Azaspiracids (AZAs) are a group of lipophilic marine biotoxins that were first discovered in blue mussels harvested in 1995 in Killary Harbour on the west coast of Ireland. At least eight people fell ill after the consumption of contaminated mussels and developed symptoms of nausea, stomach cramps, vomiting and severe diarrhoea. Until now, eleven different analogs of these toxins have been described, with a twelfth one theoretically postulated. This paper describes the detection and identification of twenty new analogs of azaspiracid, including dihydroxy-AZAs and carboxy-AZAs, using state-of-the-art techniques including ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS). Blue mussels (Mytilus edulis) from a toxic event of the northwest coast of Ireland in 2005 were extracted and analysed using LC/MS. The mass spectra obtained from different instruments enabled identification of previously unknown analogs of azaspiracid with additional hydroxyl and carboxyl substituents. Mass fragmentation patterns of the dihydroxy-AZAs indicated the positions of these substituents to be at the C3 and C23 position. The previously theoretically postulated AZA12 was also observed in this study. Product ion spectra showed the presence of a unique fragment ion at m/z 408 for all C23-hydroxylated analogs. This fragmentation competes with the fragmentation leading to m/z 362, a fragment ion that has shown to be present in all AZAs. The novel analogs have not been seen in plankton or water samples and are believed to be metabolites of AZAs formed in mussels. All the new AZA analogs were present at low concentrations in the shellfish and it is probably safe to assume that they do not pose a risk for the shellfish consumer.

Azaspiracids (AZA) are polyether marine toxins that accumulate in various shellfish species and have been associated with severe gastrointestinal human intoxications since 1995. This toxin class has since been reported from several countries, including Morocco and much of western Europe. A regulatory limit of 160 µg AZA/kg whole shellfish flesh was established by the EU in order to protect human health; however, in some cases, AZA concentrations far exceed the action level. Herein we discuss recent advances on the chemistry of various AZA analogs, review the ecology of AZAs, including the putative progenitor algal species, collectively interpret the in vitro and in vivo data on the toxicology of AZAs relating to human health issues, and outline the European legislature associated with AZAs.

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Azaspiracids cause severe damages in the epithelium of several organs. In this study we have investigated the effects of azaspiracid-1 (AZA-1) on two epithelial cell lines. Nanomolar concentrations of AZA-1 reduced MCF-7 cell proliferation and impaired cell-cell adhesion. AZA-1 altered the cellular pool of the adhesion molecule E-cadherin by inducing a dose- and time-dependent accumulation of an E-cadherin fragment (E-cadherin-related antigen [ECRA(100)]), with a concentration inducing the half-maximal effect (EC(50)) of 0.47nM. The immunological characterization of ECRA(100) revealed that it consists of an E-cadherin molecule lacking the intracellular domain, and these data showed that the effect induced by AZA-1 in MCF-7 cells is undistinguishable from that induced by yessotoxin (YTX) in the same experimental system. A comparison of toxin effects in MCF-7 and Caco 2 cells confirmed that the effects induced by AZA-1 and YTX are undistinguishable in these cells. Treatment of fibroblasts with AZA-1 did not affect the cellular pool of N-cadherin showing that the toxin effect is cadherin-specific. A comparison of the effects induced by AZA-1, YTX, and okadaic acid on F-actin and E-cadherin in MCF-7 and Caco 2 cells showed that 1nM AZA-1 did not cause significant changes in F-actin and that accumulation of ECRA(100) did not correlate with decreased levels of F-actin under our experimental conditions. Matching our results with those available in literature, we notice that, when molecular effects induced by AZA-1 and YTX have been studied in the same in vitro systems, experimental data show that they are undistinguishable in terms of sensitive cellular parameters, effective doses, and kinetics of responses in several cell lines. The possibility that azaspiracids and YTXs might share their molecular mechanism(s) of action in defined biological settings should be considered.