F. Leira

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.

Azaspiracid-1, a potent, nonapoptotic new phycotoxin with several cell targets

This paper reports on potential cellular targets of azaspiracid-1 (AZ-1), a new phycotoxin that causes diarrhoeic and neurotoxic symptoms and whose mechanism of action is unknown. In excitable neuroblastoma cells, the systems studied were membrane potential, F-actin levels and mitochondrial membrane potential. AZ-1 does not modify mitochondrial activity but decreases F-actin concentration. These results indicate that the toxin does not have an apoptotic effect but uses actin for some of its effects. Therefore, cytoskeleton seems to be an important cellular target for AZ-1 effect. AZ-1 does not induce any modification in membrane potential, which does not support for neurotoxic effects. In human lymphocytes, cAMP, cytosolic calcium and cytosolic pH (pHi) levels were also studied. AZ-1 increases cytosolic calcium and cAMP levels and does not affect pHi (alkalinization). Cytosolic calcium increase seems to be dependent on both the release of calcium from intracellular Ca(2+) pools and the influx from extracellular media through Ni(2+)-blockable channels. AZ-1-induced Ca(2+) increase is negatively modulated by protein kinase C (PKC) activation, protein phosphatases 1 and 2A (PP1 and PP2A) inhibition and cAMP increasing agents. The effect of AZ-1 in cAMP is not extracellularly Ca(2+) dependent and insensitive to okadaic acid (OA).

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.

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.

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.

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.

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.

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.

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

Abstract 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 μg okadaic acid/g hepatopancreas, which regulates the possibility of harvesting and commercialization of contaminated mussels.

Canning process that diminishes paralytic shellfish poison in naturally contaminated mussels (Mytilus galloprovincialis)

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.