Azaspiracid Poisoning Research Articles

SoundToxins: A Research and Monitoring Partnership for Harmful Phytoplankton in Washington State.

The more frequent occurrence of marine harmful algal blooms (HABs) and recent problems with newly-described toxins in Puget Sound have increased the risk for illness and have negatively impacted sustainable access to shellfish in Washington State. Marine toxins that affect safe shellfish harvest because of their impact on human health are the saxitoxins that cause paralytic shellfish poisoning (PSP), domoic acid that causes amnesic shellfish poisoning (ASP), diarrhetic shellfish toxins that cause diarrhetic shellfish poisoning (DSP) and the recent measurement of azaspiracids, known to cause azaspiracid poisoning (AZP), at low concentrations in Puget Sound shellfish. The flagellate, Heterosigma akashiwo, impacts the health and harvestability of aquacultured and wild salmon in Puget Sound. The more recently described flagellates that cause the illness or death of cultivated and wild shellfish, include Protoceratium reticulatum, known to produce yessotoxins, Akashiwo sanguinea and Phaeocystis globosa. This increased incidence of HABs, especially dinoflagellate HABs that are expected in increase with enhanced stratification linked to climate change, has necessitated the partnership of state regulatory programs with SoundToxins, the research, monitoring and early warning program for HABs in Puget Sound, that allows shellfish growers, Native tribes, environmental learning centers and citizens, to be the "eyes on the coast". This partnership enables safe harvest of wholesome seafood for consumption in the region and helps to describe unusual events that impact the health of oceans, wildlife and humans.

The effect of temperature and salinity on growth rate and azaspiracid cell quotas in two strains of Azadinium poporum (Dinophyceae) from Puget Sound, Washington State

Azaspiracids (AZA) are novel lipophilic polyether marine biotoxins associated with azaspiracid shellfish poisoning (AZP). Azaspiracid-59 (AZA-59) is a new AZA that was recently detected in strains of Azadinium poporum from Puget Sound, Washington State. In order to understand how environmental factors affect AZA abundances in Puget Sound, a laboratory experiment was conducted with two local strains of A. poporum to estimate the growth rate and AZA-59 (both intra- and extracellular) cell quotas along temperature and salinity gradients. Both strains of A. poporum grew across a wide range of temperatures (6.7 °C to 25.0 °C), and salinities (15 to 35). Growth rates increased with increasing temperature up to 20.0 °C, with a range from 0.10 d−1 to 0.42 d−1. Both strains of A. poporum showed variable growth rates from 0.26 d−1 to 0.38 d−1 at salinities from 15 to 35. The percentage of intracellular AZA-59 in both strains was generally higher in exponential than in stationary phase along temperature and salinity gradients, indicating higher retention of toxin in actively growing cells. Cellular toxin quotas varied by strain in both the temperature and salinity treatments but were highest at the lowest growth rates, especially for the faster growing strain, NWFSC1011.Consistent with laboratory experiments, field investigations in Sequim Bay, WA, during 2016–2018 showed that A. poporum was detected when salinity and temperature became favorable to higher growth rates in June and July. Although current field data of A. poporum in Puget Sound indicate a generally low abundance, the potential of local A. poporum to adapt to and grow in a wide range of temperature and salinity may open future windows for blooms. Although increased temperatures, anticipated for the Puget Sound region over the next decades, will enhance the growth of A. poporum, these higher temperatures will not necessarily support higher toxin cell quotas. Additional sampling and assessment of the total toxicity of AZA-59 will provide the basis for a more accurate estimation of risk for azaspiracid poisoning in Puget Sound shellfish.

Amphidoma languida (Amphidomatacea, Dinophyceae) with a novel azaspiracid toxin profile identified as the cause of molluscan contamination at the Atlantic coast of southern Spain

Azaspiracids (AZA) are a group of food poisoning phycotoxins that are known to accumulate in shellfish. They are produced by some species of the planktonic dinophycean taxon Amphidomataceae. Azaspiracids have been first discovered in Ireland but are now reported in shellfish from numerous global sites thus showing a wide distribution. In shellfish samples collected in 2009 near Huelva (Spain), AZA was also found along the Andalusian Atlantic coast for the first time. Analysis using LC–MS/MS revealed the presence of two different AZA analogues in different bivalve shellfish species (Chamelea gallina, Cerastoderma edule, Donax trunculus, and Solen vagina). In a number of samples, AZA levels exceeded the EU regulatory level of 160μg AZA-1 eq. kg−1 (reaching maximum levels of >500μg AZA-1 eq. kg−1 in Chamelea gallina and >250μg AZA-1 eq. kg−1 in Donax trunculus) causing closures of some local shellfish production areas. One dinophyte strain established from the local plankton during the AZA contamination period and determined as Amphidoma languida was in fact toxigenic, and its AZA profile disclosed it as the causative species: it contained AZA-2 as the main compound and the new compound AZA-43 initially detected in the shellfish. AZA-43 had the same mass as AZA-3, but produced different collision induced dissociation (CID) spectra. High resolution mass spectrometric measurements indicated that there is an unsaturation in the H, I ring system of AZA-43 distinguishing it from the classical AZA such as AZA-1, -2, and -3. Furthermore, the Spanish strain was different from the previously reported AZA profile of the species that consist of AZA-38 and AZ-39. In molecular phylogenetics, the Andalusian strain formed a monophyletic group together with other strains of Am. languida, but ITS sequences data revealed surprisingly high intragenomic variability. The first Andalusian case of AZA contamination of shellfish above the EU regulatory limit reported here clearly revealed the risk of azaspiracid poisoning (AZP) for this area and also for the Atlantic coast of Iberia and North Africa. The present study underlines the need for continuous monitoring of AZA and the organisms producing such toxins.

Absorption and Effect of Azaspiracid-1 Over the Human Intestinal Barrier

Background: Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellates genera Azadinium and Amphidoma. These toxins cause azaspiracid poisoning (AZP), characterized by severe gastrointestinal illness in humans after the consumption of bivalve molluscs contaminated with AZAs. The main aim of the present study was to examine the consequences of human exposure to AZA1 by the study of absorption and effects of the toxin on Caco-2 cells, a reliable model of the human intestine. Methods: The ability of AZA1 to cross the human intestinal epithelium has been evaluated by the Caco-2 transepithelial permeability assay. The toxin has been detected and quantified using a microsphere-based immunoassay. Cell alterations and ultrastructural effects has been observed with confocal and transmission electron microscopy Results: AZA1 was absorbed by Caco-2 cells in a dose-dependent way without affecting cell viability. However, modifications on occludin distribution detected by confocal microscopy imaging indicated a possible monolayer integrity disruption. Nevertheless, transmission electron microscopy imaging revealed ultrastructural damages at the nucleus and mitochondria with autophagosomes in the cytoplasm, however, tight junctions and microvilli remained unaffected. Conclusion: After the ingestion of molluscs with the AZA1, the toxin will be transported through the human intestinal barrier to blood causing damage on epithelial cells.

In vitro chronic effects on hERG channel caused by the marine biotoxin azaspiracid-2

Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellate Azadinium spinosum that accumulate in many shellfish species. Azaspiracid poisoning caused by AZA-contaminated seafood consumption is primarily manifested by diarrhea in humans. To protect human health, AZA-1, AZA-2 and AZA-3 content in seafood has been regulated by food safety authorities in many countries. Recently AZAs have been reported as a low/moderate hERG channel blockers. Furthermore AZA-2 has been related to arrhythmia appearance in rats, suggesting potential heart toxicity. In this study AZA-2 in vitro effects on hERG channel after chronic exposure are analyzed to further explore potential cardiotoxicity. The amount of hERG channel in the plasma membrane, hERG channel trafficking and hERG currents were evaluated up to 12 h of toxin exposure. In these conditions AZA-2 caused an increase of hERG levels in the plasma membrane, probably related to hERG retrograde trafficking impairment. Although this alteration did not translate into an increase of hERG channel-related current, more studies will be necessary to understand its mechanism and to know what consequences could have in vivo. These findings suggest that azaspiracids might have chronic cardiotoxicity related to hERG channel trafficking and they should not be overlooked when evaluating the threat to human health.

Mussels Increase Xenobiotic (Azaspiracid) Toxicity Using a Unique Bioconversion Mechanism

Azaspiracid Poisoning (AZP) is a human toxic syndrome which is associated with the consumption of bivalve shellfish. Unlike other shellfish, mussels contain a large array of azaspiracid analogs, many of which are suspected bioconversion products. These studies were conducted to elucidate the metabolic pathways of azaspiracid (AZA1) in the blue mussel (Mytilus edulis) and revealed that the main biotransformation product was the more toxic demethyl analog, AZA3. To elucidate the mechanism of this C-demethylation, an unprecedented xenobiotic bioconversion step in shellfish, AZA1 was fed to mussels that contained no detectable azaspiracids. Triple quadrupole mass spectrometry (MS) and high resolution Orbitrap MS were used to determine the uptake of AZA1 and the toxin profiles in three tissue compartments of mussels. The second most abundant bioconversion product was identified as AZA17, a carboxyl analog of AZA3, which is a key intermediate in the formation of AZA3. Also, two pairs of isomeric hydroxyl analogs, AZA4/AZA5 and AZA7/AZA8, have been confirmed as bioconversion products for the first time. Ultra high resolution (100 k) MS studies showed that the most probable structural assignment for AZA17 is 22-carboxy-AZA3 and a mechanism for its facile decarboxylation to form AZA3 has been proposed.

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.

Chemistry, etiology, and food chain dynamics of marine toxins

Highly complex chemical structures of marine natural toxins involved in seafood poisoning were determined using small samples. The biogenetic process by which the toxins accumulate in fish and shellfish was explained by the food chain. The primary sources of the toxins were identified as dinoflagellates, bacteria, and blue-green algae. The topics related to ciguatera, puffer fish, diarrhetic shellfish poisoning, neurotoxic shellfish poisoning, azaspiracid poisoning, red tides, and Gracilaria red alga poisoning are described. (Communicated by Saburo TAMURA, M.J.A.)

Is there a risk of human poisoning by azaspiracids from shellfish harvested at the Portuguese coast?

Azaspiracid poisoning (AZP), the most recently discovered human gastrointestinal illness resulting from consumption of contaminated shellfish, so far has been found in coastal areas of northern Europe. This is the first report of a survey carried out for contamination of shellfish harvested in costal areas of Portugal for the presence of azaspiracids. The study design covered the commercial species usually more contaminated by toxins from dinoflagellates (blue mussel, common cockle, donax clam) in coastal areas representative of the NW, SW and south coasts. A method based on liquid chromatography–mass spectrometry was setup for the first time for this purpose. No azaspiracids were found on 300 samples tested between 2002 and 2003. On at least three samples a peak with a retention time matching that of AZA2 was found, never surpassing one tenth of the current EU limit. Unambiguous identification of any known AZA did not occur yet. The risk for human outbreaks of AZP seems to be very low, comparatively with amnesic shellfish poisoning (ASP), where levels close to the allowance level are found sparsely, or to diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP), where high levels and registered human outbreaks have been found in recent years.

Azaspiracid poisoning, the food-borne illness associated with shellfish consumption

Azaspiracid poisoning (AZP) is a recently discovered toxic syndrome that was identified following severe gastrointestinal illness from the consumption of contaminated mussels (Mytilus edulis). The implicated toxins, azaspiracids, are polyethers with unprecedented structural features. Studies toward total toxin synthesis revealed that the initial published structures were incorrect and they have now been revised. These toxins accumulate in bivalve molluscs that feed on toxic microalgae of the genus Protoperidinium, previously considered to be toxicologically benign. Although first identified in shellfish from Ireland, azaspiracid contamination of several types of bivalve shellfish species has now been confirmed throughout the western coastline of Europe. Toxicological studies have indicated that azaspiracids can induce widespread organ damage in mice and that they are probably more dangerous than previously known classes of shellfish toxins. The exclusive reliance on live animal bioassays to monitor azaspiracids in shellfish failed to prevent human intoxications. This was a consequence of poor sensitivity of the assay and the fact that azaspiracids are not exclusively found in the shellfish digestive glands used for toxin testing. The strict regulatory control of azaspiracids in shellfish now requires frequent testing of shellfish using highly specific and sensitive methods involving liquid chromatography-mass spectrometry.

The fragmentation pathways of azaspiracids elucidated using positive nanospray hybrid quadrupole time‐of‐flight (QqTOF) mass spectrometry

The azaspiracids, AZA1, AZA2 and AZA3, are the predominant shellfish toxins responsible for the human toxic syndrome, azaspiracid poisoning. Collision induced dissociation (CID) mass spectra were generated for azaspiracids using nano‐electrospray ionisation (ESI) with a hybrid quadrupole time‐of‐flight (QqTOF) mass spectrometer in positive mode. Six main backbone fragmentations of the polyether skeleton of azaspiracids were observed as well as multiple neutral losses of water molecules from the parent and product ions. The characteristic charge‐remote fragmentation of the carbon skeleton of azaspiracids produced nitrogenous ions. The three azaspiracids differ from one another by 14 Da due to methylation in the A‐ and E‐rings. Three fragmentation pathways, involving cleavage of the E‐ring, C27–C28 and G‐ring, gave ions that were common to all azaspiracids. Another three fragmentations involving the A‐ring, C‐ring and C19–C20, were useful for distinguishing between azaspiracid analogues. Multiple tandem ion‒trap mass spectrometry (MSn) was used to confirm the fragmentation pathways.

Studies toward the total synthesis of azaspiracids: synthesis of the FGHI ring domain

Synthesis of the FGHI ring domain of azaspiracids, the causative agents for a new type of shellfish poisoning, azaspiracid poisoning (AZP), has been achieved. The synthesis features dithiane anion–epoxide coupling for convergent fragment assembly.

The first identification of azaspiracids in shellfish from France and Spain

Incidents of human intoxications throughout Europe, following the consumption of mussels have been attributed to Azaspiracid Poisoning (AZP). Although first discovered in Ireland, the search for the causative toxins, named azaspiracids, in other European countries has now led to the first discovery of these toxins in shellfish from France and Spain. Separation of the toxins, azaspiracid (AZA1) and analogues, AZA2 and AZA3, was achieved using isocratic reversed-phase liquid chromatography coupled, via an electrospray ionisation source, to an ion-trap mass spectrometer. Azaspiracids were identified in mussels ( Mytilus galloprovincialis), 0.24 μg/g, from Galicia, Spain, and scallops ( Pecten maximus), 0.32 μg/g, from Brittany, France. Toxin profiles were similar to those found in the equivalent shellfish in Ireland in which AZA1 was the predominant toxin.

Geographical, temporal, and species variation of the polyether toxins, azaspiracids, in shellfish.

Azaspiracid Poisoning (AZP) is a new toxic syndrome that has caused human intoxications throughout Europe following the consumption of mussels (Mytilus edulis), harvested in Ireland. Shellfish intoxication is a consequence of toxin-bearing microalgae in the shellfish food chain, and these studies demonstrated a wide geographic distribution of toxic mussels along the entire western coastal region of Ireland. The first identification of azaspiracids in other bivalve mollusks including oysters (Crassostrea gigas), scallops (Pecten maximus), clams (Tapes phillipinarium), and cockles (Cardium edule) is reported. Importantly, oysters were the only shellfish that accumulated azaspiracids at levels that were comparable with mussels. The highest levels of total azaspiracids (microg/g) recorded to-date were mussels (4.2), oysters (2.45), scallops (0.40), cockles (0.20), and clams (0.61). An examination of the temporal variation of azaspiracid contamination of mussels in a major shellfish production area revealed that, although maximum toxin levels were recorded during the late summer period, significant intoxications were observed at periods when marine dinoflagellate populations were low. Although human intoxications have so far only been associated with mussel consumption, the discovery of significant azaspiracid accumulation in other bivalve mollusks could pose a threat to human health.

Food safety implications of the distribution of azaspiracids in the tissue compartments of scallops (Pecten maximus)

Azaspiracids, a new class of shellfish toxins, have been implicated in several recent incidents of human intoxications following the consumption of mussels (Mytilus edulis). A study was undertaken to examine the distribution of azaspiracid poisoning (AZP) toxins in scallops (Pecten maximus) and individual shellfish were dissected into five tissue fractions for the determination of toxin composition. Separation of the predominant azaspiracids, AZA1-3, was achieved using reversed-phase liquid chromatography with detection by positive electrospray multiple tandem mass spectrometry. The AZP toxin composition was determined in the adductor muscle (meat), gonad (roe), hepatopancreas (digestive glands), mantle and gill of scallops. Substantial differences in the AZP toxin levels between tissue compartments were observed and toxins were concentrated predominantly, about 85%, in the hepatopancreas. There was also a significant variation in the total toxin levels between individual scallops from the same sample batch and the RSD was 60% (n = 9). Interestingly, although all three AZP toxins were present in phytoplankton and mussels, AZA3 was not detected in the scallop samples examined. It was concluded that to improve food safety, only the adductor muscle and gonad of scallops should be permitted for sale to the public.

Azaspiracid poisoning, the food-borne illness associated with shellfish consumption

Azaspiracid poisoning, the food-borne illness associated with shellfish consumption

Detection of five new hydroxyl analogues of azaspiracids in shellfish using multiple tandem mass spectrometry

The polyether dinoflagellate toxins, azaspiracids, are responsible for azaspiracid poisoning (AZP), a new human toxic syndrome arising from the consumption of shellfish. To date, five azaspiracids have been isolated and fully structurally elucidated, including, AZA1, its 8-methyl and 22-demethyl analogues, AZA2 and AZA3, respectively, and two hydroxyl derivatives of AZA3, named AZA4 and AZA5. Using a recently developed method involving liquid chromatography with multiple tandem mass spectrometry (LC–MS n ), five new azaspiracids, AZA7–AZA11, have been found in mussels ( Mytilus edulis). AZA6 is a positional isomer of AZA1 and four of the new compounds are isomers with a mass of 857.5 amu. AZA7 and AZA8 are hydroxyl analogues of AZA1 while AZA9 and AZA10 are hydroxyl analogues of AZA6. AZA11 is a hydroxyl analogue of AZA2. The separation of all 11 azaspiracids was achieved using isocratic reversed phase liquid chromatography using a combination of eluent additives, trifluoroacetic acid and ammonium acetate. The ion-trap MS experiments, with electrospray ionisation, involved the fragmentation of the protonated molecule [M+H] +, trapping and fragmenting the product ions due to the loss of a water molecule [M+H–H 2O] +, together with mass spectral data analysis that included the characteristic A-ring fragmentation for each compound.

Ubiquitous ‘benign’ alga emerges as the cause of shellfish contamination responsible for the human toxic syndrome, azaspiracid poisoning

A new human toxic syndrome, azaspiracid poisoning (AZP), was identified following illness from the consumption of contaminated mussels ( Mytilus edulis). To discover the aetiology of AZP, sensitive analytical protocols involving liquid chromatography–mass spectrometry (LC–MS) were used to screen marine phytoplankton for azaspiracids. Collections of single species were prepared by manually separating phytoplankton for LC–MS analysis. A dinoflagellate species of the genus, Protoperidinium, has been identified as the progenitor of azaspiracids. Azaspiracid-1, and its analogues, AZA2 and AZA3, were identified in extracts of 200 cells using electrospray multiple tandem MS. This discovery has significant implications for both human health and the aquaculture industry since this phytoplankton genus was previously considered to be toxicologically benign. The average toxin content was 1.8 fmol of total AZA toxins per cell with AZA1 as the predominant toxin, accounting for 82% of the total.

Comparison of solid-phase extraction methods for the determination of azaspiracids in shellfish by liquid chromatography–electrospray mass spectrometry

Azaspiracids have been identified as the cause of a new toxic syndrome called azaspiracid poisoning (AZP) that has led to incidents of human intoxications throughout Europe following the consumption of mussels. Although five AZP toxins have been structurally elucidated to-date, azaspiracid (AZA1), 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3) are the predominant toxins. Separation of the three main AZP toxins was achieved using reversed-phase liquid chromatography (LC) and coupled to an electrospray ionisation source of an ion-trap mass spectrometer. Five reversed-phase (C 18) and three diol solid-phase extraction (SPE) cartridges were compared for their efficacy in the cleanup of shellfish matrix. The comparison was based on the optimum recoveries of AZA1, AZA2 and AZA3 from extracts of mussel tissues. LC–electrospray MS 3 analysis was used to quantify the AZP toxins in wash and eluate fractions in the SPE studies. Good recovery and reproducibility data were obtained for one diol SPE cartridge and two C 18 SPE cartridge types.

First evidence of an extensive northern European distribution of azaspiracid poisoning (AZP) toxins in shellfish

Azaspiracids have recently been identified as the toxins responsible for a series of human intoxications in Europe since 1995, following the consumption of cultured mussels (Mytilus edulis) from the west coast of Ireland. Liquid chromatography–mass spectrometric (LC–MS) methods have been applied in the study reported here to investigate the new human toxic syndrome, azaspiracid poisoning. Separation of azaspiracid (AZA1) and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3), was achieved using reversed-phase LC and coupled, via an electrospray ionisation source, to an ion-trap mass spectrometer. These azaspiracids have now been identified in mussels from Craster (north-east England) and Sognefjord (south-west Norway) using source collision induced dissociation-MS and multiple tandem MS detection. AZA1 was the predominant toxin and toxin profiles were similar to those found in contaminated Irish shellfish. This is the first report of the occurrence of these azaspiracids outside Ireland with the significant implications that these toxins may occur in shellfish throughout northern Europe.