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, it is found in several other animals (e.g., in blue-ringed octopuses, rough-skinned newts, and moon snails). It is also produced by certain infectious or symbiotic bacteria like Pseudoalteromonas, Pseudomonas, and Vibrio as well as other species found in symbiotic relationships with animals and plants. it has shown efficacy for the treatment of cancer-related pain in phase II and III clinical trials.
Tetrodotoxin is a sodium channel blocker. It inhibits the firing of action potentials in neurons by binding to the voltage-gated sodium channels in nerve cell membranes 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.
Its mechanism of actionselective blocking of the sodium channelwas shown definitively in 1964 by Toshio Narahashi and John W. Moore at Duke University, using the sucrose gap voltage clamp technique.
Sources in nature
Apart from their bacterial species of most likely ultimate biosynthetic origin (see below), tetrodotoxin has been isolated from widely differing animal species, including:
- species of Nassarius gastropods,
- several starfish, including Astropecten species,
- species of Chaetognatha (arrow worms),
- toads of the genus Atelopus,
- the eastern newt (Notophthalmus viridescens)
- the western or rough-skinned newts (Taricha; wherein it was originally termed "tarichatoxin"), and the identity of maculotoxin and TTX was reported in Science in 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 animals 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 animal species from which it is isolated is relatively clear; TTX-producing bacteria include Actinomyces, Aeromonas, Alteromonas, Bacillus, Pseudomonas, and Vibrio species;
{|class="wikitable plainlist"
|+Association of animals with TTX-producing bacteria
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! Animal !! Bacteria !! Ref
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| Takifugu obscurus, obscure pufferfish
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- Aeromonas sp. Ne-1
- Bacillus sp. W-3
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|-
| Astropecten polyacanthus, a starfish
| Vibrio alginolyticus
|
|-
| Four species of Chaetognatha (arrow worms)
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- V. alginolyticus
|
|-
| Species of Nemertea (ribbon worms)
| Vibrio spp.
|
|-
|}
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", although technical concerns about the approach have been raised. 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. 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 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 (VGSCs) present in mammals: tetrodotoxin-sensitive voltage-gated sodium channels (TTX-s Na<sup>+</sup> channels) and tetrodotoxin-resistant voltage-gated sodium channels (TTX-r Na<sup>+</sup> channels). Tetrodotoxin inhibits TTX-s Na<sup>+</sup> channels at concentrations of around 1–10 nM, whereas micromolar concentrations of tetrodotoxin are required to inhibit TTX-r Na<sup>+</sup> channels. Nerve cells containing TTX-r Na<sup>+</sup> channels are located primarily in cardiac tissue, while nerve cells containing TTX-s Na<sup>+</sup> 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<sup>+</sup> channels in the central nervous system makes tetrodotoxin a valuable agent for the silencing of neural activity within a cell culture.
Biosynthesis
The biosynthetic route to TTX is only partially understood. It is long known that the molecule is related to saxitoxin, and as of 2011 it is believed that there are separate routes for aquatic (bacterial) and terrestrial (newt) TTX. 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. In 2021, the first genome of a TTX-producing bacterium was produced. This "Bacillus sp. 1839" was identified as Cytobacillus 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.
Resistance
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 Na<sub>v</sub> 1.4 (skeletal muscle VGSC, "TTX-s"), are found in many such animals. 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 of convergent evolution caused by how TTX binds to the unmutated VGSC. The structure was confirmed by X-ray crystallography in 1970. Yoshito Kishi and coworkers reported the first total synthesis of racemic tetrodotoxin in 1972. M. Isobe and coworkers and J. Du Bois reported the asymmetric total synthesis of tetrodotoxin in 2003. The two 2003 syntheses used very different strategies, with Isobe's route based on a Diels-Alder approach and Du Bois's work using C–H bond activation. Since then, methods have rapidly advanced, with several new strategies for the synthesis of tetrodotoxin having been developed.
Poisoning
Toxicity
TTX is extremely toxic. The material safety data sheet for TTX lists the oral median lethal dose (LD<sub>50</sub>) for mice as 334 μg per kg. For comparison, the oral LD<sub>50</sub> of potassium cyanide for mice is 8,500 μg per kg, demonstrating that even orally, TTX is more poisonous than cyanide. 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.
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. As a result, TTX causes loss of sensation, and paralysis of muscles including the diaphragm and intercostal muscles, stopping breathing.
History
thumbnail|upright|A Chinese pharmacopoeia, 1930.
The therapeutic uses of puffer fish (tetraodon) eggs were mentioned in the first Chinese pharmacopoeia Pen-T'so Ching (The Book of Herbs, allegedly 2838–2698 BC by Shennong; 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 principal 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 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.
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. According to Fuhrman, the ship naturalists J. R. Forster and Georg Forster spent so much time drawing and describing the fish that only the roe and liver were cooked. The three men experienced numbness and weakness in their limbs, only surviving the incident due to the small amount they ingested.
Symptoms and treatment
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.