Tobacco-Specific Nitrosamine-Derived O2-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[44-(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 pot 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.