Diazepam exerts immunosuppressive effects on macrophages isolated from the head kidney of catfish (Silurus asotus).

Diazepam (DZ), a common benzodiazepine, is increasingly detected in aquatic environments, yet its immunological effects in fish remain poorly understood. We investigated the impact of DZ at concentrations ranging from 1 to 10 mg/L on head kidney-derived macrophages of Amur catfish (Silurus asotus). Cell viability was unaffected, but DZ induced dose-dependent macrophage aggregation and significantly reduced phagocytic activity. Transcriptomic profiling revealed 1053 differentially expressed genes, with suppression of immune-related pathways, including PI3K-Akt, MAPK, phagosome, and toll-like receptor signaling. Increased expression of translocator protein, a negative regulator of neuroinflammation, further supported the inhibition of the above pathways. These findings demonstrate that DZ attenuates macrophage immune functions, highlighting the immunosuppressive risks of neuroactive pharmaceuticals in aquatic systems. Our study provides mechanistic insight into the neuroimmune crosstalk triggered by DZ exposure, underscoring the need to consider psychiatric drugs as emerging contaminants of ecological concern.