Assessment Of Alterations In X-ray Irradiation-induced DNA Damage Of Glioma Cells By Using High-resolution Proton Nuclear Magnetic Resonance Spectroscopy

Objective:To study the DNA damage caused by a series does of X-ray irradiation(0,1,5,10 and 15Gy)in glioma cell lines(C6,U87 and U251)using high-resolution proton magnetic resonance(1HNMR)spectroscopy in vitro,and to preliminarily investigate its mechanism.Methods:Three glioma cell lines(C6,U87 and U251)were randomly separated into 5 groups,and exposed to 0,1,5,10 and 15Gy X-ray irradiation,respectively.OGy was designed as control group,and 1,5,10 and 15Gy were designed as treatment group.After exposure to X-ray,each groupwas separated into five parts:1)to determine the DNA damage parameters(tail length,%DNA in tail,andtail moment)by single cell gel electrophoresis assay(it also could benamed "comet assay");2)to detect the cell cycle distribution by flow cytometry;3)to detect apoptosis rates by flow cytometry;4)to calculate the colony-forming efficiencyand use themulti-targetsingle-hittingmodelto calculate thecellular radiosensitivity parameters(SF2,survival fractionat a dose of 2Gy,representing the tumor cell intrinsic radiosensitivity);5)to detectmetabolite alterations by1HNMR spectroscopy.The differences between cellular radiosensitivity parameters of cell lines were assessed by independent-sample t-test.DNA damage,cell cycle distributions,cell apoptosis rates,and the metabolite ratios caused by a series dose of X-ray irradiation were compared by one-way analysis of variance(ANOVA).Results:1.DNA damage Comet tails became longeralong with higher exposure to X-ray,which indicatedthat DNA strand breaks were induced by X-ray irradiation.Obvious changes in tail length,TDNA and tail moment were observed.Compared to the control group,the DNAdamage of each treatment groupwas increased(C6,P<0.01;U87,P<0.05;U251,P<0.01).And DNA damage increased in a dose-dependent way with the X-ray increasing.2.Cell cycle distribution Comparedwith the control group,the treatment groups of threecell lines showed cell cycle blockage.After exposure to 1 Gy X-ray,the percentage of G1 in the threecell lines was increased(C6,P<0.05;U87,P<0.01;U251,P<0.01)comparing with control group.When C6 cells were exposed to5Gy,10Gy and 15Gy,theGl phase blockage increased(1Gy vs.5Gy,P<0.05;5Gy vs.10Gy,P<0.05;10Gy vs.15Gy,P>0.05);and that of U87 and U251 deceased(U87:1Gy vs.5Gy,P<0.05;5Gy vs.10Gy,P<0.05;10Gy vs.15Gy,P>0.05.U251:1Gy vs.5Gy,P<0.O01 5Gy vs.lOGy,P<0.01;10Gy vs.15Gy,P<0.01).3.The apoptosis rates of three glioma cell linesCompared with control group,the apoptosis rates of treatment groups inncreased obviously(C6,P<0.01;U87,P<0.01;U251,P<0.01).In glioma cell lines C6 and U87,apoptosis rateswere slowly increased(C6,P<0.01;U87,P<0.05).However,in U251 cells,the apoptosis rates showed a fast initial increase(0Gy vs.1Gy,P<0.01),then a low increase(1Gy vs.5Gy,P<0.05;5Gy vs.10Gy,P<0.01;10Gy vs.15Gy,P>0.05).4.Colony-forming assay Compared with the control group,the colony forming efficiencies of treatment groups decreased(C6,P<0.01;U87,P<0.01;U251,P<0.01).The colony forming efficiencies were compared between treatment groups,and the results demonstrated as follows:1Gy vs.5Gy,each P of three cell lines<0.01;5Gy vs.10Gy,each P of C6 and U251<0.01,the P value of U87<0.05;10Gy vs.15Gy,the P value of C6<0.01,each P value of U87 and U251>0.05.5.The radiosensitivity of cells Dose-survival curves of three cell lineswere obtained and the SF2(survival fractionwith irradiation dose of 2Gy)of C6,U251 andU87were 0.557,0.456and0.148,respectively.The radiosensitivity of U87 was bigger than U251,and the radiosensitivity of U251 was bigger than C6.6.Changes of metabolite ratios The ratios of Lac/Cr and Suc/Crdecreased as the dose of X-rayexposure increased,whereas the ratios of Cho/Cr increased.Thesignificant changes occurred in the three treated glioma cell lines(C6,P<0.05;U87,P<0.05;U251,P<0.01),which were linearly correlated with DNA damage(0.783<R2<0.996).Conclusion:Metabolites ratios(Lac/Cr,Suc/Cr,Cho/Cr)detected by 1H NMR spectroscopy were linearly associated with the DNA damage,which could lead to glioma cell apoptosis,caused by X-ray irradiation.1H NMR spectroscopy has the potential to provide a noninvasive biomarker to predict DNA damage after radiotherapy of glioma cells.