MUTAGENIC AND GENOTOXIC EFFECTS OF 3 VINYL CHLORIDE-INDUCED DNA LESIONS - 1,N(6)-ETHENOADENINE, 3,N(4)-ETHENOCYTOSINE, AND 4-AMINO-5-(IMIDAZOL-2-YL)IMIDAZOLE

ABSTRACT: The mutagenic and genotoxic properties of 1,N6-ethenoadenine (epsilonAde), 3,N4-ethenocytosine (epsilonCyt), and 4-amino-5-(imidazol-2-yl)imidazole (beta) were investigated in vivo. The former two modified bases are known DNA adducts formed by the human carcinogen vinyl chloride; beta is formed by pyrimidine ring-opening of epsilonAde. Chemically synthesized deoxyhexanucleotides containing epsilonAde and beta, d[GCT(epsilonA)GC], and d[GCT(beta)GC], respectively, were described previously [Biochemistry (1987) 26, 5626-5635]. EpsilonCyt was inserted into an oligonucleotide, d[GCTAG(epsilonC)], by a mild enzymatic synthetic procedure, which avoided exposure of the base to alkaline conditions. 3,N4-Etheno-2'-deoxycytidine 3',5'-bisphosphate coupled with reasonable efficiency (30-40%) to the 3'-nucleoside of an acceptor pentamer, d(GCTAG), in a reaction catalyzed by T4 RNA ligase in the presence of ATP. Each of the three modified hexanucleotides and an unmodified control were inserted into a six-base gap positioned at a known site in the genome of bacteriophage M13-NheI. A nick was placed in the DNA strand opposite that containing the single DNA lesions, enabling the formation of singly adducted single-stranded genomes by denaturation. After transfection of the adducted phage DNAs into Escherichia coli, each of the adducts was found to be genotoxic. The most toxic lesion was beta, which reduced survival of the genome by 97%. EpsilonCyt and epsilonAde reduced survival by 90% and 65%, respectively. An examination of the surviving phage populations revealed that each of the three adducts was mutagenic. The least mutagenic lesion was epsilonAde (0.1% of the survivors were mutant), which showed primarily A --> G transitions. The epsilonAde rearrangement product, beta, was also found to induce mutations but at a 20-fold higher frequency (approximately 2%). In this case, however, mutagenesis was random, possibly because the hydrogen-bonding face of this lesion has been obliterated. EpsilonCyt induced mutations at a frequency of 1.5-2%; its mutations were mainly C --> T transitions, although targeted C --> A and -1 deletions were also detected. The possible respective roles of these three DNA lesions in the mutagenic and carcinogenic activities of vinyl chloride and related haloalkanes are discussed.