| Objective: As a new photocatalyst, anti-UV agent and photoelectriceffect agent, titanium dioxide nanoparticles(Nano-TiO2) was widely used incosmetics, antibacterial, the management of environmental pollution,anti-aging, the paint on the surface of the car and other neighborhood. Recentstudies have shown that Nano-TiO2can through the cell membrane, enter cells,induce inflammation and then lead to DNA damage after Nano-TiO2wasexposed to mice. Nano-TiO2even can get through nuclear membrane into thecells, then it directly combines with genetic material to form DNA adduct,consequently, increase the risk of cancer greatly. Different crystals and surfacecharacteristics of Nano-TiO2can cause different levels of DNA damage to thecells cultured in vitro. This is not only related to its physical characteristics butalso bound up with the mechanism of genetic toxicity of it.Although current studies are all about the characteristics and types ofDNA damage induced by Nano-TiO2, but the molecular mechanisms have notbeen clarified. So the possible mechanisms of DNA damage induced byNano-TiO2become the focus of attention.Methods:1Cell line and establish cell modelThe L02cell line was derived from China Center for Type CultureCollection (CCTCC). Cells were cultured in Dulbecco’s modified Eagle’smedium (DMEM) with10%heat inactivated fetal calf serum (FCS) at37°Cwith a5%CO2saturated humidity. The L02cells in logarithmic growth phase,exposed to0.1,1,10μg/mL Nano-TiO2(grain-size is25nm),0.1%DMEMserved as the negative control.2Morphology observation of L02cellsThe morphology change of L02cells were observed by microscope. 3The ROS levels in L02cellsThe ROS levels in L02cells were detected through loading DCFH-DAwhich served as a fluorescent probe by using flow cytometry.4DNA damage in L02cellsDNA damage was detected by the SCGE (comet assay) after Nano-TiO2treatment.5Host cell reactivation assay(HCR)The pGL3-Control plasmid was treated with CdCl2(2μmol/L) in TEbuffer at room temperature for2hours. The damaged pGL3-Control plasmidwas dissolved in TE buffer and then recovered by precipitation with ethanol.The L02cells were transiently co-transfected with damaged pGL3-Controlplasmid and pRL-TK reporter plasmid. Six hour later, the cells aftertransfection were treated with Nano-TiO2for24h. Protein concentration andthe activities of firefly-luciferase and renilla-luciferase in the specimen weredetermined. The luciferase activities were represented with fluorescenceintensity/μg protein. The ratio of firefly-luciferase and renilla-luciferaseactivities were calculated to evaluate the repairabilities of L02cells.6Real-time PCRThe Real-time RT-PCR was used to detect FOXO3a, ChK2, GADD45α,XRCC1mRNA expression.7Western blotThe FOXO3a, ChK2, GADD45α, XRCC1protein expression wasdetected by Western blot.Results:1The morphological change of L02cells after exposed to nano-TiO2The L02cells were rhombic, adherent compact growth, high refractiveindex. After Nano-TiO2exposure, some cells shrinked into circular, therefractive index decreased, the adherent ability decreased and most of cellsfloated in the culture medium.2The levels of ROS induced by Nano-TiO2of L02cellsWith the increase of concentration of the Nano-TiO2, the levels of ROS increased. After the cells were treated with0.1μg/mL Nano-TiO2for6h or24h,the ROS levels were not significantly increase, compared with thecontrol(P>0.05); After the cells were treated with1μg/mL or10μg/mLNano-TiO2for6h or24h, the ROS levels were significantly increase,compared with the control(P<0.05or P<0.01); The ROS levels in cells treatedwith1μg/mL or10μg/mL Nano-TiO2for6h were higher than that of24h(P<0.05).3DNA damage induced by Nano-TiO2detected by comet assayWith the increase of concentration of Nano-TiO2, the Olive tail momentsignificantly increased, compared with the control(P<0.05or P<0.01), andthere was a good dose-response relationship(r=0.997, P<0.05).4DNA repair induced by Nano-TiO2detected by host cell reactivationassay (HCR)With the increase of concentration of Nano-TiO2, the DNA repaircapacity significantly decreased, compared with the control(P<0.05).5FOXO3a, ChK2, GADD45α, XRCC1mRNA expression in L02cellsafter exposed to Nano-TiO2Compared with the control group, the expression level of FOXO3amRNA in all exposed groups decreased significantly along with the increaseof the concentration of Nano-TiO2(P<0.05). Compared with the control group,the expression levels of ChK2and GADD45α mRNA in1μg/mL and10μg/mLexposed groups decreased significantly (P<0.05). Compared with the controlgroup, the expression levels of XRCC1mRNA in0.1μg/mL treatment groupincreased significantly while in1μg/mL10μg/mL treatment groups decreasedsignificantly (P<0.05).6FOXO3a, ChK2, GADD45α, XRCC1protein expression in L02cellsafter exposed to Nano-TiO2After exposed to Nano-TiO2, the expression levels of FOXO3a proteindecreased, compared with the control group, the expression level ofFOXO3a protein in10μg/mL treatment group was significantly decreased(P<0.05); Compared with the control group, the expression levels of ChK2 protein in0.1μg/mL treatment group increased significantly while in1μg/mL10μg/mL treatment groups decreased significantly (P<0.05). Compared withthe control group, the expression level of GADD45α protein in all exposedgroups decreased significantly along with the increase of the concentration ofNano-TiO2(P<0.05). Compared with the control group, the expression levelsof XRCC1protein in1μg/mL and10μg/mL exposed groups decreasedsignificantly (P<0.05).Conclusion:1Nano-TiO2can produce oxidative stress and induce oxidative damagein L02cells.2Nano-TiO2can induce DNA damage and affect the DNA repairabilitiesin L02cells.3After Nano-TiO2exposure, the expression of FOXO3a and GADD45αwas inhibited, The antioxidant effect of FOXO3a was abrogated and theFOXO3a/GADD45α complex was decreased and then reduced the DNArepair.4ChK2and XRCC1expression can be induced by low concentration ofNano-TiO2, and then activitied the DNA repair. However, high concentrationof Nano-TiO2inhibited ChK2and XRCC1expression and then weakened theability of DNA repair in L02cells. |
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