Differential responses in the biotransformation systems of the oyster Crassostrea gigas (Thunberg, 1789) elicited by pyrene and fluorene: Molecular, biochemical and histological approach - Part II.

Pyrene (PYR) and fluorene (FLU) are among the sixteen priority Polycyclic Aromatic Hydrocarbons (PAH) of the United States Environmental Protection Agency and are both frequently detected in contaminated sites. Due to the importance of bivalve mollusks in biomonitoring programs and the scarce information on the biotransformation system in these organisms, the aim of this study was to investigate the effect of PYR and FLU at the transcriptional level and the enzymatic activities of some biotransformation systems in the Pacific oyster Crassostrea gigas, and to evaluate the histological effects in their soft tissues. Oysters C. gigas were exposed for 24 h and 96 h to PYR (0.25 and 0.5 µM) and FLU (0.6 and 1.2 µM). After exposure, transcript levels of cytochrome P450 coding genes (CYP1-like, CYP2-like, CYP2AU2, CYP356A1, CYP17α-like), glutathione S tranferase genes (omega GSTO-like and microsomal, MGST-like) and sulfotransferase gene (SULT-like), and the activity of ethoxyresorufin O-deethylase (EROD), Glutathione S-transferase (GST) and microssomal GST (MGST) were evaluated in gills. Histologic changes were also evaluated after the exposure period. PYR and FLU bioconcentrated in oyster soft tissues. The half-life time of PYR in water was lower than fluorene, which is in accordance to the higher lipophilicity and bioconcentration of the former. EROD activity was below the limit of detection in all oysters exposed for 96 h to PYR and FLU. The reproductive stage of the oysters exposed to PYR was post-spawn. Exposure to PYR caused tubular atrophy in digestive diverticula, but had no effect on transcript levels of biotransformation genes. However, the organisms exposed for 96 h to PYR 0.5 µM showed higher MGST activity, suggesting a protective role against oxidative stress in gills of oysters under higher levels of PYR in the tissues. Increased number of mucous cells in mantle were observed in oysters exposed to the higher FLU concentration, suggesting a defense mechanisms. Oysters exposed for 24 h to FLU 1.2 µM were in the ripe stage of gonadal development and showed higher transcript levels of CYP2AU2, GSTO-like and SULT-like genes, suggesting a role in the FLU biotransformation. In addition, after 96 h of exposure to FLU there was a significant increase of mucous cells in the mantle of oysters but no effect was observed on the EROD, total GST and MGST activities. These results suggest that PAH have different effects on transcript levels of biotransformation genes and enzyme activities, however these differences could also be related to the reproductive stage.

Differential responses in the biotransformation systems of the oyster Crassostrea gasar (Adanson, 1757) elicited by pyrene and fluorene: molecular, biochemical and histological approach - Part I.

Polycyclic aromatic hydrocarbons (PAHs) are among the main contaminants in aquatic environments. PAHs can affect organisms due to their carcinogenic, mutagenic and/or teratogenic characteristics. Depending on the PAHs, concentration, and period of exposure, biological damage can occur leading to histopathologic alterations. This study aimed to evaluate the molecular, biochemical and histological responses of the oyster Crassostrea gasar exposed to pyrene (0.25 and 0.5 µM) and fluorene (0.6 and 1.2 µM), after exposure for 24 and 96 h. Concentrations of both PAHs were quantified in the water and in oyster tissues. Transcript levels of phase I (CYP3475C1, CYP2-like, CYP2AU1 and CYP356A) and phase II (GSTO-like, MGST-like and SULT-like) biotransformation-related genes and the activities of ethoxyresorufin-O-deethylase (EROD), total and microsomal glutathione S-transferase (GST and MGST) were evaluated in the gills. Also, histological changes and localization of mRNA transcripts CYP2AU1 in gills, mantle, and digestive diverticula were evaluated. Both PAHs accumulated in oyster tissues. Pyrene half-life in water was significantly lower than fluorene. Transcript levels of all genes were higher in oysters exposed to of pyrene 0.5 µM (24 h). Only CYP2AU1 gene was up-regulated by fluorene exposure. EROD and MGST activities were higher in oysters exposed to pyrene. Tubular atrophy in the digestive diverticula and an increased number of mucous cells in the mantle were observed in oysters exposed to pyrene. CYP2AU1 transcripts were observed in different tissues of pyrene-exposed oysters. A significant correlation was observed between tubular atrophy and the CYP2AU1 hybridization signal in oysters exposed to pyrene, suggesting the sensibility of the species to this PAH. These results suggest an important role of biotransformation-related genes and enzymes and tissue alterations associated to pyrene metabolism but not fluorene. In addition, it reinforces the role of CYP2AU1 gene in the biotransformation process of PAHs in the gills of C. gasar.

Transcriptional effects in the estuarine guppy Poecilia vivipara exposed to sanitary sewage in laboratory and in situ.

The urban growth has increased sanitary sewage discharges in coastal ecosystems, negatively affecting the aquatic biota. Mangroves, one of the most human-affected coastal biomes, are areas for reproduction and nursing of several species. In order to evaluate the effects of sanitary sewage effluents in mangrove species, this study assessed the hepatic transcriptional responses of guppy fish Poecilia vivipara exposed to sanitary sewage 33% (v:v), using suppressive subtraction hybridization (SSH), high throughput sequencing of RNA (Ion-proton) and quantification of transcript levels by qPCR of some identified genes in fish kept in a sewage-contaminated environment. Genes identified are related predominantly to xenobiotic biotransformation, immune system and sexual differentiation. The qPCR results confirmed the induction of cytochrome P450 1A (CYP1A), glutathione S transferase A-like (GST A-like) methyltransferase (MET) and UDP glycosyltransferase 1A (UDPGT1A), and repression of complement component C3 (C3), doublesex and mab-3 related transcription factor 1 (DMRT1), and transferrin (TF) in the laboratory experiment. In the field exposure, the transcript levels of CYP1A, DMRT1, MET, GST A-like and UDPGT1A were higher in fishes exposed at the contaminated sites compared to the reference site. Chemical analysis in fish from the laboratory and in situ experiments, and surface sediment from the sewage-contaminated sites revealed relevant levels of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCBs) and linear alkylbenzenes (LABs). These data reinforce the use of P. vivipara as a sentinel for monitoring environmental contamination in coastal regions.

Biochemical and molecular responses in oysters Crassostrea brasiliana collected from estuarine aquaculture areas in Southern Brazil.

Biochemical and molecular responses were evaluated in oysters Crassostrea brasiliana collected from three oyster farms, at Guaratuba Bay, southern Brazil, forming a pollutant gradient: Farm 1 (reference site - farther from the urban area), Farm 2 (intermediate site) and Farm 3 (nearest to the urban area). Oxidative stress markers, DNA damage and transcript levels of CYP2AU1, CYP2-like1, CYP2-like2, SULT-like, GPx-like, SOD-like, CAT-like, GSTmicrosomal-like, GSTomega-like, FABP-like and ALAd-like genes were analyzed in the gills. The levels of polycyclic aromatic hydrocarbons, linear alkylbenzenes and polychlorinated biphenyls were also evaluated in the soft tissues of the oysters and in the sediment of the Farms. Higher GSTomega-like, CYP2AU1 and FABP-like transcript levels, GR and G6PDH activities and lipid peroxidation levels were observed in oysters from Farms 2 and 3, suggesting pollutant effects on oysters. Alterations in oxidative stress markers also suggest a response against a prooxidant condition in C. brasiliana due to pollutant effects.

Molecular and cellular effects of temperature in oysters Crassostrea brasiliana exposed to phenanthrene.

Exposure of aquatic organisms to polycyclic aromatic hydrocarbons (PAH), such as phenanthrene (PHE), may increase the production of reactive oxygen species (ROS) and cause changes in the biotransformation systems. In addition, changes in water temperature can cause adverse effects in the organisms. Estuarine species, like the oyster Crassostrea brasiliana, can adapt and tolerate temperature variation. To evaluate the influence of temperature on biological responses of C. brasiliana exposed to PHE, oysters were maintained at three temperatures (18, 24 and 32 °C) for 15 days and co-exposed afterwards to 100 µg.L-1 of PHE for 24 and 96 h. Levels of PHE in the water and oyster tissues were determined, respectively after 24 and 96 h. In addition, thermal stress, biotransformation and oxidative stress-related genes were analyzed in oyster gills, together with the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferases (GST) and levels of lipid peroxidation. Oyster accumulated significant levels of PHE. HSP70-like transcripts were affected by PHE exposure only at 32 °C. Transcript levels of cytochrome P450 isoforms (CYP2-like2 and CYP2AU1) were down-regulated in oysters exposed to PHE for 24 h at 32 °C. GSTΩ-like transcript levels were also down-regulated in the PHE-exposed group at 32 °C. After 96 h, CYP2-like2 transcripts were higher in the PHE exposed groups at 32 °C. Oysters kept at 18 °C showed higher levels of SOD-like transcripts, together with higher GST, GPx and G6PDH activities, associated to lower levels of lipoperoxidation. In general the biological responses evaluated were more affected by temperature, than by co-exposure to PHE.

Effects of phenanthrene on early development of the Pacific oyster Crassostrea gigas (Thunberg, 1789).

Phenanthnere (PHE) is a polycyclic aromatic hydrocarbon continuously discarded in the marine environment and bioavailable to many aquatic species. Although studies about PHE toxicity have been documented for adult oysters, the effects on early developmental stages are poorly characterized in bivalves. In this study, the effects of PHE (0.02 and 2.0µg.L-1) were evaluated on the embryogenesis and larval development of Crassostrea gigas. Toxicity bioassays, growth and deformities assessment, analysis of shell calcium abundance and transcript levels of genes related to xenobiotic biotransformation (CYP2AU2, CYP30C1), immune system (Cg-Tal) and tissue growth and shell formation (Ferritin, Insulin-like, Cg-Try, Calmodulin and Nacrein) were assayed in D-shape larvae after 24h of PHE exposure. At the highest concentration (2.0µg.L-1), PHE decreased the frequency of normal development (19.7±2.9%) and shell size (53.5±2.8mm). Developmental deformities were mostly related to abnormal mantle and shell formation. Lower calcium levels in oyster shells exposed to PHE 2.0µg.L-1 were observed, suggesting effects on shell structure. At this same PHE concentration, CYP30C1, Cg-Tal, Cg-Tyr, Calmodulin were upregulated and CYP2AU2, Ferritin, Nacrein, and Insulin-Like were downregulated compared to control larvae. At the lowest PHE concentration (0.02µg.L-1), it was observed a minor decrease in normal larval development (89,6±6%) and the remaining parameters were not affected. This is the first study to provide evidences that exposure to PHE can affect early oyster development at the molecular and morphological levels, possibly threatening this bivalve species.

Transcriptional changes in oysters Crassostrea brasiliana exposed to phenanthrene at different salinities.

Euryhaline animals from estuaries, such as the oyster Crassostrea brasiliana, show physiological mechanisms of adaptation to tolerate salinity changes. These ecosystems receive constant input of xenobiotics from urban areas, including polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene (PHE). In order to understand the influence of salinity on the molecular responses of C. brasiliana exposed to PHE, oysters were acclimatized to different salinities (35, 25 and 10) for 15days and then exposed to 100µgL-1 PHE for 24h and 96h. Control groups were kept at the same salinities without PHE. Oysters were sampled for chemical analysis and the gills were excised for mRNA quantification by qPCR. Transcript levels of different genes were measured, including some involved in oxidative stress pathways, phases I and II of the xenobiotic biotransformation systems, amino acid metabolism, fatty acid metabolism and aryl hydrocarbon receptor nuclear translocator putative gene. Higher transcript levels of Sulfotransferase-like gene (SULT-like) were observed in oysters exposed to PHE at salinity 10 compared to control (24h and 96h); cytochrome P450 isoforms (CYP2AU1, CYP2-like1) were more elevated in oysters exposed for 24h and CYP2-like2 after 96h of oysters exposed to PHE at salinity 10 compared to control. These results are probably associated to an enhanced Phase I biotransformation activity required for PHE detoxification under hyposmotic stress. Higher transcript levels of CAT-like, SOD-like, GSTm-like (96h) and GSTΩ-like (24h) in oysters kept at salinity 10 compared to organisms at salinities 25 and/or 35 are possibly related to enhaced ROS production. The transcription of these genes were not affected by PHE exposure. Amino acid metabolism-related genes (GAD-like (24h), GLYT-like, ARG-like (96h) and TAUT-like at 24h and 96h) also showed different transcription levels among organisms exposed to different salinities, suggesting their important role for oyster salinity adaptation, which is not affected by exposure to these levels of PHE.

Thiol oxidation of hemolymph proteins in oysters Crassostrea brasiliana as markers of oxidative damage induced by urban sewage exposure.

Urban sewage is a concerning issue worldwide, threatening both wildlife and human health. The present study investigated protein oxidation in mangrove oysters (Crassostrea brasiliana) exposed to seawater from Balneário Camboriú, an important tourist destination in Brazil that is affected by urban sewage. Oysters were exposed for 24 h to seawater collected close to the Camboriú River (CAM1) or 1 km away (CAM2). Seawater from an aquaculture laboratory was used as a reference. Local sewage input was marked by higher levels of coliforms, nitrogen, and phosphorus in seawater, as well as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), linear alkylbenzenes (LABs), and fecal steroid in sediments at CAM1. Exposure of oysters to CAM1 caused marked bioaccumulation of LABs and decreased PAH and PCB concentrations after exposure to both CAM1 and CAM2. Protein thiol oxidation in gills, digestive gland, and hemolymph was evaluated. Lower levels of reduced protein thiols were detected in hemolymph from CAM1, and actin, segon, and dominin were identified as targets of protein thiol oxidation. Dominin susceptibility to oxidation was confirmed in vitro by exposure to peroxides and hypochlorous acid, and 2 cysteine residues were identified as potential sites of oxidation. Overall, these data indicate that urban sewage contamination in local waters has a toxic potential and that protein thiol oxidation in hemolymph could be a useful biomarker of oxidative stress in bivalves exposed to contaminants. Environ Toxicol Chem 2017;36:1833-1845. © 2016 SETAC.

Exposure to phenanthrene and depuration: Changes on gene transcription, enzymatic activity and lipid peroxidation in gill of scallops Nodipecten nodosus.

Understanding the mechanism of phenanthrene (PHE) biotransformation and related cellular responses in bivalves can be an important tool to elucidate the risks of polycyclic aromatic hydrocarbons (PAHs) to aquatic organisms. In the present study it was analyzed the transcriptional levels of 13 biotransformation genes related to cytochrome P450 (CYP), glutathione S-transferase (GST), sulfotransferase (SULT), flavin-containing monooxygenase and fatty acid-binding proteins by qPCR in gill of scallops Nodipecten nodosus exposed for 24 or 96h to 50 or 200µgL(-1) PHE (equivalent to 0.28 and 1.12µM, respectively), followed by depuration in clean water for 96h (DEP). Likewise, it was quantified the activity of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), GST and levels of lipid peroxidation. Increased transcriptional levels of CYP2UI-like, CYP2D20-like, CYP3A11-like, GSTomega-like, SULT1B1-like genes were detected in organisms exposed to PHE for 24 or 96h. In parallel, GR and GPX activities increased after 96h exposure to 200µgL(-1) PHE and G6PDH activity increased after 24h exposure to 50µgL(-1) PHE. This enhancement of antioxidant and phase I and II biotransformation systems may be related to the 2.7 and 12.5 fold increases in PHE bioaccumulation after 96h exposure to 50 and 200µgL(-1) PHE, respectively. Interestingly, DEP caused reestablishment of GPX and GR activity, as well as to the transcript levels of all upregulated biotransformation genes (except for SULT1B1-like). Bioaccumulated PHE levels decreased 2.5-2.9 fold after depuration, although some biochemical and molecular modifications were still present. Lipid peroxidation levels remained lower in animals exposed to 200µgL(-1) PHE for 24h and DEP. These data indicate that N. nodosus is able to induce an antioxidant and biotransformation-related response to PHE exposure, counteracting its toxicity, and DEP can be an effective protocol for bivalve depuration after PHE exposure.

Upregulation of biotransformation genes in gills of oyster Crassostrea brasiliana exposed in situ to urban effluents, Florianópolis Bay, Southern Brazil.

The release of untreated sanitary sewage, combined with unplanned urban growth, are major factors contributing to degradation of coastal ecosystems in developing countries, including Brazil. Sanitary sewage is a complex mixture of chemicals that can negatively affect aquatic organisms. The use of molecular biomarkers can help to understand and to monitor the biological effects elicited by contaminants. The aim of this study was to evaluate changes in transcript levels of genes related to xenobiotic biotransformation in the gills of oysters Crassostrea brasiliana transplanted and kept for 24h at three areas potentially contaminated by sanitary sewage (Bücheller river, BUC; Biguaçu river, BIG; and Ratones island, RAT), one farming area (Sambaqui beach, SAM) and at one reference site (Forte beach, FOR) in the North Bay of Santa Catarina Island (Florianópolis, Brazil). Transcript levels of four cytochrome P450 isoforms (CYP2AU1, CYP3A-like, CYP356A1-like and CYP20A1-like), three glutathione S-transferase (GST alpha-like, GST pi-like and GST microsomal 3-like) and one sulfotransferase gene (SULT-like) were evaluated by means of quantitative reverse transcription PCR (qRT-PCR). Chemical analysis of the sediment from each site were performed and revealed the presence of aliphatic and polycyclic aromatic hydrocarbons, linear alkylbenzenes and fecal sterols in the contaminated areas (BUC and BIG). Water quality analysis showed that these sites had the highest levels of fecal coliforms and other parameters evidencing the presence of urban sewage discharges. Among the results for gene transcription, CYP2AU1 and SULT-like levels were upregulated by 20 and 50-fold, respectively, in the oysters kept for 24h at the most contaminated site (BUC), suggesting a role of these genes in the detoxification of organic pollutants. These data reinforce that gills possibly have an important role in xenobiotic metabolism and highlight the use of C. brasiliana as a sentinel for monitoring environmental contamination in coastal regions.

Histological responses and localization of the cytochrome P450 (CYP2AU1) in Crassostrea brasiliana exposed to phenanthrene.

Phenanthrene (PHE) is an abundant polycyclic aromatic hydrocarbon (PAH), widely distributed in aquatic environment. The aim of this study was to evaluate the histological and molecular effects in the native oyster Crassostrea brasiliana(Lamarck, 1819) exposed to 100 and 1000 µg L(-1) PHE for 1, 5 and 10 days. Histological and chemical analyses were performed to evaluate, respectively, alterations in oyster tissues and bioaccumulation. In situ hybridization (ISH) was used to assess tissue distribution of CYP2AU1, a gene formerly identified as activated by PHE exposure in this species.Quantitative polymerase chain reaction (qPCR) in mantle was carried out to validate ISH data. Oysters bioaccumulated PHE increasingly along the exposure period in both exposure concentrations. Histologic changes, like tubular atrophy in digestive diverticula (digestive gland) and increased number of mucous cells in the mantle were observed in animals exposed to PHE for 10 days. ISH showed the presence of CYP2AU1transcripts in gills, digestive diverticula, mantle, intestine and gonads, but significant differences in transcript detection by ISH between treatments occurred only in gills, mantle and intestine. A positive and significant correlation between tubular atrophy and CYP2AU1hybridization signal was observed in digestive diverticula, suggesting that this gene product might be involved in energetic metabolism in C. brasiliana. Increased mucous cells and CYP2AU1transcript levels were observed in the mantle, where the inner and middle lobes showed higher intensity of hybridization signal. Mantle should be considered as a target organ for CYP2AU1 transcript evaluation and histological alterations in biomonitoring studies. CYP2AU1 signal in female gonads was observed in all follicular cells from different gonadic stages, while in male only the spermatic follicle cells of the wall in the pre-spawning stage showed this signal. ISH was an effective technique to evaluate the effects of PHE exposure and to locate CYP2AU1 transcripts in different tissues of oyster C. brasiliana.

Effect of linear alkylbenzene mixtures and sanitary sewage in biochemical and molecular responses in pacific oyster Crassostrea gigas.

Urban effluents are rich in nutrients, organic matter, pharmaceuticals and personal care products (PPCPs), pesticides, hydrocarbons, surfactants, and others. Previous studies have shown that oysters Crassostrea gigas accumulate significant levels of linear alkylbenzenes (LABs) in sanitary sewage contaminated sites, but there is little information about its toxicological effects in marine bivalves. The aim of this study was to analyze the transcription of genes in two tissues of C. gigas exposed for 12, 24, and 36 h to LABs or sanitary sewage. Likewise, the activity of antioxidant and biotransformation enzymes was measured in oysters exposed for 36 h in all groups. Oysters exposed to LABs and oysters exposed to sanitary sewage showed different patterns of transcriptional responses. LAB-exposed oysters showed lower level of biological responses than the oysters exposed to sanitary sewage. Despite the ability of the oyster C. gigas to accumulate LABs (28-fold), the data indicate that these contaminants are not the cause for the transcriptional responses observed in oysters exposed to sanitary sewage. Possibly, the biological changes observed in the sanitary sewage-exposed oysters are associated with the presence of other contaminants, which might have caused synergistic, additive, or antagonistic effects. The results show that FABP-like and GST-ω-like messenger RNAs (mRNAs) have a rapid response in tissues of oyster C. gigas exposed to sanitary sewage, suggesting a possible protective response and a role in maintaining homeostasis of these organisms.

Expression and immunohistochemical localization of the cytochrome P450 isoform 356A1 (CYP356A1) in oyster Crassostrea gigas.

Cytochrome P450 family (CYP) is a group of proteins virtually found in all living organisms. The main role of most CYPs is to metabolize endo and xenobiotics. Most of the studies on CYP have been carried out in mammals and other vertebrates, however recently a growing interest has been devoted to the identification of CYP isoforms in invertebrates. A gene belonging to the CYP sub-family, CYP356A1, was identified in sanitary sewage-exposed Pacific oysters, Crassostrea gigas. Through heterologous expression, we produced CYP356A1 purified protein and raised a mouse polyclonal antibody. Dot blot tests showed that oysters exposed in situ for 14 days to untreated urban effluent discharges had significantly higher levels of CYP356A1 in digestive gland. Using immunohistochemical techniques we observed that the lining epithelial cells of mantle, stomach and intestine showed a strong CYP356A1 staining, but the mucus and secretory cells were negative. Digestive diverticulum parenchyma and gills lining cells showed strong CYP356A1 reaction, while the filamentary rod (connective tissue) was negative. Free cells, as hemocytes and brown cells also showed CYP356A1 immunoreactions indicating the presence of biotransformation activity in these cells. Male germ cells at early stages expressed CYP356A1 but not sperm mature cells, suggesting that this protein could be involved in the male gonadal development. This study shows the use of a specific antibody to a mollusk CYP isoform and that this protein is inducible in oysters environmentally exposed to urban sewage effluents.

Differential gene transcription, biochemical responses, and cytotoxicity assessment in Pacific oyster Crassostrea gigas exposed to ibuprofen.

Pharmaceuticals, such as anti-inflammatory nonsteroidal drugs, are frequently detected in aquatic ecosystems. Studies about the effects of these substances in nontarget organisms, such as bivalves, are relevant. The aim of this study was to evaluate the effects on antioxidant status caused by ibuprofen (IBU) in oysters Crassostrea gigas exposed for 1, 4, and 7 days at concentrations 1 and 100 µg L(-1). Levels of IBU in tissues of oysters, as well as cell viability of hemocytes, were measured. The transcription of cytochrome P450 genes (CYP2AU2, CYP356A1, CYP3071A1, CYP30C1), glutathione S-transferase isoforms (GST-ω-like and GST-π-like), cyclooxygenase-like (COX-like), fatty acid binding protein-like (FABP-like), caspase-like, heat shock protein-like (HSP70-like), catalase-like (CAT-like), and the activity of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST) were also evaluated in the gills of oysters. The highest levels of IBU were observed in animals exposed to 100 µg L(-1). A significant upregulation of CYP2AU1, CYP356A1, CYP3071A1, GST-ω-like, GST-π-like, COX-like, and FABP-like was observed in oysters exposed to IBU under different experimental conditions. Oysters exposed to 1 µg L(-1) for 7 days showed a significantly higher transcription of CYP2AU2, CYP356A1, CYP3071A1, GST-ω-like, and GST-π-like but lower GR activity. In conclusion, C. gigas exposed to environmentally relevant concentrations of IBU (1 µg L(-1)) exhibited increased transcription of certain genes and alterations on antioxidant and auxiliary enzymes, which could, in the the long term, cause damages to exposed organisms.

Changes in protein expression of pacific oyster Crassostrea gigas exposed in situ to urban sewage.

The composition and concentration of substances in urban effluents are complex and difficult to measure. These contaminants elicit biological responses in the exposed organisms. Proteomic analysis is a powerful tool in environmental toxicology by evidencing alterations in protein expression due to exposure to contaminants and by providing a useful framework for the development of new potential biomarkers. The aim of this study was to determine changes in protein expression signatures (PES) in the digestive gland of oysters Crassostrea gigas transplanted to two farming areas (LIS and RIB) and to one area contaminated by sanitary sewage (BUC) after 14 days of exposure. This species is one of the most cultivated molluscs in the world. The identified proteins are related to the cytoskeleton (CKAP5 and ACT2), ubiquitination pathway conjugation (UBE3C), G protein-coupled receptor and signal transduction (SVEP1), and cell cycle/division (CCNB3). CKAP5 showed higher expression in oysters kept at BUC in comparison with those kept at the farming areas, while ACT2, UBE3C, SVEP1, and CCNB3 were suppressed. The results suggest that these changes might lead to DNA damage, apoptosis, and interference with the immune system in oyster C. gigas exposed to sewage and give initial information on PES of C. gigas exposed to sanitary sewage, which can subsequently be useful in the development of more sensitive tools for biomonitoring coastal areas, particularly those devoted mainly to oyster farming activities.