Investigation Of Difenoconazole-induced HepG2 Cell Apoptosis And The Molecular Mechanism Of Its Interactions With CYP3A4

Difenoconazole, one of triazole compounds, is widely used for environmental fungicides in various crops. The potential toxicity of difenoconazole has caused wide public concerns among researchers in environment and toxicology, because it exists in environmental media and residues in vivo. This project mainly focused on the mechanism of toxicity of difenoconazole toward to human HepG2 cells and the potential inhibitory activity to human CYP3A4 enzyme. The main results and conclusions obtained are as follows:(1) MTT assay, western blot, flow cytometry and confocal experiment methods were combined to explore the toxicity mechanism in HepG2 cells. Our results showed that difenoconazole suppressed the viability of HepG2 cells in a concentration-dependent manner. The obvious cleavage on nuclear morphology (i.e. karyopyknosis) was also revealed. Difenoconazole can significantly regulate apoptotic proteins Caspase-8,9 and PARP expression in a dose- and time-dependent manner. The reactive oxygen species were also produced in the mitochondria and endoplasmic reticulum. In early stage of apoptosis, mitochondrial membrane potential were decreased with the increasing concentration of difenoconazole. The cytochrome C was released from the mitochondria into the cytoplasm. ER stress-related proteinsalso displayed a remarkable trend, indicating that difenoconazole induced HepG2 cell apoptosis via ER stress pathway. HepG2 cell apoptosis induced by difenoconazole is mediated through both mitochondrial ROS pathway and endoplasmic reticulum stress pathway.(2) The real-time quantitative PCR and western blot methods were used to detect the change of mRNA and protein expression of CYP3A4. Difenoconazole induced no changes of mRNA and protein expression of CYP3A4. however, it showed a potent inhibitory activity toward CYP3A4.A significant cytotoxicityof difenoconazole was revealed toward HepG2 cells at high concentrations and the molecular mechanisms of induced HepG2 cell apoptosis was elucidated. A potent inhibitory activity of difenoconazole was also observed. This project provides essential data to the risk assessment of triazole pesticides and facilitates the relevant risk management.