WASHINGTON -- Animal experiments at Duke University Medical Center show
that harmless doses of three chemicals used to protect Gulf War soldiers
from insect-borne diseases and nerve-gas poisoning are highly toxic when
used in combination, researchers reported Wednesday. They said the findings
may explain the wide array of symptoms reported by an estimated 30,000 Gulf
In studies using chickens, the researchers specifically found that two pesticides, DEET and permethrin, and the anti-nerve gas agent pyridostigmine bromide (PB) were harmless when used alone, even at three times the doses soldiers likely received. But when used in combination, the chemicals caused neurological deficits in the test animals similar to those reported by some Gulf War veterans, according to Duke pharmacologist Mohamed Abou-Donia and Tom Kurt, a toxicologist at The University of Texas Southwestern Medical Center in Dallas.
Chickens were selected over rodents as test animals because their susceptibility to neurotoxic chemicals more closely resembles that of humans, the scientists said.
The findings were prepared for presentation Wednesday at the annual meeting of the Federation of American Societies for Experimental Biology and will be published in the May issue of the Journal of Toxicology and Environmental Health.
The researchers said their findings are similar to those reported in Scotland last month and by an Israeli team last year.
Adding to those findings, the Duke and UT Southwestern scientists have developed a theory to explain why the chemical mix is dangerous. They said their results indicate the anti-nerve gas agent reduces the body's normal ability to inactivate the two pesticides, which can then travel to and damage the brain and nervous system. Such a mechanism could explain the wide array of symptoms reported by some Gulf War veterans, including memory loss, headache, fatigue, muscle and joint pain, weakness, shortness of breath and tremors, the researchers said.
"The decision to use these chemicals was made to protect soldiers from indigenous diseases in the gulf, such as malaria and leishmaniasis," said Abou-Donia, lead investigator of the study. "Without protection, there may have been thousands of deaths. But it appears that, for some veterans, the precautions prevented one set of problems and created another. Now, our task is to analyze the veterans' symptoms by investigating all the potential causes, not only for their sakes but for the welfare of future soldiers."
The Duke study is one of a three-part investigation on Gulf War illnesses organized by UT Southwestern. Co-authors of the Duke study include former Duke researcher Kenneth R. Wilmarth, now at ENVIRON Corp. in Arlington, Va.; Kurt; Karl F. Jensen of the Environmental Protection Agency at Research Triangle Park, N.C.; and Frederick W. Oehme of Kansas State University.
"Together, the three phases of our investigation may solve the mystery of some Gulf War veterans' illnesses," Kurt said. "The animal studies are an important component because they test the biological plausibility of our theory that combinations of certain chemicals can cause symptoms that are not caused by individual chemicals alone."
In the Duke study, researchers exposed healthy chickens to each of the three chemicals -- DEET, permethrin and PB -- individually and then in various combinations.
Doses of each chemical were selected prior to the study by determining the maximum amount a chicken could withstand without showing clinical signs -- a dose representing at least three times the amount soldiers likely received. DEET and permethrin were administered subcutaneously and PB was given orally.
"Even if a person was exposed to one chemical alone at three times the recommended dose, he or she would have remained healthy," Abou-Donia said. "Our first task was to demonstrate the safety of each chemical when used individually."
The chickens exposed to individual chemicals showed no outward signs of illness or debilitation, the researchers said. But chickens exposed to any two chemical combinations exhibited varying degrees of weight loss, diarrhea, shortness of breath, decreased activity, stumbling, leg weakness and a reluctance to walk, impaired flying or tremors. The combination of all three chemicals produced the most severe signs, resulting in total paralysis or death in some chickens.
A laboratory analysis of tissues in the central and peripheral nervous systems showed that multiple chemical exposure caused enlarged axons and axonal degeneration, a sign of widespread nervous system damage.
Tests also suggested that the severity of clinical signs depends on how active a particular blood enzyme is in removing the foreign chemicals from the body, the researchers said. This "scavenger" enzyme, called plasma butyrylcholinesterase (BuChE), inactivates foreign chemicals such as DEET and permethrin.
However, the scientists said there is a finite and limited amount of BuChE in the bloodstream, enough to neutralize DEET alone or permethrin alone. When multiple chemicals are present, the enzyme is unable to neutralize them all, resulting in a toxic accumulation of chemicals in the bloodstream and thus in the brain and nervous system.
Moreover, the anti-nerve gas agent PB further inhibits the action of this scavenger enzyme, BuChE. While PB's intended purpose is to temporarily shield and protect another similar enzyme, acetylcholine esterase (AChE), from nerve gas damage, it cannot distinguish between AChE and BuChE and therefore binds to both, the researchers said. So, even less BuChE is available to combat and neutralize DEET and permethrin.
"Pyridostigmine bromide actually pumps more of the other chemicals to the brain," said Abou-Donia. "While PB itself cannot cross the blood-brain barrier, it magnifies the effects of the other two chemicals by tying up the available BuChE."
Abou-Donia said an additional genetic risk factor arises in some individuals who have a faulty form of BuChE, resulting in low enzymatic activity and thus a diminished ability to inactivate drugs or pesticides. This risk factor, which affects only 3 to 4 percent of the population, may boost the toxicity of these chemicals.
"Individuals with genetic types of decreased plasma BuChe activity should be considered potentially at higher risk when exposed to PB and related compounds, and this may account for some of the more severe symptoms seen in up to 4 percent of the Gulf War veterans," said Abou-Donia. An estimated 700,000 military personnel served in the Gulf War.
In addition, soldiers who took higher-than-recommended doses of PB as an added precaution against nerve gas attacks may have caused nerve-cell overstimulation, contributing to tremors, muscle spasms and other symptoms of increased nerve-cell activity.
The research team is conducting a follow-up study analyzing blood samples from veterans with and without symptoms to determine if low enzymatic activity is associated with signs of illness.