Effects Of Long-term Low-dose Radiation And Cadmium Exposure On Human Lymphoblast Cells And The Relevant Roles Of DNA Methylation

Since the enormous medical and industrial applications of ionizing radiation, more attentions have been attracted to the risks of low-dose radiation (LDR), however, the effects and underlying mechanisms of long-term LDR are still not clear. LDR could induce biological responses and cancer risks different from that of high-dose radiation (HDR), such as hypersensitivity, genomic instability, bystander effect and adaptive response. Moreover, human health is threatened not only by radiation but also by chemical toxicant. Since the wide useage of cadmium (Cd) in industry and its persistence in environment, people have paid more attention to the influence of Cd on human health. Accumulating evidence suggest that Cd and ionizing radiation are crucial human carcinogens, but little is known about the effects of long-term LDR and long-term low-dose cadmium exposure, especially the combination effect of these two factors.It’s well known that DNA methylation, a main and most studied epigenetic event, is important for cell proliferation, development, gene expression and maintenance of genome stability and occurs in many prokaryotic and eukaryotic organisms. There are ennormous reports of DNA methylation alters in rodents and human cell lines exposed to ionising radiation. Current evidence suggests that some responses of LDR might be epigenetic in nature especially DNA methylation, but the mechanisms remain to be elucidated. The available experimental data indicates that DNA hypermethylation at the global and gene-specific levels occurres in association with cadmium-induced malignant transformation.In the present study, with the treatment of long-term LDR and long-term low-dose Cd on human lymphoblast cells (HMy2.CIR), we investigated the effects and underlying DNA methylation mechanisms in these two stresses. Moreover, we also studied the cross-adaptative response (cross-AR) of long-term LDR and long-term low-dose Cd. A deeper understanding of the molecular mechanism of long-term LDR and long-term low-dose Cd may lead to the improvement of the risk assessment and then opens up new approaches of protection. Part I Role of DNA methylation in long-term low-dose gamma-rays induced cellular effects in human lymphoblast cellsObjective:In this study, we aimed to investigate the effects of long-term exposure to low-dose y-rays on cell proliferation, radiosensitivity and adaptive response of radiosensitive human lymphoblast cells HMy2.CIR and to elucidate the role of DNA methylation involved in these processes.Methods:Cells were divided into two groups:the long-term LDR exposure group and its nonirradiated control group. HMy2.CIR cells were fractionally exposed to a low-dose of y-rays,0.032Gy, which could enhance cell proliferation,3times/per week for four weeks. Control cells were sham irradiated. After irradiation per week, micronuclei formation in binucleated cells was counted to detect DNA damage, and cell proliferation was assayed by CCK-8kit. The global genomic DNA methylation was measured by cytosine extension assay. Cell apoptosis and expression of y-H2AX were measured by flow cytometry. Gene expression of cyclinDl, PCNA, bcl-2and bax and protein expression of HP1and MeCP2were detected by real-time RT-PCR and western blotting, respectively.Results:Long-term low-dose γ-rays exposure enhanced cell proliferation, which was accompanied with up-regulation of cell cycle regulation gene cyclinD1and proliferation regulation gene PCNA in HMy2.CIR cells. In addition, long-term LDR triggered a cellular adaptive response to challenging dose (2Gy) of y-rays. Compared to unirradiated cells, the long-term LDR decreased micronuclei formation rate, apoptosis induction and y-H2AX expression after irradiation of a challenging dose of y-rays. Gene expression analysis by real-time RT-PCR revealed an increased expression of the anti-apoptotic gene bcl-2and a decreased expression of pro-apoptotic gene bax. Moreover, cytosine extension assay indicated that the global genomic DNA methylation accompanied with an increase of gene expression of DNA methyltransferases DNMT1and protein expression of methyl CpG binding protein2(MeCP2) was increased after long-term LDR exposure, while heterochromatin protein1(HP1), a marker for heterochromatin, was also increased. After the treatment of5-aza-dC, a DNA methyltransferase inhibitor, the global genomic DNA hypermethylation was decreased and the LDR-induced adaptive response was eliminated.Conclusion:The present findings indicate that long-term LDR promotes cell proliferation by up-regulating cell cycle related genes, while it induces adaptive response by increasing global genomic DNA methylation and acquisition of apoptotic resistance in HMy2.CIR cells. Part II Influence of long-term low-dose cadmium exposure on the proliferation of human lymphoblast cells and relevant molecular mechanismObjective:Cadmium (Cd) has long been considered as a non-genotoxic carcinogen due to epigenetic mechanisms. However, the relationship between the alteration of DNA methylation, expression of p16and Cd-influenced cell proliferation is still poorly understood, which was investigated here.Methods:Cells were treated with cadmium chloride (CdCl2) at concentrations of0,0.005,0.01,0.1,0.5and1μM in culture media, and divided into two groups: short-term group and long-term group. For short-term group, cells were collected after48h of Cd treatment and the long-term group cells were grown in Cd-containing medium for3months. Cell proliferation was analyzed with a cell counter and gene expression of DNMT1, DNMT3b and p16were detected by real-time RT-PCR. The global genomic DNA methylation was measured by cytosine extension assay. Micronuclei formation in binucleated cells was counted to detect DNA damage. The promoter methylation of p16gene was analyzed by the methylation-specific PCR (MSP).Results:The cell count data revealed that the cell proliferation could be increased when HMy2.CIR cells were treated with low-dose Cd for48h, and this inscrese became more significant when the cells were chronically exposed to Cd with a low concentration for3months. The mRNA expressions of DNA methyltransferases genes, DNMT1and DNMT3b, were increased in a dose-dependent manner, while the expression of p16mRNA was remarkably decreased. Further investigation showed that the level of genomic DNA methylation was increased and the CpG islands of p16promoter regions were hypermethylated in the long term Cd-exposed cells. Moreover, the DNA demethylating agent,5-aza-dC, eliminated the Cd-enhanced cell proliferation associated with overexpression of p16gene.Conclusion:The chronic exposure of low-dose Cd could induce hypermethylation at p16promoter region and hence suppress p16expression and promote cell proliferation, which might be a viable mechanism for Cd-induced cancers. Part Ⅲ Cross-adaptive response induced by long-term LDR and long-term low-dose cadmium and the relevant mechanismObjective:Human health is threatened not only by ionizing radiation but also by environmental chemical toxicants, thus the health risk caused by environmental factors should be assessed in the context of these two factors. In this study, we investigated the cross-AR of long-term LDR and long-term low-dose cadmium in radiosensitive human lymphoblast cells HMy2.CIR and elucidated the role of DNA methylation involved in these processes.Methods:The long-term LDR group were fractionally exposed to a low dose of y-rays,0.032Gy,3times/per week for four weeks, while control cells were sham irradiated. For the long-term low-dose cadmium group, cells were treated with CdCl2at concentrations of0,0.005,0.01, and0.1μM in culture media for3months. After exposure of challenging dose of cadmium or y-rays, micronuclei formation in binucleated cells was counted to detect adaptive response (AR).Results:Our data showed that long-term low-dose cadmium exposure could induce AR or cross-AR in HMy2.CIR cells toward subsequent challenge of cadmium or y-rays. When challenged by50μM CdCl2, HMy2.CIR cells exhibited significant AR. However, when the challenging dose increased to100μM, HMy2.CIR cells just showed slight and insignificant AR. Moreover, a trend of concentration-dependent change in magnitude of adaptive response (MAR) was observed. As to cadmium induced cross-AR, when challenged by2Gy y-rays, HMy2.CIR cells exhibited more significant AR. We also found that long-term LDR could induce AR or cross-AR in HMy2.CIR cells. When challenged by2Gy y-rays, the MAR was lower than that of50μM CdCl2. When challenged by100μM CdCl2, HMy2.CIR cells just showed slight and insignificant AR. After the treatment of5-aza-dC, a DNA methyltransferase inhibitor, the global genomic DNA hypermethylation was decreased and the AR and cross-AR of long-term LDR and long-term low-dose cadmium were all eliminated. Conclusion:The long-term low-dose cadmium and long-term LDR could induce AR or cross-AR in HMy2.CIR cells. The MAR in adapted cells was related to multiple factors including priming dose of cadmium and the challenging dose of cadmium or irradiation. DNA methylation was involved in the AR or cross-AR.