A role for mitochondrial dysfunction in perpetuating radiation-induced genomic instability.
Radiation-induced genomic instability (RIGI) manifests as a heritable increased rate of genetic alterations in the progeny of irradiated cells generations after the initial insult. The progeny can show an increased frequency of chromosomal translocations, deletions, mutations, micronuclei, and decreased plating efficiency. What perpetuates RIGI is unclear; however, persistently increased levels of reactive oxygen species (ROS) are frequently associated with genomically unstable clones. Furthermore, addition of free radical scavengers (e.g., DMSO, glycerol, and cationic thiol cysteamine) reduces the incidence of instability after irradiation, implicating a ROS-mediated role in RIGI induction. Because mitochondria are a major natural cellular source of ROS, we tested the hypothesis that mitochondrial dysfunction has a role in maintaining the elevated ROS levels in our irradiated, genetically unstable GM10115 Chinese hamster ovary cells. Amplex Red fluorometry measurements indicate that the relative contribution of uncoupler-sensitive mitochondrial hydrogen peroxide production to total cellular hydrogen peroxide generation is greater in unstable cells. Measurements of mitochondrial DNA levels and cell cytometric fluorescent measurements of Mitotracker Green FM indicate that differences in mitochondrial ROS production are not due to varying mitochondrial levels. However, mitochondrial respiration measured in digitonin-permeabilized cells is impaired in unstable clones. In addition, manganese superoxide dismutase, a major mitochondrial antioxidant enzyme, exhibits increased immunoreactivity but decreased enzyme activity in unstable clones, which along with decreased respiration rates may explain the increased levels of cellular ROS. These studies show that mitochondria from unstable cells are abnormal and likely contribute to the persistent oxidative stress in the unstable clones.
Kim
Grace J
GJ
Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, Maryland 21201-1559, USA. gkim002@umryland.edu
Fiskum
Gary M
GM
Morgan
William F
WF
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D042822:Genomic Instability / Q000528:radiation effects*; D006861:Hydrogen Peroxide / Q000378:metabolism; D008928:Mitochondria / Q000502:physiology*; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase / Q000378:metabolism
D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase