Metal-induced antioxidant defense in the solitary ascidian Ciona intestinalis

Antioxidant enzymes play important roles in antioxidant responses caused by metabolic process or pathogen invasion. In many organisms, it has been reported that antioxidant enzymes participate in their innate immune defense against immuno-stimulant challenges such as β-glucan and sulfated polysaccharide, LPS or viruses. Cu,Zn superoxide dismutase (SOD, EC 1.15.1.1) is one of these key enzymes, highly conserved from invertebrates to vertebrates, involved in scavenging superoxide radicals into molecular oxygen and hydrogen peroxide. However, little is known about the responses of antioxidant enzymes like Cu,Zn SOD in tunicates after exposure to environmental changes in heavy metal concentrations. The present research focuses on structural and functional studies of the Cu,Zn SOD gene in the solitary ascidian Ciona intestinalis. The gene sequence has been identified in GenBank and the amino acid sequence of the codified protein has been compared with those deduced from orthologous genes in other metazoans, both vertebrates and invertebrates, to determine if the amino acids important for catalytic activity were conserved and whether the enzyme of C. intestinalis acquired special features, relatively to the primary sequence, during its evolution. The in silico analysis was extended to the promoter regions for the presence of regulatory sequences such as antioxidant response elements (ARE), metal response elements (MRE) and xenobiotic response elements (XRE). The available sequences were used for cladistic studies, providing some interesting inference on the molecular evolution of the C. intestinalis protein. Using semi-quantitative RT-PCR technique, Cu,Zn SOD gene expression have been evaluated as a function of exposure to three different metals (Cd, Zn and Cu). For this purpose, specimens of C. intestinalis were divided into three experimental groups, which were exposed for 6, 24, 48, 72, 96 and 120 h at equimolar concentrations (10 μM) of single metal. Untreated specimens were used as controls. Cu,Zn SOD is mainly induced by Cu and Zn, metals that are components of the active site. Besides, it was provided an initial estimation on the localization of expression by in situ hybridization. The results indicate that the cells most involved in the expression of the considered genes are the hemocytes.