Figure 4

The mechanistic basis for TiO ₂ long nanobelt (NB-2)-induced toxicity to alveolar macrophages (AM) is lysosomal breakdown resulting in cathepsin B release. A, panels illustrate the concentration-dependent cell death uniquely initiated by NB-2 exposure in the C57BL/6 AM. Cell viability by trypan blue exclusion is on the right, and measured apoptosis is on the left. B, corresponding cathepsin activity in the AM media showed significant increases in cultures that were exposed to NB-2. C, This is supported by the in vivo observation that cathepsins are significantly increased in the lavage fluid of mice 24 hours following NB-2 instillation (30 μg/mouse). Panels in D, show acridine orange-stained lysosomes in unstimulated cells, NS-exposed AM, early (1hr) NB-2-exposed AM and late (4 hr) NB-2-exposed AM, respectively. The NS-exposure appears to concentrate the lysosomes in the AM, whereas the NB-2-exposure initially causes reorganization of the lysosomes and subsequently depletes the lysosomes from the AM. (E), similar patterns were visualized using a fluorescent cathepsin B substrate under identical assay conditions as in (D). The NS-exposure appears to concentrate the cathepsin B in the AM, whereas the NB-2-exposure depletes the cathepsin in the exposed AM. Data expressed as mean ± SEM. Asterisk (*) indicates P < 0.05, double asterisks (**) indicates P < 0.01, and triple asterisks (***) indicates P < 0.001 compared to baseline or control production levels.