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Tetrodotoxin and the Life Tree New Scientist 2001

AT FIRST the US federal officers thought they had stumbled upon a shipment of heroin. The suspicious package they intercepted last year, en route from Japan to a private address in the US, contained several vials packed with a white crystalline powder. But on-the-spot tests revealed that it was no narcotic. It took a while for forensic scientists at the Lawrence Livermore National Laboratory in California to identify a sample, and what they found was alarming. The powder tumed out to be tetrodotoxin (TTX)-one of the deadliest poisons on Earth. Gram for gram, TTX is 10,000 times more lethal than cyanide. Its potency is well known in East Asia, where it regularly kills diners who have braved the capricious delic acy known as fugu, or pufferfish. This neurotoxin has a terrifying modus operandi-25 minutes after exposure it begins to paralyse its victims, leaving the brain fully aware of what's happening.

Death usually results, within hours, from suffocation or heart failure. There is no antidote. But if luckless patients can hang on for 24 hours, they usually recover without further complications. It is this strange return from the dead that some believe gives birth to Haitian zombies. The Livermore team estimated that to extract the 90 milligrams of TTX discovered by the Feds, you'd need between 45 and 90 kilograms of pufferfish livers and ovaries-the animal's most deadly tissues. No one knows what use its intended recipient had in mind. In the past few years, however, the biological mystery of TTX itself has been unravelled. This is no ordinary poison. It is one of very few neurotoxins that are not protein-based. Stranger still, TTX is found in a wide range of creatures, from algae to angelfish, spanning entire kingdoms of life. It is unlikely that such a unusual molecule evolved independently in so many unrelated animals. So where does it come from? "The biogenesis of TTX has been a problem we've been trying to figure out for years," says biotoxins expert Bruce Haistead, a fellow of the California Academy of Sciences in San Francisco. Now, after decades of detective work, chemists and ecologists have tracked down the culprit. Any magic brew worth its cauldron contains eye-of newt. But you don't need occult powers to mix a deadly potion if the recipe includes the rough-skinned newt (Taricha granulosa) or the California newt (Taricha torosa). These amphibians make no secret of their toxicity. When threatened, they arch their backs, revealing a telltale red belly. Like the thorny ballooning of a frightened pufferfish or the sudden appearance of angry sapphire hoops for which the blue-ringed octopus is named, the newt's red chassis is a clear warning that predators ignore at their peril. The newt's powerful toxin was first isolated in the 1960s by Stanford University chemist Harry Mosher and his graduate student Melanchton Brown. At that time, the campus was in a rural setting, and nearby ponds provided many litres of newt spawn, which in turn yielded tiny quantities of the poison. In honour of the donor animals, the researchers named the crude concentrate that they extracted tarichatoxin. But not long after the discovery, Mosher found out that their new toxin was in fact identical to a molecule that had already been described-a toxin isolated from pufferfish and known as fugu poison or TTX. In the late 1970s, maculotoxin-the fearsome venom of the blue-ringed octopus (Hapalochlaena maculosa)-was likewise found to be TTX. Since then, the list of creatures known to possess this powerful poison has lengthened to include flatworms, parrotfish, starfish, marine nematodes, arrow worms, sea squirts, angelfish, xanthid crabs, sea stars, globefish, horseshoe crabs, ribbon worms living on cultured oysters, trumpet shells and other gastropods, the Central American toad genus Atelopus-members of which are known as harlequin frogs for their festively threatening coloration-and a species of tropical "poison arrow" frog.

The latest additions are three species of gobie fish from Taiwan. TTX has also been found in low concentrations alongside a structurally similar group of molecules called saxitoxins (STX) in the algal blooms known as red tides. When chemists first worked out the molecular structure of TTX in the 1960s, Mosher was taken aback. Nothing quite like it had ever been seen before. "The structure was an astonishing new molecular arrangement," he recalls. Mosher and his colleagues had trouble even classifying the new molecule in the standard schema of natural products. "It is not an alkaloid, steroid or carbohydrate," he says, "and it is not like any conventional amino acid. It's a low molecular weight, small molecule with a unique cage structure." Toxicologist William Haugen Light, from the California Academy of Sciences, calls it "one of nature's strangest molecules". Three nitrogen atoms are responsible for the TTX molecule's awesome body count. Together, they form what is known as a guanidinium group, which is attached to a complex arrangement of paired hydrogen and oxygen atoms. Once inside a victim's body, the poison shuts down the nervous system through its effect on neurons'sodium ion channels. The guanidinium group tenaciously attaches to the channel entrance: "The TTX molecule forms a cap or plug that reversibly binds to the mouth of the sodium channels on the nerve axon," explains Mosher, 'thereby preventing nerve conduction.' Like the wrong key jammed tightly in a lock, TTX molecules clog the channels, blocking the rapid flooding of sodium ions across the cell membrane that normally propagates nerve impulses. A victim of fugu poisoning, or a swimmer bitten by a blue-ringed octopus, first feels a tingling about the mouth. This quickly spreads to the rest of the body, bringing with it paralysis and feelings of warmth, together with waves of nausea and tremors. But because TTX cannot cross the bloodbrain barrier, the sufferer remains helplessly conscious while the peripheral nervous system shuts down. By the time TTX molecules let go of their sodium channels hours or days later, most victims have died.

Natural born killers?

How could creatures so widely spread throughout the tree of life have independently evolved this unlikely defence? The consensus is that they could not. In 1984, Mosher amd Fred Fuhrman, a chemist from Stanford University, proposed that all species with TTX may share an undiscovered microbial symbiont which accounts for their improbable molecular weaponry. The idea was championed by other researchers, most notably Japanese chemists Takeshi Noguchi at Tohaku University in Sendai and Tamao Yasumoto of Nagasaki University, who found several marine bacteria capable of producing small quantities of TTX. But critics pointed out that the amounts of toxin produced by these microbes in the lab were much too low to explain the levels of poison regularly found in pufferfish and other species that produce TIX. Worse still, in the mid-1990s the Japanese findings were called into question when it was discovered that the chromatographic signature of TTX-by which Noguchi and Yasumoto had identified the molecule-was the same as that of several other marine toxins. Many researchers came to suspect that instead of Mosher's hypothetical symbiont, a polluted food chain could explain the wide array of creatures toting TTX in their tissues. Like DDT's infamous climb up the food chain, they argued, TTX travels through a network of TTX-immune life forms that feed upon one another, becoming more concentrated in the tissue of each subsequent consumer. This was seen as the only way to explain the high concentrations of TTX in some predatory species. But just when it looked like Mosher's notion of a microbial symbiont should be scrapped, two teams of scientists on opposite sides of the globe announced that bacteria do indeed produce TTX. First was a team of American and Dutch researchers investigating the mysterious mass deaths of sea urchins in the Netherlands Antilles. In 1997, millions of the animals living just down-current of the main harbour at Curaqao lost their spines and then their lives. The team, led by Kim Ritchie of the University of North Carolina at Chapel Hill and Ivan Nagelkerken of the Carmabi Foundation in Curaqao, captured a new species of bacterium dubbed Pseudoalteromonas haloplanktis strain VL-1 from the tissues of dying urchins. In a paper published last year, they revealed the microbe's deadly weapon: TTX. Even more spectacularly, a team of biologists from Dongguk University in South Korea led by Myoung-ja Lee simultaneously provided incontrovertible proof that pufferfish owe their toxicity to a microbial biowarfare factory in their guts. Lee and his team made a culture of bacteria taken from the intestines of one of the most highly toxic species of pufferfish (Fugu vermicularis radiatus) and found their own new strain. They have preliminarily identified the new bug as a Vibrio species-the same genus of microbes that carries cholera. When tended in a Petri dish, the microbe-which they named Vibrio LM-1-also produced TTX. "This study revealed for the first time that a Vibrio strain from the highly toxic wild pufferfish produced TTX," says Lee. Researchers have yet to nail bacterial culprits in Taricha newts and other nonmarine species that produce TTX. But most scientists in the field now think that they will. "I think that the evidence now is very convincing for the bacterial origin of TTX," says Garriet Smith from the University of South Carolina who worked with Ritchie. Smith also believes that the killer bacterium that the Korean team lifted from Asian pufferfish innards is the same one that the Ritchie team found perpetrating a sea-urchin massacre in the Antilles.

'Lee and colleagues probably isolated Pseudoalteromonas, ', says Smith. He points out that the Korean team did not use generic sequence data to identify the strain as a Vibrio species, relying instead upon physical and biochemical characteristics for identification. "If their 'Vibrio' isolate was [genetically] sequenced, I suspect that it would actually be a Pseudoalteromonas," he says. Even if LM-1 and VL-1 tum out to be distinct strains, the finding that two species of bacteria carry TTX genes would not be terribly surprising. For one thing, the Vibrio and Pseudoalteromonas genera are dosely related. In addifion, bacterial genomes are highly promiscuous, containing amalgams of their own ancestral lineage and genes added to the mix by bacteria-hunting viruses called phages. Bacteria that survive a phage attack often end up carrying DNA from the virus's previous victims. It is precisely this process that gave a previously innocuous strain of Vibrio bacterium the power to transmit cholera. Mosher clearly got the microbe part right. But are TrX-producing bacteria benevolent symbionts, living in harmony with their carriers, or are they opportunistic killers? "They are undoubtedly both," concludes Smith. Most animals, including sea urchins and humans, fall victim to the microbes' deadly toxin, but others have learned to live with them. "In the case of pufferfish, they have probably been mutualistic for a very long time, enabled by a mutation in the fish's sodium ion receptor proteins to which TTX is unable to bind," he says. A,nd recent research by Kunio Yamamori at Kitasato University in japan reveals that pufferfish also have TTX-specific binding proteins that circulate alongside blood cells, mopping up loose TTX molecules. Similar proteins have been found in horseshoe crabs and xanthid crabs. In other words, although the microbes are natural born killers, they have cut a deal with some marine and semi-aquatic animals. The most likely explanation is that they exchange poisons for nutrients. "Pseudoalteromonas haloplanktis probably exists free-4iving in the water and colonises other organisms as conditions become favourable," says Smith. By setting up home in the guts of pufferfish or the salivary glands of blue-ringed octopuses, they are in a good position to take food from their host, while at the same time providing a valuable defensive weapon. What remains unclear is why the bacteria evolved TTX in the first place. "They may use these poisons to keep competing microbes away," suggests Light. But Mosher is quick to reject that explanation. He sent samples of his newt toxin to a lab specifically to test for antibacterial activity in the 1960s. "It was reported to be negative in several such tests," he says. Of course, it's possible that TTX might help bacteria fight the immunological defences of animals such as the hapless sea urchins. Nobody knows for sure. Peter Anderson, a comparative neurobiologist from the University of Florida, believes the most likely explanation is that TTX is just a spectacular by-product of mundane bacterial metabolism. Many Vibrio bacteriamanipulate sodium gradients across cell membranes to fuel the rotor-like flagella that allow them to scoot around. If Anderson is right, the poison's vicious effects are purely coincidental.

Therapeutic poisons

While some animals have evolved to tum TTX's dark powers into venom or a deterrent against attackers, humans are now learning to put the poison to good use. It has a place in the neuroscientist's tool kit as a molecular probe. Trace amounts of the poison may even be therapeutic. Stanford University neurobiologists have found that, in mice, vanishingly small doses of TTX prevent a form of epilepsy that results from severe brain trauma. Other research suggests that tiny amounts of TTX may prove useful in controlling difficult-totreat neurogenic pain-the sort associated with migraine and multiple sclerosisbecause it penetrates nerve cells. However, less benign pracfitioners may have already anticipated medical science in the exploitation of TTX. In 1982, Harvard ethnobotanist Wade Davis discovered that Haitian voodoo "zombie masters" apparently create their living-dead slaves by means of a powder containing, among other things, extract of pufferfish. Many opposed Davis's views, however, and even if he is right, zombie masters must kill far more people than they resurrect. Halstead worries that terrorists, too, may make use of TTX, and others share his concem. Smith points out that TTX-producing bacteria are easier to isolate and grow than some microbes that are already feared as potential agents of bioterrorism, including clostridium, which causes tetanus and botulism. "Conditions required to maxiniise TTX production are also published," says Smith, "so use of these bacteria by terrorists should be of some concern." Last year's US haul seems to prove that someone, somewhere, has had the same idea. 13

Bryant Furlow is a science writer based in New Mexico

Further reading: 'A tetrodotoxin-producing Vibrio strain, LM-1 " by myoung-ja Lee and others, Applied and Envlmnmental Micmbiology, vol 66, p 1698 (20M) -A tetrodotoxin-producing madne pathogen" by K'lm Ritchie and others, Natum, vol 44, p 354 (2000) "Toxicity in animals: trends In evolution?' by Dietrich Mebs, Toxicon, vol 39, p 87 (2001)