Discovery of treatment for nerve agents targeting a new metabolic pathway.

The inhibition of acetylcholinesterase is regarded as the primary toxic mechanism of action for chemical warfare agents. Recently, there have been numerous reports suggesting that metabolic processes could significantly contribute to toxicity. As such, we applied a multi-omics pipeline to generate a detailed cascade of molecular events temporally occurring in guinea pigs exposed to VX. Proteomic and metabolomic profiling resulted in the identification of several enzymes and metabolic precursors involved in glycolysis and the TCA cycle. All lines of experimental evidence indicated that there was a blockade of the TCA cycle at isocitrate dehydrogenase 2, which converts isocitrate to α-ketoglutarate. Using a primary beating cardiomyocyte cell model, we were able to determine that the supplementation of α-ketoglutarate subsequently rescued cells from the acute effects of VX poisoning. This study highlights the broad impacts that VX has and how understanding these mechanisms could result in new therapeutics such as α-ketoglutarate.

Comparison of low-level sarin and cyclosarin vapor exposure on pupil size of the Gottingen minipig: effects of exposure concentration and duration.

The current studies estimated effective (miosis) concentrations of the nerve agents' sarin (GB) and cyclosarin (GF) as a function of exposure duration in the Gottingen minipig and determined dependency of the median effective dosage (ECT50) over time. Male and female Gottingen minipigs were exposed to various concentrations of vapor GB or GF for 10, 60, or 180 min. Infrared images of the pig's pupil before, during, and after nerve agent exposure were captured digitally and pupil area was quantified. An animal was classified "positive" for miosis if there was a 50% reduction in pupil area (as compared to baseline) at any time during or after the GB or GF exposure. Maximum likelihood estimation was used on the resulting quantal data to calculate ECT50 (miosis) values, with approximate 95% confidence intervals, for each of the six gender-exposure duration groups. As a group, male minipigs were significantly more sensitive to the pupil constricting effects of GF than were female minipigs. In male minipigs, GF is approximately equipotent to GB for 60-min exposures and more potent for 10- and 180-min exposures. In the female minipig GF is slightly more potent than GB for 10-min exposures but then progressively becomes less potent over the 60- and 180-min durations of exposure. The values of the toxic load exponents were essentially independent of the model fits used: 1.32 +/- 0.18 for GB exposures and 1.60 +/- 0.22 for GF exposures. Since neither of these intervals overlaps 1, Haber's rule is not an appropriate time-dependence model for these data sets.

Tolerance to the miotic effect of sarin vapor in rats after multiple low-level exposures.

Inhibition of acetylcholinesterase (AChE) by the organophosphorous compound sarin (GB) results in the accumulation of acetylcholine and excessive cholinergic stimulation. There are few data in the literature regarding the effects of multiple low-level exposures to GB and other organophosphorous compounds via relevant routes of exposure. Therefore, the present study was undertaken, and is the first, to investigate the effect of low-level repeated whole-body inhalation exposures to GB vapor on pupil size and cholinesterase activity in the eyes and blood. Male Sprague-Dawley rats were exposed to 4.0 mg/m3 of GB vapor for 1 h on each of 3 consecutive days. Pupil size and cholinesterase activities were determined at various points throughout the exposure sequence. The results demonstrate that multiple inhalation exposures to GB vapor produce a decrease in the miotic potency of GB in rats. This tolerance developed at a dose of GB that produced no overt signs of intoxication other than miosis. AChE and butyrylcholinesterase activity did not increase throughout the exposure sequence, suggesting that the tolerance cannot be attributed to a reduced inhibitory effect of GB. A decrease in the amount of GB present in the eye occurred after the third exposure. However, this change is insufficient to explain the tolerance, as there was no corresponding increase in AChE activity. Thus, the mechanism mediating the miotic tolerance observed after multiple inhalation exposures to the nerve agent GB remains uncertain, although several possibilities can be excluded based on the results of the present study.

Chronological changes in electrolyte levels in arterial blood and bronchoalveolar lavage fluid in mice after exposure to an edemagenic gas.

Detection of acute lung injury is important if therapeutic medical countermeasures are to be used to reduce toxicity in a timely manner. Indicators of injury may aid in the eventual treatment course and enhance the odds of a positive outcome following a toxic exposure. This study was designed to investigate the effects of a toxic exposure to the industrial irritant gas phosgene on the electrolyte levels in arterial blood and bronchoalveolar lavage fluid (BALF). Phosgene is a well-known chemical intermediate capable of producing life-threatening pulmonary edema within hours after exposure. Four groups of 40 Crl:CD-1(ICR)BR male mice were exposed whole-body to either air or phosgene at a concentration x time (c x t) amount of 32-42 mg/m(3) (8-11 ppm) phosgene for 20 min (640-840 mg x min/m(3)). BALF from air- or phosgene-exposed mice was taken at 1, 4, 8, 12, 24, 48, or 72 h postexposure. After euthanasia, the trachea was excised, and 800 microl saline was instilled into the lungs. The lungs were washed 5x. Eighty microliters of BALF was placed in a cartridge and inserted into a clinical i-STAT analyzer. Na(+), Cl(-), K(+), and ionized Ca(2+) were analyzed. Arterial blood electrolyte levels were also analyzed in four additional groups of air- or phosgene-exposed mice. The left lung was removed to determine wet weight (WW), an indicator of pulmonary edema. Na(+) was significantly higher in air than in phosgene-exposed mice at 4, 8, and 12 h postexposure. Temporal changes in BALF Cl(-) in phosgene mice were not statistically different from those in the air mice. Both Ca(2+) and K(+) were significantly higher than in the air-exposed mice over 72 h, p < or = 0.03 and p < or = 0.001 (two-way analysis of variance, ANOVA), respectively. Significant changes in BALF K(+) and Ca(2+) occurred as early as 4 h postexposure in phosgene, p < or = 0.005, versus air-exposed mice. Over time, there were no significant changes in arterial blood levels of Na(+), Cl(-), or Ca(2+) for animals exposed to air versus phosgene. However, arterial K(+) concentrations were significantly higher, p < or = 0.05, than in air-exposed mice across all time points, with the highest K(+) levels of 7 mmol/L occurring at 8 h and 24 h after exposure. Phosgene caused a time-dependent significant increase in WW from 4 to 12 h, p < or = 0.025, compared with air-exposed mice. These data demonstrate that measuring blood K(+) levels 1 h after exposure along with BALF Na(+), K(+), and Ca(2+) may serve as an alternate indicators of lung injury since both K(+) and Ca(2+) follow temporal increases in air-blood barrier permeability as measured by wet weight.

The fate of antioxidant enzymes in bronchoalveolar lavage fluid over 7 days in mice with acute lung injury.

Characterization of lung injury is important if timely therapeutic intervention is to be used properly and successfully. In this study, lung injury was defined as the progressive formation of pulmonary edema. Our model gas was phosgene, a pulmonary edemagenic compound. Phosgene, widely used in industry, can produce life-threatening pulmonary edema within hours of exposure. Four groups of 40 CD-1 male mice were exposed whole-body to either air or a concentration x time (c x t) amount of 32-42 mg/m(3) (8-11 ppm) phosgene for 20 min (640-840 mg x min/m(3)). Groups of air- or phosgene-exposed mice were euthanized 1, 4, 8, 12, 24, 48, or 72 h or 7 days postexposure. The trachea was excised, and 800 micro l saline was instilled into the lungs and washed back and forth 5 times to collect bronchoalveolar lavage fluid (BALF). The antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), total glutathione (GSH), and protein were determined at each time point. Phosgene exposure significantly enhanced both GPx and GR in phosgene-exposed mice compared with air-exposed mice from 4 to 72 h, p < or = 0.01 and p < or = 0.005, respectively. BALF GSH was also significantly increased, p < or = 0.01, from 4 to 24 h after exposure, in comparison with air-exposed. BALF protein, an indicator of air/blood barrier integrity, was significantly higher than in air-exposed mice 4 h to 7 days after exposure. In contrast, BALF SOD was reduced by phosgene exposure from 4 to 24 h, p < or = 0.01, versus air-exposed mice. Except for protein, all parameters returned to control levels by 7 days postexposure. These data indicate that the lung has the capacity to repair itself within 24-48 h after exposure by reestablishing a functional GSH redox system despite increased protein leakage. SOD reduction during increased leakage may indicate that barrier integrity is affected by superoxide anion production.

Acute changes in lung histopathology and bronchoalveolar lavage parameters in mice exposed to the choking agent gas phosgene.

Phosgene (CG) is a highly irritant gas widely used industrially as a chemical intermediate for the production of dyes, pesticides, and plastics, and can cause life-threatening pulmonary edema within 24 hours of exposure. This study was designed to investigate acute changes in lung tissue histopathology and selected bronchoalveolar lavage fluid (BALF) factors over time to determine early diagnostic indicators of exposure. Three groups of 40 male mice each were exposed to 32 mg/m3 (8 ppm) CG for 20 minutes, and 3 groups of 40 control male mice were exposed to filtered room air for 20 minutes, both exposures were followed by room air washout for 5 minutes. At 1, 4.8, 12, 24, 48, and 72 hours after exposure each group of mice was euthanized and processed for histopathology, bronchoalveolar lavage or gravimetric measurements, respectively. Over time, the histopathological lesions were characterized by acute changes consisting of alveolar and interstitial edema, fibrin and hemorrhage, followed by significant alveolar and interstitial flooding with inflammatory cell infiltrates and scattered bronchiolar and terminal airway epithelial degeneration and necrosis. From 48 to 72 hours, there was partial resolution of the edema and degenerative changes, followed by epithelial and fibroblastic regeneration centered on the terminal bronchiolar areas. Bronchoalveolar lavage was processed for cell differential counts, LDH, and protein determination. Comparative analysis revealed significant increases in both postexposure lung wet/dry weight ratios, and early elevations of BALF LDH and protein, and later elevations in leukocytes. This article describes the use of histopathology to chronicle the temporal pulmonary changes subsequent to whole body exposure to phosgene, and correlate these changes with BALF ingredients and postexposure lung wet weights in an effort to characterize toxic gas-induced acute lung injury and identify early markers of phosgene exposure.

Temporal changes in respiratory dynamics in mice exposed to phosgene.

One hallmark of phosgene inhalation toxicity is the latent formation of life-threatening, noncardiogenic pulmonary edema. The purpose of this study was to investigate the effect of phosgene inhalation on respiratory dynamics over 12 h. CD-1 male mice, 25-30 g, were exposed to 32 mg/m(3) (8 ppm) phosgene for 20 min (640 mg min/m(3)) followed by a 5-min air washout. A similar group of mice was exposed to room air for 25 min. After exposure, conscious mice were placed unrestrained in a whole-body plethysmograph to determine breathing frequency (f), inspiration (Ti) and expiration (Te) times, tidal volume (TV), minute ventilation (MV), end inspiratory pause (EIP), end expiratory (EEP) pause, peak inspiratory flows (PIF), peak expiratory flows (PEF), and a measure of bronchoconstriction (Penh). All parameters were evaluated every 15 min for 12 h. Bronchoalveolar lavage fluid (BALF) protein concentration and lung wet/dry weight ratios (W/D) were also determined at 1, 4, 8, and 12 h. A treatment x time repeated-measures two-way analysis of variance (ANOVA) revealed significant differences between air and phosgene for EEP, EIP, PEF, PIF, TV, and MV, p < or =.05, across 12 h. Phosgene-exposed mice had a significantly longer mean Ti, p < or =.05, compared with air-exposed mice over time. Mice exposed to phosgene showed marked increases (approximately double) in Penh across all time points, beginning at 5 h, when compared with air-exposed mice, p < or =.05. BALF protein, an indicator of air/blood barrier integrity, and W/D were significantly higher, 10- to 12-fold, in phosgene-exposed than in air-exposed mice 4-12 h after exposure, p