OXIDATIVE DAMAGE OF MITOCHONDRIAL AND NUCLEAR-DNA INDUCED BY IONIZING-RADIATION IN HUMAN HEPATOBLASTOMA CELLS

Purpose: Since reactive oxygen species (ROS) act as mediators of radia tion-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 mitoc hondrial DNA (mtDNA) in human hepatoblastoma cells (Hep G2) depleted o f GSH prior to radiation. Methods and Materials: GSH, oxidized glutath ione (GSSG), and generation of ROS were determined in irradiated (50-5 00 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 i n a dose-dependent generation of ROS, an effect accompanied by a decre ase of reduced GSH, ranging from a 15% decrease for 50 cGy to a 25% de crease for 400 cGy and decreased GSH/GSSG from a ratio of 17 to a rati o of 7 for controls and from 16 to 6 for diethyl maleate (DEM)-treated cells, Depletion of GSH prior to radiation accentuated the increase o f ROS by 40-50%. The depletion of GSH by radiation was apparent in dif ferent subcellular sites, being particularly significant in mitochondr ia, Furthermore, depletion of nuclear GSH to 50-60% of initial values prior to irradiation (400 cGy) resulted in DNA fragmentation and apopt osis. Consequently, the survival of Hep G2 to radiation was reduced fr om 25% of cells not depleted of GSH to 10% of GSH-depleted cells. Fitt ing the survival rate of cells as a function of GSH using a theoretica l model confirmed cellular GSH as a key factor in determining intrinsi c sensitivity of Hep G2 cells to radiation. mtDNA displayed an increas ed susceptibility to the radiation-induced loss of integrity compared to nuclear DNA, an effect that was potentiated by GSH depletion in mit ochondria (10-15% intact mtDNA in GSH-depleted cells vs. 25-30% of rep leted cells), Conclusion: GSH plays a critical protective role in main taining nuclear and mtDNA functional integrity, determining the intrin sic radiosensitivity of Hep G2, Although the DNA repair is a complex p rocess that is not yet completely understood, the protective role of G SH probably does not seem to involve the repair of classical DNA damag e but may relate to modification of DNA damage dependent signaling. (C ) 1998 Elsevier Science Inc.