Low doses of chlorothalonil cause testicular impairment in mammals through the antimicrobial-like mechanism.

Chlorothalonil is a widely used fungicide that exerts activity through interacting with glutathione (GSH) and thiol-containing proteins or other nucleophilic molecules due to its electrophilicity. However, it remains little known whether chlorothalonil exerts similar actions in mammals and causes male reproductive damage. Here, we detected reduced cell viability and glycolytic impairment coupled with the redox imbalance including ROS accumulation in GC-1 cells (testicular germ cell line) treated with chlorothalonil; however, the nucleophilic antioxidant N-acetylcysteine (NAC) rescued chlorothalonil-caused cell damage. In animal experiments, exposure to chlorothalonil (30, 300, and 3000 μg/kg/d) from gestational day 10 to postnatal day (PND) 10 caused slight effects on testicular development in suckling male mice. However, extended exposure to postnatal week (PNW) 15 resulted in testicular impairment in male offspring, including germ cell apoptosis, reduced sperm count, and increased sperm tail malformation, along with reduced GSH content and altered expression of redox-related genes, and certain significant alterations even occurred in the low-dose group. Furthermore, we also observed reduced motility and redox disturbance in mouse sperm in vitro, and NAC exerted significant rescue effects. Moreover, surface plasmon resonance (SPR) analysis revealed the binding of chlorothalonil to β-actin, a sensitive target of electrophilic substances. Taken together, all data indicate that low dose of chlorothalonil caused testicular impairment in mice possibly through interacting with nucleophilic molecules. This study challenges the acceptable daily intake (20 or 30 μg/kg/d) of chlorothalonil established previously, and calls for great attention on male reproductive hazards of more electrophilic pollutants.