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.

A systems dynamics approach to the efficacy of oxime therapy for mild exposure to sarin gas.

The use of nerve agents such as sarin is as much a threat today as any other time in our history. The events in Syria in 2013 are proof of this. "The Obama administration asserted Sunday for the first time that the Syrian government used the nerve gas sarin to kill more than 1,400 people (August 21, 2013) in the world's gravest chemical weapons attack in 25 years." With these recent events clear in our mind, we must focus on the horrific nature of these chemical agents to devise a strategy that will enable first responders to counteract these insidious chemicals. This paper presents research on a physiologically based pharmacokinetic model to determine whether the current treatment protocol prescribed by the Center for Disease Control (CDC) and the US Army is effective in treating victims suffering from acute exposure symptoms. The model was used to determine what treatment should be used for victims suffering from mild exposure symptoms. The results indicate that the current CDC and US Army treatment is effective, but treatment with oxime therapy was not effective in alleviating symptoms of mild exposure. By applying these results, an effective treatment protocol was developed.

Analysis of nitromethane from samples exposed in vitro to chloropicrin by stable isotope dilution headspace gas chromatography with mass spectrometry.

Chloropicrin (trichloronitromethane) is a widely used soil fumigant and an old chemical warfare agent. The metabolism of chloropicrin is not well known in mammals but nitromethane has been shown to be one of its main metabolites. Here, a fast and simple headspace gas chromatography with mass spectrometry method was applied for the measurement of nitromethane from aqueous samples. The analytical method was validated using stable isotope labeled internal standard and a small sample volume of 260 µL. No conventional sample preparation steps were needed. The method was accurate (relative standard deviations ≤1.5%) and linear (R(2) = 0.9996) within the concentration range of 0.1-6.0 µg/mL. This method was used to measure nitromethane in in vitro incubations with human and pig liver cell fractions containing enzymes for xenobiotic metabolism, exposed to chloropicrin. The results indicate that the presence of glutathione is necessary for the formation of nitromethane from chloropicrin. Also, nitromethane was formed mostly in liver cytosol fractions, but not in microsomal fractions after the incubation with chloropicrin. Our results suggest that although nitromethane is not the unequivocal biomarker of chloropicrin exposure, this method could be applied for screening the elevated levels in humans after chloropicrin exposure.

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.

Reductive activation of type 2 ribosome-inactivating proteins is promoted by transmembrane thioredoxin-related protein.

Members of the type 2 ribosome-inactivating proteins (RIPs) family (e.g. ricin, abrin) are potent cytotoxins showing a strong lethal activity toward eukaryotic cells. Type 2 RIPs contain two polypeptide chains (usually named A, for "activity", and B, for "binding") linked by a disulfide bond. The intoxication of the cell is a consequence of a reductive process in which the toxic domain is cleaved from the binding domain by oxidoreductases located in the lumen of the endoplasmic reticulum (ER). The best known example of type 2 RIPs is ricin. Protein disulfide isomerase (PDI) was demonstrated to be involved in the process of ricin reduction; however, when PDI is depleted from cell fraction preparations ricin reduction can still take place, indicating that also other oxidoreductases might be implicated in this process. We have investigated the role of TMX, a transmembrane thioredoxin-related protein member of the PDI family, in the cell intoxication operated by type 2 RIPs ricin and abrin. Overexpressing TMX in A549 cells resulted in a dramatic increase of ricin or abrin cytotoxicity compared with control mock-treated cells. Conversely, no difference in cytotoxicity was observed after treatment of A549 cells or control cells with saporin or Pseudomonas exotoxin A whose intracellular mechanism of activation is not dependent upon reduction (saporin) or only partially dependent upon it (Pseudomonas exotoxin A). Moreover, the silencing of TMX in the prostatic cell line DU145 reduced the sensitivity of the cells to ricin intoxication further confirming a role for this enzyme in intracellular ricin activation.

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.

The acute toxicity, tissue distribution, and histopathology of inhaled ricin in Sprague Dawley rats and BALB/c mice.

Ricin is a highly toxic ribosome-inactivating protein derived from the castor bean (Ricinus communis). Due to the relative ease of producing ricin, it is characterized as a category B priority pathogen by the Center for Disease Control and Prevention. The purpose of this study was to compare the acute toxicity, associated histopathology, as well as the regional respiratory tract deposition and clearance kinetics of inhaled ricin in rats and mice using a single pure preparation. Acute toxicity was evaluated in five groups of six animals per species exposed nose-only to ricin aerosols and followed up to 7 days post-exposure. Tissues were collected for histopathology. The calculated median lethal doses (LD50s) were 0.24 µg/kg (rats) and 0.58 µg/kg (mice). Histological changes were noted in nose, larynges, trachea, lung, thymus, and spleen of both species. Pulmonary deposition in rats inhaling 94-99 ng/L ricin for 20 min (low dose) or 40 min (high dose) were 45.9 and 96 ng/g lung, respectively. Clearance was best described by a single-component negative exponential function. Estimated lung doses were 0.38 and 1.43 µg/g·h among the low and high dose rats, respectively. In mice inhaling 94 ng/L ricin for 20 min, pulmonary deposition was 91.1 ng/g lung and the estimated tissue dose was 1.72 µg/g·h. No ricin was detected in extra-respiratory tract tissue or in excreta. Results of this study demonstrate differences exist in pulmonary deposition, clearance rates, and tissue dose and histopathological changes between rats and mice inhaling ricin.

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.

Cutaneous and ocular late complications of sulfur mustard in Iranian veterans.

Although sulfur mustard (SM) has been used as a chemical warfare agent since the early twentieth century, it has reemerged in the past decade as a major threat around the world. This agent injured over 100,000 Iranians and one-third is suffering from late effects until today. Mustard affects many organs such as the skin, eyes, and lungs, as well as the gastrointestinal, endocrine, and hematopoietic system. In this study we focused on review of the late Cutaneous and ocular complications caused by exposure to SM. All studies regarding long-term ocular and cutaneous effects, which have been done on Iranian population, were collected from domestic and international sources. Pruritus is the most common complain and a malignant change is the most important lesion, which has to be considered. Also this agent is causes of chronic and delayed destructive lesions in the ocular surface and cornea, leading to progressive visual deterioration and ocular irritation.

Medical aspects of sulphur mustard poisoning.

Sulphur mustard is one of the major chemical warfare agents developed and used during World War I. Large stockpiles are still present in several countries. It is relatively easy to produce and might be used as a terroristic weapon. Sulphur mustard is a vesicant agent and causes cutaneous blisters, respiratory tract damage, eye lesions and bone marrow depression. The clinical picture of poisoning is well known from the thousands of victims during World War I and the Iran-Iraq war. In the latter conflict, sulphur mustard was heavily used and until now about 30,000 victims still suffer from late effects of the agent like chronic obstructive lung disease, lung fibrosis, recurrent corneal ulcer disease, chronic conjunctivitis, abnormal pigmentation of the skin, and several forms of cancer. Despite enormous research efforts during the last 90 years, no specific sulphur mustard antidote has been found. The prospering knowledge and developments of modern medicine created nowadays new chances to minimize sulphur mustard-induced organ damage and late effects.

Quantitation of fluoride ion released sarin in red blood cell samples by gas chromatography-chemical ionization mass spectrometry using isotope dilution and large-volume injection.

A new method for measuring fluoride ion released isopropyl methylphosphonofluoridate (sarin, GB) in the red blood cell fraction was developed that utilizes an autoinjector, a large-volume injector port (LVI), positive ion ammonia chemical ionization detection in the SIM mode, and a deuterated stable isotope internal standard. This method was applied to red blood cell (RBC) and plasma ethyl acetate extracts from spiked human and animal whole blood samples and from whole blood of minipigs, guinea pigs, and rats exposed by whole-body sarin inhalation. Evidence of nerve agent exposure was detected in plasma and red blood cells at low levels of exposure. The linear method range of quantitation was 10-1000 pg on-column with a detection limit of approximately 2-pg on-column. In the course of method development, several conditions were optimized for the LVI, including type of injector insert, injection volume, initial temperature, pressure, and flow rate. RBC fractions had advantages over the plasma with respect to assessing nerve agent exposure using the fluoride ion method especially in samples with low serum butyrylcholinesterase activity.

Skin toxicokinetics of mustard gas in the guinea pig: effect of hypochlorite and safety aspects.

Sulfur mustard (SM, mustard gas) is a chemical warfare vesicant that rapidly penetrates the skin due to its hydrophobicity. This study measured the rate of SM disappearance from the skin after topical application of the vesicant. In both fur-covered and hairless animals, the remaining toxicant levels measured 60 min after exposure to undiluted SM were 0.6% and 0.3%, respectively, of the initially applied SM amount. However, SM concentration reached 0.4% of the initial dose 3 h following exposure in female fur-covered guinea pigs. SM quantities extracted from skin of male fur-covered and hairless guinea pigs immediately after 16 min of exposure to SM vapor were 12.2 and 21.8 microg, respectively; levels declined to 1.6 and 1.7 microg at 30 and 15 min following termination of exposure of male fur-covered and hairless guinea pigs, respectively. Three swabbing treatments of undiluted SM-exposed skin with gauze pads soaked in 0.5% hypochlorite caused 68% reduction in skin SM content. Similar findings were obtained when hypochlorite was replaced by water (64% reduction). SM content in the gauze pads was 59, 38 and 25 microg, respectively, for the first, second and third decontamination processes with water. No SM was detected in the gauze pads soaked with hypochlorite. In vitro studies showed that incubation of SM with 0.5% hypochlorite at a ratio of 10:1 (v/v) did not cause SM inactivation, whereas 4% hypochlorite reduced SM levels by 17%. However, at a decontaminant:SM ratio of 1000:1, 0.5% and 4% hypochlorite reduced SM levels by 92% and 99%, respectively. These findings are important for health authorities and regulatory agencies in planning precautionary steps to be taken in case of emergency and in routine laboratory work.

Cutaneous uptake of 14C-HD vapor by the hairless guinea pig.

The hairless guinea pig (HGP) is used by our laboratory to model the human cutaneous response to sulfur mustard (HD), bis(2-chloroethylsulfide), exposure. We determined the HD content in the skin of HGP after a 7-min exposure to vapors saturated with a mixture of HD and 14C-HD. Concentration/time (CT) values in the range of 2 micrograms/cm2/min were determined by counting skin 14C disintegrations per min (dpm) in animals euthanized immediately after exposure. These values are similar to human penetration rates obtained by other investigators. A rate curve monitoring the reduction in skin 14C dpm was developed for animals euthanized between 0 and 24 hr post- exposure. This curve showed the greatest change after 1 hr. The epidermal (62%) to dermal (38%) ratio of 14C at 24 hr was measured for two animals. We saw no site preference for HD penetration among the 8 sites used. The 14C content of template adhesive tape was determined to follow HD distribution. These results contribute to a better understanding of the cutaneous response to HD in the HGP model.

Toxicity of the organophosphate chemical warfare agents GA, GB, and VX: implications for public protection.

The nerve agents, GA, GB, and VX are organophosphorus esters that form a major portion of the total agent volume contained in the U.S. stockpile of unitary chemical munitions. Congress has mandated the destruction of these agents, which is currently slated for completion in 2004. The acute, chronic, and delayed toxicity of these agents is reviewed in this analysis. The largely negative results from studies of genotoxicity, carcinogenicity, developmental, and reproductive toxicity are also presented. Nerve agents show few or delayed effects. At supralethal doses, GB can cause delayed neuropathy in antidote-protected chickens, but there is no evidence that it causes this syndrome in humans at any dose. Agent VX shows no potential for inducing delayed neuropathy in any species. In view of their lack of genotoxicity, the nerve agents are not likely to be carcinogens. The overreaching concern with regard to nerve agent exposure is the extraordinarily high acute toxicity of these substances. Furthermore, acute effects of moderate exposure such as nausea, diarrhea, inability to perform simple mental tasks, and respiratory effects may render the public unable to respond adequately to emergency instructions in the unlikely event of agent release, making early warning and exposure avoidance important. Likewise, exposure or self-contamination of first responders and medical personnel must be avoided. Control limits for exposure via surface contact of drinking water are needed, as are detection methods for low levels in water or foodstuffs.