9, A-37. . (1960). PrinMcGraw-Hill, sd Robinson, ia Orally ad- and rabbit, 7. Rowe, V, K, as determined of laboratory 697-705. ;cation, and Recommended j Chloroform. 114. 1909). ChloroJ repair. Bull. Principles and Chicago Press, % t, i a ?! 30 fio W CO CO -Cs 00 CO l Hie f\- i" ' I I " (_ | C"C i (O' -dot) TOXICOLOGY AND APPLIED PHARMACOLOGY 50, 523-531 (1979) Os 1 Acute Hepatoxicity of Vinyl Chloride and Ethylene: Modification by Trichloropropene Oxide, Diethylmaleate, and Cysteine1 R. B. CONOLLY2 AND R. J. JaEGER2 Department of Physiology, Harvard School of Public Health, Boston, Massachusetts 02115 Received January 8, 1979; accepted June 16, 1979 Acute Hepatotoxicity of Vinyl Chloride and Ethylene: Modification by Trichloro propene Oxide, Diethylmaleate, and Cysteine. Conolly, R. B,, and Jaeger, R, J. (1979). Toxicol. Appl, Pharmacol. 50, 523-531. We have reported that vinyl chloride (VC) and ethylene are acutely hepatotoxic in rats pretreated with polychlorinated biphenyl (PCB). The present study used several chemical treatments to investigate the mechanisms of acute VC and ethylene toxicity in PCB-pretreated rats. Trichloropropene oxide (TCPO) depletes hepatic glutathione (CSH) and inhibits hepatic epoxide hydrase (EH). Diethylmaleate (DEM) also depletes hepatic GSH. Cysteine is a rate-limiting precursor in hepatic GSH. synthesis. Effects of these various treatments on VC and ethylene toxicity were compared with their influence on hepatic GSH concentrations. Exposures to VC and ethylene were by inhalation at 1000 to 50,000 ppm. TCPO significantly increased VC toxicity in fasted but not in fed rats. Ethylene toxicity was not affected by TCPO. These actions of TCPO were not attributable to changes in hepatic GSH. DEM significantly lowered GSH during exposure, but did not increase the hepatotoxicity of either VC or ethylene. This suggests either that hepatic GSH concentrations are not an important determinant of the acute response to VC and ethylene or that DEM has important effects in addition to hepatic GSH depletion. Cysteine protected significantly, though incompletely, against acute VC hepatotoxicity. There was no effect of cysteine on the toxicity of ethylene. These data indicate that in PCB-treated rats, hepatic GSH and EH influence the acute hepatotoxicity of VC, but not that of ethylene. Vinyl chloride (VC) and ethylene are acutely hepatotoxic in rats pretreated with poly chlorinated biphenyl (PCB) (Conolly et al,, 1978). PCB is an inducer of the hepatic mixed-function oxidase system (MFOS) (AIvares and Kappas, 1977). VC and ethylene 1 Research supported by Grant ES-00002 from the National Institute for Environmental Health Sciences. : Present address; Imperial Chemical Industries Limited, Central Toxicology Laboratory, Alderley Park, Cheshire SK10 4TJ. England. 1 Research Career Development Awardee (ES0027) from the National Institute for Environmental Health Sciences. are not acutely hepatotoxic without such pretreatment. The demonstration of this toxicity, the structural similarity of the two compounds, and their industrial importance (Anonymous, 1978) have lead us to a com parative study of their hepatotoxic mechan isms. Initial results of these studies showed that a number of parameters--environ mental (e.g., fed-fast status, exposure tem perature) and chemical (e.g., preexposure treatment with trichloropropene oxide)-- could affect the hepatotoxicity of VC and ethylene (Conolly and Jaeger, 1977). oou-ooxxwizosit-MSOTOo o Copyright C 19TM by Academic Pres*, Inc, All ngnts oi'reproduction in any lorm reserved. Printed n Great Britain 524 TOXICITY OF VINYL CHLORIDE AND ETHYLENE In the work reported here, we studied the effects of several chemicals--trichloropropene oxide (TCPO), diethylmaleate (DEM), and cysteine--on the acute hepatotoxicity of VC and ethylene in PCB-pretreated rats. TCPO dissolved in water and dosed at 0.5 g cysteine/kg in a volume of 10.0 ml/kg, DEM* was dissolved in vehicle and dosed at 0.5 ml DEM/kg in a volume of 2.0 ml/kg. TCPO* vvas dissolved in vehicle and dosed at 0,1 ml TCPO/kg in a volume of 1,0 ml/kg. Inhalation exposures. All exposures to VC or is a potent in vitro inhibitor of epoxide, hydrase (EH) and. an in vivo depletor of hepatic GSH (Oesch and Daly, 1972). DEM is an effective depletor of hepatic GSH (Boyland and Chasseaud, 1970). Cysteine is a rate-limiting GSH precursor (Jocelyn, 1972). We investigated the influence of these treatments, TCPO, DEM, and cysteine, on hepatic GSH concentrations and the relation ship of changes in these GSH concentrations to the hepatotoxicity of VC and ethylene. Mortality, serum sorbitol dehydrogenase (SDH), and liver weight to body weight ratios were used to measure toxicity. ethylene were by inhalation. They began between 9:00 and 11:00 am and lasted 4 hr. The inhalation chambers, gases, and analytical methodology were as previously described (Conolly et al,, I97S). Biochemical assays. Determination of SDH activity used a method modified from that of Gerlach and Hiby (1974), Serum (0.3 ml or an appropriate dilution thereof), triethanolamine buffer (2.3 ml, 0,2 m, pH 7.4), and NADH (0.1 ml, 12m.vi in 0.1?; NaHC03) were mixed and incubated at 30'C for 30 min. At the end of (his incubation o( -)-fructose (0.3 ml, 4.0 m) was added. The disappearance of NADH absorbance vvas monitored at 366 nm using a Gilford 240 spectrophotometer equipped with a Gilford digital absorbance meter. Liver nonprotein sulfhydryl, expressed as GSH, was determined by the method of Jaeger et al, (1977). Beck et at. (I95S) have METHODS shown about 90% of rat hepatic nonprotein sulf hydryl to be GSH. Data presentation and analysis. Liver weight data Animals. Mali Holtzman rats.' 170-250 g. housed five or six per cage, were used. They were supplied with commercial rat chow1 and tap water ad libitum. The animals were maintained on a 12-hr light-dark cycle. In experiments with fasted rats, food was removed IS hr before VC or ethylene exposure. Fasting animals were allowed water. In all experi ments, food and water were not available during inhalation exposure. After exposure the animals were allowed water but not food. Sacrifice was by cervical transection. For GSH determinations, the rats were killed at 2 or 4 hr from the start of a 4-hr inhalation exposure. For determination of SDH activity, sacrifice was 24 hr after the start of exposure (Conolly and Jaeger, 1977), Treatments, All treatments were by gavage. PCB4 was solubilized in a vehicle of water containing 0.5 *' (v/v) Tween 30 and 0,5 % (w/v) methyl cellulose. Rats were given 100 nig PCB/kg in a volume of 2.5 ml/kg once daily between 2:00 and 5:00 pvt for 3 consecutive days. VC or ethylene exposure was on the day following the third PCB treatment. Cysteine, DEM, and TCPO were given within 0.5 hr before the start of inhalation exposure. Cysteine-HCI7 was ' Holtzman. Madison, Wis. 1 Purina Rat Chow, Ralston Purina Co.. St. Louis. Mo. are presented as grams of liver per 100 grams body weight to correct for differences in rat weights. Hepatic GSH concentrations are reported as a per centage of their respective control. The percentages of control values are based on calculation of milligrams of hepatic GSH per 100 g body weight. This trans formation corrects for injury-related changes in hepatic weight. For all liver weight and SDH values, statistical evaluation was based on the logarithmic transformation of the raw data to assure similarity of variances. Accordingly, the data are presented as geometric mean and standard error range. The effects of TCPO and cysteine on toxicity were examined at several concentrations of VC and ethylene. These effects were evaluated by two-way analysis of variance (ANOVA) (concentrations of VC or ethylene and treatment vs vehicle) (Armor and Couch, 1972). The F test vvas used to compare variance due to TCPO or cysteine treatment. The effect on toxicity of changes in VC or ethylene con centration vvas not tested as this has previously been shown to be dose responsive (Conolly et at,. 1973). Data from fed and fasted rats were treated separately in these analyses. Single concentrations of VC and ethylene were used to evaluate the effect of DEM on toxicity. The hypothesis that a difference in toxicity would exist between fed rats given DF.M and fasted * Aroclor 1254, Monsanto Chemical Co., St. Louis. Mo. 7 Reagent grade. Fisher Scientific Co,, Fair Lawn. N.J. * Minimum purity 35 %. ICN. K & K Laboratories, Plainview N.J. 'TCPO was 99"; pure. Aldrich Chemical Co., Milwaukee, Wisconsin. a, - S' i. 1 g cysteine,'kg in iissolvfit in vehicle name of 2.0 ml/kg. id dosed at 0.1 ml >sures to VC or :y began between ir. The inhalation thodolow- were as .. 1973) >n of SI it of C 30 Jpropri (2.3 03 12 mvi a ted a ion D( disapp GO ir C>O ,e CO if I at 36 equip S a Liver n determn icu vj tHC . el a!. (1933) have nonprotein sulf- Liver weight data r 100 grams body m rat vveights. eported as a perfhe percentages of tion of milligrams eight. This iransated changes in and SDH values, n the logarithmic assure similarity i are presented as :rror range. The an toxicity were 'ns of VC and ated by two-way concentrations of hide) (Armor and ised io compare e treatment. The or ethv lene cons previously been oily el n/., 1973). treated separately uons of VC and .'tfect of DEM on erence m toxicity DEM and fasted v K Laboratories, a Chemical Co.. \ ' \ I \1 CONOLLY AND JAEGER 525 rats given vehicle was evaluated by independent / test (Snedecor and Cochran, 1967). In all experiments mortality figures were tested for significance by both logit analysis (Finney. 1964) and by Fisher's exact test (Armitage, 1971). A p value of less than 0.05 was considered significant. RESULTS TCPO~Effects on the Acute Hepatotoxiciiy of Vinyl Chloride and Ethylene in PCB Pretreated Rats Table 1 summarizes the data for TCPO effects on rats exposed to VC. A NOVA of the data from fasted rats showed significant increases in liver weight and SDH. No effect of TCPO in fed rats exposed to VC was seen. Mortality was not significantly affected in these experiments. In contrast to its effect on VC toxicity, TCPO did not influence the toxicity of ethylene in either fed or fasted rats (Table 2). TCPO--Effects on Hepatic Glutathione during Vinyl Chloride or Ethylene Exposure of PCB Pretreated Rats Exposure of fasted rats to 4500 p.pm VCcaused minimal injury_io the-vehicle-treated group, but marked injury to the rats given TCPO (Table 1). Figure I shows that this concentration of VC caused significant hepatic GSH depletion in both vehicle and TCPO treated groups, but that there were no differences in GSH concentrations between groups. Figure 2 shows that TCPO treatment of fasted rats lowers hepatic GSH during exposure to 25,000 ppm ethylene. This effect of TCPO, which was significant at 2 hr but not at 4 hr, was not associated with any change in the hepatotoxic response to ethylene (Table 2). Figure 2 also shows that this concentration of ethylene, which caused severe hepatic injury (Table 2), did not itself deplete hepatic GSH. TABLE I Effect of TCPO os the Acute Hepatotoxicity of Vinyl Chloride in PCB-Pretreated Rats Vinyl chloride concentration (ppm) Fed or Vehicle Liver weight fasted or TCPO" (g/IOOy body wu* * Serum SDH activity*"*'' Mortality -- -- -- 1.000 4.500 3,000 1.000 4,500 3,000 10.000 25.000 40.000 10.000 25,000 40.000 Fed Fed Fasted Fasted Fasted Fasted Fasted Fasted Fasted Fasted Fed Fed Fed Fed Fed Fed Vehicle TCPO Vehicle TCPO Vehicle Vehicle Vehicle TCPO TCPO TCPO Vehicle Vehicle Vehicle TCPO TCPO TCPO 6.29 (5.99-6.60) 6.42 (6,31-6.54) 6.52 (6.32-6.73) 7.23 (6.99-7.4S) 6.94 (6.76-7.12) 7.13 (6.93-7.34) 10.09 (.9.20-1 l .07) 7.49 (7.34-7.66) 7.94 (7.67-3.22) 11.61 (10.30-12.49) $.13 (7.70-3.69) 10.50 (10.12-10.33) 10.53 (10.22-10.35) S.96 (3.60-9.33) 10.29 (10.11-10.47) 10.60 (10.25-10.97) 22.1 (19.9-24.6) 10.1 (3.7-11.7) 13.7 (17.6-19.9) IJ.I (13.5-17.0) 26.3 (22.9-31.4) 34.*: (29.1-40.3) 437 (233-S39) 27.2 (20.3-35.5) 133 (97.5-132) 1.633 (1333-1993) 121 (79.6-135) 772 (605-936) 1.192 (709-2001) 173 (133-224) 63S (433-942) 394 (554-1442) 0/4 0/5 0/4 0/11 0/10 0/19 3/11 0/5 1/19 7/11 1/15 3/11 5/12 1/15 3/11 6/12 "Rats were given either vehicle (1.0 ml. kg) or TCPO (0.1 ml/kg) in vehicle (1.0 ml. kg). * Effect of TCPO on liver weight in fasted rats exposed to VC significant (p = 0.00' by F test), e Values are expressed as the geometric mean and standard error range. J Effect of TCPO on SDH in fasted rats exposed to VC significant (p = 0.013 by F test). TOXICITY OF VINYL CHLORIDE AND ETHYLENE TABLE 2 Effect of TCPO on the Acute Hepatotoxicity of Etw^e hEated Rats --'''Eiiudene concentration (ppm) Fed or fasted or TCPO* (g/IOOg body wt,)*~-_ Serum SDH activity4 Mortality 6,000 12,000 13,000 25,000 6,000 12,000 13,000 25,000 10,000 25,000 50,000 10,000 25,000 50,000 Fasted Fasted Fasted Fasted Fasted Fasted Fasted Fasted Fed Fed Fed Fed Fed Fed Vehicle Vehicle Vehicle Vehicle TCPO TCPO TCPO TCPO Vehicle Vehicle Vehicle TCPO TCPO TCPO 6.3S (6.23-6.52) 7.63 (7.30-7.97) 8.66 (8.44-8.89) 8.95 (8.72-9.20) 6.78 (6.62-6.95) 7.91 (7.66-8.16) 8.17 (7.84-8.51) 9.19 (8.96-9.42) 6.73 (6.49-6.98) 7.27 (7.09-7.45) 8.04 (7.31-3.29) 6.64 (6.46-6.83) 8.28 (7.S6-8.73) 7.44 (7.32-7.56) 10,6(9.0-12.4) 137 (129-270) 499 (374-667) 833 (712-973) 21.0(17.5-25.1) 226(160-318) 399 (305-521) 865 (722-1036) 52.8 (37.3-75.0) 416 (309-559) 1,177 (1070-1295) 42.0 (32.0-55.1) 599 (409-877) 1,029 (778-1360) 0/12 0/12 0/12 1/13 0/12 0/12 0/12 0/14 0/3 0/5 1/6 0/10 0/5 2/6 ' Rats were given either vehicle (1.0 ml/kg) or TCPO (0.1 ml/kg) in vehicle (1,0 ml/kg). 4 Values are expressed as the geometric mean and standard error range. R&S 133492 OI23A HOURS FROM START 4 HR VC EXPOSURE 14,500 PPM VC) OI2 3A HOURS FROM START 4 HR ETHYLENE EXPOSURE (25,000 PPM) Fig. I. Effect of trichloropropene oxide (TCPO) on hepatic glutathione during exposure of fasted, PCBpretreated rats to 4500 ppm vinyl chloride (VC) (>i = 3). See Table I for the corresponding toxicity data. Fig. 2. Effect of trichloropropene oxide (TCPO) on hepatic glutathione during exposure of fasted, PCBpretreated rats to 25.000 ppm ethylene (n = 4). At 2 hr GSH in the TCPO-trcated group was signifi cantly lower (p<0,02). See Table 2 for the corres ponding toxicity data. Mortality 0/12 0/12 0/12 1/13 0/12 0/12 0/12 0/14 0/S 0/5 1/6 0/10 0/5 2/6 U<g). CONOLLY AND JAEGER 527 Relationship of Hepatic GSH Depletion by --s*Et_tn VC and Ethylene Hepatotoxicitv Immediately Dtfore VC or ethylene expo sure hepatic GSJj'Concgp-M-n.r;... ^-rCd and" tasted PC&-pfctreated rats were 20.27 + 0.89 and__L5.28 + 1.70 mg GSH/lOOg body wt, respectively. For both of these determinations n = 3. Fed rats given DEM and fasted rats given vehicle were sacrificed during exposure to 10,000 ppm VC or 20,000 ppm ethylene and hepatic GSH concentrations determined. Figure 3 shows that 10.000 ppm VC caused significant GSH depletion in both fed-DEM and fast-vehicle groups, and that there were no differences in hepatic GSH concentrations. Table 3 shows that fasting, but not DEM, significantly increased the toxicity of 10,000 ppm VC in PCB-pretreated rats. Furthermore, DEM did not affect the magni tude of the fed-fast difference. The data for ethylene were analogous. Hepatic GSH (Fig. 4) was significantly (p<0.0l at 2 and 4 hr) lower in the fed-DEM group than in the' HOURS FROM START A HR VC EXPOSURE (10,000 PPM VC) Fic. 3. Effect of diethylmaleate (DEM) on hepatic glutathione during exposure to 10.000 ppm vinyl chloride (VC). Fed, PCB-pretreated rats were given diethylmaleate so that their hepatic GSH during exposure to 10,000 ppm was the same as. or lower than, that of simultaneously exposed, fasted rats. Each datum point has n = 3. except for Fed-DEM, 0 hr, which has n = 2. See Table 3 for the corres ponding toxicity data. T i TE 3 ENE ^0 0 , T HR OO CD C-pO to oxide (TCPO) on e of fasted. PCB- lene in = 4). At roup was signifi2 for the corres- \ \ 1 I 1 TABLE 3 Effect of Diethylmaleate on the Acute Hepatotoxicity of Vinyl Chloride or Ethylene in Fed and Fasted PCB-Pretreated Rats Inhalation exposure (ppm) Fed or Vehicle Liver weight fasted or DEM- (g/100 g body wt.) Serum SDH activity* Mortality --, -- -- -- Vinyl chloride 10,000 10,000 10.000 10.000 Ethylene 20.000 20.000 20.000 20,000 Fed Fed Fasted Fasted Fed Fed Fasted Fasted Fed Fed Fasted Fasted Vehicle DEM Vehicle DEM Vehicle DEM Vehicle DEM Vehicle DEM Vehicle DEM 6.43 (6.23-6,53) 6.82 (6.62-7.03) 6.52 (6.32-6.73) 6.85 (6.63-7.08) 13.1 (12.0-14.3) 14.7 (11.9-18.1) 13.7 (17.6-19.9) 15.4 (14.5-16.3) 7,23 (6.92-7.66) 7.63 (7.48-7.79) 10.24 (9.19-11.40)' J 11.36 (10.43-12.31 )J 48.3 (34.1-69,3) 41.2 (32.1-52.7) 360 (351 -2106r j 950 (625-1445)J 7.70 (7.43-7.97) 3.30 (3.12-3.48) 3.26 (8.04-3.50) 9.44 (9.10-9.80) 369 (301-453) 205 (IS3-23I) 362 (303-432) 327 (263-399) 0/4 0/5 0/4 0/5 0/5 0/5 0/5 0/5 0/7 0/13 0/13 0/13 * Rais ^ere given either vehicle (2,0 nil kg) or DEM (0.5 ml.kg) m vehicle (2.0 ml/kg). * Values are expressed as the geometric mean and itandard error range, r Significantly different ip < 0.05 bv the independent i test) from Fed-Vehicle, VC-exposed. J Significantly different ip < 0.05 by the independent t test) from Fed-DEM, VC-ex posed. 3 \! * 'i# , , v , . ' - - 52S _ TOXICITY OF VINYL CHLORIDE AND ETHYLENE vCmen vC ttPOSCO * ctsr(int * vc uroue * 33 9? CP 0 12 14 HOURS FROM START A HR ETHYLENE EXPOSURE UO.OOO PPM) FtG. 4. Effect of diethylmaleate (DEM) on hepatic glutathione during exposure to 20,000 ppm ethylene. Fed, PCB-pretreated rats were given diethylmaleate so that their hepatic GSH during exposure to ethylene was significantly lower than that of simultaneously exposed, fasted rats (p<0.01 at 2 and 4 hr), (n =4). See Table 3 for the corresponding toxicity data. fasted-vehicle group during exposure to 20,000 ppm ethylene. However, in these ethylene-exposed rats, the significantly lower hepatic GSH of the DEM-treated group was not associated with any increase in toxicity (Table 3). HOURS from START 4 HR VC EXPOSURE (8,000 PP* VC) Fic. 5, Effect of cysteine on hepatic glutathione during exposure of fasted, PCB-treated rats to 3000 ppm vinyl chloride (VC). Fasted, PCB-pretreated rats were given cysteine so that their hepatic GSH (total nonprotein sulfhydryl) during exposure to vinyl chloride was the same as or greater than that of nonexposed rats [n = 6). See Table 4 for the corres ponding toxicity data. exposed to 8,000, 16,000, and 24,000 ppm ethylene. There was no effect of cysteine on the toxicity of ethylene at these concentrations. Relationship of Cysteine Treatment to Changes in the Acute Hepatotoxicity of VC and Ethylene Figure 5 shows that cysteine completely prevented depiction of hepatic GSH during exposure of fasted, PCB-pretreated rats to 8000 ppm VC. ANOVA of the data in Table 4 showed that cysteine protected against the acute hepatotoxic effects of VC in fasted rats. This effect was significant for increases in both liver weight and SDH. It should be noted, however, that this protection was not complete. Significant increases in liver weight and SDH occurred after VC exposure of cysteine-treated rats (Table 4). Mortality was not a significant factor in these experi ments. PCB-pretreated rats given cysteine were DISCUSSION Trichloropropene Oxide The increased toxicity of VC in TCPOtreated rats (Table 1) is consistent with literature reports on the biochemical effects of TCPO. It inhibits EH (Oesch and Daly, 1972) and is a substrate for GSH-S-alkylepoxytransferase (GSH-SAT) (Mukhtar and Bresnick, 1976), while VC metabolism in volves epoxide formation (Green and Hathway, 1977) and GSH conjugation (Watanabe et al,, 1976b). Figure 1 suggests that dif ferences in hepatic GSH concentrations do not explain this action of TCPO. It is likely therefore, the increased toxicity of VC in TCPO-treated rats is due to inhibition of EH. The action of TCPO in fasted, but not in fed rats might reflect a greater importance of EH 4 UfOJEO IFOI^ repatic glutathione B-trealcd rats to ed, PCB-pretreated :heir hepatic CSH in'ng exposure to treater than that of e -4 for the corres- nd 24,000 ppm f cysteine on the concentrations. vc tonsi: .hemi :sch , GS1 (Mui SJ D- cn th --A ts 05 05 y. -CCO i- CJI d netab 1- een a - ion (Watanabe gests that dif- centrations do ?0. it is likely city of VC| in libition of EH. but not in fed ortance of EH \ \ 1 CONOLLY AND MEG^r 529 TABLE 4 Effect of Cysteine on the Acute Hepatotoxicity of Vinyl Chloride in PCB*Pretreated Rats Vinyl chloride concentration (ppm) Fed or fasted Water or cysteine' Liver weight (g/100 g body wt)`,f Serum SDH activity'1' Mortality -- -- 5,000 8,000 12,000 17,000 5,000 8,000 12,000 17,000 Fasted Fasted Fasted Fasted Fasted Fasted Fasted Fasted Fasted Fasted Water Cysteine Water Water Water ' Water Cysteine Cysteine Cysteine Cysteine 6.94 (6.64-7.26) 6.74 (6.54-6.93) 8.84 (7.93-9.86) 9.21 (8.90-9.52) 11.69 (11.10-12.32) 11.12 (10.60-11.67) 6.82 (6.65-7.01) 7.S2 (7.52-8.13) 9.49 (S.71-10.34)' 9.51 (S.72-I0.3S)' 13.3 (15.4-21.7) 22.9 (19.5-27.0) 203 (107-386) 253 (203-315) 634 (573-749) 762 (469-123 S) 30.3 (26.6-34.4) 75.9 (57.9-98.1)' 423 (226-792)' 132 (90.S-194)' 0/4 0/9 0/6 1/30 0/6 2/6 1/6 2/30 1/6 0/6 * Rats were given either water (10.0 ml/kg) or cysteine (0.5 g/kg) in water (10.0 ml/kg). 1 Effect of cysteine on liver weight in VC exposed rats significantly different (p = 0.001 by F test). ' Values are expressed as the geometric mean and standard error range. ' Effect of cysteine on SDH in VC exposed rats significantly different (p = 0.002 by F test). 'Significantly different (p< 0.02 by the independent t test) from cysteine treated, not exposed. when, as in the fasted rat, hepatic GSH is depleted. TCPO did not affect ethylene toxicity (Table 2), though evidence exists that ethylene is metabolized to ethylene oxide (Ehrenberg et al., 1977; Jerie and Hall, 1978). These data suggest then that EH and GSHSAT do not act on the oxide. The lack of hepatic GSH depletion by ethylene (Fig. 2) is consistent with a noninvolvement of GSHSAT in ethylene metabolism. Alternatively, ethylene oxide may not be a toxic metabolite of ethylene. Diethylntaleate Jaeger et al. (1977) reported that DEM did not increase the hepatotoxicity of VC in phenobarbital-pretreated, normally fed rats. We have shown here the same effect of DEM in both fed and fasted rats pretreated with PCB and exposed to VC or to ethylene (Table 3). In these experiments the hepatic GSH concentrations of DEM-treated fed rats were the same as (VC, Fig. 3), or lower than (ethylene, Fig. 4), the corresponding concentrations in vehicle-treated fasted rats. These results are consistent with our other findings on ethylene but conflict with the data on VC toxicity suggesting involvement of hepatic GSH (Table 4). However, a substan tial body of evidence suggests the importance of GSH in detoxification of electrophilic VC metabolites (Green and Hathway, 1975; Watanabe et a/., 1976a,b). Hence, we suspect that hepatic GSH concentrations are in fact an important determinant of acute VC toxicity. Some action of DEM in addition to GSH depletion could explain the lack of potentiation of VC toxicity. For example, Chuang et al. (197S) have shown that DEM inhibits aryl hydrocarbon hydroxylase (cyto chrome F-448), an enzyme strongly induced by the PCB mixture Arocior 1254 used in these studies (Alvares and Kappus, 1977). Cysteine Leaf and Neuberger (1947) have shown that dietary cysteine, but not glycine or glutamic acid, directly influences hepatic GSH con centrations in rats. Waelsch and Rittenberg TOXICITY OF VINYL CHLORIDE AND ETHYLENE R&S 133496 (1942) have.^imatird-thc--Hatf:IiTe of hepatic "G5H to be 2-4 hr. It is likely, therefore, that the lack of "GSH" depletion during VC exposure of treated rats (Fig. 5) in fact represents some combination of increased hepatoto.xicity of ethylene and halogenated ethylenes after PCB pretreatment. Enciron. Health Perspect. 21, 131-135. Conolly, R. B,, Jaeger, R. J,, and Szabo, S. (1978). Acute hepatoto.xicity of ethylene, vinyl fluoride, vinyl chloride and vinyl bromide after Aroclor I2J4 concentrations of hepatic GSH and of non pretreatment. Exp. Mol. Pathol. 23, 25-33. protein sulfhydry! (i.e., cysteine). The com bination--GSH and cysteine--may not pos sess the same potential for detoxification of reactive chemical species as would the same Ehrenberg, L., Osterman-Golkar, S., Segerback. D,, Svensson, K,, and Callerman, C. J. (1977). Evaluation of genetic risks of alkylating agents. III. Alkylation of haemoglobin after metabolic con version of the ethene to ethane oxide in vivo. amount of -SH present solely as GSH. This reflects the fact that GSH, but not cysteine, is a substrate for the various GSH trans ferases. The development of liver injury in the cysteine-treated, VC-exposed group (Table Motor. Res. 45. 175-184. Finney, E, A, (1964). Stotistical Methods in Biological Assays), 2nd ed. Hafner, New York. Gerlach, U., and Hiby. W, (1974). Sorbitol dehydro genase. In Methods of Enzymatic Analysis (H. U'. Bergmeyer, ed.), Vol. 2, pp. 569-573, Academic 4) may be due to the fact that much of the Press. New York. -SH was present as cysteine rather than GSH, and hence did not possess the same potential for enzymatically mediated metabo lite detoxification. Green, T.. and Hathway, D. E. (1975). The biolo gical fate in rat of vinyl chloride in relation to its oncogenicity. Chem.-Bioi. interact. 11, 545-562. Green, T,, and Hathway, D. E. (1977). The chemistry and biogenesis of the S-containing metabolites Cysteine had no effect on the acute hepatotoxicity of ethylene in PCB-treated rats. This is further evidence that GSH does not play an important role in acute ethylene hepatotoxicity. of vinyl chloride in rats. Chem.-Biot. Interact. 17, 137-150. Jaeger, R. J.. Murphy, S. D., Reynolds. E. S.. Szabo. S.. and Moslen, M. T. (1977), Chemical modification of acute hepatotoxicity of vinyl chloride monomer in rats. Toxicol. Appl. Pharmacol. 41, 597-607. REFERENCES Jerie, P. H.. and Hall, M. A. (1978). The identifica tion of ethylene oxide as a major metabolite of Anonymous (1978). 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