5
Sarin

Sarin is a highly toxic nerve agent produced for chemical warfare. It was synthesized in 1937 in Germany in a quest for improved insecticides (Somani, 1992). Although its battlefield potential was soon recognized, Germany refrained during World War II from using its stockpiles. Sarin’s first military use did not occur until the Iran–Iraq conflict in the 1980s (Brown and Brix, 1998).

Exposure to sarin can be fatal within minutes to hours. In vapor or liquid form, sarin can be inhaled or absorbed, respectively, across the skin, eyes, or mucous membranes (Stewart and Sullivan, 1992). Because of its extreme potency, sarin is lethal to 50 percent of exposed individuals at doses of 100 to 500 mg across the skin, or 50–100 mg/min/m3 by inhalation (in an individual weighing about 70 kg) (Somani, 1992).

Sarin is a member of a class of chemicals known as organophosphorus esters (or organophosphates). There are about 200 distinct organophosphate insecticides marketed today in thousands of formulations (Klaassen et al., 1996). A few highly toxic members of this large class are chemical warfare agents, but most are insecticides (Table 5.1) (Lotti, 2000). The drug pyridostigmine bromide (PB) is pharmacologically similar to sarin and other organophosphates, but it is a member of a different chemical class, the carbamates (see Chapter 6). Both PB and sarin exert their effects by binding to and inactivating the enzyme acetylcholinesterase (AChE). The binding of sarin to AChE is irreversible, whereas the binding of PB is reversible.

Since AChE is responsible for the breakdown of the neurotransmitter acetylcholine (ACh), the inactivation of this enzyme results in a dramatic elevation of ACh levels at cholinergic synapses (Gunderson et al., 1992). The term “cho-



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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines 5 Sarin Sarin is a highly toxic nerve agent produced for chemical warfare. It was synthesized in 1937 in Germany in a quest for improved insecticides (Somani, 1992). Although its battlefield potential was soon recognized, Germany refrained during World War II from using its stockpiles. Sarin’s first military use did not occur until the Iran–Iraq conflict in the 1980s (Brown and Brix, 1998). Exposure to sarin can be fatal within minutes to hours. In vapor or liquid form, sarin can be inhaled or absorbed, respectively, across the skin, eyes, or mucous membranes (Stewart and Sullivan, 1992). Because of its extreme potency, sarin is lethal to 50 percent of exposed individuals at doses of 100 to 500 mg across the skin, or 50–100 mg/min/m3 by inhalation (in an individual weighing about 70 kg) (Somani, 1992). Sarin is a member of a class of chemicals known as organophosphorus esters (or organophosphates). There are about 200 distinct organophosphate insecticides marketed today in thousands of formulations (Klaassen et al., 1996). A few highly toxic members of this large class are chemical warfare agents, but most are insecticides (Table 5.1) (Lotti, 2000). The drug pyridostigmine bromide (PB) is pharmacologically similar to sarin and other organophosphates, but it is a member of a different chemical class, the carbamates (see Chapter 6). Both PB and sarin exert their effects by binding to and inactivating the enzyme acetylcholinesterase (AChE). The binding of sarin to AChE is irreversible, whereas the binding of PB is reversible. Since AChE is responsible for the breakdown of the neurotransmitter acetylcholine (ACh), the inactivation of this enzyme results in a dramatic elevation of ACh levels at cholinergic synapses (Gunderson et al., 1992). The term “cho-

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines linergic synapses” refers to sites throughout the body where acetylcholine exerts its actions at the synapse, or junction, between nerve cells or between nerve cells and skeletal muscles. Widespread overstimulation of muscles and nerves induced by excessive levels of acetylcholine is primarily responsible for the acute cholinergic syndrome triggered by exposure to sarin and other organophosphate (OP) nerve agents. ACUTE CHOLINERGIC SYNDROME In humans, exposure to high doses of sarin produces a well-characterized acute cholinergic syndrome featuring a variety of signs and symptoms affecting the peripheral and central nervous systems (Gunderson et al., 1992) (Table 5.2). The peripheral effects are categorized as either muscarinic or nicotinic, in reference to the type of receptor stimulated by acetylcholine. The muscarinic signs and symptoms usually appear first (Lotti, 2000), although the sequence of effects may vary according to the route of sarin’s absorption (Stewart and Sullivan, 1992). If the dose of sarin is sufficiently high, death results after convulsions and respiratory failure (Lotti, 2000). Medical management of the acute cholinergic syndrome includes mechanical ventilation and the administration of several medications (anticholinergics, anticonvulsants, and drugs that break the chemical bond between sarin and AChE) (Sidell and Borak, 1992). The acute health effects of sarin are exquisitely dependent on dose. Because the actual doses to humans under battlefield or terrorist circumstances cannot be measured or are difficult to reconstruct, they can be inferred on the basis of their acute clinical effects. A high level of sarin exposure of humans (after single or multiple exposures) is presumed to have occurred when the acute cholinergic syndrome is manifest. An intermediate-level exposure is presumed to have TABLE 5.1 Examples of Organophosphates Nerve Agents Sarin (GB) Soman (GD) Tabun (GA) Cyclosarin (GF) o-Ethyl-S-[2-(diisopropylamino)ethyl]methylphosphonothiolate (VX) Insecticides Parathion Malathion Dichlorvos Diazinon Chlorpyrifos

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines TABLE 5.2 Acute Cholinergic Syndrome Site of Action Signs and Symptoms Muscarinic Pupils Miosis, marked, usually maximal (pinpoint), sometimes unequal Ciliary body Frontal headache, eye pain on focusing, blurring of vision Nasal mucous membranes Rhinorrhea, hyperemia Bronchial tree Chest tightness, prolonged wheezing, dyspnea, chest pain, increased bronchial secretion, cough, cyanosis, pulmonary edema Gastrointestinal Anorexia, nausea, vomiting, abdominal cramps, epigastric and substernal tightness with heartburn and eructation, diarrhea, tenesmus, involuntary defecation Sweat glands Increased sweating Salivary glands Increased salivation Lacrimal glands Increased lacrimation Heart Bradycardia Bladder Frequency, involuntary micturition Nicotinic Striated muscle Easy fatigue, mild weakness, muscular twitching, fasciculations, cramps, generalized weakness or flaccid paralysis (including muscles of respiration), with dyspnea and cyanosis Sympathetic ganglia Pallor, transitory elevation of blood pressure followed by hypotension Central nervous system   Immediate (acute) effects: generalized weakness, depression of respiratory and circulatory centers with dyspnea, cyanosis, and hypotension; convulsions, loss of consciousness, and coma   Delayed (chronic) effects: giddiness, tension, anxiety, jitteriness, restlessness, emotional lability, excessive dreaming, insomnia, nightmares, headaches, tremor, withdrawal and depression, bursts of slow waves of elevated voltage on electrogram, drowsiness, difficulty concentrating, slowness of recall, confusion, slurred speech, ataxia   SOURCE: Gunderson et al., 1992. occurred when the acute cholinergic effect is limited to miosis (contraction of the pupil), rhinorrhea (an extreme type of runny nose), and depressed cholinesterase levels in the blood. Finally, low-level exposure may have occurred even though there are no immediately detectable cholinergic signs and symptoms (Brown and Brix, 1998). The health effects of low levels of sarin exposure are of

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines most interest to Gulf War veterans because of their possible exposure from demolition of Iraqi munitions at Khamisiyah, Iraq (see discussion below). POSSIBLE U.S. TROOP EXPOSURE In March 1991, during the cease-fire period, troops from the U.S. 37th and 307th Engineering Battalion destroyed enemy munitions throughout the occupied areas of southern Iraq (PAC, 1996). The large storage complex at Khamisiyah, Iraq, which contained more than 100 bunkers, was destroyed. Two sites within the complex—one of the bunkers and another site called the “pit”—contained stacks of 122-mm rockets loaded with sarin and cyclosarin (Committee on Veterans’ Affairs, 1998). U.S. troops performing demolitions were unaware of the presence of nerve agents because their detectors, which were sensitive only to lethal or near-lethal levels of nerve agents (CDC, 1999), did not sound any alarms before demolition. It was not until October 1991 that inspectors from the United Nations Special Commission (UNSCOM) first confirmed the presence of a mixture of sarin and cyclosarin at Khamisiyah (Committee on Veterans’ Affairs, 1998). At the request of the Presidential Advisory Committee (PAC), the Central Intelligence Agency (CIA) and the Department of Defense (DoD) conducted exposure modeling to determine the extent of exposure of U.S. military personnel to the nerve agents. Since there was no air monitoring at the time of the Khamisiyah demolition, various models were employed to develop estimates of ground level concentrations of sarin and cyclosarin as a function of distance and direction from the detonation sites (PAC, 1996). The CIA–DoD report integrated four different components: (1) UNSCOM reporting and intelligence summaries of the amount, purity, and type of chemical warfare agents stored at Khamisiyah; (2) the results of experiments1 performed later at Dugway Proving Ground to simulate the demolition at Khamisiyah and thus estimate the amount of sarin and cyclosarin released, the release rate, and the associated type of release (instantaneous, continuous, or fly-out); (3) a combination of dispersion models, which incorporated meteorological conditions at the time (including wind direction), to simulate the transport and diffusion of the plume in order to estimate agent concentrations downwind; and (4) unit location information to determine the position of troops in relation to the plume’s path (CIA–DoD, 1997). The result of this modeling effort is a series of geographic maps of the Khamisiyah area that overlays known troop unit locations with the projected path of the sarin–cyclosarin plume. According to the model, the plume includes two levels of potential exposure, the first is “a first-noticeable-effects” level (approximately 1 mg/min/m3), where the estimated exposure was high enough to 1   These experiments, employing a substitute chemical (triethyl phosphate) to simulate chemical warfare agent, measured agent release concentrations after replicating the rockets in the pit, terrain, original warhead design, stacking of rockets, and other relevant information.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines cause watery eyes, runny nose, tightness of chest, muscle twitching or other early signs of chemical warfare (CW) agent exposure; the second is a lower-exposure area where the estimated dosage was less than that needed to produce first noticeable effects (CIA–DoD, 1997). The CIA–DoD report estimated that approximately 10,000 U.S. troops had been located within a 25-km radius of Khamisiyah and thus might have been exposed over a period of hours to the lower exposure level (CIA–DoD, 1997). Uncertainties with the model led to DoD’s doubling these figures to 20,000 U.S. troops with possible exposure within a 50-km radius; however, the dose levels remained unaltered. The CIA–DoD findings were challenged in a U.S. Senate report (Committee on Veterans’ Affairs, 1998). The Senate report took issue with the methodology, especially the reconstruction of the pit site, the nature of the demolition, and the number of exposed troops. At the request of the Senate Committee on Veterans’ Affairs, the Air Force Technical Applications Center (AFTAC) prepared another exposure model. The AFTAC report summary—the only portion of the report made public—indicates that AFTAC used different models than those employed by CIA–DoD to simulate atmospheric chemistry (Committee on Veterans’ Affairs, 1998). The report indicated additional geographic areas of low-level exposure not modeled by CIA–DoD. Neither the AFTAC nor the CIA–DoD report appears to have undergone independent peer review. DoD is conducting a complete remodeling of the Khamisiyah demolition, which is projected to be completed by the end of 2000. This remodeling, unlike the initial effort, is expected to be peer reviewed. It incorporates improved intelligence information, improved transport and diffusion modeling, and improved knowledge of unit locations. The committee encourages DoD to complete its ongoing remodeling efforts and to publish results in the peer-reviewed literature to enable broad review and independent validation of its work. Although exposure to sarin and cyclosarin was estimated by CIA–DoD modeling, there were no medical reports by the U.S. Army Medical Corps at the time of the release that were consistent with signs and symptoms of acute exposure to sarin (PAC, 1996). Further, a 1997 survey mailed by DoD to 20,000 troops who were within a 50-km radius of Khamisiyah found that more than 99 percent of respondents (n = 7,400) reported no acute cholinergic effects (CIA– DoD, 1997). Nevertheless, low-level exposure, as noted earlier, could have occurred without producing acute cholinergic effects. Two other storage sites in central Iraq sustained damage from air attacks during the Gulf War, but chemical agent releases were too far removed from U.S. troops for exposure to have occurred (PAC, 1996). At one site (Muhammadiyat), munitions with 2.9 metric tons of sarin–cyclosarin and 1.5 metric tons of mustard gas were damaged. At the other site (Al Muthanna), munitions containing 16.8 metric tons of sarin–cyclosarin were damaged (PAC, 1996). Atmospheric modeling by the CIA and DoD determined that the nearest U.S. personnel—located 400 km away—were outside the range of contamination (PAC, 1996). In summary, exposure models indicate that sarin–cyclosarin release occurred in March 1991 as a result of U.S. demolition of a storage depot in

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Khamisiyah, Iraq. The degree of exposure of U.S. troops located within the path of a sarin–cyclosarin plume, which is being remodeled in an upcoming DoD study, is at this point presumed to be low on the basis of previous exposure modeling and in the absence of medical personnel or veterans’ reporting symptoms of an acute cholinergic syndrome. The remainder of this chapter examines the scientific literature on the adverse health effects of sarin. It begins with a discussion of the toxicology of sarin and its effects on animals. It then summarizes the modest number of published toxicology studies on cyclosarin. The chapter next proceeds to its major focus, the health effects of sarin in humans. Most, if not all, toxicological and epidemiological studies focused on the health effects of sarin, as opposed to sarin in combination with other agents. SARIN TOXICOLOGY Sarin (GB; o-isopropyl methylphosphonofluoridate) is an organophosphate ester with high potency as an anticholinesterase nerve agent. It is a clear, colorless liquid with a molecular weight of 140.11, a boiling point of 158°C, and a vapor pressure of 1.48–2.9 mm Hg at 25°C (making it highly volatile). Sarin presents a liquid and a vapor hazard. In the liquid state, sarin can rapidly penetrate skin (as well as clothing), and in the vapor state it can contact the eye directly or be inhaled into the lungs, whereupon it is rapidly absorbed (Spencer et al., 2000). Exposure of the eye to vapor, which produces pinpoint pupils (miosis) and blurring of vision, accounts for one of the earliest signs of sarin exposure (Gunderson et al., 1992; Stewart and Sullivan, 1992). Mechanisms of Acute Toxicity Inhibition of Acetylcholinesterase There is widespread agreement that the principal mechanism of toxicity after sarin exposure is by inhibition of acetylcholinesterase and consequent rise in ACh, leading to overstimulation at cholinergic synapses (Somani, 1992; Lotti, 2000; Spencer et al., 2000). These effects are dose related. The degree of inhibition of AChE in the mouse brain depends directly on the administered intravenous (i.v.) dose of sarin (Tripathi and Dewey, 1989). High doses of sarin (100 μg/kg) administered subcutaneously to rats produce a 32 percent increase in ACh levels (Flynn and Wecker, 1986). Sarin inhibits AChE by phosphorylating a serine hydroxyl on the ester portion of the active site of this enzyme.2 The phosphorylated enzyme is hydrolyzed very slowly, with a half-life of reactivation of hours to days (Gray, 1984). The 2   During its normal function, AChE hydrolyzes acetylcholine to produce choline, acetic acid, and the reactivated enzyme. The reactivated enzyme is available to bind to another acetylcholine molecule. AChE has one of the fastest turnover rates known.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines phosphorylated enzyme then can undergo a second process, called aging, by loss of an alkyl group (dealkylation). The half-life for “aging” is about 5 hours after sarin exposure (Sidell and Borak, 1992). Only during this period prior to aging can treatment with oxime therapy (e.g., pralidoxime chloride) successfully remove sarin from the enzyme and thus block the aging process. After aging has occurred, the phosphorylated enzyme (now negatively charged) is resistant to cleavage or hydrolysis and can be considered irreversibly inhibited. Recovery of AChE function occurs only with synthesis of new enzyme. Inhibition of AChE prevents the breakdown of acetylcholine, which accumulates in central and peripheral nerve synapses, leading to the acute cholinergic syndrome. Sarin also may exert its effects through other cholinergic mechanisms (unrelated to inhibition of AChE). A new line of research suggests that sarin (in picomolar concentrations) may interact directly with muscarinic ACh receptors (Rocha et al., 1998; Chebabo et al., 1999). Researchers uncovered this new mechanism by studying sarin’s ability to reduce evoked GABA (gamma-aminobutyric acid) release from hippocampal neurons. This effect of sarin is blocked by the muscarinic receptor antagonist atropine, but not by nicotinic receptor antagonists (Rocha et al., 1998). These findings suggest that sarin may interact with presynaptic muscarinic receptors, thereby reducing action potential-dependent release of GABA in the postsynaptic neuron (Chebabo et al., 1999). It is reasonable to consider that sarin acts as a muscarinic receptor antagonist inhibiting the evoked release of GABA. Reductions in the levels of GABA, which is an inhibitory neurotransmitter, may contribute to the convulsive properties of sarin. Noncholinergic Mechanisms For decades, researchers observed puzzling relationships between the extent of neurobehavioral toxicity and the degree of inhibition of AChE. For example, only sarin-induced tremor has a slight correlation with AChE inhibition in rat striatum, whereas chewing, hind-limb abduction, and convulsions have no clear correlation (Hoskins et al., 1986). Some sarin-treated rats with 90 percent inhibition of AChE in the striatum of the brain had no convulsions or hind-limb abduction, while rats with less enzyme inhibition exhibited both. From these findings, researchers have concluded that noncholinergic mechanisms may also contribute to toxicity induced by sarin and other organophosphates. The difficulty has been in disentangling which effects are mediated directly by sarin and which are secondary to its inhibition of AChE. Several studies suggest that sarin may alter the level of neurotransmitters other than ACh. In most of these studies, however, the neurotransmitter effects are seen in brain regions where there are cholinergic synapses. Significant increases in catecholamines, measured histochemically, were found in the substantia nigra pars compacta and locus coeruleus of the brain following intramus-

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines cular (i.m.) injection of sarin at one-third of the median lethal dose (LD50)3 (Dasheiff et al., 1977). Catecholamine levels in the nucleus accumbens decreased. All changes, except for the latter, returned to normal within 10 days. It is not clear whether these changes represented the direct action of sarin on enzymes related to noncholinergic neurotransmission or were secondary to the production of excessive ACh (Somani, 1992). Alternatively, stress could activate catecholamine neurons. Levels of the neurotransmitter serotonin 5-hydroxytryptamine (5-HT) were decreased, and its major metabolite (5-hydroxyindoleacetic acid, or 5-HIAA) increased, in rat striatum after subconvulsive doses of sarin. Since this effect was also seen after administration of the OP nerve agents soman and tabun, it most likely is not agent specific, but rather is a likely consequence of an acute increase of acetylcholine in the striatum (Fernando et al., 1984). Neuropathological damage in the hippocampus, dorsal thalamus, and piriform cortex was found in about 70 percent of rats within 24 hours of administering a single dose of sarin (95 μg/kg, i.m., or 1 LD50 (Kadar et al., 1995). These animals had prolonged convulsions, whereas the other 30 percent with short convulsive episodes had minimal brain damage. The authors interpreted these results to mean that convulsions may have caused the severe hypoxic damage. The neuropathology in the most affected animals continued to increase for 3 months, involving brain regions previously unaffected. The study attributed the progressive, long-term neuropathology either to delayed neurotoxicity of sarin or to secondary retrograde degeneration. It did not directly investigate potential neurochemical mechanisms underlying the neuropathology. Toxicokinetics This section discusses the absorption, distribution, metabolism, and elimination of sarin. In general, these events occur very rapidly after exposure, although there is some variability depending on the route of administration and the species studied (Somani, 1992). Most of the research reported here comes from animal studies, but where possible, human toxicokinetic studies are also reported. Absorption and Metabolism Sarin in vapor or liquid form is absorbed rapidly to produce local and systemic effects. Local effects, such as those on the eyes (e.g., miosis) and nose, are the product of sarin vapors directly interacting with AChE at the nerve endings near body surfaces (Sidell and Borak, 1992). Systemic effects, including those within the central nervous system (CNS), occur as a result of absorption of sarin into the circulation from the skin, respiratory tract, or gastrointestinal tract (Lotti, 2000). 3   LD50 is the lethal dose to half or 50 percent of the test subjects.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines The fate of sarin in the blood is a major determinant of how much sarin reaches the central nervous system and other sites of systemic toxicity. In the blood, sarin first interacts with several esterases (a class of enzymes). Some of the esterases, such as paraoxonase, hydrolyze sarin to inactive metabolites (Davies et al., 1996; Lotti, 2000). Two other blood esterases—AChE and butyrylcholinesterase (BuChE)—irreversibly bind to sarin. AChE found on the surface of red blood cells (RBCs), although chemically indistinguishable from AChE in the nervous system, has unknown physiological functions (Sidell and Borak, 1992). These esterases in the blood are often described as “false targets”—by binding irreversibly to sarin, AChE and BuChE sequester sarin in the blood, thereby preventing some or all from reaching the CNS (Spencer et al., 2000). However, esterases in the blood can be overwhelmed by high doses of sarin. The acute cholinergic syndrome occurs when RBC AChE is inhibited by 75–80 percent (Sidell and Borak, 1992). Distribution and Elimination The tissue distribution of sarin and its metabolites has been studied in rodents. In one study a single sublethal dose (80 μg/kg) of radiolabeled sarin was administered intravenously, after which tissues were examined at distinct points in time for 24 hours (Little et al., 1986). Within 1 minute, sarin was distributed to the brain (and thus crossed the blood–brain barrier), lungs, heart, diaphragm, kidneys, liver, and plasma, with the greatest concentrations found in the last three tissues. Thereafter, the concentrations in all tissues declined. Within 15 minutes, sarin concentrations declined by 85 percent, followed by a second, more gradual decline. Relatedly, within the first minute, about half of the labeled sarin was associated with the major sarin metabolite isopropyl methylphosphonic acid (IMPA). A nonextractable label was present in constant amounts in all tissues, except plasma, throughout the time course of the experiment. The kidneys are the major route of elimination of sarin or its metabolites. In the above study, Little and colleagues (1986) determined that kidneys contained the highest concentrations of sarin and its metabolites, whereas much lower concentrations of metabolite were detected in the liver. This suggests a minor role for the liver in detoxification of sarin. Shih and colleagues (1994) injected rats subcutaneously with a single dose of 75 μg/kg of sarin. They then measured excretion of the hydrolyzed metabolites, the alkylmethylphosphonic acids, which include IMPA and other methylphosphonic acids. Urinary elimination was found to be quite rapid; the terminal elimination half-life of sarin metabolites in urine was 3.7 ± 0.1 hours. Nearly all of the administered dose of sarin was retrieved from the urine in metabolite form after 2 days. Distribution, metabolism, and elimination of sarin in humans appear to resemble findings in animals. Minami and colleagues (1997) detected the sarin metabolite IMPA in urine of humans after the terrorist attack on the Tokyo subway system (see later description). They found peak levels of IMPA or methylphosphonic acid in urine 10–18 hours after exposure but did not report meta-

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines bolic rates. The levels of IMPA in urine correlated with the degree of clinical symptoms. They also found evidence of distribution of sarin to the human brain in 4 of the 12 people who died after exposure. Solubilized sarin-bound AChE from formalin-fixed cerebellar tissue of victims of the Tokyo attack contained a derivative of the sarin hydrolysis product methylphosphonic acid (MPA) (Matsuda et al., 1998). The estimated amounts of MPA ranged from 0.32 to 1.13 nmol/g tissue. Although no IMPA was found, it was assumed that IMPA had hydrolyzed to MPA in the formalin solution over 2 years of storage. Biomarkers of Exposure Biomarkers of acute sarin exposure can be detected in blood or urine. In blood, the extent of inhibition of RBC AChE is considered the best marker of acute exposure. Sarin preferentially inhibits RBC AChE more than BuChE; however, after high-level sarin exposure, complete inhibition of both esterases occurs (Sidell and Borak, 1992). Since inhibition of blood cholinesterases is a common feature of organophosphates and other anticholinesterases, this biomarker is not specific to sarin exposure. Further, its utility as a biomarker is limited to a short time after exposure, with a return to original blood esterase levels by about 1–3 months (Grob, 1963). The recovery times for blood esterases are somewhat different. BuChE is replaced after about 50 days following de novo synthesis in the liver. RBC AChE recovery is contingent upon the turnover rate of red blood cells, which is about 1 percent per day. This esterase is synthesized with the RBC (Sidell and Borak, 1992). Sensitive methods for detecting urinary metabolites as biomarkers of sarin exposure were recently developed by Japanese researchers in the aftermath of the Tokyo terrorism incident (Minami et al., 1997, 1998). Black and colleagues (1999) recently found a sensitive biomarker that can specifically identify sarin at low concentrations in human plasma. The researchers found a novel phosphonylation site, presumably from human serum albumin, at which sarin interacts with a tyrosine residue. In contrast, the biomarkers noted above are indices of sarin exposure but do not uniquely identify sarin as opposed to other CW agents. The advantage of this potentially new method is that it can directly implicate sarin at low concentrations. Animal Studies This section summarizes the toxic effects of sarin in laboratory animals. Most animal studies of sarin did not examine low-level exposure, but instead focused on lethal, near-lethal, or maximum tolerated doses (MTDs).4 These high doses produced the acute cholinergic syndrome and in many cases necessitated 4   The MTD is the highest dose used during a long-term study that will not alter the life span of the animal and slightly suppresses body weight gain (i.e., 10 percent) in a 90-day subchronic study.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines pharmacological intervention to prevent death. Although these studies enable researchers to deduce with some certainty what organ systems will not be affected by low levels of sarin (i.e., those systems that are not affected by large doses), they are not useful in distinguishing between primary damage caused by sarin and secondary damage caused by hypoxic events following convulsions. Acute Toxicity In animals, sarin is acutely toxic and fatal in microgram quantities in a matter of minutes. There is some variability depending on the species and the route of administration. Table 5.3 outlines the doses and routes of administration that produce acute lethality (within 24 hours) in the animal species tested. The LD50 in the rat and mouse are similar, with subcutaneous (s.c.), intramuscular, and intravenous doses requiring 150–180 μg/kg. Oral administration requires nearly 10 times more sarin. The hen, guinea pig, and cat are more sensitive than rats and mice, with lethal doses ranging from 16–40 μg/kg s.c. to 561 μg/kg oral. The immediate cause of death from sarin poisoning is respiratory arrest (Rickett et al., 1986). In baboons, sarin administered to the upper airway in vapor form (30 μg/kg) causes apnea within 5 minutes (Anzueto et al., 1990). Since the dose was twice the LD50, mechanical ventilation was needed to keep the animals alive. Their apnea was correlated with the absence of activity in the phrenic nerve (which projects to the diaphragm), suggesting a central effect of sarin on respiration. Respiration recovered spontaneously within 1–2 days, al- TABLE 5.3 Acute Lethality of Sarin Administered to Various Species Species, Strain Route LD50 (μg/kg) Reference Rat s.c. 158–165 Landauer and Romano, 1984; Singer et al., 1987; Somani, 1992 Mouse, CD-1 s.c. 160–170 Clement, 1991 Mouse i.m. 179 Somani, 1992 Mouse i.v. 109 Little et al., 1986; Tripathy and Dewey, 1989 Mouse, Swiss albino inhalation 600 mg/min/m3 Husain et al., 1993 Hen oral 561 Bucci et al., 1993 Hen s.c. 16.5–16.7a Gordon et al., 1983 Guinea pig s.c. 53 (divided doses) Fonnum and Sterri, 1981; Somani, 1992 Cat s.c. 30–35 Goldstein et al., 1987 NOTE: i.m. = intramuscular; i.v. = intravenous; s.c. = subcutaneous. aConverted from 0.119 μmol/kg in Ross white or Light Sussex hens.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines warfare conditions revealed that 10–20 percent reported (in the absence of actual exposure to chemical weapons) moderate to severe psychological symptoms, including anxiety, claustrophobia, and panic (Fullerton and Ursano, 1990). For vestibulocerebellar testing, Yokoyama and colleagues (1998a) used computerized posturography on sarin cases and controls. Computerized posturography is a standard means of assessing vestibular function by placing subjects in the middle of a platform and measuring how their movements displaced the platform (via pressure transducers connected from the platform to a computer). The study found significant impairment only in female cases (n = 9) who performed more poorly (with their eyes open) in their ability to maintain postural sway and their center of gravity when they moved at low frequencies (0–1 Hz) in the anterior– posterior direction. Female patients also performed more poorly in the area of sway (i.e., the area on the platform over which the test subject moves to maintain balance). None of the postural sway tests were abnormal in male cases (n = 9). The authors viewed their findings as suggestive of a gender difference in a “delayed” effect of acute sarin poisoning on the vestibulocerebellar system. Their characterization of this effect as “delayed” is questionable, since there is no evidence of this postural testing having been performed at an earlier point after sarin exposure. Thus, the effect may be chronic, rather than delayed. The Tokyo sarin experience confirms that acute exposure to sarin leads to the acute cholinergic syndrome. Sarin exposure at high levels can be fatal if cardiopulmonary compromise or convulsions ensue. Visual disturbances are frequent sequelae of the acute exposure, particularly in individuals with high-level exposure. Neurophysiological testing of a small group of asymptomatic sarin-exposed individuals does show chronic changes in visual and event-related evoked potentials and vestibulocerebellar function months after the acute syndrome has subsided. These neurophysiological data are suggestive of subtle, persistent CNS effects from sarin. Except for digit symbol test abnormalities, significant cognitive deficits were not detected. Gulf War Veterans As explained earlier in this chapter, CIA–DoD modeling determined that U.S. troops located within 25 km of the Khamisiyah weapons site demolition in March 1991 may have been exposed to low or intermediate levels of sarin (CIA–DoD, 1997). U.S. troops did not report acute cholinergic symptoms at the time, but the possibility of low-level, asymptomatic exposures cannot be discounted. In a series of studies on members of a naval battalion (n = 249) called to active duty for the Gulf War, Haley and Kurt (1997) found that veterans who believed themselves to have been exposed to chemical weapons14 were more 14   Based on self-reports about their perceptions of CW exposure, rather than any evidence of symptomatology. Their geographical and temporal location in relation to the Khamisiyah demolition site was not reported. The questionnaire was sent to participants in 1994, before DoD reported that chemical weapons exposure could have occurred.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines likely to be classified as having one of six new proposed syndromes (Haley et al., 1997; see also Chapter 2). Specifically, this syndrome—labeled by the investigators as “confusion–ataxia” or “syndrome 2”—features problems with thinking, disorientation, balance disturbances, vertigo, and impotence. This was the only syndrome of the six to have been associated with self-reported chemical weapons exposure (see Chapter 6). A follow-up study of vestibular function was performed on a subset of those veterans (n = 23) who had the highest factor scores on three of the syndromes identified in 1997 by Haley and Kurt (Roland et al., 2000). The study was designed to probe the nature of veterans’ vestibular symptoms, rather than to examine the relationship between vestibular performance and exposure in the Gulf War. Of the 23 veterans in this study, 13 exhibited syndrome 2, whereas the others exhibited syndromes 1 (impaired cognition) and 3 (arthromyoneuropathy) (see Chapter 2). Based on a new questionnaire, veterans with syndrome 2 reported dizzy spells with greater frequency and longer duration than veterans with the other two syndromes. Veterans with syndrome 3, but not syndrome 2, performed significantly differently from controls on dynamic platform posturography (a test similar to that used by Japanese researchers to identify impairment in sarin-exposed females; see Yokoyama et al., 1998a). Veterans with other syndromes also had performance decrements on some of the measures of vestibular function. The study concluded that there was both subjective and objective evidence of injury to the vestibular system in this group of Gulf War veterans with newly defined syndromes. Haley and Kurt (1997) hypothesized that these newfound chronic syndromes represent variants of OPIDN caused by exposure to various combinations of organophosphates (pesticides and nerve agents) and carbamate pesticides that inhibit cholinesterases and NTE (see Chapters 2 and 6). Genetic Susceptibility to Sarin Toxicity One of the mechanisms of sarin inactivation is by hydrolysis with the enzyme paraoxonase (PON1), an esterase found in liver and serum. The human PON1 gene has polymorphisms at positions 192 (Arg/Gln) and 55 (Leu/Met) (Furlong et al., 1993). The former accounts for three genotypes (QQ, RR, and QR) relating to the catalytic properties of two forms of an enzyme (types R and Q allozymes), which hydrolyze certain organophosphates at different rates. The R allozyme (Arg192) hydrolyzes the organophosphate paraoxon at a high rate; however, it has a low activity toward OP nerve agents such as sarin and soman (Davies et al., 1996). Lower activity means that more sarin would be bioavailable to exert its anticholinesterase effects. The Q allozyme, on the other hand, has high activity toward organophosphate nerve agents (and low activity toward paraoxon). Thus, individuals with the Q allozyme (QQ or QR) are expected to have greater hydrolysis of sarin than individuals homozygous for the R allele (RR). Since hydrolytic activity with the same genotype can vary about tenfold, it is also important to determine the level of allozyme expression—in addition to

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines the genotype—in order to characterize an individual’s PON1 status (Richter and Furlong, 1999). In Caucasian populations, the frequency of the R allele is about 0.3, but the frequency is 0.66 in the Japanese population (Yamasaki et al., 1997). This would make individuals in the Japanese population more sensitive to the toxicity of sarin, a fact that may have contributed to their morbidity and mortality after the terrorist attacks. A recent study investigated PON1 genotype and serum enzyme activity in a group of 25 ill Gulf War veterans and 20 controls (Haley et al., 1999). Ill veterans were more likely than controls to possess the R allele (QR heterozygotes or R homozygotes) and to exhibit lower enzyme activity. This study raises the possibility that the R genotype (low sarin-hydrolyzing activity) may represent a risk factor for illness in Gulf War veterans. However, because of the very small size of the study, such findings necessitate further confirmation in a larger population (Furlong, 2000) (also see Chapter 6). CONCLUSIONS The committee reached the following conclusions after reviewing the literature on sarin. The committee was unable to formulate any conclusions about cyclosarin because of the paucity of toxicological and human studies. The committee concludes that there is sufficient evidence of a causal relationship between exposure to sarin and a dose-dependent acute cholinergic syndrome that is evident seconds to hours subsequent to sarin exposure and resolves in days to months. The acute cholinergic syndrome has been recognized for decades and has been documented in human studies summarized in this chapter. This syndrome, as well as cholinergic signs and symptoms, is evident seconds to hours after exposure (see Table 5.2) and usually resolves in days to months. The syndrome and the cholinergic signs and symptoms are produced by sarin’s irreversible inhibition of the enzyme acetylcholinesterase. Inactivation of the enzyme that normally breaks down the neurotransmitter acetylcholine leads to the accumulation of acetylcholine at cholinergic synapses. Excess quantities of acetylcholine result in widespread overstimulation of muscles and nerves. At high doses, convulsions and death can occur. The committee concludes that there is limited/suggestive evidence of an association between exposure to sarin at doses sufficient to cause acute cholinergic signs and symptoms and subsequent long-term health effects. Many health effects are reported in the literature to persist after sarin exposure: fatigue, headache, visual disturbances (asthenopia, blurred vision, and narrowing of the visual field), asthenia, shoulder stiffness, and symptoms of posttraumatic stress disorder; and abnormal test results, of unknown clinical

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines significance, on the digit symbol test of psychomotor performance, EEG records of sleep, event-related potential, visual evoked potential, and computerized posturography. These conclusions are based on retrospective studies of three different exposed populations in which the acute cholinergic signs and symptoms were documented as an acute effect of exposure. The findings from those studies are based on comparisons with control populations. One population consisted of industrial workers accidentally exposed to sarin in the United States; the other two populations were civilians exposed during terrorism episodes in Japan. The health effects listed above were documented at least 6 months after sarin exposure, and some persisted up to a maximum of 3 years, depending on the study. Whether the health effects noted above persist beyond the 3 years has not been studied. The committee concludes that there is inadequate/insufficient evidence to determine whether an association does or does not exist between exposure to sarin at low doses insufficient to cause acute cholinergic signs and symptoms and subsequent long-term adverse health effects. On the basis of positive findings in a study of nonhuman primates and in studies of humans exposed to organophosphate insecticides (see Appendix E), it is reasonable to hypothesize the occurrence of long-term adverse health effects from exposure to low levels of sarin. Studies of low-level exposure of workers find that organophosphate insecticides are consistently associated with higher prevalence of neurological and/or psychiatric symptom reporting (see Appendix E). However, there are no well-controlled human studies expressly of sarin’s long-term health effects at doses that do not produce acute signs and symptoms. REFERENCES Anzueto A, deLemos RA, Seidenfeld J, Moore G, Hamil H, Johnson D, Jenkinson SG. 1990. Acute inhalation toxicity of soman and sarin in baboons. Fundam Appl Toxicol 14(4):676–687. Baker DJ, Sedgwick EM. 1996. Single fibre electromyographic changes in man after organophosphate exposure. Hum Exp Toxicol 15(5):369–375. Black RM, Harrison JM, Read RW. 1999. The interaction of sarin and soman with plasma proteins: The identification of a novel phosphonylation site. Arch Toxicol 73(2):123–126. Brown MA, Brix KA. 1998. Review of health consequences from high-, intermediate-and low-level exposure to organophosphorous nerve agents. J Appl Toxicol 18(6): 393–408. Bucci TJ, Parker RM. 1992. Toxicity Studies on Agents GB and GD (Phase 2): 90-Day Subchronic Study of GB (Sarin, Type II) in CD-Rats. Available from the National Technical Information Service. NTIS/AD-A248 618/1. Bucci TJ, Parker RM, Crowell JA, Thurman JD, Gosnell PA. 1992. Toxicity Studies on Agents GB and GD (Phase 2): 90-Day Subchronic Study of GB (Sarin, Type I) in

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines CD-Rats. Available from the National Technical Information Service. NTIS/ADA248 617/3. Bucci TJ, Parker RM, Gosnell PA. 1993. Toxicity Studies on Agents GB and GD (Phase 2): Delayed Neuropathy Study of Sarin, Type II, in SPF White Leghorn Chickens. Available from the National Technical Information Service. NTIS/AD-A257357. Burchfiel JL, Duffy FH. 1982. Organophosphate neurotoxicity: Chronic effects of sarin on the electroencephalogram of monkey and man. Neurobehav Toxicol Teratol 4(6):767–778. Burchfiel JL, Duffy FH, Van Sim M. 1976. Persistent effects of sarin and dieldrin upon the primate electroencephalogram. Toxicol Appl Pharmacol 35(2):365–379. CDC (Centers for Disease Control and Prevention). 1999. Background Document on Gulf War-Related Research for the Health Impact of Chemical Exposures During the Gulf War: A Research Planning Conference. Atlanta, GA: CDC. Chebabo SR, Santos MD, Albuquerque EX. 1999. The organophosphate sarin, at low concentrations, inhibits the evoked release of GABA in rat hippocampal slices. Neurotoxicology 20(6):871–882. CIA–DoD (Central Intelligence Agency and Department of Defense). 1997. Modeling the Chemical Warfare Agent Release at the Khamisiyah Pit. Washington, DC: CIA– DoD. Clement JG. 1991. Variability of sarin-induced hypothermia in mice: Investigation into incidence and mechanism. Biochem Pharmacol 42(6):1316–1318. Clement JG. 1992. Efficacy of various oximes against GF (cyclohexyl methylphosphonofluoridate) poisoning in mice. Arch Toxicol 66(2):143–144. Committee on Veterans’ Affairs, U.S. Senate. 1998. Report of the Special Investigation Unit on Gulf War Illnesses. 105th Congress, 2nd session. Washington, DC: U.S. Government Printing Office. S.PRT 105-39. Crowell JA, Parker RM, Bucci TJ, Dacre JC. 1989. Neuropathy target esterase in hens after sarin and soman. J Biochem Toxicol 4(1):15–20. Dasheiff RM, Einberg E, Grenell RG. 1977. Sarin and adrenergic–cholinergic interaction in rat brain. Exp Neurol 57(2):549–560. Davies DR, Holland P, Rumens MJ. 1960. The relationship between the chemical structure and neurotoxicity of alkyl organophosphorus compounds. Brit J Pharmacol 15:271–278. Davies DR, Holland P. 1972. Effect of oximes and atropine upon the development of delayed neurotoxic signs in chickens following poisoning by DFP and sarin. Biochem Pharmacol 21(23):3145–3151. Davies HG, Richter RJ, Keifer M, Broomfield CA, Sowalla J, Furlong CE. 1996. The effect of the human serum paraoxonase polymorphism is reversed with diazoxon, soman and sarin. Nature Genetics 14(3):334–336. De Bleecker JL, De Reuck JL, Willems JL. 1992. Neurological aspects of organophosphate poisoning. Clin Neurol Neurosurg 94:93–103. D’Mello GD, Duffy EA. 1985. The acute toxicity of sarin in marmosets (Callithrix jacchus): A behavioral analysis. Fundam Appl Toxicol 5(6 Pt 2):S169–S174. Duffy FH, Burchfiel JL, Bartels PH, Gaon M, Sim VM. 1979. Long-term effects of an organophosphate upon the human electroencephalogram. Toxicol Appl Pharmacol 47(1):161–176. Fernando JC, Hoskins BH, Ho IK. 1984. A striatal serotonergic involvement in the behavioural effects of anticholinesterase organophosphates. Eur J Pharmacol 98(1): 129–132.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Flynn CJ, Wecker L. 1986. Elevated choline levels in brain. A non-cholinergic component of organophosphate toxicity. Biochem Pharmacol 35(18):3115–3121. Fonnum F, Sterri SH. 1981. Factors modifying the toxicity of organophosphorous compounds including soman and sarin. Fundam Appl Toxicol 1(2):143–147. Fullerton CS, Ursano RJ. 1990. Behavioral and psychological responses to chemical and biological warfare. Mil Med 155(2):54–59. Furlong CE. 2000. PON1 status and neurologic symptom complexes in Gulf War veterans. Genome Research 10(2):153–155. Furlong CE, Costa LG, Hassett C, Richter RJ, Sundstrom JA, Adler DA, Disteche CM, Omiecinski CJ, Chapline C, Crabb JW, Humbert R. 1993. Human and rabbit paraoxonases: Purification, cloning, sequencing, mapping and role of polymorphism in organophosphate detoxification. Chem-Biol Interactions 87:35–48. Goldman M, Klein AK, Kawakami TG, Rosenblatt LS. 1988. Toxicity Studies on Agents GB (Sarin, Types I and II) and GD (Soman). Available from the National Technical Information Service. NTIS AD-A187841. Goldstein BD, Fincher DR, Searle JR. 1987. Electrophysiological changes in the primary sensory neuron following subchronic soman and sarin: Alterations in sensory receptor function. Toxicol Appl Pharmacol 91(1):55–64. Gordon J, Inns R, Johnson M, Leadbeater L, Maidment M, Upshall D, Cooper G, Rickard R. 1983. The delayed neuropathic effects of nerve agents and some other organophosphorus compounds. Arch Toxicol 52(2):71–82. Gray AP. 1984. Design and structure–activity relationships of antidotes to organophosphorus anticholinesterase agents. Drug Metab Rev 15(3):557–589. Grob D. 1963. Anticholinesterase intoxication in man and its treatment. Handbuch der Experimentellen Pharmakologie 15(Supplement, Chapter 22): 989–1027. Gunderson CH, Lehmann CR, Sidell FR, Jabbari B. 1992. Nerve agents: A review. Neurology 42(5):946–950. Haley RW, Kurt TL. 1997. Self-reported exposure to neurotoxic chemical combinations in the Gulf War. A cross-sectional epidemiologic study. JAMA 277(3):231–237. Haley RW, Kurt TL, Hom J. 1997. Is there a Gulf War syndrome? Searching for syndromes by factor analysis of symptoms. JAMA 277(3):215–222. Haley RW, Billecke S, La Du BN. 1999. Association of low PON1 type Q (type A) arylesterase activity with neurologic symptom complexes in Gulf War veterans. Toxicol Appl Pharmacol 157(3):227–233. Hartgraves SL, Murphy MR. 1992. Behavioral effects of low-dose nerve agents. In: Somani SM, ed. Chemical Warfare Agents. San Diego, CA: Academic Press. Pp. 125–154. Hoskins B, Fernando JC, Dulaney MD, Lim DK, Liu DD, Watanabe HK, Ho IK. 1986. Relationship between the neurotoxicities of soman, sarin and tabun, and acetylcholinesterase inhibition. Toxicol Lett 30(2):121–129. Husain K, Vijayaraghavan R, Pant SC, Raza SK, Pandey KS. 1993. Delayed neurotoxic effect of sarin in mice after repeated inhalation exposure. J Appl Toxicol 13(2):143–145. Husain K, Pant SC, Raza SK, Singh R, Das Gupta S. 1995. A comparative study of delayed neurotoxicity in hens following repeated administration of organophosphorus compounds. Indian J Physiol Pharmacol 39(1):47–50. Jacobson KH, Christensen MK, DeArmon IA Jr, Oberst FW. 1959. Studies of chronic exposures of dogs to GB (isopropyl methylphosphonofluoridate) vapor. Arch Ind Health 19(1):5–10.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Kadar T, Shapira S, Cohen G, Sahar R, Alkalay D, Raveh L. 1995. Sarin-induced neuropathology in rats. Hum Exp Toxicol 14(3):252–259. Klaassen CD, Amdur MO, Doull J, eds. 1996. Casarett and Doull’s Toxicology: The Basic Science of Poisons. 5th edition. New York: McGraw-Hill. Klein AK, Nasr ML, Goldman M. 1987. The effects of in vitro exposure to the neurotoxins sarin (GB) and soman (GD) on unscheduled DNA synthesis by rat hepatocytes. Toxicol Lett 38(3):239–249. Koplovitz I, Gresham VC, Dochterman LW, Kaminskis A, Stewart JR. 1992. Evaluation of the toxicity, pathology, and treatment of cyclohexylmethylphosphonofluoridate (CMPF) poisoning in rhesus monkeys. Arch Toxicol 66(9):622–628. LaBorde JB, Bates HK, Dacre JC, Young JF. 1996. Developmental toxicity of sarin in rats and rabbits. J Toxicol Environ Health 47(3):249–265. Landauer MR, Romano JA. 1984. Acute behavioral toxicity of the organophosphate sarin in rats. Neurobehav Toxicol Teratol 6(3):239–243. Little PJ, Reynolds ML, Bowman ER, Martin BR. 1986. Tissue disposition of [3H]sarin and its metabolites in mice. Toxicol Appl Pharmacol 83(3):412–419. Lotti M. 2000. Organophosphorous compounds. In: Spencer P, Schaumburg H, Ludolph A, eds. Experimental and Clinical Neurotoxicology. 2nd edition. New York: Oxford University Press. Pp. 897–925. Marrs TC, Maynard RL, Sidell FR. 1996. Chemical Warfare Agents: Toxicology and Treatment. New York: John Wiley & Sons. Matsuda Y, Nagao M, Takatori T, Niijima H, Nakajima M, Iwase H, Kobayashi M, Iwadate K. 1998. Detection of the sarin hydrolysis product in formalin-fixed brain tissues of victims of the Tokyo subway terrorist attack. Toxicol Appl Pharmacol 150(2):310–320. Metcalf DR, Holmes JH. 1969. EEG, psychological, and neurological alterations in humans with organophosphorous exposure. Ann NY Acad Sci 160:357–385. Minami M, Hui DM, Katsumata M, Inagaki H, Boulet CA. 1997. Method for the analysis of the methylphosphonic acid metabolites of sarin and its ethanol-substituted analogue in urine as applied to the victims of the Tokyo sarin disaster. J Chromatogr B Biomed Sci Appl 695(2):237–244. Minami M, Hui DM, Wang Z, Katsumata M, Inagaki H, Li Q, Inuzuka S, Mashiko K, Yamamoto Y, Ootsuka T, Boulet CA, Clement JG. 1998. Biological monitoring of metabolites of sarin and its by-products in human urine samples. J Toxicol Sci 23(Suppl 2):250–254. Moore DH. 1998. Long term health effects of low dose exposure to nerve agent. J Physiology 92(3–4):325–328. Morita H, Yanagisawa N, Nakajima T, Shimizu M, Hirabayashi H, Okudera H, Nohara M, Midorikawa Y, Mimura S. 1995. Sarin poisoning in Matsumoto, Japan. Lancet 346(8970):290–293. Murata K, Araki S, Yokoyama K, Okumura T, Ishimatsu S, Takasu N, White RF. 1997. Asymptomatic sequelae to acute sarin poisoning in the central and autonomic nervous system 6 months after the Tokyo subway attack. J Neurol 244(10):601–606. Nakajima T, Sato S, Morita H, Yanagisawa N. 1997. Sarin poisoning of a rescue team in the Matsumoto sarin incident in Japan. Occup Environ Med 54(10):697–701. Nakajima T, Ohta S, Morita H, Midorikawa Y, Mimura S, Yanagisawa N. 1998. Epidemiological study of sarin poisoning in Matsumoto City, Japan. J Epidemiol 8(1):33–41. Nakajima T, Ohta S, Fukushima Y, Yanagisawa N. 1999. Sequelae of sarin toxicity at one and three years after exposure in Matsumoto, Japan. J Epidemiol 9(5):337–343.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Nieminen SA, Lecklin A, Heikkinen O, Ylitalo P. 1990. Acute behavioural effects of the organophosphates sarin and soman in rats. Pharmacol Toxicol 67(1):36–40. Nozaki H, Hori S, Shinozawa Y, Fujishima S, Takuma K, Sagoh M, Kimura H, Ohki T, Suzuki M, Aikawa N. 1995. Secondary exposure of medical staff to sarin vapor in the emergency room. Intensive Care Med 21(12):1032–1035. NRC (National Research Council). 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents, Vol. 1: Anticholinesterases and Anticholinergics. Washington, DC: National Academy Press. NRC (National Research Council). 1985. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents, Vol. 3. Final Report. Current Health Status of Test Subjects. Washington, DC: National Academy Press. Ohbu S, Yamashina A, Takasu N, Yamaguchi T, Murai T, Nakano K, Matsui Y, Mikami R, Sakurai K, Hinohara S. 1997. Sarin poisoning on Tokyo subway. South Med J 90(6):587–593. Okumura T, Takasu N, Ishimatsu S, Miyanoki S, Mitsuhashi A, Kumada K, Tanaka K, Hinohara S. 1996. Report on 640 victims of the Tokyo subway sarin attack. Ann Emerg Med 28(2):129–135. Okumura T, Suzuki K, Fukuda A, Kohama A, Takasu N, Ishimatsu S, Hinohara S. 1998a. The Tokyo subway sarin attack: Disaster management, Part 1: Community emergency response. Acad Emerg Med 5(6):613–617. Okumura T, Suzuki K, Fukuda A, Kohama A, Takasu N, Ishimatsu S, Hinohara S. 1998b. The Tokyo subway sarin attack: Disaster management, Part 2: Hospital response. Acad Emerg Med 5(6):618–624. PAC (Presidential Advisory Committee). 1996. Presidential Advisory Committee on Gulf War Veterans’ Illnesses: Final Report. Washington, DC: U.S. Government Printing Office. Pearce PC, Crofts HS, Muggleton NG, Ridout D, Scott EAM. 1999. The effects of acutely administered low dose sarin on cognitive behaviour and the electroencephalogram in the common marmoset. J Psychopharmacol 13(2):128–135. Polhuijs M, Langenberg JP, Benschop HP. 1997. New method for retrospective detection of exposure to organophosphorus anticholinesterases: Application to alleged sarin victims of Japanese terrorists. Toxicol Appl Pharmacol 146(1):156–161. Richter RJ, Furlong CE. 1999. Determination of paraoxonase (PON1) status requires more than genotyping. Pharmacogenetics 9(6):745–753. Rickett DL, Glenn JF, Beers ET. 1986. Central respiratory effects versus neuromuscular actions of nerve agents. Neurotoxicology 7(1):225–236. Rocha ES, Chebabo SR, Santos MD, Aracava Y, Albuquerque EX. 1998. An analysis of low level doses of cholinesterase inhibitors in cultured neurons and hippocampal slices of rats. Drug Chem Toxicol 21 (Suppl 1):191–200. Roland PS, Haley RW, Yellin W, Owens K, Shoup AG. 2000. Vestibular dysfunction in Gulf War syndrome. Otolaryngol Head Neck Surg 122:319–329. Sekijima Y, Morita H, Yanagisawa N. 1997. Follow-up of sarin poisoning in Matsumoto. Ann Intern Med 127(11):1042. Senanayake N, Karalliedde L. 1987. Neurotoxic effects of organophosphorous insecticides. N Engl J Med 316(13):761–763. Shih ML, McMonagle JD, Dolzine TW, Gresham VC. 1994. Metabolite pharmacokinetics of soman, sarin and GF in rats and biological monitoring of exposure to toxic organophosphorus agents. J Appl Toxicol 14(3):195–199. Sidell FR, Borak J. 1992. Chemical warfare agents: II. Nerve agents. Ann Emerg Med 21(7):865–871.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Singer AW, Jaax NK, Graham JS, McLeod CG Jr. 1987. Cardiomyopathy in soman and sarin intoxicated rats. Toxicol Lett 36(3):243–249. Somani SM. 1992. Chemical Warfare Agents. New York: Academic Press. Spencer P, Wilson B, Albuquerque E. 2000. Sarin, other “nerve agents,” and their antidotes. In: Spencer P, Schaumburg H, Ludolph A, eds. Experimental and Clinical Neurotoxicology. 2nd edition. New York: Oxford University Press. Stewart CE, Sullivan J Jr. 1992. Military munitions and antipersonnel agents. In: Sullivan JB Jr, Krieger G, eds. Hazardous Materials Toxicology: Clinical Principles of Environmental Health. Baltimore: Williams & Wilkins. Pp. 986–1014. Suzuki J, Kohno T, Tsukagosi M, Furuhata T, Yamazaki K. 1997. Eighteen cases exposed to sarin in Matsumoto, Japan. Intern Med 36(7):466–470. Tripathi HL, Dewey WL. 1989. Comparison of the effects of diisopropylfluorophosphate, sarin, soman, and tabun on toxicity and brain acetylcholinesterase activity in mice. J Toxicol Environ Health 26(4):437–446. Willems JL, Palate BM, Vranken MA, De Bisschop HC. 1983. Proceedings of the International Symposium on Protection Against Chemical Warfare Agents. Umea, Sweden: National Defense Research Institute. Pp. 95–100. Woodall J. 1997. Tokyo subway gas attack. Lancet 350(9073):296. Worek F, Eyer P, Szinicz L. 1998. Inhibition, reactivation and aging kinetics of cyclohexylmethylphosphonofluoridate-inhibited human cholinesterases. Arch Toxicol 72(9):580–587. Yamasaki Y, Sakamoto K, Watada H, Kajimoto Y, Hori M. 1997. The Arg192 isoform of paraoxonase with low sarin-hydrolyzing activity is dominant in the Japanese. Hum Genet 101(1):67–68. Yokoyama K, Araki S, Murata K, Nishikitani M, Okumura T, Ishimatsu S, Takasu N. 1998a. A preliminary study on delayed vestibulo-cerebellar effects of Tokyo Subway sarin poisoning in relation to gender difference: Frequency analysis of postural sway. J Occup Environ Med 40(1):17–21. Yokoyama K, Araki S, Murata K, Nishikitani M, Okumura T, Ishimatsu S, Takasu N. 1998b. Chronic neurobehavioral and central and autonomic nervous system effects of Tokyo subway sarin poisoning. J Physiol Paris 92(3-4):317–323. Yokoyama K, Araki S, Murata K, Nishikitani M, Okumura T, Ishimatsu S, Takasu N, White RF. 1998c. Chronic neurobehavioral effects of Tokyo subway sarin poisoning in relation to posttraumatic stress disorder. Arch Environ Health 53(4):249–256. Young GD, Koplovitz I. 1995. Acute toxicity of cyclohexylmethylphosphonofluoridate (CMPF) in rhesus monkeys: Serum biochemical and hematologic changes. Arch Toxicol 69(6):379–383.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines CONTENTS      TOXICOLOGY   209      Structure and Mechanism of Action,   210      Animal Studies,   211      Interactions with Other Agents,   217      Genetic Susceptibility,   220      HUMAN STUDIES   222      Clinical Studies,   222      Healthy-Volunteer Studies,   231      Epidemiologic Studies,   245      CONCLUSIONS   250      Acute Effects,   250      Chronic Effects,   252      REFERENCES   253