title: γ-H2AX is a sensitive marker of DNA damage induced by metabolically activated 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.
abstract: 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.
authors.affiliation: Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan. Electronic address: ibuki@u-shizuoka-ken.ac.jp.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Ibuki
authors.affiliation: Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Shikata
authors.affiliation: Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan; National Institute of Occupational Safety and Health, Kawasaki, Japan.
authors.forename: Tatsushi
authors.initials: T
authors.lastname: Toyooka
mesh_terms: D065767:Activation, Metabolic; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D002273:Carcinogens / Q000378:metabolism*; D045744:Cell Line, Tumor; D004249:DNA Damage; D006657:Histones / Q000378:metabolism*; D006801:Humans; D009602:Nitrosamines / Q000378:metabolism*
chemical_list: D002273:Carcinogens; C468783:H2AX protein, human; D006657:Histones; D009602:Nitrosamines; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D001189:Aryl Hydrocarbon Hydroxylases; C414206:CYP2A13 protein, human
keywords: CYP2A13; CYP2A6; DSBs; Histone H2AX; NNK