Abstract
[Objective] The biological effects of ionizing radiation are classified into direct action and indirect action, thus a low LET irradiation such as X-rays mainly induce an indirect action with the production of reactive oxygen species (ROS). Mitochondria act as both a source of ROS and a target organ of ROS in this process. Although hematopoietic stem cells (HSCs) are highly radio-sensitive cells, there is still little information regarding the intracellular ROS production and the involvement of the mitochondria. The present study examined these interactions in human HSCs.
[Methods] This study used highly purified CD34+ cells prepared from human placental/umbilical cord blood. The clonogenic potential was assayed by the methyl cellulose technique containing the optimal cytokine cocktails. The cells were treated with 2’,7’-dichlorofluoresceindiacetate and Mitotracker Green FM in order to estimate the intracellular ROS production and the fluorescence intensity of mitochondria, respectively. Each assay was analyzed by flow cytometry. X irradiation was performed by an X-ray generator under conditions of 150 kV, 20 mA, 0.5-mm Al plus 0.3-mm Cu filters at a dose of 90—100 cGy/min.
[Result and Discussion] No ROS were detected in the CD34+ cells immediately after irradiation, and little ROS production was observed in the cells cultured with cytokines at 3 to 6 h after irradiation. In a similar experiment, 10 to 20-fold increases in the ROS production were observed in human monocytic U937 cells and human normal fibroblasts WI-38 cells. However, the intensity of luminescence from mitochondria observed in CD34+cells was only half that seen in both cell lines. There was a positive correlation between the surviving fractions of 4 Gy irradiated-HSCs and the luminescence of mitochondria. Therefore, the present results suggest the possibility that the ROS production was either significantly reduced or eliminated by the mechanisms associated with mitochondria in HSCs.
