Combined Effects of Lead and Chromium at Environmentally Relevant Concentrations in Zebrafish (Danio rerio) Liver: Role of Nrf2-Keap1-ARE Pathway

Abstract

The extensive industrial use of lead (Pb) and chromium (Cr) has led to their persistent release into aquatic ecosystems, posing severe ecological and toxicological challenges. While the individual toxicities of these metals are well-documented, their combined effects, particularly on toxicity mechanisms and cellular stress responses, remain inadequately understood. This study investigated the hepatotoxic effects of Pb and Cr, both individually and in combination, in zebrafish (Danio rerio), focusing on oxidative stress and the Nrf2-Keap1-ARE signaling pathway. Zebrafish were exposed to environmentally relevant concentrations of Pb (2.5, 5, and 10 ppb), Cr (0.5, 1, and 2 ppm), and their combination for 15, 30, and 60 days. Combined exposure elicited heightened oxidative stress, marked by elevated reactive oxygen species, lipid peroxidation, catalase activity, and a significant reduction in glutathione levels. Histopathological analysis revealed severe alterations, including vacuolation, sinusoidal dilation, and necrosis, with the most pronounced effects observed in the combined exposure groups. Gene expression analysis demonstrated the upregulation of oxidative stress-related genes (nrf2, ho1, nqo1, gpx1, catalase, gst, cu/znsod, mnsod, and cyp1a) and heat shock protein (hsp70) and the downregulation of keap1, and ucp2, particularly in co-treated groups. Immunofluorescence observation confirmed enhanced nuclear translocation of Nrf2, while atomic absorption spectrophotometry studies revealed significant bioaccumulation of Pb and Cr in the liver, especially during combined exposures. These findings underscore that Pb and Cr co-exposure intensifies oxidative stress and hepatotoxicity via the Nrf2-Keap1-ARE pathway, emphasizing the environmental and health risks associated with heavy metal contamination in aquatic ecosystems.