The aim of this study was to establish and validate a practical method to disperse nanoparticles in physiological solutions for biological in vitro and in vivo studies.

The in-vitro genotoxicity of nanosized TiO₂ rutile and anatase was assessed in comparison with fine TiO₂ rutile in human bronchial epithelial BEAS 2B cells using the single-cell gel electrophoresis (comet) assay and the cytokinesis-block micronucleus test. BEAS 2B cells were exposed to eight doses (1—100 μg/cm²) of titanium(IV) oxide nanosized rutile (>95%, <5% amorphous SiO₂ coating; 10 × 40 nm), nanosized anatase (99.7%; <25 nm), or fine rutile (99.9%; <5 μm) for 24, 48, and 72 h. Fine rutile reduced cell viability at lower doses than nanosized anatase, which was more cytotoxic than nanosized rutile. In the comet assay, nanosized anatase and fine rutile induced DNA damage at several doses with all treatment times. Dose-dependent effects were seen after the 48- and 72-h treatments with nanosized anatase and after the 24-, 48- (in one out of two experiments), and 72-h treatments (one experiment) with fine rutile. The lowest doses inducing DNA damage were 1 μg/cm² for fine rutile and 10 μg/cm ² for nanosized anatase. Nanosized rutile showed a significant induction in DNA damage only at 80 μg/cm² in the 24-h treatment and at 80 and 100 μg/ cm² in the 72-h treatment (with a dose-dependent effect). Only nanosized anatase could elevate the frequency of micronucleated BEAS 2B cells, producing a significant increase at 10 and 60 μg/cm ² after the 72-h treatment (no dose-dependency). At increasing doses of all the particles, MN analysis became difficult due to the presence of TiO₂ on the microscopic slides. In conclusion, our studies in human bronchial epithelial BEAS 2B cells showed that uncoated nanosized anatase TiO₂ and fine rutile TiO₂ are more efficient than SiO ₂-coated nanosized rutile TiO₂ in inducing DNA damage, whereas only nanosized anatase is able to slightly induce micronuclei.

The importance of nanotechnologies and engineered nanoparticles has grown rapidly. It is therefore crucial to acquire up-to-date knowledge of the possible harmful health effects of these materials. Since a multitude of different types of nanosized titanium dioxide (TiO₂) particles are used in industry, we explored their inflammatory potential using mouse and cell models. BALB/c mice were exposed by inhalation for 2 h, 2 h on 4 consecutive days, or 2 h on 4 consecutive days for 4 weeks to several commercial TiO₂ nanoparticles, SiO₂ nanoparticles, and to nanosized TiO₂ generated in a gas-to-particle conversion process at 10 mg/m³. In addition, effects of in vitro exposure of human macrophages and fibroblasts (MRC-9) to the different particles were assessed. SiO₂-coated rutile TiO₂ nanoparticles (cnTiO₂) was the only sample tested that elicited clear-cut pulmonary neutrophilia. Uncoated rutile and anatase as well as nanosized SiO₂ did not induce significant inflammation. Pulmonary neutrophilia was accompanied by increased expression of tumor necrosis factor-alpha (TNF-α) and neutrophil-attracting chemokine CXCL1 in the lung tissue. TiO₂ particles accumulated almost exclusively in the alveolar macrophages. In vitro exposure of murine and human macrophages to cnTiO₂ elicited significant induction of TNF-α and neutrophil-attracting chemokines. Stimulation of human fibroblasts with cnTiO₂-activated macrophage supernatant induced high expression of neutrophil-attracting chemokines, CXCL1 and CXCL8. Interestingly, the level of lung inflammation could not be explained by the surface area of the particles, their primary or agglomerate particle size, or radical formation capacity but is rather explained by the surface coating. Our findings emphasize that it is vitally important to take into account in the risk assessment that alterations of nanoparticles, e.g., by surface coating, may drastically change their toxicological potential.