Integration of network toxicology, transcriptomics and metabolomics reveals the novel neurotoxic mechanisms of diazepam and its metabolites.

Diazepam is a widely used benzodiazepine, and its presence in animal-derived food products is strictly prohibited in China. However, the occasional detection of diazepam residues in freshwater fish has raised growing concerns regarding the safety of aquatic products and potential risks to human health. This study integrated network toxicology, cytotoxicity tests, transcriptomics and metabolomics to evaluate the neurotoxic effects and underlying mechanisms associated with diazepam and its principal metabolite, nordazepam. Both compounds exhibited notable neurotoxic effects, with microglial cells (HMC3) demonstrating greater sensitivity compared to oligodendrocytes (Mo 3.13) and neurons (SH-SY5Y). Network analysis and experimental data identified STAT3, CASP3, and TSPO as core targets mediating neurotoxicity associated with withdrawal symptoms, cognitive deficits, and motor impairments. Molecular docking and dynamics simulations further confirmed stable binding to these targets. These findings suggest that current risk assessments of diazepam residues necessitate reevaluation and underscore the critical role of microglial cells in diazepam and nordazepam-induced neurotoxicity, providing valuable insights for public health and dietary exposure safety.