Synergistic induction of DNA strand breakage by cigarette tar and nitric oxide

Cigarette smoking is a major cause of human cancer at a variety of sites, although its carcinogenic mechanisms remains unestablished. Cigarette smoke can be divided into two phases, gas phase and particulate matter (tar). Both phases contain high concentrations of oxidants and free radicals, especially nitric oxide (NO) and nitrogen oxides in the gas phase and quinone/hydroquinone complex in the tar. We have found that incubation of pBR322 plasmid DNA with aqueous extracts of cigarette tar and a NO-releasing compound (diethylamine NONOate) caused synergistic induction of DNA single-strand breakage, whereas either cigarette tar alone or NO alone induced much less strand breakage. This synergistic effect of cigarette tar and NO on DNA strand breakage was prevented by high concentrations of superoxide dismutase, carboxy-PTIO (an NO-trapping agent) or N-acetylcysteine, whereas hydroxyl radical scavengers such as dimethylsulfoxide, ethanol and D-mannitol did not show inhibitory effects. Possible mechanisms for this synergistic effect mediated by cigarette tar and NO are proposed, including involvement of peroxynitrite, which is a strong oxidant and nitrating agent formed rapidly by the reaction between NO and O2.-. NO is present in the gas phase of smoke and may be formed by a constitutive or inducible NO synthase in the lung, whereas O2.- is generated by auto-oxidation of polyhydroxyaromatic compounds such as catechol and 1,4-hydroquinone present in cigarette tar. Thus, potent reactive species including peroxynitrite formed by the interaction between cigarette tar and NO may play an important role in smoking-related diseases including lung cancer.