BRIEF REVIEW OF NATURAL NONPROTEIN NEUROTOXINS
Jiri Patockaa and Ladislav Stredab
a. Military Medical Academy, Department of Toxicology, 500 01 Hradec Kralove and Faculty of Health and Social Care, Univerzity of South Bohemia, Ceske Budejovice and
b. State Office for Nuclear Safety, Department for Control of the Prohibition of Chemical Weapons, 110 00 Prague, Czech Republic
E-mail: firstname.lastname@example.org ; email@example.com
Natural toxins are chemical agents of biological origin and can be produced by all types of organisms, from microbes to higher animals. Toxins can be extremely toxic and many of them are effective at far lower dosages than are the conventional chemical agents. Toxins, as compounds of biological origin, are often classed as biological agents, but they are not infectious and are more similar to chemicals with respect to their military potential for tactical use; they should be considered to be chemical agents. Therefore the Chemical Weapons Convention (CWC) (1993) also includes toxins as chemical agents, and, specifically includes toxins in its control regime along with other highly toxic chemicals.
Neurotoxins are a group of toxins, whose highly specific effects on the nervous system of animal, including humans, by interfere with nerve impulse transmission. Neurotoxins are a varied group of compounds, both chemically and pharmacologically; they vary in chemical structures and mechanisms of action and produce very distinct biological effects. Neurotoxins may cause symptoms similar to chemical nerve agents, such as miosis, convulsions, tremor, seizures and rigid paralysis. Militarily significant neurotoxins are the topic of this review article. Each neurotoxin is briefly characterized chemically, pharmacologically and toxicologically. Their natural sources, availability, stability, and military potential are discussed.
This survey of neurotoxins includes phycotoxins: anatoxins, tetrodotoxin, saxitoxins, brevetoxins and ciguatoxin; amphibian toxins: batrachotoxins; coral toxin: palytoxin; molusca toxin: onchidal.
Anatoxins are toxins produced by cyanobacteria. Three common anatoxins have been described: (1) anatoxin-a and (2) homoanatoxin-a are secondary amines and (3) anatoxin-a(s) is a phosphate ester of a cyclic N-hydroxyguanine structure. The chemical structures of these anatoxins are given in Fig. 1. Anatoxin-a and homoanatoxin-a are postsynaptic depolarizing neuromuscular blocking agents  that bind strongly to the nicotinic acetylcholine receptor . These compounds are potent neurotoxins which cause rapid death in mammals by respiratory arrest (the mouse LD50 is approximately 250 µg/kg (microgram/kg) i.p. ). Anatoxin-a(s) is even more potent towards mice (LD50 20-40 µg/kg) and is a cholinesterase inhibitor [4,5]. Unlike anatoxin-a, anatoxin-a(s) induces hypersalivation in mammals, as well as other symptoms more typical of neurotoxicity such as diarrhea, shaking and nasal mucus discharge . All anatoxins are deadly and their military potential as a toxin weapon is very high. They are thermally labile and may be inactivated by heat. These toxins can enter the body by ingestion, injection, inhalation and through abraded skin.
Anatoxin-a and Homoanatoxin-a
Anatoxin-a is one of a group of low molecular weight neurotoxic alkaloids first described as found fresh-water cyanobacteria, Anabaena flos-aquae, from Canada . It is a small bicyclic compound, very soluble in water. The toxin can enter the body by inhalation, injection, and when exposed to highconcentrations, through the skin. The mechanism of action is not known in detail but it binds to and stimulates acetylcholine receptors andalso irreversibly inhibits the enzyme acetylcholinesterase. The symptoms of anatoxin-a poisoning resemble those of nerve agent poisoning . The toxin acts very rapidly and symptoms begin in less than 5 minutes after ingestion. Anatoxin-a is also known as Very Fast Death Factor (VFDF). The victim suffers twitching, muscle spasm, paralysis and respiratory arrest. Death may occur in minutes or hours depending on the dose. The lethal dose LD50 for mice or rats in different forms of application is between 150 and 250 µg/kg . The toxic dose in humans is not known but is estimated to be less than 5 mg for an adult male.. There is no specific treatment. Anatoxin-a is sensitive to heat, light and alkali. The detection and identification of these compound is difficult and it is possible only in a well-provided analytical laboratory. The toxin can be chemically synthesized in a racemic mixture .
Natural anatoxin-a is (+)-diastereoisomer and this stereoisomer is more toxic. Anatoxin-a’s LD50 values for mice by i.v. to, are 386 µg/kg for (+)-anatoxin-a hydrochloride and 913 µg/kg for racemic anatoxin-a hydrochloride. No deaths were observed in mice after i.p. administration of (-)-anatoxin-a hydrochloride at doses up to 73 mg/kg .
Homoanatoxin-a is structurally very similar to anatoxin-a. Homoanatoxin-a was first prepared synthetically  and later was found in the cyanobacteria Oscillatoria formosa in Norway . Its chemical structure is very similar to anatoxin-a, as well as its mechanism of toxic action and clinical toxicolgy. Lethal doses of homoanatoxin-a for mice were in the range 288-578 µg/kg with i.p. administration and 2890-5780 µg/kg by oral administration, respectively .
Anatoxin-a(s) occurs only in the species Anabaena flos-aquae. It is an acetylcholinesterase inhibitor that binds to the enzyme and renders it unable to hydrolyze the acetylcholine. Since the acetylcholine is not deactivated, the ion channel is left open, once again destroying muscle function through exhaustion. Anatoxin-a(s) is an organic phosphonate, similar in its action to synthetic organophosphonate nerve agents, such as sarin, soman or VX , whichinhibit cholinesterases by phosphorylation their active site . Anatoxin-a(s) is the only natural organophosphonate known. If given intraperitoneally to rats, it causes signs of severe cholinergic overstimulation, such as salivation, lacrimation, urinary incontinence, defecation, convulsion, fasciculation and respiratory arrest . The potent toxicity of anatoxin-a(s), LD50 from 20 to 40 µg/kg for mice via i.p., is attributed to its exceptional anticholinesterase activity. Atropine and probably oxime reactivators of cholinesterase may be used in the treatment of anatoxin-a(s) poisoning .
Onchidal is a toxic component of some poisonous marine mollusks, Onchidella binneyi, O. nigricans or O. patelloides . Chemically it is a simple lipophilic acetate ester (Fig. 2), and, although not an organophosphonate, it inhibits acetylcholinesterase. It is an irreversible inhibitor with a novel mechanism of action . However too little information is known about this interesting compound and it is difficult to predict its toxicity or its military potential. At all events onchidal will be easy to synthesize from available raw materials.
Tetrodotoxin (TTX) is a potent and rapid acting, lethal marine neurotoxin, named after the order of fish from which it is most commonly associated, the Tetraodontiformes (tetras-four and odontos-tooth), or the tetraodon pufferfish. Other marine organisms and some terrestrial poisonous aniamls also have been found to store TTX. In Japan, the puffer fish called fugu is considered a delicacy and several tens of people die each year as a consequence of eating this food. [Editor’s note: In Japan, some people value the thrill of the tingling effect a trace of TTX has in fugu.] In Japan approximately 646 cases were reported between the years of 1974 and 1983, with 179 of those cases resulting in death. Even today between 30 and 100 cases are reported annually, an obvious health problem.
TTX is an especially potent neurotoxin, specifically blocking the voltage-gated sodium channels on the surface of nerve membranes. The molecule has an unusual tricyclic structure (Fig. 3) and consists of a positively charged guanidinium group, which gives the name to this class of neurotoxins q.v., guanidinium toxins. The tetrodotoxin-Na channel binding site is extremely tight (Kd = 10-10 M). TTX mimics the hydrated sodium cation, enters the mouth of the Na+-channel peptide complex, binds to a peptide glutamate side group, among others, and then further tightens its hold when the peptide changes conformation in the second half of the binding event. Following these complex conformational changes, TTX is electrostatically attached to the opening of the Na+- gate channel.
TTX is extremely toxic. The LD50 for rats is 8 µg/kg by injection or 30 µg/kg if ingested. The toxicity for humans is also extremely high for inhaled toxin with the LD50 of about 150 µg/man (i.e., 2 µg/kg). Ingested TTX requires much higher doses (30 µg/kg) because stomach acid destroys the toxin. TTX is a slightly water soluble and heat stable, but it is sensitive to strong acids and alkalis. The toxin can enter the body by ingestion, injection, inhalation and through abraded skin. The onset of symptoms occurs within minutes when the toxin is injected or inhaled but symptoms develop more slowly (10 to 40 minutes) with ingested toxin. The first symptom of intoxication is a slight numbness of the lips and tongue, appearing between 20 minutes to three hours after eating poisonous pufferfish. The next symptom is increasing paraesthesia in the face and extremities, which may be followed by sensations of lightness or floating. Headache, epigastric pain, nausea, diarrhea, and/or vomiting may occur. Occasionally, some reeling or difficulty in walking may occur. The second stage of the intoxication is increasing paralysis. Many victims are unable to move and even sitting may be difficult. The victim suffer by weakness, dilatation of pupils, twitching, tremor and loss of muscle coordination and loss of voice. There is increasing respiratory distress. Paralysis increases and convulsions, mental impairment, and cardiac arrhythmia may occur. The victim, although completely paralyzed, may be conscious and in some cases completely lucid until shortly before death. Death usually occurs within four to six hours, with a known range of about 20 minutes to eight hours.
Kishi and his co-workers synthesized a racemic mixture of TTX in a 15-step process in 1972. Since then more than 30 synthetic works about TTX or its intermediates has been published .
As with anatoxins, the saxitoxins (STX) are neurotoxic alkaloids, which are also known as paralytic shellfish poisons (PSPs) () due to their occurence and association with seafood. The name saxitoxin is derived from the mollusk in which it was first identified, Saxidomus giganteus. During red tides (an explosive growth of phytoplanktons whose red pigments color the water), secretion of saxitoxin is especially dangerous. The dinoflagellate planktons in the ocean, particularly Protogonyaulax, Alexandrium catenella, A. minutum, A. ostenfeldii, A. tamarense, Gymnodinium catenatum and Pyrodinium bahamense var. compressum, all produce saxitoxins can be bioaccumulated by marine mollusks filter feeding upon the microalgae . Although mussels themselves are apparently unafffected by saxitoxin, mussel predators quickly develop the poison symptoms. PSP is dangerous to both humans and marine animals. It is paradoxical that as civilization has advanced, the incidences of red tides and PSP have also increased. Oceanic nutrients from pollution have increased, providing greater nutrient levels for dinoflagellate phytoplanktons, which in turn depelet the oxygen in the water resulting in eutrophication.
Saxitoxins are tricyclic compounds and their molecular skeleton are structurally related to tetrodotoxin. The most important of these are shown in Fig. 4. There are molecules with two guanidino groups with pKa’s of 11.3 and 8.2, respectively. At physiological pH then, the first-guanidino carries a positive charge, whereas the second-guanidino group is partially deprotonated. Because of this polar nature, the saxitoxin molecule readily dissolves in water and lower alcohols but is insoluble in organic solvents. It is stable in solution at neutral and acidic pH’s, even at high temperatures, but alkaline exposure oxidizes and inactivates the toxin. There are a number of STX variants generally divided into groups based on their structure or organism of origin. The single sulphated STXs are known as gonyautoxins (GTX) and B-toxins; the doubly sulphated STXs are known as C-toxins. There are also decarbamyl STXs (dcSTX) and a group of STX variantsunique to Lyngbia wollei, known as Lyngbia-wollei-toxins (LWTX) . STX was the first known and has been the most studied toxic component of paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na+ channels in nerve cells, thus casuing their neurotoxic effects. Although the toxin’s mechanism of action is well known at the molecular level, there are still many unresolved questions about its pharmacokinetics and the PSP intoxication syndrome in mammals. STXs are highly toxic, killing guinea pigs at only 5 µg/kg when injected i.m. The lethal doses for mice are very similar with varying adminstration routes:t i.p. (LD50 = 10 µg/kg), i.v. (LD50 = 3.4 µg/kg) or p.o. (LD50 = 263 µg/kg) The oral LD50 for humans is 5.7 µg/kg, therefore approximately 0.5 mg of saxitoxin is lethal if ingested and the lethal dose by injection is about ten times lower. The human inhalation toxicity of aerosolized saxitoxin is estimated to be 5 mg/min/m3. Saxitoxin can enter the body via open wounds and a lethal dose of 0.05 mg/person by this route has been suggested. Saxitoxin is 1,000 times more toxic than the potent nerve gas sarin.
Saxitoxin is a potent neurotoxin that specifically and selectively binds the sodium channels in neural cells. Thus, it physically occludes the opening of the Na+ channel and prevents any sodium cations from going in or out of the cell. Since neuronal transmittance of impulses and messages depends on the depolarization of the inside of the cell, the action potentials are stopped, impairing a variety of bodily functions, including breathing. Saxitoxin acts in a similar manner to Botulinum toxin because it is a cholinergic agonist that inhibits the release of acetylcholine at synapses in the peripheral nervous system. Human nerves are especially sensitive to the toxins and in the early stages of PSP, victims experience tingling and numbness of the mouth, tongue, face and extremities. Nausea and vomiting may accompany the above symptoms. In severe cases, the patient will exhibit advanced neurological dysfunction such as ataxia, weakness, dizziness, numbing of the lips, mouth and tongue, fatigue, difficulty breathing, and sense of dissociation followed by complete paralysis. The diaphragm and the diaphragm may stop working and death can occur after cardio-respiratory failure. Symptoms occur between 10 minutes and four hours after ingestion, depending on the dose. Inhalation of the toxin will produce a more rapid onset of symptoms, and, injection of saxitoxin may cause death in less than 15 minutes. If a victim survives for 12 hours, he has a good chance of recovering as the toxin is rapidly removed from the body. There is no specific antidote therapy, only symptomatic treatment; Mechanical ventilation has been used successfully in some cases.
Identification of saxitoxin as a cause of intoxication by virtue of clinical symptoms is not simple but is very importrant for military medicine, because faulty identification of this toxin as nerve gas poisoning may be fatal and administration of atropine would increase fatalities. The laboratory detection and identification of compound is difficult and is possible only in a well-provided analytical laboratory.
Saxitoxins are very dangerous compounds with possible military potential. They are extremely toxic, available on a large scale by extraction in high yield from cultures of toxin-producing species Protogonyaulax, relatively persistent and difficult to identify. They are white solids resistant to heat and acid but rapidly destroyed by alkaline solutions and also susceptible to oxygen. Saxitoxin has been incorporated into the Schedule 1 List of compounds for the Chemical Weapons Convention.
Batrachotoxins (BTX) are steroidal alkaloids released from the skin granular glands of tropical frogs from the genus Phyllobates . These litle and very gaily coloured creatures are known as ” dart poison” or “poison arrow” frogs because they are used as a source of -poison for coating arrows used among some South American Indians. The most important representants are batrachotoxin and homobatrachotoxin whose chemical structure is given in Fig 5. They are stable in storage and can be chemically synthesized. Batrachotoxins bind to the sodium ion channels of nerve axons and muscle cells. They inhibit closure of the channels so the neurone becomes completely depolarized and unable to transmit a signal .
Batrachotoxin and homobatrachotoxin are among the most potent of all naturally occurring nonprotein poisons. They are strong cardiotoxins, affecting ion permeability, which leads to an irreversible depolarization of nerves and muscles, arrhythmias, fibrillation, and cardiac failure . When given to animals, batrachotoxins cause loss of balance, profound weakness, convulsions and cyanosis with rapid succession. Observed symptoms in laboratory animals include strong muscle contractions, violent convulsions, salivation, dyspnoe and death, even at doses of less than 0.1 µg . At higher doses, e.g., 1 µg, death occurs in mice within one minute.The LD50 value of batrachotoxin in mice (subcutaneously) is 0.2 µg/kg, with minimal lethal doses from 0.01 to 0.02 µg/kg. The toxicity of homobatrachotoxin is only slightly less than batrachotoxin, with minimal lethal doses being about 0.04 and 0.06 µg/kg, respectively . It is about ten thousand times more toxic than sarin. If we suppose that man is at least as susceptible as mice to these compounds, the lethal dose is about 180 µg for a person. Larger animals are often more susceptible to toxins that smaller organisms, so that the lethal dose for man may be even less. Myers et al.  anticipated a lethal dose of batrachotoxin for man of only 2.0 to 7.5 µg, when administered by injection. The oral potency of batrachotoxin is much lower; therefore, Indians can eat animals captured by their darts without of risk of intoxication. In additions, the small amount of poison used is metabolized and the metabolites are not poisonous; most importantly, cooking may also destroy the toxins, although not all toxins are heat labile.
No effective antidote is known, but treatment of batrachotoxin poisoning might best be based on the paradigm for agents with similar mechanism of action, as for example aconitine, veratridine or digitalis. One of the few drugs available for this purpose may be DigiBind and a new comparable product, DigiTab, used to treat digitalis and oleander poisoning .
According to an ancient Hawaiian legend, there lived in the Hana district on Maui a man who always seemed to be busy planting and harvesting. Whenever the people in the neighborhood went fishing, upon their return, one of the group was missing. This went on for some time without the people having any explanation about the disappearances. At last the fishermen became suspicious of the man who tended his humble patch. They grabbed him, tore off his clothes and discovered on his back the mouth of a shark. They killed and burned him and threw the ashes into the sea. At the spot where this happened, so goes the legend, the limu (seaweed) became toxic. The tidepool containing the poisonous limu subsequently became kapu (sacred) to the Hawaiians. They would cover the limu with stones and were very secretive about its location. They firmly believed that disaster would strike if anyone were to attempt to gather the toxic limu (later identified as a soft coral, Palythoa toxica). This toxic moss was known as “limu make o Hana” (deadly seaweed of Hana) and from this material, Professor Paul J. Scheuer at the University of Hawaii extracted by ethanol a new substance he named palytoxin . [Editor’s note: palytoxin is not produced by the red seaweed, but by the small soft coral mistaken as seaweed.] The crude ethanol extracts of the Palythoa toxica proved to be so toxic that an accurate LD50 was difficult to determine. More recently, the toxicity has been determined to be 50-100 ng/kg i.p. in mice. The compound is an intense vasoconstrictor; in dogs, it causes death within 5 min at 60 ng/kg. By extrapolation, a toxic dose in a human would be about 4 micrograms. It is the most toxic organic substance known!
Shimizu  and Moore  published the chemical structure of palytoxin and it was prepared synthetically in 1989 [29,30]. Palytoxin is a fabulously interesting compound, with a bizarre structure and many extraordinary signs (Fig. 6). Palytoxin is a large, very complex molecule with lipophilic and hydrophilic areas. The palytoxin molecule has the longest continuous chain of carbon atoms known to exist in a natural product. In the molecule of palytoxin, C129H223N3O54, 115 of the 129 carbons are in a continuous chain.. There are 54 atoms of oxygen, but only 3 atoms of nitrogen. Another unusual structure of palytoxin is that it contains 64 stereogenic centers, which means that palytoxin can have 264 stereoisomers! Added to this, the double bonds can exhibit cis/trans isomerism, which means that palytoxin can have more than 1021 (one sextilion) stereoisomers! This staggering molecular complexity should indicate the difficult nature of designing a stereocontrolled synthetic strategy that will produce just the one correct (natural) stereocenter out of >1021 possible stereoisomers.
Palytoxin induces powerful membrane depolarization and ionic channeling [31,32]. Palytoxin is a potent hemolysin, histamine releaser, inhibitor of Na/K ATPase, and a cation ionophore . It is also a non-TPA-type tumor promoter [34,35].
Brevetoxins (BVX) are neurotoxins produced by algae called Ptychodiscus brevis (formerly Gymnodinium breve) from which the toxin name is derived. The algae proliferate during red tide incidents. Brevetoxins and related toxins are believed to have been responsible for massive fish kills from red tides in several regions. A long history of toxic microalgal blooms exists in the Gulf of Mexico, blooms that have caused massive fish kills and respiratory irritation in humans. It was later realized that the toxin in these blooms could also be passed to humans via shellfish to cause a syndrome named neurotoxic shellfish poisoning (NSP). Until cases were reported in New Zealand and Australia in the early 1990s, reports of NSP were limited to the Americas.
Victims of NSP can be misdiagnosed as suffering the fish-poisoning syndrome caused by ciguatera . Typical symptoms are tingling in the face, throat and digits, dizziness, fever, chills, muscle pains, abdominal cramping, nausea and vomiting, diarrhea, headache, reduced heart rate and pupil dilation. There have been no reported fatalities from NSP, although the toxin kills test mammals when administered by various routes, including orally.
The brevetoxins are lipophilic 10- and 11-ring polyethers with molecular weights around 900 Da . There are two classes of brevetoxins, the first contains eight 6-membered rings and two heptameric and an 8-membered ring (A type I brevetoxin, Fig. 7). The second class of brevetoxins has only 10 rings, with variation in the size of the rings ranging from five bonds to nine bonds (A type II brevetoxin, Fig. 7) .
These toxins depolarize and open voltage gated sodium (Na+) ion channels in cell walls, leading to uncontrolled Na+ influx into the cell . Brevetoxins bind to the ion channels of nerve and muscle tissue that selectively allows sodium to pass into the cell. These sodium channels open during an action potential in response to the change in the electrical potential across the cell membrane. Brevetoxins change the voltage at which this opening occurs nearer to the voltage threshold that triggers this process essentially making the sodium channel, and consequently, the affected nervous and muscular cells hyperexcitable .
Brevetoxins are unusually stable materials in the dry state. They are stable as well as in different solvents (acetone, acetonitrile, alcohol, ethyl acetate or DMSO), including water, where half-lives for active material range from 4-6 months. Solutions with a pH lower than 2 or higher than10 degrade the toxins.
Ciguatera fish poisoning was described as early as 1606 in the South Pacific island chain called New Hebrides. A similar outbreak there and in nearby New Caledonia was reported in 1774 by the famed English navigator, Captain James Cook. He described the clinical symptoms of his sick crew, symptoms that coincide with those described today. In addition, viscera from the same fishes eaten by Cook’s crew were given to pigs, causing their death.
The term ciguatera originated in the Caribbean area to designate intoxication induced by the ingestion of the marine snail Turbo pica (called cigua), first described by a Cuban ichthyologist. Today, the term is widely used to denote a particular type of fish poisoning that results from ingestion of primarily reef fishes encountered around islands in the Caribbean and the Pacific. Current information points to one of the many polyether toxins, such as ciguatoxin and related compounds, which are structurally similar to okadaic acid. Ciguatoxin is produced by the Dinoflagellate Gambierdiscus toxicus and has been isolated from the flesh and viscera of ciguatoxic fish.
Ciguatoxin is not a single compound, but a class of compounds. At present, 24 related ciguatoxins are known and these were found in different fishes from the Pacific. They are low molecular weight, lipid polyethers. They stimulate the enhancement of sodium ions through cell membranes (nerve or muscle cells). In this way, the toxins affect the cells and create the clinical symptoms seen in man. Structural formula of ciguatoxin CTx1 is given in Fig. 8.
Ciguatoxin is regarded as a neurotoxin , but the clinical symptoms of ciguatera poisoning can be classified into four broad groups: neurologic, cardiovascular, gastrointestinal and general symptoms. Symptoms usually begin within10 minutes to 12 hours, but can occur up to 36 hours after eating a poisonous fish. The disease commonly begins with nausea, vomiting and diarrhea, generalized weakness, a decreased sensation to pain or touch, unusual or painful sensations produced by ordinary stimuli, a burning or tingling of the hands and legs or around the mouth, muscle pain and temperature reversal sensation (hot things feel cold and cold things feel hot). Other less common symptoms include: chills, itching, dizziness, sweating, headache and taste disturbances (a metallic taste or fuzzy sensation).
The nausea, vomiting, and other gastrointestinal symptoms last for approximately 1 to 2 days. Weakness may last for 1 to 7 days. Neurologic symptoms such as tingling or temperature reversal generally persist for up to a week, but it is not unusual for these symptoms to periodically re-occur for a month or more. The poisoned victim may note an increased or decreased heart rate. Medical personnel may also note low blood pressure, dilated pupils, and irregular heart rhythm. These symptoms resolve in two to three days .
Examination of the clinical symptoms in patients with pufferfish, shellfish (red tide due to dinoflagellates) and polyether type toxin (ciguatoxin, okadaic acid, brevetoxin and other polyether) poisonings shows that the symptoms overlap and the causative toxins cannot be discriminated. In other words, there is no unique feature that separates the clinical effect. The temperature reversal was supposedly unique for ciguatoxin. Ciguatera fish poisoning is probably more important than any other form of seafood poisoning. Its epidemiology is complex and it is impossible to predict outbreaks. The ciguatoxins are not destroyed by cooking and, if consumed in sufficient dose, can cause symptoms persisting for weeks, months or years .
The best treatment for ciguatera poisoning during the early phase of poisoning (1-3 days after eating a toxic fish) has been mannitol infusion carried out in a physician’s office or emergency unit of the hospital. This is not a cure or antidote for ciguatoxin, but relieves many of the severe symptoms of poisoning, except for diarrhea in some patients. Some patients obtain no relief, others may present recurrence of symptoms in 24 hours. However, in the majority of the patients, mannitol infusion has been very helpful [44,45].
In some patients, long term symptoms occur or reoccur after generally eating fatty foods, seafood products and alcohol. The long term chronic symptoms such as muscle ache, joint pains and a weak, tired feeling in some patients have been successfully treated with tocainide hydrochloride (antiarrhytmics) and amitriptyline (antidepressant). Amitriptyline works best in patients showing symptoms of depression. Currently gabapentin was used as effective therapeutics of ciguatera intoxication .
The group of natural nonprotein neurotoxins is very important family of enormously toxic compounds, with unique chemical structures and strong biological effects. Therefore these compounds are very dangerous from many points of view. In examining their military potential, although they are very toxic, the ability to mass produce and weaponize these chemical is problematic. It is true that some physical measures, such as the protective mask and decontamination systems, developed for the chemical threat are, for the most part, effective against neurotoxin threats. But research to develop individual medical countermeasures to toxins is complicated by several factors. An adversary could select a number of neurotoxins for use in low-tech and relatively inexpensive weapons. Many more are potentially available through genetic engineering or chemical synthesis. It is also true that present toxin weapons could be more easily obtained and used than nuclear weapons. Many toxins actually may be more easily produced and used than conventional explosive weapons. Colorless, tasteless, odorless, small-scale aerosols may be generated relatively easily with a cheap plastic nebulizer attached to a pump or pressurized air bottle. However, it is not true that production and use of toxins as true mass casualty weapons is a trivial undertaking.
1. Carmichael WW, Biggs, DF, Gorham PR. Toxicology and pharmacological action of Anabaena flos-aquae toxin. Science 1977;187:542-544.
2. Spivak CE, Witkop B, Albuquerque EX. Anatoxin-a: a novel, potent agonist at the nicotinic receptor. Mol. Pharmacol 1980;18:384-394.
3. Devlin JP, Edwards OE, Gorham PR, Hunter NR, Pike RK, Starvick B. Anatoxin-a, a toxic alkaloid from Anabaena flos-aquae NCR-44h. Can J Chem 1977;5:1367-1371.
4. Mahmood NA, Carmichael WW. The pharmacology of anatoxin-a(s), a neurotoxin produced by the freshwater cyanobacterium Anabaena flos-aquae NRC 525-17. Toxicon 1986;24:425-434.
5. Mahmood NA, Carmichael WW. Anatoxin-a(s), an anticholinesterase from the cyanobacterium Anabaena flos-aquae NRC 525-17. Toxicon 1987;25:1221-1227.
6. Cook WO, Iwamoto GA, Schaeffer DJ, Carmichael WW, Beasley VR. Pathophysiologic effects of anatoxin-a(s) in anaesthetized rats: the influence of atropine and artificial respiration. Pharmacol Toxicol 1990;67:151-155.
7. Gorham PJ, McLachlan J, Hammer UT, Kim WK. (1964) Isolation and toxic strains of Anabaena flos-aquae (Lyngb.). Breb Verh Internat Verein Limnol 1964;15:796-804.
8. Molloy L, Wonnacott S, Gallagher T, Brough PA, Livett BG. Anatoxin-a is a potent agonist of the nicotinic acetylcholine receptor of bovine adrenal chromaffin cells. Eur J Pharmacol 1995;289:447-453.
9. Fawell JK, Mitchell RE, Hill RE, Everett DJ. The toxicity of cyanobacterial toxins in the mouse: II anatoxin-a. Hum Exp Toxicol 1999;18:168-173.
10. Koskinen AM, Rapoport H. Synthetic and conformational studies on anatoxin-a: a potent acetylcholine agonist. J Med Chem 1985;28:1301-1309.
11. Valentine WM, Schaeffer DJ, Beasley VR. Electromyographic assessment of the neuromuscular blockade produced in vivo by anatoxin-a in the rat. Toxicon 1991;29:347-357.
12. Wonnacott S, Swanson KL, Albuquerque EX, Huby NJ, Thompson P, Gallagher T. Homoanatoxin: a potent analogue of anatoxin-A. Biochem Pharmacol 1992;43:419-423.
13. Lilleheil G, Andersen RA, Skulberg OM, Alexander J. Effects of a homoanatoxin-a-containing extract from Oscillatoria formosa (Cyanophyceae/cyanobacteria) on neuromuscular transmission. Toxicon 1997;35:1275-1289.
14. Mahmood NA, Carmichael WW, Pfahler D. Anticholinesterase poisoning in dogs from a cyanobacteial (blue-green algae) bloom dominated by Anabaena flos-aquae. Ann J Vet Res 1988;49:500-503.
15. Abramson SN, Radic Z, Manker D, Faulkner DJ, Taylor P. Onchidal: a naturally occurring irreversible inhibitor of acetylcholinesterase with a novel mechanism of action. Mol Pharmacol 1989;36:349-354.
16. Cook WO, Beasley VR, Lovell RA, Dahlem AM, Hooser SB, Mahmood NA, Carmichael WW. Consistent inhibition of peripheral cholinesterases by neurotoxins from the freshwater cyanobacterium Anabaena flos-aquae: Studies of ducks, swine, mice and a steer. Environ Toxicol Chem 1989;8:915-922.
17. Noya B, Paredes MD, Ozores L, Alonso R. 5-exo radical cyclization onto 3-alkoxyketimino-1,6-anhydromannopyranoses. Efficient preparation of synthetic intermediates for (-)-tetrodotoxin. J Org Chem 2000; 65:5960-5968.
18. Gallacher S, Flynn KJ, Franco JM, Brueggemann EE, Hines HB. Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp. (Dinophyta) in culture. Appl Environ Microbiol 1997;63:239-245.
19. Onodera H, Satake M, Oshima Y, Yasumoto T, Carmichael WW: New saxitoxin analogues from the freshwater filamentous cyanobacterium Lyngbya wollei. Nat Toxins 1997;5:146-51.
20. Andrinolo D, Michea LF, Lagos N. Toxic effects, pharmacokinetics and clearance of saxitoxin, a component of paralytic shellfish poison (PSP), in cats. Toxicon 1999;37:447-464.
21. Myers CW, Daly JW, Malkin BA. Dangerously toxic new frog (Phyllobate) used by Emberá Indians of Western Colombia, with discussion of blowgun fabrication and dart poisoning. Bull Am Museum Nat History 1978;161: Article 2.
22. Warnick JE, Albuquerque EX, Onur R, Jansson SE, Daly J, Tokuyama T, Witkop B. The Pharmacology of Batrachotoxin. VIII. Structure-Activity Relationships and effect of pH. J Pharm Exp Ther 1975;193:232-245.
23. Albuquerque EX, Daly JW, Witkop B. Batrachotoxin: chemistry and pharmacology, Science 1977;172:995-1002.
24. Märki F., Witkop B.The venom of the Colombian arrow poison frog Phyllobates bicolor. Experientia 1963;19:329-338.
25. Patocka J, Schwanhaeuse WK, Marini MVP. Dart poison frogs and their toxins. ASA Newsletter 1999;74:16-18. ASANewsletter 99/995f.
26. Moore RE, Scheuer PJ. Palytoxin: a new marine toxin from a coelenterate. Science 1971;172:495-498.
27. Shimizu Y. Complete structure of palytoxin elucidated. Nature 1983;302:212.
28. Moore RE. Structure of palytoxin. Fortschr Chem Org Naturst 1985;48:81-202.
29. Armstrong RW, Beau JM, Cheon SH, Christ WJ, Fujioka H et al. Total Synthesis of Palytoxin Carboxylic Acid and Palytoxin Amide. J Amer Chem Soc 1989;111:7530-7533.
30. Mann J. Chemical synthesis. Scaling molecular Everests. Nature 1989;342:227-228.
31. Ozaki H, Tomono J, Nagase H, Urakawa N. The mechanism of contractile action of palytoxin on vascular smooth muscle of guinea-pig aorta. Japan. J. Pharmacol. 1983;33:1155-1162.
32. Lauffer L, Stengelin S, Beress L, Hucho F. Palytoxin-induced permeability changes in excitable membranes. Biochim Biophys Acta 1985;818: 55-60.
33. Habermann, E. Palytoxin acts through Na+, K+-ATPase. Toxicon 1989;27:1171-1187.Pa
34. Ohmura E, Tsushima T, Emoto N, Obuchi E, Shizume K. Effects of tumor promoters (mezerein, teleocidin and palytoxin) on growth hormone secretion from rat anterior pituitary cells cultured in monolayer. Life Sci 1987;41:691-696.
35. Kuroki D, Bignami GS, Wattenberg EV. Activation of stress-activated protein kinase/c-jun n-terminal kinase by the non-TPA-type tumor promoter palytoxin. Cancer Res 1996;56:637-644.
36. Lombet A, Bidard JN, Lazdunski M. Ciguatoxin and brevetoxins share a common receptor site on the neuronal voltage-dependent Na+ channel. FEBS Lett 1987;219:355-359.
37. Baden DG. Brevetoxins: unique polyether dinoflagellate toxins. FASEB J 1989;3:1807-1817.
38. Nakanishi K. The chemistry of brevetoxins: a review. Toxicon 1985;23:473-479.
39. Baden DG. Marine food-borne dinoflagellate toxins. Intemat Rev Cytology 1983;82: 99-150.
40. Atchison WD, Luke VS, Narahashi T, Vogel SM. Nerve membrane sodium channels (Neurotoxins – from p. 23) as the target site of brevetoxins at neuromuscular junctions. Br J Pharmacol 1986;189:731-738.
41. Pearn J. Neurology of ciguatera. J Neurol Neurosurg Psychiatry 2001;70:4-8.
42. Crump JA, McLay CL, Chambers ST. Ciguatera fish poisoning. N Z Med J 1999;112:282-283.
43. Lehane L, Lewis RJ. Ciguatera: recent advances but the risk remains. Int J Food Microbiol 2000;61:91-125.
44. Eastaugh JA. Delayed use of intravenous mannitol in ciguatera (fish poisoning). Ann Emerg Med 1996;28:105-106.
45. Mattei C, Molgo J, Marquais M, Vernoux J, Benoit E. Hyperosmolar D-mannitol reverses the increased membrane excitability and the nodal swelling caused by Caribbean ciguatoxin-1 in single frog myelinated axons. Brain Res 1999;847:50-58.
46. Perez CM, Vasquez PA, Perret CF. Treatment of ciguatera poisoning with gabapentin. N Engl J Med 2001;344:692-693.
Fig. 1. Chemical structures of anatoxins. Anatoxin-a, homoanatoxin-a, and anatoxin-a(s).
Fig. 2. Chemical structure of onchidal.
Fig. 3. Chemical structure of tetrodotoxin.
Fig. 4. Chemical structurs of saxitoxins. STX = saxitoxin, NeoSTX = neosaxitoxin, GTX = Gonyautoxin.
Fig. 5. Chemical structure of batrachotoxin.
Fig. 6. Chemical structure of palytoxin.
Fig. 7. Chemical structures of brevetoxins. Brevetoxin A, a type I brevetoxin and brevetoxin B, a type II brevetoxin.
Fig. 8. Chemical structure of ciguatoxin.
For the Professional in Government and Industry with an interest in Nuclear, Biological and Chemical Defense, Disarmament and Verification; Emergency and Disaster Medical Planning; Industrial Health and Safety; and Environmental Protection