Biochemical responses in Pacific oysters Magallana gigas (Thunberg, 1793): Tools to evaluate the environmental quality of aquaculture areas.

The discharge of sanitary sewage into the bays of the Florianópolis Metropolitan Area (Southern Brazil), has led to the contamination of oyster farms. Consequently, linear alkylbenzenes (LABs) were quantified in the sediment, and the biochemical responses in gills and digestive gland of oysters from six farms were assessed. Our findings revealed elevated levels of LABs in the sediment of the Imaruim and Serraria farms. Additionally, alterations were observed in the antioxidant enzymes: catalase, glutathione peroxidase and superoxide dismutase in both oyster tissue from the Serraria, Santo Antonio de Lisboa and Sambaqui farms. Furthermore, correlation analyses indicated strong and moderate associations between biochemical responses, organic contaminants, and certain physicochemical parameters. Consequently, our results demonstrated the activation of the antioxidant system in oysters, representing a protective response to the presence of sanitary sewage and other contaminants. Therefore, we propose the utilization of biochemical biomarkers for monitoring the environmental quality of farms.

Analysis of transcriptional responses of normalizing genes on Crassostrea brasiliana under different experimental conditions.

Bivalves show remarkable plasticity to environmental changes and have been proposed as sentinel organisms in biomonitoring. Studies related to transcriptional analysis using quantitative real-time polymerase chain reaction (qRT-PCR) in these organisms have notably increased, imposing a need to identify and validate adequate reference genes for an accurate and reliable analysis. In the present study, 9 reference genes were selected from transcriptome data of Crassostrea brasiliana to identify their suitability as qRT-PCR normalizer genes. The transcriptional patterns were analyzed in gills of oysters under 3 different conditions: different temperatures (18, 24, or 32 °C) and phenanthrene (100 µg L-1 ) combined exposure; different salinities (10, 25, or 35‰) and phenanthrene combined exposure; and 10% of diesel fuel water-accommodated fraction (diesel-WAF) exposure. Reference gene stability was calculated using 5 algorithms (geNorm, NormFinder, BestKeeper, ΔCt, RefFinder). Transcripts of ankyrin-like (ANK), glyceraldehyde 3-phosphate dehydrogenase-like (GAPDH), and α-tubulin-like (TUBA) genes showed minor changes in different temperature/phenanthrene treatment. Transcripts of ANK, ß-actin-like, and ß-tubulin-like genes showed better stability at salinity/phenanthrene treatment, and ANK, TUBA, and 28S ribosomal protein-like genes showed the most stable transcription pattern in oysters exposed to diesel-WAF exposure. The present study constitutes the first systematic analysis of reference gene selection for qRT-PCR normalization in C. brasiliana. These genes could be employed in studies using qRT-PCR analysis under similar experimental conditions. Environ Toxicol Chem 2017;36:2190-2198. © 2017 SETAC.

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.

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.

In vitro and in vivo inhibition of acetylcholinesterase and carboxylesterase by metals in zebrafish (Danio rerio).

Metals are natural components in ecosystems; however, if these elements are in excess, they can have adverse effects on living organisms. This study analyzes the interference of copper, lead, iron and cadmium in acetylcholinesterase (AChE) and carboxylesterase (CbE) activities in zebrafish. AChE was significantly inhibited in vitro by copper, iron, lead and cadmium at higher concentrations (10 and 20 mmol/L), whereas CbE was inhibited only at a concentration of 20 mmol/L. In vivo, only lead and cadmium were able to cause AChE inhibition at higher concentrations, while iron didn't cause any changes, and copper promoted an increase in AChE activity at a concentration of 0.06 mg/L. CbE activity did not change at any of the times (two and seven days) and concentrations tested, except in the case of copper exposure, which resulted in a decrease in CbE activity. Indeed, iodoacetamide treatment didn't changed AChE neither CbE activities, results which indicate that the metal inhibiting effect is probably not due to its biding to thiol groups close the active site of the enzyme. This outcome reveals that metals are important esterase inhibitors in zebrafish, and should be considered in environmental monitoring studies that use esterase inhibition as exposure biomarkers of organophosphate and carbamate pesticides.

Esterase inhibition in tadpoles of Scinax fuscovarius (Anura, Hylidae) as a biomarker for exposure to organophosphate pesticides.

PURPOSE: Organophosphate pesticides (OPs) are among the most used insecticides in agriculture, causing the inhibition of esterases like acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CbE). Pesticides can reach the aquatic environment, posing risks to non-target organisms, including tadpoles. METHODS: In this work, we characterized the activities of AChE, BChE and CbE in tadpoles of the snouted treefrog Scinax fuscovarius, and verified their in vitro sensibility to different inhibitors [phenylmethane sulfonyl fluoride (PMSF), tetra-isopropylpyrophosphamide (iso-OMPA) and the OP diazinon]. In vivo effects of diazinon and esterase recovery after 2-pyridine-aldoxime (2-PAM) treatment of the protein extract were also studied in tadpoles with distinct stages of development exposed to 1 and 3 mg/l for 2 and 7 days. RESULTS: Optimal conditions were established for AChE and CbE; BChE activity was negligible. PMSF affected esterase activities and is not recommended for homogenization buffers. Iso-OMPA treatment caused no changes in AChE and CbE activities, but diazinon inhibited these enzymes in a dose-responsive manner. In vivo, CbE activity was insensitive to diazinon in younger tadpoles, but inhibited after 2 days of exposure in more developed tadpoles. AChE activity was inhibited after 2 and 7 days of exposure, in a dose-responsive manner. Esterase reactivation by 2-PAM was obtained both in vitro and in vivo. CONCLUSIONS: (1) Tadpoles can be adequate sentinel organisms in biomonitoring studies of OP contamination; (2) AChE was more sensitive than CbE to diazinon; (3) tadpoles from earlier developmental stages seems to be less responsive to OPs; (4) AChE activity was sensitive to diazinon in both development stages, being a better OP biomarker.