Mutagenesis by exocyclic alkylamino purine adducts in Escherichia coli

Exocyclic alkylamino purine adducts, including N2-ethyldeoxyguanosine, N2-isopropyldeoxyguanosine, and N6-isopropyldeoxyadenosine, occur as a consequence of reactions of DNA with toxins such as the ethanol metabolite acetaldehyde, diisopropylnitrosamine, and diisopropyltriazene. However, there are few data addressing the biological consequences of these adducts when present in DNA. Therefore, we assessed the mutagenicities of these single, chemically synthesized exocyclic amino adducts when placed site-specifically in the supF gene in the reporter plasmid pLSX and replicated in Escherichia coli, comparing the mutagenic potential of these exocyclic amino adducts to that of O6-ethyldeoxyguanosine. Inclusion of deoxyuridines on the strand complementary to the adducts at 5′ and 3′ flanking positions resulted in mutant fractions of N2-ethyldeoxyguanosine and N2-isopropyldeoxyguanosine-containing plasmid of 1.4 ± 0.5% and 5.7 ± 2.5%, respectively, both of which were significantly greater than control plasmid containing deoxyuridines but no adduct (p = 0.04 and 0.003, respectively). The mutagenicities of the three exocyclic alkylamino purine adducts tested were of smaller magnitude than O6-ethyldeoxyguanosine (mutant fraction = 21.2 ± 1.2%, p = 0.00001) with the N6-isopropyldeoxyadenosine being the least mutagenic (mutant fraction = 1.2 ± 0.5%, p = 0.13). The mutation spectrum generated by the N2-ethyl and -isopropyldeoxyguanosine adducts included adduct site-targeted G:C → T:A transversions, adduct site single base deletions, and single base deletions three bases downstream from the adduct, which contrasted sharply with the mutation spectrum generated by the O6-ethyldeoxyguanosine lesion of 95% adduct site-targeted transitions. We conclude that N2-ethyl and -isopropyldeoxyguanosine are mutagenic adducts in E. coli whose mutation spectra differ markedly from that of O6-ethyldeoxyguanosine.