Palytoxin toxicity

In the first attempts to determine palytoxin toxicity, the erode ethanol extracts of the Paly thoa toxica proved to be so toxic that an accurate LD50 was difficult to determine. More recently, the toxicity... 

Palytoxin derivatives are exceptionally toxic to cells and tissue in vitro, with effects being recorded at nanomolar concentrations. Such in vitro studies have proved very important in identifying the effect of palytoxin on ion movements across the cell membrane involving the Na,K-ATPase, and palytoxin has proved valuable in investigating the mechanism of action of ion pnmps [ 110,111 ]. In many cases, it was shown that the toxicity of palytoxin to cells was cansed by osmotic lysis dne to interaction with the Na,K-ATPase, as shown by the ability of onabain to prevent cytotoxicity, bnt whether snch effects are relevant to the toxicity of palytoxin in vivo is donbtfnl. Effects on ion transport have never been demonstrated in vivo, and ouabain had no effect on palytoxin toxicity in vivo [93]. 

Distribution. The first indication of the occurrence of palytoxin in fish was presented in 1969 (16). The filefish Altera scripta belonging to family Monacathidae was traditionally known in Okinawa, Japan, to contain a toxic substance in the gut and, thus, to kill pigs when fed to them. The presence of fragments of Palythoa sp. in the guts and the resemblance in solubility between the fish toxin and palytoxin led the authors to a conclusion that the toxic principle in the filefish viscera was palytoxin. Incidence of human intoxication due to eating the filefish was not confirm. ... 

To ensure that the inhibition of EGF binding by palytoxin was not a consequence of cell toxicity, the effect of palytoxin on DNA synthesis in Swiss 3T3 cells was monitored. When cells were incubated in the presence of palytoxin, 10% fetal calf serum, and H-thymidine for 19.5 hr, no depression in the extent of H-thymidine incorporation into DNA was detected up to 3.7 pM palytoxin (Table I). Although 11 pM palytoxin was toxic when present for a prolonged period, under the conditions of the assays described above no toxicity was detected (Table I). When cells were incubated in the presence of palytoxin, 0.1% fetal calf serum, and H-thymidine, palytoxin did not stimulate significant incorporation of H-thymidine into DNA. Thus, although it can modulate the EGF receptor system under these conditions, palytoxin alone does not appear to be mitogenic for Swiss 3T3 cells. 

Palytoxin is hemolytic (4) and is an extremely potent toxin (7). We have shown that in rat liver cells palytoxin stimulates de-esterification of cellular lipids to liberate arachidonic acid (5). These rat liver cells metabolize this increased arachidonic acid via the cyclooxygenase pathway to produce prostaglandin (PG) I2 and lesser amounts of PGE2 and PGp2. Palytoxin acts on many cells in culture to stimulate the production of cyclooxygenase metabolites (Table I). Clearly, the myriad pharmacological effects of the arachidonic acid metabolites must be considered in any explanation of the many clinical manifestations of palytoxin s toxicity. 

Earlier studies by Wiles et al. (5) in which palytoxin was administered by various routes showed that this material was extremely toxic to rabbits, dogs, and monkeys. The effect of route of administration on toxicity varies in that intravenous (iv), intramuscular (im), and subcutaneous (sc) toxicity is high, yet intrarectal (ir) or oral (po) palytoxin is relatively ineffective. It was also observed that palytoxin... 

The toxicity of palytoxin in sue unanesthetized animal species was established as well as the toxicity of the toxin when administered by various routes. Tables I and II show the doses and numbers of animals used in this phase of the study. 

Table I. Intravenous Toxicity of Palytoxin in Several Animal Species...

Table IH. Toxicity of Palytoxin in Rats When an Initial Sublethal Dose is Followed by a Lethal Dose...

Table VI. Toxicity of Palytoxin in Monkeys (Normal) When an Initial Sublethal ...

The iv toxicity of palytoxin is shown in Table I. Rabbits and dogs appear to be the sensitive to palytoxin rats and guinea pigs appear less sensitive. Table II shows the comparative toxicity of palytoxin administered by several routes - again iv palytoxin is extremely toxic while ir and po are relatively without toxic effects. 

Palytoxin is probably one of the most potent toxins known to humans. Intravenous LD q values in the sue species that have been studied are consistently less than 0.5 ig/kg. In addition, palytoxin possesses a speed of action and other pharmacologic properties that are markedly different from those exhibited by other toxic materials. For example, when injected iv or sc, palytoxin is extremely toxic yet when given po or ir, it is relatively non-toxic. It is also very interesting that the doses of palytoxin required to kill are somewhat different in anesthetized vs. unanesthetized animals. 

The direct injection of potent vasodilatory agents such as papaverine or isosor-bide dinitrate, into the ventricles of the heart reverses the action of palytoxin in approximately one-half of the animals. These extreme measures are required because palytoxin kills quickly. Antidotes injected into the venous circulation were not able to reach the heart because the stagnation of venous blood occurs so rapidly that antidotes are simply pooled on the venous side of the circulation and never reach the heart. In these studies isosorbide dinitrate appeared to be approximately twice as effective as papaverine in reversing the toxic effects of palytoxin. 

Palytoxin is a complex marine natural product containing 71 stereochemical elements (Fig. 5). The structure of PTX was elucidated by Moore. PTX is isolated from a zoanthid (order Zoanthidea) a type of soft coral commonly found in coral reefs all around the world. These animals come in a variety of different colonizing formations and in numerous colors. They can be found as individual polyps, attached by a fleshy stolon or a mat that can be created from pieces of sediment, sand and rock (soft coral). PTX is considered to be one of the most toxic nonpeptide substances known, second only to Maitotoxin. Typical symptoms of palytoxin poisoning are angina-like chest pains, asthma-like breathing difficulties, tachycardia, unstable blood pressure, hemolysis (destruction of red blood... 

Palytoxin was isolated in 1971 in Hawaii from Limu mate o Hane ( deadly seaweed of Hana ) which had been used to poison spear points. It is one of the most toxic compounds known requiring only about 0.15 microgram per kilogram for death by injection. The complicated structure was determined a few years later. 

The X 263 chromophore is a V-(3 -hydroxypropyl)-/rau5 -3-amidoacrylamide moiety (Moore et al. 1975). This moiety accounts for the positive response of palytoxin to the ninhydrin test, while its destruction is connected to loss of toxicity, accompanied by a negative ninhydrin test (Uemura et al. 1980a). The 263-nm chromophore is sensitive to methanolic 0.05 M HCl or aqueous 0.05 M NaOH, disappearing with a half-life of 85 and 55 minutes, respectively. However, neutralization within 2 minutes regenerates palytoxin with no apparent loss in toxicity (Moore and Scheuer 1971). 

Exposure of palytoxin to both visible and UV light results in structural changes in both the 263 and 233 chromophores and can reduce its toxicity at least twentyfold in only 5 minutes in UV and 30 minutes invisible light (Hewetsonet al. 1990). 

This section will focus on the available chemical data concerning only toxic substances produced by Ostreopsis sp., shown to possess palytoxin characteristics. For reasons of convenience, toxins will be presented according to producing species. Palytoxin-like compounds have been reported for O. siamensis, O. ovata, and O. mascarenensis. The neurotoxins ostreotoxin-1 and -3 produced by O. lenticularis have not been to date characterised by use of analytical methods as palytoxin analogues, despite their reported mouse lethality and possible connection to ciguatera (Tindall et al. 1990 Mercado et al. 1994 Meunier et al. 1997). With regard to the last of the toxic species, the oidy... 

O. siamensis was first characterized as a toxin producer by Nakajima et al. (1981). Some years later, Yasumoto et al. (1987) and Holmes et al. (1988) reported the lethafity and haemolytic activity of the O. siamensis toxins. Usami et al. (1995) were the first to elucidate the structure of the major ostreocin produced by O. siamensis (strain SOA 1 from Aka island, Okinawa, Japan) and point out its structural and chemical properties resemblance to palytoxin. This major constituent was named ostreocin-D and accounted for 90% of total toxicity of extracts. None of the other (more than 10) minor ostreocins present in the O. siamensis extracts were identical to palytoxin, as initially indicated by ESl-MS (Ukena et al. 2001, 2002). New Zealand O. siamensis isolates have also been reported to produce toxins exhibiting strong haemolytic activity and mouse lethality (Rhodes et al. 2000, 2002). Recently, Penna et al. (2005) have reported the presence of toxins with strong delayed haemolytic activity in Ostreopsis cf siamensis from the NW Mediterranean Sea. This haemolytic activity was inhibited by the palytoxin antagonist ouabain, indicating the palytoxin-like nature of these toxins. 

O. ovata from Okinawa, Japan, produced a butanol-soluble compound which was lethal to mice (Nakajima et al. 1981) this was later confirmed by Yasumoto et al. (1987), who also detected slight haemolytic activity in the O. ovata cell extracts. On the other hand, crude methanol extracts of O. ovata from the Virgin Islands were found to be nontoxic to mice (Tindall et al. 1990). Summer blooms of O. ovata in the Italian coasts have been coimected to respiratory problems in swimmers and sunbathers, most probably through inhalation of toxic aerosols (Sansoni et al. 2003 Simoni et al. 2003, 2004) such problems could possibly arise from inhalation of a palytoxin-like substance (Paddle 2003). Finally, extracts of O. ovata from Brazil and the Mediterranean Sea contained substances exhibiting strong delayed haemolysis, inhibited by ouabain, and mouse lethality with symptoms typical of palytoxin (Graneli et al. 2002 Riobo et al. 2004 Penna et al. 2005). 

CME and BSF also exhibited the typieal of palytoxin delayed haemolytie aetivity. Most of the mouse toxieity and haemolytie potency were fonnd in the BSF and subseqnent analyses were carried out to identify the nature of the toxic compounds (Turquet et al. 2002 Lenoir et al. 2004). 

The BSF was further analysed with HPLC-DAD and compared to reference palytoxin from P toxica, by using a mobile phase of water acidified to pH 2.5 with trifluoroacetic acid (solvent A) and pure acetouitrile (solvent B). A linear gradient was apphed from 30% to 70% of solvent B over 45 minutes. The BSF HPLC screening revealed two distinct peaks, which were eluted at approximately 38% acetonitrile with retention times very close to that of reference palytoxin. Both peaks also showed two UV absorption maxima, at 233 and 263 mn, while the ratio between their absorbance (233 versus 263 tun) was identical to that calculated for reference palytoxin. The peaks were collected separately and the toxic compounds pmified from the BSF were called mascarenotoxin-A (McTx-A) and mas-carenotoxin-B (McTx-B) (Lenoir et al. 2004). 

The MS profiles of McTx-A and McTx-B were both very similar to the respective profile of reference palytoxin, but the estimated molecular masses (between 2500-2535 Da) were lower than that of reference palytoxin (2680 Da) or other palytoxins and ostreocin-D. Nevertheless, the MS profile and fragmentation patterns of McTx-A and McTx-B together with mouse bioassay symptomatology and delayed haemolytic activity confirm the palytoxin-like character of these compounds. Quantitative differences in the hemolytic action and mouse lethality, as well as minor deviations in the MS spectra and retention times, could be attributed to structural variations between mascarenotoxins and the reference palytoxin (Lenoir et al. 2004). This is also supported by Usami et al. (1995), who showed that small changes in the structure of palytoxin analogues can have an impact on mouse toxicity, haemolytic potency, and cytotoxicity. 

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