Abstract
[Objective] There are many evidences that indicate the importance of intracellular reactive oxygen species (ROS) in the signaling pathway of apoptosis. Our recent study showed that intracellular ROS was produced after several hours of X-irradiation, leading to the release of cytochrome c from mitochondria. In the previous meeting, we reported that the upregulation of mitochondrial function played a role in the increase of ROS production induced by X-irradiation. To further elucidate the mechanism of ROS production after X-irradiation, we analyzed the effect of X-irradiation on mitochondrial functions, particularly focusing on cellular oxygen consumption rate, in various cell lines.
[Methods] Various human or mouse cell lines (A549, HeLa, MKN45, MeWo, and NIH3T3) were used in this study. Cells were irradiated with 10 Gy of X-rays. Fluorescent intensity of DCFDA or MitoSOX Red was assessed with a flowcytometer to evaluate intracellular ROS level. Cellular oxygen consumption rate was measured by X-band ESR using LiNc-BuO as an oxygen probe. Cellular mitochondrial contents were analyzed by two methods; mitochondrial membrane contents assessed by MitoTracker Green FM and mitochondrial DNA amount measured by real-time PCR.
[Results] X-irradiation caused the increase of mitochondria-derived ROS production in all cell lines tested. When we measured cellular oxygen consumption rate as an index of mitochondrial electron transport chain function, it was enhanced in all cell lines after X-irradiation, suggesting that the upregulation of mitochondrial function after X-irradiation was an universal phenomenon in mammalian cells. We also found that X-irradiation induced the increase of cellular mitochondrial contents while it activated mitochondrial Complex I. These results suggested that the increase of the active mitochondria as well as the activation of mitochondrial electron transport chain after X-irradiation caused the enhancement of cellular respiration, leading to the consequent increase of intracellular ROS production.
