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Environmental Health Ftnpeztives Vol. 21. pp. SSS9, 1977 Comparative Mammalian Metabolism of Vinyl Chloride and Vinylidene Chloride in Relation to Oncogenic Potential by David C. Hathway* Elucidation of Uw role of vinyl chloride metabolite* in the various reaction sequent** which comprise the metabolic pathway, including the interaction of reactive metabolites with some purine and pyrimidioe residues of target-organ DNA, provides some explanation for the (oncogenic) properties associated with the original substance. Comparative investigation of the bioiogicai fate of vinylidene chloride reveals an agent of low oncogenic potential which is likely to be damaging only under special circumstances, and specks differences which suggest thst the mouse k more susceptible than the rat towards vinylidene chloride oncogenicity. The research work with which this communica- tion 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, A'-acetyW-(2-hydroxymethyl)cysteine was shown to be a major vinyl chloride metaljplite in rats, but according to the method of protective esterification that was used so a derivative either of N-acetyJ-5-(2-chloroethyl)cysteine or of N-acetyl-5-(2-hydnoxyethyl)cysteine was isolated from body fluids (/, 2). Thus, by Fischer-Speier -- -- Kr .......i c /i jY-acetyl-S-(2-hydroxyethyI)cy$teine (a) with the methanol-HCl reagent gave a mixture of N-acetyl-5-(2-chloroethyl)cysteine (b), and 5(2-chloroethyl)cysteine, and conversely, the 0-methyl ester of N-acetyl-S-(2-chloroethyl)cysteine (b) was hydrolyzed rapidly by water to that of iV*acetyl-5-(2-hydroxyethyl)cysteine (a) (2). Hence, the reversible reaction processes connecting the two substances would seem to be modulated through the intermediacy of episulfonium ion (c) and formation of this ion would in fact be rate-limiting in respect cjf the hydrolysis of 4 el (bl Cl MW WMtvwm in passing that throughout the investigations de scribed, mass spectrometry, involving electron im pact (El) and chemical ionization 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 of the 0-methyl ester of 'Imperial Chemical Industries Limited, Central Toxicology Laboratory, Alderiey Park, Cheshire SK10 4TJ, England. December 1977 <di 55 N-acetyl-5-(2-chloroethyI)cysteine. Nucleophilic attack of OH~ on the episulfonium ion would be expected to give olefin (3), and in fact, Nacetyl-5-vinylcysteine (d) (2) was recovered from the urine of vinyl chloride-treated animals whenever diazomethane esterification was used to protect 5-containing metabolites. Surprisingly, JV-acetyl-5-(2-hydroxyethyI)cysteine O-methyl ester was methylated with neu tral methanol, and the O-methyl esters of JV-acetyl5-(2-methoxy[,4C]ethyl)cysteine plus N-acetyl5-[14C]vinyl-cysteine degrade to give the volatile [,4C]S-(2-methoxyethyI) (prop-1 or 2-enyl) sulfide. Although the mechanism of formation was not inves tigated, we felt that acetaldehyde, a known dissocia tion product of 5-vinylcysteine-derived 5-vinylcysteine-5-oxide (4) might undergo concerted con densation with A/-acetyl-5-(2-methoxyethyI)cysteine leading to elimination of thermodynamically stable glyoxylate. [There is an analogy for such a concerted , condensation reaction in the work of Dabritz and Virtanen (4) on the tear-producing volatile compo nents of the onion.] The half-mustard 5-containing metabolites of vinyl chloride did not behave as mutagens in the Ames test Q). Thiodiglycollic acid is another major vinyl chloride metabolite (/). 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). 5(2-HydroxyethyI)cysteine gave 0.5% 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 [Eq. (1)] appears to include endgroup oxidation (I), amino-acid transamination (II), and oxidative decarboxylation (III), and the results of the animal feeding experiments suggest that chloroacetaldehyde (g) chloroacetic acid (h), and 5-(2-carboxymethyl)cysteine (i) might lie on a com mon pathway connecting vinyl chloride (e) with thiodiglycollic acid (j).'However, other evidence im plies that chloroacetic arid (h) does not belong to this metabolic pathway (e-j). Thus, < 0.1% has even been detected in the body fluids of any of our vinyl ch(NHjh:o,h HOjC-CH* s V* CM:NHjIC0jH mu SlCHjCOjH) (1) -CSH I CI -0 CH-CHjSCHjCHO I -CSH C-0 CMCHjSCMjCHj OH COjH NCI HHlCAHcjt SCHjCOHHj OH -OH I cI *o CHCHjSCHjCOjH NI H CHCHjSCHjCOjH NHj III ""CMj sw -H W' SCHjCOjHj <fl 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 pro duced 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. A feasible metabolic pathway for thiodiglycollic acid from chloroacetic acid and involving cysteine desulfhydrase is unacceptable. Experiments with unlabeled vinyl chloride in rats in which the cysteine-cystine pools had been labeled adequately with ,4C gave [,4C]thiodiglycollic acid, showing that a part of the C-skeleton must be derived in fact from cysteine. In rats treated with chloroacetaldehyde, the presence of thiodiglycollic acid and N-acetyl-5-(2-hydroxyethyl)cysteine, but not of chloroacetic acid, among the urinary metabolites was established by mass fragmentometry. Thus, it is probable that in vivo chloroethylene 56 Environmental Health Perspectives r i SL 066680 c to oxide (0 was formed (5) from vinyl chloride (e) and transformed spontaneously (6) into chloroacetal- dehyde (g): there is supporting evidence (7-10) for vinyl chloride epoxidation in vitro. This supposition is supported by the facts that chloroacetaldehyde affords both JV-acetyl-S-(2-hydroxyethyl)cy5teine and thiodiglycollic acid in vivo and that S-(2carboxymethyl) cysteine has been identified by mass fragmentometry amongst the hydrolytic products of an hepatic extract prepared from vinyl chloride* treated animals. Since chloroacetaldehyde and chloroethylene 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 90-D-2'-deoxy ribofur- ano$ylimidazo-[2,l*i]purine or 3/S-D-2'-deoxy ribofuranosyl-2-oxo-2,3-dihydroimidazo-[ 1,2-c]pyrimidine from deoxy adenosine or deoxy cytidine by reaction with chloroacetaldehyde (13) or chloro* ethylene oxide was readily confirmed. Recognition of the nucleoside units of DNA that were modified by reaction with active vinyl chloride metabolites in vivo provides opportunity for the construction from appropriate animal data of the corresponding dose* response, time*response relationships, in compari* son with the ones for tumor incidence/occurrence in those animals. The presence of these two imidazonucleoside derivatives has now been estab lished by mass fragmentometry (16) in the enzymic hydrolysate of modified rat-liver DNA, prepared from rats, which had been exposed chronically to vinyl chloride (250 ppm in their drinking water) for 1 year (Fig. I). A smaller proportion of the 9/9-D*2'-deoxy ribofuranosylimidazo[2,I-i]purine, than would have been expected to* have been formed, was found both in the animal experiments with vinyl chloride and in model reac tions between chloroacetaldehyde and calf thymus DNA (76). 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 pres ence of the detached purine, viz., imidazo-[2,l-i] purine. Hence, the alkylation that produces imidazo-derivative formation (with DNA) labilizes the N-purine /3-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 im portant, because in general, there is excellent agreement between the severe damaging effect of depurination to DNA and mutagenicity (17-19). Thus, in retrospect, one would suspect vinyl chloride of being mutagenic/carcinogenic. On the other hand, vinylidene chloride (k) metabolism in rats gave thiodiglycollic acid (r) and CICHCHO + DNA t modified DNA QR HC-CHCI in living rt$ r in nvo modified hepatocyte DNA -------------------------[______ <_____________ 1 HO OH HO OH , and asjociated 'depurination* Figure I, Scheme suggesting the model reaction at chloroacetaldehyde with (calf-thymus) DNA and the biotransformation of hepatocyte DNA by vinyl chloride in wVo. Both reaction processes afford 30-0-2' -deoxy ribofur* noiyl-30-oxo-2,3-dihydroimidzo-l 1,2-c jpyrimvdine (left-hand side) and 9'0-o-2`-deoxy ribofuranosylimidazo[2.1-i]purine (right-hand side). an iV-acetyl-5-cysteinyl-acetyl derivative (p) as major urinary metabolites, plus substantial amounts of chloroacetic acid (I), dithioglycollic acid (t) and thioglycollic acid (s) (20). It is probable that chloroacetic acid (1), which is a vinylidene chloride metabolite per se, lies on a major metabolic path way for vinylidene chloride, since it affords several metabolites in common with vinylidene chloride m There is a strong supposition that detoxification of chloroacetic acid (1) is effected through a gluta thione 5-acyl transferase-catalyzed reaction pro cess and ensuing degradative sequence for the re sulting carboxymethyigiutathione (n), and that this represents the principal metabolic pathway for chloroacetic acid and a major one for vinylidene chloride. Thiodiglycollic acid is the ultimate detox ification product, and previous work (?) established the biotransformation of 5-(2-carboxymethyl) cys teine (g) into that substance. A feasible metabolic pathway to thiodiglycollic acid from chloroacetic acid and involving cysteine desullhydrase is unac ceptable. In experiments (rats) with unlabeled vinylidene chloride in which the cysteine-cystine pools had been labeled with 14C, labeled thiodigly collic acid resulted, and a part of the C-skeleton of December 1977 57 SL 066681 that substance must be derived in fact from cysteine (20). Formation of a small amount of [uC]thiodiglycollic acid (t) (and hence of the intermediate ['"Clthiodiglycollic acid) (s) is reconcilable with the action of Michaelis's (21) unspecific -thionase, which would lyse a small proportion of the prepon derating [uC]thiodig!ycoliic acid. Moreover, Kolbe electrolysis (22) of one molecu lar proportion of the [uC]thiodigIyco!lic acid metabolite from [l-1'lC]l.l-dichloroethylene or [l-'^Cjchloroacetic acid gave one equivalent of 1-'CO.(2J), and this evidence is consistent with the transformation of vinylidene chloride into chioroacetic acid by a mechanism involving migra tion of one Cl atom and the loss of the other one (20.23). Hence, the metabolic pathway 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 CO- and urea are produced by the action of epoxide hydratase on 1,1-dichloroethylene oxide or by a minor oxidative pathway for chioroacetic acid. There is a strong supposition that the N-acetyl-5-cysteinylacetyl derivative (p), which is a metabolite of vinylidene chloride, but not of chioroacetic acid, may be formed in fact from 1,1-dichloroethylene oxide through the agency of glutathione 5-epoxide transferase to afford 5-glutathione acetyl chloride (m) and its subsequent reactions (20). This supposition is important, since the reactivity displayed by 1,1-dichloroethylene oxide appears to be relevant to the possible interac tion of reactive vinylidene chloride metabolites with mouse kidney DN A (Fig. 2). which is a prerequisite of tumor initiation (24). Such 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 is in progress to investigate this hypothesis. Comparative studies (25) provide clues of differ ences between rats and mice in the processing of vinylidene chloride (Table 1). Thus, in mice, the production of thiodiglycollic acid is considerably reduced and the formation of the /V-acetyl-5cysteinylacetyl metabolite is increased. The higher j3-ihionase 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 ex creted a proportion of a dose of chioroacetic acid as unchanged starting acid. Thus, in mice, the metabolic pathway from chioroacetic acid to thiodiglycollic acid seems to be readily saturable, possibly on account of an inadequacy in the reac tion catalysed by glutathione 5-acyl transferase. Under these circumstances, detoxification of 58 CtCHiCit * Figure 2. Scheme suggesting the feasible interaction ofreactive vinylidene chloride metabolites. 1,1-dichloroethylene oxide and chloroacetyl chloride, with adenosine and cytidine re spectively. Table 1. Relative proportion of products from metabolism of chloroacctie add and vinylidene chloride in rata and mice. Yield--of metaboVlit"es, 96 Mice Substrate Chioroacetic add Metabolite Chioroacetic acid Thiodiglycollic acid N-Acetyl-S-<2-cart>oxy methyl) cysteine Rats - 90 2 Yllner BKJ-DEH 6-22 - 37 30-40 40 40 Vinylidene chloride Chioroacetic acid Thiodiglycollic acid Thioglycollic acid Dithioglycollic acid N-Acetyl-5-Cysieinyl- acetyl derivative 3 37 3 J 48 3 5 20 70 1,1-dichloroethylene oxide by glutathione 5-epoxide transferase and the modification of DNA by 1.1-dichloroethylene oxide or chloroacetyl chloride would be expected to be more significant in mice than in rats. This diagnosis of species suscep tibility seems to accord with Maltoni's (24) discov ery of vinylidene chloride oncogenicity in (the kid neys of) mice. Environmental Health Perspectives i i i A i *X i > SL 066682 V perh f be dat cum" whit Th and b. G. H. 1. C vin Inti 2. C b r... 3. Og 0-6 a c1 4. Da ein d J. ' c 2 6. G C 7. J Ills 8. Ba aiv t 9. C cin bo I ( 10. i De Vinyltdene chloride emerges as an agent of low, perhaps very low, oncogenic potential, which can be damaging only in a special set of biological cir cumstances. which we have partially defined and on which work is continuing. 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. REFERENCES 1. Green, T., and Hathway, D. E. The biological fate in rats of vinyl chloride in relation to its oncogenicity. Chcm. Biol. Interact. II: 545 (197JI. 2. Green. T,. and Hathway. D. E. The chemistry and bn'penesis of 5-containing metabolites of vinyl chloride in rji> Chern. Biol, Interact. 7: 137 (1977). 3. Oc-ton, A. G.. et al. The replacement reactions of d./3 -dichlorodiethyl sulphide and of some analogues in aqueous solution: the isolation of 0-chloro-0'-hydroxydiethylsulphide. Trans. Faraday Soc. 44: 43 (1948). 4. Dabritz, E., and Virtanen, A. I. 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Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in S. typhimiiritim strains, Int. J. Cancer 13: 429 11975). 12. Malaveille C.. et al. Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacctaldehyde and chloroethanol. Biochem. Biophys. Res. Commun. 65: 36J (1975). 13. McCann. J.. et al. Mutagenicity of chloroacetaldehyde. a possible metabolic product of 1.2-dichloroethanc (ethylene dichloride), chloroethano! (ethylene chlorohydrin). vinyl chloride and cyclophosphamide. Proc. Nat. Acad. Sci. U.S.A. 72: 3190(1973). 14. Huberman, E,. Bartsch. H.. and Sachs. L. Mutation induc tion in Chinese hamster V79 cells by two vinyl chloride metabolites, chloroethylene oxide and chloroacetaldehyde. Int. J. Cancer IS: 539(1973). 15. Barrio. J. R,. Secrist, J. A., and Leonard. N, J, Fluorescent adenosine and cytidine derivatives. Biochem. Biophys. Res. Commun. 46: 597 (1972). 16. Green. T., and Hathway. O. E. Interactions of vinyl chloride with rat-liver DNA in vivo. Chem. Biol. Interact. In press. 17. Lawley. P. D.. et al. Inactivation of bacteriophage T7 by mono- and di-functional sulphur mustards in relation to moss- linking and depurination of bacteriophage DNA. J. Mol. Biol. 39: 181 (19691. 18. Roberts. J. J. Nucleic acid modifications and qiwcer. 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 EfTects of Alkylating Agents. Butterworths. London. 1966. 20. Jones. B. K.. and Hathway. D. E. The biological fate of vinylidene chloride in rats. Chem. Biol. Interact, In press. 21. Michaelis, L.. and Schubert. M. P. The reaction of iodoacctic acid on mercaptans and amides. J. Biot. Chem. 106: 331 (1934). 22. Kolbc. H. Untersuehungen uberdie Elektrolyse organischer Verbindungen. Justus Liebigs Ann. Chem. 69: 2J7 (1849), 23. Walker, G. H.. and Hathway. D- E. Electrochemical analysis of the [carboxy*IJC)aliphattC carboxylic acid me tabolites resulting from tracer molecules. Biochem. J. 167:505 (1977). 24. Maltoni. C. Proceedings of the TAPPI International Con ference. Hamburg- January 26. 1977. 25. Jones. B. K., and Hathway. D. E. Differences between mice and rats in the metabolism of vinylidene chloride. Br. J. Cancer, In press. 26. Yltner. S. Metabolism of chloroacetate-l-,-,C in the mouse. Acta Pharmacol. Toxicol. 30: 69 (1971). 3 December 1977 I' SL 066683 59