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

<div><p>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.</p></div

¹⁶O TBI causes persistent increases in DNA damage in HSCs but not in HPCs two weeks after the exposure.

<p>(A) Representative analysis of DNA damage measured in Lin^- cells by flow cytometry using γH2AX staining in BM HPCs and HSCs from control and 1.0 Gy¹⁶O TBI mice. The histograms indicate γH2AX MFI from a representative experiment. (B) The γH2AX MFI in BM HPCs and HSCs after TBI are presented as mean ± SD. (C) Sorted HPCs and HSCs from irradiated and non-irradiated mice were stained with γH2AX antibody. The numbers of foci in each cell were counted and expressed as mean ± SD. (D) The distribution of foci was expressed as the percentages of different numbers of foci in control and irradiated HSCs. The statistical significance for the difference between the control groups and each of irradiated groups is indicated by asterisks. *p<0.05, **p<0.01 by one-way ANOVA analysis.</p

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

<p>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.</p

¹⁶O TBI drives HSCs from quiescence into the cell cycle.

<p>Lin⁻cells were isolated from control (CTL) and irradiated (TBI) mice 2 weeks after 0.1, 0.25 and 1.0 Gy TBI. Cell cycle was measured by flow cytometry using Ki-67 and 7-AAD double staining in BM HPCs and HSCs from control and irradiated mice. (A-C) The percentages of G0, G1 and G2SM phases in BM HPCs, LSK cells and HSCs after TBI are presented as mean ± SD (n = 5). The distribution of cell cycle phases in HPCs, LSK cells and HSCs was analyzed by Chi-Square test as indicated by X² (HPC, X² = 19.084, p<0.05; LSK cells, X² = 6.486, p<0.05; HSCs, X² = 33.853, p<0.05). The statistical significance for the difference in each cell cycle phase between the control groups and irradiated groups is indicated by asterisks. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA analysis.</p

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

<p>(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.</p

Properties of oxygen ion irradiation.

<p>Properties of oxygen ion irradiation.</p

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

<p>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.</p

¹⁶O TBI causes sustained reduction of HSC clonogenic function.

<p>(A–C) At 2 weeks after TBI, BM-MNCs were isolated from irradiated and sham-irradiated (CTL) mice. A CFC assay was performed, and the results are presented as mean CFUs per 1x10⁵ BM-MNCs (n = 5). (D and E) Total BM cells (BMCs) were analyzed by CAFC assays, and the numbers of two and five week CAFCs were expressed as mean ± SD (n = 3 mice per group) per 1x10⁵ BMCs. The statistical significance for the difference 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.</p