Samra_2026_J.Hazard.Mater_514_142954

Lead (Pb) and copper (Cu) frequently co-occur in aquatic environments, yet their combined neurotoxic mechanisms remain unclear. Here, zebrafish (Danio rerio) larvae were exposed to environmentally relevant concentrations of Pb (10 microg/L) and Cu (20 microg/L) to assess neurobehavioral and molecular effects. Co-exposure reduced locomotor activity, altered stress-related behavioral responses, and increased developmental abnormalities. Acetylcholinesterase activity was suppressed, accompanied by elevated lipid peroxidation and disrupted antioxidant defenses. Apoptosis was activated via Bax/Bcl-2/Caspase-3 modulation, with downregulation of neurodevelopmental and neurotransmission marker genes. Ferroptosis emerged as a significant contributor to Pb + Cu neurotoxicity, as indicated by dysregulation of nrf2, keap1, and gpx4. Mitochondrial dysfunction was evident through reduced ATP, impaired biogenesis, disrupted electron transport, and excessive fission. Inflammation was mediated via NF-kappaB/p38-MAPK pathway, with upregulation of pro-inflammatory cytokines and altered anti-inflammatory markers. Western blotting confirmed activation of Nrf2/Keap1/HO-1 signaling axis, highlighting the functional role of ferroptosis in Pb- and Cu-induced neurotoxicity. Importantly, treatment with the ferroptosis inhibitor ferrostatin-1 partially alleviated oxidative damage and neurobehavioral deficits, supporting a contributory role of ferroptosis in the observed effects. Overall, these findings indicate that co-exposure to Pb and Cu induces neurotoxicity in zebrafish larvae through interconnected pathways involving oxidative stress, mitochondrial dysfunction, and ferroptosis-associated processes. This study highlights the ecological relevance of metal co-exposure and its potential risks to aquatic organisms.