ToxSci Advance Access published online on March 31, 2004
Toxicological Sciences, doi:10.1093/toxsci/kfh121
Toxicological Sciences © Society of Toxicology 2004; all rights reserved
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1 Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT, 06269
* To whom correspondence should be addressed. E-mail: Andrea.Hubbard{at}uconn.edu.
Past studies in our laboratory have shown that silica (aquartz) 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 reveal that silica exposure elicits lysosomal permeability after 1 hr 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, evidenced decreased caspase activation and apoptotic DNA fragmentation. MH-S cells, pretreated with pepstatin A, an inhibitor of lysosomal cathepsin D, demonstrated decreased caspase 9 and 3 activation as well as the percent 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 sphinogmyelinase. 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.
© 2004 Toxicological Sciences © Society of Toxicology 2004; all rights reserved.
Immunotoxicology
Silica Induced Apoptosis in Mouse Alveolar Macrophages Is Initiated by Lysosomal Enzyme Activity
2 Department of Molecular & Cell Biology, University of Connecticut, Storrs, CT, 06269
3 Department of Chemical Engineering, University of Connecticut, Storrs, CT, 06269
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