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.

中文翻译：

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铁死亡参与氟硝西泮在斑马鱼中的神经毒性


近年来，苯二氮卓类药物 （BZD） 等精神活性药物经常在水环境中被检测到，然而，人们对它们对神经系统健康的潜在影响和潜在机制的认识仍然有限。本研究评价了典型苯二氮卓类药物氟硝西泮 （FLZ，0.2 和 5 μg/L） 在斑马鱼胚胎和成人中的神经毒性，并探讨了铁死亡与 FLZ 诱导的神经毒性之间的关系。结果表明，急性暴露于 FLZ 显着抑制斑马鱼胚胎孵化并促进死亡，诱导幼虫畸形，并导致幼虫异常神经行为反应，这可能是由于铁死亡诱导。30 天亚急性暴露于 FLZ 的结果表明，它降低了成年斑马鱼的运动功能并诱导了认知障碍。脑组织的免疫荧光显示暴露于 FLZ 的斑马鱼端脑神经元减少，中脑小胶质细胞增加。脑线粒体的超微结构显示严重损伤。此外，FLZ 暴露增加了脑组织中的铁水平，降低了 GSH/GSSG 并增加了 LPO，这与铁死亡相关基因的异常表达有关。在共暴露于去铁胺 （DFO） 的挽救实验中，恢复了与铁死亡相关的运动相关参数和生化指标，提示 FLZ 可诱导铁死亡。分子对接结果表明，FLZ 与转铁蛋白具有更高的亲和力。本研究阐明了铁死亡与 FLZ 诱导的神经毒性之间的密切关系，这对于理解精神活性物质引起的生理损伤和评估环境风险具有重要意义。