Nicotine- and tar-free cigarette smoke induces cell damage through reactive oxygen species newly generated by PKC-dependent activation of NADPH oxidase

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 h 2o 2 or o 2 - generated by the xanthine/xanthine oxidase system,indicating involvement of h 2o 2 or o 2 - 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. © the japanese pharmacological society.