Cloning a new cytochrome P450 isoform (CYP356A1) from oyster Crassostrea gigas.

We have cloned the full-length cDNA of the first member of a new cytochrome P450 (CYP) family from the Pacific oyster Crassostrea gigas. This new CYP gene was obtained based on an initial 331bp fragment previously identified among the list of the differentially expressed genes in oysters exposed to untreated domestic sewage. The full-length CYP has an open reading frame of 1500bp and based on its deduced amino acid sequence was classified as a member of a new subfamily, CYP356A1. A phylogenetic analysis showed that CYP356A1 is closely related to members of the CYP17 and CYP1 subfamilies. Semi-quantitative RT-PCR was performed to analyze the CYP356A1 expression in different tissues of the oyster (digestive gland, gill, mantle and adductor muscle). Results showed slightly higher CYP356A1 expression in digestive gland and mantle, than the other tissues, indicating a possible role of the CYP356A1 in xenobiotic biotransformation and/or steroid metabolism.

Induced gene expression in oyster Crassostrea gigas exposed to sewage.

Pacific oysters, Crassostrea gigas, were exposed to untreated sewage diluted in seawater. After 48h of exposure, the expression of genes associated to biotransformation pathways (CYP356A1, GSTO, MDR, FABP and ALAS) were analyzed in gills through semi-quantitative RT-PCR. A significant induction in all genes analyzed in the sewage-exposed oysters was observed. These genes are related to phase I (CYP356A1), phase II (GSTO) and phase III (MDR) biotransformation systems, to the uptake and transport of hydrophobic ligands (FABP) and to the synthesis of prosthetic group heme (ALAS). The organisms were able to survive in contaminated conditions since protective mechanisms have been properly stimulated.

Characterization of ethoxyresorufin O-deethylase activity (EROD) in oyster Crassostrea brasiliana.

Cytochrome P450 family 1 (CYP1) is involved in polycyclic aromatic hydrocarbons (PAHs) biotransformation. PAHs can induce CYP1 protein expression and enzyme activity, the latter being usually quantified as 7-ethoxyresorufin O-deethylase activity (EROD). The aim of this study was to characterize EROD activity in the bivalve mollusk Crassostrea brasiliana. EROD activity was evaluated in cytosolic and microsomal fractions of gills, digestive gland and mantle of C. brasiliana. No EROD activity was detected in mantle, but it was present in microsomal fraction of gills and digestive gland with NADPH as coenzyme. Optima temperature and pH for EROD assay were 30°C and 7.4, respectively. EROD apparent Km (Kmapp) was 4.32µM for gills and 5.56µM for digestive gland. EROD Vmax was 337.3fmol·min-1·mg of protein-1 in gills and 297.7fmol·min-1·mg of protein-1 in digestive gland. Compared to other bivalves, a higher Kmapp and a lower Vmax was found in oyster which may suggest that oyster CYP1-like enzyme has lower affinity for substrate 7-ethoxyresorufin (7-ER) than those species. CYP1 inhibitor ellipticine (ELP) inhibited EROD activity in all tested concentrations in both tissues. The higher ELP concentration, 100µM, inhibited 78% of EROD activity in gills and 47% in digestive gland. The CYP1 inhibitors α-naphthoflavone and furafylline did not inhibited EROD activity in microsomes of both tissues. In conclusion, EROD activity can be used to determine CYP1-like activity in oysters and possibly a CYP1A1/A2-like enzyme is responsible for this catalysis.

Key metabolic pathways involved in xenobiotic biotransformation and stress responses revealed by transcriptomics of the mangrove oyster Crassostrea brasiliana.

The Brazilian oyster Crassostrea brasiliana was challenged to three common environmental contaminants: phenanthrene, diesel fuel water-accommodated fraction (WAF) and domestic sewage. Total RNA was extracted from the gill and digestive gland, and cDNA libraries were sequenced using the 454 FLX platform. The assembled transcriptome resulted in ̃20,000 contigs, which were annotated to produce the first de novo transcriptome for C. brasiliana. Sequences were screened to identify genes potentially involved in the biotransformation of xenobiotics and associated antioxidant defence mechanisms. These gene families included those of the cytochrome P450 (CYP450), 70kDa heat shock, antioxidants, such as glutathione S-transferase, superoxide dismutase, catalase and also multi-drug resistance proteins. Analysis showed that the massive expansion of the CYP450 and HSP70 family due to gene duplication identified in the Crassostrea gigas genome also occurred in C. brasiliana, suggesting these processes form the base of the Crassostrea lineage. Preliminary expression analyses revealed several candidates biomarker genes that were up-regulated during each of the three treatments, suggesting the potential for environmental monitoring.

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.

Biochemical biomarkers and hydrocarbons concentrations in the mangrove oyster Crassostrea brasiliana following exposure to diesel fuel water-accommodated fraction.

Understanding the toxic mechanisms by which organisms cope to environmental stressful conditions is a fundamental question for ecotoxicology. In this study, we evaluated biochemical responses and hydrocarbons bioaccumulation of the mangrove oyster Crassostrea brasiliana exposed for 96 h to four sublethal concentrations of diesel fuel water-accommodated fraction (WAF). For that purpose, enzymatic activities (SOD, CAT, GPx, GR, G6PDH, GST and GGT), HSP60 and HSP90 immunocontent and lipid peroxidation (LPO) levels were determined in the gill and digestive gland of oysters and related to the hydrocarbons accumulated in the whole soft tissues. The results of this study revealed clear biochemical responses to diesel fuel WAF exposure in both tissues of the oyster. The capacity of C. brasiliana to bioaccumulate aliphatic and aromatic hydrocarbons in a dose-dependent manner is a strong indication of its suitability as a model in biomonitoring programs along the Brazilian coast, which was also validated by the response of the antioxidant defenses, phase II biotransformation and chaperones. HSP60 levels and GGT activity were the most promising biomarkers in the gill, while GST and GR activities stood out as suitable biomarkers for the detection of diesel toxicity in the digestive gland. The decrease of SOD activity and HSP90 levels may also reflect a negative effect of diesel exposure regardless the tissue. The present results provide a sound preliminary report on the biochemical responses of C. brasiliana challenged with a petroleum by-product and should be carefully considered for use in the monitoring of oil and gas activities in Brazil.

Differential gene expression in Poecilia vivipara exposed to diesel oil water accommodated fraction.

Diesel fuel is a potential contaminant of estuarine and mangrove areas, particularly because it is the main fuel used in small boats and larger vessels. The aim of this work was to identify genes differentially expressed in the liver of Poecilia vivipara (Guppy) exposed to 10% diesel fuel water accommodated fraction (WAF), employing the subtractive suppressive hybridization (SSH) method. The results showed 27 differentially expressed gene fragments, 12 up-regulated and 15 down-regulated. Among the up-regulated genes were CYP1A, UDPGT1a, ABCC4, Methyltransferase and Apolipoprotein A1. Down-regulated genes included Vitellogenins, C1 Inhibitor and Complement Component 3c. The identified genes are associated with different metabolic functions like biotransformation, membrane transport and immune system, indicating the susceptibility and/or molecular responses of this organism to the toxic effects elicited by diesel fuel WSF.