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-(imidaz ol-2-yl)imidazole (beta) were investigated in vivo. The former two mod ified bases are known DNA adducts formed by the human carcinogen vinyl chloride; beta is formed by pyrimidine ring-opening of epsilonAde. Ch emically synthesized deoxyhexanucleotides containing epsilonAde and be ta, d[GCT(epsilonA)GC], and d[GCT(beta)GC], respectively, were describ ed previously [Biochemistry (1987) 26, 5626-5635]. EpsilonCyt was inse rted into an oligonucleotide, d[GCTAG(epsilonC)], by a mild enzymatic synthetic procedure, which avoided exposure of the base to alkaline co nditions. 3,N4-Etheno-2'-deoxycytidine 3',5'-bisphosphate coupled with reasonable efficiency (30-40%) to the 3'-nucleoside of an acceptor pe ntamer, d(GCTAG), in a reaction catalyzed by T4 RNA ligase in the pres ence of ATP. Each of the three modified hexanucleotides and an unmodif ied control were inserted into a six-base gap positioned at a known si te in the genome of bacteriophage M13-NheI. A nick was placed in the D NA strand opposite that containing the single DNA lesions, enabling th e 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 examinati on 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 transit ions. The epsilonAde rearrangement product, beta, was also found to in duce mutations but at a 20-fold higher frequency (approximately 2%). I n this case, however, mutagenesis was random, possibly because the hyd rogen-bonding face of this lesion has been obliterated. EpsilonCyt ind uced 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 relat ed haloalkanes are discussed.