The impact of radiation on bone quality in reduced gravity is unknown, and must be studied to understand effects of space radiation on bone health. The long-term objective of the proposed research is the development of countermeasures to prevent bone loss during missions and thus reduce fracture risk.

To define the risks associated with space radiation-induced bone loss, the following aims were studied to examine the effects of modeled space radiation using scenarios applicable for Lunar Outpost missions:

Specific Aim 1: Examine the combined effects of a modeled solar particle event and unloading on bone, and subsequent recovery during reloading. Hypothesis: Proton radiation with unloading will induce a more severe bone loss than unloading alone.

Summary of Aim 1 Findings: • Mice that were exposed to a 1 Gy dose of protons and 28-days of hindlimb suspension experienced damage that was additive. Irradiation caused a 15% decline in trabecular bone mass in normally loaded mice and a 17% decline in skeletally unloaded mice. • A long-term suppression of bone formation inhibits recovery of bone mass.

Aim 1 Future Direction: The effects of mixed radiation, modeling solar particle events and cosmic radiation, needs to be modeled with fractionated exposure during skeletal disuse. It is important to understand if skeletal disuse modifies the threshold dose at which bone loss from radiation is observable. The degree to which animal models predict the human response to space radiation (astronauts) needs to be better understood by studying cancer patients receiving radiation therapy, particularly at the periphery of the radiation field where doses are lower.

Specific Aim 2: Examine the cellular and molecular mechanisms for initiating bone loss following exposure to several types of modeled space radiation, including acute proton exposure, low-dose-rate proton exposure, and mixed radiation types (proton and HZE). Understanding underlying molecular causes is critical to developing countermeasures for radiation-induced bone loss. Hypothesis: The initiating mechanism of bone loss is initiated by osteoclast activation caused by a radiation-induced inflammatory response.

Summary of Aim 2 Findings: • The acute response to 50 cGy dose of protons or heavy ions is the activation of osteoclastic bone resorption. • Serum markers for bone resorption increase 24 hours after exposure and osteoclast numbers and eroded surface are greater at 3-days. • Osteoclast activation is temporary, reducing to baseline levels approximately 10-days after irradiation. • We identified that this bone loss is preceded by death of bone marrow cells, followed by greater expression then levels of inflammatory cytokines IL-1, IL-6 and TNFalpha. • This osteoclast-mediated acute response is not mitigated by low-dose-rate exposure to protons. • Acute, osteoclast-mediated bone loss from 50 cGy proton exposure is approximately 50% that of a 2 Gy exposure. • The acute bone loss caused by heavy ions (oxygen, silicon, neon, iron) is approximately 30% - 50% greater than that caused by protons.

Aim 2 Future Direction: A more detailed description of acute relative biological effectiveness (RBE), both at the functional and cellular level, needs to be characterized. The association of acute marrow death and osteoclast activation needs to be studied at the causation level with cell culture models.

Specific Aim 3: Test the efficacy of three countermeasures for bone loss caused by proton exposure: 1) the bisphosphonate risedronate; 2) the RANKL blocking protein osteoprotegerin, and 3) an antioxidant agent, alpha-lipoic acid. Hypothesis: Potent inhibitors of bone resorption, both zoledronate and osteoprotegerin will prevent the bone loss caused by radiation. Antioxidants will address multiple radiation-induced problems; alpha-lipoic acid decreases osteoclast differentiation and activity.

Summary of Aim 3 Findings: • The osteoclast inhibiting bisphosphonate risedronate fully prevented bone loss from a 2 Gy whole body dose of X-rays • Because of intellectual property concerns with a patent application filing, the RANKL-inhibiting protein osteoprotegerin was not able to be tested. • As a substitute for OPG, two additional bisphosphonates, pamidronate and zoledronate, were tested. Zoledronate was more effective than pamidronate. • Neither of the antioxidant compounds alpha-lipoic acid nor n-acetal cystine (NAC) reduced bone loss from radiation exposure. • The IL-1 and TNFalpha blocking compounds, IL-1ra (Kineret) and TNFbp (Enbrel), were tested as potential countermeasures. Neither protein, independently or in combination, mitigated bone loss from radiation exposure.

Aim 3 Future Directions: As an antiresorptive agent that suppresses osteoclast activity through a different mechanism than bisphosphonates, osteoprotegerin should be studied as a countermeasure (intellectual property concerns are currently being addressed). The most effective dosing regimen for risedronate and zoledronate needs to be further refined. Additionally, a myostatin inhibitor should be studied as a countermeasure that may have positive effects on both bone and muscle. Finally, countermeasures should be tested in models that combine both skeletal challenges astronauts will be exposed to: microgravity and space radiation.

Postmenopausal women receiving radiation therapy (RT) for pelvic tumors have a 65-200% increased risk of hip fracture compared to women receiving non-RT cancer treatment. It is generally accepted that RT damages local osteoblasts and vasculature resulting in a low turnover, gradual decline in bone mass. However, it has recently been observed in rodent models that ionizing radiation activates osteoclasts. To test the hypothesis that early osteoclastic resorption may cause a rapid loss of bone, changes in proximal femur strength, bone density, and mineral content were examined in women receiving RT for gynecological tumors. Eight women (age 36-71 years) with cervical (n=6), vaginal, or uterine cancer provided informed consent. CT scans were performed pre-RT and on the last day of RT (6 weeks later). Patients received 50.4 Gy over the course of 28 days. Total dose to the proximal femur was ~25.0 Gy. CT scans were used for finite element strength and volumetric quantitative CT (vQCT) analyses. Proximal femur strength was calculated for models representing a single-limb stance load (SL) and a fall load (FL) onto the posterolateral aspect of the greater trochanter. Volumetric bone mineral density (vBMD) and bone mineral content (BMC) were calculated via vQCT for trabecular (Tr), cortical (Co) and integral (Tr + Co) compartments of the proximal femur. Significance was determined by paired t-test. All patients lost proximal femur strength for both SL and FL conditions (-5%,-10% p<0.05). vBMD was reduced in both the Tr and integral (-17%,-6% p<0.01), but not the Co, compartments. BMC was reduced for all regions: Tr -24%, Co -14% and integral -16% (p <0.02). Co BMC decline is accompanied by a loss of Co, and integral volume (-14%,-10% p<0.05) indicating periosteal resorption and a thinning of the cortex. Linear regression analysis shows a greater loss of Tr BMC with decreasing age (p=0.03), but there was no correlation of age with BMD or strength changes. RT caused rapid decline of bone strength, density and mineral content in the proximal femur. Only an early activation of osteoclasts can account for this rate of loss (BMC decline >2%/wk). For context, the bone loss from 6-wks of RT is roughly equivalent to 3 years of bone loss in women due to menopause. Future studies will examine later time points to determine the degree of recovery. As more data are available, prophylactic treatment of radiation-induced bone loss with antiresorptives should be considered.

Men and Women with Lung Cancer

The large degree of bone loss demonstrated in women with gynecological tumors receiving radiation therapy (RT) led to the development of a second, retrospective clinical trial to determine if this degree of loss occurred in patients receiving radiation therapy for other types of cancer and if men also experienced a loss of bone. vBMD loss was calculated 6-months after radiation therapy. Methods: A retrospective analysis of 25 lung cancer patients was performed. Change in volumetric bone mineral density (vBMD) was examined using pre- and 6 months post-RT thoracic CT scans. Ten women (mean age of 61 years) and 15 men (72 years) were studied. Patients typically received a total RT dose of ~66 Gy in 2.0 Gy/day fractions to the tumor. The trabecular bone of 6 vertebral bodies was contoured; the average CT density was calculated for pre- and post-RT contours. Results: The average loss of thoracic vertebra vBMD for each patient was -21% (p<0.001), with no difference in the degree of loss for women (-21%, p<0.001) and men (-21%, p<0.001). The degree of loss of vBMD (-21%) at the thoracic vertebra in both men and women receiving RT for lung cancer 6-months after RT is very similar to the degree of vBMD (-17%) and BMC loss (-24%) experienced by women with gynecological tumors only 6-weeks after therapy.

• B3, MO5, N14, Acute7: The following therapies were tested: risedronate, pamidronate, zoledronate, alpha-lipoic acid, n-acetal cystine, Enbrel and Kineret. The FDA approved bisphosphonates risedronate, pamidronate and zoledronate where demonstrated to efficaciously prevent radiation-induced bone loss. Neither of the antioxidant compounds alpha-lipoic acid nor n-acetal cystine (NAC) reduced bone loss from radiation exposure.

• B11: Radiation causes an acute bone loss from osteoclast activation that persists due to long-term suppression of bone formation. Its effects are additive to the effects of skeletal disuse.

• Acute1: This osteoclast-mediated acute response is not mitigated by low-dose-rate exposure to protons. Acute, osteoclast-mediated bone loss from 50 cGy proton exposure is approximately 50% that of a 2 Gy exposure.

• Acute2: We have completed a clinical trial in women with gynecological tumors showing acute bone loss in 6-weeks after the first radiation therapy fraction. Bone loss declined in the lumbar vertebra as the distance from the target volume increased: bone loss was less with lower dose exposure. Studying cancer patients, particularly at the periphery of radiation exposure, may help predict bone loss in astronauts; for example low dose and dose rate exposure from an SPE.

• Acute3, Degen 7: Mice that were exposed to a 1 Gy dose of protons and 28-days of hindlimb suspension experienced damage that was additive. Irradiation caused a 15% decline in trabecular bone mass in normally loaded mice and a 17% decline in skeletally unloaded mice. A long-term suppression of bone formation inhibits recovery of bone mass.

• Degen1: This project initiated the development of animal models to study both the acute and degenerative effects of space radiation on bone tissue. Much more study is required to appropriately understand the effects of ionizing radiation skeletal tissue.

• Degen2: Radiation causes an acute bone loss from osteoclast activation that persists due to long-term suppression of bone formation. Its effects are additive to the effects of skeletal disuse. The acute response to 50 cGy dose of protons or heavy ions is the activation of osteoclastic bone resorption. Serum markers for bone resorption increase 24 hours after exposure and osteoclast numbers and eroded surface are greater at 3-days. Osteoclast activation is temporary, reducing to baseline levels approximately 10-days after irradiation. We identified that this bone loss is preceded by death of bone marrow cells, followed by greater expression then levels of inflammatory cytokines IL-1, IL-6 and TNFalpha.

• Degen3: There is virtually no latency period in the activation of osteoclastic bone resorption by exposure to doses of protons and heavy ions of doses as low as 50 cGy (and potentially lower).

The impact of radiation on bone quality and fracture healing in reduced gravity is unknown, and must be studied to understand effects of space radiation on bone health. The long-term objective of the proposed research is the development of countermeasures to prevent bone loss during missions and thus reduce fracture risk.

To define the risks associated with space radiation-induced bone loss, the proposed aims will examine effects of modeled space radiation using scenarios applicable for Lunar Outpost missions:

Specific Aim 1: Examine the combined effects of a modeled solar particle event and unloading on bone, and subsequent recovery during reloading. Hypothesis: Proton radiation with unloading will induce a more severe bone loss than unloading alone.

Specific Aim 2: Examine the cellular and molecular mechanisms for initiating bone loss following exposure to several types of modeled space radiation, including acute proton exposure, low-dose-rate proton exposure, and mixed radiation types (proton and HZE). Understanding underlying molecular causes is critical to developing countermeasures for radiation-induced bone loss. Hypothesis: The initiating mechanism of bone loss is initiated by osteoclast activation caused by a radiation-induced inflammatory response.

Specific Aim 3: Test the efficacy of three countermeasures for bone loss caused by proton exposure: 1) the bisphosphonate risedronate; 2) the RANKL blocking protein osteoprotegerin, and 3) an antioxidant agent, alpha-lipoic acid. Hypothesis: Potent inhibitors of bone resorption, both zoledronate and osteoprotegerin will prevent the bone loss caused by radiation. Antioxidants will address multiple radiation-induced problems; alpha-lipoic acid decreases osteoclast differentiation and activity.

Study Summary:

Postmenopausal women receiving radiation therapy (RT) for pelvic tumors have a 65-200% increased risk of hip fracture compared to women receiving non-RT cancer treatment. It is generally accepted that RT damages local osteoblasts and vasculature resulting in a low turnover, gradual decline in bone mass. However, it has recently been observed in rodent models that ionizing radiation activates osteoclasts. To test the hypothesis that early osteoclastic resorption may cause a rapid loss of bone, changes in proximal femur strength, bone density, and mineral content were examined in women receiving RT for gynecological tumors.

Eight women (age 36-71 years) with cervical (n=6), vaginal, or uterine cancer provided informed consent. CT scans were performed pre-RT and on the last day of RT (6 weeks later). Patients received 50.4 Gy over the course of 28 days. Total dose to the proximal femur was ~25.0 Gy. CT scans were used for finite element strength and volumetric quantitative CT (vQCT) analyses. Proximal femur strength was calculated for models representing a single-limb stance load (SL) and a fall load (FL) onto the posterolateral aspect of the greater trochanter. Volumetric bone mineral density (vBMD) and bone mineral content (BMC) were calculated via vQCT for trabecular (Tr), cortical (Co) and integral (Tr + Co) compartments of the proximal femur. Significance was determined by paired t-test. All patients lost proximal femur strength for both SL and FL conditions (-5%,-10% p<0.05). vBMD was reduced in both the Tr and integral (-17%,-6% p<0.01), but not the Co, compartments. BMC was reduced for all regions: Tr -24%, Co -14% and integral -16% (p <0.02). Co BMC decline is accompanied by a loss of Co, and integral volume (-14%,-10% p<0.05) indicating periosteal resorption and a thinning of the cortex. Linear regression analysis shows a greater loss of Tr BMC with decreasing age (p=0.03), but there was no correlation of age with BMD or strength changes.

RT caused rapid decline of bone strength, density and mineral content in the proximal femur. Only an early activation of osteoclasts can account for this rate of loss (BMC decline >2%/wk). For context, the bone loss from 6-wks of RT is roughly equivalent to 3 years of bone loss in women due to menopause. Future studies will examine later time points to determine the degree of recovery. As more data are available, prophylactic treatment of radiation-induced bone loss with antiresorptives should be considered.

Antioxidant Countermeasures: Oxidative stress after irradiation has been speculated to mediate radiation-induced bone loss, as some evidence points to the efficacy of a-lipoic acid. Antioxidants, including alipoic acid, have been effective in preventing bone loss in models of reduced-estrogen induced osteoporosis. We have tested the efficacy of high doses of two antioxidants in terms of preventing bone loss after irradiation (a-lipoic acid and ascorbic acid): Neither suppressed any functional bone loss.

Predisposition of Heredity: We have also performed an experiment comparing the response of 4 strains of mice to radiation-induced bone loss, with the goal of determining both improving our spaceflight model and determining if there is a hereditary/genotype predisposition to being radiation sensitive or in sensitive. We want to explore transitioning our model towards using a higher bone density strain of mouse. When combining radiation with another skeletal challenge, such as disuse, the low bone density C57BL/6 (B6) mice have very little bone remaining. BALBc mice do respond the same as B6 mice; the rate and degree of bone loss from irradiation are the same. We are in the process of analyzing data from two other strains: DBA mice are considered to be radiation resistant and C3H mice are less susceptible to disuse osteoporosis. We are potentially observing some preservation of bone moss in DBA and C3H mice, however more analysis is necessary.

Rotating wall vessel changes in osteoclast gene expression profile: We collaborated with Dr. Reddy at the Medical University of South Carolina on a project examining changes in gene expression profile in osteoclast cultures using the rotating wall vessel to model microgravity (Sambandam et al., J Cell Biochem 2010).

The impact of radiation on bone quality and fracture healing in reduced gravity is unknown, and must be studied to understand effects of space radiation on bone health. The long-term objective of the proposed research is the development of countermeasures to prevent bone loss during missions and thus reduce fracture risk.

To define the risks associated with space radiation-induced bone loss, the proposed aims will examine effects of modeled space radiation using scenarios applicable for Lunar Outpost missions:

Specific Aim 1: Examine the combined effects of a modeled solar particle event and unloading on bone, and subsequent recovery during reloading. Hypothesis: Proton radiation with unloading will induce a more severe bone loss than unloading alone.

Specific Aim 2: Examine the cellular and molecular mechanisms for initiating bone loss following exposure to several types of modeled space radiation, including acute proton exposure, low-dose-rate proton exposure, and mixed radiation types (proton and HZE). Understanding underlying molecular causes is critical to developing countermeasures for radiation-induced bone loss. Hypothesis: The initiating mechanism of bone loss is initiated by osteoclast activation caused by a radiation-induced inflammatory response.

Specific Aim 3: Test the efficacy of three countermeasures for bone loss caused by proton exposure: 1) the bisphosphonate risedronate; 2) the RANKL blocking protein osteoprotegerin, and 3) an antioxidant agent, alpha-lipoic acid. Hypothesis: Potent inhibitors of bone resorption, both zoledronate and osteoprotegerin will prevent the bone loss caused by radiation. Antioxidants will address multiple radiation-induced problems; alpha-lipoic acid decreases osteoclast differentiation and activity.

This loss of bone mass following radiotherapy has been hypothesized to occur as a result of damage to bone-forming osteoblasts and the bone vasculature itself. While previous studies typically observed atrophy as a late effect, loss of volumetric bone mineral content has been reported in cervical cancer patients five weeks post treatment and described as a low-turnover type of osteoporosis. An inhibition of osteoblasts and osteoblast progenitors from radiation exposure has been further described both in vitro and in vivo, and we have reported a long-term reduction of bone mass in irradiated mice. Despite evidence that bone loss can occur soon after irradiation, a putative increase in osteoclast activity has received little attention as a potential contributor to radiation-induced osteoporosis.

To date, pharmacological interventions to prevent bone loss caused by radiation therapy have not been employed. In fact, no animal model currently exists to identify causal mechanisms and to properly develop such therapies. Our work through the last year has identified a rapid activation of osteoclasts after radiation exposure that is prevented by treatment with risedronate.

The following aims supported by NSBRI have direct clinical relevance.

Specific Aim 2a: Radiation exposure results in an early activation of osteoclasts leading to rapid bone loss (Willey et al., Radiation Res, 2008).

Specific Aim 3: Risedronate prevents radiation-induced bone loss (Willey et al., Bone, in press).

Clinical Trials: Radiation therapy (RT) to treat cervical and lung cancers cause a decline in proximal femur strength and thoracic lumbar vertebra bone mineral density, respectively.

Procter and Gamble Pharmaceuticals awarded us an unrestricted grant to study bone loss in cervical cancer patients (PI Dr. Joyce Keyak University of California at Irvine). A finite element model of the proximal femur was developed from CT imaging taken on the last day of RT (6 weeks after starting RT) and compared to the pretreatment planning CT scan. FEA tests the strength of the femur when single-leg stance and falling loads are applied. Of the four patients studied to date, there is a significant decline in single leg stance load (p = 0.04) and a trend towards a decline in fall load (p = 0.11).

Dr. Larry Marks (Chair, Dept. of Radiation Oncology, UNC Chapel Hill) has thoracic CT scans from approximately 100 lung cancer patients before and six months after RT. The goal of the original prospective study was to examine the development of lung fibrosis. The body of 6 thoracic vertebrae from 15 of these patients have been analyzed. On average, the patients lost -21% vBMD in these six vertebrae. For women, there is a significant correlation with the degree of vBMD decline and age, with greater loss occurring with increasing age (p = 0.03). A similar trend was observed among men (p = 0.07).

The impact of radiation on bone quality in reduced gravity is unknown, and must be studied to understand effects of space radiation on bone health. The long-term objective of the proposed research is the development of countermeasures to prevent bone loss during missions and thus reduce fracture risk.

The second year of this project was very productive, with progress made on all three Aims.

Specific Aim 1: Bone loss from exposure to a 1 Gy whole-body dose of protons and skeletal loading is additive. Manuscript in preparation.

Specific Aim 2b: Exposure to a dose of <50 cGy radiation of mixed type results in both cortical and trabecular bone loss. Paper published July 2008.

Specific Aim 2c: Bone loss from exposure to ionizing radiation is a local response and is preceded by greater expression on proinflammatory cytokines.

Specific Aim 3a: Risedronate prevents radiation-induced bone loss. Publication in press for the journal Bone.

Specific Aim 3b: Ongoing tests of IL-1 receptor antagonist, TNF binding protein and ascorbic acid (vitamin C) as countermeasures to block local 1) radiation mediated inflammation, and 2) reactive oxygen species created by ionizing radiation and resulting cell death.

The impact of radiation on bone quality and fracture healing in reduced gravity is unknown, and must be studied to understand effects of space radiation on bone health. The long-term objective of the proposed research is the development of countermeasures to prevent bone loss during missions and thus reduce fracture risk.

To define the risks associated with space radiation-induced bone loss, the proposed aims will examine effects of modeled space radiation using scenarios applicable for Lunar Outpost missions:

Specific Aim 1: Examine the combined effects of a modeled solar particle event and unloading on bone, and subsequent recovery during reloading. Hypothesis: Proton radiation with unloading will induce a more severe bone loss than unloading alone.

Specific Aim 2: Examine the cellular and molecular mechanisms for initiating bone loss following exposure to several types of modeled space radiation, including acute proton exposure, low-dose-rate proton exposure, and mixed radiation types (proton and HZE). Understanding underlying molecular causes is critical to developing countermeasures for radiation-induced bone loss. Hypothesis: The initiating mechanism of bone loss is initiated by osteoclast activation caused by a radiation-induced inflammatory response.

Specific Aim 3: Test the efficacy of three countermeasures for bone loss caused by proton exposure: 1) the bisphosphonate risedronate; 2) the RANKL blocking protein osteoprotegerin, and 3) an antioxidant agent, alpha-lipoic acid. Hypothesis: Potent inhibitors of bone resorption, both zoledronate and osteoprotegerin will prevent the bone loss caused by radiation. Antioxidants will address multiple radiation-induced problems; alpha-lipoic acid decreases osteoclast differentiation and activity.

This loss of bone mass following radiotherapy has been hypothesized to occur as a result of damage to bone-forming osteoblasts and the bone vasculature itself. While previous studies typically observed atrophy as a late effect, loss of volumetric bone mineral content has been reported in cervical cancer patients five weeks post treatment and described as a low-turnover type of osteoporosis. An inhibition of osteoblasts and osteoblast progenitors from radiation exposure has been further described both in vitro and in vivo, and we have reported a long-term reduction of bone mass in irradiated mice. Despite evidence that bone loss can occur soon after irradiation, a putative increase in osteoclast activity has received little attention as a potential contributor to radiation-induced osteoporosis.

The effect of radiation on the number and activity of osteoclasts is varied in published reports, from observed decreases in osteoclast numbers, to stable numbers, to a qualitative description of an increase in the osteoclast population. A better understanding of the effects of radiation on osteoclasts needs to be addressed in order to reduce or prevent the subsequent bone atrophy and fracture risk.

To date, pharmacological interventions to prevent bone loss caused by radiation therapy have not been employed. In fact, no animal model currently exists to identify causal mechanisms and to properly develop such therapies. Our work through the last year has identified a rapid activation of osteoclasts after radiation exposure that is prevented by treatment with risedronate.

The following aims supported by NSBRI have direct clinical relevance.

Specific Aim 2a: Radiation exposure results in an early activation of osteoclasts leading to rapid bone loss.

Female C57BL/6 mice were exposed to a 2 Gy whole-body dose of x-rays, or not irradiated. A serum marker for osteoclast activity (TRAP5b) was significantly increased 1 and 3 days after exposure (50% and 14%, respectively). Histological examination of bones collect from mice 3 days after exposure revealed a significant increase osteoclast number and surface. There were no changes in serum osteocalcin (a marker for osteoblast activity) or osteoblast surface.

Specific Aim 3: Risedronate prevents radiation-induced bone loss.

Twenty-week-old female C57BL/6 mice were exposed to a 2 Gy whole-body dose of x-rays, or not irradiated. Groups (3 groups) included non-irradiated controls administered a placebo injection, irradiated mice administered a placebo injection, and irradiated mice treated with risedronate (30 ug/kg every other day. Within these three groups mice were humanely euthanized at 1, 2, and 3 weeks after exposure. There was significant bone loss of trabecular bone at three skeletal sites (proximal tibia, distal femur, and 5th lumbar spine) at 1 weeks after exposure. Risedronate prevented this bone loss entirely.

The impact of radiation on bone quality in reduced gravity is unknown, and must be studied to understand effects of space radiation on bone health. The long-term objective of the proposed research is the development of countermeasures to prevent bone loss during missions and thus reduce fracture risk.

The first year of this project was very productive, with progress made on all three Aims.

Aim 1: Bone loss from exposure to a 1 Gy whole-body dose of protons and skeletal loading is additive.

Mice were exposed to a 1 Gy dose or protons and hindlimb suspended, a model for skeletal unloading, for a duration of 4-weeks (mimicking long-term spaceflight). Despite a profound bone loss from unloading, mice exposed to both unloading and protons had significantly more bone loss than unloaded mice not exposed to radiation.

Aim 2a: Radiation exposure results in an early activation of osteoclasts leading to rapid bone loss.

Mice were exposed to a 2 Gy dose of x-rays, or not irradiated. A serum marker for osteoclast activity (TRAP5b) was significantly increased 1 and 3 days after exposure. Histological examination of bones collect from mice 3 days after exposure revealed a significant increase osteoclast number and surface. There were no changes in serum osteocalcin (a marker for bone formation) or osteoblast surface.

Aim 2b: Exposure to a dose of <50 cGy radiation of mixed type results in both cortical and trabecular bone loss.

Bones were collected from a study in which mouse brains were exposed to collimated iron radiation. These conditions resulted in exposure to a complex mixtures of charged particles transmitted by the collimator. Measured radiation doses of uncollimated secondary particles were 0.47 Gy at the proximal humerus. The proximal humerus of irradiated mice had lower trabecular bone volume fraction, lower trabecular thickness, greater cortical porosity, and lower polar moment of inertia.

Aim 3: Risedronate prevents radiation-induced bone loss.

Mice were exposed to a 2 Gy dose of x-rays, or not irradiated. Groups (3) included non-irradiated controls administered a placebo injection, irradiated mice administered a placebo injection, and irradiated mice treated with risedronate (30 ug/kg every other day). Within these three groups mice were humanely euthanized at 1, 2, and 3 weeks after exposure. There was significant bone loss of trabecular bone at three skeletal sites (proximal tibia, distal femur, and 5th lumbar spine) 1 week after exposure. Risedronate prevented this bone loss entirely.

The impact of radiation on bone quality and fracture healing in reduced gravity is unknown and must be studied to understand effects of space radiation on bone health. The long-term objective of this project is the development of countermeasures to prevent bone loss during missions and thus reduce fracture risk.

To define the risks associated with space radiation-induced bone loss, the following aims will examine effects of modeled space radiation using scenarios applicable for Lunar Outpost missions:

1. Examine combined effects of a modeled solar particle event and unloading on bone, and subsequent recovery during reloading. Hypothesis: Proton radiation with unloading will induce a more severe bone loss than unloading alone.

2. Examine initiation of osteoclast activation and subsequent bone loss following exposure to several types of modeled space radiation, including acute proton exposure, low-dose-rate proton exposure and mixed radiation types (proton and HZE). Understanding underlying molecular causes is critical to developing countermeasures for radiation-induced bone loss. Hypothesis: The initiating mechanism of bone loss is radiation-induced inflammation, increasing RANKL production due to damaged marrow, causing early osteoclast activation.

3. Test the efficacy of three countermeasures for bone loss caused by proton exposure: a) The bisphosphonate zoledronate; b) The RANKL blocking protein osteoprotegerin; and c) An antioxidant agent, -lipoic acid. Hypothesis: Potent inhibitors of bone resorption, both zoledronate and osteoprotegerin will prevent the bone loss caused by radiation. Antioxidants will address multiple radiation-induced problems; -lipoic acid decreases osteoclast differentiation and activity.