In Vivo Genotoxicity of Crystalline Silica as Evidenced by Micronuclei in Pulmonary Alveolar Macrophages: Low-Dose Study

Abstract

We have previously published results showing time-course data (Leigh et at., 1998) and a dose-response relationship between macrophage micronucleus formation and crystalline silica dose in intratracheally instilled rats at 2.5, 7.5, and 22.5 mg dosage, without an inert dust control (Wang et al., 1997). We here extend this study to low dose (0.025, 0.25, and 2.5 mg crystalline silica) with 2.5 mg TiO₂ control. Specific-pathogen-free male Wistar rats were intratracheally instilled with 0.5 ml saline, and 0.025 mg, 0.25 mg, or 2.5 mg crystalline silica (Min-U-Sil 5) and 2.5 mg TiO₂ suspended in 0.5 ml saline (5 rats in each group). Five days after instillation, rats were sacrificed and 10 ml of bronchial alveolar lavage fluid was obtained. A 100-μ1 volume was placed on slides by Cytospin centrifugation, stained with Diff-Quik, and 1000 macrophages were scored for micronuclei (defined by diameter < half main nucleus; same staining; round shape and complete separation). Micronucleus incidence was significantly elevated (p < .01) at the lowest crystalline silica dose compared with saline control. There was a dose-response relationship with crystalline silica exposure. Numbers (mean ± SEM) of micronucleated macrophages per 1000 macrophages scored were 1.5 ± 0.5 (saline), 3.3 ± 0.3 (0.025 mg crystalline silica), 7.1 ± 0.4 (0.25 mg crystalline silica), 10.1 ± 0.3 (2.5 mg crystalline silica), and 0.9 ± 0.3 (2.5 mg TiO₂). We conclude that intratracheal instillation of low doses of crystalline silica can induce micronucleus formation in alveolar macrophages in a dose-related manner. We further believe that this is not a nonspecific effect, consistent with crystalline silica being a genotoxic carcinogen.