Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection.

The growing concern about ciguatera fish poisoning (CF) due to the expansion of the microorganisms producing ciguatoxins (CTXs) increased the need to develop a reliable and fast method for ciguatoxin detection to guarantee food safety. Cytotoxicity assay on the N2a cells sensitized with ouabain (O) and veratridine (V) is routinely used in ciguatoxin detection; however, this method has not been standardized yet. This study demonstrated the low availability of sodium channels in the N2a cells, the great O/V damage to the cells and the cell detachment when the cell viability is evaluated by the classical cytotoxicity assay and confirmed the absence of toxic effects caused by CTXs alone when using the methods that do not require medium removal such as lactate dehydrogenase (LDH) and Alamar blue assays. Different cell lines were evaluated as alternatives, such as human neuroblastoma, which was not suitable for the CTX detection due to the greater sensitivity to O/V and low availability of sodium channels. However, the HEK293 Nav cell line expressing the α1.6 subunit of sodium channels was sensitive to the ciguatoxin without the sensitization with O/V due to its expression of sodium channels. In the case of sensitizing the cells with O/V, it was possible to detect the presence of the ciguatoxin by the classical cytotoxicity MTT method at concentrations as low as 0.0001 nM CTX3C, providing an alternative cell line for the detection of compounds that act on the sodium channels.

Synergistic effect of environmental food pollutants: Pesticides and marine biotoxins.

Emerging marine biotoxins such as ciguatoxins and pyrethroid compounds, widely used in agriculture, are independently treated as environmental toxicants. Their maximum residue levels in food components are set without considering their possible synergistic effects as consequence of their interaction with the same cellular target. There is an absolute lack of data on the possible combined cellular effects that biological and chemical pollutants, may have. Nowadays, an increasing presence of ciguatoxins in European Coasts has been reported and these toxins can affect human health. Similarly, the increasing use of phytosanitary products for control of food plagues has raised exponentially during the last decades due to climate change. The lack of data and regulation evaluating the combined effect of environmental pollutants with the same molecular target led us to analyse their in vitro effects. In this work, the effects of ciguatoxins and pyrethroids in human sodium channels were investigated. The results presented in this study indicate that both types of compounds have a profound synergistic effect in voltage-dependent sodium channels. These food pollutants act by decreasing the maximum peak inward sodium currents and hyperpolarizing the sodium channels activation, effects that are boosted by the simultaneous presence of both compounds. A fact that highlights the need to re-evaluate their limits in feedstock as well as their potential in vivo toxicity considering that they act on the same cellular target. Moreover, this work sets the cellular basis to further apply this type of studies to other water and food pollutants that may act synergistically and thus implement the corresponding regulatory limits taking into account its presence in a healthy diet.

Targeting Chloride Ion Channels: New Insights into the Mechanism of Action of the Marine Toxin Azaspiracid.

Azaspiracids (AZAs) are marine toxins produced by dinoflagellates belonging to the genera Azadinium and Amphidoma that caused human intoxications after consumption of contaminated fishery products, such as mussels. However, the exact mechanism for the AZA induced cytotoxic and neurotoxic effects is still unknown. In this study several pharmacological approaches were employed to evaluate the role of anion channels on the AZA effects that demonstrated that cellular anion dysregulation was involved in the toxic effects of these compounds. The results presented here demonstrated that volume regulated anion channels (VRACs) are affected by this group of toxins, and, because there is not any specific activator of VRACs besides the intracellular application of GTPγ-S molecule, this group of natural compounds could represent a powerful tool to analyze the role of these channels in cellular homeostasis. In addition to this, in this work, a detailed pharmacological approach was performed in order to elucidate the anion channels present in human HEK293 cells as well as their regulation by the marine toxins azaspiracids. Altogether, the data presented here demonstrated that the effect of azaspiracids in human cells was completely dependent on ATP-regulated anion channels, whose upregulation by these toxins could lead to regulatory volume decrease and underlie the reported toxicity of these compounds.

Subacute immunotoxicity of the marine phycotoxin yessotoxin in rats.

Yessotoxin (YTX) is a marine phycotoxin produced by dinoflagellates and accumulated in filter feeding shellfish. YTX content in shellfish is regulated by many food safety authorities to protect human health, although currently no human intoxication episodes have been unequivocally related to YTX presence in food. The immune system has been proposed as one of the target organs of YTX due to alterations of lymphoid tissues and cellular and humoral components. The aim of the present study was to explore subacute immunotoxicity of YTX in rats by evaluating the haematological response, inflammatory cytokine biomarkers and the presence of YTX-induced structural alterations in the spleen and thymus. The results showed that repeated administrations of YTX caused a decrease of lymphocyte percentage and an increase of neutrophil counts, a reduction in interleukine-6 (IL-6) plasmatic levels and histopathological splenic alterations in rats after four intraperitoneal injections of YTX at doses of 50 or 70 µg/kg that were administered every 4 days along a period of 15 days. Therefore, for the first time, subacute YTX-immunotoxicity is reported in rats, suggesting that repeated exposures to low amounts of YTX might also suppose a threat to human health, especially in immuno-compromised populations.

Cytotoxicity of goniodomin A and B in non contractile cells.

Goniodomin A is a phycotoxin produced by the dinoflagellates Alexandrium hiranoi (formerly Goniodoma pseudogoniaulax) and Alexandrium monilatum. This polyether macrolide exerts a potent antifungal effect and disturbs the actomyosin ATPase activity and the F-actin meshwork in diverse cell types. Goniodomin B is a fused acetal isomer isolated with goniodomin A with unknown activity. Histopathological changes induced by goniodomin A postulated hepatocytes as target cells. In this study both compounds induce a time and concentration dependent fall in the viability of Clone 9 rat hepatocytes. Furthermore, for both compounds, primary rat hepatocytes are almost 10 folds less sensitive than Clone 9 cells. Goniodomin A is highly effective in the nanomolar range while micromolar concentrations of goniodomin B are necessary to observe cytoxicity. Additionally, goniodomin A induced a significant increase in the F-actin and decrease in the G-actin content of Clone 9 cells but did not change the actin of primary cultured hepatocytes. However, goniodomin B could not exert significant alterations in the cytoskeleton of neither cell type. Futhermore goniodomin A as well as goniodomin B are cytotoxic to excitable cells. Both analogues triggered a time dependent decrease on viability in BE(2)-M17 human neuroblastoma cells. In this cell model goniodomin A increased the intracellular calcium and depolarized cells. We conclude that goniodomins A and B are biologically active molecules in hepatocytes and also in excitable cells BE(2)-M17. However, the analogue goniodomin B, whose activity is described in this work for the first time, is a much less potent compound.

Subacute Cardiovascular Toxicity of the Marine Phycotoxin Azaspiracid-1 in Rats.

Azaspiracids (AZAs) are marine toxins produced by Azadinium spinosum that get accumulated in filter feeding shellfish through the food-web. The first intoxication was described in The Netherlands in 1990, and since then several episodes have been reported worldwide. Azaspiracid-1, AZA-2, and AZA-3 presence in shellfish is regulated by food safety authorities of several countries to protect human health. Azaspiracids have been related to widespread organ damage, tumorogenic properties and acute heart rhythm alterations in vivo but the mechanism of action remains unknown. Azaspiracid toxicity kinetics in vivo and in vitro suggests accumulative effects. We studied subacute cardiotoxicity in vivo after repeated exposure to AZA-1 by evaluation of the ECG, arterial blood pressure, plasmatic heart damage biomarkers, and myocardium structure and ultrastructure. Our results showed that four administrations of AZA-1 along 15 days caused functional signs of heart failure and structural heart alterations in rats at doses ranging from 1 to 55 µg/kg. Azaspiracid-1 altered arterial blood pressure, tissue inhibitors of metalloproteinase-1 plasma levels, heart collagen deposition, and ultrastructure of the myocardium. Overall, these data indicate that repeated exposure to low amounts of AZA-1 causes cardiotoxicity, at doses that do not induce signs of other organic system toxicity. Remarkably, human exposure to AZAs considering current regulatory limits of these toxins may be dangerously close to clearly cardiotoxic doses in rats. These findings should be considered when human risk is estimated particularly in high cardiovascular risk subpopulations.

Detection of palytoxin-like compounds by a flow cytometry-based immunoassay supported by functional and analytical methods.

Palytoxin (PLTX) is a complex marine toxin produced by zoanthids (i.e. Palythoa), dinoflagellates (Ostreopsis) and cyanobacteria (Trichodesmium). PLTX outbreaks are usually associated with Indo-Pacific waters, however their recent repeated occurrence in Mediterranean-European Atlantic coasts demonstrate their current worldwide distribution. Human sickness and fatalities have been associated with toxic algal blooms and ingestion of seafood contaminated with PLTX-like molecules. These toxins represent a serious threat to human health. There is an immediate need to develop easy-to-use, rapid detection methods due to the lack of validated protocols for their detection and quantification. We have developed an immuno-detection method for PLTX-like molecules based on the use of microspheres coupled to flow-cytometry detection (Luminex 200™). The assay consisted of the competition between free PLTX-like compounds in solution and PLTX immobilized on the surface of microspheres for binding to a specific monoclonal anti-PLTX antibody. This method displays an IC50 of 1.83 ± 0.21 nM and a dynamic range of 0.47-6.54 nM for PLTX. An easy-to-perform extraction protocol, based on a mixture of methanol and acetate buffer, was applied to spiked mussel samples providing a recovery rate of 104 ± 8% and a range of detection from 374 ± 81 to 4430 ± 150 µg kg(-1) when assayed with this method. Extracts of Ostreopsis cf. siamensis and Palythoa tuberculosa were tested and yielded positive results for PLTX-like molecules. However, the data obtained for the coral sample suggested that this antibody did not detect 42-OH-PLTX efficiently. The same samples were further analyzed using a neuroblastoma cytotoxicity assay and UPLC-IT-TOF spectrometry, which also pointed to the presence of PLTX-like compounds. Therefore, this single detection method for PLTX provides a semi-quantitative tool useful for the screening of PLTX-like molecules in different matrixes.

Diarrhetic effect of okadaic acid could be related with its neuronal action: Changes in neuropeptide Y.

Okadaic acid (OA) and dinophysistoxins (DTXs) are a group of marine toxins that cause diarrheic shellfish poisoning (DSP) in humans and animals. These compounds are produced by dinoflagellates of the Prorocentrum and Dinophysis genera and can accumulate in filter-feeding bivalves, posing a serious health risk for shellfish consumers. The enteric nervous system (ENS) plays a crucial role in the regulation of the gastrointestinal tract. In addition, neuropeptides produced by ENS affects the epithelial barrier functions. In the present work we used a two-compartment human coculture model containing the SH-SY5Y neuroblastoma cell line and polarized colonic epithelial monolayers (Caco-2) to study the OA intestinal permeability. First, we have determined OA cytotoxicity and we have found that OA reduces the viability of SH-SY5Y in a dose-dependent way, even though DTX1 is 4 to 5 times more potent than OA. Besides DTX1 is 15 to 18 orders of magnitude more potent than OA in decreasing transepithelial electrical resistance (TEER) of caco-2 cells without inducing cytotoxicity. Permeability assays indicate that OA cross the monolayer and modulates the neuropeptide Y (NPY) secretion by neuroblastoma cells. This NPY also affects the permeability of OA. This offers a novel approach to establish the influence of OA neuronal action on their diarrheic effects through a cross talk between ENS and intestine via OA induced NPY secretion. Therefore, the OA mechanisms of toxicity that were long attributed only to the inhibition of protein phosphatases, would require a reevaluation.

Acute cardiotoxicity evaluation of the marine biotoxins OA, DTX-1 and YTX.

Phycotoxins are marine toxins produced by phytoplankton that can get accumulated in filter feeding shellfish. Human intoxication episodes occur due to contaminated seafood consumption. Okadaic acid (OA) and dynophysistoxins (DTXs) are phycotoxins responsible for a severe gastrointestinal syndrome called diarrheic shellfish poisoning (DSP). Yessotoxins (YTXs) are marine toxins initially included in the DSP class but currently classified as a separated group. Food safety authorities from several countries have regulated the content of DSPs and YTXs in shellfish to protect human health. In mice, OA and YTX have been associated with ultrastructural heart damage in vivo. Therefore, this study explored the potential of OA, DTX-1 and YTX to cause acute heart toxicity. Cardiotoxicity was evaluated in vitro by measuring hERG (human èter-a-go-go gene) channel activity and in vivo using electrocardiogram (ECG) recordings and cardiac damage biomarkers. The results demonstrated that these toxins do not exert acute effects on hERG channel activity. Additionally, in vivo experiments showed that these compounds do not alter cardiac biomarkers and ECG in rats acutely. Despite the ultrastructural damage to the heart reported for these toxins, no acute alterations of heart function have been detected in vivo, suggesting a functional compensation in the short term.

Hapalindoles from the cyanobacterium fischerella: potential sodium channel modulators.

Hapalindoles make up a large group of bioactive metabolites of the cyanobacterial order Stigonematales. 12-epi-Hapalindole E isonitrile, 12-epi-hapalindole C isonitrile, 12-epi-hapalindole J isonitrile, and hapalindole L from Fischerella are acutely toxic for insect larvae; however, the biochemical targets responsible for the biological activities of hapalindoles are not understood. We describe here the electron impact mass spectra of these four hapalindole congeners; their structures were confirmed by nuclear magnetic resonance spectroscopy. In combination with the presented mass spectra of (15)N-labeled species and their retention times on a gas chromatography capillary column, a rapid and reliable determination should be possible in future research. The bioactivity of these hapalindoles was tested on mammalian cells focusing on their effects in the BE(2)-M17 excitable human neuroblastoma cell line. The fluorescent dye Alamar Blue was applied to monitor cytotoxicity, fura-2 to evaluate changes in the cytosolic calcium concentrations, and bis-oxonol to detect effects on membrane potential. Data showed that the hapalindoles did not affect cell viability of the neuroblastoma cells, even when they were incubated for 72 h. Neither depolarization nor initiation of calcium influx was observed in the cells upon hapalindole treatment. However, the data provide evidence that hapalindoles are sodium channel-modulating neurotoxins. They inhibited veratridine-induced depolarization in a manner similar to that of neosaxitoxin. Our data suggest hapalindoles should be added to the growing number of neurotoxic secondary metabolites, such as saxitoxins and anatoxins, already known in freshwater cyanobacteria. As stable congeners, hapalindoles may be a risk in freshwater ecosystems or agricultural water usage and should therefore be considered in water quality assessment.

In vitro chronic effects on hERG channel caused by the marine biotoxin azaspiracid-2.

Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellate Azadinium spinosum that accumulate in many shellfish species. Azaspiracid poisoning caused by AZA-contaminated seafood consumption is primarily manifested by diarrhea in humans. To protect human health, AZA-1, AZA-2 and AZA-3 content in seafood has been regulated by food safety authorities in many countries. Recently AZAs have been reported as a low/moderate hERG channel blockers. Furthermore AZA-2 has been related to arrhythmia appearance in rats, suggesting potential heart toxicity. In this study AZA-2 in vitro effects on hERG channel after chronic exposure are analyzed to further explore potential cardiotoxicity. The amount of hERG channel in the plasma membrane, hERG channel trafficking and hERG currents were evaluated up to 12 h of toxin exposure. In these conditions AZA-2 caused an increase of hERG levels in the plasma membrane, probably related to hERG retrograde trafficking impairment. Although this alteration did not translate into an increase of hERG channel-related current, more studies will be necessary to understand its mechanism and to know what consequences could have in vivo. These findings suggest that azaspiracids might have chronic cardiotoxicity related to hERG channel trafficking and they should not be overlooked when evaluating the threat to human health.

Evaluation of the intestinal permeability and cytotoxic effects of cylindrospermopsin.

Cylindrospermopsin is a freshwater and widespread cyanotoxin considered hazardous for human health. Climate change and eutrophication are the main factors influencing the increasing presence of cylindrospermopsin producers that can contaminate human and animal drinking waters, leading to a rise in ecological and human risk. In order to reach the bloodstream and thus the target receptor, an orally administered drug must first cross the intestinal barrier. The goal of this study was to examine the cylindrospermopsin intestinal permeability and its cellular effects on intestinal and hepatic cells. We explored the human intestinal permeability of cylindrospermopsin by performing in vitro permeation studies across the Caco-2 cell monolayer. Cell permeability data indicated a limited passage of the toxin through the intact intestinal epithelium in a time and concentration dependent way. Cylindrospermopsin induced neither damage on the integrity of the monolayer nor cytotoxicity in tests performed with Caco-2 even at micromolar concentration. Opposite, when hepatic Clone 9 cells were exposed to cylindrospermopsin, a noticeable cytotoxicity was observed being more marked at the higher concentrations used. In addition, this cell line showed alterations in reduced glutathione content due to cylindrospermopsin over time. Meanwhile glutamate cysteine ligase levels, the first rate-limiting enzyme of the glutathione route, showed a significant increase. Therefore our results indicate that cylindrospermopsin cytotoxicity is unrelated to protein inhibition or a decrease of reduced glutathione levels in Clone 9 cells.

In vivo arrhythmogenicity of the marine biotoxin azaspiracid-2 in rats.

Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellate Azadinium spinosum that accumulate in several shellfish species. Azaspiracid poisoning episodes have been described in humans due to ingestion of AZA-contaminated seafood. Therefore, the contents of AZA-1, AZA-2 and AZA-3, the best-known analogs of the group, in shellfish destined to human consumption have been regulated by food safety authorities of many countries to protect human health. In vivo and in vitro toxicological studies have described effects of AZAs at different cellular levels and on several organs, however, AZA target remains unknown. Very recently, AZAs have been demonstrated to block the hERG cardiac potassium channel. In this study, we explored the potential cardiotoxicity of AZA-2 in vivo. The effects of AZA-2 on rat electrocardiogram (ECG) and cardiac biomarkers were evaluated for cardiotoxicity signs besides corroborating the hERG-blocking activity of AZA-2. Our results demonstrated that AZA-2 does not induce QT interval prolongation on rat ECGs in vivo, in spite of being an in vitro blocker of the hERG cardiac potassium channel. However, AZA-2 alters the heart electrical activity causing prolongation of PR intervals and the appearance of arrhythmias. More studies will be needed to clarify the mechanism by which AZA-2 causes these ECG alterations; however, the potential cardiotoxicity of AZAs demonstrated in this in vivo study should be taken into consideration when evaluating the possible threat that these toxins pose to human health, mainly for individuals with pre-existing cardiovascular disease when regulated toxin limits are exceeded.

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.

Human muscarinic acetylcholine receptors are a target of the marine toxin 13-desmethyl C spirolide.

Spirolides are a group of cyclic imine marine toxins recently described. Although no human intoxication has been related to their presence in shellfish yet, the possible toxicological consequences to human health are actually unknown. The elucidation of the spirolide mechanism/s of action would help to estimate the threat to human consumers. Previous toxicological studies in mice suggested the involvement of acetylcholine receptors. In this work, the effects of the 13-desmethyl C spirolide on the activity and the expression of muscarinic acetylcholine receptors (mAChR) were analyzed using a human neuroblastoma cell model. The 13-desmethyl C spirolide inhibited the acetylcholine-induced calcium signal with a reduction of the maximum response to acetylcholine in the presence of the toxin. The 13-desmethyl C spirolide also reduced binding of the mAChR specific antagonist [(3)H]QNB to neuroblastoma cells. The effect of the 13-desmethyl C spirolide persisted after toxin removal and was inhibited by protection of the primary binding site with high concentrations of atropine suggesting an interaction of the spirolide with the orthologous binding site of mAChR. Moreover, the toxin induced a change in the characteristics of the membrane-associated M3 mAChRs, although it did not alter the total levels of M3 mAChR protein. The 13-desmethyl C spirolide targets mAChRs causing a reduction of function, a decrease of specific antagonist binding to mAChRs, and alteration of membrane-bound receptors that might have important toxicological implications.

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.

Ostreocin-D impact on globular actin of intact cells.

Ostreocin-D, discovered in the past decade, is a marine toxin produced by dinoflagellates. It shares structure with palytoxin, a toxic compound responsible for the seafood intoxication named clupeotoxism. At the cellular level, the action sites and pharmacological effects for ostreocin-D are still almost unknown. Previously, we demonstrated that these toxins change the filamentous actin cytoskeleton, which is essential for multiple cellular functions. However, nothing has yet been reported about what happens with the unpolymerized actin pool. Here (i) the effects induced by ostreocin-D on unpolymerized actin, (ii) the Ca2+ role in such a process, and (iii) the cytotoxic activity of ostreocin-D on the human neuroblastoma BE(2)-M17 cell line are shown for the first time. Fluorescently labeled DNase I was used for staining of monomeric actin prior to detection with both laser-scanning cytometry and confocal microscopy techniques. Cellular viability was tested through a microplate metabolic activity assay. Ostreocin-D elicited a rearrangement of monomeric actin toward the nuclear region. This event was not accompanied by changes in its content. In addition, the presence or absence of external Ca2+ did not change these results. This toxin was also found to cause a decrease in the viability of neuroblastoma cells, which was inhibited by the specific blocker of Na+/K+-ATPase, ouabain. All these responses were comparable to those obtained with palytoxin under identical conditions. The data suggest that ostreocin-D modulates the unassembled actin pool, activating signal transduction pathways not related to Ca2+ influx in the same way as palytoxin.

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.

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 Ca(2+) 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 Ca(2+) 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.

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.