A. Morales et al., OXIDATIVE DAMAGE OF MITOCHONDRIAL AND NUCLEAR-DNA INDUCED BY IONIZING-RADIATION IN HUMAN HEPATOBLASTOMA CELLS, International journal of radiation oncology, biology, physics, 42(1), 1998, pp. 191-203
Citations number
63
Categorie Soggetti
Oncology,"Radiology,Nuclear Medicine & Medical Imaging
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.