An assessment of transcriptional changes in porcine skin exposed to bromine vapor.

Bromine is an industrial chemical that can cause severe cutaneous burns. This study was a preliminary investigation into the effect of cutaneous exposure to bromine vapor using a weanling swine burn model and microarray analysis. Ventral abdominal sites were exposed to a mean calculated bromine vapor concentration of 0.69 g L(-1) for 10 or 20 min. At 48 h postexposure, total RNA from skin samples was isolated, processed, and hybridized to Affymetrix GeneChip Porcine Genome Arrays. Expression analysis revealed that bromine vapor exposure for 10 or 20 min promoted similar transcriptional changes in the number of significantly modulated probe sets. A minimum of 83% of the probe sets was similar for both exposure times. Ingenuity pathways analysis revealed eight common biological functions among the top 10 functions of each experimental group, in which 30 genes were commonly shared among 19 significantly altered signaling pathways. Transcripts encoding heme oxygenase 1, interleukin-1ß, interleukin 2 receptor gamma chain, and plasminogen activator inhibitor-1 were identified as common potential therapeutic targets for Phase II/III clinical trial or FDA-approved drugs. The present study is an initial assessment of the transcriptional responses to cutaneous bromine vapor exposure identifying molecular networks and genes that could serve as targets for developing therapeutics for bromine-induced skin injury.

Temporal effects in porcine skin following bromine vapor exposure.

Bromine is an industrial chemical that causes severe cutaneous burns. When selecting or developing effective treatments for bromine burns, it is important to understand the molecular mechanisms of tissue damage and wound healing. This study investigated the effect of cutaneous bromine vapor exposure on gene expression using a weanling swine burn model by microarray analysis. Ventral abdominal sites were exposed to a mean calculated bromine vapor concentration of 0.51 g/L for 7 or 17 min. At 6 h, 48 h, and 7 days post-exposure, total RNA from skin samples was isolated, processed, and analyzed with Affymetrix GeneChip® Porcine Genome Arrays (N = 3 per experimental group). Differences in gene expression were observed with respect to exposure duration and sampling time. Ingenuity Pathways Analysis (IPA) revealed four common biological functions (cancer, cellular movement, cell-to-cell signaling and interaction, and tissue development) among the top ten functions of each experimental group, while canonical pathway analysis revealed 9 genes (ARG2, CCR1, HMOX1, ATF2, IL-8, TIMP1, ESR1, HSPAIL, and SELE) that were commonly shared among four significantly altered signaling pathways. Among these, the transcripts encoding HMOX1 and ESR1 were identified using IPA as common potential therapeutic targets for Phase II/III clinical trial or FDA-approved drugs. The present study describes the transcriptional responses to cutaneous bromine vapor exposure identifying molecular networks and genes that could serve as targets for developing therapeutics for bromine-induced skin injury.

Gene expression analysis of bromine-induced burns in porcine skin.

Bromine is an industrial chemical that is irritating to the skin and causes cutaneous burns. An important factor in selecting or developing an effective treatment is to understand the underlying molecular mechanisms of tissue damage and wound healing. This study used a weanling swine burn model and microarray analysis to evaluate the effect of exposure length and sampling times on the transcriptional changes in response to cutaneous bromine injury. Ventral abdominal sites (N=4/treatment group) were exposed to 600microL undiluted bromine for 45 s or 8 min. At 24 h and 7d post-exposure, total RNA from skin samples was isolated, processed, and hybridized to Affymetrix GeneChip Porcine Genome Arrays. Expression analysis revealed that bromine exposure duration appeared to have less effect on the transcript changes than the sampling time. The percent transcripts changed at 24h were similar (30%) whether having a 45 s or 8 min bromine exposure; percent transcripts changed at 7d were also similar (62%) regardless of exposure length. However, only 13-14% of the transcripts were similar when comparing samples analyzed at 24h and 7d. Ingenuity Pathways Analysis (IPA) revealed six common biological functions among the top 10 functions of each experimental group, while canonical pathway analysis revealed 11 genes that were commonly shared among 24 significantly altered signaling pathways. Additionally, there were 11 signaling pathways in which there were no commonly shared transcripts. The present study is an initial assessment of the transcriptional responses to cutaneous bromine exposure identifying molecular networks and genes that could serve as targets for developing therapeutics for bromine-induced skin injury.

Microarray analysis of mouse ear tissue exposed to bis-(2-chloroethyl) sulfide: gene expression profiles correlate with treatment efficacy and an established clinical endpoint.

Bis-(2-chloroethyl) sulfide (sulfur mustard; SM) is a potent alkylating agent. Three treatment compounds have been shown to limit SM damage in the mouse ear vesicant model: dimercaprol, octyl homovanillamide, and indomethacin. Microarrays were used to determine gene expression profiles of biopsies taken from mouse ears after exposure to SM in the presence or absence of treatment compounds. Mouse ears were topically exposed to SM alone or were pretreated for 15 min with a treatment compound and then exposed to SM. Ear tissue was harvested 24 h after exposure for ear weight determination, the endpoint used to evaluate treatment compound efficacy. RNA extracted from the tissues was used to generate microarray probes for gene expression profiling of therapeutic responses. Principal component analysis of the gene expression data revealed partitioning of the samples based on treatment compound and SM exposure. Patterns of gene responses to the treatment compounds were indicative of exposure condition and were phenotypically anchored to ear weight. Pretreatment with indomethacin, the least effective treatment compound, produced ear weights close to those treated with SM alone. Ear weights from animals pretreated with dimercaprol or octyl homovanillamide were more closely associated with exposure to vehicle alone. Correlation coefficients between gene expression level and ear weight revealed genes involved in mediating responses to both SM exposure and treatment compounds. These data provide a basis for elucidating the mechanisms of response to SM and drug treatment and also provide a basis for developing strategies to accelerate development of effective SM medical countermeasures.

Preferential expression of matrix metalloproteinase-9 in mouse skin after sulfur mustard exposure.

Matrix metalloproteinases (MMPs), a class of enzymes responsible for the degradation of extracellular matrix proteins, play important roles in inflammatory and immune responses. In skin, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are normally inactive but can be expressed during tissue injury. Both degrade collagen IV and other critical components of the basement membrane zone that separates the epidermis from the dermis. The expression of MMP-2 and -9 was studied in sulfur mustard (SM)-exposed ear skin from mice to determine their role in tissue vesicant injury. Punch biopsies of mouse ears were collected between 6 and 168 h after exposure to 97.5 mM (0.08 mg) SM diluted in CH(2)Cl(2). They were examined histologically and assayed for MMP-2 and -9 expression by gelatinase activity assays, real-time reverse transcriptase-polymerase chain reaction and Western blot analysis. A time-related increase in overall gelatinase activity was observed in SM-treated ears. At 168 h after SM exposure, the relative levels of MMP-9 mRNA were increased 27-fold and MMP-9 protein 9-fold when compared with the control (CH(2)Cl(2) treated) ears. In contrast, there were no observable increases in the MMP-2 mRNA or protein levels between treated and control ears. These observations suggest the differential expression of MMP-2 and -9 during the cutaneous response to SM injury and suggest a role for MMP-9 in SM-induced injury.

Microarray analysis of gene expression in murine skin exposed to sulfur mustard.

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 > or =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.

Time- and dose-dependent analysis of gene expression using microarrays in sulfur mustard-exposed mice.

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.