A Brief History and Overview of Tetrodotoxin (TTX)
Erick Auyoung
SID 12979691
May 6, 1999
MCB 165-Molecular Neurobiology and Neurochemistry
Term Paper
Keywords: sodium channel, neurotoxin, action potential, biological effects
Abstract
The marine neurotoxin tetrodotoxin (TTX) is primarily found in Japanese puffer fish, but has also been isolated in animals from four different phyla. After isolation of crystalline TTX, experiments became more detailed, as scientists became interested in using TTX as a selective sodium channel blocker for use in other research investigations. The mechanism of the TTX-sodium channel interaction is still being debated, although the trapped-ion mechanism seems to be widely accepted now. This mechanism was concluded after experimentation with mutated sodium channels and with TTX chemical analogs. TTX's effects on animals center on its sodium channel-blocking ability, as action potentials are inhibited and the nervous system is impaired. This leads to major symptoms, involving respiratory failure and death in many cases of human TTX poisoning. A new treatment for TTX poisoning is being developed, but precautionary measures have to be taken when dealing with fish possibly tainted with TTX. The latest discoveries in this field are the isolation of TTX in cultured puffer fish, computer models showing the mechanism of sodium channel interactions in detail, and the appearance of 4-AP as a possible antidote for TTX poisoning.
The neurotoxin tetrodotoxin (TTX) has been known for centuries, but only until the mid-20th century has the structure of this highly unusual compound been deduced, and the mechanism for its neurotoxic effects are still being studied and debated today. Most people are only familiar with tetrodotoxin's effects in food poisoning with Japanese puffer fish, but scientists are much more familiar with the chemical, which is especially helpful in research on the voltage-gated sodium channel. Current studies on TTX center on finding a suitable antidote for the toxin and in determining the role of sodium channels in various bodily functions. Tetrodotoxin has come full circle, from poisoning ancient people unfortunate enough to ingest toxic fish to helping us understand more about our own bodies while fueling research to save future lives.
History
Tetrodotoxin was first isolated in 1950 (Fuhrman 1986), but its effects have been known for centuries. The first studies all centered on researching poisonous fish, mostly from the order Tetraodontiformes, which have been known to be poisonous since ca. 2500 BC in Egypt, and from about the same time in China, due to their value as food. (Fuhrman 1986) European explorers to the Far East also described poisonings by puffer fish, or fugu, and they relayed this information back to Europe, which caused continental scientists to flock to Japan to begin experimental tests on the mysterious fugu fish. Charles Remy carried out the first scientific experiments on fugu in 1882, as he described the symptoms of poisoning and determined that the majority of the toxin was located in the gonads of the fish. (Fuhrman 1986) Research continued by Japanese scientists educated in European universities, and Takahashi and Inoko compiled the first major study on the pharmacology of TTX in 1889. They detailed the respiratory failure of poisoned patients and the resistance of the fish to their own toxin, but the researchers were unable to purify the toxin. (Fuhrman 1986) The first isolation of a toxin came in 1911 by Tahara, but this was only about 40ure tetrodotoxin. (Fuhrman 1986) Pre-World War II experiments centered on tarichatoxin, a poisonous substance from the Taricha torosa California newt with a TTX concentration of about 1%. (Fuhrman 1986) Twitty and Tatum's experiment in 1932 showed tarichatoxin produced hypertension, respiratory paralysis and nerve unresponsiveness in cats. (Fuhrman 1986) Initial hypotheses concerning tarichatoxin poisoning were all based on the assumption that the toxin selectively inhibits glucose metabolism by brain tissue. Quastel's experiment in 1941 showed that tarichatoxin inhibited oxygen consumption in brain cells produced by electrical stimulation, and he linked it to the influx of sodium ions, suggesting disruption of the voltage-gated sodium channels. (Fuhrman 1986) However, further experiments were put on hold due to the war and the primitive technology available, which hampered more detailed investigations.
The next big breakthroughs in TTX research came in rapid succession. Turner and Fuhrman's experiment on frog nerve axons in 1946 concluded that tarichatoxin reduced action potentials, but that the toxic effects were completely reversible if the toxin was washed out. (Fuhrman 1986) Crystalline TTX was finally isolated in 1950 by Yokoo, and a Stanford University research group isolated crystalline tarichatoxin in 1962 (Fuhrman 1986), after decades of numerous failed attempts due to the complicated structure and unusual chemical properties of the toxin. Scientists raced to deduce the chemical structure of the crystalline TTX, and it was finally identified in 1964. (Fuhrman 1986) The effects of TTX were pinpointed to a block of preganglionic cholinergic and somatic motor nerves, which were unable to depolarize, and thus no action potentials could be fired. (Fuhrman 1986) Narahashi's research in 1960 led to a more detailed mechanism of TTX action by showing that tetrodotoxin blocked the action potential of muscle fibers by inhibiting the increase in conductance associated with the movement of sodium ions across cell membranes. (Fuhrman 1986) The mid-1960s also brought together the separate fields of tarichatoxin and tetrodotoxin research, as TTX was isolated in the Taricha genus and Notophthalmus viridescens species of newt in the United States, which tarichatoxin researchers happened to be working with. (Fuhrman 1986)
Research since the 1970s has mainly centered on isolating TTX in various species, finding antidotes for TTX poisoning, further explanations for the mechanism of tetrodotoxin's sodium channel-blocking capabilities, and the use of TTX-resistant and TTX-sensitive sodium channels in experiments for other fields. The high-affinity selective binding of TTX to sodium channels has proved to be a useful marker for cell researchers. (Kao 1986; WHO 1984) Our knowledge of tetrodotoxin has markedly expanded in the last 50 years, and this research is now mostly benefiting sodium channel researchers, as TTX is the most important and most widely used tool for selectively blocking sodium channels.
Distribution of Tetrodotoxin in Nature
Tetrodotoxin is widely distributed in nature, in both marine and terrestrial animals, but most of these species have only recently been identified. The eventual discovery of TTX in four different phyla was highly unexpected. Puffer fish of the order Tetraodontiformes were the first species determined to contain TTX, but not all Tetraodon fish toxins are TTX, as 34 of the 40 fish in the order have toxins similar in structure to TTX, but are far less potent. (Fuhrman 1986) In 1964, only Taricha genus newts and puffer fish were known to have TTX. (Fuhrman 1986) However, frogs were soon discovered to contain TTX, as Mosher discovered the toxin in the Atelopus varius and Atelopus chiriquiensis species found in Central America. (Fuhrman 1986) Discoveries continued in many marine species, and TTX has now been isolated in at least 14 families. There appear to be few similarities between the species, which are highly varied. Crabs of the Xanthidae family, starfish of the Astropectinidae family, the Australian blue-ringed octopus (Hapalochlaena maculosa and H. lunata), and at least three families (Buccinidae, Bursidae, Terebridae) of marine snails all contain TTX, but bear little resemblance to each other. Most species have only been singled out through fatal interactions with unsuspecting humans, as TTX was identified in the octopus through fatal bites to humans, and in mollusks like the Japanese ivory shell, Babylonia japonica, through fatal human poisonings. (WHO 1984) Among fish, six different families have now been identified, including the families for puffer fish, porcupine fish, and gobies. (Fuhrman 1986) Ingestion of the Indo-Pacific goby, Ctenogobius criniger, has been reported as the cause for several fatal human TTX poisonings. (Yang et al. 1995)
The appearance of TTX in four separate phyla (Chordata, Mollusca, Echinodermata, Arthropoda) has led scientists to question the origins of the toxin, which is unlikely to have evolved independently four separate times. The source of TTX has been widely debated. The TTX content of animals fluctuates according to regional, seasonal, and individual differences, suggesting that the toxin is not genetically inherited, but is somehow due to the organism's surroundings. (Yasumoto 1986) The 1984 Shimizu study showed that cultivated puffer fish lack TTX, but that they accumulated the toxin when fed toxic puffer fish livers, which led to a conclusion suggesting the toxin's origins are from the food chain. (Yasumoto 1986) Shimizu further hypothesized that, due to the apparent lack of in situ biosynthesis of tetrodotoxin in these animals, various bacteria produced the TTX, and that it was passed up the food chain, resulting in the wide variety of tetrodotoxin-containing species and of the habitats in which these animals live. (Shimizu 1986) However, Yasumoto's study found that fish species most similar to puffer fish accumulated TTX, while fish highly dissimilar to fugu did not contain the toxin, including species living in the same area as poisonous fish, which seemed to contradict Shimizu's research. (Yasumoto 1986) This led scientists to further investigate the puzzling findings, and Matsumura conducted a study in 1995 to examine Shimizu's conclusions. Matsumura's experiment showed that TTX is also present in cultured puffer fish, although in much smaller concentrations than in wild fugu. Using new technology unavailable to Shimizu in 1984, he found that cultured fugu concentrated their TTX in the skin and muscle, while the toxin was predominantly isolated in the ovary and liver of wild fugu. (Matsumura 1996) This result was odd, as the body muscles of Tetraodontidae family fish are usually free of TTX, with the notable exception of Lagocephalus lunaris lunaris, which has fatal amounts of TTX in its muscle (WHO 1984), and speculation continues as to the reasons behind the differences in TTX distribution within poisonous fish. Matsumura's cultured fish averaged approximately 70 nanograms of toxin per gram of fish, significantly less than in wild fugu, but still measurable (Matsumura 1996), which put earlier hypotheses and conclusions in dispute, and has led to current ongoing research into the origins of tetrodotoxin in nature.
Mechanism of Tetrodotoxin-Sodium Channel Interactions
The mechanism of TTX-sodium channel interactions are still being hotly debated, and hypotheses fall into and out of favor as new research papers are published and old theories are called into question. The most popular initial hypotheses of the TTX binding mechanism were based on unproved assumptions. One popular theory, which persisted for many decades until new experiments disproved it, hypothesized that the guanidium end of the TTX molecule lodges itself inside the sodium channel while binding to an interior receptor site, and the toxin's carbon backbone acts like a plug. (Kao 1986) The mechanism currently in favor is the "trapped ion" mechanism, in which tetrodotoxin binds to a receptor site on the extracellular side of a sodium channel, preventing ions from flowing through the pathway. The external mouth of the sodium channel is funnel-shaped, and TTX can block the channel by binding to a site near the narrow end of the pore. (Kirsch et al. 1994) Recent experiments show that the extracellular cations of calcium and magnesium selectively compete with and inhibit TTX binding (Kirsch et al. 1994), and that cation binding promotes the closed state of the channel, which has less affinity for TTX binding than an open channel. (Conti et al. 1996) However, stimulated channels will bind and release calcium ions constantly, which leaves many windows of opportunity for TTX to bind to open channels. Once tetrodotoxin binds to a sodium channel, it cannot be pried out by calcium, and the channel will remain blocked indefinitely. (Conti et al. 1996) Resting channels with closed gates and calcium bound to the extracellular receptor are the least likely to be affected by TTX, while hyperpolarized, overactive channels without a bound cation are the most likely to be blocked by TTX, indicating voltage-dependent conformations of the protein channel have different affinities for TTX binding. (Conti et al. 1996) However, the experiment also showed that cations trapped inside the channel between a bound TTX molecule and a closed inner channel gate will stimulate TTX release from the channel receptor binding site, although the reasons for this observation are not well understood. (Conti et al. 1996)
Mutation of the sodium channel also produces insights into the mechanism of tetrodotoxin binding. Experiments show that Cys373 in domain I of the cardiac sodium channel protein is integral for TTX binding, and that substitution of this cysteine residue will lower the channel's affinity for TTX binding. (Kirsch et al. 1994) Further mutations, which were done to show differences in the mechanisms of TTX and saxitoxin (a neurotoxin found in shellfish with the same lethal effects as tetrodotoxin) binding, once thought to be roughly similar. However, channel receptor mutations have shown the two neurotoxins to be different in their binding mechanism, and they are not interchangeable. Kirsch's experiment mutated a cysteinyl sulfhydryl on the channel near the binding site for the toxins, adjacent to the mouth of the pore. The addition of a positively charged group to the sulfhydryl reduced the pore's conductance by 87%, but this was prevented by tetrodotoxin, which actually increased the channel's conductance by pulling the charged group out of the funnel opening, while saxitoxin did not have this capability. (Kirsch et al. 1994) On the other hand, the addition of a negatively charged group to the sulfhydryl inhibited saxitoxin binding, but did not prevent tetrodotoxin binding. (Kirsch et al. 1994) The toxin-receptor complex was found to assume different conformations when bound with saxitoxin and tetrodotoxin, which further led the researchers to conclude that the mechanisms for binding were very different. (Kirsch et al. 1994)
Penzotti elaborated on Kirsch's work by further mutations of the channel protein, facilitated by computer models interpreting the experimental data. Other residues on the channel protein were mutated, and these mutations led to different binding affinities for saxitoxin and tetrodotoxin. Tyr401 was found to be essential for tetrodotoxin binding, while saxitoxin favored more extracellular residues, such as Glu758 and Asp1532. (Penzotti et al. 1998) The computer models showed that tetrodotoxin heavily favors binding to domain I of the protein, while saxitoxin favors no domain. The TTX closely packs with the domain I P-loop, and the Tyr401 residue is adjacent to the C-4, C-5, C-7, and C-8 groups of the TTX molecule. (Penzotti et al. 1998) The C-9 and C-10 hydroxyl groups were shown to be interacting with Glu758, the C-11 hydroxyl associates with Glu403, and the guanidium group of N-1, N-2, and N-3 is in immediate contact with Glu755. (Penzotti et al. 1998) The strong binding affinity with domain I gives researchers information on the mechanism of TTX binding, and is useful in making mutant sodium channels insensitive to TTX for use in other experiments. However, these mechanisms are still hypotheses, and some researchers still debate the conclusions drawn from these experiments.
Chemistry of Tetrodotoxin and Tetrodotoxin Analogs
Many analogs of tetrodotoxin have been synthesized and are currently being studied, as these modifications are useful for researchers in studying the mechanism of TTX's interaction with sodium channels and other structure-activity relationships. TTX is a highly unusual-looking molecule, with a unique intramolecular hemilactal bond, and is classified as an aminohydroquinazoline compound with a molecular mass of 319. (WHO 1984) TTX extracted from the viscera of fugu is a colorless crystal that is slightly soluble in water, but very soluble in dilute acid. (WHO 1984) It does not dissolve in any neutral organic solvent, such as acetone or DMSO, so it cannot be extracted from aqueous solutions, which accounts for the numerous failed attempts to isolate TTX. (Mosher 1986) Crystalline TTX is also stable at neutral pH and in weak acids, but it is unstable at pH levels above 8.5 or below 3. (Mosher 1986; WHO 1984) So far, scientists have been unable to produce a complete synthesis of this complex molecule, so puffer fish are still the main source for TTX for experiments. (Mosher 1986)
Structural analogs of TTX have been made since the 1970s, mostly with limited alterations in order to study the biological effects of various substitutions. The nitrogenous guanidium group is highly vulnerable to attack, although it is always protonated and positively charged at physiological pH, and has a pKa of 11.6. (Kao 1986; WHO 1984) Changes to this group have to be radical, considering the limitations in modifying this structure, and these changes also usually result in the inactivation of TTX. (Kao 1986) Therefore, the role of the guanidium group in the mechanism for sodium channel binding must be inferred from research done on other substituent groups, as changes to this ring structure also ordinarily result in drastic changes in the rest of the molecule.
On the other side of the guanidium group is the C-6 group, which has also been extensively studied. Modifications to the -CH2OH substituent reduce the potency of TTX, but they are still somewhat toxic. (Kao 1986) Nortetrodotoxin, the substitution of an -OH group to C-6, is only 0.08 to 0.25 times as potent as tetrodotoxin. (Kao 1986)
C-4 analogs, which alter the carbon substituents adjacent to the guanidium group, also have reduced potency. Deoxytetrodotoxin (the C-4 -OH group is replaced by -H) is less than 0.15 times as potent, while 4-epitetrodotoxin (the -OH group is moved from an equatorial to an axial configuration) is only 0.4 times as potent as unaltered TTX. (Kao 1986) An unusual modification to TTX involves the replacement of both the C-4 and C-9 -OH groups with an oxygen bridge, resulting in anhydrotetrodotoxin, which is only 0.02 times as potent as TTX, (Kao 1986) showing that these hydroxyl groups are important for channel blockage, which was later confirmed by Penzotti's study.
The C-10 analogs have unusual properties, as the -OH group equilibrates with an unprotonated form, making a zwitterion that is less potent than the cationic form. (Kao 1986) The potency varies with the pH, and relative potencies are in agreement with the abundance of protonated C-10 groups, with the pH 7.8 form being 2.7 times as potent as pH 8.8 tetrodotoxin. (Kao 1986) This indicates that the -OH group of C-10 is important in hydrogen binding with the sodium channel receptor site, which is also backed by Penzotti's experiment.
Modifications to the C-11 end, however, have little effect on receptor binding, so most toxic activity is retained. (Mosher 1986) All of these TTX analogs help to back up theories of the sodium channel binding mechanism through different structure-activity potency relationships.
Biological Effects of Tetrodotoxin
The effects of tetrodotoxin on biological functions are highly varied, but are all due to its sodium channel blocking interactions. The first known effects were described in humans poisoned by eating toxic puffer fish, but the first detailed experiments were done on laboratory animals. Current research involves rats, after original experiments with tarichatoxin were performed on cats and dogs. (Fuhrman 1986) TTX-induced muscle paralysis in rats was shown to result in decreased duration of action potentials after hyperpolarization, and this was attributed to the lack of positively-charged sodium ions flowing back inside the axon due to TTX blockage of sodium channels. (Gardiner and Seburn 1997) TTX-treated muscles also showed less twitch and tetanic (muscle rigidity) forces, and fatigued more quickly than non-TTX-treated muscles. (Gardiner and Seburn 1997) Further experimentation showed that these muscles had less of a prominent mitochondrial marker, succinate dehydrogenase, which indicated that less metabolic activity is taking place, and the decrease in available ATP for TTX-treated muscles explains their diminished capabilities. (Gardiner and Seburn 1997) . Sublethal doses of TTX administered to rats also decreased brain stem auditory evoked potentials (BAEPs), and impaired the auditory and visual processing centers of the brain and central nervous system in a highly selective manner, while only slightly impairing other systems. (Moore et al. 1996)
In humans, TTX effects in the body are rather simple, but they precipitate a complex set of symptoms. Ingested TTX selectively blocks sodium channels, inhibiting the generation of action potentials and axonal transmissions in motor, sensory, and autonomic nerves in the peripheral nervous system, preventing cell depolarization and subsequent neurotransmitter release. (Ludwig et al. 1995; Yang et al. 1995; Lysko et al. 1993) This blockage of nerve cell function generates symptoms characteristic of marine neurotoxin poisoning. The onset of symptoms generally occurs between 10 and 45 minutes after ingestion of the toxin, but can be delayed for as long as three hours, depending on differences in the individual and the amount of TTX consumed. (WHO 1984) Paraesthesia (the loss of sensation or feeling) begins to appear quickly in the face and extremities, followed by sensations of numbness throughout the rest of the body. (WHO 1984) This numbness is normally accompanied by diarrhea, nausea, vomiting, ataxia (loss of muscle coordination), dizziness, vertigo, and headache. (WHO 1984; Yang et al. 1995) Later, more serious symptoms predominate, especially if a large dose of TTX was ingested. Respiratory problems start to occur within two or three hours, with dyspnea (shallow rapid respiration) worsening until emergency medical care is given or death occurs. (WHO 1984; Yang et al. 1995) Cyanosis (skin turning blue and livid) follows as oxygen becomes more deficient in the body. Respiratory failure eventually leads to convulsions and cardiac arrhythmia, and the patient normally suffocates to death, usually within six to eight hours of the initial poisoning. (WHO 1984; Yang et al. 1995) Victims normally retain consciousness until shortly before death, and TTX poisoning is considered to be highly painful and traumatic. (WHO 1984)
However, poisoned patients have been diagnosed with both hypotension and hypertension, which has led to hypotheses regarding the contradictory nature of TTX effects. (WHO 1984; Yang et al. 1995) Hypotension was ordinarily much more common, but Yang's study of 17 TTX poisoning cases included 8 cases involving hypertension. Hypotension is theorized to be due to a post-ganglionic blockade of sympathetic nerves innervating vascular smooth muscle, resulting in extreme parasympathetic symptoms. Yang postulated that the hypertension cases could be due to an exaggerated response to sympathetic stimuli, as TTX blocks axonal transmission, leaving autonomic effectors intact. The parasympathetic nervous system is then paralyzed slightly earlier than the sympathetic nervous system, and the temporary imbalance between their effects on peripheral blood vessels causes them to tightly constrict. (Yang et al. 1995)
Prevention and Treatment of Tetrodotoxin Poisoning
Tetrodotoxin is one of the most poisonous non-protein substances known to man, and TTX-poisoned patients are in an immediate life-threatening situation due to the severe symptoms and high mortality rate (almost 60%) of tetrodotoxin poisoning (WWW 1). Although the incidence of TTX poisoning is rare, it still represents a significant public health threat in many parts of the world, and most poisoned individuals die, including approximately 50 per year in Japan. (Moore et al. 1996; WWW 1) The increase in world trade has led to the shipment and sale of mislabeled toxic fish to countries where TTX poisoning is unknown. (WHO 1984) The most toxic members of the Tetraodontidae family of fugu, sunfishes, and porcupine fishes, are caught off the coasts of China and Japan, where they are considered a delicacy, and Japanese sushi chefs must obtain a special government license to prepare poisonous puffer fish (WWW 2). However, frozen fish flesh from fugu-infested regions needs to be taken with special care and must be rigorously inspected to prevent the mixing of poisonous and non-poisonous fish. The safety criteria for edible fugu is about 2.2 micrograms of toxin per gram of flesh, which is well above the levels for cultured fugu, (Matsumura 1996) although even tiny amounts of ingested wild fugu can result in severe consequences, with a fatal dose estimated to be between three and six ounces.
Treatment of TTX-poisoned patients must begin immediately. Diagnosis of tetrodotoxin poisoning must be made quickly, so emergency room personnel have to be trained to notice the characteristic symptoms of neurotoxin poisoning. In California, three cases of TTX poisoning were treated with intravenous hydration, activated charcoal, and pumping of the stomach. (WWW 1) Although these methods for treating TTX poisoning are currently in use in hospitals worldwide, scientists are hard at work investigating other chemicals for use as antidotes. One such drug, still in the developmental stages, is 4-aminopyridine (4-AP). A 1995 study showed that 4-AP restored normal breathing and heart rate within 30 minutes of administration to poisoned laboratory animals at the point of weakest cardiorespiratory performance. (Chang et al. 1995) TTX caused disruptions in the central respiratory frequency controller mechanism in these animals, reversible through administration of 4-AP, and had significant central and peripheral nervous system pharmacological actions. (Chang et al. 1995) Experiments continued on 4-AP, and it was found to be a potassium channel blocker that reversed the symptoms of TTX poisoning. (Benton et al. 1996) The optimal therapeutic dose was found to be 2 milligrams per kilogram of body weight for the experimental animals. (Benton et al. 1996) Research on 4-AP is now focused on perfecting the drug to optimize its effects for use as an antidote for human TTX poisoning.
Conclusion
Scientists have now unlocked many mysteries surrounding the marine neurotoxin tetrodotoxin. Many sources of TTX have been discovered, a highly plausible theory regarding the mechanism of the TTX-sodium channel interaction has been put forth, chemical properties of the molecule have been determined, and an antidote for TTX poisoning is nearing perfection. However, many more questions remain to be answered. The reasons behind the varied organisms containing TTX remain a mystery, along with the biosynthesis of the molecule. The exact mechanism of sodium channel interaction and the reasons behind the diverse symptoms of TTX poisoning still pique the curiosity of researchers around the world. Scientists are still interested in understanding why certain treatments for TTX poisoning work and what pathways are used by antidotes. Other scientists are testing to see if TTX can be used to treat other nervous system ailments by harnessing its sodium channel-blocking ability to good use. Although these and many other questions remain regarding the TTX molecule itself, TTX has become a useful tool for researchers studying the voltage-gated sodium channel, and tetrodotoxin's importance in many biological experiments makes it a chemical that will be cited in many papers published well into the 21st century.
References
1.) Benton, B.J. et al. Abstract: 4-aminopyridine antagonizes the lethal effects of saxitoxin and tetrodotoxin. Toxicon 34(3): 335 (1996).
2.) Chang, F.C.T. et al. Abstract: 4-aminopyridine reverses the sublethal effects of saxitoxin and tetrodotoxin. FASEB Journal 9(4): A664 (1995).
3.) Conti, F. et al. Use dependence of tetrodotoxin block of sodium channels: A revival of the trapped-ion mechanism. Biophysical Journal 71(3): 1295-1312 (1996).
4.) Fuhrman, F.A. Tetrodotoxin, Tarichatoxin, and Chiriquitoxin: Historical Perspectives. Chapter 1 (pp 1-13) in Tetrodotoxin, Saxitoxin, and the Molecular Biology of the Sodium Channel (Volume 479) (C.Y. Kao and S.R. Levinson editors). New York Academy of Sciences (1986).
5.) Gardiner, P.F. and Seburn, K.L. The effects of tetrodotoxin-induced muscle paralysis on the physiological properties of muscle units and their innervating motoneurons in rat. Journal of Physiology 499(1): 207-216 (1997).
6.) Kao, C.Y. Structure-Activity Relations of Tetrodotoxin, Saxitoxin, and Analogues. Chapter 6 (pp 52-65) in Tetrodotoxin, Saxitoxin, and the Molecular Biology of the Sodium Channel (Volume 479) (C.Y. Kao and S.R. Levinson editors). New York Academy of Sciences (1986).
7.) Kirsch, G.E., Alam, M., & Hartmann, H.A. Differential effects of sulfhydryl reagents on saxitoxin and tetrodotoxin block of voltage-dependent sodium channels. Biophysical Journal 67(6): 2305-2315 (1994).
8.) Ludwig, M., Callahan, M.F., & Morris, M. Effects of tetrodotoxin on osmotically stimulated central and peripheral vasopressin and oxytocin release. Neuroendocrinology 62(6): 619-627 (1995).
9.) Lysko, P.G. et al. Abstract: Neuroprotective effects of tetrodotoxin in cultured cerebellar neurons and in gerbil global brain ischemia. Society for Neuroscience 19(1): 286 (1993).
10.) Matsumura, K. Tetrodotoxin concentrations in cultured puffer fish, Fugu rubripes. Journal of Agricultural and Food Chemistry 44(1): 1-2 (1996).
11.) Moore, E.L. et al. Abstract: Effects of tetrodotoxin on auditory and visual evoked potentials in the rat. Society for Neuroscience 22(2): 1066 (1996).
12.) Mosher, H.S. The Chemistry of Tetrodotoxin. Chapter 4 (pp 32-42) in Tetrodotoxin, Saxitoxin, and the Molecular Biology of the Sodium Channel (Volume 479) (C.Y. Kao and S.R. Levinson editors). New York Academy of Sciences (1986).
13.) Penzotti, J.L. et al. Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule. Biophysical Journal 75(6): 2647-2657 (1998).
14.) Shimizu, Y. Chemistry and Biochemistry of Saxitoxin Analogues and Tetrodotoxin. Chapter 3 (pp 24-30) in Tetrodotoxin, Saxitoxin, and the Molecular Biology of the Sodium Channel (Volume 479) (C.Y. Kao and S.R. Levinson editors). New York Academy of Sciences (1986).
15.) WHO: World Health Organization. Environmental Health Criteria 37, Aquatic (Marine and Freshwater) Biotoxins. World Health Organization, Geneva (1984).
16.) Yang, C.-C. et al. An outbreak of tetrodotoxin poisoning following gastropod mollusc consumption. Human and Experimental Toxicology 14(5): 446-450 (1995).
17.) Yasumoto, T. et al. Interspecies Distribution and Possible Origin of Tetrodotoxin. Chapter 5 (pp 44-50) in Tetrodotoxin, Saxitoxin, and the Molecular Biology of the Sodium Channel (Volume 479) (C.Y. Kao and S.R. Levinson editors). New York Academy of Sciences (1986).
WWW 1.) Tetrodotoxin Poisoning Associated with Eating Puffer Fish Transported from Japan-California, 1996. Morbidity and Mortality Weekly Report 45(19) (1996).
WWW 2.) Tetrodotoxin.
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