Role of Mitochondria in Silica-Induced Apoptosis of Alveolar Macrophages: Inhibition of Apoptosis by Rhodamine 6g and N-acetyl-L-cysteine

Abstract

Induction of apoptosis by silica in alveolar macrophages (AM) may be a critical step in silica-induced lung injury and pulmonary fibrosis. This study investigated the mechanism(s) through which silica induces apoptosis in AM and their production of proinflammatory cytokines. Using N-acetyl-L-cysteine (NAC) for glutathione (GSH) synthesis and removal of reactive oxygen species (ROS), and rhodamine 6G (R6G) to inhibit the mitochondrial-dependent function, this study found that silica-induced apoptosis of rat AM in primary culture is mitochondria dependent and exhibits a mechanism involving ROS generation, increased mitochondrial release of cytochrome c, and the activation of caspase 9, but not caspase 8, activity. Silica-induced apoptosis was accompanied by a lowering of intracellular and mitochondrial GSH (mGSH) and was blocked by pretreatment of cells with NAC or R6G. When cells were exposed to silica and then treated with either NAC or R6G, silica-induced apoptosis was not affected by the blocking agent. In addition, R6G, which inhibited cellular ATP production and mitochondrial ROS generation, had no effect on apoptosis induced by exogenous hydrogen peroxide or superoxide. Pretreatment of cells with NAC or R6G also inhibited silica-induced production of interleukin (IL)-1β and tumor necrosis factor (TNF)-α, but the inhibition of these cytokines with agents known to block their secretion did not protect cells from silica-induced apoptosis. Data indicate that silica-induced apoptosis is mediated through mitochondrial generation of ROS, which may be inhibited by pretreatment of cells with R6G that prevents ROS generation, or with NAC that maintains a high level of mGSH. The secretion of IL-1β and TNF-α by silica-exposed AM was markedly inhibited by NAC and R6G, suggesting that the production of these cytokines is also ROS dependent.