Project description:Sulfur mustard (SM) is a potent alkylating agent. We are developing medical countermeasures to reduce the injury caused by SM exposure. Screening in the mouse ear vesicant model has identified three effective compounds: dimercaprol (British anti-lewisite), indomethacin, and octyl homovanillamide (OHV). To identify gene expression changes that correlate with compound efficacy we used oligonucleotide microarrays to compare gene expression profiles in vehicle-exposed skin, SM-exposed skin, and skin pretreated with each compound before SM exposure. Mice were topically exposed on the inner surface of the right ear to SM alone or pretreated for 15 min with one of the compounds and then exposed to SM. Left ears were vehicle-exposed. Ear tissue was harvested 24 hr later for ear weight determination (an endpoint indicating compound efficacy). The exposure groups were: methylene chloride (sulfur mustard vehicle); ethanol (drug vehicle); 0.08 mg sulfur mustard; 6.25 mg dimercaprol 15 min before 0.08 mg sulfur mustard; 1.34 mg indomethacin 15 min before 0.08 mg sulfur mustard; 0.6 mg octylhomovanillamide 15 min before 0.08 mg sulfur mustard; 6.25 mg dimercaprol alone; 1.34 mg indomethacin alone; 0.6 mg octylhomovanillamide alone. RNA was extracted from the tissues and used to generate oligonucleotide microarray probes. Principal component analysis of the gene expression data revealed partitioning of the samples based on drug treatment and SM exposure. Vehicle-exposed mouse ears clustered away from the other treatment groups. SM-exposed mouse ears pretreated with dimercaprol or OHV clustered more closely with vehicle-exposed ears, while SM-exposed mouse ears pretreated with indomethacin clustered more closely with SM-exposed ears. This clustering of the samples is supported by the ear weight data, in which the indomethacin group has ear weights closer to the SM-exposed group, whereas the dimercaprol and OHV groups have ear weights closer to the vehicle-exposed group. Correlation coefficients were calculated for each gene based on the correlation between gene expression level and ear weight. These data provide the basis for understanding what gene expression changes are important in the development of effective SM medical countermeasures. Experiment Overall Design: Exposure of mouse ears to sulfur mustard alone, sulfur mustard preceded by drug treatment, or vehicle compounds. Naive controls were also included. Biological replicates of at least n=3 were examined for each exposure condition.

Project description:Bis-2-chloroethyl sulfide (sulfur mustard, SM) is a potent alkylating agent and vesicant. Exposure to SM results in activation of numerous signaling cascades, including mitogen-activated protein kinase (MAPK) signaling pathways. These pathways include the Erk, p38, and JNK pathways, which are involved in cell growth, inflammation, and stress signaling. However, the precise roles of these pathways in SM toxicity have not been fully elucidated. We used Western blotting and microarray analysis to examine the activation and role of each pathway following SM exposure in primary human epidermal keratinocytes. Western blotting revealed increased phosphorylation of p38 and JNK following SM exposure; however, phosphorylation of Erk was equivocal, suggesting that growth conditions may impact activation of Erk by SM. We used pharmacologic inhibitors to target each MAPK and then compared the gene expression profiles to identify SM-induced gene networks regulated by each MAPK. Cells were pretreated with 10 µM SB 203580 (p38 inhibitor), PD 98059 (Erk inhibitor), or SP 600125 (JNK inhibitor) 60 minutes before exposure to 200 µM SM. Cells were harvested at 1h, 4h, and 8h post-exposure, and RNA was extracted for synthesis of microarray probes. Probes were hybridized to Affymetrix U133 Plus 2.0 arrays for gene expression profiling. Analysis of variance was performed to identify genes significantly modulated due to pharmacologic inhibition in SM-exposed cells. Pathway mapping confirmed alterations in SM-induced Erk, JNK, and p38 MAPK signaling due to pharmacologic inhibition. SM-induced expression of IL-8, IL-6, and TNF-alpha was decreased by p38 MAPK inhibition, but not by inhibition of other MAPKs. Based on the number of significant pathways mapped to each MAPK in the presence and absence of inhibitors, the p38 MAPK pathway appeared to be the MAPK pathway most responsive to SM exposure. Interestingly, pathway mapping of the microarray data identified potential cross-talk between MAPK signaling pathways and other pathways involved in SM-induced signaling. Mining of these results will increase our understanding of the role of MAPK pathways in SM-induced signal transduction and may identify potential therapeutic targets for medical countermeasure development.

Project description:Sulfur mustard (SM) is a potent alkylating agent. We are developing medical countermeasures to reduce the injury caused by SM exposure. Screening in the mouse ear vesicant model has identified three effective compounds: dimercaprol (British anti-lewisite), indomethacin, and octyl homovanillamide (OHV). To identify gene expression changes that correlate with compound efficacy we used oligonucleotide microarrays to compare gene expression profiles in vehicle-exposed skin, SM-exposed skin, and skin pretreated with each compound before SM exposure. Mice were topically exposed on the inner surface of the right ear to SM alone or pretreated for 15 min with one of the compounds and then exposed to SM. Left ears were vehicle-exposed. Ear tissue was harvested 24 hr later for ear weight determination (an endpoint indicating compound efficacy). The exposure groups were: methylene chloride (sulfur mustard vehicle); ethanol (drug vehicle); 0.08 mg sulfur mustard; 6.25 mg dimercaprol 15 min before 0.08 mg sulfur mustard; 1.34 mg indomethacin 15 min before 0.08 mg sulfur mustard; 0.6 mg octylhomovanillamide 15 min before 0.08 mg sulfur mustard; 6.25 mg dimercaprol alone; 1.34 mg indomethacin alone; 0.6 mg octylhomovanillamide alone. RNA was extracted from the tissues and used to generate oligonucleotide microarray probes. Principal component analysis of the gene expression data revealed partitioning of the samples based on drug treatment and SM exposure. Vehicle-exposed mouse ears clustered away from the other treatment groups. SM-exposed mouse ears pretreated with dimercaprol or OHV clustered more closely with vehicle-exposed ears, while SM-exposed mouse ears pretreated with indomethacin clustered more closely with SM-exposed ears. This clustering of the samples is supported by the ear weight data, in which the indomethacin group has ear weights closer to the SM-exposed group, whereas the dimercaprol and OHV groups have ear weights closer to the vehicle-exposed group. Correlation coefficients were calculated for each gene based on the correlation between gene expression level and ear weight. These data provide the basis for understanding what gene expression changes are important in the development of effective SM medical countermeasures. Keywords: ordered

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Comparison of transcriptional responses to sulfur mustard, nitrogen mustard, and half mustard exposure in epidermal keratinocytes

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