Silica-Induced Caspase Activation in Mouse Alveolar Macrophages Is Dependent upon Mitochondrial Integrity and Aspartic Proteolysis

Abstract

Although silica has been documented to cause apoptotic cell death, the cellular pathways leading to caspase activation have not been extensively investigated. Here we demonstrate in a mouse macrophage cell line (MH-S cells) that alpha-quartz silica exposure (12.5 mug/cm2 to 50 mug/cm2) elicited activation of both caspase 3 and caspase 9, whereas anatase titanium dioxide (TiO2), a non-fibrogenic particle, did not. Silica exposure in vitro also induced apoptosis after 6 h, as measured by the appearance of subdiploid cell fragments in a flow cytometric analysis. Exposure to TiO 2 did not elicit significant apoptosis. Silica-induced apoptosis and caspase 3 activation were, in part, caspase 9 dependent, as determined by their sensitivity to either a general caspase inhibitor (Z-VAD-FMK) or a specific caspase 9 inhibitor (Z-LEHD-FMK). Silica exposure in vitro also elicited significant mitochondrial depolarization after 2 and 6 h of exposure. Cyclosporin A, an inhibitor of the mitochondrial permeability pore, partially decreased mitochondrial depolarization, caspase 3 activation, and caspase 9 activation, suggesting a role for mitochondrial dysfunction in these events. Pepstatin A, an inhibitor of cathepsin D, also decreased mitochondrial depolarization, caspase 3 activation, and caspase 9 activation, whereas leupeptin, an inhibitor of cathepsin B, had no effect. These data suggest that short-term silica exposure in vitro induces both caspase 3 and caspase 9 activity, which appears to participate in apoptosis. Activation of these caspases seems to be dependent, in part, on aspartic proteolysis and loss of mitochondrial integrity.

Silica binding and toxicity in alveolar macrophages

“…A variety of inhibitors of the lysosomal processing resulted in reduced caspase activation and apoptosis [106]. This study concluded that the apoptotic signaling pathway involved lysosomal leaking, cathepsin D and acidic sphingomyelinase, which preceded the mitochondrial depolarization and caspase 3 and 9 activation (reported earlier by the same group) caused by silica exposure [105]. It was further determined that ROS was not the cause of the lysosomal permeability [106].…”

“…The silica particle cannot be broken down in the lysosomal compartment, and the resulting failure can lead to a loss of membrane integrity in the process. Some researchers believe that the failed attempt at silica digestion in the AM leads to caspase activation and apoptosis [105,106]. A model illustrating this hypothesis can be found in Figure 4.…”

The Phagocytosis of Crystalline Silica Particles by Macrophages

“…Previous data demonstrated that exposure of cultured macrophages to silica leads to the appearance of a number of markers for cell death (13,14,(24)(25)(26). Propodium iodide staining was used as a marker for cell death to determine if other particle types used in uptake assays were toxic.…”

“…Although the interaction of silica with cells has been intensively studied, the molecular mechanisms underlying this toxicity are not well understood (8). There is evidence that silica causes the production of free radicals and reactive oxygen species (9)(10)(11) and that some cells die due to activation of the apoptotic cascade (12)(13)(14). However, the actual mechanism of silica-induced cell death is still unclear.…”

Safety Evaluation Study of Nanomaterials Aimed at Promoting Their Acceptance by Society

“…For example, exposure of cells or animals to carbon nanotubes, TiO2 nanoparticles or silver nanoparticles have been reported to induce cytotoxicity and in‰amma-tion (4-16). However, other researchers have reported that carbon nanotubes and TiO2 nanoparticles do not induce harmful eŠects (17)(18)(19). Thus, despite intensive research eŠorts, investigations of biological and/or cellular responses to NMs are often inconsistent and even contradictory.…”