Document bVwQXx6GNkGmZRvGzN4mVgMO

^931 Chem.-Biol. Interaction*. 17 (1977) 137--150 Elsevier/North'Holland Scientific Publishers, Ltd. 137 THE CHEMISTRY AND BIOGENESIS OF THE S-CONTAINING METABOLITES OF VINYL CHLORIDE IN RATS T. GREEN and D.E. HATHWAY Imperial Chemical Industrie* Limited, Centra/ Toxicology Laboratory. Alderley Park. Cheshire SK10 4TJ (Great Britain) (Received October 20th, 1976) (Accepted January 25th, 1977) SUMMARY - In order to determine whether vinyl chloride yields chloroethylene oxide in vivo, the biogenesis of the various urinary S-containing metabolites in rats has been investigated. jV-Acetyl-S-(2-hydroxyethyl)cysteine is a major vinyl chloride metabolite in rats, but according to the method of protective esterification that is used, so either Ar-acetyl-S-(2-chloroethyl)cysteine or N-acetyi-S-(2-hydroxyethyl)cysteine may be isolated from the body fluids. N-Acetyl-5-vinylcysteine is a second related metabolite. These S-containing vinyl chloride metabolites are not mutagenic in S. typhimurium. Neutral methanol methylates N-acetyl-S(2*hydroxyethyl)cysteine. N-Acetyl-S-(2-methoxyethyl)cysteine plus N-acetyliS-vinylcysteine degrade to give the volatile S-(2-methoxyethyl)(prop-l or 2-enyl)sulphide. Administration of several vinyl chloride metabolites and closely related compounds to rats shows that chloroacetaldehyde and S-(carboxymethyl)cysteine, but not chloroacetic acid, lie on a pathway or pathways connect ing vinyl chloride with thiodiglycollic acid. The fact (a) that chloroacetal dehyde affords both thiodiglycollic acid and N-acetyl-S-(2-hydroxyethyl)- cysteine in the animal and (b) that S-(carboxymethyl)cysteine has been iden tified amongst the hydrolytic products from an hepatic extract prepared from vinyl chloride-treated animals is consistent with the formation of chlo roacetaldehyde, and with the reaction of chloroethylene oxide or chloro acetaldehyde with glutathione in the presence of a glutathione S-epoxide transferase to give the identified S-containing metabolites. '/ *7//77 Abbreviations: GC, gas chromatograph. RSV 0012134 138 INTRODUCTION The way in which various S-containing metabolites of vinyl chloride are formed in vivo ought to provide evidence for the epoxidation which has been suggested by in vitro studies [1--4]. This supposition has caused us to inves tigate again the derivation of these substances, because in a previous paper [5], where the Fischer--Speier esterification with butan-l-ol had been used for stabilising the S-containing vinyl chloride metabolites, it became appar ent (see Ref. 6) that such derivative formation may be less suitable for pro tecting these substances in comparison with amino acids, and, e.g. 2-chloroethyl sulphides would be expected to be highly reactive in water [7]. The present paper describes the results obtained and their possible inter pretation. Poster material (no. 04-7-433) about some preliminary results was exhibited at the 10th International Congress of Biochemistry that was held in Hamburg during July 25--31, 1976. MATERIALS AND METHODS Chemicals Bromoethanol and L-cysteine were condensed with Na and liquid NH3 to give S-(2hydroxyethyl)-L-cysteine [8], which crystallized from 92% ethanol with a m.p. of 202*C (decomp.). (Carson and Wong [8] gave m.p. of 189-- 189.5*C). (Found: C, 36.5; H, 6.8; N, 8.4; S, 19.4%; CsHmOjNS requires C. 36.4; H, 6.7; N, 8.5; S, 19.4%). 5-(2-Hydroxyethyl)-L-cysteine on heating in reagent hydrochloric acid (38%) gave 5-(2-chloroethyl)-L-cysteine hydrochlo ride (m.p. 182*C), and from these 2 compounds, N-acetyliS-(2-hydroxyethyl)-L-cysteine and JV-acetyl-S-(2-chloroethyl)-L-cysteine were prepared [8]. The mass spectra of all 4 substances were in agreement with the pro posed structures, and that of the O-methyl ester derivative ofAr-acetyl-5-(2hydroxyethyi)-L-cysteine is shown in Fig. 1, but that of the O-butyl ester of AT-acetyliS-(2-chloroethyI)-L-cysteine was recorded previously [5].' S-vinyl-L-cysteine was prepared from trichloroethylene by 2-stage synthe sis: trichloroethylene and L-cysteine were condensed with Na and liquid NHj to give S-(l,2-dichlorovinyl)cysteine, which was dechlorinated with Alamalgam to give S-vinyl-L-cysteine [9]. Without further purification, the crude S-vinyl-L-cysteine was acetylated in boiling acetic anhydride. The structure of W-acetyl-S-vinyl-L-cysteine was confirmed by the mass spectrum, and that of the O-methyl ester derivative is shown in Fig. 1. S-Carboxymethyl-L-cysteine was prepared [10] from chloroacetic acid plus L-cysteine hydrochloride, and the structure of the crystalline product (m.p. 175--176*C, decomp.) was confirmed by mass spectrometry. Chloroethylene oxide was prepared pure by the method of Rannug et al. [11] *. Purity was assessed by gas chromatography, by which chloroethylene * Under chemical conditions, chloroethylene oxide hat been found to react with cysteine to gi** 4-carboxy-2-chloromethy!thi2olidine, thus involving intermediate formation of S-(2-chloro-l-hydroxyethyl)cysteine but it is very unlikely that the analogous intermedi ate would have been formed from glutathione in vivo in the presence of glutathione S-epoxide transferaae, namely S-<2-hydroxyalkyl)glutathione alkyl-epoxide-lyase (E.C. 4.4.1,71. RSV 0012135 i 100 --i SO-i 60- S 5 40 - Ct OOCHi3, * hOCHjCMjSCHjCH NHCOCHj 139 20M-COOCHj ill iJl i }irl | M-HjO M-H^O , -CHjOH *i*T r'lTr n T rVri n fi i i i i--m 100 200 l 0 l 100 -t 80 60 2 40- COOCH CHj'CHSCHjCH NHC0CH, M* 203 M-C00CH 20- M*CH^OH M* i i i i I i [ i i i r~ r~t i i i i r 100 200 Fig. 1. Mass spectrum of: (a) the O-methyl ester of N-acetyl-S-(2-hydroxyethyl) cysteine; (b) the O-methyJ ester of Ar*acetyl-5-vinylcysterne. oxide was readily separable from its chloroacetaldehyde transformation product and from the ethylene oxide starting material. In the mass spectrom* eter, chloroethylene oxide is converted into chloroacetaldehyde. RSV 0012136 140 Radioactive chemicals (14C]vinyl chloride with a specific activity of 0.46 mCi/mmole, and with a radiochemical purity exceeding 99.0% was synthesized as previously outlined [5]. L-[U-,4C]cysteine hydrochloride with a specific activity of 24.5 mCi/ mmole was obtained from the Radiochemical Centre, Amersham, Bucks. (Great Britain). Experiments with animals Adult male rats (approx. 2 months old, 200 g body weight) were used [Alderley Park strain (Wistar-derived), specific pathogen-free], and kept on a standard pellet diet. For the purpose of identifying urinary metabolites [5], groups of 4 rats were administered intragastrically. (a) [14C]vinyI chloride (100 mg/kg; 10 ^Ci) in com-oil solution. (b) Chloroacetaldehyde (50 mg/kg) as an aq. soln. (c) S-(2-hydroxyethylFL-cysteine (500 mg/kg) as an aq, soln. (d) S-(carboxymethyl)-L-cysteine (250 mg/kg) in com-oil suspension, and a 24-h collection of urine was made. Control animals were used in experi ments (b)--(d). (e) Two rats were each given repeated daily i.p. injections of an aq. soln. of L-[U-l4C]cysteine hydrochloride for 5 days (total dose, approx. 50 pCi). 30 min after the final injection, each animal was given a single dose of vinyl chloride (100 mg/kg, in corn-oil solution by stomach tube, and the 24-h urine was collected. (f) Two rats were each given a single intragastric dose of [ l4C]vinyl chloride (450 mg/kg; 15 ^Ci) as a corn-oil solution. The animals, which were killed by cervical dislocation after 45 min, were dissected rapidly and their livers removed. Measurement of radioactivity An automated and computerized Intertechnique Model SL30 Liquid Scin tillation Spectrometer was used for measurement of >4C, making use of stan dard channels-ratio quench-correction curves. Liquid samples were mixed with standard scintillator and radio-assayed direct. Systematic separation of the urinary metabolites into fractions of chemically similar substances Urine from each group of rats, (a)--(e), was separated into 3 fractions by anion-exchange chromatography in a manner analogous to that described by Green and Hathway [5]. The 3 N acetic acid fractions and the 3 N HC1 frac tions were evaporated separately to dryness under reduced pressure, and the different residues were methylated with an ethereal solution of diazometh ane. Methanolic solutions of the resulting O-methyl esters were analysed in the gas chromatograph--mass spectrometer system and by mass fragmentom- RSV 0012137 141 etry. The following objectives were'pursued with the various residues from the different groups of rats (a)--<e). (a) Both the 3 N acetic acid and 3 N HC1 fractions were investigated for [,4C] vinyl chloride metabolites. (b)The 3 N acetic acid fraction was analysed by GC--mass spectrometry, as well as by mass fragmentometry, for the jV-acetyl-S'-(2-hydroxyethyI)cysteine metabolite (of vinyl chloride), and the 3 N HC1 fraction was investigated for the presence of thiodiglycollic acid and chioroacetic acid by GC--mass spectrometry. (c) and (d) The 3 N HC1 fractions were analysed quantitatively for thiodi glycollic acid by GC--mass spectrometry. (e) In the experiment in which unlabelled vinyl chloride was dosed to rats in which the cysteine--cystine pools had been labelled adequately with l4C, the 3 N HC1 fraction was examined in the GC--mass spectrometer for the presence of the thiodiglycollic acid metabolite and in the gas chromatograph for apeak of [MCJthiodigiycoilic acid. Sysfemafic separation of the hepatic metabolites into fractions of chemically similar substances The combined livers from the 2 animals (f) (see above) were homogenized in 10 times the volume of extracting solution, which was prepared fresh by mixing 3 vols. of ice-cold ethanol with 1 vol. of ice-cold KH2P04 buffer (24 mmole; pH 5.5) containing EDTA (0.1 mmole) [12]. The resulting homoge nate was centrifuged at 10 000 g in a refrigerated head for 15 min, and the supernatant was removed and evaporated under reduced pressure to 5 ml. This concentrate, which contains glutathione and glutathione-related sub stances, was adjusted to pH 10--11 and applied to a column of IRA-410 anion-exchange resin in the CHjCO: cycle, which was successively washed with deionized water (500 ml) and eluted with 3 N acetic acid [5]. Bulked fractions containing significant amounts of >4C were evaporated to dryness under reduced pressure, and a solution of the residae, which was formed by heating in 0.4 ml of formic acid (90%) for 5 min at 100C, was mixed with 4 ml of 2 N HC1, and heated for 2 h at 100 C [13]. An evaporate, prepared from the resulting hydrolysate, was successively esterified with diazomethane and acylated with trifluoroacetic anhydride in methylene chloride solu tion. The derivativized sample was examined both by means of GC--mass spectrometry and by mass fragmentometry. Gas chromatography Fractions containing radioactive metabolites were examined with a Pye Model 104 instrument that was equipped with flame-ionization detection and that was coupled to an E.S.I. Nuclear 504 Radiogas detector. The col umn effluent was split in the ratio of 10 : 1 between the Radiogas detector and the flame-ionization detector. This gas chromatograph was fitted with glass columns (1.5 m long X 4 mm internal diameter), which were packed either with 6% (w/w) of OV-101 on Gas Chrom Q (80--100 mesh size) or RSV 0012138 142 Fig. 2. Mass fragmentogram of the O-methyl ester of N-acetyl-S-(2-hydroxyethyl)cysteine from the urine of chioroacetaldehyde-treated rats (the gains from m/e 144, m/e 162 and m/e 203 are in the ratio 1:10: 10). A represents the injection point for a methanolic solution of the standard sample and B that for the 3 N acetic acid fraction (for details, see Materials and Methods, derived from urinary material. The iatLer shows ail 3 ions with identical relative intensities and retention time as the standard. RSV 0012139 143 Fig. 3. Mut fragmentogram of the O-dimethyl ester of the N-trifluoroacetyl derivative of S*(carboxymethyl)cysteme from the urine of chloroacetaldehyde-treated rats (the gains for m/e 393, m/e 271 and m/e 244 are in the ratio 1 ; 1 : 1). A represents the infection point for a solution of the standard sample and B that for the 3 N acetic acid fraction (for details, see Materials and Methods) derived from urinary material. The latter shows all 3 ions with identical relative iatensitiea and retention time as the standard. RSV 0012140 144 with 10% (w/w) of SP2330 on Supeleoport (100--120 mesh size). All of the columns were operated at a 30 ml/rain flow rate of a (95 : 5 v/v) A--CO, mixture. Radioactive peaks were located, and the corresponding samples were anal ysed by GC--mass spectrometry, using the same columns under identical operating conditions. Fractions containing unlabelled metabolites were examined directly by GC--mass spectrometry, using the previously-described columns. Fractions of chloroethylene oxide were tested in the gas chromatograph by using a column (2.7 m long x 2 mm internal diameter) which had been packed with 20% (w/w) of OV-275 on Chromosorb W-AW and which was run at 50 C at 30 ml/min flow rate of He. Under these conditions, chloro ethylene oxide had a retention time of 2.2 min, chloroacetaldehyde of 3.6 min and ethylene oxide of 0.5 min. GC--mass spectrometry An LKB2091 GC--mass-spectrometer system was used for the E.I. spectra and a Dupont 21-491B GC--mass spectrometer system for C.I. spectra. Mass fragmentometry In the case of the methyl ester of /V-acetyl-S-(2-hydroxyethyl)cysteine (Fig. 2), 3 selected ions of known m/e value were m/e 203 corresponding to M--HjO, m/e 162 corresponding to M--COjCHj and m/e 144 corresponding to M--HjO--COjCHj. The 6% OV-101 column was used and run at 195C. Under these conditions, the methyl ester of /V-acetyl-S-(2-hydroxyethyl>cysteine had a retention time of 5.6 min. In the case of the dimethyl ester of the N-trifluoroacetyl derivative of S-(carboxymethyl)cysteine (Fig. 3), the selected ions were m/e 303 corre sponding to the molecular ion, m/e 271 corresponding to M--CH3OH and m/e 244 corresponding to M--C02CH3. The column, used in the gas chroma tograph, was one (2.7 m longx 2 mm internal diameter) which was packed with 20% (w/w) of OV-275 on Chromosorb W-AW and which was run at 250C. Under these conditions, the dimethyl ester of the /V-trifluoroacetyl derivative of .S-(carboxymethyl)cysteine has a retention time of 5.4 min (Fig. 3). Mutagenicity test The mutagenic potential of $-(2-hydroxyethyl)cysteme and of S-(2-chlo- roethyl)cysteine was assessed by the method of Amesetal. [14] using S. typhimurium strains TA1535, TA1538, TA98 and TA100. RESULTS AND DISCUSSION jV-Acetyl-S-(2-hydroxyethyl)cysteine is a major vinyl chloride metabolite in rats, but dependent upon the method of derivative formation that was used so either A/-acetyl-5-(2-chloroethyl)cysteine (b) (Fig. 4) or jV-acetyl-S- RSV 0012141 ci CHINHAclCOjH 145 Fig. 4. Scheme for the interrelationship of some S-cont*ining vinyl chloride metabolities. (2-hydroxyethyl)cysteine (a) wasisolated from the body fluids of vinyl chlo ride-treated animals. Thus, e.g. the O-methyl ester of Ar-acetyl-5-(2-chloroethyl)cysteine (b) was obtained if the Fischer--Speier reaction with methanol was employed for the purpose of esterification, whereas the O-methyl ester of N-acetyl-S*(2-hydroxyethyl)cysteine (a) was produced if diazomethane was used instead. The mass spectrum of the O-methyl ester of Ar-acetyliS-(2chloroethyl)cysteine (b) was identical with that of the authentic substance [8]. It is noteworthy that the molecular ion (M* = 239) was absent from the conventional mass spectrum. Similarly, the mass spectrum (Fig. la) of the O-methyl ester of #-acetyl-S-(2-hydroxyethyl)cysteine (a) (Fig. 4) was iden tical with that of authentic material (8], and again, the molecular ion (M* = 221) was absent from the conventional mass spectrum, but the (M + 1)* ion could be recognized in the mass spectrum, when the chemical ionization source was used in the GC--mass spectrometer system. Furthermore, treat ment of the O-methyl ester of Ar-acetyl-5-(2-hydroacyethyl)cysteine (a) with the methanol--HCl reagent furnished a mixture of the O-methyl esters of AT-acetyl-S-(2-chloroethyl)cystein (b) and 5-(2-chloroethyl)cystein of which the mass spectra were identical with those of the authentic substances [5]. Conversely, the O-methyl ester of AT-acetyl-S-(2-chloroethyl)cystein (b) was hydrolysed rapidly by water to the O-methyl ester of N-acetyl-S-(2-hyciroxyethyl )cysteine (a). There is a strong supposition that the reversible reaction processes con necting the substances, Ar-acetyl-S-(2-hydroxyethyl)cysteine (a) and N-&cetyliS-(2-chloroethyl)cysteine (b), are modulated through the intermediacy of episulphonium ion (c) (Fig. 4), and on this basis, e.g. the formation of (c) would be rate-determining in respect of the hydrolysis of (b). Moreover, nucleophilic attack of hydroxyl ion on the episulphonium ion (c) would be expected to afford olefine through ^-elimination [15]. In fact, asmall yield of a second product, //-acetyliS-vinylcysteine (d), was recovered from the urine of [l4C] vinyl chloride-treated animals, whenever diazomethane esterifi RSV 00Lai^2 146 cation was used for protecting the Containing metabolites. The mass spec trum of the O-methyl ester of JV-acetyl-S-vinylcysteine was identical with that of authentic material [9]. In this case, the molecular ion (M* * 203) was produced by electron impact (Fig. lb). Manipulation of the S-containing metabolites of vinyl chloride revealed them to be highly reactive substances with a specialized chemistry. Thus, the reaction of the O-raethyl ester of AT-acetyI-S-(2-hydroxyethyl)cysteme with methanol at neutral pH is noteworthy. While the molecular ion (M* - 235) was absent from the conventional mass spectrum, the (M+l)* ion could be recognized in the mass spectrum, when chemical ionization was used as the ion source. In another situation, (i) the loss of label from thin-layer plates containing a mixture of the O-methyl esters of N-acetyl-S-(2-raethoxy[,4C]ethyl)cysteine and AT-acetyl-S-[ ,4C] vinylcysteine, and (ii) the presence of ,4C in the methanol solvent peak of the GC trace of the 2 labelled compounds, indicate their ready transformation under mild conditions of reaction. A volatile product has been identified by mass spectrometry as either [,4C]S(2-methoxyethyl)(prop-l-or 2-enyl)sulphide. Whilst the mechanism of for mation has not been investigated, it is felt that acetaldehyde, a known dissociation product of S-vinylcysteine-derived S-vinylcysteine-S-oxide [9] might be involved in a concerted condensation reaction with N-acetyl-5(2-methoxy[ l4Q ethyi)-cysteine, (leading to possible elimination of glyoxylate). Since the S-containing vinyl chloride metabolites that generate the episulphonium ion (c) (Fig. 4) do not behave as mutagens in S. typhimurium strains, their further investigation was considered to be unrewarding. At this stage in the work, it seemed important to investigate the biogenesis in the mammal of the S-containing vinyl chloride metabolites. Initially, we sought to determine whether S-(2-hydroxyethyl)cysteine would serve as a source of thiodiglycollic acid, and we were able to isolate the authentic sub stance [5] in 0.5% theoretical yield from the urine of rats dosed with-(2hydroxyethyljcysteine. This result seemed to be highly significant, because of the previously-described instability of that compound under exceedingly mild conditions of reactions. Furthermore, the metabolic pathway (Fig. 5) concerned, which appears to include end-group oxidation, amino acid trans amination and oxidative decarboxylation, admits of other sources for thiodi- 0H 1 (I) ------ `K CHlNHjlCOjH (II) HOrc*^ ------- h^c-ch2 si s1 CHINHjICCjH 2SfCHjCOjHI Fig. 5. Scheme suggesting the biotransformation of S-(2-hydroxyethyl)cysteine into thiodiglycoilic acid. (I) End-group oxidation; (II) transamination; (III) oxidative decarboxyla tion. RSV 0012143 147 glycollic acid formation. The fact that high yields (Table 1) of thiodiglycollic acid (j) (Fig. 6) were obtained in rats, which were dosed separately with chloroacetaldehyde (g), chloroacetic acid (h) and S-(carboxymethyl)cysteine (i) (Fig. 6) suggested that these compounds might Lie on a common metab olic pathway connecting vinyl chloride (e) with thiodiglycollic acid 0) possibly with the other S-containing metabolites, namely N-acetyl-S-(2-hydroxyethyl)cysteine and N-acetyl-S-vinylcysteine. Nevertheless, further experimental evidence suggests that the inclusion of chloroacetic acid (h) (Fig. 6) on this metabolic pathway may be incorrigible. Thus, (I) very little chloroacetic acid, <0.1% of the dose, has ever been detected in the body fluids of any of our vinyl chloride-treated rats, and this fact implies either a high rate of turnover for (h) or the possibility that this substance is not a major vinyl chloride metabolite. The latter supposition now seems the more likely, particularly since relatively large amounts of chloroacetic acid have been found [16] in the body fluids of rats, treated with vinylidene chloride, and in those animals, thiodiglycollic acid accounted for an even higher proportion of the dose than in parallel experiments with vinyl chloride. (2) A feasible metabolic pathway of thiodiglycollic acid from chloroacetic acid and involving cysteine desulphydrase [E.C. 4.4.1.1] has now been shown to be unacceptable in experiments with unlabelled vinyl chloride in rats, in which the cysteine--cystine pools had been labelled ade quately with [14C]. The formation of labelled thiodiglycollic acid showed that a part of the C-skeleton must be derived in fact from cysteine. (3) More over in rats treated with chloroacetaldehyde, the presence of thiodiglycollic acid (j) (Fig. 6) and of N-acetyl-S-(2-hydroxyethyl)cysteine, but not of chlo roacetic acid (h), amongst the urinary metabolites has been established by mass fragmentometry (Fig. 2). Hence, there is a strong supposition that in rats chloroethylene oxide (f) (Fig. 6) is formed from -vinyl chloride [17] and transformed spontaneously into chloroacetaldehyde (g) [18]; there is supporting evidence {1--3] for vinyl chloride epoxidation in vitro. The biological interrelationship of gluta thione S-epoxide transferase [EC 4.4.1.7] and substrate epoxide is well TABLE 1 RELATIVE PROPORTIONS OF THIODIGLYCOLLIC ACID IN THE URINE OF RATS DOSED INTRAGASTRICALLY WITH VARIOUS METABOLITES OF VINYL CHLO RIDE Vinyl chloride metabolites Yield of thiodiglycollic acid (% of the dose) S-<2-hydroxyethyl)cysteine Chloroacetaldehyde Chloroacetic acid S-(carboxyrneihyl)cystoine 0.5 9.2 31.0 20.0 RSV 0012144 148 < HjC-CHCI CM <F ^c-cn & <9) CICHjCHO C-0 I CH-CHjSCHjCHO NH I (h) -ClCHjCOOH / +GSH / / 1/ / CC^H CHCHjSCHjCHj NHIAO OH -OH OH* COjH CHCHjSCHjCOjH NHj li > ^CH2 COgH C-CHjSCH^COjH 0 -H4 p.r ^C-CHj SCC^COjjHJj (j> Fig. 6. Scheme for the biogenesis of S-conteining rinyl chloride mcttboiit. known, and the fact that chloroacetaldehyde affords in vivo both thiodiglycollic acid and JV-acetyl-5-(2-hydroxyethyI)cysteme infers that vinyl chloride metabolism involves a reaction between glutathione and chloroethylene oxide or chloroacetaldehyde (Fig. 6). This suppostion is supported by the identification, by mass fragmentometry, of S-(carboxymethyl)cysteine (i) (Fig. 6) amongst the hydrolytic products prepared from an hepatic extract from vinyl chloride-treated animals. Since chloroacetaldehyde and chloro ethylene oxide are mutagens in S. typhimurium strains [19--21] and in RSV 0012145 149 Chinese hamster V79 cells [22], both of these substances may contribute substantially to vinyl chloride carcinogenicity in mammals. However, it ought to be explained that the proposed enzymic reaction between glutathione and chloroethylene oxide, catalysed by glutathione S-epoxide transferase, would, give a product, which is unlikely to be stable in the isolation procedure. In fact, the reaction product of the terminal cysteinyl residue undergoes ring-closure (on hydrolysis) to afford 1,4-thiaz-lene-6-carboxylic acid. The foregoing evidence suggests a scheme (Fig. 6) which is economical in respect of primary biotransformations, for the biogenesis of vinyl chloride S-containing metabolites. It now (cf. ref. 5) seems unlikely that glutathione reacts directly with vinyl chloride (e) (Fig. 6) as well as with chloroethylene oxide (f) or its chloroacetaldehyde (g) transformation product, and present work provides convincing evidence for a metabolic pathway, involving chlo roethylene oxide and chloroacetaldehyde, for vinyl chloride in the living mammal. ACKNOWLEDGEMENTS We thank Miss C. Pamela Hudson for her unstinting help and splendid technical assistance. We should also like to thank our colleague, Dr. J.A. Styles, for testing the mutagenicity of S-containing vinyl chloride metab olites. REFERENCES 1 A.G. Salmon, Cytochrome P450 and the metabolism of vinyl chloride. Cancer Lett., 2 (1976)109. 2 A. Barbin, H. Bresil, A. Croisy, P. Jaequignon, C. Malaveille, R. Montesano and H. Bartach, Liver microtome-mediated formation of alkylating agents from vinyl bro mide and vinyl chloride, Biochem. Biophys. Res. Commun., 67 (1975) 596. 3 H. Bartach and R. Montesano, Mutagenic and carcinogenic effects of vinyl chloride, Mutat. Res., 32 (1975)93. 4 H. Kappus, H.M. Bolt, A. Buchter and W. Bolt, Liver microsomal uptake of [14C] vinyl chloride and transformation to protein alkylating metabolites in vitro, Toxicol. Appl. Pharmacol., 37 (1976) 461. 5 T. Green and D.E. Hithwiy, The biological fate in rats of vinyl chloride in relation to its oncogenicity, Chem.-Biol. 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Cancer, 15 (1975) 539. RSV 0012147