Sub-chronic exposure to diazepam induces hepatic metabolism dysfunction associated with oxidative stress in zebrafish (Danio rerio).

Diazepam (DZP), one of the most widely prescribed benzodiazepines (BZDs), is commonly used clinically to treat anxiety and epilepsy by reducing neuronal excitability. However, its potential ecological toxicity remains poorly understood. In this study, adult female zebrafish were exposed to environmentally relevant concentrations of DZP for 28 days, and hepatic responses were evaluated through integrated biochemical, transcriptional, and untargeted metabolomics analyses. DZP exposure altered antioxidant enzyme activities and increased oxidative stress markers, indicating disruption of hepatic redox homeostasis. Significant alterations were also observed in triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and glucose (Glu). These physiological alternations were accompanied by modified expression of genes associated with glucose and lipid metabolism. Untargeted metabolomics analysis further revealed extensive metabolic reprogramming after DZP exposure. KEGG enrichment analysis indicated significant disturbances in energy and amino acid metabolism, with histidine metabolism identified the most prominently affected pathway. Alterations in metabolites associated with glutathione synthesis, membrane phospholipids, and purine metabolism further suggested widespread metabolic dysregulation in the liver following DZP exposure. Collectively, these findings demonstrate that environmentally relevant DZP exposure disrupts hepatic metabolism and induces oxidative stress in zebrafish, providing new insights into the ecotoxicological effects of DZP on aquatic organisms.