Exposure to multiple ion beams, broadly representative of galactic cosmic rays, causes perivascular cardiac fibrosis in mature male rats.

Long-duration space exploratory missions to the Earth's moon and the planet Mars are actively being planned. Such missions will require humans to live for prolonged periods beyond low earth orbit where astronauts will be continuously exposed to high energy galactic cosmic rays (GCRs). A major unknown is the potential impact of GCRs on the risks of developing degenerative cardiovascular disease, which is a concern to NASA. A ground-based rat model has been used to provide a detailed characterization of the risk of long-term cardiovascular disease from components of GCRs at radiation doses relevant to future human missions beyond low earth orbit. Six month old male WAG/RijCmcr rats were irradiated at a ground-based charged particle accelerator facility with high energy ion beams broadly representative of GCRs: protons, silicon and iron. Irradiation was given either as a single ion beam or as a combination of three ion beams. For the doses used, the single ion beam studies did not show any significant changes in the known cardiac risk factors and no evidence of cardiovascular disease could be demonstrated. In the three ion beam study, the total cholesterol levels in the circulation increased modestly over the 270 day follow up period, and inflammatory cytokines were also increased, transiently, 30 days after irradiation. Perivascular cardiac collagen content, systolic blood pressure and the number of macrophages found in the kidney and in the heart were each increased 270 days after irradiation with 1.5 Gy of the three ion beam grouping. These findings provide evidence for a cardiac vascular pathology and indicate a possible threshold dose for perivascular cardiac fibrosis and increased systemic systolic blood pressure for complex radiation fields during the 9 month follow up period. The development of perivascular cardiac fibrosis and increased systemic systolic blood pressure occurred at a physical dose of the three ion beam grouping (1.5 Gy) that was much lower than that required to show similar outcomes in earlier studies with the same rat strain exposed to photons. Further studies with longer follow up periods may help determine whether humans exposed to lower, mission-relevant doses of GCRs will develop radiation-induced heart disease.

Correction: Lenarczyk et al. T Cells Contribute to Pathological Responses in the Non-Targeted Rat Heart following Irradiation of the Kidneys. Toxics 2022, 10, 797.

In the original publication [...].

Galactic cosmic ray simulation at the NASA space radiation laboratory - Progress, challenges and recommendations on mixed-field effects.

For missions beyond low Earth orbit to the moon or Mars, space explorers will encounter a complex radiation field composed of various ion species with a broad range of energies. Such missions pose significant radiation protection challenges that need to be solved in order to minimize exposures and associated health risks. An innovative galactic cosmic ray simulator (GCRsim) was recently developed at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). The GCRsim technology is intended to represent major components of the space radiation environment in a ground analog laboratory setting where it can be used to improve understanding of biological risks and serve as a testbed for countermeasure development and validation. The current GCRsim consists of 33 energetic ion beams that collectively simulate the primary and secondary GCR field encountered by humans in space over the broad range of particle types, energies, and linear energy transfer (LET) of interest to health effects. A virtual workshop was held in December 2020 to assess the status of the NASA baseline GCRsim. Workshop attendees examined various aspects of simulator design, with a particular emphasis on beam selection strategies. Experimental results, modeling approaches, areas of consensus, and questions of concern were also discussed in detail. This report includes a summary of the GCRsim workshop and a description of the current status of the GCRsim. This information is important for future advancements and applications in space radiobiology.

Effects of photon irradiation in the presence and absence of hindlimb unloading on the behavioral performance and metabolic pathways in the plasma of Fischer rats.

Introduction: The space environment astronauts experience during space missions consists of multiple environmental challenges, including microgravity. In this study, we assessed the behavioral and cognitive performances of male Fisher rats 2 months after sham irradiation or total body irradiation with photons in the absence or presence of simulated microgravity. We analyzed the plasma collected 9 months after sham irradiation or total body irradiation for distinct alterations in metabolic pathways and to determine whether changes to metabolic measures were associated with specific behavioral and cognitive measures. Methods: A total of 344 male Fischer rats were irradiated with photons (6 MeV; 3, 8, or 10 Gy) in the absence or presence of simulated weightlessness achieved using hindlimb unloading (HU). To identify potential plasma biomarkers of photon radiation exposure or the HU condition for behavioral or cognitive performance, we performed regression analyses. Results: The behavioral effects of HU on activity levels in an open field, measures of anxiety in an elevated plus maze, and anhedonia in the M&M consumption test were more pronounced than those of photon irradiation. Phenylalanine, tyrosine, and tryptophan metabolism, and phenylalanine metabolism and biosynthesis showed very strong pathway changes, following photon irradiation and HU in animals irradiated with 3 Gy. Here, 29 out of 101 plasma metabolites were associated with 1 out of 13 behavioral measures. In the absence of HU, 22 metabolites were related to behavioral and cognitive measures. In HU animals that were sham-irradiated or irradiated with 8 Gy, one metabolite was related to behavioral and cognitive measures. In HU animals irradiated with 3 Gy, six metabolites were related to behavioral and cognitive measures. Discussion: These data suggest that it will be possible to develop stable plasma biomarkers of behavioral and cognitive performance, following environmental challenges like HU and radiation exposure.

T Cells Contribute to Pathological Responses in the Non-Targeted Rat Heart following Irradiation of the Kidneys.

Heart disease is a significant adverse event caused by radiotherapy for some cancers. Identifying the origins of radiogenic heart disease will allow therapies to be developed. Previous studies showed non-targeted effects manifest as fibrosis in the non-irradiated heart after 120 days following targeted X-irradiation of the kidneys with 10 Gy in WAG/RijCmcr rats. To demonstrate the involvement of T cells in driving pathophysiological responses in the out-of-field heart, and to characterize the timing of immune cell engagement, we created and validated a T cell knock downrat on the WAG genetic backgrou nd. Irradiation of the kidneys with 10 Gy of X-rays in wild-type rats resulted in infiltration of T cells, natural killer cells, and macrophages after 120 days, and none of these after 40 days, suggesting immune cell engagement is a late response. The radiation nephropathy and cardiac fibrosis that resulted in these animals after 120 days was significantly decreased in irradiated T cell depleted rats. We conclude that T cells function as an effector cell in communicating signals from the irradiated kidneys which cause pathologic remodeling of non-targeted heart.

Radiation biology workforce in the United States.

In recent decades, the principal goals of participants in the field of radiation biologists have included defining dose thresholds for cancer and non-cancer endpoints to be used by regulators, clinicians and industry, as well as informing on best practice radiation utilization and protection applications. Importantly, much of this work has required an intimate relationship between "bench" radiation biology scientists and their target audiences (such as physicists, medical practitioners and epidemiologists) in order to ensure that the requisite gaps in knowledge are adequately addressed. However, despite the growing risk for public exposure to higher-than-background levels of radiation, e.g. from long-distance travel, the increasing use of ionizing radiation during medical procedures, the threat from geopolitical instability, and so forth, there has been a dramatic decline in the number of qualified radiation biologists in the U.S. Contributing factors are thought to include the loss of applicable training programs, loss of jobs, and declining opportunities for advancement. This report was undertaken in order to begin addressing this situation since inaction may threaten the viability of radiation biology as a scientific discipline.

Effects of 5-Ion Beam Irradiation and Hindlimb Unloading on Metabolic Pathways in Plasma and Brain of Behaviorally Tested WAG/Rij Rats.

A limitation of simulated space radiation studies is that radiation exposure is not the only environmental challenge astronauts face during missions. Therefore, we characterized behavioral and cognitive performance of male WAG/Rij rats 3 months after sham-irradiation or total body irradiation with a simplified 5-ion mixed beam exposure in the absence or presence of simulated weightlessness using hindlimb unloading (HU) alone. Six months following behavioral and cognitive testing or 9 months following sham-irradiation or total body irradiation, plasma and brain tissues (hippocampus and cortex) were processed to determine whether the behavioral and cognitive effects were associated with long-term alterations in metabolic pathways in plasma and brain. Sham HU, but not irradiated HU, rats were impaired in spatial habituation learning. Rats irradiated with 1.5 Gy showed increased depressive-like behaviors. This was seen in the absence but not presence of HU. Thus, HU has differential effects in sham-irradiated and irradiated animals and specific behavioral measures are associated with plasma levels of distinct metabolites 6 months later. The combined effects of HU and radiation on metabolic pathways in plasma and brain illustrate the complex interaction of environmental stressors and highlights the importance of assessing these interactions.

Irradiation of the kidneys causes pathologic remodeling in the nontargeted heart: A role for the immune system.

Cardiac disease is a frequent and significant adverse event associated with radiotherapy for cancer. Identifying the underlying mechanism responsible for radiation injury to the heart will allow interventions to be developed. In the present study, we tested if local kidney irradiation results in remodeling of the shielded, nontargeted heart. One kidney, two kidneys, or the total body of male WAG and Dahl SS rats were irradiated with 10 Gy of X-rays. Local kidney irradiation resulted in systemic hypertension, increased BUN, infiltration of T lymphocytes, natural killer cells, and macrophages into the renal cortex and medulla, and renal fibrosis. Local irradiation of kidneys in WAG rats resulted in remodeling in the nontargeted heart after 120 days, manifested by perivascular fibrosis and increased interventricular septal thickness, but was not seen in Dahl SS rats due to a high baseline level of fibrosis in the sham-irradiated animals. Genetic depletion of T cells mitigated the nephropathy after local kidney irradiation, indicating a role for the immune system in mediating this outcome. Local kidney irradiation resulted in a cascade of pro-inflammatory cytokines and low-molecular weight metabolites into the circulation associated with transmission of signals resulting in pathologic remodeling in the nontargeted heart. A new model is proposed whereby radiation-induced cardiac remodeling in susceptible animals is indirect, with lower hemi body organs such as the kidney exporting factors into the circulation that cause remodeling outside of the irradiated field in the shielded, nontargeted heart. This nontargeted effect appears to be mediated, in part, by the immune system.

Effects of Six Sequential Charged Particle Beams on Behavioral and Cognitive Performance in B6D2F1 Female and Male Mice.

The radiation environment astronauts are exposed to in deep space includes galactic cosmic radiation (GCR) with different proportions of all naturally occurring ions. To assist NASA with assessment of risk to the brain following exposure to a mixture of ions broadly representative of the GCR, we assessed the behavioral and cognitive performance of female and male C57BL/6J × DBA2/J F1 (B6D2F1) mice two months following rapidly delivered, sequential 6 beam irradiation with protons (1 GeV, LET = 0.24 keV, 50%), 4He ions (250 MeV/n, LET = 1.6 keV/µm, 20%), 16O ions (250 MeV/n, LET = 25 keV/µm 7.5%), 28Si ions (263 MeV/n, LET = 78 keV/µm, 7.5%), 48Ti ions (1 GeV/n, LET = 107 keV/µm, 7.5%), and 56Fe ions (1 GeV/n, LET = 151 keV/µm, 7.5%) at 0, 25, 50, or 200 cGy) at 4-6 months of age. When the activity over 3 days of open field habituation was analyzed in female mice, those irradiated with 50 cGy moved less and spent less time in the center than sham-irradiated mice. Sham-irradiated female mice and those irradiated with 25 cGy showed object recognition. However, female mice exposed to 50 or 200 cGy did not show object recognition. When fear memory was assessed in passive avoidance tests, sham-irradiated mice and mice irradiated with 25 cGy showed memory retention while mice exposed to 50 or 200 cGy did not. The effects of radiation passive avoidance memory retention were not sex-dependent. There was no effect of radiation on depressive-like behavior in the forced swim test. There was a trend toward an effect of radiation on BDNF levels in the cortex of males, but not for females, with higher levels in male mice irradiated with 50 cGy than sham-irradiated. Finally, sequential 6-ion irradiation impacted the composition of the gut microbiome in a sex-dependent fashion. Taxa were uncovered whose relative abundance in the gut was associated with the radiation dose received. Thus, exposure to sequential six-beam irradiation significantly affects behavioral and cognitive performance and the gut microbiome.

Age at Exposure to Radiation Determines Severity of Renal and Cardiac Disease in Rats.

Radiotherapy with sparsely ionizing photons is a cornerstone of successful cancer treatment. Age at time of exposure to radiation is known to influence biological outcomes for many end points. The effect of dose and age at exposure upon the occurrence of radiogenic cardiovascular disease is poorly understood. The goal of this work was to determine the response of maleWAG/RijCmcr rats at 6 months of age to gamma rays, and at 6 months or 6 weeks of age to X rays, using clinically relevant biomarkers of cardiovascular disease and kidney injury. Overall, there were significant radiation-induced effects on the levels of bicarbonate (P=0.0016), creatinine (P=0.0002), calcium (P = 0.0009), triglycerides (P = 0.0269) and blood urea nitrogen, albumin, protein, AST, alkaline phosphatase, total cholesterol and HDL (all P < 0.0001). Of those variables with a significant radiation-dose effect, there were significant modifications by age at time of exposure for bicarbonate (P = 0.0033), creatinine (P = 0.0015), AST (P = 0.0040), total cholesterol (P = 0.0006) and blood urea nitrogen, calcium, albumin, protein, alkaline phosphatase and HDL (all P < 0.0001). Cardiac perivascular collagen content was significantly increased in rats that were 8.0 Gy X-ray irradiated at 6 weeks of age (P < 0.047) but not at 6 months of age. While systemic blood pressure was elevated in both cohorts after 8.0 Gy X-ray irradiation (compared to agematched sham-irradiated controls), the magnitude of the increase above baseline was greater in the younger rats (P < 0.05). These findings indicate that dose and age at time of irradiation determine the timeline and severity of cardiac and renal injury.

Combined Effects of Three High-Energy Charged Particle Beams Important for Space Flight on Brain, Behavioral and Cognitive Endpoints in B6D2F1 Female and Male Mice.

The radiation environment in deep space includes the galactic cosmic radiation with different proportions of all naturally occurring ions from protons to uranium. Most experimental animal studies for assessing the biological effects of charged particles have involved acute dose delivery for single ions and/or fractionated exposure protocols. Here, we assessed the behavioral and cognitive performance of female and male C57BL/6J × DBA2/J F1 (B6D2F1) mice 2 months following rapidly delivered, sequential irradiation with protons (1 GeV, 60%), 16O (250 MeV/n, 20%), and 28Si (263 MeV/n, 20%) at 0, 25, 50, or 200 cGy at 4-6 months of age. Cortical BDNF, CD68, and MAP-2 levels were analyzed 3 months after irradiation or sham irradiation. During the dark period, male mice irradiated with 50 cGy showed higher activity levels in the home cage than sham-irradiated mice. Mice irradiated with 50 cGy also showed increased depressive behavior in the forced swim test. When cognitive performance was assessed, sham-irradiated mice of both sexes and mice irradiated with 25 cGy showed normal responses to object recognition and novel object exploration. However, object recognition was impaired in female and male mice irradiated with 50 or 200 cGy. For cortical levels of the neurotrophic factor BDNF and the marker of microglial activation CD68, there were sex × radiation interactions. In females, but not males, there were increased CD68 levels following irradiation. In males, but not females, there were reduced BDNF levels following irradiation. A significant positive correlation between BDNF and CD68 levels was observed, suggesting a role for activated microglia in the alterations in BDNF levels. Finally, sequential beam irradiation impacted the diversity and composition of the gut microbiome. These included dose-dependent impacts and alterations to the relative abundance of several gut genera, such as Butyricicoccus and Lachnospiraceae. Thus, exposure to rapidly delivered sequential proton, 16O ion, and 28Si ion irradiation significantly affects behavioral and cognitive performance, cortical levels of CD68 and BDNF in a sex-dependent fashion, and the gut microbiome.

Detrimental Effects of Helium Ion Irradiation on Cognitive Performance and Cortical Levels of MAP-2 in B6D2F1 Mice.

The space radiation environment includes helium (4He) ions that may impact brain function. As little is known about the effects of exposures to 4He ions on the brain, we assessed the behavioral and cognitive performance of C57BL/6J × DBA2/J F1 (B6D2F1) mice three months following irradiation with 4He ions (250 MeV/n; linear energy transfer (LET) = 1.6 keV/μm; 0, 21, 42 or 168 cGy). Sham-irradiated mice and mice irradiated with 21 or 168 cGy showed novel object recognition, but mice irradiated with 42 cGy did not. In the passive avoidance test, mice received a slight foot shock in a dark compartment, and latency to re-enter that compartment was assessed 24 h later. Sham-irradiated mice and mice irradiated with 21 or 42 cGy showed a higher latency on Day 2 than Day 1, but the latency to enter the dark compartment in mice irradiated with 168 cGy was comparable on both days. 4He ion irradiation, at 42 and 168 cGy, reduced the levels of the dendritic marker microtubule-associated protein-2 (MAP-2) in the cortex. There was an effect of radiation on apolipoprotein E (apoE) levels in the hippocampus and cortex, with higher apoE levels in mice irradiated at 42 cGy than 168 cGy and a trend towards higher apoE levels in mice irradiated at 21 than 168 cGy. In addition, in the hippocampus, there was a trend towards a negative correlation between MAP-2 and apoE levels. While reduced levels of MAP-2 in the cortex might have contributed to the altered performance in the passive avoidance test, it does not seem sufficient to do so. The higher hippocampal and cortical apoE levels in mice irradiated at 42 than 168 cGy might have served as a compensatory protective response preserving their passive avoidance memory. Thus, there were no alterations in behavioral performance in the open filed or depressive-like behavior in the forced swim test, while cognitive impairments were seen in the object recognition and passive avoidance tests, but not in the contextual or cued fear conditioning tests. Taken together, the results indicate that some aspects of cognitive performance are altered in male mice exposed to 4He ions, but that the response is task-dependent. Furthermore, the sensitive doses can vary within each task in a non-linear fashion. This highlights the importance of assessing the cognitive and behavioral effects of charged particle exposure with a variety of assays and at multiple doses, given the possibility that lower doses may be more damaging due to the absence of induced compensatory mechanisms at higher doses.

Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change.

Exposure to a small number of high-energy heavy charged particles (HZE ions), as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/µm) in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/µm) at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis.

Sex- and dose-dependent effects of calcium ion irradiation on behavioral performance of B6D2F1 mice during contextual fear conditioning training.

The space radiation environment includes energetic charged particles that may impact behavioral and cognitive performance. The relationship between the dose and the ionization density of the various types of charged particles (expressed as linear energy transfer or LET), and cognitive performance is complex. In our earlier work, whole body exposure to (28)Si ions (263 MeV/n, LET=78keV/µm; 1.6 Gy) affected contextual fear memory in C57BL/6J × DBA2/J F1 (B6D2F1) mice three months following irradiation but this was not the case following exposure to (48)Ti ions (1 GeV/n, LET=107keV/µm; 0.2 or 0.4 Gy). As an increased understanding of the impact of charged particle exposures is critical for assessment of risk to the CNS of astronauts during and following missions, in this study we used (40)Ca ion beams (942 MeV/n, LET=90keV/µm) to determine the behavioral and cognitive effects for the LET region between that of Si ions and Ti ions. (40)Ca ion exposure reduced baseline activity in a novel environment in a dose-dependent manner, which suggests reduced motivation to explore and/or a diminished level of curiosity in a novel environment. In addition, exposure to (40)Ca ions had sex-dependent effects on response to shock. (40)Ca ion irradiation reduced the response to shock in female, but not male, mice. In contrast, (40)Ca ion irradiation did not affect fear learning, memory, or extinction of fear memory for either gender at the doses employed in this study. Thus (40)Ca ion irradiation affected behavioral, but not cognitive, performance. The effects of (40)Ca ion irradiation on behavioral performance are relevant, as a combination of novelty and aversive environmental stimuli is pertinent to conditions experienced by astronauts during and following space missions.