γ-H2AX is a sensitive marker of DNA damage induced by metabolically activated 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a nicotine-derived nitrosamine, is a potent pulmonary carcinogen present in tobacco smoke. DNA adducts induced by metabolically activated NNK cause carcinogenesis; however, the DNA adducts are difficult to detect in cultured cells because of low intrinsic metabolic enzyme activity. In this study, we indirectly detected NNK-induced DNA adducts via the phosphorylation of histone H2AX (γ-H2AX) in A549 human lung adenocarcinoma epithelial cells. NNK treatment dose-dependently induced γ-H2AX. This γ-H2AX induction was suppressed by ataxia telangiectasia mutated inhibitors, suggesting that DNA double-strand breaks (DSBs) are formed during replication and repair of DNA adducts; however, DSBs could not be directly detected by biased sinusoidal field gel electrophoresis (BSFGE). CYP2A13-overexpressing cells showed prolonged γ-H2AX induction compared with control cells, and DSBs could be detected by BSFGE in CYP2A13-overexpressing cells as a clear migration of double-stranded DNA. These findings suggest that γ-H2AX is a sensitive marker of DNA adducts and provides a possible system for genotoxicity screening of chemicals such as NNK, which need metabolic activation to induce DNA damage.