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-1beta (IL-1beta), 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-alpha (TNF-alpha) 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-1beta, and MMP-9 and modest changes in TNF-alpha 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.

Honey bees readily associate an odor with sucrose reinforcement, and the response generalizes to other odors as a function of structural similarity to the conditioned odor. Recent studies have shown that a portion of odor memory is consolidated in the antennal lobes (AL), where first-order synaptic processing of sensory information takes place. The AL and/or the sensory afferents that project into them show staining patterns for the enzyme nitric oxide synthase, which catalyzes the release of the gaseous transmitter nitric oxide (NO). The results show that pharmacological blockade of NO release impairs olfactory discrimination only when release is blocked before conditioning. Blockade of GABAergic transmission disrupts discrimination of similar but not dissimilar odorants, and does so when the block occurs before condition or before testing. These results show that GABA and NO regulate the specificity of associative olfactory memory in the AL.

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 Cutaneous and Ocular Toxicology Downloaded from informahealthcare.com by University of Montreal on 12/01/14For personal use only.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), macrophage inflammatory protein-2 (MIP-2), MIP-1a, tumor necrosis factor-a, and interleukin (IL)-1b following exposure to SM when compared to unexposed controls. A significant time-dependent increase was observed in MCP-1, MIP-1a, and IL-1b over the 24 hr time period. At 24 hr postexposure, skin treated with the anti-inflammatory drug olvanil showed a significant decrease in SM-induced edema. Additionally, mRNA levels of MCP-1, MIP-2, and IL1b were decreased when compared to skin exposed to SM alone. In this study, we identified molecular biomarkers at the mRNA level, up-regulated in skin exposed to SM, which can be partially suppressed by olvanil. Further characterization of the mRNA and protein expression patterns of proinflammatory biomarkers may enable the use of other classes of anti-inflammatory drugs or therapeutic treatments against SM dermal injury.

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information SPONSOR/MONITOR'S ACRONYM(S) USAMRICD SPONSOR/MONITOR'S REPORT NUMBER(S)USAMRICD-TR-01-04 DISTRIBUTION / AVAILABILITY STATEMENTApproved for public release; distribution unlimited SUPPLEMENTARY NOTES ABSTRACTVesicants, such as sulfur mustard (HD), pose an insidious threat to the respiratory tract. Exposure causes acute lung injury characteristic of an inflammatory response. Nuclear Factor-KAPPA B (NF-DB) is a redox sensitive transcription factor important in regulating genes involved in the inflammation. This study evaluated levels of NF-DB following exposure to HD. Male rats were euthanized at 3,6, or 24 hr following intravenous HD or isopropanol (IPA controls) exposure. Nuclear protein samples extracted from pulmonary tissue were analyzed using an electrophoretic mobility shift assay. Bronchoalveolar lavage fluid (BALF) protein concentrations and lactate dehydrogenase (LDH) were determined to indicate lung injury. NF-DB in HD lungs was not detected at a level significantly above that found in IPA samples. LDH was higher than in the IPA-treated group, p<.013, at 24 h. Protein concentrations were higher at 24 h compared with 3 and 6 h, p<.05. In conclusion, i.v. HD causes lung injury at 24 h as measured by BALF protein and LDH low-level inflammatory response related to time but not to dose. This model may be useful in developing anti-inflammatory treatments against HD exposure. These data suggest that alterations in NF-DB may not play a role in HD-induced lung injury in rodents intravenously exposed to HD. SUBJECT TERMSnuclear factor-AAP/M B; sulfur mustard; HD; pulmonary tissue; electrophoretic mobility shift assay, bronchoalveolar lavage fluid, lactate dehydrogenase, protein