Effect of mouse strain and gender on LD(50) of yessotoxin.

The aim of the present study was to determine whether the intraperitoneal LD(50) for yessotoxin (YTX) in mice varies with strain or gender. Thirty-six male and 36 female mice, of body weight 16-20 g, from each of the strains ICR (CD-1), Swiss (CFW-1) and NMRI were employed. They were not fasted before YTX treatment. At each dose, nine mice were injected with YTX solutions at 1.0 mL/20 g body weight, and observed for 24h. Symptoms and time to death were recorded. Within each mouse strain and gender arm, the study was performed as a basic four level Response Surface Pathway designed trial with nine mice at each dose level. YTX was isolated from a culture of Protoceratium reticulatum. The LD(50) values for female and male mice, respectively, were estimated as 380 and 462 microg/kg for the ICR, 269 and 328 microg/kg for the Swiss, and 314 and 412 microg/kg for the NMRI strains. The increases in LD(50) from female to male mice were found to be 22% for ICR, 22% for Swiss and 31% for NMRI. The largest difference in LD(50) among mouse strains was detected between the ICR and Swiss strains, where the deviation was 41% in both females and males. The difference between mouse strains was found significant (p = 0.03). For all three strains, females were more susceptible than males, with a difference in LD(50) of 1.2-1.3-fold. The largest difference between the least- and most-susceptible strain was 1.4-fold for both females and males. The largest difference in LD(50), 1.7-fold, was observed between female Swiss and male ICR mice. The difference between genders was not significant (p = 0.12). These results indicate that other factors, like handling of the animals, and the source and handling of the toxin, may significantly influence the outcome of studies on acute toxicity since the reported differences in LD(50) vary by a factor of about seven.

Relative toxicity of dinophysistoxin-2 (DTX-2) compared with okadaic acid, based on acute intraperitoneal toxicity in mice.

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.

Diarrhetic shellfish poisoning by okadaic acid esters from Brown crabs (Cancer pagurus) in Norway.

In 2002 several hundred people were taken ill after eating self-harvested brown crabs (Cancer pagurus) in the southern part of Norway. The symptoms were similar to diarrhetic shellfish poisoning (DSP) although with a somewhat delayed onset. This happened at the same time as an unusual early bloom of Dinophysis acuta had lead to high amounts of DSP toxins in blue mussels (Mytilus edulis) in the same area. The proposed cause of the intoxication was that crabs had accumulated toxins by eating blue mussels. Analyses of crab material from the area revealed very little free toxin in the form of okadaic acid (OA). However, after alkaline hydrolysis of the material, the amounts of OA found in the crabs were above the toxic level. MS/MS analysis of a sample from one intoxication episode indicated presence of the 14:0, 16:1, 16:0 and 18:1 fatty acid esters of okadaic acid. Esterified OA constituted more than 90% of total identified DSP toxins in crabs, indicating that not only esterified toxin from mussels was accumulated, but also that appreciable transfer of OA to OA-esters occurred in the crabs.

Yessotoxins in Norwegian blue mussels (Mytilus edulis): uptake from Protoceratium reticulatum, metabolism and depuration.

The Protoceratium reticulatum cell density at Flodevigen reached a maximum of 2200 cells/L on 16 May 2001. The levels of yessotoxins (YTXs) in blue mussels (Mytilus edulis) at the same site increased sharply by 14 May and peaked on 28 May, after which they steadily declined. No other algal species present showed a similar pattern of correspondence. Together with the recent finding that Norwegian strains of P. reticulatum produce YTXs, these results indicate that P. reticulatum causes yessotoxin (YTX) contamination of shellfish in Norway, and that only relatively low cell densities are necessary for this to occur. The mussels from Flodevigen were analyzed by LC-MS for YTX, 45-hydroxyYTX, carboxyYTX, and a new yessotoxin believed to be 45-hydroxycarboxyYTX, and by ELISA for YTXs. The seasonal variations in toxin content versus time measured by the two methods were qualitatively very similar, although the response in the ELISA was 3-9 times higher due to the antibodies detecting other YTXs that were not detected by the LC-MS method. Changes in the LC-MS profile for YTXs, and in the ratio of YTXs by LC-MS to YTXs by ELISA with time, were consistent with extensive metabolism of YTX in the mussels. Kinetic analysis of the LC-MS data showed an initial half-life of 20 days for YTX, and for YTX+45-hydroxyYTX, in the mussels. Similar analysis of the ELISA data gave a half-life of 24 days for YTXs. The depuration rate remained consistent over a 3-month period during which the temperature remained at 13-16 degrees C.

Tissue distribution, effects of cooking and parameters affecting the extraction of azaspiracids from mussels, Mytilus edulis, prior to analysis by liquid chromatography coupled to mass spectrometry.

This study used liquid chromatography coupled to mass spectrometry to identify some parameters important in the analysis of azaspiracids. The first aspect was the distribution of azaspiracids within mussels, in particular the content in the digestive gland as compared to the remaining tissues. In our study, azaspiracids accumulated in the digestive gland, similar to other lipophilic toxins. The ratio of toxin in the digestive gland compared to the whole mussel was on average circa 5, both for a bulk sample collected in Norway in 2004 and for 28 samples from Ireland collected over 3 years (2001-2003). These results may justify the practise to only analyse the digestive gland, a step considered necessary to achieve adequate detection limits for azaspiracids both in the mouse bioassay and other analytical techniques. Steaming of mussels as a sample pre-treatment was found to be another parameter affecting the result. Azaspiracids concentrated indirectly, i.e. through the loss of water or juice from the matrix. The cooked shellfish tissues had a concentration of azaspiracids 2-fold higher than the uncooked shellfish, both for whole flesh and for digestive gland tissue. This finding is of particular importance since it may affect the maximum guidance level at which shellfish may be allowed for human consumption. Finally, parameters affecting the extraction efficiency were studied, including the nature of the extraction solvent, the sample-to-solvent ratio and replicate extraction. The largest differences were observed between different solvents and between different sample-to-solvent ratios, while the effect of replicate extraction was minimal if large sample-to-solvent ratios were used. Duplicate extraction using 100% methanol was found to be the best combination of parameters.

Comparison of ELISA and LC-MS analyses for yessotoxins in blue mussels (Mytilus edulis).

Blue mussels (Mytilus edulis) collected from Flødevigen Bay, Norway, in 2001 and 2002 were analysed for yessotoxins (YTXs) by ELISA and yessotoxin (YTX), 45-hydroxyYTX, and carboxyYTX by LC-MS. Results from the two methods were compared to evaluate the ELISA. The response in the ELISA was 3-13 times higher than LC-MS, probably due to the antibodies binding to other YTX analogues not included in the LC-MS analysis. Nevertheless, the correlation between ELISA and LC-MS was good, with r2 values> or =0.8. The results indicate that the ELISA is a reliable method for estimating the total level of YTXs in mussels, and are consistent with extensive metabolism of algal YTXs in mussels. YTX was a minor component in the blue mussels at all times compared to 45-hydroxyYTX and especially carboxyYTX, except when the P. reticulatum bloom occurred. The results also indicate the presence of significant amounts of YTX analogues in addition to those measured by LC-MS. All samples below 4 mg/kg by ELISA were below the current EU regulatory limit of 1 mg/kg by LC-MS. Therefore, we propose using ELISA as a screening tool with a cut-off limit at 4 mg/kg for negative samples, whereas samples above this limit would be reanalyzed by LC-MS.

Isolation and identification of (44-R,S)-44,55-dihydroxyyessotoxin from Protoceratium reticulatum, and its occurrence in extracts of shellfish from New Zealand, Norway and Canada.

44,55-Dihydroxyyessotoxin (1) was isolated from extracts of Protoceratium reticulatum and identified by analysis of its one- and two-dimensional NMR and mass spectra. In addition, LC-MS methods revealed the presence of compounds tentatively identified as (44-R,S)-44,55-dihydroxy-41a-homoyessotoxin (2) and (44-R,S)-44,55-dihydroxy-9-methyl-41a-homoyessotoxin (3). LC-MS analyses indicate that 1 is a constituent of P. reticulatum in New Zealand and Norway, and it was present in three species of mussels from New Zealand, Norway, and Canada.

Isolation of a 1,3-enone isomer of heptanor-41-oxoyessotoxin from Protoceratium reticulatum cultures.

The 1,3-enone isomer (1) of heptanor-41-oxoyessotoxin (2) was isolated from extracts of Protoceratium reticulatum during large-scale production of yessotoxin (4). We found that 2 readily isomerizes to 1 in the presence of dilute ammonia and present evidence for the existence of 40-epi-2 (3) that also isomerizes to 1. 1-3 were detected by LC-MS methods both in extracts of P. reticulatum cultures and in mussels contaminated with yessotoxins. The isomerization of 2 and 3 into 1 occurs so readily that purification on basic alumina needs to be conducted carefully. No toxic effects were recorded in mice injected intraperitoneally with 1 at a dose of 5,000 microg/kg.

Study of possible combined toxic effects of azaspiracid-1 and okadaic acid in mice via the oral route.

Toxins from the okadaic acid (OA) and azaspiracid (AZA) group cause considerable negative health effects in consumers when present in shellfish above certain levels. The main symptoms, dominated by diarrhoea, are caused by damage to the gastrointestinal (GI) tract. Even though OA and AZAs exert toxicity via different mechanisms, it is important to find out whether they may enhance the health effects if present together since they act on the same organs and are regulated individually. In this study, the main issue was the possibility of enhanced lethality in mice upon combined oral exposure to OA and AZA1. In addition, pathological effects in several organs and effects on absorption from the GI tract were studied. Although the number of mice was small due to low availability of AZA1, the results indicate no additive or synergistic effect on lethality when AZA1 and OA were given together. Similar lack of increased toxicity was observed concerning pathological effects that were restricted to the GI-tract. OA and AZA1 were absorbed from the GI-tract to a very low degree, and when given together, uptake was reduced. Taken together, these results indicate that the present practice of regulating toxins from the OA and AZA group individually does not present an unwanted increased risk for consumers of shellfish.

Pinnatoxins and spirolides in Norwegian blue mussels and seawater.

Fast-acting cyclic imines belonging to the pinnatoxin and pteriatoxin group of toxins were originally identified in shellfish of the genera Pinna and Pteria in Japan, after food poisoning events in China linked to consumption of Pinna spp. Recently, a range of new and known pinnatoxin analogs has been identified in shellfish, sediment, and seawater samples from Australia and New Zealand. Although the structurally closely-related spirolide toxins are better known, and have a worldwide distribution including Norway and other parts of Europe, the presence of pinnatoxins has not been reported in European waters or shellfish. Here we report results from a survey of Norwegian blue mussels for the presence of pinnatoxins and spirolides, by LC-MS/MS analysis of extracts obtained as part of Norway's routine monitoring programme for regulated algal toxins during late autumn and early winter 2009. Spirolides and pinnatoxin G were widespread (pinnatoxin G (1), spirolide C (2), iso-spirolide C (3), 13-desmethylspirolide C (4), 13,19-didesmethylspirolide C (5), and 20-methylspirolide G (6) were detected in 69%, 13%, 60%, 22%, 33%, and 77%, respectively, of the shellfish samples) and, although levels were generally low, concentrations of up to 115 µg/kg of pinnatoxin G (1) and 226 µg/kg of 13-desmethylspirolide C (4) were detected. We also analyzed stored extracts from passive sampling disks deployed as part of a separate study in autumn 2007. All the stored extracts contained 20-methylspirolide G (which predominated at most locations), most contained pinnatoxin G (73%) and 13,19-didesmethylspirolide C (67%), but iso-spirolide C (36%) and 13-desmethylspirolide C (52%) were also detected in many of the samples. These results suggest that pinnatoxins may be much more widespread than previously suspected, and indicate that they or related compounds could be responsible for sporadic incidents of rapid-onset symptoms during mouse bioassays of shellfish in Europe and elsewhere. The toxicological significance of these levels of pinnatoxins and spirolides is at present unclear. However, although pinnatoxins appear to be less toxic than spirolides by intraperitoneal injection in the mouse bioassay, recently published preliminary toxicological data indicate that pinnatoxins may be as much as an order of magnitude more toxic than spirolides by oral ingestion via food.

Combined oral toxicity of azaspiracid-1 and yessotoxin in female NMRI mice.

For many years, the presence of yessotoxins (YTXs) in shellfish has contributed to the outcome of the traditional mouse bioassay and has on many occasions caused closure of shellfisheries. Since YTXs do not appear to cause diarrhoea in man and exert low oral toxicity in animal experiments, it has been suggested that they should be removed from regulation. Before doing so, it is important to determine whether the oral toxicity of YTXs is enhanced when present together with shellfish toxins known to cause damage to the gastrointestinal tract. Consequently, mice were given high doses of YTX, at 1 or 5 mg/kg body weight, either alone or together with azaspiracid-1 (AZA1) at 200 µg/kg. The latter has been shown to induce damage to the small intestine at this level. The combined exposure caused no clinical effects, and no pathological changes were observed in internal organs. These results correspond well with the very low levels of YTX detected in internal organs by means of LC-MS/MS and ELISA after dosing. Indeed, the very low absorption of YTX when given alone remained largely unchanged when YTX was administered in combination with AZA1. Thus, the oral toxicity of YTX is not enhanced in the presence of sub-lethal levels of AZA1.

Sub-lethal dosing of azaspiracid-1 in female NMRI mice.

The main aim of this study was to examine absorption and pathological effects of a single sub-lethal dose of the marine biotoxin azaspiracid-1 (AZA1) in mice after oral intubation. When the mice received AZA1 at doses of 100, 200 or 300 µg/kg body weight (b.w.), the toxin was absorbed dose-dependently. Highest concentrations after 24 h were detected in kidneys, spleen and lungs, followed by liver and heart. Only trace amounts were seen in the brain. After seven days, the toxin level had dropped significantly in all organs except for the kidneys. The amount of toxin absorbed was highest in the liver, followed by kidneys, lungs, spleen and heart and the total amount of toxin in the internal organs analysed after 24 h was estimated to be only about 2% of the total amount given for all three dose groups. Pathological changes were only detected in the upper part of the small intestine (duodenum), consisting of mild cellular detachment in the tips of the villi, expansion of the crypts and necrotic changes in lamina propria. In a previous study very long persistence of damage to the gastrointestinal tract by repeated exposures to AZA toxins was reported. In our study, full recovery from the pathological changes was observed seven days after a single exposure to AZA1 at the doses applied.

Discovery of fatty acid ester metabolites of spirolide toxins in mussels from Norway using liquid chromatography/tandem mass spectrometry.

Cultured mussels sampled in the spring of 2002 and 2003 from Skjer, a location in the Sognefjord, Norway, tested positive in the mouse bioassay for lipophilic toxins. In a previous report, it was established that a number of spirolides, cyclic imine toxins produced by the phytoplankton Alexandrium ostenfeldii, were present in the mussels and were responsible for the observed toxicity. The main toxin proved to be a new compound named 20-methyl spirolide G. In subsequent studies, a delayed onset of spirolide-like symptoms in the mouse bioassay exceeding the usual time limit of 20 min was observed in some samples, with symptoms and death appearing as long as 45-50 min after injection. It is well known that shellfish can extensively metabolize other toxins, such as okadaic acid and the dinophysistoxins, to fatty acid acyl esters and it is also known that a delayed onset of toxic symptoms with such metabolites can occur. Analyses performed with liquid chromatography/tandem mass spectrometry (LC/MS/MS) have revealed a complex mixture of esters of 20-methyl spirolide G in the contaminated mussels. Precursor ion scanning has delineated the range of fatty acid esters involved, while product ion scanning has provided information on structure. Identity was also supported through reaction of 20-methyl spirolide G with palmitic anhydride, which produced a derivative with a retention time and spectrum identical with one putative metabolite, 17-O-palmitoyl-20-methyl spirolide G.

Detection and identification of spirolides in norwegian shellfish and plankton.

Mussels sampled in the spring of 2002 and 2003 from Skjer, a location in Sognefjord, Norway, tested positive in the mouse bioassay for lipophilic toxins. The symptoms, which included cramps, jumping, and short survival times (as low as 4 min), were not characteristic of toxins previously observed in Norway. A survey of the algae present at the aquaculture sites showed that the toxicity correlated with blooms of Alexandrium ostenfeldii. Up to 2200 cells/L were found at the peak of one bloom. In Canadian waters, this alga is known to be a producer of the cyclic imine toxins, spirolides. Analysis of mussel extracts from Skjer in the spring of 2002 and 2003, using liquid chromatography tandem mass spectrometry, revealed the presence of several new spirolides. The same compounds were also found in algal samples dominated by A. ostenfeldii, which had been sampled from Skjer in February 2003. A large-scale extraction of mussel digestive glands and chromatographic fractionation of the extracts allowed the isolation and structure elucidation of the main spirolide, 20-methyl spirolide G, with a molecular weight of 705.5. This is the first confirmed occurrence of spirolides in mussels and plankton from Norway.

Isolation of 41a-homoyessotoxin and the identification of 9-methyl-41a-homoyessotoxin and nor-ring A-yessotoxin from Protoceratium reticulatum.

41a-homoyessotoxin (1), a new analogue of yessotoxin (7), was isolated from extracts of Protoceratium reticulatum grown in culture. In addition, 9-methyl-41a-homoyessotoxin (2) and a nor-ring A-yessotoxin (3) were identified as the major components of a mixed fraction. The structural information was initially obtained from LC-UV and LC-MS3 chromatograms, with subsequent definitive structure determination by NMR, LC-MS/MS, and high-resolution MS. Full 1H and indirectly detected 13C NMR assignments for all but two carbon atoms were obtained from ca. 100 microg of 1. Full 1H NMR assignments for 2 and 3 and identification of three new heptanor-41-oxo-analogues of 3 (4-6) during LC-MS3 analysis of a fraction containing 1-3 and 7 are also reported.