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Does Pfiesteria Produce a Toxin?
Researchers Track Elusive Chemical

By Erica Goldman

Peter Moeller in his lab - photo by Peter Moeller
Over seven years, NOAA's Peter Moeller has worked hard to pin down Pfiesteria's mysterious toxin (molecule pictured at right). His team found that the toxin forms when Pfiesteria binds copper to sulfur, a pathway that unleashes a fierce and potentially destructive free radical cascade in the process. Photographs by Peter Moeller.

Scientists know that Pfiesteria feeds voraciously on blood and tissue — not to mention other algae — but is it toxic? Does it produce a toxin that it releases in the water, a poison that not only stuns microscopic prey but that might kill fish and possibly sicken people?

Showing whether Pfiesteria produces a toxin has proved no simple matter. Time and time again over the years, different labs reported finding toxic activity in the water of their Pfiesteria cultures. But the substance would seemingly vanish before chemists could lay their hands on it. Frustratingly elusive, the suspected toxin appeared either nonexistent or a master of deception.

Although they still don't know what role, if any, it played in specific fish kills, scientists have now caught a toxic culprit — at least part of it. Earlier this year, a team led by natural products chemist Peter Moeller of the National Oceanic and Atmospheric Administration's (NOAA) National Ocean Service in Charleston, South Carolina identified a compound with toxic activity produced by Pfiesteria. They published their findings in the January 2007 issue of Environmental Science & Technology.

Moeller's team found that the toxic compound made by Pfiesteria contains copper and sulfur and that it produces free radicals, highly reactive and unstable atoms or groups of atoms sometimes described as a sort of molecular welding torch. In animal tissues, free radicals can wreak havoc on cells and may speed the course of cancer, cardiovascular disease, and age-related diseases.

The newly identified compound produced by Pfiesteria represents the first time a free radical mechanism of toxicity has been identified in the marine environment. In the terrestrial world, Moeller notes, there are several examples. Scientists have linked a food contaminant called ochratoxin, a free radical compound produced by a few species of fungi, to kidney damage, birth defects, and cancer. Researchers have also discovered that bleomycin, a compound with free radical properties produced by the bacterium Streptomyces verticillus, can be used as a powerful chemotherapy agent.

How does Pfiesteria produce this free radical toxin? And why? Moeller explains that copper could hold the key to both the "how" and the "why." Copper is toxic to phytoplankton, even in small amounts. Pfiesteria binds copper tightly to sulfur, locking it up, but in doing so produces a toxic chemical reaction (see The Copper Connection).

Moeller suspects that the Pfiesteria's toxic compound forms as a direct response to copper in the environment and may have evolved to protect the dinoflagellate from cellular harm.

To determine why any given harmful algal species produces a toxin is quite tricky, says Alan Lewitus, a long-time Pfiesteria researcher and now a program manager for NOAA who helps to administer the Ecology and Oceanography of Harmful Algal Blooms (ECOHAB) grant program. According to Lewitus and others, while Pfiesteria may have evolved the ability to make this chemical in order to remove metals from its environment, the resulting toxin may also play an important role in inhibiting competitors or predators. This would make it a trait that evolution would select for in a seemingly purposeful manner.

"It's funny how nature and evolution work," Lewitus says.

Delicate Detective Work

Determining precisely how Pfiesteria produces this free radical toxin is still a long way off. Just identifying this unstable compound took Moeller's lab seven years of painstaking work. Seven years is actually about average for a discovery in natural products chemistry, but the work itself proved quite tricky, Moeller explains, because the toxin degraded so dynamically — practically¬ in real time.

"When you're dealing with free radicals, you're dealing with a moving target," says Moeller. "I like to jokingly invoke the Heisenberg uncertainty principle — once you pin down one aspect of it, the rest has changed. That is what free radicals¬ do."

Moeller's team worked under red light to slow the toxin's disappearing act long enough to study it. Moeller knew from previous work with free radicals that white light makes them even less stable. By removing most of the wavelengths that make up white light, the researchers gained a little more time, an extra day or two, to prepare the samples for the Nuclear Magnetic Resonance (NMR) spectrometer, an instrument that analyzes a molecule's structure using a strong magnetic field.

Early in the game the team suspected that a metal such as copper was involved in the molecule's structure. Mass spectral analysis, which identifies molecular structure based on mass and charge, kept showing a substance that was many times heavier than what the NMR data predicted. Moeller implicated metals early on because the substance appeared very water soluble and not lipophilic (fat-loving), both properties of metals. In addition, the mass of the compound fluctuated, a characteristic common to metal complexes.

But the larger community of harmful algal bloom (HAB) researchers was skeptical. "They kept telling me, �No, no we don't have metals. Those things don't exist. They are not important,'" Moeller says. "So I let it go for a couple of years, looking for different things. But we kept isolating the same substances. Every time."

So the team moved forward and performed a process called sublimation. Taking samples from a Pfiesteria bloom in the Neuse River in North Carolina, they removed the water and under a vacuum evaporated the salts that were left. What they found was more copper than could be accounted for in natural seawater. That gave a pretty good clue that they were looking for copper in the structure of the toxin.

To actually characterize the structure of this mystery molecule, Moeller grew Pfiesteria in mass culture to provide toxic extracts for further analyses. To start this culture he took Neuse River Pfiesteria cells and cultured them in seawater from the open ocean, rather than from shallow estuarine water where the dinoflagellate is typically active. Why? Because ocean water is less complicated to analyze chemically. The open ocean of the Gulf Stream is not as rich in organic and other contaminating matter, making it easier and faster to purify the compound of interest¬.

But in the end, choosing to culture Pfiesteria from the open ocean may have made things more complicated for Moeller. In Moeller's proposed structure, he suggests that the molecule contains ligands — atoms or smaller organic molecules that act like counterweights to stabilize the copper atom. These ballast-like units are small in Moeller's proposed structure, but in the organic soup of an estuary, one might suspect that they would be much bigger and more varied.

Moeller has taken criticism on this point and he admits that the exact nature of these molecular counterweights remains unknown. In his view, if they turn out to be longer than he's predicting — as might be the case in estuarine waters — the toxin might prove even more potent. This is because longer ligands could stabilize free radicals better, and the more time these free radicals hang around the more toxic they could become. He's careful to add that "we still don't know what this will mean in the wild."

Fueling Debate

The discovery by Moeller and his team answers one important question about arguably the world's most notorious dinoflagellate. Pfiesteria can produce a compound that exhibits toxic activity.

Other questions still remain. Has the burden of proof been met to definitively identify the Pfiesteria toxin? Could Pfiesteria produce this toxin in sufficient quantities — and would the toxin linger long enough — to cause fish kills? Do other marine organisms make free radical toxins?

Robert Gawley for one, a prominent organic chemist from the University of Arkansas in Fayetteville who has also studied Pfiesteria, believes that a new toxin has not yet been identified. "The commonly accepted criteria for chemical characterization of new compounds have not been met by Moeller's evidence," he writes in an email.

Moeller knows that there is still work to be done and a lot of questions to be answered. Free radical toxins are not common and they are new to the community of harmful algal bloom (HAB) researchers. But, he says, "I believe that we've stumbled upon a very real and very important mechanism."

NOAA's Lewitus agrees. "I think this work by Moeller puts to rest the question about whether Pfiesteria can potentially produce a toxin. I think that this is a discovery that will allow the field to move forward," he says. Until now, the toxin question had created a research bottleneck, Lewitus says, and this discovery will free up researchers to move on to the question of what environmental relevance this toxin might have.

Lewitus does not know yet whether the discovery will pave the way for a spate of research proposals on the environmental significance of this Pfiesteria toxin. He'll be one of the first to find out, though, because in his role at ECOHAB he'll see many of the proposals on harmful algae as they start to come in during the next grant competition.

Other researchers remain skeptical of the whole concept of toxic Pfiesteria. Al Place, a biologist at the University of Maryland Biotechnology Institute in Baltimore who studies the toxic dinoflagellate Karlodinium, raises the point that until this new compound can be identified in nature and until a dose-response curve for it has been developed in the laboratory — an experimentally verified relationship that shows an increasing response to increasing amounts of the chemical — we won't know what significance this molecule may have. Place also doesn't think that the free radical toxin could have remained in the water long enough to have killed fish. Instead, he suspects that Karlodinium, which co-occurred with Pfiesteria during the blooms, was more likely the culprit (see The Case for a Toxic Culprit).

As for Moeller, he hopes that other scientists will soon explore whether Pfiesteria's free radical toxin could have played a role in large-scale fish kills. Free radical mechanisms of toxicity are new to the marine environment, and research will likely require a departure from more conventional approaches, like dose-response curves, he explains. And Moeller recognizes that working with this highly unstable chemical system will be quite difficult. He himself plans to explore whether other marine organisms, in addition to Pfiesteria, employ metals to produce free radical toxins. He suspects that this may be a common mechanism that dinoflagellates use to protect themselves from metals in their environment. But he hopes to move out of the Pfiesteria business for good. He says, "I've only got so much blood to give."

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