RESUMO
Acetylcholinesterase (AChE; EC 3.1.1.7) from aquatic organisms have been used to evaluate the exposure of specimens to pesticides and heavy metals at sublethal levels in environmental samples. AChE of Mytella charruana was extracted to characterize its physicochemical and kinetic properties as well as the effect of organophosphate (dichlorvos, diazinon, chlorpyrifos, methyl-parathion and temephos), carbamates (carbaryl, carbofuran and aldicarb), benzoylureas (diflubenzuron and novaluron), pyrethroid (cypermethrin) and juvenile hormone analog - JHA (pyriproxyfen) and the effect of metal ions: Hg2+, Cd2+, Pb2+, As3+, Cu2+ and Zn2+, in order to evaluate the potential of the enzyme as biomarker. The optimum pH of M. charruana AChE was 8.5 and the maximum activity peak occurred at 48 °C, being highly thermostable maintaining 97.8% of its activity after incubation at 60 °C. The Michaelis-Menten constants (km) for the substrates acetylthiocholine and propionylthiocholine were 2.8 ± 1.26 and 4.94 ± 6.9 mmol·L-1, respectively. The Vmax values for the same substrates were 22.6 ± 0.90 and 10.2 ± 4.94 mU·mg-1, respectively. Specific inhibition results suggest an AChE presenting active site with dimensions between those of AChE and butyrylcholinesterase (BChE). The IC20 values related to the effect of the pesticides on the enzyme showed higher inhibitory power of temephos (0.17 µmol·L-1), followed by aldicarb (0.19 µmol·L-1) and diflubenzuron (0.23 µmol·L-1). Metal ions inhibited M. charruana enzyme in the following order: Hg2+ > Pb2+ > Cd2+ > As3+ > Cu2+ > Zn2+. These data suggest that the enzyme showed potential as in vitro biomarker of the exposure to temephos, mercury, zinc and copper.
Assuntos
Acetilcolinesterase/metabolismo , Bivalves/enzimologia , Monitoramento Ambiental , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Acetilcolinesterase/genética , Animais , Biomarcadores/metabolismo , Metais Pesados , PraguicidasRESUMO
An in-situ experiment was performed to study metabolic responses of the freshwater mussel Diplodon chilensis to water contaminated by leachates from an open dump and cattle activity, in order to analyze both the effects of those contaminants on aquatic environments and the potential use of a native bivalve to evaluate the effects of anthropic influence and eutrophication. Bivalves from a reference site were cage-transplanted to a control site (site A) and to a temporal water pond (site B) over 30 and 60 periods. Water quality analyses revealed that the site B was affected by anthropogenic influence. Mussel's hemocytes from site B showed 50% lower reactive oxygen species production and 130% higher lysosomal membrane stability in the site B mussels. In addition, no oxidative stress was evident in gills, despite the elevated copper and iron concentrations recorded in the site B water samples (CuB = 0.3350 ± 0.0636 mg. L-1vs. CuA = 0.0045 ± 0.0007 mg. L-1; FeB = 3.8650 ± 0.4031 mg. L-1vs. FeA = 0.0365 ± 0.0049 mg. L-1). In contrast, the adductor muscle accumulated more Fe (~10-20-fold) than the gills and showed signs of oxidative stress, e.g. superoxide dismutase activity and TBARS levels were increased by 10% were 34%, respectively, in the site B compared with the site A after 60 days of exposure. Additionally, the adductor muscle showed signs of anaerobic metabolism activation. Cu is accumulated in gills from both sites' individuals, at 60 days, in concordance with the increase in the activity of the cu-containing enzyme cytochrome-c-oxidase. There was a reduction in the overall condition and digestive gland index in bivalves exposed at site B, associated with diminished levels of lipid and protein contents. Metal-pollution and eutrophication affects D. chilensis metabolism and is associated to tissue-specific exposure, anaerobic metabolism and general energetic condition depletion.
Assuntos
Bivalves/efeitos dos fármacos , Eutrofização , Metais Pesados/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Bivalves/enzimologia , Bivalves/metabolismo , Bovinos , Cobre/metabolismo , Água Doce , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Metais Pesados/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Poluentes Químicos da Água/metabolismo , Qualidade da ÁguaRESUMO
Cellulolytic enzymes have been studied in several organisms, such as insects, molluscs and other organisms, which can have enzymes endogenously produced or by symbiotic microorganisms. These enzymes are responsible for breaking down the cellulosic material upon which these organisms feed, probably with the aim of assimilating the sugars and nutrients. As Teredinidae bivalves grown in mangrove trees, this study aimed to measure endo-ß-1,4-glucanase activity in different organs and its content. Endo-ß-1,4-glucanase activity was detected in different organs of the Teredinidae bivalves, including gills and digestive organs tissues and its content. Moreover, organisms such as teredinids grow up inside wood and this process could perhaps be related to creating growth space. All the endoglucanase extracts, from organs tissues and contents, showed maximum activity at 40 °C. The maximum activity was observed at pH 5.5 for all the extracts, except for intestine tissue, which maximum was at pH 6. Moreover, some of the extracts showed a different profile of the activity as a pH influence, suggesting different distribution of enzymes over the digestive system of the teredinids. The results suggested that the endo-ß-1,4-glucanase from Teredinidae could be applied in process that requires low temperature, such as, simultaneous saccharification and fermentation, since it presents lower optimum temperature in comparison to enzymes from terrestrial microorganisms.
Assuntos
Bivalves/enzimologia , Celulase/metabolismo , Animais , Bivalves/química , Celulase/química , Estabilidade Enzimática , Trato Gastrointestinal/química , Trato Gastrointestinal/enzimologia , Temperatura Alta , Concentração de Íons de HidrogênioRESUMO
This work describes purification of a protease from the visceral mass of the mussel Mytella charruana as well as evaluation of its ability to hydrolyze milk casein to generate antimicrobial peptides. The enzyme showed pI of 4.1 and a single polypeptide band of 83.1â¯kDa after SDS-PAGE. Sequence similarities with tropomyosin and myosin from mollusks were detected. The protease showed a trypsin-like activity with optimal temperature of 40⯰C and stability in a wide pH range (3.0-9.0). Km was 4.28⯱â¯0.34â¯mM of the synthetic substrate N-benzoyl-dl-arginyl-ρ-nitroanilide, whereas Vmax was 0.056⯱â¯0.001â¯nmolâ¯min-1. The enzyme hydrolyzed casein, and the hydrolysate inhibited the growth of Escherichia coli, Micrococcus luteus, Bacillus subtilis, and Klebsiella pneumoniae at a minimal inhibitory concentration of 5.0⯵gâ¯mL-1. In conclusion, the visceral mass of M. charruana contains a trypsin-like protease that can generate peptides from casein that have a bacteriostatic effect.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Bivalves/enzimologia , Peptídeos/farmacologia , Serina Endopeptidases/metabolismo , Animais , Antibacterianos/química , Antifúngicos/química , Caseínas/química , Caseínas/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos/metabolismo , Serina Endopeptidases/isolamento & purificação , Temperatura , Vísceras/enzimologiaRESUMO
The Ribeira de Iguape River (Southeast Brazil) is metal contaminated by mining activities. Despite it has been cataloged as "in via of restoration" by the literature, this basin is still a sink of pollution in some segments of the fluvial system. This study aimed to assess the sediment quality in the lower part of the RIR basin. The employed approach was based on biological responses of the freshwater clam Corbicula fluminea after 7-day exposure bioassays using as the reference site the Perequê Ecological Park. Toxic responses (burial activity and lethality) and biochemical biomarkers (GST, GR, GPx, LPO, MTs, AChE and DNA damage) were evaluated and then integrated with metal bioavailability and chemical concentrations to address the sediment quality in the area through the weight-of-evidence approach. A multivariate analysis identified linkages between biological responses and contamination. Results pointed that, despite being below the benchmarks of the US Environmental Protection Agency, there is slight metal contamination in the lower part of the basin which induces oxidative stress in C. fluminea; other toxic responses were sometimes attributed to As and Cr bioaccumulation. The sediment quality values (TEL-PEL values in mg/kg) were calculated for the current study for As (0.63-1.31), Cr (3.5-11.05), Cs (1.0-1.17), Cu (6.32-7.32), Ni (6.78-7.46), Ti (42.0-215), V (1.77-8.00). By comparison with other international guidelines, the sediment quality of the lower basin of the Vale de Ribeira does not identify a significant environmental risk.
Assuntos
Sedimentos Geológicos/química , Mineração , Poluentes Químicos da Água/metabolismo , Animais , Biomarcadores/metabolismo , Bivalves/enzimologia , Bivalves/metabolismo , Brasil , Água Doce , Estresse Oxidativo , Poluentes Químicos da Água/toxicidadeRESUMO
The triangle sail mussel, Hyriopsis cumingii, is the most important freshwater pearl mussel in China. However, the mechanisms underlying its chitin-mediated shell and nacre formation remain largely unknown. Here, we characterized a chitin synthase (CS) gene (HcCS1) in H. cumingii, and analyzed its possible physiological function. The complete ORF sequence of HcCS1 contained 6903 bp, encoding a 2300-amino acid protein (theoretical molecular mass = 264 kDa; isoelectric point = 6.22), and no putative signal peptide was predicted. A myosin motor head domain, a CS domain, and 12 transmembrane domains were found. The predicted spatial structures of the myosin head and CS domains were similar to the electron microscopic structure of the heavy meromyosin subfragment of chicken smooth muscle myosin and the crystal structure of bacterial cellulose synthase, respectively. This structural similarity indicates that the functions of these two domains might be conserved. Quantitative reverse transcription PCR results showed that HcCS1 was present in all detected tissues, with the highest expression levels detected in the mantle. The HcCS1 transcripts in the mantle were upregulated following shell damage from 12 to 24 h post-damage, and they peaked (approximately 1.5-fold increase) at 12 h after shell damage. These findings suggest that HcCS1 was involved in shell regeneration, and that it might participate in shell and nacre formation in this species via chitin synthesis. HcCS1 might also dynamically regulate chitin deposition during the process of shell and nacre formation with the help of its conserved myosin head domain.
Assuntos
Exoesqueleto/metabolismo , Bivalves/genética , Quitina Sintase/genética , Quitina/biossíntese , Nácar/metabolismo , Sequência de Aminoácidos , Animais , Bivalves/classificação , Bivalves/enzimologia , Galinhas , Quitina Sintase/química , Quitina Sintase/metabolismo , Água Doce , Expressão Gênica , Glucosiltransferases/química , Glucosiltransferases/genética , Ponto Isoelétrico , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Subfragmentos de Miosina/química , Subfragmentos de Miosina/genética , Fases de Leitura Aberta , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
We used immunocytochemical and fluorometric techniques to show that gill cells of two marine invertebrates, the crab Neohelice granulata (osmoregulator) and the clam Mesodesma mactroides (osmoconformer), increase the expression of membrane transporters [Na(+)/K(+)-ATPase and aquaporin (AQP1)] after whole-animals exposure (96 h) to sublethal concentrations of copper in water of salinity 30 ppt, when both clams and crabs are isosmotic with respect to the environmental medium. A plausible interpretation of our findings is that this increased expression in membrane transporters may serve as an attempt to ameliorate the deleterious effects of copper on the mechanisms involved in ion and volume regulation in gill cells.
Assuntos
Aquaporina 1/biossíntese , Bivalves/efeitos dos fármacos , Braquiúros/efeitos dos fármacos , Cobre/toxicidade , ATPase Trocadora de Sódio-Potássio/biossíntese , Poluentes Químicos da Água/toxicidade , Animais , Bivalves/enzimologia , Bivalves/metabolismo , Braquiúros/enzimologia , Braquiúros/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Monitoramento Ambiental , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Brânquias/metabolismo , Imuno-Histoquímica , Microscopia de Fluorescência , Osmose , SalinidadeRESUMO
The objective of this work was to evaluate the potential use of the bivalve Tivela mactroides, as a Caribbean sentinel species. Organisms were collected from a relatively clean sandy beach, and were exposed to a gradient of petroleum hydrocarbon-polluted sediments for periods of 3, 4 and 6 days, after which their activity levels of biotransformation enzymes were measured. NADPH-cytochrome c reductase, glutathione transferase and cytochrome b5 activities increased according to total hydrocarbon concentration, whereas NADH-cytochrome ferricyanide reductase activity decreased. Catalase activity was constant while superoxide dismutase activity was inhibited. The results indicated that T. mactroides may serve as a sentinel species. However, further studies are recommended on the influence of biotic and abiotic variables on basal activities of enzymes, and their responses to other pollutants.
Assuntos
Bivalves/metabolismo , Exposição Ambiental , Monitoramento Ambiental/métodos , Hidrocarbonetos/toxicidade , Poluição por Petróleo , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores/metabolismo , Bivalves/efeitos dos fármacos , Bivalves/enzimologia , Sedimentos Geológicos/química , Hidrocarbonetos/análise , Poluição por Petróleo/análise , Venezuela , Poluentes Químicos da Água/análiseRESUMO
Free radicals are extremely reactive and produce damage and modify cell functions. Furthermore, superoxide dismutase and catalase are believed to play a key role in the enzymatic defence of the cells. Indeed, some authors have argued that reduced free-radical damage could explain increased longevity. Margaritifera margaritifera is one of the longest-lived animals in the world (up to 100-200 years). Furthermore, this organism may serve as a useful model for gerontologists interested in exploring the mechanisms that promote long life and the slowing of senescence. The present study estimated for the first time individual enzymatic activity for superoxide dismutase isozymes (Cu,Zn-SOD and Mn-SOD) and catalase in tissue preparations of gills, digestive glands and mantles of two natural populations of M. margaritifera. Superoxide dismutase activities showed significant differences in the tissues analysed of specimens from the same river and in specimens from different rivers for the same tissue. Catalase activity levels also showed significant variation, but differences among tissues, within tissues or between rivers were of relatively little interest. We failed to find any relationship between individual enzymatic activities and the age estimated for each mussel. Indeed, the wide variation found in activity levels can be principally interpreted as an adaptation to the unpredictable and changing nature of freshwater natural habitats.