RESUMO
Cation fluxes appear to play a key role in palytoxin-induced signal. There are other cellular targets that have not been described as well as the biochemical signaling cascades that transmit palytoxin-stimulated signals remain to be clarified. Since modifications of cations, mainly calcium, are generally associated to cell death or apoptosis, we wanted to further evaluate the effect of palytoxin on cell death. Then, in vitro cytotoxic effects of palytoxin were characterized on human neuroblastoma cells. By using several techniques, we studied markers of cell death and apoptosis, such as cell detachment, mitochondrial membrane potential, caspases, DNA damage, LDH leakage, propidium iodide uptake, F-actin depolymerization and inhibition of cellular proliferation. Results show that palytoxin triggers a series of toxic responses; it inhibits cell proliferation, induces cell rounding, detachment from the substratum and F-actin disruption. Among the apoptotic markers studied we only detected fall in mitochondrial membrane potential. Neither caspases activation nor chromatin condensation or DNA fragmentation were observed in palytoxin-treated cells.
Assuntos
Acrilamidas/toxicidade , Apoptose/efeitos dos fármacos , Neuroblastoma/metabolismo , Acrilamidas/farmacologia , Actinas/efeitos dos fármacos , Actinas/metabolismo , Caspases/efeitos dos fármacos , Caspases/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Venenos de Cnidários , Fragmentação do DNA/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacosRESUMO
Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate assay: 4-methylumbelliferyl phosphate and fluorescein diphosphate (FDP). In this report, we present the results obtained using a new fluorogenic substrate of PP-2A, the compound dimethylacridinone phosphate (DDAO). A linear relationship between PP-2A concentration and DDAO-induced fluorescence was observed. Okadaic acid (0.0157-9.43 nM)-dependent inhibition of phosphatase activity showed similar results using FDP and DDAO. Recovery percentages obtained with FDP and DDAO in spiked mussel samples (both raw and canned) were very similar and reproducible. Comparative analysis of DSP-contaminated mussel samples by HPLC and FDP/DDAO-PP-2A showed a good correlation among all methods, thus demonstrating that DDAO can be used as a fluorogenic substrate to quantify okadaic acid and related toxins in bivalve molluscs with optimum reliability.
Assuntos
Acridinas/farmacologia , Bivalves , Fluorometria/métodos , Himecromona/análogos & derivados , Toxinas Marinhas/análise , Fosfoproteínas Fosfatases/química , Frutos do Mar , Animais , Bivalves/química , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/farmacologia , Fluoresceína/metabolismo , Himecromona/metabolismo , Indicadores e Reagentes/metabolismo , Toxinas Marinhas/metabolismo , Toxinas Marinhas/farmacologia , Ácido Okadáico/farmacologia , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
The solubility of solids and liquids in supercritical CO2 with added entrainers was modeled with a modified version of the equation of Chrastil to include the effect of entrainers. By considering the formation of the solute-entrainer-solvent complexes an equation is obtained which predicts an exponential increase of solubility with fluid density and/or entrainer concentration. The correlating model was tested by non-linear regression through a computerized iterative process for several systems where an entrainer was present. Four experimental parameters are easily regressed from experimental data, hence the corresponding properties of components such as chemical potentials or critical parameters are not needed. Instead of its simplicity, this thermodynamical model provided a good correlation of the solubility enhancement in the presence of entrainer effect.
Assuntos
Dióxido de Carbono/química , Modelos Químicos , Solubilidade , TermodinâmicaRESUMO
Okadaic acid (OA) and dinophysistoxin-2, two of the main diarrhetic shellfish toxins, can be determined by high-performance liquid chromatography coupled to fluorimetry as pyrenacyl esters. Toxin fluorescent derivatives were obtained after quantitative derivatization with 1-bromoacetylpyrene in acetonitrile. An efficient improvement in the silica gel clean-up procedure of the pyrenacyl derivatives is reported. The clean-up cartridge is washed with hexane-dichloromethane (1:1, v/v), dichloromethane-ethyl acetate (8:2, v/v), and finally the pyrenacyl esters were eluted with dichloromethane-methanol (9:1, v/v). We compare this procedure with other methods already described. Good results were obtained with mussels, scallops and clams. The clean-up procedure showed good robustness when checked against silica and solvents activity. Using samples of mussel hepatopancreas with an OA concentration ranging from 0 to 2 micrograms OA/g hepatopancreas, the inter-assay relative standard deviation ranged from 5.5 to 12.6%.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Corantes Fluorescentes/química , Toxinas Marinhas/análise , Moluscos , Pirenos/química , Frutos do Mar , Animais , Ritmo Circadiano , Diarreia/induzido quimicamente , Doenças Transmitidas por Alimentos/etiologia , Toxinas Marinhas/química , Ácido Okadáico/análise , Ácido Okadáico/química , Piranos/análise , Piranos/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de FluorescênciaRESUMO
A modification of the high-performance liquid chromatographic method with fluorimetric detection method for the determination of diarrhetic shellfish poisoning toxins was developed to completely avoid the use of dangerous chlorinated solvents. The method was validated for the toxin okadaic acid (OA) over a period of 6 months where 12 calibrations were performed and 72 samples were analyzed. Analysis of toxic and non-toxic mussels, clams and scallops demonstrated its selectivity. Linearity was observed in the tested range of interest for monitoring purposes of edible shellfish, from the limit of detection (0.3 microg OA/g hepatopancreas) to 13 microg OA/g hepatopancreas. Intra-assay precision of the method was 7% RSD at the quantification limit (0.97 microg OA/g hepatopancreas at S/N=10). Accuracy was tested in triplicate recovery experiments from OA-spiked shellfish where recovery ranged from 92 to 106% in the concentration range of 0.8 to 3.6 microg OA/g hepatopancreas. Useful information on critical factors affecting calibration and reproducibility is also reported. Good correlation (R=0.87) was observed between the results of the method and those of the method of Lee, after the analysis of 45 samples of mussels from the galician rias.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fluorometria/métodos , Venenos de Moluscos/química , Ácido Okadáico/análise , Calibragem , Compostos Clorados/química , Diarreia/etiologia , Humanos , Piranos/análise , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Frutos do Mar , Solventes/químicaRESUMO
We have studied different biochemical indicators of apoptosis in okadaic acid-treated normal human lung fibroblasts (NHLF). Apoptosis was identified by fluorimetric microplate measurements of DNA content, caspase-3 activation and changes in mitochondrial and plasma membrane after 1-48-h treatments with 1-1000 nM okadaic acid. Cells exposed to okadaic acid showed activation of caspase-3, decreased DNA content (<50% of controls at >100 nM okadaic acid after 12 h of incubation) and translocation of phosphatidylserine to the outer leaflet of the plasma membrane, as indicated by the increase in Merocyanine 540 fluorescence after 4 h of incubation with more than 250 nM okadaic acid. Decreased mitochondrial membrane potential (53-98% of controls) was observed with MitoTracker Red CMXRos in all cases, which indicated an active role of mitochondria during the early phase of apoptosis. However, reactive oxygen species were significantly reduced in okadaic acid-treated fibroblasts (50-70% of controls at 1000 nM after 3 h of incubation), which indicates that ROS cannot be considered as a hallmark of apoptosis in okadaic acid-treated cells. These results provide evidence of apoptotic events induced by okadaic acid in NHLF, which can be detected by means of sensitive and reliable fluorimetric microplate assays.
Assuntos
Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Ácido Okadáico/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Caspase 3 , Caspases/biossíntese , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Fragmentação do DNA , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Precursores Enzimáticos/biossíntese , Fibroblastos/metabolismo , Corantes Fluorescentes , Fluorimunoensaio , Fluorometria , Humanos , Pulmão/citologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Apoptotic changes induced by okadaic acid and yessotoxin in BE(2)-M17 neuroblastoma cells have been evaluated and quantified by combining classical methods and fast and sensitive fluorimetric microplate assays. The phosphatase inhibitor okadaic acid induced rapid time- and dose-dependent apoptotic changes in this cell line, which were evident after 1h at concentrations equal or higher than 500 nM. Decreased mitochondrial membrane potential by okadaic acid (IC(50)=350 nM at 1h) was followed by cell detachment (IC(50)=400 nM at 1h), changes in total nucleic acids content (50% of controls after 1h with 1000 nM okadaic acid), caspase-3 activation (3- to 4-fold increase at 6h) and increased Annexin-V binding (1.5-fold at 6h). Yessotoxin induced similar changes in BE(2)-M17 cells, although significant differences were found in the time-course and degree of apoptotic events induced by this phycotoxin, indicating a lower potency for yessotoxin when compared with okadaic acid. This is the first report on apoptogenic activity of yessotoxin.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Éteres Cíclicos/toxicidade , Venenos de Moluscos/toxicidade , Ácido Okadáico/toxicidade , Oxocinas , Anexina A5/metabolismo , Caspase 3 , Caspases/biossíntese , Adesão Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , DNA de Neoplasias/análise , DNA de Neoplasias/biossíntese , Relação Dose-Resposta a Droga , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Neuroblastoma/patologia , RNA Neoplásico/análise , RNA Neoplásico/biossíntese , Células Tumorais CultivadasRESUMO
This is the first report of a fluorimetric microplate assay to assess glucose uptake and metabolism in eukaryotic cells. The assay was carried out incubating normal human lung fibroblasts in the wells of microtiter trays with a fluorescent D-glucose derivative, 2-N-7-(nitrobenz-2-oxa-1,3-diazol-4-yl)amino-2-deoxy-D-glucose (2-NBDG). This dye could be incorporated by glucose transporting systems in living cells. Substrate uptake was determined by analysing the data obtained with a fluorescence microplate reader. Variables studied in the development of the assay included dye concentration and incubation period. We found that this cell assay is very sensitive, reproducible, provides fast results and graphical display of data. It requires small sample volumes and allows handling of a large number of samples simultaneously. Okadaic acid was used to assess this microplate assay in the field of cytotoxicity. This diarrhetic shellfish toxin is a tumour promoter and a specific inhibitor of protein phosphatases 1 and 2A. The exposition of cells to okadaic acid (0.1 nM-1 microM) at different time intervals causes a decrease in intracellular glucose (40-50% over controls). Results obtained with okadaic acid are the starting point to evaluate application of the method to routine toxicity probes.
Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Desoxiglucose/análogos & derivados , Desoxiglucose/metabolismo , Fibroblastos/metabolismo , Glucose/metabolismo , Pulmão/metabolismo , Microquímica/métodos , Carcinógenos/toxicidade , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/toxicidade , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Corantes Fluorescentes/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Ácido Okadáico/toxicidade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The diarrhogenic activity of the marine toxin okadaic acid (OA) has been associated to its actin-disrupting effect, which could reflect the loosening of tight junctions in vivo. In this report, we present results obtained using a fluorimetric microplate assay for quantitative measurements of OA-induced changes on F-actin pools in BE(2)-M17 cells. The proposed method shows important advantages over classical methods in terms of rapidity, sensitivity (less than 5000 cells per well) and reproducibility, thus providing a very useful tool for studying F-actin levels in living cells. Results obtained demonstrate a time- and dose-dependent decrease of F-actin pools (IC(50)=100 nM at 1 h) in OA-treated cells, which was partly counteracted by TPA, H89, forskolin, wortmannin, ionomycin and orthovanadate at early stages, but remained unaffected after 24 h of incubation. Cells exposed for 1 h to 1 nM OA showed a slight increase of F-actin pools (1.5-fold), which was blocked by genistein and lavendustin A, thus suggesting a role for tyrosine kinases-dependent pathways in OA-induced polymerization at low concentrations. These results suggest direct interactions of Ser/Thr protein phosphatases with actin-binding proteins in the regulation of actin polymerization, thus indicating that disruption of cytoskeletal structure may be a key mechanism of OA-induced diarrhea.
Assuntos
Citofotometria/métodos , Citoesqueleto/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neurônios/efeitos dos fármacos , Ácido Okadáico/farmacologia , Sulfonamidas , Actinas/efeitos dos fármacos , Actinas/metabolismo , Androstadienos/farmacologia , Colforsina/farmacologia , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Genisteína/farmacologia , Humanos , Ionomicina/farmacologia , Isoquinolinas/farmacologia , Neuroblastoma , Neurônios/metabolismo , Fenóis/farmacologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Vanadatos/farmacologia , WortmaninaRESUMO
Changes in toxin profile and total toxicity levels of paralytic shellfish poison (PSP)-containing mussels were monitored during the standard canning process of pickled mussels and mussels in brine using mouse bioassays and high-performance liquid chromatography. Detoxification percentages for canned mussel meat exceeded 50% of initial toxicity. Total toxicity reduction did not fully correspond to toxin destruction, which was due to the loss of PSP to cooking water and packing media of the canned product. Significant differences in detoxification percentages were due to changes in toxin profile during heat treatment in packing media. Toxin conversion phenomena should be determined to validate detoxification procedures in the canning industry.
Assuntos
Bivalves/parasitologia , Dinoflagellida/metabolismo , Conservação de Alimentos/métodos , Toxinas Marinhas/análise , Animais , Cromatografia Líquida de Alta Pressão , Temperatura Alta , Toxinas Marinhas/toxicidade , Camundongos , ParalisiaRESUMO
Commission Regulation (EC) N° 2074/2005 recognises the biological method as the reference method for Paralytic Shellfish Poisoning (PSP) toxins detection in molluscs. It was amended by Commission Regulation (EC) N° 1664/2006 that accepted the so-called Lawrence method as an alternative to the reference method. The goal of this study was to compare AOAC Official Methods of Analysis 959.08 (Biological method) and 2005.06 (Prechromatographic Oxidation and Liquid Chromatography with fluorescence detection) in samples with different toxin profiles. The influence of extraction solvent in the total samples toxicity was also evaluated. A total of 40 samples including mussels, clams, scallops, razor-clams, cockles, oysters and barnacles were analysed by both official methods. Samples were selected with Alexandrium and Gymnodinium toxic profiles, from different origin and including several presentations: fresh, frozen, canned and boiled. Acetic and hydrochloric acid extractions were performed in all samples and the extracts were simultaneously analysed by both methods. Most samples were naturally contaminated and two samples were spiked. Comparison of both official methods, mouse bioassay (MBA) with HCl extraction and Liquid Chromatography with fluorescence detection (HPLC-FLD) with acetic acid extraction, led to an 85% of consistent results regarding compliance with legal limit, including samples below and above it. The linear correlation coefficient was r² = 0.69 and the paired t test (two tails, α = 0.05) indicated that there were not significant differences among both sets of data. Nevertheless, toxicity differences were found in several samples. In 15 out of 18 shellfish with a Gymnodinium toxic profile, higher toxicity levels were obtained by MBA. This fact was more evident in 7 samples, partially related to the lack of standards and the impossibility of analysing dc-NEO, C1, 2 and GTX6 at the beginning of the study. However, other factors concerning the extraction and SPE clean-ups steps may also contribute. By contrast, 9 samples presented a much higher total toxicity by HPLC-FLD than by MBA. These higher results obtained by HPLC-FLD could not only be due to the use of the highest toxicity equivalency factor (TEF) for isomers oxidated into products that coelute when total toxicity of these samples were calculated. Further analyses of results obtained by HPLC-FLD and by MBA with both extracts were done separately.
Assuntos
Bioensaio/métodos , Cromatografia Líquida de Alta Pressão/métodos , Saxitoxina/análise , Saxitoxina/toxicidade , Frutos do Mar/análise , Frutos do Mar/normas , Animais , União Europeia , Camundongos , Estrutura Molecular , Saxitoxina/química , Especificidade da EspécieRESUMO
This review is an up-to-date compilation of the available literature, including scientific papers, reviews, and EFSA's opinions, on toxicity and risk assessment data on the main marine biotoxins of importance in the European Union, including the legislated ones and the ones of recent appearance which are not legislated. Information about the hazard identification and hazard characterisation of okadaic acid, dynophysistoxins, pectenotoxins, yessotoxins, azaspiracids, domoic acid, saxitoxins, tetrodotoxins, brevetoxins, ciguatoxins, cyclic imines and palytoxins is reviewed and presented in the form of a collection of risk assessments. It is concluded that the importance of having an appropriate exposure assessment reiterates the urgency of establishing a database with representative and comparable data on exposure to food items possiby containing marine biotoxins. It is also concluded that a revision of the present regulation of marine biotoxins in the EU legislation could be considered, as some regulated toxins have been shown not to pose a risk for EU's population (as yessotoxin) and some non regulated toxins have been shown to be harmful and/or to occur in the EU (as tetrodotoxin, palytoxin, and some cyclic imines) while they are not regulated.
Assuntos
Contaminação de Alimentos/análise , Toxinas Marinhas/toxicidade , Animais , Organismos Aquáticos/efeitos dos fármacos , Organismos Aquáticos/metabolismo , Exposição Ambiental , União Europeia , Análise de Alimentos/legislação & jurisprudência , Contaminação de Alimentos/legislação & jurisprudência , Contaminação de Alimentos/prevenção & controle , Humanos , Toxinas Marinhas/análise , Medição de RiscoRESUMO
Several recombinant antibodies against one of the most potent marine toxins, Palytoxin (PlTX), were obtained using two naive human semi-synthetic phage display libraries (Tomlinson I and J) as an effective method for generating specific anti-toxin single-chain variable fragment (scFv) antibodies. After four rounds of panning and selection on free palytoxin adsorbed immunotubes, individual clones were isolated, sequenced and characterized by Enzyme-Linked Immunosorbent Assay (ELISA). Four phage-antibody clones specifically recognized the toxin. A competitive ELISA assay was optimized with one of these phage antibodies giving a very reproducible standard curve with a linear regression (R(2)=0.9945), showing a working range of 0.0005-500ngmL(-1). Several spiked shellfish samples were analysed by competitive ELISA to determine the accuracy of the assay, with a mean recovery rate of 90%. This study demonstrates that phage display libraries provide a valuable system for the easy and rapid generation of specific antibody fragments directed against difficult antigenic targets, such as free small molecules. Large-scale, low-cost production of anti-palytoxin scFv antibodies in Escherichia coli (E. coli) is an exciting prospect for the development of rapid and simple detection methods. Our results suggest that anti-palytoxin phage antibodies could be a valuable tool with competitive ELISA to detect palytoxin in natural shellfish samples.
Assuntos
Acrilamidas/imunologia , Anticorpos Monoclonais/isolamento & purificação , Venenos de Cnidários/imunologia , Toxinas Marinhas/imunologia , Acrilamidas/análise , Animais , Anticorpos Monoclonais/imunologia , Bivalves/química , Clonagem Molecular , Técnicas de Química Combinatória , Ensaio de Imunoadsorção Enzimática , Contaminação de Alimentos/análise , Humanos , Toxinas Marinhas/análise , Biblioteca de Peptídeos , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Frutos do Mar/análiseRESUMO
Palytoxin isolated from the genus Palythoa is the most potent marine toxin known. The aim of the present study was to quantify palytoxin-induced cellular injury in the human intestinal cell line Caco-2. Cellular damage was measured by evaluating cell proliferation, cell membrane permeability, cell morphology and apoptotic markers. Furthermore, changes in F-actin were studied after exposure of cells to increasing amounts of palytoxin. The results show that cell proliferation decreased in a concentration-dependent manner with a mean IC(50) value of about 0.1 nM. A noticeable increase of cell detachment correlated with cell rounding and F-actin depolymerization was observed in palytoxin-treated cells. Moreover LDH was released from the cells in a dose and time dependent manner, although under these conditions there was no propidium iodide uptake. On the other hand, palytoxin impaired mitochondrial activity but other apoptotic markers, such as DNA fragmentation or caspases activation, were not observed. The results obtained in this paper suggest that the effects of palytoxin in Caco-2 cells were very potent and unspecific, since a primary necrosis and a secondary apoptosis seem to occur under these conditions.
Assuntos
Acrilamidas/toxicidade , Venenos de Cnidários , Enterócitos/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Acrilamidas/metabolismo , Actinas/metabolismo , Apoptose/efeitos dos fármacos , Células CACO-2 , Caspases/metabolismo , Adesão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA , Relação Dose-Resposta a Droga , Enterócitos/metabolismo , Enterócitos/patologia , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , L-Lactato Desidrogenase/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Necrose/induzido quimicamenteRESUMO
The search for alternative methods to the mouse bioassay (MBA) has intensified over recent years. The present work analyzes seven different species of shellfish (clams, small scallops, small clams, mussels, oysters, cockles, and edible whelks) in fresh, frozen boiled, and canned presentations using liquid chromatography-mass spectrometry (LC-MS/MS), and the results are compared with the same samples analyzed through MBA. The toxins studied were OA, DTX1, DTX2, YTX, PTX2, and AZA1, which are legislated in the EU, and SPX1, which is not regulated yet. Consistent results between LC-MS/MS and MBA were found in 69% of the samples, whereas 26% of MBA showed "false-positive" results with respect to the toxins analyzed. No "false negatives" were observed. The possibility of LC-MS/MS as an alternative or complementary technique to MBA is discussed.
Assuntos
Cromatografia Líquida , Toxinas Marinhas/análise , Espectrometria de Massas , Moluscos/química , Frutos do Mar/análise , Animais , Bioensaio , Manipulação de Alimentos , Lipídeos/química , Toxinas Marinhas/química , Camundongos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.
Assuntos
Bicarbonatos/farmacologia , Cálcio/metabolismo , Exocitose , Mastócitos/metabolismo , Animais , Bicarbonatos/metabolismo , Calcimicina/farmacologia , Cálcio/antagonistas & inibidores , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Liberação de Histamina/fisiologia , Ionóforos/farmacologia , Cinética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Tapsigargina/farmacologia , Fatores de Tempo , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Okadaic acid (OA) is a tumour promoter that induces apoptosis in several cell models. Following previous findings, the objective of this work was to elucidate the pathways involved in OA-triggered apoptosis in BE(2)-M17 cells by using a combination of pharmacological agents and apoptosis-related assays. OA-induced apoptosis involves disruption of F-actin cytoskeleton, activation of caspase-3, collapse of mitochondrial membrane potential, DNA fragmentation and decreased levels of monomeric Bcl-2 and Bax proteins. All the agents tested were unable to obliterate changes in F-actin levels, caspase-3 activation or DNA fragmentation, but all of them prevented OA-induced decrease of mitochondrial potential and changes in Bax/Bcl-2 levels. Taken together, these results demonstrate that collapse of mitochondrial membrane potential is accessory in the execution of apoptosis, which is directly dependent on cytoskeletal changes. Mitochondrial changes are mediated by complex associations among the Bcl-2 proteins. Cytochrome c release from mitochondria is a late event, occurring 24 h after OA exposure. Moreover, okadaic acid triggers activation of upstream caspases resembling the extrinsic pathway of apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Neuroblastoma/tratamento farmacológico , Ácido Okadáico/toxicidade , Actinas/efeitos dos fármacos , Actinas/metabolismo , Caspase 3 , Caspases/biossíntese , Linhagem Celular Tumoral , Citocromos c/metabolismo , Citoesqueleto/metabolismo , Fragmentação do DNA , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Neuroblastoma/patologia , Preparações Farmacêuticas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2RESUMO
The LUC gene coding for Photinus pyralis firefly luciferase was cloned in different yeast episomal plasmids in order to assess its possibilities as an in vivo reporter gene. Activity of the enzyme in transformed cells in vivo was measured by following light emission and assay conditions optimized in intact cells, with regard to oxygen concentration, temperature, cell concentration in assay mixtures and external ATP concentration. Among the factors tested, light emission was drastically influenced by the external pH in the assay (which resulted in a ten-fold amplification signal) and by substrate permeability. The growth phase of the cells was also important for the level of activity detected. Cloning of firefly luciferase gene under the control of different yeast-regulated promoters (ADH1, GAL1-10) enabled us to measure their strength which correlated well with previously described data. We conclude that firefly luciferase is an adequate gene reporter for the in vivo sensitive determination of gene expression and promoter strength in yeast.
Assuntos
Besouros/enzimologia , Expressão Gênica , Luciferases/genética , Saccharomyces cerevisiae/genética , Trifosfato de Adenosina/farmacologia , Animais , Detergentes/farmacologia , Concentração de Íons de Hidrogênio , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
The progressive increase of DSP toxic episodes in shellfish in the last few years has generated a series of criticisms centered on the suitability of the mouse bioassay as a reference method to regulate the harvest of mussels from the growth area. The observation time in injected mice is currently fixed in 12 h by the actual Spanish rules. The revision of this time period and the lack of a established DSP toxin threshold which permits the commercialization of mussels contaminated under certain levels, are some of the actual demands from the industry. In this study, the results obtained in a comparative study of DSP toxic mussels are shown using the HPLC method and the mouse bioassay. Based on these results, we consider feasible the reduction to 5 h of the observation times of the mouse bioassay currently established in the actual legislation, as well as the establishment of a DSP-toxin threshold of 2 micrograms okadaic acid/g hepatopancreas, which regulates the possibility of harvesting and commercialization of contaminated mussels.
Assuntos
Bioensaio/métodos , Cromatografia Líquida de Alta Pressão/métodos , Éteres Cíclicos/análise , Toxinas Marinhas/análise , Animais , Bivalves/química , Fígado/química , Toxinas Marinhas/toxicidade , Camundongos , Ácido Okadáico , Pâncreas/química , Taxa de SobrevidaRESUMO
The role of external HCO3- in pHi regulation in rat mast cells was studied with BCECF. In a HCO3--free medium cells undergo an alkalinization after the addition of 40 mM HCO3Na. This alkalinization is unaffected by pH. In a Na+-free medium, the addition of 20 mM HCO3Na induced a higher alkalinization than 20 mM HCO3K. Amiloride (1 mM), a Na+/H+ exchanger inhibitor, inhibited by 45% the alkalinization induced by HCO3Na, but it did not change that induced by HCO3K. The anion exchanger inhibitor DIDS reduced 20% the alkalinization induced by both salts. An alkalinization of 0.085 units is observed after the addition of 20 mM HCO3K, even when these exchangers are inhibited (in the absence of Na+ and presence of DIDS). We conclude that the Na+/H+ exchanger and the Cl-/HCO3- exchangers are alkalinizing mechanisms that regulate pHi in these cells. Also, there is some HCO3--dependent, Na+-independent mechanism responsible for the intracellular alkalinization.