Tobacco-specific nitrosamine-derived O ²-alkylthymidines are potent mutagenic lesions in SOS-induced escherichia coli

To investigate the biological effects of the O ²-alkylthymidines induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)- 1-butanone (NNK), we have replicated a plasmid containing O ²- methylthymidine (O ²-Me-dT) or O ²-[4-(3-pyridyl-4-oxobut-1- yl]thymidine (O ²-POB-dT) in Escherichia coli with specific DNA polymerase knockouts. High genotoxicity of the adducts was manifested in the low yield of transformants from the constructs, which was 2-5% in most strains but increased 2-4-fold with SOS. In the SOS-induced wild type E. coli, O ²-Me-dT and O ²-POB-dT induced 21% and 56% mutations, respectively. For O ²-POB-dT, the major type of mutation was T → G followed by T → A, whereas for O ²-Me-dT, T → G and T → A occurred in equal frequency. For both lesions, T → C also was detected in low frequency. The T → G mutation was reduced in strains with deficiency in any of the three SOS polymerases. By contrast, T → A was abolished in the pol V ^- strain, while its frequency in other strains remained unaltered. This suggests that pol V was responsible for the T → A mutations. The potent mutagenicity of these lesions may be related to NNK mutagenesis and carcinogenesis.