2° MARBEF TRAINING COURSEON

CHEMICAL METHODS IN MARINE ECOLOGYMICROORGANISMS

NAPLES 9° 12° SEPTEMBER 2008

Mediterranean Mediterranean harmful algal eventsharmful algal events

ERNESTO FATTORUSSOUNIVERSITA’ DI NAPOLI FEDERICO II

2° MARBEF TRAINING COURSEON

CHEMICAL METHODS IN MARINE ECOLOGYMICROORGANISMS

NAPLES 9° 12° SEPTEMBER 2008

Mediterranean Mediterranean harmful algal eventsharmful algal events

ERNESTO FATTORUSSOUNIVERSITA’ DI NAPOLI FEDERICO II

2 µm2 µm

SEM coccolitoforide

99 toxic species over ca 5 000 marine microalgae 99 toxic species over ca 5 000 marine microalgae IOC LIST OF TOXIC MARINE MICROALAGAE IOC LIST OF TOXIC MARINE MICROALAGAE

http://www.bi.ku.dk/IOChttp://www.bi.ku.dk/IOC

ToxicPlankton Mussels

Nutrients

The explosive growth of toxic plankton is sometimes clearly The explosive growth of toxic plankton is sometimes clearly intertwined with changes in weather conditions.intertwined with changes in weather conditions.

Important contributing causes may also be found in variations in Important contributing causes may also be found in variations in upwelling, temperature, transparency, turbulence or salinity of upwelling, temperature, transparency, turbulence or salinity of seawater, as well as in concentration of dissolved nutrients, seawater, as well as in concentration of dissolved nutrients, wind and surface illuminationwind and surface illumination

Proliferations of harmful algae Proliferations of harmful algae may may

mostly happen as massive "red mostly happen as massive "red tides" tides"

or blooms of cells or blooms of cells capable of discoloring seawatercapable of discoloring seawater

THE MARINE FOOD CHAIN

Harmful algal toxins can find their way through theHarmful algal toxins can find their way through thedifferent levels of the food chain ending upon the different levels of the food chain ending upon the

table table of unaware consumersof unaware consumers

Well documented is the Well documented is the process through which process through which shellfish can shellfish can accumulate marine accumulate marine biotoxins in their edible biotoxins in their edible tissues from harmful tissues from harmful algaealgae

Digestive Gland

Toxic

Plankton

People

Time

Toxic Incident

ShellfishH

• In addition to those toxins In addition to those toxins passed along to consumerspassed along to consumers through the food chain, through the food chain, particular phytoplankton particular phytoplankton blooms can directly affect blooms can directly affect humans accidentally humans accidentally exposed to toxic algae exposed to toxic algae through swimming or through swimming or breathing aerosols. breathing aerosols.

Harmful algal Harmful algal proliferations can also proliferations can also induce severe ecological induce severe ecological disruption through disruption through widespread killing of sea widespread killing of sea life including marine life including marine mammals and seabirdsmammals and seabirds

O O

O

O

HO

O

H

O

MeH

H

HO

OH

Me

H

O

O

O

NHMe

Me

Me

H

H

H

Me

Azaspiracid

O

OO

HO

O

O

Me

O

O

OO

O

O

Me Me MeH

O

Brevetoxin-1

N

COOH

H

H

COOH

Me

COOHH3C

Domoic acid

TOXINS CLASS

MAIN ALGAL SOURCE

TOXICITY STRUCTURE OF A REPRESENTATIVE

Azaspiracids Protoperidinium crassipes

Neurotoxic

Brevetoxins Karenia brevis Neurotoxic

Ciguatoxins Gambierdiscus toxicus

O O

O

O

HO

O

H

O

MeH

H

HO

OH

Me

H

O

O

O

NHMe

Me

Me

H

H

H

Me

Azaspiracid

O

OO

HO

O

O

Me

O

O

OO

O

O

Me Me MeH

O

Brevetoxin-1

TABLE 1. ALGAL TOXINS

TOXINS CLASS

MAIN ALGAL SOURCE

TOXICITY STRUCTURE OF A REPRESENTATIVE

Domoic Acid

Pseudo-nitzschia species

Amnesic

Gymnodimines

Karenia selliformis

Okadaic acids

Dinophysis species

Prorocentrum lima

Diarrhetic Tumor

Promoter

N

COOH

H

H

COOH

Me

COOHH3C

Domoic acid

TABLE 1. ALGAL TOXINS

TOXINS CLASS

MAIN ALGAL

SOURCE

TOXICITY STRUCTURE OF A REPRESENTATIVE

Palytoxins Ostreopsis species

Neurotoxic Tumor Promoter

Pectenotoxins Dinophysis species

Diarrhetic

OO

O

O

O O

O O

OO

MeOH

Me

OH

Me

OH

Me

Me

Me

O

CH3

Pectenotoxin-2

TOXINS CLASS

MAIN ALGAL SOURCE

TOXICITY STRUCTURE OF A REPRESENTATIVE

Saxitoxins Alexandrium species

Paralytic

Spirolides Alexandrium ostenfeldii

Yessotoxins Protoceratium reticulatum

O

OO

N

Me

Me

HO

MeOH

Me

O

Me

O

Me

Spirolide DesMeC

O

O

O

O

O

O

OO

O

O

NaO3SO

NaO3SO

H H H H

H

HH

H

OH

H H HH HMe

Me

MeH

H

O

MeHO

H

H

H

Me

Me

Yessotoxin

TOXICITY TESTSTOXICITY TESTS

• Poisonous seafood neither looks nor tastes Poisonous seafood neither looks nor tastes different from uncontaminated seafood, and different from uncontaminated seafood, and cooking or other culinary treatment do not cooking or other culinary treatment do not destroy toxins. destroy toxins.

• Therefore, fish and shellfish farms need to Therefore, fish and shellfish farms need to run costly monitoring programs for checking run costly monitoring programs for checking the occurrence of toxins . the occurrence of toxins .

• Regular tests for toxins in seafood products Regular tests for toxins in seafood products need to be attentively carried out. need to be attentively carried out.

• At the moment, the most common test is the At the moment, the most common test is the mouse bioassay. mouse bioassay.

Mouse Bioassay for Toxins

• Intraperitoneal (IP) Intraperitoneal (IP) injection of extract of injection of extract of shell-fish digestive shell-fish digestive glands into 18-20 g glands into 18-20 g mousemouse

• Measurement time to Measurement time to death; one mouse death; one mouse unit = amount that unit = amount that kills mouse in 24 kills mouse in 24 hourshours

• A validated method A validated method accepted by several accepted by several countriescountries

• Rapid and inexpensive

• Poor reproducibility and long assay time

No information on toxins profile

HARMFUL ALGAL TOXINSHARMFUL ALGAL TOXINS HARMFUL ALGAL TOXINSHARMFUL ALGAL TOXINS

The extent of harmful algal blooms has The extent of harmful algal blooms has changed considerably over the last changed considerably over the last decades. decades.

If in the past only a few regions were If in the past only a few regions were affected in scattered locations, now affected in scattered locations, now virtually every coastal state is virtually every coastal state is threatened by toxic outbreaks.threatened by toxic outbreaks.

SaxitoxinsSaxitoxins

Okadaic Okadaic

acidsacids

YessotoxinsYessotoxins

Domoic Domoic

acidsacids

PectenotoxiPectenotoxi

ss

PalytoxinsPalytoxins

SpirolidesSpirolidesMediterranean Basin

N

N NH

NH

NH2

R4

R1

H2NOH

R2

OH

R3

R1

HOHHHOHOHHHOHOH

R2

HHHOSO3HOSO3HOHHOH

R3

HHOSO3HOSO3HOHHOHH

R4

STXNEOGTX2GTX3GTX1GTX411OH-STX11OH-STX11OH-NEO11OH-NEO

dcSTXdcNEOdcGTX2dcGTX3dcGTX1dcGTX411OH-dcSTX11OH-dcSTX11OH-dcNEO11OH-dcNEO

GTX5, B1GTX6, B2C1C2C3C4

doSTX

doGTX2doGTX3

O NH2

O

O NHSO3

OOH H

Carbamoylsaxitoxins

N-Sulfocarbamoylsaxitoxins

Decarbamoylsaxitoxins

Deoxydecarbamoylsaxitoxins

STX = saxitoxinNEO = neosaxitoxinGTX = gonyautoxin

SAXITOXINSSAXITOXINS

Currently over 29 congeners of saxitoxin are known Currently over 29 congeners of saxitoxin are known

SAXITOXINSSAXITOXINS• These compounds - responsible for the These compounds - responsible for the

syndrome known as Paralytic Shellfish syndrome known as Paralytic Shellfish Poisoning (PSP) - are toxic at submicromolar Poisoning (PSP) - are toxic at submicromolar concentrations and represent a severe public concentrations and represent a severe public health risk. health risk.

• A number of dinoflagellate species are A number of dinoflagellate species are known to produce saxitoxins: known to produce saxitoxins: AlexandriumAlexandrium (formerly (formerly Gonyaulax Gonyaulax or or Protogonyaulax),Gymnodinium Protogonyaulax),Gymnodinium and and Pyrodinium spp.Pyrodinium spp.

• By far, most cases of human PSP By far, most cases of human PSP intoxications are caused by the consumption intoxications are caused by the consumption of shellfish that have accumulated saxitoxins of shellfish that have accumulated saxitoxins after filtration of toxic marine dinoflagellates.after filtration of toxic marine dinoflagellates.

SAXITOXINSSAXITOXINS• PSP symptoms develop fairly rapidly, within PSP symptoms develop fairly rapidly, within

0.5 to 2 hours after ingestion of shellfish, 0.5 to 2 hours after ingestion of shellfish, depending on the amount of toxin consumed depending on the amount of toxin consumed

• In humans the peripheral nervous system is In humans the peripheral nervous system is affected, with symptoms ranging from affected, with symptoms ranging from tingling of the tongue and lips, followed by a tingling of the tongue and lips, followed by a numbness spreading towards the numbness spreading towards the extremities, to vomiting, pain, diarrhea, loss extremities, to vomiting, pain, diarrhea, loss of coordination and breathing difficulty. In of coordination and breathing difficulty. In severe cases, ataxia, muscle weakness, and severe cases, ataxia, muscle weakness, and respiratory paralysis can occur. respiratory paralysis can occur.

• In unusual cases, because of the weak In unusual cases, because of the weak hypotensive action of the toxins, death may hypotensive action of the toxins, death may occur from cardiovascular collapse despite occur from cardiovascular collapse despite respiratory support.respiratory support.

OKADAIC ACIDSOKADAIC ACIDS

O

OHO

O

O

O O

OOH

OR3

OH

OH

R1

O

R2

R1 R2 R3

CH3 H H Okadaic acid (OA)

CH3 CH3 H Dinophysistoxin 1 (DTX1) H CH3 H Dinophysistoxin 2 (DTX2)

Okadaic acid (OA) is the parent compound Okadaic acid (OA) is the parent compound of a group of polyether toxins – of a group of polyether toxins –

consisting of at least eight congeners consisting of at least eight congeners

OKADAIC ACIDSOKADAIC ACIDS

• Okadaic acid (OA) -the parent compound of a Okadaic acid (OA) -the parent compound of a group of polyether toxins – responsible for group of polyether toxins – responsible for the Diarrhetic Shellfish Poisoning (DSP) the Diarrhetic Shellfish Poisoning (DSP) syndrome, associated with seafood syndrome, associated with seafood consumption and characterized by an acute consumption and characterized by an acute gastrointestinal disturbancegastrointestinal disturbance

• The toxins are produced both by several The toxins are produced both by several Dinophysis Dinophysis species including species including Dinophysis Dinophysis acuta, D. fortii, D. acuminata, D. norvegica, D. acuta, D. fortii, D. acuminata, D. norvegica, D. mitra, mitra, and and D. caudataD. caudata, and by benthic , and by benthic species such as species such as Prorocentrum limaProrocentrum lima. .

OKADAIC ACIDSOKADAIC ACIDS• Oral assumption of OAs can lead to Oral assumption of OAs can lead to

gastrointestinal distresses, often beginning gastrointestinal distresses, often beginning within 30 minutes after consumption of within 30 minutes after consumption of contaminated shellfish. contaminated shellfish.

• The main DSP symptom is represented by The main DSP symptom is represented by diarrhea, often associated with other diarrhea, often associated with other disturbances such as nausea, vomiting and disturbances such as nausea, vomiting and abdominal pain. abdominal pain.

• No human casualty has been to date reported No human casualty has been to date reported in literature due to any case of DSP in literature due to any case of DSP poisoning, although sometimes considerable poisoning, although sometimes considerable morbidity has resulted in hospitalizationmorbidity has resulted in hospitalization

TUMOR PROMOTION OF OKADAIC ACID

The Fujiki group first found the potent activityThe Fujiki group first found the potent activityof Okadaic acid to promote tumor formation of Okadaic acid to promote tumor formation and extensively carried out relevant studies and extensively carried out relevant studies

Okadaic acid in drinking water, 10 Okadaic acid in drinking water, 10 g per g per rat rat

per day from weeks 9-55 of the per day from weeks 9-55 of the experiment, experiment,

significantly enhanced devolopment of significantly enhanced devolopment of neoplastic changes in the glandular neoplastic changes in the glandular

stomachstomachThe mechanism of action is based on the inhibition The mechanism of action is based on the inhibition of Protein Phosphatase 2Aof Protein Phosphatase 2A

PECTENOTOXINSPECTENOTOXINS

O

O

O

O

O O

O O

OO

MeOH

Me

OH

Me

OH

Me

Me

Me

O

R

1 7

R C-7 C-7

CH2OH R Pectenotoxin 1 (PTX1) R Pectenotoxin 2 seco acid (PTX2SA)

CH3 R Pectenotoxin 2 (PTX2) S 7-epi-Pectenotoxin 2 seco acid (PTX2SA)

CHO R Pectenotoxin 3 (PTX3)

CH2OH S Pectenotoxin 4 (PTX4)

COOH R Pectenotoxin 6 (PTX6)

COOH S Pectenotoxin 7 (PTX7)

The strucutre of Pectenotoxins resembles that The strucutre of Pectenotoxins resembles that of okadaic acid. Unlike okadaic acid, the carboxyl of okadaic acid. Unlike okadaic acid, the carboxyl moiety in many pectenotoxins is in a form of moiety in many pectenotoxins is in a form of macrocyclic lactone macrocyclic lactone

PECTENOTOXINSPECTENOTOXINS• Only the genus Only the genus DinophysisDinophysis (e.g. (e.g. D. acutaD. acuta, , D. D.

fortiifortii, , D. acuminataD. acuminata, and , and D. caudataD. caudata) has ) has been implicated in contamination of been implicated in contamination of shellfish with PTXs shellfish with PTXs

• Pectenotoxins have been grouped at the Pectenotoxins have been grouped at the beginning together with DSP toxins because beginning together with DSP toxins because of their co-extractability with okadaic acids. of their co-extractability with okadaic acids. However, animal studies have indicated that However, animal studies have indicated that pectenotoxins are much less toxic than the pectenotoxins are much less toxic than the latter ones through oral route and that they latter ones through oral route and that they do not induce diarrhea.do not induce diarrhea.

PECTENOTOXINSPECTENOTOXINS• Since PTXs often co-occur with other Since PTXs often co-occur with other

phycotoxins in shellfish, no toxic episodes phycotoxins in shellfish, no toxic episodes in humans could be unequivocally related to in humans could be unequivocally related to them and therefore there is no information them and therefore there is no information about their toxicity to humans. about their toxicity to humans.

• It has been shown that PTXs are potently It has been shown that PTXs are potently cytotoxic and cause necrosis to cytotoxic and cause necrosis to hepatocytes. Nothing is known of the hepatocytes. Nothing is known of the chronic toxicology of PTXs or the potential chronic toxicology of PTXs or the potential implications to public health in the long implications to public health in the long term.term.

YESSOTOXINSYESSOTOXINS

O

O

O

O

O

O

OO

O

O

R1

R2

H H H H

H

HH

H

OH

H H HH HMe

Me

Me

H

H

OH

Me

H

R3

Me( )n

R1 R2 R3 n

Yessotoxin (YTX) 45-Hydroxyyessotoxin (45-hydroxyYTX)

Homoyessotoxin (homoYTX)

45-Hydroxyhomoyessotoxin (45-OHhomoYTX)

Carboxyyessotoxin (carboxyYTX)

Carboxyhomoyessotoxin(carboxyhomoYTX)

Noroxohomoyessotoxin (NoroxohomoYTX)

Noroxoyessotoxin (NoroxoYTX)

Me OH

Me

OH

45

OSO3H OSO3H 1

OSO3H OSO3H 1

OSO3H OSO3H 2

OSO3H OSO3H 2

OSO3H OSO3H 1

OSO3H OSO3H 2

OSO3H OSO3H 2

OSO3H OSO3H 2

Me OH

Me OH

OHMe OH

COOH

Me OHCOOH

H

OMe

OMe

OH

45

O

O

O

O

O

O

OO

O

O

HO

HO3SO

HO3SO

H H H HMe HH

H

H H HH HMe

Me

Me

H

H

HOSO3H

H

H

Me

OH

Adriatoxin (ATX)

YESSOTOXINSYESSOTOXINS• The first species reported as a YTX The first species reported as a YTX

producer is the dinoflagellate producer is the dinoflagellate Protoceratium Protoceratium reticulatumreticulatum. However, more recent studies . However, more recent studies have shown that yessotoxin is also have shown that yessotoxin is also produced by the closely related species produced by the closely related species Lingulodinium polyedrumLingulodinium polyedrum and and Gonyaulax Gonyaulax spiniferaspinifera

• YTX is highly toxic towards mice when YTX is highly toxic towards mice when administered after i.p. injection, while its administered after i.p. injection, while its oral toxicity is at least tenfold lower .oral toxicity is at least tenfold lower .

YESSOTOXINSYESSOTOXINS• Yessotoxins were at beginning included Yessotoxins were at beginning included

within the Diarrhetic Shellfish Poisoning within the Diarrhetic Shellfish Poisoning (DSP) group because they are not (DSP) group because they are not distinguishable from okadaic acids on the distinguishable from okadaic acids on the basis of the mouse bioassay-based basis of the mouse bioassay-based standard procedure. However, their toxic standard procedure. However, their toxic activities are significantly different; in fact, activities are significantly different; in fact, YTXs do not induce diarrhea, whereas their YTXs do not induce diarrhea, whereas their cardiotoxic effects have been demonstrated cardiotoxic effects have been demonstrated in mice after intraperitoneal (i.p.)in mice after intraperitoneal (i.p.)

• It has been proposed that YTX may interact It has been proposed that YTX may interact with calcium channels inducing an uptake with calcium channels inducing an uptake of calcium in human lymphocytesof calcium in human lymphocytes

DOMOIC ACIDDOMOIC ACID

N

COOH

H

H

COOH

CH3

COOHH3C

N

COOH

H3C

H

COOH

CH3

COOHH

N

COOH

H

COOH

CH3

N

COOH

H

COOH

CH3

N

COOH

H

COOH

CH3

H3C

COOH

H3C

COOH

HOOC

CH3

Domoic acid

C5'-diastereomer

isodomoic acid Fisodomoic acid E

isodomoic acid D

Three geometrical isomers, isodomoic acids D, E, and F and Three geometrical isomers, isodomoic acids D, E, and F and the C5’-diastereomer have been found in small amountsthe C5’-diastereomer have been found in small amounts in both the producing diatom and shellfish tissuein both the producing diatom and shellfish tissue

DOMOIC ACIDDOMOIC ACID• Domoic acid (DA) is produced by Domoic acid (DA) is produced by Pseudo-nitzschia Pseudo-nitzschia

pungens pungens f.f. multiseries multiseries. Further species of . Further species of Pseudo-Pseudo-nitzschianitzschia ( (P. australisP. australis, , P. pseudo-delicatissimaP. pseudo-delicatissima, , P. P. galaxiaegalaxiae) have been found to produce domoic acid ) have been found to produce domoic acid

• It is the causative toxin of Amnesic Shellfish It is the causative toxin of Amnesic Shellfish Poisoning (ASP) syndrome. The clinical symptoms Poisoning (ASP) syndrome. The clinical symptoms of ASP include abdominal cramps, vomiting, of ASP include abdominal cramps, vomiting, diarrhea, incapacitating headaches, disorientation diarrhea, incapacitating headaches, disorientation and short-term memory loss. In the worst-case and short-term memory loss. In the worst-case scenario patients are victims of seizures, coma, scenario patients are victims of seizures, coma, profuse respiratory secretions, unstable blood profuse respiratory secretions, unstable blood pressure and also death.pressure and also death.

DOMOIC ACIDDOMOIC ACID• Domoic acid binds predominantly to N-

methyl-D-aspartate (NMDA) receptors in the central nervous system, thus causing depolarization of neurons.

• As a consequence, the permeability to calcium ions increases, thus inducing cell dysfunction and even death. This provokes lesions in the regions of the hippocampus responsible for learning and memory processes.

SPIROLIDESSPIROLIDES

R1 R2

H CH3 2,3

CH3 H 2,3

H CH3 CH3 H CH3 CH3 2,3

CH3 CH3

R1 R2

H CH3 2,3

H CH3

AdesMeCBdesMeDCD

EF

R

HCH3

GMeG

O

N

O

OO

OH

O

OH

R2

R1

32

O

N

O

OO

O

OHR

O

O

O

OO

OH

O

OH

R2

R1

32

NH2

Spirolides contain a spiro-linked tricyclic ether ring Spirolides contain a spiro-linked tricyclic ether ring system system

and an unusual 7-membered spiro-linked cyclic and an unusual 7-membered spiro-linked cyclic imine moietyimine moiety

SPIROLIDESSPIROLIDES• The causative micro-organism producing spirolide The causative micro-organism producing spirolide

toxins has been identified as the dinoflagellate toxins has been identified as the dinoflagellate Alexandrium ostenfeldiiAlexandrium ostenfeldii

• Spirolides induce a fast lethal toxic effect when i.p Spirolides induce a fast lethal toxic effect when i.p administered to both mice and rats, and are less administered to both mice and rats, and are less toxic by oral administration. Spirolide toxicity on toxic by oral administration. Spirolide toxicity on humans still remains unknown, although gastric humans still remains unknown, although gastric distress and tachycardia were diagnosed in distress and tachycardia were diagnosed in shellfish consumers in coincidence with the shellfish consumers in coincidence with the occurrence of spirolides in mollusks. occurrence of spirolides in mollusks.

• The unique cyclic imine moiety seems to be the The unique cyclic imine moiety seems to be the pharmacophore responsible for the fast-acting pharmacophore responsible for the fast-acting syndrome induced in mice when spirolides are i.p. syndrome induced in mice when spirolides are i.p. administered (LD50 = 40 administered (LD50 = 40 g/kg)g/kg)

PALYTOXINSPALYTOXINS

O

OH

O

O

OH

OOH

HOOH

OHMe

O

HO

OHO

MeO

OH

OO

OHHO

O

O

HN

O

NH

H2N

OH

HO

OH

OH

OH

OH

R6

OH

OH

OH

OHOHOH

R5

OH

OH

Me

R3 OH

OH

OHOH

OH Me OH

OH

OH

R2

R1

Me

OH

HO

OH

OH

OH

Palytoxin

R1 R2 R3 R4 R5

Me MeOH OHH

H OH HH HOstreocin-D

R4

( )n

n

1

1

2

3

Homopalytoxin

Bishomopalytoxin

Me MeOH OHH

Me MeOH OHH

Neopalytoxin - MeOH OHH1

Deoxypalytoxin Me MeOH OHH1

A moiety =

O

HN

O

NH

HO O( )n

R6

OH

OH

OH

OH

OH

H

A moiety

B moiety

PALYTOXINSPALYTOXINS• Palytoxin is a complex polyhydroxylated Palytoxin is a complex polyhydroxylated

water-soluble compound and one of the water-soluble compound and one of the most potent non-protein marine toxins most potent non-protein marine toxins known so far. It was first isolated in 1971 by known so far. It was first isolated in 1971 by the soft coral the soft coral Palythoa toxicaPalythoa toxica

• Although palytoxin has been isolated Although palytoxin has been isolated worldwide from zoanthids belonging to the worldwide from zoanthids belonging to the genus genus PalythoaPalythoa, its origin has long been , its origin has long been controversial. Recently, dinoflagellates controversial. Recently, dinoflagellates belonging to the genus belonging to the genus Ostreopsis Ostreopsis have have been shown as biogenetic origin of been shown as biogenetic origin of palytoxins. palytoxins.

PALYTOXINSPALYTOXINS• Palytoxin exhibits high toxicity in mammals with Palytoxin exhibits high toxicity in mammals with

intravenous LD50 ranging between 25 and 450 intravenous LD50 ranging between 25 and 450 ng/kg. Toxicity by intra-gastric administration is ng/kg. Toxicity by intra-gastric administration is significantly lower with an LD50 > 40 significantly lower with an LD50 > 40 g/kg.g/kg.

• Some fatal human poisonings attributed to Some fatal human poisonings attributed to palytoxin have been reported worldwide. palytoxin have been reported worldwide. Symptoms of intoxication include Symptoms of intoxication include vasoconstriction, hemorrhage, ataxia, muscle vasoconstriction, hemorrhage, ataxia, muscle weakness, ventricular fibrillation, pulmonary weakness, ventricular fibrillation, pulmonary hypertension, ischemia, and death. hypertension, ischemia, and death.

• Palytoxin binds to the Na+, K+-ATPase and Palytoxin binds to the Na+, K+-ATPase and converts this pump into an open channel .converts this pump into an open channel .

• In the late ‘80s, palytoxin was also identified as a In the late ‘80s, palytoxin was also identified as a skin tumor promoter. It has benn classified as askin tumor promoter. It has benn classified as a non-TPA-type tumor promoternon-TPA-type tumor promoter