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Br. J. Cancer (1081) 44, 597 B904 HATH BRIT MN823 P 597 J CANCER &?37 Letter to the Editor JAN 4 12 ^ IArA MECHANISMS OF VINYL CHLORIDE CARCINOGENICITY/MUTAGENICITY Sm,--The present letter sets out to show that the various donxyrihonucleoside (and ribonucleoside) analogues. whieh have been isolated from the chemical, non-enzymic reaction (Hathway, 1980) between the ulti mate vinyl chloride metabolites and target, organ DNA (and RNA) components in vivo. appear to be biologically significant, and oatible with ensuring ._umaoeaicitv and carcinogenicity. JT-fetST Ureen & Hathway (1978) (see also Hathway, 1977) produced strong evidence in the form, of published mass fragmentograms for the presence of the itmdazo-cyclization products of deoxyadenosine (dA) and deoxycytidine (dC) (viz. 9-(/3-D-2'-deoxyribofur- anosyl)imidazo[2,l-i]purine (etheno-dA) and 1 - (/iLD-2'-deoxyriboturftnosyl)inudazo[l,2-cJpyrimid-2(lH)-one (etheno-dO)) in chromato graphic fractions of the enzymic hydrolysates of the modified liver DNA of surviving rats which hod been exposed chronically to long term vinyl chloride in their drinking water (250 pt/10*). Out of the large group of animals which had been exposed to vinyl chloride in this way, there was a high incidence of rats that died with liver haemangiosarcoma (E.A.R.C. Monographs, 1979) during the 2-year experiment. It was implicitly inferred (Green & Hathway. 1978) that these results indicated a causa) relationship; i.e. that forma tion of etheno-dA and etheno-dC may repre sent pro-mutagenic lesions in the extra-hepa tocellular liver-tissue DNA ofanimals exposed to vinyl chloride. Furthermore, formation of etheno-dA and etheno-dC (a eiuo and in model experiments nf the reaction of vinyl chloride-derived chloroethylene oxide or its rearrangement product, chloroacetaldehyde, with calf-thymus DNA implied a common reaction mechanism. In both cases (Fig. 1), initial (Sn2) alkylation occurred at the m09t nucleophilic ring-nitrogen (N-l of the dA residues and 2V-3 of the dC ones), followed successively by loss of the elements of water with ring-closure between the oxo-group and the amino-group belonging to (7-6 of the dA residues (and of C-4 of the dC ones) and by proton loss (Hathway & Kol&r, 1980). At the same time as the foregoing. Laib & Bolt (1977, 1978) provided chromatographic evidence for the presence of the correspond ingly modified ribonuclcosides in fractions of the enzymic hydrolysate of the liver RNA of rats which had been exposed to a large, single radioactive dose of HC-vinvl chloride, and they confirmed their results with incubations of rat-liver microsomea, l*C-vinyl chloride and the appropriate polynucleotide, fortified with NADPH. From the time-courae experi ments. these authors (1978) showed relative persistence of etheno-C compared with otheno-A in rat-liver DNA, suggesting its greater biological significance. They com mented on the contrast between the pattern of RNA nucleoside analogues and that of DNA in which the dG residues (see below) were said to be the principal target for alkylation, but they have not published their experimental evidence. At that time too, Osterman.Golkar ei al. (1977) showed the chromatographic separa tion, after N&BH4 treatment, of 7-N-(2hydroxyethyl)guanine from the acid hydro lysate of the liver DNA of mice, exposed acutely to 14C-vinyl chloride, and they COPYRIGHT fll H K LEWIS ANO CO LT O LONDON* WC1E 6 6N . 1. AP00016921 598 LETTER TO THE EDITOR attributed this result to formation of 7-.N-<2oxoethyl)dG residues, but they did not look in their DNA hydrolysates for otheno-dA and etheno-dC, which, on account of the method of exposure, may have been absent from their material or below the limits of detection. Subsequently. Bolt et al. (1981) suggested that the Swedish workers' aldehyde may exist as a 0-membered cyclic hemi-acetal (Fig. 2) in DNA. Consideration of molecular models show; (i) that the imidazole ring in vinyl chloride nucleoside analogues is co-planar or almost co-pl&nar with the rest of the molecule, and this observation finds support in X-ray crystallography (Wang et at., 1074. 1976): (it) that the imidazole ring in these nucleoside analogues shields 2 normal hydrogen bonding positions (Hathwav & Kolar. 1980); (iii) that in the case of etheno-dC (or cthenoC), the second ring confers on the cytosine residue the dimensions of adenine, with the result that etheno-dC would be expected to simulate dA nucleosidos/nucleotidcs in repli cation (Hall et al., 1981: Barbin et al., 1981) and etheno-C the A ones in transcription (Spengler & Singer. 1981): (iv) that the mis- incorporation envisaged in these biological processes would be facilitated by complexformation involving base pairing (Fig. 3) of protonated molecular species, which were invoked (Topal & Fresco. L978) to extend the Watson-Crick concept for complementary base pairing; (v) that the relatively bulky imidazole ring resembles an alkyl substituent and effectively blocks one of the available base-pairing sites. When DNA-like polymers, poly(dA-dT) and poly(dC-dG). that were pre-treated with chloroacetaldehyde, were used as templates for E. coli DNA polymerase 1 in an in vitro assay (Hall et al., 1981) replication was decelerated, and increased levels of non- complementary nucleosides were incorpor ated. (Although DNA repair has never been studied per se, it is not entirely ignored in this work, as DNA polymerase 1 belongs to the repair system.) With the modified (dA-dT) templates, 1 dGMJ? was incorporated for every ~60 etheno-dA residue* present, but no misincorporation of dCMP occurred, and with the poly(dC-dG) templates. 1 misinoorporation of dAMP or dTMP occurred respect ively in the presence of --30 or 80 ethcno-dC residues. The principal miscoding* of etheno- AP00016922 LETTER TO THE EDITOR 599 dA may represent a potential pro-mutagenic lesion, which would he expected to lead to (dA-dT)-(dC-dG) transveraiona. and simi larly those of etheno-dC would possibly Induce (dC-dG)-(dA-dT} tranaversiong. Near est-neighbour analysis with modified poly(dC-dG) templates showed that 90% of the misincorporations. of say A. occurred opposite cytosine (or etheno-cytosine). but a small number of errors (-'10%) occurred opposite guanine bases, which may be due to the sus pected formation of the cyclic hemi-acetal of dG (Fig. 2) in the modified poly(dC-dG) tem plates. However, work on the kinetics and selectivity of the reaction of chloroacctaldehvde with some tRMTA constituents shows that it is probable that no nucleoside ana logues other than the imidazo-cyclization products me formed (Biernat el ah. 1978). Induction of (dA-dT)-(dC-dG) and (dCdG)-(dA-dT) transvendons from etheno-dA and etheno-dC are consistent with the fact that chloroethyleneoxide. chloroacetaldehyde and metabolically activated vinyl chloride induce_ base-pair-substitution mutations (Rannug7et ai.. 1U74: Malaveille et al., LQfS: McCann et ai. 1975: Phillips et at.. 1980). but not frame-shift mutations, in Salmonella typhimurium strains. It follows that the mechanism of vinyl chloride carcinogenicity/ mutagenicity has been studied more intens ively than that of any other human carcin ogen. D. E. Hathway l.C.l. Limited. Central Toxicology Laboratory, Alderley Park, Cheshire SKIQ 42V. During our attack on the vinyl chloride problem. I discusawl various aspects with Dr Hclmuth Bartsch (International Agency for Research on Cancer, Lyon), Professor Hermann Bolt (Institut fllr Pharmakologie dor Univermtat, Main*). Pro fessor Dietrich Honachlcr (Institut flir Pharma* kologie und Toxtkolagie dec Universit&t, Wurzburg). Drs James and Elizabeth Miller (MaArdle Labora tory for Cancer Research, University of Wisconsin. Madison) and Dr Ro.y Rsffhill (Paterson Laboratory. Christie Hospital & Holt Radium Institute. Man chester), to whom I xhould like to express my best thanks, REFERENCES Barbin, A.. Bartsch, H., Leconte, P. & Raomak, M. (1981) Studies ore the miscoding properties of 1 .ya-etlienoadenine and 3,.V*.Bthenoeytosme, DNA reaction products of vinyl chloride metabolites during in vitro synthesis. Nucleic Acid* Re*., 9, 375. Biernat. J., Ciehiolka, J,, GARxrcitr, P., Adamiak. R. \V.. Krevzosiak, W. J. 1 Wtswioaowsict, M. 41 (1978) New observations concerning the rhloroacetaldehyde reaction with some tRNA eonstituents, stable intermediates, kinetics and selectivity of the reaction. Nucleic Acid* Rt*., 5, 789 Bolt, H. M.. Filsrn, J. G. * Laib. R. J. (1081) Convalent binding of Italoethylene*. 2nd Int. Symp. Bioloffieul Rencticc Intermediate*. Ed. Snyder etui. New York: Plenum Press. (In press). Clreek, T. <fc Kathwav. D. E. (1978) Interactions of vinyl chloride with rat-liver DNA in vivo. Chem. fiiol. Internee.. 22, 211. Hall. J. A.. Saffkill. R.. Green. T. fe Hathway, I). E. (1081) The induction of errors during in vitro DNAsynthesis following vliloruacetaldohydotreatment of polyfdA riT) end poiy(dC-dG) tem plates. t'nrcinogeneeii, 2. 141, Hathway. L). E. (1077) Comparative mammalian metabolism of vinyl chloride and vitiylidene chloride in relation to oncogenic potential. Environ, He/dth Perepetl.. 21, 58. Hathway. I). E. (10B0) I'Uo importance of (nonentymic) chemical reaction procv*sos to the fate of foreign compounds in mammals. Chem. Hoc. Rev., 9, 83. Hathway. D. E- St Kolab, 0. F. (I960) Mechanisms of reaction between the ultimate chemical carcino gens and nucleic acid. Cftein. Hoc. Rev., 9, 241. [ARC Monograph* (1979) oh the Evaluation of ike Carcinogenic RUk* of Chemical* to Human*. Home monomers, plastics and synthetic elastomers, and acrolein. Int, Agency Rt*. Cancer, 19, 377. Lacs. R. J. St Bolt. H. M. (1977) Alkylation of RNA by vinyl chloride metabolites in oilro and in vivo-. Formation of l..Y8-etlionoa<lcnonirie. Toxicology, 8, 186. Lacs, R. J. St Bolt, H, M. (1978) Formation of .ethenocytidine moieties in RNA by vinyl chloride metabolites i vitro and in vivo. Arch. Toxicol., 39, 235. McCann, J., Simmon. V., STRjsrrwiBaeR. D. A Ames. U, X. (1975) Mutagenicity of c-hlor- acetaldehyrle, a possible metabolic product of 1,2-dichloroethane, chloroethanol. vinyl chloride and cyclophosphamiile. Prac. Xntl A end. Sci., V.S.A., 73, 3190. Malaveille, C.. Bartsch. H.. Barbin. A., Camus, A. M. A Montssano. R. (1975) Mutagenicity of vinyl chloride, cltloroethylene oxide, chloro- acetaldeliydeand chloroethonol. Btachtm. Biophye, Re*. Commun., 63, 383. OfiTBRMAN-OOLKAB. 8,, HULTMARK, D,, ScORBBaCK, D.. Calleman. C. J.. G6the. R. St Ehrknbero. C, A. (1977) Alkylation of DMA and protein* in mice exposed to vinyl chloride. Riockem. Biophy*. Re*. Commun., 76, 259. Phillips, R. A.. Zakler, S. A. Sc Garro. A. J. 11080) Detection of mutagen-inducod lesions in isolated DNA usings new Rnciilu* aubtiti* transformationbased assay. Afutnt. Re*., 74, 207. Rannl'o. U.. Johansson. A.. Rambl, C. St W'aoht- meister. 0. A. (1974) Tho mutagenicity of vinyl chloride after metabolic activation. Ambio, 3, 194. Hpenoler, S. fc Bixoer, B. (1961) Transcriptional errors and ambiguity resulting from the presence of l.iV^-ethenoadenosine or it.iV^-ethonocytidine in polyribonucleotides. Nucleic Acid* Re*., 9, 365. Total, M. D. A Fresco. J. R. (1078) Complementary base pairing and the origin of xubutitution mutations. .Vnlwre, 263, 285. APOOO16923 frZ69l000dV 'Urt'i '8b "'OS' wuojr'.'is ju>i,>scoon(4 ui[i jo xuouoojoiui jiiim[ouj ptiv hm<iimujui!|> v>i|i jn Apius v :o|>uo]i|.')aiVAi| ouipu.i'.i ou.n|j.i-r.Vfc j<j JU|n.U]ni_u jtiiTt [tnH.vi, i l9i(i]I .1 I "liM V '>[ T `oiUMr^ -] h V `ox'Wt fid? 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