Introduction
 

        Tetrodotoxin (anhydrotetrodotoxin 4-epitetrodotoxin, TTX, CAS number [4368-28-9]) is a heat-stable marine neurotoxin, named after an order of fish from which it is commonly associated, the Tetraodontiformes, or the tetraodon pufferfish.  Members of this order include the fahaka puffer (Tetraodon fahaka), the Congo puffer (Tetraodon miurus), and the giant mbu puffer (Tetraodon mbu).  Pufferfish from the genus Fugu (F. flavidus, F. poecilonotus, and F. niphobles), Arothron (A. nigropunctatus), Chelonodon (Chelonodon spp.), and Takifugu (Takifugu rubripes) also store TTX and related analogs in their tissues.  Other marine animals that store TTX are the Australian blue-ringed octopus (Hapaloclaena maculosa).₍₁₎
        Fish poisoning by consumption of members of the order Tetraodontiformes is one of the most violent intoxications from marine organisms.  The gonads, liver, intestines, and skin of pufferfish can contain levels of tetrodotoxin sufficient to produce rapid and violent death, although the flesh of many pufferfish may not be dangerously toxic.  Evidence of recent reports point toward a bacterial origin of this family of toxins from strains of the family Vibrionaceae, Pseudomonas spp., and Photobacterium phosphoreum.₍₂₎

        All humans are susceptible to tetrodotoxin poisoning. Tetrodotoxin poisoning may be avoided by not consuming pufferfish or other animal species containing tetrodotoxin. Most other animal species known to contain tetrodotoxin are not usually consumed by humans. Poisoning from tetrodotoxin is of major public health concern primarily in Japan, where "fugu" is a traditional delicacy. It is prepared and sold in special restaurants where trained and licensed individuals carefully remove the viscera to reduce the danger of poisoning. Importation of pufferfish into the United States is not generally permitted, although special exceptions may be granted. There is potential for misidentification and/or mislabeling, particularly of prepared, frozen fish products. ₍₁₎

Signs and Symptoms
 

        Ten to fifteen minutes after ingestion, paresthesias begins, usually as tingling of the tongue and inner surface of the mouth.  Other common symptoms include vomiting, lightheadedness, dizziness, feelings of doom, and weakness. Other manifestations include salivation, muscle twitching, diaphoresis, pleuritic chest pain, dysphagia, aphonia, and convulsions.  Severe poisoning is indicated by hypotension, bradycardia, depressed corneal reflexes, and fixed dilated pupils.₍₃₎  The secondary stage of the intoxication is increasing paralysis. Many victims often are unable to move; even sitting may be difficult. There is increasing respiratory distress.  Speech is affected, and the victim usually exhibits dyspnea, cyanosis, and hypotension.  Paralysis increases and convulsions, mental impairment, and cardiac arrythmia may occur.  The victim, although completely paralyzed, may be conscious and in some cases completely lucid shortly before death.  Death usually occurs within 4-6 hours, within a known range of about 20 minutes to 8 hours.₍₁₎
 

Structure

Tetrodotoxin consist of a positively charged Guanidinium group (outlined in blue) and a Pyrimidine ring (shown in red) with additional fused ring systems  (contain hydroxyl groups which help stabilize the tetrodotoxin-sodium channel binding complex at an aqueous interface).

        The structure of tetrodotoxin is as follows:  ₍₄₎
 
 

           And a 3-Dimensional Molecular View of Tetrodotoxin:  ₍₂₎
 
 

            Some properties of tetrodotoxin:
 

Mode of Action

        Tetrodotoxin is one of the most potent molecules known to selectively block the voltage-sensitive sodium channels of excitable tissues.  As a result, tetrodotoxin inhibits or reduces the chances of an action potential to be produced. The toxin inhibits nerve conduction, but does not depolarize the membrane and, more specifically, it eliminates or blocks inward movement of sodium, although the movement of potassium is unaffected.  Tetrodotoxin is complex in structure by small molecule standards and contains a guanidinium moiety.  The guanidinium ion is able to enter cells via the sensitive Na⁺ channels.  It is likely that the imidazole ring is the part of the molecule that lodges in the channel leaving the rest of the molecule blocking its outer mouth.  Their association and dissociation is independent of whether the channels is open or closed. ₍₉₎  The blocking action is highly specific: sodium movement is blocked only when the toxin is placed on the outer membrane surface, while no effect is noted when the toxin is placed inside the axon. ₍₅₎₍₇₎ The TTX-Na Channel binding site is extremely tight (Kd = 10-10 nM).  TTX mimics the hydrated sodium cation, enters the mouth of the Na+-Channel peptide complex, binds to a peptide glutamate side group, among others, and then further tightens it hold when the peptide changes confirmation in the second half of the binding event.  Following complex conformational changes, TTX is further electrostatically attached to the opening of the Na+ gate channel (2d event occurs in vivo as the dehydration of the aqueo-sodium complex). ₍₂₎

        Tetrodotoxin's tenacious hold on the Na+-Channel complex is further demonstrated by the occupancy time of Tetrodotoxin v. hydrated-Na+ at the complex.  Hydrated sodium reversibly binds on a nanosecond time-scale, whereas tetrodotoxin binds and remains on the order of tens of seconds.  With the bulk of the tetrodotoxin molecule denying sodium the opportunity to enter the channel, sodium movement is effectively shut down, and the action potential along the nerve membrane ceases.  A single milligram or less of tetrodotoxin - an amount that can be placed on the head of a pin, is enough to kill an adult. ₍₂₎

        Tetrodotoxin is highly polar and hydroscopic and only sparingly soluble in acidified water. Tetrodotoxin has a pK of 8.5; the hydroxyl group on C-4 masks the pK of the guanidinium group. The masking of one pK by the hydroxyl group in tetrodotoxin could have a significant cellular effect. Tetrodotoxin should be able to penetrate the lipid sheath in the nonionic form, and yet, because of the masking effect, a sufficient concentration of the active cationic form would be available for effecting action (mimicking) as a local anesthetic. In terms of structure, the hemilactal link (oxygen bridge between C-5 and C-10) is essential for Tetrodotoxin's biological toxicity ; the derivative in which the link is missing (tetrodonic acid) is completely inactive.  With tetrodotoxin, hypotension and action on the central nervous system always accompanies paralysis. Tetrodotoxin is a unique hypothermic agent as well as a potent emitic agent. The former action is thought to be exerted on the hypothalamus; the latter is believed to be exerted on the medullary chemoreceptor trigger zone.  ₍₅₎

        The Puffer fish has a mutation in the protein sequence of the sodium channel pump found on the cell membranes, this sodium channel is critical for cellular signaling pathways (e.g. transmission of impulses and the mediation of many cell functions). This point mutation in the amino acid sequence compared to the sequence in man makes these fish highly resistant to tetradotoxin poisoning, as a result tetradotoxin does not recognise the channel in pufferfish and therefore does not bind to it and block it. The puffer fish store high concentrations of tetratodoxin in various organs. However, the species differ widely in terms their resistance to tetradotoxin. ₍₉₎
 

Treatment

   Prehospital Care ₍₈₎

                - Prehospital care should include careful attention to the ABC's.
           - Patients may require endotracheal intubation for oxygenation and airway protection in the setting of muscle weakness and vomiting.
           - Cardiac dysfunction may require IV intervention with fluids, pressors, and antiarrhythmics
           - Severely poisoned patients may be very weak, have difficulty speaking, and be unable to give history. Thus, clues from the
                        environment and bystanders are very important.

   Emergency Department ₍₈₎

                - Patient care in the emergency department should focus initially on the ABC's.
           - The airway should be secure before frank respiratory failure or aspiration occurs.
           - An IV should be started early in the event acute arrhythmics or vasopressors are needed.
           - Removal of the toxin from the intestinal tract should be done by the usual toxicologic modalities. The use of nasogastric or orogastric
                        lavage is of theoretical benefit due to the rapid presentation of many patients. The administration of activated charcoal (with or
                        without a cathartic) is recommended for all symptomatic patients.
           - If vomiting has occurred, gastric lavage is not indicated.
           - Careful monitoring of vital signs and oxygenation should be started in the emergency department, as patients can suddenly
                        decompensate. All alterations in vital signs should be treated aggressively.
           - Further treatment focuses on supporting cardiovascular function until the toxin is eliminated from the body.
           - No specific antidote has been tested in humans. An animal study using monoclonal antibodies against tetrodotoxin has been done.
                        Monoclonal antibodies were shown to be life saving in mice treated both before and after the ingestion of a lethal dose of
                        tetrodotoxin. Further studies are needed to document efficiency in humans.

   Medication ₍₈₎

            No drug has been shown to reverse the effects of tetrodotoxin. Treatment is symptomatic. Anticholinesterase drugs have been
                    proposed as a treatment option but have not been tested adequately.

            Drug Category: GI Decontaminants- Empirically used to minimize the absorption of the toxin. This is only beneficial if administered
                            within 1-2 hours of ingestion of tetrodotoxin.
 
 

            Drug Category- Cholinergic agents- These medications may be useful in reversing the neurological complications of the venom,
                            however, they should not be substitute for airway management.
 
 
 

Some interesting information for the mind

        In Haitian folklore, a zombie is someone that is reanimated shortly after death by a bokor or voodoo witch doctor. The bokor typically robs the fresh grave, makes the zombie and sells the new walking dead into slavery. At least that's one version. Another version of this myth is that the victim is targeted by the bokor who poisons them with a powder that will slow their heart rate, blood pressure, and respiratory rate to the point that they would appear dead. The bokor then revives the poisoned individual after removing him from his/her fresh grave. ₍₆₎
 
 

References

1)  USDA Center for Food Safety & Applied Nutrition: Foodborne Pathogenic Microorganisms and Natural Toxins Handbook
          http://vm.cfsan.fda.gov/~mow/chap39.html

2)   http://www.chm.bris.ac.uk/motm/ttx/ttx.htm

3)  MMWR May 17,1996; Vol.45/No.19  pp.390

4)   http://neuroscience.about.com/science/neuroscience/library/g/t/bl-tetrodotoxin.htm

5)  Martin, Dean F.; Padilla, George M.; Marine Pharmacognosy: Action of Marine Biotoxins at the Cellular Level; Academic Press Inc. 1973
            pp. 11,13,17,19,21,24-27,30,41,85-86,89-90,96,102-103

6)   http://neuroscience.about.com/science/neuroscience/library/weekly/aa000619Zombie.htm

7)  Journal of Neuroscience Research; Vol.54, Issue 4, Nov 15 1998,  pp.433-443

8)  http://www.emedicine.com/emerg/topic576.htm

9)  http://neuroscience.about.com/science/neuroscience/gi/dynamic/offsite.htm?site=http%3A%2F%2Ffugu.hgmp.mrc.ac.uk%2Ffugu%2Fpffp%2Ftoxin.html