Document Qkx4DR2qO665oyrEZb6EDo4qR

#>4' V tV7 A DOMINANT L f 1 H \ l SIUDY IN MALT KATS A! I r K KI PLAT U) l XPOSURLS TO VINYL CHLORIDE OR VtNYLIDLNL CHLORIDE Robert D. Short, fan L. Minor, Joseph M. Winston, Cheng-Chun Lee Pharmacology and foxicology, Midwest Research Institute, Kansas City, Missouri Male < n tuts wire e\poicJ 6 hr/day for S davs/wk to 0, SO, 250, or 1,000 ppm at wn\i (hinrUly m 55 ppm ot linyhdene ihhiridv. Starting on week 11 at e\posurt, '!, ( males were muted with untreated icmules. There u us no evideme or cither !'r,-i',,p<anUiti'>n lots or postimplunulmn hi's in priijnant females that resulhil tr< //;.>, mating'. Consequently, it was tom hided that then exposures did no' product ,/r1 minal mutation, as manifested by a dominant lethal ettect, in male rats. INTRODUCTION Vinyl chloride (VC) and vinylidene chloride (VDC) arc structurally related monomers used in the synthesis of plastics. VC is carcinogenic in both humans (C.recch and Johnson, 1974) and animals (Viola et al., 1971; M.ilumi and Lefcmine, 1975; Lee et al., 1977). In addition, both VC and VIK ate mutagenic in a variety of microbial systems (Loprieno ct al., 1976; LKirlsch ct al., 1975). Epidemiological studies suggest that VC produces germinal mutations, as manifested by increased fetal loss, in humans (Infante ct al., 1976). However, there was no evidence of a dominant lethal mutation in male mice exposed to VC for 5 days and sequentially mated with untreated females (Anderson et al., 1976). The purpose of this study was to determine whether repeated exposures to VC or VDC, for 11 wk, produced germinal mutations of the dominant lethal type in male rats. ME I HODS < D iais (Charles River Breeding Laboratories, North Wilmington, Vu'-v.n ipiv'I ts) were used in this study. Adult mates weighing 180-200 g 'u'h- exposed t> hr/day for 5 d<iys/wk to 0, 50, 250, or 1,000 ppm of VC *' ^ ppm of VDC (Lee et al., 1977). All rats were given free access to thi' mtiori gratefully ,i.knowledge ihe comju-ieni technical assistance of Bretl Ferguson and ' r, \ Kvi;o<-sts Ini refjnnis should he sent to Robert D. Short, Pharmacology and Toxicology, V|'l >s> >,i Kr mmh h InstiMile, 4.!5 Volkcr Boulevard, Kansas t ily, Missouri 64 J1U. htornal of toxicology and Environmental Health, 3:^65-96#, 1*37 7 *"pyright )077 by Hemisphere Publishing Corporation S. 1 Environmental Health Perspectives Vol. 21, pp. yvuj 77 1977. URL 18779 COMPARATIVE MAMMALIAN METABOLISM OF VINYL AND VINYLIDENE CHLORIDES IN RELATION TO ONCOGENIC POTENTIAL'**' * by David E Hathvay 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, Alderlcy Park, Cheshire SK10 ATJ, England. + Lecture delivered in the Symposium on Compar.i t i vc Metabolism and Toxicity of Vinyl Chloride Related Compounds (NIEHS), held at Bethesda, .. i 2 The research work with which this communication is concerned is based on the idea that knowledge ot 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-acetyi-S-(2-hydroxymethyi)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 raethylacion, URL 13780 W-acetyl-*S-(2-chloroethyl)cysteine was obtained, and with diazomethane, N-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 (Vmethyl ester of N-acetyl-S-(2-hydroxyethyl)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-acetyl-S-(2-chloroethyl)cysteine (b) was hydrolysed rapidly by water to that of W-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 oi this ion would in fact be rate-limiting in respect of the hydro) ysi s> of N-<ieetyl-S-(2-chloroethyi)cysteine . Nucleophilic attack of OH on the episulphonium ion would be expected to give olefin (3), and in fail. N .i.i-1 v I S v i nv I vr.f i no (0 wmi nu'ovnVil I nun Hie mine of vinyl J URL 18781 chloride-treated animals whenever diazomethane esterification was used to protect ^-containing metabolites. Surprisingly, N-acetyl-S-(2-hydroxyethyl)cysteine O-methyl ester was methylated with neutral methanol, and the O-methyl esters of N-acetyl--(2-raethoxy [^C]e thy 1) cysteine plus N-acetyl-S-[^C]vinyl-cysteine degrade to give the volatile [1^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 Sj-vinylcysteinederived S^vinylcysteine-S-oxide (4) might undergo concerted condensation with N-acetyl-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 & 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). Fig. 2 near here In order to determine whether vinyl chloride yielded chloroethylene oxide iii vivo, the biogenesis of several vinyl chloride metabolites and related compounds were investigated in rats (2). -(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 (Fig. 2) appears to include end-group oxidation, amino-acid transamination and oxidative decarboxylation, and the results of 1 In leedinr experiments suggest that chloroace t .il dehyde (g) (Fig. 3), 4 Fig. 3 near here chloroacetic acid (h), and S^(2-carboxymethyl)cysteine (i) might lie on a common 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) 40.1% 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 cysteine- cystine pools had been labelled adequately with gave [1^C]thiodiglycollic 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 (5) 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 the fact (A) that chloroacetaldehyde affords both IJ-acetyl--(2-hydroxyethyl) cysteine and thiodiglycollic acid in vivo and (B) that (2-carboxymethyl) cysteine has been identified by mass fragmentometry amongst the hydrolytic product fi of an hepatic extract prepared from vinyl chloride-treated animals. URL 18782 5 .m il 8783 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 30-D-ribofuranosylimidazo-[2,l-i]purine or 60-D-ribofuranosyl-5-oxo-5,6-dihydroimidazo-[1,2-cJpyrimidine from adenosine or cytidine by reaction with chloroacetaldehyde (15) or chloroethylene oxide was readily confirmed. The presence of these two iraidazo-nucleoside 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 near here proportion of the 30-D-ribofuranosylimidazo-(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. imida20-[2,l-i]purine. Hence, the alkylation that produces imidazo-derivative formation (with DNA) labilizes the N^ purine 6-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). Thus, in retrospect, one would suspect vinyl chloride of being mutagenic/ carcinogenic. 6 URL 18784 On the other hand* vinylidene chloride (a) (Fig. 5) metabolism in Fig. 5 rats gave thiodiglycollic acid (g) and an N-acetyl-S-cysteiny1-acetyl near here derivative (e) as major urinary metabolites, plus substantial amounts of chloroacetic acid (b), dithioglycollie 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 Che principal metabolic pathway for chloroacetic acid and a major one for vinylidene chloride. Thiodiglycollic acid is the ultimate detoxification product, and previous work (2) established the biotransformation of S.-(2,,*carboxyiDethyl) cysteine (f) into that substance. A feasible metabolic pathway to thiodiglycollic 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 14C, labelled thiodiglycollic 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 [*4C]dithioglycollic acid (j) (and hence of the intermediate [^4C]thioglycollic acid) (h) is reconcilable with the action of Michaelis's (21) unspecific B"thionase, which would lyse a small proportion of the preponderating {^4c]thioglycollic acid (v\ infra) 7 URL 18785 Moreover, Kolbc electrolysis (22) of one molecular proportion of the Jthiodiglycollie acid metabolite from [l-*^C]l,1-dichloroethylene or [l-^C]chloroacctic acid gave 1 equivalent of ^COj^S), 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--*cysteinylactyl 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 S^epoxide transferase to afford ^-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. 8 URL 18786 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 thiodiglycollie acid is considerably reduced and the formation of the N-acetyl-S-cysteinyl3cetyl metabolite is increased. The higher B-thionose 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 S^acyl transferase. Under these circumstances, detoxification of 1,1-dichloroethylene oxide by glutathione S-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 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. 9 ACKNOWLEDGEMENT The author is indebted ter 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. URL 18787 REFERENCES 1. Green, T., and Hathway, D.E., The biological fate in rats of vinyl chloride in relation to its oncogenicity, Chem.-Biol. Interactions 11: 545 (1975). 2. Green, T., and Hathway, D.E., The chemistry and biogenesis of ^-containing metabolites of vinyl chloride in rats, Chem-Biol. Interactions, _7: 137 (1977). 3. Ogston, A.G. et al., The replacement reactions of 6S'"dichlorodiethyl sulphide and of some analogues in aqueous solution: the isolation of 8-chloro-0f-hydroxydiethylsulphide, Trans. Faraday Soc., 44: 45 (1948). If 4. Dabritz, E. and Virtanen, A.il., jS-Vinyl-cystein-S^oxyd, ein Homologes M zur Vorstufe der tranentreibenden Substanz der Zwiebel, Chem. Ber., 98: 781 (1965). URL 18788 5. Van Duuren, B.L., On the possible mechanism of carcinogenic action of vinyl chloride, Ann. New 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, 194 (1974). 8. Barbin, A., et^ al_., Liver-microsome mediated formation of alkylating agents from vinyl bromide and vinyl chloride, Biochem. Biophys. Res. 11 68m W ) 9. Greim, H., e_t_ , 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., 2} 109 (1976). 11. Bartsch, H., 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. Com., 65: 363 (1975). 13. McCann, J., et _al., Mutagenicity of chloroacetaldehyde, a possible metabolic product of 1,2-dichloroethane (ethylene dichloride), chloroethanol (ethylene chlorohydrini vinyl chloride and cyclophosphamide, Proc. Hat. 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., Secrist, J.A., and Leonard, N.J., Fluorescent adenosine and cytidine derivatives, Biochem. Biophys. Res. Comm., 46: 597 (1972). LI 16. Green, T., and Hathway, D.E., Interactions of vinyl chloride with ratliver DNA iii vivo, Chem.Biol. Interactions, in the press. 17. Lavley, P.D., et al., Inactivation of bacteriophage T7 by mono-and di-functional sulphur mustards in relation to cross-linking 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, Buttervorths, London, 1966. 20. Jones, B.K. and Hathvay, D.E., The biological fate of vinylidene chloride in rats, Chem-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). If 22. Kolbe, H., Untersuchungen uber die Elektrolyse organischer Verbindungen, Justus Liebigs Annalen der Chemie, 69: 257 (1849). 23. Walker, 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. URL 18790 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. 26. Yllner, S., Metabolism of chloroacetate-l" 14 C in the mouse, Acta pharmacol. et toxicol., 30: 69 (1971). c_ 7i--3 C--OJ (a) OH Hx l/H XC-C H^l XH SR +OH -*---------------- -OH' (c) HoC-CHo I/ s I CH. 40 -Cl' CH(NHAc)C02H -H + Hv RS^ /H ^H (d) (b) Cl IH C-C H^l x H SR r-j URL 1379 Fig. 1 Scheme for the interrelationship of some S-containing vinyl chloride metabolites. OH I h2c-ch2 s I CHo m CH(NH2)C02H ho2c-ch2 s I CHo (II) CH(NHo)C09H ho2c-ch2 s I CHo (III) C-COoH li ' 0 S(CH2C02 61.81 W Fig. 2. Scheme suggesting the biotransformation of S_-(2-hydroxyethyl) cysteine into thiodiglycollic acid. (I) End-group oxidation; (II) transamination; (III) oxidative decarboxylation. (e) <n <g> tf1 in) C!CH2COOH / / +GSH '/ l / / / URL 18794 COoH I2 chch2sch2ch2 NH(Ac) OH -OH +OH CH' R-sr i `CHr COoH \2 CHCH2SCH2C02H mz (i) CO?H I2 c-ch5sch,co9h II 2 2 2 0 R-S\ T-CH, H/ 1 S(CH2C02H)2 (j) Fig. 3 Scheme for the biogenesis of S-containing vinyl chloride met nbolites CiCHjHO Modified X>NA t in YlYO `Modified/ it^doi^U DMA 1 URL 18795 HOCH.0 HO OH atid &$$ocide,d `dz^riiidioU' 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 (B-D-1 i bo I ui .-nio:;y 1 - 5-oxo-l), 6~il i hydroi in i chi 7.o- [ I , 2-c ]pyr imi d inu ( ! e I i -li.im! side) and 3ft-l)-r i bolurnnosy 1 imi dazo- [2,1 -i ]purirte H2C=CC12 (a) aCKjCOgK r lion c-co,u i (co2h)2 i C02 1 C0(lfHj)2 (HOgCCHCHgSCHgCOjH\ 'OH 1 s(ch2co2h)2 11 HSCHgCOgH ' i (SCH2C02H), (*) oo (J) URL 18796 Fig. 5 Scheme for vinylidene chloride metabolism in rats. Fig. 6. Scheme suggesting the feasible interaction of reactive vinylidene chloride metabolites, 1,1-dichloroethylene oxide and chloroacetyl chloride, with adenosine and cytidine respectively. METABOLISM (I) of Chloroacc 1 tc acrid Metabolitc Chloroacetic acid Rats - ThiwJjgJycolHc acid 90 N-Acetyl-S- (2-carbojcynicthyl) cysteine 2 Yllncr 6-22% 37 40 Mice BKJ-DEU 30-40 40 Chloroacetic acid Thiodiglycollic acid Thioglycolii c acid Dithioglycollic acid N-Acetyl-S-cysteinylacetyl derivative (2) of Vinylidene chloride 337 3 35 5 20 48 70 URL 10798 ig. 7. The relative proportions of products resulting from the metabolism (1) of chloroacetic acid and (2) of vinylidene chloride in rats and mice. i