Embryonic diazepam exposure induces innate immune toxicity in zebrafish (Danio rerio)

Diazepam (DZP), a benzodiazepine anxiolytic drug and positive allosteric modulator of GABAA receptors, is increasingly detected in aquatic ecosystems. Its rise in environmental prevalence is likely attributable to its expanded clinical use, poorly controlled consumption and insufficient removal during wastewater treatment processes, thereby raising concerns regarding its potential ecological impacts. While previous research has predominantly focused on the neurobehavioral effects of DZP, its immunotoxic effects on aquatic organisms remain poorly characterized. In this study, zebrafish (Danio rerio) embryos were exposed to DZP concentrations ranging from environmentally relevant levels (5 and 10 μg/L) to higher doses (100 and 1000 μg/L) for 120 h post-fertilization. DZP exposure significantly delayed embryo hatching at 48 and 54 h and compromised the pathogen resistance of the resultant larvae against Aeromonas hydrophila infection. Furthermore, DZP disrupted immune homeostasis by altering the activities of key immune-related enzymes and modulating expression profiles of cytokines. Molecular docking analysis identified robust binding interactions between DZP and core proteins within the MAPK signaling pathway, indicating that DZP may suppress inflammatory responses and induce immune dysfunction via MAPK signaling. These findings highlight the immunotoxic potential of DZP during critical developmental stages and warrant further investigation into its ecological impacts.