Lethal palytoxin

Palytoxin isolated from zoanthids Palythoa spp. is known to be extremely lethal (0.5 fig/kgy mouse, i.p.) and to have the most complicated natural product structure (Figure 4) ever elucidated (13,14). The toxin was later revealed to be a potent tumor promoter (15). Yet, health risks due to the toxin have remained unclear, as the zoathids are the most unlikely organisms to be regarded as foodstuff. Recently, however, data are being compiled to indicate the wide distribution of this toxin among marine biota. 

Palytoxin (PTX) is one of the most potent marine toxins known and the lethal dose (LD q) of the toxin in mice is 0.5 Mg/kg when injected i.v. The molecular structure of the toxin has been determined fully (1,2). PTX causes contractions in smooth muscle (i) and has a positive inotropic action in cardiac muscle (4-6). PTX also induces membrane depolarization in intestinal smooth (i), skeletal (4), and heart muscles (5-7), myelinated fibers (8), spinal cord (9), and squid axons (10). PTX has been demonstrated to cause NE release from adrenergic neurons (11,12). Biochemical studies have indicated that PTX causes a release of K from erythrocytes, which is followed by hemolysis (13-15). The PTX-induced release of K from erythrocytes is depress by ouabain and that the binding of ouabain to the membrane fragments is inhibited by PTX (15). 

The LD p of pal oxin in female Swiss Albino mice 24 hours following intra-peritoneal injection is 5 x 10 mg/kg (5). The immune sera also neutralized palytoxin s lethal effects. As shown in Figure 3, 11/12 mice were killed by palytoxin (1 X 10 mg/kg), whereas 0/12 and 0/11 mice were killed by palytoxin when injected intraperitoneally in the presence of the immune serum. None of the protected mice showed any signs of distress. 

This chapter is concerned therefore with studies of the mechanism of action of palytoxin, how it produces its lethal effects, as well as attempts to develop an antidote for this form of poisoning. 

Protection Studies. Animals were given a sublethal dose of palytoxin followed at various time intervals by a lethal dose. Control and treatment data for each route of administration and species studied are given in the appropriate table (Tables III, IV, V, and VI). 

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

Protection Studies. The responses of rats and rabbits given a sublethal dose of palytoxin followed by lethal challenges are shown in Tables III and IV. 

Figure 2. Effect of treatment with isosorbide dinitrate in reversing the otherwise lethal effect of palytoxin. (Reproduced from Ref. 10.)...

Rats showed an increase tolerance to im palytoxin at 4 and at 24 hr when the initial dose had been given ir (0/8 vs. 6/12 mortalities). An initial oral po dose also provided some protection against lethal iv challenge, 1/5 vs 6/6 mortalities at 24 hr, however, after these times protection was lost. 

Figure 4. Effect of pretreatment with hydrocortisone (50 mg/kg) on response of animal to a lethal injection of palytoxin (0.01 /ig/kg).

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... 

Producing species Toxin Considered Palytoxin analogue Molecular weight Chemical formula Mouse lethality (LD50 ip) Reference... 

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). 

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. 

Marine toxins may be developed from marine organisms. Examples include saxitoxin, tetrodotoxin, palytoxin, brevetoxins, and microcystin. Saxitoxin is a sodium-channel blocker and is most toxic by inhalation compared to the other routes of exposure. Saxitoxin and tetrodotoxin are similar in mechanical action, toxicity, and physical attributes. They can be lethal within a few minutes when inhaled. It has not yet been chemically synthesized efficiently, or easily created in large quantities from natural sources. Palytoxin is produced from soft coral and is highly toxic. It is, however, difficult to produce or harvest from nature. 

Palytoxin is one of the most poisonous nonprotein substances known to date. The lethal doses of palytoxin in rats, mice, guinea pigs, rabbits, dogs, and monkeys range between 0.03 and 0.45 ftg/kg by intravenous administration [1], By extrapolation, a toxic dose in a human would be between... 

Nakajima et al. [67] were the first to characterize 0. siamensis as a toxin producer. The lethality and hemolytic activity of the O. siamensis toxins was reported some years later [80,84]. Usami et al. [23] first elucidated the structme of the major ostreocin produced by 0. siamensis (strain SOA 1 from Aka island, Okinawa, Japan) and pointed out its structural and chemical properties resemblance to palytoxin. This major constituent, accounting for 90% of the total extracts toxicity. 

In conclusion, normally, low concentrations of palytoxin are sufficient to produce a massive increase in the permeability of cells to cations. Palytoxin stimulates sodium influx and potassimn efflux, and thus produces depolarization of the membrane in several cellular systems. In excitable systems, palytoxin-stimiflated depolarization can modulate calcium channel activity, resulting in a rise in the intracellular calcimn concentration, which can afterwards regulate calcium-dependent pathways and their related events. In muscle, depolarization causes calcium release and contraction. In vivo, the depolarization caused by the toxin can produce vasoconstriction, which can be lethal. In other systems, palytoxin-regulated events may not require an increase in the intracellular calcimn concentration. Whether the high cytotoxicity of palytoxin is merely a consequence of its disruption of the ionic enviromnent of the cell remains to be elucidated. 

Animals given a sublethal dose of palytoxin intravenously or intramuscularly were partially protected against a subsequent lethal intravenous dose [90]. Hydrocortisone afforded protection when administered before the palytoxin [90], while vasodilators, injected directly into the heart, gave a degree of protection against the toxicity of intravenous palytoxin [90]. 

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