Date of Graduation

1999

Document Type

Thesis

Degree Type

MS

College

School of Medicine

Committee Chair

Vince Castranova

Committee Member

Richard Dey

Committee Member

Mark Reasor

Committee Member

Xiangling Shi

Committee Member

Knox Van Dyke

Abstract

Occupational exposure to crystalline silica has been associated with lung damage and fibrosis. Although previous studies have demonstrated that silica induces apoptosis both in vitro and in vivo, its role in the pathogenesis of silicosis remains unknown. The purpose of the present study was to: 1) characterize silica-induced apoptosis in vitro; 2) establish a temporal relationship between silica exposure, the induction of apoptosis, and lung inflammation/damage; and 3) investigate the role of apoptosis in the differential susceptibilities of two mouse strains to silica. First, experiments were undertaken to characterize the apoptotic response of rat alveolar macrophages in vitro. Primary alveolar macrophages were isolated from healthy animals and exposed for various times to various concentrations of crystalline silica. Apoptosis was measured with a commercially available ELISA kit, which specifically detects DNA fragmentation, and was confirmed by morphological analysis. Results from these experiments indicate that the apoptotic response in vitro is both time and dose dependant, and that the ELISA kit can be employed to quantify apoptosis. Next, we conducted an inhalation study. Rats were exposed to a silica aerosol of 15 mg/m3 (6 hours/day, 5 days/week) and apoptosis of bronchoalveolar lavage cells (BALC) was determined after 5, 10, 16, 20, 30, 41, 79 and 116 days of exposure. At various exposure times, cellular and tissue markers of inflammation, damage, and fibrosis were measured. Silica induced an inflammatory response in the lung characterized by an increase in polymorphonuclear leukocytes and lung damage. Although these markers of response were elevated to a stable level from 5-41 days, then increased dramatically from 41-116 days of exposure. Only at 116 days of exposure was fibrosis evident. The induction of apoptosis was evident after 5 days of exposure, increased steadily until 30 days of exposure, but did not increased further from 41 days to 116 days of exposure. These data are consistent with the hypothesis that when apoptosis is increasing rapidly inflammation and damage are somewhat controlled. However, at times when apoptosis no longer increases inflammation and damage escalate rapidly and fibrosis is initiated. Studies of quartz induced pulmonary fibrosis in rodents indicate variable sensitivities among strains. This prompted us to investigate the role that apoptosis may play in the susceptibility to pulmonary fibrosis. Mice from two strains with different susceptibilities to quartz-induced fibrosis were given a single intratracheal instillation of silica and cellular and tissue markers of inflammation and damage were monitored along with apoptosis of bronchoalveolar lavage cells at 1 day, 1 week, 1 month, and 3 months post exposure. Results from this study show that resistant mice exhibited lower levels of inflammation and damage and higher levels of apoptosis than susceptible mice at 3 months post exposure. These data along with those from the inhalation study suggest that apoptosis may play a protective role in controlling pulmonary inflammation and limiting damage in response to silica which result in fibrosis.

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