okadaic-acid and dinophysistoxin-2

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

For the purpose of assessing human health exposure, it is necessary to characterize the toxins present in a given area and their potential impact on commercial species. The goal of this research study was: (1) to screen the prevalence and concentrations of lipophilic toxins in nine groups of marine invertebrates in the northwest Iberian Peninsula; (2) to evaluate the validity of wild mussels (

Topics: Animals; Bivalvia; Chromatography, Liquid; Humans; Marine Toxins; Mytilus; Okadaic Acid; Shellfish; Shellfish Poisoning; Tandem Mass Spectrometry

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

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

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

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

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

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 produced by the dinoflagellate genus

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

Okadaic acid (OA), Dinophysistoxins (DTX1 and DTX2) and their acyl-derivatives (DTX3) are marine toxins responsible for the human diarrhetic shellfish poisoning. To date the amount of toxins ingested from consumption of shellfish has been considered equal to the amount of toxins available for uptake by the human body. The aim of this study is to assess the OA, DTX2 and DTX3 fractions released from raw and steamed mussels and cockles into the digestive fluids (bioaccessibility) using a static in vitro digestion model. Higher bioaccessibility was found in mussels (86 ± 4%) than in cockles (59 ± 9%). A significant reduction of ester derivatives of OA and an increase of OA were observed in the bioaccessible fraction of mussel samples, suggesting that DTX3 undergo conversion into their more toxic parent compounds during human digestion. However, similar increase of DTX2 and reduction of the respective acyl derivatives was not observed. Steaming lead to significant reduction of OA and analogues bioaccessibility in both species even though increased concentrations of toxins are obtained after this treatment. Risk assessment based solely on DSP toxins occurrence in seafood can conduct to an overestimation of the exposure and lead to more conservative regulatory measures.

Topics: Animals; Bivalvia; Carcinogens; Chromatography, Liquid; Esters; Fatty Acids; Humans; In Vitro Techniques; Marine Toxins; Mass Spectrometry; Okadaic Acid; Pyrans; Shellfish; Steam

Topics: Administration, Oral; Animals; Body Weight; Chromatography, Liquid; Eating; Feces; Female; Intestines; Lethal Dose 50; Liver; Marine Toxins; Mice; Okadaic Acid; Pyrans; Tandem Mass Spectrometry; Toxicity Tests

The effect of canning in pickled sauce and autoclaving on weight, toxin content, toxin concentration and toxicity of steamed mussels was studied. Weight decreased by 25.5%. Okadaic acid (OA) and DTX2 content of mussel meat decreased by 24.1 and 42.5%, respectively. The estimated toxicity of the mussel remained nearly unchanged (increased by 2.9%). A part of the toxins lost by the mussels was leached to the sauce but the remaining part should have been thermally degraded. DTX2 underwent more degradation than OA and, in both toxins, free forms more than conjugated ones. This process, therefore, cannot be responsible for the large increments of toxicity of processed mussels -relative to the raw ones-sometimes detected by food processing companies. The final product could be monitored in several ways, but analysing the whole can content or the mussel meat once rehydrated seems to be the most equivalents to the raw mussel controls.

Topics: Algorithms; Animals; Bivalvia; Condiments; Estuaries; Food Contamination; Food Inspection; Food Preservation; Food, Preserved; Harmful Algal Bloom; Hot Temperature; Humans; Marine Toxins; Okadaic Acid; Pyrans; Shellfish; Shellfish Poisoning; Spain

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

This study explores the effect of laboratory and industrial steaming on mussels with toxin concentrations above and below the legal limit. We used mild conditions for steaming, 100 °C for 5 min in industrial processing, and up to 20 min in small-scale laboratory steaming. Also, we studied the effect of heat on the toxin concentration of mussels obtained from two different locations and the effect of heat on the levels of dinophysistoxins 3 (DTX3) in both the mussel matrix and in pure form (7-O-palmitoyl okadaic ester and 7-O-palmytoleyl okadaic ester). The results show that the loss of water due to steaming was very small with a maximum of 9.5%, that the toxin content remained unchanged with no concentration effect or increase in toxicity, and that dinophysistoxins 3 was hydrolyzed or degraded to a certain extent under heat treatment. The use of liquid-certified matrix showed a 55% decrease of dinophysistoxins 3 after 10 min steaming, and a 50% reduction in total toxicity after treatment with an autoclave (121 °C for 20 min).

Topics: Animals; Bivalvia; Food Contamination; Hot Temperature; Hydrolysis; Okadaic Acid; Pyrans; Steam; Sterilization

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

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

Azaspiracids (AZAs) are lipophilic biotoxins produced by marine algae that can contaminate shellfish and cause human illness. The European Union (EU) regulates the level of AZAs in shellfish destined for the commercial market, with liquid chromatography-mass spectrometry (LC-MS) being used as the official reference method for regulatory analysis. Certified reference materials (CRMs) are essential tools for the development, validation, and quality control of LC-MS methods. This paper describes the work that went into the planning, preparation, characterization, and certification of CRM-AZA-Mus, a tissue matrix CRM, which was prepared as a wet homogenate from mussels (Mytilus edulis) naturally contaminated with AZAs. The homogeneity and stability of CRM-AZA-Mus were evaluated, and the CRM was found to be fit for purpose. Extraction and LC-MS/MS methods were developed to accurately certify the concentrations of AZA1 (1.16 mg/kg), AZA2 (0.27 mg/kg), and AZA3 (0.21 mg/kg) in the CRM. Quantitation methods based on standard addition and matrix-matched calibration were used to compensate for the matrix effects in LC-MS/MS. Other toxins present in this CRM at lower levels were also measured with information values reported for okadaic acid, dinophysistoxin-2, yessotoxin, and several spirolides.

Topics: Animals; Calibration; Chromatography, Liquid; Marine Toxins; Mollusk Venoms; Mytilus edulis; Okadaic Acid; Oxocins; Pyrans; Reference Standards; Spiro Compounds; Tandem Mass Spectrometry

The effect of industrial steaming on mussels that had been naturally exposed to DSP toxins for a long time was studied using LC-MS/MS. The estimated toxicity increased with steaming by a percentage that cannot be explained by weight loss. The estimated toxin content per mussel increased substantially with the treatment, which can only be explained by an incorrect estimation by the technique (at the extraction or analytical level) or by the presence of unknown derivatives or analogues. Direct alkaline hydrolysis of the mussel meat yielded more toxin than the standard hydrolysis (hydrolysis of the methanolic extracts), suggesting that extraction was, at least in part, responsible for the increase of toxin content. In situations as the one described in this work, it can be expected that mussels with toxicities well below the regulatory limit could easily surpass that level after industrial steaming, thus producing important losses for food processors.

Topics: Animals; Bivalvia; Chromatography, Liquid; Cooking; Food Handling; Marine Toxins; Okadaic Acid; Pyrans; Shellfish; Shellfish Poisoning; Steam; Tandem Mass Spectrometry

Seasonal dynamics and the distribution of dinophysistoxin-3 (DTX-3) in organs of mussel Crenomytilus grayanus, collected in 2013 in Peter the Great Bay, Sea of Japan, were determined. Active toxin forms (OA, DTX-1/2) concentrated in digestive glands (60-80%), whereas non-active 7-O-acyl derivatives (DTX-3) were more abundant in edible soft tissues (more than 80%). Possible mechanism of fast removing of DTX-3 from the digestive gland and accumulation of DTX-3 in the soft tissues is supposed.

Topics: Animals; Chromatography, High Pressure Liquid; Japan; Marine Toxins; Mytilidae; Okadaic Acid; Pyrans; Shellfish; Tissue Distribution

Lipophilic marine toxins pose a serious threat for consumers and an enormous economic problem for shellfish producers. Synergistic interaction among toxins may play an important role in the toxicity of shellfish and consequently in human intoxications. In order to study the toxic profile of molluscs, sampled during toxic episodes occurring in different locations in Galicia in 2014, shellfish were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), the official method for the detection of lipophilic toxins. The performance of this procedure was demonstrated to be fit for purpose and was validated in house following European guidelines. The vast majority of toxins present in shellfish belonged to the okadaic acid (OA) group and some samples from a particular area contained yessotoxin (YTX). Since these toxins occur very often with other lipophilic toxins, we evaluated the potential interactions among them. A human neuroblastoma cell line was used to study the possible synergies of OA with other lipophilic toxins. Results show that combination of OA with dinophysistoxin 2 (DTX2) or YTX enhances the toxicity triggered by OA, decreasing cell viability and cell proliferation, depending on the toxin concentration and incubation time. The effects of other lipophilic toxins as 13-desmethyl Spirolide C were also evaluated in vitro.

Topics: Animals; Atlantic Ocean; Bivalvia; Cell Line, Tumor; Cell Survival; Chromatography, High Pressure Liquid; Drug Synergism; Food Contamination; Food Inspection; Humans; Hydrophobic and Hydrophilic Interactions; Limit of Detection; Molecular Structure; Mollusk Venoms; Neurons; Okadaic Acid; Oxocins; Pyrans; Shellfish; Spain; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

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

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

Here, we report different lipophilic toxins (LTs) detected by LC-MS/MS in Mediterranean mussels (Mytilus galloprovincialis) collected through 2012 in Todos Santos Bay, northwest Baja California, Mexico. The concentration of okadaic acid (OA), dinophysistoxin 2 (DTX2), and pectenotoxin 2 (PTX2) reached 500 μg kg(-1) during July and increased to 1647 μg kg(-1) in October. These toxins were associated with the presence of Dinophysis fortii and Dinophysis acuminata and a strong stratification of the water column. Other LTs present were yessotoxins, with a maximum concentration of 1080 μg kg(-1) in June. Cyclic imines (13-desmethyl spirolide and gymnodimine) and azaspiracid 1 were also detected in the mussels but at low concentrations. Diarrhetic toxins concentrations evaluated by LC-MS/MS were compared with the results of two mouse bioassay protocols. Positive results were obtained with both MBA protocols in several samples that presented toxicities below 160 μg OA-eq kg(-1), as estimated by LC-MS/MS results whereas other samples returned negative MBA results in samples with concentrations above this level. Therefore, analytical methods need to be applied to confirm the presence of regulated LTs. This is the first report of LTs in mussels cultivated in Mexico. The occurrence of these toxins represents an emerging problem in the region.

Topics: Animals; Chromatography, Liquid; Lipids; Macrolides; Marine Toxins; Mice; Mice, Inbred ICR; Mytilus; Okadaic Acid; Pyrans; Tandem Mass Spectrometry

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

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

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

To determine the relative toxicity and effects on the cell cycle of okadaic acid and dinophysistoxin-2 in primary hepatocyte cultures.. Cytotoxicity was determined by the MTT method, caspase-3 activity and lactate dehydrogenase release to the medium. The cell cycle analysis was performed by imaging flow cytometry and the effect of the toxins on cell proliferation was studied by quantitative PCR and confocal microscopy.. We show that dinophysistoxin-2 is less toxic than okadaic acid for primary hepatocytes with a similar difference in potency as that observed in vivo in mice after intraperitoneal injection. Both toxins induced apoptosis with caspase-3 increase. They also inhibited the hepatocytes cell cycle in G1 affecting diploid cells and diploid bi-nucleated cells. In proliferating hepatocytes exposed to the toxins, a decrease of p53 gene expression as well as a lower protein level was detected. Studies of the tubulin cytoskeleton in toxin treated cells, showed nuclear localization of this molecule and a granulated tubulin pattern in the cytoplasm.. The results presented in this work show that the difference in toxicity between dinophysistoxin-2 and okadaic acid in cultured primary hepatocytes is the same as that observed in vivo after intraperitoneal injection. Okadaic acid and dinophysistoxin-2 arrest the cell cycle of hepatocytes at G1 even in diploid bi-nucleated cells. p53 and tubulin could be involved in the cell cycle inhibitory effect.

Topics: Analysis of Variance; Animals; Blotting, Western; Caspase 3; Cell Cycle; Cell Proliferation; Flow Cytometry; Hepatocytes; L-Lactate Dehydrogenase; Microscopy, Confocal; Okadaic Acid; Pyrans; Rats; Real-Time Polymerase Chain Reaction; Tetrazolium Salts; Thiazoles

Okadaic acid is one of the toxins responsible for the human intoxication known as diarrhetic shellfish poisoning, which appears after the consumption of contaminated shellfish. The main diarrhetic shellfish poisoning toxins are okadaic acid, dinophysistoxin-1, -2, and -3. In vivo, after intraperitoneal injection, dinophysistoxin-2 is approximately 40% less toxic than okadaic acid in mice. The cytotoxic and genotoxic effect of okadaic acid varies very significantly in different cell lines, so similar responses could be expected for dinophysistoxin-2. In order to determine whether this was the case, we studied the effect of okadaic acid and dinophysistoxin-2 in two hepatic cell lines (HepG2 and Clone 9). The cytotoxicity of these toxins, as well as their effects on the cell cycle and its regulation on both cell lines, were determined. Okadaic acid and dinophysistoxin-2 resulted to be equipotent in clone 9 cultures, while okadaic acid was more potent than dinophysistoxin-2 in HepG2 cell cultures. Both toxins had opposite effects on the cell cycle; they arrested the cell cycle of clone 9 cells in G2/M inducing aberrant mitosis while arresting the cell cycle of HepG2 in G0/G1. When the effect of the toxins on p53 subcellular distribution was studied, p53 was detected in the nuclei of both cell types. The effect of the toxins on the gene expression of cyclins and cyclin-dependent kinases was different for both cell lines. The toxins induced an increase in gene expression of cyclins A, B, and D in clone 9 cells while they induced a decrease in cyclins A and B in HepG2 cells. They also induced a decrease in cyclin-dependent kinase 1 in HepG2 cells.

Topics: Animals; CDC2 Protein Kinase; Cell Cycle; Cell Cycle Checkpoints; Cyclins; Gene Expression Regulation; Hep G2 Cells; Humans; Injections, Intraperitoneal; Liver; Mitosis; Okadaic Acid; Pyrans; Rats; Tumor Suppressor Protein p53

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

Topics: Enzyme Inhibitors; Molecular Conformation; Okadaic Acid; Phosphoprotein Phosphatases; Pyrans; Stereoisomerism; Structure-Activity Relationship

Contamination of shellfish from the Portuguese coast with diarrhetic shellfish poisoning (DSP) toxins is a recurrent event, with most of the commercial bivalves contaminated with high percentages of esters of okadaic acid (OA) and dinophysistoxin-2 (DTX2). This report describes the quantification of DSP toxins in unhydrolysed and hydrolysed extracts of several cockle and mussel samples naturally contaminated and the evaluation of their cytotoxicity profiles in V79 cells. The quantification of the acyl esters in the shellfish samples involved the cleavage of the ester bond through alkaline hydrolysis and the release of the parent toxins OA and DTX2. Unhydrolysed and hydrolysed extracts were then analyzed by liquid chromatography (LC) coupled with mass spectrometry (MS) for the detection and quantification of DSP toxins. The cytotoxicity of the analysed extracts was evaluated using the MTT reduction assay and compared with the cytotoxicity presented by different concentrations of OA standard (1-100 nM). OA exhibited marked cytotoxic effects and decreased cell viability in a dose dependent mode, with an IC₅₀ of 27 nM. The cytotoxicity pattern of unhydrolysed extracts was clearly dependent on the concentration of free toxins. Moreover, the cytotoxicity of the esterified toxins present was revealed after their conversion into free toxins by alkaline hydrolysis. For the hydrolysed extracts of cockles and mussels, the cytotoxicity presented was mainly related to the concentration of OA and DTX2.

Topics: Animals; Bivalvia; Cell Survival; CHO Cells; Chromatography, Liquid; Cricetinae; Cricetulus; Diarrhea; Dose-Response Relationship, Drug; Esters; Food Contamination; Formazans; Humans; Hydrogen-Ion Concentration; Hydrolysis; Inhibitory Concentration 50; Marine Toxins; Mass Spectrometry; Molecular Structure; Okadaic Acid; Pyrans; Shellfish; Shellfish Poisoning; Tetrazolium Salts; Tissue Extracts

Portuguese bivalves are recurrently contaminated with okadaic acid (OA) and dinophysistoxin-2 (DTX2), found mainly in esterified forms. Throughout the years different conditions have been reported in the literature for releasing the parent toxins through an alkaline hydrolysis step, in order to simplify their detection by HPLC-FLD or LC-MS. In order to clearly understand toxin stability and reaction end-point the binominous temperature/time course and base concentration were studied using naturally contaminated bivalve samples. The results showed a strong temperature dependence of the reaction. At 60 degrees C and 70 degrees C the hydrolysis was fast, and 40min were sufficient for maximal recovery of OA and DTX2, while at 40 degrees C and 50 degrees C it was only complete after 100min and 60min, respectively. At room temperature the reaction was slow and incomplete even after 2h. Stability of OA and DTX2 in semi-purified bivalve matrix at 70 degrees C for 2h was demonstrated. Concentrations of sodium hydroxide lower than 2.5M, corresponding to a final incubation concentration of 0.23M, resulted in incomplete release of parent toxins, demonstrating that high concentrations are needed when taking into account the dilution in the supernatant extract.

Topics: Alkalies; Animals; Chromatography, High Pressure Liquid; Dinoflagellida; Esters; Hydrolysis; Kinetics; Marine Toxins; Mass Spectrometry; Okadaic Acid; Pyrans; Shellfish; Sodium Hydroxide; Temperature

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

During the last few years the occurrence of a high percentage of esters of diarrhetic shellfish poisoning (DSP) toxins has been observed in shellfish from the Portuguese coast. Most of the commercial bivalves contain DSP toxins in ester forms, either acyl derivatives of okadaic acid (OA) or of dinophysistoxin-2 (DTX-2). The stability of these toxins in shellfish tissues and in raw methanol extracts was investigated in two different naturally contaminated species, mussel and carpet shell, over a 4-week period. The results for both species revealed that DSP toxins were more stable in tissue than in raw methanol extracts. Losses of DSP toxins were seen in the first 2 weeks and were more than 30%, but after that a period of stabilization was observed. The decrease was due probably from losses of esters of OA and DTX-2, the free toxins were stable over the period studied. The extraction most commonly used for chemical and biochemical assays relied on methanolic extraction with aqueous 80% methanol. In this work we have tested the extraction solvent on the extractability of DSP toxins from several naturally contaminated species. A single dispersive extraction with methanol, with solvent ratios of 70%, 80%, 90% and 100%, were tested. After alkaline hydrolysis of esterified toxins and clean-up with hexane and dichloromethane, the samples were analysed by liquid chromatography-mass spectrometry (LC-MS). The recovery of DSP toxins increased with increasing percentages of methanol up to 90%. A decrease in recovery with 100% methanol was observed probably due to problems during the liquid-liquid partitioning.

Topics: Animals; Bivalvia; Chromatography, Liquid; Esters; Food Contamination; Humans; Marine Toxins; Okadaic Acid; Portugal; Pyrans; Shellfish; Shellfish Poisoning; Time Factors

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

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

Diarrheic shellfish poisoning (DSP) is a recurrent gastrointestinal illness in Morocco, resulting from consumption of contaminated shellfish. In order to develop a rapid and reliable technique for toxins detection, we have compared the results obtained by a commercial immunoassay-"DSP-Check" kit" with those obtained by LC-MS. Both techniques are capable of detecting the toxins in the whole flesh extract which was subjected to prior alkaline hydrolysis in order to detect simultaneously the esterified and non esterified toxin forms. The LC-MS method was found to be able to detect a high level of okadaic acid (OA), low level of dinophysistoxin-2 (DTX2), and surprisingly, traces of azaspiracids 2 (AZA2) in mussels. This is the first report of a survey carried out for azaspiracid (AZP) contamination of shellfish harvested in the coastal areas of Morocco. The "DSP-Check" kit was found to detect quantitatively DSP toxins in all contaminated samples containing only OA, provided that the parent toxins were within the range of detection and was not in an ester form. A good correlation was observed between the two methods when appropriate dilutions were performed. The immunoassay kit appeared to be more sensitive, specific and faster than LC-MS for determination of DSP in total shellfish extract.

Topics: Animals; Bivalvia; Chromatography, Liquid; Data Collection; Diarrhea; Enzyme-Linked Immunosorbent Assay; Fisheries; Food Contamination; Furans; Marine Toxins; Mass Spectrometry; Morocco; Okadaic Acid; Pyrans; Reproducibility of Results; Shellfish Poisoning

An expedient synthesis of both axially and equatorially C35 methyl substituted spiroketals representing the C28-C38 domain of the potent and selective protein serine/threonine phosphatase inhibitor dinophysistoxin-2 (DTX-2) was developed to enable comparative stereochemical analyses and a stereochemically correct total synthesis of DTX-2. Comparison of proton and carbon NMR data of the synthetic diastereomers with those published for DTX-2 indicates that DTX-2 possesses the (30S *,34R *,35S *)-relative configuration with an axial C35 methyl substituent.

Topics: Enzyme Inhibitors; Furans; Magnetic Resonance Spectroscopy; Marine Toxins; Molecular Structure; Okadaic Acid; Phosphoric Monoester Hydrolases; Protein Structure, Tertiary; Pyrans; Spiro Compounds; Stereoisomerism

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

Using high-performance liquid chromatography with mass spectrometry, the influence of conventional steaming and another heat treatment on the level of okadaic acid and dinophysistoxin-2 in mussels (Mytilus edulis) was investigated. Concentration increases correlated with water loss during steaming, and increased distribution of okadaic acid and dinophysistoxin-2 from the digestive glands to the remainder tissues was observed as a result of the processes examined. This suggests that the analysis of whole flesh tissues, as opposed to dissected digestive glands, is more appropriate for regulatory purposes, particularly if cooked samples are being analysed. A systematic heat treatment experiment showed that while okadaic acid group toxins are stable during cooking processes, degradation does occur in mussel tissues after prolonged exposure to high temperatures. The findings of the studies reported here have importance in terms of the methodology applied in regulatory phycotoxin monitoring programmes. Therefore, options for sample pretreatment are discussed.

Topics: Animals; Chromatography, High Pressure Liquid; Cooking; Digestive System; Hot Temperature; Marine Toxins; Mass Spectrometry; Mytilus edulis; Okadaic Acid; Pyrans; Time Factors; Tissue Distribution

When substituting the mouse bioassay for lipophilic marine algal toxins in shellfish with analytical methods, science based factors of relative toxicity for all analogues that contribute to health risk to consumers are necessary. The aim of this paper is to establish the relative intraperitoneal toxicity of dinophysistoxin-2 (DTX-2) compared with okadaic acid (OA). The study was performed as an open, randomised parallel group trial with a four level response surface design within each of the two parallels. In accordance with the response surface design model, the LD50 for DTX-2 and OA was 338 and 206 microg/kg, respectively. By use of common regression analysis, the LD50 of DTX-2 and OA were estimated to 352 microg/kg and 204 microg/kg, respectively. The deviations between the LD50 estimates by the two methods was 4% for DTX-2 and less than 1% for OA. Taken together, these results indicate that the relative toxicity of DTX-2 is about 0.6, compared to OA. Results from the PP2A assay correspond very well with the results obtained by the mouse bioassay. The IC50 concentrations for DTX-2 and OA were 5.94 and 2.81 ng/mL, respectively. This indicates that OA is about twice as toxic as DTX-2. Since inhibition of PP2A is acknowledged as the main mechanism of toxicity of the OA group toxins, this supports the establishment of a relative toxicity factor of DTX-2 of 0.6 compared with OA.

Topics: Animals; Dinoflagellida; Enzyme Inhibitors; Female; Inhibitory Concentration 50; Injections, Intraperitoneal; Marine Toxins; Mice; Mice, Inbred Strains; Okadaic Acid; Phosphoprotein Phosphatases; Pyrans; Shellfish

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

Marine biotoxins from microalgae can accumulate in shellfish and lead to poisoning of human consumers as well as fish, marine mammals and sea birds. Toxicological assessment of the toxins and development of analytical methods require large amounts of high-purity toxins and their metabolites. Although these toxins can be obtained in limited amounts from contaminated shellfish or from microalgal cultures, difficulties arise when the toxin-producing microalga is difficult to culture or its identity is not known. To circumvent this problem, we have developed a large-scale method for solid-phase extraction (SPE) of lipophilic biotoxins from natural microalgal blooms in seawater. To enhance subsequent purification of toxins adsorbed on the column, we included a filtration step to release the toxins from the cells while removing insoluble compounds and cellular debris. The efficacy of the method was illustrated by extraction and purification of okadaic acid and dinophysistoxin-2 from a high-density Dinophysis acuta bloom in Spain and from a mixed bloom containing low densities of D. acuta in Norway. Isolation of the toxins adsorbed on the SPE column was simple and efficient, and results obtained so far indicate that the method is potentially applicable to a wide range of microalgal toxins such as azaspiracids, pectenotoxins, spirolides and microcystins. The method should also be useful for harvesting toxins from large-scale microalgal cultures, and for bioprospecting for and isolation of bioactive natural products from marine and freshwater environments.

Topics: Eukaryota; Marine Toxins; Molecular Structure; Norway; Okadaic Acid; Pyrans; Seawater; Spain

The aim of this work was to shed light on the anatomical distribution of diarrhetic shellfish poisoning (DSP) toxins in the mussel Mytilus galloprovincialis and to determine any possible changes undergone during the depuration process. To this end, the distribution of two DSP toxins--okadaic acid and DTX2--and some of their derivatives were studied by means of HPLC/MS at different stages of the depuration process. Mussels were collected from mussel farms located in the Galician Rías and they were collected under three types of circumstances: (a) while ingesting toxic phytoplankton cells; (b) 1 week after the toxic cells had disappeared from the water; and (c) after ca. 2 months of depuration. Additionally, in case (b), the distribution among tissues was checked every week over a depuration period of 35 days in the laboratory. DSP toxins were only detected in non-visceral tissues when the extracts were concentrated 20-fold and, even in these cases, the concentrations found were very low. When the maximum possible contribution of non-visceral tissues was computed, taking into account the technique's detection limits and tissue weight, no relevant contribution to the toxin burden of non-visceral tissues was found at any stage of depuration, with the maximum possible contributions usually below 7%. The concentrated samples analysed showed that the actual contribution in all the cases studied was, in fact, less than 1% of the total toxin burden. These findings suggest that (1) when analytical methods are used to monitor DSP toxic mussels, non-visceral tissues should be assumed to be free of toxins in order to precisely compute the toxin concentration of the whole mass of edible tissues and (2) when studying the accumulation kinetics of DSP toxins, transference from the digestive gland to other tissues should not be taken into account, as the other tissues do not contain relevant amounts of DSP toxins.

Topics: Animals; Diarrhea; Marine Toxins; Mytilus; Okadaic Acid; Pyrans; Shellfish Poisoning; Tissue Distribution

Different dinophysistoxin's profiles have been found repeatedly amongst some offshore bivalve molluscs. Species such as the clam, Spisula solida, esterify dinophysistoxins to a great extent, and contain always more okadaic acid (OA) than dinophysistoxin-2 (DTX2). In contrast, the clam Donax trunculus has a much higher percentage of non-esterified toxins, and often contains more DTX2 than OA. A detoxification experiment with D. trunculus and S. solida showed that the higher percentage of DTX2 in Donax was due to this toxin being eliminated more slowly than OA. The ester analogues of OA and DTX2 were eliminated faster than free OA and free DTX2 in D. trunculus. As D. trunculus esterifies OA to a greater extent than DTX2, the greater proportion of the free form of DTX2 explains why there is a gradual increase in total DTX2 over time. This slow elimination of free toxins contributes to wild D. trunculus specimens being, on average, six times more toxic than S. solida specimens on the Portuguese south coast. The commercial shellfish species more often monitored along the coast of Portugal between 2003 and 2004 (eight species in total) were examined for DTX2 content. The maximal percentage of total DTX2 in the total DSP toxins (OA + DTX2) was 40% in all species (clams, razors, cockles, oysters) except D. trunculus and Mytilus galloprovinciallis, in which it reached 70-90%. This phenomenon is seen only when contamination is due to the microalga Dinophysis acuta. The other important DSP-producer, Dinophysis acuminata, causes contamination only with OA. Current data suggest that D. acuta along the Portuguese coast always produces OA and DTX2 in a fixed ratio of 60:40. When this alga is dominant, contamination of bivalves occurs in a OA/DTX2 ratio of 60:40. In bivalves in which these toxins are esterified to a great extent, this profile is maintained due to the parallel elimination of OA and DTX2 esters at similar rates. However, in species with lower esterification, the relative proportion of OA and DTX2 rapidly decreases due to the selective retention of free DTX2. Elimination of pectenotoxin-2 seco acids (PTX2sa), the main pectenotoxin form found in both offshore species studied, followed an exponential decay. The half-lives of PTX2sas found in D. trunculus and S. solida were similar to that found in a previous study with the cockle, Cerastoderma edule, but longer than found in the blue mussel M. galloprovinciallis.

Topics: Animals; Bivalvia; Furans; Macrolides; Marine Toxins; Oceans and Seas; Okadaic Acid; Portugal; Pyrans; Time Factors

In bivalve mollusks from the Portuguese coast contaminated by diarrhetic shellfish poisoning (DSP), most of the parent toxins, okadaic acid (OA) or dinophysistoxin-2 (DTX2), are found esterified, and toxicity assessment is only performed after an alkaline hydrolysis step to recover the parent molecules in their free form. The presence of 7-O-acyl esters with fatty acids (FAs) has already been confirmed previously in Mytilus galloprovincialis and Donax trunculus samples. This paper reports the presence of acyl esters in a wider range of estuarine and offshore bivalve species found by direct analysis in LC-MS. The total of acyl esters found in each species represented the percentages commonly found by hydrolysis in those species in previous years, justifying the majority of the esters commonly found in shellfish. This implies that any diol esters remaining after digestion of toxic microalgae would represent only a minor contribution to the ester's contents. Esters with C14:0, C16:0, C16:1, C20:5 and C22:6 FAs were the most abundant, followed by esters with C18:0, C18:1, C18:2, C18:3 and C18:4. This is the first report of OA and DTX2 esters with odd FAs: C15:0, C17:0, C17:1, and probably a branched FA: iso-C16:0. Esters with iso-C16:0 where found in high percentages particularly in two species of estuarine clams, where they represented 13-34% of total esters found. Esters were also found in plankton, predominantly with C16:0. Total esters in plankton were not higher than 10%, not enough to justify per se the high levels found in bivalves.

Topics: Animals; Chromatography, Liquid; Esters; Fatty Acids; Hydrolysis; Marine Toxins; Mass Spectrometry; Mytilus; Okadaic Acid; Plankton; Portugal; Pyrans; Shellfish; Species Specificity

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

Diarrhetic shellfish poisoning (DSP) toxins of algal origin are frequent contaminants of coastal waters and seafood. The potential risk for human health due to the continuous presence of these toxins in food has not been clearly established. We have used cerebellar primary cultures to investigate the effects of the DSP toxin dinophysistoxin-2 (DTX-2) on central nervous system neurons and glial cells. Exposure to DTX-2 produced neurotoxicity at concentrations starting at 2.5 nM, characterized first by disintegration of neurites and later by cell death. DTX-2-induced neurodegeneration required long exposures (at least 20 h), involved DNA fragmentation and condensation and fragmentation of chromatin, typical hallmarks of apoptosis, and required the synthesis of new proteins. The concentration that reduced by 50% the maximum neuronal survival after 24 h exposure to DTX-2 (EC50(24)) was approximately 8 nM. Morphology and viability of glial cells remained unaffected up to at least 15 nM DTX-2. Higher concentrations of the toxin caused strong shrinkage of glial cell bodies and retraction of processes, and a significant reduction of glial cell viability. Glial toxicity by DTX-2 involved typical apoptotic condensation and fragmentation of chromatin. Compared to neurons, the effect on glial cells was a much shorter process, and extensive glial degeneration and death occurred after 7 h exposure to DTX-2 (EC50(7) approximately 50 nM; EC50(24) approximately 30 nM). Although further experiments are needed to confirm these toxic actions in vivo, our in vitro data suggest that chronic exposure to amounts of DSP toxins below the current safety regulatory limits may represent a risk for human health that should be taken into consideration.

Topics: Animals; Apoptosis; Astrocytes; Bivalvia; Cell Survival; Cells, Cultured; Cerebellum; DNA Fragmentation; Electrophoresis, Agar Gel; Marine Toxins; Neurites; Neurons; Neurotoxicity Syndromes; Okadaic Acid; Oxidative Stress; Pyrans; Rats

Different toxin profiles of dinophysistoxins and pectenotoxins have been reported before between blue mussel and other bivalve species, such as common cockle, razor clam, clams, etc. Comparison of toxins present in plankton in mussel growing areas and in cockle growing areas, respectively, showed there was no particular incidence of dinophysistoxin-2 (DTX2) in plankton from mussel growing areas that could account for the higher percentage of DTX2 in relation to okadaic acid (OA) found in mussels; or of pectenotoxin-2 in cockle growing areas that could explain the higher levels of pectenotoxin-2 seco acid (PTX2sa) found in cockles. A detoxification experiment between mussels and cockles showed the higher percentage of DTX2 in mussels was due to slower elimination of this toxin in relation to OA; while the lower levels of PTX2sa were due to quicker elimination by mussels than by cockles. The slower elimination of DTX2 explains why in late summer and autumn this toxin gradually accumulate in mussels throughout the entire coast, while other bivalves species have a lower percentage of DTX2, very close to the 3:2 OA:DTX2 ratio found in natural plankton assemblages when Dinophysis acuta predominates. In the clam Donax spp., DTX2 concentration also tends to build up in relation to OA, this being made up predominantly by free DTX2 while esterified DTX2 is found only in trace levels (similarly to what is found in mussel for DTX2). We hypothesise that the esterified forms of OA and DTX2 are more easily eliminated than the free forms, by all shellfish species. The free forms are more difficult to eliminate. This is particularly notable in these two species that present a very low conversion of DTX2 into acyl esters. The high pool of free toxins is partially responsible for these two species (mussel and Donax clams) being the sentinel species for DSP contamination throughout the Portuguese coast. Esters of OA and DTX2 were found in a plankton sample where D. acuta was the predominant toxic species found. The nature of the esters remains to be elucidated. The boiling of these DTX2 esters seems to favour the rearrangement of the parent molecule to the DTX2 isomer, DTX2i, recoverable after alkaline hydrolysis. The isomerization was also observed with DTX2 esters present in mussel, but thus not appear to occur with the same extent with free DTX2.

Topics: Animals; Bivalvia; Decontamination; Digestive System; Dinoflagellida; Environmental Monitoring; Esterification; Furans; Macrolides; Marine Toxins; Mollusca; Okadaic Acid; Organ Size; Pyrans; Species Specificity

Okadaic acid (OA) and dinophysistoxin-2 (DTX2) were confirmed by liquid chromatography with mass spectrometry detection both in extracts of digestive glands and edible parts of Portuguese shellfish. No dinophysistoxin-1 was found even in highly contaminated samples examined. However, only in blue mussel (Mytilus edulis) were these two parent toxins commonly found in a free form. Usually they were found largely esterified in all remaining shellfish species-common cockle (Cerastoderma edule), peppery furrow shell (Scrobicularia plana), carpet shell (Venerupis pullastra), oyster (Crassostrea japonica), razor clam (Ensisspp.), and clam (Ruditapes decussata). Oysters were the least toxic. In mussels esterified OA did not surpass 50% of the total OA found in edible parts, while DTX2 esterification rates were usually much lower. In remaining shellfish species usually more than 95% of the total OA was found esterified, while free DTX2 was rarely found. Also ratios of total DTX2/total OA were higher in mussels than in the remaining species examined. From all these species commercially exploited at the northern coast, mussels and cockles contained the highest levels of DSP toxins, thus representing the highest theoretical health risk. The previous association of DTX2 with the dinoflagellate Dinophysis acuta was confirmed with selective MS detection; while OA was the only parent diarrhoeic toxin found associated with Dinophysis acuminata.

Topics: Animals; Bivalvia; Chromatography, Liquid; Esterification; Marine Toxins; Mass Spectrometry; Okadaic Acid; Pyrans; Shellfish; Species Specificity; Tissue Extracts

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

Okadaic acid and dinophysistoxin-2 have been found yearly in Portuguese shellfish. Their presence was correlated with the occurrence of Dinophysis spp: Dinophysis acuminata has until now been found to be responsible only for OA contamination, while Dinophysis acuta contributes with OA and the rare diarrhetic toxin DTX2. Differences in toxicity levels may reflect different cell toxicities and different non-toxic phytoplankton availability as food source to shellfish.

Topics: Animals; Diarrhea; Dinoflagellida; Marine Toxins; Okadaic Acid; Pyrans; Seasons; Shellfish

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

The rare diarrhoeic shellfish poisoning (DSP) toxin, dinophysistoxin-2 (DTX-2), which is an okadaic acid (OA) isomer, has been isolated from a marine phytoplankton biomass that consisted mainly of Dinophysis acuta. Using a large double plankton net (length 5.9 m), bulk phytoplankton samples were collected off the south-west coast of Ireland and extracted with methanol and chloroform. Liquid chromatography coupled with ionspray mass spectrometry and tandem mass spectrometry (LC-MS, LC-MS-MS) showed the sample contained DTX-2 and OA, at a concentration of 80 pg/cell and 60 pg/cell, respectively. Flash chromatography using silica, sephadex LH20 and C18-silica, followed by preparative reversed-phase LC, separated DTX-2 from OA. The efficiency of the separation procedures was substantially improved by the use of a bioscreen to detect DSP toxins in eluate fractions and the application of a new derivatisation procedure for the chromatographic elucidation of toxin profiles with fluorimetric detection (LC-FLD). Thus, 1/1000th aliquots of eluate fractions were assayed using protein phosphatase-2A for the presence of inhibitory compounds. Positive fractions were further analysed for DSP toxins by LC-FLD following derivatisation using the hydrazine reagent, luminarine-3. The identity and purity of the free isolated DTX-2 was confirmed using flow injection analysis (FIA) and liquid chromatography (FIA-MS, LC-MS and LC-MS-MS).

Topics: Animals; Diarrhea; Fluorometry; Gas Chromatography-Mass Spectrometry; Hydrazines; Marine Toxins; Okadaic Acid; Phosphoprotein Phosphatases; Phytoplankton; Protein Phosphatase 2; Pyrans; Shellfish; Stereoisomerism

Liquid chromatography (HPLC) was used to search for esters of DSP toxins in Portuguese bivalves. Hexane-soluble derivatives of okadaic acid (OA) and dinophysistoxin-2 (DTX-2) were found. Presumably they are acyl derivatives, globally known as 'dinophysistoxin-3' (DTX-3). In certain instances DTX-3 content surpassed 50% of the total amount of DSP toxins. A human diarrhetic poisoning (DSP) incident with Donax clams (Donax trunculus) harvested at Fuzeta (Algarve coast) was confirmed where the apolar (DTX-3 type) and other esters remaining in the polar aqueous methanol layer were implicated. The percentage of acyl esters of OA was always higher than those of DTX-2. An enzymic mechanism for the conversion of OA and DTX-2 seems to be implicated in some kind of detoxification process because the percentage of esters increases with the toxin amount ingested by the bivalve and there is some degree of selectivity as DTX-2 seems more difficult to acylate. These findings pose a problem for the current assay methods used to detect DSP because mainly they are able to detect the parent toxins but not their esters. The current bioassay method [Le Baut, C., Bardin, B., Bardouil, M., Bohec, M., Masselin, P., Truquet, P., 1990. Etude de la decontamination de moules toxiques. Rapport IFREMER DERO-90-02 MR. 21 pp.] used in Portugal includes a hexane washing step that prevents interference from free fatty acids. However, it cannot detect the presence of acyl derivatives because they are highly soluble in hexane.

Topics: Chemistry Techniques, Analytical; Chromatography, High Pressure Liquid; Diarrhea; Enzyme Inhibitors; Fluorescence; Humans; Marine Toxins; Okadaic Acid; Portugal; Pyrans; Shellfish; Solubility

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

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

A new analogue of okadaic acid (OA), the toxin mainly responsible for diarrhetic shellfish-poisoning (DSP) phenomena in Europe, has been isolated from toxic phytoplankton (Dinophysis acuta) collected in Irish waters. Fluorimetric LC analyses of the extracts of bulk phytoplankton samples using derivatisation with 9-anthryldiazomethane (ADAM) showed a complex toxin profile, with peaks corresponding to OA and dinophysistoxin-2 (DTX-2) as well as a third unidentified compound. This minor unidentified component was isolated by chromatographic techniques such as normal-phase chromatography, gel permeation on Sephadex, solid-phase extraction and reversed-phase separations. Ionspray mass spectrometry (MS) was used for structural investigation on this compound due to the very small amount of isolated material. Flow injection analysis (FIA)-MS of the isolated compound gave positive-ion mass spectrum dominated by the protonated molecule, [M + H]+, at signal m/z 805, whereas the deprotonated molecule [M - H]- was observed in the negative-ion spectrum at signal m/z 803, thus indicating the molecular weight of 804 for the new toxin, the same as OA and its known isomers, DTX-2 and DTX-2B. Collision-induced dissociation (CID) as obtained by positive and negative tandem mass spectrometry (MS-MS) showed a fragmentation pattern for the new compound which was very similar to that of OA, DTX-2 and DTX-2B. Ionspray microLC-MS of a mixture containing the compound under investigation together with OA analogues showed the compound eluted after OA, DTX-2, DTX-2B and before DTX-1. All the chromatographic and mass spectrometric data indicated the compound to be another OA isomer and it was therefore coded DTX-2C. To the best of our knowledge this is the first report on the isolation of a new compound related to DSP toxins from natural communities of toxic phytoplankton.

Topics: Anthracenes; Chromatography, High Pressure Liquid; Diarrhea; Fluorescent Dyes; Fluorometry; Food Analysis; Foodborne Diseases; Marine Toxins; Mass Spectrometry; Okadaic Acid; Phytoplankton; Pyrans

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

Diarrhoetic shellfish poisoning (DSP) in Europe is due mainly to the presence of the dinoflagellate toxin, okadaic acid (OA). However, analysis of cultivated mussels (Mytilus edulis) from southwest Ireland revealed that an isomer of OA, dinophysistoxin-2, was the major toxin present during DSP episodes. Using fluorimetric HPLC, following derivatisation with 9-anthryldiazomethane, both OA and DTX-2 were found in shellfish during a prolonged toxic episode in 1991. However, examination of similar mussel cultivation locations in 1994 showed that DTX-2 was even more predominant. During this DSP period, OA levels were less than 0.7 microgram/g, whereas maximum DTX-2 levels of 6.3 micrograms/g hepatopancreas were recorded. This toxicity in shellfish occurred soon after high cell counts of Dinophysis acuta were observed. As well as large seasonal variability in toxin levels in rope cultured mussels, substantial variations were also observed, both horizontally and vertically, within the water column.

Topics: Animals; Bivalvia; Chromatography, High Pressure Liquid; Diarrhea; Dinoflagellida; Enzyme Inhibitors; Ethers, Cyclic; Ireland; Marine Toxins; Okadaic Acid; Pyrans; Reference Standards; Seawater; Shellfish Poisoning; Stereoisomerism

Two types of low polar derivatives of OA and dinophysitoxins have been reported in shellfish or in phytoplankton: 7-0-acyl esters containing a fatty acyl group attached through the 7-OH group and diol esters in which the carboxylic group of the toxins has been esterified. These compounds cannot be directly detected by liquid chromatography and fluorimetric detection as 9-anthryldiazomethane derivatives, owing in the first case to their low polarity and high molecular weight, and in the second case because they have the carboxylic group esterified. All of them must be hydrolysed before derivatization to be detected as Adam derivatives of the corresponding non-acylated toxins. In the Lee procedure, after extraction of the shellfish digestive glands with 80% methanol, a liquid-liquid partition with a non-polar solvent such as hexane is carried out in order to remove non-polar lipids. The presence of non-polar toxins was investigated in Spanish mussels and confirmed in the hexane layer, usually discarded in conventional extraction procedures, by analysis of the alkaline hydrolysis products. A preferred solubilization of these toxins in a non-polar solvent like hexane is reported. The inclusion of a hydrolytic step of the hexane extract in the general procedure is suggested in order to monitor the contribution of non-polar diarrhoetic shellfish poisons (DSPs) to the total DSP shellfish toxicity. This is the first report of DSPs other than OA and DTX2 in Spanish mussels.

Topics: Animals; Bivalvia; Carcinogens; Chromatography, High Pressure Liquid; Chromatography, Liquid; Diarrhea; Dinoflagellida; Esters; Ethers, Cyclic; Hydrogen-Ion Concentration; Hydrolysis; Marine Toxins; Mass Spectrometry; Okadaic Acid; Pyrans; Shellfish Poisoning; Solvents; Spain; Structure-Activity Relationship

The rare diarrhetic shellfish toxin, dinophysistoxin-2 (DTX-2), was isolated from the digestive glands of mussels (Mytilus edulis). This was achieved by chromatography on silica and Sephadex LH-20 followed by reversed-phase solid phase extraction and semi-preparative high-performance liquid chromatography (HPLC) with an Ultremex C18 column. Using 1-bromoacetylpyrene (BAP), as a precolumn derivatisation reagent, the diarrhetic shellfish toxins, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and DTX-2, were determined by HPLC with fluorimetric detection. Derivatisation using BAP was compared with 9-anthryldiazomethane (ADAM) and, although the latter exhibited a four-fold better sensitivity, the BAP method gave fewer artefact peaks from reagent decomposition. The limits of detection of OA and DTX-2 were 0.4 ng on-column using BAP, which permits this method to be used for the regulatory control of these toxins in shellfish.

Topics: Animals; Bivalvia; Chromatography, High Pressure Liquid; Dinoflagellida; Indicators and Reagents; Marine Toxins; Okadaic Acid; Pyrans; Pyrenes; Reproducibility of Results; Spectrometry, Fluorescence

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