Clothianidin (CLO) is a high‐frequently detected neonicotinoid pesticide in fruits and vegetables, whose exposure security deserves attention. This study evaluated the apoptotic toxicity of CLO on Caco‐2 cells at doses of 100 nM, 10 μM, and 1 mM. After exposure, CLO induced to a remarkable change of signaling proteins that participated in the process of cell apoptosis, including caspase 3, cleaved‐caspase 3, and caspase 9. CLO treatment further induced a decrease of mitochondrial membrane potential and increased the protein level of cytochrome C. Reactive oxygen species (ROS) and intracellular Ca2+ were also found elevated, indicating an oxidative damage caused by CLO treatment. Moreover, the production of ROS occurred in advance of Ca2+ elevation, since inhibiting ROS production could recover the elevation of Ca2+ induced by CLO exposure. The protein level of metabolic enzyme cytochrome P450 3A4 (CYP3A4) was downregulated after the treatment of CLO. Molecular docking simulation indicated that CLO had good binding characteristics with CYP3A4. Amino acid sites Arg105, Arg130, and Leu373 in CYP3A4, and nitro group and chlorothiazole group in CLO structure might be the potential binding action target. These results indicated that CLO exposure could induce an apoptotic effect on Caco‐2 cells, possibly acting through combining and inhibiting its metabolic enzyme CYP3A4, and then leading to oxidative stress and mitochondrial damage. Thus, CLO exposure might be a potential risk factor for human intestinal health.

中文翻译：

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噻虫胺暴露通过线粒体氧化损伤诱导细胞凋亡


噻虫胺 （CLO） 是一种在水果和蔬菜中经常检测到的新烟碱类杀虫剂，其暴露安全性值得关注。本研究评估了 CLO 在 100 nM、10 μM 和 1 mM 剂量下对 Caco-2 细胞的凋亡毒性。暴露后，CLO 诱导参与细胞凋亡过程的信号转导蛋白发生显着变化，包括 caspase 3 、 cleaved-caspase 3 和 caspase 9。CLO 处理进一步诱导线粒体膜电位降低，细胞色素 C 蛋白水平升高。活性氧 （ROS） 和细胞内 Ca2+ 也升高，表明 CLO 处理引起氧化损伤。此外，ROS 的产生发生在 Ca2 + 升高之前，因为抑制 ROS 的产生可以恢复 CLO 暴露诱导的 Ca2 + 升高。CLO 处理后代谢酶细胞色素 P450 3A4 （CYP3A4） 蛋白水平下调。分子对接模拟表明，CLO 与 CYP3A4 具有良好的结合特性。CYP3A4 中的氨基酸位点 Arg105 、 Arg130 和 Leu373 以及 CLO 结构中的硝基和氯噻唑基团可能是潜在的结合作用靶点。这些结果表明，CLO 暴露可以诱导对 Caco-2 细胞的凋亡作用，可能通过结合和抑制其代谢酶 CYP3A4 发挥作用，然后导致氧化应激和线粒体损伤。因此，CLO 暴露可能是人类肠道健康的潜在风险因素。