Ferroptosis involvement in the neurotoxicity of flunitrazepam in zebrafish

Abstract

The occurrence of psychoactive drugs such as benzodiazepines (BZDs) has been frequently observed in water environments, however, there is still limited understanding regarding their potential impact on neurological health and underlying mechanisms. This study evaluates the neurotoxicity of the typical BZD drug flunitrazepam (FLZ, 0.2 and 5 μg/L) in zebrafish embryos and adults, and investigates the relationship between ferroptosis and FLZ-induced neurotoxicity. The results indicate that acute exposure to FLZ significantly inhibits zebrafish embryo hatching and promotes death, induces larval deformities, and leads to abnormal neurobehavioral responses in larvae, likely due to ferroptosis induction. Results from a 30-day subacute exposure to FLZ show that it decreases motor function and induces 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.