TETRODOTOXIN
Introduction
Tetrodotoxin (TTX) is a potent toxin produced by an array of marine species as a means of immobilising their prey. This chemical species is capable of blocking voltage-sensitive sodium channels, making it one of the most lethal neurotoxins found in the marine environment. However, while this compound is potentially lethal on ingestion, it seems to hold potential therapeutic applications.
Structure and Function
TTX was named after the Tetraodontidae pufferfish family, from which it was initially isolated. TTX is also found in numerous other marine animals including species of octopus such as the blue ringed octopus, some species of amphibians and various crab species such as Atergatis floridus (floral egg crab).TTXs function in nature is to immobilise and attack prey (Tamele et al 2019). TTX, is one of the most potent marine toxins known. As a matter of fact TTX has been responsible for numerous, occasionally fatal, human intoxication incidents linked particularly to the consumption of pufferfish which is considered a delicacy known as “fugu” in Japan. Until the beginning of the 21st century, TTX was commonly found in tropical waters and was not regarded as a hazard in temperate areas such as the Mediterranean sea and Europe. However, in the early 2000s, a known TTX vector of the pufferfish species known as Lagocephalus sceleratus, has been increasingly recorded in the Eastern Mediterranean sea, due to its invasion through the Suez canal. In fact, the species manages to establish itself in these habitats and gradually spread to other Mediterranean coastal waters including Italy, Croatia, Libya, Tunisia, Malta and Spain. TTX poisoning was also recorded in Europe with Mifsud et al., (2019) recording the first cases of TTX poisoning in Malta which resulted from ingesting octopus which harboured toxic TTX.
TTX is a crystalline, weakly basic, colourless organic substance. It is also water soluble and heat stable, therefore it is not destroyed by heat processing. On the contrary, TTX increases its toxic effect when heated (Katikou et al, 2022). The molecular formula of tetrodotoxin is C11H17O8N3, It has a cage-like rigid structure and it is a heterocyclic, organic perhydro quinozoline amine molecule. Its structure was elucidated by R. B. Woodward in 1964, and is depicted in Figure 1. Tetrodotoxin is a highly polar molecule, and this is attributed to the large number of -OH groups and -NH groups present within its structure (Woodward, 1964). TTX possesses a Guanidinium group which is a molecular group consisting of HNC(NH₂)₂ and a pyrimidine ring. This pyrimidine ring contains five additional fused ring systems.
To date, at least 30 structural analogues have been described, possessing varying degrees of toxicity. These different analogues are altogether referred to as tetrodotoxins (TTXs) but vary in origin and function. Yotsu-Yamashita et al, (2013) have isolated and determined various structural analogs of TTX found in pufferfish. The characterisation of analogues is important since it may be useful in predicting biosynthetic pathways of TTX. In pufferfish TTX analogous were classified into four groups:
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Analogs chemically equivalent to TTX which are 4-epiTTX having the same molecular formula as TTX and 4,9-anhydroTTX. These are almost identical to TTX, and are in equilibrium with each other.
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Deoxy analogs, 5-deoxyTTX, 5,11-dideoxyTTX 11-deoxyTTX, 6,11-dideoxyTTX, and 5,6,11-trideoxyTTX, which contain less oxygen molecules than TTX.
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11-CH2OH oxidised analog (11-oxoTTX), which contains a CHO group of C11 rather than a CH2OH group.
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C11 lacking analogs (11-norTTX-6(S)-ol and 11-norTTX-6(R)-ol, which have CH2OH groups on C11 replaced with H and OH respectively.
Figure 1
General Structure of TTX showing pyrimidine and guanidinium groups. Created using marvinsketch version 22.9 and adapted from Katikou et al, (2022)
Figure 2
Analogues of TTX found in pufferfish by Yotsu-Yamashita et al, (2013).
Mechanism of Action in humans
TTX possesses the ability to block voltage-gated sodium channels (VGSCs), making it a powerful neurotoxin. VGSCs are members of the ion channel protein superfamily and play an essential role in both neuronal as well as in non-neuronal functions since it is responsible for the initiation and propagation of action potentials in excitable cells.
An action potential is defined as a rapid rise, followed by a rapid decrease in the membrane potential of a neuron, in response to a stimulus (Hammond, 2015), as illustrated by the figure 3. The events of an action potential are divided into the following 5 steps: (1) When a stimulus occurs, depolarisation occurs within the neuron (Hammond, 2015). (2) This involves a rapid rise in membrane potential, which opens sodium channels in the cellular membrane, resulting in a significant input of sodium ions. (3) Membrane repolarization is caused by the sudden inactivation of sodium channels, as well as a significant outflow of potassium ions through voltage-gated potassium channels (Moini et al., 2021). (4) Hyperpolarization involves a decrease in membrane potential, and occurs due to potassium ion efflux and potassium channel closure. Following hyperpolarisation, (5) the membrane potential will return back to its resting state i.e. the membrane potential returns back to that prior to the stimulus (Hammond, 2015; Moini et al., 2021).
When VGSCs are blocked by TTX, the initiation and propagation of an action potential is inhibited. This results in a lack of communication between sensory neurons which link the brainstem and the airways, and results in respiratory complications. Hence, TTX can potentially cause primary blockage of the brainstem, somatic motor, sensory, and autonomic neurons (Katikou et al., 2022).
When TTX enters the system, the positively charged imine group (NH2+) in the guanidinium group becomes attracted to the sodium binding site within the voltage-gated sodium channels on the surface of neurons (Man et al., 2010). The guanidinium group makes the TTX slightly permeable to sodium channels, and hence the TTX molecule is allowed to migrate deep into the VGSC pore. This results in the blocking of the sodium channel by TTX. (Fozzard & Lipkind., 2010). Since TTX is a significantly larger molecule than the Na+ ion, it inhibits the Na+ ion from binding, halting Na+ transport. Furthermore, the hydroxyl groups aid in stabilising this interaction of TTX to sodium channels. The structure of TTX is vital for sodium channel binding, as it mimics a hydrated sodium cation, specifically, the guanidine functional group mimics sodium ion by binding to the side groups of glutamate residues within the sodium channel pore. This interaction further tightens its association with the channel and renders the channel to function incorrectly.
TTX binding to sodium channels lasts longer than normal hydrated sodium, hence impeding this ion to flow freely through the channels (Bane et al., 2014).This results in an accumulation of sodium ions outside the neuronal cell, and a depletion of sodium ions inside the neuronal cells (Katikou et al., 2022). Low sodium ion concentration in neurons, also known as hyponatraemia, results in brain swelling and possibly coma, which are both possible effects of TTX poisoning (Katikou et al, 2022).
Figure 3
Graph showing the change in membrane potential upon stimulus and initiation of an action potential (Created with BioRender.com).
Figure 4
Tetrodotoxin inhibiting the passage of sodium ions
Symptoms upon Exposure
Typically symptoms of TTX poisoning occur within 10-45 minutes from exposure, however, if mild toxicity occurs, symptoms may take up to 3 or more hours to manifest. Exposure can occur due to ingestion of various pufferfish and blowfish species including Lagocephalus sceleratus, Arothron hispidus, and Tetrodon nigroviridis. A number of these fish are considered to be a delicacy in Asian culture and are thus eaten intentionally. Major toxicity may occur unless the fish is properly prepared for human consumption (Shibamoto & Bjeldanes, 1993).
Symptoms of TTX poisoning from consumption includes salivation with numbness and tingling in and around the mouth, as well as nausea and vomiting (Pufferfish Poisoning., 2022). TTX is known to cause symptoms indicative of brain injury, such as dilated pupils, and areflexia (the absence of deep tendon reflexes), early in the course of poisoning. Moreover, prolonged exposure to TTX may even result in loss of consciousness, paralysis and respiratory failure, which may ultimately result in death (Katikou et al, 2022)
Treatment
Unfortunately, there is yet no cure for TTX poisoning. If symptoms of exposure are identified, physicians are to induce vomiting in the patient as soon as possible. If TTX poisoning results in paralysis and loss of consciousness, the patient should be aided by artificial respirators (Hyponatremia - Symptoms and causes., 2022; Pufferfish Poisoning., 2022). If enough supportive therapy is delivered before cardiac arrest, the prognosis of TTX poisoning in humans is generally excellent. If a patient survives for more than 24 hours, he or she is likely to recover if no other life-threatening disorders are present (Katikou et al., 2022).
Therapeutic Uses of TTX in Medicine
Pain is the unpleasant sensory and emotional experience associated with stimuli that cause tissue damage. The perception of pain has the ability to fulfil a protective role by warning us of any harm and in order to prolong survival, harm should be avoided or treated (Nieto et.al, 2012).
Analgesics treat various types of pain. The neurotoxin TTX can be considered as an example of an analgesic. TTX exhibits therapeutic properties, especially to treat cancer-related pain, neuropathic pain, and visceral pain. Furthermore, TTX can potentially treat a variety of medical ailments, including heroin and cocaine withdrawal symptoms, spinal cord injuries, brain trauma, and some kinds of tumours (Bucciarelli et.al, 2021).
Cancer related pain
Pain arising from cancer is one of the most critical symptoms indicating the presence of the disease, and cancer pain tends to intensify as the stage of cancer advances. More than one-third of cancer patients suffer from moderate to severe cancer pain. Cancer pain has various causes, including cancer itself, treatments such as chemotherapy, and comorbidities not directly related to cancer, such as constipation.
Using TTX as an analgesic to treat cancer-related pain has been tested in both animal models and humans. The efficacy of TTX injections in the treatment of mild to severe cancer pain showed promise for the future use of TTX in such a setting since it has the ability to provide pain relief in the absence of serious side effects in humans (Bucciarelli et.al, 2021; Nieto et.al, 2012).
Heroin and Cocaine Addiction
Heroin withdrawal syndrome and subsequent relapse is a major issue for the successful treatment of heroin addiction. The therapeutic potential of TTX in addiction is supported by studies in laboratory animals. Most of these studies have involved microinjections of TTX into specific brain regions. Since TTX affects the brain and both heroin and cocaine addiction cause an effect on the brain, then it was concluded that TTX can be used as a treatment for both (Shi et.al, 2009).
Even after long periods of abstinence, drug-associated environmental cues or exposure to stressors can induce drug use in recovering addicts. More studies showed that intramuscular injection of TTX significantly reduced cue-induced anxiety and craving in abstinent recovering heroin addicts, with no significant effect on blood pressure or heart rate. This shows that TTX can be used to prevent relapse (Bucciarelli et.al, 2021).
Conclusion
Whilst this potent toxin is well known for its detrimental effect, tetrodotoxin ingestion and poisoning are not unheard of. While no known antidote is available for TTX poisoning, this neurotoxin has obtained promising results in patients for the treatment of pain. To fully elucidate the potential clinical uses of this neurotoxin, more appropriately powered clinical trials possessing a larger number of patients must be performed.
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Bucciarelli, G. M., Lechner, M., Fontes, A., Kats, L. B., Eisthen, H. L., & Shaffer, H. B. (2021). From poison to promise: The evolution of tetrodotoxin and its potential as a therapeutic. Toxins, 13(8), 517. https://doi.org/10.3390/toxins13080517
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