Combined ion beam irradiation platform and 3D fluorescence microscope for cellular cancer research.

To improve particle radiotherapy, we need a better understanding of the biology of radiation effects, particularly in heavy ion radiation therapy, where global responses are observed despite energy deposition in only a subset of cells. Here, we integrated a high-speed swept confocally-aligned planar excitation (SCAPE) microscope into a focused ion beam irradiation platform to allow real-time 3D structural and functional imaging of living biological samples during and after irradiation. We demonstrate dynamic imaging of the acute effects of irradiation on 3D cultures of U87 human glioblastoma cells, revealing characteristic changes in cellular movement and intracellular calcium signaling following ionizing irradiation.

RABiT-III: an Automated Micronucleus Assay at a Non-Specialized Biodosimetry Facility.

Micronuclei, detected through the cytokinesis-block micronucleus assay, are valuable indicators of ionizing radiation exposure, especially in short-term lymphocyte cultures. The peripheral human blood lymphocyte assay is recognized as a prime candidate for automated biodosimetry. In a prior project at the Columbia University Center for Radiological Research, we automated this assay using the 96-well ANSI/SLAS microplate standard format and relied on established biotech robotic systems named Rapid Automated Biodosimetry Tool (RABiT). In this study, we present the application of a similar automated biotech setup at an external high-throughput facility (RABiT-III) to implement the same automated cytokinesis-block micronucleus assay. Specifically, we employed the Agilent BRAVO liquid-handling system and GE IN Cell Analyzer 6000 imaging system in conjunction with the PerkinElmer Columbus image data storage and analysis system. Notably, this analysis system features an embedded PhenoLOGIC machine learning module, simplifying the creation of cell classification algorithms for CBMN assay image analysis and enabling the generation of radiation dose-response curves. This investigation underscores the adaptability of the RABiT-II CBMN protocol to diverse RABiT-III biotech robotic platforms in non-specialized biodosimetry centers. Furthermore, it highlights the advantages of machine learning in rapidly developing algorithms crucial for the high-throughput automated analysis of RABiT-III images.

Immune Response following FLASH and Conventional Radiation in Diffuse Midline Glioma.

PURPOSE: Diffuse midline glioma (DMG) is a fatal tumor traditionally treated with radiation therapy (RT) and previously characterized as having a noninflammatory tumor immune microenvironment (TIME). FLASH is a novel RT technique using ultra-high dose rate that is associated with decreased toxicity and effective tumor control. However, the effect of FLASH and conventional (CONV) RT on the DMG TIME has not yet been explored. METHODS AND MATERIALS: Here, we performed single-cell RNA sequencing (scRNA-seq) and flow cytometry on immune cells isolated from an orthotopic syngeneic murine model of brainstem DMG after the use of FLASH (90 Gy/sec) or CONV (2 Gy/min) dose-rate RT and compared to unirradiated tumor (SHAM). RESULTS: At day 4 post-RT, FLASH exerted similar effects as CONV in the predominant microglial (MG) population, including the presence of two activated subtypes. However, at day 10 post-RT, we observed a significant increase in the type 1 interferon α/ß receptor (IFNAR+) in MG in CONV and SHAM compared to FLASH. In the non-resident myeloid clusters of macrophages (MACs) and dendritic cells (DCs), we found increased type 1 interferon (IFN1) pathway enrichment for CONV compared to FLASH and SHAM by scRNA-seq. We observed this trend by flow cytometry at day 4 post-RT in IFNAR+ MACs and DCs, which equalized by day 10 post-RT. DMG control and murine survival were equivalent between RT dose rates. CONCLUSIONS: Our work is the first to map CONV and FLASH immune alterations of the DMG TIME with single-cell resolution. Although DMG tumor control and survival were similar between CONV and FLASH, we found that changes in immune compartments differed over time. Importantly, although both RT modalities increased IFN1, we found that the timing of this response was cell-type and dose-rate dependent. These temporal differences, particularly in the context of tumor control, warrant further study.

Sex and dose rate effects in automated cytogenetics.

Testing and validation of biodosimetry assays is routinely performed using conventional dose rate irradiation platforms, at a dose rate of approximately 1 Gy/min. In contrast, the exposures from an improvised nuclear device will be delivered over a large range of dose rates with a prompt irradiation component, delivered in less than 1 µs, and a protracted component delivered over hours and days. We present preliminary data from a large demographic study we have undertaken for investigation of age, sex and dose rate effects on dicentric and micronucleus yields. Our data demonstrate reduced dicentric and micronucleus yields at very high dose rates. Additionally, we have seen small differences between males and females, with males having slightly fewer micronuclei and slightly more dicentrics than females, at high doses.

Modeling and countering the effects of cosmic radiation using bioengineered human tissues.

Cosmic radiation is the most serious risk that will be encountered during the planned missions to the Moon and Mars. There is a compelling need to understand the effects, safety thresholds, and mechanisms of radiation damage in human tissues, in order to develop measures for radiation protection during extended space travel. As animal models fail to recapitulate the molecular changes in astronauts, engineered human tissues and "organs-on-chips" are valuable tools for studying effects of radiation in vitro. We have developed a bioengineered tissue platform for studying radiation damage in individualized settings. To demonstrate its utility, we determined the effects of radiation using engineered models of two human tissues known to be radiosensitive: engineered cardiac tissues (eCT, a target of chronic radiation damage) and engineered bone marrow (eBM, a target of acute radiation damage). We report the effects of high-dose neutrons, a proxy for simulated galactic cosmic rays, on the expression of key genes implicated in tissue responses to ionizing radiation, phenotypic and functional changes in both tissues, and proof-of-principle application of radioprotective agents. We further determined the extent of inflammatory, oxidative stress, and matrix remodeling gene expression changes, and found that these changes were associated with an early hypertrophic phenotype in eCT and myeloid skewing in eBM. We propose that individualized models of human tissues have potential to provide insights into the effects and mechanisms of radiation during deep-space missions and allow testing of radioprotective measures.

Comparison of Isolated Lymphocyte and Whole Blood-Based CBMN Assays for Radiation Triage.

Following a mass-casualty nuclear/radiological event, there will be an important need for rapid and accurate estimation of absorbed dose for biological triage. The cytokinesis-block micronucleus (CBMN) assay is an established and validated cytogenetic biomarker used to assess DNA damage in irradiated peripheral blood lymphocytes. Here, we describe an intercomparison experiment between two biodosimetry laboratories, located at Columbia University (CU) and Health Canada (HC) that performed different variants of the human blood CBMN assay to reconstruct dose in human blood, with CU performing the assay on isolated lymphocytes and using semi-automated scoring whereas HC used the more conventional whole blood assay. Although the micronucleus yields varied significantly between the two assays, the predicted doses closely matched up to 4 Gy - the range from which the HC calibration curve was previously established. These results highlight the importance of a robust calibration curve(s) across a wide age range of donors that match the exposure scenario as closely as possible and that will account for differences in methodology between laboratories. We have seen that at low doses, variability in the results may be attributed to variation in the processing while at higher doses the variation is dominated by inter-individual variation in cell proliferation. This interlaboratory collaboration further highlights the usefulness of the CBMN endpoint to accurately reconstruct absorbed dose in human blood after ionizing radiation exposure.

Biomarker integration for improved biodosimetry of mixed neutron + photon exposures.

There is a persistent risk of a large-scale malicious or accidental exposure to ionizing radiation that may affect a large number of people. Exposure will consist of both a photon and neutron component, which will vary in magnitude between individuals and is likely to have profound impacts on radiation-induced diseases. To mitigate these potential disasters, there exists a need for novel biodosimetry approaches that can estimate the radiation dose absorbed by each person based on biofluid samples, and predict delayed effects. Integration of several radiation-responsive biomarker types (transcripts, metabolites, blood cell counts) by machine learning (ML) can improve biodosimetry. Here we integrated data from mice exposed to various neutron + photon mixtures, total 3 Gy dose, using multiple ML algorithms to select the strongest biomarker combinations and reconstruct radiation exposure magnitude and composition. We obtained promising results, such as receiver operating characteristic curve area of 0.904 (95% CI: 0.821, 0.969) for classifying samples exposed to ≥ 10% neutrons vs. < 10% neutrons, and R2 of 0.964 for reconstructing photon-equivalent dose (weighted by neutron relative biological effectiveness) for neutron + photon mixtures. These findings demonstrate the potential of combining various -omic biomarkers for novel biodosimetry.

Variable Dose Rates in Realistic Radiation Exposures: Effects on Small Molecule Markers of Ionizing Radiation in the Murine Model.

Novel biodosimetry assays for use in preparedness and response to potential malicious attacks or nuclear accidents would ideally provide accurate dose reconstruction independent of the idiosyncrasies of a complex exposure to ionizing radiation. Complex exposures will consist of dose rates spanning the low dose rates (LDR) to very high-dose rates (VHDR) that need to be tested for assay validation. Here, we investigate how a range of relevant dose rates affect metabolomic dose reconstruction at potentially lethal radiation exposures (8 Gy in mice) from an initial blast or subsequent fallout exposures compared to zero or sublethal exposures (0 or 3 Gy in mice) in the first 2 days, which corresponds to an integral time individuals will reach medical facilities after a radiological emergency. Biofluids (urine and serum) were collected from both male and female 9-10-week-old C57BL/6 mice at 1 and 2 days postirradiation (total doses of 0, 3 or 8 Gy) after a VHDR of 7 Gy/s. Additionally, samples were collected after a 2-day exposure consisting of a declining dose rate (1 to 0.004 Gy/min) recapitulating the 7:10 rule-of-thumb time dependency of nuclear fallout. Overall similar perturbations were observed in both urine and serum metabolite concentrations irrespective of sex or dose rate, with the exception of xanthurenic acid in urine (female specific) and taurine in serum (VHDR specific). In urine, we developed identical multiplex metabolite panels (N6, N6,N6-trimethyllysine, carnitine, propionylcarnitine, hexosamine-valine-isoleucine, and taurine) that could identify individuals receiving potentially lethal levels of radiation from the zero or sublethal cohorts with excellent sensitivity and specificity, with creatine increasing model performance at day 1. In serum, individuals receiving a 3 or 8 Gy exposure could be identified from their pre-irradiation samples with excellent sensitivity and specificity, however, due to a lower dose response the 3 vs. 8 Gy groups could not be distinguished from each other. Together with previous results, these data indicate that dose-rate-independent small molecule fingerprints have potential in novel biodosimetry assays.

International Comparison Exercise for Biological Dosimetry after Exposures with Neutrons Performed at Two Irradiation Facilities as Part of the BALANCE Project.

In the case of a radiological or nuclear event, biological dosimetry can be an important tool to support clinical decision-making. During a nuclear event, individuals might be exposed to a mixed field of neutrons and photons. The composition of the field and the neutron energy spectrum influence the degree of damage to the chromosomes. During the transatlantic BALANCE project, an exposure similar to a Hiroshima-like device at a distance of 1.5 km from the epicenter was simulated, and biological dosimetry based on dicentric chromosomes was performed to evaluate the participants ability to discover unknown doses and to test the influence of differences in neutron spectra. In a first step, calibration curves were established by irradiating blood samples with 5 doses in the range of 0-4 Gy at two different facilities in Germany (Physikalisch-Technische Bundesanstalt [PTB]) and the USA (the Columbia IND Neutron Facility [CINF]). The samples were sent to eight participating laboratories from the RENEB network and dicentric chromosomes were scored by each participant. Next, blood samples were irradiated with 4 blind doses in each of the two facilities and sent to the participants to provide dose estimates based on the established calibration curves. Manual and semiautomatic scoring of dicentric chromosomes were evaluated for their applicability to neutron exposures. Moreover, the biological effectiveness of the neutrons from the two irradiation facilities was compared. The calibration curves from samples irradiated at CINF showed a 1.4 times higher biological effectiveness compared to samples irradiated at PTB. For manual scoring of dicentric chromosomes, the doses of the test samples were mostly successfully resolved based on the calibration curves established during the project. For semiautomatic scoring, the dose estimation for the test samples was less successful. Doses >2 Gy in the calibration curves revealed nonlinear associations between dose and dispersion index of the dicentric counts, especially for manual scoring. The differences in the biological effectiveness between the irradiation facilities suggested that the neutron energy spectrum can have a strong impact on the dicentric counts.

Machine learning approach for quantitative biodosimetry of partial-body or total-body radiation exposures by combining radiation-responsive biomarkers.

During a large-scale radiological event such as an improvised nuclear device detonation, many survivors will be shielded from radiation by environmental objects, and experience only partial-body irradiation (PBI), which has different consequences, compared with total-body irradiation (TBI). In this study, we tested the hypothesis that applying machine learning to a combination of radiation-responsive biomarkers (ACTN1, DDB2, FDXR) and B and T cell counts will quantify and distinguish between PBI and TBI exposures. Adult C57BL/6 mice of both sexes were exposed to 0, 2.0-2.5 or 5.0 Gy of half-body PBI or TBI. The random forest (RF) algorithm trained on ½ of the data reconstructed the radiation dose on the remaining testing portion of the data with mean absolute error of 0.749 Gy and reconstructed the product of dose and exposure status (defined as 1.0 × Dose for TBI and 0.5 × Dose for PBI) with MAE of 0.472 Gy. Among irradiated samples, PBI could be distinguished from TBI: ROC curve AUC = 0.944 (95% CI: 0.844-1.0). Mouse sex did not significantly affect dose reconstruction. These results support the hypothesis that combinations of protein biomarkers and blood cell counts can complement existing methods for biodosimetry of PBI and TBI exposures.

Validation of a High-Throughput Dicentric Chromosome Assay Using Complex Radiation Exposures.

Validation of biodosimetry assays is routinely performed using primarily orthovoltage irradiators at a conventional dose rate of approximately 1 Gy/min. However, incidental/ accidental exposures caused by nuclear weapons can be more complex. The aim of this work was to simulate the DNA damage effects mimicking those caused by the detonation of a several kilotons improvised nuclear device (IND). For this, we modeled complex exposures to: 1. a mixed (photons + IND-neutrons) field and 2. different dose rates that may come from the blast, nuclear fallout, or ground deposition of radionuclides (ground shine). Additionally, we assessed whether myeloid cytokines affect the precision of radiation dose estimation by modulating the frequency of dicentric chromosomes. To mimic different exposure scenarios, several irradiation systems were used. In a mixed field study, human blood samples were exposed to a photon field enriched with neutrons (ranging from 10% to 37%) from a source that mimics Hiroshima's A-bomb's energy spectrum (0.2-9 MeV). Using statistical analysis, we assessed whether photons and neutrons act in an additive or synergistic way to form dicentrics. For the dose rates study, human blood was exposed to photons or electrons at dose rates ranging from low (where the dose was spread over 32 h) to extremely high (where the dose was delivered in a fraction of a microsecond). Potential effects of cytokine treatment on biodosimetry dose predictions were analyzed in irradiated blood subjected to Neupogen or Neulasta for 24 or 48 h at the concentration recommended to forestall manifestation of an acute radiation syndrome in bomb survivors. All measurements were performed using a robotic station, the Rapid Automated Biodosimetry Tool II, programmed to culture lymphocytes and score dicentrics in multiwell plates (the RABiT-II DCA). In agreement with classical concepts of radiation biology, the RABiT-II DCA calibration curves suggested that the frequency of dicentrics depends on the type of radiation and is modulated by changes in the dose rate. The resulting dose-response curves suggested an intermediate dicentric yields and additive effects of photons and IND-neutrons in the mixed field. At ultra-high dose rate (600 Gy/s), affected lymphocytes exhibited significantly fewer dicentrics (P < 0.004, t test). In contrast, we did not find the dose-response modification effects of radiomitigators on the yields of dicentrics (Bonferroni corrected P > 0.006, ANOVA test). This result suggests no bias in the dose predictions should be expected after emergency cytokine treatment initiated up to 48 h prior to blood collection for dicentric analysis.

A machine learning method for improving the accuracy of radiation biodosimetry by combining data from the dicentric chromosomes and micronucleus assays.

A large-scale malicious or accidental radiological event can expose vast numbers of people to ionizing radiation. The dicentric chromosome (DCA) and cytokinesis-block micronucleus (CBMN) assays are well-established biodosimetry methods for estimating individual absorbed doses after radiation exposure. Here we used machine learning (ML) to test the hypothesis that combining automated DCA and CBMN assays will improve dose reconstruction accuracy, compared with using either cytogenetic assay alone. We analyzed 1349 blood sample aliquots from 155 donors of different ages (3-69 years) and sexes (49.1% males), ex vivo irradiated with 0-8 Gy at dose rates from 0.08 Gy/day to ≥ 600 Gy/s. We compared the performances of several state-of-the-art ensemble ML methods and found that random forest generated the best results, with R2 for actual vs. reconstructed doses on a testing data subset = 0.845, and mean absolute error = 0.628 Gy. The most important predictor variables were CBMN and DCA frequencies, and age. Removing CBMN or DCA data from the model significantly increased squared errors on testing data (p-values 3.4 × 10-8 and 1.1 × 10-6, respectively). These findings demonstrate the promising potential of combining CBMN and DCA assay data to reconstruct radiation doses in realistic scenarios of heterogeneous populations exposed to a mass-casualty radiological event.

Ultra-high dose rate FLASH irradiator at the radiological research accelerator facility.

The Radiological Research Accelerator Facility has modified a decommissioned Varian Clinac to deliver ultra-high dose rates: operating in 9 MeV electron mode (FLASH mode), samples can be irradiated at a Source-Surface Distance (SSD) of 20 cm at average dose rates of up to 600 Gy/s (3.3 Gy per 0.13 µs pulse, 180 pulses per second). In this mode multiple pulses are required for most irradiations. By modulating pulse repetition rate and irradiating at SSD = 171 cm, dose rates below 1 Gy/min can be achieved, allowing comparison of FLASH and conventional irradiations with the same beam. Operating in 6 MV photon mode, with the conversion target removed (SuperFLASH mode), samples are irradiated at higher dose rates (0.2-150 Gy per 5 µs pulse, 360 pulses per second) and most irradiations can be performed with a single very high dose rate pulse. In both modes we have seen the expected inverse relation between dose rate and irradiated area, with the highest dose rates obtained for beams with a FWHM of about 2 cm and ± 10% uniformity over 1 cm diameter. As an example of operation of the ultra-high dose rate FLASH irradiator, we present dose rate dependence of dicentric chromosome yields.

Cross-platform validation of a mouse blood gene signature for quantitative reconstruction of radiation dose.

In the search for biological markers after a large-scale exposure of the human population to radiation, gene expression is a sensitive endpoint easily translatable to in-field high throughput applications. Primarily, the ex-vivo irradiated healthy human blood model has been used to generate available gene expression datasets. This model has limitations i.e., lack of signaling from other irradiated tissues and deterioration of blood cells cultures over time. In vivo models are needed; therefore, we present our novel approach to define a gene signature in mouse blood cells that quantitatively correlates with radiation dose (at 1 Gy/min). Starting with available microarray datasets, we selected 30 radiation-responsive genes and performed cross-validation/training-testing data splits to downselect 16 radiation-responsive genes. We then tested these genes in an independent cohort of irradiated adult C57BL/6 mice (50:50 both sexes) and measured mRNA by quantitative RT-PCR in whole blood at 24 h. Dose reconstruction using net signal (difference between geometric means of top 3 positively correlated and top 4 negatively correlated genes with dose), was highly improved over the microarrays, with a root mean square error of ± 1.1 Gy in male and female mice combined. There were no significant sex-specific differences in mRNA or cell counts after irradiation.

Biofluid Metabolomics and Lipidomics of Mice Exposed to External Very High-Dose Rate Radiation.

High-throughput biodosimetry methods to determine exposure to ionizing radiation (IR) that can also be easily scaled to multiple testing sites in emergency situations are needed in the event of malicious attacks or nuclear accidents that may involve a substantial number of civilians. In the event of an improvised nuclear device (IND), a complex IR exposure will have a very high-dose rate (VHDR) component from an initial blast. We have previously addressed low-dose rate (LDR, ≤1 Gy/day) exposures from internal emitters on biofluid small molecule signatures, but further research on the VHDR component of the initial blast is required. Here, we exposed 8- to 10-week-old male C57BL/6 mice to an acute dose of 3 Gy using a reference dose rate of 0.7 Gy/min or a VHDR of 7 Gy/s, collected urine and serum at 1 and 7 d, then compared the metabolite signatures using either untargeted (urine) or targeted (serum) approaches with liquid chromatography mass spectrometry platforms. A Random Forest classification approach showed strikingly similar changes in urinary signatures at 1 d post-irradiation with VHDR samples grouping closer to control samples at 7 d. Identical metabolite panels (carnitine, trigonelline, xanthurenic acid, N6,N6,N6-trimethyllysine, spermine, and hexosamine-valine-isoleucine-OH) could differentiate IR exposed individuals with high sensitivity and specificity (area under the receiver operating characteristic (AUROC) curves 0.89-1.00) irrespective of dose rate at both days. For serum, the top 25 significant lipids affected by IR exposure showed slightly higher perturbations at 0.7 Gy/min vs. 7 Gy/s; however, identical panels showed excellent sensitivity and specificity at 1 d (three hexosylceramides (16:0), (18:0), (24:0), sphingomyelin [26:1], lysophosphatidylethanolamine [22:1]). Mice could not be differentiated from control samples at 7 d for a 3 Gy exposure based on serum lipid signatures. As with LDR exposures, we found that identical biofluid small molecule signatures can identify IR exposed individuals irrespective of dose rate, which shows promise for more universal applications of metabolomics for biodosimetry.

Biofluid Metabolomics of Mice Exposed to External Low-Dose Rate Radiation in a Novel Irradiation System, the Variable Dose-Rate External 137Cs Irradiator.

An important component of ionizing radiation (IR) exposure after a radiological incident may include low-dose rate (LDR) exposures either externally or internally, such as from 137Cs deposition. In this study, a novel irradiation system, VAriable Dose-rate External 137Cs irradiatoR (VADER), was used to expose male and female mice to a variable LDR irradiation over a 30 d time span to simulate fall-out-type exposures in addition to biofluid collection from a reference dose rate (0.8 Gy/min). Radiation markers were identified by untargeted metabolomics and random forests. Mice exposed to LDR exposures were successfully identified from control groups based on their urine and serum metabolite profiles. In addition to metabolites commonly perturbed after IR exposure, we identified and validated a novel metabolite (hexosamine-valine-isoleucine-OH) that increased up to 150-fold after LDR and 80-fold after conventional exposures in urine. A multiplex panel consisting of hexosamine-valine-isoleucine-OH with other urinary metabolites (N6,N6,N6-trimethyllysine, carnitine, 1-methylnicotinamide, and α-ketoglutaric acid) achieved robust classification performance using receiver operating characteristic curve analysis, irrespective of the dose rate or sex. These results show that in terms of biodosimetry, dysregulated energy metabolism is associated with IR exposure for both LDR and conventional IR exposures. These mass spectrometry data have been deposited to the NIH data repository via Metabolomics Workbench with study IDs ST001790, ST001791, ST001792, ST001793, and ST001806.

Cytogenetic Damage of Human Lymphocytes in Humanized Mice Exposed to Neutrons and X Rays 24 h After Exposure.

Detonation of an improvised nuclear device highlights the need to understand the risk of mixed radiation exposure as prompt radiation exposure could produce significant neutron and gamma exposures. Although the neutron component may be a relatively small percentage of the total absorbed dose, the large relative biological effectiveness (RBE) can induce larger biological DNA damage and cell killing. The objective of this study was to use a hematopoietically humanized mouse model to measure chromosomal DNA damage in human lymphocytes 24 h after in vivo exposure to neutrons (0.3 Gy) and X rays (1 Gy). The human dicentric and cytokinesis-block micronucleus assays were performed to measure chromosomal aberrations in human lymphocytes in vivo from the blood and spleen, respectively. The mBAND assay based on fluorescent in situ hybridization labeling was used to detect neutron-induced chromosome 1 inversions in the blood lymphocytes of the neutron-irradiated mice. Cytogenetics endpoints, dicentrics and micronuclei showed that there was no significant difference in yields between the 2 irradiation types at the doses tested, indicating that neutron-induced chromosomal DNA damage in vivo was more biologically effective (RBE ∼3.3) compared to X rays. The mBAND assay, which is considered a specific biomarker of high-LET neutron exposure, confirmed the presence of clustered DNA damage in the neutron-irradiated mice but not in the X-irradiated mice, 24 h after exposure.

Effects of Acute and Chronic Exposure to a Mixed Field of Neutrons and Photons and Single or Fractionated Simulated Galactic Cosmic Ray Exposure on Behavioral and Cognitive Performance in Mice.

During space missions, astronauts experience acute and chronic low-dose-rate radiation exposures. Given the clear gap of knowledge regarding such exposures, we assessed the effects acute and chronic exposure to a mixed field of neutrons and photons and single or fractionated simulated galactic cosmic ray exposure (GCRsim) on behavioral and cognitive performance in mice. In addition, we assessed the effects of an aspirin-containing diet in the presence and absence of chronic exposure to a mixed field of neutrons and photons. In C3H male mice, there were effects of acute radiation exposure on activity levels in the open field containing objects. In addition, there were radiation-aspirin interactions for effects of chronic radiation exposure on activity levels and measures of anxiety in the open field, and on activity levels in the open field containing objects. There were also detrimental effects of aspirin and chronic radiation exposure on the ability of mice to distinguish the familiar and novel object. Finally, there were effects of acute GCRsim on activity levels in the open field containing objects. Activity levels were lower in GCRsim than sham-irradiated mice. Thus, acute and chronic irradiation to a mixture of neutrons and photons and acute and fractionated GCRsim have differential effects on behavioral and cognitive performance of C3H mice. Within the limitations of our study design, aspirin does not appear to be a suitable countermeasure for effects of chronic exposure to space radiation on cognitive performance.

Small Molecule Responses to Sequential Irradiation with Neutrons and Photons for Biodosimetry Applications: An Initial Assessment.

Mass casualty exposure scenarios from an improvised nuclear device are expected to be far more complex than simple photons. Based on the proximity to the explosion and potential shielding, a mixed field of neutrons and photons comprised of up to approximately 30% neutrons of the total dose is anticipated. This presents significant challenges for biodosimetry and for short-term and long-term medical treatment of exposed populations. In this study we employed untargeted metabolomic methods to develop a biosignature in urine and serum from C57BL/6 mice to address radiation quality issues. The signature was developed in males and applied to samples from female mice to identify potential sex differences. Thirteen urinary (primarily amino acids, vitamin products, nucleotides) and 18 serum biomarkers (primarily mitochondrial and fatty acid ß oxidation intermediates) were selected and evaluated in samples from day 1 and day 7 postirradiation. Sham-irradiated groups (controls) were compared to an equitoxic dose (3 Gy X-ray equivalent) from X rays (1.2 Gy/min), neutrons (∼1 Gy/h), or neutrons-photons. Results showed a time-dependent increase in the efficiency of the signatures, with serum providing the highest levels of accuracy in distinguishing not only between exposed from non-exposed populations, but also between radiation quality (photon exposures vs. exposures with a neutron component) and in between neutron-photon exposures (5, 15 or 25% of neutrons in the total dose) for evaluating the neutron contribution. A group of metabolites known as acylcarnitines was only responsive in males, indicating the potential for different mechanisms of action in baseline levels and of neutron-photon responses between the two sexes. Our findings highlight the potential of metabolomics in developing biodosimetric methods to evaluate mixed exposures with high sensitivity and specificity.