Tetrodotoxin

Tetrodotoxin, also known as "tetrodox" and frequently abbreviated as TTX, sometimes colloquially referred to as "zombie powder" by those who practice Vodou, is a potent neurotoxin with no known antidote. There have been successful tests of a possible antidote in mice, but further tests must be carried out to determine efficacy in humans. Tetrodotoxin blocks action potentials in nerves by binding to the voltage-gated, fast sodium channels in nerve cell membranes, essentially preventing any affected nerve cells from firing by blocking the channels used in the process. The binding site of this toxin is located at the pore opening of the voltage-gated Na+ channel. Its name derives from Tetraodontiformes, the name of the order that includes the pufferfish, porcupinefish, ocean sunfish or mola, and triggerfish, several species of which 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.

Its mechanism of action, selective blocking of the sodium channel, was shown definitively in 1964 by Toshio Narahashi and John 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). Tarichatoxin and maculotoxin were shown to be identical to tetrodotoxin in 1964 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.

Biochemistry
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 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. 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.

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.

Poisoning
Tetrodotoxin is roughly 10 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 death due to respiratory failure. Toxicity varies between species and at different seasons and geographic localities, and the flesh of many pufferfish may not usually be dangerously toxic. It is not always fatal; however, at near-lethal doses, it can leave a person in a state of near-death for several days, while the person continues to be conscious. For this reason, tetrodotoxin has been alleged to be an ingredient in Haitian voodooism and the closest actual manifestation to zombieism in the physical world, an idea that was popularized by Harvard-trained ethnobotanist Wade Davis in a 1983 paper, and in his 1985 book, The Serpent and the Rainbow. However, 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.

Toxicity
The Material Safety Data Sheet for tetrodotoxin 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 a 75 kg person. 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 person.

History
The first recorded cases of tetrodotoxin poisoning were from the logs of Captain James Cook from 7 September 1774. He 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 killing themselves.

The toxin was first isolated and named in 1909 by Japanese scientist Dr. Yoshizumi Tahara.

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 sialorrhea, 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 to 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, then 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, however a monoclonal antibody specific to tetrodotoxin has been developed by USAMRIID and was shown to be effective for reducing lethality in murine tests.

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. 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. However, 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.

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 play a key role in 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.

Food analysis
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.

Regulation
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.