Diarrhetic Shellfish Poisoning Toxins Research Articles

Sulfated diesters of okadaic acid and DTX-1: Self-protective precursors of diarrhetic shellfish poisoning (DSP) toxins

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

The Mechanism of Diarrhetic Shellfish Poisoning Toxin Production in Prorocentrum spp.: Physiological and Molecular Perspectives.

Diarrhetic shellfish poisoning (DSP) is a gastrointestinal disorder caused by the consumption of seafood contaminated with okadaic acid (OA) and dinophysistoxins (DTXs). OA and DTXs are potent inhibitors of protein phosphatases 2A, 1B, and 2B, which may promote cancer in the human digestive system. Their expression in dinoflagellates is strongly affected by nutritional and environmental factors. Studies have indicated that the level of these biotoxins is inversely associated with the growth of dinoflagellates at low concentrations of nitrogen or phosphorus, or at extreme temperature. However, the presence of leucine or glycerophosphate enhances both growth and cellular toxin level. Moreover, the presence of ammonia and incubation in continuous darkness do not favor the toxin production. Currently, studies on the mechanism of this biotoxin production are scant. Full genome sequencing of dinoflagellates is challenging because of the massive genomic size; however, current advanced molecular and omics technologies may provide valuable insight into the biotoxin production mechanism and novel research perspectives on microalgae. This review presents a comprehensive analysis on the effects of various nutritional and physical factors on the OA and DTX production in the DSP toxin-producing Prorocentrum spp. Moreover, the applications of the current molecular technologies in the study on the mechanism of DSP toxin production are discussed.

Development of Certified Reference Materials for Diarrhetic Shellfish Poisoning Toxins, Part 2: Shellfish Matrix Materials.

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.

Development of Certified Reference Materials for Diarrhetic Shellfish Poisoning Toxins, Part 1: Calibration Solutions.

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.

The seasonal dynamics of diarrhetic shellfish poisoning toxins in the digestive gland of Crenomytilus grayanus (Dunker, 1853) analyzed by high-performance liquid chromatography with fluorometric detection

The seasonal dynamics of diarrhetic shellfish poisoning (DSP) toxins in the digestive gland of the mussel Crenomytilus grayanus (Dunker, 1853) from Peter the Great Bay in the Sea of Japan have been studied. Toxins in the form of 4-bromomethyl-7-methoxycoumarin derivatives have been analyzed using high-performance liquid chromatography (HPLC) with fluorometric detection. This method allows the determination of the quantitative and qualitative composition of individual toxins. All the C. grayanus samples contained DSP toxins, but their concentration varied substantially during the year. The highest concentration was recorded from the mussels collected in May; the lowest concentration was recorded in February. The toxins were represented predominantly by dinophysistoxin-2; in July-September and December, okadaic acid was detected. An assumption has been made about the relationship between the potential producers of DSP toxins in the waters of the Peter the Great Bay and the dynamics of these toxins in tissues of C. grayanus. The HPLC method, as an alternative to the enzyme-linked immunosorbent assay, was used to assess the quantitative and qualitative composition of DSP toxins in seafood for the first time in the Russian Federation. This method can be recommended for the routine monitoring of toxins conducted by the controlling organizations of the Russian Federal Service for Veterinary and Phytosanitary Surveillance.

Bloom of Dinophysis spp. dominated by D. sacculus and its related diarrhetic shellfish poisoning (DSP) outbreak in Alfacs Bay (Catalonia, NW Mediterranean Sea): Identification of DSP toxins in phytoplankton, shellfish and passive samplers

We report a comprehensive study of a diarrhetic shellfish poisoning (DSP) outbreak in a Mediterranean coastal embayment, integrating phytoplankton identification and quantification data, with toxin analysis by LC–MS/MS of shellfish samples (mussels and oysters), phytoplankton concentrates, and solid phase adsorbing toxin tracking (SPATT) devices. Alfacs Bay was proposed as a model for the study of semi-enclosed coastal embayments and it is the most important shellfish farming area in Catalonia (Spain, NW Mediterranean Sea). The bay suffered a ban for bivalve harvesting that lasted 78 days, from late winter to the end of the spring of 2012, due to a harmful algae bloom of the toxic dinoflagellate Dinophysis sacculusthat fluctuated in three abundance peaks, reaching maximum abundance of 2200 cell/L. Mussels sampled during the bloom contained okadaic acid (OA, maximum 592 μg/kg, 3.7 times the maximum permitted level (MPL) set by the European Commission), and pectenotoxin-2 (PTX2, maximum 61 μg/kg). Oysters also contained OA and PTX2, but always below the MPL. Toxin content per cell measured from phytoplankton concentrates in filters was most likely overestimated (up to 461 pg OA/cell and 668 pg PTX2/cell), but showed an increase of cell toxicity towards the end of the bloom. OA-diol esters were not detected in phytoplankton aggregates. Concentration of DSP toxins in SPATT devices were up to 94 ng OA/g resin⋅day, 42 ng PTX2/g resin⋅day and 7 ng PTX2-sa/g resin⋅day. The applied monitoring strategy, based on the measurement of Dinophysis cell abundance in the water column, was effective to protect public health and did not extended the regulatory closure unnecessarily. Concentration of DSP in SPATT bags followed Dinophysis abundance dynamics, thus SPATTs were confirmed as a valuable research tool for Dinophysis blooms.

Effect of the industrial canning on the toxicity of mussels contaminated with diarrhetic shellfish poisoning (DSP) toxins

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.

Application of rapid test kits for the determination of Diarrhetic Shellfish Poisoning (DSP) toxins in bivalve molluscs from Great Britain

Four commercial rapid screening methods for Diarrhetic Shellfish Poisoning were applied to the analysis of naturally contaminated shellfish samples from GB. The performance of each kit was assessed through comparison with the reference LC-MS/MS method on a range of both positive and negative bivalve mollusc samples. A quantitative PP2A protein phosphatase assay was the only assay to show the complete absence of false negative results. It showed a fair correlation with LC-MS/MS but with an overall overestimation of sample toxicity together with some indications of interference from sample matrix, most notably within oyster species. A quantitative competitive ELISA also gave a fair correlation with LC-MS/MS, with no evidence of toxicity overestimation and with a good response to samples containing little or no DST's, although one false negative was recorded. The two qualitative lateral flow assays both provided a high percentage agreement with the LC-MS/MS results and there were no indications of false positive results, although both kits also returned one false negative result. The false negative results returned by the three assays were all associated with samples containing high proportions of DTX2, a toxin which occurs commonly in UK shellfish. The scanners provided with both lateral flow assays were easy to use and the provision of numerical results enables a semi-quantitative assessment of toxicities which would significantly benefit the end user. Whilst key differences exist between the proposed assays they are all rapid, do not require expensive equipment and the work here has provided some evidence for suitability for indicative testing for some species of bivalve shellfish from GB. Further work is required however using a larger number of test kit batches on a greater number of samples, particularly for those containing high proportions of DTX2.

Analysis of diarrhetic shellfish poisoning toxins and pectenotoxin-2 in the bottlenose dolphin (Tursiops truncatus) by liquid chromatography–tandem mass spectrometry

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 2.8ng/g or less in tissue samples and 0.7ng/ml or less in urine.

Detection of diarrhetic shellfish poisoning toxins using high-sensitivity human cancer cell-based impedance biosensor

Diarrhetic shellfish poisoning (DSP) toxin is a dangerous contamination in seafood worldwide that can threaten human health and fishing. A large number of animal bioassays and chemical analytical methods are employed for DSP toxin detection. However, these toxin detection methods are low-throughput and high-cost which hamper their wide applications. In this study, HeLa and HepG2 cell lines were selected as the sensitive elements to establish the CIBs for monitoring the cytotoxicity induced by a representative DSP toxin, okadaic acid (OA). The limit of detection (LOD) of HeLa- and HepG2-based biosensors are 10.2μg/L and 3.3μg/L, respectively, which are both lower than the conventional cell-based assay (21.2μg/L of HeLa cells and 9.8μg/L of HepG2 cells). The half maximal inhibitory concentration (IC50) values of OA in HeLa and HepG2 cells which were obtained from CIB (49.9±4.9 and 39.2±4.3μg/L) are both lower than Cell Counting Kit-8 assay (CCK8) (62.7±7.1 and 45.8±6.7μg/L). Besides, CIB measurement presents good correlation with mouse bioassay (MBA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS). In summary, all the results indicate that the CIB technology had great potential to be an effective complement in DSP toxins detection.

Bioaccumulation of trace metals in mussel (Mytilus galloprovincialis) from Mali Ston Bay during DSP toxicity episodes.

The Croatian National Monitoring Program revealed the presence of Diarrhetic Shellfish Poisoning (DSP) toxicity in Mediterranean blue mussel (Mytilus galloprovincialis) from breeding farms in southern Adriatic Sea through January to June 2011. The mouse bioassay tests (MBA; at the time the official method for DSP toxins) were accompanied by atypical symptomatology in the animals and this caused doubts about the assay results. Consequently, in parallel studies reported here, the concentration of Cd, Cr, Cu, Ni, Pb and Zn in soft tissue of DSP positive and negative mussels samples was determined. Cd, Cr, Zn and Ni show higher values in approximately 75% of the DSP positive samples, whereas for Pb and Cr the values were 26% and 34%, respectively. This trend was unchanged during the whole observation period.

Nitrogenous nutrients promote the growth and toxicity of Dinophysis acuminata during estuarine bloom events.

Diarrhetic Shellfish Poisoning (DSP) is a globally significant human health syndrome most commonly caused by dinoflagellates within the genus Dinophysis. While blooms of harmful algae have frequently been linked to excessive nutrient loading, Dinophysis is a mixotrophic alga whose growth is typically associated with prey availability. Consequently, field studies of Dinophysis and nutrients have been rare. Here, the temporal dynamics of Dinophysis acuminata blooms, DSP toxins, and nutrients (nitrate, ammonium, phosphate, silicate, organic compounds) were examined over four years within two New York estuaries (Meetinghouse Creek and Northport Bay). Further, changes in the abundance and toxicity of D. acuminata were assessed during a series of nutrient amendment experiments performed over a three year period. During the study, Dinophysis acuminata blooms exceeding one million cells L-1 were observed in both estuaries. Highly significant (p<0.001) forward stepwise multivariate regression models of ecosystem observations demonstrated that D. acuminata abundances were positively dependent on multiple environmental parameters including ammonium (p = 0.007) while cellular toxin content was positively dependent on ammonium (p = 0.002) but negatively dependent on nitrate (p<0.001). Nitrogen- (N) and phosphorus- (P) containing inorganic and organic nutrients significantly enhanced D. acuminata densities in nearly all (13 of 14) experiments performed. Ammonium significantly increased cell densities in 10 of 11 experiments, while glutamine significantly enhanced cellular DSP content in 4 of 5 experiments examining this compound. Nutrients may have directly or indirectly enhanced D. acuminata abundances as densities of this mixotroph during experiments were significantly correlated with multiple members of the planktonic community (phytoflagellates and Mesodinium). Collectively, this study demonstrates that nutrient loading and more specifically N-loading promotes the growth and toxicity of D. acuminata populations in coastal zones.

Effect of the industrial steaming on the toxicity, estimated by LC–MS/MS, of mussels exposed for a long time to diarrhetic shellfish poisoning (DSP) toxins

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.

Characterization and comparison of toxin-producing isolates of Dinophysis acuminata from New England and Canada.

Following the identification of the first toxic isolate of Dinophysis acuminata from the northwestern Atlantic, we conducted detailed investigations into the morphology, phylogeny, physiology, and toxigenicity of three isolates from three sites within the northeastern U.S./Canada region: Eel Pond and Martha's Vineyard, Massachusetts, and the Bay of Fundy. Another isolate, collected from the Gulf of Mexico, was grown under the same light, temperature, and prey conditions forcomparison. Despite observed phenotypic heterogeneity, morphometrics and molecular evidence classified the three northwestern Atlantic isolates as D.acuminata Claparède & Lachmann, whereas the isolate from the Gulf of Mexico was morphologically identified as D. cf. ovum. Physiological and toxin analyses supported these classifications, with the three northwestern Atlantic isolates being more similar to each other with respect to growth rate, toxin profile, and diarrhetic shellfish poisoning (DSP) toxin content (okadaic acid + dinophysistoxin 1/cell) than they were to the isolate from the Gulf of Mexico, which had toxin profiles similar to those published for D. cf. ovum F. Schütt. The DSP toxin content, 0.01-1.8pg okadaic acid (OA) + dinophysistoxin (DTX1) per cell, of the three northwestern Atlantic isolates was low relative to other D.acuminata strains from elsewhere in the world, consistent with the relative scarcity of shellfish harvesting closures due to DSP toxins in the northeastern U.S. and Canada. If this pattern is repeated with the analyses of more geographically and temporally dispersed isolates from the region, it would appear that the risk of significant DSP toxin outbreaks in the northwestern Atlantic is low to moderate. Finally, the morphological, physiological, and toxicological variability within D.acuminata may reflect spatial (and/or temporal) population structure, and suggests that sub-specific resolution may be helpful in characterizing bloom dynamics and predicting toxicity.

Evolving to the optoelectronic mouse for phycotoxin analysis in shellfish.

Despite ethical and technical concerns, the in vivo method, or more commonly referred to mouse bioassay (MBA), is employed globally as a reference method for phycotoxin analysis in shellfish. This is particularly the case for paralytic shellfish poisoning (PSP) and emerging toxin monitoring. A high-performance liquid chromatography method (HPLC-FLD) has been developed for PSP toxin analysis, but due to difficulties and limitations in the method, this procedure has not been fully implemented as a replacement. Detection of the diarrhetic shellfish poisoning (DSP) toxins has moved towards LC-mass spectrometry (MS) analysis, whereas the analysis of the amnesic shellfish poisoning (ASP) toxin domoic acid is performed by HPLC. Although alternative methods of detection to the MBA have been described, each procedure is specific for a particular toxin and its analogues, with each group of toxins requiring separate analysis utilising different extraction procedures and analytical equipment. In addition, consideration towards the detection of unregulated and emerging toxins on the replacement of the MBA must be given. The ideal scenario for the monitoring of phycotoxins in shellfish and seafood would be to evolve to multiple toxin detection on a single bioanalytical sensing platform, i.e. 'an artificial mouse'. Immunologically based techniques and in particular surface plasmon resonance technology have been shown as a highly promising bioanalytical tool offering rapid, real-time detection requiring minimal quantities of toxin standards. A Biacore Q and a prototype multiplex SPR biosensor have been evaluated for their ability to be fit for purpose for the simultaneous detection of key regulated phycotoxin groups and the emerging toxin palytoxin. Deemed more applicable due to the separate flow channels, the prototype performance for domoic acid, okadaic acid, saxitoxin, and palytoxin calibration curves in shellfish achieved detection limits (IC20) of 4,000, 36, 144 and 46 μg/kg of mussel, respectively. A one-step extraction procedure demonstrated recoveries greater than 80% for all toxins. For validation of the method at the 95% confidence limit, the decision limits (CCα) determined from an extracted matrix curve were calculated to be 450, 36 and 24 μg/kg, and the detection capability (CCβ) as a screening method is ≤10 mg/kg, ≤160 μg/kg and ≤400 μg/kg for domoic acid, okadaic acid and saxitoxin, respectively.

Evaluation of okadaic acid, dinophysistoxin-1 and dinophysistoxin-2 toxicity on Neuro-2a, NG108-15 and MCF-7 cell lines

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.

P-glycoprotein expression in Perna viridis after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins

Bivalves naturally exposed to toxic algae have mechanisms to prevent from harmful effects of diarrhetic shellfish poisoning (DSP) toxins. However, quite few studies have examined the mechanisms associated, and the information currently available is still insufficient. Multixenobiotic resistance (MXR) is ubiquitous in aquatic invertebrates and plays an important role in defense against xenobiotics. Here, to explore the roles of P-glycoprotein (P-gp) in the DSP toxins resistance in shellfish, complete cDNA of P-gp gene in the mussel Perna viridis was cloned and analyzed. The accumulation of okadaic acid (OA), a main component of DSP toxins, MXR activity and expression of P-gp in gills of P. viridis were detected after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins in the presence or absence of P-gp inhibitors PGP-4008, verapamil (VER) and cyclosporin A (CsA). The mussel P. viridis P-gp closely matches MDR/P-gp/ABCB protein from various organisms, having a typical sequence organization as full transporters from the ABCB family. After exposure to P. lima, OA accumulation, MXR activity and P-gp expression significantly increased in gills of P. viridis. The addition of P-gp-specific inhibitors PGP-4008 and VER decreased MXR activity induced by P. lima, but had no effect on the OA accumulation in gills of P. viridis. However, CsA, a broad-spectrum inhibitor of ABC transporter not only decreased MXR activity, but also increased OA accumulation in gills of P. viridis. Together with the ubiquitous presence of other ABC transporters such as MRP/ABCC in bivalves and potential compensatory mechanism in P-gp and MRP-mediated resistance, we speculated that besides P-gp, other ABC transporters, especially MRP might be involved in the resistance mechanisms to DSP toxins.

Influence of body weight of mice on the susceptibility to okadaic acid, a diarrhetic shellfish poisoning toxin

The mouse bioassay (MBA) for diarrhetic shellfish poisoning (DSP) toxins has been widely used in many countries of the world. However, different body weight ranges of mice are designated to be used in the Japanese official method and European Union procedure. In this study we investigated whether and to what extent the body weights of the mice affect the susceptibility to DSP toxins. A lethal dose of okadaic acid, one of the representative DSP toxins, was injected intraperitoneally into mice of five different body weight range groups, from 14 to 24 g. The mice were observed until 24 h after injection. The lethality was 100% in the 14–15 and 16–17 g groups, 80% in the 19–20 g group, 50% in the 21–22 g group, and 40% in the 23–24 g group, with significant differences. Survival analysis indicated a relationship between body weights of mice and susceptibility to okadaic acid. These results would be quite useful not only for the MBA, but also to improve understanding of the biological responses to DSP toxins.

Cloning and expression analysis of P-glycoprotein gene in Crassostrea ariakensis

P-glycoprotein (P-gp), as an ATP-binding cassette transporter, can transport a variety of substrates across the cell membrane in human and animals, even in aquatic animals. Multixenobiotic resistance phenomenon (MXR) related to the P-gp has been identified and characterized in several aquatic organisms, however, the nature of this transporter and the structural basis for the resistance are still poorly understood. In this study, to provide an insight into the roles of P-gp in resistance of bivalve against environmental pollution and toxins, complete cDNA of P-gp gene in Crassostrea ariakensis was cloned and analyzed. The accumulation of okadaic acid (OA), a main component of diarrhetic shellfish poisoning (DSP) toxins, and expression of P-gp in gills, digestive glands and mantle of C. ariakensis, were detected upon exposure to Prorocentrum lima, a known producer of DSP toxins. The complete sequence of oyster P-gp showed a high identity to MDR/P-gp/ABCB proteins from other species and contained some classical features of ABCB transport proteins. Upon exposure to P. lima for 72h, the OA level increased 2.5-fold in gills and 6.4-fold in digestive glands of C. ariakensis. Meanwhile, the expression of P-gp mRNA increased 2.4-fold in gills and decreased slightly in digestive glands. No significant difference in P-gp protein expression was found by western blot in all of the tissues analyzed in P. lima-exposed oysters. Taking all of our results into account, we speculate that P-gp likely plays a role in the response to DSP toxins in C. ariakensis.

The binding of okadaic acid analogs to recombinant OABP2.1 originally isolated from the marine sponge Halichondria okadai

The binding between [24-3H]okadaic acid (OA) and a recombinant OA binding protein OABP2.1 was examined using various OA analog, including methyl okadaate, norokadanone, 7-deoxy OA, and 14,15-dihydro OA, 7-O-palmitoyl DTX1, to investigate the structure activity relationship. Among them, 7-O-palmitoyl DTX1, which is one of the diarrhetic shellfish poisoning (DSP) toxins identified in shellfish, displayed an IC50 for [24-3H]OA binding at 51±6.3nM (Mean±SD). In addition, a synthetic compound, N-pyrenylmethyl okadamide, exhibited its IC50 at 10±2.9nM (Mean±SD). These results suggested that the recombinant OABP2.1 and the N-pyrenylmethyl okadamide might be core substances in a novel assay for the DSP toxins.