Study On The Changes Of Mitochondrial Gene Expressions In Human Cells Induced By ⁶⁰Co γ-rays

Objective:Mitochondrial DNA (mtDNA) is the only extranuclear genetic material; the genes coded by mtDNA participate in cell oxidative phosphorylation and energy metabolism. It is found that radiation can induce changes in expression levels of mitochondrial coded genes, but results of present studies are not in complete agreement. On the other hand, when accidental exposure or terrorist events occur, high-throughput biomarkers will be urgent needed in order to estimate the bio-radiation exposure dose of the mass casualty. Therefore, we explore the alterations of the expression level of mitochondrial genes in human lymphoblastoid cells and other cells after irradiation at different time points. We expect to provide some scientific evidence for the problem of rapid dose estimation in the field of radiation biology.Methods:1. Cell lines were irradiated with 60Coγ-rays at dose rate of 1 Gy/min. The human lymphoblastoid cells were cultured for 4h,12h,24h,48h and 72h, respectively after being irradiated of 0.5-15Gy absorbed dose in order to detect the dose-effect relationship between the dose and transcriptional level changes of 7 mitochondrial genes. While the translation expression level changes of these genes were analyzed at the same 5 post-irradiation time-points after being irradiated of 0-18Gy in order to explore the dose-effect relationship. What's more, 3 and 8Gy were chosen to explore the alteration of gene transcriptional levels in 5 cell lines from 4 tissues at 24h after being irradiated.2. Changes at transcriptional level of mitochondrial genes were detected by real-time PCR. Meanwhile, changes at translation level of mitochondrial genes were detected by Western Blot.Results:1. At the transcriptional level,7 mitochondrial gene expression level was increased in human lymphoblastoid cells 4-24h after irradiation. The expression of ND3 gene showed a good dose-effect relationship in the range of 3-10Gy at 4h after irradiation and in the range of 3-15Gy at 24h after being irradiated; The expression levels of Cyt b were significantly enhanced at 24h post-irradiation and showed a certain dose-effect relationship within 0-15Gy; The expression of COXⅠgene showed a certain dose-effect relationship within 0-5Gy at 12h post-irradiation while there was a good dose-response relationship for COXⅢwithin a dose range of 3-10Gy at 24h post-irradiation; finally, the expression levels of ATPase6 and ATPase8 genes were enhanced with dose, and unfolded a linear relationship in the range of 3-15Gy at 24h post-irradiation.2. At the translation level, COXⅡgene expression levels were significantly increased 24-72h after being irradiated by 60Coγ-rays.Particularly at two post-irradiation timepoints,48h and 72h, COXⅡgene expression level correlated with radiation dose. But at the five time-points of 4-72h irradiated by 60Coγ-rays, there showed no significant change for both ND3 and ATPase8.3. The trend of expression levels of transcription and translation of COXⅡ,ND3 and ATPase8 gene were generally identical at the time-point of 24h after irradiation and they began to show difference after that.4. In human liver cells,7 mitochondrial gene expression levels were significantly increased at 24h after irradiated with 3 and 8Gy 60Co y-rays, and the expression levels of 3 COX genes (COXⅠ, COXⅡand COXⅢ) were linearly correlated with the dose. The gene expression of other four tumor cells showed no significant change 24h after irradiated with 3 and 8Gy Coγ-rays.Conclusion:1. At the transcriptional level,24h after irradiation by 60Coγ-rays can be the best time-point to study the radiation-induced changes of mitochondrial gene expression in human lymphocytes, and 7 mitochondrial genes have their own different optimal induction dose.2. At the transcriptional level, ND3 gene with dose range of 3-10Gy at 4h post-irradiation, COXⅠgene within dose range of 0～5Gy at 12h post-irradiation, and as well COXⅢgene within 3-10Gy and ND3,ATPase6 as well as ATPase8 within 3-15Gy at 24h after irradiation showed obvious dose-response relationships.3. At the translation level, radiation can induce COXⅡgene expression level significantly increased 24-72h after irradiation in human lymphocytes, and there showed good response relationships at the time-point of 48h and 72h.4. The radiation-induced changes of mitochondrial gene expression between the mRNA and protein levels in human lymphoblastoid cells are not identical.5. The expression of mitochondrial genes in human liver cell lines significantly increased 24h after being irradiated. The expression of mitochondrial genes in A549 slightly increased and there was no significant alteration of mitochondrial genes expressions in the other tumor cell lines 24h after being irradiated.