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ToxSci Advance Access published online on March 17, 2006
Toxicological Sciences, doi:10.1093/toxsci/kfj161
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© The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received November 24, 2005
Accepted March 2, 2006

Carcinogenicity

4-Nitroquinoline 1-Oxide Forms 8-Hydroxydeoxyguanosine in Human Fibroblasts Through Reactive Oxygen Species

Yaeno Arima 1, Chikako Nishigori 2 *, Toru Takeuchi 3, Shigenori Oka 4, Kanehisa Morimoto 5, Atsushi Utani 1, and Yoshiki Miyachi 1

1 Department of Dermatology, Kyoto University Graduate School of Medicine 54 Kawahara-Cho, Shogoin, Sakyo-Ku, Kyoto 606-8507, Japan
2 Department of Dermatology, Kyoto University Graduate School of Medicine 54 Kawahara-Cho, Shogoin, Sakyo-Ku, Kyoto 606-8507, Japan; Division of Dermatology, Clinical Molecular Medicine Kobe University Graduate School of Medicine 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe 650-0017, Japan
3 Department of Social and Environmental Medicine Osaka University Graduate School of Medicine 2-2 Yamadaoka 565-0871, Japan; Department of Environmental Medicine Kagoshima University Graduate School of Medical and Dental Sciences 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan
4 Research & Development Center, Nagase & Co., Ltd. 2-2-3 Murotani, Nishi-Ku, Kobe 651-2241, Japan
5 Department of Social and Environmental Medicine Osaka University Graduate School of Medicine 2-2 Yamadaoka 565-0871, Japan

* To whom correspondence should be addressed.
Chikako Nishigori, E-mail: chikako{at}med.kobe-u.ac.jp


  Abstract

4-Nitroquinoline 1-oxide (4NQO) is thought to elicit its carcinogenicity by producing DNA adducts after being metabolized to 4-hydroxyaminoquinoline 1-oxide, which form 8-hydroxydeoxyguanosine (8OHdG), oxidative damage. To determine whether reactive oxygen species (ROS) are involved in the generation of 8OHdG by 4NQO, we used high-performance liquid chromatography and immunohistochemistry to measure the levels of 8OHdG in normal human fibroblasts treated with 4NQO. The extent of ROS induced by 4NQO was determined by using fluorescent probes to detect ROS, electron paramagnetic resonance spectrometry using a cell-free system, and measurement of intracellular glutathione (GSH) levels. In fibroblasts, 4NQO dose-dependently increased 8OHdG levels. Hydrogen peroxide (H2O2) and superoxide were detected in cells treated with 4NQO by using dichlorofluorescin diacetate and hydroethidine, respectively,. The addition of catalase to culture medium reduced 8OHdG levels and the intensity of dichlorofluorescin fluorescence, while 4NQO generated hydroxyl radicals in the cell-free system. These findings suggest that 4NQO treatment leads to formation of superoxide, H2O2 and hydroxyl radicals, resulting in the production of a substantial amount of 8OHdG in DNA. Neither the level of 8OHdG nor that of GSH had returned to the basal level 24 h after removal of 4NQO even at a concentration as low as 1 µM. Our results suggest that generation of ROS and depletion of GSH in cells are also important factors for the generation of 8OHdG by 4NQO. This paper describes practical and sensitive ways to detect ROS and 8OHdG and discusses a new functional pathway to elicit genotoxicity.

Keywords: 4NQO; 8OHdG; reactive oxygen species; human fibroblasts; glutathione.
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