Document mm3nbm0KBxL2a2dkNwDa9MDwJ

y , 0 Box No 6 Bessemer Road Welwyn Garden City Hertfordshire Telephone Welwyn Garden 23400 (STD Code 07073) STD Code from London Area 96 Telex 264251 Iclplast Welwyn SEP 1 l 1977 Imperial Chemical Industries Limited Plastics Division Mr R W Wheeler, Jr Union Carbide Corporation South Charieston Plant PO Box 8004 South Charleston W Va 25303 USA R. ' W EELER 1R Your rel Our ref JS/JAP/DSO-107 Tel ext 3162 Date 8 September 1977 Dear Mr Wheeler VCM METABOLISM You may care to have the enclosed publication by Green and Hathway in Chera-Biol Interactions (1977) pages 137 - 150. I also enclose a copy of the paper Dr Hathway gave at Bethesda, Maryland, on 2 - 4 May, 1977 this is due to be published in Sfcvironaental Perspectives in the near future. Yours sincerely J Stafford Division Manager Health and Environment Protection EMC ASI 000012425 Cham.Biol. Interactions, 17 (1977) 137-150 EUovu-r/Norlh Holland Scientific Publishers, Ltd. 137 THE CHEMISTRY AND BIOGENESIS OF THE S-CONTAINING METABOLITES OF VINYL CHLORIDE IN RATS T. tJRKfc'N and U K IIATIJWAY Imperial Chemical Imhistrie* Limited, Central Toxicology Laboratory, Alderlcy Park, Cheshire SKIO 4TJ (Great Britam) (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. N-AcetyI-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 N-acetyl-S-(2-chloroethyl)cysteine or jV-aeetyl-S-(2-hydroxyethyl)cysteine may be isolated from the body fluids. N-Acetyl-S-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. Ar'Acetyl^5-{2-methoxyethyl)cysteine plus N~acetyl-S-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* roacotaldohyde, and with the reaction of chloroethylene oxide or chloro* acetaldehyde with glutathionr in the presence of a glutathione S-epoxide transferase to give the identified S-containing metabolites. Abbrcvutions: GC, (.mi; chromatograph. . 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 9f these substances, because in a previous paper [5], where the Fischei--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-chloro* ethyl 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-cysteinc were condensed with Na and liquid NH3 to give S-(2hydroxyethyI)-L-cysteino 18], which crystallized from 92% ethanol with a m.p. of 202C (decomp.). (Carson and Wong [8] gave m.p. of 189-- 189.5C). (Found: C, 36.5; H, 6.8; N, 8.4; S, 19.4%; CsHM03NS requires C, 36.4; H, 6.7; N, 8.5; S, 19.4%). S-(2-Hydroxyethyl)-L-cysteine on heating in reagent hydrochloric acid (38%) gave S-(2-chloroethyl)-L-cysteine hydrochlo ride (m.p. 182C), and from these 2 compounds, Af*acetyl-S-(2-hydroxyethyl)-L-cysteine and N-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 of N-acetyl-S-(2* hydroxyethyl)-L*cysteine is shown in Fig. 1, but that of the O-butyl ester of N*acetyl-S-(2-chloroethyl)-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 NH3 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 N-acetyl-S-vinyl-L-cysteine was confirmed by the mass spectrum, and that of the O-methyl ester derivative is shown in Fig. 1. 5-Carboxymethyl-L-cystcine was prepared [10] from chloroacetic acid plus L-cysteine hydrochloride, and the structure of the crystalline product (m.p. 175--1768C, 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 has been found to react with cysteine to give 4<arboxy-2-chloromethylthiazolidine, thus involving intermediate formation of 5-(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 transferase, namely S-(2-hydroxyalkyl)glutathione alkyl-epoxide-lyase [E.C. 4.4.1,7). ASI 000012427 139 Fiy. 1 Mass spectrum of: (a) the O-meihyl ester of A'-cetylnS (2 hydro*yethy!) cyiteine; (b) the Omethyl ester of .VacetyKS'-vinylcysteinc. 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. ASI 000012428 Radioactive chemicals [,4C]vinyl chloride with a specific activity of 0.46 mCi/mmole, and with a radiochemical purity exceeding 99.0% was synthesized as previously outlined [51. 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 (YVistar-dcrived), 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) [,4C]vinyl chloride (100 mg/kg; 10 pCi) in corn-oil solution. (b)Chloroacetaldehyde (50 mg/kg) as an aq. soln. (c) S-(2-hydroxyethyl)-L-cysteine (500 mg/kg) as an aq. soln. (d) S-(carboxymethyl)-L-cysteine (250 mg/kg) in corn-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 pCi) 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 l4C, 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* ASI 000012429 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 [MC]vinyl chloride metabolites. (b) The 3 N acetic acid fraction was analysed by GC--mass spectrometry, as well as by mass fragmentometry, for the N-acetyl-$-(2-hydroxyethyl)cysteine metabolite (of vinyl chloride), and the 3 N HC1 fraction was investigated for the presence of thiodiglycollic acid and chloroacetic acid by GC--mass spectrometry. (c) and (d) The 3 N I1C1 fractions were analysed quantitatively for thiodi glycollic acid by (1C-mass spectrometry. (e) In the experiment in which unlabclled 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 a peak of [ l4C]thiodiglycollic acid. Systematic 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 KH:P04 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 CHjCOJ 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 residue, 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 diazometh ane 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 Jong 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 ASI 000012430 142 Fit!- 2- Mass frajjmentogram of the O-methyl ester of A/-acetyl-S-(2-hydroxyethyl)cysteine from the urine of chloroacetaldehyde-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 mcthanolic 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 intensities and retention time as the standard. ASI 000012431 143 Fiu. 3. Maas fragmeniogram of the O-dimcthyl ester of the TV-trifluoroacetyl derivative of A'-fcarhoxymothyljcysteine from the urine of chloroacetaldehyde-treated rats (the gains for m/e 303. m/e 271 and m/c 244 are in the ratio 1 : 1 : 1). A represents the injection 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 intensities and retention time as the standard. ASI 000012432 144 with 10% (w/w) of SP2330 on Supelcoport (100--120 mesh size). All of the columns were operated at a 30 ml/min 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 5U"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 A/-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--C02CHj and m/e 144 corresponding to M--H:0--C02CHj. The 6% OV-101 column was used and run at 195C. Under these conditions, the methyl ester of A/-acetyl-S-(2-hydroxyethyl)* cysteine had a retention time of 5.6 min. In the case of the dimethyl ester of the A/-trifluoroacetyl derivative of 5-(carboxymethyl)cysteine (Fig. 3), the selected ions were m/e 303 corre sponding to the molecular ion, m/e 271 corresponding to M--CHjOH and m/e 244 corresponding to M--C02CH3. The column, used in the gas chroma tograph, was one (2.7 m long X 2 mm internal diameter) which was packed with 20% (w/w) of OV-275 on Chromosorb W-AW and which was run at 250 C. 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 S-(2*hydroxyethyl)cysteine and of S-(2-chlo- roethyl)cysteine was assessed by the method of Ames et al. [14] usingS. typhimuriutn strains TA1535, TA1538, TA98 and TA100. RESULTS AND DISCUSSION A/-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-S-(2-chloroethyl)cysteine (b) (Fig. 4) or A-acetyl-S- ASI 000012433 145 (i) OH \M 'C< SR OH -OH 1C) <1* -Cl CHINHACICO^H -H (b) Cl nc-c'h H'l VH SR H"^c-c ,,H RS ^H (dl Fig. 4. Scheme for the interrelationship of come S-containing vinyl chloride meUbolitiec. (2-hydroxyethyl)cystcine (a) was isolated from the body fluids of vinyl chlo ride-treated animals. Thus, e.g. the O-methyl ester of AT-acetyl-S-(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-hydroxyethyI)cysteine (a) was produced if diazoraethane was used instead. The mass spectrum of the O-methyl ester of Af*acetyl-S*(2* chloroethyl)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 JV-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 lV-acetyl-S-(2*hydroxyethyl)cysteine (a) with the methanol--HC1 reagent furnished a mixture of the O-methyl esters of W-acetyl-S-(2-chloroethyl)cystein (b) and S*(2-chloroethyl)cystein of which the mass spectra were identical with those of the authentic substances [5], Conversely, the O-methyl ester of A/'-acetyl-5-(2-chloroethyl)cystein (b) was hydrolysed rapidly by water to the O-methyl ester of N-acetyl-S-(2-hydroxy- ethyl)cysteine (a). There is a strong supposition that the reversible reaction processes con necting the substances, 7V-acetyl-S-(2-hydroxyethyl)cysteine (a) and N-acetyl-S-(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, a small yield of a second product, JV-acetyl-S-vinylcysteine (d), was recovered from the urine of [,4C]vinyl chloride-treated animals, whenever diazomethane esterifi- ASI 000012434 il'IG cation was used for protecting the S-containing metabolites. The mass spec trum of the O-methyl ester of N-acetyl-S-vinylcysteine was identical with that of authentic material [9]. In this case, the molecular ion (W = 203) was produced by electron impact (Fig. lb). Manipulation of the S-containing metabolites of vinyl chloride revealed them to he highly reactive substances with a specialized chemistry. Thus, the reaction of the O-methyl cspT of /V-acetyl-S-(2-hydroxyethyl)cystc`ine 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 A/-acetyl-S-(2-methoxy[,4CJethyl)cysteine and (V-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 Af-acetyl-S(2-methoxyl'4C]ethyl)-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 S-(2hydroxyethyl)cysteine. 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- i ni nm HjC-CHj -------------- HO^C-CHj ------------ HC^C-CHj ----------------- SICHjCO^ s ss C1 MZ 1ch2 c' h, CHINHjICO^H CHINHjICOjH C-COjH 0 Fits. 5. Scheme suggesting the biotransformation of S-(2-hydroxyethyl)cysteine into thiodigtyeollic acid. (I) End-group oxidation; (II) transamination; (III) oxidative decarboxyla tion. ASI 000012435 147 glycollic acid formation. The fact that high yields (Table I) of thiodiglycollic acid (j) (Fig. 6) were obtained in rats, which were dosed separately with chloroacetaidehyde (g), chloroacetic acid (h) and S-(carboxymethyl)cysteine (i) (Fig. 6) suggested that these compounds might lie on a common metabolic pathway connecting vinyl chloride (e) with thiodiglycollic acid 0) and possibly with the other S-containinga metabolites, namely N-acetyl-S-(2-hy* droxyelhyOcysteine and Af-acctyl-S-vinylcysteine. Nevertheless, further experimental evidence suggests that the inclusion of ehloniaeetie and (h) (Fig. (!) on this meLabolic pathway may he incorrigible. Tlius, (I) very little chlorouceLic acid, sO.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 [l4C]. 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 chloroacetaidehyde, the presence of thiodiglycollic acid (j) (Fig. 6) and of A/-acetyl-5-(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 chloroacetaidehyde (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 TABl.K I RELATIVE PROPORTIONS OF THIODIGLYCOLLIC ACID IN THE URINE OF RATS DOSED INTRACASTKICALLY WITH VARIOUS METABOLITES OF VINYL CHLO RIDE Vinyl diioridi' mt'l.ilniliUM Yirid of ihiodiulycollic acid (% of the done) S-( i-liydroxyiatiyHi-ysli-inr Cliloronci'laldi'hydr Chloruaectic acid S (i-arboxyrro'thyOcyiU'ini: 0.5 9.2 31.0 20.0 148 (*) HjC-CHCI Ml C? -H,C-CH & (gl -CICHjCHO I 00 1 CH-CHjSCHjCHO NI H Ih) -CICHjCOOH / / / tCSH / / / / / COjH CHCHjSC^CI^ NHlAd OH -OH tOH* CO,H CI H`CHjSC^COjH NHj <i> ^CHj CZH C-CH^SCH^COjH 0 -H 4 R-Sn .C'CH; Hy SICHjCOjH^ (j) Fig. 6. Scheme for the biogenesis of S-containing vinyl chloride metabolites. known, and the fact that chloroacetaldehyde affords in vivo both thiodiglycollic acid and JV-acetyl-S-(2-hydroxyethyl)cysteine 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 fragmentornetry, of i'-(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 ASI 000012437 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-l* enc-G-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 P4G0 and the metabolism of vinyl chloride, Cancer Lett., 2 (197G)109. 2 A. Barbin, H. Bresil, A. Croisy, P. Jacquignon, C. Malaveille, R. Montesano and M. Bartsch, Liver microsome-mediated formation of alkylating agents from vinyl bro mide and vinyl chloride, Biochem. Biophys. Res. Commun., 67 (1975) 596. 3 H. Bartsch and R. Montesano, Mutagenic and carcinogenic effects of vinyl chloride, Mutat. Res., 32 (1975) 93. -1 H. Knppus, 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. Hathway, The biological fate in rats of vinyl chloride in relation to its oncogenicity, Chem.-Biol. 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Johnson, Preparation of deproteimsed tissue extracts for chromatography and .4 assay of compounds related to glutathione, J. Chromalogr., 23 (196ti) 47*1. 13 S.U. Gurani, U.S. Kumta and M.B. Sahasrabudhe, Influence of formic acid on the hydrolysis of tissue proteins; a new and rapid method of hydrolysis of proteins, Biochim. Biophys. Acta, 16 (1955) 553. 14 B.N. Ames. J. McCann and E. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutat. Res., 31 (1975) 347. 15 A.G. Ogston, E.R. Holiday, J.S.L. Philpot and L.A. Stocken, The replacement reac tions of A5,-dichlorodiethylsulphide and of some analogues in aqueous solution: the isolation of (Ichloro-^'-hydroxydielhylsulphidc, Trans. Faraday Soc., 44 (1945) 45. 16 R.K. Jones and D.E. Hath way, The biological fate of vinylidene chloride in rats, in press. 17 B.L Van Duurcn, On the possible mechanism of carcinogenic action of vinyl chloride, Ann., N.Y. Acad. Sci,, 246 (1975) 258. 13 H. Gross und J. Freiburg, Zur Existcnz von Chloriithylenoxid, J. Prakt. Chcm., 311 (1969) 506. 19 H. Bartsch. C. Malaveille and R. Montcsano, Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in S. typhimurium strains, Int. J. Cancer, 15 (1975) 4 29. 20 C- Malaveille, H. Bartsch, A. Barb*in, A.M. Camus, R. Montesano, A. Croisy and P. Jacquignon, Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacetaldehyde and chloroethanol, Biochem. Biophys. Res. Commun., 63 (1975) 363. 21 J. McCann. V. Simon, D< Streitwieser and B.N. Ames, Mutagenicity of chloroacetal dehyde, a possible metabolic product of 1,2-dichloroethane (ethylene dichloride), chloroethanol (ethylene chlorohydrin), vinyl chloride and cyclophosphamide, Proc. Natl. Acad. Sci. USA, 72 (1975) 3190. 22 E. Hubeman, H. Bartsch and L. Sachs, Mutation induction in Chinese hamster V79 cells by two vinyl chloride metabolites, chloroethylene oxide and chloroacetaldehyde, Int. J. Cancer, 15 (1975) 539. ASI 000012439 Environmencal Health Perspectives Vol. 2i* PP- 1977. COMPARATIVE MAMMALIAN METABOLISM OF VINYL AND VINYLIDENE CHLORIDES IN RELATION TO ONCOGENIC POTENTIAL* * by David E Hachway Elucidation of the role of vinyl chloride metabolites in the various reaction sequences which comprise the metabolic pathway, including the interaction of reactive metabolites with some purine and pyrimidine residues of target-organ DNA, provides some explanation for the (oncogenic) properties associated with the original substance. Comparative investigation of the biological fate of vinylidene chloride reveals (a) an agent of low oncogenic potential which is likely to be damaging only under special circumstances, and (b) species differences which suggest that the mouse is more susceptible than the rat towards vinylidene chloride oncogenicity. * Imperial Chemical Industries Limited, Central Toxicology Laboratory, Alderloy Park, Cheshire SK10 4TJ, England. * Lecture delivered in the Symposium on Comparative Metabolism and Toxin* ty of Vinyl Chloride Related Compounds (NIEHS), held at Bethesda, ASI 000012440 2 The research work with which this communication is concerned is based on the idea that knowledge of the biology of the reactive metabolites of chemical carcinogens in the mammal, including the precise nature of the chemical changes to the DNA of the nucleus, ought to give an insight into the (oncogenic) properties of the parent compounds. In tracer studies, iN-acetyl-S-(2-hydroxymethyl)cysteine was shown to be a major vinyl chloride metabolite in rats, but according to the method of protective esterification that was used so a derivative either of N^acetyl-S^ (2-chloroethyl)cysteine or of N-acetyl-S_-(2-hydroxyethyl)cysteine was isolated from body fluids (1, 2). Thus, by Fischer-Speier methylation, N-acetyl*'S-(2-chloroethyl)cysteine was obtained, and with diazomethane, W-acetyl-S"(2"hydroxyethyl)cysteine. It might be stated en passant that throughout the investigations described, mass spectrometry, involving E.I. and chemical ionisation sources and multiple-ion detection and all combinations of these facilities, was used extensively both for product identification and analysis and for the purposes of detection. Treatment Fig. 1 of the O-methyl ester of N-acetyl--(2-hydroxyethy1)cysteine (a) (Fig. 1) near here with the methanol-HCL reagent gave a mixture of N-acetyl-S-(2-chloroethyl) cysteine (b), and -(2-chloroethyl)cysteine, and conversely, the O-methyl ester of N-acetyi-,S-(2-chloroethyl) cysteine (b) was hydrolysed rapidly by water to that of N-acetyl-S^(2-hydroxyethyl)cysteine (c) (2). Hence, the reversible reaction processes connecting the two substances would seem to be modulated through the intermediacy of episulphonium ion (c) and formation of this ion would in fact be rate-limiting in respect of the hydrolysis of N-acetyl-S-(2-chloroethyl)cysteine. Nucleophilic attack of OH on the episulphonium ion would be expected to give olefin (3), and in file i , N t y l-S-vinyl rysleinc (d) wnu recovered from the urine of vinyl ASI 000012441 J chloride-treated animals whenever diazomethane esterification was used to protect ^-containing metabolites. Surprisingly, N-acetyl--(2-hydroxyethyl)cysteine O-methyl ester was methylated with neutral methanol, and the O^methyl esters of N>-acetyl"S^(2-raethoxy[^`^C]cthyl)cysteine plus N-acetyl-S-[^C)vinyl-cysteine 14 degrade to give the volatile [ C]S-(2-methoxyethyl)(prop-1 or 2-enyl) sulphide. Although the mechanism of formation was not investigated, we felt that acetaldehyde, a known dissociation product of S^vinylcysteinederived S^vinylcysteine-S-oxide (4) might undergo concerted condensation with N-acetyl--(2-methoxyethyl)cysteine leading to elimination of thermodynamically stable glyoxylate. [There is an analogy for such a concerted condensation reaction in the work of Dabritz & Virtanen (4) on the tear-producing volatile components of the onion.) The half-mustard ^-containing metabolites of vinyl chloride did not behave as mutagens in the Ames test (2). Thiodiglycollic acid is another major vinyl chloride metabolite (1). (i Fig. 2 near here In order to determine whether vinyl chloride yielded chloroethylene oxide in vivo, the biogenesis of several vinyl chloride metabolites and related compounds were investigated in rats (2). S_-(2-Hydroxyethyl) cysteine gave 0.5Z of the authentic thiodiglycollic acid, and this result was seen to be highly significant, because of the instability (v. supra) of the starting material under exceedingly mild conditions of reaction. The metabolic pathway concerned (Fig. 2) appears to include end-group oxidation, amino-acid transamination and xidative decarboxylati n, and the results > .......... ii'i'iiim' pimiiriintinfs Riiv'vest fh.it' chloroacctaldehyde (g) (lig. 3), 000012442 4 Fig. 3 chloroacetic acid (h), and -(2-carboxymethyl)cysteine (i) night lie on a near here conmon pathway connecting vinyl chloride (e) with thiodiglycollic acid (j). However, other evidence implies that chloroacetic acid (h) does not belong to this metabolic pathway (e-j) (Fig.3). Thus, (i) d.0.\% has even been detected in the body fluids of any of our vinyl chloride-treated animals. Either there is a high rate of turn-over or this compound is not a major vinyl chloride metabolite. The latter possibility seems more likely, since relatively large amounts are produced in vinylidene chloride metabolism, and in those animals, thiodiglycollic acid accounts for an even greater proportion of the dose than in parallel experiments with vinyl chloride. (ii) A feasible metabolic pathway for thiodiglycollic acid from chloroacetic acid and involving cysteine desulphhydrase is unacceptable. Experiments with unlabelled vinyl chloride in rats in which the cysteinecystine pools had been labelled adequately with 14C gave -[14CJthiodiglycollic acid, showing that a part of the C-skeleton must be derived in fact from cysteine. (iii) In rats treated with chloroacetaldehyde, the presence of thiodiglycollic acid and N-acetyl-S-(2-hydroxyethyl)cysteine, but not of chloroacetic acid, amongst the urinary metabolites was established by mass fragmentometry. Thus, it is probable that in vivo chloroethylene oxide (f) (Fig.3) was formed (3) from vinyl chloride (e) and transformed spontaneously (6) / into chloroacetaldehyde (g); there is supporting evidence (7-10) for ^ vinyl chloride epoxidation ^in vitro. This supposition is supported by ihc fact (A) that chloroacetaldehyde affords both N-acetyl-j[-(2-hydroxyethyl) cysteine and thiodiglycollic acid in vivo and (B) that S_-(2-carboxymethyl) cysLcine Imu been identified by mass rugnien Lome try amongst the hydrolytic liroilurlii of mi lu-pat ir extract pivparod from vinyl chloride-treated animuls. A S I OOOOI2443 Since chloroacetaldehyde and c'nloroethylene oxide are mutagenic in the Ames test (11-13) and in Chinese hamster V79 cells (14), they may be relevant to vinyl chloride carcinogenicity. Respective formation of 3B-D-ribofuranosylimidazo-[2,l-iJpurine or 6B-D-ribofuranosyl-5-oxo-5,6-dihydroimidazo-[l,2-c]pyrimidine from adenosine or cytidine by reaction with chloroacetaldehyde (15) or chloroethylene oxide was readily confirmed. The presence of these two imidazo-nucleosidc derivatives has now been established by mass fragmentometry (16) in the enzymic hydrolysate of modified rat-liver DNA, prepared from rats, which had been exposed chronically to vinyl chloride Fig. 4 (250 ppm in their drinking water) for 1 year (Fig. 4). A smaller icar here proportion of the 38"D-ribofuranosyliniidazo-[2,l-i]purine, than would have been expected to have been formed, was found both in the animal experiments with vinyl chloride and in model reactions between chloroacetaldehyde and calf-thymus DNA (16). This observation is consistent with some degree of DNA depurination brought about by the reaction of vinyl chloride, and in our model experiments, we have found evidence for the presence of the detached purine. Viz, imidazo-[2,1-iJpurine. Hence, the alkylation that produces imidazo-derivative formation (with DNA) labilizes the N^ purine B~glycoside linkage, which leads to. depurination. The gap so produced might then be filled by various bases, resulting in 'mispairing' during DNA replication. These results are very important, because in general, there is excellent agreement between the severe damaging effect of depurination to DNA and mutagenicity (17-19). Tim:*, iit roi iumpire.t, otic would suspect vinyl chloride of bciug mutagenic/ carcinogenic. ASI 00001244* 6 On the other hand, vinylidene chloride (a) (Fig. 5) metabolism in Fig. 5 rats gave thiodiglyeollic acid (g) and an W-acetyl-ji-cysteinyl-acetyl near here derivative (e) as major urinary metabolites, plus substantial amounts of chloroacetic acid (b), dithioglycollic acid (j) and thioglycollic acid (h) (20). It is probable that chloroacetic acid (b), which is a vinylidene chloride metabolite per se, lies on a major metabolic pathway for vinylidene chloride (Fig. 5), since it affords several metabolites in common with vinylidene chloride (20). There is a strong supposition that detoxification of chloroacetic acid (b) is effected through a glutathione S^acyl transferase catalysed reaction process and ensuing degradative sequence for the resulting carboxymethylglutathione (d), and that this represents the principal metabolic pathway for chloroacetic acid and a major one for vinylidene chloride. Thiodiglyeollic acid is the ultimate detoxification product, and previous work (2) established the biotransformation of S-(2-carboxymethyl) cysteine (f) into that substance. A feasible metabolic pathway to thiodiglyeollic acid from chloroacetic acid and involving cysteine desulphhydrase is unacceptable. In experiments (rats) with unlabelled vinylidene chloride in which the cysteine-cystine pools had been labelled with "C, labelled thiodiglyeollic acid resulted, and a part of the C-skeleton of that substance must be derived in fact from cysteine (20). Formation of a small amount of [^Cjdithioglycollic acid (j) (and hence of the intermediate [l*C]thioglycollic acid) (h) is reconcilable with the action of Michaelis's (21) unspecific 6-thionase, which would lyse a small proportion of the preponderating [^Cjthioglycollic acid (v. infra) ASI 000012445 i 7 Moreover, Kolbe electrolysis (22) of one molecular proportion of the [1^C]thiodiglycollic acid metabolite from [l-^C]l,l-dichloroethylene or 4 [l-^C]chloroacetic acid gave 1 equivalent of ^002(23), and this evidence is consistent with the transformation of vinylidene chloride into chloroacetic acid by a mechanism involving migration of one Cl_ atom and the loss of the other one (20, 23). Hence, the metabolic pathway (Fig. 5), which was tentatively proposed for the biotransformation of vinylidene chloride into thiodiglycollic acid does in fact operate in rats. It is equivocal whether the very small amounts of CO2 and urea are produced by the action of epoxide hydratase on 1,1-dichloroethylene oxide or by a minor oxidative pathway for chloroacetic acid. There is a strong supposition that the N-acetyl-S-eysteinylactyl derivative (e) (Fig.5), which is a metabolite of vinylidene chloride, but not of chloroacetic acid, may be formed in fact from 1,1-dichloroethylene oxide through the agency of glutathione -epoxide transferase to afford S-glutathione acetyl chloride (c) and its subsequent reactions (20). This supposition is important, since the reactivity displayed by 1,1-dichloroethylene oxide (v..supra) appears to be relevant to the possible interaction of reactive vinylidene chloride metabolites with mouse-kidney Fig. 6 DNA (Fig. 6), which is a prerequisite of tumour initiation (24). Such near here interaction would be analogous to that of vinyl chloride with rat-liver DNA in vivo, which forms imidazo derivatives with some nucleoside residues (16). Further work in progress to investigate this hypothesis. ASI 000012446 8 Fig. 7 near here Comparative studies (25) provide clues of differences between rats and mice in the processing of vinylidene chloride (Fig. 7). Thus, in mice, the production of thiodiglycollic acid is considerably reduced and the formation of the N^acetyl-S^cysteinylacetyl metabolite is increased. The higher 8-thiomase activity in mice than in rats accounts for the greater conversion of thiodiglycollic acid into dithioglycollic acid via thioglycollic acid in the former species of animal. Yllner's (26) mice excreted a proportion of a dose of chloroacetic acid as unchanged starting acid. Thus, in mice, the metabolic pathway from chloroacetic acid to thiodiglycollic acid seems to be readily saturable, possibly on account of an inadequacy in the reaction catalysed by glutathione -acyl transferase. Under these circumstances, detoxification of 1,1-dichloroethylene oxide by glutathione ^-epoxide transferase and the modification of DNA by 1,1-dichloroethylene oxide or ehloroacetyl chloride would be expected to be more significant in mice than in rats. This diagnosis of species susceptibility seems to accord with Maltoni's (24) discovery of vinylidene chloride oncogenicity in (the kidneys of) mice. Vinylidene chloride emerges as an agent of low, perhaps very low, oncogenic potential, which can be damaging only in a special set of biological circumstances, which we have partially defined (v. supra) and on which work is continuing. ASI 000012447 9 acknowledgement The author is indebted to*his colleagues Messrs T Green, and B K Jones, Drs A G Salmon and P L Batten, and Mr G H Walker for their invaluable contributions and help. ASI 000012448 REFERENCES 1. Green, T., and Hathway, D.E., The biological fate in rats of vinyl chloride in relation to its oncogenicity, Chem.-Biol. Interactions U: 545 (1975). 2. Green, T., and Hathvay, D.E., The chemistry and biogenesis of ^-containing metabolites of vinyl chloride in rats, Chem-Biol. Interactions, 7_: 137 (1977). 3. Ogston, A.G. e_t al_., The replacement reactions of BS'-dichlorodiethyl sulphide and of some analogues in aqueous solution: the isolation of B-chloro-B'-hydroxydiethylsulphide, Trans. Faraday Soc., 44: 45 (1948). tv 4. Dabrit2, E. and Virtanen, A.'jI., -Vinyl-cystein-j[-oxyd, ein Homologes Vf zur Vorstufe der tranentreibenden Substanz der Zwiebel, Chem. Ber., 98: 781 (1965). 5. Van Duuren, B.L., On the possible mechanism of carcinogenic action of vinyl chloride, Ann. Nev York Acad. Sci., 246: 258 (1975). 6. Cross, H., and Freiburg, J., Zur Existenz von Chlorathylenoxid, J. prakt. Chem., 311: 506 (1969). 7. Runnug, U., et al., The mutagenicity of vinyl chloride after metabolic activation, AMBIO, _3,: 194 (1974). ASI 000012449 8. Baibin, A., ct_ nl_., Livcr-microsomc mediated formation of alkylating agents from vinyl bromide and vinyl chloride, Biochcm. Biophys. Res. 11 9. Greim, H., et_ al^., Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation, Biochem, Pharmacol., 24: 2013 (1975). 10. Salmon, A.G., Cytochrome P450 and the metabolism of vinyl chloride. Cancer Lett., 2i 109 (1976). 11. Bartsch, H.t Malaveille, C., and Montesano, R., Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in S. typhimurium strains, Int. J. Cancer, 15: 429 (1975). 12. Malaveille C., et al.. Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacetaldehyde and chloroethanol, Biochem, Biophys. Res. Coma., 65: 363 (1975). 13. McCann, J., e al., Mutagenicity of chloroacetaldehyde, a possible metabolic product of 1,2-dichloroethane (ethylene dichloride), chloroethanol (ethylene chlorohydrin} vinyl chloride and cyclophosphamide, Proc. Nat. Acad. Sci. USA, 72: 3190 (1975). 14. Huberman, E., Bartsch, H., and Sachs, L., Mutation induction in Chinese hamster V79 cells by two vinyl chloride metabolites, chloroethylene oxide and chloroacetaldehyde, Int. J. Cancer, 15: 539 (1975). 15. Barrio, J.R., S crist, J.A., and Leonard, N.J., Fluorescent adenosine and cytidine derivatives, Biochem. Biophys. Res. Comm., 46: 597 (1972). ASI 000012450 u 16. Green, T., and Hathway, D.E., Interactions of vinyl chloride with ratliver DNA in vivo, Chem.Biol. Interactions, in the press. * 17. Lawley, P.D., et^ al., Inactivation of bacteriophage T7 by mono-and di-functional sulphur mustards in relation to cross-linking and dnpurination of bacteriophage DNA, J. Mol. Biol., 39: 181 (1969). 18. Roberts, J.J., Nucleic acid modifications and cancer. In: 'Biology of Cancer', E. J. Ambrose and F.J.C. Roe, Eds., Halstead Press, Chichester, 2nd ed., 1975. 19. Loveless, A., Genetic and Allied Effects of Alkylating Agents, Butterworths, London, 1966. 20. Jones, 8.K, and Hathway, D.E., The biological fate of vinylidene chloride in rats, Chenr-Biol. Interactions, in the press. 21. Michaelis, L., and Schubert, M.P., The reaction of iodoacetic acid on mcrcaptans and amides, J, Biol. Chem., 106: 331 (1934). 22. Kolbe, H., Untersuchungen liber die Elektrolyse organischer Verbindungen, Justus Liebigs Annalen der Chemie, 69: 257 (1849). 23. Ualker, G.H. and Hathway, D.E., Electrochemical analysis of the [carboxy-*^C]aliphatic carboxylic acid metabolites resulting from tracer molecules, Biochem. J., in the press. ASI 000012451 13 24. Maltoni, C., Proceedings of the TAPPI International Conference* held in Hamburg on January 26, 1977. 25. Jones, B.K. and Hathway, D.E., Differences between mice and rats in the metabolism of vinylidene chloride, Brit. J. Cancer, in the press. 14 26. Yliner, S., Metabolism of chloroacetate-1- C in the mouse, Acta Pharmacol, et toxicol., 30: 69 (1971). j ASI <>00012452 (a) OH Hv l/H ^c-c H^l XH SR +0H - -- -OH' (c) HoC-CH, W -tCI' s -Cl' I CHo I CH(NHAc)C02H H' Hx ^H >< RS^ XH (d) (b) Cl I /H C-0 H^l x H SR Fig. 1 Schema for the intorreUtionship of some S-containing vinyl chloride metabolites. ASI 000012453 OH I h2c-ch2 (l) S I CH? i1 CH(NH2)C02H ho2c-ch2 I S (1!) I CHo |L CH(NH2)C02H ho2c-ch2 (Ml) s I CHo 1 C-C0?H II 1 $(CH2C02H Fig. 2. Scheme suggesting the biotransformation of -(2-hydroxyethyl) cysteine into thiodiglycollic acid. (I) End-group oxidation; (IX) transamination; (III) oxidative decarboxylation. ASI 000012454 (e) it) CCl I co chch2sch2ch2 NH OH I (g) (hi ------- *cic;i2cgok //// / / / / / / / +GSH / / / / / / / t / / / / chch2sch2co2h 1 CO?H I2 chch2sch2ch2 NH(Ac) OH -OH 40H CH, r-s: ^CH. -H R-S C'CHj H/ CO?H I2 CHCH2SCH2C02H nh2 (i) CO.H I2 c-ch2sch2co2h 0 S(CH2C02H)2 <j> Fiy. 3 Scheme for the biogenesis of ^-containing vinyl chloride me.tnbol i tes ASI 000012455 CiCH^HO +&NA Dfi ~ ii> Imtiq '('its J tidifiU 3)NA in vivo froiifi'Zcl' it^dot^U MA mio, K HO OH anl (Lssociitbb '<Lej>uvincite o?> Fig. 4 Scheme suggesting the model reaction of chloroacetaldehyde with (calf-thymus) DNA and the biotransformation of hcpatocyte DNA by vinyl chloride in vivo. Both reaction processes afford f>B-l)-ribtl ui-.'inosyl-5"oxo-5t6-diliydroiinidaao-(l ,2-c]pyrimidine (}< ft -Ii.iihI aide) and .'Ip-D-rihofurationy] imiilaxo-(2,1 -i Jpurine ASI 000012456 H^CClg (a) Clj V-/C1 Ac (e) CXCIuCCl *= 0 CXC^CO^ I (*) H0iI2C-C02U (C02H)o 1` C02 i oo{\m2)2 (f) I S(CH2C02H)2 i HSCHgCOgH i (SCU2C02II)2 () w (5) Fig. 5 Scheme for vinylidene chloride metabolism in rats. ASI 000012457 9 Fig. 6. Scheme suggesting the feasible interaction of reactive vinylidcne chloride metabolites, 1,1-dichloroethylene oxide and chloroacetyl chloride, with adenosine and cytidine u respectively. ASI 000012458 METABOLISM (1) of Chloroacetic acid Metaholito Chloroacetic acid Rats - Thiodiglycollic acid 90 N-Acctyl-S-(2-carboxymethyl)cysteine 2 Y1Iner 6-222 37 40 Mice BK.J-DEH 30-40 40 Chloroacetic acid Thiodiglycollic acid Thioglycolli c acid Dithioglycollic acid N-Acetyl-S.-cysteinylacetyl derivative (2) of Vinylidene chloride 3 37 3 35 5 20 48 70 7. The relative proportions of products resulting from the metabolism (1) of chloroacetic acid and (2) of vinylidene chloride in rats and mice. * ASI 000012459