We previously examined the role of apoptosis in lung pathology due to silica, a highly toxic particle and demonstrated that apoptotic cells and degradation products significantly accumulate as injury develops. In this study we test the hypothesis that titanium dioxide (TiO2), a particle with no known specific toxicity, induces apoptosis and accumulation of apoptotic degradation products. To test this, male, F344 rats were given intratracheal instillations (IT) of 0.4, 4 and 40 mg of TiO2. At 1 day, 1 week and 4 weeks after instillation rats were sacrificed, lungs preserved by intra-tracheal instillation of fixative. Sections were processed for TdT labeling of apoptotic nuclei with a fluorescent indicator and counter-stained to label non-apoptotic nuclei. The number of apoptotic cell nuclei per lung was measured by morphometric methods. Additional sections were stained with Sirius Red to detect areas of collagen accumulation. Apoptotic cells in the saline instilled lungs were minimal at 1 and 4 weeks (0.2 0.2 and 0.3 0.4 million cells per lung, mean SE). One week after IT, apoptotic cells per lung were 1.9 0.1, 4.4 0.4 and 13.3 2 million in the 0.4 mg, 4 and 40 mg groups respectively. At 4 weeks the number of apoptotic cells was increased to 5.2 0.5, 9.5 2.0 and 24.1 3.1 million in the 0.4, 4 and 40 mg TiO2 groups. Sirius Red staining of lung sections demonstrated areas with significant connective tissue accumulations in response to the IT of TiO2 in both the 4 and 40 mg TiO2 groups at 4 weeks of exposure. Sirius Red staining of collagen in saline and 0.4 mg TiO2 groups was normal at 1 and 4 weeks. Our results demonstrate that high levels of inert, non-toxic dusts produce significant numbers of apoptotic cells and products. At high levels of lung burden the induction of apoptosis is associated with development of fibrotic foci. The results suggest that injury from the accumulated apoptotic products may be responsible for injury observed in high lung burden exposures to particulates such as TiO2 which have no specific toxicity.
The Toxicologist. Society of Toxicology 41st Annual Meeting and ToxExpo, March 17-21, 2002, Nashville, Tennessee