The Effects Of Low Dose Radiation On DNA Methylation Of Bone Marrow, Liver And Spleen

As the radiation medical applicated in clinical diagnosis and treatmentand the development of nuclear power, radiation damage is also graduallyincreased. Although the research about biological effect of radiationincreased year by year in recent years, but many focus on high doseradiation than low dose radiation, This makes people lacking adequatecomprehension and understanding of low dose radiation effects. Low doseradiation do not cause obvious damage, but do not mean that low doseradiation have no danger to the body.Radiation can damage many tissues and organs of the body, Such ashematopoietic of bone marrow, liver of digestive systems and spleen of theimmune system, these all are sensitive to radiation. Previous study haveshown radiation what damage DNA of bone marrow mononuclear cells(BMNC), increase Apoptosis and decrease bone marrow proliferation ability, cause the reduction of bone marrow hematopoietic stem cell,eventually lead the happening of the blood diseases, such as leukemia. Atthe same time, radiation also can damage liver tissues.Gridley found that nomatter what type of radiation leaded to immune suppression and abnormalof spleen cells.Epigenetics is the gene expression changes that does not involve DNAsequence changes. The main regulating way including DNA methylation,histone modification, chromosome remodeling and non-coding RNA. Atpresent study, people pay more attention to DNA methylation, what iscatalyzed by DNA methyltransferases(DNMT), which make the activity ofS-methyl from S-adenosylmethionine to cytosine, then cytosine come into5-methyl cytosine.DNA methylation occurs on cytosine residues andmainly concentrate on CpG dinucleotide sites. The CpG in mammals existsin two forms, one is dispersed in the DNA, the other is located near thetranscriptional regulation regions of the CpG island. It not only can bestable or lock chromosomal status of the resting position, but also areessential to the development of gene imprinting, X chromosomeinactivation and suppression of retrotransposons original ofanimal.In1989,Kalinich found that methylated cytosine of cell lines canrestore cytosine. In1998, Tawa also reported that radiation can lead thereduction of gene-wide methylation degree. In2013, Antwih reported thatradiation can affect DNA methylation.However, these studies adopt high-dose radiation, the relation between low-dose radiation and DNAmethylation remain unclear.2004, Kovalchuk found that low dosesradiation can leaded changes of DNA methylation levels, and had atissue-specificity and organ-specificity, But the report about therelationship between low doses radiation and DNA methylation is not veryclear. Our study through observe biological effects of low doses radiation inmice, especially the effect of DNA methylation, then offer a new researchidea for radiation protection.Objective: Through study the peripheral blood cell, BMNCproliferation and apoptosis, liver and spleen histologic structure, spleenindex, DNA methylation level of whole gene and the influence of DNAmethyltransferase expression of liver, spleen and BMNC of acute lowdose radiation on mice, and then observe the biological effects of acutelow dose radiation on mice.Methods:1. The C57BL/6mice were randomly divided into5groups,25rats ineach group, the normal group (without any treatment); radiation group(According to extraction time after radiation divided into1,3,7and14dgroup).The radiation group was radiated by linear accelerator about0.39min, the total dose was1Gy, X ray absorption dose rate was300cGy/min,the effective area is25cm2and the focus-rats distance is100cm. then themice were killed by cervical dislocation after collecting blood at different time points, extracted BMNC, liver and spleen according to conventionalmethods.2. peripheral blood cell was detected by conventional methods.3. The BMNC proliferation ability was detected by CCK8.4. The BMNC apoptosis rate was detected by Flow cytometry.5. The Liver biopsy were prepared and detected by microscope.6. The spleen index was calculated and spleen biopsy were preparedand detected by microscope.7. The liver, spleen and BMNC DNA methylation level of whole genewere detected by High performance liquid chromatograph.8. The DNMT1, DNMT3A and DNMT3B expression of BMNC weredetected by western blot.9. The DNMT1, DNMT3A and DNMT3B expression of liverwere detected by western blot.10. The DNMT1, DNMT3A and DNMT3B expression of spleenwere detected by western blot.Result:1. Compared with normal group, the hair luster, activity, eatingand drinking as same as normal mice, the excrement was also normal,and no mice died in the radiation group.2. Effects of low dose radiation on peripheral blood cell:Compared with normal group, white blood cell and platelet count were significantly reduced in radiation group（P<0.05）, While white bloodcells dropped to the lowest in the1d, and platelet fell to the lowest inthe3d and still not back to normal in the14d.3. Effects of low dose radiation on BMNC proliferation ability:Compared with normal group, BMNC proliferation ability wasincreased in radiation group(P <0.05) and back to normal in the14d.4. Effects of low dose radiation on BMNC apoptosis rate:Compared with normal group, the BMNC apoptosis rate began to risein the3d in radiation group and back to normal in the14d.5. Effects of low dose radiation on liver histological structure:Compared with normal group, the hepatic lobule of radiation groupwas integrated, pathological changes tend to occur near the central vein,liver cell edema and cavitation, partial liver cells necrosis, some cellssteatosis and blood sinus was squeezed narrowed. Especially in the3dand7d, the14d is slightly better, but not back to normal.6. Effects of low dose radiation on spleen index and spleenhistological structure: Compared with normal group, spleen index in1dwas significantly reduced in radiation group (P <0.05), and back tonormal in7d. Light microscopy showed that between white pulp andred pulp had a clear demarcation and lymphocytes were closelyarranged in normal group, the spleen of radiation group occurredcongestion, the small blood vessels was hyaline degeneration and some vascular endothelial cell was swelled,especially in the3d group,gradually improved in7d and returned to normal in14d.7. Effects of low dose radiation on DNA methylation level ofwhole gene: Compared with normal group, the DNA methylation levelof whole gene of BMNC, liver and spleen were decreased in radiationgroup (P <0.05), BMNC and liver respectively back to normal in14dand3d, but the spleen has not yet returned to normal in14d.8. Effects of low dose radiation on DNA methyltransferase of BMNC:Compared with normal group, the expression of DNMT1and DNMT3Awere significantly increased in radiation group(P <0.05), but not yet backto normal in the14d. The expression of DNMT3B was also significantlyincreased in radiation group (P<0.05), but has returned to normal in the7d.9.Effects of low dose radiation on DNA methyltransferase of liver:Compared with normal group, the expression of DNMT1and DNMT3Awere significantly increased in radiation group(P <0.05), but not yet backto normal in the14d. Compared with normal group,The expression ofDNMT3B was also significantly increased in radiation group (P<0.05), buthas returned to normal in the7d.10.Effects of low dose radiation on DNA methyltransferase of spleen:Compared with normal group, the expression of DNMT1and DNMT3A inradiation group were significantly decreased (P <0.05),DNMT3B was not detected. the expression of DNMT1had not returned to normal in14d andDNMT3B has returned to normal in the7d.Conclusion：1）1Gy x ray can increase the apoptosis rate of BMNCand proliferation ability of mice, decrease the DNA methylation level andincrease expression level of DNA methyltransferase.2）1Gy x ray can canlead to disorder of liver and spleen tissues structure of mice, decrease theDNA methylation level and lead disorder of the expression level of DNAmethyltransferase of liver and spleen, and DNA methyltransferaseexpression changes have tissue and organ specificity.