リシン (Ricin) は、トウゴマ（ヒマ）の種子から抽出されるタンパク質である。ヒマの種子に毒性があることは古くから知られていたが、1888年にエストニアのスティルマルク (en) が種子から有毒なタンパク質を分離し、リシンと名付けた。
1978年9月7日、ロンドンでブルガリア出身の作家ゲオルギー・マルコフが倒れ、4日後に死亡した。このニュースを聞いて驚いたパリ在住のウラジミール・コストフは何者かに傘の先端で突かれた2週間前の経験から直ちに病院で検査した結果、リシンが封入された約1mmの白金-イリジウム合金の弾丸が発見された（厚着をしていたため体内深くまで弾丸が打ち込まれなかった）。その後マルコフの体内からも同種の弾丸が発見され、被害者2名とも共産政権だったブルガリアからの亡命者であったことから、KGBかブルガリア秘密警察 (STB) による犯行と考えられた。リシン入りの弾丸を傘に偽装した空気銃により暗殺を行ったのである。
2013年の5月にニューヨーク市のマイケル・ブルームバーグ市長（当時）および彼が支援する団体「不法な銃に反対する市長たち（Mayors Against Illegal Guns）」のもとに送られてきた手紙からリシンが検出された。先のオバマ大統領の事件と同一犯として、2013年6月にテキサス州の女性が容疑者として逮捕された。
テトロドトキシン (tetrodotoxin, TTX) は化学式C11H17N3O8で表され、ビブリオ属やシュードモナス属などの一部の真正細菌によって生産されるアルカロイドである。一般にフグの毒として知られるが、他にアカハライモリ、ツムギハゼ、ヒョウモンダコ、スベスベマンジュウガニなど幾つかの生物もこの毒をもっている。習慣性がないため鎮痛剤として医療に用いられる。分子量319.27、CAS登録番号 [4368-28-9]。語源はフグ科の学名 (Tetraodontidae) と毒 (toxin) の合成語である。
Ricin ( reye-sin) is a highly toxic, naturally occurring lectin (a carbohydrate-binding protein) produced in the seeds of the castor oil plant, Ricinus communis. A dose of purified ricin powder the size of a few grains of table salt can kill an adult human. The median lethal dose (LD50) of ricin is around 22 micrograms per kilogram of body weight if the exposure is from injection or inhalation (1.78 milligram for an average adult).  Oral exposure to ricin is far less toxic as some of the poison is inactivated in the stomach. An estimated lethal oral dose in humans is approximately 1 milligram per kilogram.
Ricin is classified as a type 2 ribosome-inactivating protein (RIP). Whereas type 1 RIPs are composed of a single protein chain that possesses catalytic activity, type 2 RIPs, also known as holotoxins, are composed of two different protein chains that form a heterodimeric complex. Type 2 RIPs consist of an A chain that is functionally equivalent to a type 1 RIP, covalently connected by a single disulfide bond to a B chain that is catalytically inactive, but serves to mediate transport of the A-B protein complex from the cell surface, via vesicle carriers, to the lumen of the endoplasmic reticulum (ER). Both type 1 and type 2 RIPs are functionally active against ribosomes in vitro; however, only type 2 RIPs display cytotoxicity due to the lectin-like properties of the B chain. In order to display its ribosome-inactivating function, the ricin disulfide bond must be reductively cleaved.
Ricin is synthesized in the endosperm of castor oil plant seeds. The ricin precursor protein is 576 amino acid residues in length and contains a signal peptide (residues 1–35), the ricin A chain (36–302), a linker peptide (303–314), and the ricin B chain (315–576). The N-terminal signal sequence delivers the prepropolypeptide to the endoplasmic reticulum(ER) and then the signal peptide is cleaved off. Within the lumen of the ER the propolypeptide is glycosylated and a protein disulfide isomerasecatalyzes disulfide bond formation between cysteines 294 and 318. The propolypeptide is further glycosylated within the Golgi apparatus and transported to protein storage bodies. The propolypeptide is cleaved within protein bodies by an endopeptidase to produce the mature ricin protein that is composed of a 267 residue A chain and a 262 residue B chain that are covalently linked by a single disulfide bond.
The quaternary structure of ricin is a globular, glycosylated heterodimer of approximately 60–65 kDa. Ricin toxin A chain and ricin toxin B chain are of similar molecular weights, approximately 32 kDa and 34 kDa, respectively.
- Ricin toxic A chain (RTA) is an N-glycoside hydrolase composed of 267 amino acids. It has three structural domains with approximately 50% of the polypeptide arranged into alpha-helices and beta-sheets.The three domains form a pronounced cleft that is the active site of RTA.
- Ricin toxic B chain (RTB) is a lectin composed of 262 amino acids that is able to bind terminal galactose residues on cell surfaces. RTB forms a bilobal, barbell-like structure lacking alpha-helices or beta-sheets where individual lobes contain three subdomains. At least one of these three subdomains in each homologous lobe possesses a sugar-binding pocket that gives RTB its functional character.
While other plants contain the protein chains found in ricin, both protein chains must be present in order to produce toxic effects. For example, plants that contain only protein chain A, such as barley, are not toxic because without the link to protein chain B, protein chain A cannot enter the cell and do damage to ribosomes.
Ricin B chain binds complex carbohydrates on the surface of eukaryoticcells containing either terminal N-acetylgalactosamine or beta-1,4-linked galactose residues. In addition, the mannose-type glycans of ricin are able to bind cells that express mannose receptors. RTB has been shown to bind to the cell surface on the order of 106-108 ricin molecules per cell surface.
The profuse binding of ricin to surface membranes allows internalization with all types of membrane invaginations. The holotoxin can be taken up by clathrin-coated pits, as well as by clathrin-independent pathways including caveolae and macropinocytosis. Intracellular vesiclesshuttle ricin to endosomes that are delivered to the Golgi apparatus. The active acidification of endosomes is thought to have little effect on the functional properties of ricin. Because ricin is stable over a wide pH range, degradation in endosomes or lysosomes offers little or no protection against ricin. Ricin molecules are thought to follow retrograde transport via early endosomes, the trans-Golgi network, and the Golgi to enter the lumen of the endoplasmic reticulum (ER).
For ricin to function cytotoxically, RTA must be reductively cleaved from RTB in order to release a steric block of the RTA active site. This process is catalysed by the protein PDI (protein disulphide isomerase) that resides in the lumen of the ER. Free RTA in the ER lumen then partially unfolds and partially buries into the ER membrane, where it is thought to mimic a misfolded membrane-associated protein. Roles for the ER chaperones GRP94, EDEM and BiP have been proposed prior to the 'dislocation' of RTA from the ER lumen to the cytosol in a manner that utilizes components of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. ERAD normally removes misfolded ER proteins to the cytosol for their destruction by cytosolic proteasomes. Dislocation of RTA requires ER membrane-integral E3 ubiquitin ligasecomplexes, but RTA avoids the ubiquitination that usually occurs with ERAD substrates because of its low content of lysine residues, which are the usual attachment sites for ubiquitin. Thus, RTA avoids the usual fate of dislocated proteins (destruction that is mediated by targeting ubiquitinylated proteins to the cytosolic proteasomes). In the mammalian cell cytosol, RTA then undergoes triage by the cytosolic molecular chaperones Hsc70 and Hsp90 and their co-chaperones, as well as by one subunit (RPT5) of the proteasome itself, that results in its folding to a catalytic conformation, which de-purinates ribosomes, thus halting protein synthesis.
RTA has rRNA N-glycosylase activity that is responsible for the cleavage of a glycosidic bond within the large rRNA of the 60S subunit of eukaryotic ribosomes. RTA specifically and irreversibly hydrolyses the N-glycosidic bond of the adenine residue at position 4324 (A4324) within the 28S rRNA, but leaves the phosphodiester backbone of the RNA intact. The ricin targets A4324 that is contained in a highly conserved sequence of 12 nucleotides universally found in eukaryotic ribosomes. The sequence, 5’-AGUACGAGAGGA-3’, termed the sarcin-ricin loop, is important in binding elongation factors during protein synthesis. The depurination event rapidly and completely inactivates the ribosome, resulting in toxicity from inhibited protein synthesis. A single RTA molecule in the cytosol is capable of depurinating approximately 1500 ribosomesper minute.
Within the active site of RTA, there exist several invariant amino acid residues involved in the depurination of ribosomal RNA. Although the exact mechanism of the event is unknown, key amino acid residues identified include tyrosine at positions 80 and 123, glutamic acid at position 177, and arginine at position 180. In particular, Arg180 and Glu177 have been shown to be involved in the catalytic mechanism, and not substrate binding, with enzyme kinetic studies involving RTA mutants. The model proposed by Mozingo and Robertus, based on X-ray structures, is as follows:
- Sarcin-ricin loop substrate binds RTA active site with target adenine stacking against tyr80 and tyr123.
- Arg180 is positioned such that it can protonate N-3 of adenine and break the bond between N-9 of the adenine ring and C-1’ of the ribose.
- Bond cleavage results in an oxycarbonium ion on the ribose, stabilized by Glu177.
- N-3 protonation of adenine by Arg180 allows deprotonation of a nearby water molecule.
- Resulting hydroxyl attacks ribose carbonium ion.
- Depurination of adenine results in a neutral ribose on an intact phosphodiester RNA backbone.
Ricin is very poisonous if inhaled, injected, or ingested. It can also be poisonous if dust contacts the eyes or if it is absorbed through damaged skin. It acts as a toxin by inhibiting protein synthesis. It prevents cells from assembling various amino acids into proteins according to the messages it receives from messenger RNA in a process conducted by the cell's ribosome (the protein-making machinery)—that is, the most basic level of cell metabolism, essential to all living cells and thus to life itself. Ricin is resistant, but not impervious, to digestion by peptidases. By ingestion, the pathology of ricin is largely restricted to the gastrointestinal tract, where it may cause mucosal injuries. With appropriate treatment, most patients will make a full recovery.
Because the symptoms are caused by failure to make protein, they may take anywhere from hours to days to appear, depending on the route of exposure and the dose. When ingested, gastrointestinal symptoms can manifest within 6 hours; these symptoms do not always become apparent. Within 2 to 5 days of exposure to ricin, effects of ricin on the central nervous system, adrenal glands, kidneys, and liver appear.
Ingestion of ricin causes pain, inflammation, and hemorrhage in the mucous membranes of the gastrointestinal system. Gastrointestinal symptoms quickly progress to severe nausea, vomiting, diarrhea, and difficulty swallowing (dysphagia). Hemorrhage causes bloody feces (melena) and vomiting blood (hematemesis). The low blood volume (hypovolemia) caused by gastrointestinal fluid loss can lead to organ failure in the pancreas, kidney, liver, and GI tract and progress to shock. Shock and organ failure are indicated by disorientation, stupor, weakness, drowsiness, excessive thirst (polydipsia), low urine production (oliguria), and bloody urine (hematuria).
Symptoms of ricin inhalation are different from those caused by ingestion. Early symptoms include a cough and fever.
When skin or inhalation exposure occur, ricin can cause an allergy to develop. This is indicated by edema of the eyes and lips; asthma; bronchial irritation; dry, sore throat; congestion; skin redness (erythema); skin blisters (vesication); wheezing; itchy, watery eyes; chest tightness; and skin irritation.
An antidote has been developed by the UK military, although it has not yet been tested on humans. Another antidote developed by the U.S. military has been shown to be safe and effective in lab mice injected with antibody-rich blood mixed with ricin, and has had some human testing.
Symptomatic and supportive treatments are available for ricin poisoning, but there is no antidote for ricin available for humans. Existing treatments emphasize minimizing the effects of the poison. Possible treatments include intravenous fluids or electrolytes, airway management, assisted ventilation, or giving medications to remedy seizures and low blood pressure. If the ricin has been ingested recently, the stomach can be flushed by ingesting activated charcoal or by performing gastric lavage. Survivors often develop long-term organ damage. Ricin causes severe diarrhea and vomiting, and victims can die of circulatory shock or organ failure; inhaled ricin can cause fatal pulmonary edema or respiratory failure. Death typically occurs within 3–5 days of exposure.
Although there is no antidote currently available for ricin poisoning, vaccination is possible by injecting an inactive form of protein chain A.This vaccination is effective for several months due to the body's production of antibodies to the foreign protein. In 1978 Bulgarian defector Vladimir Kostov survived a ricin attack similar to the one on Georgi Markov, probably due to his body's production of antibodies. When a ricin-laced pellet was removed from the small of his back it was found that some of the original wax coating was still attached. For this reason only small amounts of ricin had leaked out of the pellet, producing some symptoms but allowing his body to develop immunity to further poisoning.
The seeds of Ricinus communis are commonly crushed to extract castor oil. As ricin is not oil-soluble, little is found in the extracted castor oil.The extracted oil is also heated to more than 80 °C to denature any ricin that may be present. The remaining spent crushed seeds, called variously the "cake", "oil cake", and "press cake", can contain up to 5% ricin. While the oil cake from coconut, peanuts, and sometimes cotton seeds can be used as either cattle feed and/or fertilizer, the toxic nature of castor beans precludes their oil cake from being used as feed unless the ricin is first deactivated by autoclaving. Accidental ingestion of Ricinus communis cake intended for fertilizer has been reported to be responsible for fatal ricin poisoning in animals.
Deaths from ingesting castor plant seeds are rare, partly because of their indigestible seed coat, and because the body can, although only with difficulty, digest ricin. The pulp from eight beans is considered dangerous to an adult. Rauber and Heard have written that close examination of early 20th century case reports indicates that public and professional perceptions of ricin toxicity "do not accurately reflect the capabilities of modern medical management".
Most acute poisoning episodes in humans are the result of oral ingestion of castor beans, 5–20 of which could prove fatal to an adult. However, swallowing castor beans rarely proves to be fatal unless the bean is thoroughly chewed. The survival rate of castor bean ingestion is 98%.In 2013 a 37-year-old female in the United States survived after ingesting 30 beans. Victims often manifest nausea, diarrhea, fast heart rate, low blood pressure, and seizures persisting for up to a week. Blood, plasma, or urine ricin or ricinine concentrations may be measured to confirm diagnosis. The laboratory testing usually involves immunoassay or liquid chromatography-mass spectrometry.
|This section needs additional citations for verification. (March 2014) |
Although no approved therapeutics are currently based on ricin, it does have the potential to be used in the treatment of tumors, as a "magic bullet" to destroy targeted cells. Because ricin is a protein, it can be linked to a monoclonal antibody to target cancerous cells recognized by the antibody. The major problem with ricin is that its native internalization sequences are distributed throughout the protein. If any of these native internalization sequences are present in a therapeutic agent, the drug will be internalized by, and kill, untargeted non-tumorous cells as well as targeted cancerous cells.
Modifying ricin may sufficiently lessen the likelihood that the ricin component of these immunotoxins will cause the wrong cells to internalize it, while still retaining its cell-killing activity when it is internalized by the targeted cells. However, bacterial toxins, such as diphtheria toxin, which is used in denileukin diftitox, an FDA-approved treatment for leukemia and lymphoma, have proven to be more practical. A promising approach for ricin is to use the non-toxic B subunit (a lectin) as a vehicle for delivering antigens into cells, thus greatly increasing their immunogenicity. Use of ricin as an adjuvant has potential implications for developing mucosal vaccines.
In the U.S., ricin appears on the select agents list of the Department of Health and Human Services, and scientists must register with HHS to use ricin in their research. However, investigators possessing less than 100 mg are exempt from regulation.
It is classified as an extremely hazardous substance in the United States as defined in Section 302 of the U.S. Emergency Planning and Community Right-to-Know Act (42 U.S.C. 11002), and is subject to strict reporting requirements by facilities that produce, store, or use it in significant quantities.
The United States investigated ricin for its military potential during World War I. At that time it was being considered for use either as a toxic dust or as a coating for bullets and shrapnel. The dust cloud concept could not be adequately developed, and the coated bullet/shrapnel concept would violate the Hague Convention of 1899 (adopted in U.S. law at 32 Stat. 1903), specifically Annex §2, Ch.1, Article 23, stating "... it is especially prohibited ... [t]o employ poison or poisoned arms".World War I ended before the United States weaponized ricin.
During World War II the United States and Canada undertook studying ricin in cluster bombs. Though there were plans for mass productionand several field trials with different bomblet concepts, the end conclusion was that it was no more economical than using phosgene. This conclusion was based on comparison of the final weapons, rather than ricin's toxicity (LCt50 ~40 mg·min/m3). Ricin was given the military symbolW or later WA. Interest in it continued for a short period after World War II, but soon subsided when the U.S. Army Chemical Corpsbegan a program to weaponize sarin.
The Soviet Union also possessed weaponized ricin. There were speculations that the KGB used it outside the Soviet bloc; however, this was never proven.
Given ricin's extreme toxicity and utility as an agent of chemical/biological warfare, it is noteworthy that the production of the toxin is rather difficult to limit. The castor bean plant from which ricin is derived is a common ornamental and can be grown at home without any special care.
Under both the 1972 Biological Weapons Convention and the 1997 Chemical Weapons Convention, ricin is listed as a schedule 1 controlled substance. Despite this, more than 1 million tonnes of castor beans are processed each year, and approximately 5% of the total is rendered into a waste containing negligible concentrations of undenatured ricin toxin.
Ricin is several orders of magnitude less toxic than botulinum or tetanus toxin, but the latter are harder to come by. Compared to botulinum or anthrax as biological weapons or chemical weapons, the quantity of ricin required to achieve LD50 over a large geographic area is significantly more than an agent such as anthrax (tons of ricin vs. only kilogram quantities of anthrax). Ricin is easy to produce, but is not as practical or likely to cause as many casualties as other agents. Ricin is easily denatured by temperatures over 80 °C (175 °F) meaning many methods of deploying ricin would generate enough heat to denature it. Once deployed an area contaminated with ricin remains dangerous until the bonds between chain A or B have been broken, a process that takes two or three days. In contrast, anthrax spores may remain lethal for decades. Jan van Aken, a German expert on biological weapons, explained in a report for The Sunshine Project that Al Qaeda's experiments with ricin suggest their inability to produce botulinum or anthrax.
A biopharmaceutical company called Soligenix, Inc. has licensed an anti-ricin vaccine called RiVax™ from Vitetta et al. at UT Southwestern. The vaccine is safe and immunogenic in mice, rabbits, and humans. It has completed two successful clinical trials.
Ricin has been involved in a number of incidents. In 1978, the Bulgariandissident Georgi Markov was assassinated by Bulgarian secret policewho surreptitiously shot him on a London street with a modified umbrellausing compressed gas to fire a tiny pellet contaminated with ricin into his leg. He died in a hospital a few days later and his body was passed to a special poison branch of the British Ministry of Defence (MOD) that discovered the pellet during an autopsy. The prime suspects were the Bulgarian secret police: Georgi Markov had defected from Bulgaria some years previously and had subsequently written books and made radio broadcasts that were highly critical of the Bulgarian communist regime. However, it was believed at the time that Bulgaria would not have been able to produce the pellet, and it was also believed that the KGB had supplied it. The KGB denied any involvement, although high-profile KGB defectors Oleg Kalugin and Oleg Gordievsky have since confirmed the KGB's involvement. Earlier, Soviet dissident Aleksandr Solzhenitsyn also suffered (but survived) ricin-like symptoms after an encounter in 1971 with KGB agents.
Ten days before the attack on Georgi Markov, Bulgarian defector, Vladimir Kostov survived an attack similar to the one against Markov. Kostov was standing on an escalator of the Paris metro when he felt a sting in his lower back above the belt of his trousers. He developed a fever, but recovered. After Markov's death the wound on Kostov's back was examined and a ricin-laced pellet identical to the one used against Markov was removed.
Several terrorists and terrorist groups have experimented with ricin and caused several incidents of the poisons being mailed to U.S. politicians. For example, on May 29, 2013 two anonymous letters sent to New York City Mayor Michael Bloomberg contained traces of it. Another was sent to the offices of Mayors Against Illegal Guns in Washington DC. A letter containing ricin was also alleged to have been sent to American PresidentBarack Obama at the same time. An actress, Shannon Richardson, was later charged with the crime, to which she pleaded guilty that December. On July 16, 2014, Richardson was sentenced to 18 years in prison plus a restitution fine of $367,000.
Ricin has often been used as a plot device, such as in the television series Breaking Bad (Season 2, Season 4 and Season 5).
The popularity of Breaking Bad inspired several real-life criminal cases involving ricin or similar substances. Kuntal Patel from London attempted to poison her "controlling and selfish" mother with abrin after the latter interfered with her marriage plans. Daniel Milzman, a 19-year-old former Georgetown University student, was charged with manufacturing ricin in his dorm room, as well as the intent of "[using] the ricin on another undergraduate student with whom he had a relationship". Mohammed Ali from Liverpool, England was convicted after attempting to purchase 500 mg of Ricin over the dark web from an undercover FBI agent. He was sentenced, on 18 September 2015, to 8 years' imprisonment.
- マウス経口 LD50 0.01 mg/kg
- マウス皮下 LD50 0.0085 mg/kg
シアン化ナトリウムの神経毒に対し、テトロドトキシンは 1 µM 濃度以上で神経保護が発現する。ベラトリジンの神経毒に対するテトロドトキシの神経保護作用は IC50=30 nM 。
Tetrodotoxin (TTX) is a potent neurotoxin. Its name derives from Tetraodontiformes, an order that includes pufferfish, porcupinefish, ocean sunfish, and triggerfish; several of these species carry the toxin. Although tetrodotoxin was discovered in these fish and found in several other aquatic animals (e.g., in blue-ringed octopuses, rough-skinned newts, and moon snails), it is actually produced by certain infecting or symbiotic bacteria like Pseudoalteromonas, Pseudomonas, and Vibrioas well as other species found in the animals .
Tetrodotoxin inhibits the firing of action potentials in nerves by binding to the voltage-gated sodium channels in nerve cellmembranes and blocking the passage of sodium ions (responsible for the rising phase of an action potential) into the nerve cell (in layman terms, it prevents the nervous system from carrying messages and prevents muscles from flexing).
Its mechanism of action, selective blocking of the sodium channel, was shown definitively in 1964 by Toshio Narahashi and John W. Moore at Duke University, using the sucrose gap voltage clamp technique.
Apart from their bacterial species of most likely ultimate biosynthetic origin (see below), tetrodotoxin has been isolated from widely differing animal species, including:
- various pufferfish species,
- certain angelfish,
- several species of the blue-ringed octopus, including Hapalochlaena maculosa (where it was called "maculotoxin"),
- species of Niotha gastropods,
- species of genus Naticidae (moon snails),
- several starfish, including Astropecten species,
- several species of xanthid crabs.
- species of Chaetognatha (arrow worms),
- species of Nemertea (ribbon worms),
- a polyclad flatworm,
- land planarians of the genus Bipalium,
- toads of the genus Atelopus, and
- western, rough-skinned newts of the genus Taricha(wherein it was originally termed "tarichatoxin"),
- The Eastern newt (Notophthalmus viridescens)
Tarichatoxin was shown to be identical to TTX in 1964 by Mosher et al, and the identity of maculotoxin and TTX was reported in Sciencein 1978, and the synonymity of these two toxins is supported in modern reports (e.g., at Pubchem and in modern toxicology textbooks) though historic monographs questioning this continue in reprint.
The toxin is variously used by metazoans as a defensive biotoxin to ward off predation, or as both a defensive and predatory venom (e.g., in octopuses, chaetognaths, and ribbon worms). Even though the toxin acts as a defense mechanism, some predators such as the common garter snake have developed insensitivity to TTX, which allows them to prey upon toxic newts.
The association of TTX with consumed, infecting, or symbiotic bacterial populations within the metazoan species from which it is isolated is, as of 2016, relatively clear; presence of TTX-producing bacteria within a metazoan'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. As Lago et al. note, "there is good evidence that uptake of bacteria producing TTX is an important element of TTX toxicity in marine metazoans that present this toxin." TTX-producing bacteria include Actinomyces, Aeromonas, Alteromonas, Bacillus, Pseudomonas, and Vibrio species; in the following animals, specific bacterial species have been implicated:
- Vibrio species including Vibrio alginolyticus, from the puffer fish, Fugu vermicularis,
- Vibrio alginolyticus, from the starfish species Astropecten polyanthus,
- Aeromonas species from the puffer fish, Takifugu obscures,
- both Vibrio, Pseudomonas, and Aeromonas species from gastropod Niotha clathrata,
- Alteromonas, Bacillus, Pseudomonas, and Vibrio species from the blue-ringed octopus species Hapalochlaena macula,
- Vibrio species, including Vibrio alginolyticus again, in arrow worms, phylum Chaetognatha, and
- Vibrio species, again, in ribbon worms, phylum Nemertea.
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," 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"—although the correlation has been extended to most but not all metazoans in which the toxin has been identified. 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), using PCR methods, although technical concerns about the approach have been raised. 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-free Fugu niphobles puffers fed on TTX-containing diets saw TTX in the livers of the fishes increase to toxic levels. 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. Nevertheless, without clear biosynthetic pathways (not yet found in metazoans, but shown for bacteria), it remains uncertain whether it is simply via bacteria that each metazoan accumulates TTX; whether the quantities can be sufficiently explained by ingestion, this plus colonization, or some other mechanism.
|This section needs additional citations for verification. (April 2016) |
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, thereby blocking the passage of sodium ions into the nerve cell (which is ultimately necessary for nerve conduction); 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 nM, while the TTX-r Na+ channels bind TTX with low micromolaraffinity. 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.
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. 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.
In 1964 a team of scientists led by Robert B. Woodward at Harvard University elucidated the structure of tetrodotoxin. The structure was confirmed by X-ray crystallography in 1970. Yoshito Kishi and coworkers at Nagoya University, Nagoya, Japan, (now at Harvard University) reported the first total synthesis of D,L-tetrodotoxin in 1972. M. Isobe and coworkers at Nagoya University, Japanand J. Du Bois et al. at Stanford University, U.S., reported the asymmetrictotal 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. Since then, methods have rapidly advanced, with several new strategies for the synthesis of tetrodotoxin having been developed.
TTX is extremely toxic. The Material Safety Data Sheet for TTX lists the oral median lethal dose (LD50) for mice as 334 μg per kg. For comparison, the oral LD50 of potassium cyanide for mice is 8.5 mg per kg, demonstrating that even orally, TTX is more poisonous than cyanide. TTX is even more dangerous if injected; the amount needed to reach a lethal dose by injection is only 8 μg per kg in mice.
The toxin can enter the body of a victim by ingestion, injection, or inhalation, or through abraded skin.
Poisoning occurring as a consequence of consumption of fish from the order Tetraodontiformes is extremely serious. The organs (e.g. liver) of the pufferfish can contain levels of tetrodotoxin sufficient to produce the described paralysis of the diaphragm and corresponding 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.
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. As a result, TTX causes loss of sensation, and paralysis of voluntary muscles including the diaphragm and intercostal muscles, stopping breathing.
The therapeutic uses of puffer fish (tetraodon) eggs were mentioned in the first Chinese pharmacopea (Pen-T’ so Ching, The Book of Herbs, allegedly 2838–2698 BC by Shénnóng Běn Cǎo Jīng; but a 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 principle use was “to arrest convulsive diseases”. In the Pen-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 for tetraodon) were also recognized as both toxic and (at the right dose) could be used to prepare 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, noting that tetrodotoxin is slightly water-soluble, and soluble at 1 mg/mL in slightly acidic solutions.
The German physician Engelbert 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. There is also evidence from other sources that knowledge of such toxicity was widespread throughout southeast Asia and India.
The first recorded cases of TTX poisoning affecting Westerners are from the logs of Captain James Cook from 7 September 1774. 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. It was one of the agents studied by Japan's Unit 731, which evaluated biological weapons on human subjects in the 1930s.
The diagnosis of pufferfish poisoning is based on the observed symptomatology and recent dietary history.
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. Paresthesia of the lips and tongue is followed by developing paresthesia in the extremities, hypersalivation, sweating, headache, weakness, lethargy, incoordination, tremor, paralysis, cyanosis, aphonia, dysphagia, and seizures. The gastrointestinal symptoms are often severe and include nausea, vomiting, diarrhea, and abdominal pain; death is usually secondary to respiratory failure. There is increasing respiratory distress, speech is affected, and the victim usually exhibits dyspnea, cyanosis, mydriasis, 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, which generally occurs within 4 to 6 hours (range ~20 minutes to ~8 hours). However, some victims enter a coma.
If the patient survives 24 hours, recovery without any residual effects will usually occur over a few 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 proposed as a treatment option but have not been tested adequately".
No antidote has been developed and approved for human use, but a primary research report (preliminary result) indicates that a monoclonal antibody specific to tetrodotoxin is in development by USAMRIID that was effective, in the one study, for reducing toxin lethality in tests on mice.
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
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.voodoo preparations, in so-called zombie poisons, where subsequent careful analysis has repeatedly called early studies into question on technical grounds, and have failed to identify the toxin in any preparation, such that discussion of the matter has 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.”:748
In Haiti, tetrodotoxin is thought to have been used in
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.
- Fugu as a food
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.
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 has been investigated as a possible treatment for cancer-associated pain. Early clinical trials demonstrate significant pain relief in some patients.
In addition to the cancer pain application mentioned, mutations in one particular TTX-sensitive Na+ channel are associated with some migraineheadaches, although it is unclear as to whether this has any therapeutic relevance for most people with migraine.
Tetrodotoxin has been used clinically to relieve the headache associated with heroin withdrawal.
|The examples and perspective in this article deal primarily with the United States and do not represent a worldwide view of the subject. (February 2017)|
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 500 mg are exempt from regulation.
Tetrodotoxin serves as a plot device for characters to fake death, as in the films Miami Vice (1985), Hello Again (1987), The A-Team (2010) and Captain America: The Winter Soldier (2014), and in episodes of Nikita, MacGyver Season 7, Episode 6, where the antidote is datura stramonium leaf, CSI: NY (Season 4, episode 9 "Boo") and Chuck. In Law Abiding Citizen (2009) its paralysis is presented as a method of assisting torture. The toxin is used as a weapon in Covert Affairs. In episode 16 of Dragon Ball, the characters are inadvertently poisoned by a puffer fish soup. In season 2 episode 11 of The Simpsons, Homer ingests an improperly cut Fugu and is given 22 hours to live.
In the sci-fi series Orphan Black, a half organic, half mechanical "maggot bot" engineered by Evie Cho as a vector for gene therapy delivery to patients, makes use of tetrodotoxin as a defence mechanism to protect the device against tampering.
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, tetrodotoxin has been alleged to result in zombieism, and has been suggested as an ingredient in Haitian Vodoupreparations. This idea first appeared in the 1938 non-fiction book Tell My Horse by Zora Neale Hurston in which there were multiple accounts of purported tetrodotoxin poisoning in Haiti by a voodoo sorcerer called the Bokor. These stories were later popularized by Harvard-trained ethnobotanist Wade Davis in his 1985 book and Wes Craven's 1988 film, both titled The Serpent And The Rainbow. But this theory has been dismissed by the scientific community since the 1990s based on analytical chemistry-based tests of multiple preparations and review of earlier reports (see above).
In the 2007 remake of The Wizard of Gore, a mind control drug, referred to as Tetrodotoxin, is used by Montag the Magnificent during his performances in order to create his gory illusions.