Nicotine- and Tar-Free Cigarette Smoke Induces Cell Damage Through Reactive Oxygen Species Newly Generated by PKC-Dependent Activation of NADPH Oxidase

Abstract

We examined cytotoxic effects of nicotine/tar-free cigarette smoke extract (CSE) on C6 glioma cells. The CSE induced plasma membrane damage (determined by lactate dehydrogenase leakage and propidium iodide uptake) and cell apoptosis {determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction activity and DNA fragmentation}. The cytotoxic activity decayed with a half-life of approximately 2 h at 37°C, and it was abolished by N-acetyl-l-cysteine and reduced glutathione. The membrane damage was prevented by catalase and edaravone (a scavenger of •OH) but not by superoxide dismutase, indicating involvement of •OH. In contrast, the CSE-induced cell apoptosis was resistant to edaravone and induced by authentic H2O2 or O2− generated by the xanthine/xanthine oxidase system, indicating involvement of H2O2 or O2− in cell apoptosis. Diphenyleneiodonium [NADPH oxidase (NOX) inhibitor] and bisindolylmaleimide I [BIS I, protein kinase C (PKC) inhibitor] abolished membrane damage, whereas they partially inhibited apoptosis. These results demonstrate that 1) a stable component(s) in the CSE activates PKC, which stimulates NOX to generate reactive oxygen species (ROS), causing membrane damage and apoptosis; 2) different ROS are responsible for membrane damage and apoptosis; and 3) part of the apoptosis is caused by oxidants independently of PKC and NOX.[Supplementary methods and Figure: available only at http://dx.doi.org/10.1254/jphs.11166FP] Keywords:: cigarette smoke extract (CSE), reactive oxygen species (ROS), NADPH oxidase (NOX), apoptosis, protein kinase C (PKC)