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(2)O(2) or O(2)(-) generated by the xanthine/xanthine oxidase system, indicating involvement of H(2)O(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.
Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan.
Hiroshi
H
Asano
Takahiro
T
Horinouchi
Yosuke
Y
Mai
Osamu
O
Sawada
Shunsuke
S
Fujii
Tadashi
T
Nishiya
Masabumi
M
Minami
Takahiro
T
Katayama
Toshihiko
T
Iwanaga
Koji
K
Terada
Soichi
S
Miwa
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000473:pathology; D005910:Glioma / Q000378:metabolism*; D006207:Half-Life; D006861:Hydrogen Peroxide / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012907:Smoking / Q000009:adverse effects*; D014026:Nicotiana / Q000737:chemistry
D016877:Oxidants; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D011493:Protein Kinase C; D010100:Oxygen