Revised Manuscript Received August 24, 1993® abstract: The mutagenic and genotoxic properties of l,./V6-ethenoadenine (eAde), 3, iV4-ethenocytosine (eCyt), and 4-amino-5-(imidazol-2-yl) imidazole (/3) were investigated in vivo. The former two modified bases are known DNA adducts formed by the human carcinogen vinyl chloride;/3 is formed by pyrimidine ring-opening of eAde. Chemically synthesized deoxyhexanucleotides containing eAde and (3, d [GCT-(eA) GC], and d [GCT (/3) GC], respectively, were described previously [Biochemistry (1987) 26, 5626-5635]. eCyt was inserted into an oligonucleotide, d [GCTAG (eC)], by a mild enzymatic synthetic procedure, which avoidedexposure of the base to alkaline conditions. 3, iV4-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 M13-7V/2eI. 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 intoEscherichia coli, each of the adducts was found to be genotoxic. The most toxic lesion was/3, which reduced survival of the genome by 97%. eCyt and eAde 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 eAde (0.1% of the survivors were mutant), which showed primarily A->• G transitions. The eAde rearrangement product,/3, was also found to induce mutations but at a 20-fold higher frequency (~ 2%). In this case, however, mutagenesis was random, possibly because the hydrogen-bonding face of this lesion has been obliterated. eCyt induced mutations at a frequency of 1.5—2%; its mutations were mainly C—