Natural Toxicants: Tetrodotoxin

NATURAL TOXICANTS

Tetrodotoxin K Campbell and S Haughey, Queen’s University Belfast, Belfast, UK r 2014 Elsevier Inc. All rights reserved.

Glossary Biosensor An analytical tool that detects an analyte through the conversion of a biological response into an electrical signal with a physicochemical detector. Neurotoxin A toxic component that targets specifically nerve cells or neurons primarily by interacting with membrane proteins such as ion channels.

History and Background Tetrodotoxin (CAS number 4368-28-9) is a powerful, low molecular weight (B319 Dalton (Da)), naturally occurring neurotoxin. It is named from the Teleost fish order Tetrodontiformes from which the toxin was first isolated and described. This fish species received their name because they have four very strong teeth that almost fuse together to form a beak-like structure, which they use to chew and crack shells open to get food. Tetrodontid fish, which include puffer fish or fugu, are long established as being toxic (Figure 1). There is evidence from the early Egyptians (fifth dynasty, ca. 2500 BC), Chinese herbal medical writings (ca. 200 BC), the logs of Captain James Cook (1774), and other historical texts (e.g., Kaempfer’s History of Japan) that there was an awareness and knowledge of the toxicity associated with these fish. The first report of formal research into the pharmacology of tetrodotoxin was by Charles Remy in 1883 who described the symptoms of tetrodotoxin poisoning and documented the high concentrations of tetrodotoxin present

(a)

Dog faced puffer fish

(b)

Regulation A principle, rule, or law designed to control or govern conduct. Tetrodotoxin Potent neurotoxin produced by bacteria. Toxicity The degree by which something is poisonous to an organism.

in the gonads of puffers. Tetrodotoxin was formally named in 1909 by Dr Yoshizmi Tahara who isolated and prepared a crude extract from puffer fish. Pure crystalline tetrodotoxin was not isolated until 1950 when Yokoo isolated tetrodotoxin from the ovaries of Fugu rubripes and described it as spheroidine after a genus of puffer fish. The nomenclature of tetrodotoxin was corroborated in 1952 when Tsuda and Kawamura isolated an identical toxin using chromatographic methods. Since the 1960s, the chemistry, pharmacology, and synthesis of tetrodotoxin have been the subject of a voluminous body of work and reviews. The complete low molecular weight (319 Da) structure of tetrodotoxin was first described in 1964 at the Natural Products Symposium of the International Union of Pure and Applied Chemistry by a total of four different laboratory groups including Tsuda, Goto, Woodward, and Mosher. It is important to note that while three of these groups had been working on toxin isolated from puffer fish, the Mosher group was reporting on compound, they named tarichatoxin, isolated from eggs of the newt Taricha torosa (Figure 2).

Jewel puffer fish

(c)

Porcupine puffer fish

Figure 1 Examples of puffer fish.

Encyclopedia of Human Nutrition, Volume 2

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Natural Toxicants: Tetrodotoxin

Figure 2 California newt (Taricha torosa). Figure 4 Shewanella alga.

Hapaloclaena maculosa

Carcinoscorpius rotundicauda

Nassarius species

Xanthid crab

Figure 3 Examples of other species known to contain tetrodotoxin.

Occurence of Tetrodotoxin Tetrodotoxin now has been found in a wide genre of species. Other marine organisms have been found to store tetrodotoxin and include the Australian blue-ringed octopus (Hapaloclaena maculosa, which uses tetrodotoxin as a toxin for capturing prey), parrot fish, triggerfish, goby, angelfish, boxfish (Ostracion spp.), tobies, porcupine fish, molas or ocean sunfish, globefish, seastars, starfish (Astropecten scoparius), xanthid crabs (Eriphia spp.), a horseshoe crab (Carcinoscorpius rotundicauda), two Philippine crabs (Zosimus aeneus and Atergatis floridus), a number of marine snails, flatworms, sea squirts, several nemerteans (ribbonworms), and several species of Chaetognatha (arrow worms), which use tetrodotoxin as a venom for prey, molluscs (Nassarius spp. and the Japanese trumpet shell Boshubora), and marine algae (Jania spp.). Terrestrial organisms include the Harlequin frogs (Atelopus spp.), Costa Rican frog (Atelopus chiriquiensis), three species of California newt (Taricha spp.), and members of the Salamandridae (salamanders). The number of species found to contain tetrodotoxin continues to grow (Figure 3). It is unlikely that these tetrodotoxin-bearers possess a common gene that codes for tetrodotoxin production. The ecologic environments of tetrodotoxin-bearing animals seem to have no common factor other than being closely related to an aquatic system. Bacteria, omnipresent organisms that commonly inhabit the aquatic system, are implicated as the primary source of tetrodotoxin. The bacteria believed to be involved are Shewanella alga (Figure 4), Vibrio species, Alteromonas species, and Pseudomonas species and their proposed mechanism of tetrodotoxin accumulation in marine animals was described by Noguchi and Arakawa using the flow chart (Figure 5).

Therefore, the tetrodotoxin of puffer fish is not endogenous (produced by the puffer fish itself), but exogenous (taken from outside and accumulated) via the food chain. It has been suggested that the puffer fish accrue tetrodotoxin as a biological defense agent. There appears to be a symbiotic association between tetrodotoxin-producing bacteria and higher organisms, which offers distinct advantages to both partners. The bacteria have a host as a safe place to live, eat, and reproduce whereas the host uses the toxin for predation or defense or both. The normal visual defense mechanism for slow-swimming and clumsy pufferfishes is their ability to inflate to several times their normal size by swallowing air when threatened, and tetrodotoxin may be an inadvertent weapon. Generally, they are left alone by predators.

Synthesis of Tetrodotoxin In 1972, the first total synthesis of D,L-tetrodotoxin was described by Yoshito Kishi and co-workers at Nagoya University, Nagoya, Japan. In 2003, Isobe and co-workers at Nagoya University and Du Bois and co-workers at Stanford University, USA, reported the asymmetric total synthesis of tetrodotoxin by two different synthetic approaches. Isobe’s synthesis was based on a Diels–Alder approach and Du Bois’s synthesis involved C–H bond activation.

Structure and Mode of Action Tetrodotoxin is known as a guanidinium toxin as it consists of a positively charged guanidinium group made up of three nitrogen atoms incorporated as part of a pyrimidine ring with additional fused ring systems.

Natural Toxicants: Tetrodotoxin

Vibrio alginolyticus Shewanella alga, S. putrefaciens Alteromonas tetraodonis etc.

TTX producing marine bacteria

TTX dissolved in seawater or adsorbed on and precipitated with dead planktonic cells, etc.

Small zooplankton Detritus feeder

Flatworm Ribbonworm Arrowworm Xanthid crab Small gastropods Skeleton shrimp

Starfish

(Parasitism or symbiosis)

(Parasitism or symbiosis)

TTX in sediment

279

Pufferfish Tropical goby Large gastropods

: Food chain : Parasitism/symbiosis; decomposition Flow chart: Mechanism of tetrodotoxin accumulation Figure 5 Flow diagram of mechanism of tetrodotoxin accumulation. Reproduced from Noguchi T and Arakawa O (2008) Tetrodotoxin – Distribution and accumulation in aquatic organisms, and cases of human intoxication. Marine Drugs 6: 220–242.

NH2 + H2N NH2 Guanidinium group

The ring systems, of which there are five in total, contain hydroxyl groups. Modifications of the parent compound can occur at five sites designated R1–R5 and the main modification at these sites are primarily hydrogen to hydroxyl substitutions. The chemical structure of tetrodotoxin and its derivatives are highlighted (Figure 6). Tetrodotoxin is an extremely potent neurotoxin, specifically blocking voltage-gated sodium channels on the surface of nerve membranes. The flow of sodium ions into nerve cells is a necessary step in the conduction of nerve impulses in excitable nerve fibers and along axons. Normal axon cells have high concentrations of K þ ions and low concentrations of Na þ ions and have a negative potential. Stimulation of the axon results in an action potential, which arises from a flow of Na þ ions into the cell and the generation of a positive membrane potential. Propagation of this depolarization along the nerve terminal presages all other events. The Na þ ions flow through the cellular membrane employing the sodiumion channel, a channel that is selective for sodium ions over potassium ions by an order of magnitude. The sodium channel itself is made up of a single peptide chain with four repeating units with each unit consisting of six trans-membrane helices. The trans-membrane pore is formed when the four units fold into a cluster with the center of the cluster being the pore. Tetrodotoxin acts by competing with the hydrated sodium cation for the sodium channel. It is proposed that the binding occurs through the positively charged guanidinium group of the tetrodotoxin molecule and negatively charged carboxylate groups on side chains in the mouth of the channel. The

guanidinium group fits into the external orifice of sodium channels, but the rest of the molecule is too large to penetrate the channels, acting like a cork in a bottle. This results in plugging the sodium channels from outside preventing the flow of sodium ions into the channel, effectively shutting down sodium movement; thus, the conduction of nerve impulses along nerve fibers and axons ceases. Tetrodotoxin is quite specific in blocking the sodium-ion channel (and, therefore, the flow of sodium ions) while having no effect on potassium ions. Binding to the channel is relatively tight (Kd ¼ 1010 nM). The hydrated sodium ion binds reversibly on a nanosecond time-scale, whereas tetrodotoxin is bound for tens of seconds. The different tetrodotoxin derivatives display different binding affinities to the channel based on the changes in their chemical structure (Figure 7). The Japanese tiger puffer fish, F. rubripes, has been the subject of intensive genetic sequencing studies. A single point mutation in the amino acid sequence of the sodium-ion channel in this species causes it to be immune from being bound and blockaded by tetrodotoxin. Changing the amino acid sequence of any protein alters its structure, and, therefore, its function. Such spontaneous mutations occur frequently in animal populations. Most such changes are neutral or disadvantageous to an organism’s survival, but occasionally one confers a selective advantage. In the case of F. rubripes, one tiny change enabled the fish to incorporate tetrodotoxin-producing bacteria into its tissues and use the toxin to its own advantage.

Toxicity The intravenous and oral median lethal doses for mice are reported as 7.3 and 334 mg kg1, respectively, in mice. On the presumption that these doses are comparable for humans, 0.5 and 25 mg of tetrodotoxin would be expected to kill a 75 kg

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Natural Toxicants: Tetrodotoxin

OH R1 10

HO + 2 H2N

9 H O H 4 R2 O N 3 5 H 4a 1 N 8a 7 H R5 6 8 H H

R4

R3

H R1

R2

TTX 4-epi TTX 6-epi TTX 11-deoxyTTX 11-oxoTTX TTX-11-carboxylic acid

H OH H H H H

OH H OH OH OH OH

11-norTTX-6,6-diol 11-norTTX-6(S)-ol 11-norTTX-6(R)-ol

H H H

OH OH OH

OH OH H

chiriquitoxin

H

OH

OH

12 13 11-CH(OH)CH(NH3+)COO– R S

TTX-8-O-hemisuccinate

H

OH

OH

11-CH2OH

H + H2N

O OH

H N

H O

–OOC

+ H2N

O H

N H HO

CH2OH H

R3

OH OH

11-CHO 11-COO– OH H OH

OH OH OH

1′ 2′ 3′ 4′ OOC(CH2)2COO–

+ H2N H CH2OH

H

tetrodonic acid

H H N

H O OH H

N H HO

CH3 H

H CH

H

O H

OH H OH

H

OH

HO H

N H HO

4,9-anhydroTTX

R5 OH OH OH OH OH OH

O

H N

OH

H

R4

11-CH2OH OH 11-CH2OH OH 11-CH2OH OH 11-CH3 OH

H

H H

5,6,11-trideoxyTTX

Figure 6 Structure of tetrodotoxin and analogues. Reproduced from Yotsu-Yamashita M, Sugimoto A, Takai A, and Yasumoto T (1999) Effects of specific modifications of several hydroxyls of tetrodotoxin on its affinity to rat brain membrane. Journal of Pharmacology and Experimental Therapeutics 289(3): 1688–1696.

Na+.OH2

Tetrodotoxin

Ion channel

Ion channel

Membrane

Figure 7 Illustration of tetrodotoxin binding to the sodium ion channel.

person by injection and through consumption. Commonly, reports of a human mortal dose of tetrodotoxin are 1–2 mg with the victim eventually dying from respiratory paralysis.

Tetrodotoxin is 10–100 times as lethal as black widow spider venom (depending on the species) when administered to mice, and more than 10 000 times deadlier than cyanide. It has a similar toxicity as saxitoxin, which causes paralytic shellfish poisoning and also blocks the sodium channel – both are found in the tissues of puffer fish. A recently discovered naturally occurring congener of tetrodotoxin has proven to be four to five times as potent as tetrodotoxin. Except for a few bacterial protein toxins, only palytoxin, a bizarre molecule isolated from certain zoanthideans (small, colonial, marine organisms resembling sea anemones) of the genus Palythoa, and maitotoxin, found in certain fishes associated with ciguatera poisoning, are known to be significantly more toxic than tetrodotoxin. Palytoxin and maitotoxin have potencies nearly 100 times that of tetrodotoxin and saxitoxin, and all four toxins are unusual in being nonproteins.

Natural Toxicants: Tetrodotoxin

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Clinical Manifestations The first symptoms occur between 15 min and several hours postingestion of tetrodotoxin-containing food. Almost all toxicity is caused by the ingestion of fugu, but other species of animals have been shown to produce tetrodotoxin (e.g., California newt, parrot fish, and blue-ringed octopus). A death from ingestion of tetrodotoxin from a California newt has been documented. A recent report on toxicity found that initial symptoms may occur up to 20 h after ingestion. Initial symptoms include lip and tongue paresthesias, followed by facial and extremity paresthesias and numbness. Salivation, nausea, vomiting, and diarrhea with abdominal pain develop early. Motor dysfunction with weakness, hypoventilation (may be from dysfunction of central and peripheral nervous systems), and speech difficulties then develop. A rapid ascending paralysis occurs over 4–24 h. Extremity paralysis precedes bulbar paralysis, which is followed by respiratory muscle paralysis. Deep tendon reflexes are preserved early in the course of paralysis. Finally, cardiac dysfunction with hypotension and dysrhythmias (bradycardia), central nervous system (CNS) dysfunction (e.g., coma), and seizures develop. Patients with severe toxicity may have deep coma, fixed nonreactive pupils, apnea, and loss of all brain stem reflexes. Death can occur within 4–6 h. Typically, death occurs from respiratory muscle paralysis and respiratory failure.

Prognosis Mortality rates are difficult to establish though estimates as high as 200 cases per year with mortality approaching 50% have been reported in spite of good supportive care. Symptoms may last several days even in nonlethal ingestions, but prognosis is reported to be good if the patient survives the first 24 h. Tetrodotoxin is absorbed with activated charcoal. The treatment is symptomatic and supportive with special attention to airway management and cardiac support. Only a few cases have been reported in the US, and outbreaks in countries outside the Indo-Pacific area are rare.

Hazard Characterization Since 1958 in Tokyo, it is required that specially trained fish cutters or chefs have a license to process and prepare puffer fish. It can take up to 11 years to become a fully competent fugu chef commencing with a 3-year apprenticeship in separating edible and inedible parts. Fugu may be prepared only in kitchens used solely for this purpose. Remarkably, even the waste from fugu preparation must be disposed off in hermetically sealed containers handled by specially trained companies as homeless people may scavenge the bins for food. However, there is no centralized regulation and it is difficult to say for sure who is licensed and who is not with only 19 of Japan’s 47 prefectures requiring chefs to pass an exam to obtain a fugu license (Figure 8). The Food and Drug Administration (FDA) advises consumers in the USA to eat puffer fish (fugu, bok, blowfish,

Figure 8 Licensed chef in Japan preparing fugu from puffer fish.

globefish, swellfish, balloonfish, or sea squab) only from two recommended sources. The sources are imported puffer fish that have been processed and prepared by specially trained and certified fish cutters in the city of Shimonoseki, Japan, and puffer fish caught in the mid-Atlantic coastal waters of the US, typically between Virginia and New York. Puffer fish are imported into the US two to three times per year for special occasions, by only one approved New York importer, Wako International, under an FDA/Japanese Government agreement. This is the only acceptable source of imported puffer fish. A number of the reported poisoning incidences, however, arise from recreational fisherman whereby the fish is mis- or unidentified as being potentially poisonous and consumed by families in rural fishing areas.

Risk Management Japan is currently the only country to have a regulatory level established for tetrodotoxin in puffer fish at 2000 mg kg–1 of fish. In the USA, it is illegal to import puffer fish but problems do arise due to mislabeling of imported products. The unpublished action limit in other countries for tetrodotoxin is the same as that for paralytic shellfish poisoning toxins, saxitoxin, at 800 mg kg–1 of fish. Below these levels, the fish is deemed fit or safe for consumption. A total of 22 species of puffer fish, all belonging to the Tetraodontidae family, are currently registered as tetrodotoxin poisoning. The levels found in puffer fish vary depending not only on the species but also in the different organs with the dispersal appearing to be species-specific. In marine species, the liver and ovary commonly have the highest toxicity, followed by intestines and skin. Muscles and testis are normally nontoxic or weakly toxic and are regarded as edible by the Japanese Ministry of Health, Labor, and Welfare. The liver generally displays very high toxicity during the year except in the spawning season, at which time the ovary becomes highly toxic through the buildup of tetrodotoxin transferred from the liver. One theory is that tetrodotoxin in the eggs spawned from the ovary could play a role in protecting the eggs from predators. Similarly, when toxic puffer fish are threatened, their bodies swell to two or three times their usual size and tetrodotoxin is postulated to

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Natural Toxicants: Tetrodotoxin

be excreted from their skin to fend off their attacker. Levels of 160 000 mg kg–1 have been reported in the liver. To reduce poisoning incidents, particularly in Asia, recreational fishermen and consumers should be instructed through food safety campaigns about which puffer fish is edible or inedible. It is believed that this may help official authorities to regulate the capture and consumption of toxic puffer fish species in China in order to avoid the risk of lethal poisoning.

Methods of Analysis In Japan, puffer fish intended for human consumption are tested using the mouse bioassay. However, the mouse bioassay shows low precision and also requires a continuous supply of mice, which is unethical. Tetrodotoxin can also be tested by high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), capillary zone electrophoresis, gas chromatography–mass spectrometry (GC–MS), and liquid chromatography–mass spectrometry (LC–MS). These methods are accurate, but may be extremely slow as they require extensive sample clean up extraction steps and, therefore, cannot allow rapid screening when large numbers of sample analysis are required and the instrumentation can be expensive. Biosensors have been highlighted as alternatives to current Regulatory Methods for marine biotoxins including (1) the use of an antitetrodotoxin specific antibody as the biological reporter and amperometric detection with a screen-printed electrode in an indirect competition assay; (2) differential pulse voltametry with screen-printed electrode in the form of a competition immunoassay; (3) a tissue biosensor has been developed with frog bladder membranes, which have a high concentration of Na þ channels and, therefore, a Na þ specific electrode measures the transport of Na þ through the membrane and its dose-dependent inhibition by tetrodotoxin; (4) tetrodotoxin biosensor based on inhibition of cell function has been designed using murine spinal cord neuronal networks cultured on microelectrode arrays where the biological response is monitored as extracellular potentials; and (5) several enzyme-linked immunosorbent assays have been developed as well for the detection of this toxin, some of them preparing the ground for the development of the electrochemical antibody-based biosensors. More recent methods for tetrodotoxin detection have been employed using surface plasmon resonance and fluidic force discrimination that achieve low levels of sensitivity.

See also: Safety of Food and Beverages: Seafood

Further Reading Fozzard HA and Lipkind GM (2010) The tetrodotoxin binding site is within the outer vestibule of the sodium channel. Marine Drugs 8: 219–234. Hanifin CT (2010) The chemical and evolutionary ecology of tetrodotoxin (TTX) toxicity in terrestrial vertebrates. Marine Drugs 8: 577–593. Hwang DF and Noguchi T (2007) Tetrodotoxin poisoning. Advances in Food Nutrition Research 52: 141–236. Jang J, Lee J, and Yotsu-Yamashita M (2010) LC/MS analysis of tetrodotoxin and its deoxy analogs in the marine puffer fish Fugu niphobles from the Southern Coast of Korea, and in the brackishwater puffer fishes Tetraodon nigroviridis and Tetraodon biocellatus from Southeast Asia. Marine Drugs 8: 1049–1058. Noguchi T and Arakawa O (2008) Tetrodotoxin – Distribution and accumulation in aquatic organisms, and cases of human intoxication. Marine Drugs 6: 220–242. Rodriguez P, Alfonso A, Vale C, et al. (2008) First toxicity report of tetrodotoxin and 5,6,11-trideoxyTTX in the trumpet shell Charonia lampas lampas in Europe. Analytical Chemistry 80: 5622–5629. Saoudi M, Abdelmouleh A, and Feki AE (2010) Tetrodotoxin: A potent marine toxin. Toxin Reviews 29: 60–70. Tao J, Wei WJ, Nan L, et al. (2010) Development of competitive indirect ELISA for the detection of tetrodotoxin and a survey of the distribution of tetrodotoxin in the tissues of wild puffer fish in the waters of south-east China. Food Additives and Contaminants: Part A 10.1080/19440049.2010.504237. Vilarino N, Fonfria ES, Louzao MC, and Botana LM (2009) Use of biosensors as alternatives to current regulatory methods for marine biotoxins. Sensors 9: 9414–9443. Yotsu-Yamashita M, Sugimoto A, Takai A, and Yasumoto T (1999) Effects of specific modifications of several hydroxyls of tetrodotoxin on its affinity to rat brain membrane. The Journal of Pharmacology and Experimental Therapeutics 289: 1688–1696.

Relevant Websites http://en.diagnosispro.com/disease_information-for/pufferfish-fugu-tetrodotoxinpoisoning/18720.html DiagnosisPro. http://www.encyclopedia.com/topic/Tetrodotoxin.aspx Encyclopedia.com. http://emedicine.medscape.com/article/818763-diagnosis Medscape Reference. http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=11174599 NIH’s PubChem. http://www.steadyhealth.com/encyclopedia/Tetrodotoxin SteadyHealth.com. http://www.cfs.gov.hk/english/multimedia/multimedia_pub/ multimedia_pub_fsf_09_01.html The Centre for Food Safety: Government of the Hong Hong Special Administrative Region. http://www.chm.bris.ac.uk/motm/ttx/ttx.htm The University of Bristol: School of Chemistry. http://www.fda.gov/Food/FoodSafety/FoodborneIllness/ FoodborneIllnessFoodbornePathogensNaturalToxins/BadBugBook/ucm070842.htm US Food and Drug Administration (FDA). http://en.wikipedia.org/wiki/Tetrodotoxin Wikipedia tetrodotoxin page.