Development of chronic lung injury and pulmonary fibrosis in mice following acute exposure to nitrogen mustard

Objective: Sulfur mustards are toxic agents used as a chemical warfare in the twentieth century. Exposure to nitrogen mustards (NM), their more water-soluble analogs, is associated with respiratory, dermatological, neurological, and systemic symptoms whose severity depends on dose and length of contact. Long-term effects of acute inhalation have been related to the development of chronic lung injury and pulmonary fibrosis whose precise mechanisms and potential antidotes are yet to be discovered. Materials and methods: We have developed a model of NM-induced pulmonary fibrosis by intratracheally instilling mechlorethamine hydrochloride into C57Bl/6J male mice. Results and Discussion: Following mechlorethamine exposure, strong early and milder late inflammatory responses were observed. Initially, the number of white blood cells and levels of protein and pro-inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) increased, followed by increases in the number of macrophages and the levels of transforming growth factor-beta (TGF-beta), a pro-fibrotic mediator. Analysis of lung homogenates revealed increased phosphorylation of pro-fibrotic biomarkers, serine/threonine-selective protein kinases (p-ERK), and heat shock protein 90 (P-HSP90) at 10 and 30 days after exposure. Total collagen expression and deposition of extracellular matrix proteins also increased. Lung function measurements demonstrated the presence of both obstructive and restrictive disease in agreement with evidence of increased lower airway peribronchial collagen deposition and parenchymal fibrosis. Conclusions: We conclude that the mouse represents a useful model of NM-induced acute lung injury and chronic pulmonary fibrosis, the latter driven by the overexpression of TGF-beta, p-ERK, and P-HSP90. This model may prove useful in the pre-clinical development of antidotes and other countermeasures.