Studies On The Oxidative Stress And DNA Damage To Mice Treated With Nano-TiO₂and DDT

Objective: Nanoparticle titanium dioxide（Nano-TiO₂） is a kind ofnano-materials, and as a new photocatalyst, anti-uv agent and agent ofphotoelectric effect are widely used in the process of wastewater treatment. Inrecent years, a large number of studies have found that Nano-TiO₂can affectbody’s process of oxidative stress, leading to inflammation response, DNAdamage and lipid peroxidation, etc., harmful to human body health. DDT is akind of organochlorine pesticides and persistent organic pollutants （pops）common in the environment. It has many characteristics, such as persistent,accumulation, transference and high toxicity. Nano-TiO₂displays superadsorption to DDT after entering the environment, and uses photocatalyticoxidation degradation technology biodegrading it fully into non-toxic materialunder mild conditions. In process of degradation, Nano-TiO₂will impact onhuman health or not, and the existing research literature is less. Therefore thestudy intent to establish animal model treated with Nano-TiO₂and DDT inmice, to observe the changes of oxidative stress conditions in mice, thedetection of cell DNA damage and related genes serine/threonine proteinkinase （Akt） change in expression, and then discusses the Nano-TiO₂andDDT combinational damage effects and its mechanism of action, as to providetheoretical basis for safe application of Nano-TiO₂.Methods:1Establishment of animal model:72healthy KunMing mouse wererandomly divided into1control group and8treatment groups,8each group,male and female half. Respectively gave2g/kg body weight of50nm and120nm TiO₂,30/60mg/kg of DDT and combinated dose of Nano-TiO₂andDDT to mice via gastrointestinal tract, while distilled water （ddH2O） and cornoil as solvent controls. Lavaged for7days in a row, and put the animals to death. The tissues were collected based on detection of the related project.2Histopathological observation: made liver and kidney tissuepathological slices, then observed the pathology changes of struture aftertreated with Nano-TiO₂and DDT separately or in combination.3Determination of the contents of Ti in mice: used microwave digestionand inductively coupled plasma mass spectrometry （ICP-MS） method to detectthe Ti contents in liver, kidney, cortex, and hippocampus tissues of mice aftertreated with Nano-TiO₂and DDT separately or in combination.4Determination of intracellular ROS levels: dichlorodihydrf luoresceindiacetate （DCFH-DA） as a fluorescence probe, used flow cytometry detectiontechnology to detect intracellular reactive oxygen species （ROS） level in liver,kidney,cortex and hippocampus tissue cells of mice after treated withNano-TiO₂and DDT separately or in combination.5Determination of tissue oxidative damage index: used the kits to detectMDA content and SOD, GSH-Px vitality change in serum, liver, kidney,cortex and hippocampus tissues of mice after treated with Nano-TiO₂andDDT separately or in combination.6Determination of DNA damage: used single cell gel electrophoresisexperiment （SCGE） to detect OTM value change in liver, kidney, cortex andhippocampus tissue cells of mice after treated with Nano-TiO₂and DDTseparately or in combination.7Determination of Akt gene mRNA in expression: used polymerasechain reaction （PCR） method to detect the serine/threonine protein kinases（Akt） gene mRNA expression level change in liver, kidney, cortex andhippocampus tissue cells of mice after treated with Nano-TiO₂and DDTseparately or in combination.Results:1The effects of Nano-TiO₂and DDT on the general status of miceMice body weight increased, with no significant difference betweengroups （P>0.05）. In TiO₂-50+DDT60group individual mice displayd slighttremor and symptoms of panic gait. Compared to control group, in TiO₂and DDT combination treatmentgroups and DDT60individual treatment group, the liver organ coefficientincreased significantly （P<0.05）, the effect of combination was greater thanNano-TiO₂or DDT separately, and with the increase of DDT dose, organcoefficient increased （P<0.05）, but the particle size of TiO₂had little effect. Inother organs （kidney, cortex, hippocampus）, organ coefficient in differenttreatment groups showed no statistically significant difference （P>0.05）.2Liver and kidney tissue morphology change in mice after treated withNano-TiO₂or DDT separately and in combinationIn liver, treated with Nano-TiO₂or DDT separately, there was expansionand congestion in central venous of lobular, along with the expansion of bileduct, liver blood sinus, some cells in vacuoles degeneration and cytoplasmloose light dye. Treated with Nano-TiO₂and DDT, damage degree wasaggravating, particularly in the TiO₂-50+DDT60group appearing a largenumber of water samples; And kidney pathology observation found thattreated with Nano-TiO₂or DDT separately, mild edema of the renal cellsappeared with cloudy swelling, thickening, glomeruli swelling and bleeding.Treated with Nano-TiO₂and DDT, damage degree was aggravating, anddisordered arrangement of renal tubular cells, renal interstitial hyperemia,edema, hemorrhage, lymphocytic invasion and local point necrosis appeared.3The effects of Nano-TiO₂and DDT on Ti contents in tissue of miceCompared to control group, in groups treated with TiO₂separately andgroups with TiO₂and DDT, Ti content was significantly increased （P<0.05） inliver, kidney, cortex and hippocampus of mice; In groups treated with TiO₂and DDT （TiO₂-120+DDT30group in liver and TiO₂-50+DDT30group inkidney） there was no significant difference from the relevant groups treatedwith TiO₂separately, while the rest of the groups were significantly higherthan the relevant groups treated with TiO₂separately （P<0.05）; With thedecrease of the particle size of the TiO₂, Ti content had a tendency to increase;And DDT had no significant effect on the accumulation of Ti and Ti contentsin some organ raise relevantly. Two factors analysis of variance indicated no interaction （P>0.05） after treated with Nano-TiO₂and DDT.4The effects of Nano-TiO₂and DDT on ROS levels in cells of miceCompared to control group, Nano-TiO₂and DDT could makeintracellular ROS levels elevated in liver, kidney, cortex, and hippocampus,and the effect of combination was greater than that of bothindependend（P<0.05）; With the increase of DDT dose, intracellular ROSlevels increased correspondingly; With the decrease of the particle size of theTiO₂, ROS levels had a rising trend but the trend that liver, kidney andhippocampus intracellular ROS levels increase with the increase of DDT dosewas not obvious. Two factors analysis of variance indicated interaction（P<0.05） only in cortex after treated with Nano-TiO₂and DDT.5The effects of Nano-TiO₂and DDT on MDA、SOD、GSH-Px in tissueof miceCompared to control group, the Nano-TiO₂and DDT could make MDAcontents elevated in serum, liver, kidney, cortex, and hippocampus, and theeffect of combination was greater than that of both independend（P<0.05）;With the increase of DDT dose, MDA contents increased correspondingly;With the decrease of the particle size of the TiO₂, MDA contents had a risingtrend but the trend that serum and liver MDA contents increase with decreaseof the particle size of the TiO₂was not obvious. Two factors analysis ofvariance indicated interaction （P<0.05） only in hippocampus after treated withNano-TiO₂and DDT.Compared to control group, the Nano-TiO₂and DDT can make SODvitality decrease in serum, liver, kidney, cortex, and hippocampus, and theeffect of combination was greater than that of both independend（P<0.05）;With the increase of DDT dose, SOD vitality decreased correspondingly; Withthe decrease of the particle size of the TiO₂, SOD vitality had a drop trend butthe trend that serum, kidney and hippocampus SOD vitality decrease withdecrease of the particle size of the TiO₂was not obvious. Two factors analysisof variance indicated interaction （P<0.05） only in cortex after treated withNano-TiO₂and DDT. Compared to control group, the Nano-TiO₂and DDT can make GSH-Pxvitality decrease in serum, liver, kidney, cortex, and hippocampus, and theeffect of combination was greater than that of both independend （P<0.05）;With the increase of DDT dose, GSH-Px vitality decreased correspondingly;With the decrease of the particle size of the TiO₂, GSH-Px vitality had a droptrend but the trend in kidney and cortex was not obvious. Two factors analysisof variance indicated interaction （P<0.05） only in serum and hippocampusafter treated with Nano-TiO₂and DDT.6The effects of Nano-TiO₂and DDT on DNA damage in cell of miceCompared to control group, the Nano-TiO₂and DDT can lead to DNAdamage in liver, kidney, cortex, and hippocampus, and the effect ofcombination was greater than that of both independend（P<0.05）; With theincrease of DDT dose and the decrease of the particle size of the TiO₂, thedegree of DNA damage increased correspondingly. But there was nosignificant difference between TiO₂-120+DDT30and TiO₂-120+DDT0groupin kidney（P>0.05）. Two factors analysis of variance indicated interaction（P<0.05） in liver, cortex, and hippocampus cells after treated with Nano-TiO₂and DDT.7The effects of Nano-TiO₂and DDT on Akt gene mRNA expressionOn the determination of Akt gene family Akt1/Akt2/Akt3mRNAexpression in liver, kidney, cortex and hippocampus, it found that Nano-TiO₂and DDT can inhibit the Akt gene expression and the function of combinationtreatment was greater than two separate. With the decrease of the Nano-TiO₂particle size, inhibitory effects had been strengthen, but DDT inhibitory effectwas not obvious. In liver and kidney, the inhibition of the expression of Akt2and Akt3was greater then Akt1gene significantly, and Akt1/Akt3/Akt2geneexpression in brain tissue had the corresponding decline （P<0.05）. Two factorsanalysis of variance showed no interaction （P>0.05） after treated withNano-TiO₂and DDT. Conclusions:1It made liver and kidney of mice appear a certain degree of pathologicalchanges after treated with Nano-TiO₂or DDT separately and in combination.2Ti distributed in tissue of liver, kidney, cortex and hippocampus aftertreated with Nano-TiO₂separately and in combination with DDT.3It caused serum, liver, kidney, cortex, and hippocampus of miceoxidative damage and antioxidant ability damage after treated with Nano-TiO₂or DDT separately and in combination.4It caused DNA damage and related gene Akt expression drop of cellsafter treated with Nano-TiO₂or DDT separately and in combination.5With TiO₂particle size reduced and DDT dose increased, the damageeffects was strengthen, but the specific interaction between TiO₂and DDT andtheir concrete form need further research.6With Nano-TiO₂particle size reduced, the damage effects to the micewas strengthen, but the relationship between TiO₂particle size and its damageeffects need further research.