Oxidative damage of mitochondrial and nuclear DNA induced by ionizing radiation in human hepatoblastoma cells
Oxidative damage of mitochondrial and nuclear DNA induced by ionizing radiation in human hepatoblastoma cells
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DOI：
10.1016/s0360-3016(98)00185-0
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发表时间：
1998-08-01
影响因子：
7
通讯作者：
Fernández-Checa, JC
中科院分区：
文献类型：
--
作者：
Morales, A;Miranda, M;Fernández-Checa, JC
目的：由于活性氧（ROS）作为辐射诱导细胞损伤的介质，我们的研究目的是确定电离辐射对肝细胞还原型谷胱甘肽（GSH）的调节，以及辐射前GSH耗竭的人肝母细胞瘤细胞（Hep G2）中细胞核和线粒体DNA（mtDNA）的存活和完整性的影响。GSH，氧化型谷胱甘肽（GSSG），并在照射（50-500 cGy）的Hep G2细胞的ROS的产生进行了测定。在辐射前GSH耗竭的细胞中评估克隆存活率、核DNA片段化和mtDNA的完整性。Hep G2细胞的辐射（50-400 cGy）导致ROS的剂量依赖性产生，这种效应伴随着还原型GSH的减少，范围从50 cGy的15%降低到400 cGy的25%降低，对照的GSH/GSSG从17降低到7，马来酸二乙酯（DEM）处理的细胞从16降低到6，在辐射之前GSH的耗尽使ROS增加40- 50%。在不同的亚细胞部位，GSH的辐射消耗是明显的，特别是在线粒体中。此外，核GSH消耗到初始值的50-60%辐射前（400 cGy）导致DNA断裂和凋亡。因此，Hep G2对辐射的存活率从未耗尽GSH的细胞的25%降低到GSH耗尽细胞的10%。使用理论模型拟合细胞存活率作为GSH的函数，证实细胞GSH是决定Hep G2细胞对辐射的内在敏感性的关键因素。与核DNA相比，线粒体DNA显示出对辐射诱导的完整性丧失的敏感性增加，线粒体中的GSH耗尽增强了这种效应（GSH耗竭细胞中10-15%完整mtDNA对25-30%完整mtDNA），结论：GSH在维持细胞核和mtDNA功能完整性、决定Hep G2的固有放射敏感性、虽然DNA修复是一个复杂的过程，尚未完全理解，GSH的保护作用似乎不涉及经典的DNA损伤的修复，但可能涉及DNA损伤依赖性信号的修饰。(C)1998年爱思唯尔科学公司
Purpose: Since reactive oxygen species (ROS) act as mediators of radiation-induced cellular damage, the aim of our studies was to determine the effects of ionizing radiation on the regulation of hepatocellular reduced glutathione (GSH), survival and integrity of nuclear and mitochondrial DNA (mtDNA) in human hepatoblastoma cells (Hep G2) depleted of GSH prior to radiation.Methods and Materials: GSH, oxidized glutathione (GSSG), and generation of ROS were determined in irradiated (50-500 cGy) Hep G2 cells. Clonogenic survival, nuclear DNA fragmentation, and integrity of mtDNA were assessed in cells depleted of GSH prior to radiation.Results: Radiation of Hep G2 cells (50-400 cGy) resulted in a dose-dependent generation of ROS, an effect accompanied by a decrease of reduced GSH, ranging from a 15% decrease for 50 cGy to a 25% decrease for 400 cGy and decreased GSH/GSSG from a ratio of 17 to a ratio of 7 for controls and from 16 to 6 for diethyl maleate (DEM)-treated cells, Depletion of GSH prior to radiation accentuated the increase of ROS by 40-50%. The depletion of GSH by radiation was apparent in different subcellular sites, being particularly significant in mitochondria, Furthermore, depletion of nuclear GSH to 50-60% of initial values prior to irradiation (400 cGy) resulted in DNA fragmentation and apoptosis. Consequently, the survival of Hep G2 to radiation was reduced from 25% of cells not depleted of GSH to 10% of GSH-depleted cells. Fitting the survival rate of cells as a function of GSH using a theoretical model confirmed cellular GSH as a key factor in determining intrinsic sensitivity of Hep G2 cells to radiation. mtDNA displayed an increased susceptibility to the radiation-induced loss of integrity compared to nuclear DNA, an effect that was potentiated by GSH depletion in mitochondria (10-15% intact mtDNA in GSH-depleted cells vs. 25-30% of repleted cells),Conclusion: GSH plays a critical protective role in maintaining nuclear and mtDNA functional integrity, determining the intrinsic radiosensitivity of Hep G2, Although the DNA repair is a complex process that is not yet completely understood, the protective role of GSH probably does not seem to involve the repair of classical DNA damage but may relate to modification of DNA damage dependent signaling. (C) 1998 Elsevier Science Inc.