56Fe Radiation Research Articles

Multi-Domain Touchscreen-Based Cognitive Assessment of C57BL/6J Female Mice Shows Whole-Body Exposure to 56Fe Particle Space Radiation in Maturity Improves Discrimination Learning Yet Impairs Stimulus-Response Rule-Based Habit Learning.

Astronauts during interplanetary missions will be exposed to galactic cosmic radiation, including charged particles like 56Fe. Most preclinical studies with mature, “astronaut-aged” rodents suggest space radiation diminishes performance in classical hippocampal- and prefrontal cortex-dependent tasks. However, a rodent cognitive touchscreen battery unexpectedly revealed 56Fe radiation improves the performance of C57BL/6J male mice in a hippocampal-dependent task (discrimination learning) without changing performance in a striatal-dependent task (rule-based learning). As there are conflicting results on whether the female rodent brain is preferentially injured by or resistant to charged particle exposure, and as the proportion of female vs. male astronauts is increasing, further study on how charged particles influence the touchscreen cognitive performance of female mice is warranted. We hypothesized that, similar to mature male mice, mature female C57BL/6J mice exposed to fractionated whole-body 56Fe irradiation (3 × 6.7cGy 56Fe over 5 days, 600 MeV/n) would improve performance vs. Sham conditions in touchscreen tasks relevant to hippocampal and prefrontal cortical function [e.g., location discrimination reversal (LDR) and extinction, respectively]. In LDR, 56Fe female mice more accurately discriminated two discrete conditioned stimuli relative to Sham mice, suggesting improved hippocampal function. However, 56Fe and Sham female mice acquired a new simple stimulus-response behavior and extinguished this acquired behavior at similar rates, suggesting similar prefrontal cortical function. Based on prior work on multiple memory systems, we next tested whether improved hippocampal-dependent function (discrimination learning) came at the expense of striatal stimulus-response rule-based habit learning (visuomotor conditional learning). Interestingly, 56Fe female mice took more days to reach criteria in this striatal-dependent rule-based test relative to Sham mice. Together, our data support the idea of competition between memory systems, as an 56Fe-induced decrease in striatal-based learning is associated with enhanced hippocampal-based learning. These data emphasize the power of using a touchscreen-based battery to advance our understanding of the effects of space radiation on mission critical cognitive function in females, and underscore the importance of preclinical space radiation risk studies measuring multiple cognitive processes, thereby preventing NASA’s risk assessments from being based on a single cognitive domain.

Characterization of the Secondary Neutron Field Produced in a Thick Aluminum Shield by 1 GeV/u 56Fe Ions Using TLD-Based Ambient Dosimeters

The neutron ambient dose equivalent induced by galactic cosmic ray-like (1 GeV/u 56Fe) radiation stopped in a thick aluminum shield was measured at different angles with GSI neutron ball, the standard TLD (thermoluminescent dosimeters) based neutron dosimeter for area monitoring at the GSI facility. In order to measure reliably at large angles, a modified version of the GSI ball, including a set of three more sensitive TLD600H/700H cards, instead of one standard TLD600/700, cards was used. The modified GSI balls were calibrated in neutron reference fields of 241Am-Be(alpha,n) available at the Physikalisch-Technische Bundesanstalt (PTB). The neutron ambient dose equivalent was measured at 5 different angles (15, 40, 90, 115 and 130 degree) with respect to the beam direction and compared to the calculated detector response and ambient neutron dose equivalent results from FLUKA simulations. The dosimeter readings were corrected for signal contributions coming from secondary charged particles. An agreement within 15 % was found between the measured and calculated GSI ball response and an agreement within 30 % was found between experiments and calculated neutron dose equivalents.

Abstract 3728: Inverse effect of 28Si and 56Fe radiation on intestinal tumorigenesis vs. carcinogenesis in APC1638N/+ mice

Abstract Space radiation is a major risk factor for gastrointestinal (GI) cancer in astronauts and is a matter of concern. Earlier we have reported higher relative effects for intestinal tumorigenesis in APC1638N/+ mice after exposure to different heavy ion radiation spanning a range of LET values. The purpose of the current study was to histologically grade intestinal tumors into adenoma vs. adenocarcinoma and assess relative effects of different heavy ion radiation on tumorigenesis vs carcinogenesis. Tumors from male APC1638N/+ mice 150 d after exposure to whole-body sham, γ (0.3 keV/μm), 12C (13 keV/μm), 28Si (70 keV/μm), or 56Fe (148 keV/μm) radiation were harvested, fixed, paraffin embedded, and sectioned. Radiation doses were 0.1 Gy, 0.5 Gy and a equitoxic dose of 2.0 Gy γ-rays. H&E stained tumor sections from at least 50 tumors per study group were used for histological grading into adenoma and adenocarcinoma. Hotspot mutations for p53 and Kras genes were assessed using targeted PCR followed by amplicon sequencing in 20 tumors per study group. Our previous published data showed that among the three LETs tested relative intestinal tumorigenic effects (relative to γ-rays) peaked at a LET of 70 keV/μm (28Si) with 13 keV/μm (12C) showing the least effect and 148 keV/μm (56Fe) showing an intermediate effect. In contrast, the current study demonstrates that the frequency of adenocarcinoma peaked after 56Fe with the least effect after 12C and an intermediate effect after 28Si. Relative to γ-rays, the carcinoma frequencies were 42.4+0.9, 13.2+0.61 and 8.0+1.14 higher after 56Fe at doses of 0.1, 0.5 and 2.0 Gy respectively. Whereas the relative carcinoma frequencies of 34.2+1.35, 14.0+0.95 and 9.0+2.25 were observed after 28Si at doses of 0.1, 0.5 and 2.0 Gy respectively. While Kras mutation analysis of tumors did not show any difference among radiation types, p53 mutation analysis demonstrate a higher number of mutations in exon 5 in 56Fe relative to 28Si samples. Collectively, our data show that while LET-dependent tumorigenic effects followed a parabolic shape, carcinogenic effects followed a more linear-like pattern with highest adenocarcinoma percentage and frequency at 148 keV/μm (56Fe). Although inverse effects of LET on tumorigenesis vs. carcinogenesis could in part be attributed to the beams’ differential physical properties, our mutation data on p53 supports differential effects of the beams on tumor suppressor mutation. Citation Format: Santosh Kumar, Shubhankar Suman, Bhaskar V.s. Kallakury, Bo-Hyun Moon, Albert J. Fornace, Kamal Datta. Inverse effect of 28Si and 56Fe radiation on intestinal tumorigenesis vs. carcinogenesis in APC1638N/+ mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3728.

Abstract 3729: Heavy-ion space radiation exposure is a potential risk factor for gastrointestinal tumorigenesis even at extremely low doses

Abstract Heavy-ion radiation (HZE)-induced carcinogenesis is a major concern in astronauts planning to undertake long-term deep space exploration, such as a mission to the Mars. Due to high-LET (linear energy transfer) characteristics of HZE ions present in deep space environment, the Mars mission is expected to substantially enhance gastrointestinal cancer risk in astronauts, relative to low-LET radiation. Previously, using three different mouse models of human colorectal cancer (APCmin/+, APC1638N/+ and IL10-/- mouse) we have unequivocally demonstrated a significantly higher risk of intestinal and colonic tumorigenesis after exposure to energetic heavy ions. This study aims to obtain quantitative GI-tumorigenesis data at space relevant doses (5-50 cGy) of heavy ions covering a broad-spectrum of linear energy transfer (2-148 keV/μm), relative to γ-radiation. The APC1638N/+ mouse was used due to its best signal-to-noise ratio among all previously studied mouse models of human gastrointestinal cancer. Both female and male APC1638N/+ mice (6-8 weeks, n=20) were whole-body exposed to sham-radiation, γ-rays, 4He (2 keV/μm), 12C (13 keV/μm), 16O (22 keV/μm), 28Si (69 keV/μm), and 56Fe (148 keV/μm) -ion radiation at 5, 10, and 50 cGy dose at the NASA Space Radiation Laboratory (NSRL) in Brookhaven National Laboratory. Mice were euthanized at 150 d after radiation exposure and intestinal and colon tumor frequency and size were scored and analyzed as a function of dose, LET, and gender. The highest increase in tumor frequency was observed after 28Si followed by 56Fe, 16O, 12C, and 4He radiation, and male preponderance for tumorigenesis was evident for each radiation type and dose. At 50 cGy dose, no significant difference in tumorigenesis was observed between γ and 4He. However, at lower doses (5 and 10 cGy) significantly higher tumorigenesis was noted after 4He exposure. Furthermore, calculation of relative biological effectiveness (RBE) for tumorigenesis showed the highest value with 28Si and lower doses showed greater RBE relative to higher doses. No statistical difference in tumorigenesis pattern was evident between 16O and 12C at all studied doses. In addition no significant change in tumorigenesis pattern was evident between 28Si and 56Fe in female mice. Analysis showed greater tumorigenesis per unit of radiation (per cGy) at lower doses suggesting radiation-induced tumorigenesis reaching a saturation point at higher doses. The lack of understanding on radiation quality effects is one of the major uncertainties in space radiation exposure-associated cancer risk prediction. Using a broad-spectrum of HZE-ions we have demonstrated the dependence of gastrointestinal tumorigenesis on radiation quality even at very low doses, which has implications in cancer risk prediction for astronauts and also for accessing secondary cancer risks after particle radiotherapy. Citation Format: Shubhankar Suman, Santosh Kumar, Bo-Hyun Moon, Jerry Angdisen, Bhaskar VS Kallakury, Albert J. Fornace, Kamal Datta. Heavy-ion space radiation exposure is a potential risk factor for gastrointestinal tumorigenesis even at extremely low doses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3729.

Abstract 3736: Space radiation-induced decline in gut autophagy and expansion of both mitotic and senescent population denotes an aging phenotype with enhanced cancer risk

Abstract Exposure to space radiation is known to alter a myriad of cellular processes that could potentially affect astronauts’ health during and after long duration’s space missions. Due to high-LET (linear energy transfer) characteristics of energetic heavy ions (HZE) present in space, astronauts are predicted to be at higher risk for gastrointestinal cancer development. Proliferative epithelial cells in gastrointestinal (GI) tissue are highly radiosensitive and in our previous studies we have demonstrated a persistent stress phenotype in crypt cells after heavy ion space radiation exposure. However, uncertainty remains in our understanding of how heavy ions induce chronic stress and inflammation is linked to higher cancer risk. The purpose of the current study was to assess late effects of heavy ion (56-Fe) on the autophagy process that is required to recycle damaged cellular components and its role in HZE-induced gastrointestinal carcinogenesis. C57BL6/J mice were exposed to 1.6 Gy of 56-Fe radiation at NASA Space Radiation Laboratory (NSRL). Mice were euthanized 2-month after radiation and intestinal tissues were analyzed for oxidative stress and associated DNA damage, and alterations in antioxidant response and autophagy signaling. Further, cells underdoing mitosis, autophagy and stress-induced senescence were also scored and alterations in cell signaling pathways with established role in carcinogenesis were evaluated. Energetic heavy ions exposure resulted in a long-term increase in intracellular reactive oxygen species (ROS), accompanied with decreased expression of key antioxidant genes (Gpx, SOD and catalase). Increase in both mitochondrial and cytosolic ROS was evident as both loss of mitochondrial membrane potential (MMP) and increase in prooxidant Nox1 expression was found. Paradoxical increases in phospho-histone-3 positive (mitotic) and p16 positive (senescent) cells were found, however decreases in autophagy positive (LC3B) cells was noted in HZE-exposed mouse intestine. Upregulation of upstream mTOR-PI3K-AKT signaling and p62 were noticed in long-term suppression of autophagy (Atg12, LC3B and Beclin-1) after HZE-exposure. Here, we show that space radiation downregulated autophagy processes, which are an important cellular mechanism involved in suppression of carcinogenesis. When considered along with upregulation of proliferative pathways and increased oxidative stress, our data suggest increased risk of chronic GI diseases including cancer due to late persistence of damaged organelles in cells after energetic heavy ion radiation exposures. Collectively, this study demonstrates a unique phenomenon of heavy ion-induced suppression of autophagy concurrent with increases in both mitotic and senescent cells indicating a premature aging phenotype with enhanced cancer risk. Citation Format: Kamal Datta, Shubhankar Suman, Bo-Hyun Moon, Albert J. Fornace. Space radiation-induced decline in gut autophagy and expansion of both mitotic and senescent population denotes an aging phenotype with enhanced cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3736.

Exposure to ≤15 cGy of 600 MeV/n 56Fe Particles Impairs Rule Acquisition but not Long-Term Memory in the Attentional Set-Shifting Assay.

On future missions into deep space, astronauts will be required to work more autonomously than on previous missions, and thus their ability to perform executive functions could be critical to mission success. In this study, we determined the effect that ≤15 cGy of 600 MeV/n 56Fe particles has on attentional set-shifting (ATSET) performance of ∼10 month-old (at the time of irradiation) male Wistar rats that had been prescreened for their ability to perform the task. Exposure to 1-15 cGy of 56Fe particles leads to a significant impairment in compound discrimination (CD) performance. Should similar effects occur in astronauts, an impaired ability to execute CD would result in a decreased ability to identify and maintain focus on relevant aspects of the task being performed. The use of rats that had been prescreened for ATSET performance helped to establish that working memory of the rules for the food reward remained intact (for at least 100 days) even after 15 cGy irradiation with 600 MeV/n 56Fe particles, but that 56Fe radiation exposure affected associative cue learning/acquisition rather than an intrinsic inability to perform the CD tasks. Our data suggest that declarative memory, and the ability to transitively infer established rules, also remained intact in the irradiated rats. Thus, should similar effects occur in astronauts, 56Fe-induced CD performance deficits may only be manifested in scenarios where astronauts are required to transitively apply their knowledge to solve problems that they have not previously encountered; nevertheless, potentially one-third of astronauts may not be able to perform event-critical tasks correctly. The implication of this data, from a probabilistic risk assessment perspective, is that cognitive performance studies that use naïve rodents, thus requiring task rule acquisition as well as task performance, are likely to overestimate the risk of 56Fe-induced cognitive deficits.

Space radiation triggers persistent stress response, increases senescent signaling, and decreases cell migration in mouse intestine

Proliferative gastrointestinal (GI) tissue is radiation-sensitive, and heavy-ion space radiation with its high-linear energy transfer (high-LET) and higher damaging potential than low-LET γ-rays is predicted to compromise astronauts' GI function. However, much uncertainty remains in our understanding of how heavy ions affect coordinated epithelial cell migration and extrusion, which are essential for GI homeostasis. Here we show using mouse small intestine as a model and BrdU pulse labeling that cell migration along the crypt-villus axis is persistently decreased after a low dose of heavy-ion 56Fe radiation relative to control and γ-rays. Wnt/β-catenin and its downstream EphrinB/EphB signaling are key to intestinal epithelial cell (IEC) proliferation and positioning during migration, and both are up-regulated after 56Fe radiation. Conversely, factors involved in cell polarity and adhesion and cell-extracellular matrix interactions were persistently down-regulated after 56Fe irradiation-potentially altering cytoskeletal remodeling and cell extrusion. 56Fe radiation triggered a time-dependent increase in γH2AX foci and senescent cells but without a noticeable increase in apoptosis. Some senescent cells acquired the senescence-associated secretory phenotype, and this was accompanied by increased IEC proliferation, implying a role for progrowth inflammatory factors. Collectively, this study demonstrates a unique phenomenon of heavy-ion radiation-induced persistently delayed IEC migration involving chronic sublethal genotoxic and oncogenic stress-induced altered cytoskeletal dynamics, which were seen even a year later. When considered along with changes in barrier function and nutrient absorption factors as well as increased intestinal tumorigenesis, our in vivo data raise a serious concern for long-duration deep-space manned missions.

Abstract 4163: Space radiation-induced persistent DNA damage and alterations in cytoskeletal remodeling pathways affect intestinal epithelial cell migration in mice

Abstract Lombardi Comprehensive Cancer Center, and Department of Biochemistry and Molecular & Cellular Biology Georgetown University, Washington, DC 20057, USA. Heavy ion space radiation is considered as a risk factor of gastrointestinal (GI) cancer and perturbed intestinal epithelial cell (IEC) migration has been implicated in GI carcinogenesis. The purpose of the current study was to assess rate of IEC migration as well as dissect key IEC migration signaling events after exposure to heavy ion 56Fe irradiation and compare the results to sham- and γ-irradiation. Male C57BL/6J mice were exposed to 0.5 Gy of either γ or 56Fe radiation and control mice were sham irradiated. IEC migration distance was measured 7 and 60 d after exposure using BrdU pulse labeling. We also analyzed cell polarity, microtubule dynamics, and adhesion dynamics using immunoblots and immunofluorescence. Measure of IEC migration rate showed a greater decrease after 56Fe relative to γ radiation at both the time points. At the molecular level, compared to sham and γ radiation, 56Fe radiation exhibited significantly greater impairment of cell adhesion dynamics evidenced by altered integrins and Rock1 as well as cytoskeletal dynamics evidenced by altered MLCK expression. Similarly, perturbed cell polarity, tight junction, and microtubule dynamics was revealed by the deregulation of Par3, Claudin1, Scribble, Occludin, ZO-1, phospho-Tau and MAP1B. Also, the Wnt/β-catenin and EphB/EphrinB pathways critical for IEC migration consistently showed greater deregulation at both the time points tested after 56Fe radiation relative to γ -rays. Taken together, our molecular findings correlate with the BrdU pulse labeling data, and demonstrate greater effects of heavy ion 56Fe on IEC migration relative to γ -rays. In summary, our results have demonstrated for the first time that radiation can disrupt cell migration dynamics through alteration of signaling molecules involved in crucial cellular processes and these results have implications for heavy ion space radiation-induced gastrointestinal homeostatic deregulation and carcinogenesis. Citation Format: Santosh Kumar, Shubhankar Suman, Albert Fornace, Kamal Datta. Space radiation-induced persistent DNA damage and alterations in cytoskeletal remodeling pathways affect intestinal epithelial cell migration in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4163.

MMR Deficiency Does Not Sensitize or Compromise the Function of Hematopoietic Stem Cells to Low and High LET Radiation

One of the major health concerns on long‐duration space missions will be radiation exposure to the astronauts. Outside the earth's magnetosphere, astronauts will be exposed to galactic cosmic rays (GCR) and solar particle events that are principally composed of protons and He, Ca, O, Ne, Si, Ca, and Fe nuclei. Protons are by far the most common species, but the higher atomic number particles are thought to be more damaging to biological systems. Evaluation and amelioration of risks from GCR exposure will be important for deep space travel. The hematopoietic system is one of the most radiation‐sensitive organ systems, and is highly dependent on functional DNA repair pathways for survival. Recent results from our group have demonstrated an acquired deficiency in mismatch repair (MMR) in human hematopoietic stem cells (HSCs) with age due to functional loss of the MLH1 protein, suggesting an additional risk to astronauts who may have significant numbers of MMR deficient HSCs at the time of space travel. In the present study, we investigated the effects gamma radiation, proton radiation, and 56Fe radiation on HSC function in Mlh1+/+ and Mlh1‐/‐ marrow from mice in a variety of assays and have determined that while cosmic radiation is a major risk to the hematopoietic system, there is no dependence on MMR capacity. stem cells translational medicine 2018;7:513–520

Protective effect of mitochondrial-targeted antioxidant MitoQ against iron ion 56Fe radiation induced brain injury in mice

Exposure to iron ion 56Fe radiation (IR) during space missions poses a significant risk to the central nervous system and radiation exposure is intimately linked to the production of reactive oxygen species (ROS). MitoQ is a mitochondria-targeted antioxidant that has been shown to decrease oxidative damage and lower mitochondrial ROS in a number of animal models. Therefore, the present study aimed to investigate role of the mitochondrial targeted antioxidant MitoQ against 56Fe particle irradiation-induced oxidative damage and mitochondria dysfunction in the mouse brains. Increased ROS levels were observed in mouse brains after IR compared with the control group. Enhanced ROS production leads to disruption of cellular antioxidant defense systems, mitochondrial respiration dysfunction, altered mitochondria dynamics and increased release of cytochrome c (cyto c) from mitochondria into cytosol resulting in apoptotic cell death. MitoQ reduced IR-induced oxidative stress (decreased ROS production and increased SOD, CAT activities) with decreased lipid peroxidation as well as reduced protein and DNA oxidation. MitoQ also protected mitochondrial respiration after IR. In addition, MitoQ increased the expression of mitofusin2 (Mfn2) and optic atrophy gene1 (OPA1), and decreased the expression of dynamic-like protein (Drp1). MitoQ also suppressed mitochondrial DNA damage, cyto c release, and caspase-3 activity in IR-treated mice compared to the control group. These results demonstrate that MitoQ may protect against IR-induced brain injury.

Whole-Body Exposure to 28Si-Radiation Dose-Dependently Disrupts Dentate Gyrus Neurogenesis and Proliferation in the Short Term and New Neuron Survival and Contextual Fear Conditioning in the Long Term.

Astronauts traveling to Mars will be exposed to chronic low doses of galactic cosmic space radiation, which contains highly charged, high-energy (HZE) particles. 56Fe-HZE-particle exposure decreases hippocampal dentate gyrus (DG) neurogenesis and disrupts hippocampal function in young adult rodents, raising the possibility of impaired astronaut cognition and risk of mission failure. However, far less is known about how exposure to other HZE particles, such as 28Si, influences hippocampal neurogenesis and function. To compare the influence of 28Si exposure on indices of neurogenesis and hippocampal function with previous studies on 56Fe exposure, 9-week-old C57BL/6J and Nestin-GFP mice (NGFP; made and maintained for 10 or more generations on a C57BL/6J background) received whole-body 28Si-particle-radiation exposure (0, 0.2 and 1 Gy, 300 MeV/n, LET 67 KeV/μ, dose rate 1 Gy/min). For neurogenesis assessment, the NGFP mice were injected with the mitotic marker BrdU at 22 h postirradiation and brains were examined for indices of hippocampal proliferation and neurogenesis, including Ki67+, BrdU+, BrdU+NeuN+ and DCX+ cell numbers at short- and long-term time points (24 h and 3 months postirradiation, respectively). In the short-term group, stereology revealed fewer Ki67+, BrdU+ and DCX+ cells in 1-Gy-irradiated group relative to nonirradiated control mice, fewer Ki67+ and DCX+ cells in 0.2 Gy group relative to control group and fewer BrdU+ and DCX+ cells in 1 Gy group relative to 0.2 Gy group. In contrast to the clearly observed radiation-induced, dose-dependent reductions in the short-term group across all markers, only a few neurogenesis indices were changed in the long-term irradiated groups. Notably, there were fewer surviving BrdU+ cells in the 1 Gy group relative to 0- and 0.2-Gy-irradiated mice in the long-term group. When the short- and long-term groups were analyzed by sex, exposure to radiation had a similar effect on neurogenesis indices in male and female mice, although only male mice showed fewer surviving BrdU+ cells in the long-term group. Fluorescent immunolabeling and confocal phenotypic analysis revealed that most surviving BrdU+ cells in the long-term group expressed the neuronal marker NeuN, definitively confirming that exposure to 1 Gy 28Si radiation decreased the number of surviving adult-generated neurons in male mice relative to both 0- and 0.2-Gy-irradiated mice. For hippocampal function assessment, 9-week-old male C57BL/6J mice received whole-body 28Si-particle exposure and were then assessed long-term for performance on contextual and cued fear conditioning. In the context test the animals that received 0.2 Gy froze less relative to control animals, suggesting decreased hippocampal-dependent function. However, in the cued fear conditioning test, animals that received 1 Gy froze more during the pretone portion of the test, relative to controls and 0.2-Gy-irradiated mice, suggesting enhanced anxiety. Compared to previously reported studies, these data suggest that 28Si-radiation exposure damages neurogenesis, but to a lesser extent than 56Fe radiation and that low-dose 28Si exposure induces abnormalities in hippocampal function, disrupting fear memory but also inducing anxiety-like behavior. Furthermore, exposure to 28Si radiation decreased new neuron survival in long-term male groups but not females suggests that sex may be an important factor when performing brain health risk assessment for astronauts traveling in space.

Changes in the Hippocampal Proteome Associated with Spatial Memory Impairment after Exposure to Low (20 cGy) Doses of 1 GeV/n 56Fe Radiation.

Exposure to low (∼20 cGy) doses of high-energy charged (HZE) particles, such as 1 GeV/n 56Fe, results in impaired hippocampal-dependent learning and memory (e.g., novel object recognition and spatial memory) in rodents. While these findings raise the possibility that astronauts on deep-space missions may develop cognitive deficits, not all rats develop HZE-induced cognitive impairments, even after exposure to high (200 cGy) HZE doses. The reasons for this differential sensitivity in some animals that develop HZE-induced cognitive failure remain speculative. We employed a robust quantitative mass spectrometry-based workflow, which links early-stage discovery to next-stage quantitative verification, to identify differentially active proteins/pathways in rats that developed spatial memory impairment at three months after exposure to 20 cGy of 1 GeV/n 56Fe (20/impaired), and in those rats that managed to maintain normal cognitive performance (20/functional). Quantitative data were obtained on 665-828 hippocampal proteins in the various cohorts of rats studied, of which 580 were expressed in all groups. A total of 107 proteins were upregulated in the irradiated rats irrespective of their spatial memory performance status, which included proteins involved in oxidative damage response, calcium transport and signaling. Thirty percent (37/107) of these "radiation biomarkers" formed a functional interactome of the proteasome and the COP9 signalosome. These data suggest that there is persistent oxidative stress, ongoing autophagy and altered synaptic plasticity in the irradiated hippocampus, irrespective of the spatial memory performance status, suggesting that the ultimate phenotype may be determined by how well the hippocampal neurons compensate to the ongoing oxidative stress and associated side effects. There were 67 proteins with expression that correlated with impaired spatial memory performance. Several of the "impaired biomarkers" have been implicated in poor spatial memory performance, neurodegeneration, neuronal loss or neuronal susceptibility to apoptosis, or neuronal synaptic or structural plasticity. Therefore, in addition to the baseline oxidative stress and altered adenosine metabolism observed in all irradiated rats, the 20/impaired rats expressed proteins that led to poor spatial memory performance, enhanced neuronal loss and apoptosis, changes in synaptic plasticity and dendritic remodeling. A total of 46 proteins, which were differentially upregulated in the sham-irradiated and 20/functional rat cohorts, can thus be considered as markers of good spatial memory, while another 95 proteins are associated with the maintenance of good spatial memory in the 20/functional rats. The loss or downregulation of these "good spatial memory" proteins would most likely exacerbate the situation in the 20/impaired rats, having a major impact on their neurocognitive status, given that many of those proteins play an important role in neuronal homeostasis and function. Our large-scale comprehensive proteomic analysis has provided some insight into the processes that are altered after exposure, and the collective data suggests that there are multiple problems with the functionality of the neurons and astrocytes in the irradiated hippocampi, which appear to be further exacerbated in the rats that have impaired spatial memory performance or partially compensated for in the rats with good spatial memory.

Space radiation exposure persistently increased leptin and IGF1 in serum and activated leptin-IGF1 signaling axis in mouse intestine.

Travel into outer space is fraught with risk of exposure to energetic heavy ion radiation such as 56Fe ions, which due to its high linear energy transfer (high-LET) characteristics deposits higher energy per unit volume of tissue traversed and thus more damaging to cells relative to low-LET radiation such as γ rays. However, estimates of human health risk from energetic heavy ion exposure are hampered due to lack of tissue specific in vivo molecular data. We investigated long-term effects of 56Fe radiation on adipokines and insulin-like growth factor 1 (IGF1) signaling axis in mouse intestine and colon. Six- to eight-week-old C57BL/6J mice were exposed to 1.6 Gy of 56Fe ions. Serum and tissues were collected up to twelve months post-irradiation. Serum was analyzed for leptin, adiponectin, IGF1, and IGF binding protein 3. Receptor expressions and downstream signaling pathway alterations were studied in tissues. Irradiation increased leptin and IGF1 levels in serum, and IGF1R and leptin receptor expression in tissues. When considered along with upregulated Jak2/Stat3 pathways and cell proliferation, our data supports the notion that space radiation exposure is a risk to endocrine alterations with implications for chronic pathophysiologic changes in gastrointestinal tract.

Effects of Proton and Combined Proton and (56)Fe Radiation on the Hippocampus.

The space radiation environment contains protons and (56)Fe, which could pose a significant hazard to space flight crews during and after missions. The space environment involves complex radiation exposures, thus, the effects of a dose of protons might be modulated by a dose of heavy-ion radiation. The brain, and particularly the hippocampus, may be susceptible to space radiation-induced changes. In this study, we first determined the dose-response effect of proton radiation (150 MeV) on hippocampus-dependent cognition 1 and 3 months after exposure. Based on those results, we subsequently exposed mice to protons alone (150 MeV, 0.1 Gy), (56)Fe alone (600 MeV/n, 0.5 Gy) or combined proton and (56)Fe radiations (protons first) with the two exposures separated by 24 h. At one month postirradiation, all animal groups showed novel object recognition. However, at three months postirradiation, mice exposed to either protons or combined proton and (56)Fe radiations showed impaired novel object recognition, which was not observed in mice irradiated with (56)Fe alone. The mechanisms in these impairments might involve inflammation. In mice irradiated with protons alone or (56)Fe alone three months earlier, there was a negative correlation between a measure of novel object recognition and the number of newly born activated microglia in the dentate gyrus. Next, cytokine and chemokine levels were assessed in the hippocampus. At one month after exposure the levels of IL-12 were higher in mice exposed to combined radiations compared with sham-irradiated mice, while the levels of IFN-γ were lower in mice exposed to (56)Fe radiation alone or combined radiations. In addition, IL-4 levels were lower in (56)Fe-irradiated mice compared with proton-irradiated mice and TNF-α levels were lower in proton-irradiated mice than in mice receiving combined radiations. At three months after exposure, macrophage-derived chemokine (MDC) and eotaxin levels were lower in mice receiving combined radiations. The levels of MDC and eotaxin correlated and the levels of MDC, but not eotaxin, correlated with the percentage of newly born activated microglia in the blades of the dentate gyrus. Finally, hippocampal IL-6 levels were higher in mice receiving combined radiations compared with mice receiving (56)Fe radiation alone. These data demonstrate the sensitivity of novel object recognition for detecting cognitive injury three months after exposure to proton radiation alone, and combined exposure to proton and (56)Fe radiations, and that newly-born activated microglia and inflammation might be involved in this injury.

Abstract 816: Persistent effects of radiation on intestinal stem cells: implications for colorectal carcinogenesis

Abstract Adult stem cells, similar to somatic cells, are also subject to a similar array of insults and are therefore presumed to accrue radiation damage. Unlike somatic cells, however, lesions arising in the stem cell compartment are propagated both to daughter stem cells and to downstream lineages through the processes of self-renewal and differentiation. The impact of damage accrued in individual stem cells can thus have major ramifications for all levels of the developmental hierarchy leading to cancer initiation and promotion in human as well as in animal models of human colorectal cancer (CRC) such as in APCMin/+. Differentiation of intestinal stem cell (ISC) and ordered migration of differentiated cells along crypt-villus axis is essential for maintenance of intestinal homeostasis and provides a model system to study stem cell alterations after radiation. Furthermore, considering that tumors in animal models of CRC almost exclusively develop in the small intestine and increased intestinal tumor frequency was radiation quality dependent, we investigated persistent effects of radiation quality on ISC as well as on differentiated intestinal epithelial cells in mice. Mice (6 to 8 week; C57BL/6J) were exposed to γ (0.5 and 2 Gy) and 56-Fe (0.5 and 1.6 Gy) radiation, samples were collected 7 d and 2 m after radiation exposure, and carcinogenic precursor events such as persistent oxidative stress and DNA damage were assessed. Higher persistently increased oxidative stress and oxidative DNA damage was observed after 56-Fe relative to γ radiation. However, increased oxidative stress did not increase cell death implying that the oxidative stress was sub-lethal. Autophagy, a self-cannibalistic process of cell involved in removing damaged cell and cell constituents, was consistently downregulated in a radiation quality dependent manner even 2 m after exposure suggesting persistence of damage-bearing cells. We believe radiation-induced autophagy downregulation and sub-lethal oxidative stress is working in tandem to trigger proliferative signaling such as PI3K/Akt and mTOR in intestinal cells. Taken together our results provide insight into radiation quality dependent persistent cancer-related molecular events, which could have altered phenotypic behavior of ISC such as proliferation, differentiation, and migration. Note: This abstract was not presented at the meeting. Citation Format: Kamal Datta, Shubhankar Suman, Santosh Kumar, Albert J. Fornace. Persistent effects of radiation on intestinal stem cells: implications for colorectal carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 816. doi:10.1158/1538-7445.AM2015-816

Heavy-ion (56Fe) irradiation leads to impaired aortic relaxation prior to atherosclerotic plaque formation in ApoE-/- mice

Terrestrial radiation exposure is a well-established risk factor for cardiovascular disease. For example, coronary artery disease and stroke are both well-established adverse effects of therapeutic radiation, especially for breast and head-and-neck cancers [ 1]. Similarly, atomic bomb survivors were significantly more likely to die of cardiovascular disease than their countrymen [ 2]. Even radiation technologists, prior to 1950 (when regulations governing shielding and occupational exposure were less rigorous), had an increased risk of clinically significant atherosclerosis [ 3]. Although the character of the radiation in interplanetary space is very different from that encountered on Earth, there is concern that exposure to this cosmic radiation might pose a similar risk for astronauts. Decreased endothelium-dependent vasodilation is thought to predispose humans to the development of structural vascular changes that precede development of atherosclerosis. Therefore, in this study, we examined the effect of 56Fe, an important component of cosmic radiation, on vascular relaxation. At the NASA Space Radiation Laboratory at Brookhaven National Laboratory, 10-week-old apoE−/− mice (an age at which there is little atherosclerotic plaque in the descending aorta) were exposed to 2.6 Gy 56Fe, a dose in the range that was shown previously to accelerate the development of atherosclerotic plaques in this mouse model at 13 weeks post-exposure [ 4]. The mice were then transferred to the University of Alabama at Birmingham, where they were housed under standard conditions and fed a normal diet. At 4–5 weeks post-irradiation, aortic rings were isolated and endothelial-dependent relaxation in irradiated mice was compared with that of age-matched controls from un-irradiated apoE mice and from un-irradiated wild-type mice of the same C57BL/6 genetic background. Aortic relaxation was not significantly different between un-irradiated apoE−/− mice and un-irradiated wild-type C57BL/6 mice, consistent with previous studies. 56Fe radiation, however, resulted in significantly impaired relaxation in response to acetylcholine. This suggests that heavy-ion radiation exposure leads to impairment of normal vascular reactivity prior to the appearance of atherosclerotic plaques, suggesting that it may be a driving force, rather than a consequence, of radiation-induced accelerated atherosclerosis.

Neuronal stress following exposure to 56Fe particles and the effects of antioxidant-rich diets

Exposing young rats to particles of high energy and charge (HZE particles), a ground-based model for exposure to cosmic rays, enhances indices of oxidative stress and inflammation and disrupts the functioning of neuronal communication in critical regions of the brain [ 1]. These changes in neuronal function are similar to those seen in ageing [ 2, 3]. Although there is some recovery of function after exposure to 56Fe particles, particularly in changes observed 36 h following irradiation, long-term changes (75 days) have been observed, suggesting subcellular damage. Consequently, oxidative stress and inflammation induced by radiation could affect downstream events, such as changes in behavior and gene expression. Therefore, berry fruits high in antioxidant and anti-inflammatory activity, such as blueberries and strawberries, may prevent the occurrence of neurochemical and behavioral changes that occur if fed prior to radiation [ 4].Rats were exposed to 56Fe (1000 MeV/n; 1.5 Gy) particles at the NASA Space Radiation Laboratory at Brookhaven National Laboratory; other rats served as non-irradiated controls. The animals were fed either a control or a 2% blueberry or strawberry diet 8 weeks prior to radiation. Rats were then either euthanized at 36 h (short term) or 30 days following irradiation (long term). Before and after the irradiation, the animals were housed at USDA Human Nutrition Research Center on Aging at Tufts University, Boston.The results of the experiments indicate that: (1) 56Fe exposure caused significant differential, neurochemical changes in critical regions of the brain, such as hippocampus, striatum, frontal cortex and cerebellum, particularly long term. (2) Neurochemical changes resulted in the disruption of autophagy, increased inflammation and increased oxidative stress protein markers. (3) Antioxidant-rich berry diets significantly reduced the accumulation of toxic cellular debris in critical regions of the brain, primarily at the 30 days post-irradiation time-point. (4) Susceptibility to inflammation, autophagy dysregulation, and oxidative stress were proportional to the levels of antioxidant enzymes in the respective brain regions. (5) Exposure to 56Fe radiation may cause the accumulation of disease-related proteins such as PHF-Tau, which has been implicated in the pathogenesis of Alzheimer's disease.Irradiation with 56Fe, which causes substantial build-up of toxic cellular debris in critical regions of the brain, may overwhelm the innate antioxidant enzyme defense system [ 5]. Therefore, berry diets high in antioxidants may be used to counter these damaging effects by reducing oxidative stress and inflammation, and activating neuronal housekeeping, in addition to boosting endogenous antioxidant enzymes.This paper was presented at the NASA Session at Heavy Ion in Therapy and Space Radiation Symposium 2013.

56Fe ion irradiation enhances angiogenesis and other inter-cellular determinants of carcinogenesis risk

In the assessment of radiogenic cancer risk from space flight, it is imperative to consider effects not only on the creation of cancer cells (initiation) but also on cell–cell interactions that play an important and often decisive role in the promotion and progression phases. Autopsy results confirm that most adults carry fully malignant tumors that are held in check at a small size and will never become symptomatic [ 1, 2]. This introduces the possibility that cosmic radiation may significantly influence cancer risk through alteration of the bottleneck inter-tissue interactions responsible for maintaining this dormant state. One such bottleneck is the growth limitation imposed by the failure of the tumor to induce blood vessels (angiogenesis). Other deciding events are the ability of a tumor to proliferate and invade. We have previously shown that proton radiation, the most prevalent radiation in space, has a suppressive effect on all three of these functional responses. It down-regulates angiogenic genes like VEGF and HIF-1α and impairs cell invasion and tumor growth [ 3]. We decided to test these responses after 56Fe irradiation, an HZE radiation type present in the cosmic environment with presumably high carcinogenic potential [ 4].Human microvascular endothelial cells (HMVEC) and normal human dermal fibroblast (NHDF) cells were irradiated with different doses of 56Fe ion radiation (1 GeV/n) at Brookhaven National Laboratory and RNA was extracted 6 h later. Genomic-wide array analysis was done on the isolated RNA through the Agilent Platform. It was observed that several pro-angiogenic genes like VEGF, IL-6 and HIF-1α were significantly up-regulated after treatment with 56Fe ion radiation (Fig. 1). These results were also confirmed at the mRNA and protein levels with the human and murine lung cancer lines, A549 and LLC, respectively. Additional verification of modulation of these key genes was also observed when lungs of C57BL/6 mice treated with 56Fe ion radiation showed an increase in VEGF and MMP9 mRNA and protein expression 6 h post-irradiation (Fig. 2). Cell invasion was shown to be increased by 56Fe ion radiation in various cell types, including fibroblast, tumor and endothelial progenitor cells. 56Fe ion irradiation also modulated functional processes crucial to angiogenesis. It enhanced the ability of untargeted (bystander) endothelial cells to invade and proliferate in response to factors produced by targeted fibroblast or cancer cells in vitro. Results also carry over to in vivo. C57BL/6 mice exposed to whole-body irradiation with 0.2 Gy dose of 56Fe and injected subcutaneously with LLC tumor cells showed a significant augmentation in tumor growth and growth rate in the irradiated group. Additionally, nude mice exposed to whole-body 56Fe radiation and injected intravenously with A549 cancer cells 3 h post-irradiation demonstrated a significant enhancement in lung colonization capacity when compared with the sham-irradiated control mice injected.These results together suggest cell and tissue-level responses to 56Fe irradiation may act to overcome major cancer progression-level bottlenecks including those related to angiogenesis, cell proliferation and invasion. This is of significant concern for cancer risk estimations pertinent to NASA as achieving these cancer hallmark processes can make the difference between a radiation-induced cancer cell progressing to a clinically detectable cancer in astronauts or not. In conclusion, we demonstrate a strong radiation quality dependence for space radiation carcinogenesis risk manifested through influences on intercellular interactions in the progression phase of carcinogenesis. Fig. 1.Heatmaps of selected differentially regulated major angiogenesis genes after proton and 56Fe ion radiation in HMVECs and NHDF. Cells were treated with either 0, 0.5, 1 or 2 Gy of proton radiation or 0, 0.2, 0.4 or 1 Gy of 56Fe ion dose. Among the major regulated genes were VEGF, HIF-1A and IL-6; they were down-regulated by proton radiation and up-regulated by iron radiation. Fig. 2.Immunofluorescence images of lungs of C57BL/6 mice treated with 0, 0.2 or 1 Gy of 56Fe ion dose and stained 6 h later. Pro-angiogenic factors VEGF and MMP9 were increased in mice that received the 56Fe ion treatment.

Long-Term Differential Changes in Mouse Intestinal Metabolomics after γ and Heavy Ion Radiation Exposure

Tissue consequences of radiation exposure are dependent on radiation quality and high linear energy transfer (high-LET) radiation, such as heavy ions in space is known to deposit higher energy in tissues and cause greater damage than low-LET γ radiation. While radiation exposure has been linked to intestinal pathologies, there are very few studies on long-term effects of radiation, fewer involved a therapeutically relevant γ radiation dose, and none explored persistent tissue metabolomic alterations after heavy ion space radiation exposure. Using a metabolomics approach, we report long-term metabolomic markers of radiation injury and perturbation of signaling pathways linked to metabolic alterations in mice after heavy ion or γ radiation exposure. Intestinal tissues (C57BL/6J, female, 6 to 8 wks) were analyzed using ultra performance liquid chromatography coupled with electrospray quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) two months after 2 Gy γ radiation and results were compared to an equitoxic 56Fe (1.6 Gy) radiation dose. The biological relevance of the metabolites was determined using Ingenuity Pathway Analysis, immunoblots, and immunohistochemistry. Metabolic profile analysis showed radiation-type-dependent spatial separation of the groups. Decreased adenine and guanosine and increased inosine and uridine suggested perturbed nucleotide metabolism. While both the radiation types affected amino acid metabolism, the 56Fe radiation preferentially altered dipeptide metabolism. Furthermore, 56Fe radiation caused upregulation of ‘prostanoid biosynthesis’ and ‘eicosanoid signaling’, which are interlinked events related to cellular inflammation and have implications for nutrient absorption and inflammatory bowel disease during space missions and after radiotherapy. In conclusion, our data showed for the first time that metabolomics can not only be used to distinguish between heavy ion and γ radiation exposures, but also as a radiation-risk assessment tool for intestinal pathologies through identification of biomarkers persisting long after exposure.

Therapeutic and space radiation exposure of mouse brain causes impaired DNA repair response and premature senescence by chronic oxidant production

Despite recent epidemiological evidences linking radiation exposure and a number of human ailments including cancer, mechanistic understanding of how radiation inflicts long-term changes in cerebral cortex, which regulates important neuronal functions, remains obscure. The current study dissects molecular events relevant to pathology in cerebral cortex of 6 to 8 weeks old female C57BL/6J mice two and twelve months after exposure to a γ radiation dose (2 Gy) commonly employed in fractionated radiotherapy. For a comparative study, effects of 1.6 Gy heavy ion 56Fe radiation on cerebral cortex were also investigated, which has implications for space exploration. Radiation exposure was associated with increased chronic oxidative stress, oxidative DNA damage, lipid peroxidation, and apoptosis. These results when considered with decreased cortical thickness, activation of cell-cycle arrest pathway, and inhibition of DNA double strand break repair factors led us to conclude to our knowledge for the first time that radiation caused aging-like pathology in cerebral cortical cells and changes after heavy ion radiation were more pronounced than γ radiation.