**3. Proton radiation and hematopoietic stem cells**

During spaceflight missions outside low Earth orbit, there remains the possibility of astronauts receiving damaging doses of space radiation. Because the dosedepth distribution of SPE spectrum protons is relatively low [32], during an SPE the skin and organs near the surface of the skin will receive higher doses than deeper organs such as the bone marrow. In addition, compared to traditional radiation therapy with X-rays (photons) or electrons, proton therapy has potential benefit for clinical cancer treatment because of its favorable distribution of the radiation dose, leading to selectively increased radiation dose to the cancerous tissues while lowering the dose to normal tissues [33, 34]. However, the hematopoietic system is highly sensitive to ionizing radiation, and exposure to even a relatively low dose of SPE may still be able to result in substantial damage to the system [35, 36]. Therefore, understanding the biological effects of proton radiation is immediately needed.

One of the characteristics of the radiation-induced hematopoietic syndrome is a decline in blood cell counts, resulting from radiation-induced cell killing in circulating blood cells and suppression of hematopoietic stem and progenitor cells in the bone marrow [37–39]. Studies have reported that whole-body exposure to protons causes acute effects on the hematopoietic system in animal models [40–45]. The decreased WBCs, lymphocytes, and neutrophils were detected starting at 4 hours after 0.25–3 Gy proton radiation, with the lowest numbers observed on day 4 [43, 44]. The reduction in WBCs and lymphocytes was still evident in mice after exposure to 2.0 Gy of protons (230 MeV) [41]. This might be due to the high sensitivity of lymphocytes to proton radiation, which is consistent with the data from γ-radiation [46]. Two Gy of proton radiation induced the acute decrement of peripheral blood cells, which was shown completely recovered in the long term. The dynamic changes of peripheral blood counts might result in ignoring the negative effects of proton radiation on hematopoietic system. However, the abnormalities of splenic WBCs and lymphocytes were still detected at more than 100 days after low dose of proton radiation [47]. Taken together, proton radiation has not only acute injury but also long-term harmful effects on the hematopoietic system.

It has been well established that exposure to a significant dose of total-body γ-irradiation (TBI) induces not only the acute radiation hematopoietic syndrome but also long-term bone marrow injury [48, 49]. The acute radiation hematopoietic syndrome induced by γ-irradiation is primarily attributed to the induction

**83**

**Table 1.**

*Acute and long-term effects of proton irradiation on hematopoietic cells.*

*Space Radiation-Induced Hematopoietic Stem Cell Injury*

known about the effects of proton irradiation [35, 36].

of apoptosis of HPCs, while γ-irradiation-induced long-term BM suppression is mainly ascribed to the persistent damage to HSCs. While the effects of γ-irradiation on the hematopoietic system have been extensively documented, much less is

radiation and examined the acute and long-term effects on BM HSPCs at 2 and 22 weeks after proton radiation, respectively (**Table 1**). Results showed that exposure of mice to 1.0 Gy of proton radiation resulted in a significant decrease in the number of WBCs and PLTs from peripheral blood 2 weeks after the exposure [50]. It was demonstrated that 1.0 Gy of oxygen ion radiation (16O TBI) significantly decreased the cell counts of peripheral blood leukocytes when measured 2 weeks after exposure in male C57BL/6 mice [51]. Interestingly, the decrease of peripheral blood cell counts was not observed 2 weeks after 0.5 Gy proton TBI or 0.1 and 0.25 Gy 16O TBI [51]. The threshold dose of protons (50 or 70 MeV) to induce a decline in WBC counts in female ICR outbred mice was previously estimated to be between 0.25 and 0.5 Gy [43]. The threshold dose identified from this previous study is lower than the result from our study. It may have resulted in part from the use of different strains of mice, the time when the mice were studied after radiation or the difference in energies of protons between the two studies. For example, the linear energy transfer values for 50, 70, and 150 MeV protons would be 1.26, 0.96, and 0.55 keV/μm [52]. Because of the dose-depth distribution of SPE protons, only relatively large or higher-energy spectrum SPEs may lead to BM exposure to these doses of protons in astronauts. However, the doses of SPEs that can cause significant HSPC damage have been observed, raising the possibility that astronauts might experience reductions in circulating blood cell counts and BM HSPC damage if they encounter such an SPE. The dose-depth distribution in mice exposed to protons is different from that in humans [1]. It was found that a dose of 0.5 Gy protons (150 MeV) significantly reduced hematopoietic stem/progenitor cell function. The effects of protons at this energy and at doses below 0.5 Gy are unknown. Since the dose to the blood-forming tissues of human subjects will likely be low, future studies need to examine the hematopoietic effects of proton doses below 0.5 Gy.

Previously, mice were exposed to two different doses (0.5 and 1.0 Gy) of proton

Proton TBI can acutely induce the decrease of all lineages of peripheral blood cells, since all lineages of blood cells are generated from hematopoietic stem cells through their differentiation into various lineages of progenitors. We have shown that the exposure to both 0.5 and 1.0 Gy proton TBI damaged not only HPCs but also LSK cells, leading to the defect in their numbers and function, which were supported by the decreased abilities to form in vitro colonies including BFU-E,

*DOI: http://dx.doi.org/10.5772/intechopen.88914*

*Beyond LEO - Human Health Issues for Deep Space Exploration*

**3. Proton radiation and hematopoietic stem cells**

and potentially provide lifelong hematopoiesis. The dominant HSCs have ability to protect the whole blood system against different stress conditions [30]. Under sublethal irradiation, HSCs have been long-term damaged, which can be easily ignored in clinic because of normal cell counts from the bone marrow and peripheral blood. Damaged HSCs induced by photon-irradiation have impaired self-renewing ability, leading to bone marrow failure and death [31]. However, whether low doses of space radiation trigger long-term HSC damage remains unknown. Comparing to HSCs, MPPs and HPCs have limit or lack self-renewal ability even though they are proliferating populations. The property of MPPs and HPCs with proliferation provides a beneficial role in case of normal hematopoiesis and stress hematopoiesis. For example, in case of blood loss or infection, MPP and HPC quickly proliferate to meet the requirement of mature cell production, trying to maintain normal hematopoiesis. Under radiotherapy and chemotherapy, MPPs and HPCs can be easily depleted with acute myelosuppression because of their proliferating feature. This will lead to HSC activation, proliferation, and differentiation to reestablish MPP and HPC populations and rebuild hematopoiesis, which might result in HSC exhaustion.

During spaceflight missions outside low Earth orbit, there remains the possibility of astronauts receiving damaging doses of space radiation. Because the dosedepth distribution of SPE spectrum protons is relatively low [32], during an SPE the skin and organs near the surface of the skin will receive higher doses than deeper organs such as the bone marrow. In addition, compared to traditional radiation therapy with X-rays (photons) or electrons, proton therapy has potential benefit for clinical cancer treatment because of its favorable distribution of the radiation dose, leading to selectively increased radiation dose to the cancerous tissues while lowering the dose to normal tissues [33, 34]. However, the hematopoietic system is highly sensitive to ionizing radiation, and exposure to even a relatively low dose of SPE may still be able to result in substantial damage to the system [35, 36]. Therefore, understanding the biological effects of proton radiation is immediately needed. One of the characteristics of the radiation-induced hematopoietic syndrome is a decline in blood cell counts, resulting from radiation-induced cell killing in circulating blood cells and suppression of hematopoietic stem and progenitor cells in the bone marrow [37–39]. Studies have reported that whole-body exposure to protons causes acute effects on the hematopoietic system in animal models [40–45]. The decreased WBCs, lymphocytes, and neutrophils were detected starting at 4 hours after 0.25–3 Gy proton radiation, with the lowest numbers observed on day 4 [43, 44]. The reduction in WBCs and lymphocytes was still evident in mice after exposure to 2.0 Gy of protons (230 MeV) [41]. This might be due to the high sensitivity of lymphocytes to proton radiation, which is consistent with the data from γ-radiation [46]. Two Gy of proton radiation induced the acute decrement of peripheral blood cells, which was shown completely recovered in the long term. The dynamic changes of peripheral blood counts might result in ignoring the negative effects of proton radiation on hematopoietic system. However, the abnormalities of splenic WBCs and lymphocytes were still detected at more than 100 days after low dose of proton radiation [47]. Taken together, proton radiation has not only acute

injury but also long-term harmful effects on the hematopoietic system.

It has been well established that exposure to a significant dose of total-body γ-irradiation (TBI) induces not only the acute radiation hematopoietic syndrome but also long-term bone marrow injury [48, 49]. The acute radiation hematopoietic syndrome induced by γ-irradiation is primarily attributed to the induction

**82**

of apoptosis of HPCs, while γ-irradiation-induced long-term BM suppression is mainly ascribed to the persistent damage to HSCs. While the effects of γ-irradiation on the hematopoietic system have been extensively documented, much less is known about the effects of proton irradiation [35, 36].

Previously, mice were exposed to two different doses (0.5 and 1.0 Gy) of proton radiation and examined the acute and long-term effects on BM HSPCs at 2 and 22 weeks after proton radiation, respectively (**Table 1**). Results showed that exposure of mice to 1.0 Gy of proton radiation resulted in a significant decrease in the number of WBCs and PLTs from peripheral blood 2 weeks after the exposure [50]. It was demonstrated that 1.0 Gy of oxygen ion radiation (16O TBI) significantly decreased the cell counts of peripheral blood leukocytes when measured 2 weeks after exposure in male C57BL/6 mice [51]. Interestingly, the decrease of peripheral blood cell counts was not observed 2 weeks after 0.5 Gy proton TBI or 0.1 and 0.25 Gy 16O TBI [51]. The threshold dose of protons (50 or 70 MeV) to induce a decline in WBC counts in female ICR outbred mice was previously estimated to be between 0.25 and 0.5 Gy [43]. The threshold dose identified from this previous study is lower than the result from our study. It may have resulted in part from the use of different strains of mice, the time when the mice were studied after radiation or the difference in energies of protons between the two studies. For example, the linear energy transfer values for 50, 70, and 150 MeV protons would be 1.26, 0.96, and 0.55 keV/μm [52]. Because of the dose-depth distribution of SPE protons, only relatively large or higher-energy spectrum SPEs may lead to BM exposure to these doses of protons in astronauts. However, the doses of SPEs that can cause significant HSPC damage have been observed, raising the possibility that astronauts might experience reductions in circulating blood cell counts and BM HSPC damage if they encounter such an SPE. The dose-depth distribution in mice exposed to protons is different from that in humans [1]. It was found that a dose of 0.5 Gy protons (150 MeV) significantly reduced hematopoietic stem/progenitor cell function. The effects of protons at this energy and at doses below 0.5 Gy are unknown. Since the dose to the blood-forming tissues of human subjects will likely be low, future studies need to examine the hematopoietic effects of proton doses below 0.5 Gy.

Proton TBI can acutely induce the decrease of all lineages of peripheral blood cells, since all lineages of blood cells are generated from hematopoietic stem cells through their differentiation into various lineages of progenitors. We have shown that the exposure to both 0.5 and 1.0 Gy proton TBI damaged not only HPCs but also LSK cells, leading to the defect in their numbers and function, which were supported by the decreased abilities to form in vitro colonies including BFU-E,


**Table 1.** *Acute and long-term effects of proton irradiation on hematopoietic cells.* CFU-GM, and CFU-GEMM [50]. The in vivo functional defect of hematopoietic stem and progenitor cells after proton exposure will be further investigated through bone marrow transplantation in future studies. We have also reported that a 1.0 Gy dose of 16O TBI significantly decreased peripheral blood counts and BM HSPCs 2 weeks after the exposure [51]. Therefore, the mechanisms of space radiationinduced acute damage to the hematopoietic system should be investigated further.

In our previous studies, we firstly show that exposure to proton radiation causes long-term hematopoietic injury at 22 weeks after the exposure [53]. Our data provide the first direct evidence that exposure of mice to 1.0 Gy dose of proton radiation results in not only a sustained reduction in the frequency of BM HSCs but also in the long-term inhibition of HSCs clonogenic function to form BFU-E, CFU-GM, and CFU-GEMM colonies in vitro (**Table 2**). In contrast, the number and frequency of HPCs returned to normal levels at 22 weeks postradiation. Another question that needs to be addressed is whether proton radiation-induced HSC damage leads to hypoplastic syndrome after hematopoietic stress. Myeloid leukemia could be induced by low and/or moderate doses of γ-irradiation and 56Fe heavy ion radiation in mice [54, 55]. It has been shown that proton radiation induced minor myeloid leukemia but have high possibility to induce hepatocellular adenoma and malignant lymphoma in CBA mice [56], which is in contrast with the effects of γ-irradiation, showing that 3 Gy of γ-irradiation caused around 25% of CBA mice developing acute myeloid leukemia. These differences are due to the differential biological effectiveness between proton and γ-irradiation. To closely mimic space environment, investigators used minipig animal models to expose to electron solar particle event (eSPE) [57]. Comparing to eSPE, proton solar particle events (pSPE) have stronger negative effect on the numbers of peripheral WBCs, lymphocytes, neutrophils, and monocytes with a factor of 2.79. These data suggest that different hematopoietic populations have differential radiosensitivity to proton irradiation.

Protons have a higher linear energy transfer and are denser ionizing radiation than photon. Therefore, protons deposit high energy at the end of their range termed "Bragg peak" and cause heavily damage to the target tissues, cells, and molecules. Compared to γ-irradiation, proton radiation causes larger γ-H2AX foci [58], leads to hypermethylated DNA [59], has different transcriptome profiles [60], and modulates different signaling pathways [61]. More detailed investigation into what unique biological effects proton radiation has is called for to instruct proton studies.

Because an SPE contains protons of multiple energies below 150 MeV, some groups have developed cell culture or animal models of exposure to broad energy spectra of protons to better simulate an SPE. Previously, some differences were


**85**

*Space Radiation-Induced Hematopoietic Stem Cell Injury*

induced damage to HPCs and LSK cells in a mouse model.

hematopoietic cell response to space radiation.

found in peripheral blood cell counts between mice exposed to one-energy protons (230 MeV, 2 Gy) and mice exposed to SPE-like protons at the same dose level [41]. Exposure of minipigs to SPE-like protons at a skin dose as low as 0.5 Gy (estimated dose to the BM: 0.42 Gy) caused decrement in peripheral blood cell counts up to at least 2 weeks after exposure [45]. Hence, further studies with SPE-like proton exposures at low doses are warranted. Since we used only two radiation doses (0.5 and 1 Gy), we were not able to prepare dose-response curves for the effects observed. In one of our separate studies, mice were irradiated with fully modulated beams containing particles from 0 energy to 150 MeV and a uniform dose versus depth profile. Doses of protons were 0.1, 0.25, and 0.5 Gy. Bone marrow cells were collected at 2 weeks after irradiation and examined as described in the current manuscript. We found a dose-dependent decrease in LSK cells, together with an increase in ROS levels and apoptosis in these cells (data not shown). In summary, our study has demonstrated that acute exposures to medium doses of proton TBI

Radiation-induced cell damage might be mediated by induction of apoptosis, DNA damage, and oxidative stress [48, 62]. We therefore assessed those parameters in HPCs and LSK cells 2 weeks after proton exposure [50]. Our data indicate that HPCs and LSK cells may respond differently to proton radiation. Exposure to 1.0 Gy protons resulted in an increase in cellular apoptosis in HPCs. Irradiated LSK cells, on the other hand, showed both increased apoptosis and oxidative stress. Neither of the two cell types showed enhanced DNA damage or cell cycling 2 weeks after proton exposure. Importantly, LSK cells in mice bone marrow from acute and long-term proton exposure cause the significant induction of oxidative stress [50, 53]. In the previous studies, it has been reported that the long-term increase in ROS production in LSK cells was observed after 16O radiation and γ-rays [48, 63, 64]. For example, increasing levels of ROS production were detected at 2 months after 6.0 Gy of total-body γ-irradiation, which might be related to irradiation-induced DNA damage, leukemia, and senescence in irradiated hematopoietic stem and progenitor cells. Both proton and gamma radiation may induce residual negative effects on the bone marrow, which might be mediated by overproduction of chronic reactive oxidative stress in HSCs [63, 65, 66]. Taken together, present data indicate that irradiation-induced oxidative stress in HSCs might be a critical factor in the

Reactive oxygen species (ROS) plays an important role in determining the fate of normal stem cells. Low levels of ROS are required for stem cells to maintain their quiescence and self-renewal capacities. Increases in ROS production cause stem cell proliferation, differentiation, apoptosis, and cell death, leading to their exhaustion. Regulating ROS production in stem cells is important to maintain tissue homeostasis and repair damaged area during the life span of an organism. It has been reported that the levels of ROS were closely related to the proper functional hematopoietic stem cells. There are multiple different ways for ROS production in cells, such as mitochondria oxidative phosphorylation, glycolysis, NADPH oxidases (NOXs) enzyme, peroxisomal and cytochrome P450 metabolism, and so on. Mitochondria oxidative phosphorylation is not a major source to generate ROS in hematopoietic stem cells under homeostasis. This is because (1) HSCs locate bone marrow hypoxic niche with low levels of oxygen; (2) HSCs have small amount of mitochondrial and immature mitochondrial; (3) HSCs have high level of pimonidazole, which is a hypoxia marker; and (4) HSCs have capacity to response to hypoxia by increasing hypoxia-inducible factor 1α (HIF-1α) expression [67]. Subsequently, increasing levels of HIF-1α benefit HSCs to use anaerobic glycolysis, instead of mitochondrial oxidative phosphorylation, to produce energy along with reducing ROS production. Previous studies have shown that increasing expression of NOX enzyme might

*DOI: http://dx.doi.org/10.5772/intechopen.88914*

**Table 2.** *Induction of cellular apoptosis, ROS, and DNA damage after proton irradiation.*

#### *Space Radiation-Induced Hematopoietic Stem Cell Injury DOI: http://dx.doi.org/10.5772/intechopen.88914*

*Beyond LEO - Human Health Issues for Deep Space Exploration*

CFU-GM, and CFU-GEMM [50]. The in vivo functional defect of hematopoietic stem and progenitor cells after proton exposure will be further investigated through bone marrow transplantation in future studies. We have also reported that a 1.0 Gy dose of 16O TBI significantly decreased peripheral blood counts and BM HSPCs 2 weeks after the exposure [51]. Therefore, the mechanisms of space radiationinduced acute damage to the hematopoietic system should be investigated further. In our previous studies, we firstly show that exposure to proton radiation causes

long-term hematopoietic injury at 22 weeks after the exposure [53]. Our data provide the first direct evidence that exposure of mice to 1.0 Gy dose of proton radiation results in not only a sustained reduction in the frequency of BM HSCs but also in the long-term inhibition of HSCs clonogenic function to form BFU-E, CFU-GM, and CFU-GEMM colonies in vitro (**Table 2**). In contrast, the number and frequency of HPCs returned to normal levels at 22 weeks postradiation. Another question that needs to be addressed is whether proton radiation-induced HSC damage leads to hypoplastic syndrome after hematopoietic stress. Myeloid leukemia could be induced by low and/or moderate doses of γ-irradiation and 56Fe heavy ion radiation in mice [54, 55]. It has been shown that proton radiation induced minor myeloid leukemia but have high possibility to induce hepatocellular adenoma and malignant lymphoma in CBA mice [56], which is in contrast with the effects of γ-irradiation, showing that 3 Gy of γ-irradiation caused around 25% of CBA mice developing acute myeloid leukemia. These differences are due to the differential biological effectiveness between proton and γ-irradiation. To closely mimic space environment, investigators used minipig animal models to expose to electron solar particle event (eSPE) [57]. Comparing to eSPE, proton solar particle events (pSPE) have stronger negative effect on the numbers of peripheral WBCs, lymphocytes, neutrophils, and monocytes with a factor of 2.79. These data suggest that different hematopoietic populations have differential radiosensitivity to proton irradiation. Protons have a higher linear energy transfer and are denser ionizing radiation than photon. Therefore, protons deposit high energy at the end of their range termed "Bragg peak" and cause heavily damage to the target tissues, cells, and molecules. Compared to γ-irradiation, proton radiation causes larger γ-H2AX foci [58], leads to hypermethylated DNA [59], has different transcriptome profiles [60], and modulates different signaling pathways [61]. More detailed investigation into what unique biological effects proton radiation has is called for to instruct proton studies. Because an SPE contains protons of multiple energies below 150 MeV, some groups have developed cell culture or animal models of exposure to broad energy spectra of protons to better simulate an SPE. Previously, some differences were

**84**

**Table 2.**

*Induction of cellular apoptosis, ROS, and DNA damage after proton irradiation.*

found in peripheral blood cell counts between mice exposed to one-energy protons (230 MeV, 2 Gy) and mice exposed to SPE-like protons at the same dose level [41]. Exposure of minipigs to SPE-like protons at a skin dose as low as 0.5 Gy (estimated dose to the BM: 0.42 Gy) caused decrement in peripheral blood cell counts up to at least 2 weeks after exposure [45]. Hence, further studies with SPE-like proton exposures at low doses are warranted. Since we used only two radiation doses (0.5 and 1 Gy), we were not able to prepare dose-response curves for the effects observed. In one of our separate studies, mice were irradiated with fully modulated beams containing particles from 0 energy to 150 MeV and a uniform dose versus depth profile. Doses of protons were 0.1, 0.25, and 0.5 Gy. Bone marrow cells were collected at 2 weeks after irradiation and examined as described in the current manuscript. We found a dose-dependent decrease in LSK cells, together with an increase in ROS levels and apoptosis in these cells (data not shown). In summary, our study has demonstrated that acute exposures to medium doses of proton TBI induced damage to HPCs and LSK cells in a mouse model.

Radiation-induced cell damage might be mediated by induction of apoptosis, DNA damage, and oxidative stress [48, 62]. We therefore assessed those parameters in HPCs and LSK cells 2 weeks after proton exposure [50]. Our data indicate that HPCs and LSK cells may respond differently to proton radiation. Exposure to 1.0 Gy protons resulted in an increase in cellular apoptosis in HPCs. Irradiated LSK cells, on the other hand, showed both increased apoptosis and oxidative stress. Neither of the two cell types showed enhanced DNA damage or cell cycling 2 weeks after proton exposure. Importantly, LSK cells in mice bone marrow from acute and long-term proton exposure cause the significant induction of oxidative stress [50, 53]. In the previous studies, it has been reported that the long-term increase in ROS production in LSK cells was observed after 16O radiation and γ-rays [48, 63, 64]. For example, increasing levels of ROS production were detected at 2 months after 6.0 Gy of total-body γ-irradiation, which might be related to irradiation-induced DNA damage, leukemia, and senescence in irradiated hematopoietic stem and progenitor cells. Both proton and gamma radiation may induce residual negative effects on the bone marrow, which might be mediated by overproduction of chronic reactive oxidative stress in HSCs [63, 65, 66]. Taken together, present data indicate that irradiation-induced oxidative stress in HSCs might be a critical factor in the hematopoietic cell response to space radiation.

Reactive oxygen species (ROS) plays an important role in determining the fate of normal stem cells. Low levels of ROS are required for stem cells to maintain their quiescence and self-renewal capacities. Increases in ROS production cause stem cell proliferation, differentiation, apoptosis, and cell death, leading to their exhaustion. Regulating ROS production in stem cells is important to maintain tissue homeostasis and repair damaged area during the life span of an organism. It has been reported that the levels of ROS were closely related to the proper functional hematopoietic stem cells. There are multiple different ways for ROS production in cells, such as mitochondria oxidative phosphorylation, glycolysis, NADPH oxidases (NOXs) enzyme, peroxisomal and cytochrome P450 metabolism, and so on. Mitochondria oxidative phosphorylation is not a major source to generate ROS in hematopoietic stem cells under homeostasis. This is because (1) HSCs locate bone marrow hypoxic niche with low levels of oxygen; (2) HSCs have small amount of mitochondrial and immature mitochondrial; (3) HSCs have high level of pimonidazole, which is a hypoxia marker; and (4) HSCs have capacity to response to hypoxia by increasing hypoxia-inducible factor 1α (HIF-1α) expression [67]. Subsequently, increasing levels of HIF-1α benefit HSCs to use anaerobic glycolysis, instead of mitochondrial oxidative phosphorylation, to produce energy along with reducing ROS production. Previous studies have shown that increasing expression of NOX enzyme might

contribute to γ-irradiation-induced ROS production in HSCs [66], which was supported by increasing NOX4 expression, rather than other isoforms of NOXs in γ-ray irradiated HSCs [66]. Diphenyliodonium (a selective NOX inhibitor) treatment can partially restore the functional impairment in γ-ray irradiated HSCs by decreasing irradiation-induced ROS production in HSCs [66]. For space radiation circumstance, the higher level of NOX4 expression was observed in proton-irradiated HSCs than that in unirradiated HSCs. These data indicated that NOX enzyme, especially NOX4, might be involved in the induction of ROS production in proton-irradiated HSCs. The importance of ROS production in space radiation-induced HSC injury should further be assessed by using NOX4 inhibitor or other antioxidants, such as N-acetylcysteine and gamma-tocotrienol in future studies. There are some other potential unanswered questions including (1) whether these chronically oxidativestressed HSCs induced by proton radiation experience senescence, (2) whether these space-irradiated HSCs have chromosomal instability, and (3) whether the chromosomal aberrant HSCs after space radiation result in the leukemia development, which was evidenced in mice after γ-irradiation exposure [38, 65, 68].

One of HSC properties is its self-renewal ability, which is sustained via its slow cycling and quiescence. By using BrdU-chasing assay and H2B-GFP mice model, it has been shown that dominant HSCs divide only once every 145 days, which ensures self-renewal capacity along with providing whole life blood homeostasis and avoiding HSC exhaustion [69]. It was reported that loss of FOXO3a resulted in increasing ROS production and accelerating HSC cycling, which is along with the defect of HSC self-renewal capacity and the exhaustion of HSCs. N-acetylcysteine (NAC), an antioxidant, can protect FOXO3a mutant HSCs from oxidative stress and restore HSC dormancy. The same phenotype was also seen in the case of loss of Bmi-1 and TSC1 in mice [70–72], which is due to increasing ROS production and cycling in HSCs. Upon proton radiation, data have shown that there were far fewer HSCs in G0 phase and higher numbers of HSCs in G1 phase than nonirradiated controls. Proton radiation-induced HSC cycling is consistent with upregulation of positive cell cycle regulators cyclin D1 and cyclin D3. Although HSC proliferation might compensate for the decreased number of HSCs after proton radiation, it will be at risk of loss self-renewal of HSCs.

Additionally, it has demonstrated that the persistent increase of DNA damage in proton-irradiated HSCs, but not in HPCs, was associated with proton radiationinduced ROS production in HSCs. Unrepaired DNA damage in proton-irradiated HSCs might negatively affect HSC self-renewal, proliferation, and differentiation, leading to long-term functional damage in HSCs. Taken together, these findings provide strong evidence showing that proton TBI induces not only acute hematopoietic injury but also long-term BM suppression and HSC damage. These detrimental effects of proton radiation on hematopoietic cells are closely related to the induction of oxidative stress in irradiated HSCs. The proton exposure-induced acute and long-term hematopoietic damage might be ameliorated through using antioxidants, which should be investigated in the future.
