Document DMYVy54nN1m9xeJ8e0eXZGxZB

R&S 109639 r 310-MEDICAL RESEARCH DOCUMENT DESCRIPTION FORM 6368 69 _ 76 y '~t y G ^0 Duplicate in all cards:--?> year as-1961- File lumber [Right justify [Numeric only] Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA, preceeded by.one blank space 1______ y 20 -21AO 41 60 . 2-lVtLin(l5C,he.e.t- J~C- Lv5A5$^e -hi'tiiaomotta. u. 7JP 3 , (2___ 77 ' 78 Sub-Index Code 61 62 * pu }12" pT~ Title of Report: end with space-hyphen-hyphen-space. Follow with Index Terms, separated from each other with .comma-space. Avoid other punctuation; do not abbreviate. i -> 61 62 Source (Journal, Vol., Number, Pages,Date) 12 - C r - - Brief Summary 12 10 SUMMARY: 61 62.31 32 / 61 62 61 62 63 64 /f7f toxicology and applied pharmacology 49, 505-515 (1979) \ 7^ oop24e Formation and Inactivation of a Chemically Reactive Metabolite of Vinyl Chloride1 D. Pessayre,2 J. C. Wandscheer, V. Descatoire, J. Y. Artigou, AND J. P. BENHAMOU XJniti de Recherches de Physiopatkologie Hipatique (JNSERM), Hopital Beaujon, 92118 Clichy, France Received December 18,1978; accepted March 17,1979 Formation arid 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 ("C]vinyl chloride was incubated with hepatic microsomes, a l4C-Iabeled 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 7M50 and glutathione. Pretreatment of the animals with DDT or phenobarbital (1) in creased in vitro irreversible binding to microsomal proteins measured in tftp presence, but not in the absence, of TCPO and increased the in vivo loss of cytochrome 7*450 during inhala tion of vinyl chloride, but (2) decreased the in vitro irreversible binding to 10,000g super natant proteins and the in vivo loss of glutathione during inhalation of vinyl chloride. These results are consistent with the views that (1) vinyl chloride is activated by cytochrome P450 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 P45Q destruction is related to the formation rate of the epoxide, 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 (chloroethylene) 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 (Torkelson plastic (Berk er al., 1976). In workers et al., 1961; Lester et al., 1963; Viola et al., exposed to vinyl chloride, angiosarcomas of 1971; Maltoni and Lefemine, 1975; Prodan the liver and/or hepatic fibrosis have been et al., 1975). Several carcinogenic and/or hepatotoxic 1 This work was supported by Grant 021 R 07 from Facuhe de Medecine Xavier-Bichat (Universite de Paris VII), and ATP 58-78-90 from Institut National dc la Sante et de ia Recherche Mrfdtcale (INSERM). 1 Recipient of a fellowship from the Fonds d'Etudes du Corps Medical Hospitalier. compounds are transformed by the hepatic microsomal mixed-function oxidase system (MFOS) into chemically reactive metabolites that (t) covalently bind in vitro to micro somal proteins, (2) covalently bind in vivo 505 0OAI-0OgX/TO/09O50J-t i sozoo/o Copyright & 1979 by Academic Press, Inc- All right* of reproduction in any form reserved. Printed in Great Britain * ' -vyi- y' 506 PESSAYRE ET AL. . to tissue proteins, and (3) may destroy hepatic 10,000?; microsomal pellets were prepared by centri cytochrome .P-450 and deplete hepatic gluta fuging 3 ml of the 10,000? supernatant with 9.5 ml thione (Mitchell and Jollow, 1975; Mazel and Pessayre, 1976). In several reports, it has been shown that vinyl chloride may produce one 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?. or the other of the above-mentioned effects Transaminases, glutathione, cytochrome P-450. (Barbin et at., 1975; Reynolds et at., 1975; Scrum glutamic-pyruvic transaminase (SGPT) activity 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 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 et at., 1978). However, a global under method of Ellman (1959) in animals sacrificed at standing of the mechanisms for, and the 10-12 am. Microsomal cytochrome F-450 was 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 measured by the method of Omura and Sato (1964). Irreversible binding in vitro, [,4C]Vinyl chloride (specific activity, 1 mCi/mmol), labeled on the two carbon atoms and dissolved in methanol (I mmol/ ml), was purchased from Commissariat k l'Energie animals. Atomique, Saclay, France; its radiopurity, checked by gas-liquid chromatography, was higher than 99%. METHODS (uC]Viny! chloride (5 /tmol, 5 pCi) in 5 p\ of methanol was added, in 25-ml vials, to 1 ml of ice-cold 0.154 m Animals and treatments. Male Sprague-Dawley KCI, 0.01 m Na*- K*-phosphate buffer, pH 7.4, rats, weighing 160-200 g, were obtained from Charles containing microsomes or 10,000? supernatant . River France (Elbeuf, France). Rats were allowed from 125 mg "of liver and the following reduced water and food (Autoclave 113, UAR) ad libitum. nicotinamide adenine dinucleotide ph.osphate Some animals received one of the following pretreat (NADPH)-generating system; NADP (0.4?mol), ments: (1) cobaltous chloride (CoCIi -61-1,0), 35 mgj glucose 6-phosphate (fipmol), glucose 6-phosphate kg sc twice daily for 3 days; (2) 2-diethylaminoethyl- dehydrogenase (3 enzyme units), and MgClj (6 ^mol). 2,2-diphenylvalerate hydrochloride (SKF 525-A), 75 . The vials were capped and the mixture was incubated mg/kg, ip; (3) l,I,l-trichloro-2,2-bis(p-chlorophenyl) at 37 for 15 min in a Gallenkamp shaking incubator. ethane (DDT); 200 mg/kg ip; or (4) phenobarbetal, In- some vials, the NADPH-generating system was 100 mg/kg ip daily for 5 days. Pretreated animals were exposed to vinyl chloride or sacrificed (1) 18 hr after the last dose of CoClj, (2) 15 min after the single dose of SKF 525-A, (3) 8 days after the single dose of DDT, or (4) 24 hr after the omitted; in other vials, various substances were added; other vials were flushed with a 90% CO-10% Oj atmosphere, capped, and incubated. Radioactivity irreversibly bound to microsomal or 10,000? super natant proteins was measured exactly as previously last dose of phenobarbital. Six rats at a time were described (Allemand et al,, 1978); Proteins 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 of exposure; it must be noted that the proportion of vinyl chloride in the precipitated with trichloroacetic acid (TCA), dried, washed three times with TCA, and repeatedly ex tracted with various solvents (methanol, heptane, and ether); no detectable radioactivity was found in the three last solvent phases; UC material remaining on the proteins was then counted in an Intertechnique ABAC SL 40 scintillation counter. atmosphere inhaled by the rats did not reach 5% Irreversible binding in vivo. [,4C]Viny( chloride immediately. (6,25 mg, 100/miol, 100 pC\) was administered ip in Preparation of tissue homogenates and tissue frac 250 pt of methanol; rats were sacrificed 4 hr later; tions. Rats were stunned; blood was drawn from the tissue fragments were homogenized in 3 vol of inferior vena cava; the liver, the kidneys, the spleen, 0.154 m KCI, 0.01 m Na'K`-phosphate buffer, and the sartorius muscle were removed. Blood pH 7.4, l4C-Labe!ed material irreversibly bound to samples and tissue fragments were homogenized with tissue proteins was measured as previously described a glass-Teflon Potter-EIvehjem homogenizer in 3 vol (Allemand et al,, 1978) and summarized above. of 0.154 m KCI, 0.01m Na'iC'-phosphate buffer, Statistical analysis. Student's / test (two-tailed) for pH 7.4. The crude homogenates were centrifuged at independent data was used as a test of significance IM. 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. In 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 CoCI2, 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 P-450 decreased linearly with time during inhalation of vinyl chloride in control rats (Fig. 1); the decrease was negligible or lower in rats treated with CoCl2 or SfCF 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 CoCI2, SKF 525-A, DDT, or phenobarbital (Fig. 3). In vivo binding. After administration of [,4C]vinyl chloride, a I4C-labeled material was irreversibly bound to whole tissue pro teins (Table 1); 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 CoCl2 than in control rats (Table 1). In vitro binding. When (14C]vinyl chloride was incubated under air with hepatic microsomes and a NADPH-generating system, a radioactive material- became irreversibly Effrtfur* to 5% ehtond* hours Fig. 1. Effects of vinyl chloride inhalation on hepatic microsomal cytochrome P-450. Cytochrome P-450 concentrations were measured in animals sacrificed after various lengths of exposure to a 5% vinyl chloride-95 % air atmosphere. Results are means SD for at least six rats. E*po%ur to $ % vinyl chlorid* hours Fig. 2. Effects of vinyl chloride inhalation on hepatic glutathione. Glutathione concentrations were measured in animals sacrificed after various lengths of exposure to a 5% vinyl chloride-95 % air atmo sphere. Results arc means SD for at least six rats. 508 PESSAYRE ET AL. bound to microsomal proteins (Fig. 4), whereas no detectable binding occurred under identical conditions with microsomes from blood, kidney, spleen, or muscle (data 15 7W CaCl} SKF MM U)-A prttrvatrt tarbttai Fto. 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 /M50 concentra tion in rats not exposed, and in rats exposed, to a 5% vinyl chloride-95 % air 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 1 hr. not shown). Binding to hepatic microsomal proteins was negligible when the NADPHgenerating system was omitted (Fig. 4); binding was decreased when the incubation was made under a 90% CO-10 % Oj atmos phere or in the presence of SKF 525-A (Fig. 4). Similar findings were obtained when [I4C]vinyl chloride was incubated with hepatic 10,000g supernatant and the re labeled material irreversibly bound to 10,000g supernatant proteins was measured (Fig. 4). In vitro binding to hepatic microsomal proteins was decreased by addition to the incubation mixture of glutathione or cysteine, but not 5-methyl glutathione or glycine (Fig. 5); it was not increased by diethylmaleate (5 raM) (data not shown). Addition of diethylmaleate (5 mM) 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). Addition of 0.5 mM 1,1,1-trichloropropene 2,3-oxide (TCPO) to the incubation mixture 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 treated with DDT or phenobarbital, respec tively (Table 2). . Treatment of the animals with CoClj or SKF 525-A decreased (1) in vitro binding to 10,000g supernatant proteins, (2) in vitro TABLE 1 Vinyl Chloride Material Irreversibly Bound to Whole Tissue Proteins in VU)<s` Vinyl chloride material irreversibly bound to proteins (nmol-4 hr"*`g tissue"1) Liver Blood Kidney Spleen Muscle Control rats CoCVTreated rats 6910 165* 244 53* 22!0 5 2* 22 5 4 1* 2.0 0.9 0.30.1* * t'*C]Vinyl chloride (100 pmol; 100 pCi) in 250 of methanol was administered ip to control rats and to rats treated with CoCl,; 4 hr later, the animals were sacri ficed and the amount of "C-Iabelcd material irreversibly bound to whole tissue proteins was measured; results are the meansSD of four rats. `Significantly different from that in control rats,p<0.00l. bin wit bin the R&S 109644 uer- ene ure ;rovas ing ats ec- or : to itro Bosol lyitim Fro. 4. Irreversible binding of a vinyl chloride material to proteins in vitro. In the basal system, ["CJvinyl chloride (5 mM) was incubated under air with microsomes or 10,000supernatant from 125 mg of liver from non-pretreated rats and with a NADPH-gencrating system; in some flasks, the NADPH-generating system was omitted; other flasks were incubated under a 90% CO-IO% 02 atmosphere; in other flasks. 5KF 525-A (5 mM) was added to the incubation mixture; After 15 min of incubation, the amount of vinyl chloride material irreversibly bound to microsomal or 10,000^ supernatant proteins was measured; columns and bars represent means and SD for three experiments. a from that in tho baiol ,y,t m. P<O.COl 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 mM) 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 mM 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 m.M TCPO (Table 2). Treatment of the animals with DDT or with phenobarbital (1) decreased in vitro binding to I0,000g 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 /'-450 after inhalation of vinyl chloride for 18 hr was (1) not correlated (r = 0,077) with ! * ' =]1 ;)' ti fi 510 PESSAYRE ET AL, TABLE 2 Vinyl Chloride Material Irreversibly Bound to Protstns in Vitro` Vinyl chloride material irreversibly bound to proteins (nmol-15 min-l-g liver-1) 10,000^ supernatant Microsomes without added TCPO* Microsomes with added TCPO* Control rats CoCl,-Treated rats SKF 525-A-Treated rats DDT-Treated rats Phenobarbital-Treated rats 14 1 22* I02* 81* 8 2* 24+7 iy 13 44 235 258 33 3* 92* 189* 546f* 75 26*-' ` [,4C]Vinyl chloride (5 mM) was incubated under air with 10,000,? supernatant or microsomes from 125 mg of liver and with a NADPH-generating system for 15 min; the amount of vinyl chloride material irreversibly bound to 10,000? supernatant or microsomal proteins was measured; results arc meansSO of at least sis rats. *2//l of acetone either pure or containing I,I,l-trichloropropene-2,3-oxide (TCPO) (final concentration in the incubate, 0.5 mM) was added to the incuba tion mixture. * Significantly different from that bound to microsomes without added TCPO, p<0.05. * Significantly different from that in control rats, p < 0.05. _ in vitro binding to 10,000g 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 (1) well correlated (r -- 0.891) with in vitro binding to 10,000g 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 [MC]vinyl 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 Kappus et al. (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 with 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 macromolecule may result from two main reactions; (I) the spontaneous reaction between a chemically reactive compound and an already formed macromolecule, or (2) the % b- I X t-., -1 Fit bind! super [,4C]' with non-p systen added glutat of vin` superr bars r enzyr comp (Maz. reacti it is 1 systen NAD bindir the c cherni* chloru bound i v; . negnd to 'hows [id to 'omal '.ibited imilar t.'i al. t the oteins loride uy the VINYL CHLORIDE ACTIVATION-INACTIVATION afi-< 511 a3- aoJ 100 Vinyl tMoftd# mottrinl inmniWf bound to microsomal pnrtpn* jmmHS rm<r' % Fig. 7. Correlation between loss of cytochrome P-450 and irreversible binding to microsomal proteins in citro. The loss of hepatic microsomal cytochrome so P-450 after inhalation of a 5 % vinyl chloride-95 V, air atmosphere for IS hr (Fig. 3) is plotted against the amount of vinyl chloride material that became irre versibly bound to microsomal proteins when 5 mM 1,1,1 -trichloropropene-2,3-oxidc (TCPO) was added to the incubation mixture (Table 2). The drawn line is Qasei *yt#ro With mdiottl*hoyUl- the least-squares regression line. Fig. 6. Effects of diethylmaleate on the irreversible binding of a vinyl chloride material to IQ.OOOg supernatant proteins in citro. In the basal system, ('`CJvinyl chloride (5 mM) was incubated under air with 10,000" supernatant from 125 mg of liver from non-pretreated rats and with a NADPH-generating system; in other flasks 5 mM diethylmaleate was added. After 15 min of incubation, the amount of glutathione in the incubation mixture and the amount of vinyl chloride material irreversibly bound to lO.OOOg 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 macromolecule (Mazel and Pessayre, 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-generating 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 [,4C]vinyl Vinyl chlonrf* maiaml irreversibly bound to 10.000 supernatant proteins nmol 15 nun*' g livir '* Fig. 8. Correlation between loss of glutathione and irreversible binding to lO.OOOg supernatant proteins in citro. The loss of hepatic glutathione after inhala tion of a 5% vinyl chloridc-95< air atmosphere for 1 hr (Fig. 3) is plotted against the amount of vinyl chloride material that became irreversibly bound to 10,000^ supernatant proteins (Table 2). The drawn line is the least-squares regression line. R&S 109646 512 PESSAYRE ET AL. chloride to non-pretreated rats, a radio active material became irreversibly bound to hepatic proteins; binding was markedly decreased by CoCl2 (Table 1). These findings suggest that a chemically reactive metabolite is also formed in vivo. Covalent binding in extrahepatic organs. After ip administration of [14C]vinyl chloride, substantial amounts of metabolite were covalently bound to whole blood, kidney, and spleen proteins (Table I), whereas no detectable covalent binding occurred when microsomes from these organs were incu bated with [l4C]vinyl chloride. These findings would suggest that the chemically reactive metabolite formed in the hepatocytes may partly leave the liver and bind to proteins in other tissues. However, it is not excluded that extrahepatic organs may also form some small amounts of the reactive metabolite that were undetected in our in vitro system. Protective role of glutathione. Inhalation of vinyl chloride decreased hepatic glutathione in control rats but not in CoCl2-treated rats (Fig. 2), which suggests that glutathione was depleted by a metabolite. It has been shown that vinyl chloride is metabolized to cysteine adducts and other sulfur-containing metabolites in non-pretreated rats (Green and Hathway, 1975; Watanabe et al., 1976). Several chemically reactive metabolites have been shown to covalently bind to, and thus deplete, hepatic glutathione (Maze! and Pessayre, 1976). These observations suggest that glutathione was depleted (Fig. 2) because it was consumed by a reactive metabolite of vinyl chloride. Addition of exogenous glutathione de creased (Fig. 5), and removal of endogenous glutathione by diethyl maleate increased (Fig. 6), the in vitro covalent binding to hepatic proteins. These findings are consistent with the view that the chemically reactive metabolite of vinyl chloride may bind either to glutathione or to proteins and that binding to glutathione decreases the amount of metabolite available for binding to proteins. Proposed metabolism of vinyl chloride. It is generally hypothesized (Barbin et al., 1975; Reynolds et al., 1975; Bolt et al., 1976; Kappus et al., 1976; Reynolds et al., 1976; Osterman-Golkar et al., 1977) that vinyl chloride (chloroethylenc) is oxidized by the MFOS into vinyl chloride epoxide (chloroethylene oxide); spontaneous rearrangement of this epoxide leads to chloroacetaidehyde which is oxidized into chloroacetic acid. Although both vinyl chloride epoxide and, to a lesser extent, chloroacetaidehyde are chemically reactive metabolites, evidence has been presented suggesting that in vivo alkylation of proteins in mice was mainly due to the epoxide rather than to the aldehyde (Osterman-Golkar et al., 1977). Protective rote of epoxide hydrase. This inducible microsomal enzyme transforms reactive epoxides into inactive dihydrodiols (Oesch, 1972). Addition of TCPO (0.5 ihm) to the incubation mixture increased in vitro covalent binding to microsomal proteins (Table 2). TCPO has two effects: (1) It decreases glutathione concentrations, and (2) it . inhibits epoxide hydrase (Oesch and Daly, 1972). It is unlikely that the effects of TCPO (Table 2) were related to reduced glutathione concentration in the incubate because there is little glutathione left in washed microsomes and because addition of diethyl maleate (5 ihm) to the incubation mixture did not increase in vitro covalent binding to microsomal proteins of normal or of phenobarbital-treated rats. Rather, it is likely that T^PO inhibited the inactivation of vinyl chloride epoxide by epoxide hydrase (Kappus et al., 1976). This interpretation is further supported by the observation that addition of TCPO to the incubation mixture increased binding much more with induced microsomes, which have increased activities of epoxide hydrase (Oesch, 1972), than with microsomes from control rats (Table 2). Effects of various treatments on covalent binding, cytochrome P-450, and glutathione. Treatment of the animals with CoCl2 or SKF 525-A decreased in vitro covalent binding to hepatic proteins (Table 2) and the loss (Fig. -i Thes that rate bindi gluta Mi treat! J Wher chror bind! the a I; [I obser (1976 if tree indue hydra forma rate o inacti' added protei. (Table ! or phe cytoch tion oi tions 1 et al., if cyto epoxid if glut fractio: escapee has difi view, i be relat while g It to the r | sol. In i protein: epoxide in vitro epoxide betweer. loss of 975; 976; 976; inyl the oronent lyde icid. and, are - has vivo due iiyde This ,'rms Jiols itim) vitro icins i) It and and ts of uced > bate ft in in of ation alent irmal , it is ation drase on is that xture Juced vities with alent Hone. 1. or alent d the VINYL CHLORIDE ACT1VATION-INACTIYATION 513 loss of cvtochrome P-450 and of glutathione vinyl chloride, in variously treated animals (Fig. 3) after inhalation of vinyl chloride. (Fig. 7). In vitro covalent binding to 10,000g These findings arc consistent with the view supernatant proteins may be a good in vitro that these treatments decreased the formation estimate for the release rate of the epoxide in rate of the reactive epoxide and, thus, its the incubation mixture. A good correlation binding to cytochrome P-450 and to hepatic was found between this binding and the loss glutathione. of glutathione after inhalation of vinyl More complex effects were observed after chloride (Fig. 8). treatment with DDT or phenobarbital. (1) Whereas these treatments increased cyto chrome .P-450, they did not increase covalent GENERAL COMMENTS binding to microsomal proteins measured in This work confirms and extends the the absence of TCPO (Table 2). A similar previously held view that vinyl chloride is observation was made by Kappus et al. activated in the liver into a chemically (1976). This discrepancy might be understood reactive epoxide that covalently binds to if treatment with DDT or phenobarbital had hepatic proteins and might be responsible for induced both cytochrome .P-450 and epoxide the hepatotoxic and carcinogenic effects of hydrase, resulting in both an increased chronic inhalation of vinyl chloride (Kappus formation rate and an increased inactivation et al., 1976). In the past few years, it has been rate of the reactive epoxide. Indeed, when the recognized that a vast array of chemicals, inactivation of the epoxide was inhibited by including a number of widely used drugs, added TCPO, treatment of the animals with is activated to unstable intermediates in DDT or phenobarbital now markedly in rodents and probably in man (Mitchell and creased covalent binding to microsomal Jollow, 1975; Mazel and Pessayre, 1976; proteins as compared to that in control rats Pessayre et al., 1977, 1978; Pessayre and (Table 2). (2) Whereas treatments with DDT Benhamou, 1978). Indeed the question now or phenobarbital increased the destruction of is not so much how these compounds may cytochrome .P-450, they decreased the deple produce liver necrosis or. cancer but rather tion of glutathione (Fig. 3). Similar observa why they do not do so more often. This study tions have been made previously (Reynolds exemplifies several mechanisms that may et al., 1975). This finding could be explained protect the hepatocytes: (1) Destruction of if cytochrome P-450 were destroyed by the cytochrome P-450 may prevent further epoxide it just forms or has just formed and metabolism and toxicity; in induced animals, if glutathione were depleted by only that higher initial concentrations of cytochrome fraction of the formed epoxide that has P-450 are associated with a higher destruction escaped inactivation by epoxide hydrase and rate of cytochrome P-450. (2) Microsomal has diffused in the cytosol; according to this epoxide hydrase is closely associated with view, cytochrome P-450 destruction would cytochrome P-450 and may inactivate be related to the formation rate of the epoxide, epoxides shortly after they are formed while glutathione depletion would be related (Oesch, 1972); induction of cytochrome to the release rate of the epoxide in the cyto P-450, which increases the rate of formation sol. In vitro covalent binding to microsomal of the epoxide, is associated with induction of proteins measured in the presence of the epoxide hydrase (Oesch, 1972), which in epoxide hydrase inhibitor may be the best creases the inactivation rate of the epoxide. in vitro estimate for the formation rate of the (3) Finally, the epoxide may bind to cytosolic epoxide. A striking correlation was found glutathione which is offered as an alternative between this in vitro covalent binding and the target. Eventually, only a tiny fraction of the loss of cytochrome P-450 after inhalation of formed epoxide does bind to macromolecules. 514 PESSAYRE ET AL. In induced animals, destruction of cyto chrome /M50 during inhalation of vinyl chloride was increased, whereas depletion of glutathione was decreased. These observa tions suggest the need for a compartmen Kappus, H., Bolt, H. M,, Buchter, A,, and Bolt, W. (1976). Liver microsomal uptake of [l4C]vinyl chloride and transformation to protein al'->lating metabolites in vitro. Toxicol. Appl. Pharmacol. 37, 461-471. Lester, D,, Greenberg. L. A., and Adams, W. R. talized analysis of the effects of the chemically reactive metabolite. As a possible interpreta tion of these findings, it is suggested that cytochrome /M50 may be destroyed by the epoxide it just forms or has just formed and (1963), Effects of single and repeated exposures of humans and rats to vinyl chloride. Amer. Ind. Hyg. Ass. J. 24, 265-275. Maltoni, C., and Lefemine, G. (1975). Carcino genicity bioassays of vinyl chloride: Current results Ann. N. Y. Acad, Scl. 246, 195-218, that, accordingly, cytochrome P-450 destruc Mazel, P., and Pessayre, D. (1976). Significance of 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 hydrase and has diffused rtabolite-mediated toxicides in the safety evalua te on of drugs and chemicals. In Advances in Modern Toxicology, Vol. 1, Part 1: New Concepts in Safety Evaluation (M. A. Mehlman, R. E. Shapiro, and H. Blumenthal, cds.), pp. 307-343, Hemisphere Publ. Co., New York. in the cytosol. REFERENCES MrTCHELL, J. R., AND Jollow, D. J. (1975). Metabolic activation of drugs to toxic substances. Gastro enterology 68, 392-410. Oesch, F. (1972). Mammalian epoxide hydrases: Allemand, H,, Pessayre. D,, Descatoire, V., Degott, C. Feldmann, G., and Benhamou, J.-P. (1978). 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