RESUMO
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⯵M) and fluorene (0.6 and 1.2⯵M), after exposure for 24 and 96â¯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⯵M (24â¯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.
Assuntos
Crassostrea/citologia , Crassostrea/genética , Fluorenos/toxicidade , Pirenos/toxicidade , Animais , Biotransformação/efeitos dos fármacos , Crassostrea/efeitos dos fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Sistema Digestório/efeitos dos fármacos , Fluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Poluentes Químicos da Água/toxicidadeRESUMO
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.
Assuntos
Crassostrea/efeitos dos fármacos , Crassostrea/metabolismo , Citotoxinas/toxicidade , Ecotoxicologia , Ibuprofeno/toxicidade , Transcrição Gênica/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Antioxidantes/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Crassostrea/citologia , Crassostrea/genética , Relação Dose-Resposta a Droga , Brânquias/citologia , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Hemócitos/citologia , Hemócitos/efeitos dos fármacosRESUMO
Pesticides and heavy metals were analyzed in sentinel Crassostrea gigas oysters placed in six aquaculture sites close to a contaminated agricultural region. Each site was sampled twice. Tests revealed the presence of organochlorine (OC) pesticides in the oysters at concentrations varying from 31.8 to 72.5 µg/kg for gamma-hexachlorocyclohexane (γ-HCH); from 1.2 to 3.1 µg/kg for dichlorodiphenyldichloroethylene (4,4-DDE); from 1.6 to 2.3 µg/kg for endosulfan I; and from 1.4 to 41.2 µg/kg for endosulfan II, as well as heavy metals in concentrations that exceeded Mexican tolerance levels (405.5 to 987.8 µg/g for zinc; 4.2 to 7.3 µg/g for cadmium; and 7.2 to 9.9 µg/g for lead). Significant levels of DNA damage in oyster hemocytes were also detected. There was a significant, positive correlation between genotoxic damage and concentration of nickel or the presence of endosulfan II. Cellular viability evaluated by cytotoxic analyses was found to be high at 80%. Marked inhibition in activity of acetylcholinesterase (AChE ) and induction of glutathione S-transferase (GST) activity was noted. Data demonstrated a significant relation between AChE activity inhibition and presence of endosulfan II, γ-HCH, copper, lead, and 4,4-DDE, as well as between AChE and GST activity at different sites.
Assuntos
Crassostrea/química , Dano ao DNA , Metais Pesados/análise , Mutagênicos/análise , Resíduos de Praguicidas/análise , Praguicidas/análise , Poluentes Químicos da Água/análise , Animais , Aquicultura , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inibidores da Colinesterase/análise , Inibidores da Colinesterase/farmacologia , Ensaio Cometa , Crassostrea/citologia , Crassostrea/efeitos dos fármacos , Crassostrea/crescimento & desenvolvimento , Indução Enzimática/efeitos dos fármacos , Contaminação de Alimentos , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hemócitos/citologia , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Metais Pesados/farmacologia , Mutagênicos/farmacologia , Resíduos de Praguicidas/farmacologia , Praguicidas/farmacologia , Vigilância de Evento Sentinela , Frutos do Mar/análise , Frutos do Mar/normas , Poluentes Químicos da Água/farmacologia , Abastecimento de Água/análiseRESUMO
In response to the need for more sensitive and rapid indicators of environmental quality, sublethal effects on the lowest levels of biological organization have been investigated. The ecological relevance of these responses assumes a prevailing role to assure effectiveness as indicator of ecological status. This study aimed to investigate the linkages between biomarker responses of caged bivalves and descriptive parameters of macrobenthic community structure. For this purpose a multi-level environmental assessment of marine and estuarine zones was performed in São Paulo coast, Brazil. Multivariate analysis was applied to identify linkages between biological responses and ecological indices, as well as to characterizing the studied stations. Individuals of the marine mussel Perna perna caged along Santos Bay showed signs of oxidative stress, lysosomal membrane destabilization, histological alterations and reduced embryonic development. The estuarine oyster Crassostrea rhizophorae caged along Santos Port Channel showed alterations on biotransformation enzymes and antioxidant system, DNA damage and lysosomal membrane destabilization. The benthic community analysis showed reduced richness and diversity in the same areas of the Santos bay and estuary where biomarker responses were altered. Our results revealed that xenobiotics are inducing physiological stress, which may lead to changes of the benthic community structure and deterioration of the ecological status over time. Integrating biomarker responses and ecological indexes improved certainty that alterations found at community level could be related to xenobiotic as stressors, which was very useful to improve the discriminatory power of the environmental assessment.
Assuntos
Biodiversidade , Crassostrea/efeitos dos fármacos , Perna (Organismo)/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Baías , Biomarcadores/metabolismo , Brasil , Crassostrea/citologia , Crassostrea/fisiologia , Monitoramento Ambiental , Estuários , Perna (Organismo)/citologia , Perna (Organismo)/fisiologiaRESUMO
Most experimental procedures on molluscs are done after acclimatization of wild animals to lab conditions. Similarly, short-term acclimation is often unavoidable in a field survey when biological analysis cannot be done within the day of sample collection. However, acclimatization can affect the general physiological condition and particularly the immune cell responses of molluscs. Our aim was to study the changes in the hemocyte characteristics of the Pacific oyster Crassostrea gigas and the carpet shell clam Ruditapes decussatus acclimated 1 or 2 days under emersed conditions at 14 ± 1 °C and for 1, 2, 7, or 10 days to flowing seawater conditions (submerged) at 9 ± 1 °C, when compared to hemolymph withdrawn from organisms sampled in the field and immediately analyzed in the laboratory (unacclimated). The hemocyte characteristics assessed by flow cytometry were the total (THC) and differential hemocyte count, percentage of dead cells, phagocytosis, and reactive oxygen species (ROS) production. Dead hemocytes were lower in oysters acclimated both in emersed and submerged conditions (1%-5%) compared to those sampled in the field (7%). Compared to oysters, the percentage of dead hemocytes was lower in clams (0.4% vs. 1.1%) and showed a tendency to decrease during acclimatization in both emersed and submerged conditions. In comparison to organisms not acclimated, the phagocytosis of hemocytes decreased in both oysters and clams acclimated under submerged conditions, but was similar in those acclimated in emersed conditions. The ROS production remained stable in both oysters and clams acclimated in emersed conditions, whereas in submerged conditions ROS production did not change in both the hyalinocytes and granulocytes of oysters, but increased in clams. In oysters, the THC decreased when they were acclimated 1 and 2 days in submerged conditions and was mainly caused by a decrease in granulocytes, but the decrease in THC in oysters acclimated 2 days in emersed conditions was caused by a decrease in hyalinocytes and small agranular cells. In clams, the THC was significantly lower in comparison to those not acclimated, regardless of the conditions of the acclimatization. These findings demonstrate that hemocyte characteristics were differentially affected in both species by the tested conditions of acclimatization. The phagocytosis and ROS production in clams and phagocytosis in oysters were not different in those acclimated for 1 day under both conditions, i.e. emersed and submerged, and those sampled in the field (unacclimated). The THC was significantly affected by acclimatization conditions, so the differences between clams and oysters should be considered in studies where important concentrations of hemocytes are required. The difference in the immune response between both species could be related to their habitat (epifaunal vs. infaunal) and their ability of resilience to manipulation and adaptation to captivity. Our results suggest that functional characteristics of hemocytes should be analyzed in both oysters and clams during the first 1 or 2 days, preferably acclimated under emersed rather than submerged conditions.
Assuntos
Aclimatação/fisiologia , Bivalves/citologia , Crassostrea/citologia , Hemócitos/fisiologia , Animais , Bivalves/fisiologia , Contagem de Células Sanguíneas/veterinária , Crassostrea/fisiologia , Citometria de Fluxo , Hemócitos/química , Fagocitose/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Especificidade da EspécieRESUMO
Protozoan parasites of the genus Perkinsus are considered important pathogens responsible for mass mortalities in several mollusk species worldwide. In the present study we describe for the first time a parasite of the genus Perkinsus infecting the mangrove oyster Crassostrea rhizophorae from the Brazilian coast. Prevalence of this parasite was low in the Pacoti River estuary (Ceará, northeast Brazil) and absent in oysters from southern Brazil. Oyster gill and rectum tissues incubated in Ray's fluid thioglycollate medium (RFTM) revealed the presence of spherical hypnospores (5 to 55 microm diam.). Histological analysis showed the occurrence of typical signet-ring trophozoites and schizonts (3 to 6 microm diam.) infecting connective tissues of several organs and digestive epithelia. PCR assays specific to the genus Perkinsus, followed by cloning and sequencing of the internal transcribed spacer (ITS) region of the ribosomal ribonucleic acid (rRNA) gene complex, confirmed a close phylogenetic relationship between Brazilian Perkinsus sp. and P. beihaiensis infecting Chinese oysters.
Assuntos
Alveolados/isolamento & purificação , Crassostrea/microbiologia , Infecções Protozoárias em Animais/epidemiologia , Alveolados/genética , Animais , Oceano Atlântico/epidemiologia , Brasil/epidemiologia , Crassostrea/citologia , Filogenia , Infecções Protozoárias em Animais/parasitologiaRESUMO
Evaluation of sperm motility is the single most widely used parameter to determine semen quality in mammals and aquatic species. While a good indicator for fresh sperm viability, post-thaw motility is not always effective at predicting fertilizing ability. Techniques using fluorescent dyes can assess functionality of mammalian sperm, but have not been widely applied in aquatic organisms. The eastern oyster Crassostrea virginica is an important mollusk in the United States, and cryopreservation protocols have been developed to preserve sperm and larvae to assist research and hatchery production. In this study, protocols were developed to assess sperm cell membrane integrity and mitochondrial function by flow cytometry and to assess viability of eggs by fluorescence microscopy. The fluorescent dyes SYBR 14 and propidium iodide (PI) (to assess membrane integrity) and rhodamine 123 (R123) (to assess mitochondrial membrane potential) were used to evaluate the quality of thawed oyster sperm previously cryopreserved with different cryoprotectant and thawing treatments. Membrane integrity results were correlated with motility of thawed sperm and mitochondrial membrane potential with fertilizing ability. Fluorescein diacetate (FDA) was used to assess cytotoxicity of cryoprotectant solutions and post-thaw damage to oyster eggs. The results indicated that membrane integrity (P=0.004) and thawing treatments (P=0.04), and mitochondrial membrane potential (P=0.0015) were correlated with motility. Fertilizing ability was correlated with cryoprotectant treatments (P=0.0258) and with mitochondrial membrane potential (P=0.001). The dye FDA was useful in indicating structural integrity of fresh and thawed eggs. Exposure of eggs, without freezing, to dimethyl sulfoxide yielded higher percentages of stained eggs and fertilization rate than did exposure to propylene glycol (P=0.002). Thawed eggs were not stained with FDA (<1%) and larvae were not produced.