Task Progress:
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This project tested the central hypothesis that exposures to and high atomic number, high energy (HZE) particles induce distinct redox-modulated biochemical changes that perturb essential signaling networks leading to persistent oxidative stress and progression towards malignancy in both irradiated and non-irradiated tissues. Further, these effects depend on the linear energy transfer (LET) of the radiation and are mitigated by prior exposure to protons. The experimental design relied on exposing groups of middle-aged CBA/CaJ male mice (9–10-month-old, to simulate age of active astronauts) to low/moderate mean absorbed doses of isovelocity 1 GeV/u protons, oxygen, silicon, or calcium ions, with respective average LET values of ~0.24, 14, 44, and 88 keV/µm. The radiation was delivered to the whole body or head only. A different set of mice were exposed to 137Cs gamma rays as reference radiation. Control mice were sham treated in parallel. Various redox modulated changes, inflammatory responses and histological structural changes were examined as a function of time after irradiation (2 weeks and up to 18 months). Cancer incidence was also evaluated in targeted and non-targeted organs.
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.
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Abstracts for Journals and Proceedings
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Moore L, Guo J, Oettinger J, Hu T, de Toledo SM, Azzam EI. "Increased mortality and degenerative effects in tissues of middle-aged CBA/CaJ mice exposed to low mean absorbed doses of energetic protons or heavy ions." Presented at the 63rd Annual Meeting of the Radiation Research Society, Grand Fiesta Americana Coral Beach, Cancun, Mexico, October 14-18, 2017. Abstract Book. 63rd Annual Meeting of the Radiation Research Society, Grand Fiesta Americana Coral Beach, Cancun, Mexico, October 14-18, 2017. , Oct-2017
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Abstracts for Journals and Proceedings
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Guo J, Hu T, Moore L, Chidambaram S, Khurana, Colangelo N, de Toledo SM, Azzam EI. "Short- and Long-Term Modulation of Hematopoietic Cells’ Abundance in Mice Exposed to Space Radiation." Presented at the 2018 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 22-25, 2018. Abstract Book. 2018 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 22-25, 2018. , Jan-2018
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Abstracts for Journals and Proceedings
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Yurkow E, Adler D, Colangelo N, Domogauer J, Moore L, Guo J, de Toledo SM, Howell RW, Azzam EI. "Long-Term Effect of Heavy Ion Radiations on Radiodensitometric Characteristics of Murine Bone." 2018 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 22-25, 2018. Abstract Book. 2018 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 22-25, 2018. , Jan-2018
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Abstracts for Journals and Proceedings
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Azzam EI. "Biological changes and health effects in rodents exposed to space radiation." Presented at “Life Sciences Related to Space,” Committee on Space Research (COSPAR) 2018 42nd Scientific Assembly, Pasadena, CA, July 14-22, 2018. Abstract Book. Committee on Space Research (COSPAR) 2018 42nd Scientific Assembly, Pasadena, CA, July 14-22, 2018. , Jul-2018
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Abstracts for Journals and Proceedings
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Leung CN, DeToledo SM, Howell RW, Azzam EI, Howell DM. "Effects of Heavy Ion Whole Body Irradiation on Population and Cytotoxicity of Natural Killer Cells." Presented at the 64th Annual Meeting of the Radiation Research Society, Chicago, IL, September 23-26, 2018. Abstract Book. 64th Annual Meeting of the Radiation Research Society, Chicago, IL, September 23-26, 2018. , Sep-2018
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Abstracts for Journals and Proceedings
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Hu T, Guio J, Moore L, Domogauer JD, Colangelo NW, Chidambaram S, Howell RW, DeToledo SM, Azzam EI. "Early and late effects of space radiation on abundance of hematopoietic cells and the bone marrow microenvironment." Presented at the 64th Annual Meeting of the Radiation Research Society, Chicago, IL, September 23-26, 2018. Abstract Book. 64th Annual Meeting of the Radiation Research Society, Chicago, IL, September 23-26, 2018. , Sep-2018
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Abstracts for Journals and Proceedings
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Havati S, Rabin BM, de Toledo SM, Shibata M, Acioglu C, Elkabes S, Baisre A, Azzam EI. "Late biochemical and histological changes in the brain of rats exposed to low fluences of HZE particles." Presented at the 2019 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 22-25, 2019. Abstract Book. 2019 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 22-25, 2019. , Jan-2019
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Abstracts for Journals and Proceedings
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Azzam EI. "Radiation biology and the potential health effects at low dose and low dose rates: a glance at the past and a glimpse of the future" Presented at the 65th Annual Meeting of the Radiation Research Society, San Diego, CA, November 3-6, 2019. Abstract Book. 65th Annual Meeting of the Radiation Research Society, San Diego, CA, November 3-6, 2019. , Nov-2019
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Abstracts for Journals and Proceedings
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Da Silva A, Yu H, Guo J, Hu T, Moore L, Colangelo NW, Domogauer JD, Leung CN, Howell DM, Rogers M, Adler D, Buckendahl P, Yurkow E, de Toledo SM, Howell RW, Azzam EI. "Chronic oxidative stress and inflammatory responses in organs of mice exposed to space radiation." Presented at the 2020 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 27-30, 2020. Abstract Book. 2020 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 27-30, 2020. , Jan-2020
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Abstracts for Journals and Proceedings
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Da Silva A, Yu H, Xavier L, Azzam E. "Delayed onset of oxidative stress in kidney of mice exposed to space radiation." Presented at the 2021 NASA Human Research Program Investigators’ Workshop, Virtual, February 1-4, 2021. Abstract Book. 2021 NASA Human Research Program Investigators’ Workshop, Virtual, February 1-4, 2021. , Feb-2021
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Abstracts for Journals and Proceedings
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Da Silva A, Yu H, Simpson M, de Toledo SM, Howell RW, Azzam E. "Oxidative stress, inflammation, and metabolic alterations in kidney of mice exposed to space radiation." Presented at the 67th Annual Meeting of Radiation Research Society, Virtual Meeting, October 3-6, 2021. Abstract Book. 67th Annual Meeting of Radiation Research Society, Virtual Meeting, October 3-6, 2021. , Oct-2021
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Articles in Peer-reviewed Journals
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Buonanno M, De Toledo SM, Howell RW, Azzam EI. "Low-dose energetic protons induce adaptive and bystander effects that protect human cells against DNA damage caused by a subsequent exposure to energetic iron ions." J Radiat Res. 2015 May;56(3):502-8. http://dx.doi.org/10.1093/jrr/rrv005 ; PubMed PMID: 25805407; PubMed Central PMCID: PMC4426929 , May-2015
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Articles in Peer-reviewed Journals
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Zakaria AM, Colangelo NW, Meesungnoen J, Azzam EI, Plourde M, Jay-Gerin JP. "Ultra-high dose-rate, pulsed (FLASH) radiotherapy with carbon ions: Generation of early, transient, highly oxygenated conditions in the tumor environment." Radiat Res. 2020 Dec 1;194(6):587-93. https://doi.org/10.1667/RADE-19-00015.1 ; PMID: 32853343; PMCID: PMC7856087
, Dec-2020
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Articles in Peer-reviewed Journals
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Preston RJ, Rühm W, Azzam EI, Boice JD, Bouffler S, Held KD, Little MP, Shore RE, Shuryak I, Weil MM. "Adverse outcome pathways, key events, and radiation risk assessment." Int J Radiat Biol. 2021;97(6):804-14. https://doi.org/10.1080/09553002.2020.1853847 ; PMID: 33211576 , Jun-2021
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Articles in Peer-reviewed Journals
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Buonanno M, Gonon G, Pandey BN, Azzam EI. "The intercellular communications mediating radiation-induced bystander effects and their relevance to environmental, occupational, and therapeutic exposures." Int. J. Radiat. Biol. 2022 May 27. Review. https://doi.org/10.1080/09553002.2022.2078006 PMID: 35559659 PMCID: PMC9809126 , May-2022
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Articles in Peer-reviewed Journals
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Leung CN, Howell DM, de Toledo SM, Azzam EI, Howell RW. "Late effects of heavy-ion space radiation on splenocyte subpopulations and NK cytotoxic function." Front. Astron. Space Sci. 2022 Nov 7. https://doi.org/10.3389/fspas.2022.949432 , Nov-2022
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