In Vitro And In Vivo Toxicity Of TiO₂Particles

Objectives:To clarify the health effects and underlying mechanisum of titanium dioxide （TiO₂） particles tohuman being, in vivo and in vitro toxicities of TiO₂nanoparticles （TiO₂NPs） and TiO₂fine-particles （TiO₂NPs） were evaluated in mice and JB6cells, respectively. The antagonistic effectsof NAC on the cytotoxicity induced by TiO₂NPs were further explored.Methods:In vivo experiments: Mice were injected intravenously with a single dose of TiO₂NPs and TiO₂FPs at varying doses. The median lethal dose （LD50） was calculated by Horn’smethod. Animal mortality, blood biochemistry, hematology, genotoxicity and histopathologywere investigated14days after treatment.In vitro experiments: FITC labelled TiO₂NPs were used to localize the TiO₂NPs in cells.Apoptosis induced by TiO₂NPs and TiO₂FPs were compared using nuclei stain （Hoechst）.Fluorescent dyes of H2DCFDA and DHE were used to compare the oxidative stress caused byTiO₂NPs and TiO₂FPs. Luciferase assay was used to check the AP-1expression. Induction ofC-jun and P53protein expression by TiO₂NPs and TiO₂FPs was compared using western-blot.The carcinogenecity mechanisum of TiO₂NPs and TiO₂FPs was also explored.To investigate antagonistic effect of NAC on the oxidative stress induced by TiO₂NPs:20nMNAC was added before cells treated with TiO₂NPs, and then the inhibition of reactive oxygenspecies （ROS） induced by TiO₂NPs was detected. In addition, the antagonistic effect of NACon AP-1gene expression was also investigated. Results:In vivo experiments: The LD50s of TiO₂NPs in female and male mice were948mg/kg and1392mg/kg, respectively, indicating female mice are more sensitive to the toxicity. Certaindegree damages in liver, lungs and kidneys induced by TiO₂NPs were observed in a dose-dependent manner. However, no obvious genetic toxicity was observed in TiO₂NPs treatedmice. LD50of TiO₂FPs was114.9mg/kg. TiO₂FPs treatment induced similar pathologicaleffects in mice liver, lungs and kidneys.In vitro experiments: TiO₂NPs can penetrate cell membrane, but can’t pass through the nuclearmembrane. Hoechst staining showed that TiO₂NPs induced more cell apoptosis than TiO₂FPs.H2DCFDA, DHE and Hoechst staining showed TiO₂NPs can produce more ROS than TiO₂FPs at the same dose. Both TiO₂NPs and TiO₂FPs can increase the AP-1luciferase expression,and TiO₂NPs showed much higher induction than TiO₂FPs.Western-blot shows that at25μg/cm2TiO₂NPs can increase expression of C-jun protein, anddecrease P53pression.The addition, NAC showed ROS generation inhibition and AP-1luciferase expressiondownregulation.Conclusions:A single dose intravenous injection of TiO₂NPs and TiO₂NPs in mice could cause acutetoxicities in different organs such as in liver, lungs and kidneys etc, indicating that TiO₂NPsand TiO₂FPs may have potential hazard to human health. No significant hematological orgenetic toxicities were observed in this study. TiO₂NPs can penetrate cell membrane, and canupregulate the expression of certain cancer genes. TiO₂NPs shows higher toxicity than TiO₂FPs, which may be caused by oxidative stress in cells. NAC shows antagonistic effects on TiO₂NPs induced oxidative stress injury in culture cells.