8 research outputs found
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-1ā3 (<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 ā¼14 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<sup>-1</sup>), <i>H</i> = Kruskal-Wallis test statistic, <i>df</i> = degrees of freedom. Brackets denote result of the Dunnās 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 (Ā± 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<sup>-1</sup> and per biovolume attograms (ag) CTX3C eq. Ī¼m<sup>-3</sup>. Numbers in parentheses in the data cells of the last three columns = coefficient of variation. Correlation coefficients (R<sup>2</sup>) 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<sup>-1</sup> d<sup>-1</sup>) 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 LCāMS Response and In Vitro Toxicity of Azaspiracids <b>7</b>ā<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. AZA7ā10, <b>7</b>ā<b>10</b>, were isolated from shellfish and
their structures, previously proposed on the basis of only LCāMS/MS
data, were confirmed by NMR spectroscopy. Purified AZA4ā6, <b>4</b>ā<b>6</b>, and <b>7</b>ā<b>10</b> were accurately quantitated by qNMR and used to assay cytotoxicity
with Jurkat T lymphocyte cells for the first time. LCāMSĀ(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 EC<sub>50</sub> values,
with the potencies for each analogue being: AZA6 > AZA8 > AZA1
> AZA4 ā AZA9 > AZA5 ā AZA10. This data contributes
to the understanding of the structureāactivity 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 Ī¼g of <b>3</b> and 250 Ī¼g 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 structureāactivity 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