Changes of Reactive Oxygen and Nitrogen Species and Mitochondrial Functioning in Human K562 and HL60 Cells Exposed to Ionizing Radiation

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Abstract

Free radicals generated by mitochondria are candidates for mediating long-lasting effects of radiation on cells, including genetic instability. To better understand the significance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in these long-term effects we assayed ROS and RNS levels, the mitochondrial membrane potential and mass, and the frequency of DNA strand breaks, apoptosis and necrosis in human leukemic cells (K562 and HL60) after 12 Gy of X irradiation. An increase in intracellular ROS level was observed immediately post-irradiation, and about 24 h later a second increase of ROS was accompanied by increase in nitrogen oxide, mitochondrial potential and mitochondrial mass in both cell types. The second peak of ROS level was partially inhibited by rotenone, an inhibitor of mitochondrial complex I, in K562 but not in HL60 cells suggesting that the sources of ROS differed in the two cell types. The frequency of DNA breaks showed kinetics similar to ROS levels, with a sharp peak immediately after irradiation and a second increase 24 and 48 h later, which was significantly higher in K562 cells. Forty-eight hours after irradiation an increase in the frequency of apoptotic cells was observed in both cell lines, which became larger and statistically significant in K562 cells after inhibition of mitochondrial complex I. Our results show that ionizing radiation activates cellular processes which produce long-lasting ROS and RNS radicals, which may have different sources in different cell types and could participate in cellular signaling networks important for radiosensitivity and mode of cell death.

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