Pfiesteria 'Fesses Up.

A New Clue to Its Toxicity

Like Houdini, the mysterious dinoflagellate Pfiesteria piscicida hasn't been inclined to surrender the secrets of how it works. But with enough creative poking and prodding, something was likely to give, and it has. Researchers from the National Oceanic and Atmospheric Administration's National Ocean Service and from North Carolina State University in Raleigh sought to identify the receptor targeted by the still unknown P. piscicida toxin or toxins [EHP 109:457-462]. In doing so, they have found a vital clue that may reveal how the microbe's toxin attacks people and wildlife.

Pfiesteria has come under increasing scrutiny in the past decade after a series of outbreaks in at least two U.S. states killed more than a billion fish and caused a variety of serious human health problems. Research in the past few years has begun to breach the wall of mystery surrounding the dinoflagellate, which morphs through several life stages--only some of which are toxic to humans or fish--during its complex life cycle. No major Pfiesteria-related fish kills were confirmed in the United States in 1998, 1999, or 2000, although there were several cases where Pfiesteria was the prime suspect in outbreaks of fish lesions and some human health problems. Excess nutrients such as nitrogen and phosphorus remain the chief suspects in boosting toxic Pfiesteria outbreaks.

Building on techniques and information developed by themselves and others, the team has discovered that the toxin behaves as an agonist of the P2X7 receptor, a receptor commonly found in the immune system of people and some wildlife. The toxin may trigger a chronic cascade of inflammatory responses, an overreaction that can quickly kill some fish and likely causes the acute and chronic human health problems associated with Pfiesteria exposure, which include memory loss, learning impairment, breathing difficulty, nausea, lethargy, and skin lesions.

The team used a process of elimination to pin down which receptor was being affected by the Pfiesteria toxin. Using [GH.sub.4][C.sub.1] rat pituitary cells, the team evaluated a number of substances for their ability to increase or inhibit levels of engineered bioluminescence in the cells. They found that adenosine-5'-triphosphate (ATP), a ubiquitous energy source for enzyme reactions and a messenger for certain cellular functions, bonded to an unknown receptor and increased bioluminescence. But after researchers boosted ATP to a certain concentration, it inhibited bioluminescence and began to kill cells. The same test with a purified solution of P. piscicida toxin showed a similar pattern, suggesting that the toxin could act like ATP. (Additional testing eliminated the possibility that the suspected toxin actually is ATP because the toxin did not mimic the energy-related functions of ATP, only its messenger functions.)

Additional bioluminescence tests with other ATP mimics, antagonists, and the toxin helped researchers narrow the candidates to several P2X receptors, then to the P2X7 receptor. That receptor is present in several components of the human immune system, including macrophages, mast cells, microglia in the central nervous system, and pituitary cells. The series of tests did not eliminate the possibility that the toxin may affect other receptors, too.

Given that the compound is not ATP and other dues unveiled during this study--that the compound works in a way different from all other known compounds and that it appears to have a unique chemical structure--the researchers suspect that the Pfiesteria toxin likely is in a little known or even undiscovered class of compounds. According to John S. Ramsdell, a team member and chief of the National Ocean Service's Coastal Research Branch, they also suspect that the toxin works in concert with another unknown substance, perhaps the Pfiesteria cell itself, that first breaches the defenses of a fish or person. The Pfiesteria toxin then takes advantage of the breach in some way and does its damage.