CHOLINESTERASE INHIBITORS AND MULITSYMPTOM ILLNESSES
Numerous studies of Gulf War veterans have identified an association between self-reported multisymptom illness and self-reported exposures to several cholinesterase-inhibiting agents, including the drug pyridostigmine bromide (PB), cholinesterase- and noncholinesterase-inhibiting pesticides, and the cholinesterase-inhibiting nerve gases, sarin and cyclosarin. This appendix reviews some of the known health effects that can result from these potential exposures and addresses the evidence potentially linking those exposures to multisymptom illness, including what is often called Gulf War illness. This appendix discusses how Gulf War veterans might have been exposed to cholinesterase inhibitors, the physiologic and toxicologic actions of these chemicals, and reviews some of the studies that have attempted to link various chemical agents to symptoms indicative of cholinesterase inhibition of veterans.
When a person chooses to move a body part such as a finger, the brain sends a signal first down through the spinal cord, then from the spinal cord out the nerves of the arm and into the hand. The nerves carry an impulse down their length somewhat like an electrical current is conducted down a wire. This impulse reaches the muscles of the finger and the muscle contracts in response and the finger moves. These nerves, which carry the signal, are long nerves called motor neurons. Despite the analogy, the conduction of the impulse differs from electricity moving through a wire in several respects. First, the impulse moves along the nerve as a result of the nerve cell changing its internal charge from negative to positive along its length in a wave like fashion. Second, when a nerve connects with the muscle it does not transmit the electrical signal directly, like electricity crossing a wired junction, but instead the end of the nerve fiber releases a chemical (called a neurotransmitter). This chemical moves across a narrow space (called a synapse) between the nerve and the muscle and binds to molecules on the surface of the muscle that are concentrated around the synapse. When the neurotransmitter binds to these molecules, the molecules cause a change in the membrane of the muscle cell, which causes the muscle to contract. This same phenomenon of nerve impulse and chemical transmitter crossing a synapse occurs when nerves connect to other nerves, and when nerves connect to glands, such as the salivary glands. In these situations, the neurotransmitter crossing the synapse results in the continuation of the nerve impulse or the secretion of the gland.
There are several neurotransmitters in the body, with acetylcholine being one of the most common. Neurons that use acetylcholine as a neurotransmitter are referred to as cholinergic. The enzyme cholinesterase plays an essential role in terminating the chemical signal between a cholinergic neuron and the nerve, muscle, or gland that is being stimulated. Acetylcholinesterase (AChE) acts to quickly metabolize acetylcholine into choline and acetic acid, rendering it
inactive. It is critical for the normal function of the nervous system. When AChE is inhibited, acetylcholine can accumulate causing overstimulation of the cholinergic junctions and organs controlled by cholinergic neurons. Tissues innervated by cholinergic neurons include muscles (both smooth and voluntary); glands such as salivary, pancreas, and lachrymal; and certain parts of the brain. Thus inhibition of cholinesterase can cause overactivity of a wide variety of bodily functions. This overactivity is characteristic of poisoning by cholinesterase inhibitors (Bardin et al., 1994).
EXPOSURES TO CHOLINESTERASE INHIBITORS AND OTHER PESTICIDES DURING THE GULF WAR
Pesticides are defined by the federal Fungicide Insecticide Fumigant Rodenticide Act as any substance that kills, repels, or mitigates a pest. Under this definition, insect repellents such as diethyltoluamide (DEET) would be considered pesticides. Several types of pesticides were used in the Gulf War theater, among the most common were organophosphates, carbamates, and pyrethroids.
Organophosphate pesticides are chemicals in wide use as insecticides in agricultural and nonagricultural applications. They were initially developed by German scientists prior to World War II. They range in toxicity from very mildly toxic to extremely toxic and are toxic by mouth, inhalation, or dermal absorption. Most organophosphate pesticides require activation by an enzyme system known as the cytochrome P450 system. This system oxidizes a portion of the molecule making it much more toxic than the parent compound. It is this active molecule that binds to the cholinesterase molecule, blocking the action of the enzyme and leading to the accumulation of the neurotransmitter acetylcholine. This accumulation of acetylcholine and the overstimulation of the nerves, glands, and muscles that are innervated by cholinergic neurons, lead to the signs and symptoms seen in organophosphate poisonings. The organophosphate binds to the active site and slowly forms a covalent bond with the enzyme, thus irreversibly inhibiting the cholinesterase enzyme. The enzyme is replaced by the body over time.
Carbamates are a group of chemicals that, like organophosphates, are commonly used as insecticides. They also inhibit cholinesterase. The inhibition they cause tends to be less long lasting than inhibition with organophosphates, because unlike organophosphates, they do not go through a permanent bonding (covalent bonding) with the cholinesterase molecule. They are short acting, and the enzyme reactivates when the concentration of the carbamate in the system is reduced.
Permethrin is a low to moderate toxicity pesticide in the class of pesticides known as pyrethroids. This class is derived from natural pesticides, which are found in chrysanthemums. Permethrin alters the function of nerve ion gates by destabilizing the neuronal ion balance. This instability results in neurons firing more easily and in uncoordinated neuronal discharges. These uncoordinated discharges are largely responsible for the insecticidal effect of the chemical.
The most serious consequence of overexposure to pyrethroids is seizure activity. This however, is rare in humans as the toxicity of pyrethroids is far less for warm-blooded animals than for cold-blooded ones. Acute exposure in humans is generally characterized by burning or irritating sensations in the fingers and lips.
DEET is a liquid insect repellent that can be applied to the skin or clothing. Despite the wide use of the chemical, there have been relatively few reports of systemic toxicity. Children appear to be most at risk for DEET toxicity, particularly when it is used excessively.
Illnesses associated with DEET include contact dermatitis and eye irritation. DEET is efficiently absorbed across the gut and skin. High blood levels can lead to encephalopathy, which can have severe long-term consequences including flaccid paralysis and areflexia. Fatal poisonings have been reported. No studies of chronic low-level exposure to DEET in humans were identified by the Update committee.
Illness Associated with Cholinesterase Inhibitors and Pesticides
Acute illness due to cholinesterase inhibition from chemicals such as organophosphates, carbamates, PB, or sarin manifests with symptoms and signs resulting from toxic effects on the central and peripheral nervous system. Central nervous system (CNS) symptoms and signs include confusion, headache, loss of consciousness, and, in severe overexposures, death due to respiratory center depression. Peripheral symptoms and signs include salivation, lacrimation, blurry vision, excessive bronchial secretions, sweating, weakness, muscle fasciculations, diarrhea, and loss of bladder control. Severe cholinesterase inhibition can result in death primarily because of respiratory failure. This failure results from a combination of bronchial secretions, bronchial constriction, weakened respiratory muscle function, and inhibited respiratory drive.
Symptoms and signs resulting from chronic low-level exposure to cholinesterase inhibitors are less well defined and some physiological accommodation can occur, leading to the amelioration of some symptoms. Persistent signs and symptoms after a severe acute poisoning have been more frequently studied. Several studies have identified persistent neurobehavioral changes lasting years after recovery from severe organophosphate pesticide poisoning (Delgado et al., 2004; London et al., 1998; Rosenstock et al., 1991; Savage et al., 1988; Steenland et al., 1994; Wesseling et al., 2002).
In addition to acute toxic reactions, two other syndromes have been identified with overexposure to organophosphate pesticides: intermediate syndrome and organophosphate-induced delayed polyneuropathy (OPIDP). Intermediate syndrome occurs after an acute severe organophosphate intoxication and manifests as severe weakness, generally showing up 48 hours after the acute illness begins. This weakness can lead to respiratory failure if there is no intervention to support respiration (De Bleecker et al., 1993). OPIDP results from exposure to a select group of organophosphate chemicals that are capable of binding to and inhibiting a neuronal enzyme known as neuropathy target esterase. This syndrome usually appears about 2 weeks after the poisoning. It is characterized by weakness in the extremities and is understood to be due to a degenerative neuropathy of long motor neurons (Lotti and Morreto, 2005).
While in the Gulf War theater, servicemembers might have had exposure to three distinct groups of cholinesterase inhibitors: sarin or cyclosarin nerve gas, PB, and organophosphate and/or carbamate pesticides. Other common pesticide exposures were to insect repellents containing DEET and permethrin. Pesticides were used to control fleas, flies, and other insects. PB was used as a prophylactic antidote for exposure to nerve gas, which was known to have been previously used by Iraqi forces.
The most complete documentation of the use of pesticides, cholinesterase inhibitors, and PB pills by Gulf War veterans was conducted by the RAND Corporation for the Department of Defense (DoD). The RAND report Pesticide Use During the Gulf War: A Survey of Gulf War
Veterans (Fricker et al., 2000) collected information by phone survey in 1999 from 2005 randomly sampled veterans from the estimated 469,047 Gulf War veterans who were believed to be on the ground in theater during Operations Desert Shield and Desert Storm in 1990-1991. Fricker et al. found that about 66% of respondents reported use of pesticides in some form. Veterans reported using pesticides during the Gulf War in a variety of ways, including personal use—for example, application of lotions, sprays, and personal use of flea collars, and field use—for example, broadcast applications in eating areas or use of pest strips in eating areas or toilet facilities. The survey focused on the form of pesticide used because most respondents were unable to provide the names of the pesticides they used or saw others use.
Fricker et al. (2000) imputed the active ingredients from the form of the pesticide reportedly used and other information provided by the respondents. It was necessary to impute the ingredients because most of the respondents could not recall the name, color, or smell of the pesticides they used or saw used. The investigators estimated that 50% of veterans applied DEET in some form, 6% used permethrin, and 3% used a sulfur pesticide. A small percentage of veterans, an estimated 13,000, used tick or flea collars and were able to correctly identify those products. The survey investigators were able to identify the active ingredients in pesticides used in field use of pesticides with even less certainty than pesticides used in personal use applications. They imputed that possible active ingredients in aerosols were alethrin, permethrin, resmethrin, phenothrin (all pyrethroids), chlorpyrifos and malathion (both organophosphates), propoxure (a carbamate), and DEET (a hydrocarbon repellent).
PB is a pharmaceutical cholinesterase inhibitor used widely in the treatment of myasthenia gravis, an autoimmune neuromuscular condition, and a vascular condition known as orthostatic hypotension. In both cases, the mechanism of the drug is to reversibly inhibit the enzyme cholinesterase, which allows the accumulation of acetylcholine leading to overstimulation of the acetylcholine receptors. In the case of myasthenia gravis, these receptors are voluntary muscle receptors, which due to the disease are decreased in concentration. In the case of orthostatic hypotension, the increase in acetylcholine stimulates receptors that increase venous blood return and increase blood pressure. It is provided as a small white tablet, taken orally.
Symptoms of Toxicity from PB Overexposure.
Pyridostigmine bromide when taken in excess produces symptoms similar to those produced by overexposure to organophosphate or carbamate pesticides. These symptoms are due to the excess of acetylcholine in the body. Excess secretions such as saliva, bronchial secretions, sweating, and tearing may be seen. Muscles may fibrillate and become weak. Diarrhea, nausea, frequent or uncontrolled urination, as well as confusion or irritability may occur. Other symptoms include blurred vision and pupillary constriction, bronchospasm, and, in severe cases, pulmonary edema (Medline Plus, 2008). In terms of long-term effects of PB, the Food and Drug Administration has summarized the existing knowledge and concluded that despite a long history of PB being used in the treatment of myasthenia gravis no evidence of long-term health effects has emerged to date (FDA, 2009).
PB tablets were provided by the DoD to military personnel in the Gulf War theater as a prophylactic against cholinesterase-inhibiting nerve gas. The theory behind the use of PB for such prophylaxis is that by reversibly inhibiting a portion of the acetylcholine receptors in the body, a sufficient percentage of the receptors will be protected and will reactivate so as to permit
survival in the case of a nerve gas exposure. The RAND survey reported that about 50% of the Gulf War deployed personnel in the Army and Marine Corps/Navy used PB pills. Of those who used the pills, 95% of whom reported taking 3 or fewer pills per day for fewer than 30 days out of the month. The median use was 20 pills per 30-day period. It was also noted that those self-reporting the most PB use also tended to self-report more frequent use of pesticides.
The nerve gases sarin and cyclosarin are extremely potent cholinesterase-inhibiting chemicals. Sarin, also known as GB for German B, and cyclosarin, also known as GF, were first developed by German scientists near the start of World War II.
Sarin and cyclosarin have a potency far exceeding that of pesticidal cholinesterase inhibitors. The acute effects are effectively those of overexposure to organophosphate or carbamate pesticides. Long-term effects in humans have been identified among survivors of the sarin attacks in Japan. Chronic symptoms such as forgetfulness, depressed mood, and loss of interest or apathy were more common in victims of sarin toxicity than controls up to 10 years after their acute illness (Yanagisawa et al., 2006). Some neuropsychological performance measures were also seen to be worse among sarin victims compared with controls 3 years after acute exposure (Nishiwaki et al., 2001), and PTSD symptoms were common in a very small sample of previously poisoned victims (Ohtani et al., 2004). There are very few events that permit study of the long-term effects of sarin exposure, and no human studies exclusively of cyclosarin were found in the literature. However, the persistent abnormalities seen in sarin victims that last long after the acute toxicity has resolved appear to be similar to the long-term neurobehavioral effects seen after acute intoxication from organophosphate pesticides (Delgado et al., 2004; London et al., 1998; Rosenstock et al., 1991; Savage et al., 1988; Steenland et al., 1994; Wesseling et al., 2002).
Khamisiyah was a munitions storage site that was destroyed by US forces during Operation Desert Storm in March 1991. Servicemembers involved in the demolition were unaware that the bunker also contained the sarin and cyclosarin. A plume of nerve gas was released from the demolition that drifted in a decreasing concentration gradient away from the site on the prevailing winds (see Volume 4, Chapter 2, for a more detailed discussion of the Khamisiyah plume modeling).
The DoD attempted to determine the number and identity of the troops potentially exposed to nerve gas from the Khamisiyah demolition. The DoD modeled potential exposures in 1997 and 2000. The approaches used by the DoD on each occasion have come under strong criticism by a variety of critics, including the General Accounting Office, which conducted an independent investigation of the Khamisiyah modeling efforts by the DoD (GAO, 2004). Although the number of people exposed in each model did not differ significantly, the location of those troops did.
The time during which nerve gas may have been released from Khamisiyah is uncertain because there were aerial attacks as well as ground detonations of explosives at this site. No reports of acute symptoms among potentially exposed military personnel have been linked to the nerve gas relesases from the Khamisiyah demolition. However, very low-level exposure to sarin or cyclosarin may cause symptoms indistinguishable from systemic viral illnesses or mild bacterial illnesses and thus the symptoms might be confused with other illnesses commonly seen in an unhygienic theater of war. Only one peer-reviewed study was identified that used symptom reports from 5555 Gulf War army veterans. The symptom reports were obtained by a mailed
survey prior to the DoD’s release of information about possible nerve gas exposure from the Khamisiyah demolition (Page et al., 2005a). This study found that the 1898 veterans who were potentially exposed to the Khamisiyah plume did not have higher rates of 48 symptoms queried compared with 3336 nonexposed veterans. Of the 31 medical conditions about which respondents were asked, the potentially exposed veterans reported slightly higher rates of only two: enteritis (risk ratio [RR] 1.19, 95% confidence interval [CI] 1.0-1.43) and colitis (RR 1.39, 95% CI 1.14-1.70) in the preceding 12 months. The analysis adjusted for age, sex, race, rank, marital status, and unit component; potential exposure was based on the 2000 DoD plume modeling (Page et al., 2005b).
LONG-TERM EFFECTS OF LOW DOSES OF CHOLINESTERASE INHIBITORS
Whether long-term, low-dose (nonintoxicating) exposures to cholinesterase inhibitors results in CNS illness has not been determined (Keifer and Firestone, 2007). Organophosphate-exposed workers in California who showed reduced cholinesterase levels during routine monitoring had no decrease in neuropsychological function on 27 neurological and neuropsychological tests compared with controls (Ames et al., 1995). Neurological and neuropsychological tests of South African workers exposed to organophosphate pesticides also found no association between history of chronic organophosphate exposure and test performance; however, previous organophosphate poisoning was predictive of lowered test performance (London et al., 1998). Chronic neurologic effects have been studied in another group of organophosphate-exposed workers: sheep dippers. The most highly exposed sheep dippers showed an increase in neurological symptoms and decreases in neuropsychological test performance and peripheral sensory function (Pilkington et al., 2001; Stephens et al., 1995, 1996).
The US Agricultural Health Study, the largest cohort study of pesticide-exposed workers to date, found self-reported retinal degeneration to be associated with fungicide exposure (Kamel et al., 2007). Bessler et al. (2006), studying the same cohort, a history of an acute exposure to a high concentration of pesticide was significantly associated with self-reported, doctor-diagnosed depression in men and women. The self-reported, doctor-diagnosed depression was also associated with the highest level of self-reported lifetime, cumulative pesticide exposure in men but not in women (Besseler et al., 2008). This study is unique in identifying an association between chronic high levels of nonintoxicating exposure and depression. However, the study did not find a dose-response effect across the three levels of lifetime cumulative exposure and considered cumulative exposure to all pesticides in its exposure classification, not just those that inhibited cholinesterase. The Agricultural Health Study also found increased self-reports of Parkinson’s disease to be associated with increased use of pesticides (Kamel et al., 2007). This finding reinforced other studies that found an increased risk of Parkinsonism among rural residents, farm residents, and pesticide-exposed populations. Costello et al. (2009) recently found a strong association between subjects objectively assessed for the presence of Parkinsonism and exposure to pesticides as indicated by historically confirmed residence in proximity to sprayed agricultural land in California (Costello et al., 2009).
STUDIES IN GULF WAR POPULATIONS
Several studies of Gulf War veterans have found an association between self-reported multisystem illness and self-reported exposure to pesticides, nerve gas, or PB, or some combination of them. Haley and Kurt first reported this association in 1997. Some 249 veterans of a naval construction battalion (Seabees) responded to a mailed questionnaire about health symptoms and exposure including pesticide and PB tablet use and nerve gas-related events. Principal component analysis was used to reduce symptoms to three syndromes: syndrome 1 “impaired cognition,” syndrome 2 “confusion-ataxia,” and syndrome 3 “arthromyoneuropathy.” Work in security during the war was significantly associated with the risk of syndrome 1 (RR 5.4, 95% CI 1.8-16). Syndrome 2 was associated with reports of experiencing a nerve gas attack (RR 8.0, 95% CI 2.3-25.9) or being in sector 7 during a specified period, an area and time of potential exposure to nerve gas drift. Risk of syndrome 2 also increased with the scale of adverse effects to PB. Syndrome 3 was found to be associated with the amount of insect repellent that veterans reported using on their skin and with the scale of symptoms they reported in response to taking PB tablets. The risk for syndromes 2 or 3 was not associated with the amount of PB tablets consumed.
Ishoy et al. (1999a,b) conducted a cross-sectional interview and examination-based study of 686 (83.6%) of Danish personnel who were deployed to the Gulf on a peacekeeping mission after the end of hostilities. A sex-, age-, and profession-matched control group of 231 individuals (57.8% of 400 potential participants) who could have been but were not deployed to the Gulf were included for comparison. There was no adjustment for potential confounders in the analysis, and Gulf War veterans were significantly older, taller, heavier, more likely male, and had higher diastolic blood pressure than the nondeployed counterparts. Since their deployment to the gulf, the Danish peacekeepers were significantly more likely to have a wide variety of symptoms including headaches, blurry vision, numbness or tingling of hands of feet, balance difficulties, depression and concentration problems, fatigue, sleep difficulty, nightmares, nervousness and agitation, and difficulty pronouncing words correctly. A significant difference was found between the two groups for undiagnosed skin problems as well. Only minor differences were found between the groups for hematological parameters (Ishoy et al., 1999a). Focusing on gastrointestinal difficulties in the same cohort, Ishoy et al. (1999b) found that using a bivariate analysis, 15 of 24 exposures were associated with one or more gastrointestinal symptoms, including diarrhea and rumbling of the stomach. In logistic regression analysis, three factors remained significant, including burning waste/manure, use of insecticides against cockroaches, and low physical activity. Two other factors approached significance: tooth brushing with water contaminated with chemicals, and bathing or drinking contaminated water (fumes, oil, chemicals). As the number of these self-reported exposures increased, the risk for ongoing multisymptom illness increased (trend test not reported). The authors pointed out that in reviewing the Hill criteria for causation, the associations they found to be strongest were biologically plausible and were consistent with general medical knowledge, specifically the association between exposure to waste and infection-related gastrointestinal symptoms and inadequate vector control (cockroaches), resulting in microbial contamination and infectious gastrointestinal illnesses. It is important to point out that, except for a few people, the Danish study participants were not present in the gulf during combat operations and served primarily as support personnel and peacekeeping forces.
In a further analysis of the Danish cohort, Suadicani et al. (1999) examined the association between self-reported exposures and neuropsychological symptoms. While most self-reported exposures were significantly associated with neuropsychological symptoms on bivariate analysis, in multiple linear regression models only a few remained significant. The authors found that threats such as “observing colleagues or friends being threatened with arms or shot at” remained the only psychosocial exposure predictive of neuropsychological symptoms after controlling for demographics and potential confounders. Job demand factors such as “occasional or frequent demand of high degrees of concentration, dissatisfaction with the physical work environmental, and unwillingness from immediate superiors to listen to one’s problems were significant predictors of neuropsychological symptoms in a multivariable model.” Among self-reported physical factors that remained significantly associated with neuropsychological factors after adjusting for confounders in a logistic model were “bathing in or drinking contaminated water, exposure to depleted uranium, contact with dead animals, and burning of waste or manure.” An analysis of the prevalence of clusters of neuropsychological symptoms showed that physicochemical factors interacted with psychological factors in predicting symptoms; however, the physicochemical factor alone was not predictive of symptoms. Insecticides and use of lotions or sprays, which were significant in bivariate analysis, were no longer significant in the multivariate modeling. No information was provided on PB, presumably because it was not used by peacekeepers after the conflict. The authors demonstrated that the number of psychosocial factors reported by the participants increased so did the likelihood that the participant suffered from three to five neuropsychological symptoms.
Nisenbaum et al. (2000) provided a self-administered questionnaire on symptoms and Gulf War deployment stressors to four Air Force units on base in January-March of 1995. Using the Centers for Disease Control and Prevention (CDC) definition of chronic multisymptom illness (Fukuda et al., 1998), the authors classified cases as mild to severe depending on the severity of the case-defining symptoms. They conducted logistic regression analyses for possible risk factors to compare severe disease with no disease and mild-moderate disease with no disease. The response rate was not clearly defined in the report, although unit participation rates were provided in the Fukuda paper. Of those 1163 Gulf War veterans who participated, 1002 had complete data and remained in the final analysis. Several self-reported deployment stressor (exposures) were found to be significantly associated with severe illness, including belief that biological or chemical weapons were used, PB use, and regular insect repellent use. Having sustained injuries that required medical attention was associated only with severe illness, and an interaction term of PB use and insect repellent use was not significant.
Spencer et al. (2001) conducted a nested case-control study based on responses to a mailed survey of a random sample of 2343 of 8603 Washington and Oregon Gulf War veterans. The response rate of locatable veterans was 55% overall. Cases were defined as having reported at least one of three symptom types (fatigue, cognitive/psychological, or musculoskeletal). Controls had none of the three symptom types or had symptoms that were explained by existing medical conditions. Cases and controls were examined with a history, screening physical, a neurological examination, a 4-hour neurobehavioral battery, and laboratory tests. The results were reviewed by a clinical determination committee composed of appropriate medical specialists. Exposures were assessed by a self-reported 142-item list that was reduced by elimination of redundant factors and cluster analysis to 9 factors representing a total of 42 exposure variables. The authors found that on the basis of multivariate modeling, the greatest risks for unexplained illness were sun-related exposure, the presence of a medical condition that
the individual sought medical care for while in theater, and conditions of combat. No association was found for self-reported PB and/or insect repellent use. Exposure to oil-well fires was also not significantly associated with being a case.
Cherry et al. (2001) conducted a cross-sectional study of self-reported symptoms and exposures among 7971 UK Gulf War veterans. They had good participation with an 85.5% response rate. Principal component analysis of 94 symptoms resulted in seven factors: psychological, neurological, peripheral, respiratory, gastrointestinal, concentration, and appetite. These factors were developed using data from about half the cohort and were tested on the other half of the cohort for validation. Severity scores were derived using a Likert-like scale, and a manikin drawing was used to identify the extent of symptoms, such as tingling and pain. Using multilinear regression with adjustment for potential confounders, symptom severity scores were associated with the number of inoculations, days handling pesticides, and days exposed to burning oil wells. Regression of exposure against the seven factors found a statistically significant association between the “peripheral” factor (thought to be related to peripheral neuropathy) and the number of inoculations and the number of days handling pesticides. Days handling pesticides also associated with the “neurological” factor. Reported number of days of use of insect repellents was associated with peripheral, respiratory, and appetite factors.
Information derived from the manikins included numbness and tingling (possibly related to peripheral neuropathy) and widespread pain (thought to possibly be functional). Handling of pesticides and use of insect repellent were associated with numbness and tingling, and use of insect repellent, seeking medical care while in the gulf, and reporting side effects from PB were significantly associated with widespread pain. The authors discuss potential bias in detail and conclude, on the basis of their data, that pesticides or inoculations cannot be shown to be responsible for peripheral neuropathy in these veterans. They do suggest that objective measures for ill health are available and should be applied to this cohort (Cherry et al., 2001).
Gray et al. (2002) reported on a cross-sectional mail survey of 17,599 US deployed and nondeployed Seabees. Response rates were 64% overall with 56% of Gulf War deployed, 30% of other deployed, and 15% of nondeployed veterans responding. Significantly more self-reported multisymptom diseases and also more isolated symptoms were reported by Gulf War deployed veterans compared with nondeployed and other deployed veterans. Gulf War deployed Seabees were significantly more likely to report having more of all 33 symptoms and conditions asked when compared with the other two groups on the basis of logistic regression analyses that controlled for age, sex, race/ethnicity, and duty status. Deployed Seabees were also significantly more likely to report suffering from a wide variety of disorders than either nondeployed or other deployed, including digestive disorders, chronic fatigue syndrome (CFS), posttraumatic stress disorder (PTSD), multiple chemical sensitivity, irritable bowel syndrome, and skin rash. Deployed Seabees also reported more depression and were more likely to report being in fair or poor health than the other two groups. This study explored associations between meeting the definition for Gulf War illness and self-reported exposures. Depending on the multivariate analytic approach, there were weak associations between Gulf War illness and the following:
being exposed to fumes from munitions,
drinking contaminated water,
being exposed to sandstorms,
seeing someone killed,
contact with dead animals,
having food poisoning in one’s unit,
use of PB,
use of ciprofloxacin,
use of gas masks,
drinking from a desert bag,
receiving botulism toxin,
being female, and
On the basis of multilinear analysis, no association was found between Gulf War illness and receipt of several other vaccines, being exposed to oil-well fire smoke or jet fuel use in tents, wearing a flea collar, wearing insect repellent-treated uniforms, seeing dead bodies, eating local food or suffering food poisoning, or having direct combat exposure.
Wolfe et al. (2002) surveyed 2949 US Army soldiers from Fort Devens, Massachusetts, who had been deployed to the Persian Gulf; 1290 responded with self-reports of symptoms and exposures. A respondent was classified as having met the CDC criteria for multisymptom illness if they had at least one symptom in two of three symptom clusters: fatigue, mood and cognition, and musculoskeletal. Approximately 60% of respondents met the criteria for multisymptom illness. The authors found that after controlling for demographic factors, exposure to oil-well fires, the smell of chemicals in the air, having a heater in one’s tent, using a clinic while in the gulf, receiving an anthrax shot, and consuming PB pills showed a relationship with self-reported multisymptom illness (Wolfe et al., 2002).
Mahan et al. (2005) analyzed the responses of 5555 army veterans to the National Health Survey of Gulf War Era Veterans (Kang et al., 2000). This survey was collected on a sample of 15,000 troops deployed to the gulf and 15,000 troops who were not deployed there. The survey was mailed before some of the Gulf War veterans received notification of possible nerve gas exposure from the Khamisiyah detonation. Some 11,441 veterans responded to the questionnaire. Respondents were classified as either exposed or unexposed to the plume based on their unit locations and the 2000 DoD modeling of the drift plume. The authors controlled for age, sex, unit composition, when comparing the prevalence of 47 self-reported symptoms in the two groups of veterans. No significant differences in symptom prevalence were found except for severe wheezing and joint swelling, both of which were more common among the unexposed respondents. Among the 31 self-reported illnesses, colitis and enteritis were reported by a greater percentage of exposed veterans compared with unexposed veterans. There was no significant difference in measures of impairment such as being limited in work by one’s health, and no differences in self-ratings of health status. Exposed and unexposed veterans reported similar numbers of medical office visits and hospitalizations. The investigators found no difference in the self-reports of symptom-based conditions such as PTSD and CFS between the two groups. The results remained the same regardless of whether exposure was based on the 1995 plume model or the 2000 plume model (Mahan et al., 2005).
The Update committee has also evaluated selected studies of the effects of potential Gulf War intoxicants on animals. While this was not within its original charge, and a comprehensive examination of all of the pertinent literature was beyond its expertise, the committee nonetheless felt that this effort has merit, both for defining potential health hazards of Gulf War service and in designing potential future investigations. Accordingly, members of the committee reviewed the animal studies listed in Table 6 of the VA Research Advisory Committee’s report (RAC, 2008), focusing on two questions. First, do data from those animal studies demonstrate that stress can amplify the effects of intoxicants or drugs such as PB, in either the peripheral or the CNS? Second, do these animal studies document long-term adverse effects from short-term exposures to these agents?
With respect to the first question, perhaps the two most salient observations are studies by Friedman et al. (1996) and Abdel-Rahman et al. (2002). Friedman’s group examined the inhibition of brain AChE levels in FVB/N mice without and with stress (induced by forced swimming). They reported that the forced swimming stress paradigm was associated with enhanced inhibition of brain AChE by intraperitoneal pyridostigmine (0.1 mg/kg), concurrently with diminished integrity of the blood-brain barrier (measured by enhanced entry of Evans Blue dye and plasmid DNA). Independently of stress, intraperitoneal pyridostigmine also increased the level of RNA encoding brain c-fos and AChE. In hippocampal slice cultures, pyridostigmine enhanced expression of c-fos (but not synaptophysin) and produced a concomitant increase in evoked CA1 electrical activity. These investigators proposed that by altering the permeation properties of the blood–brain barrier, stress can amplify the CNS effects of peripherally delivered pyridostigmine, a compound with a quarternary amine thought to not normally enter the CNS. This model might explain how doses of pyridostigmine that do not normally produce CNS symptoms—such as nervousness, headaches, drowsiness, attention deficits, subnormal cognition—can trigger such symptom complexes. Aspects of the Friedman study are reinforced by the finding of Abdel-Rahman et al. (2002) that in rats stress imposed by restricting movement enhances neural effects of three simultaneously administered toxins: pyridostigmine (1.3 mg/kg/d), DEET (40 mg/kg/d), and permethrin (0.13 mg/kg/d). When superimposed on a stress paradigm, these compounds reduced the integrity of the blood-brain barrier in several regions (cingulate cortex, dentate gyrus of the hippocampus, lateral dorsal nucleus of the thalamus, and the dorsomedial nucleus of the hypothalamus); in each region, there was evidence of neuronal death and astrogliosis.
The model developed in the Friedman report has obvious relevance to understanding how brief exposures to low doses of intoxicants might have unexpected adverse effects on the CNS in Gulf War veterans. It is of interest, therefore, that Lallement et al. (1998, 2001) who also investigated the impact of stress on brain permeation by pyridostigmine, reached somewhat different conclusions. In the Lallement studies, stress was induced by heat rather than exercise in guinea pigs. Using two methods to significantly increase core temperatures, no evidence of loss of integrity of the blood-brain barrier was found. The authors urged caution in translating the results of the Friedman report to Gulf War phenomena in humans (Lallement et al., 1998). In a follow-up study of pyridostigmine administered subcutaneously to guinea pigs over 6 days, only rare and focal evidence of disruption of the blood-brain barrier was found (Lallement, 2001). In studies of CD-1 and FVB/N mice, Grauer et al. (2000) were not able to demonstrate brain permeation by pyridostigmine (0.4 mg/kg, intramuscular or intraperitoneal), using two
paradigms of stress (swimming and cold), a result that directly contradicts the conclusions of the Friedman report. While Grauer et al. did not explicitly evaluate properties of the blood-brain barrier, they inferred that it must have remained intact in the absence of any effect of peripheral pyridostigmine on brain AChE levels. This view is in alignment with a report from Telang et al. (1999) that radiolabeled pyridostigmine failed to permeate the brains of FVB/N mice, even after swim-induced stress.
A second important question that animal studies might help answer is whether short-term exposures to intoxicants can lead to long-term consequences for the CNS. Of interest in this context is the report by van Helden that a 5-hour exposure to sarin induces changes in the power spectrum of electroencephalograms (EEGs; plots of the intensity of EEG activity as a function of EEG frequency) that can be detected 1 year after exposure. Moreover, the change in the EEG occurs at a much lower dose than the appearance of miosis, which is among the most subtle clinical manifestations of sarin exposure (van Helden et al., 2004).
These findings are broadly consistent with earlier reports from Burchfiel and Duffy (1982) who found long-term changes in the EEGs of rhesus monkeys after a single high dose of sarin. High doses of sarin have been shown to result in derangements of the EEG, visual-evoked potentials, and event-related potentials as seen in human following the sarin incidents in Japan (Yanagisawa et al., 2006). What is less clear is whether the severity and intensity of putative sarin exposures of Gulf War veterans approaches those of the Japanese cases or the animal studies.
The Update committee finds that these animal studies address but do not resolve the questions about whether in the context of stress, low-level intoxicants that are otherwise well tolerated may exert adverse effects on the CNS; and whether brief exposures to some intoxicants can exert long-lasting (albeit subtle) neurotoxic effects on the brain. Because the animal data on these points are inconsistent or contradictory, the greatest need is for further investigation of these issues in the context of the symptoms of some Gulf War veterans. Specifically, investigations can focus on whether there are factors that confer heightened sensitivity to stress and intoxicants on some veterans such that they experience an increased adverse response to the brief exposures? Are there clinical tools that help define objective findings that correlate with peripheral and central Gulf War symptom complexes? Will subtle but quantitative measures of cortical function, brain oscillators, or central autonomic parameters—using techniques such as functional MRI, polysomnography, event-evoked potentials—reveal persistent abnormalities that have thus far eluded definition? In the committee’s view, these questions are among the set of issues that merit further analysis in the effort to understand and treat Gulf War illness.
GENETIC SUSCEPTIBILITY TO CHOLINESTERASE INHIBITORS
Multisymptom illness is striking for the lack of uniformity of symptoms among deployed veterans and that only a minority of the deployed cohort is symptomatic. To the extent that multisymptom illness in Gulf War veterans might be a consequence of exposure to toxins such as cholinesterase inhibitors, a potential explanation for this nonuniformity is the high degree of polymorphism in the proteins that detoxify a wide range of intoxicants. Foremost among these are the paroxonases, three esterases that metabolize oxidized lipids (PON1, 2, and 3). PON1 also metabolizes the highly toxic oxon forms of organophosphate insecticides, such as chlorpyrifos and diazinon (Aldridge, 1953; Furlong et al., 1989). Naturally occurring genetic variations in the genes encoding the paroxonases determine the expression levels and functions of the PON
proteins. For example, polymorphisms in the PON1 gene include the coding variants L55M and Q192R, as well as regulatory polymorphisms C-108T and A-162G (Brophy et al., 2001; Costa et al., 2005; Davies et al., 1996). There are also functionally significant polymorphisms in the butyrylcholinesterase (BuChE) gene that reduce BuChE levels and activity (Jensen et al., 1995). Variants in these genes have been implicated as determinants of normal aging and in various disorders, such as neurodegenerative and cardiovascular diseases (Benmoyal-Segal et al., 2005). Several studies have incriminated PON1 polymorphisms in ALS (reviewed by Wills et al., 2009).
Golomb (2008) reviewed studies found through a search of the National Library of Medicine’s PubMed database, using the key words “Gulf War,” “epidemiology,” and “acetylcholinesterase inhibitors.” She concluded that these studies—combined with veterans’ self-reports of exposures in the Gulf War theatre (including those cited earlier in this report), and studies of people occupationally exposed to cholinesterase inhibitors—supported, through “triangular evidence,” a causative association between exposure to cholinesterase inhibitors during deployment in the Gulf War and Gulf War illness. However, after a review of the primary sources cited by Golomb, the Update committee believes that more study is needed before any clear association can be inferred between Gulf War illness (multisymptom illness) and any inherited variability in PON1 or BChE genes.
An initial association between rare PON1 genotypes and multisymptom illness was first suggested by Haley et al. (1999) in a study of 25 symptomatic US Gulf War veterans, but the finding failed to reach statistical significance (p = 0.08 before correction for multiple comparisons). The authors also reported that the rare genotypes had reduced functional activity (Haley et al., 1999). In contrast to Haley et al., Mackness et al. (2000), in a larger study of 152 UK veterans with multisymptom illness, could find no association between genotype status and multisymptom illness and, furthermore, no function-genotype association for PON1 could be identified either in veterans with multisymptom illness or in controls. Although Mackness et al. (2000) did initially report an association of symptoms of Gulf War illness with reduced PON1 activity, in a follow-up study apparently with a different UK veteran cohort, Hotopf et al. (2003) concluded that reduced PON1 activity was found in deployed veterans regardless of whether they had symptoms of multisymptom illness. Because reduced PON1 activity may accompany states of chronic inflammation, the author postulated that inflammation might explain the reduced activity in deployed soldiers.
Golomb (2008) cites a US Army report (Lockridge, 1999) that states that rare genetic variants of BChE are present at a statistically increased frequency in Gulf War veterans with multisymptom sufferers. A second Department of Defense study (Sastre and Cook, 2004) found no evidence of any genotype-function correlations with BChE but, like the Lockridge study, did find an association between multisymptom illness symptoms and carriers of rare BChE genotypes who also self-reported PB exposure. However, the numbers of carriers of rare genotypes were small in both these studies—11 out of 226 veterans in Lockridge (1999) and 28 out of 304 veterans in Sastre and Cook (2004).
Thus, the Update committee concludes that current evidence raises the possibility of an association between multisymptom illness and genotypes of both BChE and PON1, but no definitive conclusions can be drawn. These data may represent an extremely important clue to the etiology of chronic symptoms seen both in the Gulf War veterans and in some civilian populations. The committee strongly recommends that well-designed studies be undertaken and replicated to more robustly test the hypothesis that naturally occurring variants in detoxifying
enzymes for AChE inhibitors may be susceptibility factors for the development of myltisumptom illness.
Human studies of Gulf War veterans have consistently shown increased symptom reports among a portion of the deployed veterans compared with nondeployed veterans (see Chapter 4). Unfortunately, objective exposure information is generally lacking, and most studies have depended upon self-reports of both exposure and illness and symptoms. No consistent relationship is apparent between specific exposures and symptoms across these studies. Studies of genetic differences in metabolizing or buffering toxins and their relationship to multisymptom illness may hold promise for understanding multisymptom illness given that genes represent an unbiased measure of susceptibility.
THE ROLE OF CHOLINESTERASE INHIBITORS AND PESTICIDES IN MULTISYMPTOM ILLNESS
While pesticide and PB use appear to have been widespread during the Gulf War, there is no evidence that Gulf War veterans suffered acute toxicity from either nerve gas or pesticides on a wide scale during and after the conflict. Except for an association with Parkinsonism, there remains substantial doubt as to whether long-term illness results from low-level chronic exposure to pesticides, including cholinesterase inhibitors. The Agricultural Health Study, the largest study of a pesticide-exposed cohort to date, has confirmed what had been reported from smaller studies of acute pesticide exposures, that is, CNS disorders may persist after acute exposures cease. Two groups of people with chronic, nonintoxicating pesticide exposures have been studied. Studies of UK sheep dippers with organophosphate exposures and one study of pesticide applicators in Iowa and North Carolina have found persistent long-term CNS effects from chronic exposure to high levels of pesticides (Beseler et al., 2008). In the latter study, no dose-response relationship was seen, and pesticides were not restricted to cholinesterase inhibitors. Low-level chronic pesticide exposure has been implicated in Parkinsonism in multiple studies and a causal association with a specific mixture of pesticides (herbicides and fungicides, neither of which are cholinesterase inhibitors) during a vulnerable period of life may explain why stronger associations with pesticide use were not found earlier and more frequently (Costello et al., 2009).
Animal studies have provided mixed results in terms of central and persistent effects of exposure to cholinesterase inhibitors and pesticides, and many questions remain. These questions include: whether the blood-brain barrier can be breached by PB during stress and whether persistent changes in peripheral or CNS function can be induced by exposure to cholinesterase inhibitors at levels comparable to those experienced by Gulf War veterans.
Studies of Gulf War veterans have not used uniform descriptions of possible exposures in theater and the exposures assessments are based almost exclusively on self-reports by veterans, often years after deployment, making it difficult to compare studies. The same is true for symptom reporting by the veterans. The terminology used by the researchers for the veterans’ symptoms can differ markedly as is evident from the factor analysis studies described in the section on “Multisymptom Illnesses” in Chapter 4.
There have been several studies that have found associations between self-reported exposures to pesticides, nerve gas, PB, and mixtures thereof, and self-reported symptoms and multisymptom illness. However, several well-designed studies have concluded that no association exists for such exposures, and other stress-related and environmental factors appear to be more important. Due to the publicity surrounding multisymptom illness and its possible
associations with environmental exposures stimulated by early studies of Gulf War veterans, obtaining unbiased results through studies using self-reports of symptoms and exposures seems unlikely at this point. Studies of genetic differences, such as polymorphisms in genes responsible for metabolic inactivation of toxins (PON1 variants) or buffering of toxic load (BChE levels), may hold promise given the objective nature of these potential modifying factors and their likely effect on biologically active doses in exposed subjects.
Given the scarcity of evidence supporting the association of chronic multisymptom illness with low-level pesticide exposure in chronically exposed populations such as the Agricultural Health Study population; the lack of clear, persistent CNS effect in animal studies of cholinesterase inhibitors and pesticides; the potential for bias in studies that base conclusions on self-reports of illness and exposure; and the lack of sufficient weight of evidence from studies of Gulf War veterans; the Update committee finds insufficient support for the conclusion that pesticides, PB, insect repellents, or combinations thereof are responsible for multisymptom illness seen in Gulf War veterans.
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