Evidence for the systemic diffusion of (2-chloroethyl)-ethyl-sulfide, a sulfur mustard analog, and its deleterious effects in brain.

Sulfur mustard, a chemical warfare agent known to be a vesicant of skin, readily diffuses in the blood stream and reaches internal organs. In the present study, we used the analog (2-chloroethyl)-ethyl-sulfide (CEES) to provide novel data on the systemic diffusion of vesicants and on their ability to induce brain damage, which result in neurological disorders. SKH-1 hairless mice were topically exposed to CEES and sacrificed at different time until 14 days after exposure. A plasma metabolomics study showed a strong systemic impact following a self-protection mechanism to alleviate the injury of CEES exposure. This result was confirmed by the quantification of specific biomarkers in plasma. Those were the conjugates of CEES with glutathione (GSH-CEES), cysteine (Cys-CEES) and N-acetyl-cysteine (NAC-CEES), as well as the guanine adduct (N7Gua-CEES). In brain, N7Gua-CEES could be detected both in DNA and in organ extracts. Similarly, GSH-CEES, Cys-CEES and NAC-CEES were present in the extracts until day14. Altogether, these results, based on novel exposure markers, confirm the ability of vesicants to induce internal damage following dermal exposure. The observation of alkylation damage to glutathione and DNA in brain provides an additional mechanism to the neurological insult of SM.

In vitro human skin permeation and decontamination of 2-chloroethyl ethyl sulfide (CEES) using Dermal Decontamination Gel (DDGel) and Reactive Skin Decontamination Lotion (RSDL).

This study compared the efficiency for in vitro human skin decontamination using DDGel and RSDL. Experiments were performed using in vitro human skin models, in which skin was mounted onto Flow-Through diffusion cells. The mass of 14-C CEES removed from skin surface after decontamination was quantitated by measuring radioactivity with a liquid scintillation spectrometer. Both decontaminants removed more than 82% of CEES from skin. DDGel skin decontamination significantly reduced toxicant amount when compared to RSDL. Mean CEES remaining in stratum corneum (SC), viable epidermis, dermis, and systemic absorption of DDGel and RSDL were, 0.12 and 0.55% (P < 0.01), 0.31 and 0.95% (p < 0.01), 1.08 and 2.92% (p < 0.05), 3.13 and 6.34% (p < 0.05), respectively. DDGel showed higher decontamination efficiency (twice decontamination efficacy factor, DEF) than RSDL and efficiently removed chemicals from the skin surface, importantly back-extracted from the SC, and significantly reduced both chemical penetration into skin and systemic absorption. Thus, DDGel can offer a potential as a next generation skin decontamination platform technology for military and civilian applications.

Dermatotoxicology of sulfur mustard: Historical perspectives from World War I.

Sulfur mustard has been used as a chemical warfare agent for the past century. After its introduction by the Germans in World War I, investigators quickly began studying its impact on the human body including its deleterious effects on skin. This review focuses on two groups in particular who conducted experiments from 1917 to 1918: the United States Army at the American University Experiment Station Laboratories and Torald Sollmann at Western Reserve University. Through this work, these researchers proved far ahead of their time by anticipating dermatologic phenomena not described in the literature until later in the twentieth century. These include regional variation of percutaneous penetration, effect of vehicle on penetration and predicting immunologic contact urticaria. The work conducted by these researchers set the groundwork for much of twentieth century dermatotoxicology.

Sulfur mustard skin lesions: A systematic review on pathomechanisms, treatment options and future research directions.

Sulfur mustard (SM) is a chemical warfare, which has been used for one hundred years. However, its exact pathomechanisms are still incompletely understood and there is no specific therapy available so far. In this systematic review, studies published between January 2000 and July 2017 involving pathomechanisms and experimental treatments of SM-induced skin lesions were analyzed to summarize current knowledge on SM pathology, to provide an overview on novel treatment options, and to identify promising targets for future research to more effectively counter SM effects. We suggest that future studies should focus on (I) systemic effects of SM intoxication due to its distribution throughout the body, (II) removal of SM depots that continuously release active compound contributing to chronic skin damage, and (III) therapeutic options that counteract the pleiotropic effects of SM.

Biological effects of adipocytes in sulfur mustard induced toxicity.

Sulphur mustard (2,2'-dichloroethyl sulfide; SM) is a vesicant chemical warfare agent whose mechanism of acute or chronic action is not known with any certainty and to date there is no effective antidote. SM accumulation in adipose tissue (AT) has been originally verified in our previous study. To evaluate the biological effect caused by the presence of abundant SM in adipocyte and assess the biological role of AT in SM poisoning, in vitro and in vivo experiments were performed. High content analysis revealed multi-cytotoxicity in SM exposed cells in a time and dose dependent manner, and adipocytes showed a relative moderate damage compared with non-adipocytes. Cell co-culture model was established and revealed the adverse effect of SM-exposed adipocyte supernatant on the growth of co-cultured cells. The pathological changes in AT from 10mg/kg SM percutaneously exposed rats were checked and inflammation phenomena were observed. The mRNA and protein levels of inflammation-related adipokines secreted from AT in rats exposed to 1, 3 and 10mg/kg doses of SM were determined by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays. The expressions of proinflammatory and anti-inflammatory adipokines together promoted the inflammation development in the body. The positive correlations between AT and serum adipokine levels were explored, which demonstrated a substantial role of AT in systemic inflammation responding to SM exposure. Thus, AT is not only a target of SM but also a modulator in the SM toxicity.

The percutaneous toxicokinetics of Sulphur mustard in a damaged skin porcine model and the evaluation of WoundStat™ as a topical decontaminant.

This study used a damaged skin, porcine model to evaluate the in vivo efficacy of WoundStat™ for decontamination of superficial (non-haemorrhaging), sulphur mustard-contaminated wounds. The dorsal skin of 12 female pigs was subjected to controlled physical damage and exposed to 10 µL 14 C-radiolabelled sulphur mustard (14 C-SM). Animals were randomly assigned to either a control or a treatment group. In the latter, WoundStat™ was applied 30 s post exposure and left in situ for 1 h. Skin lesion progression and decontaminant efficacy were quantified over 6 h using a range of biophysical measurements. Skin, blood and organ samples were taken post mortem for histopathological assessment, 14 C-SM distribution and toxicokinetic analyses. Application of SM to damaged skin without decontamination was rapidly followed by advanced signs of toxicity, including ulceration and decreased blood flow at the exposure site in all animals. WoundStat™ prevented ulceration and improved blood flow at the exposure site in all decontaminated animals (n = 6). Furthermore, significantly smaller quantities of 14 C-SM were detected in the blood (45% reduction), and recovered from skin (70% reduction) and skin surface swabs (99% reduction) at 6 h post-challenge. Overall, the distribution of 14 C-SM in the internal organs was similar for both groups, with the greatest concentration in the kidneys, followed by the liver and small intestine. WoundStat™ significantly reduced the amount of 14 C-SM recovered from the liver, a key organ for SM metabolism and detoxification. This study demonstrates that WoundStat™ is a suitable product for reducing the ingress and toxicity of a chemical warfare agent. Copyright © 2017 John Wiley & Sons, Ltd.

Accumulation of intact sulfur mustard in adipose tissue and toxicokinetics by chemical conversion and isotope-dilution liquid chromatography-tandem mass spectrometry.

Sulfur mustard (SM) is a powerful vesicant and one of the most harmful chemical warfare agents. Although having been studied for a long time, it is still difficult to fully elucidate the mechanisms of SM poisoning, and there is no effective antidote or specific treatment for SM injury. The investigations on toxicokinetics and tissue distribution of SM will help to understand its toxicity and provide a theoretical basis for pretreatment and therapy of SM poisoning. But the metabolic trajectory or fate of intact SM in vivo remains unclear, and there are insufficient experimental data to elucidate, due to its high reactivity and difficulty in biomedical sample analysis. In this paper, a sensitive method for the detection and quantification of intact SM in blood or tissues using isotope-dilution LC-MS/MS coupled with chemical conversion was developed. By transforming highly reactive SM into stable derivative product, the real concentration of intact SM in biological samples was obtained accurately. The toxicokinetics and tissue distribution studies of intact SM in rats were successfully profiled by the novel method after intravenous (10 mg/kg) or cutaneous administration (1, 3 and 10 mg/kg). The SM level in blood with peak time at 30-60 min determined in cutaneous exposure experiment was found much higher than previously reported, and the mean residence time in blood extended to 1-1.5 h. A significant accumulation of intact SM was observed in adipose tissues, including the perirenal fat, epididymal fat, subcutaneous fat and brown fat, in which the concentrations of SM were at least 15 times greater than those in non-adipose tissues in cutaneous exposed rats. The recovery of SM in body fat was calculated as 3.3 % of bioavailable SM (the bioavailability after cutaneous exposure was evaluated as 16 %). Thus, the adipose tissue was important for SM distribution and toxicity, which may pioneer a new model for both the prevention and treatment of SM exposure.

Simultaneous determination of sulfur mustard and related oxidation products by isotope-dilution LC-MS/MS method coupled with a chemical conversion.

Sulfur mustard (SM) is a highly reactive alkylating vesicant with high toxicity and complicated metabolism, the in vivo profile of its oxidation metabolism is not still fully known and urgently needs to be clarified well. In this work, an isotope-dilution high performance liquid chromatography-tandem mass spectrometric method coupled with chemical conversion was developed for the simultaneous quantification of SM and its oxidation products, i.e., mustard sulfoxide (SMO) and mustard sulfone (SMO2). The accurate measurement of SM and its oxidation products with high reaction activity was achived via the method of chemical conversion of 2-(3,5-bis(mercaptomethyl)phenoxy) acetic acid into stable derivative products. Method validation was performed in whole blood matrix, the linear range of the method was between 0.2 and 1000µg/L with correlation coefficients (r(2))>0.99, and the lower limits of quantification for SM, SMO and SMO2 were 1, 1, 0.2µg/L, respectively. The validated method was successfully applied to a toxicokinetics research of SM and its oxidation products after SM dermal exposed rats in a single dose. All three target analytes were found in whole blood samples from poisoned rats, and significant time-dependent responses were also observed. Among them, SMO2 with relatively high toxicity was identified and quantified in vivo for the first time, while SMO was the major product in whole blood and some of them continued to be oxidized to SMO2in vivo. These results give a direct experimental evidence to support that a large amount of SM is converted into the corresponding SMO and SMO2, and these oxidation products might cause potential combined toxic effects.

Structural, morphological, and functional correlates of corneal endothelial toxicity following corneal exposure to sulfur mustard vapor.

PURPOSE: Sulfur mustard (SM) is a highly reactive vesicant that causes severe ocular injuries. Following exposure to moderate or high doses, a subset of victims develops a chronic injury known as mustard gas keratopathy (MGK) involving a keratitis of unknown etiopathogenesis with secondary keratopathies such as persistent epithelial lesions, corneal neovascularization, and progressive corneal degeneration. This study was designed to determine whether SM exposure evokes acute endothelial toxicity and to determine whether endothelial pathologies were specifically observed in MGK corneas as opposed to healed corneas. METHODS: Corneas of New Zealand white rabbits were exposed to SM vapor, and the corneal endothelium was evaluated at 1 day and 8 weeks using scanning electron microscopy (SEM), transmission electron microscopy (TEM), in vivo confocal microscopy (IVM), and fluorescent microscopy. Barrier function was measured by uptake of a fluorescent dye injected into the anterior chamber. RESULTS: A centripetal endothelial injury at 1 day was observed by SEM, TEM, IVM, and fluorescent microscopy. In vivo confocal microscopy revealed additional cytotoxicity between 1 and 13 days. In contrast to healed corneas, which appeared similar to sham-exposed naive eyes at 8 weeks, MGK corneas exhibited significant evidence of continued pathological changes in the endothelium. CONCLUSIONS: Endothelial toxicity occurs at the right time and with the appropriate pathophysiology to contribute to MGK. Based on these findings, we propose a model that explains the relationships among SM dose, the biphasic progression, and the various clinical trajectories of corneal SM injury and that provides a mechanism for temporal variations in MGK onset. Finally, we discuss the implications for the management of SM casualties.

Tissue factor pathway inhibitor prevents airway obstruction, respiratory failure and death due to sulfur mustard analog inhalation.

UNLABELLED: Sulfur mustard (SM) inhalation causes airway injury, with enhanced vascular permeability, coagulation, and airway obstruction. The objective of this study was to determine whether recombinant tissue factor pathway inhibitor (TFPI) could inhibit this pathogenic sequence. METHODS: Rats were exposed to the SM analog 2-chloroethyl ethyl sulfide (CEES) via nose-only aerosol inhalation. One hour later, TFPI (1.5mg/kg) in vehicle, or vehicle alone, was instilled into the trachea. Arterial O2 saturation was monitored using pulse oximetry. Twelve hours after exposure, animals were euthanized and bronchoalveolar lavage fluid (BALF) and plasma were analyzed for prothrombin, thrombin-antithrombin complex (TAT), active plasminogen activator inhibitor-1 (PAI-1) levels, and fluid fibrinolytic capacity. Lung steady-state PAI-1 mRNA was measured by RT-PCR analysis. Airway-capillary leak was estimated by BALF protein and IgM, and by pleural fluid measurement. In additional animals, airway cast formation was assessed by microdissection and immunohistochemical detection of airway fibrin. RESULTS: Airway obstruction in the form of fibrin-containing casts was evident in central conducting airways of rats receiving CEES. TFPI decreased cast formation, and limited severe hypoxemia. Findings of reduced prothrombin consumption, and lower TAT complexes in BALF, demonstrated that TFPI acted to limit thrombin activation in airways. TFPI, however, did not appreciably affect CEES-induced airway protein leak, PAI-1 mRNA induction, or inhibition of the fibrinolytic activity present in airway surface liquid. CONCLUSIONS: Intratracheal administration of TFPI limits airway obstruction, improves gas exchange, and prevents mortality in rats with sulfur mustard-analog-induced acute lung injury.

N-acetyl-L-cysteine protects against inhaled sulfur mustard poisoning in the large swine.

CONTEXT: Sulfur mustard is a blister agent that can cause death by pulmonary damage. There is currently no effective treatment. N-acetyl-L-cysteine (NAC) has mucolytic and antioxidant actions and is an important pre-cursor of cellular glutathione synthesis. These actions may have potential to reduce mustard-induced lung injury. OBJECTIVE: Evaluate the effect of nebulised NAC as a post-exposure treatment for inhaled sulfur mustard in a large animal model. MATERIALS AND METHODS: Fourteen anesthetized, surgically prepared pigs were exposed to sulfur mustard vapor (100 µg.kg⁻¹), 10 min) and monitored, spontaneously breathing, to 12 h. Control animals had no further intervention (n = 6). Animals in the treatment group were administered multiple inhaled doses of NAC (1 ml of 200 mg.ml⁻¹ Mucomyst™ at + 30 min, 2, 4, 6, 8, and 10 h post-exposure, n = 8). Cardiovascular and respiratory parameters were recorded. Arterial blood was collected for blood gas analysis while blood and bronchoalveolar lavage fluid were collected for hematology and inflammatory cell analysis. Urine was collected to detect a sulfur mustard breakdown product. Lung tissue samples were taken for histopathological and post-experimental analyses. RESULTS: Five of six sulfur mustard-exposed animals survived to 12 h. Arterial blood oxygenation (PaO2) and saturation levels were significantly decreased at 12 h. Arterial blood carbon dioxide (PaCO2) significantly increased, and arterial blood pH and bicarbonate (HCO3⁻) significantly decreased at 12 h. Shunt fraction was significantly increased at 12 h. In the NAC-treated group all animals survived to 12 h (n = 8). There was significantly improved arterial blood oxygen saturation, HCO3⁻ levels, and shunt fraction compared to those of the sulfur mustard controls. There were significantly fewer neutrophils and lower concentrations of protein in lavage compared to sulfur mustard controls. DISCUSSION: NAC's mucolytic and antioxidant properties may be responsible for the beneficial effects seen, improving clinically relevant physiological indices affected by sulfur mustard exposure. CONCLUSION: Beneficial effects of nebulized NAC were apparent following inhaled sulfur mustard exposure. Further therapeutic benefit may result from a combination therapy approach.

Simultaneous quantification of seven plasma metabolites of sulfur mustard by ultra high performance liquid chromatography-tandem mass spectrometry.

Sulfur mustard (SM) is a hazardous chemical warfare agent that has been used in several military conflicts. SM is also considered as a major threat to civilians because of its existing stockpiles and easy production. Analysis of exposure biomarkers in biological samples collected from suspected victims is a useful tool for early diagnosis of SM poisoning. In this study, a sensitive and rapid quantitative method with ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for simultaneous determination of seven SM plasma biomarkers, including its oxidative, hydrolysis and ß-lyase metabolites. A simple one-step protein precipitation with acetonitrile-methanol (4:1) was used for sample preparation. A full validation was conducted with respect to specificity, linearity, recovery, matrix effect, precision, accuracy and stability. The lower limits of quantification for the seven metabolites ranged from 0.01µgL(-1) to 5µgL(-1). The intraday relative standard deviation was less than 7.0%, and the interday deviation was less than 9.1%. The recoveries varied in the range from 82.8% to 118%. This method has been successfully applied to a toxicokinetic study for obtaining the plasma profiles of seven metabolites in SM-exposed rats, following a single subcutaneous dose of 3.3mgkg(-1). All the targeted compounds were detected in rat plasma. bis-ß-Chloroethyl sulfoxide (SMO), thiodiglycol (TDG), thiodiglycol sulfoxide (TDGO), 1,1'-sulfonylbis-[2-S-(N-acetylcysteinyl)ethane (SBSNAE), 1,1'-sulfonylbis-[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) were found to be the major metabolites in rat plasma. The time windows for the detection of these metabolites were varied in the range of 5min to 48h after exposure. The method provides a useful tool for short-term diagnosis of SM poisoning.

Induction of neuronal damage in guinea pig brain by intratracheal infusion of 2-chloroethyl ethyl sulfide, a mustard gas analog.

Intratracheal infusion of 2-chloroethyl ethyl sulfide (CEES), a mustard gas analog and a chemical warfare agent is known to cause massive damage to lung. The purpose of this study was to determine whether intratracheal CEES infusion causes neuronal damage. Histological, immunohistochemical, and Western blot studies indicated that CEES treatment caused dose-dependent increases in blood cell aggregation, microglial cell number, microglial activation, and brain inflammation. In addition, an increased expression of α-synuclein and a decreased expression of the dopamine transporter were observed. The results indicate that intratracheal CEES infusion is associated with changes in brain morphology mediated by an increase in α-synuclein expression, leading to neurotoxicity in a guinea pig model. These changes may be mediated by oxidative stress. Furthermore, the present study indicates for the first time that intratracheal infusion of a single dose of CEES can cause neuroinflammation, which may lead to neurological disorders in later part of life.

Protection against 2-chloroethyl ethyl sulfide (CEES)-induced cytotoxicity in human keratinocytes by an inducer of the glutathione detoxification pathway.

Sulfur mustard (SM or mustard gas) was first used as a chemical warfare agent almost 100years ago. Due to its toxic effects on the eyes, lungs, and skin, and the relative ease with which it may be synthesized, mustard gas remains a potential chemical threat to the present day. SM exposed skin develops fluid filled bullae resulting from potent cytotoxicity of cells lining the basement membrane of the epidermis. Currently, there are no antidotes for SM exposure; therefore, chemopreventive measures for first responders following an SM attack are needed. Glutathione (GSH) is known to have a protective effect against SM toxicity, and detoxification of SM is believed to occur, in part, via GSH conjugation. Therefore, we screened 6 potential chemopreventive agents for ability to induce GSH synthesis and protect cultured human keratinocytes against the SM analog, 2-chloroethyl ethyl sulfide (CEES). Using NCTC2544 human keratinocytes, we found that both sulforaphane and methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) stimulated nuclear localization of Nrf2 and induced expression of the GSH synthesis gene, GCLM. Additionally, we found that treatment with CDDO-Me elevated reduced GSH content of NCTC2544 cells and preserved their viability by ~3-fold following exposure to CEES. Our data also suggested that CDDO-Me may act additively with 2,6-dithiopurine (DTP), a nucleophilic scavenging agent, to increase the viability of keratinocytes exposed to CEES. These results suggest that CDDO-Me is a promising chemopreventive agent for SM toxicity in the skin.

Uptake, tissue distribution, and excretion of 14C-sulfur mustard vapor following inhalation in F344 rats and cutaneous exposure in hairless guinea pigs.

Sulfur mustard (SM), a vessicating agent, has been used in chemical warfare since 1918. The purpose of this study was to quantitate SM vapor deposition, tissue distribution, and excretion following intratracheal inhalation in rats and cutaneous exposure in guinea pigs. 14C-SM vapors for inhalation studies were generated by metering liquid 14C-SM into a heated J tube. Vapors were transported via carrier air supplemented with oxygen and isoflurane to an exposure plenum. Anesthetized rats with transorally placed tracheal catheters were connected to the plenum port via the catheter hub for exposure (approximately 250 mg 14C-SM vapor/m(3); 10 min). For dermal exposure, 3 Teflon cups (6.6 cm(2) exposure area per cup) were applied to the backs of each animal and vapors (525 mg 14C-SM/m(3); 12 min) were generated by applying 6 µl 14C-SM to filter paper within each cup. Animals were euthanized at selected times up to 7 d postexposure. SM equivalents deposited in rats and guinea pigs were 18.1 ± 3 µg and 29.8 ± 5.31 µg, respectively. Inhaled SM equivalents rapidly distributed throughout the body within 2 h postexposure, with the majority (>70%) of material at that time located in carcass and pelt. In guinea pigs, >90% of deposited SM equivalents remained in skin, with minor distribution to blood and kidneys. Urine was the primary route of excretion for both species. Results indicate inhaled SM is rapidly absorbed from the lung and distributed throughout the body while there is limited systemic distribution following cutaneous exposure.

Dermal and ocular exposure systems for the development of models of sulfur mustard-induced injury.

Sulfur mustard (SM) is a chemical threat agent for which the effects have no current treatment. Due to the ease of synthesis and dispersal of this material, the need to develop therapeutics is evident. The present article details the techniques used to develop SM laboratory exposure systems for the development of animal models of ocular and dermal injury. These models are critical to enable evaluation of SM injury and therapeutics against that injury. Iterative trials were conducted to optimize dermal and ocular injury models in guinea pigs and rabbits respectively. The goal was a homogeneous and diffuse ocular and dermal injury that compares to the human injury. Dermal exposures were conducted by either a flow-past or static vapor cup system. Ocular exposures were conducted by a static exposure system. Ocular and dermal exposures were conducted with vaporized SM. Vapor concentrations increased with time in the dermal and ocular exposure systems but were stable with varying amounts of applied SM. A dermal deposition estimation study was also conducted. Deposited volumes increased with exposure time.

Development and validation of efficient stable isotope dilution LC-HESI-MS/MS method for the verification of ß-lyase metabolites in human urine after sulfur mustard exposure.

As a part of the project for screening unequivocal biomarkers after sulfur mustard exposure, a quantitative method for the determination of ß-lyase metabolites 1,1'-sulfonylbis-[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) was validated. Full validation was conducted according to the FDA guidelines for method validation using pooled human urine as a sample matrix. The metabolites were extracted from urine with an optimized sample preparation procedure using ENV+ solid phase extraction cartridge with reduced volume of sample and solvents. Metabolites were detected by improved and faster liquid chromatography-heated electrospray ionization-tandem mass spectrometry (LC-HESI-MS/MS) method with two transitions of each chemical using non-buffered eluents, post-column splitter and higher flow-rate. These provided over five times faster analysis than previously published method providing 4.5 min/sample cycle time, to achieve up to 200 samples per day (24 h). Quantification was performed using deuterium labelled internal standard of SBMSE. The method was linear over the concentration range of 5-200 ng/ml (average correlation coefficients were R(2)=0.997 and R(2)=0.989) for both ß-lyase metabolites, SBMSE and MSMTESE, respectively. The average retention times for SBMSE and MSMTESE were 1.96±0.01 min and 3.24±0.03 min (n=54). Calculated limits of detection were 4 ng/ml for both SBMSE and MSMTESE, respectively. Lower limits of quantification were 10 ng/ml and 11 ng/ml for SBMSE and MSMTESE, respectively. This validated method was successfully used in the First Confidence Building Exercise on Biomedical Samples Analysis organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). Identification criteria for analysing unequivocal biomarkers of sulfur mustard with LC-MS/MS after alleged use is discussed and proposed based on the validation and exercise results.

A comparison of sulfur mustard and heptane penetrating a dipalmitoylphosphatidylcholine bilayer membrane.

In the present molecular dynamics simulations we study the chemical warfare agent sulfur mustard (bis(2-chloroethyl) sulfide) and the alkane heptane inserted into a dipalmitoylphosphatidylcholine (DPPC) bilayer, a generic model for a biological membrane. We investigate the diffusion, the orientation, the preferred positioning, and the end-to-end distance of the solutes within the membrane as well as the corresponding coupling times. We compare results of equilibrium simulations and simulation at different external forces, which drag the solutes through the membrane. These properties lead to a general comparison of the rotational and translational behaviors of the two solutes during the penetration of the membrane. We show that sulfur mustard, due to its atomic charge polarization, its bigger flexibility and its smaller molecular volume, is the faster moving molecule within the membrane. In last consequence, we show that this leads to different limits for the transport mechanism as observed in these simulations. For heptane the hindrance to penetrate into the membrane is significantly higher than for sulfur mustard. In contrast to heptane molecules, which spend the most of the time penetrating the tail groups, sulfur mustard needs more time to escape the tail group-head group interface of the membrane.

The skin reservoir of sulphur mustard.

Studies of the percutaneous reservoir of sulphur mustard (HD) formed during absorption carried out during WWI and WWII are inconclusive. More recent studies have indicated that a significant amount of unreacted HD remains in human epidermal membranes during percutaneous penetration studies in vitro. The present study investigated the nature and persistence of the HD reservoir formed during in vitro penetration studies using dermatomed slices of human and pig skin (0.5mm thick). Amounts of (14)C-HD that (a) penetrated, (b) remained on the surface, (c) were extractable from and (d) remained in the skin after extraction were estimated by liquid scintillation counting (confirmed using GC-MS analysis). The results demonstrated that there is a reservoir of HD in human and pig skin for up to 24 h after contamination of the skin surface in vitro with liquid agent. At least some of this reservoir could be extracted with acetonitrile, and the amounts of extracted and unextracted HD exceed the amount required to produce injury in vivo by at least 20 fold. The study demonstrated the presence of a reservoir whether the skin was covered (occluded) or left open to the air (unoccluded). The study concluded that the extractable reservoir was significant in terms of the amount of HD required to induce a vesicant response in human skin. The extractable reservoir was at least 20 times the amount required per cm(2) estimated to cause a response in all of the human population, as defined by studies carried out in human volunteers during the 1940s.

Mustard gas: imminent danger or eminent threat?

OBJECTIVE: To increase awareness of the widespread environmental prevalence of the chemical warfare agent mustard gas, examine the acute and chronic toxic effects to exposed humans, and discuss medical treatment guidelines for mustard gas exposures. DATA SOURCES: Literature retrieval of medical case reports and clinical studies was accomplished using PubMed and the Cochrane Database (1919-March 2007). Search terms included mustard, mustard gas, sulfur mustard, chemical warfare, blister agents, vesicants, and war gas. Historical information and current events were accessed through military field manuals and Internet searches. STUDY SELECTION AND DATA EXTRACTION: All articles in English identified from the data sources were evaluated. Adult and pediatric populations were included in the review. DATA SYNTHESIS: Mustard gas and other chemical weapons are feared for their use as weapons of terror; however, the major threat of mustard gas lies elsewhere. Tons of this chemical agent were produced for war, then subsequently buried in landfills, disposed of at sea, or left to decay in storage facilities. There are documented and anecdotal reports of chemical weapon burial sites and ocean dumps across the globe spanning from the Arctic Circle to Australia. Numerous accidental exposures have occurred over the past decade. Mustard gas is corrosive to the skin, eyes, and respiratory tract. Extensive exposures can also affect other organ systems. Its ability to cause harm to multiple organ systems at extremely low doses in virtually any environmental condition makes it an extremely dangerous agent. Immediate decontamination of people exposed to mustard gas liquids and vapors is paramount. Supportive care and long-term followup is necessary for exposed persons. Research is under way to find antidotes or treatment methods for mustard gas exposure, but currently there are no definitive treatment guidelines. CONCLUSIONS: Mustard gas is a weapon, but also a prevalent environmental threat. Recognizing the immense environmental presence of mustard gas disposal sites and the signs and symptoms of exposure will help speed treatment to those accidentally or purposefully exposed.