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.

Threats of nuclear terrorism coupled with potential unintentional ionizing radiation exposures have necessitated the need for large-scale response efforts of such events, including high-throughput biodosimetry for medical triage. Global metabolomics utilizing mass spectrometry (MS) platforms has proven an ideal tool for generating large compound databases with relative quantification and structural information in a short amount of time. Determining metabolite panels for biodosimetry requires experimentation to evaluate the many factors associated with compound concentrations in biofluids after radiation exposures, including temporal changes, pre-existing conditions, dietary intake, partial- vs. total-body irradiation (TBI), among others. Here, we utilize a nonhuman primate (NHP) model and identify metabolites perturbed in serum after 7.2 Gy TBI without supportive care [LD_70/60, hematologic (hematopoietic) acute radiation syndrome (HARS) level H3] at 24, 36, 48 and 96 h compared to preirradiation samples with an ultra-performance liquid chromatography quadrupole time-of-flight (UPLC-QTOF) MS platform. Additionally, we document changes in cytokine levels. Temporal changes observed in serum carnitine, acylcarnitines, amino acids, lipids, deaminated purines and increases in pro-inflammatory cytokines indicate clear metabolic dysfunction after radiation exposure. Multivariate data analysis shows distinct separation from preirradiation groups and receiver operator characteristic curve analysis indicates high specificity and sensitivity based on area under the curve at all time points after 7.2 Gy irradiation. Finally, a comparison to a 6.5 Gy (LD_50/60, HARS level H2) cohort after 24 h postirradiation revealed distinctly increased separations from the 7.2 Gy cohort based on multivariate data models and higher compound fold changes. These results highlight the utility of MS platforms to differentiate time and absorbed dose after a potential radiation exposure that may aid in assigning specific medical interventions and contribute as additional biodosimetry tools.

Hypofractionated radiotherapy is a new and highly effective mode of radiation therapy. For this study we used biologically equivalent dose (BED), the dose required to give the same log cell kill as the schedule being studied. BED has been widely accepted to transform its dose to conventionally fractionated ones. However, actual differential effects beyond the clone-forming ability between hypofractionation and conventional radiation treatment remain unknown. We hypothesize that hypofractionation has some advantages over conventional treatment in in vitro radiobiology, excluding influences of the tumor microenvironment in angiogenesis and potential immune-stimulatory effects. For this study, two non-small cell lung cancer (NSCLC) cell lines with different α/β values were chosen: A549 (α/β = 12.4) and H460 (α/β = 2.95). We designed the following two fractionation regimens with equal BED: A549-HRT (10 Gy/1 fraction) and A549-CRT (16 Gy/8 fractions) as well as H460-HRT (10 Gy/1 fraction) and H460-CRT (26 Gy/13 fractions). After irradiation, we performed cell counting, MTT assay, flow cytometry analysis of apoptosis and cell cycle, immunocytofluorescence of γ-H2AX and Hoechst 33258, and senescence-associated β-galactosidase assay to identify differential effects. Glucose consumption and lactic acid production per cell were tested using glucose and lactate assays. Two weeks postirradiation, we collected early-passage cells of the colony cells after both conventional and fractionated irradiations for further investigation. Then, we used the side population (SP) assay, cell-counting assay and Transwell assay to test the proliferation and invasion capability, the MTT assay to identify the drug resistance of cisplatin, pemetrexed and docetaxel, the Western blot assay to test the stem cell-related proteins of NANOG, CD133, OCT4, SOX2, BMI1 and KLF4. After irradiation, the total cell count and cell viability in both cell lines gradually decreased in a similar manner. However, more senescent, necrotic cells and apoptotic cells were found in the conventionally-treated cells at an early time point postirradiation. Contrarily, a higher percentage of G₂/M cell cycle arrest and more γ-H2AX foci were found in the cell lines that received hypofractionated treatment. Glucose consumption and lactic acid production per cell were lower in the cell lines that received hypofractionated irradiation. Early-passage cells in the conventional-treated cell line showed more SP cells with higher expressions of NANOG, OCT4 and BMI1 proteins. Early-passage cells in the conventional-treated cell line also revealed higher proliferative ability, drug resistance and invasion ability. Although we detected some radiobiological differences between the two fractionation treatments, there was no obvious advantage for hypofractionation in the early days postirradiation. However, there were some advantages for hypofractionation compared to conventional treatment in early-passage cells in vitro, which may partially contribute to its clinical advantages. Moreover, the damage to healthy tissue should also be addressed to fully elucidate the implications of radiotherapy addressed in this work.

The RTGene study was focused on the development and validation of new transcriptional biomarkers for prediction of individual radiotherapy patient responses to ionizing radiation. In parallel, for validation purposes, this study incorporated conventional biomarkers of radiation exposure, including the dicentric assay. Peripheral blood samples were taken with ethical approval and informed consent from a total of 20 patients undergoing external beam radiotherapy for breast, lung, gastrointestinal or genitourinary tumors. For the dicentric assay, two samples were taken from each patient: prior to radiotherapy and before the final fraction. Blood samples were set up using standard methods for the dicentric assay. All the baseline samples had dicentric frequencies consistent with the expected background for the normal population. For blood taken before the final fraction, all the samples displayed distributions of aberrations, which are indicative of partial-body exposures. Whole-body and partial-body cytogenetic doses were calculated with reference to a 250-kVp X-ray calibration curve and then compared to the dose to blood derived using two newly developed blood dosimetric models. Initial comparisons indicated that the relationship between these measures of dose appear very promising, with a correlation of 0.88 (P = 0.001). A new Bayesian zero-inflated Poisson finite mixture method was applied to the dicentric data, and partial-body dose estimates showed no significant difference (P > 0.999) from those calculated by the contaminated Poisson technique. The next step will be further development and validation in a larger patient group.

Inbred strains of mice differ in susceptibility to both radiation-induced and bleomycin-induced pulmonary fibrosis and these traits have been mapped to a common locus on chromosome 6 which harbors genes of natural killer cell function. To investigate this putative locus of fibrosis susceptibility we assessed the fibrotic response of chromosome-6 consomic mice (B6.6A), and of mice deficient for natural killer cells, C57BL/6J Ly49A transgenic mice, after each of thoracic irradiation and bleomycin treatment via osmotic minipump. Thoracic irradiation resulted in less than 15% survival at 26 weeks in parental strain C57BL/6J and A/J mice, due to the development of pneumonitis with fibrosis in C57BL/6J (B6) mice, and pneumonitis in A/J mice. One hundred percent of consomic B6.6A mice survived at 26 weeks after thoracic irradiation, and developed a fibrosis level similar to that of fibrosis-resistant A/J mice, after irradiation (P = 0.38) or bleomycin challenge (P = 0.32). C57BL/6J Ly49A transgenic mice were confirmed through flow cytometric analysis to be deficient in NK cells, but the post-irradiation survival of these mice was not significantly different from that of wild-type littermate mice (P = 0.64). Extent of pulmonary fibrosis by histological examination did not differ between C57BL/6J Ly49A transgenic mice and wild-type littermate mice in response to either irradiation (P = 0.14) or bleomycin treatment (P = 0.62). We conclude that chromosome 6 genes, but not NK cells, contribute to the susceptibility to both radiation-induced and bleomycin-induced pulmonary fibrosis of C57BL/6J mice.

In this study, we investigated the effects of low-to-moderate doses of radiation in mice, given our limited understanding of the health risks associated with these exposures. Here, we demonstrate the different responses of the CD2F1 mouse hematopoietic system to low-to-moderate (0.5, 1, 3 or 5 Gy) doses of gamma radiation. After 3 and 5 Gy of ⁶⁰Co total-body irradiation (TBI), mouse blood cell counts were decreased and maintained below baseline up to 28–42 days. In contrast, after 0.5 Gy TBI, lymphocyte and monocyte counts increased, and peaked from day 3 to day 14. Radiation doses at 0.5 and 1 Gy did not cause cell death or T-cell subpopulation changes in spleen and thymus, whereas the clonogenicity of mouse bone marrow (BM) progenitor cells was significantly suppressed on the first day after 0.5–5 Gy TBI, and these low levels were maintained up to 42 days. Although a transient recovery in total colony forming units (CFUs) was shown in mouse BM at days 14 and 21 after 0.5 Gy TBI, the early-stage multipotential progenitor colonies (CFU-GEMM) remained at a significantly low level compared to those of the sham-irradiated (0 Gy) controls. Consistently, the level of stem cell factor (SCF) in BM cells was decreased after low-to-moderate TBI. Serum from individual mice was collected after irradiation and 23 cytokines/chemokines were measured; massive releases of cytokines and chemokines were observed at day 3 postirradiation in a dose-dependent manner. When human hematopoietic CD34⁺ cells were cultured with the serum collected from mice irradiated at different doses, a significant decrease of CFU-GEMM colonies in the CD34⁺ cells was observed. Our data suggest that low-to-moderate doses of radiation induced cellular responses that are cell type-dependent. The early stage multipotential progenitor cells in mouse BM were the most sensitive cells even to low-dose irradiation compared to spleen and thymic cells, and 0.5 Gy TBI induced hematopoietic cell injury from day 1 to the end of our experiment, day 42 postirradiation. Radiation-induced decrease of SCF in mouse BM and increase in circulating pro-inflammatory factors may be responsible for the enhanced sensitivity of hematopoietic progenitor cells to radiation.

Age at exposure is a critical factor that influences the risk of radiation-induced leukemia. Accumulating evidence suggests that ionizing radiation can induce genomic instability and promote leukemogenesis in hematopoietic stem cells (HSCs); however, the influence of age on this phenomenon has not been elucidated. In this study, infant (1-week-old) or adult (14-week-old) C3H/He mice received sham or 4 Gy whole-body irradiation, and bone marrow cells were transplanted to recipients at day 1 or 60 postirradiation. Twelve days after bone marrow transplant, we analyzed the radiation-induced genomic instability by scoring the frequency of DNA damage and micronucleus formation in colony-forming units-spleen (CFU-Ss). We observed significant increases in DNA damage and micronucleus formation in CFU-Ss of the 4 Gy irradiated adult cells transplanted at day 1 or 60 postirradiation. However, the frequency of DNA damage focus and micronucleus formation in CFU-Ss of 4 Gy irradiated infant cells transplanted at day 1 or 60 postirradiation was relatively decreased. Quantitative differences in the reactive oxygen species and cells expressing inducible nitric oxide synthase in CFU-Ss suggested that age-dependent radiation-induced genomic instability may result from chronic oxidative stress by pro-inflammatory states in HSC descendants after radiation exposure.

We investigated the performance of several commonly used fluorescent dyes after exposure to a simulated Europa mission total ionizing radiation dose of 300 krad (3 kGy) applied using a ⁶⁰Co source. Dyes irradiated in aqueous solution or as lyophilized powders were evaluated for absorbance and emission spectra, quantum yield, and where appropriate, ability to label cells or nucleic acids. Although some dyes showed significant increase or decrease in quantum yield with the dose, their spectra and cell-labeling properties remained essentially unchanged after irradiation in powder form. Irradiation in aqueous solution led to significantly greater changes, including a large blue shift in the DNA intercalator propidium iodide. These results suggest that many fluorescent probes are appropriate for use in astrobiological missions to Europa, but that SYTO9 and propidium iodide should be used with caution or not mixed with each other, as is commonly done in “Live/Dead” labeling applications.

Since the peripheral serotoninergic pathway is involved in the development of radiation-induced nausea and vomiting, referred to as radiation sickness, serotonin 5-HT₃ receptor antagonists are used as a preventive measure, although patients still suffer from these symptoms. Glutamate is known as the excitatory neurotransmitter and is involved in various autonomic symptoms. We investigated the effect of radiation on glutamate release in rats, as measured by in vivo brain microdialysis, and the effects of glutamate receptor antagonists on radiation-induced pica, which can be used as a behavioral assessment of radiation sickness in rats. A microdialysis probe was inserted into the hypothalamus of rats that received 4 Gy total-body irradiation (TBI) with or without pretreatment of 5-HT₃ receptor antagonist (granisetron, 0.1 mg/kg, i.p.), and dialysates were collected for 3 h after TBI and subjected to HPLC assay of glutamate. In addition, rats were intracerebroventricularly injected with NMDA receptor antagonist (MK-801: 3 μg/rat) or AMPA receptor antagonist (CNQX: 1 μg/rat) before TBI, and radiation-induced pica was determined. An increase in glutamate release was observed within 1 h postirradiation. The increased glutamate release was suppressed by granisetron. We also found that CNQX, but not MK-801, effectively inhibited radiation-induced pica. These results indicate that the hypothalamic glutamatergic system contributes to radiation-induced pica through the AMPA receptors.

Increasingly, the risk of a radiological or nuclear public health emergency is a major concern for the U.S. government. To address a potential incident and ensure that the U.S. Government is prepared to respond to any civilian or military casualties that could result, the U.S. Department of Health and Human Services (HHS), together with the Department of Defense, has been charged with the development of medical countermeasures (MCMs) to treat individuals experiencing acute and delayed injuries that can result from exposure to radiation. With limited research and development budgets, and the high costs associated with bringing promising approaches from the bench through advanced product development activities, and ultimately, to regulatory approval, the U.S. Government places a priority on repurposing drugs that have already been commercialized for other indications in humans. To address the benefits and challenges of repurposing licensed products for a radiation indication, the National Institute of Allergy and Infectious Diseases convened a workshop with participants from U.S. Government agencies and industry, as well as academic subject matter experts. Topics included U.S. Government efforts (e.g., funding, regulatory, stockpiling and innovative ways to make drugs available for study), as well as the unique regulatory and other challenges faced when repurposing branded or generic drugs.

The risk of a radiological or nuclear public health emergency is a major growing concern of the U.S. government. To address a potential incident and ensure that the government is prepared to respond to any subsequent civilian or military casualties, the U.S. Department of Health and Human Services and the Department of Defense have been charged with the development of medical countermeasures (MCMs) to treat the acute and delayed injuries that can result from radiation exposure. Because of the limited budgets in research and development and the high costs associated with bring promising approaches from the bench through advanced product development activities, and ultimately, to regulatory approval, the U.S. government places a priority on repurposing products for which there already exists relevant safety and other important information concerning their use in humans. Generating human data can be a costly and time-consuming process; therefore, the U.S. government has interest in drugs for which such relevant information has been established (e.g., products for another indication), and in determining if they could be repurposed for use as MCMs to treat radiation injuries as well as chemical and biological insults. To explore these possibilities, the National Institute of Allergy and Infectious Diseases (NIAID) convened a workshop including U.S. government, industry and academic subject matter experts, to discuss the challenges and benefits of repurposing products for a radiation indication. Topics covered included a discussion of U.S. government efforts (e.g. funding, stockpiling and making products available for study), as well unique regulatory and other challenges faced when repurposing patent protected or generic drugs. Other discussions involved lessons learned from industry on repurposing pre-license, pipeline products within drug development portfolios. This report reviews the information presented, as well as an overview of discussions from the meeting.