Tetrodotoxin and Maculotoxin

1. Tetrodotoxin and Maculotoxin Skye King and Azadeh Fotouhie

2. Outline • Molecular Discussion of Tetrodotoxin and Maculotoxin • Biochemistry of Tetrodotoxin and Maculotoxin • Origin, Habitat, and Geographical Distribution • Scenario: Blue-Ringed Octopus vs. Diver • Tetrodotoxin Poisoning and Mechanism • Maculotoxin Poisoning and Mechanism • Symptoms of Tetrodotoxin and Maculotoxin Poisoning • Treatment of TTX and Maculotoxin poisoning • Sources

3. Tetrodotoxin: Structure • C11H17N3O8 • Tetrodotoxin is a solid, colorless crystal, and a potent poison or toxin. • When heated above 428° F, the solid darkens. • It is also known as anhydrotetrodotoxin 4-epitetrodotoxin, or tetrodonic acid. • LD50: 5.0-8.0 μg/kg • Molecular mass: 319.28 u

4. Maculotoxin • Maculotoxin is the venom version of tetrodotoxin. They are both highly toxic to animals and to humans and target the neural pathway. The effects of maculotoxin have been found to be identical to those of tetrodotoxin and are mentioned interchangeably by researchers for the purposes of discussion. • Have different sources in nature

5. Biochemistry of Tetrodotoxin and Maculotoxin • TTX and Maculotoxin are known as neurotoxins; target sodium channels in neurons • Because of the shape of the molecule, it can perfectly plug the opening of the ion channels located on the neurons, prevented ion flow; structure fits function • Bind to Site 1 of the voltage-gated Na+ channel in neurons. • The Na+ channel becomes temporarily non-functional • No Na+ influx into the cell; no action potentials produced and transferred to other neurons or cells in the body; no information transfer • Other toxins like saxitoxin and conotoxin bind to this site as well

6. Voltage-Gated Na+ Channel TTX and Maculotoxin bind to the lumen of the Na+ channel (extracellular side) and prevent sodium influx into the cell. From http://openwetware.org/wiki/BIO254:AP

7. Biochemistry of Tetrodotoxin and Maculotoxin • Due to extensive studies of tetrodotoxin and maculotoxin, two types of voltage-gated Na+ channels have been discovered in humans: • Tetrodotoxin-sensitive channels: TTX and maculotoxin bind to this site with an affinity of 5-15 nanomolar. • Tetrodotoxin-resistant channels: TTX and maculotoxin bind to this site with significantly lower affinity

8. Biochemistry of Tetrodotoxin and Maculotoxin • Tetrodotoxin-sensitive channels are found throughout the body and in central nervous system • Tetrodotoxin-resistant channels are found in neurons located in the cardiac tissue

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10. Sources of Tetrodotoxin and Maculotoxin • Tetrodotoxin can be found in various animal species: western newts (Taricha), parrotfish, toads (Atelopus), certain starfish, certain angelfish, Chaetognatha, nemerteans, xanthid crabs, pufferfish, poison arrow frogs, and flatworms. It is usually found in specific parts or tissues of the organisms. For example, in pufferfish, TTX is found in high concentrations in the gonads, liver, intestines, and skin. • Maculotoxin is the venom version of tetrodotoxin and it is found in the salivary glands of the blue-ringed octopus (Hapalochlaena)

11. Sources of Tetrodotoxin and Maculotoxin • The toxicity of the compounds depends on • Species in which it is found and species which is victimized • Different seasons • Different geographic locations • The individual organism carrying the toxin

12. A typical scenario of maculotoxin poisoning…….. …….It was in the midst of summer, and the sun glimmered upon ocean surface as the group of scuba divers explored the hidden depths. It was then that the young, adventurous diver strayed too far from the group. He had noticed something in the distance….a strange octopus he had never before seen…. …….as foolishness grasps him and ignorance sparkles in his eyes, he decides to capture this octopus and show it to his fellow scuba divers….he smiles with triumph as he traps it between his hand and a rock jutting out of the ground. Reaching forth to grab it, he feels the octopus brush against his hand and sees a drop of blood dissipate into the water…..

13. A typical scenario of maculotoxin poisoning……..continued. …realizing he has been bitten by the octopus, panic-stricken he looks around for the others, in vain….he attempts to swim to the surface to find his fellow divers, only to realize that he has drifted too far from the group….. ….As the toxin begins to set in, unbeknownst to him, he feels the urge to swim somewhere, anywhere, to rest and ease his weak limbs….the journey to shore is a difficult one. Numb, weak, and almost paralyzed, he reaches safety….safety from the outside world; inside, he is dying….

14. Tetrodotoxin Poisoning • Tetrodotoxin poisoning can occur through eating fish that is improperly prepared, or through the contamination of other food products. • A common reason for tetrodotoxin poisoning is the consumption of the pufferfish, which contains very high levels of the toxin in its skin and internal organs.

15. Tetrodotoxin Poisoning • Pufferfish contains enough tetrodotoxin in its skin and organs to cause death in those that consume it; samples of pufferfish contain from 0.5 to 30 mg of tetrodotoxin per kg of wet tissue (LD50 is 5.0-8.0 μg/kg). • In Japan, specially trained and licensed chefs prepare a traditional dish served at special restaurants, called fugu. Fugu is prepared from pufferfish, with small amounts of tetrodotoxin in it to cause slight symptoms of tingling and numbness, without causing serious symptoms.

16. Fugu in Japan Fugu is a traditional dish of carefully prepared pufferfish, considered a delicacy in Japan

17. Tetrodotoxin Poisoning • The first noted case of TTX poisoning is known to have occurred on the ship of Captain James Cook. It is recorded that his crew ate a local tropical fish and fed the remains to the pigs on the ship. The morning after that, the crew members were all inflicted with symptoms of tetrodotoxin poisoning and all of the pigs were found dead. Clearly, the remains that were given to the pigs contained high concentrations of tetrodotoxin and killed them. • Tetrodotoxin was first isolated in 1909 by the Japanese scientist, Dr. Yoshizumi Tahara. • TTX is common in pufferfish of the Atlantic Ocean , Gulf of Mexico, and Gulf of California.

18. Maculotoxin Poisoning • Maculotoxin is found mostly in the blue-ringed octopus. • This octopus has two poison glands that can secrete poison into the saliva. One type is used to obtain food and is effective in capturing crabs as prey; the other is extremely toxic and is known as maculotoxin. • When threatened, the octopus uses this toxin as a defense mechanism. The main method of poisoning humans and other animals is not very well known. We are unsure as to whether it is through the bite, or mere secretion that the octopus defends itself.

19. Maculotoxin Poisoning • The blue-ringed octopus warns its predator when it is ready to attack. It has blue rings on its skin, which have evolved a symbiotic relationship with the maculotoxin-producing bacteria that live in the salivary glands of the octopus. The bacteria make use of the good living conditions and, in turn, produce a toxin to help the octopus defend itself. • The blue rings of the octopus change shape and flash to warn predators of their danger.

20. Maculotoxin Poisoning • An adult blue-ringed octopus weighing 25 g has enough toxin to paralyze and kill 10 adult humans. • Due to this reputation, they are being killed by fearful humans. • In other cases, in Australia, world-leading venom industries harvest the octopus to make use of its venom. • This raises concern about the possible increase in casualties due to the increasing interaction between humans and the octopus; in addition, members of the blue-ringed octopus species are decreasing with time.

21. Symptoms and Effects of Tetrodotoxin and Maculotoxin Poisoning • One significant effect that the blockage of the Na+ channel has on the body is that when Na+ does not enter the contractile cells of the heart, or myocytes, the heart can no longer contract as it should. This paralysis of the myocytes causes death in the individual poisoned.

22. Symptoms and Effects of Tetrodotoxin and Maculotoxin Poisoning • Once the Na+ channel is blocked by the toxin, action potentials can no longer be produced and information does not travel between the CNS and the body. Hence, paralysis of muscles occurs, followed by a multitude of symptoms.

23. Symptoms and Effects of Tetrodotoxin and Maculotoxin Poisoning • During the First Stage of Poisoning: • Numbness, paresthesia (tingling of lips and tongue), facial paresthesia, extremity paresthesia and numbness, headache, light-headedness, feeling that one is floating, sweating, dizziness, salivation, vomiting, nausea, diarrhea, abdominal pain, motor dysfunction, weakness, speech difficulty • Typically starts 15-30 minutes after exposure to the toxin, but can be delayed up to four hours

24. Symptoms and Effects of Tetrodotoxin and Maculotoxin Poisoning • During the Second Stage of Poisoning: • Increased paralysis throughout body, paralysis in respiratory muscles, difficulty breathing, abnormal heart rhythms, hypotension, fixed and dilated pupils, coma, seizures, respiratory arrest, death. • Death can even occur 20 minutes after exposure, or 24 hours after exposure. Usually the second stage symptoms are seen in the firs 4-8 hours

25. Treatment • There is no antidote for tetrodotoxin or maculotoxin due to the quick action and high binding affinities of the toxins • Therapy is mostly supportive for tetrodotoxin poisoning. Respiratory support is essential and must be implemented; in addition, alpha adrenergic agonists and intravenous fluids can combat hypotension.

26. Treatment • After being bitten by the blue-ringed octopus, pressure must be applied to the wound and rescue breathing must be given and continued until the victim can breathe again. • In the hospital, the toxin can be washed out while giving respiratory assistance. Children are more susceptible and thus must be given medical attention first. Victims that can live through the first 24 hours usually recover completely.

27. Sources • Chew, S.K., Goh, C.H., Wang, K.W., Mah, P.K., and Tan, B.Y. 1983. Pufferfish (Tetrodotoxin) Poisoning: Clinical Report and Role of Anti-cholinesterase in Therapy. Singapore Medical Journal. 24(3): 168-171. • Ahasan, H.A.M.N., Mamun, A.A., Karim, S.R., Bakar, M.A., Gazi, E.A., and Bala, C.S. 2004. Paralytic Complications of Pufferfish (Tetrodotoxin) Poisoning. Singapore Medical Journal. 45(2): 73. • Freeman, S.E. and Turner, R.J. 1970. Maculotoxin, A Potent Toxin Secreted by Octopus Maculosis Hoyle. Toxicol. Appl. Pharmacol. 16(3): 681-690. • Dulhunty, A. and Gage, P.W. 1971. Selective Effects of An Octopus Toxin on Action Potentials. Journal of Physiology. 218(2): 433-445. • 1. Xanthid Crab: <http://www.hawaiisfishes.com/inverts/true_crabs/red_crab-Tina.jpg> • 2. Starfish: http://www7.nationalgeographic.com/ngm/0505/feature1/online_extra.html • 3. Parrotfish: http://www.junglewalk.com/animal-pictures/660/Parrotfish-12636.jpg • 4. Blue Poison Arrow Frog: http://www.livingunderworld.org/anura/database/dendrobatidae/dendrobates/auratus/index.shtml • 5. Chaetognatha: <http://artikler.uwphoto.no/100/esv080cd16.jpg • 6. Wesern Newt: http://www.livingunderworld.org/caudata/database/salamandridae/taricha/ • 7. Blue-ringed Octopus: <http://helium.vancouver.wsu.edu/~lindblad/blueringedoctopus.html>