Modeling Radiation-Induced Epithelial Cell Injury in Murine Three-Dimensional Esophageal Organoids.

Esophageal squamous cell carcinoma (ESCC) is a deadly consequence of radiation exposure to the esophagus. ESCC arises from esophageal epithelial cells that undergo malignant transformation and features a perturbed squamous cell differentiation program. Understanding the dose- and radiation quality-dependence of the esophageal epithelium response to radiation may provide insights into the ability of radiation to promote ESCC. We have explored factors that may play a role in esophageal epithelial radiosensitivity and their potential relationship to ESCC risk. We have utilized a murine three-dimensional (3D) organoid model that recapitulates the morphology and functions of the stratified squamous epithelium of the esophagus to study persistent dose- and radiation quality-dependent changes. Interestingly, although high-linear energy transfer (LET) Fe ion exposure induced a more intense and persistent alteration of squamous differentiation and 53BP1 DNA damage foci levels as compared to Cs, the MAPK/SAPK stress pathway signaling showed similar altered levels for most phospho-proteins with both radiation qualities. In addition, the lower dose of high-LET exposure also revealed nearly the same degree of morphological changes, even though only ~36% of the cells were predicted to be hit at the lower 0.1 Gy dose, suggesting that a bystander effect may be induced. Although p38 and ERK/MAPK revealed the highest levels following high-LET exposure, the findings reveal that even a low dose (0.1 Gy) of both radiation qualities can elicit a persistent stress signaling response that may critically impact the differentiation gradient of the esophageal epithelium, providing novel insights into the pathogenesis of radiation-induced esophageal injury and early stage esophageal carcinogenesis.

NF-κB in the Radiation Response of A549 Non-Small Cell Lung Cancer Cells to X-rays and Carbon Ions under Hypoxia.

Cellular hypoxia, detectable in up to 80% of non-small cell lung carcinoma (NSCLC) tumors, is a known cause of radioresistance. High linear energy transfer (LET) particle radiation might be effective in the treatment of hypoxic solid tumors, including NSCLC. Cellular hypoxia can activate nuclear factor κB (NF-κB), which can modulate radioresistance by influencing cancer cell survival. The effect of high-LET radiation on NF-κB activation in hypoxic NSCLC cells is unclear. Therefore, we compared the effect of low (X-rays)- and high (12C)-LET radiation on NF-κB responsive genes' upregulation, as well as its target cytokines' synthesis in normoxic and hypoxic A549 NSCLC cells. The cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h, followed by irradiation with 8 Gy X-rays or 12C ions, maintaining the oxygen conditions until fixation or lysis. Regulation of NF-κB responsive genes was evaluated by mRNA sequencing. Secretion of NF-κB target cytokines, IL-6 and IL-8, was quantified by ELISA. A greater fold change increase in expression of NF-κB target genes in A549 cells following exposure to 12C ions compared to X-rays was observed, regardless of oxygenation status. These genes regulate cell migration, cell cycle, and cell survival. A greater number of NF-κB target genes was activated under hypoxia, regardless of irradiation status. These genes regulate cell migration, survival, proliferation, and inflammation. X-ray exposure under hypoxia additionally upregulated NF-κB target genes modulating immunosurveillance and epithelial-mesenchymal transition (EMT). Increased IL-6 and IL-8 secretion under hypoxia confirmed NF-κB-mediated expression of pro-inflammatory genes. Therefore, radiotherapy, particularly with X-rays, may increase tumor invasiveness in surviving hypoxic A549 cells.

Hypoxia Modulates Radiosensitivity and Response to Different Radiation Qualities in A549 Non-Small Cell Lung Cancer (NSCLC) Cells.

Hypoxia-induced radioresistance reduces the efficacy of radiotherapy for solid malignancies, including non-small cell lung cancer (NSCLC). Cellular hypoxia can confer radioresistance through cellular and tumor micro-environment adaptations. Until recently, studies evaluating radioresistance secondary to hypoxia were designed to maintain cellular hypoxia only before and during irradiation, while any handling of post-irradiated cells was carried out in standard oxic conditions due to the unavailability of hypoxia workstations. This limited the possibility of simulating in vivo or clinical conditions in vitro. The presence of molecular oxygen is more important for the radiotoxicity of low-linear energy transfer (LET) radiation (e.g., X-rays) than that of high-LET carbon (12C) ions. The mechanisms responsible for 12C ions' potential to overcome hypoxia-induced radioresistance are currently not fully understood. Therefore, the radioresistance of hypoxic A549 NSCLC cells following exposure to X-rays or 12C ions was investigated along with cell cycle progression and gene expression by maintaining hypoxia before, during and after irradiation. A549 cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h and then irradiated with X-rays (200 kV) or 12C ions (35 MeV/n, LET ~75 keV/µm). Cell survival was evaluated using colony-forming ability (CFA) assays immediately or 24 h after irradiation (late plating). DNA double-strand breaks (DSBs) were analyzed using γH2AX immunofluorescence microscopy. Cell cycle progression was determined by flow cytometry of 4',6-diamidino-2-phenylindole-stained cells. The global transcription profile post-irradiation was evaluated by RNA sequencing. When hypoxia was maintained before, during and after irradiation, hypoxia-induced radioresistance was observed only in late plating CFA experiments. The killing efficiency of 12C ions was much higher than that of X-rays. Cell survival under hypoxia was affected more strongly by the timepoint of plating in the case of X-rays compared to 12C ions. Cell cycle arrest following irradiation under hypoxia was less pronounced but more prolonged. DSB induction and resolution following irradiation were not significantly different under normoxia and hypoxia. Gene expression response to irradiation primarily comprised cell cycle regulation for both radiation qualities and oxygen conditions. Several PI3K target genes involved in cell migration and cell motility were differentially upregulated in hypoxic cells. Hypoxia-induced radioresistance may be linked to altered cell cycle response to irradiation and PI3K-mediated changes in cell motility and migration in A549 cells rather than less DNA damage or faster repair.

Residual radiation risk disparities across sex and race or ethnic groups for lifetime never-smokers on lunar missions.

Missions to the Earth's moon are of scientific and societal interest, however pose the problem of risks of late effects for returning crew persons, most importantly cancer and circulatory diseases. In this paper, we discuss NSCR-2022 model risk estimates for lunar missions for US racial and ethnic groups comparing never-smokers (NS) to US averages for each group and sex. We show that differences within groups between men and women are reduced for NS compared to the average population. Race and ethnic group dependent cancer and circulatory disease risks are reduced by 10% to 40% for NS with the largest decrease for Whites. Circulatory disease risks are changed by less than 10% for NS and in several cases modestly increased due to increased lifespan for NS. Asian-Pacific Islanders (API) and Hispanics NS are at lower risk compared to Whites and Blacks. Differences between groups are narrowed for NS compared to predictions for average populations, however disparities remain especially for Blacks and to a lesser extent Whites compared to API or Hispanic NS groups.

Preoperative stereotactic radiosurgery as neoadjuvant therapy for resectable brain tumors.

PURPOSE: Stereotactic radiosurgery (SRS) is a method of delivering conformal radiation, which allows minimal radiation damage to surrounding healthy tissues. Adjuvant radiation therapy has been shown to improve local control in a variety of intracranial neoplasms, such as brain metastases, gliomas, and benign tumors (i.e., meningioma, vestibular schwannoma, etc.). For brain metastases, adjuvant SRS specifically has demonstrated positive oncologic outcomes as well as preserving cognitive function when compared to conventional whole brain radiation therapy. However, as compared with neoadjuvant SRS, larger post-operative volumes and greater target volume uncertainty may come with an increased risk of local failure and treatment-related complications, such as radiation necrosis. In addition to its role in brain metastases, neoadjuvant SRS for high grade gliomas may enable dose escalation and increase immunogenic effects and serve a purpose in benign tumors for which one cannot achieve a gross total resection (GTR). Finally, although neoadjuvant SRS has historically been delivered with photon therapy, there are high LET radiation modalities such as carbon-ion therapy which may allow radiation damage to tissue and should be further studied if done in the neoadjuvant setting. In this review we discuss the evolving role of neoadjuvant radiosurgery in the treatment for brain metastases, gliomas, and benign etiologies. We also offer perspective on the evolving role of high LET radiation such as carbon-ion therapy. METHODS: PubMed was systemically reviewed using the search terms "neoadjuvant radiosurgery", "brain metastasis", and "glioma". ' Clinicaltrials.gov ' was also reviewed to include ongoing phase III trials. RESULTS: This comprehensive review describes the evolving role for neoadjuvant SRS in the treatment for brain metastases, gliomas, and benign etiologies. We also discuss the potential role for high LET radiation in this setting such as carbon-ion radiotherapy. CONCLUSION: Early clinical data is very promising for neoadjuvant SRS in the setting of brain metastases. There are three ongoing phase III trials that will be more definitive in evaluating the potential benefits. While there is less data available for neoadjuvant SRS for gliomas, there remains a potential role, particularly to enable dose escalation and increase immunogenic effects.

Metal Ions Modify In Vitro DNA Damage Yields with High-LET Radiation.

Cu2+ and Co2+ are metals known to increase DNA damage in the presence of hydrogen peroxide through a Fenton-type reaction. We hypothesized that these metals could increase DNA damage following irradiations of increasing LET values as hydrogen peroxide is a product of the radiolysis of water. The reaction mixtures contain either double- or single-stranded DNA in solution with Cu2+ or Co2+ and were irradiated either with X-ray, carbon-ion or iron-ion beams, or they were treated with hydrogen peroxide or bleomycin at increasing radiation dosages or chemical concentrations. DNA damage was then assessed via gel electrophoresis followed with a band intensity analysis. DNA damage was the greatest when DNA in the solution with either metal was treated with only hydrogen peroxide followed by the DNA damage of DNA in the solution with either metal post irradiation of low-LET (X-Ray) or high-LET (carbon-ion and iron-ion), respectively, and demonstrated the least damage after treatment with bleomycin. Cu2+ portrayed greater DNA damage than Co2+ following all experimental conditions. The metals' effect caused more DNA damage and was observed to be LET-dependent for single-strand break formation but inversely dependent for double-strand break formation. These results suggest that Cu2+ is more efficient than Co2+ at inducing both DNA single-strand and double-strand breaks following all irradiations and chemical treatments.

Short Double-Stranded DNA (≤40-bp) Affects Repair Pathway Choice.

To repair ionizing radiation (IR)-induced double strand breaks (DSBs), mammalian cells primarily use canonical non-homologous end-joining (cNHEJ), the homologous recombination (HR) pathway, and the alternative non-homologous end-joining (aEJ) as a backup. These pathways function either compensatively or competitively. High linear energy transfer (LET) compared to low-LET IR kills more cells at the same doses by inhibiting only cNHEJ, but not HR or aEJ. The mechanism remains unclear. The activation of each repair pathway requires the binding of different proteins to DNA fragments of varying lengths. We previously observed an increased generation of small DNA fragments (≤40 bp) in cells following high-LET IR compared to low-LET IR, suggesting that short DNA fragments were one of the major factors interfering with cNHEJ. To provide direct evidence, here we compare the efficiencies of cNHEJ, HR, or aEJ in repairing DSBs containing 30- or 60-bp fragments in vitro and in cells. We show that only cNHEJ but not HR or a-EJ was inefficient for repairing DSBs with 30-bp fragments compared to 60-bp ones, which strongly supports our hypothesis. These results not only enhance our understanding of the DSB repair pathway choice but also hold potential benefits for protection against high-LET IR-induced damage or improving high-LET radiotherapy.

DNA Damage Response and Repair in Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is an approach to the radiotherapy of solid tumors that was first outlined in the 1930s but has attracted considerable attention recently with the advent of a new generation of neutron sources. In BNCT, tumor cells accumulate 10B atoms that react with epithermal neutrons, producing energetic α particles and 7Li atoms that damage the cell's genome. The damage inflicted by BNCT appears not to be easily repairable and is thus lethal for the cell; however, the molecular events underlying the action of BNCT remain largely unaddressed. In this review, the chemistry of DNA damage during BNCT is outlined, the major mechanisms of DNA break sensing and repair are summarized, and the specifics of the repair of BNCT-induced DNA lesions are discussed.

Compact portable sources of high-LET radiation: Validation and potential application for galactic cosmic radiation countermeasure discovery.

Implementation of a systematic program for galactic cosmic radiation (GCR) countermeasure discovery will require convenient access to ground-based space radiation analogs. The current gold standard approach for GCR simulation is to use a particle accelerator for sequential irradiation with ion beams representing different GCR components. This has limitations, particularly for studies of non-acute responses, strategies that require robotic instrumentation, or implementation of complex in vitro models that are emerging as alternatives to animal experimentation. Here we explore theoretical and practical issues relating to a different approach to provide a high-LET radiation field for space radiation countermeasure discovery, based on use of compact portable sources to generate neutron-induced charged particles. We present modeling studies showing that DD and DT neutron generators, as well as an AmBe radionuclide-based source, generate charged particles with a linear energy transfer (LET) distribution that, within a range of biological interest extending from about 10 to 200 keV/µm, resembles the LET distribution of reference GCR radiation fields experienced in a spacecraft or on the lunar surface. We also demonstrate the feasibility of using DD neutrons to induce 53BP1 DNA double-strand break repair foci in the HBEC3-KT line of human bronchial epithelial cells, which are widely used for studies of lung carcinogenesis. The neutron-induced foci are larger and more persistent than X ray-induced foci, consistent with the induction of complex, difficult-to-repair DNA damage characteristic of exposure to high-LET (>10 keV/µm) radiation. We discuss limitations of the neutron approach, including low fluence in the low LET range (<10 keV/µm) and the absence of certain long-range features of high charge and energy particle tracks. We present a concept for integration of a compact portable source with a multiplex microfluidic in vitro culture system, and we discuss a pathway for further validation of the use of compact portable sources for countermeasure discovery.

Fluorescence in situ hybridization analysis of chromosomal aberrations in mouse splenocytes at one- and two-months after total body exposure to iron-56 (Fe) ion particles or X-rays.

High atomic number and energy (HZE) particles such as iron-56 (Fe) ions are a major contributor to health risks in long-term manned space exploration. The aim of this study is to understand radiation-induced differential genotoxic effects between HZE particles and low linear energy transfer (LET) photons. C57BL/6J Jms female mice of 8 weeks old were exposed to total body irradiation of accelerated Fe-particles with a dose ranging from 0.1 to 3.0 Gy or of X-rays with a dose ranging from 0.1 to 5.0 Gy. Chromosomal aberrations (CAs) in splenocytes were examined by fluorescence in situ hybridization at 1- and 2-months after exposure. Clonal expansions of cells with CAs were found to be induced only by X-rays but not by Fe-particles. Dose-dependent increase in the frequencies of stable-type CAs was observed at 1- as well as 2-months after exposure to both radiation types. The frequencies of stable-type CAs in average were much higher in mice exposed to X-rays than those to Fe-particles and did not change significantly between 1- and 2-months after exposure to both radiation types. On the other hand, the frequencies of unstable-type CAs induced by X-rays and Fe-particles were not much different, and they appeared to decrease with time from 1- to 2-months after exposure. These results suggested that larger fraction of stable-type CAs induced by Fe-particles might be non-transmissible than those by X-rays because of some associating lethal alterations on themselves or on other chromosomes in the same cells and that these cells might be removed by 1-month after Fe-TBI. We also demonstrated that exposure to Fe-particles induced insertions at relatively higher frequency to other stable-type CAs than X-rays. Our findings suggest that insertions can be used as indicators of past exposure to high-LET particle radiation.

Exosome secretion and cellular response of DU145 and PC3 after exposure to alpha radiation.

Exosomes are spherical membrane nanovesicles secreted from cells, and they play an important role in tumor immune response, metastasis, angiogenesis, and survival. Studies investigating exosomes isolated from cells exposed to photon radiation commonly used in conventional radiotherapy demonstrate the influence of this type of radiation on exosome characteristics and secretion. There is currently no research investigating the effects of densely ionizing particles such as protons and alpha radiation on exosomes. Thus we have evaluated the cellular response of human prostate cancer cells exposed to 0, 2, and 6 Gy of alpha radiation emitted from the Am-241 source. Irradiated PC3 and DU145 cell lines, characterized by differences in radiosensitivity, were studied using apoptosis, LDH, and IL-6 assays. Additionally, the corresponding concentration and size of isolated exosomes were measured using NTA. We found that exposure to ionizing radiation resulted in gross changes in viability and cell damage. There were increased amounts of apoptotic or necrotic cells as a function of radiation dose. We demonstrated that irradiated PC3 cells secrete higher quantities of exosomes compared to DU145 cells. Additionally, we also found no statistical difference in exosome size for control and irradiated cells.

Total body proton and heavy-ion irradiation causes cellular senescence and promotes pro-osteoclastogenic activity in mouse bone marrow.

Low-LET photon radiation-induced persistent alterations in bone marrow (BM) cells are well documented in total-body irradiated (TBI) rodents and also among radiotherapy patients. However, the late effects of protons and high-LET heavy-ion radiation on BM cells and its implications in osteoclastogenesis are not fully understood. Therefore, C57BL6/J female mice (8 weeks; n = 10/group) were irradiated to sham, and 1 Gy of the proton (0.22 keV/µm), or high-LET 56Fe-ions (148 keV/µm) and at 60 d post-exposure, mice were sacrificed and femur sections were obtained for histological, cellular and molecular analysis. Cell proliferation (PCNA), cell death (active caspase-3), senescence (p16), osteoclast (RANK), osteoblast (OPG), osteoblast progenitor (c-Kit), and osteoclastogenesis-associated secretory factors (like RANKL) were assessed using immunostaining. While no change in cell proliferation and apoptosis between control and irradiated groups was noted, the number of BM megakaryocytes was significantly reduced in irradiated mice at 60 d post-exposure. A remarkable increase in p16 positive cells indicated a persistent increase in cell senescence, whereas increased RANKL/OPG ratio, reductions in the number of osteoblast progenitor cells, and osteocalcin provided clear evidence that exposure to both proton and 56Fe-ions promotes pro-osteoclastogenic activity in BM. Among irradiated groups, 56Fe-induced alterations in the BM cellularity and osteoclastogenesis were significantly greater than the protons that demonstrated a radiation quality-dependent effect. This study has implications in understanding the role of IR-induced late changes in the BM cells and its involvement in bone degeneration among deep-space astronauts, and also in patients undergoing proton or heavy-ion radiotherapy.

Human mesenchymal stromal cells maintain their stem cell traits after high-LET particle irradiation - Potential implications for particle radiotherapy and manned space missions.

The influence of high-linear energy transfer (LET) particle radiation on the functionalities of mesenchymal stromal cells (MSCs) is largely unknown. Here, we analyzed the effects of proton (1H), helium (4He), carbon (12C) and oxygen (16O) ions on human bone marrow-MSCs. Cell cycle distribution and apoptosis induction were examined by flow cytometry, and DNA damage was quantified using γH2AX immunofluorescence and Western blots. Relative biological effectiveness values of MSCs amounted to 1.0-1.1 for 1H, 1.7-2.3 for 4He, 2.9-3.4 for 12C and 2.6-3.3 for 16O. Particle radiation did not alter the MSCs' characteristic surface marker pattern, and MSCs maintained their multi-lineage differentiation capabilities. Apoptosis rates ranged low for all radiation modalities. At 24 h after irradiation, particle radiation-induced ATM and CHK2 phosphorylation as well as γH2AX foci numbers returned to baseline levels. The resistance of human MSCs to high-LET irradiation suggests that MSCs remain functional after exposure to moderate doses of particle radiation as seen in normal tissues after particle radiotherapy or during manned space flights. In the future, in vivo models focusing on long-term consequences of particle irradiation on the bone marrow niche and MSCs are needed.

Mitigation of late cardiovascular effects of oxygen ion radiation by γ-tocotrienol in a mouse model.

PURPOSE: While there is concern about degenerative tissue effects of exposure to space radiation during deep-space missions, there are no pharmacological countermeasures against these adverse effects. γ-Tocotrienol (GT3) is a natural form of vitamin E that has anti-oxidant properties, modifies cholesterol metabolism, and has anti-inflammatory and endothelial cell protective properties. The purpose of this study was to test whether GT3 could mitigate cardiovascular effects of oxygen ion (16O) irradiation in a mouse model. MATERIALS AND METHODS: Male C57BL/6 J mice were exposed to whole-body 16O (600 MeV/n) irradiation (0.26-0.33 Gy/min) at doses of 0 or 0.25 Gy at 6 months of age and were followed up to 9 months after irradiation. Animals were administered GT3 (50 mg/kg/day s.c.) or vehicle, on Monday - Friday starting on day 3 after irradiation for a total of 16 administrations. Ultrasonography was used to measure in vivo cardiac function and blood flow parameters. Cardiac tissue remodeling and inflammatory infiltration were assessed with histology and immunoblot analysis at 2 weeks, 3 and 9 months after radiation. RESULTS: GT3 mitigated the effects of 16O radiation on cardiac function, the expression of a collagen type III peptide, and markers of mast cells, T-cells and monocytes/macrophages in the left ventricle. CONCLUSIONS: GT3 may be a potential countermeasure against late degenerative tissue effects of high-linear energy transfer radiation in the heart.

Effects of dietary aspirin on high-LET radiation-induced prostaglandin E2 levels and gastrointestinal tumorigenesis in Apc1638N/+ mice.

Inevitable exposure to high-LET ionizing radiation (IR) present in galactic cosmic radiation (GCR) could enhance gastrointestinal (GI) cancer incidence among astronauts undertaking deep space exploration and GI-cancer mortality has been predicted to far exceed NASA's limit of < 3% REID (Radiation exposure-induced death) from cancer. Therefore, the development of countermeasure agents against high-LET radiation-induced GI cancer is needed to safeguard astronauts during and after an outer space mission. The cyclooxygenase-2/prostaglandin E2 (COX2/PGE2) mediated activation of pro-inflammatory and oncogenic signaling has been reported to play an important role in persistent inflammation and GI-tumorigenesis after high-LET radiation exposure. Therefore, aspirin, a well-known inhibitor of the COX/PGE2 pathway, was evaluated as a potential countermeasure against 28Si-induced PGE2 and tumorigenesis in Apc1638N/+, a murine model of human GI-cancer. Animals were fed either standard or aspirin supplemented diet (75, 150, or 300 mg/day of human equivalent dose) starting at the age of 4 weeks and continued till the end of the study, while mice were exposed to 28Si-ions (300 MeV/n; 69 keV/µm) at the age of 8 weeks. Serum PGE2 level, GI tumor size (>2mm2), number, and cluster (>5 adjoining tumors) were analyzed at 150 days post-exposure. Aspirin led to a significant reduction in PGE2 in a dose-dependent manner but did not reduce 28Si-induced GI tumorigenesis even at the highest (300 mg/day) dose. In summary, this study suggests that aspirin could reduce high-LET IR-induced pro-inflammatory PGE2 levels, however, lacks the ability to reduce high-LET IR-induced GI tumorigenesis in Apc1638N/+ mice.

Strong Shift to ATR-Dependent Regulation of the G2-Checkpoint after Exposure to High-LET Radiation.

The utilization of high linear-energy-transfer (LET) ionizing radiation (IR) modalities is rapidly growing worldwide, causing excitement but also raising concerns, because our understanding of their biological effects is incomplete. Charged particles such as protons and heavy ions have increasing potential in cancer therapy, due to their advantageous physical properties over X-rays (photons), but are also present in the space environment, adding to the health risks of space missions. Therapy improvements and the protection of humans during space travel will benefit from a better understanding of the mechanisms underpinning the biological effects of high-LET IR. There is evidence that high-LET IR induces DNA double-strand breaks (DSBs) of increasing complexity, causing enhanced cell killing, owing, at least partly, to the frequent engagement of a low-fidelity DSB-repair pathway: alternative end-joining (alt-EJ), which is known to frequently induce severe structural chromosomal abnormalities (SCAs). Here, we evaluate the radiosensitivity of A549 lung adenocarcinoma cells to X-rays, α-particles and 56Fe ions, as well as of HCT116 colorectal cancer cells to X-rays and α-particles. We observe the expected increase in cell killing following high-LET irradiation that correlates with the increased formation of SCAs as detected by mFISH. Furthermore, we report that cells exposed to low doses of α-particles and 56Fe ions show an enhanced G2-checkpoint response which is mainly regulated by ATR, rather than the coordinated ATM/ATR-dependent regulation observed after exposure to low doses of X-rays. These observations advance our understanding of the mechanisms underpinning high-LET IR effects, and suggest the potential utility for ATR inhibitors in high-LET radiation therapy.

Comparison of signaling profiles in the low dose range following low and high LET radiation.

During space travel astronauts will be exposed to a very low, mixed field of radiation containing different high LET particles of varying energies, over an extended period. Thus, defining how human cells respond to these complex low dose exposures is important in ascertaining risk. In the current study, we have chosen to investigate how low doses of three different ion's at various energies uniquely change the kinetics of three different phospho-proteins. A normal hTERT immortalized fibroblast cell line, 82-6, was exposed to a range of lower doses (0.05-0.5 Gy) of radiation of different qualities and energies (Si 1000 MeV/u, Si 300 MeV/u, Si 173 MeV/u, Si 93 MeV/u, Fe 1000 MeV/u, Fe 600 MeV/u, Fe 300 MeV/u, Ti 300 MeV/u, Ti 326 MeV/u, Ti 386 MeV/u), covering a wide span of LET's. Exposed samples were analyzed for the average intensity of signal as a fold over the geometric mean level of the sham controls. Three phospho-proteins known to localize to DNA DSBs following radiation (γH2AX, pATF2, pSMC1) were studied. The kinetics of their response was quantified by flow cytometery at 2 and 24 h post exposure. These studies reveal unique kinetic patterns based on the ion, energy, fluence and time following exposure. In addition, γH2AX phosphorylation patterns are uniquely different from phospho-proteins known to be primarily phosphorylated by ATM. This latter finding suggests that the activating kinase(s), or the phosphatases deactivating these proteins, exhibit differences in their response to various radiation qualities and/ or doses of exposure. Further studies will be needed to better define what the differing kinetics for the kinases activated by the unique radiation qualities plays in the biological effectiveness of the particle.

Carbon Ion Therapy: A Modern Review of an Emerging Technology.

Radiation therapy is one of the most widely used therapies for malignancies. The therapeutic use of heavy ions, such as carbon, has gained significant interest due to advantageous physical and radiobiologic properties compared to photon based therapy. By taking advantage of these unique properties, carbon ion radiotherapy may allow dose escalation to tumors while reducing radiation dose to adjacent normal tissues. There are currently 13 centers treating with carbon ion radiotherapy, with many of these centers publishing promising safety and efficacy data from the first cohorts of patients treated. To date, carbon ion radiotherapy has been studied for almost every type of malignancy, including intracranial malignancies, head and neck malignancies, primary and metastatic lung cancers, tumors of the gastrointestinal tract, prostate and genitourinary cancers, sarcomas, cutaneous malignancies, breast cancer, gynecologic malignancies, and pediatric cancers. Additionally, carbon ion radiotherapy has been studied extensively in the setting of recurrent disease. We aim to provide a comprehensive review of the studies of each of these disease sites, with a focus on the current trials using carbon ion radiotherapy.

Benchmarking risk predictions and uncertainties in the NSCR model of GCR cancer risks with revised low let risk coefficients.

We report on the contributions of model factors that appear in projection models to the overall uncertainty in cancer risks predictions for exposures to galactic cosmic ray (GCR) in deep space, including comparisons with revised low LET risks coefficients. Annual GCR exposures to astronauts at solar minimum are considered. Uncertainties in low LET risk coefficients, dose and dose-rate modifiers, quality factors (QFs), space radiation organ doses, non-targeted effects (NTE) and increased tumor lethality at high LET compared to low LET radiation are considered. For the low LET reference radiation parameters we use a revised assessment of excess relative risk (ERR) and excess additive risk (EAR) for radiation induced cancers in the Life-Span Study (LSS) of the Atomic bomb survivors that was recently reported, and also consider ERR estimates for males from the International Study of Nuclear Workers (INWORKS). For 45-y old females at mission age the risk of exposure induced death (REID) per year and 95% confidence intervals is predicted as 1.6% [0.71, 1.63] without QF uncertainties and 1.64% [0.69, 4.06] with QF uncertainties. However, fatal risk predictions increase to 5.83% [2.56, 9.7] based on a sensitivity study of the inclusion of non-targeted effects on risk predictions. For males a comparison using LSS or INWORKS lead to predictions of 1.24% [0.58, 3.14] and 2.45% [1.23, 5.9], respectively without NTEs. The major conclusion of our report is that high LET risk prediction uncertainties due to QFs parameters, NTEs, and possible increase lethality at high LET are dominant contributions to GCR uncertainties and should be the focus of space radiation research.

Necessities in the Processing of DNA Double Strand Breaks and Their Effects on Genomic Instability and Cancer.

Double strand breaks (DSBs) are induced in the DNA following exposure of cells to ionizing radiation (IR) and are highly consequential for genome integrity, requiring highly specialized modes of processing. Erroneous processing of DSBs is a cause of cell death or its transformation to a cancer cell. Four mechanistically distinct pathways have evolved in cells of higher eukaryotes to process DSBs, providing thus multiple options for the damaged cells. The homologous recombination repair (HRR) dependent subway of gene conversion (GC) removes IR-induced DSBs from the genome in an error-free manner. Classical non-homologous end joining (c-NHEJ) removes DSBs with very high speed but is unable to restore the sequence at the generated junction and can catalyze the formation of translocations. Alternative end-joining (alt-EJ) operates on similar principles as c-NHEJ but is slower and more error-prone regarding both sequence preservation and translocation formation. Finally, single strand annealing (SSA) is associated with large deletions and may also form translocations. Thus, the four pathways available for the processing of DSBs are not alternative options producing equivalent outcomes. We discuss the rationale for the evolution of pathways with such divergent properties and fidelities and outline the logic and necessities that govern their engagement. We reason that cells are not free to choose one specific pathway for the processing of a DSB but rather that they engage a pathway by applying the logic of highest fidelity selection, adapted to necessities imposed by the character of the DSB being processed. We introduce DSB clusters as a particularly consequential form of chromatin breakage and review findings suggesting that this form of damage underpins the increased efficacy of high linear energy transfer (LET) radiation modalities. The concepts developed have implications for the protection of humans from radon-induced cancer, as well as the treatment of cancer with radiations of high LET.