Cu/Zn- and Mn-superoxide dismutase (SOD) from the copepod Tigriopus japonicus: Molecular cloning and expression in response to environmental pollutants Bo-Mi Kim a,1 , Jae-Sung Rhee b,1 , Gyung Soo Park c , Jehee Lee d , Young-Mi Lee e,⇑ , Jae-Seong Lee a,b,⇑ a Department of Chemistry, The Research Institute for Natural Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea b Department of Molecular and Environmental Bioscience, Graduate School, Hanyang University, Seoul 133-791, South Korea c Department of Marine Biotechnology, College of Liberal Arts and Sciences, Anyang University, Ganghwa 417-833, South Korea d Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju 690-756, South Korea e Department of Green Life Science, College of Convergence, Sangmyung University, Seoul 110-743, South Korea article info Article history: Received 12 February 2011 Received in revised form 8 April 2011 Accepted 17 April 2011 Available online 7 May 2011 Keywords: Copepod Tigriopus japonicus Superoxide dismutase Heavy metals Benzo[a]pyrene 4-Nonylphenol abstract Superoxide dismutase (SOD) is an important antioxidant enzyme which catalyzes conversion of superox- ide 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 mito- chondrial 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 sil- ver; 0, 10, 25, 50, and 100 lgL À1 ) and industrial chemicals (benzo[a]pyrene, 4-nonylphenol, and tribu- tyltin; 0, 1, 5, 10, and 20 lgL À1 ) for 96 h. 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 0 -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 (50 lgL À1 and 100 lgL À1 ). 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 5 lgL À1 concentration, while TBT decreased its expression at high concentrations (10 lgL À1 and 20 lgL À1 ). 4-NP increased both TJ-SODs mRNA level at 1 lgL À1 concentration, and then inhibited its expression from 5 lgL À1 concentration to a lower level than the control. This finding sug- gests that TJ-Cu/Zn-SOD and TJ-Mn-SOD would be an inducible gene upon exposure to heavy metals and B[a]P, and could be used as a potential biomarker for the risk assessment of these environmental pol- lutants. 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. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Environmental pollutants released from anthropogenic resources (i.e. industrial chemicals and water wastes) enter aquatic animals via food-uptake, epidermis, and gills, and tend to accumu- late in their body. Pollutants have been a great concern in aquatic systems because of their adverse effects on development, 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.04.043 ⇑ Corresponding authors. Address: Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea. Fax: +82 2 2299 9450 (J.-S. Lee). E-mail addresses: ymlee70@smu.ac.kr (Y.-M. Lee), jslee2@hanyang.ac.kr (J.-S. Lee). 1 These two authors equally contributed to this manuscript. Chemosphere 84 (2011) 1467–1475 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere