Document 65pO8n5MoynvvMwRLrGXO5JZR

0.Ti5 -ysr -HI ^ Enxironmfnta] Health Perspectives Vol. 2I,pp.5SS9,j977 Comparative Mammalian Metabolism of Vinyl Chloride and Vinylidene Chloride in Relation to Oncogenic Potential by David E. Hathway* Elucid*tioo of the role of vinyl chloride metabolite* In the varioai reaction aequcncoe which comprise the metabolic pathway, including the interaction of reactive metabolites with some purine and pyrimidine rwMws of target-organ DNA, proridw some explanation far the (oucogtnic) properties associated with the original substance. Comparative farssrigattop of tbe biological fate of vinylidene chloride reveals an agent of low oncogenic potential which is likely to be damaging only under special circumstances, and species differences which suggest that the mouse Is 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 ofthe 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. N-acetyl-5-(2-hydroxymethyl)cysteine was shown to be a m^jor vinyl chloride metabolite in rats, but according to the method of protective esterification that was used so a derivative either of jV-acetyl-5-(2-chloroethyI)cysteine or of N-acetyl-S-(2-hydroxyethyl)cysteine was isolated from body fluids (1, 2). Thus, by Fischer-Speier mtthylation.N-acetyl-S -(2-chloroethyl)cysteine was obtained, and with diazomethane, Af-acetylS-(2-hydroxycthyI)cysteine. It might be stated 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-methyI ester of `Imperial Chemical Industries Limited, Centra! Toxicology Laboratory, Aidcriey Park, Cheshire SKlO 4T3. England. December 1977 iV-acetyI-S-(2-hydroxyethyl)cysteine (a) with the methanol-HCl reagent gave a mixture of Ar-acetyl-5-(2-chIoroethyl)cysteine (b), and 5(2-chloroethyl)cysteine, and conversely, the D-methyl ester of AJ-acetyi-5-(2-chloroethyl)- cysteine (b) was hydrolyzed rapidly by water to that of N-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 of the hydrolysis of la) OH V kv % ** +0H* -OH' (c) 2Jh2 C5l1 H2, <b> Cl <r -c r -. HH-x^cl -cI.^^H SR CHINHAdCOgH -H+ Hx .C-C RS'" VH (dl 55 AP00008317 t i N*acetyl-S-(2-chloroethyl)cysteine. Nucleophilic attack of OH~ on the episulfonium ion would be expected to give olefin CJ), and in fact, Nacetyl-S-vinylcysteine (d) (2) was recovered from Ihe urine of vinyl chloride-treated animals whenever diazomethane esterification was used to protect S-containing metabolites. Surprisingly, N-acetyl-5-(2-hydroxyethyl)cy- steine O-methyl ester was methylated with neu tral methanol, and the 0-methyl esters of N-acetyl5-(2-methoxy[,4C]ethyl)cysteine plus N-acetyl5-[14C]vinyl-cysteine degrade to give the volatile [MC]S-(2-methoxyethyl) (prop-1 or 2-enyl) sulfide. Although the mechanism offormation was not inves tigated, we felt that acetaldehyde, a known dissocia tion product of 5-vinylcysteine-derived 5-vinyl1 cysleine-5-oxide (0 might undergo concerted con densation with N-acetyI-S-(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 and Virtanen (*) 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-Hydroxyethyl)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 (111), and the results of the animal feeding experiments suggest that chioroacetaldehyde (g) chloroacetic acid (h), and 5-(2-carboxymethy])cysteine (i) might lie on a com mon pathway connecting vinyl chloride (e) with thiodiglycollic acid (j). However, other evidence im plies that chloroacetic acid (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 r ID illi-av-o* --iUL! SIC^CC^W |"a (1) UJ HjC-CHCI in <> ,*MH i.o \ / (jW-CHjSCHjCHO NH (hi CICHjCOOH /i / +GSH // / C-0 ljHCHjSCH2CH2 NI H OT NHtXH -OH OH *)H* '--CH, CHCHjSCHjCO^H CHCH^SCHjCOjH NH, <i> CC^H ft'CM2SCM2CC^H 0 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 vinytidene 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 |MC]thiodiglycolIic acid, showing that a part of the C-skeleton must be derived in fact from cysteine. In rats treated with chioroacetaldehyde. the presence of thiodiglycollic acid and N-acetyl-S-(2-hydroxyethyljcysteine, 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 AP00008318 I i oxide (f) was formed (5) from vinyl chloride (e) and transformed spontaneously (6) into chloroacetal- dehyde (g); there is supporting evidence (7-70) for vinyl chloride epoxidation in vitro. This supposition is supported by the facts that chloroacetaldehyde affords both M-acetyl-S-(2-hydroxythyl)cysteme and thiodiglycollic add in vivo and that 5-(2carboxymethyl) cysteine has been identified by mass fragmentometry amongst the hydrolytic products of an hepatic extract prepared from vinyl chloridetreated animals. Since chloroacetaldehyde and chloroethylene oxide are mutagenic in the Ames test (77-/3) and in Chinese hamster V79 cells (/*), they may be relevant to vinyl chloride carcinogenicity. Respective formation of 9/8-D-2'-deoxy ribofur- anosy!imidazo-[2,l-i]purine or 30-D-2'-deoxy ribofuranosyl-2-oxo-2,3*dihydroimidazo-[l,2-c]pyrimidine from deoxy adenosine or deoxy cytidine by reaction with chloroacetaldehyde {15) or chloro ethylene oxide was readily confirmed. Recognition of the nucleoside units of DN A that were modified by reaction with active vinyl chloride metabolites in vivo provides opportunity for the construction from appropriate animal data of the corresponding doseresponse, 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 (]6) in the enzymic hydrolysate of modified rat-liver DNA, prepared from rats, which had been exposed chronically to vinyl chloride (230 ppm in their drinking water) for 1 year (Fig. 1). A smaller proportion of the 9/8-D-2'-deoxy ribofuranosylimidazo- [2,I-t]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 C1CHCHO -I DNA modified DNA HC-CHC1 in living rats in vivo modified hepaloeyle DNA HO OH HO OH and associated 'depurination* Figure. 1. Scheme suggesting tVie mode) reaction of chloroacetaldehyde with Icalf-ihymusl DNA and the biotransformation ofhepatocyte DNA by vinyl chloride in vivo. Both reaction processes afford 3/3-D-2'-deoxy ribofuranosyl-3/J-oxo-2,3-dihydroimidazo- [ 1,2 -clpyrimidine (left-hand side} and 9'-P-2'-deoxy ribofuranosylimidazo[2,1-Qpurine (right-hand side). an W-acetyl-J-cysteinyJ-acetyl derivative (p) as major urinary metabolites, plus substantial amounts of chloroacetic acid (1). dithioglycollic acid (t) and thioglycollic acid (s) {20). It is probable that chloroacetic acid (I), 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 (20). There is a strong supposition that detoxification of chloroacetic acid 0) is effected through a gluta thione 5-acyl transferase-catalyzed reaction pro cess and ensuing degradative sequence for the re sulting carboxymethylglutathione (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 S-(2-carboxymethyl) cys teine (g) into that substance. A feasible metabolic pathway to thiodiglycollic acid from chloroacetic acid and involving cysteine desulfhydrase is unac ceptable. In experiments (rats) with unlabeled vinylidene chloride in which the cysteine-cystine pools had been labeled with l4C, labeled thiodiglycollic acid resulted, and a part of the C-skeleton of December 1977 57 AP00008319 T that substance must be derived in fact from cysteine (2d). Formation of a small amount of [1*C]thiodiglycollic acid (t) (and hence of the intermediate [,4CJthiodigIycollic acid) (s) is reconcilable with the action of Michaelis's Ql) unspecific 0-thionase, which would lyse a small proportion of the prepon derating [l4Cjthiodig1ycollic acid. Moreover, Kolbe electrolysis (22) of one molecu lar proportion of the [l4C]thiodrglycollic acid metabolite from M-,*C]l.I-dichloroethyIene or [l-HC]chloroacetic acid gave one equivalent of MCO*(2Jh and this evidence is consistent with the transformation of vinylidene chloride into chloroacetic 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 C02 and urea are produced by the action of epoxide hydratase on l.l-dichloroethylene oxide or by a minor oxidative pathway for chloroacetic acid. There is a strong supposition that the Ar-acetyl-5-cysteinylacetyl derivative (p), which is a metabolite of vinylidene chloride, but not of chloroacetic acid, may be formed in fact from l.l-dichloroethylene oxide through the agency of glutathione 5-epoxide transferase to afford 5-giutathione acetyl chloride (m> and its subsequent reactions (20). This supposition is important, since the reactivity displayed by l.l-dichloroethylene oxide appears to be relevant to the possible interac tion of reactive vinylidene chloride metabolites with mouse kidney DNA (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 imida2o 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 l). Thus, in mice, the production of thiodiglycollic acid is considerably reduced and the formation of the A/.acety)-5cysteinylacetyl metabolite 3s increased. The higher -thionase activity in mice than in rats accounts for the greater conversion of thiodiglycollic acid into dithioglycollic acid vin thioglycollic acid in the former species of animal. YUner's (26) mice ex creted 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 reac tion catalysed by glutathione 5-acyl transferase. Under these circumstances, detoxification of 58 Figure 2. Scheme suggesting the feasible interaction ofreactive vinylidene chloride metabolites, l.l-dichloroethylene oxide and cMoroacetyl chloride, with adenosine and eytidine re* spcctively. Table 1. Relative proportion of products from metabolism of chloroacetic add and vinylidene chloride in rats and mice. Yield of metabolites. Sfr Mice Substrate Chloroacetic acid Metabolite Rats Chloroacetic arid Thiodiglycollic acid A,-Acety|,5-<2-carbox)1- methyl) cysteine 90 2 Ytlner BKJ-DEH 6-22 37 30-40 40 40 Vinylidene chloride Chloroacetic arid Thiodiglycollic arid Thioglycollic arid Dithioglycollic arid A'-Aeetyl-S-cysteinyl- acetyl derivative 3 37 3 S 43 3 3 20 70 l.l-dichloroethylene oxide by glutathione 5-epoxide transferase and the modification of DNA by l.l-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 AP00008320 Vinylidene 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 Hath* ay. D. E. The biological fate in rats of vinyl chloride in relation to its oncogenicity. Chem. Biol. Interact. N: 545 <19751. 2. Green. T.. and Halfway. D. E. The chemistry and biogenesis of ^-containing metabolites of vinyl chloride in rais. Chem. Biol. Interact. 7: 137 11977). 3. Ogston. A. G.. et al. The replacement reactions of ./3'-dich)orodiethyi sulphide and of some analogues fn aqueous solution: the isolation of -chloro-'-hydroxydiethylsulpbide. Trans. Faraday Soc. 44: 4? (1948). 4. Dabriu, E., and Virtanen, A. I. S-Vinyl-eystefn-J-oxid. ein Homologet zur Vorsiufe der tranenireibenden Substanz der Zwiebel. Chem. Ber. 96:781 (1965). 5. Van Duuren. B. L. On the possible mechanism of car cinogenic action of vinyl chloride. Ann. N.Y. Acad. Sci. 246: 258 (J975). 6. Gross. H,. and Freiburg. J. Zur Existenz von Chlorathylenoxid. J. Prakt. Chem. ?11: 506 (19691. 7. Rannug. U., el at. The mutagenicity of vinyl chloride after metabolic activation. Ambio 3: 194 (1974). 8. Barbin. A., et al. Liver-microsome mediated formation of alkylating agents from vinyl bromide and vinyl chloride. Biochem. Biophys. Res. Commun. 67: 596 119751. 9. Gretm. H.. cl al. Mutagenicity in vitro and potential car cinogenicity of chlorinated ethylenes as a function of meta bolic oxirane formation. Biochem. Pharmacol. 24: 201? (1975). 10. Salmon. A. G. Cytochrome P450 and the metabolism of vinyl chloride. Cancer Letters 2: 109 <19761. M. Barlach. H.. Malaveille.C.. and Momesano. R. Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in S. nphimurium strains, Int. J. Cancer 15:429 11975). 12. Malaveille C.. et a). Mutagenicity of vinyl chloride, chioroethylene oxide, chloroacetaidehyde and chlo- loethanoi. Biochem. Biophys. Res. Commun. 65: 363 (1975). 1?. McCann. J.. et al. Mutagenicity of chloroacetaidehyde. a possible metabolic product of 1.2-dichloroethane (ethylene dichloride), chloroethanol (ethylene chlorohydrin). vinyl chloride and cyclophosphamide. Proc. Nat. Acad, Sci. U.S.A. 72:3190(1975). 14. Huberman. E.. Bartsch. H.. and Sachs. L. Mutation induc tion in Chinese hamster V79 cells by two vinyl chloride metabolites, chioroethylene oxide and chloroacetaidehyde. Int. J, Cancer 15; 539 (1975). 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. D, 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 roustajds in relation to crosslinking and depurinadon 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. B. K.. and Hathway. D. E. The biological fate of vinylidene chloride in rats, Chem. Biol. Interact. In press. 2). Michaelis. L.. and Schubert. \f. P. The reaction ofiodoscetic acid on mercaptans and amides. J. Biol. Chem. 106: 331 (1934). 22. kolbe. H. Untersuehungen liber die Elektrolyse organischer Verbindungen. Justus Liebigs Ann. Chem. 69: 257 tl&49l. 23- Walker. G. H.. and Hathway. D. E. Electrochemical analysis of the [carboxy-^Claliphatic carboxylic acid me tabolites resulting from tracer molecules. Biochem. J. 167:505 (19771. 24. Maltoni. C. Proceedings of the TAPPl 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. YMner. S. Metabolism of chloroacetate-l-HC in the mouse. Acla Pharmacol. Toxicol. 30: 69 (1971). December 1977 59 AP00008321