Dana Anderson

Inflammatory effects of inhaled sulfur mustard in rat lung

Inhalation of sulfur mustard (SM), a bifunctional alkylating agent that causes severe lung damage, is a significant threat to both military and civilian populations. The mechanisms mediating its cytotoxic effects are unknown and were investigated in the present studies. Male rats Crl:CD(SD) were anesthetized, and then intratracheally intubated and exposed to 0.7-1.4mg/kg SM by vapor inhalation. Animals were euthanized 6, 24, 48h or 7days post-exposure and bronchoalveolar lavage fluid (BAL) and lung tissue collected. Exposure of rats to SM resulted in rapid pulmonary toxicity, including focal ulceration and detachment of the trachea and bronchial epithelia from underlying mucosa, thickening of alveolar septal walls and increased numbers of inflammatory cells in the tissue. There was also evidence of autophagy and apoptosis in the tissue. This was correlated with increased BAL protein content, a marker of injury to the alveolar epithelial lining. SM exposure also resulted in increased expression of markers of inflammation including cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNFα), inducible nitric oxide synthase (iNOS), and matrix metalloproteinase-9 (MMP-9), each of which has been implicated in pulmonary toxicity. Whereas COX-2, TNFα and iNOS were mainly localized in alveolar regions, MMP-9 was prominent in bronchial epithelium. In contrast, expression of the anti-oxidant hemeoxygenase, and the anti-inflammatory collectin, surfactant protein-D, decreased in the lung after SM exposure. These data demonstrate that SM-induced oxidative stress and injury are associated with the generation of cytotoxic inflammatory proteins which may contribute to the pathogenic response to this vesicant.

Evaluation of protease inhibitors and an antioxidant for treatment of sulfur mustard-induced toxic lung injury

Sulfur mustard (SM)-induced lung injury has been associated with protease activation, oxidative injury and inflammatory response culminating in tissue necrosis. The protease inhibitors aprotinin and ilomastat and the antioxidant trolox were evaluated for efficacy in ameliorating SM-induced lung injury. Anesthetized spontaneously breathing rats (N=6-8/group) were intratracheally intubated and exposed to 1.4 mg/kg SM (0.35 mg SM in 0.1 ml of ethanol) or ethanol alone by vapor inhalation for 50 min. At 1 min before the exposure rats were treated with one of the following: intravenous aprotinin, 4.4 mg/kg; intraperitoneal (ip) ilomastat, 25mg/kg; or ip trolox, 500 microg/kg. Aprotinin-treated animals received supplemental 2.2mg/kg doses at 1 min and 6h post-exposure (PE). A whole body plethysmograph system was used to monitor pulmonary function (PF) parameters for 1h before exposure (baseline), and from 5-6 and 23-24h post-exposure. SM inhalation caused significant increases in several PF parameters, including tidal volume, peak inspiratory flow, peak expiratory flow, end expiratory pause and enhanced pause. Consistent with the reported development of SM-induced pathology, these changes were minimal at the 5-6-h time and significant at the 23-24-h timepoint. At the later time it is known from previous work that airways are becoming obstructed with loose cellular debris, damaged cells and exudate, which contributed to the changes in PF parameters. Treatment with aprotinin or ilomastat eliminated these PF changes, yielding results comparable with controls for each of these parameters. Lung lavage fluid analysis showed that SM caused a significant increase in total protein (TP) and in the cytokines IL-1alpha and IL-13. Aprotinin treatment prevented the increases in TP and IL-1alpha production, ilomastat prevented the increased production of IL-13, and trolox treatment did not significantly prevent the SM-related increases in TP, IL-1alpha or IL-13. Histopathologic examination of lung tissue 24h post-exposure showed minimal alveolar effects caused by SM, while damage to bronchiolar regions was much more severe due to the highly reactive nature of SM. While aprotinin and ilomastat both alleviated the PF perturbations, surprisingly only aprotinin reduced the observed pathology, both grossly and histologically. These early results indicate that treatment with aprotinin and to a lesser extent ilomastat reduces some of the direct inflammatory response and damage associated with SM-induced lung injury. This research was supported by the Defense Threat Reduction Agency - Joint Science and Technology Office, Medical S&T Division.

Sulfur mustard induces apoptosis in lung epithelial cells via a caspase amplification loop

Sulfur mustard (SM [bis-(2-chloroethyl) sulfide]) is a chemical warfare agent that causes skin blisters presumably due to DNA alkylation and cross-links. We recently showed that SM also induces apoptotic death in cultured normal human bronchial/tracheal epithelial (NHBE) cells and small airway epithelial cells (SAEC) in vitro. In this process, caspases-8 and -3, but not caspase-9, were strongly activated; this suggests a death receptor pathway for apoptosis. We now show that rat lungs were induced to undergo apoptosis in vivo following exposure of rats to SM by inhalation. Further study of the mechanism of apoptosis due to SM was performed with cultured NHBE cells and SAEC using tetrapeptide inhibitors of caspases-3, and -8. Inhibition of caspase-8 drastically reduced the activation of caspase-3 and almost eliminated that of caspase-9. Moreover, caspase-3 inhibition markedly reduced the activation of caspase-8 and also almost completely inhibited activation of caspase-9. These results suggest a death receptor pathway of apoptosis that utilizes a feedback amplification mechanism involving an activated death receptor complex that leads to the activation of caspase-9 via a caspase-3 pathway. These results may be important for the design of inhibitors of these pathways for therapeutic intervention to attenuate SM injury in respiratory tract lesions.

Airway tissue plasminogen activator prevents acute mortality due to lethal sulfur mustard inhalation

RATIONALEnSulfur mustard (SM) is a chemical weapon stockpiled today in volatile regions of the world. SM inhalation causes a life-threatening airway injury characterized by airway obstruction from fibrin casts, which can lead to respiratory failure and death. Mortality in those requiring intubation is more than 80%. No therapy exists to prevent mortality after SM exposure. Our previous work using the less toxic analog of SM, 2-chloroethyl ethyl sulfide, identified tissue plasminogen activator (tPA) an effective rescue therapy for airway cast obstruction (Veress, L. A., Hendry-Hofer, T. B., Loader, J. E., Rioux, J. S., Garlick, R. B., and White, C. W. (2013). Tissue plasminogen activator prevents mortality from sulfur mustard analog-induced airway obstruction. Am. J. Respir. Cell Mol. Biol. 48, 439-447). It is not known if exposure to neat SM vapor, the primary agent used in chemical warfare, will also cause death due to airway casts, and if tPA could be used to improve outcome.nnnMETHODSnAdult rats were exposed to SM, and when oxygen saturation reached less than 85% (median: 6.5u2009h), intratracheal tPA or placebo was given under isoflurane anesthesia every 4u2009h for 48u2009h. Oxygen saturation, clinical distress, and arterial blood gases were assessed. Microdissection was done to assess airway obstruction by casts.nnnRESULTSnIntratracheal tPA treatment eliminated mortality (0% at 48u2009h) and greatly improved morbidity after lethal SM inhalation (100% death in controls). tPA normalized SM-associated hypoxemia, hypercarbia, and lactic acidosis, and improved respiratory distress. Moreover, tPA treatment resulted in greatly diminished airway casts, preventing respiratory failure from airway obstruction.nnnCONCLUSIONSntPA given via airway more than 6u2009h after exposure prevented death from lethal SM inhalation, and normalized oxygenation and ventilation defects, thereby rescuing from respiratory distress and failure. Intra-airway tPA should be considered as a life-saving rescue therapy after a significant SM inhalation exposure incident.