Study Of Two Crystalline Phases Of TiO₂-NPs Affect Genetic Stability And The Mechanisms On Human Hepatocytes

Titanium dioxide nanoparticles（TiO₂-NPs）are often used in cosmetics,pharmaceuticals,coatings,food additives,and other daily products due to their unique physicochemical properties.As exogenous substances,they display toxic effects and potential health risks.The physicochemical properties,such as particle size,surface charge,surface modification,and crystal shape,are closely related to the genotoxicity of TiO₂-NPs.Rutile and anatase are two commonly used crystalline phases of TiO₂-NPs.Whether the two crystalline phases of TiO₂-NPs would exhibit different genotoxic effects on cells under the same exposure conditions and the underlying mechanisms have not been systematically reported.The relationships between the genotoxic effects of TiO₂-NPs and their two crystalline phases,and the underlying molecular mechanisms were discussed from the potential uptake and molecular mechanisms,the possible oxidative stress effects,and the effects on cellular senescence and genetic stability of TiO₂-NPs on human cells in vitro.The rutile TiO₂-NPs（TiO₂-NPs-R）and anatase TiO₂-NPs（TiO₂-NPs-A）of similar size were selected for the study.Human hepatocytes L-02 and QGY were exposed to two different crystalline TiO₂-NPs for 48h.The UV absorption ratio Ae/A₀and the lateral light scattering angle（SSC）ratio SSCe/SSC₀ were used to evaluate the degree and the efficiency of TiO₂-NPs entry into cells,respectively.The transcript expression levels of endocytosis-associated caveolin-1 gene（CAV-1）,Clathrin heavy chain gene（CLTC）,and oxidative regulation-related gene（Nrf-2）were measured by relative quantitative qPCR;the cellular oxidative stress level was evaluated by intracellular reactive oxygen species（ROS）and malondialdehyde（MDA）,a marker of lipid peroxidation;telomere length（TL）was measured by standard curve RT-qPCR;genome instability（GIN）and cell death represented by apoptosis and necrosis were evaluated by cytoplasmic division blocking micronucleome assay（CBMN-Cyt）;cellular senescence was evaluated byβ-galactosidase in situ staining.Results showed that:（1）TiO₂-NPs-A was more likely to sediment and enter into cells via endocytosis than TiO₂-NPs-R.TiO₂-NPs-A induced significantly higher SSCe/SSC₀ values in L-02and QGY cells than TiO₂-NPs-R（P<0.001）,suggesting that TiO₂-NPs-A was more likely to enter into cells.TiO₂-NPs-A exposure significantly induced upregulation of CAV-1,CLTC in QGY cells and CAV-1 in L-02 cells（P<0.01）,while TiO₂-NPs-R exposure significantly induced downregulation of CAV-1 and upregulation of CLTC in QGY cells（P<0.01）.（2）TiO₂-NPs-A significantly induced oxidative stress effects in cells compared to TiO₂-NPs-R.TiO₂-NPs-A and TiO₂-NPs-R significantly elevated ROS levels and upregulation of Nrf-2 in L-02 cells（P<0.01）.TiO₂-NPs-A induced significantly higher MDA levels in L-02 cells than TiO₂-NPs-R（P<0.001）;TiO₂-NPs-A induced stronger ROS levels and upregulation of Nrf-2 in QGY cells than TiO₂-NPs-R（P<0.01）.（3）TiO₂-NPs-A exhibited higher genotoxic effects than TiO₂-NPs-R.In L-02 cells,TiO₂-NPs-A and TiO₂-NPs-R significantly induced TL shortening,GIN,senescence,and cell death（P<0.05）,TiO₂-NPs-A induced significantly higher cell death than TiO₂-NPs-R（P<0.01）.In QGY cells,TiO₂-NPs-R significantly induced elevated GIN and cell death（P<0.05）.TiO₂-NPs-A induced higher intensity of the above events than TiO₂-NPs-R in QGY cells（P<0.05）,and induced TL shortening and cell senescence in QGY cells.In conclusion,crystallographic differences have an important influence in the genotoxicity induced by TiO₂-NPs on L-02,QGY cells.TiO₂-NPs-A was more genotoxic than TiO₂-NPs-R,which be related to the fact that TiO₂-NPs-A was easier sediment and increased cellular endocytic capacity to enter cells,thus leading to stronger oxidative stress and causing TL shortening,increased GIN,and accelerated cellular senescence and cell death.This study provided scientific evidences on the correlation between the crystalline phases of TiO₂-NPs and genotoxicity of cells,which may further help to explore the safety application of TiO₂-NPs and prevent the potential negative health effects of TiO₂-NPs.