Mixture Effects of Lead and Chromium in Zebrafish (Danio rerio) Brain: Involvement of Nrf2-Keap1-ARE Pathway

Lead (Pb) and chromium (Cr) are prevalent aquatic contaminants capable of inducing oxidative and neurological disturbances; however, the consequences of their combined exposure on brain oxidative homeostasis remain poorly understood. The present study examined the progressive oxidative, molecular, and histopathological effects of chronic Pb and Cr exposure, individually and in combination, in zebrafish (Danio rerio), with particular emphasis on modulation of the Nrf2-Keap1-ARE pathway. Adult male zebrafish were exposed to environmentally relevant concentrations of Pb (2.5, 5, and 10 ppb), Cr (0.5, 1, and 2 ppm), and corresponding mixed exposures for 15, 30, and 60 days. Prolonged exposure caused progressive reductions in the brain somatic index, accompanied by marked oxidative imbalance characterized by increased reactive oxygen species generation, elevated lipid peroxidation, enhanced catalase activity, and reduced glutathione. Cr exposure elicited comparatively greater oxidative and molecular responses than Pb, while co-exposure consistently produced the most severe alterations. Histopathological analysis revealed vacuolar degeneration, edema, tectal disorganization, meningeal deformation, and increased mononuclear cells with surrounding clear spaces within the optic tectum. Molecular analysis demonstrated significant upregulation of nrf2, nqo1, ho1, hsp70, cyp1a, gst, gpx, cat, mnsod, cu/znsod, and ache, along with suppression of keap1 and ucp2, indicating activation of antioxidant defense and stress-responsive pathways. Enhanced Nrf2 immunoreactivity further confirmed activation of the Nrf2-mediated protective response in exposed brain tissue. Together, these results exhibited that prolonged co-exposure to environmentally relevant Pb and Cr concentrations promoted progressive oxidative, molecular, and histopathological alterations in the zebrafish brain, accompanied by activation of the Nrf2-Keap1-ARE signaling pathway.