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.

Comparative biochemical and molecular responses of biotransformation and antioxidant systems in three species of Crassostrea (Sacco, 1897) oysters exposed to chrysene.

Chrysene (CHR) is among the most persistent polycyclic aromatic hydrocarbons (PAH) in water and a priority compound for pollutants monitoring, due to its carcinogenic, mutagenic and genotoxic potential. Aquatic animals exposed to CHR may present alterations of biomarkers involved in the biotransformation and oxidative stress-related parameters. The aim of this study was to investigate differences in antioxidant and biotransformation (phase I and II) systems of Crassostrea gigas, C. gasar and C. rhizophorae and its effects resulting from CHR exposure. Adult oysters of these species were exposed to 10 µg L-1 of CHR for 24 h and 96 h. In gills, the transcripts CYP1-like, CYP2-like, CYP2AU1-like, GSTO-like, MGST-like, SULT-like were evaluated after 24 h of exposure. The activity of SOD, CAT, GPx, GR and G6PDH were analyzed in gills and digestive glands after 96 h of exposure. CHR bioaccumulated in tissues. Differences in the remaining levels of CHR in water after 96 h were observed in aquaria containing C. gigas or C. gasar oysters and may be associated to the different filtration rates between these species. Downregulate of biotransformation genes were observed in gills of C. gasar (CYP2AU1-like and GSTO-like) and C. rhizophorae (CYP1-like1, CYP2-like, MGST-like and SULT-like), suggesting that biotransformation responses may be species-specific. Differential activity of antioxidant enzymes were observed in gills and digestive gland of oysters exposed to CHR. Biochemical responses suggested that C. gigas and C. gasar are more responsive to CHR. Differential responses observed among the three Crassostrea species can be related to evolutionary differences, ecological niches and adaptation to environment.

Microplastics and linear alkylbenzene levels in oysters Crassostrea gigas driven by sewage contamination at an important aquaculture area of Brazil.

The levels of linear alkylbenzenes (LABs) and the occurrence of microplastics (MPs) in the oysters Crassostrea gigas were evaluated in six farming areas in southern Brazil. The results revealed higher concentrations of LABs in oyster tissue from the Serraria (1977 ± 497.7 ng g-1) and Imaruim (1038 ± 409.9 ng g-1) sites. Plastic microfibers were found in oysters from all locations with values from 0.33 to 0.75 MPs per oyster (0.27-0.64 MPs per gram) showing the ubiquitous presence of this contaminant in the marine environment, which could be considered a threat to farming organisms. In addition, elements such as Ti, Al, Ba, V, Rb, Cr, and Cu were found in the chemical composition of the microfibers, suggesting MPs as vectors of inorganic compounds. A positive correlation between LABs and thermotolerant coliforms suggests that sewage discharges are the main source of contamination in these oysters cultured for human consumption. The present study highlights the need for efficient wastewater treatment plants and the implementation of depuration techniques in oysters from farming areas.

Molecular changes in oysters Crassostrea gigas (Thunberg, 1793) from aquaculture areas of Santa Catarina Island bays (Florianópolis, Brazil) reveal anthropogenic effects.

Anthropogenic activities in coastal regions cause risks to the environmental and human health. Due to the carcinogenic and mutagenic potential, polycyclic aromatic hydrocarbons (PAH) are considered priority for monitoring. Most of the Brazilian production of Crassostrea gigas oysters are placed in the Bays of Santa Catarina Island. The aim of this study was to evaluate molecular responses (phase I and II of biotransformation and antioxidant defense) of C. gigas from six oyster farming areas potentially contaminated by sanitary sewage in Florianópolis Metropolitan (SC, Brazil): Santo Antônio de Lisboa, Sambaqui, Serraria, Caieira, Tapera, Imaruim. We evaluated the transcript levels of CYP1A1-like, CYP2-like, CYP2AU2-like, CYP356A1, GSTA1A-like, GSTO.4A-like, SULT-like, SOD-like and CAT-like by qRT-PCR. Only oysters from Caieira showed levels of thermotolerant coliforms allowed by the law. Chemicals analyses in soft tissues of oysters showed low to average levels of PAH in all monitored areas. Enhanced transcript levels of phase I (CYP1A1-like, CYP3564A1-like, CYP2-like and CYP2AU2-like) were observed in oysters from Serraria and Imaruí, suggesting higher biotransformation activity in these farming areas. Regarding phase II of biotransformation, GSTO.4A-like was up-regulated in oysters from Imaruí compared to Caieira and Santo Antônio de Lisboa. An upregulation of SOD-like and CAT-like were observed in oysters from Imaruí and Serraria, suggesting that oysters from these sites are facing higher prooxidant conditions compared to other areas. By integrating the biological and chemical data it is suggested that human-derived contaminants are affecting the oyster metabolism in some farming areas.

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.

Short-term spatiotemporal biomarker changes in oysters transplanted to an anthropized estuary in Southern Brazil.

Estuarine ecosystems are increasingly being affected by pollution caused by anthropogenic activities. In this study, Crassostrea gasar oysters were transplanted and maintained for seven days at three sites (S1, S2, and S3) in the Laguna Estuarine System (LES)-situated in southern Brazil-that has been exposed to multiple anthropic stresses. On the basis of the concentrations of metal and organic pollutants in oysters, we identified marked spatial variations in pollutant levels, with S3 showing the highest concentration of Ag, Fe, Ni, Zn, and total polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and linear alkylbenzenes (LABs), followed by S2 and S1. Along with the concentrations of pollutants, a set of biomarkers was analyzed. Oysters maintained at S3 showed enhanced protective defenses in gills, as observed by the increased levels of superoxide dismutase (SOD-like) and heat shock protein 90 (HSP90-like) transcripts and catalase (CAT) activity, concomitant with reduced lipid peroxidation (MDA) levels. Decreased antioxidant activities together with increased MDA levels are indicative of the digestive gland being more susceptible to pollutant-induced oxidative damage. Oysters transplanted into LES showed lower levels of cytochrome P450 transcripts (CYP356A1-like and CYP2AU1), and decreased glutathione S-transferase (GST) enzyme activity, suggesting lower biotransformation capacity. By integrating information regarding the concentration of metal and organic pollutants with that of molecular as well as biochemical biomarkers, our study provides novel insights into pollutant exposure and the potential biological impacts of such exposure on estuarine organisms in southern Brazil.

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.

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.

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.

A light in the darkness: new biotransformation genes, antioxidant parameters and tissue-specific responses in oysters exposed to phenanthrene.

Phenanthrene (PHE), a major component of crude oil, is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in aquatic ecosystems, and is readily bioavailable to marine organisms. Understanding the toxicity of PAHs in animals requires knowledge of the systems for xenobiotic biotransformation and antioxidant defence and these are poorly understood in bivalves. We report, for the first time, new transcripts and tissue-specific transcription in gill and digestive gland from the oyster Crassostrea brasiliana following 24h exposure to 100 and 1000µgL(-1) PHE, a model PAH. Six new cytochrome P450 (CYP) and four new glutathione S-transferase (GST) genes were analysed by means of quantitative reverse transcription PCR (qRT-PCR). Different antioxidant endpoints, including both enzymatic and non-enzymatic parameters, were assessed as potential biomarkers of oxidative stress. GST activity was measured as an indicator of phase II biotransformation. Rapid clearance of PHE was associated with upregulation of both phase I and II genes, with more pronounced effects in the gill at 1000µgL(-1) PHE. After 24h of exposure, PHE also caused impairment of the antioxidant system, decreasing non-protein thiols and glutathione levels. On the other hand, no change in antioxidant enzymes was observed. PHE treatment (100µgL(-1)) significantly decreased GST activity in the gill of exposed oysters. Both CYP and GST were transcribed in a tissue-specific manner, reflecting the importance of the gill in the detoxification of PAHs. Likewise, the antioxidant parameters followed a similar pattern. The data provide strong evidence that these genes play key roles in C. brasiliana biotransformation of PHE and highlight the importance of gill in xenobiotic metabolism.

Basal and 3-methylcholanthrene-induced expression of cytochrome P450 1A, 1B and 1C genes in the Brazilian guppy, Poecilia vivipara.

In fish there are four cytochrome P450 (CYP1) subfamilies: CYP1A, CYP1B, CYP1C, and CYP1D. Here we cloned Poecilia vivipara CYP1A, with an inferred amino acid sequence 91% identical to CYP1A from the killifish Fundulus heteroclitus, another member of the Cypriniformes, and an important model in ecotoxicology. In addition, we examined the expression of CYP1A, CYP1B1, and CYP1C1 by qPCR in liver, gill, and intestine of adult P. vivipara injected with 3-methylcholanthrene (3-MC) or held in clean water (control group) for 24h. All three tissues examined showed basal expression of the three CYP1 genes. CYP1A was most strongly expressed in the liver, while CYP1B1, and CYP1C1 were most strongly expressed in the gill and intestine respectively. 3-MC induced CYP1A, CYP1B1, and CYP1C1 significantly (20-120-fold) in the three organs, consistent with the regulation of CYP1A, CYP1B1 and CYP1C1 via the aryl hydrocarbon receptor. Validation of CYP1 gene biomarkers in fish collected from a contaminated urban mangrove environment was confirmed with significant induction of CYP1A and CYP1C1 in gills (10-15-fold) and CYP1B1 in liver (23-fold), relative to fish from a control site. The responsiveness of these CYP1 genes indicates P. vivipara is suitable as a model for environmental toxicology studies and environmental assessment in Brazil.