Document GKXJJyNQp2D1qqqqrz939gvOn

VM-U$)j f '*&$ 2Z.W* ISSN 0041-008X This Number-Completes Volume 49 Volume 49, Number 3 _____ July 1979 Toxicology and Applied Pharmacology Founding Editors Frederick Coulston 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 Academic Press New York and London TOXICOLOGY AND APPLIED PHARMACOLOGY 49, 505-513 (1979) Formation and Inactivation of a Chemically Reactive Metabolite of Vinyl Chloride1 D. Pessayre,1 J. C. Wandscheer, V. Descatoire, J. Y. Artigou, AND J. P. BENHAMOU Unite de Recherches de Physiopathologie Hipatique (INSERM), Hdpital Beaujon, 92118 Cllchy, France Received December 18, 1978; accepted March 17, 1979 Formation and Inactivation of a Chemically Reactive Metabolite of Vinyl Chloride. Pessayre, D., Wandscheer, J. C., Descatoire, V., Artigou, J. Y., and Benhamou, J. P. (1979). Toxicol. Appl. Pharmacol. 49, 505-515. The mechanisms responsible for, and pro tecting against, metabolic activation of vinyl chloride were investigated in the rat. When [1JC]vinyI chloride was incubated with hepatic microsomes, a ''C-labeled material became irreversibly bound to microsomal proteins; binding required NADPH. was decreased by CO. SKF 525-A, or glutathione, and was increased by l,l,l-trichloropropene-2,3-oxide (TCPO). Inhalation of a 5% vinyl chloride atmosphere decreased hepatic cytochrome P-450 and glutathione. Pretreatment of the animals with DDT or phenobarbital (I) in creased in vitro irreversible binding to microsomal proteins measured in the presence, but not in the absence, of TCPO and increased the in vivo loss of cytochrome P-450 during inhala tion of vinyl chloride, but (2) decreased the in vitro irreversible binding to 10.000c super natant protein; and the in vivo loss of glutathione during inhalation of vinyl chloride. These results are consistent with the views that (I) vinyl chloride is activated by cytochrome P-450 into its chemically reactive epoxide, (2) the epoxide may be inactivated by epoxide hydrase or by binding to glutathione, (3) pretreatment with DDT or phenobarbital in creases both the formation rate of the epoxide and its inactivation rate by epoxide hydrase, and (4) cytochrome P-450 destruction is related to the formation rate of the epo.xide, whereas glutathione depletion seems related to only that fraction of the formed epoxide which has escaped inactivation by microsomal epoxide hydrase and has diffused in the cytosol. Vinyl chloride (chloroetln lene) is massively observed (Berk et al.. 1976). Hepatic used in the manufacture of plastics: Poly angiosarcomas and liver lesions have been merization of this monomer leads to polyvinyl reproduced in experimental animals chroni chloride, the most widely used synthetic cally exposed to vinyl chloride (Torkclson plastic (Berk et al., I b)76). Tn workers et al.. 1961; Lester et al.. 1963; Viola et a/.. exposed to vinvl chloride, angiosarcomas of 1971: Maltoni and Lelemine, 1975; Prodan the liver and/or hepatic fibrosis have been et al., 1975). Several carcinogenic and.or hepatotoxic 1 This work was supported by Gram 021 R 07 from Faeulie de Medecine Xavier-Bichat (UnivcrMte de Paris VII), and ATP 5X-7X-90 from Institut National Je la Same et de la Recherche Medicate (INSERM I. ; Recipient of a fellowship from the Funds d'Etudcs du Corps Medical Hospitalier. compounds are transformed by the hepatic microsomal mixed-function oxidase system (MFOS) into chemically reactive metabolites that (1) covalently bind in vitro to micro somal proteins. (2) covalently bind in vivo 505 omi-nnsix '> tvn>?oj-iisoy.oo o fMint * Hn. AcJJcmic Prcsi, lnc4 All n^nij ui rtfjvtujutiiwn m jnv iurm reserved. Pnrud m Grji ttnum R&S 133523 506 PESSAYRE ET Al. to tissue proteins, and (3) may destroy hepatic cytochrome /M50 and deplete hepatic gluta thione (Mitchell and Jollow, 1975; Mazel and Pessayre, 1976). In several reports, it has been shown that vinyl chloride may produce one or the other of the above-mentioned effects (Barbin et at., 1975; Reynolds et at., 1975; Bolt et at., 1976; Kappus et at., 1976; Reynolds et at., 1976; Guengerich and Strickland, 1977; Ivanetich et at., 1977; Osterman-Golkar et at., 1977; Watanabe et at., 197S). However, a global under standing of the mechanisms for. and the interplay between, these various effects was hampered by the lack of a comprehensive work studying all these parameters both in non-pretreated and in variously pretreated animals. METHODS Animals and treatments. Male Sprague-Dawley rats, weighing 160-200 g. were obtained from Charles River France (Elbeuf. France), Rats were allowed water and food (Autoclave 113. UAR) ad libitum. Some animals received one of the following pretreatments: (I) cobalious chloride (CoCl..-6H^O). 35 mg,' kg sc twice daily for 3 days; (2) 2-diethylaminocihyl2,2-diphenylvalerate hydrochloride (SKF 525-A), 75 mg. kg. ip; (3) l,l,l-trich!oro-2.2-bis(/?-chlorophenyl) ethane (DDT); 200 mg, kg ip; or (4) phenobarbital, 100 mg. kg ip daily for 5 days. Pretrcated animals were exposed to vinyl chloride or sacrificed (I) IS hr after the last dose of CoC)., (2) 15 min after the single dose of SKF 525-A, (3) S days after the single dose of DDT. or (4) 24 hr after the last dose of phenobarbital. Six rats at a lime were placed in a 15-liter chamber that was continuously gassed with 2 liters,-min of a 95% air-5 % vinyl chloride atmosphere. Vinyl chloride (99.99%) was purchased from Merck Company, Darmstadt, Federal Republic of Germany. Animals were sacri ficed after various lengths or exposure; it must be noted that the proportion of vinyl chloride in the atmosphere inhaled by the rats did not reach 5% immediately. Preparation of tissue homogenates and tissue frac tions. Rats were stunned; blood was drawn from the inferior xena cava: the liver, the kiJneys. the spleen, and the sartorius muscle were removed, tllood samples and tissue fragments were homogenized with a glass-Tcilon Pottcr-EKehjcm homogeni/er in 3 vol of 0,154 \t KCI. 0.01 vt Na'K -phosphate buffer, pH 7.4. The crude homogenates were centrifuged at 10,000,?; microsomal pellets were prepared by centri fuging 3 ml of the 10,000? supernatant with 9.5 ml of 0.154 m KCI, 0.01 m Na"K*-phosphate buffer, pH 7.4, at 100.000?. The microsomal pellets were resuspended in the phosphate buffer and centrifuged again at 100,000?. Transaminases, glutathione, cytochrome P-450. Serum glutamic-pyruvic transaminase (SGPT) activity was measured by the method of Reitman and Frankel (1957). Some liver fragments were placed in Bouin's solution, embedded in paraffin, and stained with hematoxylin and eosin. Hepatic glutathione was determined in the whole liver homogenate by the method of Ellman (1959) in animals sacrificed at 10-12 am. Microsomal cytochrome P-450 was measured by the method of Omura and Sato (1964). Irreversible binding in vitro. [''CJVinyl chloride (specific activity, 1 mCi/mmol), labeled on the two carbon atoms and dissolved in methanol (1 mntolf ml), was purchased from Commissariat a FEncrgie Atomique. Saclay, France: its radiopurity, checked by gas-liquid chromatography, was higher than 99%. [uC]Viny! chloride (5 /trnol. 5 ;tCi) in 5 /tl of methanol was added, in 25-ml'vials. to 1 ml of ice-cold 0.154 m KCI, 0.01 m Na' K'-phosphate buffer. pH 7.4, containing microsomes or 10.000? supernatant from 125 mg of liver and the following reduced nicotinamide adenine dinucieotide phosphate (NADPH(-generating system: NADP (0.4 nmol), glucose 6-phosphate (6 ;/mol). glucose 6-phosphate dehydrogenase (3 enzyme units), and MgCI; (6 /miol). The vials were capped and the mixture was incubated at 37` for 15 min in a Gallenkamp shaking incubator. In some vials, the NADPH-generaiing system was omitted: in other vials, various substances were added; other vials were tlushed with a 90% CO-10% O. atmosphere, capped, and incubated. Radioactivity irreversibly bound to microsomal or 10,000? super natant proteins was measured exactly as previously described (Allcmand et at., 197S): Proteins were precipitated with trichloroacetic acid (TCA), dried, washed three limes with TCA. and repeatedly ex tracted with various solvents (methanol, heptane, and ether); no detectable radioactivity was found in the three last solvent phases: 'JC material remaining on the proteins was then counted in an liucrteehnique ABAC SL 40 scintillation counter. Irreversible binding in viva. ["C)Vinyl chloride (6.25 mg. 100;imol. 100/iCi) was administered ip in 250 l of methanol; rats were sacrificed 4 hr later: tissue fragments were homogenized in 3 vol of 0,154 \i KCI. 0.01 vi Na K -phosphjte buffer. pH 7.4. "C-Labelod material irreversibly bound to iis-ue proteins was measured as previously described (.Allcmand et of. 1973) and summarized above. Statiitival onalvas. Student's t lest (iwo-taded) (or independent data was used as a test of significance pared by centriant with 9.5 nil osphate buffer, lal pellets uere and centrifuged chrome P-tSO. (SGPT) activity tun and Franke! I laced in Bouirt's d stained with alutathione was ogenate by the tls sacrificed at le P-450 was ind Sato (1964). "]Vinyl chloride led on the two .hanol (I mmol:' trial a l'Energie | arity, checked by gher than 99 s'. 5 ;<l of methanol ice-cold 0.154 m I bulTer, pH 7.4. | O.t* supernatant ( Rowing reduced ide phosphate DP (0.4/(mol), ose 6-phosphate MgCI. (6 //mol), re was incubated taking incubator, ting system was | lubstances were a 90,; CO-IO",, ed. Rudioactiv ity r 10,000;' iuper| tly as previously ; Proteins were id (TCA), dried, d repeatedly ex- Inol. heptane, and vinyl chloride ACTIVATION-INACTIVATION 507- between means. The relationship between parameters was assessed by linear regression analysis and calcu lation of the coefficient of correlation (r). RESULTS Vinyl chloride inhalation,Tn animals sacri ficed after an 18-hr exposure to a 5% vinyl chloride atmosphere, SGPT was unchanged and histological examination did not show hepatic necrosis in control rats or in rats treated with CoCL. SKF 525-A, DDT, or phenobarbital (data not shown). The effects of inhalation of vinyl chloride on hepatic cytochrome P-450 and glutathione are shown in Figs. 1-3. Hepatic microsomal cytochrome /'-450 decreased linearly with time during inhalation of vinyl chloride in control rats (Fig, I); the decrease was negligible or lower in rats treated with CoCL or SKF 525-A and was higher in rats treated with phenobarbital or DDT (Fig. 3). Hepatic glutathione rapidly decreased during the first hour of exposure, reaching a constant low value after 6 hr of exposure in control rats (Fig. 2); the decrease was negligible or lower in rats treated with CoCL, SKF 525-A, DDT. or phenobarbital (Fig. 3). In vivo binding. After administration of [,4C]vinyl chloride, a 14C-labe!ed material was irreversibly bound to whole tissue pro teins (Table I); binding to blood, kidney, and spleen proteins was about 33% of that to liver proteins: binding to muscle proteins was about 3% (Table 1). Binding in all organs tested was less in rats treated with CoCL than in control rats (Table 1). Jn vitro binding. When [,4C]vinyl chloride was incubated under air with hepatic microsomes and a NADPH-eenerating system, a radioactive material became irreversibly t itwo ,t of significance 03 4 Ei poiun (o 5% vinyl chldfifli 18 sau'! Fio. I. EiTccin of vinyl chloride inhalation on hepatic microsomal cytochrome P-450. Cytochrome P-450 concentrations were measured in animals -jcruieod after various lengths of exposure to a 5'',. Mini chloride -95 air atmosphere. Results are moans ;.SO for at least sis rats. 0 1)4 <03tv'* to 5 Chltji'id* U Fig. 7. EiTcets of vinyl chloride Inhalation on hepatic glutathione Glutaihione concentrations were measured in animals sacrificed after various lengths of exposure to a 5", vinyl ehlorido-45",, air atmo sphere. Results are means rSD for ai least uv rats. s 133525 50S PESSAYRE ET AL. bound to microsomal proteins (Fig. 4), not shown). Binding to hepatic microsomal whereas no detectable binding occurred proteins was negligible when the NADPH- under identical conditions with microsomes generating system was omitted (Fig. 4); from blood, kidney, spleen, or muscle (data binding was decreased when the incubation was made under a 90% CO-IO% 02 atmos phere or in the presence of SKF 525-A (Fig. 4). Similar findings were obtained when [uC]vinyl chloride was incubated with hepatic lO.OOOg supernatant and the 1JC- labeled material irreversibly bound to lO.OOOg supernatant proteins was measured (Fig. 4). In vitro binding to hepatic microsomal 3S* proteins was decreased by addition to the incubation mixture of glutathione or cysteine, but not S-methyl glutathione or glycine (Fig. 5); it was not increased by diethyl- - maleate (5 m.xt) (data not shown). Addition of diethylmaleate (5 m.M) to the 10,000g supernatant incubation mixture decreased endogenous glutathione in the medium and increased in vitro binding to 10,000g super natant proteins (Fig. 6). CaClj non phiftQ- borfntal Addition of 0.5 mM 1,1,1-trichloropropene 2,3-oxide (TCPO) to the incubation mixture Fjc. 3. Effects of various treatments on the toss of cytochrome P-450 and that of glutathione. Cyto chrome P-450 lost is the difference between the mean for hepatic microsomal cytochrome P-450 concentra increased in vitro covalent binding to micro somal proteins (Table 2); whereas binding was increased by only 37 % in control rats, binding was increased by 134 and 200% in rats tion in rats not exposed. and in rats exposed. to a 5,, vinyl chloride-95air atmosphere for 18 hr. the former and the latter rats being simultaneously pre treated and sacrificed. Hepatic glutathione lost was similarly calculated after exposure to vinyl chloride for I hr. treated with DDT or phcnobarbital, respec tively (Table 2). Treatment of the animals with CoCI- or SKF 525-A decreased (l) in vitro binding to 10,000g supernatant proteins, (2) in vitro TABLE I Vinyl Chloride Material Irrevershily Bound to Whole Tissue Proteins in I Yen' Vinyl chloride material irreversibly bound to proteins (nmol-4 hr"' g tissue'1) Liscr Blood Kidney Spleen Muscle Control rats CoCL-Trcated rats 69 * 10 16-5* 24 4 5r3" 22 10 5 2" 22 i 5 4-1* 2.0 0.9 o.3o.r * [,JC]Vinyl chloride (100/imol: IOO;<Ci)in 250 ;il of methanol was administered ip to control rats and to rats treated with CoCI.: 4 hr later, ihe animals were sacri ficed and the amount of "C-labeled material irreversibly bound to whole tissue proteins was measured: results are the meansr SD of four rats. * Significantly different from that in control rats, p < 0.001. nicrosomal NADPH- (Fig. 4); incubation , 02 atmosKF" 525-A : obtained ibat< i tl i to 1 id (1 JO (/) mien :ion or c> or i 03 03 CrJo1 03 by d ). Ad he It decreased tedium and OOOg super- loropropene ion mixture ig to microbinding was rats, binding in rats >ital, respec- :h Cod, or i binding to (2) in vitro d I* VINYL-CHLORIDE ACTIVATION-INACTIVATION 509 Sosol Without N40PM With CO Witn SKF 525-A Fig. 4. Irreversible binding of a vinyl chloride material to proteins in vitro. In the basal system. ['`CJvinyl chloride (5 mxi) was incubated under air with microsomes or 10,000c supernatant from 125 mg of liver from non-pretreated rats and with a NADPH-generating system: in some flasks, the NADPH-generating system was omitted; other flasks were incubated under a 90% CO-IO% O. atmosphere; in other flasks, SK.F 525-A (5 mw) was added to the incubation mixture. After 15 min of incubation, the amount of vinyl chloride material irreversibly bound to microsomal or lO.OOOg supernatant proteins was measured; columns and bars represent means and SD for three experiments. diff*rnt fram ihot m th* bdtQl P<Q,001 gtufa<hiori Fig. 5. Effects of SH compounds on the irreversible binding of a vinyl chloride material to micro somal proteins in vitro. In the basal system, [uC]vinyl chloride (5 nisi) was incubated under air with microsomes from 125 mg of liver from non-pretreated rats and with a NADPH-generating system: in other flasks, a 5 mst concentration of various compounds was added to the incubation mixture. Columns and bars represent means and SD for three experiments. binding to microsomal proteins measured without added TCPO, and (3) in vitro binding to microsomal proteins measured in the presence of 0.5 mxi TCPO (Table 2). Treatment of the animals with DDT or with phenobarbital (I) decreased in vitro binding to IO.OOOg supernatant proteins, (2) did not change binding to microsomal proteins measured without TCPO. and (3) increased binding to microsomal proteins measured in the presence of TCPO (Table 2). The loss of hepatic microsomal cytochrome P-450 after inhalation of vinyl chloride for 18 hr was (I) not correlated (r = 0.077) with 510 PESSAYRE ET AL. TABLE 2 Vinyl Chloride Material Irreversibly Bound to Proteins in Vitro* Vinyl chloride material irreversibly bound to proteins (nmol -15 min"`-g liver'1) 10,000 supernatant Microsomes Microsomes without with added TCPO* added TCPO* Control rats CoCl.-Treated rats SKF 525-A-Treated rats DDT-Treated rats Phenobarbital-Treated rats 14+1 22* 102* 8+1* 3 2* 24 + 7 7 + 3* I34* 23 5 25 8 33 + 3* 9 + 2* 18 9* 54 + 6*-* 75:6r-* * [,JCJVinyl chloride (5 m\() was incubated under air with 10,000? supernatant or microsomes from 125 mg of liver 2nd with a NADPH-generating system for 15 min; the amount of vinyl chloride material irreversibly bound to 10,000c supernatant or microsomal proteins was measured; results are meansSD of at least six rats. * 2 ii\ of acetone either pure or containing l.l.I-trichloropropene-2.j-oxidc (TCPO) (final concentration in the incubate, 0.5 m.w) was added to the incuba tion mixture. r Significantly different from that bound to microsomes without added TCPO, p< 0.05. * Significantly different from that in control rats, p < 0.05. 31 9 (/> CP CP in $ in vitro binding to lO.OOOg supernatant proteins, (2) better correlated (r = 0.771) with in vitro binding to microsomal proteins measured without added TCPO, and (3) strikingly correlated (r = 0.972) with in vitro covalent binding to microsomal proteins measured in the presence of TCPO (Fig. 7). The loss of hepatic glutathione after inhalation of vinyl chloride for 1 hr was (I) well correlated (r = 0.891) with in vitro binding to lO.OOOg supernatant proteins (Fig. 8). (2) poorly correlated (r = 0.560) with in vitro binding to microsomal proteins measured without added TCPO. and (3) not correlated (r -- 0.061) with in vitro binding to microsomal proteins measured in the presence of TCPO. DISCUSSION Metabolic activation in the liver into a chemically reactive metabolite. In vitro, when [IJC]vinvl chloride was incubated under air with rat liver microsomes but without a NADPH-generating system, there was neg ligible radioactivity irreversibly bound to microsomal proteins (Fig. 4), which shows that vinyl chloride itself does not bind to proteins. In vitro binding to microsomal proteins required NADPH and was inhibited by CO or SKF 525-A (Fig. 4). Similar results were obtained by Kuppus ei at. (1976). These findings suggest that the radioactive material that bound to proteins in vitro (Fig. 4) was not vinyl chloride itself but a metabolite of it formed by the MFOS. The bound metabolite could not be removed by repeated washings and repeated extractions w ith solvents of various polarities; this apparently irreversible binding suggests that the metabolite was attached to proteins by a covalent bond. A covalent bond between a drug molecule, or a metabolite of it. and a tissue inacromolccule mav result from two main reactions: (I) the spontaneous reaction between a chemically reactive compound and an alreadv formed macromolecule, or (2) the VINYL CHLORIDE ACTIVATION-INACTIVATION 51 1 re was negbound to . hich shows lot bind to microsomal as inhibited 4). Similar spus el al. t that the to proteins :yl chloride mcd by the Id nd i' F ns tc me of CO CcOn ro co If tus 1 pc 1 or (2) the Bawl system With <Ji*thyt- Vmyi cnlofide mutnjl *rrvrsibly Suynd ja micratamef jftjmni (vnoJ IS rour' | Jw* Ftc.. 7. Correlation between loss of cytochrome P-450 and irreversible binding to microsomal proteins hi vitro. The loss of hepatic microsomal cytochrome P-450 after inhalation of 3 5", vinyl chloride-95";; air atmosphere for IS hr (Fig. 3) is plotted against the amount of vinyl chloride material that became irrcversibly bound to microsomal proteins when 5 mst l,l.l-lrichloropropenc*2.3-oxide (TCPO) was added to the incubation mixture (Table 2). The drawn line is the least-squares regression line. FtC. 6. EtTects of diethylmaleate on the irreversible binding of a vinyl chloride material to lO.OOOt.' supernatant proteins in vitro. In the basal system. (uC]vinyl chloride (5 mvt) was incubated under air with lO.OOO.c supernatant from 125 mg of liver from non-pretreated rats and with a NADPH-gcnerating system; in other tlasks J mvt diethylmaleate was added. After 15 min of incubation, the amount of glutathione in the incubation mixture and the amount of vinyl chloride material irreversibly hound to lO.OOOe supernatant proteins were measured. Columns and bars represent means and SD for three experiments. enzyme-mediated incorporation of a stable compound into a built-up inacromolecule (Mazel and Pessayrc, 1976). While the latter reaction might occur to some extent in vivo. it is highly unlikely to occur in an in vitro system restricted to microsomes and a NADPH-gcnerating system. Thus, covalent binding to microsomal proteins was probably the consequence of the formation of a chemically reactive metabolite of vinyl chloride which reacted with, and covalently bound to. microsomal proteins. In vivo, after ip administration of [,JC]vinyl non orctreaied / o . CuCl - / / / / SKF 525-4 / .1 Shsnotiaibilal DDT o ViflV1 * JuPt*oj|j(lt $ To ' ' '7s ifftiQfy IQ '0 OOO (J nmol IJ min 1 g ` F10. S, Correlation between loss of glutathione and irreversible binding to 10.000c supernatant proteins in vim'. The loss of hepatic glutathione after inhala tion of a 5",, vinyl chloride 95" t air atmosphere for I hr (Fig. 3) is plotteJ against the amount of vinyl chloride material that became irreversibly bound to lO.OlllV supernatant proteins 1 Table 2). The drawn line Is the least-squares regression line. 529 512 PESSAYRE et al. chloride to non-preireated rats, a radio generally hypothesized (Barbin et al., 1975; active material became irreversibly bound to Reynolds et al., 1975; Bolt et al., 1976; hepatic proteins; binding was markedly Kappus et al., 1976; Reynolds et al., 1976; decreased by CoCl; (Table 1). These findings Osterman-Golkar et al., 1977) that vinyl suggest that a chemically reactive metabolite chloride (chloroethylene) is oxidized by the is also formed in vivo. MFOS into vinyl chloride epoxide (chloro Covalent binding in extrahepatic organs. ethylene oxide); spontaneous rearrangement After ip administration of [IJCJvinyl chloride, of this epoxide leads to chloroacetaldehyde substantial amounts of metabolite were which is oxidized into chloroacetic acid. covalently bound to whole blood, kidney, Although both vinyl chloride epoxide and, and spleen proteins (Table 1), whereas no to a lesser extent, chloroacetaldehyde are detectable covalent binding occurred when chemically reactive metabolites, evidence has microsomes from these organs were incu been presented suggesting that in vivo bated with [lJC]vinyI chloride. These findings alkylation of proteins in mice was mainly due would suggest that the chemically reactive to the epoxide rather than to the aldehyde metabolite formed in the hepatocytes may (Osterman-Golkar et al., 1977). partly leave the liver and bind to proteins in Protective role of epoxide hydrase. This other tissues. However, it is not excluded inducible microsomal enzyme transforms that extrahepatic organs may also form some reactive epoxides into inactive dihydrodiols small amounts of the reactive metabolite that (Oesch, 1972). Addition of TCPO (0.5 mst) were undetected in our in vitro system. to the incubation mixture increased in vitro Protective role of glutathione. Inhalation of covalent binding to microsomal proteins vinyl chloride decreased hepatic glutathione (Table 2). TCPO has two effects; (1) It in control rats but not in CoCI,-treated decreases glutathione concentrations, and rats (Fig. 2), which suggests that glutathione (2) it inhibits epoxide hydrase (Oesch and was depleted by a metabolite. It has been Daly, 1972). It is unlikely that the effects of shown that vinyl chloride is metabolized to TCPO (Table 2) were related to reduced cysteine adducts and other sulfur-containing glutathione concentration in the incubate metabolites in non-pretreated rats (Green and because there is little glutathione left in Hath wav, 1975; Watanabe et at., 1976). washed microsomes and because addition of Several chemically reactive metabolites have diethylmaleate (5 m.vt) to the incubation been shown to covalently bind to. and thus mixture did not increase in vitro covalent deplete, hepatic glutathione (Mazel and binding to microsomal proteins of normal Pessayre, 1976). These observations suggest or of phenobarbiial-treated rats. Rather, it is that glutathione was depleted (Fig. 2) because likely that TCPO inhibited the inactivation it vvas consumed by a reactive metabolite of of vinyl chloride epoxide by epoxide hydrase vinyl chloride. (Kappus ct al.. 1976). This interpretation is Addition of exogenous glutathione de further supported by the observation that creased (Fig, 5), and removal of endogenous addition of TCPO to the incubation mixture glutathione by diethyl maleate increased increased binding much more with induced (Fig. 6). the in vitro covalent binding to microsomes. which have increased activities hepatic proteins. These findings are consistent of epoxide hydrase (Oesch. 1972), than with with the view that the chemically reactive microsomes from control rati (Table 2). metabolite of vinyl chloride may bind either Effects of various treatments on covalent to glutathione or to proteins and that binding binding, cytochrome P-450, and glutathione. to glutathione decreases the amount of Treatment of the animals with CoCI, or metabolite available for binding to proteins. SKF 525-A decreased in titro covalent Proposed metabolism of vinyl chloride. It is binding to hepatic proieini (Table 2) and the VINYL CHLORIDE ACT! VATION--INACTIVATION 513 loss of cytochrome P-450 and of glutathione (Fig. 3) after inhalation of vinyl chloride. These findings are consistent with the view that these treatments decreased the formation rate of the reactive epoxide and, thus, its binding to cytochrome P-450 and to hepatic glutathione. More complex effects were observed after treatment with DDT or phenobarbital. (I) Whereas these treatments increased cyto chrome P-450, they did not increase covalent binding to microsomal proteins measured in the absence of TCPO (Table 2). A similar observation was made by Kappus et at. (1976). This discrepancy might be understood if treatment with DDT or phenobarbital had induced both cytochrome P-450 and epoxide hydrase, resulting in both an increased formation rate and an increased inactivation rate of the reactive epoxide. Indeed, when the inactivation of the epoxide was inhibited by added TCPO, treatment of the animals with DDT or phenobarbital now markedly in creased covalent binding to microsomal proteins as compared to that in control rats (Table 2). (2) Whereas treatments with DDT or phenobarbital increased the destruction of cytochrome P-450, they decreased the deple tion of glutathione (Fig. 3). Similar observa tions have been made previously (Reynolds et al., 1975). This finding could be explained if cytochrome P-450 were destroyed by the epoxide it just forms or has just formed and if glutathione were depleted by only that fraction of the formed epoxide that has escaped inactivation by epoxide hydrase and has diffused in the cytosol; according to this view, cytochrome P-450 destruction would be related to the formation rate of the epoxide, while glutathione depletion would be related to the release rate of the epoxide in the cyto sol. In vitro covalent binding to microsomal proteins measured in the presence of the epoxide hvdrase inhibitor may be the best in vitro estimate for the formation rate of the epoxide. A striking correlation was found between this in vitro covalent binding and the loss of cytochrome P-450 after inhalation of vinyl chloride, in variously treated animals (Fig. 7). In vitro covalent binding to lO.OOOg supernatant proteins may be a good in vitro estimate for the release rate of the epoxide in the incubation mixture. A good correlation was found between this binding and the loss of glutathione after inhalation of vinyl chloride (Fig. 8). GENERAL COMMENTS This work confirms and extends the previously held view that vinyl chloride is activated in the liver into a chemically reactive epoxide that covalently binds to hepatic proteins and might be responsible for the hepatotoxic and carcinogenic effects of chronic inhalation of vinyl chloride (Kappus et al., 1976). In the past few years, it has been recognized that a vast array of chemicals, including a number of widely used drugs, is activated to unstable intermediates in rodents and probably in man (Mitchell and JoIIow, 1975; Mazel and Pessayre, 1976; Pessayre et al., 1977, 197S; Pessayre and Benhamou, 197S). Indeed the question now is not so much how these compounds may produce liver necrosis or cancer but rather why they do not do so more often. This study exemplifies several mechanisms that may protect the hepatoevtes: (I) Destruction of cytochrome P-450 may prevent further metabolism and toxicity; in induced animals, higher initial concentrations of cytochrome P-450 are associated with a higher destruction rate of cytochrome P-450, (2) Microsomal epoxide hydrase is closely associated with cytochrome P-450 and may inactivate epoxides shortly after they are formed (Oesch. 1972); induction of cytochrome P-450, which increases the rate of formation of the epoxide, is associated w ith induction of epoxide hydrase (Oesch. 1972), which in creases the inactivation rate of the epoxide. (3) Finally, the epoxide may bind to cwosolie glutathione which is offered as an alternative target. Eventually, only a tiny fraction of the formed epoxide does bind to macromolecules. 514 PESSAYRE ET AL. In induced animals, destruction of cyto chrome F-450 during inhalation of vinyl chloride was increased, whereas depletion of glutathione was decreased. These observa tions suggest the need for a compartmen talized analysis of the effects of the chemically reactive metabolite*As a possible interpreta tion of these findings, it is suggested that cytochrome P-450 may be destroyed by the epoxide it just forms or has just formed and that, accordingly, cytochrome P-450 destruc tion is related to the formation rate of the epoxide, whereas glutathione would be depleted by only that fraction of the formed epoxide which has escaped inactivation by microsomal epoxide Indrase and has diffused in the cytosol. REFERENCES AlLEMAND, H., PESSAVRE, D., DFSCaTOIRE, V., Degott, C., Feldmann, G., and Benhamov. J.-P. (1978). Metabolic activation of trichloroethylene into a chemically reactive metabolite toxic to the liver. J. Phonmia.lI. Exp. Ther. 204, 714-723. Barms, A.. Biusil, H.. Ckoisy. A.. 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