Molecule of the Moment: Tetrodotoxin (TTX)

TTX, AN UNUSUAL AND HIGHLY TOXIC NATURAL PRODUCT, HAS BEEN ISOLATED FROM AN ASTONISHING NUMBER OF ORGANISMS.

TTX, AN UNUSUAL AND HIGHLY TOXIC NATURAL PRODUCT, HAS BEEN ISOLATED FROM AN ASTONISHING NUMBER OF ORGANISMS.

Name: Tetrodotoxin (TTX)

Source: Tetrodotoxin is a highly potent neurotoxin found in a surprisingly large, phylogenetically-diverse group of organisms and has a wide eco-geographical distribution. Organisms known to contain TTX include: 

1.     marine animals such as tetraodon fish (see: puffer fish, blowfish, porcupine fish, globe fish), blue-ringed octopusesmoon snails, starfish, and crabs

2.     terrestrial amphibians such as newts and frogs

3.     microscopic organisms such as algae, plankton, and bacteria

The widespread occurrence of this highly complex molecule in such phylogenetically distinct organisms has puzzled many. Which of these organisms are biosynthesizing and producing TTX? Are the higher organisms – such as fish and amphibians- sequestering TTX from their diet? Do any of these animals harbor symbiotic microbes that supply host organisms with the compound? Or - is it possible that the higher organisms have evolved to make TTX themselves?

The answer to the true source(s) of TTX appears to be quite complex. Many different types of bacteria, including both symbiotic and environmental microbes, have been found to produce TTX, though no biosynthetic genes have been identified (yet!). In order to investigate whether certain organisms sequester the TTX from their food or obtain it from other sources (symbionts, etc), scientists have conducted isolation experiments with higher organisms raised on nontoxic diets. Interestingly, certain animals, such as puffer fish, decrease toxicity during isolation (suggesting the toxicity primarily derives from a TTX-containing diet), while others, such as newts, have been found to increase their toxicity (though no TTX-producing symbionts for amphibians have been identified to date). Much of this data is inconclusive, however, and the mystery of the origin and ubiquity of this highly unusual toxin is still very much an active research area.

(a) Tetrodotoxin (TTX) has a (b) adamantane core with a (c) guanidinium Motif. 

(a) Tetrodotoxin (TTX) has a (b) adamantane core with a (c) guanidinium Motif. 

Chemical structure: TTX is a cage-like compound with a positively charged cyclic guanidinium moiety fused to a diox-adamantane skeleton (read: four connected 6-membered carbon rings with hydroxy groups attached). Several analogues have been found in Nature, though the toxicity of these derivatives is often lower. Interestingly, TTX and its derivatives are the only known natural products that incorporate the 10-atom cage architecture of adamantane, though a few synthetic drugs are known to contain this motif.

Discovery: TTX was initially identified in pufferfish, whose potent toxicity was documented as far as back as the first or second century BC (the therapeutic use of tetradon fish eggs were even described in the earliest known Chinese pharmacopeia, The Book of Herbs!). Anecdotes of pufferfish poisoning – intentional and otherwise- were recorded over the following centuries, including this fabulous description in the journals of Captain James Cook (who luckily survived the incident) in 1774:

“We were seized with the most extraordinary weakness in all our limbs attended with numbness of sensation like to that caused by exposing one’s hands and feet to a fire after having been pinched by much frost. I had almost lost the sense of feeling nor could I distinguish between light and heavy objects, a quart pot full of water and a feather was the same in my hand”

These intriguing properties of the toxic puffer fish inevitably caught the attention of scientists and in 1889, the first comprehensive study on the pharmacology of the pufferfish poison--then termed "tetrodotoxin"--was completed in Japan. Isolation of the active compound, however, was not achieved until 1950 and the molecular structure was not elucidated for another 14 years. In 1964, chemists from three independent research groups (K. Tsuda, T. Goto, and R.B. Woodward) all reported identical structural characterizations of TTX at a natural products symposium in Kyoto. 

While many organic chemistry groups around the world were racing to identify the pufferfish toxin, scientists in California were preoccupied with studying of a toxic, paralyzing substance found in Taricha newts. With the help of organic chemists, they were able to isolate sufficient quantities of the toxic substance for characterization (using a mere 1000 kilograms of newt egg clusters). Surprisingly, they found that not only the pharmacological properties of this substance were oddly similar to tetrodotoxin, but the chemical properties were as well. This finding was reported in Science in 1964 (the same year as the puffer fish reports!); both the newts and the puffer fish contained the exact same unique, highly toxic defense chemical.

Since the initial discovery in newts and tetradon fish, TTX has been identified in many several additional organisms (see: Source).

Biology: TTX’s high toxicity is due to its ability to selectively bind voltage gated sodium channels of muscle and nerve tissues. Binding of the toxin inhibits the flow of sodium ions through the channels, halting virtually any function dependent on electrical excitability of nerve and muscle tissues (think: locomotion, cognition, etc.).

Uses:

For organisms producing/containing TTX:

·       Defense. TTX is generally thought to serve as a deterrent to predation, though this has rarely been tested experimentally. The antipredation role of this toxin has been characterized, however, in Taricha newts, whose “arms race” with its often TTX-resistant predator, the garter snake, has been extensively studied by evolutionary ecologists (highly resistant garter snakes are only found near highly toxic newts, suggesting the evolutionary pressure to evolve resistance is stronger in these populations).

·       Other ecological roles. TTX has also been found to serve as a pheromone for certain pufferfish, blue-ringed octopus, and arrowworms and has been shown to be used for prey capture by flatworms. Given the sheer number of organisms containing TTX, other functions for this compound are bound to be elucidated in the future.

For humans:

·       Neurobiology. TTX has been an incredibly valuable chemical tool for the study of neurophysiology. Classic experiments carried out in the mid and latter part of 20th century with TTX and other natural toxins helped elucidate the role of ion channels in membrane physiology. These early experiments greatly facilitated the identification and characterization of ion channels, as well as the overall electrical behavior of cell membranes. In the following decades, TTX and its interaction with sodium channels have become one of the best documented toxin/receptor pairs in science (a search for "tetrodotoxin" in PubMed yields >18,000 results!).

·       Medicine. Scientists have explored the potential of the potent analgesic properties of TTX for treating migraine, withdrawal symptoms in heroin addicts, and pain in cancer patients.

Dangers: TTX is one of the most powerful neurotoxins known (over 1000x more toxic to humans than cyanide!) and has no antidote. Responses are dose dependent and symptoms include tingling of the tongue and lips, headache, vomiting, muscle weakness, and ataxia. The onset and severity of the symptoms are dose dependent but TTX ingestion can result in DEATH due to respiratory and/or heart failure.

As tetradotoxin has the potential to pose a severe threat to both human and animal health, it is listed a "select agent" by the US Dept of Health! 

Interesting fact: Despite the very serious dangers listed above, pufferfish is still consumed, primarily in Japan, where it is considered a very tasty delicacy known as fugu. Part of the appeal of fugu is said to be the sensation of oral numbness due to just the right amount of TTX blocking the sensory nerves. To make sure patrons enjoy meal without grave consequences, fugu must be properly prepared and served carefully so there is rigorous training and licensing of chefs who are permitted to do so. Despite the precautions taken by the Japanese government (and ours!), incidents of poisoning and death by miscalculated and ill-prepared fugu dishes still occur (in particular by thrill-seeking “foodies” who specifically ask for organs known to contain high levels of TTX, such as the liver, and are granted it illegally). Eat at your own risk!!!

- KLS