ToxSci Advance Access originally published online on March 31, 2004
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Toxicological Sciences 80, 34-48 (2004)
Toxicological Sciences vol. 80 no. 1 © Society of Toxicology 2004; all rights reserved.
Silica-Induced Apoptosis in Mouse Alveolar Macrophages Is Initiated by Lysosomal Enzyme Activity
Departments of * Pharmaceutical Sciences, Molecular and Cell Biology, and Chemical Engineering, University of Connecticut, Storrs, Connecticut 06269
Received November 17, 2003; accepted February 26, 2004
Past studies in our laboratory have shown that silica (-quartz) particle exposure of a mouse alveolar macrophage cell line (MH-S) elicits mitochondrial depolarization and caspase 3 and 9 activation, contributing to apoptosis. However, cellular pathways leading to these outcomes have not been extensively investigated. Initial studies revealed that silica exposure elicits lysosomal permeability after 1 h, as evidenced by leakage of FITC-conjugated dextran and acridine orange. We next evaluated a role for the lysosomal acidic compartment in apoptosis. Cells pretreated with the lysosomotropic weak base ammonium chloride, to increase lysosomal pH, showed decreased caspase activation and apoptotic DNA fragmentation. MH-S cells pretreated with pepstatin A, an inhibitor of lysosomal cathepsin D, showed decreased caspase 9 and 3 activation as well as a decreased percentage of cells that became apoptotic. DNA fragmentation and caspase 9 and 3 activation were also decreased in cells pretreated with despiramine, an inhibitor of lysosomal acidic sphingomyelinase. Silica pretreated with aluminum lactate (to blunt surface active sites) reduced caspase activation and apoptosis. Although aluminum lactate–treated silica still induced lysosomal permeability (by FITC–dextran leakage), one measure of lysosome integrity and function suggested a reduction in the extent and/or nature of lysosomal injury (by acridine orange retention). A role for reactive oxygen species (ROS) was investigated to explore another pathway for silica-induced apoptosis in addition to lysosomal enzymes; however, no role for ROS was apparent. Thus, following silica exposure, lysosomal injury precedes apoptosis, and the apoptotic signaling pathway includes cathepsin D and acidic sphingomyelinase.
Key Words: silica; apoptosis; caspase; lysosome; cathepsin; sphingomyelinase.
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