[Purpose] Recently, several studies have shown that reactive oxygen species (ROS) play an important role in the signaling pathway of apoptosis. We reported that ROS generation was increased several hours after irradiation and participated in cytochrome c release from mitochondria in A549 cells (Ogura et al., Cancer Lett., 277:54, 2009). In this study, to reveal the mechanism of radiation-induced ROS generation, we analyzed how irradiation modulates mitochondrial functions by using mitochondrial inhibitors.
[Materials and methods] Human lung carcinoma A549 cells were irradiated with 10 Gy X-rays. Intracellular ROS level was measured by flow cytometry using specific ROS fluorescence probe DCFDA. Cellular oxygen consumption and intracellular ATP level were measured to assess mitochondrial respiratory activity, and the effect of rotenone (a complex I inhibitor) and oligomycin (a F₀/F₁-ATPase inhibitor) on respective index was examined.
[Results] ROS generation was significantly increased from 6 hours after irradiation. The cellular oxygen consumption rate was risen 12 hours after irradiation, and it was suppressed by rotenone. Intracellular ATP level was elevated time-dependently up to 24 hours after irradiation, and it was suppressed by oligomycin. These results suggested that irradiation activated mitochondrial electron transport chain, and the increase of ROS generation. We investigate the further mechanisms of radiation-induced mitochondrial activation in tumor cells.