palytoxin and Hemolysis

Palytoxin (PLTX) and its analogues have been detected as seafood contaminants associated with a series of human foodborne poisonings. Due to a number of fatalities ascribed to the ingestion of PLTX-contaminated marine organisms, the development of methods for its detection in seafood has been recommended by the European Food Safety Authority (EFSA). Due to its feasibility, the spectrophotometric hemolytic assay is widely used to detect PLTX in different matrices, even though a standardized protocol is still lacking. Thus, on the basis of available assay procedures, a new standardized protocol was set up using purified human erythrocytes exposed to PLTX (working range: 3.9 × 10(-10)-2.5 × 10(-8) M) in a K(+)-free phosphate buffered saline solution, employing a 5 h incubation at 41 °C. An intra-laboratory characterization demonstrated its sensitivity (limit of detection, LOD = 1.4 × 10(-10) M and quantitation, LOQ = 3.4 × 10(-10) M), accuracy (bias = -0.8%), repeatability (RSDr = 15% and 6% for intra- and inter-day repeatability, respectively) and specificity. However, the standardized method seems not to be suitable for PLTX quantitation in complex matrices, such as mussel (Mytilus galloprovincialis) extracts, at least below the limit suggested by EFSA (30 μg PLTXs/Kg shellfish meat). Thus, the hemolytic assay for PLTX quantitation in seafood should be used only after a careful evaluation of the specific matrix effects.

Topics: Acrylamides; Animals; Bivalvia; Cnidarian Venoms; Cross Reactions; Erythrocytes; Hemolysis; Humans; Limit of Detection; Reproducibility of Results

An electrochemical sensor for palytoxin (PlTX) detection, based on a strip of eight screen-printed electrodes connected to a cost-effective and portable apparatus, is reported. Sheep erythrocytes were used to test the palytoxin detector and degree of haemolysis was evaluated by measuring release of the cytosolic lactate dehydrogenase (LDH). Percentage haemolysis and, therefore, the amount of LDH measured, by use of NADH/pyruvate and appropriate electrochemical mediators, was correlated with the concentration of the toxin. Two different electrochemical approaches were investigated for evaluation of LDH release, but only one based on the use of a binary redox mediator sequence (phenazine methosulfate in conjugation with hexacyanoferrate(III)) proved useful for our purpose. After analytical and biochemical characterization, the sensor strip was used to measure palytoxin. Sheep blood and standard solutions of PlTX were left to react for two different incubation times (24 h or 4 h), resulting in working ranges of 7 × 10(-3)-0.02 ng mL(-1) and 0.16-1.3 ng mL(-1), respectively. The specificity of the test for palytoxin was evaluated by use of ouabain, which acts in the same way as PlTX on the Na(+)/K(+)-ATPase pump. A cross-reactivity study, using high concentrations of other marine biotoxins was also conducted. Experiments to evaluate the matrix effect and recovery from mussels are discussed.

Topics: Acrylamides; Animals; Biosensing Techniques; Bivalvia; Cnidarian Venoms; Enzyme Assays; Erythrocytes; Hemolysis; L-Lactate Dehydrogenase; Marine Toxins; Sheep; Shellfish

Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 μg·kg-1 of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 μg·kg-1 in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell-1. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises.

Topics: Acrylamides; Animals; Anthozoa; Cnidarian Venoms; Dinoflagellida; France; Hemolysis; In Vitro Techniques; Marine Toxins; Mediterranean Sea; Seafood; Seawater; Sheep

In the Mediterranean Sea, blooms of Ostreopsis cf. ovata and Ostreopsis siamensis have become increasingly frequent in the last decade and O. cf. ovata was found to produce palytoxin-like compounds (putative palytoxin, ovatoxin-a, -b, -c, -d and -e), a class of highly potent toxins. The environmental conditions seem to play a key role in influencing the abundance of Ostreopsis spp. High cell densities are generally recorded in concomitance with relatively high temperature and salinity and low hydrodynamics conditions. In this study the effects of temperature and salinity on the growth and toxicity of an Adriatic O. cf. ovata isolate were investigated. The highest growth rates of the Adriatic strain were recorded for cultures grown at 20 °C and at salinity values of 36 and 40, in accordance with natural bloom surveys. Toxicity was affected by growth conditions, with the highest toxin content on a per cell basis being measured at 25 °C and salinity 32. However, the highest total toxin content on a per litre basis was recorded at 20 °C and salinity 36, since under such conditions the growth yield was the highest. O. cf. ovata had lethal effects on Artemia nauplii and juvenile sea basses, and produced haemolysis of sheep erythrocytes. A comparison between haemolysis neutralization assay and HR LC-MS results showed a good correlation between haemolytic effect and total toxin content measured through HR LC-MS. Considering the increasing need for rapid and sensitive methods to detect palytoxin in natural samples, the haemolytic assay appears a useful method for preliminary quantification of the whole of palytoxin-like compounds in algal extracts.

Topics: Acrylamides; Animals; Artemia; Biological Assay; Cell Size; Chromatography, Liquid; Cnidarian Venoms; Dinoflagellida; Erythrocytes; Fishes; Hemolysis; Marine Toxins; Mass Spectrometry; Salinity; Sheep; Temperature; Toxicity Tests

Palytoxin (PLTX) is a marine polyether toxin with a very large and complex molecule that has both lipophilic and hydrophilic areas. It presents the longest continuous carbon atoms chain known to exist in a natural product second only to maitotoxin. This toxin was first isolated from Palythoa toxica and was subsequently reported in dinoflagellates of the genus Ostreopsis. Although PLTX has so far been associated with ciguateric fish poisoning (CFP), recent evidence suggests that PLTXs should be excluded from CFP toxins. NMR and LC-MS/MS techniques have enabled the isolation of 10-15 new analogues from dinoflagellates ever since their first discovery. Literature data on biological origin, poisonings and chemistry of certain naturally occurring PLTX analogues, commonly known as ostreocins, are detailed herein. This paper reviews all reported biological and chemical analysis methods to date for this group of compounds.

Topics: Acrylamides; Animals; Biological Assay; Chemical Fractionation; Chromatography, High Pressure Liquid; Chromatography, Liquid; Cnidarian Venoms; Dinoflagellida; Electrophoresis, Capillary; Hemolysis; Immunoassay; Marine Toxins; Mass Spectrometry; Mice; Molecular Structure; Toxicity Tests

Palytoxin (PlTX) and palytoxin-like (PlTX-like) compounds in seafood have been raising scientific concern in the last years. The constant increase in record numbers of the causative dinoflagellates of the genus Ostreopsis together with the large spatial expansion of this genus has led to intensification of research towards optimization of methods for determination of PlTX presence and toxicity. In this context, identification of seafood species which could possibly contain PlTXs constitutes an important issue for public health protection. In the present paper, worldwide occurrence of PlTX-like compounds in seafood is reviewed, while potential future strategies are discussed. PlTX has been reported to be present in several species of fish, crustaceans, molluscs and echinoderms. In one occasion, PlTX has been identified in freshwater puffer fish whereas all other records of PlTXs refer to marine species and have been recorded in latitudes approximately between 43°N and 15°S. PlTX determination in seafood has relied on different methodologies (mainly LC-MS, mouse bioassay and hemolysis neutralization assay) that have evolved over time. Future recommendations include systematic screening of PlTX in those species and areas where PlTX has already been recorded implementing updated methodologies.

Topics: Acrylamides; Animals; Biological Assay; Chromatography, Liquid; Cnidarian Venoms; Demography; Dinoflagellida; Fishes; Hemolysis; Invertebrates; Marine Toxins; Mass Spectrometry; Mice; Seafood

A clone of toxic dinoflagellate Ostreopsis sp. and six specimens of a parrotfish Scarus ovifrons were collected in October 1997 at Tokushima Prefecture, Japan. Ostreopsis sp. was cultured in ESM medium for 16 days, and after rearing the cell pellet (about 4.0x10(5) cells) was extracted with 50% methanol, partitioned between an aqueous layer and 1-butanol layer, and biochemically tested. Similarly, the crude toxin from S. ovifrons was extracted, and tested. The mice injected with each 1-butanol layer from Ostreopsis sp. and S. ovifrons showed the common symptoms of convulsion, drowsiness and collapse, and died within 48 h. The lethal potency of Ostreopsis sp. was calculated to be 1.0x10(-4) MU/cell. All specimens of S. ovifrons were found to be toxic, where the highest potency was determined as 2 MU/g in muscle of one specimen. After being injected with toxins, the serum creatine phosphokinase levels of mice were found to be elevated. Toxins from Ostreopsis sp. and S. ovifrons showed delayed haemolytic activity with mouse and human erythrocytes, which was inhibited by an anti-palytoxin (PTX) antibody antibody and ouabain. Toxins from Ostreopsis sp. and S. ovifrons thus resembled each other, and strongly suggested to be PTX or its akin substance. Additionally, a considerable number of adherent Ostreopsis sp. was found in the gut contents of S. ovifrons during the heavy occurrence of Ostreopsis sp. in October 1997 at Tokushima Prefecture. From the above results, it can be strongly postulated that the dinoflagellate Ostreopsis sp. is the origin of PTX which is sequestered by the parrotfish S. ovifrons through food chain.

Topics: Acrylamides; Animals; Cnidarian Venoms; Creatine Kinase; Dinoflagellida; Dose-Response Relationship, Drug; Erythrocytes; Fishes, Poisonous; Food Chain; Foodborne Diseases; Hemolysis; Humans; Japan; Male; Mice; Mice, Inbred Strains; Muscle, Skeletal; Pacific Ocean; Tissue Extracts

Between October 30 and November 4, 2000, eleven persons were intoxicated due to ingestion of a serranid fish Epinephelus sp. in Kochi Prefecture, Japan. Their symptoms were mainly featured by severe muscle pain, low back pain, and discharge of black urine. Serum creatine phosphokinase (CPK) levels of victims were higher (700-23,800 IU/l) than normal values, and their recovery times were more than one month. Immediately after the incident, the leftovers were collected for investigation. The causative agent was identified as palytoxin (PTX) on the basis of delayed haemolytic activity which was inhibited by an anti-PTX antibody and ouabain (g-strophanthin). To our knowledge, this is the first report on palytoxin poisoning with serranid fish.

Topics: Acrylamides; Adolescent; Adult; Animals; Cnidarian Venoms; Female; Fishes, Poisonous; Foodborne Diseases; Hemolysis; Humans; Japan; Male; Mice; Middle Aged; Muscles; Perciformes; Toxicity Tests, Acute

In order to elucidate the toxin composition of the freshwater puffer in Bangladesh, about 230 specimens of Tetraodon sp. were collected from 1997 to 1999 and extracted. After partitioning the toxins between an aqueous layer and a 1-butanol layer, the toxin in the aqueous layer was characterized as paralytic shellfish poison (PSP) (data not shown), while the toxin in the 1-butanol layer was identified as palytoxin (PTX) or PTX-like substance based on the delayed haemolytic activity which was inhibited by an anti-PTX antibody and ouabain (g-strophanthin). This is the first report on the occurrence of PTX or PTX-like substance(s) in puffer fish.

Topics: Acrylamides; Animals; Bangladesh; Chromatography, High Pressure Liquid; Cnidarian Venoms; Enzyme Inhibitors; Fish Venoms; Fishes, Poisonous; Hemolysis; Humans; Mice; Ouabain; Tetrodotoxin

The presence of palytoxin or palytoxin-like compounds in fish extracts has been presented in this study. The hemolytic assay with sheep erythrocytes demonstrated the occurrence of hemolytic factors in fish extracts of Hawaiian reef fish from Barber's Point, Oahu. The rabbit anti-palytoxin inhibition assay with fish extracts and sheep erythrocytes demonstrated that palytoxin or its congener contributed to the lysis of sheep erythrocytes. From these results, it was concluded that sheep erythrocyte hemolysis was caused by palytoxin or palytoxin-like factors present in the fish extracts. Moderate correlation (R2) between mouse toxicity and sheep erythrocyte hemolysis was shown with 50 microg (R2 = 0.48) and 100 microg (R2 = 0.45) extracts. An inverse correlation of R2 = 0.64 was shown between hemolysis and MIA endpoint.

Topics: Acrylamides; Animals; Cnidarian Venoms; Erythrocytes; Fishes; Hawaii; Hemolysis; Hemolytic Plaque Technique; Immunoassay; In Vitro Techniques; Mice; Muscles; Rabbits; Sheep; Toxicity Tests, Acute

The occurrence of palytoxin or its congener in fish extracts has been presented in this study. The presences of hemolytic factors in fish extracts of Hawaiian reef fish and their implication in ciguatera poisoning have been shown by the sheep erythrocyte assay. By use of the anti-palytoxin inhibition assay with fish extracts and sheep red blood cell (RBC), it was shown that palytoxin was one of the major factors in the lysis of sheep erythrocytes. Ouabain, an antagonist of palytoxin for the Na+/K+ ATPase receptor on RBC, also showed inhibition of sheep RBC lysis by fish extracts. From these results, it was concluded that, in part, palytoxin and other palytoxin-related, hemolysin-like factors in fish extracts were responsible for sheep cell hemolysis.

Topics: Acrylamides; Animals; Ciguatoxins; Cnidarian Venoms; Erythrocytes; Fishes; Hemolysis; In Vitro Techniques; Ouabain; Sheep; Sodium-Potassium-Exchanging ATPase; Tissue Extracts

In 1994 in Madagascar a woman died after eating a sardine, Herklotsichthys quadrimaculatus. Two heads removed, respectively, from a toxic and a nontoxic fish before cooking were retrieved, kept frozen, and used for toxin analysis. The causative toxin was identified as palytoxin or its analogs based on its cytotoxicity, delayed hemolysis, neutralization with an anti-palytoxin antibody, chromatographic properties on different columns, and MS data. The gill and esophagus of the fish contained large amount of bottom sediments indicating that the fish had fed on the bottom and thus probably obtained the toxin from a benthic organism. The benthic dinoflagellate Ostreopsis siamensis that produces palytoxin and its analogs was inferred as the probable toxin source. This is the first study to shed light on clupeotoxism, a highly fatal form of human intoxication due to ingestion of clupeoid fish.

Topics: Acrylamides; Animals; Cell Survival; Chromatography, High Pressure Liquid; Cnidarian Venoms; Fatal Outcome; Female; Fishes; Foodborne Diseases; Hemolysis; Humans; Meat; Mice; Middle Aged; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrophotometry, Ultraviolet

The hemolytic action of palytoxin was exploited to develop a simple, sensitive assay with specificity based on a palytoxin neutralizing monoclonal antibody. Suspensions of murine erythrocytes incubated at 37 degrees C in round-bottom microtiter trays formed visible cell pellets which could be lysed by palytoxin. Hemolysis by palytoxin was time- and temperature-dependent, with a 24 hr detection limit of 1 pg/ml. The assay selectively detected palytoxin in a crude extract of Palythoa tuberculosa.

Topics: Acrylamides; Animals; Antibodies, Monoclonal; Cnidarian Venoms; Erythrocytes; Female; Hemolysis; Immunoenzyme Techniques; Mice; Neutralization Tests; Sensitivity and Specificity

Palytoxin stimulated arachidonic acid metabolism (in bovine aorta endothelial and smooth muscle cells, rat keratinocytes, porcine aorta endothelial cells and rat liver cells), hemolyzed rat erythrocytes and was lethal to mice when administered intraperitoneally. Serum from rabbits immunized with a conjugate in which palytoxin was covalently bound to bovine albumin through its free amino group neutralized these biologic activities of palytoxin. Ninety-nine per cent of the neutralizing activity of the immunized rabbit serum was removed after precipitation of the rabbit IgG with a goat anti-rabbit IgG.

Topics: Acrylamides; Animals; Antibody Formation; Arachidonic Acid; Arachidonic Acids; Cell Line; Cnidarian Venoms; Epoprostenol; Erythrocytes; Female; Hemolysis; Male; Mice; Neutralization Tests; Prostaglandins; Radioimmunoassay; Rats

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.

Topics: Acrylamides; Amphotericin B; Animals; Calcium; Cell Membrane Permeability; Cnidarian Venoms; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Kinetics; Osmotic Pressure; Potassium; Rats; Rubidium; Temperature

1. Palytoxin is a haemolysin. The erythrocytes from various species can be classified into a sensitive and a hardly sensitive group. The former contain potassium as their main inside cation and are arranged according to their sensitivity as hog greater than or equal to rat, mouse greater than rabbit greater than guinea-pig greater than man. The latter, comprising those from sheep and cattle, have sodium as their main inside cation. In addition, chicken erythrocytes are relatively insensitive. 2. Haemolysis of rat erythrocytes is preceded by a lag period of 1--2 h. With increasing temperature the haemolysis proceeds more quickly but reaches the same final range between 25 and 42 degrees C. The pH optimum in Britton-Robinson buffer supplemented with saline is between 7 and 8. Washing off palytoxin during the prelytic period reduces the haemolytic power. 3. The sensitivity of rat erythrocytes decreases with increase of osmolarity between 235 and 415 mosM. Accordingly, their osmotic resistance is lowered by palytoxin in a concentration-dependent manner. 4. With both rat and sheep erythrocytes, potassium loss by far precedes the haemolysis due to palytoxin. Potassium loss is measurable already after 1 min and increases with time. After 2 hours the quotient between the ED50 of haemolysis and that of potassium loss is around 200. Thus palytoxin is an unusually strong but slow haemolysin of the osmotic type. The extreme prelytic potassium loss and the correlation between susceptibility and potassium content of erythrocytes points towards the relevance of ionic fluxes.

Topics: Acrylamides; Animals; Cattle; Chickens; Cnidarian Venoms; Erythrocytes; Guinea Pigs; Hemolysis; Humans; Kinetics; Mice; Osmolar Concentration; Rabbits; Rats; Sheep; Species Specificity; Swine