Progress in accomplishing the research outlined in our investigation of oxidative stress and the cancer risk of space radiation has proceeded as planned. We have completed the irradiation of mice in March 2017, and carried out experiments related to the three Specific Aims outlined in the project. Briefly, middle-aged CBA/CaJ male mice (9-10 month old) were exposed (whole or partial body) to isovelocity 1 GeV/u protons, calcium, silicon, or oxygen ions with respective average Linear Energy Transfer (LET) values of ~0.24, 14, 44, and 88 keV/µm. A different set of mice was exposed to cesium-137 gamm rays as reference radiation (LET ~ 0.9 keV/ µm) to examine the following:
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.