Chlorpyrifos Induction of Testicular-Cell Apoptosis through Generation of Reactive Oxygen Species and Phosphorylation of AMPK

Chlorpyrifos (CPF) is the most frequently applied insecticide. Aside from effects on the neuronal cholinergic system, previous studies suggested a potential relationship between CPF exposure and male infertility; however, the molecular mechanism remains elusive. The aim of this study was to investigate the toxic effect of CPF on testicular cells and the potential mechanism via in vitro and in vivo experiments. The cytotoxic effects of CPF on mouse-derived spermatogonial cell lines (GC-1), Sertoli cell lines (TM4) and Leydig cell lines (TM3) were assessed by a CCK-8 assay, flow cytometry, a TUNEL assay, quantitative RT-PCR, and Western blotting. Exposure to CPF (10-50 μM) for 12 or 24 h resulted in significant death in all three testicular cell lines. The number of TUNEL-positive apoptotic cells were dose-dependent and increased with raised CPF concentrations. Further investigation indicated that CPF induced cell-cycle arrest and then promoted cell apoptosis. Additionally, CPF increased reactive-oxygen-species (ROS) production and lipid peroxidation (MDA) and reduced mitochondrial-membrane potential. The mechanism of cell apoptosis induced by CPF involved an increase in phosphorylated-AMP-activated-protein-kinase (p-AMPK) levels in the tested cells. In vivo, the expression of steroid-hormone-biosynthesis-related genes in testis, spleen, and lung in F0 and F1 mice were downregulated when there was intraperitoneal injection or dietary supplementation of CPF. This study provides a potential molecular mechanism of CPF-induced toxicity in testicular cells and a theoretical basis for future treatment of male infertility.