azaspiracid and dinophysistoxin-1

Low extraction efficiency (60-81%) of okadaic acid (OA) and dinophysistoxin 1 (DTX1) was obtained for 4 out of 5 shellfish species from Washington State (WA), USA, during application of a standard extraction method for determination of lipophilic marine biotoxins by LC-MS/MS as recommended by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). OA and total OA including esters, DTX1, DTX2, and total DTX including esters, azaspiracid 1, 2, and 3 (AZA1, AZA2, and AZA3), pectenotoxin 2 (PTX2), and yessotoxin (YTX) were the toxins examined. Matrix-matched standards prepared from the same control samples used for spike-and-recovery tests were employed to evaluate toxin extraction efficiency and sample clean-up procedures. We adjusted the EURLMB extraction method by either using an acidified methanol extraction or pre-cooking shellfish homogenates at 70 °C for 20 min before EURLMB extraction. Extraction efficiency was improved markedly for OA and DTX1 with both modified methods and for YTX with the pre-cooking step included. However, recoveries were lower for YTX using the acidified methanol extraction and for PTX2 in non-mussel samples with the pre-cooking step. A hexane wash was applied to clean water-diluted non-hydrolyzed samples and a hexane wash was combined with solid-phase extraction for cleaning hydrolyzed samples. Improved sample clean-up, combined with LC-MS/MS adjustments, enabled quantification of U.S. Food and Drug Administration-regulated toxins in five shellfish species from WA with acceptable accuracy using non-matrix matched calibration standards.

Topics: Alkalies; Animals; Chromatography, Liquid; Furans; Lipids; Macrolides; Marine Toxins; Methanol; Mollusk Venoms; Okadaic Acid; Oxocins; Shellfish; Spiro Compounds; Tandem Mass Spectrometry; Washington

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.

Topics: Animals; Bivalvia; China; Chromatography, Liquid; Dinoflagellida; Furans; Heterocyclic Compounds, 3-Ring; Hydrocarbons, Cyclic; Imines; Macrolides; Marine Toxins; Mollusk Venoms; Okadaic Acid; Ostreidae; Oxocins; Pyrans; Seafood; Shellfish; Spiro Compounds; Tandem Mass Spectrometry; Water Pollutants, Chemical

A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole-genome mRNA expression of undifferentiated intestinal Caco-2 cells. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish.

Topics: Caco-2 Cells; Cell Survival; Gene Expression Profiling; Gene Expression Regulation; Genome; Humans; Marine Toxins; Okadaic Acid; Pyrans; RNA, Messenger; Spiro Compounds

Some dinoflagellates can produce lipophilic marine toxins, which pose potent threats to seafood consumers. In the Bohai Sea, an important semi-closed inland sea with intensive mariculture industry in China, there is little knowledge concerning lipophilic marine toxins and their potential threats. In this study, net-concentrated phytoplankton samples were periodically collected from 5 typical mariculture zones around the Bohai Sea, including Laishan (LS), Laizhou (LZ), Hangu (HG), Qinhuangdao (QHD) and Huludao (HLD) in 2013 and 2014, and a method using high performance liquid chromatography (HPLC) coupled with a Q-Trap mass spectrometer was applied to analyze seven representative lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), azaspiracid-1 (AZA1), gymnodimine (GYM), and 13-desmethyl spirolide C (desMeC). The method had high sensitivity and repeatability, and exhibited satisfactory recoveries for most of the lipophilic marine toxins (92.1-108%) except for AZA1 (65.8-68.9%). Nearly all the lipophilic marine toxins could be detected in phytoplankton samples from the Bohai Sea. OA, DTX1 and PTX2 were predominant components and present in most of the phytoplankton samples. The maximum content of lipophilic marine toxin in phytoplankton samples concentrated from seawater (OA 464 pg L

Topics: Animals; China; Chromatography, High Pressure Liquid; Dinoflagellida; Furans; Heterocyclic Compounds, 3-Ring; Hydrocarbons, Cyclic; Hydrophobic and Hydrophilic Interactions; Imines; Macrolides; Marine Toxins; Mollusk Venoms; Okadaic Acid; Oxocins; Phytoplankton; Pyrans; Seafood; Spiro Compounds; Tandem Mass Spectrometry

A freeze-dried mussel tissue (Mytilus edulis) reference material (CRM-FDMT1) was produced containing multiple groups of shellfish toxins. Homogeneity and stability testing showed the material to be fit for purpose. The next phase of work was to assign certified values and uncertainties to 10 analytes from six different toxin groups. Efforts involved optimizing extraction procedures for the various toxin groups and performing measurements using liquid chromatography-based analytical methods. A key aspect of the work was compensating for matrix effects associated with liquid chromatography-mass spectrometry through standard addition, dilution, or matrix-matched calibration. Certified mass fraction values are reported as mg/kg of CRM-FDMT1 powder as bottled for azaspiracid-1, -2, and -3 (4.10 ± 0.40; 1.13± 0.10; 0.96 ± 0.10, respectively), okadaic acid, dinophysistoxin-1 and -2 (1.59 ± 0.18; 0.68 ± 0.07; 3.57± 0.33, respectively), yessotoxin (2.49 ± 0.28), pectenotoxin-2 (0.66 ± 0.06), 13-desmethylspirolide-C (2.70 ± 0.26), and domoic acid (126 ± 10). Combined uncertainties for the certified values include contributions from homogeneity, stability, and characterization experiments. The commutability of CRM-FDMT1 was assessed by examining the extractability and matrix effects for the freeze-dried material in comparison with its equivalent wet tissue homogenate. CRM-FDMT1 is the first shellfish matrix CRM with certified values for yessotoxins, pectenotoxins or spirolides, and is the first CRM certified for multiple toxin groups. CRM-FDMT1 is a valuable tool for quality assurance of phycotoxin monitoring programs and for analytical method development and validation. Graphical Abstract CRM-FDMT1 is a multi-toxin mussel tissue certified reference material (CRM) to aid in development and validation of analytical methods for measuring the levels of algal toxins in seafood.

Topics: Animals; Chromatography, Liquid; Freeze Drying; Furans; Kainic Acid; Macrolides; Marine Toxins; Mass Spectrometry; Mollusk Venoms; Mytilus edulis; Okadaic Acid; Oxocins; Pyrans; Reference Standards; Seafood; Spiro Compounds

Graphene is a novel carbonic material with great potentials for the use as sorbent due to its ultrahigh surface area. Herein, we report the use of graphene as sorbent in solid-phase extraction (SPE) using pipette tip as cartridge namely GPT-SPE, together with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), for the analysis of lipophilic marine toxins (LMTs), including yessotoxins (YTX), okadaic acid (OA), dinophysistoxin-1 (DTX1), gymnodimine (GYM), spirolides-1 (SPX1), pectenotoxin-2 (PTX2) and azaspiracid-1 (AZA1) in shellfish. The GPT-SPE procedure was optimized and the performance of graphene was fully validated. Results with high-sensitivity and good reproducibility was obtained and compared with that of other sorbents like C18 silica, multi-walled carbon nanotubes (MWCNTs), commercial Oasis HLB, and Strata-X for the extraction of LMTs, which showed superiority and advantages of graphene, such as good recoveries, stability and compatibility with various solvents. In order to exhibit the potentials of graphene as an excellent sorbent material, 67 mussel samples from six coastal cities of China were analyzed. OA was found to be the dominant contaminant, while YTX was also detected with low level.

Topics: Adsorption; Animals; Bivalvia; Chromatography, High Pressure Liquid; Furans; Graphite; Heterocyclic Compounds, 3-Ring; Hydrocarbons, Cyclic; Imines; Macrolides; Marine Toxins; Mollusk Venoms; Muscles; Okadaic Acid; Oxocins; Pyrans; Reproducibility of Results; Sensitivity and Specificity; Shellfish; Solid Phase Extraction; Spiro Compounds; Tandem Mass Spectrometry

Currently, there are no fast in vitro broad spectrum screening bioassays for the detection of marine toxins. The aim of this study was to develop such an assay. In gene expression profiling experiments 17 marker genes were provisionally selected that were differentially regulated in human intestinal Caco-2 cells upon exposure to the lipophilic shellfish poisons azaspiracid-1 (AZA1) or dinophysis toxin-1 (DTX1). These 17 genes together with two control genes were the basis for the design of a tailored microarray platform for the detection of these marine toxins and potentially others. Five out of the 17 selected marker genes on this dedicated DNA microarray gave clear signals, whereby the resulting fingerprints could be used to detect these toxins. CEACAM1, DDIT4, and TUBB3 were up-regulated by both AZA1 and DTX1, TRIB3 was up-regulated by AZA1 only, and OSR2 by DTX1 only. Analysis by singleplex qRT-PCR revealed the up- and down-regulation of the selected RGS16 and NPPB marker genes by DTX1, that were not envisioned by the new developed dedicated array. The qRT-PCR targeting the DDIT4, RSG16 and NPPB genes thus already resulted in a specific pattern for AZA1 and DTX1 indicating that for this specific case qRT-PCR might a be more suitable approach than a dedicated array.

Topics: Antigens, CD; Caco-2 Cells; Cell Adhesion Molecules; Gene Expression; Gene Expression Profiling; Humans; Marine Toxins; Okadaic Acid; Oligonucleotide Array Sequence Analysis; Pyrans; Reverse Transcriptase Polymerase Chain Reaction; Spiro Compounds; Transcription Factors; Tubulin