We have previously demonstrated the efficacy of dietary supplementation with a mixture of antioxidants comprised of SeM, vitamin C, vitamin E succinate, α-lipoic acid and NAC as a preventative measure prior to TBI with X rays or as a treatment after TBI in limiting hematopoietic cell depletion, promoting hemapoietic cell recovery, and improving animal survival (17
). In the present study we aimed to study the radioprotective efficacy of the same dietary antioxidants on similar end points mediated by proton TBI. When administered as a preventative measure prior to TBI, dietary antioxidant supplementation was effective in significantly limiting radiation-induced peripheral leukopenia, neutropenia and lymphopenia at 4 and 24 h after 1 Gy, whereas the antioxidants were less effective against the hematopoietic effects of 7.2 Gy of proton TBI. Supplementation with antioxidants prior to TBI also significantly limited radiation-induced bone marrow cell depletion and the decrease in spleen mass at 24 h after exposure. Furthermore, antioxidant supplementation protected against hematopoietic syndrome-induced animal mortality in a statistically significant manner when given as a treatment after radiation exposure; survival of the irradiated animals increased from 60% in the animals fed the control diet to 93% in the animals fed with the antioxidant diet. For these data, the dose-modifying factor (DMF) for antioxidant therapy (ratio of survival of animals protected by antioxidant therapy to survival of the unprotected animals) was 1.6. The antioxidant diet was less effective in increasing survival when given in a preventative fashion before TBI. Antioxidants were effective in improving animal survival only in ICR mice exposed to a dose below the calculated LD50/30
for 1 GeV/nucleon proton TBI. Preventative antioxidant supplementation was also associated with significant modulation of proton TBI-induced changes in plasma levels of the hematopoietic cytokines Flt-3L and TGFβ1 in a dose- and time-dependent fashion. Last, preventative supplementation with antioxidants was the most effective regimen at increasing the recovery of radiation-induced peripheral leukocyte depletion.
Several recent studies have established the short-term and long-term deleterious effects of sublethal (0.5–4 Gy) proton TBI on various hematopoietic cell parameters (11
). These studies in sum elucidate the potential hematopoietic risk and harm of extended human space travel, particularly in the event of SPEs. It is worthwhile noting that the aforementioned studies used clinically relevant 250 MeV/nucleon protons. To our knowledge, none of these past studies assessed either the effect of potentially lethal doses of protons on hematopoietic cells, organs and animal survival or any countermeasures (preventative or treatment) aside from shielding (16
). Older studies did assess the hematopoietic effects of proton TBI in dogs and primates along with shielding or hypoxia as countermeasures (37
). Our previous in vivo
studies with γ rays, protons or HZE particles in mice and rats suggested that dietary supplementation with antioxidants is an effective countermeasure to prevent ionizing radiation-induced decreases in plasma total antioxidant status (a marker of oxidative stress) (18
). We therefore hypothesized that dietary antioxidants would confer a protective effect against the deleterious hematopoietic effects of proton TBI in vivo
The RBE of 250 MeV/nucleon protons (or lower energies) has been estimated to range from 0.9–1.25 depending on the particular end point measured and the energy of the protons used, although a general RBE of 1.1 is conventionally proposed and used (4
). At 70 GeV/nucleon, the proton RBE was noted to be 1.6–7.6 in Chinese hamster fibroblasts and 1.04–3.8 in lymphoid cells with single-strand DNA breaks as the end point, whereas the RBE was 1.14–1.7 for survival of Chinese hamster cells (48
). We sought to investigate the effects of proton TBI on 30-day mortality resulting from the hematopoietic syndrome in ICR mice and to establish the RBE for this end point. The LD50/30
for total-body exposure to X rays in ICR mice was previously estimated to be 7.55 Gy (49
). Similarly, we found previously that 8 Gy TBI resulted in 87% mortality at 30 days in the same strain of mice (17
). In the present study we calculated the LD50/30
of 1 GeV/nucleon protons to be 6.23 Gy, corresponding to an RBE of 1.21 compared to the results observed for X rays. Our results are in agreement with previous in vivo studies and fall within the accepted 10–20% variance in RBE in the clinical setting and the conventionally accepted value of 1.1 for various proton radiation-induced biological effects (47
We found that the radioprotective effect of dietary supplementation with antioxidants on animal mortality after proton TBI occurred only at a dose less than the LD50/30
, which was 5.9 Gy (equivalent to a dose of 6.5 Gy X rays, assuming an RBE of 1.1). In contrast, our previous results using X rays indicated that antioxidants significantly increased animal survival at a total-body dose of 8 Gy (greater than the LD50/30
of 7.55 Gy). We also noticed another difference in the efficacy of dietary supplementation with antioxidants as a radioprotective countermeasure against hematopoietic injury and death induced by TBI with X rays compared to protons. Whereas antioxidants were effective at increasing animal survival when administration began at 7 days prior to or 2 h after X irradiation, dietary antioxidants were considerably more effective at increasing animal survival after proton TBI when they were administered 2 h after TBI compared to the results observed when the antioxidants were administered both before and after TBI. These data suggest that the antioxidants used in this study could be used safely as supportive therapy after proton TBI. Although the effects of the diets on animal weights were not evaluated in this study, the effects of the antioxidant diet compared to the control AIN-93G diet on animal weights and toxicity parameters were evaluated carefully in previous studies in this laboratory (50
). In those studies, no effects on animal weight or other toxic effects were attributed to the antioxidant diet in either short-term or long-term studies involving irradiated or unirradiated animals.
The self-renewal capacity or reconstitution of hematopoietic stem cells (HSC) is dependent on ataxia telangiectasia mutated (ATM)-mediated inhibition of oxidative stress generated by p38 MAPK activity, whereas proliferation of more differentiated hematopoietic progenitor cells is less sensitive to levels of p38 MAPK-derived reactive oxygen species (ROS) (51
). Treatment of adult mice lacking the ataxia telangiectasia mutated gene (Atm−/−
) with NAC or catalase not only prevents elevation in ROS but also results in partial rescue of bone marrow failure associated with increased chronic p38 MAPK-induced ROS (51
). However, it is also known that several hematopoietic cytokines that stimulate growth, differentiation and prevention of apoptosis of progenitor cells, including granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), steel factor (SF), thrombopoietin (TPO) and erythropoietin (Epo), cause rapid increases in ROS levels in quiescent progenitor cells via receptor-mediated signaling cascades (53
). Several antioxidants, including NAC, have been shown to abolish or diminish the receptor-mediated signaling of these hematopoietic cytokines (53
). In hematopoietic stem and progenitor cells, redox signaling mediated by NADPH oxidase and its regulatory proteins may be an important regulator of the critical balance between self-renewal and differentiation (55
In the present study with proton TBI, as previously observed for total-body X irradiation, dietary antioxidants were most effective in improving animal survival when administration began 2 h after radiation exposure. Although the signaling cascades common or specific to either photons or protons are not completely known, it is evident that TBI of animals results in an inflammatory state partially mediated by oxidative stress immediately after radiation exposure that can ultimately result in animal death depending on the dose delivered. Furthermore, it remains unknown as to what extent TBI-induced oxidative stress is a necessary physiological response to promote animal survival, e.g. hematopoietic cytokine-induced receptor signaling cascades. When administered 2 h after the proton TBI, the antioxidant diet would not affect the initial oxidative stress mediated by or in response to TBI, but it could have a major effect on persistent oxidative stress induced by the radiation exposure, ultimately resulting in the most effective increase in animal survival after both proton and X irradiation. In our previous studies with mice and rats exposed to γ rays, protons or HZE particles, we found that dietary antioxidant supplementation prior to TBI resulted in prevention of the radiation-induced decrease in serum total antioxidant capacity (a surrogate for oxidative stress) at 4 h after exposure (18
). Although this observation is consistent with our hypothesis that TBI depletes serum antioxidant levels or other endogenous antioxidant stores, which accounts for the efficacy of dietary antioxidant supplementation, we fully acknowledge that direct measurement of antioxidants levels is necessary to confirm this mechanism of action.
In the bone marrow, total-body X irradiation at doses of 0.5–6 Gy results in a significant decrease or complete depletion of endogenous vitamin C and vitamin E levels as early as 1 h, with the nadir at 24 h after exposure (56
). These radiation-induced changes in endogenous antioxidant vitamin levels are associated with concurrent or delayed increases in markers of oxidative stress in the bone marrow including 4-hydroxynonenal, hexanal and thiobarbituric acid-reactive substances (57
). Interestingly, sublethal TBI with 3 Gy resulted in recovery of vitamin levels in the bone marrow at 8 days after exposure, whereas there was no recovery back to normal levels after 6 Gy X irradiation (56
). From our studies as well as those of others, antioxidant supplementation prior to or after TBI likely modifies the bone marrow response to radiation exposure.
In the current study we identified another putative means by which antioxidant supplementation affects hematopoietic cell response after TBI, which is the modulation of the hematopoietic cytokines Flt-3L and TGFβ1. Several studies have shown that after exposure to ionizing radiation, blood levels of Flt-3L are a surrogate for the extent of damage to hematopoietic progenitor cells in the bone marrow (28
). Furthermore, the concentration of Flt-3L in plasma after irradiation is inversely correlated with PMN cell counts in the peripheral blood (29
). Dietary antioxidant supplementation prior to TBI resulted in significantly lower levels of plasma Flt-3L at 4 and 24 h after 1 Gy TBI compared to levels in similarly irradiated animals fed the control diet. These results not only corroborate the protective effect of antioxidants on peripheral PMN cell counts after TBI but also suggest that preventative dietary antioxidant supplementation has a protective effect on bone marrow cell depletion after 1 Gy proton TBI. Interestingly, we observed the protective effects of antioxidants after 1 Gy TBI not only in bone marrow cell counts but also in spleen mass and peripheral PMN cell and lymphocyte counts in a similar fashion and to a similar extent. Furthermore, we observed that antioxidants did not affect the increase in plasma levels of Flt-3L after 7.2 Gy TBI. These data are consistent with the lack of difference in peripheral PMN cell and lymphocyte counts regardless of diet after 7.2 Gy TBI. The extent of peripheral leukocyte depletion after proton TBI observed in our study is consistent with results obtained from previous studies (11
). Taken together, preventative dietary antioxidant supplementation is more effective at mitigating proton TBI-induced hematopoietic cell changes at 1 Gy compared to 7.2 Gy.
Proton TBI resulted in significant changes in the plasma levels of TGFβ1 that were affected by preventative dietary antioxidant supplementation in a statistically significant manner. In animals fed the control diet, plasma TGFβ1 levels exhibited a dose-dependent response to TBI in that 1 Gy TBI resulted in significantly increased levels of the hematopoietic cytokine compared to nonirradiated control animals and 7.2 Gy resulted in significantly decreased levels at 4 h after exposure. At 24 h after TBI, TGFβ1 levels returned to the levels in nonirradiated animals in both 1-Gy and 7.2-Gy animals fed the control diet. Antioxidant supplementation resulted in an increase in TGFβ1 plasma levels at 24 h after 1 Gy compared to nonirradiated controls. Plasma levels of TGFβ1 returned to those in nonirradiated animals at 24 h after 7.2 Gy TBI regardless of diet. This suggests that antioxidant supplementation potentially abolished or delayed the endogenous TGFβ1 response. The mechanism and significance of antioxidant modulation of radiation-induced plasma TGFβ1 levels are not known. However, this is likely an important means by which antioxidants also affect bone marrow cell response or recovery after TBI. Although we did not measure plasma levels of this cytokine in our previous study with X rays, we did notice a profound effect of TBI on TGFβ1 mRNA expression in the bone marrow at 4 and 24 h after exposure (17
). Our observation that proton TBI results in significant changes in TGFβ1 levels was not observed in the study by Kaijoka et al.
, who compared levels of this cytokine in proton- and γ-irradiated animals (34
). The discordance between these studies likely represents evaluation of cytokine levels at different times. Kaijoka et al. evaluated plasma TGFβ1 at 7 days after TBI (34
), and we report that by 24 h the elevation of this cytokine in the circulation returns to levels observed in nonirradiated animals.
In this study we also observed that antioxidant supplementation increased peripheral leukocyte cell recovery when given prior to sublethal or potentially lethal proton TBI. The benefits of antioxidants in improving recovery of hematopoietic cells in the periphery and bone marrow were also observed in our study with X rays (17
). Interestingly, despite a lower impact on animal survival, antioxidant supplementation before TBI resulted in the greatest improvement in hematopoietic cell recovery. These results suggest that the end point of animal survival after potentially lethal TBI is affected by many factors, including bone marrow as well as peripheral hematopoietic cell protection and recovery.
This report shows that the effects of proton TBI on hematopoietic end points, including 30-day survival, are not completely similar to the effects observed for total-body X irradiation. Some differences may be related to the higher RBE of protons compared to photons. Dietary antioxidant supplementation may be an effective countermeasure for proton-induced hematopoietic effects. However, additional studies are needed to elucidate the endogenous hematopoietic oxidative stress response to TBI and the impact and ideal timing of exogenous antioxidants on this important regulatory hematopoietic pathway.