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| Names | |
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| IUPAC name (4R,4aR,5R,6S,7S,8S,8aR,10S,12S)-2-azaniumylidene-4,6,8,12-tetrahydroxy-6-(hydroxymethyl)-2,3,4,4a,5,6,7,8-octahydro-1H-8a,10-methano-5,7-(epoxymethanooxy)quinazolin-10-olate | |
| Other names anhydrotetrodotoxin, 4-epitetrodotoxin, tetrodonic acid, TTX | |
| Identifiers | |
3D model (JSmol) | |
| ChEBI | |
| ChEMBL | |
| ChemSpider |
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| ECHA InfoCard | 100.022.236 |
| KEGG |
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| UNII | |
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| Properties | |
| C11H17N3O8 | |
| Molar mass | 319.270 g·mol−1 |
Except where otherwise noted, data are given for materials in theirstandard state (at 25 °C [77 °F], 100 kPa). | |
Tetrodotoxin (TTX) is a potentneurotoxin. Its name derives fromTetraodontiformes, anorder that includespufferfish,porcupinefish,ocean sunfish, andtriggerfish; several of these species carry the toxin. Although tetrodotoxin was discovered in these fish, it is found in several other animals (e.g., inblue-ringed octopuses,rough-skinned newts, andmoon snails). It is also produced by certain infectious orsymbioticbacteria likePseudoalteromonas,Pseudomonas, andVibrio as well as other species found in symbiotic relationships with animals and plants.[1][2]
Although it produces thousands of intoxications annually and several deaths,[3] it has shown efficacy for the treatment of cancer-related pain in phase II and III clinical trials.[4]
Tetrodotoxin is asodium channel blocker. It inhibits the firing ofaction potentials in neurons by binding to thevoltage-gated sodium channels innerve cellmembranes and blocking the passage of sodium ions (responsible for the rising phase of an action potential) into the neuron. This prevents the nervous system from carrying messages and thus muscles from contracting in response to nervous stimulation.[5]
Its mechanism of action – selective blocking of the sodium channel – was shown definitively in 1964 byToshio Narahashi andJohn W. Moore atDuke University, using thesucrose gapvoltage clamp technique.[6]
Apart from theirbacterial species of most likely ultimate biosynthetic origin (see below), tetrodotoxin has been isolated from widely differing animal species, including:[1]
Tarichatoxin was shown to be identical to TTX in 1964 by Mosher et al.,[12][13] and the identity of maculotoxin and TTX was reported inScience in 1978,[14] and the synonymity of these two toxins is supported in modern reports (e.g., atPubchem[15] and in modern toxicology textbooks[16]) though historic monographs questioning this continue in reprint.[17]
The toxin is variously used byanimals as a defensive biotoxin toward off predation, or as both a defensive and predatory venom (e.g., in octopuses,chaetognaths, andribbon worms).[18] Even though the toxin acts as a defense mechanism, some predators such as thecommon garter snake have developed insensitivity to TTX, which allows them to prey upon toxicnewts.[19]
The association of TTX with consumed, infecting, orsymbiotic bacterial populations within the animal species from which it is isolated is relatively clear;[1] presence of TTX-producing bacteria within an animal's microbiome is determined by culture methods, the presence of the toxin by chemical analysis, and the association of the bacteria with TTX production by toxicity assay of media in which suspected bacteria are grown.[2] As Lago et al. note, "there is good evidence that uptake of bacteria producing TTX is an important element of TTX toxicity in marine animals that present this toxin."[2] TTX-producing bacteria includeActinomyces,Aeromonas,Alteromonas,Bacillus,Pseudomonas, andVibrio species;[2] in the following animals, specific bacterial species have been implicated:[a]
| Animal | Bacteria | Ref |
|---|---|---|
| Takifugu obscurus, obscure pufferfish |
| [2][5] |
| Nassarius semiplicatus, a gastropod |
| [1] |
| Hapalochlaena maculosa, theSouthern blue-ringed octopus |
| [1][2][5][20] |
| Astropecten polyacanthus, a starfish | Vibrio alginolyticus | [2][5] |
| Takifugu vermicularis, a pufferfish |
| [1][2][5][21] |
| Four species ofChaetognatha (arrow worms) |
| [22] |
| Species ofNemertea (ribbon worms) | Vibrio spp. | [1][23] |
The association of bacterial species with the production of the toxin is unequivocal – Lago and coworkers state, "[e]ndocellular symbiotic bacteria have been proposed as a possible source of eukaryotic TTX by means of an exogenous pathway",[2] and Chau and coworkers note that the "widespread occurrence of TTX in phylogenetically distinct organisms... strongly suggests that symbiotic bacteria play a role in TTX biosynthesis"[1] – although the correlation has been extended to most but not all animals in which the toxin has been identified.[1][2][5] To the contrary, there has been a failure in a single case, that of newts (Taricha granulosa), to detect TTX-producing bacteria in the tissues with highest toxin levels (skin,ovaries,muscle), usingPCR methods,[24] although technical concerns about the approach have been raised.[1] Critically for the general argument,Takifugu rubripes puffers captured and raised in laboratory on controlled, TTX-free diets "lose toxicity over time", while cultured, TTX-freeTakifugu niphobles puffers fed on TTX-containing diets saw TTX in the livers of the fishes increase to toxic levels.[1] Hence, as bacterial species that produce TTX are broadly present in aquatic sediments, a strong case is made for ingestion of TTX and/or TTX-producing bacteria, with accumulation and possible subsequent colonization and production.[1] Nevertheless, without clear biosynthetic pathways (not yet found in animals, but shown for bacteria),[25] it remains uncertain whether it is simply via bacteria that each animal accumulates TTX; the question remains as to whether the quantities can be sufficiently explained by ingestion, ingestion plus colonization, or some other mechanism.[1][2][5]
Tetrodotoxin binds to what is known assite 1 of the fastvoltage-gated sodium channel.[26] Site 1 is located at the extracellular pore opening of the ion channel. Any molecule bound to this site will block sodium ions from going into the nerve cell through this channel (which is ultimately necessary for nerve conduction).Saxitoxin,neosaxitoxin, and several of theconotoxins also bind the same site.[27]
The use of this toxin as a biochemical probe has elucidated two distinct types of voltage-gated sodium channels (VGSCs) present in mammals: tetrodotoxin-sensitive voltage-gated sodium channels (TTX-s Na+ channels) and tetrodotoxin-resistant voltage-gated sodium channels (TTX-r Na+ channels). Tetrodotoxin inhibits TTX-s Na+ channels at concentrations of around 1–10 nM,[28] whereas micromolar concentrations of tetrodotoxin are required to inhibit TTX-r Na+ channels.[29] Nerve cells containing TTX-r Na+ channels are located primarily incardiac tissue, while nerve cells containing TTX-s Na+ channels dominate the rest of the body.
TTX and its analogs have historically been important agents for use as chemical tool compounds, for use in channel characterization and in fundamental studies of channel function.[30][31] The prevalence of TTX-s Na+ channels in thecentral nervous system makes tetrodotoxin a valuable agent for the silencing ofneural activity within acell culture.
The biosynthetic route to TTX is only partially understood. It is long known that the molecule is related tosaxitoxin, and as of 2011 it is believed that there are separate routes for aquatic (bacterial) and terrestrial (newt) TTX.[32] In 2020, new intermediates found in newts suggest that the synthesis starts with geranyl guanidine in the amphibian; these intermediates were not found in aquatic TTX-containing animals, supporting the separate-route theory.[33] In 2021, the first genome of a TTX-producing bacterium was produced. This "Bacillus sp. 1839" was identified asCytobacillus gottheilii using its rRNA sequence. The researcher responsible for this study has not yet identified a coherent pathway but hopes to do so in the future.[34]
Animals that accumulate TTX as a defense mechanism as well as their predators must evolve to be resistant to the effects of TTX. Mutations in the VGSC genes, especially the genes for Nav 1.4 (skeletal muscle VGSC, "TTX-s"[35]), are found in many such animals.[36] These mutations have independently arisen several times, even multiple times in different populations of the same species as seen in the garter snake. They consist of different amino acid substitutions in similar positions, a weak example ofconvergent evolution caused by how TTX binds to the unmutated VGSC.[36]
Another path to TTX resistance is toxin-binding proteins that hold onto TTX tightly enough to prevent it reaching the vulnerable VGSCs. Various proteins that bind TTX have been found in pufferfish, crabs, and gastropods. There are also proteins that bindsaxitoxin (STX), a toxin with a similar mode of action.[36]
In 1964, a team of scientists led byRobert B. Woodward elucidated the structure of tetrodotoxin.[37] The structure was confirmed byX-ray crystallography in 1970.[38]Yoshito Kishi and coworkers reported the firsttotal synthesis ofracemic tetrodotoxin in 1972.[39][40] M. Isobe and coworkers[41][42][43] and J. Du Bois reported theasymmetric total synthesis of tetrodotoxin in 2003.[44] The two 2003 syntheses used very different strategies, with Isobe's route based on aDiels-Alder approach and Du Bois's work usingC–H bond activation. Since then, methods have rapidly advanced, with several new strategies for the synthesis of tetrodotoxin having been developed.[45][46]
TTX is extremely toxic. Thematerial safety data sheet for TTX lists the oralmedian lethal dose (LD50) for mice as 334 μg per kg.[47] For comparison, the oral LD50 ofpotassium cyanide for mice is 8,500 μg per kg,[48] demonstrating that even orally, TTX is more poisonous thancyanide. TTX is even more dangerous if administered intravenously; the amount needed to reach a lethal dose by injection is 8 μg per kg in mice.[49]
The toxin can enter the body of a victim byingestion, injection, orinhalation, or through abraded skin.[50]
Poisoning occurring as a consequence of consumption of fish from the orderTetraodontiformes is extremely serious. The organs (e.g., liver) of thepufferfish can contain levels of tetrodotoxin sufficient to produce the describedparalysis of thediaphragm and corresponding death due torespiratory failure.[51] Toxicity varies between species and at different seasons and geographic localities, and the flesh of many pufferfish may not be dangerously toxic.[5]
The mechanism of toxicity is through the blockage of fast voltage-gated sodium channels, which are required for the normal transmission of signals between the body and brain.[52] As a result, TTX causes loss of sensation, and paralysis of muscles including the diaphragm and intercostal muscles, stopping breathing.[53]
The therapeutic uses of puffer fish (tetraodon) eggs were mentioned in the first Chinese pharmacopoeiaPen-T’so Ching (The Book of Herbs, allegedly 2838–2698 BC byShennong; buta later date is more likely), where they were classified as having "medium" toxicity, but could have a tonic effect when used at the correct dose. The principal use was "to arrest convulsive diseases".[30] In thePen-T’so Kang Mu (Index Herbacea or The Great Herbal by Li Shih-Chen, 1596) some types of the fish Ho-Tun (the current Chinese name fortetraodon) were also recognized as both toxic yet, at the right dose, useful as part of a tonic. Increased toxicity in Ho-Tun was noted in fish caught at sea (rather than river) after the month of March. It was recognized that the most poisonous parts were the liver and eggs, but that toxicity could be reduced by soaking the eggs.[30] (Tetrodotoxin is slightly water-soluble, and soluble at 1 mg/ml in slightly acidic solutions.)[54]
The German physicianEngelbert Kaempfer, in his "A History of Japan" (translated and published in English in 1727), described how well known the toxic effects of the fish were, to the extent that it would be used for suicide and that the Emperor specifically decreed that soldiers were not permitted to eat it.[55] There is also evidence from other sources that knowledge of such toxicity was widespread throughout southeast Asia and India.[30]
The first recorded cases of TTX poisoning affecting Westerners are from the logs ofCaptainJames Cook from 7 September 1774.[51] On that 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 survived a mild dose of tetrodotoxin, while the pigs ate the pufferfish body parts that contain most of the toxin, thus being fatally poisoned.
The toxin was first isolated and named in 1909 by Japanese scientist Dr. Yoshizumi Tahara.[2][56][51] It was one of the agents studied by Japan'sUnit 731, which evaluated biological weapons on human subjects in the 1930s.[57]
The diagnosis of pufferfish poisoning is based on the observed symptomatology and recent dietary history.[58]
Symptoms typically develop within 30 minutes of ingestion, but may be delayed by up to four hours; however, if the dose is fatal, symptoms are usually present within 17 minutes of ingestion.[51]Having pins and needles of the lips and tongue is followed by developing it in the extremities,hypersalivation,sweating, headache, weakness, lethargy,incoordination,tremor, paralysis,bluish skin,loss of voice,difficulty swallowing, andseizures. The gastrointestinal symptoms are often severe and includenausea,vomiting,diarrhoea, andabdominal pain; death is usually secondary torespiratory failure.[53][58] There is increasingrespiratory distress, speech is affected, and the victim usually exhibitsshortness of breath,excess pupil dilation, andabnormally low blood pressure. Paralysis increases, andconvulsions, mental impairment, andirregular heartbeats may occur. The victim, although completely paralysed, may be conscious and in some cases completely understandable until shortly before death, which generally occurs within 4 to 6 hours (range ~20 minutes to ~8 hours). However, some victims enter acoma.[53][59]
If the patient survives 24 hours, recovery without any aftereffects will usually occur over a few days.[58]
Therapy is supportive and based on symptoms, with aggressive early airway management.[51] If consumed, treatment can consist of emptying the stomach, feeding the victimactivated 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.[51]Alpha adrenergic agonists are recommended in addition to intravenous fluids to increase the blood pressure;anticholinesterase agents "have been proposed as a treatment option but have not been tested adequately".[59]
Noantidote has been developed and approved for human use, but a primary research report (preliminary result) indicates that amonoclonal antibody specific to tetrodotoxin is in development byUSAMRIID that was effective, in the one study, for reducing toxin lethality in tests on mice.[60]
Poisonings from tetrodotoxin have been almost exclusively associated with the consumption of pufferfish from waters of the Indo-Pacific Ocean regions, primarily because equally toxic pufferfishes from other regions are much less commonly eaten. Several reported cases of poisonings, including fatalities, nonetheless involved pufferfish from the Atlantic Ocean,Gulf of Mexico, andGulf of California. There have been no confirmed cases of tetrodotoxicity from the Atlantic pufferfish,Sphoeroides maculatus, but three studies found extracts from fish of this species highly toxic in mice. Several recent intoxications from these fishes in Florida were due tosaxitoxin, which causesparalytic shellfish poisoning with very similar symptoms and signs. The trumpet shellCharonia 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 newtTaricha granulosa on a dare.[61]
In 2009, a major scare in theAuckland Region ofNew Zealand was sparked after several dogs died eatingPleurobranchaea maculata (grey side-gilled seaslug) on beaches.[62] 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.[63]
Statistics from the Tokyo Bureau of Social Welfare and Public Health indicate 20–44 incidents offugu 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%.[64] 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.[64] From 2006 through 2009 in Japan there were 119 incidents involving 183 people but only seven people died.[65]
Only a few cases have been reported in the United States, and outbreaks in countries outside the Indo-Pacific area are rare. In Haiti, tetrodotoxin was thought to have been used invoodoo preparations, in so-calledzombie poisons. Subsequent careful analysis has however repeatedly called early studies into question on technical grounds, and failed to identify the toxin in any preparation.[66][67][68] Discussion of the matter has therefore all but disappeared from the primary literature since the early 1990s. Kao and Yasumoto concluded in the first of their papers in 1986 that "the widely circulated claim in the lay press to the effect that tetrodotoxin is the causal agent in the initial zombification process is without factual foundation."[66]: 748
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, wherefugu is a traditional delicacy. It is prepared and sold in special restaurants where trained and licensedchefs carefully remove theviscera to reduce the danger of poisoning.[69] There is potential for misidentification and mislabelling, particularly of prepared, frozen fish products.
The mouse bioassay developed forparalytic shellfish poisoning (PSP) can be used to monitor tetrodotoxin in pufferfish and is the current method of choice. AnHPLC 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 theirtrimethylsilyl derivatives by gas chromatography/mass spectrometry.[citation needed]
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.[70]
Tetrodotoxin has been investigated as a possible treatment for cancer-associated pain. Early clinical trials demonstrate significant pain relief in some patients.[71][72]
It has also been studied in relation tomigraine headaches. Mutations in one particular TTX-sensitive Na+ channel are associated with somemigraine headaches,[73] although it is unclear as to whether this has any therapeutic relevance for most people with migraine.[74]
Tetrodotoxin has been used clinically to relieve negative affects associated withheroin withdrawal.[75]
 | The examples and perspective in this articledeal primarily with the United States and do not represent aworldwide view of the subject. You mayimprove this article, discuss the issue on thetalk page, orcreate a new article, as appropriate.(February 2017) (Learn how and when to remove this message) |
In the U.S., tetrodotoxin appears on theselect agents list of theDepartment of Health and Human Services,[76] and scientists must register with HHS to use tetrodotoxin in their research. However, investigators possessing less than 500 mg are exempt from regulation.[77]
Tetrodotoxin serves as a plot device for characters to fake death, as in the filmsHello Again (1987),The Serpent and the Rainbow (1988),The A-Team (2010),Captain America: The Winter Soldier (2014), andWar (2019), and in episodes of "Jane the Virgin",Miami Vice (1985),[78]Nikita,MacGyver (season 7, episode 6, where the antidote isDatura stramonium leaf),CSI: NY (season 4, episode 9, "Boo"), andChuck. InLaw Abiding Citizen (2009) andAlex Cross (2012), its paralysis is presented as a method of assisting torture. The toxin was also referenced in "synthetic form" in season 1, episode 2, of the series "FBI". The toxin is used as a weapon in both the second season ofArcher, inCovert Affairs and in theInside No. 9 episode "The Riddle of the Sphinx".[79][80] InColumbo, episode 2 of season 7, fugu is used to kill the antagonist's victim.[citation needed] InThe Apothecary Diaries light novel, as well as the respective manga[81][82] and anime[83][84] adaptations, fugu toxin is encountered across multiple mystery arcs.
Based on the presumption that tetrodotoxin is not always fatal, but at near-lethal doses can leave a person extremely unwell with the person remaining conscious,[58] tetrodotoxin has been alleged to result inzombieism, and has been suggested as an ingredient inHaitian Vodou preparations.[85] This idea first appeared in the 1938 non-fiction bookTell My Horse byZora Neale Hurston in which there were multiple accounts of purported tetrodotoxin poisoning in Haiti by a voodoo sorcerer called thebokor.[86] These stories were later popularized byHarvard-trainedethnobotanistWade Davis[85] in his 1985book andWes Craven's 1988 film, both titledThe Serpent and the Rainbow. James Ellroy includes "blowfish toxin" as an ingredient inHaitian Vodou preparations to produce zombieism and poisoning deaths in his 2009 novelBlood's a Rover. However, this theory has been questioned by the scientific community since the 1990s based onanalytical chemistry-based tests of multiple preparations and review of earlier reports (see above).[66][67][68]
Maculotoxin, a potent neurotoxin isolated from the posterior salivary glands of the blue-ringed octopus. Hapalochlaena maculosa, has now been identified as tetrodotoxin. This is the first reported case in which tetrodotoxin has been found to occur in a venom.
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Tetrodotoxin blocks the sodium currents and is believed to have potential as a potent analgesic and as an effective agent in detoxoification from heroin addiction without withdrawal symptoms and without producing physical dependence.
