Dissociation of Intracellular Lysosomal Rupture from the Cell Death Caused by Silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388D1macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca2+, 60% of the cells were unable to exclude trypan blue. In the absence of extracellular Ca2+, however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca2+. The percentage of P388D1cells killed by silica depended on the dose and the concentration of Ca2+in the medium. Intracellular lysosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60% of the cells exposed to 150 μg silica for 3 h in the presence or absence of Ca2+showed intracellular lysosomal rupture, whereas cell death occurred only in the presence of Ca2+. Intracellular lysosomal rupture was not associated with measurable degradation of total DNA, RNA, protein, or phospholipid or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80% of P388D1macrophages against silica toxicity although lysosomal rupture occurred in 60-70% of the cells. Intracellular lysosomal rupture was prevented in 80% of the cells by pretreatment with indomethacin (5× 10-5M), yet 40-50% of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca2+. The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98% of the cells treated for 1 h in either the presence or absence of extracellular Ca2+. With the addition of 1.8 mM Ca2+, 80% of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of-Ca2+. These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death caused by silica or A23187. Cell death is dependent on extracellular Ca2+and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.