4-Nitroquinoline 1-oxide forms 8-hydroxydeoxyguanosine in human fibroblasts through reactive oxygen species
4-Nitroquinoline 1-oxide forms 8-hydroxydeoxyguanosine in human fibroblasts through reactive oxygen species
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DOI：
10.1093/toxsci/kfj161
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发表时间：
2006-06-01
影响因子：
3.8
通讯作者：
Miyachi, Yoshiki
中科院分区：
文献类型：
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作者：
Arima, Yaeno;Nishigori, Chikako;Miyachi, Yoshiki
4-硝基喹啉1-氧化物（4 NQO）被认为是通过代谢为4-羟基氨基喹啉1-氧化物后产生DNA加合物，形成8-羟基脱氧鸟苷（8 OHdG）氧化损伤而致癌的。为了确定活性氧（ROS）是否参与4 NQO产生8 OHdG，我们使用高效液相色谱法和免疫组化来测量用4 NQO处理的正常人成纤维细胞中8 OHdG的水平。通过使用荧光探针检测ROS、使用无细胞系统的电子顺磁共振光谱法和测量细胞内谷胱甘肽（GSH）水平来确定由4 NQO诱导的ROS的程度。在成纤维细胞中，4 NQO剂量依赖性地增加8 OHdG水平。用二氯荧光素二醋酸酯法和氢乙啶法分别检测4 NQO处理后细胞内过氧化氢（H2 O2）和超氧阴离子（O2）的产生。在培养基中加入过氧化氢酶降低了8 OHdG水平和二氯荧光素荧光强度，而4 NQO在无细胞系统中产生羟基自由基。这些发现表明，4 NQO处理导致超氧化物，H2 O2和羟基自由基的形成，导致在DNA中产生大量的8 OHdG。即使在低至1 μ M的浓度下，在去除4 NQO后24小时，8 OHdG和GSH的水平都没有恢复到基础水平。我们的研究结果表明，ROS的产生和细胞内GSH的耗竭也是4 NQO产生8 OHdG的重要因素。本文介绍了实用和敏感的方法来检测ROS和8 OHdG，并讨论了一个新的功能途径，引发遗传毒性。
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 1 mu M. 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.