Whole-body proton irradiation causes long-term damage to hematopoietic stem cells in mice

Abstract

Space flight poses certain health risks to astronauts, including exposure to space radiation, with protons accounting for more than 80% of deep-space radiation. Proton radiation is also now being used with increasing frequency in the clinical setting to treat cancer. For these reasons, there is an urgent need to better understand the biological effects of proton radiation on the body. Such improved understanding could also lead to more accurate assessment of the potential health risks of proton radiation, as well as the development of improved strategies to prevent and mitigate its adverse effects. Previous studies have shown that exposure to low doses of protons is detrimental to mature leukocyte populations in peripheral blood, however, the underlying mechanisms are not known. Some of these detriments may be attributable to damage to hematopoietic stem cells (HSCs) that have the ability to self-renew, proliferate and differentiate into different lineages of blood cells through hematopoietic progenitor cells (HPCs). The goal of this study was to investigate the long-term effects of low-dose proton irradiation on HSCs. We exposed C57BL/6J mice to 1.0 Gy whole-body proton irradiation (150 MeV) and then studied the effects of proton radiation on HSCs and HPCs in the bone marrow (BM) 22 weeks after the exposure. The results showed that mice exposed to 1.0 Gy whole-body proton irradiation had a significant and persistent reduction of BM HSCs compared to unirradiated controls. In contrast, no significant changes were observed in BM HPCs after proton irradiation. Furthermore, irradiated HSCs and their progeny exhibited a significant impairment in clonogenic function, as revealed by the cobblestone area-forming cell (CAFC) and colony-forming cell assays, respectively. These long-term effects of proton irradiation on HSCs may be attributable to the induction of chronic oxidative stress in HSCs, because HSCs from irradiated mice exhibited a significant increase in NADPH oxidase 4 (NOX4) mRNA expression and reactive oxygen species (ROS) production. In addition, the increased production of ROS in HSCs was associated with a significant reduction in HSC quiescence and an increase in DNA damage. These findings indicate that exposure to proton radiation can lead to long-term HSC injury, probably in part by radiation-induced oxidative stress.

FIG. 1

Proton TBI causes sustained quantitative reduction of HSCs but not HPCs. C57BL/6J mice were exposed to 1.0 Gy proton TBI or sham irradiated (CTL). Twenty-two weeks after TBI, BM cells (BMCs) were harvested from the two hind legs of individual mice for analysis. Panel A: Representative gating strategy of flow cytometric analysis for HPCs (Lin⁻Sca1⁻c-kit⁺ cells) and HSCs (Lin⁻Sca1⁺c-kit⁺ cells) in BMCs is shown. Frequencies (panel B) and total numbers (panel C) of HPCs and HSCs in BMCs from each mouse are presented as mean ± SD (CTL: n = 4 mice; TBI: n = 5). *P < 0.05 and **P < 0.01, TBI vs. CTL.

FIG. 2

Proton TBI causes sustained reduction of HSC clonogenic function. Panel A: Twenty-two weeks after 1.0 Gy TBI, total BM cells (BMCs) were harvested from control (CTL) and irradiated (TBI) mice and were analyzed by CAFC assays. The numbers of five week CAFCs were counted and expressed as mean ± SD (n = 3–4 mice per group) of CAFCs per 100,000 BMCs. *P < 0.05 TBI vs. CTL. Panels B–D: At 22 weeks after TBI, BM-MNCs were isolated from irradiated and sham-irradiated mice. A CFC assay was performed as described in Materials and Methods. The results are presented as mean CFUs per 2 × 10⁴ BM-MNCs (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001, TBI vs. CTL.

FIG. 3

Proton TBI causes persistent increases in ROS production in HSCs but not in HPCs. Lin⁻ cells were isolated from control (CTL) and irradiated (TBI) mice 22 weeks after 1.0 Gy proton TBI as described. Panel A: Representative analysis of ROS production measured by flow cytometry using DCFDA in BM HPCs and HSCs from control and irradiated mice. The numbers presented in the histograms are DCF MFI from a representative experiment. Panel B: The levels of MFI in BM HPCs and HSCs after TBI are presented as mean ± SD (CTL: n = 4; TBI: n = 5). Panel C: The expression of NOX1, NOX2 and NOX4 mRNA in HSCs from irradiated and CTL mice was measured by qRT-PCR and expressed as fold increases compared to CTL. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001, TBI vs. CTL.

FIG. 4

Proton TBI causes cell cycling in HSCs but not in HPCs. BMCs were harvested from control (CTL) and irradiated (TBI) mice 22 weeks after 1.0 Gy proton TBI as described. Panels A and B: Percentages of G₀, G₁ and SG₂M cells in HPCs and HSCs from control and irradiated mice are presented as mean ± SD (CTL: n = 4 ; TBI: n = 5). *P < 0.05 and **P < 0.01, TBI vs. CTL. Panel C: Fold changes in relative gene expression are shown for various cell cycle regulators in sorted HSCs after TBI. Data from three independent experiments using sorted HSCs pooled from four mice per group are presented as mean ± SD (n = 3). *P < 0.05 and ***P < 0.001, TBI vs. CTL.

FIG. 5

Proton TBI causes persistent DNA damage in HSCs but not that in HPCs. Lin⁻ cells were isolated from control (CTL) and irradiated (TBI) mice 22 weeks after 1.0 Gy proton TBI as described. Panel A: Representative analysis of DNA damage measured by flow cytometry using γ-H2AX staining in BM HPCs and HSCs from control and irradiated mice. The numbers presented in the histograms are γ-H2AX MFI from a representative experiment. Panel B: The levels of MFI in BM HPCs and HSCs after TBI are presented as mean ± SD (CTL: n = 4; TBI: n = 5). *P < 0.05, TBI vs. CTL.