Eva Cagide

Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells

Palytoxin is one of the most complex and biggest molecules known to show extreme acute toxicity. The dinoflagellate Ostreopsis spp., the producer organism of palytoxin, has been shown to be distributed worldwide, thus making palytoxin an emerging toxin. Rat-derived hepatocytes (Clone 9) and BE (2)-M17 human neuroblastoma cells were used to test palytoxin or palytoxin-like compounds by measuring the cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity (as low as 0.2 ng/ml). This method was useful for toxin detection in Ostreopsis extracts and naturally contaminated mussel samples. A comparative study testing toxic mussel extracts by LC (liquid chromatography)-MS/MS (tandem MS), MBA (mouse bioassay), haemolysis neutralization assay and a cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds.

Yessotoxin induces ER-stress followed by autophagic cell death in glioma cells mediated by mTOR and BNIP3.

Yessotoxin at nanomolar concentrations can induce programmed cell death in different model systems. Paraptosis-like cell death induced by YTX in BC3H1 cells, which are insensitive to several caspase inhibitors, has also been reported. This makes yessotoxin of interest in the search of molecules that target cancer cells vulnerabilities when resistance to apoptosis is observed. To better understand the effect of this molecule at the molecular level on tumor cells, we conducted a transcriptomic analysis using 3 human glioma cell lines with different sensitivities to yessotoxin. We show that the toxin induces a deregulation of the lipid metabolism in glioma cells as a consequence of induction of endoplasmic reticulum stress. The endoplasmic reticulum stress in turn arrests the cell cycle and inhibits the protein synthesis. In the three cell lines used we show that YTX induces autophagy, which is involved in cell death. The sensibility of the cell lines used towards autophagic cell death was related to their doubling time, being more resistant the cell line with the lowest proliferation rate. The involvement of mTOR and BNIP3 in the autophagy induction was also determined.

The Sodium Channel of Human Excitable Cells is a Target for Gambierol

Background: Gambierol is a polycyclic ether toxin with the same biogenetic origin as ciguatoxins. Gambierol has been associated with neurological symptoms in humans even though its mechanism of action has not been fully characterized. Methods: We studied the effect of gambierol in human neuroblastoma cells by using bis-oxonol to measure membrane potential and FURA-2 to monitor intracellular calcium. Results: We found that this toxin: i) produced a membrane depolarization, ii) potentiated the effect of veratridine on membrane potential iii) decreased ciguatoxininduced depolarization and iv) increased cytosolic calcium in neuroblastoma cells. Conclusion: These results indicate that gambierol modulate ion fluxes by acting as a partial agonist of sodium channels.

Marine toxins and the cytoskeleton: a new view of palytoxin toxicity.

Palytoxin is a marine toxin first isolated from zoanthids (genus Palythoa), even though dinoflagellates of the genus Ostreopsis are the most probable origin of the toxin. Ostreopsis has a wide distribution in tropical and subtropical areas, but recently these dinoflagellates have also started to appear in the Mediterranean Sea. Two of the most remarkable properties of palytoxin are the large and complex structure (with different analogs, such as ostreocin‐D or ovatoxin‐a) and the extreme acute animal toxicity. The Na+/K+‐ATPase has been proposed as receptor for palytoxin. The marine toxin is known to act on the Na+ pump and elicit an increase in Na+ permeability, which leads to depolarization and a secondary Ca2+ influx, interfering with some functions of cells. Studies on the cellular cytoskeleton have revealed that the signaling cascade triggered by palytoxin leads to actin filament system distortion. The activity of palytoxin on the actin cytoskeleton is only partially associated with the cytosolic Ca2+ changes; therefore, this ion represents an important factor in altering this structure, but it is not the only cause. The goal of the present minireview is to compile the findings reported to date about: (a) how palytoxin and analogs are able to modify the actin cytoskeleton within different cellular models; and (b) what signaling mechanisms could be involved in the modulation of cytoskeletal dynamics by palytoxin.

The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytes.

Background and purpose:  Okadaic acid (OA) and microcystins (MCs) are structurally different toxins with the same mechanism of action, inhibition of serine/threonine protein phosphatases (PPs). Methyl okadaate (MeOk), a methyl ester derivative of OA, was considered almost inactive due to its weak inhibition of PP1 and PP2A. Here, we have investigated the activity and potency of MeOk in hepatic cells in comparison with that of OA and MCs.

Palytoxins and cytoskeleton: An overview.

Cytoskeleton is a dynamic structure essential for a wide variety of normal cellular processes, including the maintenance of cell shape and morphology, volume regulation, membrane dynamics and signal transduction. Cytoskeleton is organized into microtubules, actin meshwork and intermediate filaments. Actin has been identified as a major target for destruction during apoptosis and is also important under pathological conditions such as cancers. Several natural compounds actively modulate actin organization by specific signaling cascades being useful tools to study cytoskeleton dynamics. Palytoxin is a large bioactive compound, first isolated from zoanthids, with a complex structure and different analogs such as ostreocin-D or ovatoxin-a. This toxin has been identified as a potent tumor promoter and cytotoxic molecule, which leads to actin filament distortion and triggers cell death or apoptosis. In this review we report the findings on the involvement of palytoxin and analogues modulating the actin cytoskeleton within different cellular models.

Cytotoxic effect of palytoxin on mussel.

Palytoxin is a large and complex polyhydroxylated molecule with potent neurotoxic activity. Dinoflagellates from the Ostreopsis genera were demonstrated to be producers of this compound and analogues. Even though initially palytoxin appearance was restricted to tropical areas, the recent occurrence of Ostreopsis outbreaks in Mediterranean Sea point to a worldwide dissemination probably related to climatic change. Those dinoflagellates can bioaccumulate in shellfish, especially in filter-feeding mollusks and have been involved in damaging effects in seafood or human toxic outbreaks. The present study describes palytoxins effect on metabolic activity of mantle and hepatopancreas cells from the mussel Mytilus galloprovincialis Lmk. Our results indicate that palytoxin is highly cytotoxic to mussel cells; unlike it happens with other toxins more common in European coasts such as okadaic acid and azaspiracid. These findings have a special significance for the marine environment and aquiculture since they are evidence for the ability of palytoxin to affect the integrity of bivalve mollusks that are not adapted to the presence of this toxin.

Cytoskeletal toxicity of pectenotoxins in hepatic cells

Pectenotoxins are macrocyclic lactones found in dinoflagellates of the genus Dinophysis, which induce severe liver damage in mice after i.p. injection. Here, we have looked for the mechanism(s) underlying this hepatotoxicity.

Production of functionally active palytoxin-like compounds by Mediterranean Ostreopsis cf. siamensis.

Background and purpose: Dinoflagellates from the genus Ostreopsis have been related to the production of palytoxin and analogues. Based on that, this paper describes functional studies of crude extracts from Ostreopsis cf. siamensis collected in the Mediterranean Sea in order to biochemically characterize their toxic compounds. Methods: We compared the effects of 5 crude dinoflagellates extracts with a commercially available palytoxin and a purified Ostreopsis ovata extract on metabolic activity, membrane potential, and cytosolic calcium levels by using fluorescent dyes. Results: All the extracts resulted to be neurotoxic. In addition, all of them induced a membrane depolarization and a calcium increment that were abolished when preincubating with ouabain, an inhibitor of the Na+/K+ pump. Conclusion: The effects observed were quite close to those induced by palytoxin and the Ostreopsis ovata extract as well, suggesting that Ostreopsis cf. siamensis is actually producing palytoxin-like compounds that are highly toxic and functionally active.

Induction of actin cytoskeleton rearrangement by methyl okadaate--comparison with okadaic acid.

Methyl okadaate is a derivative of the lipophilic polyether okadaic acid (OA), a well‐known inducer of apoptosis. OA inhibits Ser/Thr protein phosphatases (PPs), among them types 1 and 2A (PP1 and PP2A), whereas methyl okadaate lacks PP1/PP2A inhibitory activity in vitro. As progressive loss of neuronal cytoarchitecture is a major event that precedes neuronal death, in this work we studied comparatively the effects of both toxins on actin cytoskeleton organization in human neuroblastoma cells by filamentous actin (F‐actin) labeling with the specific dye Oregon Green 514 Phalloidin. Neither methyl okadaate nor OA modified the amount of F‐actin per cell. However, confocal microscopy imaging showed that methyl okadaate induced reorganization of actin cytoskeleton, loss of the typical flattened morphology and adoption of a round shape, and a reduction in the number of neurites, with a consequent loss of cell attachment. These effects were identical to those induced by OA, although methyl okadaate potency was approximately 10‐fold lower. In order to investigate the role of membrane potential and cytosolic Ca2+ concentration in morphological changes induced by these toxins, the cells were stained with bis‐(1,3‐dibutylbarbituric acid)‐trimethine oxonol and fura‐2. No toxin effect was detected on membrane potential or calcium influx, indicating that these two signals are not responsible for cytoskeletal/morphological change induction. Methyl okadaate induced an increase of Ser/Thr phosphorylation of cellular proteins detected by western blot, showing similar phosphorylation profiles to OA. Our data suggest that methyl okadaate is an active compound that shares a pharmacological target with OA that may be a Ser/Thr phosphatase, probably different from PP1 and PP2A.