Morales A, Miranda M, Sánchez-Reyes A, Biete A, Fernández-Checa J C
Instituto Investigaciones Biomédicas, August Pi i Suñer (IDIBAPS), CSIC-UB, Barcelona, Spain.
Int J Radiat Oncol Biol Phys. 1998 Aug 1;42(1):191-203. doi: 10.1016/s0360-3016(98)00185-0.
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.
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.
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).
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.
由于活性氧（ROS）是辐射诱导细胞损伤的介质，我们研究的目的是确定电离辐射对人肝癌细胞（Hep G2）在辐射前谷胱甘肽（GSH）耗竭时肝细胞内还原型谷胱甘肽的调节、核及线粒体DNA（mtDNA）的存活和完整性的影响。
测定照射（50 - 500 cGy）的Hep G2细胞中的GSH、氧化型谷胱甘肽（GSSG）及ROS的生成。评估辐射前GSH耗竭的细胞的克隆形成存活率、核DNA片段化及mtDNA的完整性。
Hep G2细胞（50 - 400 cGy）的辐射导致ROS呈剂量依赖性生成，同时伴随着还原型GSH的减少，50 cGy时减少15%，400 cGy时减少25%，GSH/GSSG比值从对照的17降至7，马来酸二乙酯（DEM）处理的细胞从16降至6。辐射前GSH的耗竭使ROS的增加加剧了40 - 50%。辐射导致的GSH耗竭在不同亚细胞部位都很明显，在线粒体中尤为显著。此外，在照射（400 cGy）前将核GSH耗竭至初始值的50 - 60%会导致DNA片段化和凋亡。因此，Hep G2细胞对辐射的存活率从未耗竭GSH细胞的25%降至GSH耗竭细胞的10%。用理论模型将细胞存活率拟合为GSH的函数，证实细胞内GSH是决定Hep G2细胞对辐射内在敏感性的关键因素。与核DNA相比，mtDNA对辐射诱导的完整性丧失更敏感，线粒体中GSH耗竭会增强这种效应（GSH耗竭细胞中完整mtDNA为10 - 15%，而补充细胞为25 - 30%）。
GSH在维持核及mtDNA功能完整性方面起关键保护作用，决定了Hep G2的内在放射敏感性。虽然DNA修复是一个尚未完全理解的复杂过程，但GSH的保护作用可能似乎不涉及经典DNA损伤的修复，而可能与DNA损伤依赖性信号传导的修饰有关。
