Molecular cloning, characterization of CAT, and eco-toxicological effects of dietary zinc oxide on antioxidant enzymes in Eisenia fetida

The full-length cDNA of catalase (EfCAT) from Eisenia fetida was cloned (GenBank accession no. JN617999). Sequence characterization revealed that EfCAT protein sequence contained proximal heme-ligand signature sequence (³⁵¹RLFSYSDTH³⁵⁹), two glycosylation sites (N₁₄₅ and N₄₃₆), the proximal active site signature (⁶¹FDRERIPERVVHAKGAGA⁷⁸), and 12 amino acids (N₁₄₅, H₁₉₁, F₁₉₅, S₁₉₈, R₂₀₀, N₂₁₀, Y₂₁₂, K₂₃₄, I₂₉₉, W₃₀₀, Q₃₀₂, and Y₃₅₅), which were identified as putative residues involved in NADPH binding. These conserved motifs and catalase signature sequences were essential for the structure and function of EfCAT. The present study also investigated the effect of the veterinary food additive zinc oxide on antioxidant processes in E. fetida, at different concentrations and exposure durations. A significant increase (by 106.0 % compared to controls) in CAT activity at 500 mg/kg was registered at day 15. The superoxide dismutase (SOD) activity at 500 mg/kg increased to the maximum value (by 44.0 %) measured at day 15. There was a significant increase in glutathione peroxidase (GPx) activity for all concentrations after 5 days. The results showed that dietary Zn (500 mg/kg) causes oxidative damage to earthworms. At early stages of earthworms exposed to ZnO, GPx is the main enzyme to impair the oxidative status; while at later stages the enzymes CAT and SOD were the main indicators of oxidative stress. The antioxidant enzymatic variations may be an adaptive response of earthworms to survive in contaminated soils.