A complex cascade of systemic and tissue-specific responses induced by exposure to ionizing radiation can lead to functional impairment over time in the surviving population. Current methods for management of survivors of unintentional radiation exposure episodes rely on monitoring individuals over time for the development of adverse clinical symptoms due to the lack of predictive biomarkers for tissue injury. In this study, we report on changes in metabolomic and lipidomic profiles in multiple tissues of nonhuman primates (NHPs) that received either 4.0 Gy or 5.8 Gy total-body irradiation (TBI) of ⁶⁰Co gamma rays, and 4.0 or 5.8 Gy partial-body irradiation (PBI) from LINAC-derived photons and were treated with a promising radiation countermeasure, gamma-tocotrienol (GT3). These include small molecule alterations that correlate with radiation effects in the jejunum, lung, kidney, and spleen of animals that either survived or succumbed to radiation toxicities over a 30-day period. Radiation-induced metabolic changes in tissues were observed in animals exposed to both doses and types of radiation, but were partially alleviated in GT3-treated and irradiated animals, with lung and spleen being most responsive. The majority of the pathways protected by GT3 treatment in these tissues were related to glucose metabolism, inflammation, and aldarate metabolism, suggesting GT3 may exert radioprotective effects in part by sparing these pathways from radiation-induced dysregulation. Taken together, the results of our study demonstrate that the prophylactic administration of GT3 results in metabolic and lipidomic shifts that likely provide an overall advantage against radiation injury. This investigation is among the first to highlight the use of a molecular phenotyping approach in a highly translatable NHP model of partial- and total-body irradiation to determine the underlying physiological mechanisms involved in the radioprotective efficacy of GT3.

Radiosensitivity differs in humans and possibly in closely related nonhuman primates. The reasons for variation in radiosensitivity are not well known. In an earlier study, we examined gene expression (GE) pre-radiation in peripheral blood among male (n = 62) and female (n = 60) rhesus macaques (n = 122), which did or did not survive (up to 60 days) after whole-body exposure of 7.0 Gy (LD_66/60). Eight genes (CHD5, CHI3L1, DYSF, EPX, IGF2BP1, LCN2, MBOAT4, SLC22A4) revealed significant associations with survival. Access to a second rhesus macaque cohort (males = 40, females = 23, total n = 63) irradiated with 5.8–7.2 Gy (LD_29-50/60) and some treated with gamma-tocotrienol (GT3, a radiation countermeasure) allowed us to validate these gene expression changes independently. Total RNA was isolated from whole blood samples and examined by quantitative RT-PCR on a 96-well format. cycle threshold (Ct)-values normalized to 18S rRNA were analyzed for their association with survival. Regardless of the species-specific TaqMan assay, similar results were obtained. Two genes (CHD5 and CHI3L1) out of eight revealed a significant association with survival in the second cohort, while only CHD5 (involved in DNA damage response and proliferation control) showed mean gene expression changes in the same direction for both cohorts. No expected association of CHD5 GE with dose, treatment, or sex could be established. Instead, we observed significant associations for those comparisons comprising pre-exposure samples with CHD5 Ct values ≤ 11 (total n = 17). CHD5 Ct values ≤ 11 in these comparisons were mainly associated with increased frequencies (61-100%) of non-survivors, a trend which depending on the sample numbers, reached significance (P = 0.03) in males and, accordingly, in females. This was also reflected by a logistic regression model including all available samples from both cohorts comprising CHD5 measurements (n = 104, odds ratio 1.38, 95% CI 1.07–1.79, P = 0.01). However, this association was driven by males (odds ratio 1.62, 95% CI 1.10–2.38, P = 0.01) and CHD5 Ct values ≤ 11 since removing low CHD5 Ct values from this model, converted to insignificance (P = 0.19). A second male subcohort comprising high CHD5 Ct values ≥ 14.4 in both cohorts (n = 5) appeared associated with survival. Removing these high CHD5 Ct values converted the model borderline significant (P = 0.051). Based on the probability function of the receiver operating characteristics (ROC) curves, 8 (12.3%) and 5 (7.7%) from 65 pre-exposure RNA measurements in males, death and survival could be predicted with a negative and positive predictive value ranging between 85–100%. An associated odds ratio reflected a 62% elevated risk for dying or surviving per unit change (Ct-value) in gene expression, considering the before-mentioned CHD5 thresholds in RNA copy numbers. In conclusion, we identified two subsets of male animals characterized by increased (Ct values ≤ 11) and decreased (Ct values ≥ 14.4) CHD5 GE copy numbers before radiation exposure, which independently of the cohort, radiation exposure or treatment appeared to predict the death or survival in males.

After nuclear scenarios, combined injuries of acute radiation syndrome (ARS) with, e.g., abdominal trauma, will occur and may require contrast-enhanced computed tomography (CT) scans for diagnostic purposes. Here, we investigated the effect of iodinated contrast agents on radiation-induced gene expression (GE) changes used for biodosimetry (AEN, BAX, CDKN1A, EDA2R, APOBEC3H) and for hematologic ARS severity prediction (FDXR, DDB2, WNT3, POU2AF1), and on the induction of double-strand breaks (DSBs) used for biodosimetry. Whole blood samples from 10 healthy donors (5 males, 5 females, mean age: 28 ± 2 years) were irradiated with X rays (0, 1 and 4 Gy) with and without the addition of iodinated contrast agent (0.016 ml contrast agent/ml blood) to the blood prior to the exposure. The amount of contrast agent was set to be equivalent to the blood concentration of an average patient (80 kg) during a contrast-enhanced CT scan. After irradiation, blood samples were incubated at 37°C for 20 min (DSB) and 8 h (GE, DSB). GE was measured employing quantitative real-time polymerase chain reaction. DSB foci were revealed by γH2AX + 53BP1 immunostaining and quantified automatically in >927 cells/sample. Radiation-induced differential gene expression (DGE) and DSB foci were calculated using the respective unexposed sample without supplementation of contrast agent as the reference. Neither the GE nor the number of DSB foci was significantly (P = 0.07–0.94) altered by the contrast agent application. However, for some GE and DSB comparisons with/without contrast agent, there were weakly significant differences (P = 0.03–0.04) without an inherent logic and thus are likely due to inter-individual variation. In nuclear events, the diagnostics of combined injuries can require the use of an iodinated contrast agent, which, according to our results, does not alter or influence radiation-induced GE changes and the quantity of DSB foci. Therefore, the gene expression and γH2AX focus assay can still be applied for biodosimetry and/or hematologic ARS severity prediction in such scenarios.

The purpose of this investigation was to characterize the natural history of a murine total-abdominal-irradiation exposure model to measure gastrointestinal acute radiation injury. Male CD2F1 mice at 12 to 15 weeks old received total-abdominal irradiation using 4-MV linear accelerator X-rays doses of 0, 11, 13.5, 15, 15.75 and 16.5 Gy (2.75 Gy/min). Daily cage-side (i.e., in the animal housing room) observations of clinical signs and symptoms including body weights on all animals were measured up to 10 days after exposure. Jejunum tissues from cohorts of mice were collected at 1, 3, 7 and 10 days after exposure and radiation injury was assessed by histopathological analyses. Results showed time- and dose-dependent loss of body weight [for example at 7 days: 0.66 (±0.80) % loss for 0 Gy, 6.40 (±0.76) % loss at 11 Gy, 9.43 (±2.06) % loss at 13.5 Gy, 23.53 (±1.91) % loss at 15 Gy, 29.97 (±1.16) % loss at 15.75 Gy, and 31.79 (±0.76) % loss at 16.5 Gy]. Negligible clinical signs and symptoms, except body weight changes, of radiation injury were observed up to 10 days after irradiation with doses of 11 to 15 Gy. Progressive increases in the severity of clinical signs and symptoms were found after irradiation with doses >15 Gy. Jejunum histology showed a progressive dose-dependent increase in injury. For example, at 7 days postirradiation, the percent of crypts, compared to controls, decreased to 82.3 (±9.5), 69.2 (±12.3), 45.4 (±11.9), 18.0 (±3.4), and 11.5 (±1.8) with increases in doses from 11 to 16.5 Gy. A mucosal injury scoring system was used that mainly focused on changes in villus morphology damage (i.e., subepithelial spaces near the tips of the villi with capillary congestion, significant epithelial lifting along the length of the villi with a few denuded villus tips). Peak levels of total-abdominal irradiation induced effects on the mucosal injury score were seen 7 days after irradiation for doses ≥15 Gy, with a trend to show a decline after 7 days. A murine multiple-parameter gastrointestinal acute-radiation syndrome severity-scoring system was established based on clinical signs and symptoms that included measures of appearance (i.e., hunched and/or fluffed fur), respiratory rate, general (i.e., decreased mobility) and provoked behavior (i.e., subdued response to stimulation), weight loss, and feces/diarrhea score combined with jejunum mucosal-injury grade score. In summary, the natural-history radio-response for murine partial-body irradiation exposures is important for establishing a well-characterized radiation model system; here we established a multiple-parameter gastrointestinal acute-radiation syndrome severity-scoring system that provides a radiation injury gastrointestinal tissue-based assessment utility.

The increased risk of acute large-scale radiological exposure for the world's population underlines the need for optimal radiation biomarkers. Ionizing radiation triggers a complex response by the genome, proteome, and metabolome, all of which have been reported as suitable indicators of radiation-induced damage in vivo. This study analyzed peripheral blood samples from total-body irradiation (TBI) leukemia patients through mass spectrometry (MS) to identify and quantify differentially regulated proteins in plasma before and after irradiation. In brief, samples were taken from 16 leukemic patients prior to and 24 h after TBI (2 × 2.0 Gy), processed with Tandem Mass Tag isobaric labelling kit (TMTpro-16-plex), and analyzed by MS. In parallel, label-free relative quantification was performed with a RP-nanoLC-ESI-MS/MS system in a Q-Exactive mass spectrometer. Protein identification was done in Proteome Discoverer v.2.2 platform (Thermo). Data is available via ProteomeXchange with identifier PXD043516. Using two different methods, we acquired two datasets of up-regulated (ratio ≥ 1.2) or down-regulated (ratio ≤ 0.83) plasmatic proteins 24 h after irradiation, identifying 356 and 346 proteins in the TMT-16plex and 285 and 308 label-free analyses, respectively (P ≤ 0.05). Combining the two datasets yielded 15 candidates with significant relation to gamma-radiation exposure. The majority of these proteins were associated with the inflammatory response and lipid metabolism. Subsequently, from these, five proteins showed the strongest potential as radiation biomarkers in humans (C-reactive protein, Alpha amylase 1A, Mannose-binding protein C, Phospholipid transfer protein, and Complement C5). These candidate biomarkers might have implications for practical biological dosimetry.

The current geopolitical context has brought the radiological nuclear risk to the forefront of concerns. High-dose localized radiation exposure leads to the development of a musculocutaneous radiation syndrome affecting the skin and subcutaneous muscles. Despite the implementation of a gold standard treatment based on an invasive surgical procedure coupled with autologous cell therapy, a muscular defect frequently persists. Targeting the modulation of the Hedgehog (Hh) signaling pathway appears to be a promising therapeutic approach. Activation of this pathway enhances cell survival and promotes proliferation after irradiation, while inhibition by Cyclopamine facilitates differentiation. In this study, we compared the effects of three antagonists of Hh, Cyclopamine (CA), Vismodegib (VDG) and Sonidegib (SDG) on differentiation. A stable cell line of murine myoblasts, C2C12, was exposed to X-ray radiation (5 Gy) and treated with CA, VDG or SDG. Analysis of proliferation, survival (apoptosis), morphology, myogenesis genes expression and proteins production were performed. According to the results, VDG does not have a significant impact on C2C12 cells. SDG increases the expression/production of differentiation markers to a similar extent as CA, while morphologically, SDG proves to be more effective than CA. To conclude, SDG can be used in the same way as CA but already has a marketing authorization with an indication against basal cell cancers, facilitating their use in vivo. This proof of concept demonstrates that SDG represents a promising alternative to CA to promotes differentiation of murine myoblasts. Future studies on isolated and cultured satellite cells and in vivo will test this proof of concept.

The development of effective uses of biodosimetry in large-scale events has been hampered by residual, i.e., legacy thinking based on strategies that scale up from biodosimetry in small accidents. Consequently, there remain vestiges of unrealistic assumptions about the likely magnitude of victims in large radiation events and incomplete analyses of the logistics for making biodosimetry measurements/assessments in the field for primary triage. Elements remain from an unrealistic focus on developing methods to use biodosimetry in the initial stage of triage for a million or more victims. Based on recent events and concomitant increased awareness of the potential for large-scale events as well as increased sophistication in planning and experience in the development of biodosimetry, a more realistic assessment of the most effective roles of biodosimetry in large-scale events is urgently needed. We argue this leads to a conclusion that the most effective utilization of biodosimetry in very large events would occur in a second stage of triage, after initially winnowing the population by identifying those most in need of acute medical attention, based on calculations of geographic sites where significant exposures could have occurred. Understanding the potential roles and limitations of biodosimetry in large-scale events involving significant radiation exposure should lead to development of the most effective and useful biodosimetric techniques for each stage of triage for acute radiation syndrome injuries, i.e., based on more realistic assumptions about the underlying event and the logistics for carrying out biodosimetry for large populations.

In the current geopolitical climate there is an unmet need to identify and develop prophylactic radiation countermeasures, particularly to ensure the well-being of warfighters and first responders that may be required to perform on radiation-contaminated fields for operational or rescue missions. Currently, no countermeasures have been approved by the U.S. FDA for prophylactic administration. Here we report on the efficacious nature of FSL-1 (toll-like receptor 2/6 agonist) and the protection from acute radiation syndrome (ARS) in a murine total-body irradiation (TBI) model. A single dose of FSL-1 was administered subcutaneously in mice. The safety of the compound was assessed in non-irradiated animals, the efficacy of the compound was assessed in animals exposed to TBI in the AFRRI Co-60 facility, the dose of FSL-1 was optimized, and common hematological parameters [complete blood cell (CBC), cytokines, and bone marrow progenitor cells] were assessed. Animals were monitored up to 60 days after exposure and radiation-induced damage was evaluated. FSL-1 was shown to be non-toxic when administered to non-irradiated mice at doses up to 3 mg/kg. The window of efficacy was determined to be 24 h prior to 24 h after TBI. FSL-1 administration resulted in significantly increased survival when administered either 24 h prior to or 24 h after exposure to supralethal doses of TBI. The optimal dose of FSL-1 administration was determined to be 1.5 mg/kg when administered prior to irradiation. Finally, FSL-1 protected the hematopoietic system (recovery of CBC and bone marrow CFU). Taken together, the effects of increased survival and accelerated recovery of hematological parameters suggests that FSL-1 should be developed as a novel radiation countermeasure for soldiers and civilians, which can be used either before or after irradiation in the aftermath of a radiological or nuclear event.

With the current volatile geopolitical climate, the threat of nuclear assault is high. Exposure to ionizing radiation from either nuclear incidents or radiological accidents often lead to major harmful consequences to human health. Depending on the absorbed dose, the symptoms of the acute radiation syndrome and delayed effects of acute radiation exposure (DEARE) can appear within hours, weeks to months. The lung is a relatively radiosensitive organ with manifestation of radiation pneumonitis as an acute effect, followed by apparent fibrosis in weeks or even months. A recently developed, first-of-its-kind murine model for partial-body irradiation (PBI) injury, which can be used to test potential countermeasures against multi-organ damage such as gastrointestinal (GI) tract and lungs was used for irradiation, with 2.5% bone marrow spared (BM2.5–PBI) from radiation exposure. Long-term damage to lungs from radiation was evaluated using µ-CT scans, pulmonary function testing, histopathological parameters and molecular biomarkers. Pulmonary fibrosis was detected by ground glass opacity observed in µ-CT scans of male and female C57BL/6J mice 6–7 months after BM2.5–PBI. Lung mechanics assessments pertaining to peripheral airways suggested fibrotic lungs with stiffer parenchymal lung tissue and reduced inspiratory capacity in irradiated animals 6-7 months after BM2.5–PBI. Histopathological evaluation of the irradiated lungs revealed presence of focal and diffuse pleural, and parenchymal inflammatory and fibrotic lesions. Fibrosis was confirmed by elevated levels of collagen when compared to lungs of age-matched naïve mice. These findings were validated by findings of elevated levels of pro-fibrotic biomarkers and reduction in anti-inflammatory proteins. In conclusion, a long-term model for radiation-induced pulmonary fibrosis was established, and countermeasures could be screened in this model for survival and protection/mitigation or recovery from radiation-induced pulmonary damage.

The Radiation and Nuclear Countermeasures Program (RNCP) at the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) was established to facilitate the development of medical countermeasures (MCMs) and diagnostic approaches for use in a radiation public health emergency. Approvals for MCMs can be very challenging but are made possible under the United States Food and Drug Administration (FDA) Animal Rule, which is designed to enable licensure of drugs or biologics when clinical efficacy studies are unethical or unfeasible. The NIAID portfolio includes grants, contracts, and inter-agency agreements designed to span all aspects of drug development and encompasses basic research through FDA approval. In addition, NIAID manages an active portfolio of biodosimetry approaches to assess injuries and absorbed radiation levels to guide triage and treatment decisions. NIAID, together with grantees, contractors, and other stakeholders with promising products, works to advance candidate MCMs and biodosimetry tools through an established product development pipeline. In addition to managing grants and contracts, NIAID tests promising candidates in our established preclinical animal models, and the NIAID Program Officers work closely with sponsors as product managers to guide them through the process. In addition, a valuable benefit for stakeholders is working with the NIAID Office of Regulatory Affairs, where NIAID coordinates with the FDA to facilitate interactions between sponsors and the agency. Activities funded by NIAID include basic research (e.g., library screens to discover new products, determine early efficacy, and delineate mechanism of action) and the development of small and large animal models of radiation-induced hematopoietic, gastrointestinal, lung, kidney, and skin injury, radiation combined injury, and radionuclide decorporation. NIAID also sponsors Good Laboratory Practice product safety, pharmacokinetic, pharmacodynamic, and toxicology studies, as well as efficacy and dose-ranging studies to optimize product regimens. For later-stage candidates, NIAID funds large-scale manufacturing and formulation development of products. The program also supports Phase 1 human clinical studies to ensure human safety and to bridge pharmacokinetic, pharmacodynamic, and efficacy data from animals to humans. To date, NIAID has supported >900 animal studies and one clinical study, evaluating >500 new/repurposed radiation MCMs and biodosimetric approaches. NIAID sponsorship led to the approval of three of the six drugs for acute radiation syndrome under the FDA Animal Rule, five Investigational New Drug applications, and 18 additional submissions for Investigational Device Exemptions, while advancing 38 projects to the Biomedical Advanced Research and Development Authority for follow-on research and development.

High-LET-type cell survival curves have been observed in cells that were allowed to incorporate ¹²⁵I-UdR into their DNA. Incorporation of tritiated thymidine into the DNA of cells has also been shown to result in an increase in relative biological effectiveness in cell survival experiments, but the increase is smaller than observed after incorporation of ¹²⁵I-UdR. These findings are explained in the literature by the overall complexity of the induced DNA damage resulting from energies of the ejected electron(s) during the decay of ³H and ¹²⁵I. Chromosomal aberrations (CA) are defined as morphological or structural changes of one or more chromosomes, and can be induced by ionizing radiation. Whether the number of CA is associated with the linear energy transfer (LET) of the radiation and/or the actual complexity of the induced DNA double-strand breaks (DSB) remains elusive. In this study, we investigated whether DNA lesions induced at different cell cycle stages and by different radiation types [Auger-electrons (¹²⁵I), β^– particles (³H), or γ radiation (¹³⁷Cs)] have an impact on the number of CA induced after induction of the same number of DSB as determined by the γ-H2AX foci assay. Cells were synchronized and pulse-labeled in S phase with low activities of ¹²⁵I-UdR or tritiated thymidine. For decay accumulation, cells were cryopreserved either after pulse-labeling in S phase or after progression to G₂/M or G₁ phase. Experiments with γ irradiation (¹³⁷Cs) were performed with synchronized and cryopreserved cells in S, G₂/M or G₁ phase. After thawing, a CA assay was performed. All experiments were performed after a similar number of DSB were induced. CA induction after ¹²⁵I-UdR was incorporated was 2.9-fold and 1.7-fold greater compared to exposure to γ radiation and radiation from incorporated tritiated thymidine, respectively, when measured in G₂/M cells. In addition, measurement of CA in G₂/M cells after incorporation of ¹²⁵I-UdR was 2.5-fold greater when compared to cells in G₁ phase. In contrast, no differences were observed between the three radiation qualities with respect to exposure after cryopreservation in S or G₁ phase. The data indicate that the 3D organization of replicated DNA in G₂/M cells seems to be more sensitive to induction of more complex DNA lesions compared to the DNA architecture in S or G₁ cells. Whether this is due to the DNA organization itself or differences in DNA repair capabilityremains unclear.

In gene expression (GE) studies, housekeeping genes (HKGs) are required for normalization purposes. In large-scale inter-laboratory comparison studies, significant differences in dose estimates are reported and divergent HKGs are employed by the teams. Among them, the 18S rRNA HKG is known for its robustness. However, the high abundance of 18S rRNA copy numbers requires dilution, which is time-consuming and a possible source of errors. This study was conducted to identify the most promising HKGs showing the least radiation-induced GE variance after radiation exposure. In the screening stage of this study, 35 HKGs were analyzed. This included selected HKGs (ITFG1, MRPS5, and DPM1) used in large-scale biodosimetry studies which were not covered on an additionally employed pre-designed 96-well platform comprising another 32 HKGs used for different exposures. Altogether 41 samples were examined, including 27 ex vivo X-ray irradiated blood samples (0, 0.5, 4 Gy), six X-irradiated samples (0, 0.5, 5 Gy) from two cell lines (U118, A549), as well as eight non-irradiated tissue samples to encompass multiple biological entities. In the independent validation stage, the most suitable candidate genes were examined from another 257 blood samples, taking advantage of already stored material originating from three studies. These comprise 100 blood samples from ex vivo X-ray irradiated (0–4 Gy) healthy donors, 68 blood samples from 5.8 Gy irradiated (cobalt-60) Rhesus macaques (RM) (LD_29/60) collected 0–60 days postirradiation, and 89 blood samples from chemotherapy-(CTx) treated breast tumor patients. CTx and radiation-induced GE changes in previous studies appeared comparable. RNA was isolated, converted into cDNA, and GE was quantified employing TaqMan assays and quantitative RT-PCR. We calculated the standard deviation (SD) and the interquartile range (IQR) as measures of GE variance using raw cycle threshold (Ct) values and ranked the HKGs accordingly. Dose, time, age, and sex-dependent GE changes were examined employing the parametrical t-test and non-parametrical Kruskal Wallis test, as well as linear regression analysis. Generally, similar ranking results evolved using either SD or IQR GE measures of variance, indicating a tight distribution of GE values. PUM1 and PGK1 showed the lowest variance among the first ten most suitable genes in the screening phase. MRPL19 revealed low variance among the first ten most suitable genes in the screening phase only for blood and cells, but certain comparisons indicated a weak association of MRPL19 with dose (P = 0.02–0.09). In the validation phase, these results could be confirmed. Here, IQR Ct values from, e.g., X-irradiated blood samples were 0.6 raw Ct values for PUM1 and PGK1, which is considered to represent GE differences as expected due to methodological variance. Overall, when compared, the GE variance of both genes was either comparable or lower compared to 18S rRNA. Compared with the IQR GE values of PUM1 and PGKI, twofold–fivefold increased values were calculated for the biodosimetry HKG HPRT1, and comparable values were calculated for biodosimetry HKGs ITFG1, MRPS5, and DPM1. Significant dose-dependent associations were found for ITFG1 and MRPS5 (P = 0.001–0.07) and widely absent or weak (P = 0.02–0.07) for HPRT1 and DPM1. In summary, PUM1 and PGK1 appeared most promising for radiation exposure studies among the 35 HKGs examined, considering GE variance and adverse associations of GE with dose.

Despite the large variety of high-voltage semiconductor components for medium and high voltage switching and pulse-forming applications as well as for high-power high-frequency generation, the use of vacuum electron tubes still prevails to a considerable degree. Due to the common design incorporating a high energy electron beam which finally is dumped into an anode or a resonator cavity, these tubes are also considered as sources of X rays produced as bremsstrahlung and characteristic radiation, which are referred to as parasitic X rays. Here three types of vacuum-electron tubes, diode, tetrode, and thyratron, with glass housings are investigated. They are predominantly operated in the high voltage range below 30 kV and are not subject to licensing laws. The measurements of the dose rate and X-ray-spectra were performed in the laboratory without complex electrical circuitry usually used in making practical measurements for occupational radiation protection. For the diode tube, where a parasitic X-ray emission is observed only in the reverse operation as a blocking diode, a broad distribution of dose rates of electrically equivalent specimens was observed. This is attributed to field emission from the electrodes. For the tetrode and the thyratron tubes, field emission from the electrodes is identified as the dominant mechanism for the generation of parasitic X rays. Thus, technical radiation protection must focus on shielding of the glass tube rather than optimization of the electrical circuitry.

Increased radiological and nuclear threats require preparedness. Our earlier work identified a set of four genes (DDB2, FDXR, POU2AF1 and WNT3), which predicts severity of the hematological acute radiation syndrome (H-ARS) within the first three days postirradiation In this study of 41 Rhesus macaques (Macaca mulatta, 27 males, 14 females) irradiated with 5.8–7.2 Gy (LD_29-50/60), including some treated with gamma-tocotrienol (GT3, a radiation countermeasure) we independently validated these genes as predictors in both sexes and examined them after three days. At the Armed Forces Radiobiology Research Institute/Uniformed Services University of the Health Sciences, peripheral whole blood (1 ml) of Rhesus macaques was collected into PAXgene^® Blood RNA tubes pre-irradiation after 1, 2, 3, 35 and 60 days postirradiation, stored at –80°C for internal experimental analyses. Leftover tubes from these already ongoing studies were kindly provided to Bundeswehr Institute of Radiobiology. RNA was isolated (QIAsymphony), converted into cDNA, and for further gene expression (GE) studies quantitative RT-PCR was performed. Differential gene expression (DGE) was measured relative to the pre-irradiation Rhesus macaques samples. Within the first three days postirradiation, we found similar results to human data: 1. FDXR and DDB2 were up-regulated, FDXR up to 3.5-fold, and DDB2 up to 13.5-fold in the median; 2. POU2AF1 appeared down regulated around tenfold in nearly all Rhesus macaques; 3. Contrary to human data, DDB2 was more up-regulated than FDXR, and the difference of the fold change (FC) ranged between 2.4 and 10, while the median fold changes of WNT3, except days 1 and 35, were close to 1. Nevertheless, 46% of the Rhesus macaques showed down-regulated WNT3 on day one postirradiation, which decreased to 12.2% on day 3 postirradiation. Considering the extended phase, there was a trend towards decreased fold changes at day 35, with median-fold changes ranging from 0.7 for DDB2 to 0.1 for POU2AF1, and on day 60 postirradiation, DGE in surviving animals was close to pre-exposure values for all four genes. In conclusion, the diagnostic significance for radiation-induced H-ARS severity prediction of FDXR, DDB2, and POU2AF1 was confirmed in this Rhesus macaques model. However, DDB2 showed higher GE values than FDXR. As shown in previous studies, the diagnostic significance of WNT3 could not be reproduced in Rhesus macaques; this could be due to the choice of animal model and methodological challenges.

In times of war, radiological/nuclear emergency scenarios have become a reemphasized threat. However, there are challenges in transferring whole-blood samples to laboratories for specialized diagnostics using RNA. This project aims to miniaturize the process of unwieldy conventional RNA extraction with its stationed technical equipment using a microfluidic-based slide (MBS) for point-of-care diagnostics. The MBS is thought to be a preliminary step toward the development of a so-called lab-on-a-chip microfluidic device. A MBS would enable early and fast field care combined with gene expression (GE) analysis for the prediction of hematologic acute radiation syndrome (HARS) severity or identification of RNA microbes. Whole blood samples from ten healthy donors were irradiated with 0, 0.5 and 4 Gy, simulating different ARS severity degrees. RNA quality and quantity of a preliminary MBS was compared with a conventional column-based (CB) RNA extraction method. GE of four HARS severity-predicting radiation-induced genes (FDXR, DDB2, POU2AF1 and WNT3) was examined employing qRT-PCR. Compared to the CB method, twice as much total RNA from whole blood could be extracted using the MBS (6.6 ± 3.2 µg vs. 12.0 ± 5.8 µg) in half of the extraction time, and all MBS RNA extracts appeared DNA-free in contrast to the CB method (30% were contaminated with DNA). Using MBS, RNA quality [RNA integrity number equivalent (RIN^e)] values decreased about threefold (3.3 ± 0.8 vs. 9.0 ± 0.4), indicating severe RNA degradation, while expected high-quality RIN^e ≥ 8 were found using column-based method. However, normalized cycle threshold (Ct) values, as well as radiation-induced GE fold-changes appeared comparable for all genes utilizing both methods, indicating that no RNA degradation took place. In summary, the preliminary MBS showed promising features such as: 1. halving the RNA extraction time without the burden of heavy technical equipment (e.g., a centrifuge); 2. absence of DNA contamination in contrast to CB RNA extraction; 3. reduction in blood required, because of twice the biological output of RNA; and 4. equal GE performance compared to CB, thus, increasing its appeal for later semi-automatic parallel field applications.

As the great majority of gene expression (GE) biodosimetry studies have been performed using blood as the preferred source of tissue, searching for simple and less-invasive sampling methods is important when considering biodosimetry approaches. Knowing that whole saliva contains an ultrafiltrate of blood and white blood cells, it is expected that the findings in blood can also be found in saliva. This human in vivo study aims to examine radiation-induced GE changes in saliva for biodosimetry purposes and to predict radiation-induced disease, which is yet poorly characterized. Furthermore, we examined whether transcriptional biomarkers in blood can also be found equivalently in saliva. Saliva and blood samples were collected in parallel from radiotherapy (RT) treated patients who suffered from head and neck cancer (n = 8) undergoing fractioned partial-body irradiations (1.8 Gy/fraction and 50–70 Gy total dose). Samples were taken 12–24 h before first irradiation and ideally 24 and 48 h, as well as 5 weeks after radiotherapy onset. Due to the low quality and quantity of isolated RNA samples from one patient, they had to be excluded from further analysis, leaving a total of 24 saliva and 24 blood samples from 7 patients eligible for analysis. Using qRT-PCR, 18S rRNA and 16S rRNA (the ratio being a surrogate for the relative human RNA/bacterial burden), four housekeeping genes and nine mRNAs previously identified as radiation responsive in blood-based studies were detected. Significant GE associations with absorbed dose were found for five genes and after the 2nd radiotherapy fraction, shown by, e.g., the increase of CDKN1A (2.0 fold, P = 0.017) and FDXR (1.9 fold increased, P = 0.002). After the 25th radiotherapy fraction, however, all four genes (FDXR, DDB2, POU2AF1, WNT3) predicting ARS (acute radiation syndrome) severity, as well as further genes (including CCNG1 [median-fold change (FC) = 0.3, P = 0.013], and GADD45A (median-FC = 0.3, P = 0.031)) appeared significantly downregulated (FC = 0.3, P = 0.01–0.03). A significant association of CCNG1, POU2AF1, HPRT1, and WNT3 (P = 0.006-0.04) with acute or late radiotoxicity could be shown before the onset of these clinical outcomes. In an established set of four genes predicting acute health effects in blood, the response in saliva samples was similar to the expected up- (FDXR, DDB2) or downregulation (POU2AF1, WNT3) in blood for up to 71% of the measurements. Comparing GE responses (PHPT1, CCNG1, CDKN1A, GADD45A, SESN1) in saliva and blood samples, there was a significant linear association between saliva and blood response of CDKN1A (R² = 0.60, P = 0.0004). However, the GE pattern of other genes differed between saliva and blood. In summary, the current human in vivo study, (I) reveals significant radiation-induced GE associations of five transcriptional biomarkers in salivary samples, (II) suggests genes predicting diverse clinical outcomes such as acute and late radiotoxicity as well as ARS severity, and (III) supports the view that blood-based GE response can be reflected in saliva samples, indicating that saliva is a mirror of the body for certain but not all genes and, thus, studies for each gene of interest in blood are required for saliva.