anhydrotetrodotoxin, 4-epitetrodotoxin, tetrodonic acid, TTX
|Molar mass||319.27 g·mol−1|
Except where noted otherwise, data is given for materials in their standard state (at 25 °C (77 °F), 100 kPa)
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Tetrodotoxin, frequently abbreviated as TTX, is a potent neurotoxin. Its name derives from Tetraodontiformes, an order that includes pufferfish, porcupinefish, ocean sunfish or mola, and triggerfish, several species that carry the toxin. Although tetrodotoxin was discovered in these fish and found in several other animals (e.g., blue-ringed octopus, rough-skinned newt, and Naticidae) it is actually produced by certain symbiotic bacteria, such as Pseudoalteromonas tetraodonis, certain species of Pseudomonas and Vibrio, as well as some others that reside within these animals.
Tetrodotoxin inhibits the firing of action potentials in nerves by binding to the voltage-gated sodium channels in nerve cell membranes and blocking the passage of sodium ions (responsible for the rising phase of an action potential) into the nerve cell.
Its mechanism of action, selective blocking of the sodium channel, was shown definitively in 1964 by Toshio Narahashi and professor John W. Moore at Duke University, using Moore's sucrose gap voltage clamp technique.
Sources in nature
Tetrodotoxin has been isolated from widely differing animal species, including western newts of the genus Taricha (where it was formerly termed "tarichatoxin"), pufferfish, toads of the genus Atelopus, several species of blue-ringed octopuses of the genus Hapalochlaena (where it was called "maculotoxin"), several sea stars, certain angelfish, a polyclad flatworm, several species of Chaetognatha (arrow worms), several nemerteans (ribbonworms) and several species of xanthid crabs. The toxin is variously used as a defensive biotoxin to ward off predation, or as both a defensive and predatory venom (the octopuses, chaetognaths and ribbonworms). Even though the toxin acts as a defense mechanism, some predators such as the Common Garter Snake have developed a resistance to TTX which allows for them to prey upon these toxic newts. Tarichatoxin and maculotoxin were shown to be identical to tetrodotoxin in 1964 (Mosher et al.) and 1978, respectively. The toxin is produced by bacteria within blue-ringed octopuses. The most common bacteria associated with TTX production are Vibrio bacteria, with Vibrio alginolyticus being the most common species. Pufferfish, chaetognaths, and nemerteans have been shown to contain Vibrio alginolyticus and TTX. The link between these facts and production of TTX in animals has not been firmly established, and there remains much debate in the literature as to whether the bacteria are truly the source of TTX in animals.
Tetrodotoxin binds to what is known as site 1 of the fast voltage-gated sodium channel. Site 1 is located at the extracellular pore opening of the ion channel. The binding of any molecules to this site will temporarily disable the function of the ion channel. Saxitoxin, neosaxitoxin and several of the conotoxins also bind the same site.
The use of this toxin as a biochemical probe has elucidated two distinct types of voltage-gated sodium channels present in humans: the tetrodotoxin-sensitive voltage-gated sodium channel (TTX-s Na+ channel) and the tetrodotoxin-resistant voltage-gated sodium channel (TTX-r Na+ channel). Tetrodotoxin binds to TTX-s Na+ channels with a binding affinity of 5-15 nanomolar, while the TTX-r Na+ channels bind TTX with low micromolar affinity. Nerve cells containing TTX-r Na+ channels are located primarily in cardiac tissue, while nerve cells containing TTX-s Na+ channels dominate the rest of the body. The prevalence of TTX-s Na+ channels in the central nervous system makes tetrodotoxin a valuable agent for the silencing of neural activity within a cell culture.
The toxin blocks the fast Na+ current in human myocytes (the contractile cells of the muscles), thereby inhibiting their contraction. By contrast, the sodium channels in pacemaker cells of the heart are of the slow variety, so action potentials in the cardiac nodes are not inhibited by the compound. Note, the SA and AV nodes do not contain Na+ channels only Ca2+ channels, only the Purkinje fibers contain Na+ channels. The myocytes in the atrium, which surround the main cardiac pacemaker, do express this fast Na+ current and therefore the electrical activity is blocked and the heart fails to beat.
Blocking of fast Na+ channels has potential medical use in treating some cardiac arrhythmias. Tetrodotoxin has proved useful in the treatment of pain (originally used in Japan in the 1930s) from such diverse problems as terminal cancer, migraines, and heroin withdrawal.
In 1964 Robert B. Woodward elucidated the structure of tetrodotoxin. Yoshito Kishi et al. Nagoya University, Nagoya, Japan, (now at Harvard University) reported the first total synthesis of D,L-tetrodotoxin in 1972. M. Isobe et al. at Nagoya University, Japan and J. Du Bois et al. at Stanford University, U.S., reported the asymmetric total synthesis of tetrodotoxin in 2003. The two 2003 syntheses used very different strategies, with Isobe's route based on a Diels-Alder approach and Du Bois's work using C-H bond activation.
TTX is extremely toxic. The toxin can enter the body by ingestion, injection, or inhalation, or through abraded skin. The mechanism of toxicity is through the blockage of fast voltage-gated sodium channels. These are required for the normal transmission of signals between the body and brain. As a result, TTX causes paralysis of voluntary muscles (including the diaphragm and intercostal muscles, stopping breathing), loss of vagal regulation of heart rate (causing it to increase to around 100bpm), and loss of sensation.
TTX is roughly 100 times more poisonous than potassium cyanide. Fish poisoning by consumption of members of the order Tetraodontiformes is extremely serious. The organs (e.g. liver) of the pufferfish can contain levels of tetrodotoxin sufficient to produce paralysis of the diaphragm and, through this mechanism, death due to respiratory failure. Toxicity varies between species and at different seasons and geographic localities, and the flesh of many pufferfish may not be dangerously toxic. It is not always fatal, but at near-lethal doses, it can leave a person in a state of near-death for several days, while the person remains conscious. For this reason, TTX has been alleged an ingredient in Haitian Vodou and the closest approximation of zombieism, an idea popularized by Harvard-trained ethnobotanist Wade Davis in a 1983 paper, and in his 1985 book, The Serpent and the Rainbow. This idea was dismissed by the scientific community in the 1980s, as the descriptions of voodoo zombies do not match the symptoms displayed by victims of tetrodotoxin poisoning, and the alleged incidents of zombies created in this manner could not be substantiated.
The Material Safety Data Sheet for TTX lists the oral median lethal dose (LD50) for mice as 334 μg per kg. Assuming the lethal dose for humans is similar, 25 milligrams (0.000881 oz) of tetrodotoxin would be expected to kill half of the group of 75 kg (165 lb) people that ingested it. The amount needed to reach a lethal dose by injection is much smaller, 8 μg per kg, or a little over one-half milligram (0.00002 oz) to kill a 75 kg (165 lb) person.
The first recorded cases of TTX poisoning were from the logs of Captain James Cook from 7 September 1774, on which date Cook recorded his crew eating some local tropic fish (pufferfish), then feeding the remains to the pigs kept on board. The crew experienced numbness and shortness of breath, while the pigs were all found dead the next morning. In hindsight, it is clear that the crew received a mild dose of tetrodotoxin, while the pigs ate the pufferfish body parts that contain most of the toxin, thus being fatally poisoned.
Zora Neale Hurston, the author of the 1938 non-fiction book Tell My Horse, reported multiple accounts of poisoning with tetrodotoxin that had taken place in Haiti. The poisoning of people was executed by a voodoo sorcerer called the Bokor. 
Symptoms and treatment
The diagnosis of pufferfish poisoning is based on the observed symptomology and recent dietary history.
Symptoms typically develop within 30 minutes of ingestion, but may be delayed by up to four hours; however, death once occurred within 17 minutes of ingestion. Paresthesia of the lips and tongue is followed by hypersalivation, sweating, headache, weakness, lethargy, incoordination, tremor, paralysis, cyanosis, aphonia, dysphagia, seizures, dyspnea, bronchorrhea, bronchospasm, respiratory failure, coma, and hypotension. Gastroenteric symptoms are often severe and include nausea, vomiting, diarrhea, and abdominal pain. Cardiac arrhythmias may precede complete respiratory failure and cardiovascular collapse.
The first symptom of intoxication is a slight numbness of the lips and tongue, appearing between 20 minutes and three hours after eating poisonous pufferfish. The next symptom is increasing paresthesia 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; even sitting may be difficult. There is increasing respiratory distress. Speech is affected, and the victim usually exhibits dyspnea, cyanosis, and hypotension. 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 4 to 6 hours, with a known range of about 20 minutes to 8 hours.
If the patient survives 24 hours, recovery without any residual effects will usually occur over several days.
Therapy is supportive and based on symptoms, with aggressive early airway management. If ingested, treatment can consist of emptying the stomach, feeding the victim activated charcoal to bind the toxin, and taking standard life-support measures to keep the victim alive until the effect of the poison has worn off. Alpha adrenergic agonists are recommended in addition to intravenous fluids to combat hypotension. Anticholinesterase agents have been used with mixed success. No antidote has been developed and approved for human use; but a monoclonal antibody specific to tetrodotoxin has been developed by USAMRIID and was shown to be effective for reducing lethality in tests on mice.
Geographic frequency of toxicity
Poisonings from tetrodotoxin have been almost exclusively associated with the consumption of pufferfish from waters of the Indo-Pacific ocean regions, but pufferfishes from other regions are much less commonly eaten. Several reported cases of poisonings, including fatalities, involved pufferfish from the Atlantic Ocean, Gulf of Mexico, and Gulf of California. There have been no confirmed cases of tetrodotoxicity from the Atlantic pufferfish, Sphoeroides maculatus, but in three studies, extracts from fish of this species were highly toxic in mice. Several recent intoxications from these fishes in Florida were due to saxitoxin, which causes paralytic shellfish poisoning with very similar symptoms and signs. The trumpet shell Charonia sauliae has been implicated in food poisonings, and evidence suggests it contains a tetrodotoxin derivative. There have been several reported poisonings from mislabelled pufferfish, and at least one report of a fatal episode in Oregon when an individual swallowed a rough-skinned newt Taricha granulosa.
In 2009, a major scare in the Auckland Region of New Zealand was sparked after several dogs died eating Pleurobranchaea maculata (grey side-gilled seaslug) on beaches. Children and pet owners were asked to avoid beaches, and recreational fishing was also interrupted for a time. After exhaustive analysis, it was found that the sea slugs must have ingested tetrodotoxin.
- Statistical factors
From 1974 through 1983, there were 646 reported cases of pufferfish poisoning in Japan, with 179 fatalities. Statistics from the Tokyo Bureau of Social Welfare and Public Health indicate 20–44 incidents of fugu poisoning per year between 1996 and 2006 in the entire country, leading to 34–64 hospitalizations and 0–6 deaths per year, for an average fatality rate of 6.8%. Of the 23 incidents recorded within Tokyo between 1993 and 2006, only one took place in a restaurant, while the others all involved fishermen eating their catch. From 2006 through 2009 in Japan there were 119 incidents involving 183 people but only 7 people died.
Only a few cases have been reported in the United States, and outbreaks in countries outside the Indo-Pacific area are rare, except in Haiti, where tetrodotoxin is thought by some believers in voodoo mythology to assist the creation of so-called zombie poisons.
Genetic background is not a factor in susceptibility to tetrodotoxin poisoning. This toxicosis may be avoided by not consuming animal species known to contain tetrodotoxin, principally pufferfish; other tetrodotoxic species are not usually consumed by humans. Poisoning from tetrodotoxin is of particular public health concern in Japan, where pufferfish "fugu" is a traditional delicacy. It is prepared and sold in special restaurants where trained and licensed chefs carefully remove the viscera to reduce the danger of poisoning. There is potential for misidentification and mislabelling, particularly of prepared, frozen fish products.
The mouse bioassay developed for paralytic shellfish poisoning (PSP) can be used to monitor tetrodotoxin in pufferfish and is the current method of choice. An HPLC method with post-column reaction with alkali and fluorescence has been developed to determine tetrodotoxin and its associated toxins. The alkali degradation products can be confirmed as their trimethylsilyl derivatives by gas chromatography/mass spectrometry.
Detection in body fluids
Tetrodotoxin may be quantified in serum, whole blood or urine to confirm a diagnosis of poisoning in hospitalized patients or to assist in the forensic investigation of a case of fatal overdosage. Most analytical techniques involve mass spectrometric detection following gas or liquid chromatographic separation.
Tetrodotoxin is being investigated as a possible treatment for cancer-associated pain. Early clinical trials demonstrate significant pain relief in some patients.
In the U.S., tetrodotoxin appears on the select agents list of the Department of Health and Human Services, and scientists must register with HHS to use tetrodotoxin in their research. However, investigators possessing less than 100 mg are exempt from regulation.
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