The p53-binding protein 1 (53BP1) is a well-known DNA damage response (DDR) factor, which is recruited to nuclear structures at the site of DNA damage and forms readily visualized ionizing radiation (IR) induced foci. Depletion of 53BP1 results in cell cycle arrest in G₂/M phase as well as genomic instability in human as well as mouse cells. Within the DNA damage response mechanism, 53BP1 is classified as an adaptor/mediator, required for processing of the DNA damage response signal and as a platform for recruitment of other repair factors. More recently, specific 53BP1 contributions to DSB repair pathway choice have been recognized and are being characterized. In this review, we have summarized recent advances in understanding the role of 53BP1 in regulating DNA DSBs repair pathway choice, variable diversity joining [V(D)J] recombination and class-switch recombination (CSR).

Exactly a century after Röntgen's discovery of X rays, I entered a university to major in radiological sciences. At that time, I felt that, despite extensive use and indispensable roles of ionizing radiation in medicine and industry, many fascinating questions have yet to be answered concerning its biological mechanisms of action, and thus I decided to get into the field of radiation research. Fifteen years have passed since I started radiobiological studies in 1998, during which time various basic tenets I initially learned in my late teens and early twenties have been challenged by recent observations. Of these, this brief overview particularly focuses on the following five different albeit non mutually exclusive questions: (i) “Is nuclear DNA the only intracellular target for radiation effects?”; (ii) “What is the significance of delayed cell death in clonogenic survival?”; (iii) “Does an irradiated cell become a cancer cell?”; (iv) “Are cataracts tissue reactions?”; and (v) “Why is high-LET radiation biologically effective?”.

Fractionated partial or whole-brain irradiation is the primary treatment for metastatic brain tumors. Despite reducing tumor burden and increasing lifespan, progressive, irreversible cognitive impairment occurs in >50% of the patients who survive >6 months after fractionated whole-brain irradiation. The exact mechanism(s) responsible for this radiation-induced brain injury are unknown; however, preclinical studies suggest that radiation modulates the extracellular receptor kinase signaling pathway, which is associated with cognitive impairment in many neurological diseases. In the study reported here, we demonstrated that the extracellular receptor kinase transcriptionally-regulated early response gene, Homer1a, was up-regulated transiently in the hippocampus and down-regulated in the cortex of young adult male Fischer 344 X Brown Norway rats at 48 h after 40 Gy of fractionated whole-brain irradiation. Two months after fractionated whole-brain irradiation, these changes in Homer1a expression correlated with a down-regulation of the hippocampal glutamate receptor 1 and protein kinase C_γ, and an up-regulation of cortical glutamate receptor 1 and protein kinase C_γ. Two drugs that prevent radiation-induced cognitive impairment in rats, the angiotensin type-1 receptor blocker, L-158,809, and the angiotensin converting enzyme inhibitor, ramipril, reversed the fractionated whole-brain irradiation-induced Homer1a expression at 48 h in the hippocampus and cortex and restored glutamate receptor 1 and protein kinase C_γ to the levels in sham-irradiated controls at 2 months after fractionated whole-brain irradiation. These data indicate that Homer1a is, (1) a brain region specific regulator of radiation-induced brain injury, including cognitive impairment and (2) potentially a druggable target for preventing it.

We hypothesized that dietary administration of the peroxisomal proliferator-activated receptor α agonist, fenofibrate, to young adult male rats would prevent the fractionated whole-brain irradiation (fWBI)-induced reduction in cognitive function and neurogenesis and prevent the fWBI-induced increase in the total number of activated microglia. Eighty 12–14-week-old young adult male Fischer 344 × Brown Norway rats received either: (1) sham irradiation, (2) 40 Gy of fWBI delivered as two 5 Gy fractions/week for 4 weeks, (3) sham irradiation dietary fenofibrate (0.2% w/w) starting 7 days prior to irradiation, or (4) fWBI fenofibrate. Cognitive function was measured 26–29 weeks after irradiation using: (1) the perirhinal cortex (PRh)-dependent novel object recognition task; (2) the hippocampal-dependent standard Morris water maze (MWM) task; (3) the hippocampal-dependent delayed match-to-place version of the MWM task; and (4) a cue strategy preference version of the MWM to distinguish hippocampal from striatal task performance. Neurogenesis was assessed 29 weeks after fWBI in the granular cell layer and subgranular zone of the dentate gyrus using a doublecortin antibody. Microglial activation was assessed using an ED1 antibody in the dentate gyrus and hilus of the hippocampus. A significant impairment in perirhinal cortex-dependent cognitive function was measured after fWBI. In contrast, fWBI failed to alter hippocampal-dependent cognitive function, despite a significant reduction in hippocampal neurogenesis. Continuous administration of fenofibrate prevented the fWBI-induced reduction in perirhinal cortex-dependent cognitive function, but did not prevent the radiation-induced reduction in neurogenesis or the radiation-induced increase in activated microglia. These data suggest that fenofibrate may be a promising therapeutic for the prevention of some modalities of radiation-induced cognitive impairment in brain cancer patients.

Ionizing space radiation causes oxidative DNA damage and triggers oxidative stress responses, and compromised DNA repair mechanisms can lead to increased risk of carcinogenesis. Young adult mice with developed innate and adaptive immune systems that harbored either a conventional intestinal microbiota (CM) or an intestinal microbiota with a restricted microbial composition (RM) were irradiated with a total dose of 1 Gy delivered by high-energy protons (2.5 GeV/n, LET = 0.2–2 keV/μm) or silicon or iron ions (850 MeV/n, LET ≈ 50 keV/μm and 1 GeV/n, LET = 150 keV/μm, respectively). Six hours after whole-body irradiation, acute chromosomal DNA lesions were observed for RM mice but not CM mice. High-throughput rRNA gene sequencing of intestinal mucosal bacteria showed that Barnesiella intestinihominis and unclassified Bacterodiales were significantly more abundant in male RM mice than CM mice, and phylotype densities changed in irradiated mice. In addition, Helicobacter hepaticus and Bacteroides stercoris were higher in CM than RM mice. Elevated levels of persistently phosphorylated γ-H2AX were observed in RM mice exposed to high-energy protons compared to nonirradiated RM mice, and they also were associated with a decrease of the antioxidant glutathione in peripheral blood measured at four weeks after irradiation. After radiation exposure, CM mice showed lower levels of γ-H2AX phosphorylation than RM mice and an increase in specific RM-associated phylotypes, indicating a down-regulating force on DNA repair by differentially abundant phylotypes in RM versus a radiation-sensitive complex CM.

Cesium-137 is a fission product of uranium and plutonium in nuclear reactors and is released in large quantities during nuclear explosions or detonation of an improvised device containing this isotope. This environmentally persistent radionuclide undergoes radioactive decay with the emission of beta particles as well as gamma radiation. Exposure to ¹³⁷Cs at high doses can cause acute radiation sickness and increase risk for cancer and death. The serious health risks associated with ¹³⁷Cs exposure makes it critical to understand how it affects human metabolism and whether minimally invasive and easily accessible samples such as urine and serum can be used to triage patients in case of a nuclear disaster or a radiologic event. In this study, we have focused on establishing a time-dependent metabolomic profile for urine collected from mice injected with ¹³⁷CsCl. The samples were collected from control and exposed mice on days 2, 5, 20 and 30 after injection. The samples were then analyzed by ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC/TOFMS) and processed by an array of informatics and statistical tools. A total of 1,412 features were identified in ESI and ESI^– modes from which 200 were determined to contribute significantly to the separation of metabolomic profiles of controls from those of the different treatment time points. The results of this study highlight the ease of use of the UPLC/TOFMS platform in finding urinary biomarkers for ¹³⁷Cs exposure. Pathway analysis of the statistically significant metabolites suggests perturbations in several amino acid and fatty acid metabolism pathways. The results also indicate that ¹³⁷Cs exposure causes: similar changes in the urinary excretion levels of taurine and citrate as seen with external-beam gamma radiation; causes no attenuation in the levels of hexanoylglycine and N-acetylspermidine; and has unique effects on the levels of isovalerylglycine and tiglylglycine.

The effects of ionizing radiation on DNA methylation are of importance due to the role that DNA methylation plays in maintaining genome stability, and the presence of aberrant DNA methylation in many cancers. There is limited evidence that radiation-sensitivity may influence the modulation of DNA methylation by ionizing radiation, resulting in a loss of methylation. The BALB/c, CBA and C57Bl/6 strains are the most commonly utilized mouse strains in radiation research and are classified as radiation sensitive (BALB/c and CBA) or radiation resistant (C57Bl/6). We present here the first direct comparison of changes in repeat element DNA methylation (L1, B1 and Intracisternal A Particle; IAP) over time in these three mouse strains after high-dose radiation exposure. Using a high-resolution melt assay, methylation of the spleen repeat elements was investigated between 1 and 14 days after whole-body irradiation with 1 Gy X rays. Our study demonstrated that rather than a loss of methylation at the elements, all strains exhibited an early increase in L1 methylation one day after irradiation. In the most radiosensitive strain (BALB/c) the increase was also detected at 6 days postirradiation. The radioresistant C57Bl/6 strain exhibited a loss of L1 methylation at 14 days postirradiation. Less extensive changes to the B1 and IAP elements were detected at various time points, and pyrosequencing revealed that the responses of the strains were influenced by sex, with the male BALB/c and CBA mice exhibiting a greater response to the irradiation. The results of our study do not support the hypothesis that the most radiosensitive strains exhibit the greatest loss of repeat element DNA methylation after exposure to high-dose radiation. While the exact mechanism and biological outcome of the changes in DNA methylation observed here are still to be elucidated, this study provides the first evidence that radiation exposure elicits time-dependent changes in the methylation of repeat elements that are influenced by the genetic background, gender and the type of repeat element investigated. Furthermore, it suggest that any induced changes may not be persistent.

FancD2 plays a central role in the human Fanconi anemia DNA damage response (DDR) pathway. Fancd2^–/– mice exhibit many features of human Fanconi anemia including cellular DNA repair defects. Whether the DNA repair defect in Fancd2^–/– mice results in radiologic changes in all cell lineages is unknown. We measured stress of hematopoiesis in long-term marrow cultures and radiosensitivity in clonogenic survival curves, as well as comet tail intensity, total antioxidant stores and radiation-induced gene expression in hematopoietic progenitor compared to bone marrow stromal cell lines. We further evaluated radioprotection by a mitochondrial-targeted antioxidant GS-nitroxide, JP4-039. Hematopoiesis longevity in Fancd2^–/– mouse long-term marrow cultures was diminished and bone marrow stromal cell lines were radiosensitive compared to Fancd2 ^/ stromal cells (Fancd2^–/– D₀ = 1.4 ± 0.1 Gy, ñ = 5.0 ± 0.6 vs. Fancd2 ^/ D₀ = 1.6 ± 0.1 Gy, ñ = 6.7 ± 1.6), P = 0.0124 for D₀ and P = 0.0023 for ñ, respectively). In contrast, Fancd2^–/– IL-3-dependent hematopoietic progenitor cells were radioresistant (D₀ = 1.71 ± 0.04 Gy and ñ = 5.07 ± 0.52) compared to Fancd2 ^/ (D₀ = 1.39 ± 0.09 Gy and ñ = 2.31 ± 0.85, P = 0.001 for D₀). CFU-GM from freshly explanted Fancd2^–/– marrow was also radioresistant. Consistent with radiosensitivity, irradiated Fancd2^–/– stromal cells had higher DNA damage by comet tail intensity assay compared to Fancd2 ^/ cells (P < 0.0001), slower DNA damage recovery, lower baseline total antioxidant capacity, enhanced radiation-induced depletion of antioxidants, and increased CDKN1A-p21 gene transcripts and protein. Consistent with radioresistance, Fancd2^–/– IL-3-dependent hematopoietic cells had higher baseline and post irradiation total antioxidant capacity. While, there was no detectable alteration of radiation-induced cell cycle arrest with Fancd2^–/– stromal cells, hematopoietic progenitor cells showed reduced G₂/M cell cycle arrest. The absence of the mouse Fancd2 gene product confers radiosensitivity to bone marrow stromal but not hematopoietic progenitor cells.

Alpha-particle radiopharmaceutical therapy (αRPT) is currently enjoying increasing attention as a viable alternative to chemotherapy for targeting of disseminated micrometastatic disease. In theory, αRPT can be personalized through pre-therapeutic imaging and dosimetry. However, in practice, given the particularities of α-particle emissions, a dosimetric methodology that accurately predicts the thresholds for organ toxicity has not been reported. This is in part due to the fact that the biological effects caused by α-particle radiation differ markedly from the effects caused by traditional external beam (photon or electron) radiation or β-particle emitting radiopharmaceuticals. The concept of relative biological effectiveness (RBE) is used to quantify the ratio of absorbed doses required to achieve a given biological response with alpha particles versus a reference radiation (typically a beta emitter or external beam radiation). However, as conventionally defined, the RBE varies as a function of absorbed dose and therefore a single RBE value is limited in its utility because it cannot be used to predict response over a wide range of absorbed doses. Therefore, efforts are underway to standardize bioeffect modeling for different fractionation schemes and dose rates for both nuclear medicine and external beam radiotherapy. Given the preponderant use of external beams of radiation compared to nuclear medicine in cancer therapy, the more clinically relevant quantity, the 2 Gy equieffective dose, EQD2(α/β), has recently been proposed by the ICRU. In concert with EQD2(α/β), we introduce a new, redefined RBE quantity, named RBE2(α/β), as the ratio of the two linear coefficients that characterize the α particle absorbed dose-response curve and the low-LET megavoltage photon 2 Gy fraction equieffective dose-response curve. The theoretical framework for the proposed new formalism is presented along with its application to experimental data obtained from irradiation of a breast cancer cell line. Radiobiological parameters are obtained using the linear quadratic model to fit cell survival data for MDA-MB-231 human breast cancer cells that were irradiated with either α particles or a single fraction of low-LET ¹³⁷Cs γ rays. From these, the linear coefficient for both the biologically effective dose (BED) and the EQD2(α/β) response lines were derived for fractionated irradiation. The standard RBE calculation, using the traditional single fraction reference radiation, gave RBE values that ranged from 2.4 for a surviving fraction of 0.82–6.0 for a surviving fraction of 0.02, while the dose-independent RBE2(4.6) value was 4.5 for all surviving fraction values. Furthermore, bioeffect modeling with RBE2(α/β) and EQD2(α/β) demonstrated the capacity to predict the surviving fraction of cells irradiated with acute and fractionated low-LET radiation, α particles and chronic exponentially decreasing dose rates of low-LET radiation. RBE2(α/β) is independent of absorbed dose for α-particle emitters and it provides a more logical framework for data reporting and conversion to equieffective dose than the conventional dose-dependent definition of RBE. Moreover, it provides a much needed foundation for the ongoing development of an α-particle dosimetry paradigm and will facilitate the use of tolerance dose data available from external beam radiation therapy, thereby helping to develop αRPT as a single modality as well as for combination therapies.

The majority of studies on lethal radiobiological damage have focused on double-strand breaks (DSBs), a type of clustered DNA damage and the evaluation of their toxicity, while other types of clustered DNA damage have received much less attention. The main purpose of this study is to evaluate the contribution of different lesions induced by ionizing radiation to the loss of plasmid DNA functionality. We employed a simple model system comprising E. coli transformed with an irradiated plasmid [pGEM-3Zf (–)] to determine the effect of DSBs and other lesions including base damage and clustered lesions on the functionality (“viability”) of the plasmid. The yields of γ-radiation-induced single-strand breaks (SSBs) and DSBs were measured by gel electrophoresis. We found that the transformation efficiency decreases with radiation dose, but this decrease cannot be explained by the formation of DSBs. For example, at doses of 500 and 700 Gy, the relative transformation efficiency falls from 100% to 53% and 26%, respectively, while only 5.7% and 9.1% of the plasmids contain a DSB. In addition, it is also unlikely that randomly distributed base lesions could explain the loss of functionality of the plasmid, since cells can repair them efficiently. However, clustered lesions other than DSBs, which are difficult to repair and result in the loss of information on both DNA strands, have the potential to induce the loss of plasmid functionality. We therefore measured the yields of γ-radiation-induced base lesions and cluster damage, which are respectively converted into SSBs and DSBs by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). Our data demonstrate that the yield of cluster damage (i.e., lesions that yield DSBs following digestion) is 31 times higher than that of frank DSBs. This finding suggests that frank DSBs make a relatively minor contribution to the loss of DNA functionality induced by ionizing radiation, while other toxic lesions formed at a much higher frequencies than DSBs must be responsible for the loss of plasmid functionality. These lesions may be clustered lesions/locally multiply damaged sites (LMDS), including base damage, SSBs and/or intrastrand and interstrand crosslinks, leading to the loss of vital information in the DNA. Using a mathematical model, we estimate that at least three toxic lesions are required for the inactivation of plasmid functionality, in part because even these complex lesions can be repaired.