A cytolytic assay for the measurement of palytoxin based on a cultured monolayer cell line

A cytolytic assay that could detect palytoxin and its congeners has been developed by the use of an established cell line grown as monolayer to replace the current hemolytic method. We used MCF-7 cells and cytolysis was measured by the release of cytosolic lactate dehydrogenase (LDH) in the buffer added to treated cells (culture supernatant). A dose-dependent increase in LDH activity in culture supernatants was detected when MCF-7 cells were exposed to palytoxin and its analogue ostreocin D. The cytolytic response induced by palytoxin and ostreocin D was specific for this group of compounds, acting on Na+/K+-ATPase, as it was prevented when cells were preincubated with ouabain. The specificity of our assay for palytoxin and its congeners was confirmed by the finding that cytolysis was not detected when MCF-7 cells were exposed to unrelated toxins such as maitotoxin, tetrodotoxin, okadaic acid, and yessotoxin, even in the case of compounds that elicit cytotoxic responses under our experimental conditions. Using extracts from biological materials after spiking with the palytoxin standard, we found a good correlation between palytoxin levels measured by our cytolytic assay and the expected values. Our cytolytic assay detected palytoxin in naturally contaminated materials, but estimates were significantly higher than the palytoxin contents determined by LC-MS, indicating that naturally contaminated materials contain biologically active palytoxin congeners. We conclude that our cytolytic assay based on the use of MCF-7 cell monolayers is a viable alternative to animal-based methods for the determination of palytoxin and its congeners in contaminated materials.     

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.     

Functional significance of phylogeographic structure in a toxic benthic marine microbial eukaryote over a latitudinal gradient along the East Australian Current

Genetic diversity in marine microbial eukaryotic populations (protists) drives their ecological success by enabling diverse phenotypes to respond rapidly to changing environmental conditions. Despite enormous population sizes and lack of barriers to gene flow, genetic differentiation that is associated with geographic distance, currents, and environmental gradients has been reported from planktonic protists. However, for benthic protists, which have reduced dispersal opportunities, phylogeography and its phenotypic significance are little known. In recent years, the East Australian Current (EAC) has intensified its southward flow, associated with the tropicalization of temperate waters. Benthic harmful algal species have been increasingly found in south‐eastern Australia. Yet little is known about the potential of these species to adapt or extend their range in relation to changing conditions. Here, we examine genetic diversity and functional niche divergence in a toxic benthic dinoflagellate, Ostreopsis cf. siamensis, along a 1,500 km north–south gradient in southeastern Australia. Sixty‐eight strains were established from eight sampling sites. The study revealed long‐standing genetic diversity among strains established from the northern‐most sites, along with large phenotypic variation in observed physiological traits such as growth rates, cell volume, production of palytoxin‐like compounds, and photophysiological parameters. Strains from the southern populations were more uniform in both genetic and functional traits, and have possibly colonized their habitats more recently. Our study reports significant genetic and functional trait variability in a benthic harmful algal species, indicative of high adaptability, and a possible climate‐driven range extension. The observed high trait variation may facilitate development of harmful algal blooms under dynamic coastal environmental conditions.     

Palytoxin found in Palythoa sp. zoanthids (Anthozoa, Hexacorallia) sold in the home aquarium trade

Zoanthids (Anthozoa, Hexacorallia) are colonial anemones that contain one of the deadliest toxins ever discovered, palytoxin (LD₅₀ in mice 300 ng/kg), but it is generally believed that highly toxic species are not sold in the home aquarium trade. We previously showed that an unintentionally introduced zoanthid in a home aquarium contained high concentrations of palytoxin and was likely responsible for a severe respiratory reaction when an individual attempted to eliminate the contaminant colonies using boiling water. To assess the availability and potential exposure of palytoxin to marine aquarium hobbyists, we analyzed zoanthid samples collected from local aquarium stores for palytoxin using liquid chromatography and high resolution mass spectrometry and attempted to identify the specimens through genetic analysis of 16S and cytochrome c oxidase 1 (COI) markers. We found four specimens of the same apparent species of zoanthid, that we described previously to be responsible for a severe respiratory reaction in a home aquarium, to be available in three aquarium stores in the Washington D.C. area. We found all of these specimens (n = 4) to be highly toxic with palytoxin or palytoxin-like compounds (range 0.5–3.5 mg crude toxin/g zoanthid). One of the most potent non-protein compounds ever discovered is present in dangerous quantities in a select species of zoanthid commonly sold in the home aquarium trade.     

Pyranocoumarins from the twigs of Mammea siamensis

Four unusual pyranocoumarins (1-4) have been isolated from the dried twigs of M. siamensis. The structures were determined by spectroscopic data, especially 1D and 2D NMR experiments.     

Microelectrode array (MEA) platform as a sensitive tool to detect and evaluate Ostreopsis cf. ovata toxicity

In the last decade, the occurrence of harmful dinoflagellate blooms of the genus Ostreopsis has increased both in frequency and in geographic distribution with adverse impacts on public health and the economy. Ostreopsis species are producers of palytoxin-like toxins (putative palytoxin and ovatoxins) which are among the most potent natural non-protein compounds known to date, exhibiting extreme toxicity in mammals, including humans. Most existing toxicological data are derived from in vivo mouse assay and are related to acute effects of pure palytoxin, without considering that the toxicity mechanism of dinoflagellates can be dependent on the varying composition of complex biotoxins mixture and on the presence of cellular components.In this study, in vitro neuronal networks coupled to microelectrode array (MEA)-based system are proposed, for the first time, as sensitive biosensors for the evaluation of marine alga toxicity on mammalian cells. Toxic effect was investigated by testing three different treatments of laboratory cultured Ostreopsis cf. ovata cells: filtered and re-suspended algal cells; filtered, re-suspended and sonicated algal cells; conditioned growth medium devoid of algal cells. The great sensitivity of this system revealed the mixture of PTLX-complex analogues naturally released in the growth medium and the different potency of the three treatments to inhibit the neuronal network spontaneous electrical activity. Moreover, by means of the multiparametric analysis of neuronal network activity, the approach revealed a different toxicity mechanism of the cellular component compared to the algal conditioned growth medium, highlighting the potential active role of the first treatment.     

Palytoxin down-modulates the epidermal growth factor receptor through a sodium-dependent pathway

Palytoxin, a non-12-O-tetradecanoylphorbol-13-acetate type tumor promoter, has been shown to inhibit epidermal growth factor (EGF) binding to both high and low affinity receptors through a protein kinase C-independent pathway. In the present paper, we have investigated the mechanism of palytoxin action in Swiss 3T3 cells. Two lines of evidence indicate that calcium is not required for palytoxin activity. First, palytoxin can induce the loss of EGF binding sites in the absence of external calcium. Second, studies with the photosensitive protein aequorin indicate that palytoxin does not cause the influx of external calcium or the release of calcium from internal stores under the conditions used in these studies. However, palytoxin action does appear to be dependent upon the presence of sodium. When extracellular sodium is replaced by either choline, Tris, or sucrose, palytoxin is unable to decrease EGF binding to either high or low affinity receptors. Studies of sodium influx indicate that palytoxin induces rapid sodium uptake and that the rate of sodium uptake is dose-dependent. Furthermore, there appears to be a direct correspondence between the extent of inhibition of EGF binding by palytoxin and the rate of sodium uptake. Finally, the palytoxin-induced inhibition of EGF binding can be mimicked by monensin, a sodium ionophore. The specificity of this sodium dependence was tested by substituting lithium, potassium, or cesium for sodium. Although lithium is an effective substitute for sodium, palytoxin can no longer inhibit EGF binding when sodium is replaced by either potassium or cesium. Marked inhibition of palytoxin action is also obtained when 5.4 mM potassium or 5.4 mM cesium are added to the sodium-containing medium. These studies suggest that palytoxin is able to down-modulate the EGF receptor through a novel mechanism involving the activation or formation of a sodium pump or channel.     

Palytoxin acidifies chick cardiac cells and activates the Na+/H+ antiporter

The cardiotoxic action of palytoxin was investigated using embryonic chick ventricular cells. Under normal ionic conditions, palytoxin produced an intracellular acidification which is partially compensated for by the Na+/H+ antiporter thereby leading to an increased rate of ethylisopropylamiloride-sensitive 22Na+ uptake. Under depolarizing membrane conditions, palytoxin produced a cellular acidification, a cellular alkalinization or no change in intracellular pH depending on the value of the extracellular pH. We propose that palytoxin acidifies cardiac cells by opening preexisting H+ conducting pathways in the plasma membrane.     

Changes in erythrocyte permeability due to palytoxin as compared to amphotericin B

Palytoxin causes within minutes a temperature-dependent K+ loss from human and rat erythrocytes which is followed within hours by haemolysis. It decreases the osmotic resistance in a concentration-dependent manner, so that osmotic influences are negligible for K+ release but considerable in haemolysis. External K+ inhibits the haemoglobin release and Rb+ inhibits the release of K+ and haemoglobin. Ca2+ (over 20 microM) and borate (over 5 microM) enhance the loss of K+ and haemoglobin. With both Ca2+ and borate present, the efficacy of palytoxin is raised about 10 000-fold. Under these conditions, about 15 palytoxin molecules per human cell trigger a 50% K+ loss over a wide range of cell concentrations. The palytoxin effect is reversible. After depletion from K+ by low concentrations of palytoxin, human cells can be refilled with K+ and resealed. The pores formed by palytoxin are small. They allow the entrance of Na+ and choline, whereas inositol is largely excluded and Ca2+, as well as sucrose and inulin, are completely excluded. Amphotericin B resembles palytoxin in its ability to cause a considerable prelytic K+ loss and to form small pores. However, it is about 1000-times weaker than palytoxin, is not inhibited by K+ or Rb+, is not activated by Ca2+ or borate, and has a negative temperature dependence. Thus palytoxin represents a novel type of cytolysin.     

Identification of purebred Crocodylus siamensis for reintroduction in Vietnam

Crocodylus siamensis, the Siamese crocodile, is a critically endangered species of freshwater crocodile previously distributed throughout much of SE Asia. Recovery plans call for reintroductions to the wild using founder individuals currently in captivity, mostly in commercial crocodile farms. On many farms C. siamensis has been intentionally hybridised with either Cuban crocodiles, C. rhombifer, or the estuarine crocodile, C. porosus, and hybrids may be difficult to distinguish morphologically. We report on the combined use of microsatellite and mtDNA genetic markers to determine the species status of potential founder individuals for reintroduction of C. siamensis. Genetic markers were used to characterise 103 captive and wild-caught individuals of C. siamensis, C. rhombifer and C. porosus in Vietnam and to distinguish purebred versus hybrid individuals. Although the microsatellite loci used had some overlap of allele sizes among species, assignment tests allowed differentiation. Four hybrids were identified, two of which had not been recognised morphologically as hybrids, and one of these was thought to be a C. siamensis suitable for reintroduction. Ten of the identified purebred C. siamensis were subsequently released into Cat Tien National Park in southern Vietnam.     

Mitogen-activated protein kinase phosphatase-3 is a tumor promoter target in initiated cells that express oncogenic Ras

We have capitalized on the unique properties of the skin tumor promoter palytoxin, which does not activate protein kinase C, to investigate alternative mechanisms by which major signaling molecules can be modulated during carcinogenesis. We report here that palytoxin activates extracellular signal-regulated kinase (ERK) through a novel mechanism that involves inactivation of an ERK phosphatase in keratinocytes derived from initiated mouse skin (308 cells). Use of U0126 revealed that palytoxin requires the ERK kinase MEK to stimulate ERK activity, although palytoxin did not activate MEK. We found that 308 keratinocytes highly express mitogen-activated protein kinase phosphatase-3 (MKP-3), which selectively inactivates ERK. Palytoxin induced the loss of MKP-3 in a manner that corresponded to increased ERK phosphorylation. Complementary studies showed that sustained expression of exogenous MKP-3 inhibited palytoxin-stimulated ERK activation. As is characteristic of initiated keratinocytes, 308 cells express activated H-Ras. To investigate whether expression of oncogenic Ras is key to palytoxin-stimulated ERK activation, we determined how palytoxin affected ERK and MKP-3 in MCF10A human breast epithelial cells and in H-ras MCF10A cells, which stably express activated H-Ras. Palytoxin did not affect ERK activity in MCF10A cells, which had no detectable MKP-3. Like 308 cells, H-ras MCF10A cells highly express MKP-3. Strikingly, palytoxin stimulated ERK activity and induced a corresponding loss of MKP-3 in H-ras MCF10A cells. These studies indicate that in initiated cells palytoxin unleashes ERK activity by down-regulating MKP-3, an ERK inhibitor, and further suggest that MKP-3 may be a vulnerable target in cells that express oncogenic Ras.     

Sodium as a mediator of non-phorbol tumor promoter action. Down-modulation of the epidermal growth factor receptor by palytoxin

Previous studies have shown that palytoxin, a non-(12-O-tetradecanoylphorbol-13-acetate)-type tumor promoter, is able to down-modulate the epidermal growth factor (EGF) receptor through a sodium-dependent pathway in Swiss 3T3 cells. A role for sodium is supported by the observation that the sodium proton exchanger monensin and the sodium-conducting ionophore gramicidin mimic palytoxin action by causing a decrease in both high and low affinity EGF binding. However, in addition to causing sodium influx, these agents can induce other cellular effects including changes in membrane polarization, intracellular pH, and macromolecular synthesis. To determine whether any of these factors might be responsible for palytoxin action in our system, we examined the role of each of them in palytoxin-induced inhibition of EGF binding. Although palytoxin depolarizes the membrane, the observation that potassium-induced depolarization of the membrane does not cause a decrease in EGF binding, in conjunction with the fact that monensin hyperpolarizes the membrane, indicates that depolarization of the membrane is not responsible for palytoxin-induced changes in the EGF receptor. An investigation of intra-cellular pH suggests that the palytoxin effects are not mediated by proton flux. In addition, nigericin-mediated changes in intracellular pH do not cause an inhibition of EGF binding. Finally, studies conducted in the presence of cycloheximide indicate that protein synthesis is not required for palytoxin action and that inhibition of EGF receptor biosynthesis does not account for palytoxin-induced loss of EGF-binding sites. These results suggest that sodium may act as a second messenger in the signal transduction mechanism by which palytoxin modulates the EGF receptor.     

FIRST EVIDENCE OF PALYTOXIN ANALOGUES FROM AN OSTREOPSIS MASCARENENSIS (DINOPHYCEAE) BENTHIC BLOOM IN SOUTHWESTERN INDIAN OCEAN1

Benthic dinoflagellates of the genus Ostreopsis Schmidt are common in tropical and subtropical water, and some species produce toxins potentially involved in human intoxication events. A benthic bloom of Ostreopsis mascarenensis Quod was observed near Rodrigues Island during a survey of benthic dinoflagellates in the southwestern Indian Ocean. The morphology of O. mascarenensis was studied by LM and SEM. Preliminary screening of a crude extract of an O. mascarenensis bloom revealed neurotoxicity in mice similar to that induced by palytoxin. After partition of the crude extract, the highest toxicity was retained in the butanol‐soluble fraction, which retained hemolytic activity suggestive of palytoxin analogues. Two new toxins, mascarenotoxin‐A and ‐B, were resolved from this fraction by HPLC coupled to a diode array detector. The closed mass spectrum profile and fragmentation pattern obtained by advanced nano–electrospray ionization quadrupole time‐of‐flight mass spectrometry between purified toxins and a reference palytoxin confirmed the mascarenotoxins as palytoxin analogues. These results were confirmed by tandem mass spectrometry with the identification of specific fragment ion m/z 327. An on‐line liquid chromatography protocol coupled to tandem mass spectrometry was developed for detection of these palytoxin analogues. The present study describes the first purification, chemical, and toxicological characterization of new palytoxin analogues isolated from a benthic bloom of O. mascarenensis. These results suggest that O. mascarenensis, which is largely distributed in the southwestern Indian Ocean, could be a source of palytoxin poisoning in this tropical area.     

Durinskia baltica (Dinophyceae), a newly recorded species and genus from China, and its systematics

A freshwater dinoflagellate was identified as Durinskia baltica (Levander) Carty & Cox by morphological characteristics,with the plate formula:Po,x,4',2a,6',5c,4s,5',2'.Durinskia was a newly recorded dinoflagellate genus for China with two anterior intercalary plates and six characteristic precingular plates.Partial sequences of the small and large subunit ribosomal DNA and internal transcribed spacer sequences for the dinoflagellate cells were obtained from field samples.Molecular phylogenetic results indicated Durinskia species could cluster into a monophyletic group,which were distinct from Peridinium species.According to morphological and molecular evidence,it was agreed that the genus Durinskia was separated from the genus Peridinium,which could be a polyphyletic group.In addition,D.baltica was an infrequent diatom-harboring dinoflagellate which was known to possess an endosymbiotic diatom or diatom-like alga.The phylogenetic analyses indicated that D.baltica had a close affinity with Peridiniopsis penardii and P.niei,common freshwater bloom-forming species in China.     

Large diameter of palytoxin-induced Na/K pump channels and modulation of palytoxin interaction by Na/K pump ligands

Palytoxin binds to Na/K pumps to generate nonselective cation channels whose pore likely comprises at least part of the pump's ion translocation pathway. We systematically analyzed palytoxin's interactions with native human Na/K pumps in outside-out patches from HEK293 cells over a broad range of ionic and nucleotide conditions, and with or without cardiotonic steroids. With 5 mM internal (pipette) [MgATP], palytoxin activated the conductance with an apparent affinity that was highest for Na(+)-containing (K(+)-free) external and internal solutions, lowest for K(+)-containing (Na(+)-free) external and internal solutions, and intermediate for the mixed external Na(+)/internal K(+), and external K(+)/internal Na(+) conditions; with Na(+) solutions and MgATP, the mean dwell time of palytoxin on the Na/K pump was about one day. With Na(+) solutions, the apparent affinity for palytoxin action was low after equilibration of patches with nucleotide-free pipette solution. That apparent affinity was increased in two phases as the equilibrating [MgATP] was raised over the submicromolar, and submillimolar, ranges, but was increased by pipette MgAMPPNP in a single phase, over the submillimolar range; the apparent affinity at saturating [MgAMPPNP] remained approximately 30-fold lower than at saturating [MgATP]. After palytoxin washout, the conductance decay that reflects palytoxin unbinding was accelerated by cardiotonic steroid. When Na/K pumps were preincubated with cardiotonic steroid, subsequent activation of palytoxin-induced conductance was greatly slowed, even after washout of the cardiotonic steroid, but activation could still be accelerated by increasing palytoxin concentration. These results indicate that palytoxin and a cardiotonic steroid can simultaneously occupy the same Na/K pump, each destabilizing the other. The palytoxin-induced channels were permeable to several large organic cations, including N-methyl-d-glucamine(+), suggesting that the narrowest section of the pore must be approximately 7.5 A wide. Enhanced understanding of palytoxin action now allows its use for examining the structures and mechanisms of the gates that occlude/deocclude transported ions during the normal Na/K pump cycle.     

An enzyme linked immunosorbent assay for detecting palytoxin-producing bacteria

A competitive ELISA using the intact toxin as a coating antigen for detecting palytoxin was developed. This immunoassay allows palytoxin (PTX) to be determined in the range of 6–250 ng/ml. In sensitivity, this determination is comparable with RIA but is three times inferior to ELISA using monoclonal antibodies. Inhibition experiments using some toxins of marine invertebrates proved the serological specificity of the palytoxin binding to antibodies. Both the indirect and competitive ELISA were used to find PTX-producing bacteria among 420 isolates of sea bacteria. It was found that gram-negative bacteriaAeromonas sp. andVibrio sp. associated with toxic samples of the soft coralPalythoa sp. produced compounds antigenically related to PTX.     

Stimulation of superoxide anion formation by the non-TPA type tumor promoters palytoxin and thapsigargin in porcine and human neutrophils

The non-12-O-tetradecanoylphorbol-13-acetate (TPA) type tumor promoters palytoxin and thapsigargin provoked a respiratory burst in porcine and human neutrophils. The amounts of oxygen consumed and superoxide anion (O2-) produced were found to be stoichiometric. Concentrations of 6.5 X 10(-8) M palytoxin and 1.2 X 10(-6) M thapsigargin were required for half-maximal stimulation to 3 X 10(6) porcine cells/ml in Hanks' solution. Combinations of palytoxin and thapsigargin, and of one non-TPA type and one TPA-type tumor promoter, had synergistic effects in stimulating O2- formation in neutrophils, suggesting that these compounds activate the cells by different signal transduction mechanisms.     

Palytoxin-induced permeability changes in excitable membranes

Palytoxin, a toxin isolated from the Caribean corrall Palythoa caribaeorum, increases the cation permeability of excitable membranes in vitro. Three membrane systems have been investigated: axonal membranes from crayfish walking leg nerves, membranes rich in nicotinic acetylcholine receptor isolated from Torpedo californica electric tissue and, for control, artificial liposomes. Ion permeability of the latter was not affected by palytoxin, but with both biological membranes an increase in cation permeability was observed at a palytoxin concentration of 0.14 microM. Palytoxin-induced cation flow through the axonal membrane was not inhibited by tetrodotoxin, indicating that the voltage-dependent sodium channels were not involved. The effect of palytoxin on the receptor-rich membranes was not blocked by alpha-bungarotoxin, a competitive antagonist of the nicotinic acetylcholine receptor, nor by triphenylmethylphosphonium, a blocker of the receptor-ion channel. But with both the axonal and the receptor-rich membranes ouabain was an inhibitor of the palytoxin-induced cation flow. Evidence is presented that it is not the (Na+ + K+)-ATPase which is affected by palytoxin as has been postulated for similar observations with non-neuronal membranes (Chhatwal, G.S., Hessler, H.-J. and Habermann, E. (1983) Naunyn-Schmiedeberg's Arch. Pharmacol. 323, 261-268).     

Cysteine-scanning mutagenesis study of the sixth transmembrane segment of the Na,K-ATPase alpha subunit

The accessibility of the residues of the sixth transmembrane segment (TM) of the Bufo marinus Na,K-ATPase alpha subunit was explored by cysteine scanning mutagenesis. Methanethiosulfonate reagents reached only the two most extracellular positions (T803, D804) in the native conformation of the Na,K-pump. Palytoxin induced a conductance in all mutants, including D811C, T814C and D815C which showed no active electrogenic transport. After palytoxin treatment, four additional positions (V805, L808, D811 and M816) became accessible to the sulfhydryl reagent. We conclude that one side of the sixth TM helix forms a wall of the palytoxin-induced channel pore and, probably, of the cation pathway from the extracellular side to one of their binding sites.     

Immunoenzyme method for detecting microbial producers of palytoxin

A competitive ELISA using the intact toxin as a coating antigen for detecting palytoxin was developed. This immunoassay allows palytoxin (PTX) to be determined in the range of 6-250 ng/ml. In sensitivity, this determination is comparable with RIA but is three times inferior to ELISA using monoclonal antibodies. Inhibition experiments using some toxins of marine invertebrates proved the serological specificity of the palytoxin binding to antibodies. Both the indirect and competitive ELISA were used to find PTX-producing bacteria among 420 isolates of sea bacteria. It was found that gram-negative bacteria Aeromonas sp. and Vibrio sp. associated with toxic samples of the soft coral Palythoa sp. produced compounds antigenically related to PTX.