In Vitro Metabolism of Azaspiracids 1–3 with a Hepatopancreatic Fraction from Blue Mussels (Mytilus edulis)

Abstract

Azaspiracids (AZAs) are a group of biotoxins produced by the marine dinoflagellates Azadinium and Amphidoma spp. that can accumulate in shellfish and cause food poisoning in humans. Of the 60 AZAs identified, levels of AZA1, AZA2, and AZA3 are regulated in shellfish as a food safety measure based on occurrence and toxicity. Information about the metabolism of AZAs in shellfish is limited. Therefore, a fraction of blue mussel hepatopancreas was made to study the metabolism of AZA1–3 in vitro. A range of AZA metabolites were detected by liquid chromatography–high-resolution tandem mass spectrometry analysis, most notably the novel 22α-hydroxymethylAZAs AZA65 and AZA66, which were also detected in naturally contaminated mussels. These appear to be the first intermediates in the metabolic conversion of AZA1 and AZA2 to their corresponding 22α-carboxyAZAs (AZA17 and AZA19). α-Hydroxylation at C-23 was also a prominent metabolic pathway, producing AZA8, AZA12, and AZA5 as major metabolites of AZA1–3, respectively, and AZA67 and AZA68 as minor metabolites via double-hydroxylation of AZA1 and AZA2, but only low levels of 3β-hydroxylation were observed in this study. In vitro generation of algal toxin metabolites, such as AZA3, AZA5, AZA6, AZA8, AZA12, AZA17, AZA19, AZA65, and AZA66 that would otherwise have to be laboriously purified from shellfish, has the potential to be used for the production of standards for analytical and toxicological studies.

Preparation of 18O-labelled azaspiracids for accurate quantitation using liquid chromatography–mass spectrometry

“…Corresponding results were obtained from analysis of the doubly labelled AZA3 at 77 h reaction time (Figures S13 and S14 ). The m / z 125.0597 ion does not appear to involve C-1, C-3, C-8, C-21, C-22, or C-23, as this ion was observed in LC–HRMS/MS spectra of known AZAs and their metabolites [ 17 , 33 ] as well as in a synthetic specimen of the C-1 alcohol analogue of AZA3 (Figure S15 ). The finding that ring-B is involved in 18 O-exchange under weakly acidic conditions, presumably via opening of the ketals at C-10 and/or C-13, provides a possible explanation of the observed instability of AZA2 under these conditions.…”

Marine natural products

“…Treatment of these three compounds with blue mussel hepatopancreas caused production of numerous other AZAs, including several new congeners as detected by MS, therefore monitoring of the three parent compounds alone may be insufficient as a food safety measure. 353 A recent study has shown that gambierone from Gamberidiscus cheloniae is not as toxic as other ciguatera components. 354 Brevetoxin PbTx-2 has differential effects against human and dinoagellate thioredoxin reductases, suggesting it is a key endogenous regulator of this enzyme in K. brevis.…”

“…Treatment of these three compounds with blue mussel hepatopancreas caused production of numerous other AZAs, including several new congeners as detected by MS, therefore monitoring of the three parent compounds alone may be insufficient as a food safety measure. 353…”

Current Research Status of Azaspiracids

“…In mussels, the expression of AZA-1-3 can lead to the production of AZA-8, AZA-12, and AZA-5 through C-23 α hydroxylation. Additionally, the double hydroxylation of AZA-67 and AZA-68 can generate AZA-1 and AZA-2 as secondary metabolites [14].…”

“…Among these, Azadinium dexteroporum is currently known to produce the highest number of AZA variants, with up to six types reported [13]. The total number of named AZA toxins and their analogs currently stands at AZA-68 [14]. (Table 2).…”