Hepatic transcriptome, transcriptional effects and antioxidant responses in Poecilia vivipara exposed to sanitary sewage.

Aquatic environments are subject to threats from multiple human activities, particularly through the release of untreated sanitary sewage into the coastal environments. These effluents contain a large group of natural or synthetic compounds referred to as emerging contaminants. Monitoring the types and quantities of toxic substances in the environment, especially complex mixtures, is an exhausting and challenging task. Integrative effect-based tools, such as biomarkers, are recommended for environmental quality monitoring programs. In this study, fish Poecilia vivipara were exposed for 24 and 96 h to raw untreated sewage diluted 33 % (v/v) in order to identify hepatic genes to be used as molecular biomarkers. Through a de novo hepatic transcriptome assembly, using Illumina MiSeq, 54,285 sequences were assembled creating a reference transcriptome for this guppy species. Transcripts involved in biotransformation systems, antioxidant defenses, ABC transporters, nuclear and xenobiotic receptors were identified and evaluated by qPCR. Sanitary sewage induced transcriptional changes in AhR, PXR, CYP2K1, CYP3A30, NQO1, UGT1A1, GSTa3, GSTmu, ST1C1, SOD, ABCC1 and SOX9 genes from liver of fish, particularly after 96 h of exposure. Changes in hepatic enzyme activities were also observed. The enzymes showed differences in fish exposed to both periods, while in the gills there was a prevalence of significant results after 96 h. The observed differences were associated to gender and/or to sewage exposure. The obtained results support the use of P. vivipara as sentinel and model organism for ecotoxicological studies and evidence the importance of understanding the differential responses associated to gender.

Integrated biomarker responses: a further improvement of IBR and IBRv2 indexes to preserve data variability in statistical analyses.

In biomonitoring and laboratory studies, it is typical to measure a battery of molecular, biochemical, and cytogenetic biomarkers to evaluate the effects of xenobiotics in biota. However, summarizing the results of several biomarkers to inform laypersons and environmental agencies is still a challenge for researchers and environmental specialists. To address this issue, researchers have developed indexes such as the Integrated Biomarker Responses (IBR) and Integrated Biomarker Response version 2 (IBRv2) to summarize all biomarkers responses into a single value. Unfortunately, these indexes do not preserve the original biological variability, which hampers subsequent statistical analyses. In this study, we created new versions of IBR and IBRv2, which preserve individual data variability and can be used in typical statistical analyses. The new Integrated Biomarker Responses individual (IBRi), Integrated Biomarker Responses version 2 individual (IBRv2i) and Weighted Integrated Biomarker Responses version 2 individual (Weighted IBRv2i) indexes correlated with the original IBR and IBRv2 indexes and were able to detect differences among experimental groups in a simulated and case studies. Using the IBRi, IBRv2i, or Weighted IBRv2i indexes is advantageous because they maintain the data variability of the experimental groups and can be analyzed using hypothesis testing statistics like any other parameter. Additionally, this approach can help translate technical scientific terminology into a more accessible language suitable for environmental governmental agencies and decision-makers.

Gender influences molecular and histological biomarkers in mature oysters Crassostrea gasar (Adanson, 1757) after pyrene exposure.

Oysters are frequently used as sentinel organisms for monitoring effects of contaminants due to their sessile, filtering habits and bioaccumulation capacity. These animals can show elevated body burden of contaminants, such as pyrene (PYR). PYR can be toxic at a molecular level until the whole oyster, which can show reproductive and behavioral changes. Considering that biologic parameters, such as gender or reproductive stage can interfere in the toxic effects elicited by contaminants uptake, the aim of this study was to evaluate some molecular and histological responses in females and males of oyster Crassostrea gasar exposed to PYR (0.25 and 0.5 µM) for 24 h at the pre-spawning stage. PYR concentrations were analyzed in water and in tissues of female and male oysters. Gene transcripts related to biotransformation (CYP3475C, CYP2-like, CYP2AU1, CYP356A, GSTO-like, GSTM-like, SULT-like), stress (HSP70), and reproduction (Vitellogenin, Glycoprotein) were quantified in gills. In addition, histological analysis and histo-localization of CYP2AU1 mRNA transcripts in gills, mantle and digestive diverticulum were carried out. Females and males in pre-spawning stage bioconcentrated PYR in their tissues. Males were more sensitive to PYR exposure. CYP2AU1 transcripts were higher in males (p < 0.05), as well as tubular atrophy was observed only in males exposed to PYR (p < 0.05). As expected, vitellogenin transcripts were lower in males (p < 0.05). Given these results, it is suggested that levels of CYP2AU1 be a good biomarker of exposure to PYR in oyster C. gasar and that it is important to consider the gender for the interpretation of biomarker responses.

Differential responses in the biotransformation systems of the oyster Crassostrea gigas (Thunberg, 1789) elicited by pyrene and fluorene: Molecular, biochemical and histological approach - Part II.

Pyrene (PYR) and fluorene (FLU) are among the sixteen priority Polycyclic Aromatic Hydrocarbons (PAH) of the United States Environmental Protection Agency and are both frequently detected in contaminated sites. Due to the importance of bivalve mollusks in biomonitoring programs and the scarce information on the biotransformation system in these organisms, the aim of this study was to investigate the effect of PYR and FLU at the transcriptional level and the enzymatic activities of some biotransformation systems in the Pacific oyster Crassostrea gigas, and to evaluate the histological effects in their soft tissues. Oysters C. gigas were exposed for 24 h and 96 h to PYR (0.25 and 0.5 µM) and FLU (0.6 and 1.2 µM). After exposure, transcript levels of cytochrome P450 coding genes (CYP1-like, CYP2-like, CYP2AU2, CYP356A1, CYP17α-like), glutathione S tranferase genes (omega GSTO-like and microsomal, MGST-like) and sulfotransferase gene (SULT-like), and the activity of ethoxyresorufin O-deethylase (EROD), Glutathione S-transferase (GST) and microssomal GST (MGST) were evaluated in gills. Histologic changes were also evaluated after the exposure period. PYR and FLU bioconcentrated in oyster soft tissues. The half-life time of PYR in water was lower than fluorene, which is in accordance to the higher lipophilicity and bioconcentration of the former. EROD activity was below the limit of detection in all oysters exposed for 96 h to PYR and FLU. The reproductive stage of the oysters exposed to PYR was post-spawn. Exposure to PYR caused tubular atrophy in digestive diverticula, but had no effect on transcript levels of biotransformation genes. However, the organisms exposed for 96 h to PYR 0.5 µM showed higher MGST activity, suggesting a protective role against oxidative stress in gills of oysters under higher levels of PYR in the tissues. Increased number of mucous cells in mantle were observed in oysters exposed to the higher FLU concentration, suggesting a defense mechanisms. Oysters exposed for 24 h to FLU 1.2 µM were in the ripe stage of gonadal development and showed higher transcript levels of CYP2AU2, GSTO-like and SULT-like genes, suggesting a role in the FLU biotransformation. In addition, after 96 h of exposure to FLU there was a significant increase of mucous cells in the mantle of oysters but no effect was observed on the EROD, total GST and MGST activities. These results suggest that PAH have different effects on transcript levels of biotransformation genes and enzyme activities, however these differences could also be related to the reproductive stage.

Differential responses in the biotransformation systems of the oyster Crassostrea gasar (Adanson, 1757) elicited by pyrene and fluorene: molecular, biochemical and histological approach - Part I.

Polycyclic aromatic hydrocarbons (PAHs) are among the main contaminants in aquatic environments. PAHs can affect organisms due to their carcinogenic, mutagenic and/or teratogenic characteristics. Depending on the PAHs, concentration, and period of exposure, biological damage can occur leading to histopathologic alterations. This study aimed to evaluate the molecular, biochemical and histological responses of the oyster Crassostrea gasar exposed to pyrene (0.25 and 0.5 µM) and fluorene (0.6 and 1.2 µM), after exposure for 24 and 96 h. Concentrations of both PAHs were quantified in the water and in oyster tissues. Transcript levels of phase I (CYP3475C1, CYP2-like, CYP2AU1 and CYP356A) and phase II (GSTO-like, MGST-like and SULT-like) biotransformation-related genes and the activities of ethoxyresorufin-O-deethylase (EROD), total and microsomal glutathione S-transferase (GST and MGST) were evaluated in the gills. Also, histological changes and localization of mRNA transcripts CYP2AU1 in gills, mantle, and digestive diverticula were evaluated. Both PAHs accumulated in oyster tissues. Pyrene half-life in water was significantly lower than fluorene. Transcript levels of all genes were higher in oysters exposed to of pyrene 0.5 µM (24 h). Only CYP2AU1 gene was up-regulated by fluorene exposure. EROD and MGST activities were higher in oysters exposed to pyrene. Tubular atrophy in the digestive diverticula and an increased number of mucous cells in the mantle were observed in oysters exposed to pyrene. CYP2AU1 transcripts were observed in different tissues of pyrene-exposed oysters. A significant correlation was observed between tubular atrophy and the CYP2AU1 hybridization signal in oysters exposed to pyrene, suggesting the sensibility of the species to this PAH. These results suggest an important role of biotransformation-related genes and enzymes and tissue alterations associated to pyrene metabolism but not fluorene. In addition, it reinforces the role of CYP2AU1 gene in the biotransformation process of PAHs in the gills of C. gasar.

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.

Molecular and cellular effects of temperature in oysters Crassostrea brasiliana exposed to phenanthrene.

Exposure of aquatic organisms to polycyclic aromatic hydrocarbons (PAH), such as phenanthrene (PHE), may increase the production of reactive oxygen species (ROS) and cause changes in the biotransformation systems. In addition, changes in water temperature can cause adverse effects in the organisms. Estuarine species, like the oyster Crassostrea brasiliana, can adapt and tolerate temperature variation. To evaluate the influence of temperature on biological responses of C. brasiliana exposed to PHE, oysters were maintained at three temperatures (18, 24 and 32 °C) for 15 days and co-exposed afterwards to 100 µg.L-1 of PHE for 24 and 96 h. Levels of PHE in the water and oyster tissues were determined, respectively after 24 and 96 h. In addition, thermal stress, biotransformation and oxidative stress-related genes were analyzed in oyster gills, together with the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferases (GST) and levels of lipid peroxidation. Oyster accumulated significant levels of PHE. HSP70-like transcripts were affected by PHE exposure only at 32 °C. Transcript levels of cytochrome P450 isoforms (CYP2-like2 and CYP2AU1) were down-regulated in oysters exposed to PHE for 24 h at 32 °C. GSTΩ-like transcript levels were also down-regulated in the PHE-exposed group at 32 °C. After 96 h, CYP2-like2 transcripts were higher in the PHE exposed groups at 32 °C. Oysters kept at 18 °C showed higher levels of SOD-like transcripts, together with higher GST, GPx and G6PDH activities, associated to lower levels of lipoperoxidation. In general the biological responses evaluated were more affected by temperature, than by co-exposure to PHE.

Candidate cytochrome P450 genes for ethoxyresorufin O-deethylase activity in oyster Crassostrea gigas.

Vertebrate cytochrome P450 1 (CYP1) enzymes metabolize endogenous and xenobiotic compounds and usually demonstrate a substrate-inducible response. Ethoxyresorufin O-deethylase activity (EROD) is a common method to quantify CYP1 enzymes activity in these organisms. Despite the absence of this gene family in protostomes, CYP1-like genes were identified in several species, even though no evolutionary relationship has been established with the vertebrate CYP1 family. In the present study, EROD activity was evaluated in microsomal fraction of gills, digestive gland and mantle of Crassostrea gigas. Enzyme activity was quantified in gills, although no activity was detected in digestive gland and mantle. EROD kinetic characterization in gills using typical Michaelis-Menten equation demonstrated an apparent Km of 1.15µM and Vmax of 229.2 fmol.min-1mg.protein -1. EROD activity was analyzed in the presence of CYP1 inhibitors, ellipticine (ELP), furafylline (FRF), clotrimazole (CTZ), α-naphthoflavone (ANF), and the non-ionic surfactant Triton X-100. CTZ inhibited EROD activity in all tested concentrations while Triton X-100 (0.5mM) caused 16% inhibition. Transcript levels of four CYP1-like genes were determined in gills, digestive gland and mantle. In general, CYP1-like genes showed higher transcript levels in gills compared to other tissues. The transcript levels of CYP1-like 1 and 2, analyzed together, positively correlated with EROD activity observed in gills, suggesting the possible involvement of these two gene products in EROD activity in this tissue. Homology models of translated CYP1-like 1 and 2 were generated based on human CYP1A1 structure and were similar to the general canonical cytochrome P450 fold. Molecular docking analysis showed that the two putative oyster CYP1-like structures have the potential to metabolize 7-ethoxyresorufin (7-ER), although the contribution of other CYP1-like genes needs to be investigated. Proteins encoded by CYP1-like 1 and 2 genes are plausible candidates for EROD activity observed in gills of C. gigas.

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.

Differential gene expression in oyster exposed to sewage.

In order to investigate the influence of domestic sewage on the gene expression of Pacific oysters Crassostrea gigas, suppression subtractive hybridization (SSH) method was employed. Oysters were sampled at a farming area and, after 10 days of acclimation in the laboratory, were exposed to untreated domestic sewage diluted 33% for 48 h. Gills of male oysters were excised for total RNA extraction. mRNA was purified and the differential gene expression was analyzed by SSH. We obtained 61 cDNA sequences but only 15 were identified, which includes fatty acid binding protein, multidrug resistance protein, omega glutathione S-transferase, cytochrome P450 isoform CYP356A1, among others. The identified genes are associated with different metabolic functions like biotransformation, membrane transport, aerobic metabolism and translational machinery, evidencing the potential toxic effects elicited by these effluents.

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.