Mechanistic insights into the stereoselective toxicity of chiral difenoconazole in HepG2 cells

Abstract

Difenoconazole, a triazole fungicide, exists in four stereoisomeric forms: (2R,4R), (2R,4S), (2S,4R), and (2S,4S)-difenoconazole. Significant differences in toxicity among these isomers were observed in HepG2 cells. The (2S,4S)-difenoconazole isomer exhibited the strongest cytotoxicity, reducing cell viability to 44.58% at 20 μg/mL, while (2S, 4R) had the weakest effect (85.47%). LDH release and oxidative stress markers (ROS and MDA) increased significantly, accompanied by elevated antioxidant enzyme activities (SOD and CAT), particularly in cells exposed to (2S,4S)- and (2R,4R)-difenoconazole. (2S, 4S) also caused severe DNA damage, cell cycle arrest (22.4% in G2/M phase), and apoptosis (24.45%), while (2S,4R)-difenoconazole had the least impact. Transcriptomic and molecular docking analyses revealed that (2S,4S)-difenoconazole affects key pathways, including p53 signaling, oxidative phosphorylation, and cell cycle regulation, with strong binding to ROS and SOD proteins. This study highlights the stereoselective toxicity of difenoconazole, providing important insights into the environmental and health risks of chiral pesticides.