Effects Of EGCG On Chronic Low Dose X-ray-radiation Induced Cellular DNA Methylation

Background:Ionizing radiation is a well recognized genotoxic agent and human carcinogen. Recently, the health hazards of low dose radiation exposure and the possible causation to carcinogenesis have received much concern. However, the biological effects may be different as a result of exposure to low or high dose radiation, which is abide by linear no threshold model. The essence may be associated with DNA methylation. At present, the domestic and international studies about epigenetic alterations induced by low dose radiation are more focused on acute exposure, and only a few researches reported that chronic low-dose radiation induced DNA methylation alteration in vivo, however it’s none in vitro. Epigallocatechin gallate (EGCG) is a monomer of natural substance tea polyphenols. Previous studies have shown that EGCG has anti-oxidation, anti-cancer and anti-radiation biological functions. EGCG can reverse DNA methylation alterations in related anti-cancer researches, however it can affect DNA methylation pathway to play the role of radiation protection has not yet covered.Objective:We investigate whether the low-dose radiation can induce DNA methylation in L-02and HUVEC cell lines or not, and the effects of EGCG on chronic low dose radiation induced DNA methylation. We demonstrate that the mechanism of chronic low-dose radiation damage from the aspect of epigenetic DNA methylation, and provide a new green target for radiation protection.Methods:Besides global DNA methylation, we evaluated the alterations in methylation status of MGMT and P53promoter region and their expression on acute and fractioned chronic LDR exposure in human normal cell lines (L-02and HUVEC). The safe concentration of EGCG in both of the L-02and HUVEC cells was assessed by using MTT assay. We explored the effects of EGCG on global DNA methylation and promoter DNA methylation status of MGMT and P53in human normal cells (L-02and HUVEC) exposed to chronic low dose X-ray-radiation.Results: 1. The effects on global DNA methylation with low-dose X-ray exposure: the global DNA methylation level of the chronic low-dose X-ray irradiation groups (5cGy/d×5d/10d) and acute low-dose group (50cGy/d×1d) were significantly lower than that of the shaded control group (0cGy) in both L-02and HUVEC cells (P<0.05).2. The effects on P53contents with low-dose X-ray exposure: the P53contents of the shaded control group (0cGy) was not significant alteration than that of the chronic low-dose X-ray irradiation groups (5cGy/d×5d/10d) and acute low-dose group (50cGy/d×1d) in both L-02and HUVEC cells (P>0.05).3. The effects on expression levels of MGMT with low-dose X-ray exposure: the mRNA expression of MGMT of the chronic low-dose X-ray irradiation groups (5cGy/d×5d/10d) were significantly down-regulated in L-02and HUVEC, compared with the shaded control group (0cGy) and the acute low-dose group (50cGy/d×1d).4. The effects on promoter’s methylation status of MGMT with low-dose X-ray exposure: the MGMT was hypermethylated in the two choronic groups (5cGy/d×5d/10d), were largely unmethylated in the acute group (50cGy/d×1d) and shaded control group (0cGy) in L-02and HUVEC cells.5. The safe concentration of EGCG:In L-02and HUVEC cell lines, there was a dose-dependent response after EGCG treatment at5000,2500,1250,313,78,20μM. The survival percentage was over85%in L-02and HUVEC cells after EGCG treatment at78μM and20μM for12h,24h,48h and72h. So we selected EGCG treatment at50,20μM for further experiments in L-02and HUVEC cells.6. The effects of EGCG on global DNA methylation with low-dose X-ray exposure (5cGy/d×10d):the global DNA methylation level of the EGCG (20M,50μM) groups were significantly higher than that of the control group (EGCG0μM) in HUVEC cells. However, in L-02cells, there were no effects on the global DNA methylation of chronic irradiation groups (P <0.05).7. The effects of EGCG on P53contents with low-dose X-ray exposure: the P53contents of the EGCG (0μM,20μM,50μM) groups and positive control group (5-Aza-CdR5μM) was not significant alteration in both L-02and HUVEC cells with low-dose X-ray exposure(5cGy/d×lOd)(P>0.05). 8. The effects of EGCG on expression levels of MGMT with low-dose X-ray exposure (5cGy/d×5d/10d): Compared with the shaded control group (EGCG0μM), the mRNA expression of MGMT of the EGCG groups (20μM,50μM) and the positive control group (5-Aza-CdR5μM) was significantly up-regulated in L-02and HUVEC.9. The effects of EGCG on promoter’s methylation status of MGMT with low-dose X-ray exposure (5cGy/d×5d/10d):Compared with the shaded control group (EGCG0μM), the MGMT was unmethylated in the EGCG groups (20μM,50μM) and he positive control group (5-Aza-CdR5μM).Conclusions:Low dose radiation contributed to changes in global DNA and MGMT promoter methylation easily, and chronic low-dose radiation exposure plays a role of a mightier inducer of epigenetic effects than the acute one-time exposure. EGCG could reverse the decreased global DNA methylation level, the promoter’s DNA methylation status of MGMT and up-regulate the expression silenced by chronic low dose radiation. Chronic low dose radiation could be a stronger inducer through accumulated abnormal DNA methylation in radiation-attributable diseases, and EGCG could be a potent natural radioprotective agent via epigenetic regulation.