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

Hiroshi Asano; Takahiro Horinouchi; Yosuke Mai; Osamu Sawada; Shunsuke Fujii; Tadashi Nishiya; Masabumi Minami; Takahiro Katayama; Toshihiko Iwanaga; Koji Terada; Soichi Miwa

doi:10.1254/jphs.11166FP

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

Hiroshi Asano, Takahiro Horinouchi, Yosuke Mai, Osamu Sawada, Shunsuke Fujii, Tadashi Nishiya, Masabumi Minami, Takahiro Katayama, Toshihiko Iwanaga, Koji Terada, Soichi Miwa

Author information

Hiroshi Asano
Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan
Takahiro Horinouchi
Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan
Yosuke Mai
Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan
Osamu Sawada
Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan
Shunsuke Fujii
Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan
Tadashi Nishiya
Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan
Masabumi Minami
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Japan
Takahiro Katayama
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Japan
Toshihiko Iwanaga
Laboratory of Histology and Cytology, Graduate School of Medicine, Hokkaido University, Japan
Koji Terada
Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan
Soichi Miwa
Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan

Keywords: cigarette smoke extract (CSE), reactive oxygen species (ROS), NADPH oxidase (NOX), apoptosis, protein kinase C (PKC)

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Supplementary material

2012 Volume 118 Issue 2 Pages 275-287

DOI https://doi.org/10.1254/jphs.11166FP

View "Advance Publication" version (February 3, 2012).

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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.

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