Clothianidin Exposure Induces Cell Apoptosis via Mitochondrial Oxidative Damage

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.