palytoxin and Neuroblastoma

Marine neurotoxins accumulate in seafood and therewith represent a threat for consumers. At the European level, the use of in vivo bioassays is banned from 2015 onwards, except for the control of production areas. Cytotoxicity in the neuro-2a assay has been shown a promising in vitro alternative. However, given that cytotoxicity may be sensitive to confounding factors the current study investigates the suitability of functional endpoints as alternatives to cytotoxicity for the detection of marine neurotoxins. Microarray analyses were performed following exposure of neuro-2a cells to three marine neurotoxins (palytoxin (PlTx), saxitoxin (STX) and tetrodotoxin (TTX)) to identify genes up- or down-regulated that can be used as biomarkers for screening purposes. In addition to microarrays, the voltage dependent fluorescent probe bisoxonol was used to assess changes in cellular membrane potential. Biomarkers based on mRNA expression were detected for PlTx but not for STX and TTX. STX and TTX decreased the fluorescence of bisoxonol while PlTx showed no effect. When using cytotoxicity as the read out the neuro-2a assay detects these three neurotoxins at similar concentrations. Therefore it is concluded that the newly investigated endpoints in the neuro-2a assay are not preferred over cytotoxicity in a suitable broad and sensitive bioassay for the detection of marine neurotoxins in real practice.

Topics: Acrylamides; Animals; Biological Assay; Biomarkers; Cell Line, Tumor; Cell Survival; Cnidarian Venoms; Endpoint Determination; Fluorescence; Gene Expression Regulation, Neoplastic; Mice; Neuroblastoma; Oligonucleotide Array Sequence Analysis; Saxitoxin; Sodium-Potassium-Exchanging ATPase; Tetrodotoxin

Marine pelagic diazotrophic cyanobacteria of the genus Trichodesmium (Oscillatoriales) are widespread throughout the tropics and subtropics, and are particularly common in the waters of New Caledonia. Blooms of Trichodesmium are suspected to be a potential source of toxins in the ciguatera food chain and were previously reported to contain several types of paralyzing toxins. The toxicity of water-soluble extracts of Trichodesmium spp. were analyzed by mouse bioassay and Neuroblastoma assay and their toxic compounds characterized using liquid chromatography coupled with tandem mass spectrometry techniques. Here, we report the first identification of palytoxin and one of its derivatives, 42-hydroxy-palytoxin, in field samples of Trichodesmium collected in the New Caledonian lagoon. The possible role played by Trichodesmium blooms in the development of clupeotoxism, this human intoxication following the ingestion of plankton-eating fish and classically associated with Ostreopsis blooms, is also discussed.

Topics: Acrylamides; Animals; Biological Assay; Cell Line, Tumor; Chromatography, Liquid; Cnidarian Venoms; Cyanobacteria; Female; Humans; Male; Mice; Neuroblastoma; Pyrans; Tandem Mass Spectrometry; Toxicity Tests

Palytoxin and related compounds are neurotoxic phycotoxins produced by benthic microalgae belonging to the genus Ostreopsis. For several years this family of phycotoxins has been posing a threat to human health since they can bioaccumulate in shellfish. With the aim of replacing current biological assays, such as the mouse or hemolytic assays, we investigated using the Neuro-2a neuroblastoma cell line to detect palytoxin and related compounds. Cell death induced by the effects of PlTX and analogues on Na+, K+-ATPase were measured using the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay for mitochondrial reductase activity as a surrogate for cell number. The specificity of the Neuro-2a cell-based assay for palytoxin detection was confirmed by using ouabain, which also acts on Na+, K+-ATPase. Pre-treatment of the Neuro-2a cells with ouabain minimizes the effects of palytoxin. The specificity of the Neuro-2a assay was confirmed by the finding that cell death was not detected when Neuro-2a cells were exposed to other phycotoxins with unrelated cellular targets. When the Neuro-2a assay was used to detect palytoxin in mussel extracts spiked with levels of palytoxin around the proposed regulatory value of 250 microg palytoxin/kg shellfish, a good correlation was observed between the levels found and the expected values. We conclude by proposing an experimental design for functional assays using the Neuro-2a cell line for the specific detection of four neurotoxic phycotoxin families: saxitoxins, brevetoxins, ciguatoxins and palytoxins.

Topics: Acrylamides; Animals; Biological Assay; Bivalvia; Cell Line, Tumor; Cell Survival; Cnidarian Venoms; Marine Toxins; Mice; Neuroblastoma

Cation fluxes appear to play a key role in palytoxin-induced signal. There are other cellular targets that have not been described as well as the biochemical signaling cascades that transmit palytoxin-stimulated signals remain to be clarified. Since modifications of cations, mainly calcium, are generally associated to cell death or apoptosis, we wanted to further evaluate the effect of palytoxin on cell death. Then, in vitro cytotoxic effects of palytoxin were characterized on human neuroblastoma cells. By using several techniques, we studied markers of cell death and apoptosis, such as cell detachment, mitochondrial membrane potential, caspases, DNA damage, LDH leakage, propidium iodide uptake, F-actin depolymerization and inhibition of cellular proliferation. Results show that palytoxin triggers a series of toxic responses; it inhibits cell proliferation, induces cell rounding, detachment from the substratum and F-actin disruption. Among the apoptotic markers studied we only detected fall in mitochondrial membrane potential. Neither caspases activation nor chromatin condensation or DNA fragmentation were observed in palytoxin-treated cells.

Topics: Acrylamides; Actins; Apoptosis; Caspases; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Shape; Chromatin; Cnidarian Venoms; DNA Fragmentation; Humans; Membrane Potential, Mitochondrial; Neuroblastoma

The suitability and sensitivity of two neural cell models, NG108-15 and Neuro-2a, to different marine toxins were evaluated under different incubation and exposure times and in the presence or absence of ouabain and veratridine (O/V). NG108-15 cells were more sensitive to pectenotoxin-2 than Neuro-2a cells. For saxitoxin, brevetoxin-3, palytoxin, okadaic acid and dinophysistoxin-1 both cell types proved to be sensitive and suitable for toxicity evaluation. For domoic acid preliminary results were presented. Setting incubation time and exposure time proved to be critical for the development of the assays. In order to reduce the duration of the assays, it was better to reduce cell time incubation previous to toxin exposure than exposure time. For palytoxin, after 24h of growth, both cell types were sensitive in the absence of O/V. When growth time previous to toxin exposure was reduced, both cell types were unsensitive to palytoxin when O/V was absent. Although dinophysistoxin-1 and okadaic acid are both phosphatase inhibitors, these toxins did not respond similarly in front of the experimental conditions studied. Both cell types were able to identify Na-channel acting toxins and allowed to quantify the effect of saxitoxin, brevetoxin-3, palytoxin, okadaic acid, dinophysistoxin-1 and pectenotoxin-2 under different experimental conditions.

Topics: Acrylamides; Animals; Cell Line, Tumor; Cnidarian Venoms; Dose-Response Relationship, Drug; Furans; Glioma; Hybrid Cells; Kainic Acid; Macrolides; Marine Toxins; Mice; Neuroblastoma; Okadaic Acid; Oxocins; Pyrans; Saxitoxin; Time Factors; Toxicity Tests

Palytoxin-induced whole-cell and single channel currents were recorded in mouse neuroblastoma cells. Palytoxin-induced single channel currents had a slope conductance of 26 pS (20-22 degrees C). Palytoxin-induced channels were permeable to sodium and potassium and slightly permeable to calcium, choline and tetramethylammonium. They did not seem to be significantly permeable to chloride or protons. Both the steady-state and the rate of the dose-dependent effects of palytoxin could be accounted for if one assumed that a palytoxin-induced channel resulted from the binding of two palytoxin molecules to a membrane receptor with respective dissociation constants of 5 nM and 10 pM. In the continued presence of low palytoxin concentrations (less than 1 nM) the effect was maintained. Higher palytoxin concentrations induced a transient and irreproducible effect. The effect of palytoxin was decreased when either external sodium was replaced by potassium or in the absence of calcium in external and/or internal media. The results suggest that ionic currents result from the binding of palytoxin molecules to a membrane receptor and that receptor-toxin complexes can be internalized.

Topics: Acrylamides; Animals; Calcium; Cell Membrane Permeability; Choline; Cnidarian Venoms; Electric Conductivity; Electrophysiology; Glioma; Hybrid Cells; Ion Channels; Mice; Neuroblastoma; Potassium; Quaternary Ammonium Compounds; Sodium; Tumor Cells, Cultured