Thomas H. Snider

Comparison of an In Vitro Skin Model to Normal Human Skin for Dermatological Research

EpiDerm™, an in vitro human skin equivalent (HSE), was compared to normal human breast skin (NHS) to morphologically and biochemically assess its feasibility for dermatological research. Intralot and interlot variability was studied in day 0, 1, 2, and 3 in vitro cultures and in day 0, 3, 5, and 7 NHS. For NHS, light microscopy (LM) at day 0 showed stratified epidermis which exhibited an increase in vacuoles and dark basal cells as storage increased to 3, 5, and 7 days. Transmission electron microscopy (TEM) revealed typical organelles in the epidermis and a convoluted basement membrane at day 0. With increased storage, vacuoles and paranuclear clefts became numerous, necrosis increased, tonofilaments became less organized, and overall cellular integrity decreased. Biochemical data showed consistent MTT and glucose utilization (GU) through day 5, while lactate production decreased to 75% by day 3. By LM, day 0 HSE consisted of a thick, compact, stratum corneum that sent projections between the stratum granulosum cells. By TEM, the configuration, organization, differentiation, distribution, and frequency of the organelles differed slightly from NHS. In addition, the basement membrane of the HSE was not completely differentiated, and the dermis was thin and acellular. Although day 1 and 2 cultures showed little change, day 3 exhibited an overall degeneration. Biochemical analysis showed GU and lactate production decreased through day 3. In conclusion, the EpiDerm™ HSE, although exhibiting slight differences, was morphologically and biochemically similar to normal human epidermis and may be a valuable model in assessing the toxicology, metabolism, or pharmacology of nonvesicating compounds. Microsc. Res. Tech., 37:172–179, 1997.

A comprehensive evaluation of the efficacy of leading oxime therapies in guinea pigs exposed to organophosphorus chemical warfare agents or pesticides.

The currently fielded pre-hospital therapeutic regimen for the treatment of organophosphorus (OP) poisoning in the United States (U.S.) is the administration of atropine in combination with an oxime antidote (2-PAM Cl) to reactivate inhibited acetylcholinesterase (AChE). Depending on clinical symptoms, an anticonvulsant, e.g., diazepam, may also be administered. Unfortunately, 2-PAM Cl does not offer sufficient protection across the range of OP threat agents, and there is some question as to whether it is the most effective oxime compound available. The objective of the present study is to identify an oxime antidote, under standardized and comparable conditions, that offers protection at the FDA approved human equivalent dose (HED) of 2-PAM Cl against tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and VX, and the pesticides paraoxon, chlorpyrifos oxon, and phorate oxon. Male Hartley guinea pigs were subcutaneously challenged with a lethal level of OP and treated at approximately 1 min post challenge with atropine followed by equimolar oxime therapy (2-PAM Cl, HI-6 DMS, obidoxime Cl₂, TMB-4, MMB4-DMS, HLö-7 DMS, MINA, and RS194B) or therapeutic-index (TI) level therapy (HI-6 DMS, MMB4-DMS, MINA, and RS194B). Clinical signs of toxicity were observed for 24 h post challenge and blood cholinesterase [AChE and butyrylcholinesterase (BChE)] activity was analyzed utilizing a modified Ellmans method. When the oxime is standardized against the HED of 2-PAM Cl for guinea pigs, the evidence from clinical observations, lethality, quality of life (QOL) scores, and cholinesterase reactivation rates across all OPs indicated that MMB4 DMS and HLö-7 DMS were the two most consistently efficacious oximes.

Efficacy of the topical skin protectant in advanced development.

A topical skin protectant (TSP) (ICD 2289) is being developed to protect service members from exposure to chemical warfare agents (CWA). The TSP is designed for use on the skin at the overgarment closures and other vulnerable areas to enhance protection. The TSP, which is in phase II clinical studies, is a cream containing two chemically inert substances: perfluoroalkylpolyether and polytetrafluoroethylene. Animal data showed that the TSP was effective against percutaneous penetration of a blister agent, sulfur mustard (HD), by reducing the size of skin lesions and against T‐2 mycotoxin by preventing the development of erythema and edema. The insect repellent N,N‐diethyl‐m‐toluamide (DEET) reduced the TSP protection against HD regardless of the order of application on rabbit skin prior to dosing of HD. The protection was sustained when DEET was removed with a dry gauze prior to TSP application. The TSP was also effective against percutaneous exposure of nerve agents—thickened (with 5% methyl methacrylamide) soman (TGD) and VX (O‐ethyl‐S‐[2‐(diisopropylamino)ethyl]methylphosphonothioate)—by reducing the mortality rate and protecting the red blood cell acetylcholinesterase activity. The TSP was effective against VX when DEET was applied prior to TSP application. Because human efficacy studies using CWA cannot be conducted, the efficacy will be demonstrated by the level of protection against poison ivy (urushiol) contact dermatitis in humans.

Model for assessing efficacy of topical skin protectants against sulfur mustard vapor using hairless guinea pigs.

Sulfur mustard (HD; 2,2′‐dichlorodiethyl sulfide) can produce incapacitating blisters in humans following dermal exposure. Most non‐human animal models, however, do not form the large fluid‐filled blisters observed in humans. Many models, nevertheless, do produce similar damage at the dermal/epidermal junction when evaluated by histopathology. In this study, it was observed that the hairless guinea pig (HGP) exhibits similar histopathological responses following exposure to HD vapor. Two sets of HGPs were exposed percutaneously for various lengths of time to HD vapor. In one set, the HGPs were sacrificed 24 h after exposure, and skin specimens were collected and processed for histopathology. In the other set, light reflectance was measured at skin test sites 4, 5, 6 and 24 h after exposure, to assess erythema. The Nikolsky’s sign test was also performed 24 h after exposure by rotating a metal disk glued to the skin test site and inspecting the skin for loss of epidermis. Probit analysis of data indicated that the exposure durations that produced a 50% incidence of microblisters and Nikolsky’s sign were ca. 7.5 and 4.5 min, respectively. Maximum erythema was observed 6 h following a 6 min exposure. Operating parameters for assessing the efficacies of skin protectants have been characterized.

Evaluation of HemogloBind™ treatment for preparation of samples for cholinesterase analysis

Acetylcholine is an essential neurotransmitter found throughout the nervous system. Its action on postsynaptic receptors is regulated through hydrolysis by various carboxylesterases, especially cholinesterases (ChEs). The acute toxicity of organophosphate (OP) compounds is directly linked to their action as inhibitors of ChE. One widely used assay for evaluating ChE activity is a spectrophotometric method developed by Ellman et al. When the enzyme source is from tissues or, in particular, blood, hemoglobin displays a spectrophotometric peak at the same wavelength used to analyze cholinergic activity. This creates a substantial background that interferes with the Ellman’s assay and must be overcome in order to accurately monitor cholinesterase activity. Herein, we directly compare blood processing methods: classical method (1.67 ± 0.30 U/mL) and HemogloBind™ treatment (1.51 ± 0.17 U/mL), and clearly demonstrate that pretreatment of blood samples with Hemoglobind™ is both a sufficient and rapid sample preparation method for the assessment of ChE activity using the Ellman’s method.

Quantitative proteomic analysis of the brainstem following lethal sarin exposure

The brainstem represents a major tissue area affected by sarin organophosphate poisoning due to its function in respiratory and cardiovascular control. While the acute toxic effects of sarin on brainstem-related responses are relatively unknown, other brain areas e.g., cortex or cerebellum, have been studied more extensively. The study objective was to analyze the guinea pig brainstem toxicology response following sarin (2×LD50) exposure by proteome pathway analysis to gain insight into the complex regulatory mechanisms that lead to impairment of respiratory and cardiovascular control. Guinea pig exposure to sarin resulted in the typical acute behavior/physiology outcomes with death between 15 and 25min. In addition, brain and blood acetylcholinesterase activity was significantly reduced in the presence of sarin to 95%, and 89%, respectively, of control values. Isobaric-tagged (iTRAQ) liquid chromatography tandem mass spectrometry (LC-MS/MS) identified 198 total proteins of which 23% were upregulated, and 18% were downregulated following sarin exposure. Direct gene ontology (GO) analysis revealed a sarin-specific broad-spectrum proteomic profile including glutamate-mediated excitotoxicity, calcium overload, energy depletion responses, and compensatory carbohydrate metabolism, increases in ROS defense, DNA damage and chromatin remodeling, HSP response, targeted protein degradation (ubiquitination) and cell death response. With regards to the sarin-dependent effect on respiration, our study supports the potential interference of sarin with CO2/H(+) sensitive chemoreceptor neurons of the brainstem retrotrapezoid nucleus (RTN) that send excitatory glutamergic projections to the respiratory centers. In conclusion, this study gives insight into the brainstem broad-spectrum proteome following acute sarin exposure and the gained information will assist in the development of novel countermeasures.

Toxicity and median effective doses of oxime therapies against percutaneous organophosphorus pesticide and nerve agent challenges in the Hartley guinea pig

Anticholinesterases, such as organophosphorus pesticides and warfare nerve agents, present a significant health threat. Onset of symptoms after exposure can be rapid, requiring quick-acting, efficacious therapy to mitigate the effects. The goal of the current study was to identify the safest antidote with the highest therapeutic index (TI = oxime 24-hr LD50/oxime ED50) from a panel of four oximes deemed most efficacious in a previous study. The oximes tested were pralidoxime chloride (2-PAM Cl), MMB4 DMS, HLö-7 DMS, and obidoxime Cl2. The 24-hr median lethal dose (LD50) for the four by intramuscular (IM) injection and the median effective dose (ED50) were determined. In the ED50 study, male guinea pigs clipped of hair received 2x LD50 topical challenges of undiluted Russian VX (VR), VX, or phorate oxon (PHO) and, at the onset of cholinergic signs, IM therapy of atropine (0.4 mg/kg) and varying levels of oxime. Survival was assessed at 3 hr after onset clinical signs. The 3-hr 90th percentile dose (ED90) for each oxime was compared to the guinea pig pre-hospital human-equivalent dose of 2-PAM Cl, 149 µmol/kg. The TI was calculated for each OP/oxime combination. Against VR, MMB4 DMS had a higher TI than HLö-7 DMS, whereas 2-PAM Cl and obidoxime Cl2 were ineffective. Against VX, MMB4 DMS > HLö-7 DMS > 2-PAM Cl > obidoxime Cl2. Against PHO, all performed better than 2-PAM Cl. MMB4 DMS was the most effective oxime as it was the only oxime with ED90 < 149 µmol/kg against all three topical OPs tested.

A dynamic system for delivering controlled bromine and chlorine vapor exposures to weanling swine skin.

Abstract Context: Assessing the hazards of accidental exposure to toxic industrial chemical (TIC) vapors and evaluating therapeutic compounds or treatment regimens require the development of appropriate animal models. Objective: The objective of this project was to develop an exposure system for delivering controlled vapor concentrations of TICs to the skin of anesthetized weanling pigs. Injury levels targeted for study were superficial dermal (SD) and deep dermal (DD) skin lesions as defined histopathologically. Materials and methods: The exposure system was capable of simultaneously delivering chlorine or bromine vapor to four, 3-cm diameter exposure cups placed over skin between the axillary and inguinal areas of the ventral abdomen. Vapor concentrations were generated by mixing saturated bromine or chlorine vapor with either dried dilution air or nitrogen. Results: Bromine exposure concentrations ranged from 6.5 × 10−4 to 1.03 g/L, and exposure durations ranged from 1 to 45 min. A 7-min skin exposure to bromine vapors at 0.59 g/L was sufficient to produce SD injuries, while a 17-min exposure produced a DD injury. Chlorine exposure concentrations ranged from 1.0 to 2.9 g/L (saturated vapor concentration) for exposures ranging from 3 to 90 min. Saturated chlorine vapor challenges for up to 30 min did not induce significant dermal injuries, whereas saturated chlorine vapor with wetted material on the skin surface for 30–60 min induced SD injuries. DD chlorine injuries could not be induced with this system. Conclusion: The vapor exposure system described in this study provides a means for safely regulating, quantifying and delivering TIC vapors to the skin of weanling swine as a model to evaluate therapeutic treatments.

Acute toxicity of phorate oxon by oral gavage in the Sprague-Dawley rat

The oral toxicity of phorate oxon (PHO), with emphasis on gender- and age-related effects, was characterized in the Sprague-Dawley rat. The oral LD50 (95% fiducial limits) for PHO in corn oil was 0.88 (0.79, 1.04) mg/kg in males and 0.55 (0.46, 0.63) mg/kg in females with a probit slope of 15. Females had higher baseline blood cholinesterase titers, but males were significantly more tolerant. Younger rats generally had lower absolute cholinesterase blood titers. However as PHO challenges increased, baseline-normalized cholinesterase inhibition was independent of age and gender. Butyrylcholinesterase (BChE) and especially acetylcholinesterase (AChE) in brains of younger females were affected more than that in either males or older females. In summary, while female rats, especially older females, had higher titers relative to males, female rats were more susceptible in terms of absolute cholinesterase inhibition and 24-hr lethality data, but the differences were not observed when titers were normalized to baseline levels.

Evaluating the broad-spectrum efficacy of the acetylcholinesterase oximes reactivators MMB4 DMS, HLö-7 DMS, and 2-PAM Cl against phorate oxon, sarin, and VX in the Hartley guinea pig

HighlightsMMB4 DMS and HLö‐7 DMS demonstrate superior qualities against CWNA and pesticides.They both provide significant PR against all three OPs compared to atropine controls.Significance was not readily observed across the board when compared against 2‐PAM Cl.The ED50 values of MMB4 DMS was lower than other oximes against all three Ops. ABSTRACT Organophosphorus (OP) compounds, including pesticides and chemical warfare nerve agents (CWNA), are threats to the general population as possible weapons of terrorism or by accidental exposure whether through inadvertent release from manufacturing facilities or during transport. To mitigate the toxicities posed by these threats, a therapeutic regimen that is quick‐acting and efficacious against a broad spectrum of OPs is highly desired. The work described herein sought to assess the protective ratio (PR), median effective doses (ED50), and therapeutic index (TI = oxime 24‐h LD50/oxime ED50) of MMB4 DMS, HLö‐7 DMS, and 2‐PAM Cl against the OPs sarin (GB), VX, and phorate‐oxon (PHO). All OPs are representative of the broader classes of G and V chemical warfare nerve agents and persistent pesticides. MMB4 DMS and HLö‐7 DMS were previously identified as comparative efficacy leads warranting further evaluations. 2‐PAM Cl is the U.S. FDA‐approved standard‐of‐care oxime therapy for OP intoxication. Briefly, PRs were determined in male guinea pigs by varying the subcutaneously (SC) delivered OP dose followed then by therapy with fixed levels of the oxime and atropine (0.4 mg/kg; administered intramuscularly [IM]). ED50s were determined using a similar approach except the OP dose was held constant at twice the median lethal dose (2 × LD50) while the oxime treatment levels were varied. The ED50 information was then used to calculate the TI for each OP/oxime combination. Both MMB4 DMS and HLö‐7 DMS provided significant protection, i.e., higher PR against GB, VX, and PHO when compared to atropine controls, but significance was not readily demonstrated across the board when compared against 2‐PAM Cl. The ED50 values of MMB4 DMS was consistently lower than that of the other oximes against all three OPs. Furthermore, based on those ED50s, the TI trend of the various oximes against both GB and VX was MMB4 DMS > HLö‐7 DMS > 2‐PAM Cl, while against PHO, MMB4 DMS > 2‐PAM Cl > HLö‐7 DMS.