Improved Isolation Procedure for Azaspiracids from Shellfish, Structural Elucidation of Azaspiracid-6, and Stability Studies

Azaspiracids are a group of lipophilic polyether toxins produced by the small dinoflagellate <i>Azadinium spinosum</i>. They may accumulate in shellfish and can result in illnesses when consumed by humans. Research into analytical methods, chemistry, metabolism, and toxicology of azaspiracids has been severely constrained by the scarcity of high-purity azaspiracids. Consequently, since their discovery in 1995, considerable efforts have been made to develop methods for the isolation of azaspiracids in sufficient amounts and purities for toxicological studies, in addition to the preparation of standard reference materials. A seven-step procedure was improved for the isolation of azaspiracids-1–3 (<b>1</b>, <b>2</b>, and <b>3</b>) increasing recoveries 2-fold as compared to previous methods and leading to isolation of sufficiently purified azaspiracid-6 (<b>6</b>) for structural determination by NMR spectroscopy. The procedure, which involved a series of partitioning and column chromatography steps, was performed on 500 g of <i>Mytilus edulis</i> hepatopancreas tissue containing ∼14 mg of <b>1</b>. Overall yields of <b>1</b> (52%), <b>2</b> (43%), <b>3</b> (43%), and <b>6</b> (38%) were good, and purities were confirmed by NMR spectroscopy. The structure of <b>6</b> was determined by one- and two-dimensional NMR spectroscopy and mass spectrometry. The stability of <b>6</b> relative to <b>1</b> was also assessed in three solvents in a short-term study that demonstrated the greatest stability in aqueous acetonitrile

Results of a Kruskal-Wallis nonparametric one factor ANOVA for differences in CTX toxicity among <i>Gambierdiscus</i> and <i>Fukuyoa</i> species.

<p><i>Gambierdiscus excentricus</i> and <i>G</i>. <i>silvae</i> were excluded from the analysis because only a single clone was examined. Abbreviations: n = sample size, M = median toxicity (fg CTX3C eq. cell^-1), <i>H</i> = Kruskal-Wallis test statistic, <i>df</i> = degrees of freedom. Brackets denote result of the Dunn’s follow up test. The statistic is designed to estimate median toxicities to determine if the species partitioned into distinct groups.</p

Representative plots showing the long-term steady state growth of the <i>Gambierdiscus</i> and <i>Fukuyoa</i> isolates achieved in this study.

<p>Exponential growth was achieved by acclimating cells to optimal temperature, light and nutrient conditions and maintained in exponential growth phase by periodic dilution with nutrient rich media.</p

The species, strain designations, isolate locations, replicate growth rates and toxicities of the <i>Gambierdiscus</i> and <i>Fukuyoa</i> strains examined in this study.

<p>The citations in the species column indicate where the species was described. The reference(s) under the strain designation indicate other publications where the strain has been studied. Many of the strains analyzed for CTX-like activity in this study were also assayed for maitotoxicity in separate investigations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185776#pone.0185776.ref032" target="_blank">32</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185776#pone.0185776.ref033" target="_blank">33</a>]. The strain growth rates (± standard deviation) were determined from triplicate, independent cultures started for each isolate. Mean species growth rates and average toxicities were determined by averaging all replicate culture data for a given species. Toxicity was normalized both as femtograms (fg) CTX3C equivalents [eq.] cell^-1 and per biovolume attograms (ag) CTX3C eq. μm^-3. Numbers in parentheses in the data cells of the last three columns = coefficient of variation. Correlation coefficients (R²) for the time versus cell number relationships used to the calculate growth rates for each of the cultures exceeded 0.98.</p

Toxin production rates.

<p>This figure shows the estimated toxin production (fg CTX3C eq. cell^-1 d^-1) rate for each species.</p

Passive Sampling and High Resolution Mass Spectrometry for Chemical Profiling of French Coastal Areas with a Focus on Marine Biotoxins

Passive samplers (solid phase adsorption toxin tracking: SPATT) are able to accumulate biotoxins produced by microalgae directly from seawater, thus providing useful information for monitoring of the marine environment. SPATTs containing 0.3, 3, and 10 g of resin were deployed at four different coastal areas in France and analyzed using liquid chromatography coupled to high resolution mass spectrometry. Quantitative targeted screening provided insights into toxin profiles and showed that toxin concentrations and profiles in SPATTs were dependent on the amount of resin used. Between the three amounts of resin tested, SPATTs containing 3 g of resin appeared to be the best compromise, which is consistent with the use of 3 g of resin in SPATTs by previous studies. MassHunter and Mass Profiler Professional softwares were used for data reprocessing and statistical analyses. A differential profiling approach was developed to investigate and compare the overall chemical diversity of dissolved substances in different coastal water bodies. Principal component analysis (PCA) allowed for spatial differentiation between areas. Similarly, SPATTs retrieved from the same location at early, medium, and late deployment periods were also differentiated by PCA, reflecting seasonal variations in chemical profiles and in the microalgal community. This study used an untargeted metabolomic approach for spatial and temporal differentiation of marine environmental chemical profiles using SPATTs, and we propose this approach as a step forward in the discovery of chemical markers of short- or long-term changes in the microbial community structure

Structure Elucidation, Relative LC–MS Response and In Vitro Toxicity of Azaspiracids <b>7</b>–<b>10</b> Isolated from Mussels (Mytilus edulis)

Azaspiracids (AZAs) are marine biotoxins produced by dinoflagellates that can accumulate in shellfish, which if consumed can lead to poisoning events. AZA7–10, <b>7</b>–<b>10</b>, were isolated from shellfish and their structures, previously proposed on the basis of only LC–MS/MS data, were confirmed by NMR spectroscopy. Purified AZA4–6, <b>4</b>–<b>6</b>, and <b>7</b>–<b>10</b> were accurately quantitated by qNMR and used to assay cytotoxicity with Jurkat T lymphocyte cells for the first time. LC–MS­(MS) molar response studies performed using isocratic and gradient elution in both selected ion monitoring and selected reaction monitoring modes showed that responses for the analogues ranged from 0.3 to 1.2 relative to AZA1, <b>1</b>. All AZA analogues tested were cytotoxic to Jurkat T lymphocyte cells in a time- and concentration-dependent manner; however, there were distinct differences in their EC₅₀ values, with the potencies for each analogue being: AZA6 > AZA8 > AZA1 > AZA4 ≈ AZA9 > AZA5 ≈ AZA10. This data contributes to the understanding of the structure–activity relationships of AZAs

Isolation, Structure Elucidation, Relative LC-MS Response, and in Vitro Toxicity of Azaspiracids from the Dinoflagellate <i>Azadinium spinosum</i>

We identified three new azaspiracids (AZAs) with molecular weights of 715, 815, and 829 (AZA33 (<b>3</b>), AZA34 (<b>4</b>), and AZA35, respectively) in mussels, seawater, and <i>Azadinium spinosum</i> culture. Approximately 700 μg of <b>3</b> and 250 μg of <b>4</b> were isolated from a bulk culture of <i>A. spinosum</i>, and their structures determined by MS and NMR spectroscopy. These compounds differ significantly at the carboxyl end of the molecule from known AZA analogues and therefore provide valuable information on structure–activity relationships. Initial toxicological assessment was performed using an in vitro model system based on Jurkat T lymphocyte cytotoxicity, and the potencies of <b>3</b> and <b>4</b> were found to be 0.22- and 5.5-fold that of AZA1 (<b>1</b>), respectively. Thus, major changes in the carboxyl end of <b>1</b> resulted in significant changes in toxicity. In mussel extracts, <b>3</b> was detected at low levels, whereas <b>4</b> and AZA35 were detected only at extremely low levels or not at all. The structures of <b>3</b> and <b>4</b> are consistent with AZAs being biosynthetically assembled from the amino end