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Green turtles, Chelonia mydas, have been included in biomonitoring efforts given its status as an endangered species. Many studies, however, rely on samples from stranded animals, raising the question of how death affects important biochemical and molecular biomarkers. The goal of this study was to investigate post mortem fluctuations in the antioxidant response and metabolism of carbohydrates in the liver of C. mydas. Liver samples were obtained from six green turtles which were submitted to rehabilitation and euthanized due to the impossibility of recovery. Samples were collected immediately after death (t = 0) and at various time intervals (1, 2, 3, 4, 5, 6, 12, 18 and 24 h post mortem), frozen in liquid nitrogen and stored at -80 °C. The activities of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH) were analyzed, as were the levels of lipid peroxidation, glycogen concentration, RNA integrity (RNA IQ) and transcript levels of carbonic anhydrase and pyruvate carboxylase genes. Comparison between post mortem intervals showed a temporal stability for all the biomarkers evaluated, suggesting that changes in biochemical and molecular parameters following green turtle death are not immediate, and metabolism may remain somewhat unaltered up to 24 h after death. Such stability may be associated with the overall lower metabolism of turtles, especially under an oxygen deprivation scenario such as organismal death. Overall, this study supports the use of biomarkers in sea turtles sampled within a period of 24 h post mortem for biomonitoring purposes, though it is recommended that post mortem fluctuations of particular biomarkers be evaluated prior to their application, given that proteins may show varying degrees of susceptibility to proteolysis.

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Acetaminophen (paracetamol) (PAR) is one of the most popular non-steroidal anti-inflammatory drugs (NSAIDs) with analgesic and antipyretic properties consumed worldwide and often detected in the aquatic environment. Due to the fact that PAR induces oxidative stress in mammals, the aim of this study was to evaluate if similar effects were observed in oysters Crassostrea gigas, given their economic and ecological importance and worldwide distribution. Oysters were exposed for 1, 4 and 7 days to two different sublethal PAR concentrations (0, 1 and 100µgL-1). Cell viability, DNA damage in hemocytes and enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH) and glutathione S-transferases (GST) were evaluated in oyster gills. In addition, changes at transcriptional level of Cu/Zn superoxide dismutase (SOD), catalase-like (CAT-like), cytochrome P450 genes (CYP30C1, CYP2AU2, CYP3071A1, CYP356A1), glutathione S-transferase isoforms (GST-ω and GST-π-like), cyclooxygenase (COX), fatty acid binding proteins-like (FABP-like), and caspase genes were evaluated in oyster gills and digestive gland. No changes in cell viability and DNA damage were observed in oysters exposed to both PAR concentrations. Similarly, no significant changes were detected in the major antioxidant enzymes (except for auxiliary enzyme GR) in oyster gills, suggesting that changes in GR activity are enough to counteract a potential oxidative stress in C. gigas gills under these experimental conditions. Furthermore, changes at transcriptional level are concentration and tissue dependent. PAR elicited an inhibition of CYP30C1, CYP3071A1 and FABP-like transcripts highlighting their role in drug metabolism, transport and detoxification of PAR in the gills. GST transcript levels were type, tissue and concentration-dependent. GST-π-like was down-regulated in oyster gills exposed to the lowest PAR concentration and up-regulated in the digestive gland of oysters exposed to the highest PAR concentration. However, GST-ω transcript levels were lower only in oysters digestive gland exposed to the lowest PAR concentration. Therefore, changes at transcriptional level were more sensitive to assess the exposure to PAR at environmental relevant concentrations.

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Nanotechnology is a rapid field of development with the enhancement of the production of different types of nanoparticles (NPs) applied in several industrial and commercial applications which increase the risk of their presence in the aquatic environment. Ag NPs have a wide application in everyday life products. However, there is concern about the exposure effects on aquatic organisms to these NPs. Therefore, this study aims to assess gene transcription alterations in mussels Mytilus galloprovincialis gills exposed for 2 weeks to Ag NPs (42 ± 10 nm, 10 µg.L(-1)). The genes were selected based on previous biomarkers and proteomic results and included superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST), caspase 3/7-1 (CAS), cathepsin L (CATH), heat-shock protein 70 (HSP 70), cytochrome P450 4YA (CYP 4YA), the elongation factor (EF1), actin and α- tubulin. No significant changes in gene transcription profiles were observed after exposure of M. galloprovincialis to Ag NPs for 15 days. The lack of significant gene transcription responses is in light with previous results obtained for mussels exposed to these NPs and may be related to the fact that enzyme kinetics and relative abundance of proteins (increase of antioxidant enzymes and metalllothioneins (MTs) with the time of exposure) do not always directly reflect their relative mRNA levels. Nevertheless, their overall expression maintenance may signify that, at end of the exposure period (15 days), the transcription of the respective genes is no longer required, pointing out to a possible adaptation effect to nanoparticles or due to the levels of Ag NPs accumulated in this tissue at this exposure time. This study highlights that gene transcription application and role as an additional and/or alternative end point approach is important to understand the mode of action of these emergent contaminants in aquatic organisms. However, in future studies, the time window needs to be adjusted, as genes are likely to respond earlier to the nanoparticle exposure.

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Active pharmaceutical ingredients (APIs) are emergent environmental contaminants widely detected in surface waters as result of incomplete waste water treatment plant (WWTP) removal processes and improper disposal. The assessment of potential effects of APIs on non-target organisms is still scarce since besides presenting multiple chemical structures, properties and modes of action, these compounds occur as complex mixtures. This study comprises a 15-day exposure of mussels Mytilus galloprovincialis to mixtures (at environmentally relevant nominal concentrations) of non-steroidal inflammatory drugs ibuprofen (IBU) and diclofenac (DCF) (250 ng L(-1) each) and selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLX) (75 ng L(-1)) (MIX 1) along with the addition of classical pro-oxidant copper (Cu) (5 µg L(-1)) (MIX 2). The goals included the assessment of oxidative stress, neurotoxic and endocrine effects on this sentinel species applying both a multibiomarker and gene expression (here and later gene expression is taken as synonym to gene transcription, although it is acknowledged that it is also affected by, e.g. translation, and mRNA and protein stability) analysis approaches. The results revealed a swifter antioxidant response in digestive glands than in gills induced by MIX 1, nevertheless the presence of Cu in MIX 2 promoted a higher lipid peroxidation (LPO) induction. Neither mixture altered acetylcholinesterase (AChE) activity, while both triggered the formation of vitellogenin-like proteins in females confirming the xenoestrogenic effect of mixtures. All these results varied with respect to those obtained in previous single exposure essays. Moreover, RT-PCR analysis revealed a catalase (CAT) and CYP4Y1 gene expression down- and upregulation, respectively, with no significant changes in mRNA levels of genes encoding superoxide dismutase (SOD) and glutathione-S-transferase (GST). Finally, this study highlights variable tissue and time-specific biomarker responses and gene expression alterations, which along with several interactions between each mixture component on each biomarker confirm the susceptibility of mussels to API mixtures.

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The West Indian manatee Trichechus manatus is threatened with extinction in Brazil, and this study focused on nondestructive blood samples analyzed for metals, polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs), as well as biochemical and hematological biomarkers. Studied manatees were kept at Projeto Peixe-Boi headquarters in Pernambuco State, and at two natural areas in estuaries where they are released to the wild. Manatees kept at the natural estuary in Paraiba State have blood concentrations of Al, Pb, Cd, Sn that are 11, 7, 8 and 23 times greater, respectively, than the concentrations found in blood of animals from the same species in Florida, USA. An inhibition of butyrylcholinesterase in manatees kept at the two reintroduction sites in Alagoas and Paraiba States indicated possible exposure of the animals to cholinesterase inhibitor insecticides. PCBs and OCPs were not detected. Results from this study will help delineate conservation efforts in the region.

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This study aimed to provide the first biomonitoring integrating biomarkers and bioaccumulation data in São Paulo coast, Brazil and, for this purpose, a battery of biomarkers of defense mechanisms was analyzed and linked to contaminants' body burden in a weigh-of-evidence approach. The brown mussel Perna perna was selected to be transplanted from a farming area (Caraguatatuba) to four possibly polluted sites: Engenho D'Água, DTCS (Dutos e Terminais do Centro-Oeste de São Paulo) oil terminal (Sao Sebastiao zone), Palmas Island, and Itaipu (It; Santos Bay zone). After 3 months of exposure in each season, mussels were recollected and the cytochrome P4501A (CYP1A)- and CYP3A-like activities, glutathione-S-transferase and antioxidants enzymes (catalase, glutathione peroxidase, and glutathione reductase) were analyzed in gills. The concentrations of polycyclic aromatic hydrocarbons, linear alkylbenzenes, and nonessential metals (Cr, Cd, Pb, and Hg) in whole tissue were also analyzed and data were linked to biomarkers' responses by multivariate analysis (principal component analysis-factor analysis). A representation of estimated factor scores was performed to confirm the factor descriptions and to characterize the studied stations. Biomarkers exhibited most significant alterations all year long in mussels transplanted to It, located at Santos Bay zone, where bioaccumulation of organic and inorganic compounds was detected. This integrated approach using transplanted mussels showed satisfactory results, pointing out differences between sites, seasons, and critical areas, which could be related to land-based contaminants' sources. The influence of natural factors and other contaminants (e.g., pharmaceuticals) on biomarkers' responses are also discussed.

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Nile tilapia Oreochromis niloticus at 9 days post-hatch were exposed in semi-static experiments to the carbamate insecticide carbofuran, which is applied in agricultural systems in Brazil. Although the molecular mechanism of carbofuran toxicity is well known, a detailed understanding of the ecological mechanisms through which carbofuran effects can propagate towards higher levels of biological organization in fish is incomplete. Mortality rates were quantified for larvae exposed for 96 h to 8.3, 40.6, 69.9, 140, 297 and 397 µg/L carbofuran, and the LC(50) 96 h was 214.7 µg/L. In addition, the biochemical biomarker cholinesterase inhibition and behavioral biomarkers related to vision, swimming, prey capture and predator avoidance were quantified in individual larvae, as well as their growth in weight. The behavioral parameters were quantified by analysis of digitally recorded videos of individual larvae within appropriate experimental setups. The activity of the enzyme cholinesterase decreased after exposure to carbofuran with a lowest observed effects concentration (LOEC) of 69.9 µg/L. Visual acuity deficits were detected after carbofuran exposure with a LOEC of 40.6 µg/L. Swimming speed decreased with carbofuran exposure, with a LOEC of 397.6 µg/L. The number of attacks to prey (Daphnia magna nauplii) decreased in larvae exposed to carbofuran, with a LOEC of 397.6 µg/L. Growth in weight was significantly reduced in a dose dependent manner, and all carbofuran groups exhibited a statistically significant decrease in growth when compared to controls (p<0.05). The number of predator attacks necessary to capture larvae decreased after exposure to carbofuran, and the LOEC was 69.9 µg/L. These results show that exposure of sensitive early life stages of tilapia O. niloticus to sublethal concentrations of carbofuran can affect fundamental aspects of fish larval ecology that are relevant to recruitment of fish populations, and that can be better understood by the application of behavioral biomarkers.

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The initial sampling in the Marine Monitoring Program (MOMAM), coordinated by the Ministry of Marine Affairs (IEAPM), was performed along the southeast coast of Brazil. Orthopristis ruber samples were collected at Guanabara, Sepetiba and Ilha Grande Bays. Microsomal CYP1A levels and cytosolic cholinesterase (ChE), catalase (CAT) and glutathione S-transferase (GST) activities were measured in the liver of these fish according to established procedures. CAT activity and CYP1A content were significantly higher (P < or = 0.05) in fish caught at Guanabara Bay, which might be due to higher levels of peroxisome proliferators and Ah receptor agonists, respectively, at this site compared to the other sites. Also, lower GST activity was observed in fish from this site, which may possibly be related to the presence of oxidative-stress inducing compounds.

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The aim of this work was to characterize the cholinesterases from gills of Crassostrea rhizophorae in order to use them as biomarkers. Gills were homogenized and then centrifuged (9,000 x g, 4 degrees C, 30 min). S9 and Triton X-100 S9 treated (TX S9) fractions were employed as enzyme source. Km(ap) and Vmax were estimated, using acetylthiocholine iodide as substrate. Inhibition assays were performed with iso-OMPA and eserine. The Km(ap) for S9 and TX S9 fractions were 0.05 and 0.06 mM, whereas the Vmax were 1.92 and 5.84 nmol/min/mg protein. respectively. No inhibition was detected when the samples were incubated with iso-OMPA, suggesting the presence of acetylcholinesterases (AChE) in oyster gill homogenates. Sensitivity to eserine inhibition of AChE in the gills of oysters is intermediate when compared with other aquatic species.

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