Introduction
In Japan fugu or puffer fish, is a long established delicacy, in spite of its known potential for toxicity.
Tetrodotoxin (TTX) is the naturally occurring toxin that is mainly responsible for the risk to consumers. In fact, over 20 species of puffer fish have been found to harbour the toxin. TTX is both water soluble and heat stable so cooking does not negate its toxicity; rather it increases toxic effect . Consequently, great care must be taken by specially trained chefs to ensure that the hazardous parts of the fish (specifically the liver, ovaries and skin) are excised before serving; nevertheless human intoxications and some fatalities have been linked to the consumption of puffer fish.
The toxin was first discovered in 1909 by Dr. Yoshizumi Tahara from the ovaries of globefish, but puffer fish have been known to be toxic to humans for a long time. TTX is a very potent neurotoxin that is found in a variety of marine and also in some terrestrial species. Its toxicity is often emphasised by referring to the fact that it is over a thousand times more toxic to humans than cyanide; TTX has no known antidote.
There is some conjecture as to the origin of TTX in puffer fish. It has been shown that cultured non-toxic puffer fish when fed on a diet containing TTX become toxic and toxic puffer fish when fed on a TTX-free diet become non-toxic. It has also been demonstrated that the source of TTX in puffer fish is an endo-symbiotic bacteria that naturally inhabits the gut of the animal. This may be explained by the hypothesis that puffer fish could initially acquire the TTX producing bacteria via the food web and that these bacteria then persist in the fish.
Structures
The name TTX was coined by Dr. Yoshizumi Tahara in 1909. He isolated TTX from the ovaries of globefish. TTX is a heterocyclic, organic perhydroquinozolineamine molecule (aminoperhydroquinazolone). Its structure was elucidated by R. B. Woodward in 1964.
Structurally TTX consists of a guanidinium moiety connected to a highly oxygenated carbon skeleton that possesses a 2,4-dioxaadamantane portion containing five hydroxyl groups. TTX co-exists with its naturally occurring analogues. There have been 30 structural analogues of TTX reported to date and the degree of toxicity varies with structure. One of the major problems for studying these analogues is the lack of commercially available standards. A number of researcher groups have synthesized some of the analogues of TTX on a laboratory scale but availability is severely limited.
Biochemistry and toxicity
TTX is a sodium channel blocker. Binding of TTX to voltage gated sodium channel results from the interaction between the positively charged guanidine group on the TTX with the negatively charged carboxylate groups on the side chains in the mouth of the sodium channel. TTX binding prevents diffusion of sodium ions through the sodium channels. This in turn prevents depolarization and propagation of action potentials in nerve cells leading to the loss of sensation.
Biotransformation of TTX inside the human or mammalian body is yet to be widely investigated. In the puffer fish body, TTX enters the liver first after ingestion. Then it is transferred mainly to the skin in the male and the reproductive organs in the female.
In humans the onset and severity of the symptoms of TTX poisoning after ingestion is dose dependent. Initial symptoms include tingling (paresthesias) of the tongue and lips, followed by or concurrent with headache and vomiting, which may progress to muscle weakness and ataxia. In severe cases death may occur due to respiratory and/or heart failure.
Resistance to TTX in TTX Bearing Organisms
Many researchers have been intrigued as to how TTX bearing organisms are themselves resistant to the toxic effects of TTX. The reason is because in these animals the aromatic amino acid chain in the p-loop region of domain I in the sodium channels is replaced by a non-aromatic amino acid and this prevents the sodium channels in these species from being blocked.
Resistance to TTX can also be related to the presence of a TTX-binding protein present in some species of shore crab, puffer fish and gastropod.
Clinical Study
There are four grades of TTX poisoning described by Fukuda and Tani, 1941.
*Grade 1: perioral numbness and paresthesia (sensation of tingling, tickling, prickling, pricking, or burning of a person’s skin), with or without gastrointestinal symptoms.
*Grade 2: lingual numbness (numbness of face and other areas), early motor paralysis and incoordination, slurred speech with normal reflexes.
*Grade 3: generalized flaccid paralysis (reduced muscle tone without other obvious cause), respiratory failure, aphonia (the inability to produce voice due to disruption of the recurrent laryngeal nerve), and fixed/dilated pupils (conscious patient).
*Grade 4: severe respiratory failure and hypoxia (inadequacy of oxygen), hypotension (low blood pressure), bradycardia (resting heart rate of under 60 beats per minute), cardiac dysrhythmias (irregular heartbeat) and unconsciousness may occur.
The grade of TTX poisoning depends upon the amount of TTX ingested, the time after ingestion of TTX, the hydration state of body and the general health status of the victim prior to intoxication.
During the Bangladesh outbreak of TTX poisoning in 2008, the onset of symptoms was observed within 30 min of ingestion of puffer fish in 66% of the total number of cases, within 31–60 min in 24% of cases, within 61–90 min in 7% of cases and within 91–120 min in 2% of cases. The poisoning symptoms decreased gradually over 8–28 h after ingestion of the contaminated puffer fish with no residual side effects.
TTX can be found in blood within less than 24 h after its ingestion. But it can be found in urine after 4 days from the time of ingestion. Therefore, it is important to collect urine and blood samples from affected patients within 24 h after ingestion for clinical diagnosis.
The victims of the Bangladesh outbreaks in April 2008 ingested less than 50–200 g of puffer fish. The victims who died had ingested more than 100 g of TTX contaminated puffer fish.
Treatment
There is presently no antidote available for TTX poisoning. Some people have tried to make use of an anti-cholinesterase drug for treating TTX. During the large TTX incident in Bangladesh, 21 victims were given anticholinesterases; neostigmine and atropine but it wasn’t seen to improve their condition. This is because anti-cholinesterases reverse the blocking action at neuromuscular junction at the motor end plates only. Whereas TTX blocks sodium channels of motor neurons and muscle membranes.
Currently, the only treatment for TTX poisoning is to provide the victim with respiratory support until the TTX is excreted completely. Endotracheal intubation can be provided to facilitate ventilation of the lungs. Mechanical ventilation may also be provided. During the TTX poisoning outbreak in Israel, patients were given respiratory support and recovered within 4 days
In the case of early stage TTX poisoning victims are given activated charcoal in order to help the adsorption of TTX to prevent its absorption through the stomach. Gastric lavage (the passage of a tube via the mouth or nose down into the stomach followed by sequential administration and removal of small volumes of liquid) can be performed in TTX poisoning in order to reduce its severity. This procedure should be performed within 60 min after ingestion of TTX. But there are some risks associated with gastric lavage treatment such as laryngospasm (involuntary muscular contraction of the laryngeal cords), hypoxia (inadequacy of oxygen), bradycardia (a resting heart rate of under 60 beats per minute), epistaxis (nosebleed), hyponatremia (reduced levels of sodium in the blood), hypochloremia (reduced levels of chloride ions in blood), water intoxication or mechanical injury to the stomach.
Intravenous fluids are also given in order to maintain fluid-electrolyte balance in the body during TTX poisoning. An antiemetic is given which is effective against vomiting and nausea. Haemodialysis might also be useful for the treatment of TTX poisoning.
Antibodies against TTX have been used successfully in vivo. Xu et al., 2005, synthesized antibody against TTX. This antibody was able to neutralise the toxic effect of TTX both in vitro and in vivo. A monoclonal antibody for TTX (anti-TTX) is available commercially from Hawaii Biotech, Inc. However, studies on the efficacy of this monoclonal antibody in vivo have not been published, but this may herald the advent of a new type of approach to the treatment of TTX poisoning in the future.
Application of TTX in the Medical Field
Some researchers are trying to make use of the analgesic activity of TTX to treat various types of pains such as severe cancer pain. A low dose of TTX has also been shown to help in reducing cue-induced increases in heroin craving and associated anxiety.
Conclusions
In areas where TTX occurs with regularity, it is important that rapid analytical methods are deployed for the analysis of clinical samples, most especially blood and urine in suspected poisoning victims. LC-MS (Liquid chromatography-Mass spectrometry) methodologies are particularly appropriate to detect TTX and its analogues in clinical samples with the speed required in such cases. Though there is still no commercially available antidote to TTX, it may dictate the course of medical treatment, especially for those with compromised renal function.
A report in the wildlife section of “The Times” newspaper dated 11 May 2013 revealed that a red scorpion fish had been caught in the Celtic Sea off the coast of Ireland and England. This is clear evidence that exotic and toxic marine species can travel and may be commonly found in cooler European waters in the future. If migration trends like this are to continue and there are predictions that they are likely (in view of global warming), it may be prudent to carry out surveillance of susceptible marine species, algae and seawater in European territory for TTX and other toxins associated with warmer regions.
References
How C.-K., Chern C.-H., Huang Y.-C., Wang L.-M., Lee C.-H. Tetrodotoxin poisoning. 2003. Science Direct
Ahasan H.A.M.N., Mamun A.A., Karim S.R., Bakar M.A., Gazi E.A., Bala C.S. Paralytic complications of puffer fish (tetrodotoxin) poisoning. 2004. Singapore Medical Journal
Vaishali Bane, Mary Lehane, Madhurima Dikshit, Alan O’Riordan, and Ambrose Furey. Tetradotoxin : chemistry, toxicity, source distribution and detection. 2014 PubMed
Maruta S., Yamaoka K., Yotsu-Yamashita M. Two critical residues in p-loop regions of puffer fish Na+ channels on TTX sensitivity. 2008. Science Direct
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