Cu/Zn- and Mn-superoxide dismutase (SOD) from the copepod Tigriopus japonicus: Molecular cloning and expression in response to environmental pollutants

Superoxide dismutase (SOD) is an important antioxidant enzyme which catalyzes conversion of superoxide to oxygen and hydrogen peroxide in aerobic organisms. Here, we cloned and sequenced the full-length cDNA and genomic DNA of two SODs from the copepod, Tigriopus japonicus: copper/zinc SOD (TJ-Cu/Zn-SOD) and manganese SOD (TJ-Mn-SOD). To define whether TJ-Mn-SOD is a cytosolic or a mitochondrial protein, a phylogenetic analysis was performed. The genomic structure of both TJ-SOD genes was determined with the promoter region sequences. In order to investigate their potential role in response to environmental pollutants, T. japonicus were treated with heavy metal (copper, zinc, and silver; 0, 10, 25, 50, and 100μgL^-1) and industrial chemicals (benzo[α]pyrene, 4-nonylphenol, and tributyltin; 0, 1, 5, 10, and 20μgL^-1) for 96h. Subsequently, the TJ-Cu/Zn-SOD and TJ-Mn-SOD mRNA level was measured with quantitative real-time RT-PCR along with total SOD activity. The deduced amino acid residues of TJ-Cu/Zn-SOD and TJ-Mn-SOD possessed evolutionary conserved domains that are required for metal binding and Cu/ZnSOD-conserved signature sequences. The phylogenetic analysis revealed that TJ-Mn-SOD was closely clustered to mitochondrial Mn-SOD of another copepod, Lepeophtheirus salmonis. TJ-Cu/Zn-SOD gene had four exons and three introns, while the TJ-Mn-SOD gene consisted of two exons interrupted by one intron. In the 5′-flanking region of TJ-Cu/Zn-SOD and TJ-Mn-SOD, we observed several transcription regulatory elements such as p53, XRE, MRE, and ERE-half sites. In the response to heavy metals, Cu, Zn, and Ag, both TJ-Cu/Zn-SOD and TJ-Mn-SOD transcript levels along with enzyme levels were significantly increased at high concentrations (50μgL^-1 and 100μgL^-1). Particularly, in the Cu- and Ag-exposed group, the expression of TJ-Mn-SOD mRNA was regulated more sensitively than the TJ-Cu/Zn-SOD mRNA level, indicating that the chemical susceptibility would be not correlated with the form of chemicals. B[a]P treatment showed a significant increase in the expression of both TJ-SODs mRNA level and enzyme level from 5μgL^-1 concentration, while TBT decreased its expression at high concentrations (10μgL^-1 and 20μgL^-1). 4-NP increased both TJ-SODs mRNA level at 1μgL^-1 concentration, and then inhibited its expression from 5μgL^-1 concentration to a lower level than the control. This finding suggests that TJ-Cu/Zn-SOD and TJ-Mn-SOD would be an inducible gene upon exposure to heavy metals and B[α]P, and could be used as a potential biomarker for the risk assessment of these environmental pollutants. This is the first report to elucidate response of SOD to environmental pollutants in copepods. Therefore, this study would give a clue to better understand the mode of action of antioxidant genes and enzymes under oxidative stress in marine invertebrates.