Low doses of oxygen ion irradiation cause long-term damage to bone marrow hematopoietic progenitor and stem cells in mice

Abstract

During deep space missions, astronauts will be exposed to low doses of charged particle irradiation. The long-term health effects of these exposures are largely unknown. We previously showed that low doses of oxygen ion (16O) irradiation induced acute damage to the hematopoietic system, including hematopoietic progenitor and stem cells in a mouse model. However, the chronic effects of low dose 16O irradiation remain undefined. In the current study, we investigated the long-term effects of low dose 16O irradiation on the mouse hematopoietic system. Male C57BL/6J mice were exposed to 0.05 Gy, 0.1 Gy, 0.25 Gy and 1.0 Gy whole body 16O (600 MeV/n) irradiation. The effects of 16O irradiation on bone marrow (BM) hematopoietic progenitor cells (HPCs) and hematopoietic stem cells (HSCs) were examined three months after the exposure. The results showed that the frequencies and numbers of BM HPCs and HSCs were significantly reduced in 0.1 Gy, 0.25 Gy and 1.0 Gy irradiated mice compared to 0.05 Gy irradiated and non-irradiated mice. Exposure of mice to low dose 16O irradiation also significantly reduced the clongenic function of BM HPCs determined by the colony-forming unit assay. The functional defect of irradiated HSCs was detected by cobblestone area-forming cell assay after exposure of mice to 0.1 Gy, 0.25 Gy and 1.0 Gy of 16O irradiation, while it was not seen at three months after 0.5 Gy and 1.0 Gy of γ-ray irradiation. These adverse effects of 16O irradiation on HSCs coincided with an increased intracellular production of reactive oxygen species (ROS). However, there were comparable levels of cellular apoptosis and DNA damage between irradiated and non-irradiated HPCs and HSCs. These data suggest that exposure to low doses of 16O irradiation induces long-term hematopoietic injury, primarily via increased ROS production in HSCs.

Fig 1. Peripheral blood cell counts were comparable between non-irradiated and irradiated mice at three months after ¹⁶O exposure.

C57BL/6J mice were exposed to 0.05 Gy, 0.1 Gy, 0.25 Gy and 1.0 Gy doses of ¹⁶O irradiation or were sham irradiated as a control (CTL). The cell counts in peripheral blood were determined three months after radiation exposure. (A-C) The numbers of WBC, lymphocytes, monocytes, neutrophils, RBC, Hb, platelet (PLT) and mean platelet volume (MPV) in irradiated mice are presented as means ±SD (n = 5), and comparable to those in non-irradiated mice.

Fig 2. ¹⁶O TBI caused reductions in percentages and numbers of HPCs, LSK cells and HSCs at three months after exposure.

HPCs (Lin^-Sca1^-c-kit^- cells), LSK cells (Lin^-Sca1⁺c-kit⁺cells) and HSCs (Lin^-Sca1⁺c-kit⁺CD150⁺CD48^- cells) in BM were measured three months after 0.05 Gy, 0.1 Gy, 0.25 Gy, and 1.0 Gy ¹⁶O TBI. The frequencies (panel A) and numbers (panel B) of HPCs, LSK cells and HSCs from total bone marrow cells in each mouse are presented as means ±SD (n = 5). The statistical significance for differences between the control group (CTL) and each of the irradiated groups is indicated by asterisks. *p<0.05, **p<0.01, ***p<0.001 as determined by one-way ANOVA, followed by Tukey-Kramer test for individual comparisons.

Fig 3. ¹⁶O TBI caused the reduction of the clonogenic function in HPCs and HSCs.

(A) BM-MNCs were isolated from irradiated and non-irradiated (CTL) mice three months after ¹⁶O TBI and a CFU assay was performed. Results are presented as mean CFUs per 1x10⁵ BM-MNCs (n = 5). (B) Total BM cells (BMCs) were analyzed by CAFC assays, and the numbers of five-week CAFCs are expressed as means ± SD (n = 3 mice per group) per 1x10⁵ BMCs. The statistical significance for differences between the control group and each of the irradiated groups is indicated by asterisks. *p<0.05, **p<0.01 as determined by one-way ANOVA, followed by Tukey-Kramer test for individual comparisons.

Fig 4. No changes were detected in DNA damage and apoptosis in HPCs, LSK cells and HSCs at three months after ¹⁶O TBI.

(A) Lin^- cells were stained with an anti-γH2AX antibody and analyzed by flow cytometry. Data are presented as mean fluorescence intensity (MFI). (B) Isolated Lin^- cells were stained with Annexin V to determine cellular apoptosis. Percentages of Annexin V positive cells are presented as means ± SD (n = 5).

Fig 5. ¹⁶O TBI caused an increase in ROS production in LSK cells and HSCs three months after the exposure.

(A) Lin^- cells were used to measured ROS production by staining with DCFDA and analyzed by flow cytometry. The DCF mean fluorescence intensity (MFI) in BM HPCs and HSCs are presented as means ± SD (n = 5). (B) Fold changes in relative gene expression for several antioxidant genes in sorted HPCs (left panel) and HSCs (right panel) from 1.0 Gy of ¹⁶O TBI and non-irradiated mice. (C) Lin^- cells were isolated and cell cycling was measured by cytometry using Ki-67 and 7-AAD double staining in HPCs and HSCs from control (CTL) and irradiated mice. The statistical significance for differences between the control group and each of the irradiated groups is indicated by asterisks. *p<0.05, **p<0.01, ***p<0.001 as determined by one-way ANOVA, followed by Tukey-Kramer test for individual comparisons.