Rapid detection of ciguatoxins in Gambierdiscus and Fukuyoa with immunosensing tools.

Consumption of seafood contaminated with ciguatoxins (CTXs) leads to a foodborne disease known as ciguatera. Primary producers of CTXs are epibenthic dinoflagellates of the genera Gambierdiscus and Fukuyoa. In this study, thirteen Gambierdiscus and Fukuyoa strains were cultured, harvested at exponential phase, and CTXs were extracted with an implemented rapid protocol. Microalgal extracts were obtained from pellets with a low cell abundance (20,000 cell/mL) and were then analyzed with magnetic bead (MB)-based immunosensing tools (colorimetric immunoassay and electrochemical immunosensor). It is the first time that these approaches are used to screen Gambierdiscus and Fukuyoa strains, providing not only a global indication of the presence of CTXs, but also the ability to discriminate between two series of congeners (CTX1B and CTX3C). Analysis of the microalgal extracts revealed the presence of CTXs in 11 out of 13 strains and provided new information about Gambierdiscus and Fukuyoa toxin profiles. The use of immunosensing tools in the analysis of microalgal extracts facilitates the elucidation of further knowledge regarding these dinoflagellate genera and can contribute to improved ciguatera risk assessment and management.

Discovery of gymnodimine fatty acid ester metabolites in shellfish using liquid chromatography/mass spectrometry.

RATIONALE: Gymnodimines (GYMs) are fast-acting toxins that belong to the cyclic imine group, a subclass of lipophilic marine toxins. GYMs are considered to be emerging toxins but have not yet been linked to incidents of human poisoning, Limited knowledge on the metabolism of GYMs means that a proper risk assessment has not been possible and caution must be taken when establishing the relevance of GYMs in terms of food safety of marine products. METHODS: A series of mass spectrometric experiments involving precursor and product ion scans, selected reaction monitoring (SRM), and high-resolution mass spectrometry (MS) were used to detect and confirm 10-O-acyl esters of gymnodimine-A (1). RESULTS: We have detected for the first time the presence of a range of acyl ester derivatives of GYMs in shellfish samples from the Gulf of Gabes, Tunisia. The MS fragmentation pathways of 1 and its esters were also elucidated. Partial synthesis of a palmitic acid ester of 1 facilitated confirmation of identity and calibration of SRM analyses. Evidence of acyl ester metabolites of gymnodimine-B and -C was also obtained. CONCLUSIONS: A semi-quantitative analysis indicated that the majority of GYMs present in the sample were in the acylated form (>90%), suggesting that these compounds must not be neglected when trying to understand the risks associated with GYMs. There is a clear need for toxicology studies on these esters and assessment of bio-availability to humans.

The implementation of liquid chromatography tandem mass spectrometry for the official control of lipophilic toxins in seafood: single-laboratory validation under four chromatographic conditions.

We performed a comprehensive study to assess the fit for purpose of four chromatographic conditions for the determination of six groups of marine lipophilic toxins (okadaic acid and dinophysistoxins, pectenotoxins, azaspiracids, yessotoxins, gymnodimine and spirolides) by LC-MS/MS to select the most suitable conditions as stated by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). For every case, the elution gradient has been optimized to achieve a total run-time cycle of 12 min. We performed a single-laboratory validation for the analysis of three relevant matrices for the seafood aquaculture industry (mussels, pacific oysters and clams), and for sea urchins for which no data about lipophilic toxins have been reported before. Moreover, we have compared the method performance under alkaline conditions using two quantification strategies: the external standard calibration (EXS) and the matrix-matched standard calibration (MMS). Alkaline conditions were the only scenario that allowed detection windows with polarity switching in a 3200 QTrap mass spectrometer, thus the analysis of all toxins can be accomplished in a single run, increasing sample throughput. The limits of quantification under alkaline conditions met the validation requirements established by the EURLMB for all toxins and matrices, while the remaining conditions failed in some cases. The accuracy of the method and the matrix effects where generally dependent on the mobile phases and the seafood species. The MMS had a moderate positive impact on method accuracy for crude extracts, but it showed poor trueness for seafood species other than mussels when analyzing hydrolyzed extracts. Alkaline conditions with EXS and recovery correction for OA were selected as the most proper conditions in the context of our laboratory. This comparative study can help other laboratories to choose the best conditions for the implementation of LC-MS/MS according to their own necessities.

Towards the standardisation of the neuroblastoma (neuro-2a) cell-based assay for ciguatoxin-like toxicity detection in fish: application to fish caught in the Canary Islands.

The ouabain/veratridine-dependent neuroblastoma (neuro-2a) cell-based assay (CBA) was applied for the determination of the presence of ciguatoxin (CTX)-like compounds in ciguatera-suspected fish samples caught in the Canary Islands. In order to avoid matrix interferences the maximal concentration of wet weight fish tissue exposed to the neuro-2a cells was set at 20 mg tissue equivalent (TE) ml(-1) according to the sample preparation procedure applied. In the present study, the limit of quantification (LOQ) of CTX1B equivalents in fish extract was set at the limit of detection (LOD), being defined as the concentration of CTX1B equivalents inhibiting 20% cell viability (IC(20)). The LOQ was estimated as 0.0096 ng CTX1B eq.g TE(-1) with 23-31% variability between experiments. These values were deemed sufficient even though quantification given at the IC(50) (the concentration of CTX1B equivalents inhibiting 50% cell viability) is more accurate with a variability of 17-19% between experiments. Among the 13 fish samples tested, four fish samples were toxic to the neuro-2a cells with estimations of the content in CTX1B g(-1) of TE ranging from 0.058 (± 0.012) to 6.23 (± 0.713) ng CTX1B eq.g TE(-1). The high sensitivity and specificity of the assay for CTX1B confirmed its suitability as a screening tool of CTX-like compounds in fish extracts at levels that may cause ciguatera fish poisoning. Species identification of fish samples by DNA sequence analysis was conducted in order to confirm tentatively the identity of ciguatera risk species and it revealed some evidence of inadvertent misidentification. Results presented in this study are a contribution to the standardisation of the neuro-2a CBA and to the risk analysis for ciguatera in the Canary Islands.

Detection and quantification of maitotoxin-like compounds using a neuroblastoma (Neuro-2a) cell based assay. Application to the screening of maitotoxin-like compounds in Gambierdiscus spp.

SK&F 96365 was used in a Neuroblastoma (Neuro-2a) cell based assay to determine the production of maitotoxin-like (MTX-like) compounds in two strains of Gambierdiscus spp. A 2.5 hour assay was effective for the detection of the MTX-induced toxic effects with a concentration that inhibited 50% cell viability (IC(50)) equivalent to 3.38 nM MTX. Evidence was found for the production of MTX-like compounds in both Gambierdiscus strains studied at concentrations of 404 and 36.7 nmoles MTX equivalence per 10(6) cells. The assay is proposed as an efficient approach to the detection and quantification of MTX-like compounds in Gambierdiscus spp.

Evidence of okadaic acid production in a cultured strain of the marine dinoflagellate Prorocentrum rhathymum from Malaysia.

Protein phosphatase inhibition assay (PPIA), Neuroblastoma cell-based assay (Neuro-2a CBA) and LC-MS/MS analysis revealed for the first time the production of okadaic acid (OA) by a Prorocentrum rhathymum strain. Low amounts of OA were detected by LC-MS/MS analysis. Inhibition of PP2A activity and a weak toxicity to the Neuro-2a CBA were also observed.

Cell-based assay coupled with chromatographic fractioning: a strategy for marine toxins detection in natural samples.

Cell-based assays (CBA) have been proposed for the evaluation of toxicity caused by marine toxins in natural samples (fish, shellfish and microalgae). However, their application has been hindered due to the interferences present in biological matrices that may cause cellular response and interfere in toxicity evaluation. This work reviews in an extensive introduction the use of CBA for toxicity evaluation of marine toxins. Afterwards, the coupling of chromatographic fractioning with neuroblastoma Neuro-2a CBA is presented to enhance the applicability of CBA for complex matrices. Examples of application are provided for mussel samples (Mytilus galloprovincialis) and microalgae (Gambierdiscus sp.), and the results demonstrated the great potential of the combined strategy for reliable toxicological evaluation without ethical concern. Fractioning of an equivalent of 72 mg eq mL(-1) of mussel sample allowed the identification of non-toxic and toxic fractions whereas only 2.5mg eq mL(-1) of non-purified mussel sample was responsible for 20% of cell mortality. Furthermore, the application of CBA allowed selectively distinguishing between ciguatoxin-like and other unspecific toxicity in Gambierdiscus sp. extract.

Extrusion of earthworm coelomocytes: comparison of the cell populations recovered from the species Lumbricus terrestris, Eisenia fetida and Octolasion tyrtaeum.

Coelomocytes were extruded from three earthworm species: Lumbricus terrestris, Eisenia fetida and Octolasion tyrtaeum. Featuring a simple low-vacuum holding device, the proposed methodology allows the recovery of cells with minimum risk of contamination by faecal material. The viability of O. tyrtaeum coelomocytes was highly reproducible (average 93%), with an average yield of 0.92 x 10(6) viable cells per earthworm. Cell viability for L. terrestris and E. fetida averaged approximately 68% but the cell yields were higher (respectively 1.67 x 10(6) and 1.28 x 10(6)). Large inter-individual differences in cell yields were observed with L. terrestris. Flow cytometric analyses indicated species to species differences in cell populations. Coelomocytes from E. fetida were the smallest with approximately 57% of the total viable cells recovered being monitored between 2 and 10 microns. Large granulated cells (> or = 20 microns) were detected in fairly large proportions in L. terrestris and O. tyrtaeum (approximately 52 and approximately 96%, respectively) while they were less abundant in E. fetida (approximately 9%). Using the vital dye neutral red to assess functional integrity, average cellular uptakes were significantly higher for L. terrestris and O. tyrtaeum than for E. fetida (2.94, 2.66 and 0.64 micrograms/2 x 10(5) cells, respectively). In summary, the extrusion methodology herein described is applicable for the recovery of coelomocytes from a wide range of earthworm sizes and species. Moreover, this study strengthens the fact that extruded coelomocytes could be used for the evaluation of cell dysfunction and/or cell death following an in vitro and/or in vivo treatment.