Monitoring Exposure to Five Chemical Warfare Agents Using the Dried Urine Spot Technique and Liquid Chromatography-Mass Spectrometry/Mass Spectrometry-In Vivo Determination of Sarin Metabolite in Mice.

Dried urine spot (DUS) is a micro-sample collection technique, known for its advantages in handling, storage and shipping. It also uses only a small volume of urine, an essential consideration in working with small animals, or in acute medical situations. Alkyl-phosphonic acids are the direct and indicative metabolites of organophosphorus chemical warfare agents (OP-CWAs) and are present in blood and urine shortly after exposure. They are therefore crucially important for monitoring casualties in war and terror scenarios. We report here a new approach for the determination of the metabolites of five CWAs in urine using DUS. The method is based on a simple and rapid sample preparation, using only 50 µL of urine, spotted and dried on DBS paper, extracted using 300 µL methanol/water and analyzed via targeted LC-MS/MS. The detection limits for the five CWAs, sarin (GB), soman (GD), cyclosarin (GF), VX and RVX in human urine were from 0.5 to 5 ng/mL. Recoveries of (40-80%) were obtained in the range of 10-300 ng/mL, with a linear response (R2 > 0.964, R > 0.982). The method is highly stable, even with DUS samples stored up to 5 months at room temperature before analysis. It was implemented in a sarin in vivo exposure experiment on mice, applied for the time course determination of isopropyl methylphosphonic acid (IMPA, sarin hydrolysis product) in mice urine. IMPA was detectable even with samples drawn 60 h after the mice's (IN) exposure to 1 LD50 sarin. This method was also evaluated in a non-targeted screening for multiple potential CWA analogs (LC-Orbitrap HRMS analysis followed by automatic peak detection and library searches). The method developed here is applicable for rapid CWA casualty monitoring.

A review of chemical warfare agents linked to respiratory and neurological effects experienced in Gulf War Illness.

Over 40% of veterans from the Persian Gulf War (GW) (1990-1991) suffer from Gulf War Illness (GWI). Thirty years since the GW, the exposure and mechanism contributing to GWI remain unclear. One possible exposure that has been attributed to GWI are chemical warfare agents (CWAs). While there are treatments for isolated symptoms of GWI, the number of respiratory and cognitive/neurological issues continues to rise with minimum treatment options. This issue does not only affect veterans of the GW, importantly these chronic multisymptom illnesses (CMIs) are also growing amongst veterans who have served in the Afghanistan-Iraq war. What both wars have in common are their regions and inhaled exposures. In this review, we will describe the CWA exposures, such as sarin, cyclosarin, and mustard gas in both wars and discuss the various respiratory and neurocognitive issues experienced by veterans. We will bridge the respiratory and neurological symptoms experienced to the various potential mechanisms described for each CWA provided with the most up-to-date models and hypotheses.

Comparative effects of scopolamine and phencynonate on organophosphorus nerve agent-induced seizure activity, neuropathology and lethality.

The efficacy of anticonvulsant therapies to stop seizure activities following organophosphorus nerve agents (NAs) has been documented as being time-dependent. We utilized the guinea pig NA-seizure model to compare the effectiveness of phencynonate (PCH) and scopolamine (SCP) when given at the early (at time of seizure onset) or late (40 min after seizure onset) phase of seizure progression. PCH possesses both anticholinergic and anti-NMDA activities, while SCP is a purely anti-muscarinic compound. Animals with cortical electrodes were pretreated with pyridostigmine bromide 30 min prior to exposure to a 2.0 x LD50 subcutaneous dose of a NA (GA, GB, GD, GF, VR, or VX), followed one min later with atropine sulfate and 2-PAM. At either early or late phase, animals were treated with either PCH or SCP and the 24-h anticonvulsant ED50 doses were determined. When administered at seizure onset, PCH, and SCP were both effective at terminating seizure activity against all NAs, with ED50 values for SCP generally being lower. At the 40 min time, ED50 values were obtained following GA, GD, GF, and VR challenges for SCP, but ED50 value was obtained only following GD for PCH, indicating a superior efficacy of SCP. When seizure activity was controlled, a significant improvement in weight loss, neuropathology, and survival was observed, regardless of treatment or NA. Overall, these results demonstrate the differing efficacies of these two similarly structured anticholinergic compounds with delayed administration and warrant further investigation into the timing and mechanisms of the seizure maintenance phase in different animal models.

Theoretical Studies Applied to the Evaluation of the DFPase Bioremediation Potential against Chemical Warfare Agents Intoxication.

Organophosphorus compounds (OP) are part of a group of compounds that may be hazardous to health. They are called neurotoxic agents because of their action on the nervous system, inhibiting the acetylcholinesterase (AChE) enzyme and resulting in a cholinergic crisis. Their high toxicity and rapid action lead to irreversible damage to the nervous system, drawing attention to developing new treatment methods. The diisopropyl fluorophosphatase (DFPase) enzyme has been considered as a potent biocatalyst for the hydrolysis of toxic OP and has potential for bioremediation of this kind of intoxication. In order to investigate the degradation process of the nerve agents Tabun, Cyclosarin and Soman through the wild-type DFPase, and taking into account their stereochemistry, theoretical studies were carried out. The intermolecular interaction energy and other parameters obtained from the molecular docking calculations were used to construct a data matrix, which were posteriorly treated by statistical analyzes of chemometrics, using the PCA (Principal Components Analysis) multivariate analysis. The analyzed parameters seem to be quite important for the reaction mechanisms simulation (QM/MM). Our findings showed that the wild-type DFPase enzyme is stereoselective in hydrolysis, showing promising results for the catalytic degradation of the neurotoxic agents under study, with the degradation mechanism performed through two proposed pathways.

Design, synthesis, and characterization of novel, nonquaternary reactivators of GF-inhibited human acetylcholinesterase.

The goal of this research was to identify structurally novel, non-quaternarypyridinium reactivators of GF (cyclosarin)-inhibited hAChE that possess the capacity to mediate in vitro reactivation of GF-inhibited human acetylcholinesterase (hAChE). New compounds were designed, synthesized and assessed in GF-inhibited hAChE assays. Structure activity relationships for AChE binding and reactivation of GF-inhibited hAChE were developed. Lead compounds from two different chemical series, represented by compounds 17 and 38, displayed proficient in vitro reactivation of GF-inhibited hAChE, while also possessing low inhibition of native enzyme.

Chiral separation of G-type chemical warfare nerve agents via analytical supercritical fluid chromatography.

Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G-type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical-scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(-) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(-) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents.

Efficacy and pharmacokinetic/pharmacodynamic study of 1,1'-methylenebis{4-[(hydroxyimino)methyl] pyridinium} dimethanesulfonate in guinea pigs and rhesus macaques exposed to cyclosarin.

Male Hartley guinea pigs and male rhesus macaques were used to determine an efficacious dose of 1,1'-methylenebis{4-[(hydroxyimino)methyl] pyridinium} dimethanesulfonate (MMB4 DMS) that would result in 80% survival, 24 hours following a single exposure to cyclosarin (GF). The pharmacokinetic/pharmacodynamic relationship between acetylcholinesterase activity and MMB4 plasma concentrations relative to survival was evaluated. Guinea pigs and non-human primates (NHPs) were concurrently administered MMB4 DMS (guinea pigs: 0, 10, 30, or 40 mg/kg, intramuscular [IM] and NHPs: 0.1, 1, 5, 10, or 20 mg/kg, IM), atropine, and diazepam following a 3 × median lethal dose (LD50) GF challenge. Clinical observations were evaluated using a quality-of-life (QOL) scoring system. All GF-exposed animals exhibited typical signs of nerve agent poisoning immediately following challenge. In guinea pigs, 24-hour survival was 0%, 50%, 90%, and 90% for 0, 10, 30, and 40 mg/kg MMB4 DMS groups, respectively. In addition, nearly all animals surviving to 24 hours were clinically normal, with many in the 30 and 40 mg/kg MMB4 DMS dose group observed as normal by 4 hours post-challenge. In NHPs, survival was 100% for all treatment groups, with all animals noted as clinically normal by 48 hours. Following treatment with atropine/MMB4 DMS/diazepam, NHPs exhibited dose- and temporal-related decreases in incidence and duration of the clinical signs of toxicity. The QOL scores improved with increasing MMB4 DMS dose in both species. The estimated ED80s were 25.5 mg/kg MMB4 DMS (human equivalent dose [HED] of 5.5 mg/kg) and ≤ 0.1 mg/kg (HED of 0.03 mg/kg) in guinea pigs and NHPs, respectively.

In vivo acetylcholinesterase reactivation in male guinea pigs and rhesus macaques following cyclosarin exposure and treatment with 1,1'-methylenebis{4-[(hydroxyimino)methyl] pyridinium} dimethanesulfonate.

Acetylcholinesterase (AChE) reactivation studies were conducted in guinea pigs (GPs) and nonhuman primates (NHPs) to determine the 1,1'-methylenebis{4-[(hydroxyimino)methyl] pyridinium} dimethanesulfonate (MMB4 DMS) dose that reactivated at least 20% of blood AChE within 15 minutes following cyclosarin (GF) dosing (used as the criterion for efficacy). Male GPs and male rhesus macaques (NHPs) were pretreated with atropine 15 minutes prior to GF administration (1 × median lethal dose [LD50]) and MMB4 DMS 15 minutes following GF administration. The GP survival was 5 of 8, 8 of 8, 8 of 8, and 6 of 8 for the 0.75, 3.0, 6.0, or 12.0 mg/kg MMB4 DMS treatment groups, respectively. In NHPs, survival was 6 of 6 at 0.5, 1.2, 3.0, or 9.3 mg/kg MMB4 DMS, respectively, 24 hours post-challenge, with the majority of animals noted as clinically normal by 24 hours. Pharmacokinetic/pharmacodynamic modeling revealed that 1.8 mg/kg in GPs or 0.013 mg/kg in NHPs would result in an average 20% reactivation; human equivalent doses were calculated as 0.39 mg/kg (based on GP data) and 0.004 mg/kg (based on NHP data). The model suggested that MMB4 plasma concentrations of 1000 ng/mL and AChE reactivation of 80% would be most effective. Although a 0.5 mg/kg MMB4 DMS dose in NHPs resulted in 100% survival and an average of 78% AChE reactivation, adverse effects associated with GF administration were still observed 24 hours post-challenge (tremors, mydriasis, and weakness were observed in 3 of 6 animals). In comparison, 6 of 6 animals treated with 1.2 mg/kg MMB4 DMS were observed as clinically normal 24 hours post-challenge.

Highly efficient cyclosarin degradation mediated by a ß-cyclodextrin derivative containing an oxime-derived substituent.

The potential of appropriately substituted cyclodextrins to act as scavengers for neurotoxic organophosphonates under physiological conditions was evaluated. To this end, a series of derivatives containing substituents with an aldoxime or a ketoxime moiety along the narrow opening of the ß-cyclodextrin cavity was synthesized, and the ability of these compounds to reduce the inhibitory effect of the neurotoxic organophosphonate cyclosarin on its key target, acetylcholinesterase, was assessed in vitro. All compounds exhibited a larger effect than native ß-cyclodextrin, and characteristic differences were noted. These differences in activity were correlated with the structural and electronic parameters of the substituents. In addition, the relatively strong effect of the cyclodextrin derivatives on cyclosarin degradation and, in particular, the observed enantioselectivity are good indications that noncovalent interactions between the cyclodextrin ring and the substrate, presumably involving the inclusion of the cyclohexyl moiety of cyclosarin into the cyclodextrin cavity, contribute to the mode of action. Among the nine compounds investigated, one exhibited remarkable activity, completely preventing acetylcholinesterase inhibition by the (-)-enantiomer of cyclosarin within seconds under the conditions of the assay. Thus, these investigations demonstrate that decoration of cyclodextrins with appropriate substituents represents a promising approach for the development of scavengers able to detoxify highly toxic nerve agents.

Counteracting poisoning with chemical warfare nerve agents.

Phosphylation of the pivotal enzyme acetylcholinesterase (AChE) by nerve agents (NAs) leads to irreversible inhibition of the enzyme and accumulation of neurotransmitter acetylcholine, which induces cholinergic crisis, that is, overstimulation of muscarinic and nicotinic membrane receptors in the central and peripheral nervous system. In severe cases, subsequent desensitisation of the receptors results in hypoxia, vasodepression, and respiratory arrest, followed by death. Prompt action is therefore critical to improve the chances of victim's survival and recovery. Standard therapy of NA poisoning generally involves administration of anticholinergic atropine and an oxime reactivator of phosphylated AChE. Anticholinesterase compounds or NA bioscavengers can also be applied to preserve native AChE from inhibition. With this review of 70 years of research we aim to present current and potential approaches to counteracting NA poisoning.

Effect of seven newly synthesized and currently available oxime cholinesterase reactivators on cyclosarin-intoxicated rats.

Seven new oxime-based acetylcholinesterase reactivators were compared with three currently available ones (obidoxime, trimedoxime, HI-6) for their ability to lessen cholinesterase inhibition in blood and brain of cyclosarin-treated rats. Oximes were given at doses of 5% their LD(50) along with 21 mg/kg atropine five min before the LD(50) of cyclosarin (120 ug/kg) was administered. Blood and brain samples were collected 30 minutes later. The greatest difference between acetylcholinesterase inhibition in blood of cyclosarin-treated rats was found after administration of HI-6 (40%), compared to 22% for trimedoxime and 6% for obidoxime. Only two of the seven newly synthesized oximes had any effect (K203 at 7%, K156 at 5%). Effective oximes against cyclosarin-inhibited plasma butyrylcholinesterase were HI-6 (42%), trimedoxime (11%), and K156 (4%). The oximes were less effective in brain than in blood, with reactivation values for HI-6 30% against acetylcholinesterase and 10% against butyrylcholinesterase. Values for newly synthesized oximes were less than 10% for K206, K269 and K203.

Methylation protocol for the retrospective detection of isopropyl-, pinacolyl- and cyclohexylmethylphosphonic acids, indicative markers for the nerve agents sarin, soman and cyclosarin, at low levels in soils using EI-GC-MS.

A practical and efficient protocol for the derivatization and detection by GC-EI-MS of isopropyl-, pinacolyl- and cyclohexylmethylphosphonic acids, key diagnostic degradation products of the nerve agents sarin, soman and cyclosarin respectively, in six different types of soil matrices is presented. The method involves the in situ conversion of the phosphonic acids to their respective methyl esters using trimethyloxonium tetrafluoroborate when present in the soils at low levels (10 µg g-1) without any prior extractions or soil preparation. The soils employed in our study were Nebraska EPA soil, Georgia soil, silt, Virginia type A soil, regular sand and Ottawa sand and were chosen for their vast differences in composition and physical features. Appealing attributes of the protocol include its rapidity (t < 30 min), mildness (ambient temperature), and practicality that includes the production of the phosphonic methyl esters that can be easily detected by GC-EI-MS and corroborated with the instrument's internal NIST spectral library or the Organisation for the Prohibition of Chemical Weapons (OPCW) central analytical database (OCAD v.21_2019). The overall efficacy of the protocol was then tested on a soil sample featured in the 44th OPCW PT that our laboratory participated in. After preparing the soil so as to give pinacolyl methylphosphonic acid at a 5 µg g-1 concentration, the acid was successfully methylated and detected by GC-EI-MS. The protocol's performance mirrors that of the universally employed diazomethane protocol but accomplishes this without any of the explosive hazards and time consuming reagent preparation commonly associated with it.

Some benefit from non-oximes MB408, MB442 and MB444 in combination with the oximes HI-6 or obidoxime and atropine in antidoting sarin or cyclosarin poisoned mice.

The effect of three newly developed bispyridinium non-oxime compounds (MB408, MB442, and MB444) on the therapeutic efficacy of a standard antidotal treatment (atropine in combination with the oxime HI-6 or obidoxime) of acute poisoning by two nerve agents (sarin and cyclosarin) in mice was studied. The therapeutic efficacy of atropine in combination with an oxime with or without one of the bispyridinium non-oximes was evaluated by determination of the 24 h LD50 values of the nerve agents studied and by measurement of the survival time after supralethal poisoning. Addition of all tested non-oximes increased the therapeutic efficacy of atropine in combination with an oxime against sarin poisoning; however, the differences were not significant. The non-oximes also positively influenced the number of surviving mice 6 h after supralethal poisoning with sarin. In the case of cyclosarin, they were also slightly beneficial in the treatment of acute poisoning. The higher dose of MB444 was able to significantly increase the therapeutic efficacy of standard antidotal treatment of poisoning with cyclosarin. The benefit of each bispyridinium non-oxime compound itself was obviously dose-dependent. In summary, the addition of MB compounds to the standard antidotal treatment of acute nerve agent poisoning was beneficial for the antidotal treatment of sarin or cyclosarin poisoning, although their benefit at 24 h after poisoning was not significant, with the exception of the higher dose of MB444 against cyclosarin.

Recent research on Gulf War illness and other health problems in veterans of the 1991 Gulf War: Effects of toxicant exposures during deployment.

Veterans of Operation Desert Storm/Desert Shield - the 1991 Gulf War (GW) - are a unique population who returned from theater with multiple health complaints and disorders. Studies in the U.S. and elsewhere have consistently concluded that approximately 25-32% of this population suffers from a disorder characterized by symptoms that vary somewhat among individuals and include fatigue, headaches, cognitive dysfunction, musculoskeletal pain, and respiratory, gastrointestinal and dermatologic complaints. Gulf War illness (GWI) is the term used to describe this disorder. In addition, brain cancer occurs at increased rates in subgroups of GW veterans, as do neuropsychological and brain imaging abnormalities. Chemical exposures have become the focus of etiologic GWI research because nervous system symptoms are prominent and many neurotoxicants were present in theater, including organophosphates (OPs), carbamates, and other pesticides; sarin/cyclosarin nerve agents, and pyridostigmine bromide (PB) medications used as prophylaxis against chemical warfare attacks. Psychiatric etiologies have been ruled out. This paper reviews the recent literature on the health of 1991 GW veterans, focusing particularly on the central nervous system and on effects of toxicant exposures. In addition, it emphasizes research published since 2008, following on an exhaustive review that was published in that year that summarizes the prior literature (RACGWI, 2008). We conclude that exposure to pesticides and/or to PB are causally associated with GWI and the neurological dysfunction in GW veterans. Exposure to sarin and cyclosarin and to oil well fire emissions are also associated with neurologically based health effects, though their contribution to development of the disorder known as GWI is less clear. Gene-environment interactions are likely to have contributed to development of GWI in deployed veterans. The health consequences of chemical exposures in the GW and other conflicts have been called "toxic wounds" by veterans. This type of injury requires further study and concentrated treatment research efforts that may also benefit other occupational groups with similar exposure-related illnesses.

Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity.

The antidotal treatment of organophosphates (OP) nerve agents (NA) poisoning is based on anticholinergics (e.g. atropine) combined with oxime reactivators (e.g. 2PAM) of acetylcholinesterase (AChE). This treatment is symptomatic and does not degrade the OP. New small-molecule OP scavengers were developed as bifunctional hybrids. Their molecular design was based on combining a nucleophile that directly degrades OP with a moiety that reactivates OP-inhibited AChE. The OP degrading moiety is either benzhydroxamic acid (BHA) or 4-pyridinehydroxamic acid (4PHA) coupled via (CH2)n, (n = 1 or 3) to 2PAM. Three newly synthesized oxime-hydroxamate hybrids: 2PAMPr4PHA, 2PAMMeBHA and 2,4-DiPAMMeBHA were found to detoxify sarin, cyclosarin and soman in solution at 3-10-fold faster rate than 2PAM and to reactivate OP-AChE in vitro. 2PAMPr4PHA displayed 18-fold faster reactivation than 2-PAM of cyclosarin-inhibited HuAChE (kr = 3.6 × 102 vs. 0.2 × 102 M-1min-1, respectively, 37 °C). These hybrids inhibited AChE reversibly, IC50 = 16-48 µM, thereby decreasing the inhibition rates by OPs. The LD50 (im) of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA are >568, 508 and >506 µmol/kg in rats and 144, 203 and >506 µmol/kg in guinea pigs. The rate of blood ChE recovery by the hybrids administered either pre- or post-exposure to 0.8xLD50 sarin was comparable or faster than 2PAM. Antidotal efficacy of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA administered with atropine, as pre-treatment to sarin in rats (im), yielded protection ratios (PR) 11.6, 11.5 and 4.7, respectively, vs. 5.5 with 2PAM. Post-treatment against various OPs in rats and guinea-pigs yielded PRs higher or similar to that of 2 PAM. Our in vivo data indicates that some hybrids may serve as efficient small molecule scavengers for mitigating the toxicity of OP NAs.

Effects of low-level sarin and cyclosarin exposure on white matter integrity in Gulf War Veterans.

BACKGROUND: We previously found evidence of reduced gray and white matter volume in Gulf War (GW) veterans with predicted low-level exposure to sarin (GB) and cyclosarin (GF). Because loss of white matter tissue integrity has been linked to both gray and white matter atrophy, the current study sought to test the hypothesis that GW veterans with predicted GB/GF exposure have evidence of disrupted white matter microstructural integrity. METHODS: Measures of fractional anisotropy and directional (i.e., axial and radial) diffusivity were assessed from the 4T diffusion tensor images (DTI) of 59 GW veterans with predicted GB/GF exposure and 59 "matched" unexposed GW veterans (mean age: 48 ± 7 years). The DTI data were analyzed using regions of interest (ROI) analyses that accounted for age, sex, total brain gray and white matter volume, trauma exposure, posttraumatic stress disorder, current major depression, and chronic multisymptom illness status. RESULTS: There were no significant group differences in fractional anisotropy or radial diffusivity. However, there was increased axial diffusivity in GW veterans with predicted GB/GF exposure compared to matched, unexposed veterans throughout the brain, including the temporal stem, corona radiata, superior and inferior (hippocampal) cingulum, inferior and superior fronto-occipital fasciculus, internal and external capsule, and superficial cortical white matter blades. Post hoc analysis revealed significant correlations between higher fractional anisotropy and lower radial diffusivity with better neurobehavioral performance in unexposed GW veterans. In contrast, only increased axial diffusivity in posterior limb of the internal capsule was associated with better psychomotor function in GW veterans with predicted GB/GF exposure. CONCLUSIONS: The finding that increased axial diffusivity in a region of the brain that contains descending corticospinal fibers was associated with better psychomotor function and the lack of significant neurobehavioral deficits in veterans with predicted GB/GF exposure hint at the possibility that the widespread increases in axial diffusivity that we observed in GW veterans with predicted GB/GF exposure relative to unexposed controls may reflect white matter reorganization after brain injury (i.e., exposure to GB/GF).

In vitro toxicokinetic studies of cyclosarin: molecular mechanisms of elimination.

The toxicokinetics of in vitro elimination of highly toxic cyclosarin (GF) in biological systems revealed striking stereoselective differences in the range of 0.01µM to 1mM GF. While weak concentration dependency was detected for elimination of the toxic (-)-enantiomer indicating catalytic processes, elimination of less toxic (+)-GF followed unusual kinetics with relatively high concentration dependency. Fast initial GF binding in human heparinised plasma increased only at lower initial GF concentrations while (+)-GF binding strongly increased with decreasing GF concentration. In displacement experiments it was shown for the first time that GF binding on plasma components in rats and mice plasma was reversible. Investigations with human plasma require further methodical improvement. GF elimination by diisopropylfluorophosphatase (DFPase) wildtype as phosphotriesterase (PTE) model showed some similarities compared to human heparinised plasma. Impact of human serum albumin is negligible. When comparing kinetics of GF elimination with metabolite formation (fluoride and cyclohexyl methyl phosphonic acid, CHMPA), marked differences were detected. From the results a model was postulated illustrating possible steps of molecular mechanisms of GF interaction with plasma proteins including high affine fast initial binding followed by formation of metastable phosphonylated plasma proteins with subsequent hydrolysis and release of metabolites.

Effects of low-level sarin and cyclosarin exposure on hippocampal subfields in Gulf War Veterans.

BACKGROUND: More than 100,000 US troops were potentially exposed to chemical warfare agents sarin (GB) and cyclosarin (GF) when an ammunition dump at Khamisiyah, Iraq was destroyed during the 1991 Gulf War (GW). We previously reported reduced hippocampal volume in GW veterans with suspected GB/GF exposure relative to matched, unexposed GW veterans estimated from 1.5T magnetic resonance images (MRI). Here we investigate, in a different cohort of GW veterans, whether low-level GB/GF exposure is associated with structural alterations in specific hippocampal subfields, estimated from 4T MRI. METHODS: The Automatic Segmentation of Hippocampal Subfields (ASHS) technique was used to quantify CA1, CA2, CA3 and dentate gyrus (DG), and subiculum (SUB) subfields volumes from high-resolution T2-weighted images acquired on a 4T MR scanner in 56 GW veterans with suspected GB/GF exposure and 56 "matched" unexposed GW veterans (mean age 49±7 years). RESULTS: GB/GF exposed veterans had smaller CA2 (p=0.003) and CA3/DG (p=0.01) subfield volumes compared to matched, unexposed GW veterans. There were no group difference in total hippocampal volume, quantified with FreeSurfer, and no dose-response relationship between estimated levels of GB/GF exposure and total hippocampal or subfield volume. CONCLUSIONS: These findings extend our previous report of structural alterations in the hippocampi of GW veterans with suspected GB/GF exposure to volume changes in the CA2, CA3, and DG hippocampal subfields in a different cohort of GW veterans with suspected GB/GF exposure.

Acute and Long-Term Impact of Chemical Weapons: Lessons from the Iran-Iraq War.

Chemical weapons have given the human experience of warfare a uniquely terrifying quality that has inspired a general repugnance and led to periodic attempts to ban their use. Nevertheless, since ancient times, toxic agents have been consistently employed to kill and terrorize target populations. The evolution of these weapons is examined here in ways that may allow military, law enforcement, and scientific professionals to gain a perspective on conditions that, in the past, have motivated their use - both criminally and as a matter of national policy during military campaigns. Special emphasis is placed on the genocidal use of chemical weapons by the regime of Saddam Hussein, both against Iranians and on Kurdish citizens of his own country, during the Iran-Iraq War of 1980-88. The historical development of chemical weapons use is summarized to show how progressively better insight into biochemistry and physiology was adapted to this form of warfare. Major attributes of the most frequently used chemical agents and a description of how they affected military campaigns are explained. Portions of this review describing chemical-casualty care devote particular focus to Iranian management of neurotoxic (nerve) agent casualties due to the unique nature of this experience. Both nerve and blistering "mustard" agents were used extensively against Iranian forces. However, Iran is the only nation in history to have sustained large-scale attacks with neurotoxic weapons. For this reason, an understanding of the successes and failures of countermeasures to nerve-agent use developed by the Iranian military are particularly valuable for future civil defense and military planning. A detailed consideration of these strategies is therefore considered. Finally, the outcomes of clinical research into severe chronic disease triggered by mustard-agent exposure are examined in the context of the potential of these outcomes to determine the etiology of illness among US and Allied veterans of the 1991 Persian Gulf War.

Elimination pathways of cyclosarin (GF) mediated by ß-cyclodextrin in vitro: pharmacokinetic and toxicokinetic aspects.

Cyclodextrins (CD) are promising small molecular scavengers showing favourable degradation of extremely toxic organophosphorus compounds (OP) such as tabun (GA), soman (GD) or cyclosarin (GF). For ß-CD derivatives as potential OP antidotes with low intrinsic toxicity it is of great interest to completely understand the modes of interaction of both compounds in terms of OP detoxification. The mechanisms of CD action are not completely understood which prompted us to investigate the interactions of GF and ß-cyclodextrin (ß-CD) as model compounds. Using positive electrospray ionization mass spectrometry (ESI/MS), the formation of covalent conjugates of ß-CD with O-cyclohexylmethylphosphonate (CHMP) residue was detected for the first time and was examined in vitro. With a newly developed LC-MS method the formation of O-cyclohexylmethylphosphonic acid (CHMPA) (i.e. GF hydrolysis) and covalent CHMP-ß-CD conjugates was analyzed. Compared to water, tris(hydroxymethyl)aminomethane (TRIS) reduced the formation of covalent conjugates but amplified formation of CHMPA. Depending on experimental conditions the degradation of GF by ß-CD may be preferably catalytic or stoichiometric. For illustrating different possible reaction pathways a scheme was established that could support the idea of ß-CD acting as an artificial enzyme. These results provide an important insight into the ß-CD mediated detoxification pathways of GF.