- Combined effects of okadaic acid and pectenotoxin-2, 13-desmethylspirolide C or yessotoxin in human intestinal Caco-2 cells. [Journal Article]
- CChemosphere 2019 Apr 05; 228:139-148
- Lipophilic phycotoxins are secondary metabolites produced by phytoplanktonic species. They accumulate in filtering shellfish and can cause human intoxications. Humans can be exposed to combinations o…
Lipophilic phycotoxins are secondary metabolites produced by phytoplanktonic species. They accumulate in filtering shellfish and can cause human intoxications. Humans can be exposed to combinations of several phycotoxins. The toxicological effects of phycotoxin mixtures on human health are largely unknown. Published data on phycotoxin co-exposure show that okadaic acid (OA) is simultaneously found with pectenetoxin-2 (PTX-2), 13-desmethylspirolide C (also known as SPX-1), or yessotoxin (YTX). Therefore, the aim of this study was to examine the effects of three binary mixtures, OA/PTX-2, OA/SPX-1 and OA/YTX on human intestinal Caco-2 cells. A multi-parametric approach for cytotoxicity determination was applied using a high-content analysis platform, including markers for cell viability, oxidative stress, inflammation, and DNA damage. Mixtures effects were analyzed using two additivity mathematical models. Our assays revealed that OA induced cytotoxicity, DNA strand breaks and interleukin 8 release. PTX-2 slightly induced DNA strand breaks, whereas SPX-1 and YTX did not affect the investigated endpoints. The combination of OA with another toxin resulted in reduced toxicity at low concentrations, suggesting antagonistic effects, but in increased effects at higher concentrations, suggesting additive or synergistic effects. Taken together, our results demonstrated that the cytotoxic effects of binary mixtures of lipophilic phycotoxins could not be predicted by additivity mathematical models. In conclusion, the present data suggest that combined effects of phycotoxins may occur which might have the potential to impact on risk assessment of these compounds.
- Contamination status of lipophilic marine toxins in shellfish samples from the Bohai Sea, China. [Journal Article]
- EPEnviron Pollut 2019 Feb 28; 249:171-180
- Lipophilic marine toxins in shellfish pose significant threats to the health of seafood consumers. To assess the contamination status of shellfish by lipophilic marine toxins in the Bohai Sea, nine s…
Lipophilic marine toxins in shellfish pose significant threats to the health of seafood consumers. To assess the contamination status of shellfish by lipophilic marine toxins in the Bohai Sea, nine species of shellfish periodically collected from five representative aquaculture zones throughout a year were analyzed with a method of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), homo-yessotoxin (homo-YTX), azaspiracids (AZA2 and AZA3), gymnodimine (GYM), and 13-desmethyl spirolide C (13-DesMe-C), were detected in more than 95 percent of the shellfish samples. Toxins PTX2, YTX, 13-DesMe-C and GYM were predominant components detected in shellfish samples. Scallops, clams and mussels accumulated much higher level of lipophilic marine toxins compared to oysters. Toxin content in shellfish samples collected from different sampling locations showed site-specific seasonal variation patterns. High level of toxins was found during the stages from December to February and June to July in Hangu, while from March to April and August to September in Laishan. Some toxic algae, including Dinophysis acuminata, D. fortii, Prorocentrum lima, Gonyaulax spinifera and Lingulodinium polyedrum, were identified as potential origins of lipophilic marine toxins in the Bohai Sea. The results will offer a sound basis for monitoring marine toxins and protecting the health of seafood consumers.
- Devastating farmed abalone mortalities attributed to yessotoxin-producing dinoflagellates. [Journal Article]
- HAHarmful Algae 2019; 81:30-41
- A large dinoflagellate bloom in Walker Bay (South Africa) in January 2017 impacted 3 land-based abalone farms resulting in the death of several million animals. Satellite-derived images of Chl-a from…
A large dinoflagellate bloom in Walker Bay (South Africa) in January 2017 impacted 3 land-based abalone farms resulting in the death of several million animals. Satellite-derived images of Chl-a from the Ocean and Land Colour Imager (OLCI) on board the European Space Agency Sentinel-3 A showed bloom initiation in late December 2016 and dispersal in mid-February 2017. The bloom was dominated by two dinoflagellate species identified by light microscopy as Gonyaulax spinifera (Claparède & Lachmann) Diesing, 1866 and Lingulodinium polyedrum (Stein) Dodge, 1989. These morphologically based identifications were confirmed by phylogenetic analysis using partial sequences of the large subunit rDNA of both dinoflagellates. The appearance of yessotoxins (YTX) in abalone clearly coincided with increases in dinoflagellate concentrations. Yessotoxins in both the plankton and abalone were dominated by the two analogues homo-YTX and 45-hydroxy-YTX. The absence of toxins in a clonal culture of L. polyedrum implicated G. spinifera as the likely source of YTX. Toxin concentrations were found to be highest in the gills which showed the most significant pathology, including severe, generalized disruption of the gill epithelium characterized by degeneration and necrosis of epithelial cells accompanied by a modest inflammatory response. Some farms undertook pre-emptive or emergency harvesting to reduce financial losses.
- [Yessotoxin: risk assessment for public health. Justification of regulations of content in seafood]. [Review]
- VPVopr Pitan 2018; 87(3):18-29
- Yessotoxin and its derivatives (about 90) are isolated from algae belonging to the species Protoceratium reticulatum, Gonyaulax cf. Spinifera, Lingulodinium polyedrum and from invertebrate organisms …
Yessotoxin and its derivatives (about 90) are isolated from algae belonging to the species Protoceratium reticulatum, Gonyaulax cf. Spinifera, Lingulodinium polyedrum and from invertebrate organisms that feed on these algae. Previously yessotoxin have been associated with the group of diarrheal toxins. Later studies of the possible impact of yessotoxin on the activity of alkaline phosphatase allowed to exclude them from this group. Yessotoxin causes a violation of calcium entry in the cells, which, in turn, effects the calcium-calmodulin system and thus influences into homeostasis of the organism as a whole. It was shown that yessotoxin induces a biphasic change in the concentration of adenosine monophosphate, an initial increase with a subsequent relative decrease, within some minutes after adding the toxin to the lymphocytes cell culture. Yessotoxin has effects on immune system; which is manifested in an increase of cytokines level, by inducing the expression of the genes encoding them. Yessotoxin have impact into processes of cell adhesion via E-cadherin and, thus, could be an important factor in the development of Alzheimer's disease. It has been established that yessotoxin caused the development of apoptosis. In those cases all three mechanisms of cell death took place - apoptosis, paraptosis and autophagy. Yessotoxin's acute toxicity doses according to different data are from 100 to 500-750 μg per 1kg of body weight. Yessotoxin's acute reference dose (ARfD) - 25 μg/kg of body weight per day. The results of the analysis of yessotoxin level in shellfish meat showed that none of the studied samples contained more than 3.75 mg yessotoxin equivalents/kg shellfish meat. This level has been adopted by the European Union as the maximum acceptable level of yessotoxin in shellfish meat (EU Regulation N 786/2013). Presented data on the mechanism of action, toxicity and prevalence of yessotoxins make it necessary to establish regulations of their content in seafood, placed on the markets of the Eurasian Economic Union.
- A Strategy to Replace the Mouse Bioassay for Detecting and Identifying Lipophilic Marine Biotoxins by Combining the Neuro-2a Bioassay and LC-MS/MS Analysis. [Journal Article]
- MDMar Drugs 2018 Dec 12; 16(12)
- Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regu…
Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.
- Determination of lipophilic marine toxins in fresh and processed shellfish using modified QuEChERS and ultra-high-performance liquid chromatography-tandem mass spectrometry. [Journal Article]
- FCFood Chem 2019 Jan 30; 272:427-433
- A simple QuEChERS method coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to improve the extraction efficiency of lipophilic marine toxins…
A simple QuEChERS method coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to improve the extraction efficiency of lipophilic marine toxins (yessotoxins, dinophysistoxins, okadaic acid, azazspiracids, and spirolides) in fresh and processed shellfish products. The proposed method included freezing and dispersive solid-phase extraction with graphene oxide as the sorbent to clean complex matrices containing lipids (e.g., free fatty acids) and pigments. Quantification was performed using matrix-matched calibration curves. Recoveries were 85%-117.4% and the relative standard deviation for precision was less than 10% for marine toxins in fresh and processed shellfish products. The limits of detection (signal-to-noise = 3) and quantification (signal-to-noise = 10) were 0.10-1.47 and 0.32-4.92 μg/kg, respectively. The validated QuEChERS method, coupled with UPLC-MS/MS, was applied successfully to determine lipophilic marine toxins in fresh and processed shellfish samples.
- Effects of salinity variation on growth and yessotoxin composition in the marine dinoflagellate Lingulodinium polyedra from a Skagerrak fjord system (western Sweden). [Journal Article]
- HAHarmful Algae 2018; 78:9-17
- The marine dinoflagellate Lingulodinium polyedra is a toxigenic species capable of forming high magnitude and occasionally harmful algal blooms (HABs), particularly in temperate coastal waters throug…
The marine dinoflagellate Lingulodinium polyedra is a toxigenic species capable of forming high magnitude and occasionally harmful algal blooms (HABs), particularly in temperate coastal waters throughout the world. Three cultured isolates of L. polyedra from a fjord system on the Skagerrak coast of Sweden were analyzed for their growth characteristics and to determine the effects of a strong salinity gradient on toxin cell quotas and composition. The cell quota of yessotoxin (YTX) analogs, as determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), ranged widely among strains. For two strains, the total toxin content remained constant over time in culture, but for the third strain, the YTX cell quota significantly decreased (by 32%) during stationary growth phase. The toxin profiles of the three strains differed markedly and none produced YTX. The analog 41a-homo-YTX (m/z 1155), its putative methylated derivative 9-Me-41a-homo-YTX (m/z 1169) and an unspecified keto-YTX (m/z 1047) were detected in strain LP29-10H, whereas strain LP30-7B contained nor-YTX (m/z 1101), and two unspecified YTX analogs at m/z 1159 and m/z 1061. The toxin profile of strain LP30-8D comprised two unspecified YTX analogs at m/z 1061 and m/z 991 and carboxy-YTX (m/z 1173). Strain LP30-7B cultured at multiple salinities (10, 16, 22, 28 and 34) did not tolerate the lowest salinity (10), but there was a statistically significant decrease (by 21%) in toxin cell quota between growth at the highest versus lower permissible salinities. The toxin profile for strain LP30-7B remained constant over time for a given salinity. At lower salinities, however, the proportion of the unspecified YTX analog (m/z 1061) was significantly higher, especially with respect to nor-YTX (m/z 1101). This study shows high intra-specific variability in yessotoxin composition among strains from the same geographical region and inconsistency in toxin cell quota under different environmental regimes and growth stages in culture. This variation has important implications for the kinetics of YTX production and food web transfer in natural bloom populations from diverse geographical regions.
- Single-Cell Tracking of A549 Lung Cancer Cells Exposed to a Marine Toxin Reveals Correlations in Pedigree Tree Profiles. [Journal Article]
- FOFront Oncol 2018; 8:260
- Long-term video-based tracking of single A549 lung cancer cells exposed to three different concentrations of the marine toxin yessotoxin (YTX) reveals significant variation in cytotoxicity, and it co…
Long-term video-based tracking of single A549 lung cancer cells exposed to three different concentrations of the marine toxin yessotoxin (YTX) reveals significant variation in cytotoxicity, and it confirms the potential genotoxic effects of this toxin. Tracking of single cells subject to various toxic exposure, constitutes a conceptually simple approach to elucidate lineage correlations and sub-populations which are masked in cell bulk analyses. The toxic exposure can here be considered as probing a cell population for properties and change which may include long-term adaptation to treatments. Ranking of pedigree trees according to a measure of "size," provides definition of sub-populations. Following single cells through generations indicates that signaling cascades and experience of mother cells can pass to their descendants. Epigenetic factors and signaling downstream lineages may enhance differences between cells and partly explain observed heterogeneity in a population. Signaling downstream lineages can potentially link a variety of observations of cells making resulting data more suitable for computerized treatment. YTX exposure of A549 cells tends to cause two main visually distinguishable classes of cell death modalities ("apoptotic-like" and "necrotic-like") with approximately equal frequency. This special property of YTX enables estimation of correlation between cell death modalities for sister cells indicating impact downstream lineages. Hence, cellular responses and adaptation to treatments might be better described in terms of effects on pedigree trees rather than considering cells as independent entities.
- Lipophilic marine biotoxins SERS sensing in solutions and in mussel tissue. [Journal Article]
- TTalanta 2018 Sep 01; 187:47-58
- To detect and recognise three structurally related marine biotoxins responsible for the diarrheic shellfish poisoning (DSP) symptom, namely okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysis…
To detect and recognise three structurally related marine biotoxins responsible for the diarrheic shellfish poisoning (DSP) symptom, namely okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) respectively, as well as the structurally different yessotoxin (YTX), we developed a novel surface-enhanced micro-Raman scattering (micro-SERS) approach to investigate for the first time their micro-SERS signalling in solution and jointly analysed them in conjunction with the normal and toxic mussel tissue. YTX provided the main SERS feature surprisingly similar to DTX-1 and DTX-2, suggesting similar molecular adsorption mechanism with respect to the AgNPs. A fingerprint SERS band at 1017 cm-1 characteristic for the C-CH3 stretching in DTX-1 and DTX-2 and absent in OA SERS signal, allowed direct SERS discrimination of DTX-1,2 from OA. In acid form or as dissolved potassium salt, OA showed reproducible SERS feature for 0.81 μM to 84.6 nM concentrations respectively, while its ammonium salt slightly changed the overall SERS signature. The inherently strong fluorescence of the shellfish tissue, which hampers Raman spectroscopy analysis, further increases when toxins are present in tissue. Through SERS, tissue fluorescence is partially quenched. Artificially intoxicated mussel tissue with DSP toxins and incubated with AgNPs allowed direct SERS evidence of the toxin presence, opening a novel avenue for the in situ shellfish tracking and warning via micro-SERS. Natural toxic tissue containing 57.91 μg kg-1 YTX (LC-MS confirmed) was micro-SERS assessed to validate the new algorithm for toxins detection. We showed that a portable Raman system was able to reproduce the lab-based SERS results, being suitable for in situ raw seafood screening. The new approach provides an attractive, faster, effective and low-cost alternative for seafood screening, with economic, touristic and sustainable impact in aquaculture, fisheries, seafood industry and consumer trust.
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- Yessotoxin detection in bivalve molluscs: A case study from coastal mussel farms (Sardinia, Italy). [Journal Article]
- IJItal J Food Saf 2017 Oct 20; 6(4):7015
- This work reports the first communication relating to the presence of yessotoxins in Mytilus galloprovincialis from coastal mussel farms (Sardinia, western Mediterranean) detected during 2008 and 201…
This work reports the first communication relating to the presence of yessotoxins in Mytilus galloprovincialis from coastal mussel farms (Sardinia, western Mediterranean) detected during 2008 and 2013 through a monitoring programme. The paper emphasizes how the changes both in yessotoxin permitted limits and used methods, established by legislation, have influenced the interpretation of the obtained results. Consequently, the samples that resulted negative during 2008 would have been positive until August 2013 and negative from September 2013 up to now, and the samples that were positive in 2013 would have been positive in 2008 and negative nowadays, according to Regulation currently in force. Regular monitoring of biotoxins demonstrated that, although yessotoxins have been rarely present in the past in Sardinia, they may cause toxicity in shellfish. So, it's important to keep up on legislation's changing and laboratory methods.