No abstract

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.

Azaspiracids (AZA) are polyether marine dinoflagellate toxins that accumulate in shellfish and represent an emerging human health risk. Although human exposure is primarily manifested by severe and protracted diarrhea, this toxin class has been shown to be highly cytotoxic, a teratogen to developing fish, and a possible carcinogen in mice. Until now, AZA's molecular target(s) has not yet been determined. Using three independent methods (voltage clamp, channel binding assay, and thallium flux assay), we have for the first time demonstrated that AZA1, AZA2, and AZA3 each bind to and block the hERG (human ether-à-go-go related gene) potassium channel heterologously expressed in HEK-293 mammalian cells. Inhibition of K+ current for each AZA analogue was concentration-dependent (IC50 value range: 0.64 - 0.84 μM). The mechanism of hERG channel inhibition by AZA1 was investigated further in Xenopus oocytes where it was shown to be an open state-dependent blocker and, using mutant channels, to interact with F656 but not with Y652 within the S6 transmembrane domain that forms the channel's central pore. AZA1, AZA2, and AZA3 were each shown to inhibit [3H]dofetilide binding to the hERG channel and thallium ion flux through the channel (IC50 value range: 2.1 – 6.6 μM). AZA1 did not block K+ current of the closely related EAG1 channel. Collectively, these data suggest that the AZAs physically block the K+ conductance pathway of hERG1 channels by occluding the cytoplasmic mouth of the open pore. Although the concentrations necessary to block hERG channels are relatively high, AZA-induced blockage may prove to contribute to the toxicological properties of the AZAs.

Since azaspiracid-1 (AZA1) was identified in 1998, the number of AZA analogues has increased to over 30. The development of an LC-MS method using a neutral mobile phase led to the discovery of isomers of AZA1, AZA2, and AZA3, present at ~2-16% of the parent analogues in phytoplankton and shellfish samples. Under acidic mobile phase conditions, isomers and their parents are not separated. Stability studies showed that these isomers were spontaneous epimerization products whose formation is accelerated with the application of heat. The AZA1 isomer was isolated from contaminated shellfish and identified as 37-epi-AZA1 by nuclear magnetic resonance (NMR) spectroscopy and chemical analyses. Similar analysis indicated that the isomers of AZA2 and AZA3 corresponded to 37-epi-AZA2 and 37-epi-AZA3, respectively. The 37-epimers were found to exist in equilibrium with the parent compounds in solution. 37-epi-AZA1 was quantitated by NMR, and relative molar response studies were performed to determine the potential differences in LC-MS response of AZA1 and 37-epi-AZA1. Toxicological effects were determined using Jurkat T lymphocyte cells as an in vitro cell model. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that 37-epi-AZA1 elicited a lethal response that was both concentration- and time-dependent, with EC50 values in the subnanomolar range. On the basis of EC50 comparisons, 37-epi-AZA1 was 5.1-fold more potent than AZA1. This data suggests that the presence of these epimers in seafood products should be considered in the analysis of AZAs for regulatory purposes.