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Sulfur mustard (SM) inhalation causes debilitating pulmonary injury in humans which progresses to fibrosis. Herein, we developed a rat model of SM toxicity which parallels pathological changes in the respiratory tract observed in humans. SM vapor inhalation caused dose (0.2–0.6 mg/kg)-related damage to the respiratory tract within 3 days of exposure. At 0.4–0.6 mg/kg, ulceration of the proximal bronchioles, edema and inflammation were observed, along with a proteinaceous exudate containing inflammatory cells in alveolar regions. Time course studies revealed that the pathologic response was biphasic. Thus, changes observed at 3 days post-SM were reduced at 7–16 days; this was followed by more robust aberrations at 28 days, including epithelial necrosis and hyperplasia in the distal bronchioles, thickened alveolar walls, enlarged vacuolated macrophages, and interstitial fibrosis. Histopathologic changes were correlated with biphasic increases in bronchoalveolar lavage (BAL) cell and protein content and proliferating cell nuclear antigen expression. Proinflammatory proteins receptor for advanced glycation end product (RAGE), high-mobility group box protein (HMGB)-1, and matrix metalloproteinase (MMP)-9 also increased in a biphasic manner following SM inhalation, along with surfactant protein-D (SP-D). Tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS), inflammatory proteins implicated in mustard lung toxicity, and the proinflammatory/profibrotic protein, galectin (Gal)-3, were upregulated in alveolar macrophages and in bronchiolar regions at 3 and 28 days post-SM. Inflammatory changes in the lung were associated with oxidative stress, as reflected by increased expression of heme oxygenase (HO)-1. These data demonstrate a similar pathologic response to inhaled SM in rats and humans suggesting that this rodent model can be used for mechanistic studies and for the identification of efficacious therapeutics for mitigating toxicity.

Sulfur mustard (SM) is a bifunctional alkylating agent that causes severe injury to the respiratory tract. This is accompanied by an accumulation of macrophages in the lung and the release of the proinflammatory cytokine, tumor necrosis factor (TNF)α. In these studies, we analyzed the effects of blocking TNFα on lung injury, inflammation and oxidative stress induced by inhaled SM. Rats were treated with SM vapor (0.4 mg/kg) or air control by intratracheal inhalation. This was followed 15–30 min later by anti-TNFα antibody (15 mg/kg, i.v.) or PBS control. Animals were euthanized 3 days later. Anti-TNFα antibody was found to blunt SM-induced peribronchial edema, perivascular inflammation and alveolar plasma protein and inflammatory cell accumulation in the lung; this was associated with reduced expression of PCNA in histologic sections and decreases in BAL levels of fibrinogen. SM-induced increases in inflammatory proteins including soluble receptor for glycation end products, its ligand, high mobility group box-1, and matrix metalloproteinase-9 were also reduced by anti-TNFα antibody administration, along with increases in numbers of lung macrophages expressing TNFα, cyclooxygenase-2 and inducible nitric oxide synthase. This was correlated with reduced oxidative stress as measured by expression of heme oxygenase-1 and Ym-1. Together, these data suggest that inhibiting TNFα may represent an efficacious approach to mitigating acute lung injury, inflammatory macrophage activation, and oxidative stress induced by inhaled sulfur mustard.

Chemical exposure emergencies posing a serious threat to the health and survival of civilians can be caused by terrorism events or industrial accidents. The National Institutes of Health (NIH) deve...

Lewisite (LEW), a potent arsenical vesicating chemical warfare agent, poses a continuous risk of accidental exposure in addition to its feared use as a terrorist weapon. Ocular tissue is exquisitely sensitive to LEW and exposure can cause devastating corneal lesions. However, detailed pathogenesis of corneal injury and related mechanisms from LEW exposure that could help identify targeted therapies are not available. Using an established consistent and efficient exposure system, we evaluated the pathophysiology of the corneal injury in New Zealand white rabbits following LEW vapor exposure (at 0.2 mg/L dose) for 2.5 and 7.5 min, for up to 28 day post-exposure. LEW led to an increase in total corneal thickness starting at day 1 post-exposure and epithelial degradation starting at day 3 post-exposure, with maximal effect at day 7 postexposure followed by recovery at later time points. LEW also led to an increase in the number of blood vessels and inflammatory cells but a decrease in keratocytes with optimal effects at day 7 postexposure. A significant increase in epithelial-stromal separation was observed at days 7 and 14 post 7.5 min LEW exposure. LEW also caused an increase in the expression levels of cyclooxygenase-2, IL-8, vascular endothelial growth factor, and matrix metalloproteinase-9 at all the study time points indicating their involvement in LEW-induced inflammation, vesication, and neovascularization. The outcomes here provide valuable LEW-induced corneal injury endpoints at both lower and higher exposure durations in a relevant model system, which will be helpful to identify and screen therapies against LEW-induced corneal injury.

Phosgene is classified as a chemical warfare agent, yet data on its short-duration high concentration toxicity in a nose-only exposure rat model is sparse and inconsistent. Hence, an exposure system for short term/high concentration exposure was developed and characterized. Herein, we report the median lethal concentration (LC(50)) for a 10-min nasal exposure of phosgene in a 24-h rat survival model. Male Wistar rats (Envigo) weighing 180-210 g on the day of exposure, were exposed to phosgene gas via nose-only inhalation using a system specifically designed to allow the simultaneous exposure and quantification of phosgene. After 24 h, the surviving rats were euthanized, the lung/body mass ratio determined, and lung tissues analyzed for histopathology. Increased terminal airway edema in the lungs located primarily at the alveoli (resulting in an increased lung/body mass ratio) coincided with the observed mortality. An LC(50) value of 129.2 mg/m(3) for a 10-min exposure was determined. Furthermore, in agreement with other highly toxic compounds, this study reveals a LC(50) concentration value supportive of a non-linear toxic load model, where the toxic load exponent is > 1 (n(e) = 1.17). Thus, in line with other chemical warfare agents, phosgene toxicity is predicted to be more severe with short-duration, high-concentration exposures than long-duration, low-concentration exposures. This model is anticipated to be refined and developed to screen novel therapeutics against relevant short-term high concentration phosgene exposures expected from a terrorist attack, battlefield deployment, or industrial accident.

Phosgene Oxime (CX), an urticant or nettle agent categorized as a vesicant, is a potential chemical warfare and terrorist weapon. Its exposure can result in widespread and devastating effects including high mortality due to its fast penetration and ability to cause immediate severe cutaneous injury. It is one of the least studied chemical warfare agents with no effective therapy available. Thus, our goal was to examine the acute effects of CX following its cutaneous exposure in SKH-1 hairless mice to help establish a relevant injury model. Results from our study show that topical cutaneous exposure to CX vapor causes blanching of exposed skin with an erythematous ring, necrosis, edema, mild urticaria and erythema within minutes after exposure out to 8h post-exposure. These clinical skin manifestations were accompanied with increases in skin thickness, apoptotic cell death, mast cell degranulation, myeloperoxidase activity indicating neutrophil infiltration, p53 phosphorylation and accumulation, and an increase in COX-2 and TNFα levels. Topical CX-exposure also resulted in the dilatation of the peripheral vessels with a robust increase in RBCs in vessels of the liver, spleen, kidney, lungs and heart tissues. These events could cause a drop in blood pressure leading to shock, hypoxia and death. Together, this is the first report on effects of CX cutaneous exposure, which could help design further comprehensive studies evaluating the acute and chronic skin injuries from CX topical exposure and elucidate the related mechanism of action to aid in the identification of therapeutic targets and mitigation of injury.

Nitrogen mustard (NM) is a bifunctional alkylating agent that is highly reactive in the skin causing extensive tissue damage and blistering. In the present studies, a modified cutaneous murine patch model was developed to characterize NM-induced injury and to evaluate the efficacy of an indomethacin pro-drug in mitigating toxicity. NM (20 µmol) or vehicle control was applied onto 6 mm glass microfiber filters affixed to the shaved dorsal skin of CD-1 mice for 6 min. This resulted in absorption of approximately 4 µmol of NM. NM caused localized skin damage within 1 d, progressing to an eschar within 2–3 d, followed by wound healing after 4–5 d. NM-induced injury was associated with increases in skin thickness, inflammatory cell infiltration, reduced numbers of sebocytes, basal keratinocyte double stranded DNA breaks, as measured by phospho-histone 2A.X expression, mast cell degranulation and increases in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Wound healing was characterized by epidermal hyperplasia and marked increases in basal cells expressing proliferating cell nuclear antigen. A novel indomethacin-anticholinergic prodrug (4338) designed to target cyclooxygenases and acetylcholinesterase (AChE), was found to markedly suppress NM toxicity, decreasing wound thickness and eschar formation. The prodrug also inhibited mast cell degranulation, suppressed keratinocyte expression of iNOS and COX-2, as well as markers of epidermal proliferation. These findings indicate that a novel bifunctional pro-drug is effective in limiting NM mediated dermal injury. Moreover, our newly developed cutaneous patch model is a sensitive and reproducible method to assess the mechanism of action of countermeasures.

Sulfur mustard (2,2′-dichlorodiethyl sulfide, SM) is a chemical warfare agent that generates an inflammatory response in the skin and causes severe tissue damage and blistering. In earlier studies, we identified cutaneous damage induced by SM in mouse ear skin including edema, erythema, epidermal hyperplasia and microblistering. The present work was focused on determining if SM-induced injury was associated with alterations in mRNA and protein expression of specific cytokines and chemokines in the ear skin. We found that SM caused an accumulation of macrophages and neutrophils in the tissue within one day which persisted for at least 7 days. This was associated with a 2–15 fold increase in expression of the proinflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor α at time points up to 7 days post-SM exposure. Marked increases (20–1000 fold) in expression of chemokines associated with recruitment and activation of macrophages were also noted in the tissue including growth-regulated oncogene α (GROα/CXCL1), monocyte chemoattractant protein 1 (MCP-1/CCL2), granulocyte-colony stimulating factor (GCSF/CSF3), macrophage inflammatory protein 1α (MIP1α/CCL3), and IFN-γ-inducible protein 10 (IP10/CXCL10). The pattern of cytokines/chemokine expression was coordinate with expression of macrophage elastase/MMP12 and neutrophil collagenase/MMP8 suggesting that macrophages and neutrophils were, at least in part, a source of cytokines and chemokines. These data support the idea that inflammatory cell-derived mediators contribute to the pathogenesis of SM induced skin damage. Modulating the infiltration of inflammatory cells and reducing the expression of inflammatory mediators in the skin may be an important strategy for mitigating SM-induced cutaneous injury.

Dermal exposure to the vesicant sulfur mustard causes marked inflammation and tissue damage. Basal keratinocytes appear to be a major target of sulfur mustard. In the present studies, mechanisms mediating skin toxicity were examined using a mouse skin construct model and a full-thickness human skin equivalent (EpiDerm-FTTM). In both systems, administration of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide (CEES, 100–1000 µM) at the air surface induced mRNA and protein expression of heat shock proteins 27 and 70 (Hsp27 and Hsp70). CEES treatment also resulted in increased expression of caveolin-1, the major structural component of caveolae. Immunohistochemistry revealed that Hsp27, Hsp70 and caveolin-1 were localized in basal and suprabasal layers of the epidermis. Caveolin-1 was also detected in fibroblasts in the dermal component of the full thickness human skin equivalent. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that Hsp27 and Hsp70 were localized in caveolae. Treatment of mouse keratinocytes with filipin III or methyl-β-cyclodextrin, which disrupt caveolar structure, markedly suppressed CEES-induced Hsp27 and Hsp70 mRNA and protein expression. CEES treatment is known to activate JNK and p38 MAP kinases; in mouse keratinocytes, inhibition of these enzymes suppressed CEES-induced expression of Hsp27 and Hsp70. These data suggest that MAP kinases regulate Hsp 27 and Hsp70; moreover, caveolae-mediated regulation of heat shock protein expression may be important in the pathophysiology of vesicant-induced skin toxicity.

Thioredoxin reductase (TrxR) is a selenocysteine-containing flavoprotein that catalyzes the NADPH-dependent reduction of oxidized thioredoxin and plays a key role in regulating cellular redox homeostasis. In the present studies we examined the effects of 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant, on TrxR in lung epithelial cells. We speculated that vesicant-induced alterations in TrxR contribute to oxidative stress and toxicity. Treatment of human lung A549 epithelial cells with CEES resulted in a time- and concentration-dependent inhibition of TrxR. Using purified rat liver TrxR, we demonstrated that only the reduced enzyme was inhibited and that this inhibition was irreversible. The reaction of TrxR with iodoacetamide, which selectively modifies free thiol or selenol on proteins, was also markedly reduced by CEES, suggesting that CEES induces covalent modification of the reduced selenocysteine-containing active site in the enzyme. This was supported by our findings that recombinant mutant TrxR in which selenocysteine was replaced by cysteine, was marked less sensitive to inhibition by CEES, and that the vesicant preferentially alkylated selenocysteine in the C-terminal redox motif of TrxR. TrxR also catalyzes quinone redox cycling, a process that generates reactive oxygen species. In contrast to its inhibitory effects on thioredoxin reductase activity, CEES was found to stimulate redox cycling. Taken together, these data suggest that sulfur mustard vesicants target TrxR and that this may be an important mechanism mediating oxidative stress and tissue injury.

Epithelial cell migration during wound healing is regulated in part by enzymatic processing of laminin-332 (formerly LN-5), a heterodimer formed from alpha, beta, and gamma polypeptide chains. Under static conditions, laminin-332 is secreted into the extracellular matrix as a proform and has two chains processed to smaller forms, allowing it to anchor epithelial cells to the basement membrane of the dermis. During incisional wounding, laminin gamma2 chains in particular are processed to smaller sizes and function to promote epithelial sheet migration over the wound bed. The present study examines whether this same function occurs following chemical injury. The mouse ear vesicant model (MEVM) was used to follow the pathology in the ear and test whether processed laminin-332 enhances epithelial cell migration. Skin biopsies of sulfur mustard (SM) exposed ears for several time points were analyzed by histology, immunohistochemistry, real-time PCR, and Western blot analysis. SM exposure greatly increased mRNA levels for laminin-gamma2 in comparison to the other two chains. Protein production of laminin-gamma2 was upregulated, and there was an increase in the processed forms. Protein production was in excess of the amount required to form heterotrimeric laminin-332 and was associated with the migrating epithelial sheet, suggesting a potential role in wound healing for monomeric laminin-gamma2.

Vesicants including sulfur mustard (SM) and nitrogen mustard (NM) are bifunctional alkylating agents that cause skin inflammation, edema and blistering. This is associated with alterations in keratinocyte growth and differentiation. Endogenous cannabinoids, including N-arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoyl glycerol (2-AG), are important in regulating inflammation, keratinocyte proliferation and wound healing. Their activity is mediated by binding to cannabinoid receptors 1 and 2 (CB1 and CB2), as well as peroxisome proliferator-activated receptor alpha (PPARα). Levels of endocannabinoids are regulated by fatty acid amide hydrolase (FAAH). We found that CB1, CB2, PPARα and FAAH were all constitutively expressed in mouse epidermis and dermal appendages. Topical administration of NM or SM, at concentrations that induce tissue injury, resulted in upregulation of FAAH, CB1, CB2 and PPARα, a response that persisted throughout the wound healing process. Inhibitors of FAAH including a novel class of vanillyl alcohol carbamates were found to be highly effective in suppressing vesicant-induced inflammation in mouse skin. Taken together, these data indicate that the endocannabinoid system is important in regulating skin homeostasis and that inhibitors of FAAH may be useful as medical countermeasures against vesicants.

The thioredoxin (Trx) system, which consists of Trx and thioredoxin reductase (TrxR), is a major cellular disulfide reduction system important in antioxidant defense. TrxR is a target of mechlorethamine (methylbis(2-chloroethyl)amine; HN2), a bifunctional alkylating agent that covalently binds to selenocysteine/cysteine residues in the redox centers of the enzyme, leading to inactivation and toxicity. Mammalian Trx contains two catalytic cysteines; herein, we determined if HN2 also targets Trx. HN2 caused a time- and concentration-dependent inhibition of purified Trx and Trx in A549 lung epithelial cells. Three Trx cross-linked protein complexes were identified in both cytosolic and nuclear fractions of HN2-treated cells. LC-MS/MS of these complexes identified both Trx and TrxR, indicating that HN2 cross-linked TrxR and Trx. This is supported by our findings of a significant decrease of Trx/TrxR complexes in cytosolic TrxR knockdown cells after HN2 treatment. Using purified recombinant enzymes, the formation of protein cross-links and enzyme inhibition were found to be redox status-dependent; reduced Trx was more sensitive to HN2 inactivation than the oxidized enzyme, and Trx/TrxR cross-links were only observed using reduced enzyme. These data suggest that HN2 directly targets catalytic cysteine residues in Trx resulting in enzyme inactivation and protein complex formation. LC-MS/MS confirmed that HN2 directly alkylated cysteine residues on Trx, including Cys32 and Cys35 in the redox center of the enzyme. Inhibition of the Trx system by HN2 can disrupt cellular thiol-disulfide balance, contributing to vesicant-induced lung toxicity.

Sulfur mustard (SM) is a chemical warfare agent that causes skin damage characterized by inflammation and blistering. We investigated leukocyte infiltration into mouse skin after SM exposure. The d...

The chemical warfare agent sulfur mustard [bis-(2-chloroethyl)-sulfide; SM] produces a delayed inflammatory response followed by blister formation in skin of exposed individuals. Studies are underway evaluating the efficacy of pharmacological compounds to protect against SM skin injury. Microarray analysis provides the opportunity to identify multiple transcriptional biomarkers associated with SM exposure. This study examined SM-induced changes in gene expression in skin from mice cutaneously exposed to SM using cDNA microarrays. Ear skin from five mice, paired as SM-exposed right ear and dichloromethane vehicle-exposed left ear at six dose levels (0.005, 0.01, 0.02, 0.04, 0.08, and 0.16 mg; 6 mM to 195 mM range), was harvested at 24 h post-exposure. SM-induced gene expression was analyzed using cDNA microarrays that included 1,176 genes. Genes were selected on the basis of all mice (N = 5) in the same dose group demonstrating a ≥2-fold increase or decrease in gene expression for the SM-exposed tissue compared to the dichloromethane vehicle control ear tissue at all six SM doses. When skin exposed to all six concentrations of SM was compared to controls, a total of 19 genes within apoptosis, transcription factors, cell cycle, inflammation, and oncogenes and tumor suppressors categories were found to be upregulated; no genes were observed to be downregulated. Differences in the number and category of genes that were up- or down-regulated in skin exposed to low (0.005–0.01 mg) and high (0.08–0.16 mg) doses of SM were also observed. The results of this study provide a further understanding of the molecular responses to cutaneous SM exposure, and enable the identification of potential diagnostic markers and therapeutic targets for treating SM injury. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:289–299, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20043

Sulfur mustard [bis(2‐chloroethyl)sulfide, SM] is a chemical warfare agent that penetrates the skin rapidly and causes extensive blistering. Using the mouse ear vesicant model (MEVM), we evaluated the effect of topically applied anti‐inflammatory agents (octyl homovanillamide and heptyl isovanillamide) on ear edema formation and gene expression following SM exposure. Relative ear weight and real‐time reverse transcriptase polymerase chain reaction of GM‐CSF, IL‐1β, and IL‐6 were used to evaluate the effects of octyl homovanillamide and heptyl isovanillamide. Both vanilloids significantly reduced SM‐induced edema. At the single dose and number of animals/group tested, octyl homovanillamide produced a trend of reduced mRNA levels; however, the reduction was not significant for GM‐CSF, IL‐1β, or IL‐6. Heptyl isovanillamide significantly reduced (p ≤ 0.05) GM‐CSF, IL‐1β, and IL‐6 mRNA levels. These results show that octyl homovanillamide and heptyl isovanillamide reduce skin edema and heptyl isovanillamide si...

Cutaneous exposure to sulfur mustard [bis(2-chloroethyl) sulfide (SM)] produces a delayed inflammatory skin response that is followed by severe dermal injury. Assessment of anti-inflammatory therapies against SM-induced skin injury has mainly relied on qualitative histopathological evaluation. The goal of this study was to identify proinflammatory biomarkers in the hairless mouse vesicant model that could be used as additional indicators of SM-induced skin injury for evaluating anti-inflammatory treatment. SM-induced inflammation was determined at 2, 6, and 24 hr postexposure by changes in edema. Ribonuclease protection assay (RPA) was used to determine changes in gene expression of inflammatory mediators. At 2, 6, and 24 hr postexposure, a time-dependent increase in edema was observed in SM-exposed skin, which was significant at 6 and 24 hr when compared to unexposed controls. Ribonuclease protection assay analysis revealed a two-fold or greater increase in monocyte chemoattractant protein-1 (MCP-1), mac...

Publisher Summary This chapter deals with the cutaneous actions of sulfur mustard (SM) and discusses the basic mechanism of action and mediators involved. While stable in lipophilic solvents, SM has a half life of only 24 min at room temperature in aqueous physiological solutions, since it rapidly reacts with water to form thiodiglycol and HCl. This rapid activation of SM in an aqueous environment also allows it to react with small molecules of biological interest, as well as proteins, carbohydrates, lipids, RNA, and DNA. The initial reaction involves the formation of a cyclic ethylene sulfonium ion, which readily targets reactive groups on skin components, including sulfhydryls, phosphates, ring nitrogens, and carboxyl groups. As a bifunctional alkylating agent, SM forms monofunctional adducts and intra- and intermolecular crosslinks. Since it is so reactive and indiscriminate in its molecular targets, SM affects many pathways and is cytotoxic on many levels. There are three independent mechanisms of cytotoxicity alkylation of DNA/POLY(ADP-RIBOSE) or polymerase (PARP) activation, reactions with glutathione/lipid peroxidation, and reactions with glutathione/calcium homeostasis. Countermeasure research to date has focused primarily on the development of therapeutics, but little attention has been given to the use of advances in drug delivery, or pharmaceutics. Drug delivery by encapsulation of active moieties in nanoparticles has been increasingly used for chemotherapeutic agents such as doxorubicin. However, with the exception of barrier creams, nanoparticle-mediated cutaneous SM detoxification has largely been limited to decontamination

Cutaneous exposure to sulfur mustard [bis(2-chloroethyl) sulfide; SM] produces a delayed inflammatory skin response and severe tissue injury. Pig skin has organ similarities to human skin that is characterized by the content and types of epidermal lipids, the density of hair follicles and presence of sweat glands, which together afford penetration of topically applied compounds, complex inflammatory responses, and subsequent wound healing. The goal of this study was to identify in vivo proinflammatory biomarkers of the SM porcine skin injury within 72 h after SM challenge, using the weanling pig model. Changes in gene expression of inflammatory mediators were examined at 3, 6, 24, 48, and 72 h, using subtraction library analyses and by quantitation of selected transcripts by reverse transcription–polymerase chain reaction (RT–PCR). Sequence analysis of subtraction libraries identified up-regulation of IL-8 at 24, 48, and 72 h. No other specific proinflammatory gene transcripts were isolated from the libraries. Specific transcript RT–PCR analysis showed increased production of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and matrix metalloproteinase-9 (MMP-9, gelatinase B) mRNA levels in response to SM exposure. Tumor necrosis factor-α (TNF-α) expression was only slightly increased and no change in the levels of expression was observed for monocyte chemoattractant protein-1 and MMP-2. This study identifies the main proinflammatory mediators involved in SM-induced skin injury in a weanling pig model. The results suggest transcriptional activity in the inflammatory response proteins IL-8, IL-6, IL-1β, and MMP-9 and modest changes in TNF-α that together produce inflammation and contribute to the pathogenesis of SM dermatotoxicity. Therefore, drugs preventing SM-induced inflammation should be prime candidates for medical intervention to lessen collateral inflammation associated with tissue destruction. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:263–272, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10050

The CD1-haired mouse and the SKH-hairless mouse are two animal models that have been used to evaluate sulfur mustard (HD) exposure and protection in our laboratory. In a recent study we observed that a substance P inhibitor protected the haired mouse ear against an HD solution, but the same drug was not successful in protecting the hairless mouse against HD vapor. This experiment prompted us to compare HD exposures between these models. We determined the (14)C content in the skin after exposures to HD containing (14)C-HD. Rate curves were generated for applications of (1) HD in methylene chloride to the haired mouse ear; (2) HD in methylene chloride to the hairless mouse dorsal skin; and (3) saturated HD vapor to the hairless mouse dorsal skin for 6 min. The curves showed a reduction in (14)C disintegrations per min in animals euthanized 0 to 2 h postexposure. The largest percentage of decrease of (14)C content in skin occurred within 30 min of HD challenge for all exposures. An 8-mm skin-punch biopsy and a 14-mm annular skin section surrounding the region of the 8-mm skin punch were taken from the hairless mouse dorsal skin exposed to HD in methylene chloride. The ratio of the (14)C content in the 8-mm skin punch to that in the surrounding 14-mm annular skin section was 7.3, demonstrating that the HD application spreads beyond the initially biopsied site. A concentration/time value of 6.3 mug/cm(2)/min was determined by counting skin (14)C disintegrations per minute in animals euthanized immediately after exposure to saturated HD vapor. Determinations of the amount of HD showed that similar quantities of HD, 0.4 mg, were detected on each model. These results contribute to a better quantitative understanding of HD application in the haired and hairless mouse models.

Sulfur mustard (bis(2-chloroethyl) sulfide, SM) is a highly reactive bifunctional alkylating agent inducing edema, inflammation, and the formation of fluid-filled blisters in the skin. Medical countermeasures against SM-induced cutaneous injury have yet to be established. In the present studies, we tested a novel, bifunctional anti-inflammatory prodrug (NDH 4338) designed to target cyclooxygenase 2 (COX2), an enzyme that generates inflammatory eicosanoids, and acetylcholinesterase, an enzyme mediating activation of cholinergic inflammatory pathways in a model of SM-induced skin injury. Adult SKH-1 hairless male mice were exposed to SM using a dorsal skin vapor cup model. NDH 4338 was applied topically to the skin 24, 48, and 72 hr post-SM exposure. After 96 hr, SM was found to induce skin injury characterized by edema, epidermal hyperplasia, loss of the differentiation marker, keratin 10 (K10), upregulation of the skin wound marker keratin 6 (K6), disruption of the basement membrane anchoring protein laminin 322, and increased expression of epidermal COX2. NDH 4338 post-treatment reduced SM-induced dermal edema and enhanced skin re-epithelialization. This was associated with a reduction in COX2 expression, increased K10 expression in the suprabasal epidermis, and reduced expression of K6. NDH 4338 also restored basement membrane integrity, as evidenced by continuous expression of laminin 332 at the dermalepidermal junction. Taken together, these data indicate that a bifunctional anti-inflammatory prodrug stimulates repair of SM induced skin injury and may be useful as a medical countermeasure.

The mouse ear vesicant model (MEVM) provides a quantitative edema response as well as histopathological and biochemical endpoints as measurements of inflammation and tissue damage following exposure to the chemical warfare agent sulfur mustard (HD). In the MEVM, several topically applied anti-inflammatory agents provided a significant degree of protection against HD-induced edema and dermal-epidermal separation. This study evaluated the protective effects of three of these pharmacological compounds when administered systemically in the MEVM. Alzet osmotic pumps were used to deliver a subcutaneous dose of the appropriate anti-inflammatory agent, starting 24 h before exposure to sulfur mustard and continuing until 24 h post-exposure to HD. Twenty-four hours after pump implantation, 5 μl of a 195 mM (0.16 mg) solution of sulfur mustard (density = 1.27 g ml -1 ; MW = 159; purity = 97.5%) in methylene chloride was applied to the inner surface of the right ear of each mouse. Sulfur mustard injury in the mouse ear was measured by both edema response (fluid accumulation) and histopathological damage (necrosis, epidermal-dermal separation). The systemic administration of hydrocortisone, indomethacin and olvanil provided a significant reduction in edema (24%, 26% and 22%, respectively) from the positive control. Compared to HD-positive controls, hydrocortisone, indomethacin and olvanil caused a significant reduction in subepidermal blisters (71%, 52% and 57%, respectively) whereas only hydrocortisone produced a significant reduction in contralateral epidermal necrosis (41%). We show here that these anti-inflammatory drugs are effective when administered systemically in the MEVM.

Tissue homogenates from mouse ear skin exposed to sulfur mustard (HD, which is a military designation and probably originated from a World War I slang term 'Hun Stuff') were assayed for serine and cysteine protease activities. Enzyme activity was measured using synthetic chromogenic thioester and fluorogenic 7-amino-4-methylcoumarin (AMC) substrates. The tissue samples were obtained from animals (n = 6) at 3, 6, 12 and 24 h post-exposure from the right ear (HD exposed), whereas control samples were obtained from the left ear (treated only with dichloromethane vehicle). The samples of naive control (left and right ear) were obtained from animals that received no HD treatment (n = 3). Elastase activity was assayed with t-butyloxycarbonyl-Ala-Ala-Ala-thiobenzylester, tryptase activity with benzyloxycarbonyl-Arg-AMC and benzyloxycarbonyl-Arg-thiobenzylester, chymase activity with succinylAla-Ala-Pro-Phe-thiobenzylester and succinyl-Ala-Ala-Pro-Phe-AMC, cathepsin B activity with benzyloxycarbonyl-Arg-Arg-AMC, cathepsin H activity with Arg-AMC and calpain activity with succinyl-Leu-Tyr-AMC. The HD-exposed skin homogenates obtained at 12 and 24 h post-exposure had higher elastase activity (670% and 1900% increase) than control samples. For tryptase and calpain activities, only HD-exposed skin homogenates at 24h post-exposure showed higher activities (220% and 170% increase) when compared to the control. No differences from control were observed for HD-exposed skin obtained at 3 and 6 h post-exposure for elastase, tryptase and calpain activities. Generally, both unexposed and HD-exposed skin had distinct cathepsin B and cathepsin H enzyme activities and small chymase activity. Enzymatic assays were also performed for other serine, cysteine and metalloproteases. These data document that proteases are involved in HD skin injury and continued assessment of proteolytic activity should be useful for identifying effective antiproteases with therapeutic use in reducing or eliminating tissue injury caused by HD cutaneous exposure.

The mouse ear edema model is recognized for its usefulness in studying skin responses and damage following exposure to chemical irritants, and for evaluating pharmacological agents against chemically induced skin injury. We recently modified the mouse ear edema model for use with sulfur mustard (HD) and used this model to study the protective effect of 33 topically applied compounds comprising five pharmaceutical strategies (anti-inflammatories, protease inhibitors, scavengers/chelators, poly(ADP-ribose) polymerase (PARP) inhibitors, calcium modulators/chelators) against HD-induced dermatotoxicity. Pharmacological modulation of HD injury in mouse ears was established by a reduction in edema or histopathology (epidermal necrosis and epidermal-dermal separation) at 24 h following topical liquid HD exposure. Ten of the 33 compounds administered as single topical pretreatments up to 2 h prior to HD challenge produced significant reductions in edema. Five of these ten also produced significant reductions in histological endpoints. Three candidates (olvanil, indomethacin, hydrocortisone) showing protection at 24 h were evaluated further for 'extended protection' at 48 and 72 h after HD challenge and showed significant modulation of edema at 48 h but not at 72 h. Olvanil also showed significant reductions in histology at 48 and 72 h. Olvanil and indomethacin were shown to reduce significantly the edema at 24 h post-exposure when administered topically 10 min after HD challenge, with olvanil additionally protecting against epidermal necrosis. These results demonstrate prophylactic and treatment effects of pharmacological agents against HD-induced skin injury in an in vivo model and support the continued use of the mouse ear vesicant model (MEVM) for evaluating medical countermeasures against HD.

Cutaneous exposure to sulfur mustard (bis(2-chloroethyl) sulfide, HD), a chemical warfare agent, produces a delayed inflammatory skin response and severe tissue injury. Despite defined roles of inflammatory cytokines produced or released in response to skin-damaging chemicals, in vivo cytokine responses associated with HD-induced skin pathogenesis are not well understood. Additionally, there is little information on the in vivo temporal sequence of gene expression of cytokines postexposure to HD. The goal of these studies was to identify in vivo molecular biomarkers of HD skin injury within 24 hours after HD challenge. Gene expression of interleukin 1beta (IL-1beta), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6), and interleukin 1alpha (IL-1alpha) in the mouse ear vesicant model was examined by quantitative reverse transcription-polymerase chain reaction (RT-PCR). An increase in IL-1beta mRNA levels was first observed at 3 hours. IL-1beta, GM-CSF, and IL-6 mRNA levels were dramatically increased at 6-24 hours postexposure. IL-1alpha mRNA levels were not increased following HD exposure. Immunohistochemical studies demonstrated that IL-1beta and IL-6 protein was produced at multiple sites within the ear, including epithelial cells, inflammatory cells, hair follicles, sebaceous glands, the dermal microvasculature, smooth muscle, and the dermal connective tissue. An increase in the intensity of staining for IL-1beta, and IL-6 was observed in localized areas at 6 hours and was evident in multiple areas at 24 hours. Positive staining for GM-CSF immunoreactive protein was localized to the inflammatory cells within the dermis. The number of immunostaining cells was increased as early as 1 hour following HD exposure. These studies document an early increase in the in vivo expression of inflammatory cytokines following cutaneous HD exposure. An understanding of the in vivo cytokine patterns following HD skin exposure may lead to defining the pathogenic mechanisms of HD injury and the development of pharmacological countermeasures.

Inflammatory reactions associated with sulfur mustard (bis(2-chloroethyl)sulfide, SM)-induced skin pathology, including the release of potent proteolytic enzymes capable of destroying cutaneous connective tissue proteins, are not well defined in vivo. In this study, protease activities were measured as potential indicators of SM-induced skin injury in a hairless mouse sulfur mustard vapor exposure model. We also investigated the effects of synthetic protease inhibitors on SM-induced proteolytic activity. Skin homogenates prepared from hairless mouse dorsal skin exposed to SM vapor (1.4 g/m3) were assayed for serine and cysteine protease activities using synthetic chromogenic and fluorogenic substrates. Skin samples were obtained 24 h after SM challenge from animals exposed for either 6 or 12 min, while controls were obtained from unexposed skin. With 6 min SM-exposed skin, significantly higher elastase, tryptase, cathepsin B, cathepsin L, and calpain II enzymatic activities were detected compared ...

Sulfur mustard (bis(2-chloroethyl) sulfide (HD)), a potent cutaneous vesicant and bifunctional alkylating agent, produces significant time-dependent histopathological changes in the skin of the mouse. The right ears of male CD1 mice were exposed topically to 5.0 microl of 195 mM (0.16 mg) HD in dichloromethane and harvested at 6, 12, 18 and 24 h. The left ear control was dosed with 5.0 microl of dichloromethane. In all controls and HD-treated mouse ear, moderate immunofluorescence staining was seen at the epidermal-dermal junction with bullous pemphigoid (BP), epidermolysis bullosa acquisita (EBA) and laminin (Lam), and light staining was observed with bullous pemphigoid 180 (BP180), fibronectin (Fn) and type IV collagen (Coll IV). Mouse anti-human monoclonal antibodies for GB3, L3d and 19-DEJ-1 (Uncein) did not cross-react. In microvesicles, BP, BP180 and Fn showed areas of light focal epidermal staining and homogeneous dermal staining, and EBA, Lam and Coll IV showed moderate dermal staining. Both BP and Fn exhibited weak, inconsistent staining with time. Immunoelectron microscopy (IEM) revealed similar results, with an increase in cell damage from 6 to 24 h, which corresponded to a decrease in staining intensity. Cell proliferation, expressed as the growth fraction of proliferating cell nuclear antigen (PCNA), showed an increase in cell damage. The growth fraction was lower in the inner ear and showed time-dependent differences. The immunofluorescence and IEM results indicate that HD causes an undulating inconsistent separation in the uppermost lamina lucida with focal cleavage into the lower portion of the basal keratinocytes just above the plasma membrane. Although this pattern of separation differs from other in vivo models in which the split occurs exclusively within the lamina lucida, this should not preclude its role as a screening model to study the effects and development of specific prophylactic and therapeutic strategies.

Sulfur mustard (SM) is a bifunctional alkylating agent causing skin inflammation, edema and blistering. A hallmark of SM-induced toxicity is follicular and interfollicular epithelial damage. In the present studies we determined if SM-induced structural alterations in hair follicles and sebaceous glands were correlated with cell damage, inflammation and wound healing. The dorsal skin of hairless mice was treated with saturated SM vapor. One to seven days later, epithelial cell karyolysis within the hair root sheath, infundibulum and isthmus were apparent, along with reduced numbers of sebocytes. Increased numbers of utriculi, some with connections to the skin surface, and engorged dermal cysts were also evident. This was associated with marked changes in expression of markers of DNA damage (phospho-H2A.X), apoptosis (cleaved caspase-3), and wound healing (FGFR2 and galectin-3) throughout pilosebaceous units. Conversely, fatty acid synthase and galectin-3 were down-regulated in sebocytes after SM. Decreased numbers of hair follicles and increased numbers of inflammatory cells surrounding the utriculi and follicular cysts were noted within the wound 3–7 days post SM exposure. Expression of phospho-H2A.X, cleaved caspase-3, FGFR2 and galectin-3 was decreased in dysplastic follicular epidermis. Fourteen days after SM, engorged follicular cysts which expressed galectin-3 were noted within hyperplastic epidermis. Galectin-3 was also expressed in basal keratinocytes and in the first few layers of suprabasal keratinocytes in neoepidermis formed during wound healing indicating that this lectin is important in the early stages of keratinocyte differentiation. These data indicate that hair follicles and sebaceous glands are targets for SM in the skin.

The mouse ear inflammation model was used to establish simple endpoints of skin injury following cutaneous exposure to sulfur mustard (bis(2- chloroethyl)sulfide, HD). Mouse ear edema and histopathologic response to a single topical application of 5 muL of 8, 16, 32, 64, or 128 mg/mL HD in dichloromethane was evaluated at 12, 18, and 24 h postexposure. Edema response was determined from weighed 8 mm diameter skin punch biopsies taken from the center of exposed and control ears. Key light microscopic histopathologic changes assessed on the inner (exposed side) and outer surfaces (contralateral side) of the exposed ear included epidermal necrosis (EN) and epidermal-dermal separation (subepidermal blister, SEB). HD produced statistically significant (p

Three filamentous fungi were examined for the ability to biotransform phenanthrene to oxidative (phase I) and conjugative (phase II) metabolites. Phenanthrene metabolites were purified by high-performance liquid chromatography (HPLC) and identified by UV/visible absorption, mass, and 1H NMR spectra. Aspergillus niger ATCC 6275, Syncephalastrum racemosum UT-70, and Cunninghamella elegans ATCC 9245 initially transformed [9-(14)C]phenanthrene to produce metabolites at the 9,10-, 1,2-, and 3,4-positions. Subsequently, sulfate conjugates of phase I metabolites were formed by A. niger, S. racemosum, and C. elegans. Minor glucuronide conjugates of 9-phenanthrol and phenanthrene trans-9, 10-dihydrodiol were formed by S. racemosum and A. niger, respectively. In addition, C. elegans produced the glucose conjugates 1-phenanthryl beta-D-glucopyranoside and 2-hydroxy-1-phenanthryl beta-D-glucopyranoside, a novel metabolite. [9-(14)C]Phenanthrene metabolites were not detected in organic extracts from biotransformation experiments with the yeasts, Candida lipolytica 37-1, Candida tropicalis ATCC 32113, and Candida maltosa R-42.

The endoplasmic reticulum (ER) stress response is a cell survival pathway upregulated when cells are under severe stress. Severely damaged mouse ear skin exposed to the vesicant, sulfur mustard (bis-2-chloroethyl sulfide, SM), resulted in increased expression of ER chaperone proteins that accompany misfolded and incorrectly made proteins targeted for degradation. Time course studies with SM using the mouse ear vesicant model (MEVM) showed progressive histopathologic changes including edema, separation of the epidermis from the dermis, persistent inflammation, upregulation of laminin γ2 (one of the chains of laminin-332, a heterotrimeric skin glycoprotein required for wound repair), and delayed wound healing from 24 h to 168 h post exposure. This was associated with time related increased expression of the cell survival ER stress marker, GRP78/BiP, and the ER stress apoptosis marker, GADD153/CHOP, suggesting simultaneous activation of both cell survival and non-mitochondrial apoptosis pathways. Dual immunofluorescence labeling of a keratinocyte migration promoting protein, laminin γ2 and GRP78/BIP, showed colocalization of the two molecules 72 h post exposure indicating that the laminin γ2 was misfolded after SM exposure and trapped within the ER. Taken together, these data show that ER stress is induced in mouse skin within 24 h of vesicant exposure in a defensive response to promote cell survival; however, it appears that this response is rapidly overwhelmed by the apoptotic pathway as a consequence of severe SM-induced injury.

Sulfur mustard (SM, bis(2-chloroethyl)sulfide) is a bifunctional alkylating agent that causes dermal inflammation, edema and blistering. To investigate the pathogenesis of SM-induced injury, we used a vapor cup model which provides an occlusive environment in which SM is in constant contact with the skin. The dorsal skin of SKH-1 hairless mice was exposed to saturated SM vapor or air control. Histopathological changes, inflammatory markers and DNA damage were analyzed 1–14 days later. After 1 day, SM caused epidermal thinning, stratum corneum shedding, basal cell karyolysis, hemorrhage and macrophage and neutrophil accumulation in the dermis. Cleaved caspase-3 and phosphorylated histone 2A.X (phospho-H2A.X), markers of apoptosis and DNA damage, respectively, were increased whereas proliferating cell nuclear antigen (PCNA) was down-regulated after SM exposure. By 3 days, epithelial cell hypertrophy, edema, parakeratosis and loss of epidermal structures were noted. Enzymes generating pro-inflammatory mediators including myeloperoxidase and cyclooxygenase-2 were upregulated. After 7 days, keratin-10, a differentiation marker, was evident in the stratum corneum. This was associated with an underlying eschar, as neoepidermis began to migrate at the wound edges. Trichrome staining revealed increased collagen deposition in the dermis. PCNA expression in the epidermis was correlated with hyperplasia, hyperkeratosis, and parakeratosis. By 14 days, there was epidermal regeneration with extensive hyperplasia, and reduced expression of cleaved caspase-3, cyclooxygenase-2 and phospho-H2A.X. These findings are consistent with the pathophysiology of SM-induced skin injury in humans suggesting that the hairless mouse can be used to investigate the dermatoxicity of vesicants and the potential efficacy of countermeasures.

A preparation procedure for making multiple inflammatory biomarker measurements from the same skin tissue was assessed. The backs of euthymic hairless mice were exposed to sulfur mustard (HD) vapor for 6 min. Animals were euthanized 24 h following exposure, dorsal skin tissue was excised and 12-mm, full-thickness biopsy punches of the exposed skin sites were taken. Specimens were snap-frozen, crushed to a powder using a biopulverizer unit, solubilized in buffer and centrifuged. Supernatant was assayed for pro-inflammatory cytokines and the acute-phase reactive protein, serum amyloid P (SAP). Myeloperoxidase (MPX), which is indicative of neutrophil infiltration into the skin, was associated with the pellet fraction. Results indicate an elevation of interleukin-6, SAP and MPX in mouse skin tissue specimens 24 h following HD vapor exposure. The tissue preparation procedure allows the use of a single skin specimen to make multiple inflammatory endpoint measurements requiring different preparation processes, and it will be used in subsequent studies to characterize further the inflammatory nature of HD-exposed skin tissue.

a r t i c l e i n f o Article history: Sulfur mustard is a potent vesicant that induces in! ammation, edema and blistering following dermal exposure. To assess molecular mechanisms mediating these responses, we analyzed the effects of the model sulfur mustard vesicant, 2-chloroethyl ethyl sul" de, on EpiDerm-FT™, a commercially available full-thickness human skin equivalent. CEES (100-1000 ! M) caused a concentration-dependent increase in pyknotic nuclei and vacuolization in basal keratinocytes; at high concentrations (300-1000 ! M), CEES also disrupted keratin " lament architecture in the stratum corneum. This was associated with time-dependent increases in expression of proliferating cell nuclear antigen, a marker of cell proliferation, and poly(ADP-ribose) polymerase (PARP) and phosphorylated histone H2AX, markers of DNA damage. Concentration- and time- dependent increases in mRNA and protein expression of eicosanoid biosynthetic enzymes including COX-2, 5- lipoxygenase, microsomal PGE2 synthases, leukotriene (LT) A4 hydrolase and LTC4 synthase were observed in CEES-treated skin equivalents, as well as in antioxidant enzymes, glutathione S-transferases A1-2 (GSTA1-2), GSTA3 and GSTA4. These data demonstrate that CEES induces rapid cellular damage, cytotoxicity and in! ammation in full-thickness skin equivalents. These effects are similar to human responses to vesicants in vivo and suggest that the full thickness skin equivalent is a useful in vitro model to characterize the biological effects of mustards and to develop potential therapeutics.

Dermal exposure to sulfur mustard causes inflammation and tissue injury. This is associated with changes in expression of antioxidants and eicosanoids which contribute to oxidative stress and toxicity. In the present studies we analyzed mechanisms regulating expression of these mediators using an in vitro skin construct model in which mouse keratinocytes were grown at an air-liquid interface and exposed directly to 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. CEES (100-1000 microM) was found to cause marked increases in keratinocyte protein carbonyls, a marker of oxidative stress. This was correlated with increases in expression of Cu,Zn superoxide dismutase, catalase, thioredoxin reductase and the glutathione S-transferases, GSTA1-2, GSTP1 and mGST2. CEES also upregulated several enzymes important in the synthesis of prostaglandins and leukotrienes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-2 (mPGES-2), prostaglandin D synthase (PGDS), 5-lipoxygenase (5-LOX), leukotriene A(4) (LTA(4)) hydrolase and leukotriene C(4) (LTC(4)) synthase. CEES readily activated keratinocyte JNK and p38 MAP kinases, signaling pathways which are known to regulate expression of antioxidants, as well as prostaglandin and leukotriene synthases. Inhibition of p38 MAP kinase suppressed CEES-induced expression of GSTA1-2, COX-2, mPGES-2, PGDS, 5-LOX, LTA(4) hydrolase and LTC(4) synthase, while JNK inhibition blocked PGDS and GSTP1. These data indicate that CEES modulates expression of antioxidants and enzymes producing inflammatory mediators by distinct mechanisms. Increases in antioxidants may be an adaptive process to limit tissue damage. Inhibiting the capacity of keratinocytes to generate eicosanoids may be important in limiting inflammation and protecting the skin from vesicant-induced oxidative stress and injury.

Sulfur mustard (SM), a chemical weapon first employed during World War I, targets the skin, eyes, and lung. It remains a significant military and civilian threat. The characteristic response of human skin to SM involves erythema of delayed onset, followed by edema with inflammatory cell infiltration, the appearance of large blisters in the affected area, and a prolonged healing period. Several in vivo and in vitro models have been established to understand the pathology and investigate the mechanism of action of this vesicating agent in the skin. SM is a bifunctional alkylating agent which reacts with many targets including lipids, proteins, and DNA, forming both intra- and intermolecular cross-links. Despite the relatively nonselective chemical reactivity of this agent, basal keratinocytes are more sensitive, and blistering involves detachment of these cells from their basement membrane adherence zones. The sequence and manner in which these cells die and detach is still unresolved. Much has been discovered over the past two decades with respect to the mechanisms of SM-induced cytotoxicity and the intracellular and extracellular targets of this vesicant. In this review, the effects of SM exposure on the skin are described, as well as potential mechanisms mediating its actions. Successful therapy for SM poisoning will depend on following new mechanistic leads to develop drugs that target one or more of its sites of action.

The chemical warfare agent sulfur mustard (SM) produces blister formation with a severe inflammatory reaction in skin of exposed individuals. The development of efficacious countermeasures against SM vesication requires an understanding of the cellular and molecular mechanism of SM-induced tissue injury. This study examined SM-induced alterations in gene expression using Atlas Mouse 5K DNA microarrays (5002 genes) to identify transcriptional events associated with SM skin injury. Mice (N=3) were exposed topically to SM (0.04, 0.08, and 0.16 mg; 48.8, 97.5, and 195 mM) on the inner surface of the right ear and skin tissues were harvested at 1.5, 3, 6, and 12 h. Genes were selected based on the three mice in the same dose group demonstrating a > or =2-fold increase or decrease in gene expression for the SM-exposed tissue when compared to the dichloromethane vehicle control ear at all three doses and four time points. At the 0.04 mg SM dose, the genes observed were primarily involved in inflammation, apoptosis, and cell cycle regulation. Exposure to 0.08 mg SM increased the expression of genes related to inflammation and cell cycle regulation. Exposure to 0.16 mg SM led to a total of six genes that were changed at all observed time periods; however, these genes do not appear to be directly influential in biological mechanisms such as inflammation, apoptosis, and cell cycle regulation as was observed at the lower SM doses of 0.04 and 0.08 mg. These functional categories have been observed in previous studies utilizing both in vivo and in vitro model systems of SM-induced dermal injury, suggesting that molecular mechanisms associated with inflammation, apoptosis, and cell cycle regulation may be appropriate targets for developing prophylactic/therapeutic treatments for SM skin injury.

Sulfur mustard [bis(2-chloroethyl)sulfide; SM] is a chemical warfare agent that produces edema and blister formation with a severe inflammatory reaction. The mouse ear vesicant model for SM injury has been used to evaluate pharmacological agents for countering SM dermal injury. The vanilloid olvanil reduces SM-induced edema and mRNA expression of cytokines and chemokines, suggesting that blocking the inflammatory effects of neuropeptides, such as substance P (SP), may provide protection against SM-induced dermal injury. This study examined SP expression in mice exposed to SM (0.16 mg) on the inner surface of the right ear, with or without olvanil pretreatment at 1, 10, 30, 60, and 360 min following exposure. In naive skin, SP mRNA localization was associated with blood vessels and sebaceous glands. In SM-exposed skin, SP mRNA was also detected in perivascular dermal cells. Immunohistochemical localization of SP protein was observed in the ear skin of naive, SM-, olvanil/SM-, and vehicle-treated mice. Quantification of SP+ perivascular dermal cells revealed that SM exposure led to a significant increase (P < or = 0.05) in SP+ cells over the observed time period. Olvanil pretreatment significantly reduced (P < or = 0.05) the mean number of SP+ cells at 60 and 360 min. This study demonstrates that SP expression could provide an additional endpoint for evaluating the effectiveness of vanilloid drugs on SM-induced skin inflammation.

Previous studies have shown the antidotal efficacy of topical iodine at 15 and 30 min post-exposure to sulfur mustard (SM). Here we demonstrate efficacy at longer intervals (20, 30, 45, and 60 min, respectively, for data) using an improved topical povidone-iodine preparation termed N66, which contains steroidal and non-steroidal anti-inflammatory agents. In the mouse, N66 reduced severity of ear edema by 43, 47, 44, and 36%; ear epidermal ulceration by 74, 58, 45, and 58%; and epidermal necrosis by 54, 34, 26, and 31% at the respective time points. A similar effect was observed with encrustation. The healing marker, grade of acanthotic area, showed dramatic increases of 39.6-, 25.3-, 20.9-, and 22-fold. Severity of the dermal parameters, acute inflammation and dermal necrosis, was reduced by 63, 34, 34, and 38% and 80, 54, 54, and 59%, respectively. In guinea pig skin, topical treatment with N66 45 min post-exposure reduced the SM-induced ulceration area by 75%. The histological parameters subepidermal microblister formation, epidermal ulceration, epidermal necrosis, and encrustation were reduced by 63, 61, 41, and 41%, respectively. The healing marker, grade of acanthotic area, was elevated by 73%. N66 induced a statistically significant reduction in two dermal markers for tissue damage: acute inflammation (33%) and dermal necrosis (48%). Reduced skin damage was also observed in areas adjacent the treated sites. The pharmacologically active components of N66 showed additive effect. These findings suggest that the povidone-iodine preparation combined with anti-inflammatory agents functions as a potent antidote against skin lesions induced by SM at relatively long intervals between exposure and treatment.

In an effort to understand the pathophysiology of sulfur mustard (2,2' dichlorodiethyl sulfide, HD)-induced cutaneous lesions, a number of animal models have been used. Animal models have been and will continue to be used in the development of therapeutic strategies to protect against and/or moderate lesions, and to potentiate wound healing after HD exposure. Upon reviewing the histopathologic features seen after HD-exposure, we propose roles for different animal models in HD-research. Hematoxylin and eosin slides from protocols done originally as dose response studies for either liquid or vapor HD-exposures were examined. The animal models reported include the hairless guinea pig (HGP), weanling pig (WP), mouse ear (ME) and hairless mouse (HM). In all these animal models. HD induces subepidermal blister formation as well as epidermal cell death. The HGP appears to be the most sensitive model for epidermal necrosis. The HGP and, to a lesser degree, the HM react with a marked neutrophilic infiltrate. The ME provides a quantitative measure for HD effects and a mild inflammatory infiltrate similar to what is seen in human skin. Doses necessary to produce microblister formation in the WP are usually associated with more significant stromal and vascular changes than in other animal models. In addition to a quantitative measure of the HD effect and a mild inflammatory response, the cost, as well as the availability of specific antibodies, and DNA and RNA probes and primers gives the ME advantages for both drug screening and for the study of the pathophysiology of HD-induced cutaneous lesions. The sensitivity of the HGP and the abundant experience with vapor exposures establishes a place for this animal model in barrier cream and drug screening. The similarity of WP skin to human skin is important in the study of wound healing after HD exposure, as well as in the study of the pathophysiology of the cutaneous lesion and in more definitive therapeutic studies.

The trans -dihydrodiols produced during the metabolism of phenanthrene by Cunninghamella elegans, Syncephalastrum racemosum, and Phanerochaete chrysosporium were purified by high-performance liquid chromatography (HPLC). The enantiomeric compositions and optical purities of the trans -dihydrodiols were determined to compare interspecific differences in the regio- and stereoselectivity of the fungal enzymes. Circular dichroism spectra of the trans -dihydrodiols were obtained, and the enantiomeric composition of each preparation was analyzed by HPLC with a chiral stationary-phase column. The phenanthrene trans -1,2-dihydrodiol produced by C. elegans was a mixture of the 1 R ,2 R and 1 S ,2 S enantiomers in variable proportions. The phenanthrene trans -3,4-dihydrodiol produced by P. chrysosporium was the optically pure 3 R ,4 R enantiomer, but that produced by S. racemosum was a 68:32 mixture of the 3 R ,4 R and 3 S ,4 S enantiomers. The phenanthrene trans -9,10-dihydrodiol produced by P. chrysosporium was predominantly the 9 S ,10 S enantiomer, but those produced by C. elegans and S. racemosum were predominantly the 9 R ,10 R enantiomer. The results indicate that although different fungi may exhibit similar regioselectivity, there still may be differences in stereoselectivity that depend on the species and the cultural conditions.