2 ISJ 11: 149-162, 2014 ISSN 1824-307X RESEARCH REPORT Molecular characterization of two glutathione peroxidase genes in Mytilus galloprovincialis and their transcriptional responses to sub-chronic arsenate and cadmium exposure Q Wang1, X Ning2, Q Zhang3, F Liu3, H Wu1, Y Zhang1, J Zhao1 1Key Laboratory of Coastal Zone Environment Processes and Ecological Remediation, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, PR China 2Yantai Oceanic Environmental Monitoring Central Station of SOA, Yantai 264006, PR China 3China Agriculture University (Yantai), Yantai 264670, PR China Accepted May 16, 2014 Abstract Glutathione peroxidases (GPxs) are key enzymes in the antioxidant defense system of living organisms, and protect organisms against oxidative stresses. In this study, the full-length cDNA sequences encoding cytosolic GPx (MgcGPx) and phospholipid-hydroperoxide GPx (MgGPx4) were identified from Mytilus galloprovincialis. The mussels were exposed to 0, 1, 10, and 100 μg/L cadmium and arsenate for 30 days. The mRNA transcripts of these two genes and total GPx activity were examined in the gills and digestive gland after contaminants exposure. The mussels exposed to cadmium and arsenate responded mainly by down-regulating MgcGPx and MgGPx4 mRNA transcription in gills and up-regulating transcription in digestive gland. However, total GPx activities increased following cadmium exposure but decreased after arsenate stress in both tissues. These results suggest that MgcGPx and MgGPx4 perhaps play an important role in maintaining cellular redox homeostasis and protecting M. galloprovincialis against cadmium and arsenate toxicity. It can also be inferred that these genes have the potential to be used as molecular biomarkers for assessing cellular stress and toxicity of contaminants in this mussel. Key Words: cytosolic GPx; phospholipid-hydroperoxide GPx; arsenic; cadmium; bivalve; antioxidant enzyme Introduction In recent years, a great number of contaminants including metals, petroleum oils and organic pollutants have been discharged into the Bohai Sea, a territorial sea in North China (Ma et al., 2001). Among these contaminants, metal contaminants have been recognized as the major pollutants in coastal areas. Cadmium (Cd) has been found in high concentrations in marine environments of the Bohai Sea (Meng et al., 2004; Meng et al., 2008; Wang et al., 2009). In addition, arsenic (As) pollution also has been of great concern because a total arsenic concentration was up to 440 μg L-1 in some polluted estuarine areas of the Bohai Sea (Meng et al., 2004; Wu et al., 2013). ___________________________________________________________________________ Corresponding authors: Huifeng Wu Jianmin Zhao Key Laboratory of Coastal Zone Environment Processes and Ecological Remediation Yantai Institute of Coastal Zone Research Chinese Academy of Sciences Yantai 264003, PR China E-mail: hfwu@yic.ac.cn. E-mail: jmzhao@yic.ac.cn The pollutants including metal contaminants constitute a potential threat to marine organisms (Winston and Di Giulio, 1991). The toxic damage induced by metals and arsenic is generally associated with excessive formation of reactive oxygen species (ROS), which cause oxidative modification of the major cellular macromolecules (Leonard et al., 2004; Ventura-Lima et al., 2011). Both Cd and arsenic have been shown to be toxic to marine bivalves and affect their physiological status by causing oxidative stress (Dovzhenko et al., 2005; Wang et al., 2010; Chakraborty et al., 2010). Excessive ROS production has been found to oxidize and damage cell membrane, proteins, and nucleic acids (Hensley et al., 2000). In marine animals, the oxidative stress can be counteracted by non-enzymatic antioxidant and enzymatic antioxidant systems (Fernández et al., 2010). The key components of these enzymatic antioxidant systems include glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) (Apel and Hirt, 2004). GPx (EC 1.11.1.9 and EC 1.11.1.12) can 149 mailto:jmzhao@yic.ac.cn catalyze the reduction of H2O2 or organic hydroperoxides to water or corresponding alcohols using reduced glutathione (GSH) (Margis, 2008). They are generally classified into two subgroups as selenium-dependent GPx and non-selenium GPx, based on the presence of selenocysteine (Sec) encoded by a stop codon TGA (Arthur, 2000). At present, eight GPx isozymes have been characterized in mammals, including: cytosolic GPx (GPx1), gastrointestinal GPx (GPx2), plasma GPx (GPx3), phospholipid hydroperoxide GPx (GPx4), epididymal GPx (GPx5), olfactory epithelium GPx (GPx6), endoplasmic reticulum phospholipid hydroperoxide GPxs (GPx7 and GPx8) (Herbette et al., 2007; Nguyen et al., 2011). In contrast to other tetrameric GPxs, GPx4 is monomeric and membrane-associated, especially in membrane fractions. GPx4 is unique in its substrate specificity because it can interact with lipophilic substrates, including the peroxidized phospholipids and cholesterol, and reduce these hydroperoxides to hydroxide compounds (Thomas et al., 1990). They mainly participate in the repair of disrupted bio-membranes, which has been considered as the main defense strategy against oxidative membrane damage (Bae et al., 2009). The role of cytosolic selenium-dependent GPxs in host immune and antioxidant defense system has been well documented in many mollusks, such as Unio tumidus (Doyen et al., 2006), Crassostrea gigas (Jo et al., 2008), Chlamys farreri (Mu et al., 2010), Meretrix meretrix (Wang et al., 2011a), Venerupis philippinarum (Zhang et al., 2011; Zhang et al., 2012) and Haliotis discus hannai (Wu et al., 2010). However, GPx4s have been less characterized and their roles in antioxidant system are less studied in aquatic animals. Until recently, only a few GPx4 genes have been cloned in aquatic organisms such as hydra (Dashe et al., 2006), midge (Nair et al., 2012) and several fishes (Wang et al., 2012; Pacitti et al., 2013). To our knowledge, no GPx4 gene has been cloned from mollusks although GPx4 activity was observed in the mussel Perna perna (Almeida et al., 2005). The mussel Mytilus galloprovincialis, extensively dispersed in the Bohai Sea, has a high adaptive ability to environments and occupies a broad range of habitat types. They are apt to be exposed to pollutants and suffer from oxidative stress in esutarine and coastal area of the Bohai sea. In China, this mussel has been used as a biomonitoring organism to assess the marine environment. Therefore, it is necessary to study how the antioxidant system of the mussel responds to environmental stress. To investigate the possible role of GPxs in protecting marine mussel from oxidative damage, two GPx genes were characterized and their transcriptional responses to sub-chronic cadmium and arsenic exposures were studied. In addition, the possibility of these genes used as biomarkers was also discussed. Materials and methods Animal culture Adult animals (shell length: 5.0 - 7.0 cm) were purchased from a local mussel culture farm and acclimatized in aerated seawater (33 psu) at 20 °C for 7 days before commencement of the exposure experiment. During the acclimatization period, the mussels were fed with Phaeodactylum tricornutum and Platymonas helgolandica, and the seawater was completely renewed once every 24 h. Contaminant exposure After the acclimatization, the mussels were divided into 21 groups and cultured in polyethylene tanks with 20 L seawater, each containing 20 individuals. The exposure experiments were performed using analytical grade salts of CdCl2·2.5H2O and Na2HAsO4·12H2O (Guoyao, Shanghai, China). Stock solutions were prepared in deionized water at a concentration of 1 g L-1, which was high enough to prevent weighing errors and salinity change. The exposure experiment for each contaminant included three concentrations: 1, 10 and 100 μg L-1 (contaminant concentration, not salt concentrations). There were 3 replicates (tanks) per treatment and the other three untreated tanks were employed as the control groups. The mussels were fed as described above and the culture medium was renewed once every 24 h. The gills and digestive glands of four individuals from each tank were randomly sampled for gene expression and antioxidant enzyme activity analysis after 30 days of exposure respectively. All the samples were quickly frozen in liquid nitrogen and then transferred to a -80 °C refrigerator till use. RNA isolation, cDNA synthesis and cloning of mussel GPx genes Total RNA was extracted from gills using the TRIzol Reagent (Invitrogen, USA) following the supplier’s protocol. The extracted RNA was then treated with RQ1 RNase-Free DNase (Promega, USA) to remove DNA contamination. Single-stranded cDNAs were synthesized from one microgram of the total RNA using M-MLV reverse transcriptase (Promega, USA) according to the manufacturer’s instructions. BLAST analysis of all expressed sequence tag (EST) sequences from a cDNA library of M. gallopronvicialis (unpublished) revealed that two ESTs were highly similar to the previously identified GPx genes, respectively. The 3′ ends of MgcGPx and MgGPx4 were obtained by rapid amplification of cDNA ends (RACE) using the SMARTTM RACE cDNA amplification Kit (Clontech, USA) according to the manufacturer’s manual. Two RACE primers were designed according to these ESTs and they were included in Table 1. The PCR products were purified using agarose gel electrophoresis, cloned into the pMD-18T simple vector (Takara, Japan) and then sequenced in both directions by the Chinese National Human Genome Center (SinoGenoMax, Beijing, China). Sequence analysis The MgcGPx and MgGPx4 cDNA sequences were analyzed using the BLASTx search program (http://www.ncbi.nlm.nih.gov/blast/). The deduced amino acid sequences were analyzed using the ExPASy server (http://www.expasy.org/tools/). Multiple alignments were performed with the ClustalW program (http://www.ebi.ac.uk/clustalw/). A 150 Table 1 Primers used in this study Gene name Forward primer (5'-3') Reverse primer (5’-3’) Description MgcGPx GGAAAATGGAAACGGTGAAG TATGGGAACCTGTGACAAGAAC 3’ RACE MgGPx4 AGGAGCCTGGAACTGAAGC GTTCTGGGGATGTCAACCTG 3’ RACE β-actin GCTATCCAGGCCGTACTCT GCGGTGGTTGTGAATGAG qRT-PCR α-tubulin GACCACCCATACCACCCTT CTCCGTGAGATCGACATTC qRT-PCR 18s rRNA TCGGATTGGTGAGACTGGAT TGCTGCCTTCCTTGGATGT qRT-PCR 28s rRNA CCGAGACCGAGGATTTGCC ACCGATTCGCCACTGACCC qRT-PCR ubiquitin C ATCAACAGCGTCTCATCT GCTCAACTTCTAGCGTAAT qRT-PCR EF1 GCTGGTATCTCATCTAACG CTTCACTGTATGGTGGTTC qRT-PCR GAPDH AGGGTCCAATGAAGGGTG TTAAGAGCGATGCCAGCT qRT-PCR DDX CATCAGAAGAAGGTGGCT AACAGTTGGTCGTAGGGT qRT-PCR MgcGPx AATGCCCTTGAGCATGTTCG TGAAACTAACAGCATCGTCGC qRT-PCR MgGPx4 AGTCAGGAGCCTGGAACTGA TGCCTCCTTGTTTGTGTTTG qRT-PCR phylogenetic tree was constructed with Mega4.1 software using the neighbor-joining (NJ) method based on the alignment (Tamura et al., 2007). Bootstrap analysis was used with 1000 replicates to test the relative support for the branches produced by the NJ analysis. The selenocysteine insertion sequence (SECIS) elements were searched using the SECISearch 2.19 program (http://genome.unl.edu./SECISearch.html). Quantitative real time PCR (qRT-PCR) analysis of MgcGPx and MgGPx4 expression In order to select appropriate internal standards for qRT-PCR analysis of gene expression in mussel exposed to cadmium and arsenic, the expressions of eight housekeeping genes, i.e., β-actin, α-tubulin, elongation factor 1-α (EF1), 18S rRNA, 28S rRNA, ubiquitin C, DEAD-box RNA helicase (DDX) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined. The data were analyzed with geNorm to calculate the expression stability (M values) of potential reference genes required for accurate normalization (V values) (Dang and Sun, 2011). Hemocytes, gills, digestive gland, adductor muscle, mantle and gonad of six untreated individuals were sampled to investigate the tissue-specific distribution of MgcGPx and MgGPx4 transcripts. The tissues of digestive gland and gills were selected to analyze temporal expression profile of MgcGPx and MgGPx4 challenged by Cd and AsV, respectively. Total RNA extraction and cDNA synthesis were performed as described above. qRT-PCR was carried out in an ABI 7500 Real-time Detection System by using the SYBR ExScript qRT-PCR Kit (Takara) as described before (Wang et al., 2013). The PCR amplification was carried out in a total volume of 50 μL, containing 25 μL of 2 × SYBR Green PCR Master Mix, 20 μL of the diluted cDNA, 1 μL of each of primers (10 μmol L-1), and 3 μL of DEPC-treated water. The thermal profile for qPCR was 50 °C for 2 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All reactions were run in triplicate. Dissociation curve analysis of amplicon was performed at the end of each PCR reaction to confirm that only one PCR product was amplified and detected. The expression level of MgcGPx and MgGPx4 was analyzed using 2−ΔΔCT method (Livak and Schmittgen, 2001). The qRT-PCR primers of MgcGPx and MgGPx4 are shown in Table 1. Measurement of total GPx activity in digestive gland and gills For assays on total GPx activity, the tissues of digestive gland and gills from six mussels were weighed and homogenized individually in 10 % (w/v) 50 mM Tris-HCl buffer (pH 7.5) using a tissue grinder (Ultra Turrax IKA T10 basic, Germany) held in an ice-water cooled bath. The homogenate was centrifuged at 10,000×g (4 ℃) for 30 min (Eppendorf centrifuge 5804R, Germany). Then the supernatant was collected and kept at 4 °C until used for further analysis. The total protein concentrations were determined according to the method described by Bradford (1976). Total GPx activity was measured as described before (Wang et al., 2010). All measurements were carried out in a Magellan plate reader (Tecan, Switzerland). Statistical analysis SPSS 16.0 software (SPSS Inc., USA) was used for statistical analysis. The data were checked for homogeneity of variance before analysis. The data were analyzed by one-way analysis of variance (one-way ANOVA), followed by a Tukey's post hoc test. A probability level of p < 0.05 was considered significant. 151 http://genome.unl.edu./SECISearch.html a) b) Fig. 1 The complete nucleotide and deduced amino acid sequences of MgcGPx (A) and MgGPx4 (B). The asterisk (*) indicates the stop codon. The start and stop codons are included in a box. The selenocysteine (U) is shaded. The polyadenylation signal is underlined in bold font and the predicted SECIS element is underlined in italic font. Results Sequence analysis Two nucleotide sequences of 972 bp and 889 bp representing the complete cDNA sequence of MgcGPx and MgGPx4 were obtained by overlapping EST and the amplified fragments. Full-length cDNAs of MgcGPx and MgGPx4 were deposited in GenBank under the accession numbers of HQ891311 and JQ866922, respectively. The deduced amino acid sequences of MgcGPx and MgGPx4 were shown below and the corresponding nucleotide acid sequence in Figure 1. The complete sequence of MgcGPx cDNA 152 a) b) Fig. 2 Multiple sequence alignments of MgcGPx (A) and MgGPx4 (B) with other GPx orthologs deposited in GenBank. The black shadow region indicates positions where all sequences share the same amino acid residue. Gaps are indicated by dashes to improve the alignment. The GPx signature motif 2 elements and active site motifs are marked by frame, respectively. The catalytically important residues are indicated by asterisks. The GenBank accession numbers of GPx sequences are shown in Table 2. 153 Fig. 3 Phylogenetic tree constructed by neighbour-joining method based on the sequences of GPxs from different animals. Numbers at the forks indicate the bootstrap values (in %) out of 1,000 replicates. The sequences used to construct phylogeny trees of GPxs are shown in Table 2. Table 2 Sequences used for multiple sequence alignments and phylogenetic analysis Gene Species Common name Accession numbers GPx1 Homo sapiens human NP_000572.2 GPx1 Equus caballus horse NP_001159951.1 GPx1 Bos taurus cattle NP_776501.1 GPx1 Danio rerio zebra fish NP_001007282.2 GPx2 Homo sapiens human NP_002074.2 GPx2 Rattus norvegicus brown rat NP_899653.2 GPx3 Homo sapiens human AAP50261.1 GPx4 Rhipicephalus microplus tick ABA62390.1 GPx4 Hydra vulgaris hydroid ABC25027.1 GPx4 Xenopus laevis African clawed frog NP_001165213.1 GPx4 Salmo salar Atlantic salmon ACH86324.1 GPx4 Gallus gallus chicken AAM18080.2 GPx4 Mus musculus house mouse BAA22780.1 GPx4 Homo sapiens human CAA50793.1 GPx4 Mytilus galloprovincialis Mediterranean mussel JQ866922 GPx5 Homo sapiens human CAA06463.1 GPx6 Homo sapiens human AAY68223.1 GPx Mizuhopecten yessoensis yesso scallop ADQ92353.1 GPx Hyriopsis cumingii triangle shell mussel ACY72387.1 GPx Sepiella maindroni cuttlefish AEK48346.1 GPx Pinctada fucata pearl oyster ADC35417.1 GPx Mytilus galloprovincialis Mediterranean mussel HQ891311 154 a) b) Fig. 4 Tissue-specific mRNA expression of MgcGPx (A) and MgGPx4 (B) determined by quantitative real-time PCR. Each bar represented mean ± SE (n = 4). encoded a polypeptide of 199 amino acids with a predicted molecular weight of 22.97 kDa and a theoretical isoelectric point (pI) of 9.11. SMART program analysis revealed that MgcGPx contained a typical GSHPx domain ranging from Phe15 to Arg128. The full-length cDNA of MgGPx4 encoded a polypeptide of 170 amino acids with a predicted molecular weight of 19.48 kDa and a theoretical pI of 8.62. SMART program analysis demonstrated that MgGPx4 also contained a GSHPx domain ranging from Ile14 to Lys123. SignalP program analysis revealed that no putative signal peptide existed in the deduced amino acids of MgcGPx and MgGPx4. In addition, a 98 bp and a 93 bp SECIS element were identified in the 3’ untranslated region (UTR) of MgcGPx and MgGPx4 cDNAs, respectively. Both the SECIS elements were predicted to form a stem-loop secondary structure and belonged to the form 2 SECIS element. Multiple alignments and Phylogenetic analysis Multiple alignments revealed that the characteristic Sec (U48) residue, catalytically essential residues (Q82, W160), the typical GPx signature 2 motif 72LGFPCNQF79 and the conserved active site motif 160WNFEKF165 were well conserved in MgcGPx and other selected mollusk GPx sequences (Fig. 2A). Similarly, MgGPx4 also contained the characteristic residues (U46, Q82 and W136), the conserved 72LGFPCNQF79 motif and 136WNFTKF141 motif in its sequence (Fig. 2B). To evaluate the molecular evolutionary relationship of MgcGPx and MgGPx4 against other GPxs, the amino acid sequences of 20 representative GPxs were selected to construct the phylogenetic tree (Fig. 3). The phylogenetic tree of GPxs included three major clades: the GPx1 and GPx2 (including GPxs from mollusks) clade; the GPx3, GPx5 and GPx6 clade; and the GPx4 clade. 155 a) b) Fig. 5 Temporal mRNA expression profiles of MgcGPx (A) and MgGPx4 (B) mRNA in gill and digestive gland after Cd-exposure measured by quantitative real-time PCR. Each bar represented mean ± SE (n = 4). Significance across control was indicated with an asterisk at p < 0.05. MgcGPx firstly clustered with GPxs from the yesso scallop Mizuhopecten yessoensis and pearl oyster Pinctada fucata, then formed a mollusc subgroup with GPxs from the triangle shell mussel Hyriopsis cumingii and the cuttlefish Sepiella maindroni, and it further grouped with typical GPx1s and GPx2s from vertebrates. In the GPx4 clade, MgGPx4 firstly grouped with GPx4s from the tick Rhipicephalus microplus and the hydroid Hydra vulgaris and then clustered with GPx4s from other vertebrates including fish, amphibian, bird and mammals. The phylogenetic analysis indicated that MgcGPx was derived from a common ancestor with most mollusc GPx family and MgGPx4 was a typical member of GPx4 family. Tissue-specific distribution of MgcGPx and MgGPx4transcripts GeNorm identified β-actin as the most stable gene with a V2/3 value 0.139 less than the proposed geNorm cutoff value of 0.15, which was then followed by ubiquitin C, α-tubulin, GAPDH, DDX and EF1. Therefore, β-actin was used as the internal control for gene expression normalization. The tissue-specific distribution patterns of MgcGPx and MgGPx4 mRNA were investigated by qRT-PCR with β-actin as the internal control. For MgcGPx, MgGPx4 and β-actin genes, there was only one peak at the corresponding melting temperature in the dissociation curve analysis, indicating that the PCR was specifically amplified (data not shown). The transcripts of MgcGPx and MgGPx4 were detected in all the examined tissues, including digestive gland, gills, hemocytes, adductor muscle, mantle and gonad. The MgcGPx transcript was mainly detected in the tissue of hemcoytes, digestive gland and gills, moderately expressed in adductor muscle and gonad (Fig. 4A). A similar tissue-specific distribution pattern was also observed for MgGPx4 transcript with the highest expression level in hemocytes, moderate levels in gills and digestive gland, least level in mantle (Fig. 4B). 156 a) b) Fig. 6 Temporal mRNA expression profiles of MgcGPx (A) and MgGPx4 (B) mRNA in gill and digestive gland after AsV-exposure measured by quantitative real-time PCR. Each bar represented mean ± SE (n = 4). Significance across control was indicated with an asterisk at p < 0.05. Transcriptional response of MgcGPx and MgGPx4 in gills and digestive gland exposed to Cd After sub-chronic Cd exposure, the transcript level of MgcGPx was significantly down-regulated in gills at relative high concentrations (10 μg L-1 and 100 μg L-1), whereas the transcriptional levels of MgcGPx increased significantly in digestive gland at all exposure concentrations compared to the control (p < 0.05) (Fig. 5A). However, a significantly higher transcriptional up-regulation of MgGPx4 in digestive gland was only observed in the mussels exposed to 100 μg L-1 Cd. In gills, the mRNA expression of MgGPx4 was also significantly inhibited at the highest concentration (100 μg L-1) compared to that of the control (p < 0.05) (Fig. 5B). Transcriptional response of MgcGPx and MgGPx4 in gills and digestive gland exposed to AsV In gills, the transcription of MgcGPx was significantly inhibited at all exposure concentrations compared to the control. However, the mRNA expression level of MgcGPx in digestive gland had no significant change at all concentrations compared to the control (p < 0.05) (Fig. 6A). Similarly, the transcription level of MgGPx4 in gills significantly decreased at all concentration. The mRNA expression level of MgGPx4 was significantly higher in the digestive gland of mussels treated with higher concentrations (10 μg L-1 and 100 μg L-1) compared to the control (p < 0.05) (Fig. 6B). Alteration of total GPx activities in gills and digestive gland after Cd and AsV exposure A significant decrease in GPx activity was observed in gills of the mussels exposed to 10 μg L-1 and 100 μg L-1 Cd compared to the control (p < 0.05). In digestive gland, the GPx activity increased significantly at all exposure concentrations (Fig. 7A). The modulation in transcript level of GPx coincided with the change in total GPx activity following Cd 157 a) b) Fig. 7 Alteration of total GPx activities in gill and digestive gland after Cd (A) and AsV (B) exposure. Each bar represented mean ± SE (n = 4). Significance across control was indicated with an asterisk at p < 0.05. exposure. After sub-chronic exposure, 1 μg L-1 and 10 μg L-1 AsV significantly inhibited GPx activity in gills (p < 0.05). Arsenate also inhibited GPx activity in digestive gland, and a statistically significant effect was observed in the 100 μg L-1 treated group (Fig. 7B). There was no obvious relation between gene expression and enzymatic activity of GPx following AsV exposure in this mussel. Discussion Glutathione peroxidase enzymes are involved in the elimination of ROS produced in physiological and pathological processes. Their differential expression as compared to normal expression pattern can indicate exposure to cellular stress and adverse cellular effects (Dash et al., 2006). Thus, GPxs have been proposed as biomarkers for the monitoring of physiological stress and toxicity induced by environmental toxicants in different aquatic organisms such as mollusks and fishes (Cossu et al., 1997; Sayeed et al., 2003; Almeida et al., 2004; Zhang et al., 2004; Valavanidis et al., 2006). In some field and laboratory studies, the GPx activities in M. galloprovincialis have been already examined to assess the toxic effects of contaminants in marine environment (Cravo et al., 2009; Maria et al., 2009; Chatziargyriou and Dailianis, 2010; Gomes et al., 2012; Nahrgang et al., 2013). However, no GPx genes have been characterized from this ecotoxicologically important species so far, although the mRNA expression of MgcGPx has been examined in mussels exposed to chemical mixtures (Giuliani et al., 2013). In this study, typical mollusc GPx and GPx4 genes were cloned and characterized in M. galloprovincialis. Both amino acid sequences contained a selenocysteine encoded by a terminator codon TGA, suggesting that they belonged to the family of selenocysteine-containing proteins. Multiple sequence alignment showed that both sequences contained the conserved GPx signature motif 2 and active site motif, which strongly implied that they were new members of the 158 selenium-dependent GPx family. Phylogenetic analysis demonstrated that MgcGPx was similar to vertebrate GPx1s and GPx2s, indicating it might be a GPx1 or GPx2 isoform. Furthermore, the mollusc GPx branch was in the base of GPx1 and GPx2 branch, suggesting a parallel evolution among most mollusc GPxs and vertebrate GPx1/GPx2 (Fu et al., 2012). Tissue-specific distribution patterns of MgcGPx and MgGPx4 transcripts indicated that both transcripts were mainly expressed in hemocytes, gills and digestive gland. In bivalves, ROS production had been shown to be involved in the immune defense and detoxification process of hemocytes (Gomez-Mendikute and Cajaraville, 2003). The comparably high expression levels of MgcGPx and MgGPx4 may be associated with the high oxidative stress occurring in hemocytes. The enrichment of MgcGPx and MgGPx4 transcripts in digestive gland was probably due to the fact that digestive gland was the major metabolic site for pollutants detoxification in molluscs (Livingstone, 1991). The high transcript level of GPx in digestive gland was also reported in scallops and clams (Mu et al., 2010; Wang et al., 2011a; Zhang et al., 2011). The gill is continuously exposed to environmental stress factors such as metallic ion, salinity, toxic substance and pathogen. Therefore, the transcripts of MgcGPx and MgGPx4 might be highly transcribed in this tissue. Similar results were also found in abalone, oyster and clam (De Zoysa et al., 2008; Jo et al., 2008; Zhang et al., 2011, 2012). Cadmium and arsenic are prooxidant contaminants and exposure to them is often associated with antioxidant response in aquatic organisms (Winston and Di Giulio, 1991; Ventura-Lima et al., 2011). Recently, the change of GPx at transcriptional level has been used to evaluate the antioxidant response to contaminant stress in molluscs (Jo et al., 2008; Bigot et al., 2011, Zhang et al., 2011; Wang et al., 2012). Several studies have demonstrated that mollusc GPx activity can be modulated upon cadmium exposure (Chandran et al., 2005; Legeay et al., 2005; Wang et al., 2010; Wang et al., 2011b). However, the antioxidant response to arsenic exposure was less studied in molluscs (Chakraborty et al., 2010). In this report, a dose-response study was carried out to elucidate gene expression profiles of MgcGPx and MgGPx4 upon contaminants exposure. In the gill tissues, the expression levels of both transcripts decreased in a dose-dependent manner after Cd exposure, although a significant decrease was only observed in relative high concentration. After AsV exposure, both transcripts of MgcGPx and MgGPx4 were significantly down-regulated at all concentrations in gills, indicating that both these genes responded sensitively to arsenic exposure even at the environmentally relevant concentration. However, another study reported that GPx transcript in gills of oyster C. gigas increased as the Cd concentration (10, 50 and 100 μg L-1) and exposure time (11 day) increased. It was deduced that short term cadmium exposure might stimulate the mRNA expression of GPx to counteract the antioxidant stress in oyster (Jo et al., 2008). However, sub-chronic contaminant exposure might cause strong oxidative stress in gills which are usually constantly contacted with pollutants. The stress could dramatically lower the metabolic capacity and result in a decrease in mRNA expression of antioxidant enzymes (Zhang et al., 2004, 2011). Additionally, the tissue structure damage in gills of bivalve might result in the inhibition of GPx mRNA expression after AsV exposure (Chakraborty et al., 2010). It has also been reported that the mRNA expression of GPx4 was inhibited in fish olfactory tissue which was frequently contact with contaminant. In common carp, the mRNA expression of GPx4b was greatly down-regulated following Cd (10 mg L-1) exposure for 21 d (Hermesz and Ferencz, 2009). In Coho salmon, both low (3.7 μg L-1) and high (347 μg L-1) level Cd exposures for 24 - 48 h significantly decreased GPx4a mRNA expression in olfactory (Wang et al., 2012). In addition, negative correlations were observed between the mRNA expression levels of MgcGPx and MgGPx4 in gills and the concentration of contaminant exposure, suggesting that the mRNA expression of these genes might be used as potential biomarkers for assessing the toxicity associated with contaminant exposures. Several previous studies also proposed the use of GPx4 transcript level in aquatic animals as biomarker for monitoring environmental pollution (Dash et al., 2006; Nair et al., 2012). In digestive gland, Cd but not AsV exposure modulated the mRNA expression of MgcGPx at all concentrations, suggesting that MgcGPx was perhaps involved in the detoxification process of Cd in digestive gland. Similarly, the up-regulation of GPx mRNA expression had also been reported in digestive gland of V. philippinarum after Cd exposure (Zhang et al., 2011). However, few studies have investigated the possible role of bivalve GPx4 in the detoxification process. It was reported that GPx4 activity could be stimulated by Cd (200 μg L-1) stress in digestive gland, indicating a protective role of GPx4 against lipid peroxidation in P. perna (Almeida et al., 2004). No information is available on the expression pattern of Gpx4 in response to pollutant in mollusc so far. In the present study, the transcription of MgGPx4 was up-regulated in mussels exposed to both Cd and AsV at the high concentration (100 μg L-1). These results indicated that the transcript of MgGPx4 was modulated to protect the mussel against oxidative stress in the digestive glands of M. galloprovincialis. Similarly, the expression of GPx4 mRNA was also induced after Cd (49.2 mg L-1) and AsV (24.0 mg L-1) exposure for 6 h in hydra H. vulgaris (Dash et al., 2006). In the aquatic midge C. riparius, the expression of GPx4 mRNA was found to be modulated upon exposure to different concentrations and time durations of Cd (Nair et al., 2012). In this study, total GPx activity was also assayed to determine the correlation between the gene expression and enzymatic activity. It was found that the increase in transcript level of MgcGPx and MgGPx4 mainly coincided with the increase of total GPx activity following Cd exposure. However, the change of total GPx activity was different with the mRNA expression pattern of MgcGPx and MgGPx4 in digestive gland after AsV exposure. A different 159 sensitivity towards the inducers at the transcription level, mRNA processing, or transport and protein stability may lead to discrepancies between transcript level and enzyme activity (Okey, 1990). Moreover, MgcGPx and MgGPx4 contributed only partially to the total enzymatic activity of GPx in this mussel. The inhibition of GPx activity by arsenate might be ascribed to the fact that AsV can form complexes with GSH to facilitate the excretion and biotransformation processes of this element (Aposhian et al., 2004). Therefore, AsV might have adverse impact on the content of GSH in cells to affect the total GPx activity. In summary, we have cloned two GPx genes (MgcGPx and MgGPx4) in M. galloprovincialis. Both the encoded GPx proteins had the conserved selenocysteine residue, catalytic sites and signature motifs. The constitutive transcripts of these genes were detected in six tissues examined. The modulation of MgcGPx and MgGPx4 transcript were involved in the antioxidant responses against pollutants challenge. This work will contribute to a better understanding of detoxification systems in marine bivalves. Moreover, the expression of MgcGPx and MgGPx4 mRNA can be considered as potential molecular biomarkers for assessing contaminant toxicity and aquatic environmental quality. 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