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Home , Neurotoxin, Tetrodotoxin

Niharika Mandal et al. / Journal of Pharmacy Research 2012,5(7),3567-3570 Review Article Available online through ISSN: 0974-6943 http://jprsolutions.info : An intriguing molecule Niharika Mandal*, Samanta Sekhar Khora, Kanagaraj Mohanapriya, and Soumya Jal School of Biosciences and Technology, VIT University, Vellore-632013 Tamil Nadu, India Received on:07-04-2012; Revised on: 12-05-2012; Accepted on:16-06-2012

ABSTRACT Tetrodotoxin (TTX) is one of the most potent of biological origin. It was first isolated from puffer and it has been discovered in various arrays of organism since then. Its origin is still unclear though some reports indicate towards microbial origin. TTX selectively blocks the , inhibiting thereby, leading to respiratory . TTX is mainly caused due to consumption of puffer fish. No Known for TTX exists. Treatment is symptomatic. The present review is therefore, an effort to give an idea about the distribution, origin, structure, pharmacol- ogy, toxicity, symptoms, treatment, resistance and application of TTX.

Key words: Tetrodotoxin, Neurotoxin, Puffer fish.

INTRODUCTION One of the most intriguing biotoxins isolated and described in the twentieth cantly more toxic than TTX. Palytoxin and have potencies nearly century is the neurotoxin, Tetrodotoxin (TTX, CAS Number [4368-28-9]). 100 times that of TTX and , and all four are unusual in being A neurotoxin is a that acts specifically on usually by interact- non-proteins. Interestingly, there is also some evidence for a bacterial bio- ing with membrane proteins and channels mostly resulting in paralysis. genesis of saxitoxin, palytoxin, and maitotoxin in living the toxin TTX is a so lethal that the US Food and Administration warn acts primarily on myelinated (sheathed) peripheral nerves and does not it can to “rapid and violent ”. It is named after the order of fish appear to cross the - barrier.” from which it is most commonly associated, the . The member of this order includes the various types of puffer fish. It was first Distribution isolated in 1909 from Spheroides rubripes (puffer fish).[1] Pure crystalline A total of 22 species of puffers in the family are reported to form of TTX was isolated in 1950 from the overies of rubripes contain TTX, [8] while the closely related porcupine fish and boxfish does (puffer fish).[2] TTX was believed to occur only in puffer fish for a long not contain TTX. TTX is accumulated in the , gonads, intestine, muscle time, however the toxin has been found in a variety of animals. The toxin is and of the puffer fish.[9-10] The distribution of TTX in puffer fish varies variously used as a defensive biotoxin to ward off predation as in case of between species and at different seasons and geographic localities. Other puffer fish,[3] or as both defensive and predatory like in the case of marine organisms which contain TTX are worms such as Lineus Fuscoviridis blue ringed .[4-6] annelids like Pseudopolamilla occelata, Snails like Charonia and Niotha, Crustaceans like (Carcinoscorpius rotundicauda) as well TTX is a non protienaceous low molecular weight (319.28 amu) neuro- as Xanthid Crabs of different genera, of genus Astropecte, goby fish toxin. TTX is highly polar and hygroscopic and is only sparingly soluble in (Yongeichthys criniger) etc. Some blue-ringed of genus acidified water. It has a pk of 8.5. A single milligram or less of TTX - an Hapalochlaena contain TTX in their posterior salivary glands and soft amount that can be placed on the head of a pin, is enough to kill an . tissues.[6] Some terrestrial like Californian ( torosa) The LD50 of TTX for is 10.2µg/kg. [7] and from genus Atelopus were also reported to contain TTX.[11-12] This highly potent neurotoxin is found in a diverse array of organisms, including According to William H. Light, “Tetrodotoxin is ten times as deadly as the , , arthropods, nematodes, mollusks, fish, and am- venom of the many- of Southeast Asia. It is 10 to 100 times as phibians. The number of species containing TTX continues to grow. lethal as black widow venom (depending upon the species) when administered to mice, and more than 10,000 times deadlier than . It Origin has the same toxicity as saxitoxin which causes paralytic shellfish poison- The metabolic source of TTX is still uncertain. Until now TTX was be- ing ([both TTX and saxitoxin block the Na+ channel - and both are found in lieved to be produced by the (or the host). However, recent reports the tissues of puffer fish]) A recently discovered, naturally occurring con- point towards the bacterial origin of this toxin TTX is considered likely to gener of tetrodotoxin has proven to be four to five times as potent as TTX. be produced by marine bacteria associated with marine animals. The most Except for a few bacterial protein toxins, only palytoxin, a bizarre molecule common TTX producing bacteria are bacteria, with Vibrio isolated from certain zoanthideans (small, colonial, marine organisms re- alginolvticus being the most common species. Puffer fish,[13] sembling sea anemones) of the genus Palythoa, and maitotoxin, found in Chaetognathas,[14] and Nemerteans,[15] have been shown to contain and Vibrio certain associated with ciguatera , are known to be signifi- alginolvticus TTX. These TTX producing bacteria are primarily marine species,[16-19] although a few freshwater species have also been identified.[20] [21-23] *Corresponding author. A vast array of taxonomic groups is known to possess TTX. This is unusual for animal toxins, as they are usually very specific for a particular Niharika Mandal Research Associate group. In marine organisms the accepted hypothesis is that the TTX present School of Biosciences and Technology in metazoans results from either dietary uptake of bacterially produced [22-26] Medical Biotechnology Laboratory TTX or with TTX producing bacteria. However this hy- [21, 27-30] SMV 203, VIT University, pothesis is not acceptable for TTX present in terrestrial taxa. This is Vellore 632013,Tamil Nadu, India probably because TTX in terrestrial metazoans appears to be limited to a

Journal of Pharmacy Research Vol.5 Issue 7.July 2012 3567-3570 Niharika Mandal et al. / Journal of Pharmacy Research 2012,5(7),3567-3570 single class of vertebrates (Amphibia) with limited distributions within the Tetrodotoxin Toxicity class, unlike marine species in which TTX is found in a wide array of taxa. The first recorded case of tetrodotoxin poisoning was on 7 September Moreover, the presence of multiple analogs of TTX are common in the 1774,[1] when Captain James Cook recorded his crew eating some local TTX profiles of some TTX bearing , but are absent or a very tropic fish (puffer fish), then feeding the remains to the pigs kept on board. minor component in the TTX profiles of marine taxa or TTX producing The crew experienced numbness and , while the pigs bacteria.[31-37] Therefore, several genera of bacteria have been identified as were all found dead the next morning. It was clear that the crew received a TTX producers,[19, 26, 38] but these findings are controversial.[39-41]The pro- mild dose of tetrodotoxin, while the pigs ate the puffer fish body parts that duction of TTX in animals has not been firmly established, and there re- contain most of the toxin, thus being fatally poisoned. Tetrodotoxin toxic- mains much debate in the literature as to whether the bacteria are truly the ity mostly occurs due to the consumption of puffer fish. The organs (e.g. source of TTX in animals. liver) of the puffer fish can contain levels of tetrodotoxin sufficient to pro- duce paralysis of the diaphragm and death due to respiratory failure,[1] On Structure the other hand, puffer fish is considered as a notorious delicacy in Japan. It The structure of TTX was first elucidated in 1964 at the is prepared by chefs who are specially trained and certified by the govern- Symposium of the International Union of Pure applied Chemistry by four ment. Despite these precautions, many cases of tetrodotoxin poisoning are different Lab groups including K. Tsuda, T. Goto, R.B. Woodward and H.S. reported each year in patients ingesting puffer fish. Mosher.[42] TTX has a guanidium ion with a complex oxygenated cyclohex- ane framework with both guanidine and orth-oacid functional groups. [43-45] TTX is not always fatal, but at near-lethal doses, it can leave a person in a Numerous natural, synthetic and semi-synthetic analogs of TTX have been state of near-death for several days, while the person remains conscious. reported. Amphibians have been a plentiful source of TTX analogs.[31-32, 46- That is why, TTX has been used an ingredient in Haitian Vodou and the 48] The hemilactal forms of TTX are more common naturally occurring closest approximation of zombieism, an idea popularized by Harvard- analogs. These analogs are very potent and have equivalent or trained ethnobotanist Wade Davis in a 1983 paper, and in his 1985 book, greater than TTX itself. [47-51] The structure and analogs of TTX are shown The Serpent and the Rainbow. This idea was dismissed by the scientific in Fig1. community in the 1980s, as the descriptions of voodoo do not match the symptoms displayed by victims of tetrodotoxin poisoning, and the alleged incidents of zombies created in this manner could not be sub- The pharmacology of TTX was studied by Takahashi in 1889,[42] but, it stantiated.[58] was not until 1950’s that more detailed understanding of the pharmacology of TTX began to emerge. TTX was shown to block sodium channels in Symptoms excitable membranes.[52-54] It is found that TTX binds and blocks voltage Symptoms typically develop within 30 minutes of , but may be -10 gated sodium channels with very high specificity (Kd = 10 nM), thereby delayed by up to four hours however; death within 17 minutes of ingestion preventing the influx of sodium . The positively charged amino end of has also been recorded.[1] The first symptom of intoxication is a slight TTX forms complex electrostatic bonds with two charged rings of amino numbness of the lips and tongue.[1] followed by in face and acid residues in the outer pore of the sodium channel. [55-57] extremities, sweating, headache, weakness, lethargy, incoordination, , paralysis, , aphonia, , , dyspnea, bronchorrhea, The flow of sodium ions into nerve cells is a necessary step in the conduc- bronchospasm, respiratory failure, , and . Gastroenteric tion of nerve impulses in excitable nerve fibers and along . Normal symptoms are often severe and include , , , and cells have high concentrations of K+ ions and low concentrations of abdominal . Cardiac may precede complete respiratory Na+ ions and have a negative potential. Stimulation of the axon results in an failure and cardiovascular collapse. If the patient survives 24 hours, recov- action potential which arises from a flow of Na+ions into the and the ery without any residual effects usually occurs over several days. generation of a positive membrane potential. Propagation of this depolar- ization along the nerve terminal presages all other events. The Na+ ions Treatment flow through the cellular membrane employing the sodium , a No known antidote for TTX poisoning exists. Treatment is symptomatic channel that is selective for sodium ions over potassium ions by an order with aggressive early airway management, it includes emptying the stom- of magnitude. Tetrodotoxin is quite specific in blocking the Na+ ion channel ach, feeding the victim activated charcoal to bind the toxin, and taking stan- and therefore the flow of Na+ ions while having no effect on dard life-support measures to keep the victim alive until the effect of the K+ ions. Tetrodotoxin competes with the hydrated sodium cataion and en- poison has worn off.[1] Alpha adrenergic are recommended in addi- ters the Na+-channel where it binds. The hydrated sodium ion binds re- tion to intravenous fluids to combat hypotension. Anticholinesterase agents versibly on a nanosecond time-scale, whereas TTX is bound for tens of have been used with mixed success. A specific to seconds. With the bulk of the TTX molecule denying sodium the opportu- tetrodotoxin has been developed and was shown to be effective for reducing nity to enter the channel, sodium movement is effectively shut down, and lethality in murine tests.[59] the action potential along the nerve membrane ceases. Resistance to TTX Saxitoxin and several of the also bind the same site. Compared An obvious question is, since TTX is so lethal, then how do animals like the with saxitoxin, Tetrodotoxin is slightly less neuroactive but has more pro- puffer fish and Taricha newts that accumulate it in large concentrations in longed effects. It is less easily reversed and creates small transient potentia- their bodies remain unaffected? This is because Puffers have evolved with tion of maximal muscular contraction with subliminal doses. Tetrodotoxin a slightly different sodium channel which still allows sodium ion to pass has less effect on muscle fiber (saxitoxin can cause neuromuscular muscle through without becoming inhibited by TTX. It is due to the key substitu- weakness without hypotension); however it causes hypotension via effect tions of amino acids which modify the sodium channel thereby, reducing on the vasomotor tone through preganglionic fibers or direct action on the affinity to the toxin and allowing the channels to function normally. [60] cardiac muscle. Similar to puffer fish resistance newts have also developed resistance due to conformational change in the sodium channel.[21] Interestingly, garter (Thamnophis sirtalis) that prey on the Taricha have

Journal of Pharmacy Research Vol.5 Issue 7.July 2012 3567-3570 Niharika Mandal et al. / Journal of Pharmacy Research 2012,5(7),3567-3570 also acquired TTX resistance.[61-62] Snakes from populations that do not 11. Wakely JF, Fuhrman GJ, Fuhrman FA, Fischer HG, Mosher HS, encounter the newts in the wild, however, are poisoned when fed newts in The occurrence of tetrodotoxin (tarichatoxin) in amphibia and the the laboratory, indicating a co-evolutionary interaction between the preda- distribution of the toxin in the organs of newts (Taricha). Toxicon tor and its prey. [7] 1996, 3,195-203. 12. Yamashita MY, Mebs D, Yasumoto T, Tetrodotoxin and its ana- Application of TTX logues in extracts from the toad Atelopus oxyrhynchus (family: The use of TTX as a biochemical probe has elucidated two distinct types of Bufonidae). Toxicon, 1992, 30,1489-1492. voltage-gated sodium channels present in : the tetrodotoxin-sensi- 13. Noguchi T, Hwang DF, Arakawa O, Sugita H, Deguchi Y, Shida Y, tive voltage-gated sodium channel (TTX-s Na+ channel) and the tetrodot- Hashimoto K, Vibrio alginolyticus, a tetrodotoxin-producing bac- oxin-resistant voltage-gated sodium channel (TTX-r Na+ channel). Tetro- terium, in the intestines of the fish Fugu vermicularis dotoxin binds to TTX-s Na+ channels with a very high binding affinity, vermicularis. Marine Biology, 1987, 94,625–630. while the TTX-r Na+ channels bind TTX with very low affinity. Nerve 14. Thuesen EV, Kogure K, Bacterial production of tetrodotoxin in cells containing TTX-r Na+ channels are located primarily in cardiac tissue, four species of ”. Biological Bulletin, 176, 1989, while nerve cells containing TTX-s Na+ channels dominate the rest of the 191–194. body. The prevalence of TTX-s Na+ channels in the central nervous 15. 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Journal of Pharmacy Research Vol.5 Issue 7.July 2012 3567-3570