Field study of metal concentrations and biomarker responses in resident oysters of an estuarine system in southern Brazil.

Pollutant exposure is considered an important factor responsible for the decline in marine biodiversity of Latin American coastal ecosystems. This threat has been detected in an estuarine system in southern Brazil, which prompted an investigation into the long-term biological effects of a chronic metal contamination on resident oysters from the Laguna Estuarine System (LES). Here, we present the species- and size-specific variations of biomarker responses (catalase, glucose-6-phosphate dehydrogenase, glutathione S-transferase, and protein carbonylation) in the gills and digestive gland of Crassostrea gigas and Crassostrea gasar. In parallel, concentrations of eight metals (Al, Cd, Cr, Cu, Fe, Mn, Pb, Zn) in soft tissues were measured. Our analyses revealed that the metal levels exhibited decreasing order in both species: Zn > Fe > Al > Cu > Mn > Cd. Except for Cu and Al, metal concentrations did not differ between oyster species. Biomarker results highlighted that C. gasar presented higher antioxidant responses, whereas C. gigas showed increased biotransformation upon exposure to LES pollutants, which varied according to the tissue. However, C. gasar showed a significant higher content of protein carbonylation but was not related to metals. In our research approach, the observation of metals presence and biomarkers-related responses are considered biologically relevant from an ecotoxicological perspective and serve as a baseline for future pollution studies in estuaries of Latin America. Finally, we recommend adopting a suite of biomarkers in both C. gasar and C. gigas, regardless their size and weight, as sentinel organisms in future regional biomonitoring studies in southern Brazil.

Characterization of a fatty acid-binding protein from the Pacific oyster (Crassostrea gigas): pharmaceutical and toxicological implications.

Pharmaceuticals and their metabolites constitute a class of xenobiotics commonly found in aquatic environments which may cause toxic effects in aquatic organisms. Several different lipophilic molecules, including some pharmaceuticals, can bind to fatty acid-binding proteins (FABPs), a group of evolutionarily related cytoplasmic proteins that belong to the intracellular lipid-binding protein (iLBP) family. An oyster FABP genome-wide investigation was not available until a recent study on gene organization, protein structure, and phylogeny of Crassostrea gigas iLBPs. Higher transcript levels of the C. gigas FABP2 gene were found after exposure to sewage and pharmaceuticals. Because of its relevance as a potential biomarker of aquatic contamination, in this study, recombinant FABP2 from C. gigas (CgFABP2) was successfully cloned, expressed, and purified, and in vitro and in silico assays were performed using lipids and pharmaceuticals. This is the first characterization of a protein from the iLBP family in C. gigas. Homology modeling and molecular docking were used to evaluate the binding affinities of natural ligands (palmitic, oleic, and arachidonic acids) and pharmaceuticals (ibuprofen, sodium diclofenac, and acetaminophen). Among the tested fatty acids, CgFABP2 showed preference for palmitic acid. The selected pharmaceuticals presented a biphasic-binding mode, suggesting a different binding affinity with a preference for diclofenac. Therefore, the approach using circular dichroism and in silico data might be useful for ligand-binding screening in an invertebrate model organism.

Molecular effects of Microcystin-LA in tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus) is a freshwater phytoplanktivorous fish species reported to accumulate and tolerate large amounts of cyanotoxins such as microcystins (MCs). The present study aimed to investigate molecular responses to the acute exposure of Nile tilapia to the Microcystin-LA analogue (MC-LA). Thus, the specimens were sublethally exposed to 1000 µg kg-1 of MC-LA for 12, 24, 48, and 96 h. Gene expression of PP1, PP2A, GST, GPX and actin was analyzed by quantitative PCR. The protein abundance profile of PP2A was determined by immunoblotting, while the integrity of its biological function was assessed by a phosphatase enzymatic assay. PP2A activity was significantly and strongly reduced by MC-LA. A resulting feedback mechanism significantly increased PP2A gene expression and protein abundance in all assessed times. However, a recovery of that phosphatase activity was not observed. In this study, the observed increase in GPX gene expression was the only response that could be directly related to the unknown factors associated to the fish survival to such high dose exposure.

Stress responses in Crassostrea gasar exposed to combined effects of acute pH changes and phenanthrene.

Ocean acidification is a result of the decrease in the pH of marine water, caused mainly by the increase in CO2 released in the atmosphere and its consequent dissolution in seawater. These changes can be dramatic for marine organisms especially for oysters Crassostrea gasar if other stressors such as xenobiotics are present. The effect of pH changes (6.5, 7.0 and 8.2) was assessed on the transcript levels of biotransformation [cytochromes P450 (CYP2AU1, CYP2-like2) and glutathione S-transferase (GSTΩ-like)] and antioxidant [superoxide dismutase (SOD-like), catalase (CAT-like) and glutathione peroxidase (GPx-like)] genes, as well as enzyme activities [superoxide dismutase, (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transferases transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH)] and lipid peroxidation (MDA) in the gills of Crassostrea gasar exposed to 100 µg·L-1 of phenanthrene (PHE) for 24 and 96 h. Likewise, the PHE burdens was evaluated in whole soft tissues of exposed oysters. The accumulation of PHE in oysters was independent of pH. However, acidification promoted a significant decrease in the transcript levels of some protective genes (24 h exposure: CYP2AU1 and GSTΩ-like; 96 h exposure: CAT-like and GPx-like), which was not observed in the presence of PHE. Activities of GST, CAT and SOD enzymes increased in the oysters exposed to PHE at the control pH (8.2), but at a lower pH values, this activation was suppressed, and no changes were observed in the G6PDH activity and MDA levels. Biotransformation genes showed better responses after 24 h, and antioxidant-coding genes after 96 h, along with the activities of antioxidant enzymes (SOD, CAT), probably because biotransformation of PHE increases the generation of reactive oxygen species. The lack of change in MDA levels suggests that antioxidant modulation efficiently prevented oxidative stress. The effect of pH on the responses to PHE exposure should be taken into account before using these and any other genes as potential molecular biomarkers for PHE exposure.

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.