Enantioselective effects of dinotefuran on oxidative stress and energy metabolism in HepG2 cells

• S-(+)-dinotefuran exhibited stronger hepatotoxicity than R-(–)-enantiomer in HepG2 cells. • S-(+)-dinotefuran uniquely disrupted oxidative stress, glycolysis, and mitochondrial respiration. • Targeted metabolomics shows inhibition of key metabolites including pyruvate, malate, and aspartate • Proteomics revealed S-(+)-dinotefuran dysregulates redox homeostasis and fatty acid β-oxidation. • Molecular docking confirms tighter binding of S-(+)-dinotefuran to peroxisomal 3-ketoacyl-CoA thiolase. Dinotefuran, a chiral neonicotinoid insecticide, is widely detected in environmental and biological samples and exhibits enantioselective effects on non-target organisms. However, its potential hepatotoxic mechanisms remain largely unclear. In this study, we demonstrated that S-dinotefuran ( S -DIN) induced HepG2 cells more pronounced oxidative stress and energy metabolism disruption compared with R-dinotefuran ( R -DIN). Targeted metabolomics covering 25 key metabolites involved in energy metabolism revealed that S -DIN caused the broadest and most substantial perturbations, characterized by the accumulation of tricarboxylic acid (TCA) cycle intermediates to sustain cellular energy demands. Proteomic analysis further indicated that S -DIN triggered greater alterations in proteins associated with metabolism and oxidative stress. Mechanistically, due to its stereochemical configuration, S -DIN displayed a stronger binding affinity to 3-ketoacyl-CoA thiolase, peroxisomal, thereby initiating a cascade of oxidative damage and metabolic dysregulation. Collectively, these findings highlight the greater hepatotoxic potential of S -DIN over R -DIN and provide critical insights for the risk assessment and safe application of chiral neonicotinoid pesticides.