Sulfasalazine Enhances Copper Toxicity in <i>Danio rerio</i> Larvae: Divergent Protective Roles of Deferoxamine and Ferrostatin‐1

ABSTRACT Copper is an environmental contaminant capable of disrupting redox homeostasis and impairing antioxidant defenses in aquatic organisms, thereby increasing susceptibility to oxidative stress. The cystine/glutamate antiporter (system xc − ) plays a central role in maintaining intracellular glutathione (GSH) levels, a major antioxidant defense. This study investigated how its pharmacological inhibition by sulfasalazine (SAS) modulates copper toxicity in Danio rerio larvae and evaluated the protective effects of ferrostatin‐1 (Fer‐1) and deferoxamine (DFO). Larvae at 72 h post‐fertilization (hpf) were pre‐exposed to 10 mM SAS for 4 h, followed by 24 h exposure to 5 μM CuSO 4 with either 100 μM DFO or 5 μM Fer‐1. Behavioral assays showed that copper induced abnormal swimming, but these effects were not intensified in SAS‐copper groups. However, SAS pre‐exposure exacerbated copper toxicity, reducing survival and non‐protein thiols (NPSH) levels while increasing reactive oxygen species (ROS) and lipid peroxidation (LPO). Fer‐1 and DFO reversed most alterations; however, in SAS‐copper groups, DFO preserved NPSH and reduced ROS but increased mortality and LPO. Fer‐1 restored survival and attenuated LPO across treatments. Correlation analyses revealed inverse associations between survival, ROS and LPO, while NPSH levels negatively correlated with oxidative damage. A strong correlation between ROS and LPO underscores the central role of lipid peroxidation in copper toxicity. Our findings demonstrated that GSH depletion by SAS sensitizes larvae to copper‐induced oxidative stress, highlighting the importance of system xc − in regulating susceptibility to metal toxicity.