Study on the alleviation of reactive oxygen species-mediated flunitrazepam toxicity in zebrafish (Danio rerio) by vitamin C and its mechanism.

The frequent detection of psychoactive drugs in aquatic environments has caused various toxic effects on aquatic organisms, highlighting the urgent need to explore remediation methods and mechanisms. Against the backdrop of toxicity induced by the typical benzodiazepine (BZD) flunitrazepam (FLZ) in zebrafish, this study evaluates the mitigating effects of vitamin C (VC) on FLZ-induced embryonic developmental toxicity, larval behavioral anomalies, apoptosis, oxidative stress, and mitochondrial dysfunction at environmentally relevant concentrations through reactive oxygen species (ROS)-mediated pathways. Furthermore, molecular dynamics simulations were utilized to decipher the mechanism underlying ROS inhibition. Results demonstrated that co-exposure to 0.5 μg/L VC with FLZ (0.05 μg/L and 0.2 μg/L) significantly elevated the hatching rate of zebrafish embryos at 72 hpf and decreased the larval malformation rate at 96 hpf. In terms of physiological and biochemical indicators, VC significantly inhibited the FLZ-induced increase in ROS and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels. VC also upregulated the activity of mitochondrial uncoupling protein 2 (UCP2), a key regulator of ROS production. Molecular docking and dynamics simulations revealed that VC competitively binds to the LYS 38 and LYS 240 sites of UCP2, destabilizing FLZ-UCP2 interactions via steric hindrance and hydrogen bond competition. With the restoration of UCP2 activity, its proton leak function was enhanced, suppressing excessive ROS generation. Consequently, uqcr2b, cox4i1l, and atp5g3b were normalized, restoring ATP synthesis capacity and significantly alleviating FLZ-induced mitochondrial dysfunction. This study elucidates the mechanism by which VC counteracts ROS-mediated FLZ toxicity, providing critical insights for assessing environmental risks and formulating protective strategies against pollutants.