| Engineering titanium dioxide nanoparticles(TiO2-NPs)are widely used in the environmental,communication,medicine and materials sciences fields,and are also used as additives in food and commercial products such as toothpaste and sunscreen.TiO2-NPs can easily be taken into human body through oral ingestion or respire and are mainly accumulated in the liver and lungs.Studies have found that TiO2-NPs exposure from various sources in the environment has potential genotoxic and cytotoxic effects,but the mechanisms at the cellular and subcellular levels remain to be fully revealed.Investigating the mechanisms of TiO2-NPs genotoxicity is of great significance for the safe application of engineering NPs in the environmental,materials and commercial products fields,and to prevent the possible impacts to human health.TiO2-NPs cause oxidative stress through the production of reactive oxygen species(ROS).Telomeres,located at the ends of chromosomes,are important in maintaining the structural and functional stability of chromosomes and are particularly susceptible to ROS attack.Oxidative stress may be the important cause of abnormal changes in telomere length(TL),which in turn leads to chromosome instability(CIN)and the change of cell death parthway.However,litter attention is been paid to the possible effects and molecular mechanisms of TiO2-NPs on telomeres.Based on the main metabolism pathways and target organs of the body after TiO2-NPs exposure,our study analyzed and discussed the oxidative stress effects,and the stress effects on telomere and chromosome stability induced by TiO2-NPs from the aspects of cells,chromosomes and molecule mechinisms.(1)Effects of oxidative stress induced by TiO2-NPs on different cell types.Human telomerase-positive hepatocytes L-02,hepatoma cells QGY,lung cells HBE,and telomerase-negative skin cells BJ were used in our study.H2O2 level,one of the ROS components,was analyzed after four test cells exposed to TiO2-NPs.It was found that TiO2-NPs entered into the cells in a time-(0-72 h)and dose-(0-80?g/mL)dependent manner,which caused a significant increase of H2O2 levels in the test cells(P<0.05).We also found that TiO2-NPs induced the mRNA level of Nrf-2,a key transcriptional regulator of antioxidant enzymes,down-regulated in BJ and L-02 cells(P<0.01),up-regulated in QGY cells(P<0.01),but had no significant changes in HBE cells.20 ?g/mL antioxidant N-acetylcysteine(NAC)was added to 80 ?g/mL of Ti02-NPs for co-exposure experiments.Results showed that NAC only reduced TiO2-NPs-induced ROS levels in BJ cells(P<0.01).These results indicate that TiO2-NPs exposure causes cellular oxidative stress,while different cells have different antioxidant capacity.Up-regulation of Nrf-2 may play a positive role in enhancing the anti-stress effect induced by TiO2-NPs.(2)Effects of TiO2-NPs on TL and telomere maintenance mechanisms.Four test cells were exposed to 40 and 80 ?g/mL TiO2-NPs for 72 h,and the TLs of the cells were evaluated by RT-qPCR.Results showed that TiO2-NPs exposure resulted in significant shortening of TL in L-02,HBE and BJ cells(P<0.05),but no change of TL in QGY cells was detected.The mRNA levels of telomerase activity component hETRT and telomere-binding protein complex shelterin related to telomere maintenance were further analyzed.After Ti02-NPs exposure,the expression of hTERT was down-regulated(P<0.05),and the expression of shelterin was unchanged in HBE cells;the expression of shelterin was up-regulated(P<0.05)in BJ cells;the expression of hTERT and shelterin were down-regulated(P<0.05)in L-02 cells;the expression of hTERT and shelterin were up-regulated(P<0.05)in QGY cells.Compared with the expression of Nrf-2,we found that the expression of Nrf-2 and shelterin were consistent in hTERT expression cells,but there was no similar relationship in BJ cells without hTERT expression,which suggested that Nrf-2 positively regulate the transcription of shelterin in telomerase-positive cells.High expression of Nrf-2 enhanced the antioxidant capacity,and up-regulation of hTERT and shelterin strengthened telomere maintenance of cells.Conversely,cellular antioxidant and telomere maintenance capabilities were weakened when Nrf-2,hTERT,and shelterin were down-regulated.This might be an important way for telomeres stable in QGY cells under TiO2-NPs exposure,while shortened in L-02,HBE and BJ cells.In the co-exposure experiment of 20?g/mL NAC and 80?g/mL TiO2-NPs,NAC significantly inhibited the TL shortening of HBE,L-02 and BJ cells induced by TiO2-NPs(P<0.01),suggesting that the maintenance of antioxidation capabilities is conducive to the stability of TL.(3)Effects of TiO2-NPs on genetic stability.The cytokinesis-block micronucleus cytome assay(CBMN-Cyt)was used to evaluate the effects of TiO2-NPs exposure on CIN and cell death(apoptosis and necrosis)associated with TL abnormalities.Results showed that 40 and 80 ?g/mL TiO2-NPs exposure significantly induced CIN in L-02,HBE,and BJ cells(P<0.05),but did not affect CIN in QGY cells;TiO2-NPs exposure induced necrosis(P<0.05),and the TNF-a and RIP3 transcription levels associated with necrosis were significantly increased(P<0.05)in four test cells.When cells co-exposed to 20?g/mL NAC and 80?g/mL TiO2-NPs,we found that NAC significantly decreased the high CIN(P<0.05)and cell death induced by TiO2-NPs(P<0.05).Theses imply that CIN induced by TiO2-NPs in normal cells is associated with TL shortening,and the anti-stress ability of cancer cells and normal physiological cells are different;high-doses TiO2-NPs induce cell necrosis through TNF-a-RIP3 pathway;antioxidant NAC can prevent and alleviate the associated genotoxicity induced by TiO2-NPs.(4)Cytoxicity and genotoxicity of TiO2-NPs at environmental exposure concentrations.TL,CIN and cell death of L-02,HBE,and QGY cells after exposure of 1 and 4?g/mL TiO2-NOs,similar to the exposure concentration of the population,for 12 days were also evaluated.The long-term exposure in low concentration is the characteristic of population exposed to TiO2-NPs.Results showed that 1 ?g/mL TiO2-NPs exposure increased the TL of HBE(P<0.001)but not of L-02,and induced CIN and cell death increase in L-02(P<0.05)but not in HBE.4?g/mL TiO2-NPs exposure decreased the TL and increased the CIN and cell death in both HBE and L-02(P<0.05).Significant TL shortening(P<0.001)and cell death increase(P<0.05)but not the CIN were observed in QGY after exposed to 1 and 4?g/mL TiO2-NPs.These results believed that the TL,CIN and cell death of different types of cell have different sensitivities to low-dose TiO2-NPs.In order to reasonably propose the exposure limit and safety threshold of TiO2-NPs,the toxicity evaluation of TiO2-NPs should involve cells from different tissues and organs.In conclusion,TiO2-NPs exposure resulted in a significant increase in ROS,causing cellular oxidative stress.Under this circumstance induced by TiO2-NPs,the up-regulation of Nrf-2,hTERT and shelterin were important factors for cells to increase antioxidant capacity and maintain TL stability.If TiO2-NPs exposure induced down-regulation of Nrf-2 and shelterin,and hTERT non-expression/down-regulation,it could damage the antioxidant capacity and telomere maintenance of cell,leading to telomeres shortening and CIN increase.Exposed to high doses of TiO2-NPs could up-regulate the TNF-a-RIP3 pathway and induce necrosis.The negative effects of the genetic and redox balance induced by TiO2-NPs can accelerate cell senescence,which become essential mechanisms of TiO2-NPs to affect human health and increase the risks of genetic-environmental related diseases.Moderate antioxidant intervention can effectively prevent the genotoxicity of TiO2-NPs exposure. |
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