Genotoxicity of inhaled nanosized TiO2 in mice

► Mice inhaled nanosized TiO2 anatase–brookite (0.8, 7.2, or 28.5mg/m3) for 5 days. ► A dose-dependent deposition of Ti in lung tissue was seen. ► No increase in DNA damage was observed in lung epithelial cells. ► No induction of micronuclei was detected in blood polychromatic erythrocytes. ► A clear pulmonary neutrophilia was, however, present at 28.5mg/m3 TiO2. ► Systemic TiO2 doses were much lower than in previous positive oral and i.p. studies. In vitro studies have suggested that nanosized titanium dioxide (TiO2) is genotoxic. The significance of these findings with respect to in vivo effects is unclear, as few in vivo studies on TiO2 genotoxicity exist. Recently, nanosized TiO2 administered in drinking water was reported to increase, e.g., micronuclei (MN) in peripheral blood polychromatic erythrocytes (PCEs) and DNA damage in leukocytes. Induction of micronuclei in mouse PCEs was earlier also described for pigment-grade TiO2 administered intraperitoneally. The apparent systemic genotoxic effects have been suggested to reflect secondary genotoxicity of TiO2 due to inflammation. However, a recent study suggested that induction of DNA damage in mouse bronchoalveolar lavage (BAL) cells after intratracheal instillation of nanosized or fine TiO2 is independent of inflammation. We examined here, if inhalation of freshly generated nanosized TiO2 (74% anatase, 26% brookite; 5 days, 4h/day) at 0.8, 7.2, and (the highest concentration allowing stable aerosol production) 28.5mg/m3 could induce genotoxic effects in C57BL/6J mice locally in the lungs or systematically in peripheral PCEs. DNA damage was assessed by the comet assay in lung epithelial alveolar type II and Clara cells sampled immediately following the exposure. MN were analyzed by acridine orange staining in blood PCEs collected 48h after the last exposure. A dose-dependent deposition of Ti in lung tissue was seen. Although the highest exposure level produced a clear increase in neutrophils in BAL fluid, indicating an inflammatory effect, no significant effect on the level of DNA damage in lung epithelial cells or micronuclei in PCEs was observed, suggesting no genotoxic effects by the 5-day inhalation exposure to nanosized TiO2 anatase. Our inhalation exposure resulted in much lower systemic TiO2 doses than the previous oral and intraperitoneal treatments, and lung epithelial cells probably received considerably less TiO2 than BAL cells in the earlier intratracheal study.