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Cu/Zn- and Mn-superoxide dismutase ( SOD) from the copepod Tigriopus japonicus: Molecular cloning and expression in response to environmental pollutants
► Identification of cDNA and genomic DNA of two
SODs from the copepod. ► Identification of transcription regulatory elements in
SOD promoters. ► Confirmation of response to heavy metals (Cu, Ag, Zn) on
SOD transcripts. ► Confirmation of response to endocrine disrupting chemicals on
SOD transcripts. ► Elucidation on response of SOD to environmental pollutants in copepod.
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 100
μg
L
−1) and industrial chemicals (benzo[α]pyrene, 4-nonylphenol, and tributyltin; 0, 1, 5, 10, and 20
μg
L
−1) 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′-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
μg
L
−1 and 100
μg
L
−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 5
μg
L
−1 concentration, while TBT decreased its expression at high concentrations (10
μg
L
−1 and 20
μg
L
−1). 4-NP increased both
TJ-SODs mRNA level at 1
μg
L
−1 concentration, and then inhibited its expression from 5
μg
L
−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.