ToxSci Advance Access originally published online on March 17, 2006
Toxicological Sciences 2006 91(2):382-392; doi:10.1093/toxsci/kfj161
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4-Nitroquinoline 1-Oxide Forms 8-Hydroxydeoxyguanosine in Human Fibroblasts through Reactive Oxygen Species
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* Department of Dermatology, Kyoto University Graduate School of Medicine, Sakyo-Ku, Kyoto 606-8507, Japan; Division of Dermatology, Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Chuo-Ku, Kobe 650-0017, Japan; Department of Social and Environmental Medicine, Osaka University Graduate School of Medicine, Suita 565-0871, Japan; Department of Environmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8520, Japan; and ¶ Research and Development Center, Nagase & Co., Ltd., Nishi-Ku, Kobe 651-2241, Japan
Received November 24, 2005; accepted March 2, 2006
4-Nitroquinoline 1-oxide (4NQO) is thought to elicit its carcinogenicity by producing DNA adducts after being metabolized to 4-hydroxyaminoquinoline 1-oxide, which forms 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 1µM. 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.
Key Words: 4NQO; 8OHdG; reactive oxygen species; human fibroblasts; glutathione.