Erythrocytic nuclear abnormalities and apoptotic gene dysregulation in zebrafish organs: Individual and combined genotoxic effects after lead and chromium exposure

Abstract

Lead (Pb) and chromium (Cr) are among the most extensively studied heavy metals due to their widespread presence and hazardous impacts on aquatic life. Pb and Cr exhibit strong genotoxic potential in aquatic environments by inducing DNA damage through overproduction of reactive oxygen species, resulting in oxidative stress, DNA strand breaks, and chromosomal aberrations. Disruption of nuclear integrity, impaired DNA repair pathways, and apoptosis activation further exacerbate their toxicity. However, most studies focus on their individual effects, often overlooking their combined genotoxic effects across multiple organs using a multi-endpoint approach under chronic, environmentally relevant exposure conditions. This study investigates the individual and combined genotoxic effects of Pb and Cr in adult male zebrafish (Danio rerio), a widely accepted ecotoxicological model, focusing on the blood, liver, gut, brain, and kidney as target tissues. Zebrafish were exposed to environmentally relevant concentrations of Pb (2.5, 5, and 10 ppb), Cr (0.5, 1, and 2 ppm), and their mixtures for 15, 30, and 60 days. Genotoxicity was assessed through erythrocytic nuclear anomalies assay, DNA fragmentation via agarose gel electrophoresis, apoptotic gene expression (p53, bax, bcl-2, caspase-9, and caspase-3) using qRT-PCR, and metal bioaccumulation by atomic absorption spectrophotometry. Results demonstrated a concentration- and time-dependent increase in nuclear anomalies, particularly micronuclei, blebbed, lobed, and notched nuclei, with the combined group showing the highest frequency. DNA fragmentation analysis confirmed significant genotoxicity, particularly in the liver and gut. Gene expression profiling revealed significant upregulation of pro-apoptotic genes (p53, bax, caspase-9, and caspase-3) and downregulation of bcl-2, indicating apoptosis induction. These findings underlined the synergistic and apoptotic effects of Pb and Cr, highlighting the importance of assessing chemical mixtures in aquatic toxicology. The study also offers mechanistic insights into tissue-specific toxicity, emphasizing the efficacy of zebrafish as a robust model for environmental risk assessment.