Biochemical and molecular biomarkers and their association with anthropogenic chemicals in wintering Manx shearwaters (Puffinus puffinus).

Anthropogenic pollution poses a threat to marine conservation by causing chronic toxic effects. Seabirds have contact throughout their lives with pollutants like plastic, metals, polychlorinated biphenyls (PCBs), and organochlorine pesticides such as hexachlorocyclohexanes (HCHs). We assessed 155 Manx shearwaters (Puffinus puffinus) stranded along the Brazilian coast, analyzing associations between organic pollutants, plastic ingestion, biomarkers (transcript levels of aryl hydrocarbon receptor, cytochrome P450-1A-5 [CYP1A5], UDP-glucuronosyl-transferase [UGT1], estrogen receptor alpha-1 [ESR1], and heat shock protein-70 genes) and enzymes activity (ethoxy-resorufin O-deethylase and glutathione S-transferase [GST]). Plastic debris was found in 29 % of the birds. The transcription of UGT1 and CYP1A5 was significantly associated with hexachlorobenzene (HCB) and PCBs levels. ESR1 was associated with HCB and Mirex, and GST was associated with Drins and Mirex. While organic pollutants affected shearwaters more than plastic ingestion, reducing plastic availability remains relevant as xenobiotics are also potentially adsorbed onto plastics.

Emerging and legacy contaminants on the Brazilian southern coast (Santa Catarina): A multi-biomarker approach in oysters Crassostrea gasar (Adanson, 1757).

Coastal environments, such as those in the Santa Catarina State (SC, Brazil), are considered the primary receptors of anthropogenic pollutants. In this study, our objective was to evaluate the levels of emerging contaminants (ECs) and persistent organic pollutants (POPs) in indigenous Crassostrea gasar oysters from different regions of SC coast in the summer season (March 2022). Field collections were conducted in the São Francisco do Sul, Itajaí, Florianópolis and Laguna coastal zones. We analyzed the bioaccumulation levels of 75 compounds, including antibiotics (AB), endocrine disruptors (ED), non-steroidal anti-inflammatory drugs (NSAIDs), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and pesticides. Furthermore, we assessed biomarker responses related to biotransformation, antioxidant defense, heat shock protection and oxidative damage in oysters' gills. Prevalence of ECs was observed in the central and southern regions, while the highest concentrations of POPs were detected in the central-northern regions of SC. Oysters exhibited an induction in biotransformation systems (cyp2au1 and cyp356a1, sult and GST activity) and antioxidant enzymes activities (SOD, CAT and GPx). Higher susceptibility to lipid peroxidation was observed in the animals from Florianópolis compared to other regions. Correlation analyses indicated possible associations between contaminants and environmental variables in the biomarker responses, serving as a warning related to climate change. Our results highlight the influence of anthropogenic activities on SC, serving as baseline of ECs and POPs levels in the coastal areas of Santa Catarina, indicating more critical zones for extensive monitoring, aiming to conserve coastal regions.

Biochemical responses of the mussel Perna perna after exposure to environmental concentrations of the UV filter Benzophenone-3.

Personal care products, such as UV filters, are frequently present in aquatic ecosystems, but studies on their impact on marine organisms are still scarce. Here we addressed the effects of benzophenone-3 (BP-3) on the antioxidant status of Perna perna mussels exposed to concentrations of 0.1 and 3 µg.L-1 for 72 h and 7 days. Glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PDH) activity and lipoperoxidation (MDA) were evaluated in the gills. A significant reduction (p < 0.05) in the activity of G6PDH and GPx was observed after exposure for 7 days to 0.1 µg.L-1. However, no significant differences were observed in GST activity and MDA levels, independently of the exposure time. Principal component analysis (PCA) showed an association of BP-3 highest concentration with GR and MDA at 72 h and only with GR at 7 days of exposure. Similarly, the integrated biomarker response (IBR) demonstrated GR and MDA alterations. In conclusion, environmentally relevant concentrations of BP-3 altered antioxidant and auxiliary enzymes, which could cause long-term damage to P.perna mussels. The need to implement more efficient techniques in wastewater treatment systems is pointed out, especially in summer, when UV filters are used more frequently and abundantly.

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.

How does habitat influence metabolism? Clues from biomarker response and contaminant profile in Tursiops truncatus (Montagu, 1821) subspecies living in parapatry.

In western South America (WSA) two subspecies of bottlenose dolphin are recognized: Tursiops truncatus gephyreus, predominantly found in estuaries and river mouths, and Tursiops truncatus truncatus, occurring along the continental shelf. Despite a partial spatial overlap, both subspecies are considered to occupy different habitats and ecological niches. In the present study, chemical analyzes as well as biochemical and molecular biomarkers were used to investigate the influence of niche partitioning over metabolic pathways associated with the detoxification of persistent organic pollutants (POPs), antioxidant metabolism, immune activity and lipid metabolism in Tursiops truncatus subspecies living in parapatry. Overall, the profile and levels of bioaccumulated PCBs, pesticides and PBDEs were similar between groups, with a greater variety of pesticides, such as γ-HCHs, heptachlor, oxychlordane and o,p'DDT, detected in T. truncatus gephyreus. Multivariate analysis of variance (MANOVA) and non-metric multidimensional scaling (NMDS) results indicated that glutathione reductase (GR) and superoxide dismutase (SOD) enzymatic activities were higher in coastal dolphins, as were the mRNA levels of metallothionein 2A (MT2A), interleukin-1α (IL-1α), ceramide synthase 3 (CERS3) and fatty acid elongase (ELOVL4). In parallel, mRNA levels of fatty acid synthase complex 1 (FASN 1) were higher in oceanic dolphins. These findings suggest that, due to their occurrence in coastal habitats, T. truncatus gephyreus is more exposed to environmental pollutants and pathogenic microorganisms. Likewise, niche partitioning may influence lipid biosynthesis, possibly due to differences on feeding habits, reflecting in an enhanced long chain ceramides biosynthesis in T. truncatus gephyreus. Collectively, these data reinforce the need to address habitat specificities in conservation efforts, since distinct groups can be facing different anthropogenic pressures in WSA.

Fipronil impairs the GABAergic brain responses of Nile Tilapia during the transition from normoxia to acute hypoxia.

γ-aminobutyric acid (GABA) is one of the main neurotransmitters involved in the adaptation processes against the damage that hypoxia can cause to the brain. Due to its antagonist action on GABA receptors, the insecticide fipronil can turn the fish more susceptible to the negative effects of hypoxia. This study aimed to understand better if fipronil affects these GABAergic responses of Tilapia ahead to hypoxia. Oreochromis Niloticus (Nile Tilapia) were exposed for 3 and 8 h to fipronil (0.0, 0.1, and 0.5 µg.L-1 ) under normoxia (dissolved O2 > 6 mg.L-1 ) and moderate hypoxia (dissolved O2 < 2 mg.L-1 ) conditions. Briefly, hypoxia caused opposite effects on the gene transcription of the evaluated ionotropic and metabotropic GABA receptors. Unexpectedly, we obtained reduced HIF1A mRNA and brain GABA levels, mostly in the first 3 h of the experiment, for the hypoxic group compared with the normoxia one. Besides that, we also demonstrated that the insecticide fipronil impairs the brain GABAergic signaling of a hypoxia-tolerant fish during the transition from a normoxic to an acute hypoxic state. Thus, these results predict the relevant impact on the brain metabolic adaptations of fishes exposed to such stressful conditions in an aquatic environment, as well as the effects of fipronil in the GABAergic responses to hypoxia, which in turn may have ecological and physiological significance to hypoxia-tolerant fishes exposed to this insecticide.

Effects of 4-n-nonylphenol in liver of male and female viviparous fish (Poecilia vivipara).

4-n-Nonylphenol (NP) is one of the most toxic alkylphenols found in the environment. To evaluate the transcriptional effects of NP in the viviparous fish Poecilia vivipara, a hepatic transcriptome and qPCR analysis of genes were carried out. Guppies separated by sex were injected with two doses of NP (15 µg/g and 150 µg/g) or peanut oil (control). After 24 h, analysis of transcriptional level of Aryl Hydrocarbon Receptor (AhR), Estrogen Nuclear Receptor Alpha (ESR1), Pregnane X Receptor (PXR), Cytochromes P450 (CYP1A, CYP2K1 and CYP3A30), Glutathione S-transferase A3 and Mu 3 (GSTa3 and GSTMu3), SRY-Box Transcription Factor 9 (SOX9), Vitellogenin-1 (VIT), ATP Binding Cassette Subfamily C Member 1 (ABCC1), Multidrug Resistance-Associated Protein 2 (MRP2) and UDP Glucuronosyltransferase Family 1 Member A1 (UGT1A1) was evaluated. 205,046 transcripts were assembled and protein prediction resulted in 203,147 predicted peptides. In females, no significant changes were detected in the transcription of some phase I biotransformation and ABC transporter genes. AhR, PXR, GSTa3 and SOX9 genes where higher in the lower dose group (15 µg/g) compared to control. In male fish, no changes were observed in the transcript levels of the nuclear receptors, in endocrine disruption and phase I biotransformation genes. GSTa3 showed lower transcription in fish treated with both doses. ABCC1 was higher in guppies treated with the lower dose while MRP2 showed less transcripts. This short-term and low-dose exposure to NP caused changes that could serve as early indicators of deleterious processes. These results indicate P. vivipara as a good sentinel in biomonitoring programs.

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.

Influence of temperature on growth, development and thyroid metabolism of American bullfrog tadpoles (Lithobates catesbeianus) exposed to the herbicide tebuthiuron.

The influence of temperature (25 and 32 °C) on the negative effects of the herbicide tebuthiuron (TBU, 0, 10, 50 and 200 ng.L-1, 16 days) on thyroid function and metamorphosis of Lithobates catesbeianus tadpoles was evaluated. Metamorphosis was accelerated by TBU exposure at 25 ºC, but delayed at 32 ºC with considerable losses of body mass. T3 and T4 levels were not altered. The highest TBU concentrarion at 25 ºC increased TRâ€¯ß and DIO3 transcript levels, which is consistent with development acceleration in tadpoles. At 32 ºC TRâ€¯ß transcript levels were lower than the values recorded at 25 ºC, and those tadpoles exposed to the highest TBU concentration presented increased diameter of thyroid follicles compared to controls at same temperature. This study evidences that TBU at environmentally realistic concentrations is able to disrupt thyroidogenesis in bullfrog tadpoles, impairing their development. These effects are influenced by temperature.

Characterisation of UDP-glucuronosyltransferase activity in sea turtle Chelonia mydas.

Uridine diphosphate glucuronosyltransferase (UGT) enzymes conjugate many lipophilic chemicals, such as drugs, environmental contaminants, and endogenous compounds, promoting their excretion. The complexity of UGT kinetics, and the location of enzyme active site in endoplasmic reticulum lumen, requires an accurate optimisation of enzyme assays.In the present study, we characterised UGT activity in liver microsomes of green turtles (Chelonia mydas), an endangered species. The conditions for measuring UGT activity were standardised through spectrofluorimetric methods, using the substrates 4-methylumbelliferone (4-MU) and uridine diphosphate glucuronic acid (UDPGA) at 30 °C and pH 7.4.The green turtles showed UGT activity at the saturating concentrations of substrates of 250 µM to 4-MU and 7 mM to UDPGA. The alamethicin, Brij®58, bovine serum albumin (BSA), and magnesium increased UGT activity. The assay using alamethicin (22 µg per mg of protein), magnesium (1 mM), and BSA (0.25%) reached the highest Vmax (1203 pmol·min-1mg·protein-1). Lithocholic acid and diclofenac inhibited UGT activity in green turtles.This study is the first report of UGT activity in the liver of green turtles and provides a base for future studies to understand the mechanisms of toxicity by exposure to contaminants in this charismatic species.

Integrated biomarker responses in oysters Crassostrea gasar as an approach for assessing aquatic pollution of a Brazilian estuary.

The Laguna Estuarine System (LES), southern Brazil, suffers impacts from anthropogenic activities, releasing contaminants into the ecosystem. This study evaluated changes in biochemical and molecular biomarkers and contaminants concentrations in oysters Crassostrea gasar transplanted and kept for 1.5 and 7 days at three potentially contaminated sites (S1, S2, and S3) at LES. Metals varied spatiotemporally; S1 exhibited higher Ag and Pb concentrations, whereas Cd was present in S3. S2 was a transition site, impacted by Ag, Pb, or Cd, depending on the period. Organic contaminants concentrations were higher before transplantation, resulting in the downregulation of biotransformation genes transcripts levels. Phase II-related genes transcripts and metals showed positive correlations. Decreased levels of HSP90-like transcripts and antioxidant enzymes activity were related to increased pollutant loads. Integrated biomarker response index (IBR) analysis showed S1 and S3 as the most impacted sites after 1.5 and 7 days, respectively. Regardless of the scenario, LES contaminants pose a significant threat to aquatic biota.

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.

Metal bioaccumulation, oxidative stress and antioxidant responses in oysters Crassostrea gasar transplanted to an estuary in southern Brazil.

The present study assessed the spatial and temporal variations on metal bioaccumulation and biochemical biomarker responses in oysters Crassostrea gasar transplanted to two different sites (S1 and S2) at the Laguna Estuarine System (LES), southern Brazil, over a 45-days period. A multi-biomarker approach was used, including the evaluation of lipid peroxidation (MDA) levels, and antioxidant defense enzymes (CAT, GPx, GR and G6PDH) and phase II biotransformation enzyme (GST) in the gills and digestive gland of oysters in combination with the quantification of Al, Cd, Cu, Pb, Fe, Ni and Zn in both tissues. The exposed oysters bioaccumulated metals, especially Al, Cd and Zn in gills and digestive gland, with most prominent biomarker responses in the gills. Results showed that GPx, GR and G6PDH enzymes offered an increased and coordinated response possibly against metal (Zn, Ni, Cd and Cu) contamination in gills. GST was inversely correlated to Cd levels, being its activity significantly lowered over the 45-d exposure periods at S2. On contrary, in digestive gland GST was slightly positively correlated to Cd, revealing a compensatory mechanism between tissues to protect oysters' cells against oxidative damages, since MDA levels also decreased. CAT also appeared to be involved in the cellular protection against oxidative stress, being increased in gills. However, CAT was negatively correlated to Al levels, which might suggest a possible inhibitory effect of this metal in the gills of C. gasar. Differences between tissues were evident by the Integrative Biomarker Responses version 2 (IBRv2) indexes, which showed different pattern between tissues when studying the sites and exposure periods separately. This study provided evidence for the effectiveness of using a multi-biomarker approach in oyster C. gasar to monitor estuarine metal pollution.

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.

Effects of phenanthrene on early development of the Pacific oyster Crassostrea gigas (Thunberg, 1789).

Phenanthnere (PHE) is a polycyclic aromatic hydrocarbon continuously discarded in the marine environment and bioavailable to many aquatic species. Although studies about PHE toxicity have been documented for adult oysters, the effects on early developmental stages are poorly characterized in bivalves. In this study, the effects of PHE (0.02 and 2.0µg.L-1) were evaluated on the embryogenesis and larval development of Crassostrea gigas. Toxicity bioassays, growth and deformities assessment, analysis of shell calcium abundance and transcript levels of genes related to xenobiotic biotransformation (CYP2AU2, CYP30C1), immune system (Cg-Tal) and tissue growth and shell formation (Ferritin, Insulin-like, Cg-Try, Calmodulin and Nacrein) were assayed in D-shape larvae after 24h of PHE exposure. At the highest concentration (2.0µg.L-1), PHE decreased the frequency of normal development (19.7±2.9%) and shell size (53.5±2.8mm). Developmental deformities were mostly related to abnormal mantle and shell formation. Lower calcium levels in oyster shells exposed to PHE 2.0µg.L-1 were observed, suggesting effects on shell structure. At this same PHE concentration, CYP30C1, Cg-Tal, Cg-Tyr, Calmodulin were upregulated and CYP2AU2, Ferritin, Nacrein, and Insulin-Like were downregulated compared to control larvae. At the lowest PHE concentration (0.02µg.L-1), it was observed a minor decrease in normal larval development (89,6±6%) and the remaining parameters were not affected. This is the first study to provide evidences that exposure to PHE can affect early oyster development at the molecular and morphological levels, possibly threatening this bivalve species.

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.

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.

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.

Upregulation of biotransformation genes in gills of oyster Crassostrea brasiliana exposed in situ to urban effluents, Florianópolis Bay, Southern Brazil.

The release of untreated sanitary sewage, combined with unplanned urban growth, are major factors contributing to degradation of coastal ecosystems in developing countries, including Brazil. Sanitary sewage is a complex mixture of chemicals that can negatively affect aquatic organisms. The use of molecular biomarkers can help to understand and to monitor the biological effects elicited by contaminants. The aim of this study was to evaluate changes in transcript levels of genes related to xenobiotic biotransformation in the gills of oysters Crassostrea brasiliana transplanted and kept for 24h at three areas potentially contaminated by sanitary sewage (Bücheller river, BUC; Biguaçu river, BIG; and Ratones island, RAT), one farming area (Sambaqui beach, SAM) and at one reference site (Forte beach, FOR) in the North Bay of Santa Catarina Island (Florianópolis, Brazil). Transcript levels of four cytochrome P450 isoforms (CYP2AU1, CYP3A-like, CYP356A1-like and CYP20A1-like), three glutathione S-transferase (GST alpha-like, GST pi-like and GST microsomal 3-like) and one sulfotransferase gene (SULT-like) were evaluated by means of quantitative reverse transcription PCR (qRT-PCR). Chemical analysis of the sediment from each site were performed and revealed the presence of aliphatic and polycyclic aromatic hydrocarbons, linear alkylbenzenes and fecal sterols in the contaminated areas (BUC and BIG). Water quality analysis showed that these sites had the highest levels of fecal coliforms and other parameters evidencing the presence of urban sewage discharges. Among the results for gene transcription, CYP2AU1 and SULT-like levels were upregulated by 20 and 50-fold, respectively, in the oysters kept for 24h at the most contaminated site (BUC), suggesting a role of these genes in the detoxification of organic pollutants. These data reinforce that gills possibly have an important role in xenobiotic metabolism and highlight the use of C. brasiliana as a sentinel for monitoring environmental contamination in coastal regions.