Role of Ascorbic Acid in Mitigating Oxidative Stress and Mutagenicity Induced by Heavy Metals Toxicity in <i>Heteropneustes fossilis</i> . L

Environmental contamination by hazardous metals poses significant risks to aquatic ecosystems and human health. Heavy metals such as cadmium (Cd), mercury (Hg), and lead (Pb) induce oxidative stress (OS) through the excessive generation of reactive oxygen species (ROS), resulting in behavioural, biochemical, genetic, and histological damage. To evaluate the protective efficacy of ascorbic acid (AA) against acute Cd, Hg, and Pb toxicity in Heteropneustes fossilis using an integrated behavioural, biochemical, oxidative stress, and genotoxicity assessment framework, focusing on its antioxidant properties and ability to scavenge free radicals, we used an integrated behavioural, biochemical, oxidative stress, and genotoxicity assessment framework. Adult H. fossilis were randomly allocated into treatment groups (n = 10/group) and exposed for 96 h to Cd (50.4 mg/L), Hg (0.70 mg/L), Pb (280.04 mg/L), AA (100 mg/L), or metal + AA combinations. Blinded observers recorded behavioural and histological outcomes. Biochemical and oxidative stress markers (SOD, CAT, GSH, MDA, AChE) were quantified using validated assays. Data were tested for normality (Shapiro-Wilk), outliers, and homogeneity of variance, followed by a one-way ANOVA with post hoc multiple comparisons (Tukey). Results are reported as mean ± SEM. AChE activity showed a significant increase in Cd- and Hg-exposed groups (p < 0.05), confirming neurotoxicity, whereas AA co-treatment restored values toward control levels, likely through its free radical scavenging activity. Antioxidant enzymes (SOD, CAT, and GSH) were significantly reduced across the Cd, Hg, and Pb groups (p < 0.01), while MDA levels were elevated, indicating lipid peroxidation. No significant differences were observed between the control, AA alone, and HMs + AA groups for most biochemical markers, indicating that AA plays a protective role by mitigating oxidative damage. Micronucleus test results showed a marked increase in micronuclei frequency in all HMs groups (p < 0.001), with a significant reduction after AA co-exposure, suggesting genoprotective effects. Histopathological alterations in gills, liver, kidney, and brain were pronounced in metal-exposed fish but minimal when co-treated with AA. Ascorbic acid significantly mitigates heavy metal-induced neurotoxicity, oxidative stress, genotoxicity, and tissue damage in H. fossilis. These findings support AA as a potent bioactive antioxidant capable of reducing acute heavy metal toxicity in fish, with potential relevance for other aquatic species.