Biomedical subjects

F Leira

Publications and source records attributed to F Leira.

11 recordsLinked to original sources

PubMed2003

Cytoskeletal disruption is the key factor that triggers apoptosis in okadaic acid-treated neuroblastoma cells.

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.

Actins↗

PubMed2003

Development of a F actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins.

A new cytotoxicity assay for detection and quantitation of diarrhetic shellfish toxins (DSP) is presented. This assay is based upon fluorimetric determination of F-actin depolymerization induced by okadaic acid (OA)-class compounds in the BE(2)-M17 neuroblastoma cell line. No interferences were observed with other marine toxins such as saxitoxin, domoic acid, or yessotoxin, thus indicating a good specificity of the assay as expected by the direct relationship between protein phosphatase inhibition and cytoskeletal changes. The proposed method is rapid (<2h) and shows a linear response in the range of 50-300 nM OA. The detection limit of the assay for crude methanolic extracts of bivalves lies between 0.2 and 1.0 microg OA per gram of digestive glands, depending on the type of samples (fresh or canned), thus being similar to that of the mouse bioassay. The performance of this assay has been evaluated by comparative analysis of 32 toxic mussel samples by the F-actin assay, mouse bioassay, HPLC and PP2A inhibition assay. Results obtained by the F-actin method showed no differences with HPLC and significant correlation with PP2A inhibition assay (r(2)=0.71). No false negative results were obtained with this new cell assay, which also showed optimum reproducibility.

Actins↗

PubMed2002

Characterization of distinct apoptotic changes induced by okadaic acid and yessotoxin in the BE(2)-M17 neuroblastoma cell line.

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.

Annexin A5↗

PubMed2002

Fluorescent microplate cell assay to measure uptake and metabolism of glucose in normal human lung fibroblasts.

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.

4-Chloro-7-nitrobenzofurazan↗

PubMed2001

A fluorimetric method based on changes in membrane potential for screening paralytic shellfish toxins in mussels.

To prevent the consumption of bivalves contaminated with paralytic shellfish poisoning (PSP), toxin levels in seafood products are estimated by using the official mouse bioassay. Because of the limitations of this bioassay other methods of monitoring toxins are clearly needed. We have developed a test to screen for PSP toxins based on its functional activity; the toxins bind to the voltage-gated Na+ channels and block their activity. The method is a fluorimetric assay that allows quantitation of the toxins by detecting changes in the membrane potential of human excitable cells. This assay gives an estimate of toxicity, since each toxin present in the sample binds to sodium channels with an affinity which is proportional to its intrinsic toxic potency. The detection limits for paralytic shellfish toxins were found to be 1 ng saxitoxin equivalents/ml compared to the regulatory limit threshold of 400 ng/ml (equivalent to 80 microg/100 g) used in most countries. Our results indicate that this fluorescent assay is a specific, very sensitive, rapid, and reliable method of monitoring PSP toxin levels in samples from seafood products and toxic algae.

Animals↗

PubMed2001

Apoptotic events induced by the phosphatase inhibitor okadaic acid in normal human lung fibroblasts.

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.

Apoptosis↗

PubMed2000

Development and validation of a high-performance liquid chromatographic method using fluorimetric detection for the determination of the diarrhetic shellfish poisoning toxin okadaic acid without chlorinated solvents.

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.

Calibration↗

PubMed2000

Characterization of 9H-(1,3-dichlor-9, 9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP).

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.

Acridines↗

PubMed1997

A fluorescent microplate assay for diarrheic shellfish toxins.

A fluorescent enzyme inhibition assay for okadaic acid using 4-methylumbelliferyl phosphate and fluorescein diphosphate as substrates for the enzyme phosphatase 2A was developed. In the inhibition assay, performed in a microtiter plate, the PP2A was inhibited by adding okadaic acid and the resulting fluorescence enhancement derived from enzymatic hydrolysis of the substrate was quantified in a fluorescence plate reader. The measurable range of okadaic acid was 3.2 to 3200 pg/ml with an IC50 = 0.1 nM. The detection limit of okadaic acid was 2.56 pg/well in buffer solutions and 12.8 ng/g hepatopancreas in shellfish extracts. The coefficient of variation (CV, n = 22) for each point ranged from 18.80 to 37.90% (mean 28.35%). The proposed method is very convenient, rapid, and sensitive by using the enzyme inhibition assay system and fluorescent reaction as a detection system. This work demonstrates that the fluorescent assay can be used to quantify the amount of okadaic acid in shellfish samples and also is valid for very dilute samples, such as phytoplankton samples.

Animals↗

PubMed1996

Determination of DSP toxins: comparative study of HPLC and bioassay to reduce the observation time of the mouse bioassay.

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.

Animals↗

PubMedDate not supplied

Study of cytoskeletal changes induced by okadaic acid in BE(2)-M17 cells by means of a quantitative fluorimetric microplate assay.

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.

Actins↗