Studies On The Oxidative Stress And DNA Damage In Hepg2Cells Treated With Nano-tiO₂and Ddt

Objective：Nanometer titanium dioxide(nano-TiO₂)is a nano-materials,with UV resistant, antibacterial, self-cleaning, anti-aging effects, can bewidely used in cosmetics, functional fibers, plastics, ink, paint, paint, fineceramics. Normally people finds nano-TiO₂is relatively low toxic compounds,but recently a number of studies show that nano-TiO₂may produce damageeffect to human health. DDT is white crystals and organic chlorine pesticide,In the half of the twentieth Century, DDT prevent and control agriculturalpests,diseases such as typhoid harm reducing malaria played great role. ButDDT is very difficulty to degradated in the environment and possibleaccumulat in the fat. The method of nano-TiO₂as catalysts to photocatalyticdegradation DDT, due to in the light of the condition, DDT degradedcompletely non-toxic inorganic get increasingly the attention of people.In process of nano-TiO₂degradation DDT, will hurt to human body or not,the existing research literatures is less. Therefore, there has very importantpractical value of people research on combined effects of nano-TiO₂and DDTto assess the human health effects. This paper through observation antioxidantfunction and DNA damage effects of different particle size of nano-TiO₂andDDT combined exposure in HepG₂cells, discussion the toxicological effect ofdifferent particle size of nano-TiO₂and DDT, provide scientific basis inbiological safety evaluation of nano-TiO₂.Methods:1Establishment of cell model:the good condition of HepG₂cells,adding1μg/mL50nmTiO₂or120nmTiO₂,0.1,1,10μmol/L DDT, and the abovenano-TiO₂and DDT combined effects, DMSO (1mL/L) as solvent control,exposure time is24hours.2The observation of morphology: through biological microscope observat the morphology change of HepG₂cells after exposed.3The determination of cell activity: detect the cell survival effects of HepG₂cells by using MTT method after exposed.4The determination of oxidative damage: detect the MDA content,SOD andGSH-Px activity, evaluation the influence of oxidative damage in HepG₂cellsafter exposed.5The determination of ROS level: using flow cytometry detection techniqueto detect the ROS levels with DCFH-DA as a fluorescent probe in HepG₂cellsafter exposed..6The determination of DNA damage: detect the damage of DNA by using theSCGE, for evaluate the effect of DNA damage in HepG₂cells after exposed.7The changes of mRNA expression on DNA repair gene: using the PCRdetection technology measure the MTH1gene mRNA expression, evaluationthe effect of DNA damage in HepG₂cells after exposed.Results:1The effects of Nano-TiO₂and DDT on morphology of HepG₂cellsObservat the results by biological microscope: normal HepG₂cells arespindle, adherent compact growth, high refractive index,proliferation. Afterthe effects of Nano-TiO₂and DDT, the number of cells decreased, cellshrinkage circular,the refractive index decreases, the adherent ability drops,most floating in the culture fluid.2The effects of Nano-TiO₂and DDT on survival rate of HepG₂cellsThe cell survival rate of exposed groups reduced, except DDT0.1μmol/Lgroup, the differences were statistically significant (P <0.05or P <0.01).When DDT acting alone,with the concentration increasing, the survivalrate reduced gradually, the differences were statistically significant (P <0.05orP <0.01), dose-response relationship,correlation coefficient "r" is-0.9309.When DDT0,0.1,1,10μmol/L, the survival rate of50nmTiO₂grouplower than120nmTiO₂group, but no significant difference (P>0.05).The survival rate of combined effect lower than alone effect, thedifference of survival rate in DDT10μmol/L has statistics significance (P <0.05), other groups showed no significant difference (P>0.05).3The effects of Nano-TiO₂and DDT on oxidative damage of HepG₂cells3.1The changes of MDA contentThe MDA content of all exposed groups rised, except DDT0.1μmol/Lgroup, the differences were statistically significant (P <0.05or P <0.01).When DDT acting alone, with the concentration increasing, the MDAcontent rised gradually, the differences were statistically significant (P <0.05or P <0.01), dose-response relationship, correlation coefficient "r" is0.9729.When DDT0,0.1,1μmol/L, the MDA content of50nmTiO₂grouphigher than120nmTiO₂group,the differences were statistically significant(P <0.05or P <0.01); When DDT10μmol/L, the MDA content of50nmTiO₂group higher than120nmTiO₂group, but no significant difference(P>0.05).The MDA content of combined effect higher than alone effect, thedifferences were statistically significant (P <0.05or P <0.01). By two factorialanalysis, the combined effect of Nano-TiO₂and DDT appear interaction(P <0.05).3.2The changes of SOD activityThe SOD activity of all exposed groups reduced, the differences werestatistically significant (P <0.05or P <0.01).When DDT acting alone,with the concentration increasing, the SODactivity reduced gradually, the differences were statistically significant(P <0.05or P <0.01), dose-response relationship, correlation coefficient "r" is-0.8476.When DDT0,0.1,1,10μmol/L, the SOD activity of50nmTiO₂grouplower than120nmTiO₂group, the differences were statistically significant(P <0.05).The SOD activity of combined effect higher than alone effect, thedifferences were statistically significant (P <0.05or P <0.01). By two factorialanalysis, the combined effect of Nano-TiO₂and DDT appear interaction(P <0.05).3.3The changes of GSH-PXactivity The GSH-PXactivity of all exposed groups reduced, exceptDDT0.1μmol/L group, the differences were statistically significant (P <0.05orP <0.01).When DDT acting alone,with the concentration increasing,the GSH-PXactivity reduced gradually, the differences were statistically significant(P <0.05or P <0.01), dose-response relationship,correlation coefficient "r" is-0.8571.When DDT0,0.1,1,10μmol/L, the GSH-PXactivity of50nmTiO₂group lower than120nmTiO₂group, the differences were statisticallysignificant (P <0.05).The GSH-PXactivity of combined effect reduce compared with aloneeffect, the difference was statistically significant (P <0.05). By two factorialanalysis, the combined effect of Nano-TiO₂and DDT appear interaction(P <0.05).4The effects of Nano-TiO₂and DDT on ROS level of HepG₂cellsThe ROS level of all exposed groups rised, except DDT0.1μmol/L group,the differences were statistically significant (P <0.05or P <0.01).When DDT acting alone,with the concentration increasing,the ROS levelrised gradually, the differences were statistically significant(P <0.05orP <0.01), dose-response relationship,correlation coefficient "r" is0.9971.When DDT1,10μmol/L, the ROS level of50nmTiO₂group higher than120nm TiO₂group,the differences were statistically significant (P <0.05orP <0.01); When DDT0,0.1μmol/L, the ROS level of50nmTiO₂grouphigher than120nmTiO₂group, but no significant difference (P>0.05).The ROS level of combined effect higher than alone effect, thedifferences were statistically significant (P <0.05or P <0.01). By twofactorial analysis, the combined effect of Nano-TiO₂and DDT appearinteraction (P <0.05).5The effects of Nano-TiO₂and DDT on DNA damage of HepG₂cellsThe olive tail moment of all exposed groups increased,the differenceswere statistically significant (P <0.05or P <0.01). When DDT acting alone, with the concentration increasing, the olive tailmoment increased gradually, the differences were statistically significant(P <0.05or P <0.01), dose-response relationship, correlation coefficient "r" is0.8416.When DDT0,0.1,10μmol/L, the olive tail moment of50nmTiO₂grouplonger than120nmTiO₂group, the differences were statistically significant(P <0.05or P <0.01); When DDT1μmol/L, the olive tail moment of50nmTiO₂group longer than120nmTiO₂group, but no significant difference(P>0.05).The olive tail moment of combined effect longer than alone effect, thedifferences were statistically significant (P <0.05or P <0.01). By two factorialanalysis, the combined effect of Nano-TiO₂and DDT appear interaction(P <0.05).6The effects of Nano-TiO₂and DDT on MTH1mRNA level expression ofHepG₂cellsThe expression level of mRNA in all exposed groups rised, except50nmTiO₂group and120nmTiO₂group, the differences were statisticallysignificant (P <0.05orP <0.01).When DDT acting alone, with the concentration increasing,the expressionlevel of mRNA rised gradually, the differences were statistically significant(P <0.05or P <0.01), dose-response relationship, correlation coefficient "r" is0.9218.When DDT1μmol/L, the expression level of mRNA in50nmTiO₂grouphigher than120nmTiO₂group, the differences were statistically significant(P <0.05or P <0.01); When DDT0,0.1,10μmol/L, the expression level ofmRNA in50nmTiO₂group higher than120nmTiO₂group,but no significantdifference (P>0.05).The expression level of mRNA in combined effect higher than aloneeffect, the differences were statistically significant (P <0.05or P <0.01). Bytwo factorial analysis, the combined effect of Nano-TiO₂and DDT appearinteraction (P <0.05). Conclusion:1The effect of Nano-TiO₂and DDT can make damage and survival ratereduce in HepG₂cells.2The effect of Nano-TiO₂and DDT can make damage on antioxidantfunction and produces oxidative stress in HepG₂cells.3The effect of Nano-TiO₂and DDT can induce damage on DNA inHepG₂cells.4With the Nano-TiO₂particle size reducing, the injury has increasedafter exposed.5Nano-TiO₂and DDT combined exposure compared to Nano-TiO₂orDDT aloned exposure, the effect of damage tended to increase, showinteraction, but people need to further research of the action is synergisticeffects or not.