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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., Competing Interests: EMCH and CPC: Leica Microsystems and Applied Scientific Instrumentation (ASI) (P, R)., (© 2024 Optica Publishing Group.)

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 R 2 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., (© 2023. The Author(s).)

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., (© 2022. The Author(s).)

Standard dosimetry protocols exist for highly penetrating photon and particle beams used in the clinic and in research. However, these protocols cannot be directly applied to shallow penetration MeV-range ion beams. The Radiological Research Accelerator Facility has been using such beams for almost 50 years to irradiate cell monolayers, using self-developed dosimetry, based on tissue equivalent ionization chambers. To better align with the internationally accepted standards, we describe implementation of a commercial, NIST-traceable, air-filled ionization chamber for measurement of absorbed dose to water from low energy ions, using radiation quality correction factors calculated using TRS-398 recommendations. The reported dose does not depend on the ionization density in the range of 10-150 keV/μm.

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 β 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., (©2021 by Radiation Research Society. All rights of reproduction in any form reserved.)

Purpose: To study the agreement between proton microdosimetric distributions measured with a silicon-based cylindrical microdosimeter and a previously published analytical microdosimetric model based on Geant4-DNA in-water Monte Carlo simulations for low energy proton beams., Methods and Material: Distributions for lineal energy ( y ) are measured for four proton monoenergetic beams with nominal energies from 2.0 MeV to 4.5 MeV, with a tissue equivalent proportional counter (TEPC) and a silicon-based microdosimeter. The actual energy for protons traversing the silicon-based microdosimeter is simulated with SRIM. Monoenergetic beams with these energies are simulated with Geant4-DNA code by simulating a water cylinder site of dimensions equal to those of the microdosimeter. The microdosimeter response is calibrated by using the distribution peaks obtained from the TEPC. Analytical calculations for y ¯ F and y ¯ D using our methodology based on spherical sites are also performed choosing the equivalent sphere to be checked against experimental results., Results: Distributions for y at silicon are converted into tissue equivalent and compared to the Geant4-DNA simulated, yielding maximum deviations of 1.03% for y ¯ F and 1.17% for y ¯ D . Our analytical method generates maximum deviations of 1.29% and 3.33%, respectively, with respect to experimental results., Conclusion: Simulations in Geant4-DNA with ideal cylindrical sites in liquid water produce similar results to the measurements in an actual silicon-based cylindrical microdosimeter properly calibrated. The found agreement suggests the possibility to experimentally verify the calculated clinical y ¯ D with our analytical method., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Although radiation is widely used to treat cancers, resistance mechanisms often develop and involve activation of DNA repair and inhibition of apoptosis. Therefore, compounds that sensitize cancer cells to radiation via alternative cell death pathways are valuable. We report here that ferroptosis, a form of nonapoptotic cell death driven by lipid peroxidation, is partly responsible for radiation-induced cancer cell death. Moreover, we found that small molecules activating ferroptosis through system x c - inhibition or GPX4 inhibition synergize with radiation to induce ferroptosis in several cancer types by enhancing cytoplasmic lipid peroxidation but not increasing DNA damage or caspase activation. Ferroptosis inducers synergized with cytoplasmic irradiation, but not nuclear irradiation. Finally, administration of ferroptosis inducers enhanced the antitumor effect of radiation in a murine xenograft model and in human patient-derived models of lung adenocarcinoma and glioma. These results suggest that ferroptosis inducers may be effective radiosensitizers that can expand the efficacy and range of indications for radiation therapy.

In the possible event of a detonation of an improvised nuclear device (IND), the immediate radiation would consist of both photons (gamma rays) and neutrons. Since neutrons generally have a high relative biological effectiveness (RBE) for most physiological end points, it is important to understand the effect that neutrons would have on the biodosimetry methods that are being developed for medical triage purposes. We previously compared the transcriptomic response in human blood after neutron and photon irradiation. In this study, we analyzed the effect of mixed-field-neutron-photon radiation on gene expression responses in human peripheral blood, to elucidate the neutron contribution in the setting of a radiation exposure from an IND detonation. We used four combinations of mixed neutron-photon exposures, with increasing percentages of neutrons, to a cumulative dose of 3 Gy. The mixed-field exposures consisted of 0%, 5%, 15% and 25% of neutrons, where 0% corresponds to 3 Gy of pure X rays. A maximum neutron exposure, corresponding to 83% neutrons (0.75 Gy) was also used in the study. Increases were observed in both the number and expression level of genes, with increasing percentages of neutrons from 0% to 25% in the mixed-field exposures. Gene ontology analysis showed an overall predominance of TP53 signaling among upregulated genes across all exposures. Some TP53 regulated genes, such as EDA2R , GDF15 and VWCE , demonstrated increased expression with increasing neutron percentages in mixed-field exposures. Immune response, specifically natural-killer-cell mediated signaling, was the most significant biological process associated with downregulated genes. We observed significant suppression of T-cell-mediated signaling in mixed-field exposures, which was absent in the response to pure photons. In this first study investigating gene expression in human blood cells exposed to mixed neutron-photon fields similar to an actual IND explosion, we have identified a number of genes responding to the 3 Gy dose that showed increasing expression as the neutron percentage increased. Such genes may serve as better indicators of the expected biological damage than a report of total physical dose, and thus provide more relevant information for treating physicians.

Heightened threats for nuclear terrorism using improvised nuclear devices (IND) necessitate the development of biodosimetry assays that could rapidly assess thousands of individuals. However, the radiation exposures from an IND may be complex due to mixed fields of neutrons and photons (γ-rays), shielding from buildings, and proximity to the epicenter among others. In this study we utilized lipidomics to analyze serum samples from mice exposed to various percentages of neutrons and X-rays to a total dose of 3 Gy. Triacylglycerides, phosphatidylserines, lysophosphatidylethanolamines, lysophosphatidylcholines (LPCs), sphingolipids, and cholesteryl esters all showed delayed increases at day 7 compared to day 1 after irradiation, while diacylglycerides decreased in mixed field exposures and phosphatidylcholines (PCs) remained largely unchanged. Individual lipid molecules with a high degree of unsaturation exhibited the highest fold changes in mixed fields compared to photons alone. More importantly, the increased ratio of LPCs to PCs of each irradiation group compared to control could be used as a radiation biomarker and highlights the existence of a pro-inflammatory phenotype. The results showed that even a small percentage of neutrons in a mixed field can lead to high biological responses with implications for accurate biodosimetry, triage and medical managements of exposed populations.

Background: Radiation exposure due to the detonation of an improvised nuclear device remains a major security concern. Radiation from such a device involves a combination of photons and neutrons. Although photons will make the greater contribution to the total dose, neutrons will certainly have an impact on the severity of the exposure as they have high relative biological effectiveness., Results: We investigated the gene expression signatures in the blood of mice exposed to 3 Gy x-rays, 0.75 Gy of neutrons, or to mixed field photon/neutron with the neutron fraction contributing 5, 15%, or 25% of a total 3 Gy radiation dose. Gene ontology and pathway analysis revealed that genes involved in protein ubiquitination pathways were significantly overrepresented in all radiation doses and qualities. On the other hand, eukaryotic initiation factor 2 (EIF2) signaling pathway was identified as one of the top 10 ranked canonical pathways in neutron, but not pure x-ray, exposures. In addition, the related mTOR and regulation of EIF4/p70S6K pathways were also significantly underrepresented in the exposures with a neutron component, but not in x-ray radiation. The majority of the changed genes in these pathways belonged to the ribosome biogenesis and translation machinery and included several translation initiation factors (e.g. Eif2ak4, Eif3f), as well as 40S and 60S ribosomal subunits (e.g. Rsp19, Rpl19, Rpl27). Many of the differentially downregulated ribosomal genes (e.g. RPS19, RPS28) have been causally associated with human bone marrow failure syndromes and hematologic malignancies. We also observed downregulation of transfer RNA processes, in the neutron-only exposure (p < 0.005). Ingenuity Pathway Analysis (p < 0.05) of differentially expressed genes predicted significantly suppressed activity of the upstream regulators c-Myc and Mycn, transcription factors known to control ribosome biogenesis., Conclusions: We describe the gene expression profile of mouse blood following exposure to mixed field neutron/photon irradiation. We have discovered that pathways related to protein translation are significantly underrepresented in the exposures containing a neutron component. Our results highlight the significance of neutron exposures that even the smallest percentage can have profound biological effects that will affect medical management and treatment decisions in case of a radiological emergency.

The increased threat of terrorism across the globe has raised fears that certain groups will acquire and use radioactive materials to inflict maximum damage. In the event that an improvised nuclear device (IND) is detonated, a potentially large population of victims will require assessment for radiation exposure. While photons will contribute to a major portion of the dose, neutrons may be responsible for the severity of the biologic effects and cellular responses. We investigated differences in response between these two radiation types by using metabolomics and lipidomics to identify biomarkers in urine and blood of wild-type C57BL/6 male mice. Identification of metabolites was based on a 1 Gy dose of radiation. Compared to X rays, a neutron spectrum similar to that encountered in Hiroshima at 1-1.5 km from the epicenter induced a severe metabolic dysregulation, with perturbations in amino acid metabolism and fatty acid β-oxidation being the predominant ones. Urinary metabolites were able to discriminate between neutron and X rays on day 1 as well as day 7 postirradiation, while serum markers showed such discrimination only on day 1. Free fatty acids from omega-6 and omega-3 pathways were also decreased with 1 Gy of neutrons, implicating cell membrane dysfunction and impaired phospholipid metabolism, which should otherwise lead to release of those molecules in circulation. While a precise relative biological effectiveness value could not be calculated from this study, the results are consistent with other published studies showing higher levels of damage from neutrons, demonstrated here by increased metabolic dysregulation. Metabolomics can therefore aid in identifying global perturbations in blood and urine, and effectively distinguishing between neutron and photon exposures.

The detonation of an improvised nuclear device would produce prompt radiation consisting of both photons (gamma rays) and neutrons. While much effort in recent years has gone into the development of radiation biodosimetry methods suitable for mass triage, the possible effect of neutrons on the endpoints studied has remained largely uninvestigated. We have used a novel neutron irradiator with an energy spectrum based on that 1-1.5 km from the epicenter of the Hiroshima blast to begin examining the effect of neutrons on global gene expression, and the impact this may have on the development of gene expression signatures for radiation biodosimetry. We have exposed peripheral blood from healthy human donors to 0.1, 0.3, 0.5 or 1 Gy of neutrons ex vivo using our neutron irradiator, and compared the transcriptomic response 24 h later to that resulting from sham exposure or exposure to 0.1, 0.3, 0.5, 1, 2 or 4 Gy of photons (X rays). We identified 125 genes that responded significantly to both radiation qualities as a function of dose, with the magnitude of response to neutrons generally being greater than that seen after X-ray exposure. Gene ontology analysis suggested broad involvement of the p53 signaling pathway and general DNA damage response functions across all doses of both radiation qualities. Regulation of immune response and chromatin-related functions were implicated only following the highest doses of neutrons, suggesting a physiological impact of greater DNA damage. We also identified several genes that seem to respond primarily as a function of dose, with less effect of radiation quality. We confirmed this pattern of response by quantitative real-time RT-PCR for BAX, TNFRSF10B, ITLN2 and AEN and suggest that gene expression may provide a means to differentiate between total dose and a neutron component.

Background: In the event of an improvised nuclear device detonation, the prompt radiation exposure would consist of photons plus a neutron component that would contribute to the total dose. As neutrons cause more complex and difficult to repair damage to cells that would result in a more severe health burden to affected individuals, it is paramount to be able to estimate the contribution of neutrons to an estimated dose, to provide information for those making treatment decisions., Results: Mice exposed to either 0.25 or 1 Gy of neutron or 1 or 4 Gy x-ray radiation were sacrificed at 1 or 7 days after exposure. Whole genome microarray analysis identified 7285 and 5045 differentially expressed genes in the blood of mice exposed to neutron or x-ray radiation, respectively. Neutron exposure resulted in mostly downregulated genes, whereas x-rays showed both down- and up-regulated genes. A total of 34 differentially expressed genes were regulated in response to all ≥1 Gy exposures at both times. Of these, 25 genes were consistently downregulated at days 1 and 7, whereas 9 genes, including the transcription factor E2f2, showed bi-directional regulation; being downregulated at day 1, while upregulated at day 7. Gene ontology analysis revealed that genes involved in nucleic acid metabolism processes were persistently downregulated in neutron irradiated mice, whereas genes involved in lipid metabolism were upregulated in x-ray irradiated animals. Most biological processes significantly enriched at both timepoints were consistently represented by either under- or over-expressed genes. In contrast, cell cycle processes were significant among down-regulated genes at day 1, but among up-regulated genes at day 7 after exposure to either neutron or x-rays. Cell cycle genes downregulated at day 1 were mostly distinct from the cell cycle genes upregulated at day 7. However, five cell cycle genes, Fzr1, Ube2c, Ccna2, Nusap1, and Cdc25b, were both downregulated at day 1 and upregulated at day 7., Conclusions: We describe, for the first time, the gene expression profile of mouse blood cells following exposure to neutrons. We have found that neutron radiation results in both distinct and common gene expression patterns compared with x-ray radiation.

We present the complete construction methodology for an anatomically accurate mouse phantom made using materials which mimic the characteristics of tissue, lung, and bone for radiation dosimetry studies. Phantoms were constructed using 2 mm thick slices of tissue equivalent material which was precision machined to clear regions for insertion of lung and bone equivalent material where appropriate. Images obtained using a 3D computed tomography (CT) scan clearly indicate regions of tissue, lung, and bone that match their position within the original mouse CT scan. Additionally, radiographic films are used with the phantom to demonstrate dose mapping capabilities. The construction methodology presented here can be quickly and easily adapted to create a phantom of any specific small animal given a segmented CT scan of the animal. These physical phantoms are a useful tool to examine individual organ dose and dosimetry within mouse systems that are complicated by density inhomogeneity due to bone and lung regions.

The radiation sciences are increasingly interdisciplinary, both from the research and the clinical perspectives. Beyond clinical and research issues, there are very real issues of communication between scientists from different disciplines. It follows that there is an increasing need for interdisciplinary training courses in the radiological sciences. Training courses are common in biomedical academic and clinical environments, but are typically targeted to scientists in specific technical fields. In the era of multidisciplinary biomedical science, there is a need for highly integrated multidisciplinary training courses that are designed for, and are useful to, scientists who are from a mix of very different academic fields and backgrounds. We briefly describe our experiences running such an integrated training course for researchers in the field of biomedical radiation microbeams, and draw some conclusions about how such interdisciplinary training courses can best function. These conclusions should be applicable to many other areas of the radiological sciences. In summary, we found that it is highly beneficial to keep the scientists from the different disciplines together. In practice, this means not segregating the training course into sections specifically for biologists and sections specifically for physicists and engineers, but rather keeping the students together to attend the same lectures and hands-on studies throughout the course. This structure added value to the learning experience not only in terms of the cross fertilization of information and ideas between scientists from the different disciplines, but also in terms of reinforcing some basic concepts for scientists in their own discipline.

A soft x-ray microbeam using proton-induced x-ray emission (PIXE) of characteristic titanium (K(α) 4.5 keV) as the x-ray source has been developed at the Radiological Research Accelerator Facility (RARAF) at Columbia University. The proton beam is focused to a 120 μm × 50 μm spot on the titanium target using an electrostatic quadrupole quadruplet previously used for the charged particle microbeam studies at RARAF. The proton induced x-rays from this spot project a 50 μm round x-ray generation spot into the vertical direction. The x-rays are focused to a spot size of 5 μm in diameter using a Fresnel zone plate. The x-rays have an attenuation length of (1/e length of ~145 μm) allowing more consistent dose delivery across the depth of a single cell layer and penetration into tissue samples than previous ultra soft x-ray systems. The irradiation end station is based on our previous design to allow quick comparison to charged particle experiments and for mixed irradiation experiments.

The array of microbeam cell-irradiation systems, available to users at the Radiological Research Accelerator Facility (RARAF), Center for Radiological Research, Columbia University, is expanding. The HVE 5MV Singletron particle accelerator at the facility provides particles to two focused ion microbeam lines: the sub-micron microbeam II and the permanent magnetic microbeam (PMM). Both the electrostatic quadrupole lenses on the microbeam II system and the magnetic quadrupole lenses on the PMM system are arranged as compound lenses consisting of two quadrupole triplets with "Russian" symmetry. Also, the RARAF accelerator is a source for a proton-induced x-ray microbeam (undergoing testing) and is projected to supply protons to a neutron microbeam based on the (7)Li(p, n)(7)Be nuclear reaction (under development). Leveraging from the multiphoton microscope technology integrated within the microbeam II endstation, a UV microspot irradiator - based on multiphoton excitation - is available for facility users. Highlights from radiation-biology demonstrations on single living mammalian cells are included in this review of microbeam systems for cell irradiation at RARAF.