Chlorpyrifos-mediated mitochondrial calcium overload induces EPC cell apoptosis via ROS/AMPK/ULK1

Chlorpyrifos (CPF) is a typical organophosphate insecticide known to has serious toxicological effects on aquatic animals and causes many environmental contamination problems. To assess the effects of CPF on the epithelioma papulosum cyprini (EPC) cells of the common carps from the point of calcium ion (Ca²⁺) transport, the CPF-exposed EPC models were primarily established, and both AO/EB staining and Annexin V/PI assay with flow cytometry analysis were subsequently implemented to identify that CPF-induced EPC cell apoptosis, in consistent with the up-regulated expression of BAX, Cyt-c, CASP3 and CASP9, and down-regulated BCL-2 expression. Then, Mag-Fluo-4 AM, Fluo-4 AM and Rhod-2 AM staining probes were co-stained with ER-Tracker Red and Mito-Tracker Green applied to image cellular Ca²⁺ flux, illuminating Ca²⁺ depleted from ER and flux into mitochondria, resulting in ER stress and mitochondrial dysfunction. Additionally, 2-Aminoethyl Diphenylborinate (2-APB), 4-Phenylbutyric acid (4-PBA) and Dorsomorphin (Compound C) were performed as the inhibitor of Ca²⁺ transition, ER stress and AMPK phosphorylation, suggesting CPF-mediated Ca²⁺ overload triggered ER stress. And the over-generation of Mito-ROS intensified oxidative stress, promoting the phosphorylation of AMPK and deteriorating cell apoptotic death. The results of this study demonstrated Ca²⁺ overload-dependent mitochondrial dysfunction engages in the CPF-induced apoptosis, providing a novel concept for investigating the toxicity of CPF as environmental pollution on aquatic organisms.