Document Bke2mdj31nMMejqDZwo4Gj6L

Environmental Health Perspectives Vnl. 21. pp. 15-59. 1977 Comparative Mammalian Metabolism of Vinyl Chloride and Vinyiidene Chloride in Relation to Oncogenic Potential by David E. Hathway* Elucidation of the role of tin} 1 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 vinyiidene 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 vinyiidene 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, N-acetyl-5-(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-acetyliS'-(2-chloroethyl)cysteine or of /V-acetyl-5-(2-hydroxyethyl)cysteine was isolated from body fluids (/, 2). Thus, by Fischer-Speier methylation,iV-acetyl-5-(2-chloroethyl)cysteirie was obtained, and with diazomethane, /V-acetyl'S-(2-hydroxyethyl)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 O-methyl ester of iV-acetyl-5-(2-hydroxyethyl)cysteine (a) with the methanol-HCl reagent gave a mixture of /V-acetyl-5-(2-chIoroethyI)cysteine (b), and S(2-chloroethyl)cysteine, and conversely, the O-methyl ester of JV-acetyl-S-(2-chloroethyl)cysteine (b) was hydrolyzed rapidly by water to that of /V-acetyl-S-(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-limitmg- irr respect of the hydrolysis of (a) OH %y H^l nH SR +0H' -- -OH' *. (c) (b) H.C-CH, \1 S1 Cl 1M *" --*cr - H"l '"H -cr SR CH, I` CHlNHAcICO^ -H+ "Imperial Chemical Industries Limited, Central Toxicology Laboratory. Aiderley Park, Cheshire SKI0 4TJ, England. December 1V77 (d) 55 SL 097446 r% X JV-acetyl--<2-chloroethyl)cysteine. Nucleophilic attack of OH- on the episulfonium ion would be expected--to'give olefin (2), and in fact. N- acetyl-5-vinylcysteine (d) (2) was recovered from the urine of vinyl chloride-treated animals whenever diazomethane esterification was used to protect S-containing metabolites. Surprisingly, AT-acetyl-5-(2-hydroxyethyl)cysteine O-methyl ester was methylated with neu tral methanol, and the 0-methyl esters of N-acetylS-(2-methoxy[14C]ethyl)cysteine plus N-acetyfS-[14C]vinyl-cysteine degrade to give the volatile [14C]S-(2-methoxyethyl) (prop-1 or 2-enyl) sulfide. Although the mechanism of formation was not inves tigated, we felt that acetaldehyde, a known dissocia tion pnoduct of 5-vinylcysteine-deci'ved ^5-vinylcysteine-5-oxide (?) might undergo concerted con-' densation with N-acetyl-5-(2-methoxyeihyl)cy$teine 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 S-containing metabolites of vinyl chloride did not behave as mutagens in the Ames test (2). 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)3 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 S-(2-carboxymethyl)cysteine (i) might lie on a com mon pathway connecting vinyl chloride (e) with thiotfaKycottic 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 OH HjC-CHj sI CH'NHjICOjH (III H0jC-Ch2 5I Ch NHjlCOjK C-OO.H SICHjCOjHI U) CHCHzSCHzCH2 mi OH CHCHjSCHjCOjH m1 CI 0l,H CHCHjSCH2CHz NHIAcI oh -OH *OH COf CHCHjJCHjCOjH lil r-s'6<ih* CO^ C'CH^SCHjCO^H 0 -H' R-Sv ;c-CHj H' SICHjCOjHIj lj) 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 ``C gave [uCjthiodiglycollic 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 (V-acetyl-S-fi-hydroxy- ethyflcysteine, bui not of chloroacetic acid, among the urinary metabolites was established by mass fragmentometry. Thus, it is probable that in vivo chloroethylene Environmental Health Perspectives HIP" . iipppygm'"!) SL 097447 oxide (f) was formed (5) from vinyl chloride (e) and CICHCHO HC = CHCI transformed spontaneously (<5) into chloroacetal- fDNA in living nils deh>de <g): there is supporting evidence (7-/0) for vin\I chloride epoxidation in vitro. This supposition is supported by the facts that chloroacetaidehyde affords both <V-acetyl-5-(2-Jiydfoxyethyl)cysteine and thiodiglyccllic acid in vivo and that 5-(2- modified DNA in m o modified carboxymethyl) cysteine has been identified by mass , hepuiocyte DNA fragmentometry amongst the hydrolytic products of an hepatic extract prepared from vinyl chlcride- tteated animals. Since chloroacetaidehyde and chloroethjlene 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 9/3-d-2'-deoxy ribofur- anosylimidazo-[2,1 -iJpurine or 3/?-D-2'-deoxy ribofuranosyl-2-oxo-2,3-dihydroimidazo-[l,2-c]py- rimidine from deoxy adenosine or deoxy cytidine by reaction with chloroacetaidehyde (15) or chloro- ethylene oxide was readily confirmed. Recognition of the nucleoside units of DNA that were modified HO OH HO OH and associated by reaction with active vinyl chloride metabolites in `depurination1 vivo provides opportunity for the construction from appropriate animal data of the corresponding dose- Figure I. Scheme suggesting the model reaction of response, time-response relationships, in compari chloroacetaidehyde with (calf-thymus) DNA and the bio 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 transformation of hepatocyte DNA by vinyl chloride in vivo. Both reaction processes afford 3/3-D-2'-deoxy ribofura nosy 1-3 /3-oxo-2,3-dihydroimidazo-[ 1,2-c]pyrimidine (left-hand side) and 9'0-D-2'-deoxy ribofuranosylimidazo[2,l-i]purine (right-hand side). hydrolysate of modified rat-liver DNA, prepared from rats, which had been exposed chronically to an /V-acetyl-S-cysteinyl-acetyl derivative (p) as vinyl chloride (250 ppm in their drinking water) major urinary metabolites, plus substantial amounts for I year (Fig. 1). A smaller proportion of of chloroacetic acid (1), dithioglycollic acid (t) and the 9/3-D-2'-deoxy ribofuranosylimidazo- thioglycollic acid (s) (20). It is probable that [2,l-i]purine, than would have been expected to chloroacetic acid (1), which is a vinylidene chloride have been formed, was found both in the animal metabolite per se, lies on a major metabolic path experiments with vinyl chloride and in model reac way for vinylidene chloride, since it affords several tions between chloroacetaidehyde and calf thymus metabolites in common with vinylidene chloride DNA' (16). This observation is consistent with Q0). some degree of DNA depurination brought about There is a strong supposition that detoxification by the reaction of vinyl chloride, and in our model of chloroacetic acid (1) is effected through a gluta experiments, we have found evidence for the pres thione 5-acyl transferase-catalyzed reaction pro ence of the detached purine, viz., imidazo-[2,l-i] cess and ensuing degradative sequence for the re purine. Hence, the alkylation that produces sulting carboxymethylglutathione (n), and that this imidazo-derivative formation (with DNA) labilizes represents the principal metabolic pathway for the N^purine /3-glycoside .linkage, which leads to chloroacetic acid and a major one for vinylidene depurination. The gap so produced might then be chloride. Thiodiglycollic acid is the ultimate detox filled by various bases* resulting in "mispairing" ification product, and previous work (2) established during DNA replication. These results are very im the biotransformation of 5-(2-carboxymethyl) cys portant, because in general, there is excellent teine (g) into that substance. A feasible metabolic agreement between the severe damaging effect of pathway to thiodiglycollic acid from chloroacetic depurination to DNA and mutagenicity (17-19). acid and involving cysteine desulfhydrase is unac- Thus, in retrospect, one would suspect vinyl ceptable. In experiments (rats) with- unlabeled chloride of being mutagenic/carcinogenic. vinylidene chloride in which the cysteine-cystine On the other hand, vinylidene chloride (k) pools had been labeled with MC, labeled thiodigly metabolism in rats gave thiodiglycollic acid (r) and collic acid resulted, and a part of the C-skeleton of December 1977 57 SL 097448 that substance must be derived in fact from cysteine (20). Formation of a small amount of [l4C]thiodiglycollic actd (t) (and hence of the intermediate [HC]thiodiglycollic acid) (s) is reconcilable with the action of Michaelis's (21) unspecific /3-thionase, which would lyse a small proportion of the prepon derating [,4C]thiodiglycolIic acid. Moreover, Kolbe electrolysis (22) of one molecu lar proportion of the [l4Clthiodiglycollic acid metabolite from [ l-14C] 1.1-dichloroethylene or [l-,4C]ch!oroacetic acid gave one equivalent of ,4CO..(22), 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 CO, 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 iV-acetyl-5-cysteinylacetyl derivative (p), 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 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 w ith 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-Scysteinytacetyl metabolite is increased. The higher /3-thionase 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 chloroacetic acid as unchanged starting acid. Thus, in mice, the metabolic pathway from chlorohcetic 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 CtCHiUl o Figlrl 2. Scheme sucjiesting the feasihle interaction of reactive wnvlidene chloride metabolites, l.l-dichloructhylene oxide and ehloroucetyI chloride, with adenosine and cytidine re spectively . Table 1. Reljtiie proportion of products from metabolism of chloroacetic acid and vinylidene chloride in rats and mice. Yield of metabolites. 9? Substrate Chloroacetic acid Metabolite Rats Chloroacetic acid Thiodiglycollic acid X - Xcetv l-5-(2-carboxy- methyl) cysteine - 90 T Vinvhdene chloride C hloioacetic acid 1 hiodigly collie acid ThsoeUcollie acid Dithioglycollic acid X -Acetyl-X-cysteinyl- acetyi derivative 3 37 3 5 48 Mice X liner BKJ-DEH Cr-22 _ 37 30-40 40 40 3 5 20 70 I ,l-dichloroethylene oxide by glutathione 5-epoxide transferase and the modification of DNA by 1.1-dichlorocthylene oxide or chloroacetyl chloride would be expected to be more significant in mice than in rats. ThN 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 SL 097449  Vin> liiiene chloride emerges as an agent of low, perhaps very low, oncogenic potential, which can be damaging only in a special set of biological circum>tances. which we hjjve partially defined and on which work is continuing. The author k indebted to his colleagues Messrs T Green, .nid B. k, Jones. Drs. A, G. Salmon and P L Batten, and Mr. G. H. Walker tor their invaluable eontubutions and help REFERENCES 1. Green. T . and Hathway. D. E. 1 he biological fate in rats of xinyl chloride in relation to its oncogenicity. Chem. Biol. Interact. II: 545 (1975). 2. Green, T.. and Hathway. D. E. The chemistry and biogenesis of i'-conlaining metabolites of vinyl chloride m rats. Chem. Biol. Inteiact. 7: 137 (1977). 3. Ogston. A. G.. et al. The replacement reactions of (3,/J -dichlorodiethyl sulphide and of some analogues in aqueous solution: the isolation of d-chloro-0-hydroxydiethylsulphide. Trans. Faraday Soc, 44- 45 (1948). 4. Diibritz, E.. and Virtanen, A. I. 5-Vinyl-cystein-S-oxid, ein Homologes zur Vorstufe der triinentreibenden Substaru derZwiebel. Chem. Ber. 98: 781 (1963). 5. Van Duuren. B. L. On the possible mechanism of car cinogenic action of vinvl chloride. Ann-. N.Y. Acad. Sci. 246:258 (1975), 6. Gross, H.. and Freiburg. J. Zur F.xistenz von Chlorathylenoxid. J. Prakt. Chem. 311: 506 (1969). 7. Rannug. U.. et al. The mutagenicity of vinyl chloride after metabolic activation. Ambio 3: 194 (1974).. 8. Barbin, A., et al. IJser-microsome mediated formation of alkylating agents from vinyl bromide and vinyl chloride. Biochem. Biophys. Res. Commun. 67: 596 (1975). 9. Greim. H., et al. Mutagenicity m vitro and potential car cinogenicity of chlorinated ethylcnes as a function of meta bolic oxirane formation. Biochem. Pharmacol. 24: 2013 (1975). 10. Salmon, A. G. Cytochrome P450 and the metabolism ot vinyl chloride. Cancer Letters 2: 109 (1976). 11 Bartsch. H., Malaveille. C . and Montesano. R. Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in S. ttfilwnttriiim strains, Int. J. Cancer 15: 429(1975). 12 Malaveille C . et al. Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacetaldchyde and chloroethunol. Biochem, Riophvs. Res Commun. 65, 363 (PCS). It McCann. J , et al. Mutagenicity of chloroacctaldehyde, a possible metabolic product of 1,2-dichloroethane (ethylene dichloride). chloioethanol (ethylene chlorohydrin), vinyl chlonde and cyclophosphamide Proc Nat, Acad. Set. L.S.A. 72: 3190 (1975). 14 Huberman. F.. 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 15: 539 (1975). 15 Barrio. J. R.. Secrist. J. A., and Leonard. N. J. Fluorescent adenosine and c\tidine 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. Lauley, P. D., et al. Inactivation of bacteriophage T7 by mono- and di-functional sulphur mustards in relation to crosslinking and depurination 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. 21. Michaelis. L., and Schubert. M. P. The reaction of iodoace- tic acid on mercaptans and amides. J. Biol. Chem. 106: 331 (1934). 22. Kolbe. H. Untersuchungen iiberdie Elektrolyse organischer Verbindungen. Justus Liebigs Ann. Chem. 69: 237 (1849), 23. Walker. G. H., and Hathway, D. E. Electrochemical analysis of the [carboxy*'*C]aliphatic 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 xinylidene chloride. Br. J. Cancer In press. 26. Yllner, S. Metabolism of chloroacetate-l-'*C in the mouse. Acta Pharmacol. Toxicol. 30: 69 (1971). CrfUMtflfc v.UbWI December 1977 59 SL 097450