Ferroptosis involvement in the neurotoxicity of flunitrazepam in zebrafish

In recent years, psychoactive drugs such as benzodiazepines (BZDs) have been frequently detected in water environments, however, there is still limited understanding regarding their potential impact on neurological health and underlying mechanisms. This study evaluated the neurotoxicity of the typical BZD drug flunitrazepam (FLZ, 0.2 and 5 μg/L) in zebrafish embryos and adults, and investigated the relationship between ferroptosis and FLZ-induced neurotoxicity. The results indicated that acute exposure to FLZ significantly inhibited zebrafish embryo hatching and promotes death, induced larval deformities, and led to abnormal neurobehavioral responses in larvae, likely due to ferroptosis induction. Results from a 30-day subacute exposure to FLZ showed that it decreased motor function and induced cognitive impairment in adult zebrafish. Immunofluorescence of brain tissues revealed a reduction in neurons in the telencephalon and an increase in microglia in the mesencephalon of the zebrafish exposed to FLZ. The ultrastructure of brain mitochondria showed serious damage. Besides, FLZ exposure increased iron levels, reduced GSH/GSSG and increased LPO in brain tissue, which is related to the abnormal expression of genes associated with ferroptosis. In the rescue experiments with co-exposure to deferoxamine (DFO), the motor-related parameters and biochemical indexes related to ferroptosis were restored, suggesting that FLZ can induce ferroptosis. The molecular docking results indicated that FLZ had a higher affinity with transferrin. This study elucidates the close relationship between ferroptosis and FLZ-induced neurotoxicity, which is significant for understanding the physiological damage caused by psychoactive substances and assessing environmental risks.