One fish, Two fish, Red fish, Blow fish… Nick Lowe & Emily Bauer

Introduction History Source Mechanism of Action Chemistry Case Studies

Tetrodotoxin Deadly toxin that blocks voltage activated Na + Channels.

The Puffer Fish Tetrodotoxin(TTX) is named after the order of fish from which it is most commonly associated, the Tetraodontiformes or the tetraodon pufferfish.

The deadly blowfish has been feared for thousands of years. Discovery The tomb of an Egyptian Pharoah was engraved with the blowfish’s image. The Bible warned against eating fish without fins and scales, like the Red Sea puffer. The structure of tetrodotoxin was determined by Robert Woodward in 1964.

Eating the Honorable Fugu Fugu's trade volume is rising because of people's curiosity in eating the toxic fish. Fugu has been eaten in China and Japan for thousands of years. Fugu is one of the most expensive foods in Japan. A single fish served in a restaurant, it can bring in $200.

Fugu chefs in Japan must pass a test and eat the fugu he/she has prepared. There are over 1,500 fugu restaurants in Tokyo. Only 25% of the applicants pass the test. A Fugu Chef

Voodoo + Tetrodotoxin=? Voodoo is the predominant religion in Haiti Many believe in the existence of zombies Two explainations 1. Religious 2. Scientific Does Voodoo or drugs make zombies?

In ’83, ethnobotonist Wade Davis reported existence of “Zombies” in Haiti Named one of National Geographic’s “Explorers for the Millennium” Many remain skeptical of his findings Stresses “psychobiological” aspect of his hypothesis The Serpent and The Rainbow

Is there experimental proof of Zombies? “Zombie” powder given to rats in the lab of Leon Roizin Some rats appeared “comatose” Certain rats immobilized for 24 hrs, then recovered Similar response in monkey RESULTS NEVER PUBLISHED!!!!!!

Animals That Contain Tetrododoxin California newt Taricha salamander Parrotfish Frogs of the genus Atelopus Starfish Blue-ringed octopus Angelfish Triggerfish Goby Cod Xanthid crabs Boxfish Porcupine fish Globefish Horseshoe crab Marine snails

What makes Tetrodotoxin Bacteria inside these animals make the toxin. These bacterial species, include strains of the family Vibrionaceae, Pseudomonas sp., and Photobacterium phosphoreum. Supporting Evidence Puffer fish grown in culture do not produce tetrodotoxin. The blue-ringed octopus accummulates tetrodotoxin in a special salivary gland and infuses its prey with toxin by bite. Xanthid crabs contain tetrodotoxin. Tetrodotoxin in algae species Jania is produced by a bacteria species Alteromas. Puffer fish do not have the genes coding for the synthesis tetrodotoxin molecules.

The TTX-producing bacteria and host organisms offer advantages to both partners. The bacteria get a safe place to live, eat, and reproduce The hosts use the toxin for predation or defense. The blue-ringed octopuses employ TTX as a potent venom for securing prey. Newts arch their backs, revealing a red-and-black surface, a common warning sign in nature.

Why aren’t host organisms effected? A single point mutation in the amino acid sequence of the sodium-ion channel makes it immune from being bound by TTX. Tetrodotoxin will not recognize the channel in these organisms. The only known predators resistant to this toxin is the common garter snake.

Toxicity "Weight-for-weight, tetrodotoxin is ten times as deadly as the venom of the many-banded krait of Southeast Asia. It is 10 to 100 times as lethal as black widow spider venom when administered to mice, and more than 10,000 times deadlier than cyanide.” William H. Light One blow fish is enough to kill more than 30 people. The estimated lethal dose for an adult, is one to two milligrams.

Action Tetrodotoxin acts directly on the electrically active sodium channel in nerve tissue. It blocks diffusion of sodium through the sodium channel. This prevents the firing of action potentials in nerve cells. Tetrodotoxin acts on both the central and the peripheral nervous systems, ultimately paralyzing muscles. Respiratory arrest is the cause of death.

Action potential The flow of Na₊ ions into the axon leads the axon to become positively charged. The inside grows increasingly more positive.

Within the channel are two types of charged particles forming the gates that control the diffusion of Na+. Sodium Channels openclosed The gate closes at polarization and opens at depolarization.

Depolarization As Na+ ions flow into the axon the action potential is initiated.

Repolarization The membrane of the axon is repolarized by the closing of Na+ channels and the opening of K+ channels.

Transmission

Muscle contraction

When Na+ Channels are blocked an action potential can not be initiated and muscles can not contract. This is important for things like… Breathing

Effect on the Heart Tetrodotoxin can block neural and skeletal muscle Na+ channels at 10 nM. It takes 100 times more to block cardiac channels. Cardiac muscle skeletal muscle Na + channels differ in structure and protein composition.

Symptoms of TTX poisoning Symptoms occur within 30 min Initial tingling sensation in mouth Followed by high fever, headache,vomiting, lightheadedness, dizziness, feelings of doom, anxiety, and weakness Elevated blood pressure Bleeding lesions may occur over much of the body Respiratory arrest

SAXITOXIN PROCAINE Structures TETRODOTOXIN TTX and STX inhibit Na + channels. Both bind the outer enzyme complex. Produce similar physiological responses. Local anesthetics are also selective for some Na+ channels.

Biosynthesis of TTX

Synthesis of Tetrodotoxin

Isolation of TTX Extracted from the gonads, liver, intestines, and skin of pufferfish

Tetrodotoxin Mechanism Guanidinium moiety present on molecule + charge at physiological pH Selective, complete, and reversible binding

These toxins bind to same sites on sodium channel Binding Conformations of Tetrodotoxin and Saxitoxin TetrodotoxinSaxitoxin

Tetrodotoxin binds to the outside of the sodium channel on a one-to-one basis.

Uses of Tetrodotoxin Tetrodotoxin is used as a tool because of its selective blocking of the voltage-gated Na+-channel. It was used to isolate channels, and identify the structures of Na+ channels. Researchers have synthesized a labeled 3H- tetrodotoxin and a photoactivatable form which may help locate the tetrodotoxin-binding site. In diluted from it is also used as a painkiller for victims of neuralgia, arthritis and rheumatism.

Antidotal Evidence In 1998, three chefs in southern California collapsed soon after eating small amounts of puffer fish brought back from Japan by a co-worker. Two women nearly died after eating puffer fish in a Los Angeles restaurant. In Australia, several people recently died soon after being bitten by the blue-ringed octopuses. A 69-year-old woman was admitted to the Casualty Dept. for food poisoning. Prognosis was bleak…

+ = In July 1979, a 29-year-old college student in Oregon suddenly collapsed at a party and later died. TTX

Thanks! Have a great day any questions?

References Booth, William, “Voodoo Science,” Science. (1988) Hille, Beritle, Ionic Channels of Excitable Mebranes. (1992)