Isabel R. Ares

Actin cytoskeleton of rabbit intestinal cells is a target for potent marine phycotoxins.

SUMMARY Biotoxins produced by harmful marine microalgae (phycotoxins) can be accumulated into seafood, representing a great risk for public health. Some of these phycotoxins are responsible for a variety of gastrointestinal disturbances; however, the relationship between their mechanism of action and toxicity in intestinal cells is still unknown. The actin cytoskeleton is an important and highly complicated structure in intestinal cells, and on that basis our aim has been to investigate the effect of representative phycotoxins on the enterocyte cytoskeleton. We have quantified for the first time the loss of enterocyte microfilament network induced by each toxin and recorded fluorescence images using a laser-scanning cytometer and confocal microscopy. Our data show that pectenotoxin-6, maitotoxin, palytoxin and ostreocin-D cause a significant reduction in the actin cytoskeleton. In addition, we found that the potency of maitotoxin, palytoxin and ostreocin-D to damage filamentous actin is related to Ca2+ influx in enterocytes. Those results identify the cytoskeleton as an early target for the toxic effect of those toxins.

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.

Lactone Ring of Pectenotoxins: a Key Factor for their Activity on Cytoskeletal Dynamics

Background: Pectenotoxins are a group of natural products from marine origin that can accumulate in shellfish and intoxicate humans. Recently, novel homologues such as pectenotoxin-11 (PTX-11) and pectenotoxin-2 seco acid (PTX-2SA) have been identified. Their toxic potential towards experimental animals has been evaluated however their interaction with cellular systems is almost unknown. This is the first report showing (i) the biological activity of PTX-11 and PTX-2SA on actin cytoskeleton and morphology of living cells and (ii) the structure- activity relationship for this family of toxic compounds. Methods: Fluorescent phalloidin was utilized to quantify and visualize any modification in polymerized actin. Fluorescence values were obtained with laser-scanning cytometer and cells were imaged through confocal microscopy. For structure-activity evaluations, pectenotoxin-1 (PTX-1) and pectenotoxin-2 (PTX-2) was also analyzed. Results: Data showed that PTX-11 triggered a remarkable depolymerizing effect on actin cytoskeleton and also modifications in the shape of cells. In contrast, PTX-2SA did not evidence the same effects. Conclusion: Our findings point out that (i) the actin cytoskeleton is a common target for PTX-11, PTX-2 and PTX-1, but not for PTX-2SA, and (ii) this difference in activity is related to the presence or absence of an intact lactone ring in their structures.

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.

The cytoskeleton, a structure that is susceptible to the toxic mechanism activated by palytoxins in human excitable cells

Palytoxin is a marine toxin responsible for a fatal type of poisoning in humans named clupeotoxism, with symptoms such as neurologic disturbances. It is believed that it binds to the Na+/K+‐ATPase from the extracellular side and modifies cytosolic ions; nevertheless, its effects on internal cell structures, such as the cytoskeleton, which might be affected by these initial events, have not been fully elucidated. Likewise, ostreocin‐D, an analog of palytoxin, has been only recently found, and its action on excitable cells is therefore unknown. Therefore, our aim was to investigate the modifications of ion fluxes associated with palytoxin and ostreocin‐D activities, and their effects on an essential cytoskeletal component, the actin system. We used human neuroblastoma cells and fluorescent dyes to detect changes in membrane potential, intracellular Ca2+ concentration, cell detachment, and actin filaments. Fluorescence values were obtained with spectrofluorymetry, laser‐scanning cytometry, and confocal microscopy; the last of these was also used for recording images. Palytoxin and ostreocin‐D modified membrane permeability as a first step, triggering depolarization and increasing Ca2+ influx. The substantial loss of filamentous actin, and the morphologic alterations elicited by both toxins, are possibly secondary to their action on ion channels. The decrease in polymerized actin seemed to be Ca2+‐independent; however, this ion could be related to actin cytoskeletal organization. Palytoxin and ostreocin‐D alter the ion fluxes, targeting pathways that involve the cytoskeletal dynamics of human excitable cells.

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.

Impact of the pectenotoxin C-43 oxidation degree on its cytotoxic effect on rat hepatocytes.

The metabolism of toxins that have accumulated in fish and shellfish is considered a detoxification process, as happens with pectenotoxins (PTXs) in the Japanese scallop Patinopecten yessoensis. PTXs are macrolactones that display hepatotoxicity in mice, principally by capping or sequestering actin, their molecular target. PTX-2, which is considered to be the parental compound, oxidizes progressively to PTX-1, PTX-3, and PTX-6 in the Japanese scallop. In this study, we observed that PTX-1, PTX-6, and PTX-9 induce dose-dependent damage in the actin cytoskeleton and in the viability of primary cultured rat hepatocytes. In Clone 9 rat hepatocytes, PTX-1 and PTX-9 also affect the morphology of cells, but surprisingly, PTX-6 induced no effect. In accordance with this lack of activity, the actin cytoskeleton of CaCo-2 cells, another epithelial cell line, is not affected by PTX-6. In conclusion, the order of cytotoxicity of the analogues is PTX-2 > PTX-1 > PTX-6 >PTX-9. From a structure-activity perspective, the increase in the level of oxidation of the PTX molecule on C-43 decreases its cytotoxicity. Furthermore, PTX-6 is not able to induce effects on immortal cells while retaining its toxicity against primary cultured cells, whereas PTX-9, a 7-S-isomer, is active in both cellular models. The different cytotoxicities exerted by PTX-6 on cell lines and primary cells could be determined by the presence of a carboxylic acid group on C43 of the PTX molecule.

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.

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.