8-Oxoguanine DNA glycosylase 1 (OGG1) from the copepod Tigriopus japonicus: molecular characterization and its expression in response to UV-B and heavy metals.

8-Oxoguanine DNA glycosylase 1 (EC 3.2.2.23) is encoded by OGG1 gene and plays a key role in removing 8-oxo-7,8-dihydroguanine (8-oxoG) base in DNA lesion by reactive oxygen species (ROS). To identify and characterize OGG1 gene (TJ-OGG1) in the copepod Tigriopus japonicus, the full-length cDNA sequence, genomic structure, and promoter region was analyzed. In addition, to investigate transcriptional change of TJ-OGG1 mRNA under oxidative stress conditions, T. japonicus were exposed to environmental oxidative inducers, H(2)O(2), UV-B, and heavy metals (Cd, Cu, and Zn), respectively. The full-length cDNA of TJ-OGG1 gene was 1708 bp in length, encoding 343 amino acid residues. The deduced amino acid sequences of TJ-OGG1 showed a 56% similarity with human. Two conserved motifs (HhH and PVD loop) and two conserved residues (lysine and aspartic acid) in active sites were also observed. TJ-OGG1 genome structure contained six exons and five introns and putative transcription factor binding sites such as Nrf-2, p53, ERE-half sites, and XRE were detected on the promoter region. TJ-OGG1 mRNA level was increased at approximately three-fold (P<0.05) at 1mM and approximately 4-fold (P<0.01) at 10mM of H(2)O(2), respectively. UV-B enhanced the expression of TJ-OGG1 mRNA at 15kJ/m(2) (P<0.05) and more (P<0.001). In a time-course experiment, TJ-OGG1 gene was highly transcribed within 12h after exposure of 10 kJ/m(2) (P<0.01) and 20 kJ/m(2) (P<0.001). The expression of TJ-OGG1 mRNA after exposure to Cu and Cd for 96 h was significantly up-regulated at 0.1 μg/L and then remarkably reduced in a dose-dependent manner. Their transcript levels did not change at low dose (0.1 and 1 μg/L) but were dose-dependently down-regulated at high dose (10 and 100 μg/L). These findings suggest that H(2)O(2), UV-B, and heavy metals induce oxidative stress and generate oxidatively damaged DNA. Consequently, the enhanced TJ-OGG1 gene expression would be associated with active involvement of TJ-OGG1 gene in DNA repair process as a cellular protection mechanism. This is the first report on the cloning and characterization of OGG1 gene in aquatic animals. This study is helpful for a better understanding of the molecular mechanisms of cellular protection against various environmental oxidative stress inducers such as UV-B and heavy metals in aquatic invertebrates.

Kim BM ，Rhee JS ，Seo JS ，Kim IC ，Lee YM ，Lee JS ... -  《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY》

Role of crustacean hyperglycemic hormone (CHH) in the environmental stressor-exposed intertidal copepod Tigriopus japonicus.

To identify and characterize CHH (TJ-CHH) gene in the copepod Tigriopus japonicus, we analyzed the full-length cDNA sequence, genomic structure, and promoter region. The full-length TJ-CHH cDNA was 716 bp in length, encoding 136 amino acid residues. The deduced amino acid sequences of TJ-CHH showed a high similarity of the CHH mature domain to other crustaceans. Six conserved cysteine residues and five conserved structural motifs in the CHH mature peptide domain were also observed. The genomic structure of the TJ-CHH gene contained three exons and two introns in its open reading frame (ORF), and several transcriptional elements were detected in the promoter region of the TJ-CHH gene. To investigate transcriptional change of TJ-CHH under environmental stress, T. japonicus were exposed to heat treatment, UV-B radiation, heavy metals, and water-accommodated fractions (WAFs) of Iranian crude oil. Upon heat stress, TJ-CHH transcripts were elevated at 30 °C and 35 °C for 96 h in a time-course experiment. UV-B radiation led to a decreased pattern of the TJ-CHH transcript 48 h and more after radiation (12 kJ/m(2)). After exposure of a fixed dose (12 kJ/m(2)) in a time-course experiment, TJ-CHH transcript was down-regulated in time-dependent manner with a lowest value at 12h. However, the TJ-CHH transcript level was increased in response to five heavy metal exposures for 96 h. Also, the level of the TJ-CHH transcript was significantly up-regulated at 20% of WAFs after exposure to WAFs for 48 h and then remarkably reduced in a dose-dependent manner. These findings suggest that the enhanced TJ-CHH transcript level is associated with a cellular stress response of the TJ-CHH gene as shown in decapod crustaceans. This study is also helpful for a better understanding of the detrimental effects of environmental changes on the CHH-triggered copepod metabolism.

Kim BM ，Jeong CB ，Han J ，Kim IC ，Rhee JS ，Lee JS ... -  《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY》

Environmental stressors (salinity, heavy metals, H2O2) modulate expression of glutathione reductase (GR) gene from the intertidal copepod Tigriopus japonicus.

Seo JS ，Lee KW ，Rhee JS ，Hwang DS ，Lee YM ，Park HG ，Ahn IY ，Lee JS ... -  《AQUATIC TOXICOLOGY》

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 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.

Kim BM ，Rhee JS ，Park GS ，Lee J ，Lee YM ，Lee JS ... -  《-》

Cloning and expression of ecdysone receptor (EcR) from the intertidal copepod, Tigriopus japonicus.

Hwang DS ，Lee JS ，Lee KW ，Rhee JS ，Han J ，Lee J ，Park GS ，Lee YM ，Lee JS ... -  《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY》