okadaic-acid and dinophysistoxin-1

Diarrhetic Shellfish Poisoning (DSP) is a specific type of food poisoning, characterized by severe gastrointestinal illness due to the ingestion of filter feeding bivalves contaminated with a specific suite of toxins. It is known that the problem is worldwide and three chemically different groups of toxins have been historically associated with DSP syndrome: okadaic acid (OA) and dinophysistoxins (DTXs), pectenotoxins (PTXs) and yessotoxins (YTXs). PTXs and YTXs have been considered as DSP toxins because they can be detected with the bioassays used for the toxins of the okadaic acid group, but diarrhegenic effects have only been proven for OA and DTXs. Whereas, some PTXs causes liver necrosis and YTXs damages cardiac muscle after intraperitoneal injection into mice. On the other hand, azaspiracids (AZAs) have never been included in the DSP group, but they cause diarrhoea in humans. This review summarizes the origin, characterization, structure, activity, mechanism of action, clinical symptoms, method for analysis, potential risk, regulation and perspectives of DSP and associated toxins produced by marine dinoflagellates.

Topics: Animals; Dinoflagellida; Humans; Liver; Macrolides; Mice; Molecular Structure; Mollusk Venoms; Myocardium; Necrosis; Okadaic Acid; Oxocins; Pyrans; Rats; Shellfish; Shellfish Poisoning; Toxicity Tests

Poisoning events concerning diarrhetic shellfish poisons (DSPs) are increasing continually. It is extremely necessary to develop simple analysis methods for screening simultaneously different types of DSPs from food-related samples. Okadaic acid (OA) and its analogues, i.e., dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2), are the prevalent DSPs. Herein, a facile and label-free fluorescent aptasensor targeting the three DSPs was constructed with a pair of group-specific split aptamers and silver nanocluster beacon. In presence of the targets, the DNA templates attached at the ends of the split aptamers would be dragged close to trigger enhanced fluorescence signals from silver nanoclusters. The aptasensor offered high sensitivity and good selectivity, with limit of detection of 2.282 nmolL

Topics: Humans; Marine Toxins; Okadaic Acid; Poisons; Pyrans; Shellfish; Shellfish Poisoning; Silver

Topics: Dinoflagellida; Estuaries; Humans; Marine Toxins; Okadaic Acid; Shellfish Poisoning

The successful cultivation of

Topics: Bays; Ciliophora; Dinoflagellida; Japan; Marine Toxins; Okadaic Acid

Topics: Animals; Diarrhea; Humans; Lethal Dose 50; Mice; Okadaic Acid; Pyrans; Shellfish Poisoning

Okadaic acid (OA) and its main structural analogs dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) are marine lipophilic phycotoxins distributed worldwide that can be accumulated by edible shellfish and can cause diarrheic shellfish poisoning (DSP). In order to study their toxicokinetics, mice were treated with different doses of OA, DTX1, or DTX2 and signs of toxicity were recorded up to 24 h. Toxin distribution in the main organs from the gastrointestinal tract was assessed by liquid chromatography-mass spectrometry (LC/MS/MS) analysis. Our results indicate a dose-dependency in gastrointestinal absorption of these toxins. Twenty-four hours post-administration, the highest concentration of toxin was detected in the stomach and, in descending order, in the large intestine, small intestine, and liver. There was also a different toxicokinetic pathway between OA, DTX1, and DTX2. When the same toxin doses are compared, more OA than DTX1 is detected in the small intestine. OA and DTX1 showed similar concentrations in the stomach, liver, and large intestine tissues, but the amount of DTX2 is much lower in all these organs, providing information on DSP toxicokinetics for human safety assessment.

Topics: Animals; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Female; Intestines; Marine Toxins; Mass Spectrometry; Mice; Mice, Inbred C57BL; Okadaic Acid; Shellfish; Shellfish Poisoning; Stomach; Tissue Distribution; Toxicokinetics

Low extraction efficiency (60-81%) of okadaic acid (OA) and dinophysistoxin 1 (DTX1) was obtained for 4 out of 5 shellfish species from Washington State (WA), USA, during application of a standard extraction method for determination of lipophilic marine biotoxins by LC-MS/MS as recommended by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). OA and total OA including esters, DTX1, DTX2, and total DTX including esters, azaspiracid 1, 2, and 3 (AZA1, AZA2, and AZA3), pectenotoxin 2 (PTX2), and yessotoxin (YTX) were the toxins examined. Matrix-matched standards prepared from the same control samples used for spike-and-recovery tests were employed to evaluate toxin extraction efficiency and sample clean-up procedures. We adjusted the EURLMB extraction method by either using an acidified methanol extraction or pre-cooking shellfish homogenates at 70 °C for 20 min before EURLMB extraction. Extraction efficiency was improved markedly for OA and DTX1 with both modified methods and for YTX with the pre-cooking step included. However, recoveries were lower for YTX using the acidified methanol extraction and for PTX2 in non-mussel samples with the pre-cooking step. A hexane wash was applied to clean water-diluted non-hydrolyzed samples and a hexane wash was combined with solid-phase extraction for cleaning hydrolyzed samples. Improved sample clean-up, combined with LC-MS/MS adjustments, enabled quantification of U.S. Food and Drug Administration-regulated toxins in five shellfish species from WA with acceptable accuracy using non-matrix matched calibration standards.

Topics: Alkalies; Animals; Chromatography, Liquid; Furans; Lipids; Macrolides; Marine Toxins; Methanol; Mollusk Venoms; Okadaic Acid; Oxocins; Shellfish; Spiro Compounds; Tandem Mass Spectrometry; Washington

Lipophilic phycotoxins (LPs) pose significant threats to the health of marine mammals, birds, and human beings. The distribution and components of lipophilic phycotoxins contamination in subtropical area in the South China Sea are rarely known. This study systematically assessed the composition, concentration, and distribution of typical LPs in a typical subtropical bay, Daya Bay located in the South China Sea. Phytoplankton, seawater, suspended particulate matter, sediments, and shellfish samples were simultaneously collected from Daya Bay, and analyzed using liquid chromatography with tandem mass spectrometry. Okadaic acid, dinophysistoxins-1, pectenotoxins-2, yessotoxin and its derivate homo-yessotoxin, azaspiracid-2, 13-desmethyl spirolide C and gymnodimine were widely spread in multiple media in Daya Bay. Pectenotoxins-2 was the most widely distributed and highly concentrated toxin in the marine environments of Daya Bay. Toxin homo-yessotoxin was only detected in sediments and shellfish samples, and none of yessotoxin group components were found in phytoplankton and seawater, indicating that sediments were the major source of yessotoxin in shellfish. The study strongly demonstrated the lipophilic phycotoxins accumulated in shellfish are multisource, not only derived from toxigenic algae, but also from other marine media containing lipophilic phycotoxins. This study systematically distinguished multi-pathways of bioaccumulation of LPs in the marine shellfish.

Topics: Animals; Bays; China; Chromatography, Liquid; Environmental Monitoring; Furans; Heterocyclic Compounds, 3-Ring; Humans; Hydrocarbons, Cyclic; Imines; Macrolides; Marine Toxins; Mollusk Venoms; Okadaic Acid; Oxocins; Phytoplankton; Pyrans; Seafood; Seawater; Shellfish; Spiro Compounds; Tandem Mass Spectrometry

Diarrhetic shellfish poisoning (DSP) is a syndrome caused by the intake of shellfish contaminated with a group of lipophilic and thermostable toxins, which consists of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2). These toxins are potent protein Ser/Thr phosphatase inhibitors, mainly type 1 protein phosphatase (PP1) and type 2A protein phosphatase (PP2A). Different effects have been reported at the cellular, molecular and genetic levels. In this study, changes in cell survival and cell mobility induced by OA, DTX-1 and DTX-2 were determined in epithelial cell lines of the colon and colon cancer. The cell viability results showed that tumoral cell lines were more resistant to toxins than the nontumoral cell line. The results of the functional assays for testing cell migration, evaluation of cell death and the expression of proteins associated with cell adhesion showed a dual effect of toxins since in the nontumoral cell line, a greater induction of cell death, presumably by anoikis, was detected. In the tumoral cell lines, there was an induction of a more aggressive phenotype characterized by increased resistance to toxins, increased migration and increased FAK activation. In tumoral cell lines of colon cancer, OA, DTX-1/DTX-2 induce a more aggressive phenotype.

Topics: Animals; Carcinogens; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Colonic Neoplasms; Focal Adhesion Kinase 1; Humans; Inhibitory Concentration 50; Okadaic Acid; Protein Phosphatase 2

The phycotoxins, okadaic acid (OA) and dinophysistoxins 1 and 2 (DTX-1 and -2), are protein phosphatase PP2A and PP1 inhibitors involved in diarrhetic shellfish poisoning (DSP) in humans. Data on the in vivo acute toxicity of the OA-group toxins show some differences and the European Food Safety Authority (EFSA) has determined toxicity equivalent factors (TEFs) of one for the reference toxin, OA, as well as for DTX-1 and 0.6 for DTX-2. However, recent in vitro studies indicated that DTX-1 seems to be more toxic than OA. As OA was described as apoptotic and aneugenic compound, we analyzed the DNA damage responses induced by the 3 toxins through γH2AX and pH3 biomarkers on proliferative HepaRG cells using High Content Analysis. We quantitatively examined the responses for γH2AX and pH3 by benchmark dose analyzing (BMD) using PROAST software. We found that the three toxins increased both γH2AX- and pH3-positive cells populations in a concentration-dependent manner. The 3 toxins induced mitotic arrest, characteristic of aneugenic compounds, as well as DNA strand-breaks concomitantly to cytotoxicity. BMD analysis showed that DTX-1 is the most potent inducer of DNA damage, followed by OA and DTX-2. The quantitative genotoxic data provided in this study are additional findings for reconsidering the estimated TEFs of this group of phycotoxins.

Topics: Benchmarking; Biomarkers; Cell Line, Transformed; Cell Proliferation; DNA Damage; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hepatocytes; Histones; Humans; Hydrophobic and Hydrophilic Interactions; Mitosis; Mutagenicity Tests; Mutagens; Okadaic Acid; Phosphorylation; Pyrans; Software

Diarrhetic shellfish poisoning (DSP) toxins are a class of natural organic contaminants that pose a serious threat not only to marine ecosystems and fisheries but also to human health. They are widely distributed in coastal and offshore waters around the world. However, the persistence and photochemical degradation characteristics of DSP in an aqueous environment are still unclear. This study aimed to elucidate the photochemical fate of two representative DSP toxins, namely, okadaic acid (OA) and dinophysistoxin-1 (DTX1). Results showed that photo-mediated chemical reactions play a crucial role in eliminating DSP toxins in seawater. However, the degradation of OA and DTX1 was relatively slow under natural solar radiation, with a removal efficiency of 90.0% after exposure for more than 20 days. When the reaction solutions of OA and DTX1 were exposed to Hg lamp radiation, their degradation followed pseudo-first-order kinetics, and was remarkably influenced by seawater pH and metal-ion concentration. A total of 24 tentative transformation products (TPs) of OA and DTX1 were identified via liquid chromatography high-resolution mass spectrometry. C12 (C

Topics: Ecosystem; Humans; Kinetics; Marine Toxins; Okadaic Acid; Photolysis; Pyrans

Bivalve mollusks accumulate diarrhetic shellfish toxins (DSTs) from toxigenic microalgae, thus posing a threat to human health by acting as a vector of toxins to consumers. In bivalves, free forms of DSTs can be esterified with fatty acids at the C-7 site to form acyl esters (DTX3), presumably a detoxification mechanism for bivalves. However, the effects of esterification of DSTs on fatty acid metabolism in mollusks remain poorly understood. In this study, mussels (Mytilus galloprovincialis) were fed the DST-producing dinoflagellate Prorocentrum lima for 10 days followed by an additional 10-days depuration in filtered seawater to track the variation in quantity and composition of DST acyl esters and fatty acids. A variety of esters of okadaic acid (OA) and dinophysistoxin-1 (DTX1) were mainly formed in the digestive gland (DG), although trace amounts of esters also appeared in muscle tissue. A large relative amount of OA (60%-84%) and DTX1 (80%-92%) was esterified to DTX3 in the visceral mass (referred to as digestive gland, DG), and the major ester acyl chains were C16:0, C16:1, C18:0, C18:1, C20:1 and C20:2. The DG and muscle tissues showed pronounced differences in fatty acid content and composition during both feeding and depuration periods. In the DG, fatty acid content gradually decreased in parallel with increasing accumulation and esterification of DSTs. The decline in fatty acids was accelerated during depuration without food. This reduction in the content of important polyunsaturated fatty acids, especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), would lead to a reduction in the nutritional value of mussels. Enzymes involved in lipid metabolism, including acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), lipoprotein lipase (LPL) and hepatic lipase (HL), were actively involved in the metabolism of fatty acids in the DG, whereas their activities were weak in muscle tissue during the feeding period. This study helps to improve the understanding of interactions between the esterification of DSTs and fatty acid dynamics in bivalve mollusks.

Topics: Animals; Dinoflagellida; Esterification; Esters; Fatty Acids; Food Chain; Lipid Metabolism; Marine Toxins; Microalgae; Mytilus; Okadaic Acid; Seafood; Shellfish; Shellfish Poisoning

Topics: Animals; Bivalvia; Marine Toxins; New Zealand; Okadaic Acid; Phytoplankton; Risk Assessment; Shellfish; Shellfish Poisoning

Diarrheic shellfish poisoning (DSP) is caused by the consumption of shellfish contaminated with a group of phycotoxins that includes okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2). These toxins are inhibitors of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), but show distinct levels of toxicity. Aside from a difference in protein phosphatases (PP) inhibition potency that would explain these differences in toxicity, others mechanisms of action are thought to be involved. Therefore, we investigated and compared which mechanisms are involved in the toxicity of these three analogues. As the intestine is one of the target organs, we studied the transcriptomic profiles of human intestinal epithelial Caco-2 cells exposed to OA, DTX-1, and DTX-2. The pathways specifically affected by each toxin treatment were further confirmed through the expression of key genes and markers of toxicity. Our results did not identify any distinct biological mechanism for OA and DTX-2. However, only DTX-1 induced up-regulation of the MAPK transduction signalling pathway, and down-regulation of gene products involved in the regulation of DNA repair. As a consequence, based on transcriptomic results, we demonstrated that the higher toxicity of DTX-1 compared to OA and DTX-2 was consistent with certain specific pathways involved in intestinal cell response.

Topics: Animals; Caco-2 Cells; Cell Survival; Dose-Response Relationship, Drug; Humans; Intestinal Mucosa; Marine Toxins; Okadaic Acid; Shellfish Poisoning

Okadaic acid (OA) group toxins may accumulate in shellfish and can result in diarrhetic shellfish poisoning when consumed by humans, and are therefore regulated. Purified toxins are required for the production of certified reference materials used to accurately quantitate toxin levels in shellfish and water samples, and for other research purposes. An improved procedure was developed for the isolation of dinophysistoxin-2 (DTX2) from shellfish (

Topics: Animals; Biomass; Chromatography, High Pressure Liquid; Magnetic Resonance Spectroscopy; Marine Toxins; Microalgae; Mytilus edulis; Okadaic Acid; Spectrophotometry, Ultraviolet; Tandem Mass Spectrometry

Approximately 70 species of

Topics: Australia; Dinoflagellida; Marine Toxins; Okadaic Acid; Phylogeny; Pyrans; Tropical Climate

Marine algal toxins, highly toxic secondary metabolites, have significant influences on coastal ecosystem health and mariculture safety. The occurrence and environmental control factors of lipophilic marine algal toxins (LMATs) in the surface seawater of the Changjiang estuary (CJE) and the adjacent East China Sea (ECS) were investigated. Pectenotoxin-2 (PTX2), okadaic acid (OA), dinophysistoxin-1(DTX1), and gymnodimine (GYM) were detected in the CJE surface seawater in summer, with concentration ranges of not detected (ND)-105.54 ng/L, ND-13.24 ng/L, ND-5.48 ng/L, and ND-12.95 ng/L, respectively. DTX1 (ND-316.15 ng/L), OA (ND-16.13 ng/L), and PTX2 (ND-4.97 ng/L) were detected in the ECS during spring. LMATs formed a unique low-concentration band in the Changjiang diluted water (CJDW) coverage area in the typical large river estuary. PTX2, OA, and DTX1 in seawater were mainly derived from

Topics: China; Dinoflagellida; Environmental Monitoring; Estuaries; Furans; Heterocyclic Compounds, 3-Ring; Hydrocarbons, Cyclic; Imines; Macrolides; Marine Toxins; Oceans and Seas; Okadaic Acid; Phytoplankton; Pyrans; Seawater; Water Pollutants

Galicia is an area with a strong mussel aquaculture industry in addition to other important bivalve mollusc fisheries. Between 2014 and 2017, 18,862 samples were analyzed for EU regulated marine lipophilic toxins. Okadaic acid (OA) was the most prevalent toxin and the only single toxin that produced harvesting closures. Toxin concentrations in raft mussels were generally higher than those recorded in other bivalves, justifying the use of this species as an indicator. The Rías of Pontevedra and Muros were the ones most affected by OA and DTX2 and the Ría of Ares by YTXs. In general, the outer areas of the Rías were more affected by OA and DTX2 than the inner ones. The OA level reached a maximum in spring, while DTX2 was almost entirely restricted to the fall-winter season. YTXs peaked in August-September. The toxins of the OA group were nearly completely esterified in all the bivalves studied except mussels and queen scallops. Risk of intoxication with the current monitoring system is low. In less than 2% of cases did the first detection of OA in an area exceed the regulatory limit. In no case, could any effect on humans be expected. The apparent intoxication and depuration rates were similar and directly related, suggesting that the rates are regulated mainly by oceanographic characteristics.

Topics: Animals; Biological Monitoring; Bivalvia; Food Contamination; Furans; Macrolides; Marine Toxins; Mollusk Venoms; Okadaic Acid; Oxocins; Pyrans; Spain

Blooms of the dinoflagellate

Topics: Animals; Dinoflagellida; Environmental Monitoring; Furans; Harmful Algal Bloom; Macrolides; Marine Toxins; New Zealand; Okadaic Acid; Perna; Pyrans; Shellfish; Shellfish Poisoning; Water Pollutants

A novel procedure for the preparation of magnetic covalent organic frameworks (COFs) is reported. In situ functionalization of Fe3O4 with dopamine rapidly afforded amino-functionalized magnetic nanoparticles, which after decoration with a COF building block and subsequent COF growth gave access to magnetic composite mTpBD-Me2. The optimized synthesis conditions yielded crystalline and superparamagnetic material with no loss in surface area as compared to bulk COF. The composite material was employed for the first time in magnetic solid-phase extraction of marine biotoxins from seawater with high efficiency, where calculated maximum adsorption capacities of 812 mg g-1 and 830 mg g-1 were found for okadaic acid (OA) and dinophysistoxin-1 (DTX-1), respectively, corresponding to an increase of ∼500-fold for OA and ∼300-fold for DTX-1 as compared to the commonly used non-magnetic macroporous resins. Nearly quantitative desorption efficiency of both biotoxins was obtained using 2-propanol as solvent, rendering the composite materials recyclable with merely minor losses in adsorption capacity after five consecutive cycles of adsorption/desorption. In addition, retention of crystallinity after the adsorption cycles highlights the stability of the composite in seawater. These results illustrate the great efficiency of the novel material in biotoxin adsorption and show great promise for its application in environmental monitoring programs.

Topics: 2-Propanol; Adsorption; Dopamine; Ferrosoferric Oxide; Kinetics; Magnetics; Metal-Organic Frameworks; Okadaic Acid; Pyrans; Solid Phase Extraction

Lipophilic marine toxins in shellfish pose significant threats to the health of seafood consumers. To assess the contamination status of shellfish by lipophilic marine toxins in the Bohai Sea, nine species of shellfish periodically collected from five representative aquaculture zones throughout a year were analyzed with a method of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), homo-yessotoxin (homo-YTX), azaspiracids (AZA2 and AZA3), gymnodimine (GYM), and 13-desmethyl spirolide C (13-DesMe-C), were detected in more than 95 percent of the shellfish samples. Toxins PTX2, YTX, 13-DesMe-C and GYM were predominant components detected in shellfish samples. Scallops, clams and mussels accumulated much higher level of lipophilic marine toxins compared to oysters. Toxin content in shellfish samples collected from different sampling locations showed site-specific seasonal variation patterns. High level of toxins was found during the stages from December to February and June to July in Hangu, while from March to April and August to September in Laishan. Some toxic algae, including Dinophysis acuminata, D. fortii, Prorocentrum lima, Gonyaulax spinifera and Lingulodinium polyedrum, were identified as potential origins of lipophilic marine toxins in the Bohai Sea. The results will offer a sound basis for monitoring marine toxins and protecting the health of seafood consumers.

Topics: Animals; Bivalvia; China; Chromatography, Liquid; Dinoflagellida; Furans; Heterocyclic Compounds, 3-Ring; Hydrocarbons, Cyclic; Imines; Macrolides; Marine Toxins; Mollusk Venoms; Okadaic Acid; Ostreidae; Oxocins; Pyrans; Seafood; Shellfish; Spiro Compounds; Tandem Mass Spectrometry; Water Pollutants, Chemical

A scallop midgut gland certified reference material, NMIJ CRM 7520-a, was developed for validation and quality assurance during the inspection of shellfish for diarrhetic shellfish toxins. The candidate material was prepared by using naturally-toxic and nontoxic boiled midgut glands spiked with okadaic acid (OA). The homogeneity and stability of the material were found to be appropriate. For the characterization of OA and dinophysistoxin-1 (DTX1), nine participants were involved in a co-laboratory study based on the Japanese Official Testing Method, where the compounds were assayed by liquid chromatography-tandem mass spectrometry following alkaline hydrolysis. The analytical values were obtained by the standard addition method with a standard spiking solution calibrated using the standard-solution certified reference materials OA and DTX1. The certified concentrations with expanded uncertainties (coverage factor k = 2, approximate 95% confidence interval) were determined to be (0.205 ± 0.061) mg/kg for OA and (0.45 ± 0.11) mg/kg for DTX1.

Topics: Animals; Calibration; Chromatography, Liquid; Diarrhea; Humans; Intestines; Marine Toxins; Okadaic Acid; Pectinidae; Pyrans; Reference Standards; Shellfish; Shellfish Poisoning; Tandem Mass Spectrometry

A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole-genome mRNA expression of undifferentiated intestinal Caco-2 cells. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish.

Topics: Caco-2 Cells; Cell Survival; Gene Expression Profiling; Gene Expression Regulation; Genome; Humans; Marine Toxins; Okadaic Acid; Pyrans; RNA, Messenger; Spiro Compounds

An interlaboratory comparison (ILC) was organized as a measure of the analytical competency in the liquid chromatography-tandem mass spectrometry quantification of okadaic acid (OA) and dinophysistoxin-1 (DTX1) in scallop midgut gland samples. The test sample was prepared using boiled midgut glands of naturally contaminated scallops with DTX1 and its esters by spiking with OA, and homogeneity and stability of this test sample was assessed to be appropriate. Twenty laboratories participated in the ILC based on the Japanese official testing method; they submitted two sets of analytical concentrations of target analytes along with the details of their analytical protocols. For assessing these data, assigned values were established from another ILC where ten participants quantified the target analytes by the standard addition method. The mean analytical results of the former ILC showed good agreement with the assigned values, and the corresponding relative reproducibility standard deviations met the criterion of CODEX STAN 292. Meanwhile, the results of more than half of the participants were out of the uncertainty range of the assigned values; these participants were encouraged to investigate their protocols to improve their analytical capability.

Topics: Animals; Chromatography, Liquid; Intestines; Marine Toxins; Okadaic Acid; Pectinidae; Pyrans; Reproducibility of Results; Shellfish; Shellfish Poisoning; Tandem Mass Spectrometry

The mouse bioassay for diarrhetic shellfish poisoning toxins has been used worldwide. In this study, dinophysistoxin-1 (DTX-1) and okadaic acid (OA) were compared for toxicity. The lethality rate increased and the median survival time decreased in a dose-dependent manner in both DTX-1 and OA. The median lethal dose value was 150.4 µg/kg (95% confidence interval=130.1-171.2 µg/kg) for DTX-1 and 185.6 µg/kg (95% confidence interval=161.2-209.6 µg/kg) for OA. The toxicity equivalent factor 1:1 has been used for OA and DTX-1 in the EU and Japan. Thus, it may be considered that toxicity potential of DTX-1 has remained underestimated as compared to that of OA and DTX-1 might be more toxic than OA.

Topics: Animals; Biological Assay; Bivalvia; Dose-Response Relationship, Drug; Lethal Dose 50; Mice; Okadaic Acid; Pyrans; Toxicity Tests

To detect and recognise three structurally related marine biotoxins responsible for the diarrheic shellfish poisoning (DSP) symptom, namely okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) respectively, as well as the structurally different yessotoxin (YTX), we developed a novel surface-enhanced micro-Raman scattering (micro-SERS) approach to investigate for the first time their micro-SERS signalling in solution and jointly analysed them in conjunction with the normal and toxic mussel tissue. YTX provided the main SERS feature surprisingly similar to DTX-1 and DTX-2, suggesting similar molecular adsorption mechanism with respect to the AgNPs. A fingerprint SERS band at 1017 cm

Topics: Animals; Bivalvia; Hydrophobic and Hydrophilic Interactions; Marine Toxins; Mollusk Venoms; Okadaic Acid; Oxocins; Pyrans; Spectrum Analysis, Raman; Surface Properties

In recent years, detection of trace amounts of dissolved lipophilic phycotoxins in coastal waters has been possible using solid phase adsorption toxin tracking (SPATT) samplers. To explore the contribution of dissolved diarrhetic shellfish toxins (DST) to the accumulation of toxins by cultivated bivalves, mussels (

Topics: Animals; Marine Toxins; Microalgae; Mytilus; Okadaic Acid; Particulate Matter; Pyrans

Okadaic acid (OA) and the structurally related compounds dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) are marine phycotoxins that cause diarrheic shellfish poisoning (DSP) in humans due to ingestion of contaminated shellfish. In order to guarantee consumer protection, the regulatory authorities have defined the maximum level of DSP toxins as 160 µg OA equivalent kg-1 shellfish meat. For risk assessment and overall toxicity determination, knowledge of the relative toxicities of each analogue is required. In absence of enough information from human intoxications, oral toxicity in mice is the most reliable data for establishing Toxicity Equivalence Factors (TEFs).. Toxins were administered to mice by gavage, after that the symptomatology and mice mortality was registered over a period of 24 h. Organ damage data were collected at necropsy and transmission electron microscopy (TEM) was used for ultrastructural studies. Toxins in urine, feces and blood were analyzed by HPLC-MS/MS. The evaluation of in vitro potencies of OA, DTX1 and DTX2 was performed by the protein phosphatase 2A (PP2A) inhibition assay.. Mice that received DSP toxins by gavage showed diarrhea as the main symptom. Those toxins caused similar gastrointestinal alterations as well as intestine ultrastructural changes. However, DSP toxins did not modify tight junctions to trigger diarrhea. They had different toxicokinetics and toxic potency. The lethal dose 50 (LD50) was 487 µg kg-1 bw for DTX1, 760 µg kg-1 bw for OA and 2262 µg kg-1 bw for DTX2. Therefore, the oral TEF values are: OA = 1, DTX1 = 1.5 and DTX2 = 0.3.. This is the first comparative study of DSP toxins performed with accurate well-characterized standards and based on acute toxicity data. Results confirmed that DTX1 is more toxic than OA by oral route while DTX2 is less toxic. Hence, the current TEFs based on intraperitoneal toxicity should be modified. Also, the generally accepted toxic mode of action of this group of toxins needs to be reevaluated.

Topics: Administration, Oral; Animals; Body Weight; Chromatography, High Pressure Liquid; Female; Heart; Intestine, Small; Liver; Mice; Myocardium; Okadaic Acid; Protein Phosphatase 2; Pyrans; Stomach; Tandem Mass Spectrometry; Toxicity Tests

Diarrhetic shellfish toxins (DSTs) are a group of phycotoxins that include okadaic acid (OA)/dinophysistoxin (DTX) analogues. At present, detailed data on the distribution of DST is insufficient, and studies of the appropriate sample sizes are lacking. This study investigated the DST frequency distribution in scallops and mussels by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and a resampling analysis of existing data was carried out. The DST population-interval and the necessary sample size were also estimated. DSTs are localized in the scallop digestive-gland, and the DST concentrations in scallops were water-depth-dependent. DST concentrations in scallops and mussels showed normal distributions, but mussels tended to contain more DSTs than scallops. In the statistical resampling analysis of the acquired data on scallops and mussels, especially that using the bootstrap method, sample size was difficult to estimate when the DST variation was large. Although the DST population-interval could be statistically estimated from the sample standard deviation of three samples, the sample size corresponded to the risk management level, and the use of 13 or more samples was preferable. The statistical methods used here to analyze individual contents and estimate population content-intervals could be applied in various situations and for shellfish toxins other than DSTs.

Topics: Animals; Chromatography, Liquid; Gastrointestinal Tract; Gills; Gonads; Marine Toxins; Muscle, Skeletal; Mytilus edulis; Okadaic Acid; Pectinidae; Pyrans; Shellfish Poisoning; Tandem Mass Spectrometry

Diarrhetic shellfish toxins produced by the dinoflagellate genus

Topics: Animals; Bivalvia; Dinoflagellida; Environmental Monitoring; Food Contamination; Marine Toxins; Okadaic Acid; Pyrans; Scotland; Seafood; Shellfish Poisoning; Water Pollutants

Limaol (1), along with a dinophysistoxin 1 derivative and an okadaic acid (OA) derivative, was isolated from the large-scale cultivation of the benthic marine dinoflagellate Prorocentrum lima. The structure of 1 was determined by a combination of NMR spectroscopy and mass spectrometry and contained tetrahydropyran, 1,3,5,7-tetra(methylene)heptane, and octahydrospiro[pyran-2,2'-pyrano[3,2-b]pyran] moieties. The absolute configuration of 1 was completely elucidated on the basis of ROESY correlations, J-based configuration analysis, and modified Mosher's ester analysis. Limaol showed moderate cytotoxicity when compared to OA against three cancer cell lines.

Topics: Animals; Cell Line, Tumor; Dinoflagellida; Magnetic Resonance Spectroscopy; Marine Toxins; Molecular Structure; Okadaic Acid; Polyketides; Pyrans; Spiro Compounds

Many toxic secondary metabolites used for defense are also toxic to the producing organism. One important way to circumvent toxicity is to store the toxin as an inactive precursor. Several sulfated diesters of the diarrhetic shellfish poisoning (DSP) toxin okadaic acid have been reported from cultures of various dinoflagellate species belonging to the genus Prorocentrum. It has been proposed that these sulfated diesters are a means of toxin storage within the dinoflagellate cell, and that a putative enzyme mediated two-step hydrolysis of sulfated diesters such as DTX-4 and DTX-5 initially leads to the formation of diol esters and ultimately to the release of free okadaic acid. However, only one diol ester and no sulfated diesters of DTX-1, a closely related DSP toxin, have been isolated leading some to speculate that this toxin is not stored as a sulfated diester and is processed by some other means. DSP components in organic extracts of two large scale Prorocentrum lima laboratory cultures have been investigated. In addition to the usual suite of okadaic acid esters, as well as the free acids okadaic acid and DTX-1, a group of corresponding diol- and sulfated diesters of both okadaic acid and DTX-1 have now been isolated and structurally characterized, confirming that both okadaic acid and DTX-1 are initially formed in the dinoflagellate cell as the non-toxic sulfated diesters.

Topics: Animals; Dinoflagellida; Marine Toxins; Okadaic Acid; Pyrans; Shellfish Poisoning

The occurrence and seasonal variations of marine algal toxins in phytoplankton and oyster samples in Daya Bay (DYB), South China Sea were investigated. Two Dinophysis species, namely, D. caudata and D. acuminata complex, were identified as Okadaic acid (OA)/pectenotoxin (PTX) related species. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis demonstrated that 2.04-14.47 pg PTX2 per cell was the predominant toxin in single-cell isolates of D. caudata. D. acuminata was not subjected to toxin analysis. The occurrence of OAs in phytoplankton concentrates of net-haul sample coincided with the presence of D. accuminata complex, suggesting that this species is most likely an OA producer in this sea area. OA, dinophysistoxins-1 (DTX1), PTX2, PTX2sa, gymnodimine (GYM), homoyessotoxin (homoYTX), and domoic acid (DA) demonstrated positive results in net haul samples. To our best knowledge, this paper is the first to report the detection of GYM, DA, and homoYTX in phytoplankton samples in Chinese coastal waters. Among the algal toxins, GYM demonstrated the highest frequency of positive detections in phytoplankton concentrates (13/17). Five compounds of algal toxins, including OA, DTX1, PTX2, PTX2sa, and GYM, were detected in oyster samples. DA and homoYTX were not detected in oysters despite of positive detections for both in the phytoplankton concentrates. However, neither the presence nor absence of DA in oysters can be determined because extraction conditions with 100% methanol used to isolate toxins from oysters (recommended by the EU-Harmonised Standard Operating Procedure, 2015) would likely be unsuitable for this water-soluble toxin. In addition, transformation of DA during the digestion process of oysters may also be involved in the negative detections of this toxin. GYM exhibited the highest frequency of positive results in oysters (14/17). OAs were only detected in the hydrolyzed oyster samples. The detection rates of PTX and PTX2sa in oysters were lower than those in the net haul samples.

Topics: Animals; Bays; China; Chromatography, Liquid; Dinoflagellida; Environmental Monitoring; Heterocyclic Compounds, 3-Ring; Hydrocarbons, Cyclic; Imines; Kainic Acid; Marine Toxins; Okadaic Acid; Ostreidae; Phytoplankton; Pyrans; Tandem Mass Spectrometry; Water Pollutants, Chemical

Some dinoflagellates can produce lipophilic marine toxins, which pose potent threats to seafood consumers. In the Bohai Sea, an important semi-closed inland sea with intensive mariculture industry in China, there is little knowledge concerning lipophilic marine toxins and their potential threats. In this study, net-concentrated phytoplankton samples were periodically collected from 5 typical mariculture zones around the Bohai Sea, including Laishan (LS), Laizhou (LZ), Hangu (HG), Qinhuangdao (QHD) and Huludao (HLD) in 2013 and 2014, and a method using high performance liquid chromatography (HPLC) coupled with a Q-Trap mass spectrometer was applied to analyze seven representative lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), azaspiracid-1 (AZA1), gymnodimine (GYM), and 13-desmethyl spirolide C (desMeC). The method had high sensitivity and repeatability, and exhibited satisfactory recoveries for most of the lipophilic marine toxins (92.1-108%) except for AZA1 (65.8-68.9%). Nearly all the lipophilic marine toxins could be detected in phytoplankton samples from the Bohai Sea. OA, DTX1 and PTX2 were predominant components and present in most of the phytoplankton samples. The maximum content of lipophilic marine toxin in phytoplankton samples concentrated from seawater (OA 464 pg L

Topics: Animals; China; Chromatography, High Pressure Liquid; Dinoflagellida; Furans; Heterocyclic Compounds, 3-Ring; Hydrocarbons, Cyclic; Hydrophobic and Hydrophilic Interactions; Imines; Macrolides; Marine Toxins; Mollusk Venoms; Okadaic Acid; Oxocins; Phytoplankton; Pyrans; Seafood; Spiro Compounds; Tandem Mass Spectrometry

Topics: Animals; Climate Change; Dinoflagellida; Embryo, Nonmammalian; Embryonic Development; Enzyme Inhibitors; Fishes; Harmful Algal Bloom; Okadaic Acid; Pyrans; Water Pollutants, Chemical

A DTX-1-producing microalga,

Topics: Animals; Diarrhea; Dinoflagellida; Gastrointestinal Tract; Gills; Humans; Japan; Kidney; Marine Toxins; Mutagenicity Tests; Mytilidae; Oceans and Seas; Okadaic Acid; Pyrans; Seafood; Seawater; Tissue Distribution

A freeze-dried mussel tissue (Mytilus edulis) reference material (CRM-FDMT1) was produced containing multiple groups of shellfish toxins. Homogeneity and stability testing showed the material to be fit for purpose. The next phase of work was to assign certified values and uncertainties to 10 analytes from six different toxin groups. Efforts involved optimizing extraction procedures for the various toxin groups and performing measurements using liquid chromatography-based analytical methods. A key aspect of the work was compensating for matrix effects associated with liquid chromatography-mass spectrometry through standard addition, dilution, or matrix-matched calibration. Certified mass fraction values are reported as mg/kg of CRM-FDMT1 powder as bottled for azaspiracid-1, -2, and -3 (4.10 ± 0.40; 1.13± 0.10; 0.96 ± 0.10, respectively), okadaic acid, dinophysistoxin-1 and -2 (1.59 ± 0.18; 0.68 ± 0.07; 3.57± 0.33, respectively), yessotoxin (2.49 ± 0.28), pectenotoxin-2 (0.66 ± 0.06), 13-desmethylspirolide-C (2.70 ± 0.26), and domoic acid (126 ± 10). Combined uncertainties for the certified values include contributions from homogeneity, stability, and characterization experiments. The commutability of CRM-FDMT1 was assessed by examining the extractability and matrix effects for the freeze-dried material in comparison with its equivalent wet tissue homogenate. CRM-FDMT1 is the first shellfish matrix CRM with certified values for yessotoxins, pectenotoxins or spirolides, and is the first CRM certified for multiple toxin groups. CRM-FDMT1 is a valuable tool for quality assurance of phycotoxin monitoring programs and for analytical method development and validation. Graphical Abstract CRM-FDMT1 is a multi-toxin mussel tissue certified reference material (CRM) to aid in development and validation of analytical methods for measuring the levels of algal toxins in seafood.

Topics: Animals; Chromatography, Liquid; Freeze Drying; Furans; Kainic Acid; Macrolides; Marine Toxins; Mass Spectrometry; Mollusk Venoms; Mytilus edulis; Okadaic Acid; Oxocins; Pyrans; Reference Standards; Seafood; Spiro Compounds

One of the proposed mechanisms to explain why Diarrhetic Shellfish Poison (DSP) toxins are tumor promoters is founded on the capacity of these toxins to increase TNF-α secretion. Although macrophages are the principal cells in the activation of the inflammatory response, the immune profile that Okadaic acid (OA) and Dinophysistoxin-1 (DTX-1) trigger in these cells has not been fully explored. We have therefore investigated the effect of various concentrations of both toxins on the activity of several inflammatory factors. Our results demonstrate that OA and DTX-1, at sublethal doses, stimulate secretion of inflammatory factors. Nevertheless DTX-1 was more potent than OA in increasing TNF-α and IL-6 as well as their dependent chemokines KC, MCP-1, LIX, MIP-1 α, MIP-1 β and MIP-2. On the other hand, secretion of IFN-γ and the anti-inflammatory cytokines, IL-4 and IL-10, was unaffected. In addition, DTX-1 also raises matrix metalloproteinase-9 (MMP-9) activity. In this study, for the first time the effect of OA and DTX-1 over the secretion of pro-inflammatory and carcinogenic signals in macrophages are compared, showing that DTX-1 is ten times more potent that OA. The inflammatory profile produced by DTX-1 is shown for the first time. The safe limit regulation should be changed to DSP toxins zero tolerance in the shellfish to be consumed by humans.

Topics: Animals; Chemokines; Cytokines; Enzyme-Linked Immunosorbent Assay; Immunity, Innate; Interleukin-6; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Okadaic Acid; Pyrans; Toxicity Tests; Tumor Necrosis Factor-alpha

A method was developed for the simultaneous determination of okadaic acid, dinophysistoxin-1 and dinophysistoxin-2 in shellfish using ultra performance liquid chromatography with tandem mass spectrometry. Shellfish poisons in spiked samples were extracted with methanol and 90% methanol, and were hydrolyzed with 2.5 mol/L sodium hydroxide solution. Purification was done on an HLB solid-phase extraction column. This method was validated in accordance with the notification of Ministry of Health, Labour and Welfare of Japan. As a result of the validation study in nine kinds of shellfish, the trueness, repeatability and within-laboratory reproducibility were 79-101%, less than 12 and 16%, respectively. The trueness and precision met the target values of notification.

Topics: Animals; Chromatography, Liquid; Food Contamination; Methanol; Okadaic Acid; Poisons; Pyrans; Reproducibility of Results; Shellfish; Shellfish Poisoning; Sodium Hydroxide; Solutions; Tandem Mass Spectrometry

Okadaic acid (OA), a lipophilic shellfish toxin, was accurately quantified using quantitative nuclear magnetic resonance with internal standards for the development of an authentic reference standard. Pyridine and the residual proton in methanol-d4 were used as removable internal standards to limit any contamination. They were calibrated based on a maleic acid certified reference material. Thus, the concentration of OA was traceable to the SI units through accurate quantitative NMR with an internal reference substance. Signals from the protons on the oxygenated and unsaturated carbons of OA were used for quantification. A reasonable accuracy was obtained by integrating between the lower and upper (13)C satellite signal range when more than 4 mg of OA was used. The best-determined purity was 97.4% (0.16% RSD) when 20 mg of OA was used. Dinophysistoxin-1, a methylated analog of OA having an almost identical spectrum, was also quantified by using the same methodology.

Topics: Calibration; Dinoflagellida; Magnetic Resonance Spectroscopy; Marine Toxins; Okadaic Acid; Pyrans; Reference Standards; Sensitivity and Specificity; Shellfish

Okadaic acid (OA) and its analogs dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic polyethers produced by marine dinoflagellates. These toxins accumulate in shellfish and cause diarrhetic shellfish poisoning (DSP) in humans. Regulatory testing of shellfish is essential to safeguard public health and for international trade. Certified reference materials (CRMs) play a key role in analytical monitoring programs. This paper presents an overview of the interdisciplinary work that went into the planning, production, and certification of calibration-solution CRMs for OA, DTX1, and DTX2. OA and DTX1 were isolated from large-scale algal cultures and DTX2 from naturally contaminated mussels. Toxins were isolated by a combination of extraction and chromatographic steps with processes adapted to suit the source and concentration of each toxin. New 19-epi-DSP toxin analogs were identified as minor impurities. Once OA, DTX1, and DTX2 were established to be of suitable purity, solutions were prepared and dispensed into flame-sealed glass ampoules. Certification measurements were carried out using quantitative NMR spectroscopy and LC-tandem MS. Traceability of measurements was established through certified external standards of established purity. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization, with components from the measurement, stability, and homogeneity studies being propagated into final combined uncertainties.

Topics: Animals; Calibration; Chromatography, Liquid; Diarrhea; Humans; Magnetic Resonance Spectroscopy; Marine Toxins; Okadaic Acid; Pyrans; Reference Standards; Shellfish; Shellfish Poisoning; Tandem Mass Spectrometry

Okadaic acid (OA) and its analogs, dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic biotoxins produced by marine algae that can accumulate in shellfish and cause the human illness known as diarrhetic shellfish poisoning (DSP). Regulatory testing of shellfish is required to protect consumers and the seafood industry. Certified reference materials (CRMs) are essential for the development, validation, and quality control of analytical methods, and thus play an important role in toxin monitoring. This paper summarizes work on research and development of shellfish tissue reference materials for OA and DTXs. Preliminary work established the appropriate conditions for production of shellfish tissue CRMs for OA and DTXs. Source materials, including naturally incurred shellfish tissue and cultured algae, were screened for their DSP toxins. This preliminary work informed planning and production of a wet mussel (Mytilus edulis) tissue homogenate matrix CRM. The homogeneity and stability of the CRM were evaluated and found to be fit-for-purpose. Extraction and LC-tandem MS methods were developed to accurately certify the concentrations of OA, DTX1, and DTX2 using a combination of standard addition and matrix-matched calibration to compensate for matrix effects in electrospray ionization. The concentration of domoic acid was also certified. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization. The presence of other toxins in the CRM was also assessed and information values are reported for OA and DTX acyl esters.

Topics: Animals; Calibration; Chromatography, Liquid; Diarrhea; Humans; Marine Toxins; Molecular Conformation; Okadaic Acid; Pyrans; Reference Standards; Shellfish; Shellfish Poisoning; Tandem Mass Spectrometry

With the increasing number of outbreaks of food-borne diseases caused by okadaic acid (OA) and its analogue dinophysistoxin-1 (DTX-1), two key diarrhetic shellfish poison (DSP) toxins, OA and DTX-1, have become a serious threat to public health and have attracted significant public attention in China. The aim of our study was to monitor OA and DTX-1 contamination in commercially available seafood and to provide references for tracking these toxins and preventing disease outbreaks. From 2010 to 2012, 40 species were collected from six coastal cities of four inland seas in China. An enzyme-linked immunosorbent assay (ELISA) and a lateral flow immunochromatographic (LFIC) test strip were used to analyse the samples, and the results were further confirmed using a commercially available ELISA kit. The monitoring results indicated that 23 of 40 species were positive for contamination. In addition, 14 of the positive species were determined to be inedible because the content of OA and DTX-1 was above the regulatory limit. Simultaneously, we verified that the digestive glands of shellfish tended to accumulate toxin, in contrast to the flesh. The highest concentrations of OA and DTX-1 were recorded in Scapharca broughtonii, which was collected from Qing Dao, in relation to the other analysed species. Moreover, the Arca family as well as Mytilus galloprovincialis were severely contaminated by OA and its analogue. The above results indicate that some of the commercially available seafood from the coastal cities in China may be inedible due to serious marine toxin contamination. The results of this study might play an important role in protecting consumer health and safety screening of marine products.

Topics: Animals; China; Disease Outbreaks; Enzyme-Linked Immunosorbent Assay; Food Contamination; Marine Toxins; Oceans and Seas; Okadaic Acid; Pyrans; Shellfish; Shellfish Poisoning; Species Specificity

Marine dinoflagelates from the genus Dynophisis are important producers of Diarrhetic Shellfish Poisoning (DSP) toxins which are responsible for human intoxications. The present work is an approach to study the relative toxicity of DSP toxins effects on Neuro-2a, NG108-15 and MCF-7 cell-lines. Certified standards of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) were used. Our results show that the three toxins exhibit similar cytotoxicity in Neuro-2a and NG108-15 cell lines. Conversely, MCF-7 cells were the least sensitive to these toxins. DTX-1 displayed the most toxic effect in the three tested cell lines.

Topics: Cell Line; Dose-Response Relationship, Drug; Humans; Marine Toxins; MCF-7 Cells; Okadaic Acid; Pyrans

Phycotoxins are marine toxins produced by phytoplankton that can get accumulated in filter feeding shellfish. Human intoxication episodes occur due to contaminated seafood consumption. Okadaic acid (OA) and dynophysistoxins (DTXs) are phycotoxins responsible for a severe gastrointestinal syndrome called diarrheic shellfish poisoning (DSP). Yessotoxins (YTXs) are marine toxins initially included in the DSP class but currently classified as a separated group. Food safety authorities from several countries have regulated the content of DSPs and YTXs in shellfish to protect human health. In mice, OA and YTX have been associated with ultrastructural heart damage in vivo. Therefore, this study explored the potential of OA, DTX-1 and YTX to cause acute heart toxicity. Cardiotoxicity was evaluated in vitro by measuring hERG (human èter-a-go-go gene) channel activity and in vivo using electrocardiogram (ECG) recordings and cardiac damage biomarkers. The results demonstrated that these toxins do not exert acute effects on hERG channel activity. Additionally, in vivo experiments showed that these compounds do not alter cardiac biomarkers and ECG in rats acutely. Despite the ultrastructural damage to the heart reported for these toxins, no acute alterations of heart function have been detected in vivo, suggesting a functional compensation in the short term.

Topics: Animals; Cardiotoxicity; CHO Cells; Cricetinae; Cricetulus; Electrocardiography; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Female; Mollusk Venoms; Natriuretic Peptide, Brain; Okadaic Acid; Oxocins; Pyrans; Rats; Rats, Sprague-Dawley; Troponin I; Troponin T

Okadaic acid (OA) and dinophysistoxins (DTXs) are a group of marine toxins that cause diarrheic shellfish poisoning (DSP) in humans and animals. These compounds are produced by dinoflagellates of the Prorocentrum and Dinophysis genera and can accumulate in filter-feeding bivalves, posing a serious health risk for shellfish consumers. The enteric nervous system (ENS) plays a crucial role in the regulation of the gastrointestinal tract. In addition, neuropeptides produced by ENS affects the epithelial barrier functions. In the present work we used a two-compartment human coculture model containing the SH-SY5Y neuroblastoma cell line and polarized colonic epithelial monolayers (Caco-2) to study the OA intestinal permeability. First, we have determined OA cytotoxicity and we have found that OA reduces the viability of SH-SY5Y in a dose-dependent way, even though DTX1 is 4 to 5 times more potent than OA. Besides DTX1 is 15 to 18 orders of magnitude more potent than OA in decreasing transepithelial electrical resistance (TEER) of caco-2 cells without inducing cytotoxicity. Permeability assays indicate that OA cross the monolayer and modulates the neuropeptide Y (NPY) secretion by neuroblastoma cells. This NPY also affects the permeability of OA. This offers a novel approach to establish the influence of OA neuronal action on their diarrheic effects through a cross talk between ENS and intestine via OA induced NPY secretion. Therefore, the OA mechanisms of toxicity that were long attributed only to the inhibition of protein phosphatases, would require a reevaluation.

Topics: Caco-2 Cells; Cell Line, Tumor; Cell Survival; Diarrhea; Electric Impedance; Humans; Neuroblastoma; Neurons; Neuropeptide Y; Okadaic Acid; Pyrans

Okadaic acid (OA) and its derivatives, DTX-1 and DTX-2, are marine biotoxins associated with diarrhetic shellfish poisoning. Routine monitoring of these toxins relies on the mouse bioassay. However, due to the technical unreliability and animal usage of this bioassay, there is always a need for convenient and reliable alternative assay methods. A panel of monoclonal antibodies against OA was generated and the most suitable was selected for biosensor-based assay development using surface plasmon resonance. The cross reactivity of the selected antibody with DTX-1 was found to be 73%, confirming the antibody suitability for both OA and DTX detection. The OA and derivative assay was designed as an inhibition assay covering the concentrations 1-75 ng/ml, with a sensitivity of 22.4 ng/ml. The assay was highly reproducible and preliminary validation showed no matrix interference from mussel extracts and good recovery of added standard in mussel extracts, with %CV of <9.3%. This assay could provide a useful and convenient screening tool for OA and its derivatives with a comprehensive extraction protocol for shellfish monitoring programmes.

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Biological Assay; Biosensing Techniques; Bivalvia; Hydrogen-Ion Concentration; Immunoassay; Marine Toxins; Mice; Mice, Inbred BALB C; Okadaic Acid; Pyrans; Sensitivity and Specificity; Shellfish; Surface Plasmon Resonance

Toxins produced by harmful algae are associated with detrimental health effects and mass mortalities of marine mammals. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is generally used to confirm the presence of algal toxins in marine mammals. Sample preparation and LC-MS/MS methods for the determination of three diarrhetic shellfish poisoning (DSP) toxins (okadaic acid, OA; dinophysistoxin-1, DTX1; dinophysistoxin-2, DTX2) and pectenotoxin-2 (PTX2) in bottlenose dolphin (Tursiops truncatus) urine and tissue samples were evaluated using spike-and-recovery tests. Sample clean-up with either reversed-phase silica or polymeric solid-phase extraction (SPE) reduced interference of sample matrices and improved toxin recoveries, with polymeric SPE showing higher sample loading capacity. LC separation on Xbridge C18 columns using acetonitrile/water gradient elutions with ammonia as the additive was chosen for its high detectivity and sensitivity in the MS detection of DSP toxins in negative ion mode. The retention times of OA, DTX1, and DTX2, separated as negative ions, increased with LC column temperature while the retention time of PTX2, separated as the neutral molecule, was weakly affected. At the same column temperature, retention times of OA, DTX1, and DTX2 gradually increased as the mobile phases aged while the retention time of PTX2 remained unchanged; higher column temperatures resulted in a greater increase in the retention time of each DSP toxin with mobile phase aging. Average recoveries of the 4 toxins in bottlenose dolphin samples ranged from 80% to 130% with relative standard deviations of less than 15% using the LC mobile phases prepared within one week at a column temperature of 30°C or 40°C. The preferred column temperature was 30°C, as the retention times of DSP toxins were less affected by mobile phase aging at this temperature. The limit of detection of each toxin analyzed in bottlenose dolphin samples was 2.8 ng/g or less in tissue samples and 0.7 ng/ml or less in urine.

Topics: Animals; Bottle-Nosed Dolphin; Chromatography, Liquid; Diarrhea; Furans; Macrolides; Marine Toxins; Okadaic Acid; Pyrans; Shellfish Poisoning; Solid Phase Extraction; Tandem Mass Spectrometry

For the first time the presence of dinophysistoxin-1 (DTX-1) in a culture of Prorocentrum foraminosum was revealed in cells and in the culture medium. The clone was isolated from coastal waters of the Sea of Japan and identified by molecular analyses of SSU and D1/D2 regions of LSU rDNA. The concentration of DTX-1 in cells was 8.4 ± 2.5 pg/cell and, in cell-free media, 27.9 ± 14.7 µg/L. The toxin presence was confirmed by HPLC with high-resolution tandem mass-spectrometry.

Topics: Chromatography, High Pressure Liquid; Clone Cells; Culture Media; Dinoflagellida; Environmental Monitoring; Harmful Algal Bloom; Mass Spectrometry; Oceans and Seas; Okadaic Acid; Pyrans; Russia; Seawater; Species Specificity

Japanese scallops, Patinopecten yessoensis, were fed with the toxic dinoflagellate Dinophysis fortii to elucidate the relative magnitude of assimilation, accumulation, and metabolism of diarrhetic shellfish toxins (DSTs) and pectenotoxins (PTXs). Three individual scallops were separately exposed to cultured D. fortii for four days. The average cell number of D. fortii assimilated by each individual scallop was 7.7 × 10⁵. Dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2) and their metabolites were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS) and the toxin content in individual tissues (digestive gland, adductor muscle, gill, gonad, mantle, and the others), feces and the seawater medium were quantified. Toxins were almost exclusively accumulated in the digestive gland with only low levels being detected in the gills, mantles, gonads, and adductor muscles. DTX1 and PTX2 were the dominant toxins in the D. fortii cells fed to the scallops, whereas the dominant toxins detected in the digestive gland of scallops were PTX6 and esterified acyl-O-DTX1 (DTX3). In other tissues PTX2 was the dominant toxin observed. The ratio of accumulated to assimilated toxins was 21%-39% and 7%-23% for PTXs and DTXs respectively. Approximately 54%-75% of PTX2 and 52%-70% of DTX1 assimilated by the scallops was directly excreted into the seawater mainly without metabolic transformation.

Topics: Animals; Dinoflagellida; Feces; Food Chain; Furans; Gastrointestinal Tract; Gills; Gonads; Macrolides; Muscles; Okadaic Acid; Pectinidae; Pyrans; Seawater; Water Pollutants

Okadaic acid (OA) and its analogues, dinophysistoxin 1 (DTX1) and dinophysistoxin 2 (DTX2), are lipophilic and heat-stable marine toxins produced by dinoflagellates, which can accumulate in filter-feeding bivalves. These toxins cause diarrheic shellfish poisoning (DSP) in humans shortly after the ingestion of contaminated seafood. Studies carried out in mice indicated that DSP poisonous are toxic towards experimental animals with a lethal oral dose 2-10 times higher than the intraperitoneal (i.p.) lethal dose. The focus of this work was to study the absorption of OA, DTX1 and DTX2 through the human gut barrier using differentiated Caco-2 cells. Furthermore, we compared cytotoxicity parameters. Our data revealed that cellular viability was not compromised by toxin concentrations up to 1 μM for 72 h. Okadaic acid and DTX2 induced no significant damage; nevertheless, DTX1 was able to disrupt the integrity of Caco-2 monolayers at concentrations above 50 nM. In addition, confocal microscopy imaging confirmed that the tight-junction protein, occludin, was affected by DTX1. Permeability assays revealed that only DTX1 was able to significantly cross the intestinal epithelium at concentrations above 100 nM. These data suggest a higher oral toxicity of DTX1 compared to OA and DTX2.

Topics: Caco-2 Cells; Humans; Intestinal Absorption; Lethal Dose 50; Marine Toxins; Microscopy, Confocal; Okadaic Acid; Pyrans

The phycotoxin, okadaic acid (OA) and dinophysistoxin 1 and 2 (DTX-1 and -2) are protein phosphatase PP2A and PP1 inhibitors involved in diarrhetic shellfish poisoning (DSP). Data on the toxicity of the OA-group toxins show some differences with respect to the in vivo acute toxicity between the toxin members. In order to investigate whether OA and congeners DTX-1 and -2 may induce different mechanisms of action during acute toxicity on the human intestine, we compared their toxicological effects in two in vitro intestinal cell models: the colorectal adenocarcinoma cell line, Caco-2, and the intestinal muco-secreting cell line, HT29-MTX. Using a high content analysis approach, we evaluated various cytotoxicity parameters, including apoptosis (caspase-3 activation), DNA damage (phosphorylation of histone H2AX), inflammation (translocation of NF-κB) and cell proliferation (Ki-67 production). Investigation of the kinetics of the cellular responses demonstrated that the three toxins induced a pro-inflammatory response followed by cell cycle disruption in both cell lines, leading to apoptosis. Our results demonstrate that the three toxins induce similar effects, as no major differences in the cytotoxic responses could be detected. However DTX-1 induced cytotoxic effects at five-fold lower concentrations than for OA and DTX-2.

Topics: Apoptosis; Caco-2 Cells; Cell Line, Tumor; Cell Proliferation; DNA Damage; HT29 Cells; Humans; Inflammation; Intestinal Mucosa; Intestines; Okadaic Acid; Pyrans; Toxins, Biological

In the present study, okadaic acid (OA) and dinophysistoxin-1 (DTX1) were spiked into artificial seawater at low, medium and high estuarine salinities (9‰, 13.5‰ and 27‰). Passive samplers (HP20 resin) used for solid phase adsorption toxin tracking (SPATT) technology were exposed in these seawaters for 12-h periods. Adsorption curves well fitted a pseudo-secondary kinetics model. The highest initial sorption rates of both toxins occurred in the seawater of medium salinity, followed by seawater of low and high estuarine salinity. Pore volumes of micropores (<2 nm) and small mesopores (2 nm

Topics: Adsorption; Alveolata; Chromatography, High Pressure Liquid; Okadaic Acid; Polystyrenes; Porosity; Pyrans; Salinity; Seawater

The protein phosphatase inhibition assay (PPIA) is a well-known strategy for the determination of diarrheic shellfish poisoning (DSP) lipophilic toxins, which deserves better characterization and understanding to be used as a routine screening tool in monitoring programs. In this work, the applicability of two PPIAs to the determination of okadaic acid (OA), dinophysistoxin-1 (DTX-1), dinophysistoxin-2 (DTX-2), and their acyl ester derivatives in shellfish has been investigated. The inhibitory potencies of the DSP toxins on a recombinant and a wild PP2A have been determined, allowing the establishment of inhibition equivalency factors (IEFs) (1.1 and 0.9 for DTX-1, and 0.4 and 0.6 for DTX-2, for recombinant and wild PP2A, respectively). The PPIAs have been applied to the determination of OA equivalent contents in spiked and naturally contaminated shellfish samples. Results have been compared to those obtained by LC-MS/MS analysis, after application of the IEFs, showing good agreements.

Topics: Animals; Biological Assay; Bivalvia; Chromatography, High Pressure Liquid; Humans; Marine Toxins; Okadaic Acid; Protein Phosphatase 2; Pyrans; Shellfish; Tandem Mass Spectrometry

Diarrhetic shellfish poisoning is a gastrointestinal illness caused by consumption of bivalves contaminated with dinophysistoxins. We report an illness cluster in the United States in which toxins were confirmed in shellfish from a commercial harvest area, leading to product recall. Ongoing surveillance is needed to prevent similar illness outbreaks.

Topics: Animals; Aquaculture; Child, Preschool; Harmful Algal Bloom; Humans; Middle Aged; Mytilus; Okadaic Acid; Pyrans; Shellfish Poisoning; Washington

Dinophysis sacculus is associated with DSP outbreaks especially in the Mediterranean Sea and is supposed to be mildly toxic based on few toxin results from field samples. First report of LC-MS analysis of D. sacculus cultures from Galicia (NW Spain) showed moderate amounts of OA (7.8 pg cell(-1)) comparable to those found in Dinophysis acuminata from the same region, PTX2 (13.2 pg cell(-1)) and trace amounts of DTX1 (0.8 pg OA equiv. cell(-1)). The contribution of D. sacculus to DSP outbreaks in the Galician Northern Rías should not be underestimated.

Topics: Chromatography, Liquid; Dinoflagellida; Environmental Monitoring; Humans; Marine Toxins; Mass Spectrometry; Okadaic Acid; Pyrans; Shellfish Poisoning; Spain

Graphene is a novel carbonic material with great potentials for the use as sorbent due to its ultrahigh surface area. Herein, we report the use of graphene as sorbent in solid-phase extraction (SPE) using pipette tip as cartridge namely GPT-SPE, together with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), for the analysis of lipophilic marine toxins (LMTs), including yessotoxins (YTX), okadaic acid (OA), dinophysistoxin-1 (DTX1), gymnodimine (GYM), spirolides-1 (SPX1), pectenotoxin-2 (PTX2) and azaspiracid-1 (AZA1) in shellfish. The GPT-SPE procedure was optimized and the performance of graphene was fully validated. Results with high-sensitivity and good reproducibility was obtained and compared with that of other sorbents like C18 silica, multi-walled carbon nanotubes (MWCNTs), commercial Oasis HLB, and Strata-X for the extraction of LMTs, which showed superiority and advantages of graphene, such as good recoveries, stability and compatibility with various solvents. In order to exhibit the potentials of graphene as an excellent sorbent material, 67 mussel samples from six coastal cities of China were analyzed. OA was found to be the dominant contaminant, while YTX was also detected with low level.

Topics: Adsorption; Animals; Bivalvia; Chromatography, High Pressure Liquid; Furans; Graphite; Heterocyclic Compounds, 3-Ring; Hydrocarbons, Cyclic; Imines; Macrolides; Marine Toxins; Mollusk Venoms; Muscles; Okadaic Acid; Oxocins; Pyrans; Reproducibility of Results; Sensitivity and Specificity; Shellfish; Solid Phase Extraction; Spiro Compounds; Tandem Mass Spectrometry

Research performed on transgenic animals has led to numerous advances in biological research. However, using traditional retroviral methods to generate transgenic avian research models has proved problematic. As a result, experiments aimed at genetic manipulations on birds have remained difficult for this popular research tool. Recently, lentiviral methods have allowed the production of transgenic birds, including a transgenic Japanese quail (Coturnix coturnix japonica) line showing neuronal specificity and stable expression of enhanced green fluorescent protein (eGFP) across generations (termed here GFP quail). To test whether the GFP quail may serve as a viable alternative to the popular chicken model system, with the additional benefit of genetic manipulation, we compared the development, organization, structure, and function of a specific neuronal circuit in chicken (Gallus gallus domesticus) with that of the GFP quail. This study focuses on a well-defined avian brain region, the principal nuclei of the sound localization circuit in the auditory brainstem, nucleus magnocellularis (NM), and nucleus laminaris (NL). Our results demonstrate that structural and functional properties of NM and NL neurons in the GFP quail, as well as their dynamic properties in response to changes in the environment, are nearly identical to those in chickens. These similarities demonstrate that the GFP quail, as well as other transgenic quail lines, can serve as an attractive avian model system, with the advantage of being able to build on the wealth of information already available from the chicken.

Topics: Animals; Animals, Genetically Modified; Animals, Newborn; Brain Stem; Chick Embryo; Cochlea; Coturnix; Electric Stimulation; Embryo, Nonmammalian; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; Fluoxetine; Functional Laterality; GABA Antagonists; Gene Expression Regulation, Developmental; Glutamate Decarboxylase; Green Fluorescent Proteins; Humans; In Vitro Techniques; Kv1.3 Potassium Channel; Lentivirus; Male; Membrane Potentials; Microtubule-Associated Proteins; Models, Animal; Neural Pathways; Neurons; Okadaic Acid; Patch-Clamp Techniques; Picrotoxin; Pyrans; Quinoxalines; Selective Serotonin Reuptake Inhibitors; Synapsins; Transgenes; Valine

The main diarrhetic shellfish toxins, okadaic acid (OA) and dinophysistoxin-1, 2 (DTX-2, 2) were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as pyrenacyl esters in clams (Ruditapes decussatus) collected in Tunis north lagoon from January 2007 to June 2008. Sample analyses by LC-MS/MS displayed OA and related congeners (DTX-2, 2) with a highest detected level of 21 μg OA eq/kg shellfish meat for the samples of January 2007. Nevertheless, all samples were MBA negative. During the study period, potentially toxic dinoflagellate Dinophysis sacculus was recorded all year, blooming at different times. Highest concentrations were recorded during January 2007 with 4.6 × 10(4) cells per liter and 4.10(4) cells per liter in the northern and southern districts, respectively. Results show that there is no significant correlation between D. sacculus densities in water column and diarrhetic shellfish poisoning (DSP) toxins concentrations unregistered in clams. These data reveal that DSP toxicity in clams of Tunis north lagoon is low according to European regulatory limit (160 μg OA eq/kg shellfish meat). However, a potential threat, in this area, is represented by DSP toxic species as D. sacculus and provides grounds for widen and reinforcing sanitary control of the phycotoxin measures in the region.

Topics: Animals; Bivalvia; Chromatography, Liquid; Dinoflagellida; Environmental Monitoring; Epidemiological Monitoring; Marine Toxins; Okadaic Acid; Pyrans; Shellfish Poisoning; Tandem Mass Spectrometry; Tunisia; Water Pollutants, Chemical

More than 200 people in China suffered illness with symptoms of diarrhetic shellfish poisoning (DSP) following consumption of mussels (Mytilus galloprovincialis). The event occurred in the cities of Ningbo and Ningde near the East China Sea in May, 2011. LC-MS/MS analysis showed that high concentrations of okadaic acid, dinophysistoxin-1, and their acyl esters were responsible for the incidents. The total concentration was more than 40 times the EU regulatory limit of 160 μg OA eq./kg. Pectentoxin-2 and its seco-acids were also present in the mussels. Additionally, yessotoxins were found to be responsible for positive mouse bioassay results on scallop (Patinopecten yessoensis) and oyster (Crassostrea talienwhanensis) samples collected from the North Yellow Sea in June, 2010. This work shows that high levels of lipophilic toxins can accumulate in shellfish from the Chinese coast and it emphasises that adequate chemical analytical methodologies are needed for monitoring purposes. Further research is required to broaden the knowledge on the occurrence of lipophilic toxins in Chinese shellfish.

Topics: Abdominal Pain; Alveolata; Animals; China; Diarrhea; Diet; Disease Outbreaks; Food Contamination; Humans; Marine Toxins; Mollusk Venoms; Mytilus; Okadaic Acid; Ostreidae; Oxocins; Pacific Ocean; Pectinidae; Pyrans; Seafood; Shellfish Poisoning

A method for the simultaneous determination of okadaic acid (OA) and its derivatives dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2) and yesstoxin (YTX) in shellfish using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. After being extracted with methanol, the extract was cleaned-up by solid phase extraction of a Strata-X cartridge. The separation of the 4 toxins were performed on a XTerra MS C18 column (100 mm x 2.1 mm, 3.5 microm) using gradient elution of acetonitrile and water both containing ammonium formate and formic acid as eluent modifiers. The qualitative and quantitative analysis were carried out by electrospray ionization (ESI) mass spectrometry in selective reaction monitoring (SRM) mode. The OA, DTX-1 and YTX were analyzed in negative ion mode, while PTX-2 in positive ion mode. The matrix-matched external standard calibration curves were used for the quantitative analysis. The calibration curves were linear in the range of 2.0 - 200.0 microg/L for OA, DTX-1 and YTX, 1.0 - 100.0 microg/L for PTX-2, with the quantification limits of 1.0 microg/kg and 0.5 microg/kg, respectively. The average recoveries for the toxins were between 83. 1% and 105.7% with the relative standard deviations (RSD) of 3.16% - 9.29%. The proposed method is sensitive, effective and simple. It was applicable for the determination and confirmation of OA, DTX-1, PTX-2 and YTX in shellfish products. The OA, DTX-1, PTX-2 and YTX in some shellfish samples collected from Yellow Sea were found by the method.

Topics: Animals; Chromatography, Liquid; Furans; Macrolides; Mollusca; Mollusk Venoms; Okadaic Acid; Oxocins; Pyrans; Shellfish; Tandem Mass Spectrometry

Marine toxins from microscopic algae can accumulate through the food chain and cause various neurological and gastrointestinal illnesses for human health. Herein, we designed a new ultrasensitive multiplexed immunoassay protocol for simultaneous electrochemical determination of brevetoxin B (BTX-2) and dinophysistoxin-1 (DTX-1) in seafood using distinguishable metal nanocluster-labeled molecular tags as traces on bifunctionalized magnetic capture probes. To construct such a bifunctionalized probe, monoclonal mouse anti-BTX-2 (mAb(1)) and anti-DTX-1 (mAb(2)) antibodies were co-immobilized on a magnetic bead (MB-mAb(1,2)). The distinguishable metal nanoclusters including cadmium nanoclusters (CdNC) and copper nanoclusters (CuNC) were synthesized using the artificial peptides with amino acid sequence CCCYYY, which were used as distinguishable signal tags for the label of the corresponding bovine serum albumin-BTX-2 and bovine serum albumin-DTX-1 conjugates. A competitive-type immunoassay format was adopted for the online simultaneous monitoring of BTX-2 and DTX-1 on a homemade flow-through magnetic detection cell. The assay was based on the stripping voltammetric behaviors of the labeled CdNC and CuNC at the various peak potentials in pH 2.5 HCl containing 0.01 M KCl using square wave anodic stripping voltammetry (SWASV). Under optimal conditions, the multiplexed immunoassays enabled simultaneous detection of BTX-2 and DTX-1 in a single run with wide working ranges of 0.005-5 ng mL(-1) for two marine toxins. The limit of detection (LOD) and limit of quantification (LOQ) were 1.8 and 6.0 pg mL(-1) for BTX-2, while those for DTX-1 were 2.2 and 7.3 pg mL(-1), respectively. No non-specific adsorption and electrochemical cross-talk between neighboring sites were observed during a series of procedures to detect target analytes. The covalent conjugation of biomolecules onto the nanoclusters and magnetic beads resulted in good repeatability and intermediate precision down to 9.5%. The method featured unbiased identification of negative (blank) and positive samples. No significant differences at the 0.05 significance level were encountered in the analysis of 12 spiked samples, including Sinonovacula constricta , Musculista senhousia , and Tegillarca granosa , between the multiplexed immunoassay and commercially available enzyme-linked immunosorbent assay (ELISA) for analysis of BTX-2 and DTX-1.

Topics: Adsorption; Animals; Cadmium; Copper; Food Contamination; Immunoassay; Limit of Detection; Marine Toxins; Metal Nanoparticles; Okadaic Acid; Oxocins; Pyrans; Seafood

Okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2, and yessotoxin (YTX) are classes of lipophilic toxins found in marine animals. OA and DTX1 accumulation causes diarrhetic shellfish poisoning, a worldwide public health problem. Diarrhetic shellfish poisoning has not previously been reported in gastropods, which are widely consumed in Korea. Seasonal variation in marine lipophilic toxins in gastropods was investigated using liquid chromatography-tandem mass spectrometry. Eighty specimens of Neptunea cumingii, 65 specimens of Rapana venosa, and 95 specimens of Batillus cornutus were collected at the Tongyeong fish market on the southern coast of Korea between May 2009 and December 2010. OA, DTX1, and YTX were detected in meat and digestive glands in all gastropod species studied. Pectenotoxin-2 was not found in any sample tested. Lipophilic toxins were detected in the digestive glands of gastropods; no lipophilic toxin was detected in the salivary glands of the carnivorous gastropods, N. cumingii and R. venosa. The highest concentrations of OA (21.5 ng/g) and DTX1 (8.4 ng/g) were detected in the digestive glands of R. venosa, and the maximum concentration of YTX (13.7 ng/g) was found in the digestive glands of N. cumingii. The maximum toxicities in gastropod tissues were lower than the European standard for acceptable levels. The concentrations of lipophilic toxins in carnivorous gastropods showed a high degree of seasonal variation; lipophilic toxins in carnivorous gastropods were found predominantly in spring and summer. This is the first report of the occurrence of lipophilic toxins in Korean gastropods.

Topics: Animals; Consumer Product Safety; Food Contamination; Food Safety; Gastropoda; Humans; Mollusk Venoms; Okadaic Acid; Oxocins; Pyrans; Republic of Korea; Seasons; Shellfish; Species Specificity

To investigate the contamination of shellfish poisoning of mussels, the poisonous constituents in that were isolated and identified.. The mussel tissue homogenate was extracted by acetone, and then the acetone extract was partitioned between diethyl ether and water. The ether extract was fractionated by column chromatography over silica gel and further isolated by semi-preparative RP-HPLC, monitored by ultra performance liquid chromatography coupled with triple quadrupole mass spectrometry.. Four poisonous constituents were isolated. Two of them were elucidated as pectenotoxin-2 seco acid and 7-epi-pectenotoxin-2 seco acid, respectively, on the basis of mass spectral data and compared with the production of enzymatic hydrolysis of PTX-2, and others were identified as okadaic acid and dinophysistoxin-1 by UPLC-MS/MS analysis compared with standard substances.. OA, DTX-1, 7-epi-PTX-2sa and PTX-2sa had been isolated from the mussel, respectively. The concentrations of free OA, DTX-1 and total OA in which were surpassed the maximum permitted levels in EU. OA and DTX-1 were confirmed to be the main toxins responsible for this DSP outbreak.

Topics: Animals; China; Chromatography, High Pressure Liquid; Food Contamination; Furans; Macrolides; Mytilus edulis; Oceans and Seas; Okadaic Acid; Pyrans; Shellfish

Chromatographic techniques coupled to mass spectrometry is the method of choice to replace the mouse bioassay (MBA) to detect marine toxins. This paper evaluates the influence of different parameters such as toxin solvents, mass spectrometric detection method, mobile-phase-solvent brands and equipment on okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) quantification. In addition, the study compares the results obtained when a toxin is quantified against its own calibration curve and with the calibration curve of the other analogues. The experiments were performed by liquid chromatography (LC) and ultraperformance liquid chromatography (UPLC) with tandem mass spectrometry detection (MS/MS). Three acetonitrile brands and two toxin solvents were employed, and three mass spectrometry detection methods were checked. One method that contains the transitions for azaspiracid-1 (AZA-1), azaspiracid-2 (AZA-2), azaspiracid-3(AZA-3), gimnodimine (GYM), 13-desmethyl spirolide C (SPX-1), pectenotoxin-2 (PTX-2), OA, DTX-1, DTX-2, yessotoxin (YTX), homoYTX, and 45-OH-YTX was compared in both instruments. This method operated in simultaneous positive and negative ionization mode. The other two mass methods operated only in negative ionization mode, one contains transitions to detect DTX-1, OA DTX-2, YTX, homoYTX, and 45-OH-YTX and the other only the transitions for the toxins under study OA, DTX-1, and DTX-2. With dependence on the equipment and mobile phase used, the amount of toxin quantified can be overestimated or underestimated, up to 44% for OA, 46% for DTX-1, and 48% for DTX-2. In addition, when a toxin was quantified using the calibration curve of the other analogues, the toxin amount obtained is different. The maximum variability was obtained when DTX-2 was quantified using either OA or a DTX-1 calibration curve. In this case, the overestimation was up to 88% using the OA calibration curve and up to 204% using the DTX-1 calibration curve. In summary, the correct quantification of DSP toxins by MS detection depends on multiple factors. Since these factors are not taken into account in a validated protocol, these results question the convenience of having MS/MS as a reference method for protecting consumers of marine toxins, moreover if toxicity of each group is considered independently and total toxicity is not summed anymore as it is in the MBA.

Topics: Chromatography, High Pressure Liquid; Marine Toxins; Okadaic Acid; Pyrans; Tandem Mass Spectrometry

Currently, there are no fast in vitro broad spectrum screening bioassays for the detection of marine toxins. The aim of this study was to develop such an assay. In gene expression profiling experiments 17 marker genes were provisionally selected that were differentially regulated in human intestinal Caco-2 cells upon exposure to the lipophilic shellfish poisons azaspiracid-1 (AZA1) or dinophysis toxin-1 (DTX1). These 17 genes together with two control genes were the basis for the design of a tailored microarray platform for the detection of these marine toxins and potentially others. Five out of the 17 selected marker genes on this dedicated DNA microarray gave clear signals, whereby the resulting fingerprints could be used to detect these toxins. CEACAM1, DDIT4, and TUBB3 were up-regulated by both AZA1 and DTX1, TRIB3 was up-regulated by AZA1 only, and OSR2 by DTX1 only. Analysis by singleplex qRT-PCR revealed the up- and down-regulation of the selected RGS16 and NPPB marker genes by DTX1, that were not envisioned by the new developed dedicated array. The qRT-PCR targeting the DDIT4, RSG16 and NPPB genes thus already resulted in a specific pattern for AZA1 and DTX1 indicating that for this specific case qRT-PCR might a be more suitable approach than a dedicated array.

Topics: Antigens, CD; Caco-2 Cells; Cell Adhesion Molecules; Gene Expression; Gene Expression Profiling; Humans; Marine Toxins; Okadaic Acid; Oligonucleotide Array Sequence Analysis; Pyrans; Reverse Transcriptase Polymerase Chain Reaction; Spiro Compounds; Transcription Factors; Tubulin

Okadaic acid (OA) and analogues of dinophysistoxin (DTX) are key diarrheic shellfish poisoning (DSP) toxins, which possibly arouse DSP symptoms by consuming the contaminated shellfish. Because of the stable toxicity in high temperature and the long-term carcinogenicity, the outbreaks of DSP related to consumption of bivalve mollusks contaminated by DSP toxins pose a hazard to public health. Therefore, it is worth developing a fast and reliable analytical method for the detection of OA and analogues in shellfish. In this paper, an indirect competitive enzyme-linked immunosorbent assay (ELISA) (icELISA) for detecting OA and DTX-1 in seafood was developed based on monoclonal antibody (McAb).. The OA was conjugated to human immunoglobulin G (IgG) and bovine serum albumin (BSA) by the active ester method as the immune antigen and the detective antigen. The spleen cells from BALB/c mice immunized with OA-IgG were fused with SP2/0 myeloma cells. A hybridoma cell line, which secreted McAb against OA, was selected by "limiting dilution" cloning. An icELISA was developed based on immobilized conjugate (OA-BSA) competing the McAb with the free OA in seafood sample.. A hybridoma cell line, which secreted IgG1 subclass monoclonal antibody (McAb) against OA, was selected. The IC(50) of the McAb for OA and dinophytoxin-1 (DTX-1) were 4.40 and 3.89 ng/mL, respectively. Based on the McAb, an indirect competitive ELISA for detection of OA and DTX-1 in seafood was developed. The regression equation was y = 54.713x - 25.879 with a coefficient correlation of R (2) = 0.9729. The linear range and the limit of detection were 0.4-12.5 and 0.45 ng/mL, respectively. The average recovery of OA and DTX-1 spiked shellfish was 82.29% with the coefficient of variation of 7.67%.. The developed icELISA is a fast, sensitive, and convenient assay for detecting of total amount of OA and DTX-1 in seafood.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Enzyme-Linked Immunosorbent Assay; Food Analysis; Humans; Mice; Mice, Inbred BALB C; Okadaic Acid; Protein Binding; Pyrans; Seafood; Serum Albumin, Bovine; Water Pollutants, Chemical

Okadaic acid (OA) and dinophysistoxin-1 (DTX1) cause diarrheic shellfish poisoning. This article examines the biochemical interactions of the two toxins with novel okadaic acid binding proteins (OABPs) 2.1 and 2.3, originally isolated from the marine sponge Halichondria okadai. First, recombinant OABPs 2.1 and 2.3 were expressed in Escherichia coli BL21 (DE3) cells. Binding assays using [24-(3)H]OA and the recombinant OABP 2.1 or 2.3 demonstrated the dissociation constant K(d) of 1.30±0.56 nM and 1.54±0.35 nM, respectively. Binding of [24-(3)H]okadaic acid to recombinant OABP2.1 was almost equally replaced with OA and DTX1. OA-induced cytotoxicity in mouse leukemia P388 cells was inhibited in the presence of the recombinant OABPs 2.1 and 2.3 with an EC(50) of 92±8.4 nM and 87±13 nM, respectively. These results suggest that the blockage of OA-induced cytotoxicity by OABPs 2.1 and 2.3 may be involved in regulating symbiotic relationships present in the sponge H. okadai.

Topics: Animals; Carrier Proteins; Cell Line, Tumor; Marine Toxins; Mice; Okadaic Acid; Porifera; Protein Binding; Pyrans; Recombinant Proteins

An approach involving both chemical and biological methods was undertaken for the detection and quantification of the marine toxins okadaic acid (OA), dinophysistoxin-1 (DTX-1) and their respective esters in mussels from different sampling sites in Greece during the period 2006-2007. Samples were analyzed by means of a) high performance liquid chromatography with fluorometric detection (HPLC-FLD), using 9-athryldiazomethane (ADAM), as a pre-column derivatization reagent, b) liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and c) the mouse bioassay. Free OA and DTX-1 were determined by both HPLC-FLD and LC-MS/MS, while their respective esters were determined only by LC-MS/MS after alkaline hydrolysis of the samples. The detection limit (L.O.D.) and quantification limit (L.O.Q.) of the HPLC-FLD method were 0.015 microg/g HP and 0.050 microg/g HP, respectively, for OA. The detection limit (L.O.D.) and quantification limit (L.O.Q.) of the LC-MS/MS method were 0.045 microg/g HP and 0.135 microg/g HP, respectively, for OA. Comparison of results between the two analytical methods showed excellent agreement (100%), while both HPLC-FLD and LC-MS/MS methods showed an agreement of 97.1% compared to the mouse bioassay.

Topics: Animals; Biological Assay; Bivalvia; Chromatography, High Pressure Liquid; Greece; Limit of Detection; Mice; Okadaic Acid; Pyrans; Tandem Mass Spectrometry

An approach involving chemical, functional and biological techniques was taken for the detection and quantification of the marine toxin okadaic acid (OA) in mussels from Thermaikos and Saronikos Gulfs, Greece, during DSP episodes that occurred in 2006-2007. Samples were analyzed using the mouse bioassay, high performance liquid chromatography with fluorimetric detection (HPLC-FLD), using l-bromoacetylpyrene (BAP), as a precolumn derivatisation reagent, and the protein phosphatase 2A inhibition assay (PP2AIA) using a commercially available kit. Okadaic acid (OA) and its polar and non-polar esters were detected and quantified by HPLC-FLD, after hydrolysis of the samples during preparation. The detection limit of the HPLC method for OA was 5.86 microg OA/kg, which permits this method to be used for the regulatory control of these toxins in shellfish. Comparison of the results by all three methods revealed excellent consistency.

Topics: Animals; Biological Assay; Bivalvia; Chromatography, High Pressure Liquid; Diarrhea; Disease Outbreaks; Fluorometry; Foodborne Diseases; Greece; Humans; Mice; Okadaic Acid; Phosphoric Monoester Hydrolases; Pyrans; Shellfish Poisoning; Time Factors

Microalgae of the genus Dinophysis and Prorocentrum are known producers of okadaites, responsible for the human syndrome known as diarrhetic shellfish poisoning (DSP). In temperate regions, only species from the genus Dinophysis are commonly held responsible for shellfish contamination. This is probably related to the different ecological strategies of the two genera, namely the planktonic nature of Dinophysis versus the benthic/epiphytic nature of toxic Prorocentrum species. In recent years, the threat of global warming has drawn attention to the study of benthic toxic microalgae in southern European waters. Here we present results on the toxin production and toxin profile of a Prorocentrum lima strain isolated from the Portuguese coast. This strain, IO66-01, presented a mean growth rate of 0.49 divisions d(-1), not common in temperate strains, and only comparable with tropical strains. The parent toxins found were okadaic acid (OA) and dinophysistoxin-1 (DTX1). The major diol esters were D8- and D9- congeners of both OA and DTX1.

Topics: Animals; Chromatography, High Pressure Liquid; Dinoflagellida; Indicators and Reagents; Marine Toxins; Mass Spectrometry; Okadaic Acid; Portugal; Pyrans; Spectrometry, Mass, Electrospray Ionization

Okadaic acid (OA) and structurally related toxins dinophysistoxin-1 (DTX-1), and DTX-2, are lipophilic marine biotoxins. The current reference method for the analysis of these toxins is the mouse bioassay (MBA). This method is under increasing criticism both from an ethical point of view and because of its limited sensitivity and specificity. Alternative replacement methods must be rapid, robust, cost effective, specific and sensitive. Although published immuno-based detection techniques have good sensitivities, they are restricted in their use because of their inability to: (i) detect all of the OA toxins that contribute to contamination; and (ii) factor in the relative toxicities of each contaminant. Monoclonal antibodies (MAbs) were produced to OA and an automated biosensor screening assay developed and compared with ELISA techniques. The screening assay was designed to increase the probability of identifying a MAb capable of detecting all OA toxins. The result was the generation of a unique MAb which not only cross-reacted with both DTX-1 and DTX-2 but had a cross-reactivity profile in buffer that reflected exactly the intrinsic toxic potency of the OA group of toxins. Preliminary matrix studies reflected these results. This antibody is an excellent candidate for the development of a range of functional immunochemical-based detection assays for this group of toxins.

Topics: Animals; Antibodies, Monoclonal; Biosensing Techniques; Bivalvia; Enzyme-Linked Immunosorbent Assay; Food Contamination; Marine Toxins; Mice; Mice, Inbred BALB C; Okadaic Acid; Pyrans

Okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) are algal toxins that can accumulate in shellfish and cause diarrhetic shellfish poisoning. Recent studies indicate that DTX-2 is about half as toxic and has about half the affinity for protein phosphatase 2A (PP2A) as OA. NMR structural studies showed that DTX-1 possessed an equatorial 35-methyl group but that DTX-2 had an axial 35-methyl group. Molecular modeling studies indicated that an axial 35-methyl could exhibit unfavorable interactions in the PP2A binding site, and this has been proposed as the reason for the reduced toxicity of DTX-2. Statistical analyses of published data indicate that the affinity of PP2A for DTX-1 is 1.6-fold higher, and for DTX-2 is 2-fold lower, than for OA. We obtained X-ray crystal structures of DTX-1 and DTX-2 bound to PP2A. The crystal structures independently confirm the C-35 stereochemistries determined in the earlier NMR study. The structure for the DTX-1 complex was virtually identical to that of the OA-PP2A complex, except for the presence of the equatorial 35-methyl on the ligand. The favorable placement of the equatorial 35-methyl group of DTX-1 against the aromatic pi-bonds of His191 may account for the increased affinity of PP2A toward DTX-1. In contrast, the axial 35-methyl of DTX-2 caused the side chain of His191 to rotate 140 degrees so that it pointed toward the solvent, thereby opening one end of the hydrophobic binding cage. This rearrangement to accommodate the unfavorable interaction from the axial 35-methyl of DTX-2 reduces the binding energy and appears to be responsible for the reduced affinity of PP2A for DTX-2. These results highlight the potential of molecular modeling studies for understanding the relative toxicity of analogues once the binding site at the molecular target has been properly characterized.

Topics: Animals; Binding Sites; Crystallography, X-Ray; Marine Toxins; Mice; Molecular Conformation; Okadaic Acid; Protein Phosphatase 2; Pyrans; Stereoisomerism

A rapid analytical optical biosensor-based immunoassay was developed and validated for the detection of okadaic acid (OA) and its structurally related toxins from shellfish matrix. The assay utilizes a monoclonal antibody which binds to the OA group of toxins in order of their toxicities, resulting in a pseudofunctional assay. Single-laboratory validation of the assay for quantitative detection of OA determined that it has an action limit of 120 microg/kg, a limit of detection of 31 microg/kg, and a working range of 31-174 microg/kg. The midpoint on the standard matrix calibration curve is 80 microg/kg, half the current regulatory limit. Inter- and intra-assay studies of negative mussel samples spiked with various OA concentrations produced average coefficient of variation (CV) and standard deviation (SD) values of 7.9 and 10.1, respectively. The assay was also validated to confirm the ability to accurately codetect and quantify dinophysistoxin-1 (DTX-1), DTX-2, and DTX-3 from shellfish matrix. Alkaline hydrolysis was not required for the detection of DTX-3 from matrix. Excellent correlations with the data generated by the biosensor method and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were obtained using a certified reference material (R(2) = 0.99), laboratory reference material, and naturally contaminated mussel samples (R(2) = 0.97). This new procedure could be used as a rapid screening procedure replacing animal-based tests for DSP toxins.

Topics: Animals; Biosensing Techniques; Chromatography, Liquid; Immunoassay; Marine Toxins; Okadaic Acid; Pyrans; Shellfish; Tandem Mass Spectrometry

The suitability and sensitivity of two neural cell models, NG108-15 and Neuro-2a, to different marine toxins were evaluated under different incubation and exposure times and in the presence or absence of ouabain and veratridine (O/V). NG108-15 cells were more sensitive to pectenotoxin-2 than Neuro-2a cells. For saxitoxin, brevetoxin-3, palytoxin, okadaic acid and dinophysistoxin-1 both cell types proved to be sensitive and suitable for toxicity evaluation. For domoic acid preliminary results were presented. Setting incubation time and exposure time proved to be critical for the development of the assays. In order to reduce the duration of the assays, it was better to reduce cell time incubation previous to toxin exposure than exposure time. For palytoxin, after 24h of growth, both cell types were sensitive in the absence of O/V. When growth time previous to toxin exposure was reduced, both cell types were unsensitive to palytoxin when O/V was absent. Although dinophysistoxin-1 and okadaic acid are both phosphatase inhibitors, these toxins did not respond similarly in front of the experimental conditions studied. Both cell types were able to identify Na-channel acting toxins and allowed to quantify the effect of saxitoxin, brevetoxin-3, palytoxin, okadaic acid, dinophysistoxin-1 and pectenotoxin-2 under different experimental conditions.

Topics: Acrylamides; Animals; Cell Line, Tumor; Cnidarian Venoms; Dose-Response Relationship, Drug; Furans; Glioma; Hybrid Cells; Kainic Acid; Macrolides; Marine Toxins; Mice; Neuroblastoma; Okadaic Acid; Oxocins; Pyrans; Saxitoxin; Time Factors; Toxicity Tests

To explore the nutrient properties of Prorocentrum lima and biosynthesis mechanism of diarrhetic shellfish poison (DSP), the growth and activities of alkaline phosphatase of Prorocentrum lima were observed under different phosphorus sources. DSP productions were also analyzed. The maximum growth rate (micro(max)) was slightly lower under beta-sodium glycerophosphate than those under NaH2PO4 and ATP as phosphorus sources, respectively. The maximum biomass (X) under ATP was higher than those under NaH2PO4 and beta-sodium glycerophosphate as the phosphorus sources, respectively. When the concentration of NaH2PO4 was below 2 micromol/L, the activity of alkaline phosphatase increased significantly. However, the activities were much low in the all treatments when beta-sodium glycerophosphate used as phosphorus source, whereas the activities increased with the concentration of ATP when ATP used as phosphorus source. The level of okadaic acid (OA) in Prorocentrum lima at the stationary phase under beta-sodium glycerophosphate was higher than those under NaH2PO4 and ATP. These suggested that beta-sodium glycerophosphate could be utilized directly by Prorocentrum lima with lower efficiency; ATP could induce alkaline phosphatase to produce inorganic phosphate for algae. DSP production in Prorocentrum lima were different under various phosphate sources, beta-sodium glycerophosphate enhanced production of DSP. The difference in DSP production might be related with the physiological state of Prorocentrum lima.

Topics: Alkaline Phosphatase; Animals; Dinoflagellida; Glycerophosphates; Marine Toxins; Okadaic Acid; Phosphates; Phosphorus; Pyrans

Marine algal toxins of the okadaic acid group can occur as fatty acid esters in blue mussels, and are commonly determined indirectly by transformation to their parent toxins by alkaline hydrolysis. Some data are available regarding the identity of the fatty acid esters, mainly of palmitic acid (16:0) derivatives of okadaic acid (OA), dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2). Other fatty acid derivatives have been described, but with limited mass spectral data. In this paper, the mass spectral characterization of the [M-H](-) and [M+Na](+) ions of 16 fatty acid derivatives of each of OA, DTX1 and DTX2 is presented. The characteristic fragmentation of [M+Na](+) ions of OA analogues provided a useful tool for identifying these, and has not been described previously. In addition, a set of negative ion multiple reaction monitoring (MRM) methods was developed for direct determination of 16 fatty acid esters of OA, 16 fatty acid esters of DTX1 and 16 fatty acid esters of DTX2 in shellfish extracts. The MRM methods were employed to study the profiles of fatty acid esters of OA analogues in blue mussels and to compare these with fatty acid ester profiles reported for other groups of marine algal toxins.

Topics: Animals; Environmental Monitoring; Esterification; Fatty Acids; Food Analysis; Marine Toxins; Mytilus edulis; Norway; Okadaic Acid; Pyrans; Shellfish; Spectrometry, Mass, Electrospray Ionization

Diarrhetic shellfish poisoning (DSP) toxins pose a serious health risk for consumers of bivalves and other shellfish, as well as a huge economic burden for the bivalve-producing farmers. In this work, the aim was to utilize a solubilization-based protein-isolation method to produce a low-DSP toxin protein isolate from toxic blue mussels that are unsuitable for the whole shellfish market. A homogenate of whole mussel meat was solubilized at low pH (2.8) or high pH (11.1), followed by centrifugation and reprecipitation of the solubilized mussel proteins at the isoelectric pH. In a second centrifugation, precipitated proteins were collected. These processes resulted in 81 (acid solubilization) and 72% (alkaline solubilization) reduction in the initial DTX-1 toxin content of the mussel meat. No other DSP toxins were found in the protein isolates. Acid processing of mussel meat resulted in 50% reduction in the total lipid content, while alkaline treatment did not significantly affect the lipid content. The effect of citric acid and calcium chloride addition to the mussel meat-water homogenate on lipid and toxin content was also investigated. A poor correlation factor was surprisingly obtained between reductions in DTX-1 toxin and lipids in protein isolates from processed toxic mussels. Results from an analytical mass balance of the DTX-1 toxin during acid processing showed that 61% of this toxin ended up in the aqueous supernatant after the second centrifugation. The present study presents a promising alternative way of utilizing mussels for food production in periods when they are toxic.

Topics: Animals; Calcium Chloride; Citric Acid; Dinoflagellida; Food Handling; Hydrogen-Ion Concentration; Lipids; Marine Toxins; Mytilus edulis; Okadaic Acid; Proteins; Pyrans

Okadaic acid analogues are well known as protein phosphatase inhibitors and occur naturally in marine shellfish feeding on dinoflagellates of the genus Dinophysis, leading to diarrhetic shellfish poisoning of shellfish consumers. Knowledge of the correct structures for these toxins is important in understanding their toxicology, biochemistry, and biosynthesis. We have performed extensive NMR analyses on okadaic acid (1), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) obtained from natural sources. Consequently, we were able to unambiguously deduce the stereochemistries at C-35 for DTX-1 and DTX-2 based on analysis of NMR coupling constants and NOE interactions. Our results revealed that DTX-2 (3) has a stereochemistry opposite to that of DTX-1 (2) at C-35. Molecular modeling of the docking of 1-3 with protein phosphatase-1 and protein phosphatase 2A (PP2A) suggested that the reduced affinity of DTX-2 for PP2A may be due to the newly defined stereochemistry at the 35-methyl group. The implications of these findings for biosynthesis and toxicology are discussed.

Topics: Animals; Carbon Isotopes; Computational Biology; Dinoflagellida; Magnetic Resonance Spectroscopy; Marine Toxins; Models, Molecular; Molecular Conformation; Nitrogen Isotopes; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Phosphatase 2; Pyrans; Solvents

A cis-isomer of a C(8)-diol ester of okadaic acid (1) was isolated during large-scale purification of pectenotoxins (PTXs) from extracts of Dinophysis acuta collected from the west coast of South Island, New Zealand. The compound was identified by NMR spectroscopic and liquid chromatography-mass spectrometry (LC-MS) studies, and is the first reported cis-isomer of an okadaic acid C(8)-diol-ester identified in Dinophysis. The more abundant trans-C(8)-diol ester of okadaic acid (2) isolated from the same Dinophysis extract was rapidly hydrolyzed to okadaic acid in vitro by the supernatant from green-lipped mussel hepatopancreas.

Topics: Animals; Chromatography, High Pressure Liquid; Dinoflagellida; Esterification; Furans; Hepatopancreas; Hydrolysis; Macrolides; Magnetic Resonance Spectroscopy; Marine Toxins; Molecular Structure; Okadaic Acid; Pyrans; Spectrometry, Mass, Electrospray Ionization

An approach involving chemical and biological techniques was taken for the detection and quantification of the marine toxin okadaic acid (OA) in mussels from Thermaikos Gulf, Greece, during a 4-month DSP episode that occurred in 2002. Samples were analyzed using the mouse bioassay, high performance liquid chromatography (HPLC) with fluorimetric detection and an enzyme-linked immunosorbent assay (ELISA). Okadaic acid was quantifiable at three different sites of Thermaikos Gulf reaching a maximum concentration of 36 microg/g hepatopancreas. High correlation was revealed between results derived from HPLC and ELISA (R2=0.998), while 91% consistency between HPLC and the mouse bioassay results was observed.

Topics: Animals; Chromatography, High Pressure Liquid; Diarrhea; Environmental Monitoring; Enzyme-Linked Immunosorbent Assay; Food Contamination; Greece; Marine Toxins; Mice; Mytilus; Okadaic Acid; Pyrans; Shellfish Poisoning

Prorocentrum lima was isolated from the coastal Fleet lagoon, Dorset, UK in 2000 and a number of clonal cultures established. These were analyzed for okadaic acid (OA), dinophysistoxin-1 (DTX-1), DTX-2, DTX-4 and diol esters by liquid chromatography coupled to mass spectrometry. OA concentrations varied from 0.4 to 17.1pg OAcell(-1) and DTX-1 from 0.4 to 11.3pg DTX-1cell(-1); DTX-2 was not detected in these isolates. OA and DTX-1 were detected in the culture media, as a result of toxin excretion. DTX-4 and a selection of DTX-4 diol esters were identified using selected ion monitoring, although not all strains produced these compounds. Cell size and number of marginal and valve pores of each strain were observed using scanning electron microscopy. OA and DTX-1 concentrations, pigment content and changes in nitrate and phosphate concentrations in the culture media were followed during growth of one strain of P. lima in batch culture. Diarrhetic shellfish poisoning (DSP) toxins have been previously detected in shellfish cultivated in the Fleet lagoon, but in the absence of any Dinophysis sp. cells. The identification of toxic P. lima strains from the Fleet suggests that this dinoflagellate is the most probable source of occasional DSP detected in the lagoon.

Topics: Animals; Chromatography, Liquid; Dinoflagellida; England; Marine Toxins; Mass Spectrometry; Microscopy, Electron, Scanning; Okadaic Acid; Pigments, Biological; Pyrans; Seawater; Species Specificity

A rapid and simple method for confirmation of the diarrhetic shellfish poisons (DSP): okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) using fluorescence detection following derivatization with 9-chloromethylanthracene, has been established as an alternate to LC/MS. Exposure of the anthrylmethyl derivatives of OA, DTX-1 and DTX-2 to near UV light (300-400 nm) resulted in the loss of these compounds to below detection limits within 30 min, with a concurrent appearance of two additional compounds. Based on the mass spectral evidence, we propose that these newly formed compounds are the decarboxylation products of the derivatized diarrhetic shellfish poisons. UV radiation is, therefore, proposed as a rapid and simple confirmation technique for these DSP in mussel samples.

Topics: Animals; Anthracenes; Bivalvia; Chromatography, Liquid; Dinoflagellida; Marine Toxins; Mass Spectrometry; Okadaic Acid; Pyrans; Sensitivity and Specificity; Spectrometry, Fluorescence; Ultraviolet Rays

This paper describes for the first time a massive intoxication episode due to consumption of shellfish contaminated with 7-O-acyl-derivative dinophysistoxin-1, named Dinophysistoxin-3 (DTX-3). 7-O-acyl-derivative dinophysistoxin-1, a compound recently described in the literature, was found in shellfish samples collected in the Chilean Patagonia fjords. This compound does not inhibit Protein Phosphatases and also does not elicit the symptoms described for Diarrheic Shellfish Poisoning (DSP). The data showed here, give evidence of metabolic transformation of 7-O-acyl-derivative dinophysistoxin-1 (DTX-3) into Dinophysistoxin-1 (DTX-1, Methyl-Okadaic acid) in intoxicated patients. This metabolic transformation is responsible for the diarrheic symptoms and the intoxication syndrome showed by patients that consumed contaminated shellfish, which showed only the presence of 7-O-acyl-derivative dinophysistoxin-1. Patients fecal bacterial analysis for the presence of enteropathogens was negative and the mouse bioassay for DSP, performed as described for regulatory testing, was also negative. The HPLC-FLD and HPLC-MS analysis showed only the presence of DTX-3 as the only compound associated to DSP toxins in the contaminated shellfish samples. No other DSP toxins were found in the shellfish sample extracts. However, the patient fecal samples showed DTX-1 as the only DSP toxins detected in fecal. Moreover, the patient fecal samples did not show DTX-3. Since 7-O-acyl-derivative dinophysistoxin-1 (DTX-3) was the only compound associated to DSP toxins detected in the shellfish samples, an explanation for the diarrheic symptoms in the intoxicated patients would be the metabolic transformation of DTX-3 into DTX-1. This transformation should occur in the stomach of the poisoned patients after consuming 7-O-acyl-derivatives dinophysistoxin-1 (DTX-3) contaminated bivalves.

Topics: Abdominal Pain; Adolescent; Adult; Biotransformation; Chile; Diarrhea; Feces; Food Contamination; Gastric Mucosa; Humans; Marine Toxins; Mass Spectrometry; Mytilus edulis; Okadaic Acid; Pyrans; Shellfish; Shellfish Poisoning; Vomiting

Afferent activity, especially in young animals, can have profound influences on postsynaptic neuronal structure, function and metabolic processes. Most studies evaluating activity regulation of cellular components have examined the expression of ubiquitous cellular proteins as opposed to molecules that are specialized in the neurons of interest. Here we consider the regulation of two proteins (voltage-gated potassium channel subunits Kv1.1 and Kv3.1) that auditory brainstem neurons in birds and mammals express at uniquely high levels. Unilateral removal of the avian cochlea leads to rapid and dramatic reduction in the expression of both proteins in the nucleus magnocellularis (NM; a division of the avian cochlear nucleus) neurons as detected by immunocytochemistry. Uniform downregulation of Kv1.1 was reliable by 3 hours after cochlea removal, was sustained through 96 hours, and returned to control levels in the surviving neurons by 2 weeks. The activity-dependent changes in Kv3.1 appear to be bimodal and are more transient, being observed at 3 hours after cochlea removal and recovering to control levels within 24 hours. We also explored the functional properties of Kv1.1 in NM neurons deprived of auditory input for 24 hours by whole-cell recordings. Low-threshold potassium currents in deprived NM neurons were not significantly different from control neurons in their amplitude or sensitivity to dendrotoxin-I, a selective K+ channel antagonist. We conclude that the highly specialized abundant expression of Kv1.1 and 3.1 channel subunits is not permanently regulated by synaptic activity and that changes in overall protein levels do not predict membrane pools.

Topics: Animals; Animals, Newborn; Brain Stem; Cell Count; Chickens; Cochlea; Denervation; Densitometry; Functional Laterality; Immunohistochemistry; In Vitro Techniques; Kv1.1 Potassium Channel; Membrane Potentials; Neural Conduction; Neurons; Neuropeptides; Okadaic Acid; Patch-Clamp Techniques; Potassium Channels; Potassium Channels, Voltage-Gated; Pyrans; Shaw Potassium Channels; Time Factors

Inhibitors of the G2 DNA damage checkpoint can selectively sensitize cancer cells with impaired p53 tumor suppressor activity to killing by DNA-damaging drugs or ionizing radiation and have been proposed as a promising therapeutic strategy. An extract from the Northeastern Pacific marine sponge Merriamum oxeato showed G2 checkpoint inhibitory activity, and fractionation identified the known dinoflagellate toxin dinophysistoxin 1 (1) and the two novel analogues 27-O-acetylokadaic acid (2) and 27-O-acetyldinophysistoxin 1 (3) as the active compounds. The mixture of 1, 2, and 3 was extremely potent at inhibiting the G2 checkpoint (IC(50) = 1 ng/mL) and cellular protein Ser/Thr phosphatases (IC(50) = 1 ng/mL), and it radiosensitized MCF-7 breast cancer cells expressing mutated p53 at all concentrations tested. However, the mixture of 1, 2, and 3 was also very toxic to cells not exposed to DNA damage (IC(50) = 1 ng/mL), making these compounds poor candidates for therapeutic agents to augment the effectiveness of DNA-damaging therapies.

Topics: Animals; Breast Neoplasms; British Columbia; DNA Damage; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Female; Flow Cytometry; G2 Phase; Humans; Inhibitory Concentration 50; Mitosis; Molecular Structure; Okadaic Acid; Phosphoprotein Phosphatases; Porifera; Pyrans; Tumor Cells, Cultured; Tumor Suppressor Protein p53

A benthic toxic dinoflagellate identified as Prorocentrum lima (Syn. Exuviaella lima), and designated as strain PRL-1, was isolated from the coast of El Pardito (Coyote) Island in Baja California Sur, Mexico, after a fisherman poisoning incident involving consumption of liver from Lutjanus colorado, and Mycteroperca prionura fish. Purification and culturing was done in ES-Si medium, under 12:12 light/dark cycle (4 x 20 W cool-white fluorescent lamps), at 22 degrees C and constant stirring during 28 days. Whole cells were toxic to Artemia franciscana and its methanolic extract to mouse and to the marine yeast Debaryomyces hansenii. Chromatographic analysis (TLC and HPLC-MS) of such extract indicated an unusual proportion (1:2) okadaic acid (OA) and dinophysistoxin-1 (DTX-1). Estimated total toxin content by mouse bioassay (based on OA toxicity) was 19 pg/cell, a value significantly higher than that found by HPLC-MS (about 5.2 pg/cell, taking into account OA and DTX-1 only), suggesting that additional toxic components of unidentified nature are detected with the bioassay. This is the first report of a successful isolation and culturing of a toxic dinoflagellate from the Gulf of California, Mexico.

Topics: Animals; Chromatography, Thin Layer; Ciguatera Poisoning; Ciguatoxins; Diarrhea; Dinoflagellida; Foodborne Diseases; Mexico; Mice; Okadaic Acid; Pyrans; Risk Assessment; Shellfish

Okadaic acid (OA) homologues in toxic dinoflagellate Dinophysis fortii, scallops Patinopecten yessoensis and mussels Mytilus galloprovincialis collected at the same site in Mutsu Bay, Japan were determined by high-performance liquid chromatography fluorescence detection (HPLC-FLD) as their 9-anthryldiazomethane (ADAM) derivatives. Prominent toxin in the scallops and the mussels was esterified dinophysistoxin-1 (DTX3) and dinophysistoxin-1 (DTX1), respectively, although only DTX1 was detected in D. fortii. Toxin contents in the mussels were significantly higher than those in the scallops, indicating that mussels have higher potential to accumulate OA homologues than scallops.

Topics: Animals; Chromatography, High Pressure Liquid; Esterification; Mollusca; Okadaic Acid; Pyrans; Species Specificity; Spectrometry, Fluorescence

The DSP toxin composition of 19 Prorocentrum lima isolates from different locations of the Galician rias (Vigo and Pontevedra) was investigated by high performance liquid chromatography coupled with fluorimetric detection. Boiling and freeze/thaw/hydrolyse methodology were applied during extraction to detect OA, DTX1, DTX2 and their esterified derivatives. OA and DTX2 were detected in both free and esterified form, the latter always in very low amounts, whilst DTX1 was always present in the free form. This indicate that the hypothesized self-protection mechanism of toxin storage in the less active esterified forms does not seem to apply to DTX1. A slight increase in the toxin concentration per cell was found during growth, although toxin composition did not vary appreciably. Toxin production and toxin profile varied significantly depending on the isolate. Four groups of P. lima were differentiated by cluster analysis according to their toxin composition. It is noteworthy that one of the clusters comprised all the strains collected from one location characterised by its geographical isolation, whereas the other clusters consisted of isolates from different locations. The differences in the toxin profile from P. lima strains and from the DSP contaminated shellfish, together with the very good correlation between Dinophysis spp occurrence and DSP toxicity in shellfish, support that these planktonic species are the main agents responsible for DSP events in Galicia.

Topics: Animals; Chromatography, High Pressure Liquid; Cluster Analysis; Dinoflagellida; Enzyme Inhibitors; Esters; Fluorometry; Foodborne Diseases; Marine Toxins; Okadaic Acid; Pyrans; Spain; Statistics as Topic

Two kinds of diarrhoetic shellfish toxins, okadaic acid (OA) and dinophytoxin-1 (DTX-1) were determined by micellar electrokinetic capillary chromatography (MEKC) with ultraviolet detection. A detection limit of 3.25 microg/mL for both of them was achieved. The UV absorbance of these toxins measured at 200 nm showed good linearity in the range of 6.25-200 microg/mL with R = 0.992 for OA and 0.997 for DTX-1. Three kinds of shellfish (Chlamys farreri, Mytilus edulis and Ruditaps philippinarum) collected from eight locations (sampling in the intertidal zone) along the Dalian Bay sea area of China were surveyed in February and May of 2000. Results indicated that three kinds of shellfish were contaminated by OA and DTX-1. Based on per gram of hepatopancreas in February, the contamination contents ranged from 0 to 1.26 microg for OA and from 0 to 1.82 microg for DTX-1, and in May, the contents ranged from 0 to 1.45 microg for OA and 0 to 2.56 microg for DTX-1. Among the eight locations, Hei Shi Jiao and Long Wang Tang were the most contaminated areas. Of the three kinds of shellfish, Mytilus edulis was the most significant species in accumulating OA and DTX-1.

Topics: Animals; Bivalvia; China; Chromatography, Micellar Electrokinetic Capillary; Food Contamination; Marine Toxins; Molecular Structure; Mollusca; Oceans and Seas; Okadaic Acid; Pyrans; Shellfish

Protein phosphatases are involved in many cellular processes. One of the most abundant and best studied members of this class is protein phosphatase type-2A (PP2A). In this study, PP2A was purified from the mussel Mytilus chilensis. Using both SDS-PAGE and size exclusion gel filtration under denaturant conditions, it was confirmed that the PP2A fraction was essentially pure. The isolated enzyme is a heterodimer and the molecular estimated masses of the subunits are 62 and 28 kDa. The isolated PP2A fraction has a notably high p-NPP phosphatase activity, which is inhibited by NaCl. The hydrolytic p-NPP phosphatase activity is independent of the MgCl2 concentration. The time courses of the inhibition of the PP2A activity of p-NPP hydrolysis by increasing concentrations of three phycotoxins that are specific inhibitors of PP2A are shown. Inhibitions caused by Okadaic acid, dinophysistoxin-1 (DTX1, 35-methylokadiac acid) and Microcystine L-R are dose-dependent with inhibition constants (Ki) of 1.68, 0.40 and 0.27 nM respectively. Microcystine L-R, the most potent phycotoxin inhibitor of PP2A isolated from Mytilus chilensis with an IC50 = 0.25 ng/ml, showed the highest specific inhibition effect an the p-NPP hydrolisis. The calculated IC50 for DTX1 and OA was 0.75 ng/ml and 1.8 ng/ml respectively.

Topics: Animals; Bivalvia; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Marine Toxins; Microcystins; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Phosphatase 2; Pyrans

Determination of diarrhetic shellfish-poisoning (DSP) toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-6 (PTX6) was carried out by liquid chromatography (LC) followed by on-line atmospheric pressure electrospray ionization-mass spectrometric (ESI-MS) detection with a heated capillary interface. Mass spectra of authentic OA, DTXI and PTX6 standards exhibited abundant [M-H] at m/z 803, 817 and 887, respectively. Linearity of peak area obtained by selected-ion monitoring (SIM) for [M-H]- of each toxin was confirmed over a wide range of concentrations from 10 pg to 30 ng. LC-ESI-MS analysis of OA, DTX1 and PTX6 in scallops and mussels, collected at the same site (Mutsu Bay, Japan), was carried out. Scallops and mussels collected at the same site showed different toxin profiles. Although PTX6 was detected from scallops, it was not detected from mussels.

Topics: Animals; Bivalvia; Chromatography, Liquid; Diarrhea; Furans; Hydrolysis; Macrolides; Mass Spectrometry; Mollusk Venoms; Okadaic Acid; Pyrans; Reference Standards; Toxins, Biological

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.

Topics: Calibration; Chlorine Compounds; Chromatography, High Pressure Liquid; Diarrhea; Fluorometry; Humans; Mollusk Venoms; Okadaic Acid; Pyrans; Quality Control; Reference Standards; Reproducibility of Results; Shellfish; Solvents

Non-toxic scallops Patinopecten yessoensis were reared for 5 days with the toxic dinoflagellate Dinophysis fortii sampled from the sea. The total cell number ingested by five scallops during the feeding experiment was approximately 682 x 10(3). Okadaic acid (OA), dinophysistoxin-1 (DTX1) and esterified toxins of OA and DTX1 in D. fortii and scallop extracts were determined by liquid chromatography fluorescence detection (LC-FLD) as their 9-anthryldiazomethane (ADAM) derivatives. Only DTX1 was detected in D. fortii extracts used for the feeding experiment. The content of esterified DTXI in scallops fed on D. fortii was significantly higher than that of DTX1. Identification of esterified DTX1, 7-O-palmitoyl DTX1 (7-O-16:0 DTX1), in D. fortii and scallop extracts was carried out by LC equipped with an atmospheric pressure electrospray ionization-mass spectrometry (ESI-MS). Only extracts of scallops fed on D. fortii yielded a mass spectrum exhibiting abundant [M-H]- at m/z 1055 corresponding to 7-O-16:0 DTX1. These findings are the first direct evidence of the transformation of DTX1 to 7-O-acyl DTX1 in scallop tissues.

Topics: Animals; Biotransformation; Chromatography, High Pressure Liquid; Dinoflagellida; Exocrine Glands; Hydrolysis; Indicators and Reagents; Marine Toxins; Mass Spectrometry; Mollusca; Okadaic Acid; Pyrans; Spectrometry, Fluorescence

This report highlights the characteristics of an okadaic acid immunoassay with limits of detection in the subfemtomole range. Two different immunoassay formats were investigated and their characteristics compared in relation to linear ranges, limits of detection, and cross-reactivity with other seafood toxins present in water and/or mussel samples. The developed ELISA system can be manipulated to quantitatively measure total diarrhetic shellfish poisoning (DSP) content or for okadaic acid and dinophysistoxin-1 individual concentrations by variation of the format of the immunoassay. Real mussel samples were validated in percentage recovery test. Calibration curves were established, and aliquots of real samples were tested. Very good recoveries were attained, highlighting the validity of the ELISA system to accurately determine the DSP concentration in mussel samples.

Topics: Animals; Bivalvia; Calibration; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Isomerism; Okadaic Acid; Pyrans; Sensitivity and Specificity; Water

Further studies on mussel samples from Galicia, Spain, have revealed the presence of okadaic acid (OA), dinophysistoxin 2 (DTX2), and the fatty acid acyl esters of both of these toxins as the "DTX3" complex. Measurements were performed with an improved in situ method for the formation of 9-anthryldiazomethane (ADAM) derivatives followed by liquid chromatography with fluorescence detection. Base hydrolysis of DTX3 toxins gave free OA and DTX2, which were determined following ADAM derivatization. Results were confirmed by liquid chromatography/mass spectrometry analyses, and in most of the samples, free DTX2 was the most abundant toxin. However, the OA/DTX2 ratio in the DTX3 conjugated form was different, with OA being the most abundant in all cases. This difference could be due to different rates of metabolism of OA and DTX2 to the acyl esters or due to contamination of the shellfish by the two toxins at different points in time, resulting in less acyl ester formation for one toxin versus the other. The second possibility would be reasonable if two different source organisms were producing the toxins.

Topics: Animals; Bivalvia; Chromatography, Liquid; Mass Spectrometry; Okadaic Acid; Pyrans; Spectrometry, Fluorescence

A negative mode liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) method was developed to detect low concentrations of the diarrhetic shellfish poisoning (DSP) toxins okadaic acid and dinophysistoxin-1 (DTX-1). Detection relies upon monitoring the transition of negative precursor ions [M - H]- to a common fragment ion of m/z 255. Our limit of detection for okadaic acid with this method is 0.5 pg on column. LC-SRM MS has allowed us to detect persistent, low concentrations of DSP toxins from Singapore shellfish.

Topics: Animals; Bivalvia; Chromatography; Okadaic Acid; Pyrans; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization

A simplified procedure for the enzyme inhibition assay to measure okadaic acid and DTX-1 in mussels, based on the use of a commercially available enzyme preparation, is presented. The detection limit is 10 ng of toxin per g of digestive glands. Using Certified Reference Material (MUS-2), high accuracy and good precision is demonstrated for contamination levels higher than 32 ng g(-1). Twenty samples can be processed in about 9 h by one operator, at the cost of US$ 10 per sample. Some possibilities for further enhancing the sensitivity and reducing the processing time are discussed and a monitoring example is presented.

Topics: Animals; Biological Assay; Bivalvia; Environmental Monitoring; Enzyme Inhibitors; Mice; Okadaic Acid; Phosphoprotein Phosphatases; Pyrans; Reproducibility of Results; Sensitivity and Specificity

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%.

Topics: Animals; Chromatography, High Pressure Liquid; Circadian Rhythm; Diarrhea; Fluorescent Dyes; Foodborne Diseases; Marine Toxins; Mollusca; Okadaic Acid; Pyrans; Pyrenes; Reproducibility of Results; Sensitivity and Specificity; Shellfish; Spectrometry, Fluorescence

The ability of voltage-gated potassium channel alpha-subunits to form heteromultimers has complicated efforts to use toxins to characterize potassium channels in native cells. Here I investigate the effects of subunit composition on toxin blocking affinity, using three members of the Shaker subfamily of potassium channel alpha-subunits (Kv1.1, Kv1.2 and Kv1.4), which are known to form heteromultimers in vivo, in the Xenopus oocyte expression system. These subunits were coexpressed as pairs in which one member was toxin-sensitive and the other relatively insensitive. The blocking affinity of two dendrotoxins (DTX-I and delta-DTX) and a scorpion toxin (tityustoxin-Kalpha) on the resulting mixed population of channels was measured to evaluate three models of toxin block. The single subunit model, in which a single toxin-sensitive subunit renders the channel toxin sensitive, best described all of the data for the two dendrotoxins and the block of tityustoxin-Kalpha for a mixed population of channels composed of Kv1.1 and Kv1.2 subunits. However, with tityustoxin-Kalpha, the data for a mixed population of Kv1.2 and Kv1.4 subunits was fit best by a model in which the toxin interacts with all four subunits for high-affinity block. The data suggest that subunit composition of potassium channels can have a large effect on toxin block and that different toxins yield strikingly diverse results with the same pair of subunits, even when they are nearly identical in blocking affinity for the toxin-sensitive subunit.

Topics: Animals; Elapid Venoms; Electrophysiology; Female; Okadaic Acid; Oocytes; Potassium Channels; Pyrans; Scorpion Venoms; Toxins, Biological; Xenopus laevis

Mussel samples from four locations along the Norweigian coast were extracted by methods for diarrheic shellfish toxins (DST) and tested by chemical and biological methods, including histopathology. All samples had previously been found to be highly toxic in mice, with symptoms indicating the presence of non-diarrheagenic toxins in the mouse bioassay. Chemical analysis revealed that the DST okadaic acid (OA) and dinophysistoxin-1 (DTX1) were present each one in one sample, but only a minor part of the total toxicity could be attributed to these toxions. In the other two samples, OA and DTX1 were absent. Incubation of the mussel extracts from all four samples with freshly prepared hepatocytes indicated the presence of unknown toxin(s) which may not be classified within the DST complex. Purified mussel samples were given to baby mice both via intraperitoneal (i.p.) injections and by oral intubation. Oral toxicity was about 25-50 times lower than toxicity obtained by i.p. injections, a result in accordance with acute toxic properties of many toxins. Risk assessment of the unknown toxin(s) requires chemical identification, but the preliminary results obtained indicate a large margin of safety, based on the large amounts of mussel extracts necessary to yield toxic effects in the intestine and liver in experimental animals upon oral exposure versus human intake.

Topics: Administration, Oral; Animals; Bivalvia; Dinoflagellida; Injections, Intraperitoneal; Intestines; Jejunum; Liver; Marine Toxins; Mice; Norway; Okadaic Acid; Pyrans; Rats; Shellfish

A number of algal toxins were tested for the ability to induce apoptosis (regulated cell death) in primary hepatocytes from salmon and rat. The tested toxins included the liver targeting substances microcystin-LR and nodularin, substances associated with the diarrhetic shellfish poison complex (okadaic acid, dinophysistoxin-1 and pectenotoxin-1) and calyculin A. All toxins induced apoptosis in both salmon and rat hepatocytes in less than 2 h. The apoptotic changes were evident both by electron and light microscopy and were counteracted by the caspase inhibitor ZVAD-fmk and by the Ca2+/calmodulin dependent kinase II inhibitor KN-93. The salmon hepatocytes were 10-20-fold more sensitive to okadaic acid and dinophysistoxin-1 (EC50=20 nM) than rat hepatocytes and other mammalian cell lines tested. An assay was devised using hepatocyte apoptosis as parameter for detection of algal toxins. This assay was at least as sensitive as HPLC determination for okadaic acid in mussel extracts. It also detected algal toxins which do not inhibit protein phosphatases, like pectenotoxin-1. Subapoptotic concentrations of the toxins inhibited hepatocyte aggregation. Using this parameter, less than 200 pg okadaic acid could be detected. In conclusion, salmon hepatocytes in suspension culture provide a rapid and sensitive system for detection of a broad range of apoptogenic toxins.

Topics: Animals; Apoptosis; Cell Aggregation; Cells, Cultured; Eukaryota; Liver; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Pyrans; Rats

Two human cell lines have been used, HEp-2 and (de)differentiated Caco-2, derived from a larynx and a colon carcinoma, respectively, with the aim of evaluating and characterizing the cytotoxicity of okadaic acid (OA) and related toxins. Effects of OA and dinophysistoxin-1 (DTX-1) on cell viability (neutral red uptake) and on cell morphology/cytoskeleton structure have been observed in both cell lines, though at different time exposures and with different concentrations. The morphological alteration was detected earlier than the viability inhibition in HEp-2 cells with both toxins and in Caco-2 cells with DTX-1. HEp-2 cells have shown to be more sensitive than the intestinal cell line and thus possibly suitable for screening of contaminated samples, while Caco-2 cells could be used for further investigating the possible mechanisms involved in diarrhoeic shellfish poisoning (DSP) toxins.

Topics: Apoptosis; Caco-2 Cells; Cell Nucleus; Cell Survival; Coloring Agents; Cytoskeleton; Epithelial Cells; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Inhibitory Concentration 50; Laryngeal Neoplasms; Marine Toxins; Neutral Red; Okadaic Acid; Pyrans; Tubulin; Tumor Cells, Cultured

Diarrhea associated with shellfish poisoning is poorly understood. The responsible toxin, dinophysistoxin 1, has been identified as okadaic acid, a potent phosphatase inhibitor, but its effects on intestinal epithelia have not been examined. The aim of this study was to investigate the effect of okadaic acid on intestinal epithelial function, both Cl- secretion and barrier function.. Cultured human intestinal epithelial T84 cell monolayers were used. The effect of okadaic acid on these monolayers was assessed by measuring electrophysiological parameters, lactate dehydrogenase release, and 22Na+ and [3H]mannitol flux rates. Protein phosphorylation studies were performed to identify potentially involved proteins.. Okadaic acid does not directly stimulate Cl- secretion from intestinal epithelial cells. On the contrary, the response to well-characterized secretagogues is attenuated by okadaic acid. However, it does decrease transepithelial electrical resistance in a polarized fashion without inducing cytotoxicity. Sodium-mannitol flux studies suggest that the observed decrease in resistance is attributable to an increase in paracellular permeability.. Okadaic acid, the toxin responsible for diarrheic shellfish poisoning, does not stimulate Cl- secretion but increases the paracellular permeability of intestinal epithelia. This alteration in intestinal epithelial physiology may contribute to the diarrhea of shellfish poisoning.

Topics: Cell Membrane Permeability; Cells, Cultured; Electric Impedance; Enzyme Inhibitors; Gap Junctions; Humans; Intestinal Mucosa; Marine Toxins; Membrane Glycoproteins; Okadaic Acid; Phosphorylation; Pyrans

In routine monitoring of diarrhoeic shellfish poison (dinophysistoxin-1 and okadaic acid) there appeared to be an inconsistency between the mouse bioassay and existing chemical analysis based on liquid chromatography. The sample cleanup procedure has been subject to minor modifications in an effort to overcome the problem. However, further studies have appeared necessary and in this study all experimental factors that can influence the sample cleanup using solid-phase extraction columns prior to the LC analysis have been evaluated by use of experimental statistical design in order to understand the effect of the various factors and to optimize the conditions for recovery of the toxins. Based on our experiments we suggest using a solid-phase extraction silica column of 100 mg in the sample cleanup procedure and washing solvents composed of dichloromethane instead of chloroform to minimize the effect of stabilizing alcohol. It is sufficient to apply 7.5 ml hexane-dichloromethane (1:1) to the column in the first washing step and 2.5 ml of dichloromethane in the final washing. Elution is complete by use of 2.5 ml chloroform with 3.5% methanol. Toxic shellfish tested by this procedure confirmed the mouse bioassay.

Topics: Animals; Chromatography, High Pressure Liquid; Dinoflagellida; Marine Toxins; Mice; Okadaic Acid; Pyrans; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Fluorescence

Consumption of shellfish contaminated with algal toxins produced by marine dinoflagellates can lead to diarrhetic shellfish poisoning (DSP). UK legislation necessitates toxin detection by mouse bioassay but this method is non-specific and lacks sensitivity. As an alternative method, an HPLC technique has been optimized, with detection limits of 0.26 micrograms of toxin/g of shellfish hepatopancreas for both Okadaic Acid (OA) and Dinophysistoxin-1 (DTX-1). A colorimetric protein phosphatase inhibition (PPI) assay has also been optimized. This assay detects inhibition of protein phosphatase 1 (PPI gamma) by OA and DTX-1 with detection limits of 1.5 ng of total toxin/g of hepatopancreas. Contaminated shellfish from several European sources, the UK monitoring programmes and mussels associated with an outbreak of DSP poisoning in the UK, have been analyzed and assessed using the two alternative methods and a commercially available enzyme-linked immunosorbent assay (ELISA) kit. The results indicate that both the HPLC and PPI assays correlate well with each other and with the UK standard mouse bioassay. In contrast, and not withstanding its advantages of rapidity and ease, the ELISA kit did not accurately and consistently detect low toxin concentrations, although it may be useful as a screening tool.

Topics: Animals; Biological Assay; Chromatography, High Pressure Liquid; Colorimetry; Diarrhea; Enzyme-Linked Immunosorbent Assay; Europe; Foodborne Diseases; Humans; Liver; Marine Toxins; Mice; Mice, Inbred BALB C; Okadaic Acid; Pancreas; Phosphoprotein Phosphatases; Protein Phosphatase 1; Pyrans; Sensitivity and Specificity; Shellfish; United Kingdom

The reagent 9-chloromethylanthracene was evaluated for derivatization of the diarrhetic shellfish poisons, okadaic acid and dinophysistoxin-1 (DTX-1), to form fluorescent products separable by liquid chromatography. The toxins were reacted with the reagent in acetonitrile in the presence of tetramethylammonium hydroxide for 1 h at 90 degrees C. The products were purified by using two silica solid-phase extraction cartridges before being determined by reversed-phase liquid chromatography with fluorescence detection. The results are comparable to those obtained using 9-anthryldiazomethane (ADAM) for okadaic acid and DTX-1 in mussel tissue. Detection limits were estimated to be about 70-100 ng/g hepatopancreas (equivalent to 12-20 ng/g whole tissue) for each toxin.

Topics: Animals; Anthracenes; Bivalvia; Chromatography, Liquid; Diarrhea; Ethers, Cyclic; Indicators and Reagents; Marine Toxins; Okadaic Acid; Pyrans; Shellfish

Mussel aquaculture is an important industry for the Galician Rias, located in northwestern Atlantic coast of Spain. Since 1976 this region has been seriously affected by incidents of paralytic and diarrhetic shellfish poisoning (PSP and DSP). A particularly bad episode occurred in 1993, when the toxic event lasted for an unusually long period. Many people were stricken ill with unusual symptoms. In this paper we report on the chemical analysis of toxic 1993 mussel samples, using the techniques of liquid chromatography and capillary electrophoresis coupled with mass spectrometry. These analyses revealed a very complex toxin profile, with both PSP and DSP toxins present. Two DSP toxins, okadaic acid and DTX2, were observed, while the primary PSP toxins were B1 and the decarbamoylated derivatives of saxitoxin, GTX2 and GTX3. Small amounts of saxitoxin and other as yet unidentified PSP toxins were observed.

Topics: Animals; Aquaculture; Bivalvia; Carcinogens; Chromatography, High Pressure Liquid; Diarrhea; Dinoflagellida; Electrophoresis, Capillary; Gas Chromatography-Mass Spectrometry; Marine Toxins; Okadaic Acid; Paralysis; Pyrans; Shellfish Poisoning; Spain; Structure-Activity Relationship

A rapid HPLC method with fluorescence detection for the determination of okadaic acid (OA) and dinophysistoxin-1 (DTX-1) in mussels and mussel products is presented. For fluorescence labelling of OA and DTX-1, 9-anthryldiazomethane (ADAM) is used. HPLC with a column-switching system is proposed to avoid time-consuming clean-up procedures after derivatization of sample extracts with ADAM. The column-switching system as well as the chromatographic conditions and detection are described.

Topics: Animals; Bivalvia; Chromatography, High Pressure Liquid; Diarrhea; Digestive System; Indicators and Reagents; Marine Toxins; Okadaic Acid; Pyrans; Specimen Handling; Spectrometry, Fluorescence

The fluorimetric determination of okadaic acid (OA) and dinophysistoxin-1 (DTX-1), the principal toxins of diarrhetic poisoning, is reported. The digestive glands of mussels or scallops were homogenized with 2-propanol. OA and DTX-1 were extracted from the homogenate, with hexane-ethyl acetate and labelled with 2,3-(anthracenedicarboximido)ethyl trifluoromethanesulfonate in dry acetonitrile. After cleaning up by passage through a short silica gel column, the fluorescent derivatives were determined by HPLC. The derivatives were at first separated on a Develosil Ph-5 column, and only the target fraction obtained was introduced into a Develosil ODS K-5 column by a valve-switching device. Both toxins were determined in the range 2.5-500 pg, and the detection limits were 0.8 pg (OA) and 1.3 pg (DTX-1) with a signal-to-noise ratio of 3.

Topics: Animals; Bivalvia; Chromatography, High Pressure Liquid; Diarrhea; Digestive System; Indicators and Reagents; Marine Toxins; Okadaic Acid; Pyrans; Shellfish; Spectrometry, Fluorescence

Direct detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and some of their related compounds in toxic mussels (Mytilus galloprovincialis) is reported using ionspray liquid chromatography-mass spectrometry (LC-ISP-MS). This was employed to analyse diarrhoetic shellfish poisoning (DSP) toxins in mussels collected from coastal areas of the northern and southern Adriatic Sea. DTX-1 was found in some samples from both the northern and southern Adriatic and this is the first report of the unambiguous identification of this toxin in Italian mussels. The low levels found indicate that this toxin did not play a significant role in toxicity in these samples. Okadaic acid was found in all the mussels examined, although its concentration was not always sufficient to account for DSP toxicity. Furthermore, two related compounds of OA were detected in all the samples and one related DTX-1 compound was observed in some samples from the northern Adriatic. All three compounds are still to be identified, but it is possible that these substances are involved in mussel DSP toxicity in the Adriatic Sea.

Topics: Animals; Bivalvia; Carcinogens; Chromatography, High Pressure Liquid; Chromatography, Liquid; Dinoflagellida; Fish Venoms; Italy; Marine Toxins; Mass Spectrometry; Okadaic Acid; Pyrans; Reference Standards; Shellfish

The injuries and repair processes in the intestines of mice induced by dinophysistoxin 3 (DTX 3) were compared morphologically to those induced by okadaic acid (OA) and dinophysistoxin 1 (DTX 1). DTX 3 impaired intestinal villi by the oral route only, whereas OA and DTX 1 caused intestinal injury with both oral and intraperitoneal exposures. The character of the lesions caused by the 3 toxins and the recovery processes were highly similar. Within 5 min of dosing, the basal portion of the covering epithelium became homogeneous and peeled from the lamina propria, while the upper portion containing microvilli remained intact. There were two types of villous injury and recovery: 1) When the injuries were limited to the villi, new cells from the crypts moved upward and differentiated into columnar cells. 2) When injuries progressed into the glands of Lieberkuhn, clusters of crypt cells were exposed to the intestinal lumen, and in the most severe case they were completely separated. Villous fusion was often seen in the recovery process of the type 2 cases. Recovery from the injuries was almost completed within 2 days. When mice were pretreated with fusarenon-X, a mycotoxin which injuries undifferentiated crypt cells preferentially, the injury induced by OA to the intestinal crypts was exacerbated and the recovery was delayed.

Topics: Administration, Oral; Animals; Carcinogens; Cell Differentiation; Dinoflagellida; Epithelium; Ethers, Cyclic; Injections, Intraperitoneal; Intestines; Jejunum; Male; Marine Toxins; Mice; Mice, Inbred ICR; Microscopy, Electron; Microscopy, Electron, Scanning; Mycotoxins; Okadaic Acid; Pyrans; Trichothecenes

The specificity of five mouse monoclonal antibodies to okadaic acid was studied for use in an enzyme-linked immunosorbent assay of okadaic acid and its analogs. OA8-2 and OA22-22 antibodies (IgG2a-kappa), which bind more strongly to dinophysistoxin-1 and 7-O-palmitoyl-dinophysistoxin-1 than to okaic acid or 7-O-palmitoyl-okadaic acid in 50% aqueous methanol, were useful in the detection of dinophysistoxins-1 and -3. OA10-8 (IgG1-kappa), which binds more strongly to 7-O-palmitoyl-okadaic acid and 7-O-palmitoyl-dinophysistoxin-1 than to okadaic or dinophysistoxin-1 in 50% aqueous methanol, was useful in the detection of dinophysistoxin-3. OA423-3 (IgG1-kappa), which binds weakly to dinophysistoxin-1 and 7-O-palmitoyl-dinophysistoxin-1 in 20% aqueous methanol, was useful in the selective detection of okadaic acid. OA958-2 (IgG1-kappa), which binds with equal strength to each of the four toxins in methanol, was useful in the detection of all okadaic acid analogs, and the minimum detectable concentration was 30 ng/ml. OA423-3 and OA958-2 retained their binding ability in 50% acetone, ethyl ether, or benzene in methanol.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Dinoflagellida; Enzyme-Linked Immunosorbent Assay; Ethers, Cyclic; Marine Toxins; Mice; Okadaic Acid; Pyrans

The diarrhetic shellfish poisoning toxin-producing dinoflagellate, Prorocentrum lima, isolated from Nova Scotian waters, contained both okadaic acid (OA) and dinophysistoxin-1 (DTX-1) throughout its growth cycle in culture; maximum concentrations of toxins and highest OA/DTX-1 ratios occurred during the stationary phase. Cells of P. lima survived 0 degrees C for 5 weeks and recovered when brought to a higher temperature. During the cold period, some cell damage probably occurred with concomitant losses of toxins to the medium. Nitrogen concentration in the medium was used to limit growth or stress the cells physiologically, and when growth was limited, increases in toxin associated with the cells were recorded. The relative amounts of okadaic acid were always greater than dinophysistoxin-1, but the significance of these ratios remains to be determined.

Topics: Animals; Cell Count; Chromatography, High Pressure Liquid; Cold Temperature; Culture Media; Dinoflagellida; Ethers, Cyclic; Marine Toxins; Nitrogen; Okadaic Acid; Phosphoprotein Phosphatases; Pyrans; Reference Standards; Shellfish; Spectrometry, Fluorescence

Several derivatization reagents for the conversion of okadaic acid and related DSP toxins to fluorescent derivatives for analysis by liquid chromatography have been examined, viz: 9-anthryldiazomethane (ADAM), 1-pyrenyldiazomethane (PDAM), 4-diazomethyl-7-methoxycoumarin (DMMC), 4-bromomethyl-7-methoxycoumarin (BrMMC), 4-bromomethyl-7,8-benzcoumarin (BrMBC), 4-bromomethyl-7-acetoxycoumarin (BrMAC), and 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC). The ADAM reagent provides the greatest selectivity and sensitivity, but its application on a routine basis has been limited by its instability and cost. Improvement of this method was achieved through the production of ADAM in situ from the stable 9-anthraldehyde hydrazone. A detection limit of 30 ng/g hepatopancreas (equivalent to 6 ng/g whole tissue) was achieved. The other aryldiazomethane reagents were found to have insufficient reactivity. Of the bromomethylcoumarin reagents, BrDMC was found to have the greatest promise. The reagent is inexpensive and has excellent stability and purity. Quantitative derivatization may be achieved in a 2 hour reaction at 45 degrees C with N,N-diisopropylethylamine as a catalyst. Unfortunately, the lower reaction selectivity of BrDMC compared to that of ADAM limits its application to isolated toxins, plankton samples, and shellfish tissues with high levels of DSP toxins. The use of BrDMC for the determination of how toxin levels in shellfish tissues will require development of a more extensive clean-up prior to derivatization. Successful application of the ADAM and coumarin derivatization methods to real-world samples has been demonstrated.

Topics: Animals; Anthracenes; Chromatography, High Pressure Liquid; Coumarins; Dinoflagellida; Ethers, Cyclic; Fluorescent Dyes; Marine Toxins; Okadaic Acid; Pyrans; Pyrenes; Shellfish; Spectrometry, Fluorescence; Temperature; Umbelliferones

Effects of compounds of the okadaic acid type (okadaic acid, dinophysistoxin-1, calyculin A and tautomycin) on proliferation by digestive-tract epithelial cells were investigated in mice and rats. In mice, a single oral administration of these agents caused significant enhancement of BrdU labeling indices in a dose/response manner. Exceptions showing no response were limited to the pyloric mucosa for okadaic acid, the pyloric and fundic mucosa for calyculin A and the pyloric mucosa for tautomycin. Sequential analysis of labeling indices after a single oral administration of dinophysistoxin-1 revealed two peaks of cell proliferation at 18 h and 36 h in the esophagus, ileum and colon. The labeling indices of the forestomach, fundus, pylorus and jejunum, on the other hand, continuously increased from 6 h after the administration. Elevated proliferation was also observed in the skin after 30 h or after, but no effects on the liver or kidney were evident. A single oral administration of the okadaic acid type of compounds also dose-dependently enhanced cell proliferation of the rat digestive tract. These results strongly suggest that the okadaic acid class of compounds may exert promoting potential for the gastrointestinal mucosa when administered orally.

Topics: Administration, Oral; Animals; Antifungal Agents; Bromodeoxyuridine; Carcinogens; Cell Division; Dose-Response Relationship, Drug; Ethers, Cyclic; Intestinal Mucosa; Male; Marine Toxins; Mice; Mice, Inbred ICR; Okadaic Acid; Oxazoles; Pyrans; Rats; Rats, Sprague-Dawley; Spiro Compounds; Time Factors

Liquid chromatography (LC)-linked protein phosphatase 1/2A (PP-1/PP2A) bioassay was used to quantitatively identify diarrhetic shellfish toxins in marine phytoplankton (cultured and natural assemblages) and commercially available mussels. Using this approach, multiple protein phosphatase inhibitor profiles of varying composition were found in diarrhetic mussels from Holland and Canada. Based on LC elution positions and relative activity versus PP-1 and PP-2A, at least six inhibitors distinct from known diarrhetic shellfish toxins were identified and termed mussel phosphatase inhibitor (MPI) 19,22,23,25,33 and 42. The levels of these inhibitors, in okadaic acid equivalent units, varied from 100 pg to 3350 ng per g shellfish tissue. The combined levels of PP-1/2A inhibitors in all instances superseded that of okadaic acid/dinophysistoxin-1 and may contribute to the diarrhetic shellfish toxin profile of the contaminated mussels. The efficacy of LC-protein phosphatase bioassay was established for cultured phytoplankton where picogram levels of okadaic acid could be detected from microgram extracts of Prorocentrum lima. Analyses of plankton net tows from estuarine mussel culture sites in Eastern Canada revealed a heterogeneous population of protein phosphatase inhibitors, with dinophysistoxin-1 being most prevalent. This toxin was predominant for at least 2 months in mussel populations in the immediate vicinity of plankton sampling sites. The results are consistent with a hypothetical model in which marine bacteria, cyanobacteria and dinoflagellates combine to produce a variety of protein phosphatase inhibitors effective against signal transduction pathways in higher eukaryotes.

Topics: Animals; Bivalvia; Chromatography, Liquid; Diarrhea; Ethers, Cyclic; Marine Toxins; Okadaic Acid; Phosphoprotein Phosphatases; Phytoplankton; Protein Phosphatase 1; Pyrans; Rabbits

Phosphorylation by protein kinases has been established as a key factor in the regulation of cytoskeletal structure. However, little is known about the role of protein phosphatases in cytoskeletal regulation. To assess the possible functions of protein phosphatases in this respect, we studied the effects of the phosphatase inhibitors calyculin A, okadaic acid, and dinophysistoxin 1 (35-methylokadaic acid) on BHK-21 fibroblasts. Within minutes of incubation with these inhibitors, changes are seen in the structural organization of intermediate filaments, followed by a loss of microtubules, as assayed by immunofluorescence. These changes in cytoskeletal structure are accompanied by a rapid and selective increase in vimentin phosphorylation on interphase-specific sites, and they are fully reversible after removal of calyculin A. The results indicate that there is a rapid phosphate turnover on cytoskeletal intermediate filaments and further suggest that protein phosphatases are essential for the maintenance and structural integrity of two major cytoskeletal components.

Topics: Animals; Cell Line; Cricetinae; Cytoskeleton; Ethers, Cyclic; Interphase; Kinetics; Marine Toxins; Okadaic Acid; Oxazoles; Peptide Mapping; Phosphopeptides; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Kinases; Pyrans; Vimentin

Okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, induces differentiation in human MCF-7, AU-565, and MB-231 breast tumor cells. In MCF-7 cells, OA elicited within 5 min an increase in the levels of a set of phosphorylated cellular proteins, within hours expression of the early response genes junB, c-jun, and c-fos, and within days manifestation of differentiation. Differentiation was also induced by two related protein phosphatase inhibitors, but not by an inactive OA derivative or by an inhibitor that penetrates epithelial cells poorly. These results indicate that OA and related agents can induce tumor breast cell differentiation, and this induction is correlated with their ability to inhibit PPH 1 and 2A.

Topics: Antibodies, Monoclonal; Autoradiography; Blotting, Northern; Breast Neoplasms; Caseins; Cell Differentiation; Electrophoresis, Polyacrylamide Gel; Ethers, Cyclic; Female; Fluorescent Antibody Technique; Gene Expression; Genes, fos; Genes, jun; Humans; Isoenzymes; Kinetics; Marine Toxins; Microcystins; Okadaic Acid; Oligonucleotide Probes; Oxazoles; Peptides, Cyclic; Phosphates; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Pyrans; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

A novel method for the detection of acylated diarrhetic shellfish poisoning toxins is reported. Direct determination of these compounds is possible using high performance liquid chromatography coupled with ion-spray mass spectrometry. An extract, purified from the digestive glands of toxic mussels (Mytilus edulis) contaminated with okadaic acid, dinophysistoxin-1, and a recently reported analog, dinophysistoxin-2, was also shown to contain small amounts of dinophysistoxin-3, a mixture of 7-O-acyl ester derivatives of dinophysistoxin-1. In addition, acyl ester derivatives of okadaic acid and dinophysistoxin-2 were also detected by direct LC-MS analysis and confirmed by analysis of their hydrolysis products. This is the first report of the detection of other naturally occurring 7-O-acyl esters similar to dinophysistoxin-3.

Topics: Acylation; Animals; Bivalvia; Chromatography, Liquid; Chromatography, Thin Layer; Diarrhea; Ethers, Cyclic; Hydrolysis; Magnetic Resonance Spectroscopy; Marine Toxins; Mass Spectrometry; Okadaic Acid; Pyrans; Shellfish; Spectrophotometry, Ultraviolet; Vasoconstrictor Agents

An improved liquid chromatographic/mass spectrometric (LC/MS) method utilizing gradient elution and ion-spray ionization is described for the sensitive determination of okadaic acid and dinophysistoxin-1, the principal toxins implicated in cases of diarrhetic shellfish poisoning. The method was used to confirm the presence of both toxins, together with a recently identified isomer of okadaic acid, dinophysistoxin-2, in various samples of cultivated blue mussels (Mytilus edulis) from Canadian and European waters. The method provided a mass detection limit of 0.4 ng for each toxin, thus allowing detection of 40 ng per g of whole mussel tissue (or approximately 10 ng/g if only the digestive glands were used in the assay). Quantitative results obtained by LC/MS were in good agreement with those obtained by derivatization and high-performance liquid chromatography with fluorescence detection.

Topics: Animals; Bivalvia; Ethers, Cyclic; Gas Chromatography-Mass Spectrometry; Marine Toxins; Okadaic Acid; Pyrans

The standard mouse bioassay, used to assess 'diarrhoetic shellfish poison' (DSP), is based on intraperitoneal administration of toxic mussel extracts, and monitoring of survival time within a 24-hr period. Toxic effects on mice were examined for extracts of mussel samples from two different regions of south Norway known to possess toxins of specific properties. Both samples revealed an exponential pattern in the dose-response relationship. Whereas the time lag from injection to death was linearly dependent on mouse weight, the effect of weight also increased with decreased sample toxicity. When tested with doses adjusted for weight, a marked individual variation was found within all size groups of mice. The results imply that, regarding prohibition limits for distribution and sale of mussels, a certain degree of variation with regard to time should be accepted in the testing of parallel samples. On the basis of the results, a revised method for the determination of toxicity by mouse bioassay is proposed for DSP testing. The method is based on administration to two mice of size-adjusted doses of extracts, followed by a 4-hr surveillance period and a 1-hr upper limit of acceptable time variation between parallel samples. The method shows advantages regarding savings of time and money, in precision in determination of toxicity level, as well as curtailed exposure to toxin and reduced suffering of laboratory animals.

Topics: Animals; Biological Assay; Bivalvia; Body Weight; Diarrhea; Dose-Response Relationship, Drug; Ethers, Cyclic; Marine Toxins; Mice; Okadaic Acid; Pyrans

The effects of tautomycin, a protein phosphatase inhibitor, on recycling of cell surface molecules were studied with transferrin receptor (TFR) of human myeloid leukemia K562 cells and with CD4 of murine thymocytes. Tautomycin increased expression of TFR of K562 cells whereas phorbol dibutylate (PDBu) decreased it. Tautomycin inhibited PDBu-induced down-regulation of CD4 although it did not induce up-regulation. Okadaic acid also inhibited down-regulation of CD4 which was induced by PDBu. The results suggest that certain inhibitors of protein phosphatases preferentially inhibit endocytosis of cell surface molecules.

Topics: Alkaloids; Antifungal Agents; Carbazoles; CD4 Antigens; Down-Regulation; Ethers, Cyclic; Indole Alkaloids; Okadaic Acid; Phorbol 12,13-Dibutyrate; Protein Kinase C; Pyrans; Receptors, Transferrin; Spiro Compounds; Staurosporine

Okadaic acid and dinophysistoxin-1 (35-methylokadaic acid) induced hyperphosphorylation of a 58 kDa protein in primary human fibroblasts, due to inhibition of protein phosphatase 1 and 2A activities. The protein was present in the nuclear and cytosolic fractions. Its pI was 5.3. The hyperphosphorylated protein reacted with monoclonal and polyclonal anti-vimentin antibodies, but not with anti-nucleolin antibody. Phosphorylation of vimentin was stimulated in vitro by dinophysistoxin-1 dose-dependently in the presence of protein phosphatase 2A and protein kinases.

Topics: Blotting, Western; Ethers, Cyclic; Fibroblasts; Humans; Kinetics; Marine Toxins; Molecular Weight; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Phosphatase 2; Pyrans; Vimentin

Okadaic acid, dinophysistoxin-1 (35-methylokadaic acid), and calyculin A are the okadaic acid class of non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters, which do not bind to the phorbol ester receptors in cell membranes or activate protein kinase C in vitro. They have potent tumor-promoting activities on mouse skin, as strong as TPA-type tumor promoters, such as TPA, teleocidin, and aplysiatoxin. DNA samples isolated from tumors induced by dimethylbenz[alpha]anthracene and each of the okadaic acid class tumor promoters had the same mutation at the second nucleotide of codon 61 (CAA to CTA) in the c-H-ras gene. Okadaic acid receptors, protein phosphatases 1 and 2A, are present in the particulate as well as cytosolic fractions of various mouse tissues. The apparent "activation" of protein kinases by the okadaic acid class tumor promoters, after their incubation with 32P-ATP, protein kinases, and protein phosphatases, was observed. This activation was caused by inhibition of protein phosphatases 1 and 2A by the okadaic acid class tumor promoters. Treatment of primary human fibroblasts and human keratinocytes with the okadaic acid class tumor promoters induced the hyperphosphorylation of a 60-kDa protein in nuclear and cytosolic fractions, due to the inhibition of protein phosphatases. The 60-kDa protein is a proteolytic fragment of nucleolin, a major nonhistone protein and is designated as "N-60." The mechanisms of action of the okadaic acid class tumor promoters are discussed with emphasis on the inhibition of protein phosphatase activity.

Topics: Animals; Carcinogens; DNA; DNA Damage; DNA, Neoplasm; Enzyme Activation; Ethers, Cyclic; Genes, ras; Humans; Marine Toxins; Mice; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Proteins; Pyrans; Receptors, Drug; Skin; Skin Neoplasms; Structure-Activity Relationship

The serologic activities of structurally related okadaic acid derivatives have been determined. Binding of [3H]okadaic acid to rabbit anti-okadaic acid is inhibited with equal effectiveness by okadaic acid, dinophysistoxin-1, acanthifolicin, okadaic acid tetramethyl ether, and okadaic acid spiroketal II. Okadaic acid spiroketal I, which lacks the F- and G-rings of okadaic acid, inhibits serologic binding about 60 times less effectively. The F- and G-rings of okadaic acid may comprise part of the epitopes recognized by some of the polyclonal antibodies.

Topics: Animals; Antibody Specificity; Antigen-Antibody Reactions; Binding, Competitive; Ethers, Cyclic; Okadaic Acid; Pyrans; Rabbits; Radioimmunoassay; Spiro Compounds

Extracts of "diarrhetic" shellfish toxins from the edible mussel (Mytilus edulis) were tested with biological and chemical assays to determine toxin content. When tested with the standard mouse bioassay, a higher toxicity was detected in most samples compared to that revealed from detection of the diarrhea-causing substances okadaic acid and dinophysistoxin-1 by high-performance liquid chromatography. Routine extraction of toxins for the two assays was carried out with two different solvents, acetone versus aqueous methanol. Accordingly, we questioned whether the variation in results between the methods could be due to differences in chemical properties of these two solvents. When tested, the two solvent systems showed practically the same efficiency concerning the extraction of okadaic acid and dinophysistoxin-1. This demonstrated that toxins other than those causing diarrhea were present in the samples, and that the mouse bioassay was sensitive to these additional toxins. Subsequent testing of the samples with the mouse bioassay, employing both acetone and methanol extracts, revealed that at least two classes of toxins were present in the mussel samples in addition to okadaic acid and dinophysistoxin-1. It is unclear whether the shellfish toxins revealed in this study are partially from known, nondiarrhetic types, such as pectenotoxins or yessotoxins, or from unknown toxin groups exhibiting ichthyotoxic and hemolytic properties.

Topics: Animals; Bivalvia; Carcinogens; Chromatography, High Pressure Liquid; Diarrhea; Ethers, Cyclic; Marine Toxins; Mice; Okadaic Acid; Pyrans

Mussels exposed to dinoflagellates may represent a human health risk due to accumulation of a variety of algal toxins. In several parts of the world, algal toxins leading to diarrhea (diarrhetic shellfish poisons, DSP) are found in mussels for extended periods of the year. Routine monitoring of these toxins involves ip injections in mice. Chemical analytical methods have been developed for only some of the toxins in question, namely, those giving diarrhea. Other toxins in the DSP complex are not easily detected by analytical methods. In this report we show that freshly prepared hepatocytes from rats are a convenient means to differentiate between the toxins that give diarrhea and those that do not. Consequently, hepatocytes can be useful in both screening and as a tool in the process of developing analytical methods. Freshly prepared hepatocytes might be useful in combination either with the mouse bioassay or with chemical analytical methods.

Topics: Animals; Bacterial Toxins; Bivalvia; Cells, Cultured; Cyanobacteria Toxins; Diarrhea; Dose-Response Relationship, Drug; Ethers, Cyclic; L-Lactate Dehydrogenase; Liver; Macrolides; Male; Marine Toxins; Microcystins; Microscopy, Electron, Scanning; Mollusk Venoms; Okadaic Acid; Oxocins; Pyrans; Rats; Shellfish

Okadaic acid and dinophysistoxin-1 are non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoters, which enhance chemically induced tumorigenesis on mouse skin through a different mechanism from that of TPA. In the present study, we examined the promoting effects of these okadaic acid class tumor promoters on a two-stage transformation using BALB/3T3 cells which was designed to simulate in vivo two-stage carcinogenesis. Cells were treated first with a low dose of the initiator 3-methylcholanthrene (MCA) and then with a test chemical. Okadaic acid and dinophysistoxin-1 significantly enhanced the MCA-induced cell transformation. Okadaic acid tetramethyl ether, an inactive compound, did not affect the transformation of MCA-treated cells. The okadaic acid class of tumor promoters failed to induce transformation without pretreatment by MCA. Okadaic acid did not show initiating activity in the two-stage transformation assay in which cells were treated first with okadaic acid and then with TPA. These results indicate that this transformation assay with BALB/3T3 cells is useful to predict tumor-promoting activity of non-TPA-type as well as TPA-type tumor promoters, before long-term in vivo two-stage carcinogenesis experiments are carried out.

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Dinoflagellida; Ethers, Cyclic; Marine Toxins; Mice; Mice, Inbred Strains; Okadaic Acid; Pyrans

Okadaic acid and dinophysistoxin-1, the principal toxic components in diarrhetic shellfish poisoning, may be detected by high-performance liquid chromatography and fluorometric measurement as 9-anthrylmethyl esters. However, "greasy" samples may occur and the fluorescent reagent 9-anthryldiazomethane may decompose during storage, resulting in impurities that may seriously interfere with quantitative determination. Ultrasonic treatment of the samples during derivatization with 9-anthryldiazomethane was found to improve reproducibility. This may result from increased access to reactive sites on toxins by 9-anthryldiazomethane due to disruption of micelles formed by toxins and other partly hydrophobic compounds. A procedure for cleaning the derivatized samples, using a 0.1 g silica cartridge column and different eluent compositions from that reported by LEE et al. (1987), was found to facilitate chromatogram interpretation. Deoxycholic acid, a commercial available bile acid, was found to be an acceptable internal standard. The 9-anthrylmethyl esters of okadaic acid, dinophysistoxin-1 and deoxycholic acid, were stable at 4 degrees C for at least seven days when stored dry or in methanol.

Topics: Animals; Anthracenes; Bivalvia; Chromatography, High Pressure Liquid; Esters; Ethers, Cyclic; Fluorescent Dyes; Marine Toxins; Okadaic Acid; Pyrans; Reproducibility of Results; Ultrasonics

Topics: Animals; Binding, Competitive; Brain; Carcinogens; Cytosol; DNA, Neoplasm; Ethers, Cyclic; Humans; Marine Toxins; Mice; Molecular Structure; Mutation; Nuclear Proteins; Nucleolin; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphoproteins; Protein Kinases; Proteins; Proto-Oncogene Proteins p21(ras); Pyrans; Receptors, Drug; RNA-Binding Proteins; Skin; Skin Neoplasms; Spiro Compounds; Structure-Activity Relationship

Acanthifolicin (9,10-epithio-okadaic acid from Pandoras acanthifolium) inhibited protein phosphatase-1 (PP1) similarly to okadaic acid (IC50 = 20 nM and 19 nM, respectively) but was slightly less active against protein phosphatase-2A (PP2A) (IC50 = 1 nM and 0.2 nM, respectively). Methyl esterification of acanthifolicin sharply reduced its activity. PP2A was inhibited with an IC50 = 5.0 microM, whilst PP1 was inhibited less than 10% at 250 microM toxin. Okadaic acid methyl ester was similarly inactive whereas dinophysistoxin-1 (35-methyl okadaic acid) inhibited PP1/2A almost as potently as okadaic acid. Pure acanthifolicin/okadaic acid methyl ester may be useful as specific inhibitors of PP2A at 1-10 microM concentrations in vitro and perhaps in vivo. The data also indicate that a region on these toxins important for PP1/2A inhibition comprises the single carboxyl group.

Topics: Ethers, Cyclic; Marine Toxins; Molecular Structure; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Phosphatase 2; Pyrans; Spiro Compounds; Structure-Activity Relationship

Okadaic acid and dinophysistoxin-1 isolated from a black sponge, Halichondria okadai are non-12-O-tetrade-canoylphorbol 13-acetate (non-TPA)-type tumor promoters of mouse skin. Okadaic acid at concentrations of 10-100 ng/ml stimulated prostaglandin E2 production in rat peritoneal macrophages. Dinophysistoxin-1 (35-methylokadaic acid) stimulated prostaglandin E2 production as strong as okadaic acid, but okadaic acid tetramethyl ether, an inactive compound as a tumor promoter, did not. Okadaic acid at 10 ng/ml (12.4 nM) stimulated prostaglandin E2 production as strongly as TPA at 10 ng/ml (16.2 nM) 20 h after incubation. Unlike TPA-type tumor promoters, okadaic acid required a lag phase before stimulation. The duration of this lag phase was dependent on the concentration of okadaic acid. Indomethacin inhibited okadaic acid-induced preostaglandin E2 production in a dose-dependent manner, and its inhibition was more strongly observed in okadaic acid-induced prostaglandin E2 production. Cycloheximide inhibited okadaic acid-induced release of radioactivity from [3H]arachidonic acid-labeled macrophages and prostaglandin E2 production dose dependently, suggesting that protein synthesis is a prerequisite for the stimulation of arachidonic acid metabolism. These results support our idea that tumor promoters, at very low concentrations, are able to stimulate arachidonic acid metabolism in rat peritoneal macrophages.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Carcinogens; Cells, Cultured; Dinoprostone; Ethers, Cyclic; Ionophores; Kinetics; Macrophages; Marine Toxins; Okadaic Acid; Pyrans; Rats; Tetradecanoylphorbol Acetate

Dinophysistoxin-1, 35-methylokadaic acid, is a causative agent of diarrhetic shellfish poisoning. The biological activities and tumor-promoting activity of dinophysistoxin-1 were studied together with those of okadaic acid and 7-O-palmitoyl okadaic acid. Dinophysistoxin-1 is a skin irritant and induces ornithine decarboxylase in mouse skin with the same potency as okadaic acid. 7-O-Palmitoyl okadaic acid induced a lower activity than the other compounds. Dinophysistoxin-1 inhibited the specific [3H]okadaic acid binding to a particulate fraction of mouse epidermis. The binding affinities of dinophysistoxin-1 and okadaic acid to a particulate fraction were almost the same. Dinophysistoxin-1 showed a tumor-promoting activity as strong as that of okadaic acid in a two-stage carcinogenesis experiment on mouse skin. The percentages of tumor-bearing mice in the groups treated with 100 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) followed by 5 micrograms of dinophysistoxin-1, twice a week, and with DMBA followed by 5 micrograms of okadaic acid twice a week were 86.7% and 80.0% in week 30, respectively. The average number of tumors per mouse was 4.6 in the former group and 3.9 in the latter. Dinophysistoxin-1 and okadaic acid act on cells through different pathways from the 12-O-tetradecanoylphorbol-13-acetate-type tumor promoters.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cocarcinogenesis; Enzyme Induction; Ethers, Cyclic; Female; Irritants; Marine Toxins; Mice; Okadaic Acid; Ornithine Decarboxylase; Pyrans; Skin Neoplasms; Structure-Activity Relationship

Sequential ultrastructural changes were studied in mouse digestive organs after i.p. injections of dinophysistoxin-1 and pectenotoxin-1, causative agents of diarrhetic shellfish poisoning. Dinophysistoxin-1, a diarrheagenic substance, produced severe mucosal injuries in the small intestine within 1 hr after the administration of the toxin. The injuries were divided into 3 consecutive stages: extravasation of villi vessels, degeneration of absorptive epithelium and desquamation of the degenerated epithelium from the lamina propria. In contrast to dinophysistoxin-1, pectenotoxin-1, a non-diarrheagenic toxin from diarrhetic shellfish poisoning causative mussels, resulted in no abnormalities in the small intestine, but did cause characteristic liver injuries. Within 1 hr after the injection of pectenotoxin-1 numerous non-fatty vacuoles appeared in the hepatocytes around the periportal regions of the hepatic lobules. Electron microscopic observations with colloidal iron demonstrated that these vacuoles originated from invaginated plasma membranes of the hepatocytes.

Topics: Animals; Animals, Suckling; Duodenum; Female; Liver; Macrolides; Male; Marine Toxins; Mice; Mice, Inbred BALB C; Microscopy, Electron; Okadaic Acid; Pyrans