Integrating pollutant levels and biochemical biomarkers in oysters (Crassostrea rhizophorae and Crassostrea gasar) indicates anthropic impacts on marine environments along the coast of Santa Catarina state, Brazil.

This study aimed to carry out a general diagnosis of the contamination of the coastal marine environment of the Santa Catarina state (SC, Brazil) by different classes of environmental pollutants, as well as to evaluate possible adverse effects of the contaminants on biochemical biomarkers of oysters, Crassostrea gasar and Crassostrea rhizophorae. 107 chemicals were evaluated in water, sediment and oyster samples from nine sites along the coastline of SC. We also examined various biochemical biomarkers in the oysters' gills and digestive glands to assess potential effects of contaminants. In general, the northern and central regions of the littoral of SC presented higher occurrences and magnitudes of contaminants than the southern region, which is probably related to higher urbanization of center and northern areas of the littoral. The biomarker analysis in the oysters reflected these contamination patterns, with more significant alterations observed in regions with higher levels of pollutants. Our results may serve as a first baseline for future and more extensive monitoring actions and follow-up of the degree of contamination in the state, allowing for inspection actions and management of areas most affected by marine pollutants.

Biochemical and histopathological responses in Nile tilapia exposed to a commercial insecticide mixture containing dinotefuran and lambda-cyhalothrin.

The indiscriminate use of pesticides has led to an increased risk of environmental contamination and pest resistance worldwide, favoring the development of less hazardous formulations. The commercial insecticide ZEUS® (Ihara, Brazil) combining dinotefuran and lambda-cyhalothrin was recently formulated in order to meet the environmental sustainability and food security. However, little is known about the potential toxic effects of ZEUS® to aquatic species. Thus, we report, for the first time, the biochemical and histological responses in tilapia (Oreochromis niloticus) following 96 h exposure to 0.01 mg/L, 0.05 mg/L and 0.1 mg/L ZEUS®. Different biochemical endpoints, including acetylcholinesterase (AChE), gamma-glutamyltransferase (GGT) and alkaline phosphatase (ALP), were assessed as potential biomarkers of insecticide effects. Glutathione S-transferase (GST) was evaluated as a marker of phase II biotransformation, and histopathological changes were measured to indicate gill alterations following ZEUS® exposure. After 96 h exposure, ZEUS® treatment increased GST activity in the liver of fish exposed to the highest concentration, while the intermediate dose increased both renal GGT and hepatic ALP activities. These findings reflect the importance of the liver and kidneys in the detoxification of ZEUS® and highlight the need to understand further toxicity effects. Likewise, the histopathological analysis of gills provided evidence that ZEUS® caused moderate damages. Despite biomarkers alterations reported for O. niloticus following ZEUS® exposure, by comparing our findings with data on toxicity of individual compounds, the commercial ZEUS® mixture seems to present similar or even lower adverse effects on freshwater fish.

Biochemical and molecular biomarkers in integument biopsies of free-ranging coastal bottlenose dolphins from southern Brazil.

Adverse effects of exposure to persistent organic pollutants (POPs) threaten the maintenance of odontocete populations. In southern Brazil, coastal bottlenose dolphins from the Laguna Estuarine System (LES) and Patos Lagoon Estuary (PLE) were sampled using remote biopsies during the winter and summer months. Levels of bioaccumulated POPs were measured in the blubber. The activities of glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were also quantified, as were the mRNA transcript levels of aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), cytochrome P450 1A1-like (CYP1A1), metallothionein 2A (MT2A), GST-π, GPx-4, GR, interleukin 1 alpha (IL-1α), and major histocompatibility complex II (MHCII) in the skin. In general, levels of POPs were similar among sites, sexes, ages and seasons. For most animals, total polychlorinated biphenyl (ΣPCBs) levels were above the threshold level have physiological effects and pose risks to cetaceans. The best-fitting generalized linear models (GLMs) found significant associations between GR, IL-1α and GPx-4 transcript levels, SOD and GST activities, and total polybrominated diphenyl ether (ΣPBDEs) and pesticide levels. GLMs and Kruskal-Wallis analyses also indicated that there were higher transcript levels for most genes and lower GST activity in the winter. These results reinforce the need to consider the influence of environmental traits on biomarker values in wildlife assessments.

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.

Proteomic response of gill microsomes of Crassostrea brasiliana exposed to diesel fuel water-accommodated fraction.

Diesel fuel water-accommodated fraction (diesel-WAF) is a complex mixture of organic compounds that may cause harmful effects to marine invertebrates. Expression of microsomal proteins can be changed by oil exposure, causing functional alterations in endoplasmic reticulum (ER). The aim of this study was to investigate changes in protein expression signatures in microsomes of oysterl Crassostrea brasiliana (=C.gasar) gill after exposure to 10% diesel-WAF for 24 and 72 h. Protein expression signatures of gills of oysters exposed to diesel-WAF were compared to those of unexposed oysters using two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. A total of 458 protein spots with molecular weights between 30-75 kDa were detected by 2-DE in six replicates of exposed oyster proteomes compared to unexposed ones. Fourteen differentially expressed proteins (six up-regulated and eight down-regulated) were identified. They are: proteins related to xenobiotic biotransformation (cytochrome P450 6 A, NADPH-cytochrome P450 reductase); cytoskeleton (α-tubulin, ß-tubulin, gelsolin); processing and degradation of proteins pathways (thioredoxin domain-containing protein E3 ubiquitin-protein ligase MIB2); involved in the biosynthesis of glycolipids and glycoproteins (beta-1,3-galactosyltransferase 1); associated with stress responses (glutamate receptor 4 and 14-3-3 protein zeta, corticotropin-releasing factor-binding protein); plasmalogen biosynthesis (fatty acyl-CoA reductase 1), and sodium-and chloride-dependent glycine transporter 2 and glyoxylate reductase/hydroxypyruvate reductase. Different patterns of protein responses were observed between 24 and 72 h-exposed groups. Expression pattern of microsomal proteins provided a first insight on the potential diesel-WAF effects at protein level in microsomal fraction of oyster gills and indicated new potential biomarkers of exposure and effect. The present work can be a basis for future ecotoxicological studies in oysters aiming to elucidate the molecular mechanisms behind diesel-WAF toxicity and for environmental monitoring programs.

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.

Suppressive subtractive hybridization libraries prepared from the digestive gland of the oyster Crassostrea brasiliana exposed to a diesel fuel water-accommodated fraction.

Diesel fuel can cause adverse effects in marine invertebrates by mechanisms that are not clearly understood. The authors used suppressive subtractive hybridization to identify genes up- and downregulated in Crassostrea brasiliana exposed to diesel fuel. Genes putatively involved in protein regulation, innate immune, and stress response, were altered by diesel challenge. Three genes regulated by diesel were validated by quantitative real-time polymerase chain reaction. This study sheds light on transcriptomic responses of oysters to diesel pollution.

Comparison of the antioxidant defense system in Crassostrea rhizophorae and Crassostrea gigas exposed to domestic sewage discharges.

Oysters Crassostrea rhizophorae and Crassostrea gigas were kept for fourteen days at four sites in São José, SC, Brazil, chosen according to a sewage discharge contamination gradient. Enzymatic activities of CAT, GST, G6PDH and GR were evaluated in gills and digestive glands. Higher CAT activity was observed in tissues of C. rhizophorae in response to sewage contamination possibly indicating peroxisome proliferation induction. C. gigas showed elevated G6PDH activity in digestive gland, and GR in gills, after domestic sewage exposure. In conclusion, C. rhizophorae and C. gigas showed different biochemical responses after in situ exposure to domestic sewage. However, C. rhizophorae showed more significant changes in CAT suggesting that this organism could be a better monitor to this kind of effluent.