Original Contribution
Increased sensitivity to asbestos-induced lung injury in mice lacking extracellular superoxide dismutase

https://doi.org/10.1016/j.freeradbiomed.2005.09.030Get rights and content

Abstract

Asbestosis is a chronic form of interstitial lung disease characterized by inflammation and fibrosis that results from the inhalation of asbestos fibers. Although the pathogenesis of asbestosis is poorly understood, reactive oxygen species may mediate the progression of this disease. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) can protect the lung against a variety of insults; however, its role in asbestosis is unknown. To determine if EC-SOD plays a direct role in protecting the lung from asbestos-induced injury, intratracheal injections of crocidolite were given to wild-type and ec-sod-null mice. Bronchoalveolar lavage fluid (BALF) from asbestos-treated ec-sod-null mice at 24 h, 14 days, or 28 days posttreatment showed increased inflammation and total BALF protein content compared to that of wild-type mice. In addition, lungs from ec-sod-null mice showed increased hydroxyproline content compared to those of wild-type mice, indicating a greater fibrotic response. Finally, lungs from ec-sod-null mice showed greater oxidative damage, as assessed by nitrotyrosine content compared to those of their wild-type counterparts. These results indicate that depletion of EC-SOD from the lung increases oxidative stress and injury in response to asbestos.

Introduction

Asbestosis, a form of interstitial lung disease characterized by inflammation and lung fibrosis, is a chronic, debilitating disease that causes significant morbidity and mortality in affected patients [1]. Although exposure to asbestos fibers may have occurred 20 to 40 years earlier, the long latency period of the disease implies that many workers in the United States are still at risk of developing asbestosis in the upcoming years. Thus, this disease is currently and will remain a significant health problem [2], [3].

In order to better investigate the pathogenesis of this disease, animal models have been used extensively to help elucidate the underlying biochemical mechanisms responsible for the development of asbestosis (reviewed in [4]). Studies conducted in these model systems have confirmed the fibrogenic and carcinogenic properties of asbestos fibers that have been observed in cases of human exposure. Even brief exposures to asbestos fibers in animal models have resulted in the persistent presence of these fibers in the lung parenchyma where they stimulate the recruitment and activation of inflammatory cells and the proliferation of fibroblasts and type II epithelial cells [5], [6], [7], [8] (see below).

Although the molecular mechanisms underlying asbestosis are largely unknown, current evidence suggests a role for reactive oxygen species (ROS) in the pathogenesis of this disease [4], [9]. It has been previously shown that asbestos, particularly the highly fibrogenic amphibole fibers, can cause oxidative damage to the lung both directly, through hydroxyl radical formation via the Haber-Weiss reaction with fiber surface iron [10], [11], and indirectly through recruitment and activation of ROS-producing inflammatory cells [12], [13], [14]. A variety of antioxidants including manganese superoxide dismutase, catalase, and iron chelators such as deferoxamine have shown protective effects in a variety of in vitro and in vivo models of asbestos-mediated lung disease [15], [16], [17], [18].

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is a 135-kDa tetrameric enzyme that scavenges superoxide radicals in the extracellular space (reviewed in [19]). EC-SOD is expressed in especially high levels in mammalian lungs where it is bound to the extracellular matrix through a positively charged heparin/matrix-binding domain. Proteolytic cleavage of the heparin/matrix-binding domain has been associated with loss of the enzyme from the extracellular matrix under experimental conditions that mimic human interstitial lung disease, such as bleomycin treatment or hyperoxia [20], [21]. In addition, it has been shown that both bleomycin treatment and hyperoxia cause greater lung tissue damage in mice that carry a targeted disruption of the ecsod gene (ecsod-null mice) when compared to wild-type mice [22], [23]. Recent evidence from our laboratory indicates that asbestos exposure in mice results in decreased lung EC-SOD protein levels and activity. The loss of this enzyme may enhance oxidative stress and injury in this model [24].

To elucidate the role of EC-SOD in oxidative stress-associated lung disease, we have examined the type and extent of asbestos-induced pulmonary damage in ecsod-null mice. Using a model in which wild-type and ecsod-null mice are exposed intratracheally to crocidolite asbestos, we have determined that lack of EC-SOD leads to increased lung injury and exacerbates asbestos-induced lung inflammation and fibrosis. These results support the hypothesis that the loss of EC-SOD from the lung results in increased oxidative lung injury and leads to increased inflammation and fibrosis in a mouse model of asbestosis.

Section snippets

Materials

Eosin Y, phloxine B, chloramine T, methyl cellusolve, titanium dioxide, and anti-β-actin antibody were purchased from Sigma (St. Louis, MO). Anti-nitrotyrosine antibody was purchased from Molecular Probes (Eugene, OR). Mayer's hematoxylin, 10% buffered formalin, and p-dimethylaminobenzaldehyde were purchased from Fisher Scientific (Pittsburgh, PA). Clear Rite was obtained from Richard-Allan Scientific (Kalamazoo, MI). Enhanced chemiluminescence detection reagents were purchased from Amersham

Asbestos treatment results in the loss of EC-SOD from lung parenchyma

Previous studies have demonstrated a protective role for EC-SOD in a variety of lung pathologies that are associated with high levels of oxidative stress [27], [28], [29]. Since asbestosis has been associated with both increased oxidant production and decreased levels of antioxidant enzymes (reviewed in [9]), this study was conducted to determine if loss of EC-SOD from lung tissue exacerbates asbestos-induced damage. We have established a mouse model of asbestosis in which the hallmarks of

Discussion

Particle and fiber-induced lung diseases as a result of both occupational and nonoccupational exposures are a significant health concern in the United States and elsewhere. Although a detailed mechanism of action for asbestos-induced pathogenesis has not yet been elucidated, oxidant/antioxidant imbalances in the lung as a result of exposure are believed to play a key role [30]. Several studies have shown that asbestos exposure both in vitro and in vivo can lead to ROS formation and oxidative

Acknowledgment

This work is supported in part by the National Institutes of Health Grant HL63700 (T.D.O.) and an American Heart Association Established Investigator Award (T.D.O.) and National Institutes of Health Grant (NIEHS) 1F30ES01362 (R.J.T.).

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