Ana G. Cabado

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.

Calcium‐pH crosstalks in rat mast cells: cytosolic alkalinization, but not intracellular calcium release, is a sufficient signal for degranulation

The aim of this work was to study the relationship between intracellular alkalinization, calcium fluxes and histamine release in rat mast cells. Intracellular alkalinization was induced by nigericin, a monovalent cation ionophore, and by NH4Cl (ammonium chloride). Calcium cytosolic and intracellular pH were measured by fluorescence digital imaging using Fura‐2‐AM and BCECF‐AM. In rat mast cells, nigericin and NH4Cl induce a dose‐dependent intracellular alkalinization, a dose‐dependent increase in intracellular calcium levels by releasing calcium from intracellular pools, and an activation of capacitative calcium influx. The increase in both intracellular calcium and pH activates exocytosis (histamine release) in the absence of external calcium. Under the same conditions, thapsigargin does not activate exocytosis, the main difference being that thapsigargin does not alkalinize the cytosol. After alkalinization, histamine release is intracellular‐calcium dependent. With 2.5 mM EGTA and thapsigargin the cell response decreases by 62%. The cytosolic alkalinization, in addition to the calcium increase it is enough signal to elicit the exocytotic process in rat mast cells.

Tetrodotoxin, an Extremely Potent Marine Neurotoxin: Distribution, Toxicity, Origin and Therapeutical Uses.

Tetrodotoxin (TTX) is a potent neurotoxin responsible for many human intoxications and fatalities each year. The origin of TTX is unknown, but in the pufferfish, it seems to be produced by endosymbiotic bacteria that often seem to be passed down the food chain. The ingestion of contaminated pufferfish, considered the most delicious fish in Japan, is the usual route of toxicity. This neurotoxin, reported as a threat to human health in Asian countries, has spread to the Pacific and Mediterranean, due to the increase of temperature waters worldwide. TTX, for which there is no known antidote, inhibits sodium channel producing heart failure in many cases and consequently death. In Japan, a regulatory limit of 2 mg eq TTX/kg was established, although the restaurant preparation of “fugu” is strictly controlled by law and only chefs qualified are allowed to prepare the fish. Due to its paralysis effect, this neurotoxin could be used in the medical field as an analgesic to treat some cancer pains.

In-house validation of a multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes.

A wide variety of qPCR methods currently exist for Salmonella spp., Escherichia coli O157 and Listeria monocytogenes detection. These methods target several genes and use different detection chemistries, either in simplex or in multiplex formats. However, the majority of these methods have not been carefully validated, and the number of validated methods that use multiplex qPCR is even lower. The aim of the present study was to develop and validate a multiplex qPCR method from previously validated simplex qPCR primers and probes. A modified broth medium was selected and primary and secondary enrichment times were further optimized. Efficiency of the newly combined qPCR system was comprised between 91% and 108%, for simplex and multiplex analyses. A total of 152 food and environmental, natural and spiked samples, were analyzed for the evaluation of the method obtaining values above 91% that were reached for all the quality parameters analyzed. A very low limit of detection (5 cfu/25 g after enrichment) for simultaneous identification of these 3 pathogens was obtained.

Migration of BADGE (bisphenol A diglycidyl-ether) and BFDGE (bisphenol F diglycidyl-ether) in canned seafood.

Migration of potentially toxic materials used for the lining of commercial can goods remains an important issue, especially with respect to certain types of processed foods. Seafood is one type where more information is needed with respect to other ingredients used for adding value to fishery products. Most cans are internally coated with starters of resins such as bisphenol A diglycidyl-ether (BADGE) and bisphenol F diglycidyl-ether (BFDGE), both considered as toxic compounds. Several seafood products, sardines, tuna fish, mackerel, mussels, cod and mackerel eggs, were manufactured in different conditions changing covering sauce, time and temperature of storage and heat-treated for sterilization in cans. Migration kinetics of BADGE and BFDGE from varnish into canned products were evaluated by HPLC in 70 samples after 6, 12 or 18 months of storage. Results showed that there is no migration of BADGE in tuna fish, sardines, mussels or cod. However, migration of BFDGE occurs in all species, in a storage time-dependent way and content of fat, although migration of these compounds is not affected by sterilization conditions. All samples analyzed presented values lower than 9 mg BADGE/kg net product without exceeding European limits. However, concerning BFDGE migration, European legislation does not allow the use and/or the presence of BFDGE. Main migration takes place in mackerel reaching the highest values, 0.74 mg BFDGE/kg and 0.34 mg BADGE/kg net product, in red pepper sauce.

Cyclic Imines: Chemistry and Mechanism of Action: A Review

In recent years, there has been an increase in the production of shellfish and in global demand for seafood as nutritious and healthy food. Unfortunately, a significant number of incidences of shellfish poisoning occur worldwide, and microalgae that produce phycotoxins are responsible for most of these. Phycotoxins include several groups of small to medium sized natural products with molecular masses ranging from 300 to over 3000 Da. Cyclic imines (CIs) are a recently discovered group of marine biotoxins characterized by their fast acting toxicity, inducing a characteristic rapid death in the intraperitoneal mouse bioassay. These toxins are macrocyclic compounds with imine (carbon-nitrogen double bond) and spiro-linked ether moieties. They are grouped together due to the imino group functioning as their common pharmacore and due to the similarities in their intraperitoneal toxicity in mice. Spirolides (SPXs) are the largest group of CIs cyclic imines that together with gymnodimines (GYMs) are best characterized. Although the amount of cyclic imines in shellfish is not regulated and these substances have not been categorically linked to human intoxication, they trigger high intraperitoneal toxicity in rodents. In this review, the corresponding chemical structures of each member of the CIs and their derivatives are reviewed as well as all the data accumulated on their mechanism of action at cellular level.

Effects of environmental regimens on the toxin profile of Alexandrium ostenfeldii

Environmental conditions are key factors in the development of marine toxic phytoplankton. Spirolides are marine toxins with a heptacyclic imine ring responsible for the toxicity in mice. Alexandrium ostenfeldii (A. ostenfeldii) is the main producer of these toxins, although this dinoflagellate often produces toxins belonging to the paralytic shellfish poisoning (PSP) group. The present study shows the first evidence that external environmental factors can influence the toxin profile produced by the dinoflagellate A. ostenfeldii. The species investigated is indigenous to the North Atlantic coast, and their cells grew under several environmental parameters. Toxin production was measured by means of liquid chromatography-mass spectrometry (LC-MS) and the chromatograms reflect the presence of two spirolides in all cultures; one in the region m/z 692.5, corresponding to 13-desmethyl spirolide C (13-desMeC) and the other in the region m/z 678.5, which corresponds to 13,19-didesmethyl spirolide C (13,19-didesMeC). The physical parameters studied were salinity, culture media, and photoperiod. The highest amount of toxin per cell was obtained when dinoflagellates grew in F/2 and Walne medium, 28 per thousand salinity, and 24 h of light. However, the highest proportion of 13,19-didesMeC with respect to 13-desMeC was achieved in L1 medium, 33 per thousand salinity, and 14:10 h light:dark. On the contrary, the highest proportion of 13-desMeC in cells was obtained when A. ostenfeldii was cultured in F/2 medium, 28 per thousand salinity, and the same photoperiod. Therefore, from these data the optimum conditions to culture A. ostenfeldii and to obtain the highest amount of spirolide per cell are shown. In addition, these environmental conditions can be considered a tool to predict and avoid A. ostenfeldii blooms.

Decrease of marine toxin content in bivalves by industrial processes.

Harmful algal blooms cause important economical losses due to the accumulation of toxins in shellfish. Natural detoxification occurs but this mechanism is very slow in most cases. The achievement of a method for the rapid detoxification of commercial bivalves would be very interesting for the shellfish harvesting sector in order to diminish economical losses due to harvesting areas closure. In this work, four different methods easily applicable in the food industry (freezing, evisceration, ozonization and thermal processing) were studied to gain the detoxification of four species of bivalves (mussels, scallops, clams and cockles) contaminated with the three main types of toxins (ASP, DSP, PSP). Results show that for ASP a significant decrease of the toxin levels below the legal limit (20 microg/g) is achieved by using hepatopancreas ablation or combination of simple steps (evisceration and/or thermal processing/and or freezing). In our hands, PSP toxin levels are sharply decreased under the limit of detection (35 microg STX eq/100g) after a thermal processing, inducing percentages of detoxification higher than 50%. The effect of freezing on the levels of PSP is very dependent on the matrix studied. DSP toxins are not significantly reduced with none of these methods.

Influence of protein kinase C, cAMP and phosphatase activity on histamine release produced by compound 48/80 and sodium fluoride on rat mast cells

We have studied the effect of protein kinase C and protein kinase A activation, and phosphatase inhibition on two different stimuli with distinct mechanisms of action. The first stimulus is compound 48/80, and its action is mediated probably by a Gi-protein, while the other is sodium fluoride, which unspecifically activates G-proteins. We established a comparative study because the action of compound 48/80 is calcium-independent, while fluoride is strictly calcium-dependent. The activation of protein kinase C was attained with the phorbol esther 12-O-tetradecanoylphorbol-13-acetate, protein kinase A was activated by increasing cAMP levels with forskolin or rolipram, and the phosphatase activity was inhibited with okadaic acid (OA), which inhibits phosphatases type 1 and 2A. Our results show that OA enhances the response to fluoride and compound 48/80 in the absence of calcium, and we conclude that calcium has a negative feedback role on the cell response. Protein kinase A activation strongly inhibits the response to fluoride, and the results show a positive regulation of protein kinase C and a negative regulation of protein kinase A over fluoride response. As previously reported by other authors for the ionophore A23187, TPA notably potentiates the response to fluoride, which supports its possible modulatory role on extracellular calcium-dependent stimuli.

Comparison between a TaqMan Polymerase Chain Reaction Assay and a Culture Method for ctx-Positive Vibrio cholerae Detection

The main objective of the present work was to evaluate a real-time polymerase chain reaction (PCR) method to detect toxigenic Vibrio cholerae in Pangasius hypophthalmus, a freshwater fish cultured mainly in South East Asia. A FDA traditional culture method and a real-time PCR method of the ctx gene were used for detection of V. cholerae in spiked samples of pangasius fish. After an overnight enrichment of samples at 37 degrees C in alkaline peptone water, 2 cfu/25 g of fish was detected with both methods. Although both methods were very sensitive, obtaining results with culture methods may take several days, while real-time PCR takes only a few hours. Furthermore, with traditional methods, complementary techniques such as serotyping, although not available for all serogroups, are needed to identify toxigenic V. cholerae. However, with real-time PCR, toxigenic serogroups are detected in only one step after overnight enrichment.