NOTE: Extended to 1/14/2021 per NSSC information (Ed., 2/11/2020)

NOTE: Extended to 1/14/2020 per NSSC information (Ed., 3/12/19)

The groups of mice were as follows: 1: Control; 2: gamma rays: 1.5 Gy (acute, single fraction, whole body); 3: gamma rays: 3 Gy (acute, single fraction, whole body); 4: 1 GeV protons: 0.2 Gy (0.0035 Gy/min, whole body); 5: 1 GeV/u Ca: 0.2 Gy (in 3 fractions; 1 acute fraction/day; whole body); 6: 1 GeV/u Ca: 0.3 Gy (in 3 fractions; 1 acute fraction/day; whole body); 7: 1 GeV/u Ca: 0.4 Gy (in 3 fractions; 1 acute fraction/day; whole body); 8: 1 GeV/u Ca: 0.4 Gy (in 1 acute fraction; whole body); 9: 1 GeV/u Ca: 0.4 Gy (in 1 acute fraction; head only); 10: 1 GeV/u Si: 0.4 Gy (in 3 fractions; 1 acute fraction/day; whole body); 11: 1 GeV/u Si: 0.4 Gy (in 1 acute single fraction; whole body); 12: 1 GeV/u O: 0.4 Gy (in 3 fractions; 1 acute fraction/day; whole body); 13: 1 GeV/u O: 0.4 Gy (in 1 acute single fraction; whole body); 14: 1 GeV protons followed by 1 GeV/u Ca: whole body exposure to 0.2 Gy of protons delivered 24 h prior to 0.4 Gy of 1 GeV/u Ca ions delivered to the whole body; 15: 1 GeV protons followed by 1 GeV/u Ca: whole body exposure to 0.2 Gy of protons delivered 24 h prior to 0.4 Gy of 1 GeV/u Ca ions targeted to the head only.

Major highlights:

- Effects on modulation of the abundance of circulating hematopoietic cells and bone marrow stem cells. Up to 10-fold increases in circulating neutrophils were detected at two weeks in mice exposed to 20, 30, or 40 cGy of either of the heavy ions delivered in a fractionated manner (p<0.001). The groups also showed an increase in circulating monocytes (p<0.01). The mice exposed to a single bolus of 40 cGy of Ca ions did not show significant increases at 2 weeks; however, by 3 months, increases in neutrophils were detected (p<0.001). These increases in neutrophils and monocytes in circulating blood were associated with decreases in these cell subsets in bone marrow (p<0.05), suggesting mobilization out of this compartment. Common myeloid, as well as granulocyte / macrophage and megakaryocyte-erythroid progenitors were decreased (p<0.1) in bone marrow. Notably, decreases (p<0.01) in short-term hematopoietic stem cells were detected. The alterations observed at 2 weeks were associated with changes in the levels of circulating inflammatory cytokines. Furthermore, histological analyses revealed prominent interstitial lung disease in mice exposed to a single bolus of 40 cGy of heavy ions, characterized with thickened alveolar septa, pulmonary congestion, and endothelial hyperplasia. The mice exposed to the fractionated regimens presented mild lung injury.

The early response of neutrophils and monocytes in mice exposed to the energetic heavy ions returned to a normal range at 6 months after irradiation and remained in this normal range at 13 months. However, at the latter time point, the proportion (%) of circulating plasma cells, but not B cells, were increased (p<0.001) in mice exposed to 30 or 40 cGy of Ca ions delivered in a fractionated manner. The effect seems to depend on the radiation dose and delivery manner. Similar finding occurred in mice exposed to 40 cGy of silicon or oxygen ions, but not in mice exposed to gamma rays or protons. This indicates the phenotypes may be specific to high LET radiations and is suggestive of the development of a plasma cell dyscresia.

At 15-18 months after exposure to HZE particles, an impaired cytotoxic function of natural killer (NK) cells was also observed, which suggests long-term overall suppression and decreased function of these cells. There was no change in the number of red blood cells or the concentration of hemoglobin across all irradiated groups; however, there was an increase in the hematocrit percentages for animals irradiated with 40 cGy of 1 GeV/u Ca ions delivered as a single bolus to the whole body (p = 0.03). The blood of the irradiated mice was also analyzed for changes in triglycerides, cholesterol content, as well as concentration of several biomarkers.

- Long-term effects on morphological changes and radio-densitometric characteristics of bone. Consistent with an effect on bone marrow stem cells, computed tomography (micro-CT) revealed morphological change in trabecular bone at distal femur metaphysis at two weeks after exposure to the HZE ions. Trabecular bone in these mice demonstrated more rod shaped, instead of mostly plate-shaped in the control mice.

In addition, CT and micro-CT performed at 15-18 months after irradiation revealed persistent effects on bone density. Intra-group co-plots showed that the normalized bone density distributions for the mice in the 0 cGy (control) group exhibit a high degree of overlap, displaying limited variation throughout the distributions. In contrast, intra-group co-plots of the data for the heavy ion and gamma-irradiated mice revealed marked distribution heterogeneity within these test groups. Micro-CT examinations were consistent with aberrant mineralization in HZE-irradiated mice.

- Ultrasound echocardiography (effect on heart physiology). Relative to gamma-irradiated mice, there were significant changes (p<0.05) in cardiac output of mice irradiated with the higher doses of HZE particles (30, 40 cGy). A change in the left ventricular mass index was also observed (p<0.02).

- Oxidative stress and inflammation in various organs. Oxidative stress in various tissues was detected by oxyblot assay. Analyses of fibrosis, at 15-18 months after exposure to HZE particles, in lung, liver, kidney, and heart, revealed a significant persistence of inflammation (p<0.01 to p<0.001). The fibrosis was associated with increases in the levels of proteins harboring 3-nitrotyrosine and lipid peroxidation adducts (4-hydroxynonenal).

Analyses of mitochondrial stress in heart tissue was also performed. The results showed significant stress at early times after irradiation. The induced stress was significantly attenuated by 15-18 months after exposure.

At 15 months, antioxidant enzyme analyses in kidney (superoxide dismutases, catalase, glutathione-S-transferase) revealed a trend of increased activity, being significant for catalase (p<0.05). The increases, however, were not sufficient to eliminate the induced oxidative stress.

H&E (Hematoxylin and Eosin) staining showed structural changes in kidney, liver, and lung. Analyses of structural changes in brain are ongoing. Also, analyses have been performed to investigate microgliosis and astrogliosis in all the treated groups. These analyses are near completion.

Analyses of signaling pathways associated with DNA damage, oxidative stress, and inflammation were pursued in various organs.

- Pathological changes in reproductive tissues. At 15-18 months after irradiation, notable increases (p<0.0001) were observed in the size of the seminal vesicle during gross examination of mice exposed to 40 cGy of 1 GeV/u of Ca ions. In contrast, the size of the seminal vesicles in mice exposed to 150 cGy of gamma rays were similar to those observed in control mice.

- Global changes in protein expression level and S-nitrosylation. To understand the effects of whole or partial body exposure to space radiation on signal transduction mediated by S-nitrosylation, we performed mass spectrometry analyses on the cerebellum of sham-irradiated mice or cerebellum or mice that received whole body or head only exposures to 40 cGy from 1 GeV/u of calcium ions delivered acutely as a single bolus. The mice used in experiments were sacrificed 14 days after irradiation. Promptly after harvesting the cerebellums, the proteins were extracted and processed for analyses. The resulting expression patterns of proteins (general proteome) and S-nitrosylated protein (S-nitroso proteome) were analyzed by bioinformatics classification data mining tools and pathway analysis tools. The results from the protein expression studies indicate that there was no change in protein expression level of neuronal Nitric Oxide Synthase (nNOS) before and after irradiation with 40 cGy of calcium ions targeted either to the whole body or head only. The experiment did not detect the expression of the other NOSs (i.e., inducible and endothelial Nitric Oxide Synthases). Thus, the changes observed in nitrosylated protein cannot be attributed to the change in the level of NOSs. This result does not exclude a possibility where the irradiation may have affected the activity level of NOSs enzymes. However, the changes in nitrosylation observed may be due to alternative mechanisms such as transnitrosylation.

- Histological determinations of cancer incidence. Analyses of cancer incidence is in progress with the assistance of a biostatistician. Histological examinations have so far shown that relative to control, cancer incidence in middle-aged mice (9-10 months) is significantly lower than reported by others in mice exposed to HZE particles at younger age.

NOTE: Extended to 1/14/2021 per NSSC information (Ed., 2/11/2020)

NOTE: Extended to 1/14/2020 per NSSC information (Ed., 3/12/19)

In previous reports, we described our findings on the effects of space radiation on the abundance of hematopoietic cells and the bone marrow microenvironment. We also reported on tissue injury in liver and lung, and on the effects on bone density and cardiac function. During the past year, we expanded our investigation of tissue injury and analyzed oxidative stress in kidney. We also expanded our analyses of cardiac function and bone.

Kidney: Using in situ immunodetection and immunoblotting techniques, we found that the kidneys of mice exposed to energetic calcium ions 15 months earlier harbored increased levels of proteins with lipid peroxide adducts, which is suggestive of persistent inflammatory responses. These increases were highly significant when the mice were exposed to 40 cGy of Ca ions delivered either as a single bolus or in a fractionated manner. At 2 weeks after irradiation, no significant changes in lipid peroxidation were detected.

Heart: In the hearts of mice exposed to either energetic calcium ions or gamma rays, similarity in blood ejection fraction and pulmonary artery/aorta diameter ratio suggests that the left and right side’s heart functions are equivalent within groups without any sign of heart failure. However, the left ventricle (LV) dimension, volume, and output parameter showed a tendency in LV size reduction, especially in the Calcium 40 cGy (fractionated) group. Ongoing analyses are being extended to examine fibrosis in both the right and left ventricles and in the tricuspid valve, whether a possible relation with right atrium and liver enlargement exists. The mice exposed to fractionated dose of 40 cGy from energetic oxygen or silicon ions did not show significant difference within the groups. The only significant difference is in the change in fractional area where the control group exhibited higher fractional area than the silicon and gamma-irradiated groups.

Bone: Ongoing analyses of computed tomography scans of the skeletons of the mice exposed to the various types of space radiation are using artificial intelligence to gauge qualitative and quantitative changes at 15 months after irradiation.

Effect on the bone marrow niche assessed by computed tomography (micro-CT): In last year’s progress report, we described temporal changes in the relative abundance of circulating immune cells, and of their precursors in bone marrow of control mice, and mice exposed to either energetic proton or energetic heavy particles (HZE particles). The results were compared with those obtained in mice exposed in parallel to gamma rays from a cesium-137 source (acute whole-body exposure to a dose of 150 or 300 cGy). Increases up to 10-fold in circulating immune cells (neutrophils and monocytes) were detected in mice following two weeks of exposure to 20, 30, or 40 cGy of any of the heavy ions used in the study (1 GeV/nucleon calcium, silicon, oxygen). These increases were observed whether the radiation exposure was delivered in a single bolus or in a fractionated manner. Furthermore, the increases in neutrophils and monocytes in circulating blood were associated with decreased abundance of these cell subsets in bone marrow. The common myeloid progenitors, as well as the granulocyte/macrophage, and megakaryocyte-erythroid progenitors were also significantly decreased in the bone marrow, together with significant decreases in short-term hematopoietic stem cells.

During this reporting period, we have investigated the bone marrow niche by micro-CT (high resolution computerized tomography). Prominent morphological changes in trabecular bone at the distal femur metaphysis were detected at two weeks after whole body exposure of the mice to 40 cGy of 1 GeV/n oxygen ions delivered as single acute bolus. A 3-dimensional view revealed that the trabecular bone has changed from plate-shape to rod-shape in the irradiated mice supporting an effect on the bone marrow niche where decreases in the progenitor cells were detected. Analysis of the number of nodes and struts in the trabecular bone in femur is ongoing.

Plasma cells: We have expanded our analyses on the abundance of circulating hematopoietic cells. Whereas, at early times (2 weeks – 3 months) increases in neutrophils and monocytes were significant in peripheral blood of irradiated mice, the abundance of these cells at 14 months was not different from that in control mice. However, significant increases were detected in plasma cells, which is suggestive of plasma cell proliferative diseases.

Natural killer (NK) cells: Decreased NK activity of 30% and 25% when compared to 0 Gy control, respectively, were found in spleen of mice exposed to 150 cGy gamma rays (acute) or 40 cGy from energetic oxygen ions delivered in 3 fractions as measured by the chromium release assay. The percentage of splenocytes that are NK by flow cytometry in the irradiated samples were not statistically different from those in control. Together, the results suggest that both the sparsely ionizing gamma rays (acute) and densely ionizing 40 cGy oxygen ions (3 fractions) exposures can lead to long term decreases in NK activity.

2. Tissue injury

Consistent with events leading to a persistence of inflammatory responses, ongoing investigation of late injury in the various harvested mouse organs is revealing significant fibrosis in lung, liver, spleen, and heart at 15 months after whole body exposure to HZE particles. This late expression of fibrosis is associated with nitrosative stress (a process associated with inflammation) as revealed by up-regulation of 3-nitrotyrosine modified proteins. Analyses of other biomarkers of inflammation/oxidative stress were also performed on lung tissue at 2 weeks and 15 months after whole body irradiation, including TGFbeta, pSMAD2, Il-6, CYP2b10, and TNFalpha. In particular, Il-6, which participates in the inflammatory process was increased in liver at 15 months after HZE-particle irradiation.

We have pursued in situ studies of telomeric DNA double strand breaks (DNA breaks at end of chromosomes, an indicator of ageing), and analyzed inflammatory responses (reactive microgliosis and astrogliosis) in the hippocampus and striatum in brain sections of the HZE-particle-irradiated mice. We found that at 6 hours after the end of the irradiation protocol, ~2% of cells displayed few and discrete gammaH2AX foci (a marker of DNA damage) that were completely resolved by 2 weeks after irradiation. In addition, only 31% of gammaH2AX foci co-localized with telomeres, demonstrating that most DNA damage occurs in non-telomeric DNA. Surprisingly, in contrast to control animals, gammaH2AX positive cells increased with advancing age, irradiated animals did not, suggesting the possibility of radiation stimulated mechanisms that protect against age-related degenerative processes in the brain. We did not detect significant evidence of microgliosis or astrogliosis in the striatum of mice exposed to mean absorbed dose of 40 cGy of 1 GeV/nucleon calcium, silicon, or oxygen particles delivered in a fractionated manner to the whole body.

Cardiovascular: We have completed evaluation of cardiac function by echocardiography in control mice, and in heavy ion-irradiated mice (1 GeV/nucleon calcium, silicon, or oxygen particles, 20 cGy or 40 cGy delivered in a fractionated manner) or gamma-irradiated mice (150 cGy, single acute bolus). The measurements were acquired at 15 months after the radiation exposure in 8-12 mice/group. Whereas mice exposed to energetic calcium ions showed statistically significant change in left ventricular mass following exposure to fractionated doses of 20 or 40 cGy, the mice exposed to fractionated dose of 40 cGy from energetic oxygen or silicon ions did not show significant changes for this endpoint. The mice exposed to 150 cGy of gamma rays showed a significant change in the left ventricular mass, but the change was not as large as in the calcium-irradiated mice.

Classical bone morphogenetic proteins (BMPs) were originally named for their osteo-inductive properties. We have pursued analyses of BMP2 signaling in the aorta of control and calcium-irradiated mice. The results indicated decrease in BMP2 signaling inferred through phosphorylation of BMP2 downstream effectors SMAD1/5/9 proteins. This decrease in BMP2 signaling was associated as expected with an increase in TGFbeta (although, the TGFbeta increase did not reach statistical significance). These experiments are being expanded to investigate the effects of energetic silicon and oxygen ions.

Bone: Exposure to densely ionizing particles may result in modulation of bone formation, resorption, and mineralization. To characterize the effects of energetic heavy (HZE) particles on bone dynamics in living mammals, a radio-densitometric evaluation of bone was conducted 15 months following exposure of the mice to fractionated doses of energetic calcium, silicon, or oxygen ions (isovelocity 1 GeV/nucleon). Relative bone density of mice from the various treatment groups was evaluated by comparing the frequency distribution of the image voxel intensities within a range of radio-density known to encompass low and high-density bone. Six to eight mice per treatment group were imaged. Intra-group comparisons showed that the bone density distributions for the mice in the 0 cGy (control) group exhibit a high degree of overlap, displaying limited variation throughout the distributions. In contrast, the data for the irradiated mice revealed marked distribution heterogeneity within these test groups. Further micro-CT imaging of Ca-irradiated mice and their respective control showed abnormalities detected in femurs of the irradiated mice, which suggests aberrant mineralization. These scanning results are being analyzed to gain quantitative information.

In summary, our ongoing studies in mice revealed that exposure to energetic heavy particles when the mice are 10 month-old leads to both short- and long-term biological changes that can have a significant impact on health. Exposure to moderate mean absorbed doses of space radiation : i) Induces persistent oxidative stress and inflammatory effects; ii) perturbs abundance of hematopoietic cells; iii) fractionation of the dose from energetic heavy particles results in greater level of tissue damage than induced by a single bolus; iv) exposure to energetic heavy particles induces non-targeted effects; v) in contrast to our in vitro results, low dose of 1 GeV protons delivered at low dose rate does not appear to induce significant adaptive responses as measured by the endpoints analyzed so far in this study; vi) analysis of cancer induction is in progress: paraffin-embedding and sectioning of tissues is ongoing. The slides will be assessed by expert pathologists; vii) analyses of oxidative stress and inflammatory responses in non-targeted organs following head only irradiation are being initiated, together with analytical studies in mice exposed to protons prior to exposure to energetic calcium ions.

1- To assess chronic oxidative stresses, inflammatory responses, and degenerative conditions in organs that differ in their radiation sensitivity. 2- To evaluate the relative biological effectiveness of the space radiations compared to acute cesium-137 gamma rays in enhancing the rate of cancer incidence 3- To measure oxidative changes and cancer incidence in non-irradiated organs after exposure of the head to a moderate dose (0.4 Gy) of high atomic number (Z) and high energy (E) HZE particles, and to compare the observed changes with those in the targeted organ (brain). 4- To examine the protective effect of whole-body pre-exposure to a conditioning dose of 0.2 Gy of 1 GeV protons delivered at low dose-rate prior to head exposure to acute dose of 0.4 Gy of HZE particles.

At 6 h, 2 weeks, and 3, 6, and 10 months after irradiation, 5 mice or more from each of the groups described below were anesthetized and peripheral blood was collected. The mice were then perfused with saline and different organs (heart, liver, lung, kidney, bone marrow, brain, reproductive organs, eyes, femurs) were harvested for cellular, biochemical, molecular, and histological analyses, and for archiving in NASA’s Space Radiation Tissue Sharing Forum. At 15 months the remainder of the mice (60-150) in each group were sacrificed. At this latter time point, a set of live mice (n=6-12) was scanned by computed tomography and ultrasound-echocardiography.

The groups of mice were as follows: 1: Control ; 2: Gamma rays: 1.5 Gy (acute single bolus, whole body) ; 3: Gamma rays: 3 Gy (acute single bolus, whole body) ; 4: 1 GeV protons: 0.2 Gy (0.0035 Gy/min, whole body) ; 5: 1 GeV/u Ca: 0.2 Gy (in 3 fractions; 1 acute fraction/day; whole body) ; 6: 1 GeV/u Ca: 0.3 Gy (in 3 fractions; 1 acute fraction/day; whole body) ; 7: 1 GeV/u Ca: 0.4 Gy (in 3 fractions; 1 acute fraction/day; whole body) ; 8: 1 GeV/u Ca: 0.4 Gy (acute single bolus; whole body) ; 9: 1 GeV/u Ca: 0.4 Gy (acute single bolus; head only) ; 10: 1 GeV/u Si: 0.4 Gy (in 3 fractions; 1 acute fraction/day; whole body) ; 11: 1 GeV/u Si: 0.4 Gy (acute single bolus; whole body) ; 12: 1 GeV/u O: 0.4 Gy (in 3 fractions; 1 acute fraction/day; whole body) ; 13: 1 GeV/u O: 0.4 Gy (acute single bolus; whole body) ; 14: 1 GeV protons followed by 1 GeV/u Ca: whole body exposure to 0.2 Gy of protons delivered 24 h prior to 0.4 Gy of 1 GeV/u Ca ions delivered to the whole body ; 15: 1 GeV protons followed by 1 GeV/u Ca: whole body exposure to 0.2 Gy of protons delivered 24 h prior to 0.4 Gy of 1 GeV/u Ca ions targeted to the head only.

HIGHLIGHTS FROM THE RESULTS:

At 2 weeks, and 3, 6, and 15 months after irradiation, peripheral blood and bone marrow were drawn from at least 5 mice of each of the groups described above to examine alterations in cell subsets using multicolor flow cytometry. Relative percent change in specific cell populations and absolute cell counts were determined. Increases up to 10-fold in circulating neutrophils were detected at two weeks in mice exposed (whole body) to 20, 30, or 40 cGy of either of the heavy ions delivered in a fractionated manner (p<0.001). The groups also showed an increase in circulating monocytes (p<0.01). The mice exposed to a single bolus of 40 cGy of Ca ions did not show significant increases at 2 weeks; however, by 3 months, increases in neutrophils were detected (p<0.001). These increases in neutrophils and monocytes in circulating blood were associated with decreases in these cell subsets in bone marrow (p<0.05), suggesting mobilization out of this compartment. Common myeloid, as well as granulocyte / macrophage and megakaryocyte-erythroid progenitors were decreased (p<0.01) in bone marrow. Notably, decreases (p<0.01) in short-term hematopoietic stem cells were detected. The alterations observed at 2 weeks were associated with changes in the levels of circulating inflammatory cytokines (e.g., TNF-alpha, IL-1 beta, CXCL1). Furthermore, histological analyses revealed prominent interstitial lung disease in mice exposed to a single bolus of 40 cGy of heavy ions, characterized with thickened alveolar septa, pulmonary congestion, and endothelial hyperplasia. The mice exposed to the fractionated regimens presented mild lung injury.

The early response of neutrophils and monocytes in mice exposed to the energetic heavy ions returned to a normal range at 6 months after irradiation, and remained in this normal range at 14 months. However, at the latter time point, the proportion (%) of circulating plasma cells, but not B cells, were increased (p<0.001) in mice exposed to 30 or 40 cGy of Ca ions delivered in a fractionated manner. The effect seems to depend on the radiation dose and delivery manner, as it was not detected in mice exposed to 20 cGy of Ca ions delivered in a fractionated manner, nor in mice exposed to 40 cGy of Ca ions delivered in a single bolus. Similar finding occurred in mice exposed to 40 cGy of Si ions, but not in mice exposed to gamma rays or protons. This indicates the phenotypes may be specific to high LET radiations, and is suggestive of the development of a plasma cell dyscresia. Analyses of the long-term effects of exposure to 1 GeV/u oxygen ions will occur in March 2018.

Relative to control, at 15 months after irradiation of mice with energetic Ca ions, an increase in the hematocrit percentages was detected in animals irradiated with 40 cGy delivered as a single bolus to the whole body (p = 0.03). There were massive decreases in circulating triglycerides in the blood serum after irradiation with 20 or 40 cGy delivered in a fractionated manner. There were also increases in blood glucose. There were no changes in the concentration of high density lipoproteins (HDL) and low density lipoproteins (LDL) in all groups. There was an increase in alanine transaminase (ALP) in blood upon irradiation with a fractionated dose of 20 cGy delivered to the whole body.

Preliminary studies show that exposure to HZE particles leads to oxidative modification of proteins from different organs that persist over time. In addition, alterations in signaling pathways (in particular Bone Morphogenetic Protein (BMP) signaling) was detected in aorta.

Proteomic studies in cerebellums of mice exposed to 40 cGy of 1 GeV/u Ca ions: We determined the global S-nitrosylation patterns (S-nitroso proteome) using `Biotin Switch' assay coupled with mass spectrometry (MS) analyses. The resulting expression patterns of proteins (general proteome) and S-nitrosylated protein (S-nitroso proteome) are being analyzed by bioinformatics classification data mining tools and pathway analysis tools. In addition, S-nitrosylation sites will be examined by computational biology and structural bioinformatics analysis tools to obtain stereochemical and physicochemical characteristics of S-nitrosylation sites in proteins.

Computed tomography scans of bone: Intra-group co-plots showed that the normalized bone density distributions for the mice in the 0 cGy (control; n=6) group exhibit a high degree of overlap, displaying limited variation throughout the distributions. Interestingly, intra-group co-plots of the data for the 20 cGy Ca ion and 150 cGy gamma ray groups revealed marked distribution heterogeneity within these test groups.

Cardiac function evaluated by echocardiography. Vevo2100 (VisualSonics) ultrasound equipment was used to assess echocardiographic endpoints in sham-irradiated mice or mice exposed to 40 cGy of 1 GeV/u 40Ca ions delivered in 3 fractions over 3 days or in mice exposed to 150 cGy acute 137Cs gamma rays (n = 6-9/each group). Several parameters were assessed. Preliminary analyses revealed that exposure to energetic Ca ions results in significant effects on stroke volume, cardiac output, and the left ventricular mass index. This work is currently proceeding with larger sample numbers and in mice exposed to protons, silicon and oxygen ions.

Gross pathological changes: At 15 months after irradiation, preliminary studies indicate significant abnormalities (p<0.01) in seminal vesicle and orbital tissues of mice exposed 15 months earlier to energetic calcium ions delivered to the whole body or head only.

1- Chronic oxidative stresses and inflammatory responses in organs that differ in their radiation sensitivity following exposure of the mice to the different radiation types described above;

2- To evaluate the relative biological effectiveness of isovelocity protons and high atomic number and high energy (HZE) particles described above compared to acute cesium-137 gamma rays in enhancing the rate of cancer incidence;

3- To measure oxidative changes and cancer incidence in non-irradiated organs (liver, lung) after exposure of the head to an acute mean absorbed dose of 0.4 Gy of 1 GeV/u Ca, and to compare the observed changes with those in the targeted organ (brain);

4- To examine the protective effect of whole-body pre-exposure to a conditioning dose of 0.2 Gy of 1 GeV protons delivered at low dose-rate prior to head exposure to acute dose of 0.4 Gy of 1 GeV/u Ca ions.

At two weeks, and 3, 6, and 10 months after irradiation, 5 mice from each of the groups described below were sacrificed, and peripheral blood as well as different organs (heart, liver, lung, kidney, bone marrow, brain, and testes) were harvested for cellular, biochemical, molecular, and histological analyses, as well as for archiving and participation in NASA’s Space Radiation Tissue Sharing Forum. The groups of mice were as follows:

1: Control; 2: gamma rays: 1.5 Gy (acute, single fraction, whole body); 3: gamma rays: 3 Gy (acute, single fraction, whole body); 4: 1 GeV protons: 0.2 Gy (0.0035 Gy/min, whole body); 5: 1 GeV/u Ca: 0.2 Gy (in 3 fractions; 1 acute fraction/day; whole body); 6: 1 GeV/u Ca: 0.3 Gy (in 3 fractions; 1 acute fraction/day; whole body); 7: 1 GeV/u Ca: 0.4 Gy (in 3 fractions; 1 acute fraction/day; whole body); 8: 1 GeV/u Ca: 0.4 Gy (in 1 acute fraction; whole body); 9: 1 GeV/u Ca: 0.4 Gy (in 1 acute fraction; head only); 10: 1 GeV/u Si: 0.4 Gy (in 3 fractions; 1 acute fraction/day; whole body); 11: 1 GeV/u Si: 0.4 Gy (in 1 acute single fraction; whole body); 12: 1 GeV/u O: 0.4 Gy (in 3 fractions; 1 acute fraction/day; whole body); 13: 1 GeV/u O: 0.4 Gy (in 1 acute single fraction; whole body); 14: 1 GeV protons followed by 1 GeV/u Ca: whole body exposure to 0.2 Gy of protons delivered 24 h prior to 0.4 Gy of 1 GeV/u Ca ions delivered to the whole body; 15: 1 GeV protons followed by 1 GeV/u Ca: whole body exposure to 0.2 Gy of protons delivered 24 h prior to 0.4 Gy of 1 GeV/u Ca ions targeted to the head only.

PRELIMINARY RESULTS: Here we report analyses performed in unirradiated control mice and in mice exposed to either 1 GeV protons, 1 GeV/u Ca ions, or cesium-137 gamma rays 2 weeks, and 3 months earlier. We examined alterations in circulating hematopoietic cell subsets using multicolor flow cytometry. The relative percent change in specific cell populations and absolute cell counts were also determined. Significant changes were detected in cell subsets after exposure to the energetic Ca ions. Specifically, ~ 10-fold increase in neutrophils were detected at two weeks in mice exposed to 20, 30, or 40 cGy of Ca ions delivered in a fractionated manner. The groups also showed significant increase in macrophages. These increases were attenuated by 3 months. The mice exposed to a single bolus of 40 cGy of Ca ions did not show significant increases at 2 weeks; however, by 3 months, increases in neutrophils were detected. The alterations observed at 2 weeks were associated with changes in the levels of circulating inflammatory cytokines (e.g., TNF-alpha, IL-1beta, CXCl1). Furthermore, histological analyses revealed prominent interstitial lung disease in mice exposed to a single bolus of 40 cGy of Ca ions at 2 weeks, characterized with thickened alveolar septa, pulmonary congestion, and endothelial hyperplasia. The mice exposed to the fractionated regimens presented mild lung injury.

In addition to the above, our preliminary studies show that 1 GeV/u Ca ions (20 cGy administered in 3 acute fractions over 3 days), and protons (20 cGy administered in a single bolus delivered over 60 min), lead to oxidative modification of cardiac proteins detectable by Oxyblot analysis two weeks after irradiation. Analyses of oxidative stress in other tissues (liver, brain) are ongoing.

Mass spectrometry (MS) and statistical analyses to examine the effects of low and moderate mean absorbed doses of HZE particles on the modulation of SNO protein levels in mice brains two weeks following whole body irradiation with 0.5 GeV/u titanium ions performed in earlier studies are nearing completion. Protein S-nitrosylation (SNO) is a reversible post-translational modification (PTM) through the covalent addition of nitric oxide (•NO) onto cysteine thiols. Strikingly, compared to control, greater increases in global S-nitrosylation of brain proteins were detected after exposure to low (5 cGy) than moderate (50 cGy) doses. Immunoprecipitation of candidate proteins (e.g., peroxyredoxin or Prx-1) confirmed the mass spectrometry results. Furthermore, Western blot analyses revealed upregulation of inducible nitric oxide synthase (iNOS) levels in brains exposed to 5 cGy. Notably, Prx-1 and iNOS are implicated in regulation of numerous oxidative stress-responsive pathways. Studies of protein nitrosylation in the cerebellum of mice at 2 weeks after exposure to 0 or 40 cGy of 1 GeV/u Ca ions delivered in a single bolus to the whole body or to the head only are ongoing.

1- Chronic oxidative stresses and inflammatory responses in organs that differ in their radiation sensitivity

2- To evaluate the relative biological effectiveness of 1 GeV/u Ca ions compared to acute 137Cs gamma rays in enhancing the rate of cancer incidence

3- To determine the beneficial effects of a pre-exposure to low linear energy transfer (LET) protons that may minimize the harmful effects (oxidative stress, DNA damage) of a subsequent exposure to high LET Ca ions

4- To measure oxidative changes and cancer incidence in non-irradiated organs (liver, lung) after exposure of the head to an acute mean absorbed dose of 0.4 Gy of 1 GeV/u Ca, and to compare the observed changes with those in the targeted organ (brain)

5- To examine the protective effect of whole-body pre-exposure to a conditioning dose of 0.2 Gy of 1 GeV protons delivered at low dose-rate prior to head exposure to acute dose of 0.4 Gy of 1 GeV/u Ca ions

At two weeks after exposure, 5 mice from each of the groups described below were sacrificed, and peripheral blood as well as different organs (heart, liver, lung, kidney, bone marrow, and brain) were harvested for cellular, biochemical, molecular, and histological analyses.

The groups of mice were as follows: 1: Control ; 2: Gamma rays: 1.5 Gy (acute, single fraction, whole body); 3: Gamma rays: 3 Gy (acute, single fraction, whole body); 4: 1 GeV protons: 0.2 Gy (0.0035 Gy/min, whole body); 5: 1 GeV/u Ca: 0.2 Gy (in 3 fractions; 1 acute fraction/day; whole body); 6: 1 GeV/u Ca: 0.3 Gy (in 3 fractions; 1 acute fraction/day; whole body); 7: 1 GeV/u Ca: 0.4 Gy (in 3 fractions; 1 acute fraction/day; whole body); 8: 1 GeV/u Ca: 0.4 Gy (in 1 acute fraction; whole body); 9: 1 GeV/u Ca: 0.4 Gy (in 1 acute single fraction delivered to the head only); 10: 1 GeV protons followed by 1 GeV/u Ca: whole body exposure to 0.2 Gy of protons delivered 24 h prior to 0.4 Gy of 1 GeV/u Ca ions delivered to the whole body; 11: 1 GeV protons followed by 1 GeV/u Ca: whole body exposure to 0.2 Gy of protons delivered 24 h prior to 0.4 Gy of 1 GeV/u Ca ions targeted to the head only.

PRELIMINARY RESULTS:

Assays analyzing the effects of the different radiation delivery regimens described above on inflammatory cytokines, changes in abundance of immune cells, as well as redox modulated changes affecting the activity of enzymes implicated in oxidative metabolism are ongoing.

Inflammatory Cytokines: Preliminary analyses of a panel of inflammatory cytokines revealed prominent changes in the levels of several cytokines in blood plasma. The results strongly support a modulatory effects on the immune system and inflammatory responses by energetic calcium ions.

Blood Cells: Analysis of lymphoid (T, B, and NK sub-populations) and myeloid (monocytes/macrophages and neutrophils) cells in peripheral blood was performed. Among many alterations in abundance of cellular subsets, prominent changes were observed in the abundance of neutrophils and CD4+ T-helper lymphocytes, which are an essential part of the immune system. Notably, the changes reflect a significant modulating effect of fractionation of the radiation dose from calcium ions.

Biochemical Changes: Preliminary analyses of a battery of enzymes suggest that mitochondria, the major contributor to oxidative metabolism, may be particularly susceptible to space radiation-induced damage. In particular, mitochondrial Lon, an ATP-powered proteolytic machine that selectively degrades key rate limiting proteins as well as misfolded, unassembled, and oxidatively damaged proteins, is sensitively regulated by space radiation.