Robyn C. Kiser

Therapeutic approaches to dermatotoxicity by sulfur mustard I. Modulation of sulfur mustard‐induced cutaneous injury in the mouse ear vesicant model†**

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. Published in 2000 by John Wiley & Sons, Ltd.

In vitro and in vivo comparison of sulfur donors as antidotes to acute cyanide intoxication

Antidotes for cyanide (CN) intoxication include the use of sulfane sulfur donors (SSDs), such as thiosulfate, which increase the conversion of CN to thiocyanate by the enzyme rhodanese. To develop pretreatments that might be useful against CN, SSDs with greater lipophilicity than thiosulfate were synthesized and assessed. The ability of SSDs to protect mice against 2LD50 of sodium cyanide (NaCN) administered either 15 or 60 min following administration of an SSD was assessed. To study the mechanism of action of the SSD, the candidate compounds were examined in vitro for their effect on rhodanese and 3‐mercaptopyruvate sulfurtransferase (MST) activity under increasing SSD concentrations. Tests were conducted on nine candidate SSDs: ICD1021 (3‐hydroxypyridin‐2‐yl N‐[(N‐methyl‐3‐aminopropyl)]‐2‐aminoethyl disulfide dihydrochloride), ICD1022, (3‐hydroxypyridin‐2‐yl N‐[(N‐methyl‐3‐aminopropyl)]‐2‐aminoethyl disulfide trihydrochloride), ICD1584 (diethyl tetrasulfide), ICD1585 (diallyl tetrasulfide), ICD1587 (diisopropyl tetrasulfide); ICD1738 (N‐(3‐aminopropyl)‐2‐aminoethyl 2‐oxopropyl disulfide dihydrochloride), ICD1816 (3,3′‐tetrathiobis‐N‐acctyl‐l‐alanine), ICD2214 (2‐aminoethyl 4‐methoxyphenyl disulfide hydrochloride) and ICD2467 (bis(4‐methoxyphenyl) disulfide). These tests demonstrated that altering the chemical substituent of the longer chain sulfide modified the ability of the candidate SSD to protect against CN toxicity. At least two of the SSDs at selected doses provided 100% protection against 2LD50 of NaCN, normally an LD99. All compounds were evaluated using locomotor activity as a measure of potential adverse behavioral effects. Positive hypoactivity relationships were found with several disulfides but none was found with ICD1584, a tetrasulfide. Separate studies suggest that the chemical reaction of potassium cyanide (KCN) and cystine forms the toxic metabolite 2‐iminothiazolidine‐4‐carboxylic acid. An alternative detoxification pathway, one not primarily involving the sulfur transferases. may be important in pretreatment for CN intoxication. Although studies to elucidate the precise mechanisms are needed. it is clear that these newly synthesized compounds provide a new rationale for anti‐CN drugs, with fewer side‐effects than the methemoglobin formers. Copyright

Evaluation of compounds as barriers to dermal penetration of organophosphates using acetylcholinesterase inhibition.

An efficient, objective method for evaluating the efficacy of barrier compounds in preventing dermal penetration of organophosphates (OP) in rabbits was developed using time-dependent reduction in erythrocyte (RBC) acetylcholinesterase (AChE) activity as an endpoint. Anesthetized rabbits, with or without a dermal application of a mixture of high- and low-molecular-weight polyethylene glycols (mean molecular weight of 540 daltons; PEG 540), were exposed to different percutaneous doses of 3 highly toxic OP compounds. Dose-response curves were generated for RBC AChE inhibition as a function of percutaneous dose for each OP test material over time. From data generated, a single dose of each OP was selected to challenge PEG-540-protected and unprotected animals to validate the method as a means of differentiating effective from ineffective barriers to skin penetration. Data for a complete evaluation of a PEG 540 test barrier application were obtained within 4 h and anesthesia was maintained for the entire period.

Alterations of Gene Expression in Sulfur Mustard‐Exposed Skin Topically Treated with Vanilloids

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 significantly reduced cytokine mRNA expression following SM exposure. In addition to measuring edema formation, monitoring expression of biomarkers such as GM‐CSF, IL‐1β, and IL‐6 may also serve to evaluate therapeutic treatments against SM‐induced dermal 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.

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‐1β, 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.

A 7-day mouse model to assess protection from sulfur mustard (SM) skin injury

The mouse ear vesicant model (MEVM) is a screening tool used to identify protective compounds against acute sulfur mustard (SM)‐induced skin injury. It provides endpoints of edema and histopathology 24 h following a topical SM exposure to assess protection against inflammation and tissue damage. To further evaluate successful compounds, the MEVM was modified for use as a 7‐day model. Dose response studies were conducted with SM to select an optimal challenge dose for the new model. Due to severity of SM‐induced tissue damage by Day 7, edema and histopathology were determined unreliable endpoints. Therefore, a modified Draize scoring system (no damage to extensive necrosis) was incorporated as an endpoint to evaluate tissue damage out to Day 7. To aid in optimal SM dose selection, retro synthetic capsaicin (RSCAP), a protective compound in the MEVM, was evaluated as a treatment 15 min before exposure to 0.06, 0.08, and 0.16 mg SM. The RSCAP compound provided similar significant protection at Day 7 against the 0.06‐ (42% reduction) and 0.08‐mg doses (32% reduction), but was not effective against the severely necrotizing 0.16‐mg SM dose. Based on these results, an optimum SM dose of 0.08 mg was selected. Retro synthetic capsaicin and two pharmacologically inactive analogs were tested as topical treatments 15 min prior to SM challenge. The RSCAP compound significantly reduced injury, whereas the inactive analogs had no protective effect. The RSCAP also significantly reduced SM injury when administered topically 10 min after SM challenge. These data support the use of the 7‐day mouse ear vesicant treatment model (MEVTM) in evaluating candidate antivesicant compounds.

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.