James A. Blank

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.

Modulation of sulfur mustard-induced inflammation and gene expression by olvanil in the hairless mouse vesicant model

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), macrophage inflammatory protein-2 (MIP-2), MIP-1α, tumor necrosis factor-α, and interleukin (IL)-1β following exposure to SM when compared to unexposed controls. A significant time-dependent increase was observed in MCP-1, MIP-1α, and IL-1β 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 IL-1β 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.

Procedure for assessing myeloperoxidase and inflammatory mediator responses in hairless mouse skin.

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. Copyright

Neuromuscular Effects of Low Level Exposures to Sarin, Pyridostigmine, Deet, and Chlorpyrifos

A study is being initiated to investigate subtle neurobehavioral effects and neuropathology in rats due to exposure to combinations of low levels of Sarin (GB), N,N-diethyl-m-toluamide (DEET), chlorpyrifos (CPF), and pyridostigmine bromide (PB). A similar study is being initiated in rhesus monkeys to investigate neurophysiologic effects and neuromuscular pathology due to exposure to a combination of GB, DEET, CPF, and PB, along with vaccination with botulinum toxoid. A description of these studies is presented.

An Automated Method for the Analysis of Methemoglobin

Production of methemoglobin (met Hb) is effective in treating or preventing cyanide intoxication due to the affinity of cyanide for met Hb. This paper describes an automated method for measuring met Hb in mouse blood utilizing the Roche COB AS FARA centrifugal analyzer and a modified Evelyn–Malloy procedure. Blood samples were spiked with various quantities of potassium ferricyanide to produce met Hb, and automated and manual analyses of these samples were compared. Following validation of the COB AS method, two known met Hb inducers, sodium nitrite and p-aminopropiophenone (PAPP), and two sulfur donor compounds, sodium thiosulfate and ICD #1021, not known to produce met Hb were tested in vivo. Five mice per compound were injected IP and blood samples were analyzed periodically over a time period of approximately 2 h. The automated technique requires only a drop of blood, which allows repeated sampling from the same animal, and can be adapted for blood from different species. A nalyses are rapid and sensitive, and results are reproducible.

Effect of Sulfur Mustard Exposure on Human Epidermal Keratinocyte Viability and Protein Content

Sulfur mustard (HD) is a bifunctional alkylating agent that has mutagenic, cytotoxic, and vesicating properties (1–3). Although HD preferentially alkylates DNA, it is also known to modify RNA and proteins covalently (4). DNA alkylation has been postulated to be the molecular mechanism that initiates a cascade of events terminating in vesication (5). DNA alkylating agents, like HD, have been shown to cause unbalanced cell growth, a condition that occurs when cells exposed to cytotoxic drugs increase in cell volume, protein, and RNA content as cellular mass accumulates, but the cells fail to undergo cell division (6, 7). Depending on concentration, HD will produce different effects in vitro, ranging from inhibition of proliferation with unbalanced cell growth at lower concentrations to cytotoxicity at higher exposure levels (4,8). With an inhibition of proliferation occurring with HD-exposed human epidermal keratinocytes (HEKs), measures of biochemical parameters taken from cultures at extended times postexposure must be standardized to account for differences in cell number. The primary objective of these studies was to assess the feasibility of using protein as a means of standardizing data across HD treatment groups.

Acetylcholinesterase Inhibition Measurements for the Evaluation of Decontaminant Efficacy Following Percutaneous Organophosphorus Compound Exposure

A rapid and sensitive in vivo method for the evaluation of skin decontaminant efficacy following percutaneous exposure to organophosphonates (OPs) was developed using erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) inhibition in the rabbit as an end point. The level of AChE inhibition was evaluated for use as a more humane means of assessing skin decontaminant efficacy than lethality-based methods. Groups of anesthetized animals were exposed percutaneously to either of two highly toxic OPs [thickened soman (TGD) or VX], and 2 min later were treated with a known effective skin decontaminant or were untreated (negative control). Blood samples were drawn and assayed for AChE activity 5 min before TGD or VX exposure and at 30, 60, and 120 min after exposure. Percent AChE inhibition relative to preexposure levels was calculated at each postexposure time for each animal in control and treatment groups. Efficacy data based on percent AChE inhibition were compared with results from previous efficacy studies p...