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.     

Complex yessotoxins profile in Protoceratium reticulatum from north-western Adriatic sea revealed by LC-MS analysis.

While the occurrence of yessotoxin (YTX) has been reported worldwide from Protoceratium reticulatum, the biogenetic origin of some YTX analogues is still unknown, thus raising an issue whether they are metabolites of YTX formed in mussels or true products of different dinoflagellate species. Findings reported herein suggest that P. reticulatum from the north-western Adriatic sea is responsible for production, together with YTX (1), of homoYTX (2), 45-OHYTX (3), carboxyYTX (5), and noroxoYTX (7). YTX and its analogues have been determined by high performance liquid chromatography coupled with electrospray ion trap mass spectrometry (HPLC-MS and HPLC-MSn experiments). The result is the first to confirm production of these YTX analogues from this species and indicate it as a producing organism of homoYTX, 45-OHYTX, carboxyYTX, and noroxoYTX found in shellfish.     

Use of ELISA to identify Protoceratium reticulatum as a source of yessotoxin in Norway.

Picked cells of Protoceratium reticulatum collected from five locations in Norway were shown by ELISA analysis to contain yessotoxins (YTXs). The production of yessotoxin (YTX) was verified by culturing followed by LC-MS analysis of one of the Norwegian isolates. This is the first report of the biogenic origin of YTXs in Norway. The sensitivity of the ELISA method made it possible to quantitate YTXs in algal cultures, net-hauls, and in single cells of P. reticulatum. The cells picked from cultures and net-hauls contained 18-79 pg YTXs per cell. Dilution series and analyses of cells from non-YTX-producing algal species demonstrated the presence of only minimal matrix effects on the ELISA, probably attributable to the presence of salts. The sensitivity of this method makes it possible to search for other possible producers of YTXs, and might also make it possible to follow the YTXs through the food chain. This method allows, for the first time, measurement of the variability in toxin content within a population of dinoflagellate cells--rather than just the average amount of toxin per cell.     

Yessotoxins profile in strains of Protoceratium reticulatum from Spain and USA.

Seven strains of Protoceratium reticulatum isolated from Spain and the USA were cultured in the laboratory. Yessotoxins (YTXs) quantification and toxin profile determination were performed by LC-FLD and LC-MS/MS. The four Spanish strains were found to produce YTX and known YTX analogs, however, YTX was not detected in any of the three USA strains. Among the strains that produced YTXs, toxin production ranged between 2.9 and 28.6pg/cell. The YTX profile was substantially different between strains, in three out of the four Spanish strains YTX was the main toxin and in the fourth homoYTX was the prominent toxin. This work demonstrates that YTX is not always the main toxin in P. reticulatum and a high variability in YTX amounts and profile found in other locations is confirmed.     

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.     

Complex toxin profiles in phytoplankton and Greenshell mussels (Perna canaliculus), revealed by LC-MS/MS analysis.

Toxin profiles were determined in phytoplankton cell concentrates and Greenshell mussels (Perna canaliculus) exposed to a dinoflagellate bloom dominated by Dinophysis acuta and Protoceratium reticulatum. This was achieved by using a method for the simultaneous identification and quantification of a variety of micro-algal toxins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionisation (+/-) and monitoring of daughter ions in multiple reaction modes. Plankton concentrates and shellfish contained high levels of yessotoxins (YTXs) and pectenotoxins (PTXs) and low levels of okadaic acid (OA). A high proportion (>87%) of the OA in both plankton and shellfish was released by alkaline hydrolysis. An isomer of pectenotoxin 1 (PTX1i) was nearly as abundant as pectenotoxin 2 (PTX2) in the plankton and shellfish, and the latter contained high levels of their respective seco acids. DTX1, DTX2, and PTX6 were not detected. MS-MS experiments revealed that the shellfish contained several other oxygenated metabolites of YTX in addition to 45-hydroxy yessotoxin (45OH-YTX). Gymnodimine (GYM) was present in the shellfish but not plankton and it was probably the residue from a previous GYM contamination event. Unlike the other toxins, GYM was concentrated in tissues outside the digestive gland and levels did not decrease over 5 months. The depuration rates of YTX and PTXs from mussels were modelled.     

Desulfoyessotoxins from Adriatic mussels: a new problem for seafood safety control

Two new desulfated yessotoxin (YTX) analogues were isolated from a toxic batch of Adriatic mussels collected in October 2004. Their stereostructures were elucidated through extensive NMR and MS-based analysis. The finding of these desulfocarboxyhomoYTXs in shellfish poses additional problems to those institutions entitled to control seafood safety, since desulfated YTXs fail the European Union method currently in force for checking toxicity in mollusks.     

Pectenotoxins and yessotoxin from Arica Bay, North Chile as determined by tandem mass spectrometry

Lipophilic phycotoxins were measured by tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS) in size-fractionated plankton samples taken at five stations in Arica Bay, northern Chile in the southern summer 2007/2008. Pectenotoxins-2 (PTX-2), -11 (PTX-11), -2 seco acid (PTX-2sa) and yessotoxin (YTX) were identified by comparison of retention times and collision-induced mass spectra of certified standards and field sample extracts. This is the first report of PTXs and YTX from planktonic samples in Chilean coastal waters.     

Production and release of yessotoxins by the dinoflagellates Protoceratium reticulatum and Lingulodinium polyedrum in culture.

The presence of YTX was confirmed in Protoceratium reticulatum cultures and detected for the first time in Lingulodinium polyedrum cultures, mainly in the cells but also, to a lesser extent, dissolved in the culture medium. The production of yessotoxins (YTXs) by cultures of different strains of P. reticulatum and L. polyedrum was studied with liquid chromatography coupled to fluorometric detection using the dienophile reagent DMEQ-TAD and liquid chromatography-mass spectrometry. When comparing toxin production at different stages of culture growth, larger amounts of toxins were observed in the cellular fraction and in the culture medium at the last stage of the culture (day 21) in both species. Although YTX was detected in culture medium, with this study it was not possible to explain which is the release mechanism of the toxin in the medium.     

First evidence of azaspiracids (AZAs): A family of lipophilic polyether marine toxins in scallops (Argopecten purpuratus) and mussels (Mytilus chilensis) collected in two regions of Chile

Azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 following the consumption by consumers of intoxicated shellfish (Mytilus edulis). These azaspiracids have now been identified in mussels (Mytilus chilensis) and scallops (Argopecten purpuratus) from two Chilean locations. This is the first report of the occurrence of azaspiracid toxins in these species (Mytilus chilensis and Argopecten purpuratus) from Chile. The areas studied were Bahía Inglesa (III Region, 27° SL) and Chiloé Archipelago, both important scallop and mussels farming areas. Separation of azaspiracid (AZA1), azaspiracid isomer (AZA6) and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3), was achieved using reversed-phase LC and toxins were identified using a turbo electrospray ionisation (ESI) source, to a triple quadrupole mass spectrometer.In mussels, AZA1 was the predominant toxin in mussel hepatopancreas with AZA2, AZA3 and AZA6 present in approximate equivalent amounts in the remaining tissues, 20-30% of the AZA1 level. AZA2 predominated in the scallop samples with the toxin almost entirely present in the hepatopancreas (digestive gland). AZA1 was only observed in some of the scallop samples and was present at 12-15% of the AZA2 levels.Whilst the levels of AZAs in Chilean samples are below the EU regulatory limit of 160 μg/kg, it is significant that this toxin is present in Pacific Ocean species. Consequently measures should be taken by regulatory authorities to implement regular seafood monitoring to ensure safety of harvested product.     

Structural confirmation and occurrence of azaspiracids in Scandinavian brown crabs (Cancer pagurus).

In 2005 and 2006, azaspiracids were for the first time detected in brown crabs (Cancer pagurus) from the west coast of Sweden and the north and north-west coast of Norway. Azaspiracids are marine toxins that have been detected in blue mussels in Europe in recent years. On some occasions, they have been responsible for human intoxications with symptoms similar to those occurring by consumption of shellfish contaminated with okadaic acid group toxins. While the latter toxin group has been reported to accumulate in green crabs and brown crabs, azaspiracids have previously only been reported to occur in bivalve molluscs. LC-MS analysis of the hepatopancreas (HP) and roe of brown crabs revealed the presence of azaspiracid-1, -2 and -3, but only very low levels were detected in the white meat from the claws or the main shell. Mass spectral data were recorded using two different mass spectrometers, one with a triple-quadrupole mass analyzer and one with a linear ion-trap mass analyzer. The identities of the toxins were confirmed by comparing retention times and mass spectra of azaspiracid standards and the detected toxins. Levels detected ranged from 1.4 microg/kg tissue up to as much as 733 microg/kg tissue, although the majority of samples analyzed were below the suggested regulatory limit of 170 microg/kg HP. Higher levels were detected in HP compared with roe. Very little azaspiracids were detected in mussels from the same locations at the same time, and no proposed microalgal source of azaspiracids was reported in the water previous to or at the time of collection of the toxic crabs.     

Detection and identification of glycoyessotoxin A in a culture of the dinoflagellate Protoceratium reticulatum.

The toxin composition of a culture of the dinoflagellate Protoceratium reticulatum was investigated using LC-FLD, after derivatization with DMEQ-TAD (4-(2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalimylethyl)-1,2,4-triazoline-3,5-dione)). Besides yessotoxin (YTX), the new YTX analogue, glycoyessotoxin A (G-YTXA) was detected in culture medium as well as in cells. The conditions for extraction were optimized and the production profile established. Retention time of the resulting fluorescent G-YTXA adduct was identified by comparison of the appropriate standard. Additionally, both G-YTXA and the DMEQ-TAD-G-YTXA adduct were confirmed by LC-MS showing ion peaks at m/z 1273 [M-2Na+H](-) and m/z 1618 [M-2Na+H](-), respectively. The LC-MS(n) displayed a fragmentation pattern similar to that of the YTX series.     

First report on azaspiracid and yessotoxin groups detection in French shellfish.

The French Phytoplankton and Phycotoxins monitoring network (REPHY) recently found positive or dubious negative shellfish samples using lipophilic toxins mouse bioassay. These samples were analyzed by liquid chromatography (LC) in combination with mass spectrometry (MS) to detect the following toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs), yessotoxins (YTXs), spirolides (SPXs) and gymnodimines (GYMs). Over the 2006-2007 period, chemical analyses revealed various lipophilic toxin profiles according to shellfish sampling locations. In addition to OA and/or PTX-2 and their derivatives, several other compounds were found for the first time in France: (1) during the summer of 2006, AZA-1 and AZA-2 in Queen scallops (Aequipecten opercularis) from Northern Brittany; (2) during the summer of 2007, YTX and its major metabolites (45-hydroxy-YTX, homo-YTX, carboxy-YTX) in shellfish from the Mediterranean coast. Regarding YTX-group, the toxin profiles evolution in mussels during summer showed that: (i) the carboxy-YTX depuration rate was much slower than the YTX and 45-hydroxy-YTX ones; (ii) the homo-YTX concentration, which was initially very weak, increased significantly during the last depuration phase, which seems to reveal a YTX-group high metabolisation level in mussels. This paper reports for the first time on AZA and YTX-groups detection in French shellfish.     

Toxins in mussels (Mytilus galloprovincialis) associated with diarrhetic shellfish poisoning episodes in China

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

Yessotoxins: A toxicological overview

Yessotoxins (YTXs) are polyciclic ether compounds produced by phytoplanktonic dinoflagellates and accumulated in filter feeding shellfish. These toxins can be ingested by humans through contaminated seafood consumption. Initially, YTXs were classified as Diarrhetic Shellfish (DS) toxins but the biological activity of these compounds, which lack of diarrheogenic effects, differs from that of diarrheic toxins. Thus, YTXs have been recently classified as a separate group of algal toxins.Yessotoxin (YTX), homoyessotoxin and 45-hydroxy-homoyessotoxin are lethal after intraperitoneal injection to mice but not after single or repeated oral administration. The target organ seems to be the cardiac muscle cells, where these toxins induce light and electron microscopy ultrastructural changes not only after intraperitoneal injection, but also after oral exposure. On the other hand, di-desulfo-yessotoxin affects liver and pancreas, where it induces fatty degeneration. The mechanisms at the basis of the cardiac effects of YTX and homoyessotoxins are still not completely understood. No short term and chronic toxicity data are available as well as pharmacokinetic studies are lacking. Nevertheless, YTX is known to exert different in vitro activities, such as changes of intracellular calcium and cyclic AMP levels, alteration of cytoskeletal and adhesion molecules, caspases activation and opening of the permeability transition pore of mitochondria. This review reports the current knowledge on the in vivo toxicity and in vitro effects of these toxins.     

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.     

Azaspiracid poisoning (AZP) toxins in shellfish: Toxicological and health considerations

It has been almost a decade since a previously unknown human toxic syndrome, azaspiracid poisoning (AZP), emerged as the cause of severe gastrointestinal illness in humans after the consumption of mussels (Mytilus edulis). Structural studies indicated that these toxins, azaspiracids, were of a new unprecedented class containing novel structural features. It is now known that the prevalent azaspiracids in mussels are AZA1, AZA2 and AZA3, which differ from each other in their degree of methylation. Several hydroxylated and carboxylated analogues of the main azaspiracids have also been identified, presumed to be metabolites of the main toxins. Since its first discovery in Irish mussels, the development of facile sensitive and selective LC-MS/MS methods has resulted in the discovery of AZA in other countries and in other species. Mice studies indicate that this toxin class can cause serious tissue injury, especially to the small intestine, and chronic exposure may increase the likelihood of the development of lung tumours. Studies also show that tissue recovery is very slow following exposure. These observations suggest that AZA is more dangerous than the other known classes of shellfish toxins. Consequently, in order to protect human consumers, proper risk assessment and regulatory control of shellfish and other affected species is of the utmost importance.     

High levels of yessotoxin in mussels and presence of yessotoxin and homoyessotoxin in dinoflagellates of the Adriatic Sea.

Identification of YTX and homoYTX in natural phytoplankton populations containing significant amounts of Gonyaulax polyedra and determination of detailed toxin profiles of mussels (Mytilus galloprovincialis) periodically collected from two sites of the Northern Adriatic coast from February to October 1997 was performed by LC-FLD following derivatization with ADAM or DMEQ-TAD and LC-MS and LC-MS-MS. OA and YTX concentrations were recorded in the range 0.11-2.31 and 0.18-9.02 microg per g of hepatopancreas, respectively. HomoYTX was also detected both in phytoplankton and mussel samples.     

Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice.

The acute toxicity of yessotoxin (YTX), homoyessotoxin (homoYTX) and 45-hydroxy-homoyessotoxin (45-OH-homoYTX) has been studied in comparison to that of okadaic acid (OA), the main diarrhogenic toxin, both after intraperitoneal (i.p.) and oral administration. After i.p. administration, homoYTX and YTX showed similar lethality (LD(50)=444 microg/kg and 512 microg/kg), higher than that of OA (LD(50)=225 microg/kg), while 750 microg/kg of 45-OH-homoYTX did not cause death. OA induced the already known toxic signs: before death, mice were motionless and cyanotic; small intestine and liver damage were shown at post-mortem. Mice treated with YTX and homoYTX were restless and jumped before death; necroscopy did not show major changes. After oral treatment, 2 mg/kg of OA induced diarrhoea and body weight loss, causing 4/5 deaths; necroscopy and/or histology revealed degenerative lesions to small intestine, forestomach and liver (confirmed by increased plasma transaminase), but no myocardium alterations. On the contrary, the oral treatment with YTX (1 and 2 mg/kg) and its derivatives (1 mg/kg) did not cause any death or signs of toxicity, except some ultrastructural myocardiocyte alterations, adjacent to capillaries, such as cytoplasmic protrusions (YTX, 1 and 2 mg/kg), fibrillar alteration (YTX, 1 mg/kg) or mitochondria assemblage (45-OH-homoYTX). Altogether, our data show that YTX and its derivatives are less toxic than OA after acute oral and i.p. treatments, at doses which may represent up to 100 times of the possible human daily intake.     

Identification of 45-hydroxy-46,47-dinoryessotoxin, 44-oxo-45,46,47-trinoryessotoxin, and 9-methyl-42,43,44,45,46,47,55-heptanor-38-en-41-oxoyessotoxin, and partial characterization of some minor yessotoxins, from Protoceratium reticulatum.

Preparative HPLC purification of a side-fraction obtained during purification of 44,55-dihydroxyyessotoxin (6) afforded fractions containing previously unidentified yessotoxin analogues. Careful analysis of these fractions by HPLC-UV, LC-MS3, and NMR spectroscopy, revealed the identities of some of these analogues as 45-hydroxy-46,47-dinoryessotoxin (1), 44-oxo-45,46,47-trinoryessotoxin (2) and 9-methyl-42,43,44,45,46,47,55-heptanor-38-en-41-oxoyessotoxin (5). Numerous other analogues were present but could only be characterized by HPLC-UV and LC-MS3 due to their low abundance. The HPLC-UV and LC-MS3 data confirm the presence of large numbers of yessotoxin analogues, some of which may be oxidative degradation products, in extracts of Protoceratium reticulatum. Compound-1 is the first 46,47-dinoryessotoxin to be identified.