The Experimental Effect Of DNA Methylation Induced By Ionizing Radiation

[Objective]To examine the effect of tritium oxide (HTO) and cobalt-60 gamma ray on DNA methylation pattern and DNA methyltransferase 1 (Dnmt1) expression, screen out the special damage methylated genes by ionizing radiation, elaborate the mechanism of radiation damage in epgenetics, and provide new idea for the radiation protection.[Methods]1. The C57BL/6J male mice were exposed to HTO (1.85×10⁵ Bq/g,5.55×10⁵ Bq/g,16.65×10⁵ Bq/g) or the cobalt-60 gamma ray(1Gy/min, 4Gy). The amount of peripheral white blood cell (WBC) and micronucleus rate of polychromatic crythrocytes (fMNPCE) were measured. Different blood biochemical parameters were determined with automatic biochemistry analyzer. The superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents of the hepatic tissue were detected. The changes of DNA methylation pattern were detected with Methylated DNA Immunoprecipitation-chip (MeDIP-chip), and the expressions of Dnmt1 of the tissues with western blot and immunohisto-chemical streptavidin-perosidase (SP) method analysis. The expression level of Dnmt1 protein was reflected with the integrated optical density (IOD) per high power vision of the immunohisto-chemical photographs, analyzed by image-pro plus6.0.2. Exponentially growing BRL cells cultured in vitro were irradiated by cobalt-60 gamma ray (1Gy/min, 4Gy). The cell cycle was analyzed with fluorescence-activated cell sorting (FACS) in the flow cytometry (FCM), and the expressions of Dnmt1 and P53 at different time points with western blot analysis.[Results]1. fMNPCE and WBC(1) The fMNPCE in bone marrow was significantly increased in every experiments groups (P<0.05), as the initial concentration and the cumulative doses of HTO were increased; The amount of peripheral WBC was correspondingly decreased, but there was significantly different only in the high concentration group (P<0.05).(2) In the same initial concentration groups, the fMNPCE were increased continuously with increment of cumulative doses, there were significantly different from the forth day (P<0.05); The total number of peripheral WBC were continuously decreased at the second, forth, and sixth day, there were significant differences (P<0.05), at the tenth day began to recover, and had been returned to normal at the 28th day when having no significant differences compared with the control group.(3) In the irradiated group with 4Gy gamma ray, the amount of peripheral WBC was decreased significantly (P<0.05), but the fMNPCE was increased significantly (P<0.05)2. Measurement of Blood Biochemistry and Lipid Peroxidation in Hepatic Tissue(1) In the middle and high concentration groups, the AST and ALT were all significantly increased at the sixth and tenth day (P<0.05), the amount of total protein was changed in the same way as AST and ALT, and the ALP and glucose level were increased significantly in the both groups (P<0.05).(2) In the middle concentration group, the SOD activity was no significant changes, but in the high concentration group was significantly decreased (P<0.05), and in the both experiment groups, the level of MDA was significantly increased (P<0.05).3. DNA methylation pattern in Hepatic TissueWith the internal irradiation of HTO and external irradiation of cobalt-60 gamma ray, the methylation profiles of the mice in promoter regions were changed obviously as follow:(1) In the control group, there were 3922 genes with methylated CpG in promoter regions, but 4202 in the middle concentration group, 4950 in the high concentration group, and 4560 in the irradiated group.(2) In the middle concentration group, there were 1031 genes with new methylation, but 1550 in the high concentration group, and 1300 in the irradiated group. There were 746 genes with demethylation in the middle concentration group, but 518 in the high concentration group and 660 in the irradiated group. The most methylated gene was chr11 and the most demethylated was chr2.(3) For the improving the screening criteria, the obviously methylated genes in the middle concentration group were Aebp2, Sema3f, Pcdh8, Itpripl2, Dnmt3a, Nefl, Fbll1, Snrpe, Lemd1, Hoxc10, Dbf4 and Slc25a40, but in the high concentration group were Limk1, Trim71, Stac2, Inhbb, 1110034A24Rik, Rcc2, Ccdc102a, Fbxl3, Rshl2b and Cdyl2, in the irradiated group were Trim71, Sema3f, Pcdh8, Sox4, 1110034A24Rik, Limk1, Nefl and Xpr1. The obviously demethylated genes in the middle concentration group were Rest, Eif1a and Kcna7, in the high dose group were Rest, Phip and Eif1a, and in the irradiated group were Sfrs18, Dr1, Ankrd42, Nae1, Sfi1, Accn1, Srd5a1, Nsun2, Eif1a and Dusp5.4. Dnmt1 protein expression(1) Compared with the control group, as the initial injection of HTO were increased and the cumulative dose of HTO increased, the Dnmt1 protein expressions in the lung, liver, kidney were decreased. In every experiments groups, the Dnmt1 protein expressions of lung were no significantly differences (F=2.52, P>0.05), but in the middle and high concentration groups, that of liver were significantly decreased (F=20.84, P<0.05), and that of kidney were significantly decreased in every experiment groups (F=13.34, P<0.05).(2) In the groups of the same initial concentration, as the internal irradiation time went, the Dnmt1 protein expression had some characteristics in every tissue: In the lung, there were significant differences at the second and forth day (F=16.65, P<0.05), in the second day that reached to the lowest, at the tenth day recovered basically. In the liver, there were significant differences at the forth, sixth, tenth and 28th day (F=35.57, P<0.05), at the sixth day reached to a nadir, and then recovered gradually, in the kidney, there were significant differences at the second, forth, sixth, and the tenth day (F=17.31, P<0.05), it reached to a nadir at the forth day and recovered basically at the 28th, and in the brain, that reached to a nadir at the second day, recovered at the 28th day when having no significant differences compared with the control group.(3) In the irritated groups, except the lung tissue, the Dnmt1 expression level of liver, kidney, testicle and brain tissues in the external irradiation groups were significantly higher than that in the control group.5. Cell cycle, Dnmt1 protein and P53 protein of BRL cellAfter cobalt-60 gamma ray irradiation with 4Gy, G2 cell cycle arrest appeared at the forth hour and recovered at the 24th hour, the cell number of S cycle decreased and G1 cell cycle arrest appeared at the 12th hour, weren't returned to normal at the 48th hour. The expressions of Dnmt1 protein were increased at the forth and the 12th hour, and then began to recover, weren't returned to normal at the 48th hour. The expression of P53 protein was increased at the forth hour, and recovered at the 12^th hour.[Conclusions]1. Ionizing radiation can affect the methylation pattern and expression of Dnmt1, and the effect on Dnmt1 expression was of dose and time dependent, but the special damage methylated genes by ionizing radiation damage yet need further studies.2. Dnmt1 may take part in G1, G2 cell cycle arrest and DNA damage repair mediated by P53 after ionizing radiation damage.