Extensive research focuses on both understanding the fundamental underlying mechanisms and finding suitable treatments for such patients. However, major health concerns remain, such as secondary drug effects, osteonecrosis of the jaw and atypical femoral fractures, motivating continued search for osteoprotective treatments against bone loss. Space radiation is a unique type of radiation, composed of various ions and photons due to the combination of galactic cosmic rays , solar particle events , and protons and electrons trapped in the magnetosphere. Linear energy transfer of a given ion species reflects its ionization capacity and the heavier the nuclei , the more primary and secondary ionizations occur for each particle traversal. Galactic space radiation is composed of mostly low-LET species including proton and helium ions but also HZE particles, including iron. High-LET radiation increases the multiplicity of damage types on a variety of target molecules in the cell, and poses significant challenges to cellular repair processes when this complex damage occurs on the DNA23. In contrast to radiation doses experienced on the International Space Station 23, future long duration missions outside the protection of the Earth’s magnetosphere, or unshielded exposures to SPE,seedling starter pot may achieve total doses capable of causing cancellous bone loss. It is estimated that the maximum dose of exposure to space radiation over a protracted 3-year mission outside the magnetosphere would be ~2 Gy, which equals the typical single fraction dose delivered during standard clinical radiotherapy.

Initially, radiation increases the expression of pro-osteoclastogenic cytokines in both marrow cells and mineralized tissue and increases the number and activity of bone-resorbing osteoclasts. Total body irradiation increases the generation of reactive oxygen species by marrow cells1 and stimulates gene expression levels of pro-osteoclastogenic cytokines such as Rankl, Mcp1, Tnf-α and Il-6 in addition to Nfe2l229. Nfe2l2 encodes a master transcription factor that regulates the expression of more than 600 genes to help defend the cell from damaging ROS and resulting oxidative stress. We showed previously that increases in gene expression for Nfe2l2 and osteoclastogenic cytokines one day after TBI precede the manifestation of bone loss .The changes in remodeling activity caused by exposure to radiation can lead to impaired structural integrity and fragility both in animal models and radiotherapy patients. Radiation-induced bone loss resembles accelerated, age-related structural changes, particularly in rapid-turnover cancellous tissue. Over the course of three days to one month, relatively low doses of radiation can cause rapid and progressive strut thinning and removal of cancellous tissue as shown by our group and others. We hypothesized that diets or drugs capable of preventing the early increase in pro-osteoclastogenic and oxidative stress-related factors mitigate cancellous bone loss caused by both low LET and high LET radiation. To test this, we evaluated the following candidate interventions: an antioxidant diet cocktail , including 5 antioxidants reported to protect target tissues from ionizing radiation, dihydrolipoic acid , which possesses antioxidant properties4,35, Ibuprofen, an anti-inflammatory drug, to test the ability to prevent inflammation-related bone loss and Dried Plum , shown to mitigate age-related bone loss as an anti-resorptive in other disease models.

The candidate interventions first were evaluated using early gene expression markers 29 one day after TBI. Interventions were then tested for their ability to prevent radiation-induced cancellous bone loss. Thus, analysis of early changes in gene expression levels for osteoclastogenic cytokines after exposure to ionizing radiation served both to identify potentially effective interventions, and to further establish the relevance of early changes in marrow cytokine expression for cancellous bone loss. Here we report that one of our selected interventions completely prevented cancellous bone loss caused by ionizing radiation.To evaluate overall health, body weights and food consumption were monitored throughout the experiments . Candidate interventions were administrated to the mice prior to TBI using pre-feeding protocols reported to effectively protect other tissues or bone and following exposure to radiation as described in the methods . There were no significant differences in food consumption or final body weights within diet groups due to irradiation , indicating that the various diets were well tolerated. Hence, difference in body weights was not a factor for the differences observed in skeletal properties.To test candidate interventions for their ability to mitigate elevated expression levels of pro-osteoclastogenic and antioxidant genes caused by irradiation, bone marrow was recovered 24hours after exposure then analyzed by qPCR . In the control diet -fed groups, radiation exposure led to an increase in marrow cell expression of genes associated with bone resorption, including the osteoclastogenic cytokine Rankl , monocyte chemokine attractant Mcp1 , and the pro-inflammatory molecule Tnf-α . Opg, the decoy receptor for RANKL, was not within range of detection in control, sham-irradiated animals, but exhibited increased expression levels after irradiation. In addition, radiation caused a two-fold increase in expression levels of the global antioxidant transcription factor, Nfe2l2. These changes in pro-osteoclastogenic cytokine and antioxidant gene expressions were consistent with our previous findings.DP was effective in maintaining all gene expression levels comparable to controls one day after exposure to gamma irradiation . The inhibitory effects of DHLA treatment were comparable to DP except for the apparent suboptimal effect of DHLA to inhibit Tnf-α expression.

Ibuprofen only mitigated the expression of Nfe2l2, Mcp1 and Opg. Surprisingly, the AOX diet did not prevent the changes in gene expression caused by radiation, although it effectively counteracts other types of radiation damage.Since DP exhibited the most definitive protection from radiation-induced increases in expression levels of the pro-resorption genes tested in this study, we then assessed its ability to prevent associated decrements in skeletal microarchitecture. Mice were fed DP for 17 days , then exposed to 2 Gy gamma radiation and tissues were recovered 11 days later, a regimen well established to induce cancellous bone loss. Microcomputed tomography measurements were analyzed by 2-factor ANOVA and revealed main effects in percent Bone Volume/Total Volume for radiation and diet as well as interaction effects . Radiation caused a 32% decrement in BV/TV , a 25% decrease in trabecular number , and a 13% increase in trabecular separation compared to sham-irradiated controls fed the control diet . Trabecular thickness was unaffected by irradiation, consistent with our prior findings. In contrast, mice on the DP diet did not exhibit decrements after exposure to 2Gy 137Cs in any of the structural parameters, indicating potent radio protective effects of DP against cancellous bone loss. The tibial shaft was analyzed by micro-computed tomography to determine if DP diet or irradiation affected structural or material properties of cortical tissue, which mainly contribute to whole bone mechanical properties . Consistent with previous findings, irradiation did not affect cortical bone structure , nor did feeding the DP diet.In contrast to the DP diet-supplemented groups, Ibuprofen and DHLA-treated mice displayed decrements in cancellous microarchitecture similar to irradiated animals that were not provided any treatment .Because HZE and protons, comprising the major species of space radiation, exert effects that can differ from gamma radiation, we examined the effectiveness of candidate antioxidant treatments after TBI with simulated space radiation . AOX also was included to test a corollary hypothesis that an unchecked increase in pro-osteoclastogenic gene expression leads to bone loss in a space radiation model. As expected,round nursery pots irradiated animals on the control diet showed decrements in percent bone volume and other structural parameters compared to sham-treated animals on the same diet. Irradiation with simulated space radiation caused a main effect of radiation but not diet, on BV/TV . Consistent with the corollary hypothesis, AOX diet did not prevent the radiation-induced decreases in BV/TV and Tb.N , but appeared to exert a modest protective effect on Tb.Sp . No effects were observed in Tb.Th as expected . DHLA did not prevent radiation-induced bone loss , similar to the results obtained with gamma-irradiation. In contrast, DP fully preserved cancellous percent bone volume and other structural parameters after irradiation, suggesting its potential as a radiomitigator for HZE and proton exposures .

Exposure to ionizing radiation caused both a rapid increase in expression of pro-osteoclastogenic cytokines and a later decrement in cancellous BV/TV and microarchitectural integrity , consistent with our previous findings. DP was effective at reducing expression of early pro-osteoclastogenic cytokines, and an important indicator of antioxidant responses, Nfe2l2, in bone marrow. DP also completely prevented microarchitectural deficits, whereas other treatments did not. The AOX diet, which effectively mitigates morbidity caused by exposure to high doses radiation, failed to prevent effects of radiation on expression of osteoclast-related genes and subsequent bone loss. Our study demonstrates the complexity of the processes underlying bone loss caused by exposure to radiation. Results indicate that co-existence of high levels of pro-resorption, pro-inflammation, and oxidative stress-related genes in the bone marrow strongly correlated with cancellous bone loss. Treatments that failed to mitigate the alterations in these molecular markers ultimately were unsuccessful in mitigating radiation-induced decrements in skeletal microarchitecture. This suggests that preventing up-regulation of these molecular responses should be considered in the development of a rational strategy to mitigate bone loss. However, seemingly paradoxical is the observation that DHLA, which appeared nearly as effective as DP in preventing radiation-induced increases in expression of these markers, did not protect skeletal integrity. A plausible explanation for this observation is that there are other equally important determinants of bone loss apart from up-regulation of these molecular markers. In this case, DP was clearly more effective at ameliorating most of these determinants than DHLA as DP abrogated the decrements in BV/TV caused by radiation. In addition, AOX and DP diets displayed similar total antioxidant capacity, suggesting that antioxidant capacity of the diets alone, as measured by this assay, is not sufficient to protect bone from radiation. This again, is consistent with the idea that the determinants of bone loss are multi-factorial. DP is known to inhibit resorption in models of aging and ovariectomy-induced osteopenia as do other polyphenol-rich fruits , although prior to this study, radioprotective effects of dried plum were not reported. The mechanism mediating DP radioprotection is uncertain, although there is evidence that specific components including polyphenols, promote osteogenesis and prevent osteoclastogenesis. Purified dried plum polyphenols contains various polyphenols such as gallic acid, caffeoyl-quinic acids, coumaric acid and rutin. These polyphenols are known for their high antioxidant and anti-inflammatory properties. Consistent with these in vitro findings, DP diet prevented IR-induced elevation in levels of Nfe2l2 and Tnf-α in vivo compared to animals fed with controls diets. In the context of spaceflight relevance, it will be of particular interest to determine the ability of the DP diet to prevent simulated or actual microgravity-induced bone loss as musculoskeletal disuse leads to deficits in both cortical and cancellous compartments of bone4. Several in vitro studies demonstrate the potential of DP to prevent free radical damage as well as inflammatory responses in RAW 264.7 cells and MC3T31 cells. In thecontext of skeletal remodeling, purified polyphenols from DP powder inhibit bone-resorbing osteoclastogenesis in vitro by down-regulating osteoclast differentiation and expression of osteoclast-specific genes in RAW264.7 cells after treatment with lipopolysaccharide or H2O2 . In addition, DPP enhances differentiation of an osteoblast cell line in vitro both under normal conditions, as well as after treatment with TNF-α. Together these findings indicate that DP polyphenols may exert beneficial effects on remodeling by inhibiting bone resorption and/or improving bone formation. Beyond the scope of this study but topics worthy of future investigation, include a determination of the active component of plum that exert the observed protective effects. It still has to be linked directly in vivo that the dried plum polyphenols are the active compound exerting the radio-protective effects. In addition, even when purified, DP contains multiple polyphenols, and it remains uncertain whether the majority of the beneficial effects of DP are derived from a single or more complex combination of polyphenols. Moreover, the roles and properties of other bioactive compounds in plum in bone remodeling have yet to be determined. Studies conducted to date using the DP diet suggest that the combination of multiple constituents may be needed to exert full protection against radiation-induced bone loss. In addition, long duration experiments with DP and ionizing radiation would be valuable to assess whole bone mechanical properties along with structure, as cortical changes develop more slowly due to the lower metabolic activity of cortical tissue compared to cancellous tissue. Nonetheless, the relatively rapid changes in cancellous structure observed due to exposure to ionizing radiation are likely to be biologically relevant as radiation caused the removal of trabecular struts , which generally is irreversible, and DP diet prevented this strut loss.