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This study characterizes the development of a head-out inhalation exposure system for assessing respiratory toxicity of vaporized chemical agents in untreated, non-anesthetized rats. The organophosphate diisopropyl fluorophosphate (DFP) induces classical cholinergic toxicity following inhalation exposure and was utilized to validate the effectiveness of this newly designed inhalation exposure system. A saturator cell apparatus was used to generate DFP vapor at 9750, 10950, 12200, 14625 and 19500mg min/m(expn 3) which was carried by filtered nitrogen into a glass mixing tube, where it combined with ambient air before being introduced to the custom-made glass exposure chamber. Male Sprague-Dawley rats (250 300 g) were restrained in individual head-out plethysmography chambers, which acquired respiratory parameters before, during and after agent exposure. All animals were acclimated to the exposure system prior to exposure to reduce novel environment-induced stress. The LCt50, as determined by probit analysis, was 12 014mg min/m3. Weight loss in exposed animals was dose-dependent and ranged from 8 to 28% of their body weight 24 h after exposure. Increased salivation, lacrimation, urination, defecation (SLUD) and mild muscular fasciculation were observed in all DFP-exposed animals during and immediately following exposure. In all exposed animals, DFP vapor produced significant inhibition of acetylcholinesterase (AChE) activity in cardiac blood, bronchoalveolar lavage fluid (BALF), whole brain and lung tissue as well as alterations in tidal volume and minute volume. These studies have provided valuable information leading to the initiation of studies evaluating inhalational toxicity and treatments following exposure to the more lethal and potent chemical warfare nerve agents., The original document contains color images. Published in Toxicology Mechanisms and Methods, v23 n7 p537-547, 2013.

Nerve agents pose a threat to the respiratory tract with exposure that could result in acute compromised lung performance and death. The determination of toxicity by inhalation is important for the rational development of timely therapeutic countermeasures. This study was designed to deliver aerosolized dilute nerve agents in a dose-response manner to investigate the extent of lethality of nerve agents: soman, sarin, VX and VR. Male rats (240-270 g) were anesthetized intramuscularly with 10 mg/kg xylazine and 90 mg/kg ketamine. Following anesthesia, rats were intubated with a glass endotracheal tube (ET) and placed in a glove box. The ET was connected to a closed circuit nebulizer system (Aeroneb, Aerogen, Inc.) that delivered a particle size of 2.0 micro m and was in series between the ventilator and the ET. Nerve agents were delivered by a small animal ventilator set for a volume of 2.5 ml x 60-80 breaths/min. VX or VR were nebulized and delivered in concentrations ranging from 6.25-800 micro g/kg over a 10-min exposure time period. Sarin (GB) or soman (GD), 6.5-1250 micro g/kg, were delivered in a similar manner. Lethality by inhalation occurred either during the 1 0-min exposure period or less than 15 min after the cessation of exposure. Survivors were euthanized at 24 h postexposure. LCt50 estimates ( 95% confidence intervals [Cis]) were obtained from the sequential stage-wise experiments using the probit analysis. Probit analysis revealed that the LD50 for VX was 110.7 micro g/kg (CI: 73.5-166.7), VR 64.2 micro g/kg (CI: 42.1-97.8); soman (GD), 1 67 micro g/kg (CI: 90-31 0), and sarin (GB), 154 micro g/ kg (CI: 98-242), respectively. Although VR is a structural isomer of VX, the compounds appear to be markedly different in terms of toxicity when delivered by aerosol., The original document contains color images. Prepared in collaboration with 27 SOCES/CEAN Cannon Air Force Base, NM. Published in Toxicology Mechanisms and Methods, v23 n2 p127-133, 2013.

Sulfur mustard (HD) is an alkylating agent that reacts rapidly with macromolecular targets resulting in the formation of stable adducts providing depots for markers of exposure. The purpose of this study was to validate an analytical procedure for detection of HD-plasma protein adducts and to establish the utility of the method in an HD rat inhalation study. Calibration curves were prepared in human and rat plasma at six levels of HD (12.5 to 400nM). Correlation coefficients for the mean data were 0.9987 for human and 0.9992 for rat plasma. The percent coefficient of variation (%CV) derived from the mean concentration data ranged from 0.53 to 14.1% in human (n = 5) and 0.57 to 10.63% in rat (n = 6) plasma. Intraday and interday precision and accuracy studies were conducted at three concentration levels (25, 150, 300nM) to represent low, medium, and high concentrations of HD relative to those employed in the calibration curve. Precision and accuracy were assessed by determining %CV and %error, respectively. For intra- and interday studies, the %CVs and absolute % errors were less than 15%. The limits of quantitation were 20.88nM for human and 16.73nM for rat plasma. In animal studies, rats received nebulized HD at six doses. The data indicate a dose-dependent relationship between maximal plasma concentrations and dose administered (R(2) =0.9728). Results from this study indicate an accurate, precise, and sensitive method. The method was useful in determining plasma protein adduct formation in a rat inhalation model., Pub. in Journal of Analytical Toxicology, v32 p37-43, 2008. Supported in part by Defense Threat Reduction Agency.

Studies were performed to address treatment against phosgene gas. Efficacy was judged by examining post-treatment (PTx) effects on pulmonary edema (PE) i.e. lung weight gain (LWG), survival rates (SR), odds ratios (OR), and glutathione (GSH) redox state, and lipid peroxidation (LP). N-acetylcysteine (NAC), ibuprofen (lBU), aminophylline (AMIN), and isoproterenol (ISO) were studied in the isolated perfused rabbit lung model (lPRLM) and mouse model. Intratracheal (IT) NAC delivered 1 h after phosgene exposure lowered pulmonary artery pressure (Ppa), LWG, leukotrienes (LTs), LP, and oxidized GSH. Using the lPRLM, AMIN after phosgene exposure significantly reduced LP and LTs, and LWG. Ptx with ISO in the IPRLM by combined intravascular (lV) or intratracheal (IT) route 1 h after phosgene lowered Ppa, tracheal pressure, LWG, and LTs. In mice lBU was administered i.p. 20 min after phosgene at 0, 3, 9, or 15 mg/mouse. At 5 h, a second lBU injection was given. The OR was 5 for the 9/4.5 lBU group at 12 h and 13 for the 15/7.5 mg IBU. lBU enhanced mouse SR by reducing PE, LP, and GSH depletion. Treatment of phosgene injury involves early intervention that reduces LP, maintains GSH, and prevents the release of LTs responsible for PE.

Vesicants, such as sulfur mustard (HD), pose an insidious threat to the respiratory tract. Exposure causes acute lung injury characteristic of an inflammatory response. Nuclear Factor-KAPPA B (NF- square B) is a redox sensitive transcription factor important in regulating genes involved in the inflammation. This study evaluated levels of NF-square B following exposure to HD. Male rats were euthanized at 3, 6, or 24 hr following intravenous HD or isopropanol (IPA controls) exposure. Nuclear protein samples extracted from pulmonary tissue were analyzed using an electrophoretic mobility shift assay.

The effects of 2-mercaptoethanesulfonate (mesna) as a post-treatment against exposure to a lethal dose of phosgene (CG) were investigated. Mesna appears to be a potential candidate against phosgene-induced injury because it may increase sulfhydryl concentrations, which may reduce cellular injury. Mice were exposed to phosgene, 32 mg/m(3) , or air for 20 minutes. After a 5-minute washout with air, mice were injected interperitoneally (ip) with 0, 334, 834 or 1,667 mg/kg of mesna or phosphate buffered saline (PBS) at 20 minutes, 2, 5, 8 and 11 hours. Survival was assessed every 2 hours for 12 hours and at 24 hours. Lungs were removed and analyzed for wet weight, dry weight, wet/dry ratio, protein, and non-protein sulfhydryls (NPSH).