Ionizing radiation exposure induces highly lethal DNA double-strand breaks (DSBs) in all phases of the cell cycle. After DSBs are detected by the cellular machinery, these breaks are repaired by either of two mechanisms: (1) nonhomologous end joining (NHEJ), which re-ligates the broken ends of the DNA and (2) homologous recombination (HR), that makes use of an undamaged identical DNA sequence as a template to maintain the fidelity of DNA repair. DNA DSB repair must occur within the context of the natural cellular DNA structure. Among the major factors influencing DNA organization are specific histone and nonhistone proteins that form chromatin. The overall chromatin structure regulates DNA damage responses since chromatin status can impede DNA damage site access by repair proteins. During the process of DNA DSB repair, several chromatin alterations are required to sense damage and facilitate accessibility of the repair machinery. The DNA DSB response is also facilitated by hierarchical signaling networks that orchestrate chromatin structural changes that may coordinate cell-cycle checkpoints involving multiple enzymatic activities to repair broken DNA ends. During DNA damage sensing and repair, histones undergo posttranslational modifications (PTMs) including phosphorylation, acetylation, methylation and ubiquitylation. Such histone modifications represent a histone code that directs the recruitment of proteins involved in DNA damage sensing and repair processes. In this review, we summarize histone modifications that occur during DNA DSB repair processes.

Cytogenetic damage is among the few radiobiological end points that allow a precise distinction to be made between misrepaired damage, represented by exchange-type aberrations such as dicentrics and translocations, and unrepaired damage that leads to “open breaks”. This latter category includes both terminal deletions and incomplete exchanges, whose different mechanisms of formation can be recognized by multicolor fluorescence in situ hybridization (mFISH). mFISH was used to examine the yields of chromosome aberrations at the first postirradiation mitosis in human fibroblasts and lymphocytes irradiated with ¹³⁷Cs γ rays, a radiation of low-linear energy transfer (LET), and two sources of high-LET radiation: α particles from ²³⁸Pu and 1 GeV/amu ⁵⁶Fe ions. In agreement with previous studies, our results show that irrespective of radiation quality, the overall level of misrepaired damage exceeds that of unrepaired damage by a large margin. The unrepaired component of damage produced by γ rays and α particles was remarkably similar, about 5%. On that basis it is difficult to justify the popular notion that the strong LET-dependence for aberration formation is due to unrepaired DNA double-strand breaks (DSBs) that, by virtue of their complexity at the nanometer scale, are qualitatively different in nature. In marked contrast, this unrejoined component rose to about 14% after exposure to Fe ions. A closer look at the unrepaired component revealed that most of this roughly threefold difference was derived from incomplete exchanges. Despite vast differences in LET, unrejoined breaks from incomplete exchanges were far more likely to occur among exchanges that involved more than two breakpoints. We attempted to reconcile these observations in the form of a hypothesis that predicts that exchanges, irrespective of LET, should exhibit an increasing tendency for incompleteness as the number of initial breaks destined to take part in the exchange increases. This effect, we argue is not caused by the number of initial breaks per se, but instead reflects the maximum distance over which proximate breaks can interact. This adds a spatial aspect to multi-break interactions that we call “A Break Too Far”.

Radiation-induced bystander and abscopal effects, in which DNA damage is produced by inter-cellular communication, indicate mechanisms of generating damage in addition to those observed in directly irradiated cells. In this article, we show that the bone marrow of irradiated p53 ^/ mice, but not p53^–/– mice, produces the inflammatory pro-apoptotic cytokines FasL and TNF-α able to induce p53-independent apoptosis in vitro in nonirradiated p53^–/– bone marrow cells. Using a congenic sex-mismatch bone marrow transplantation protocol to generate chimeric mice, p53^–/– hemopoietic cells functioning in a p53 ^/ bone marrow stromal microenvironment exhibited greater cell killing after irradiation than p53^–/– hemopoietic cells in a p53^–/– microenvironment. Cytogenetic analysis demonstrated fewer damaged p53^–/– cells in a p53 ^/ microenvironment than p53^–/– cells in a p53^–/– microenvironment. Using the two different model systems, the findings implicate inflammatory tissue processes induced as a consequence of p53-dependent cellular responses to the initial radiation damage, producing cytokines that subsequently induce ongoing p53-independent apoptosis. As inactivation of the p53 tumor suppressor pathway is a common event in malignant cells developing in a stromal microenvironment that has normal p53 function, the signaling processes identified in the current investigations have potential implications for disease pathogenesis and therapy.

In this study, we investigated the accuracy of using off-line bioluminescence imaging (BLI) and tomography (BLT) to guide irradiation of small soft tissue targets on a small animal radiation research platform (SARRP) with on-board cone beam CT (CBCT) capability. A small glass bulb containing BL cells was implanted as a BL source in the abdomen of 11 mouse carcasses. Bioluminescence imaging and tomography were acquired for each carcass. Six carcasses were setup visually without immobilization and 5 were restrained in position with tape. All carcasses were setup in treatment position on the SARRP where the centroid position of the bulb on CBCT was taken as “truth”. In the 2D visual setup, the carcass was setup by aligning the point of brightest luminescence with the vertical beam axis. In the CBCT assisted setup, the pose of the carcass on CBCT was aligned with that on the 2D BL image for setup. For both 2D setup methods, the offset of the bulb centroid on CBCT from the vertical beam axis was measured. In the BLT-CBCT fusion method, the 3D torso on BLT and CBCT was registered and the 3D offset of the respective source centroids was calculated. The setup results were independent of the carcass being immobilized or not due to the onset of rigor mortis. The 2D offset of the perceived BL source position from the CBCT bulb position was 2.3 mm ± 1.3 mm. The 3D offset between BLT and CBCT was 1.5 mm ± 0.9 mm. Given the rigidity of the carcasses, the setup results represent the best that can be achieved with off-line 2D BLI and 3D BLT. The setup uncertainty would require the use of undesirably large margin of 4–5 mm. The results compel the implementation of on-board BLT capability on the SARRP to eliminate setup error and to improve BLT accuracy.

We show here that low-dose gamma irradiation substantially increase in extracellular superoxide anion production in oncogenically transformed cells and tumor cells but not by nontransformed cells. The transfer of only a few cells from an irradiated culture to nonirradiated control cells was sufficient for the transmission of a signal to induce superoxide anion production in the nonirradiated cells. The number of irradiated cells that was necessary for the successful induction of superoxide anion production in the nonirradiated cells depended on radiation dose. When irradiated cells were allowed to incubate for 1 h before transmission to the nonirradiated cultures, nearly all of the cells from the irradiated cell population were able to communicate the inducing signal to nonirradiated cells. siRNA-dependent knockdown and reconstitution experiments showed that TGF-β1 was sufficient to mediate the bystander effect triggered by low-dose radiation in this experimental system. A kinetic analysis demonstrated that the enhanced superoxide anion production was substantially reduced before the release of the bystander signal by activated TGF-β.

Previously, we described the radiation-induced (RI) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) effect as the increased MTT metabolization at the intermediate dose region after the irradiation of an MCF-7/6 cell monolayer with an X-ray dose gradient. We wondered if the cell monolayer at the intermediate dose region was characterized by an increased metabolic activity. In this study, we unraveled the mechanisms behind the RI MTT effect. Comparison of the MTT, sulforhodamine B (SRB), 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium (WST-8), and nitroblue tetrazolium (NBT) assays indicated that the RI MTT effect is not due to an increased cell density, but to an exclusively intracellular MTT conversion. Our results for the MTT and NBT assays after digitonin pretreatment of the irradiated cell monolayer indicated a role of the plasma membrane permeability in the RI MTT effect. Assessment of the radiation impact on the oxidative phosphorylation system by Western blot analysis, spectrophotometric measurement and Blue Native gel electrophoresis showed a dose-dependent downregulation of the oxidative phosphorylation system complexes, whereby the radiosensitivity of each complex was proportional to the number of mitochondrial DNA-encoded subunits. Further, only treatment of the irradiated cell monolayer with a cocktail and not with the individual inhibitors of complexes I, II and IV during the MTT assay prevented the RI MTT effect. In general, our results demonstrate that the RI MTT effect is not due to an increased metabolic activity, but rather to an enhanced cellular MTT entry and mitochondrial MTT conversion.

The induction of nontargeted stressful effects in cell populations exposed to low fluences of high charge (Z) and high energy (E) particles is relevant to estimates of the health risks of space radiation. We investigated the up-regulation of stress markers in confluent normal human fibroblast cultures exposed to 1,000 MeV/u iron ions [linear energy transfer (LET) ∼151 keV/μm] or 600 MeV/u silicon ions (LET ∼50 keV/μm) at mean absorbed doses as low as 0.2 cGy, wherein 1–3% of the cells were targeted through the nucleus by a primary particle. Within 24 h postirradiation, significant increases in the levels of phospho-TP53 (serine 15), p21^Waf1 (CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation were detected, which suggested participation in the stress response of cells not targeted by primary particles. This was supported by in situ studies that indicated greater increases in 53BP1 foci formation, a marker of DNA damage. than expected from the number of primary particle traversals. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure of the cell cultures to 0.2 cGy of 3.7 MeV α particles (LET ∼109 keV/μm) that targets ∼1.6% of nuclei, but not after 0.2 cGy from 290 MeV/u carbon ions (LET ∼13 keV/μm) by which, on average, ∼13% of the nuclei were hit, which highlights the importance of radiation quality in the induced effect. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cell cultures exposed to HZE particles comprise <1% of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10–20 μm. Thus, the latter are unlikely to significantly contribute to stressful effects in cells not targeted by primary HZE particles.

Ablative hypofractionated radiotherapy (HFRT) significantly improves the overall survival of inoperable non-small cell lung cancer (NSCLC) patients compared with conventional radiation therapy. However, the radiobiological mechanisms of ablative HFRT remain largely unknown. The purpose of this study was to investigate the dynamic changes of tumor vessels and perfusion during and after ablative hypofractionated radiotherapy. Lewis lung carcinoma-bearing mice were treated with sham (control) and ablative hypofractionated radiotherapy of 12 Gy in 1 fraction (12 Gy/1F) and 36 Gy in 3 fractions (36 Gy/3F). Tumor microvessel density (MVD), morphology and function were examined at different times after irradiation. The results showed that, compared to the controls the MVD and hypoxia in ablative HFRT groups decreased, which were accompanied by an increase in the number of pericytes and their coverage of vessels. Functional tests revealed that tumor hypoxia and perfusion were improved, especially in the 36 Gy/3F group. Our results revealed that ablative hypofractionated radiotherapy not only repressed MVD and hypoxia, but also increased the vascular perfusion and the number of pericyte-covered vessels, suggesting that ablative HFRT normalized the tumor vasculature.

The goal of our study was to identify a histological marker for testing countermeasures for mitigation of late radiation injury to the lung. Pulmonary fibrosis is currently the best described “late effect” in survivors of acute radiation pneumonitis. However, robust fibrosis does not develop in some rodent strains for years after a single dose of radiation to the whole thorax. We observed radiation-associated focal alveolar lesions that were rich in giant cells and macrophages containing cholesterol clefts in the lungs of irradiated WAG/RijCmcr rats. These lesions were first observed after pneumonitis, around 21 weeks after receiving a radiation dose of 13 Gy to the thorax but not until 71 weeks in unirradiated rats. The number of cholesterol clefts increased with time after irradiation through 64 weeks of observation, and at 30 weeks after 13 Gy, cholesterol clefts were associated with several indices of deterioration in lung function. The number of cholesterol clefts in irradiated lung sections were reduced by the angiotensin converting enzyme (ACE) inhibitor enalapril (25–42 mg/m²/day) from 18.7 ± 4.2/lung section to 6.8 ± 2.4 (P = 0.029), 5.2 ± 1.9 (P = 0.0051) and 6.7 ± 1.9 (P = 0.029) when the drug was started at 1 week, 5 or 15 weeks after irradiation, respectively, and continued. Similar lesions have been previously observed in the lungs of one strain of irradiated mice and in patients following radiotherapy. We propose that alveolar lesions with cholesterol clefts may be used as a histological marker of the severity of radiation lung injury and to study its mitigation in WAG/RijCmcr rats.

Significant differences exist between the physiology of the immature, neonatal lung compared to that of the adult lung that may affect acute and late responses to irradiation. Identifying these differences is critical to developing successful mitigation strategies for this special population. Our current hypothesis proposes that irradiation during the neonatal period will alter developmental processes, resulting in long-term consequences, including altered susceptibility to challenge with respiratory pathogens. C57BL/6J mice, 4 days of age, received 5 Gy whole-body irradiation. At subsequent time points (12, 26 and 46 weeks postirradiation), mice were intranasally infected with 120 HAU of influenza A virus. Fourteen days later, mice were sacrificed and tissues were collected for examination. Morbidity was monitored following changes in body weight and survival. The magnitude of the pulmonary response was determined by bronchoalveolar lavage, histological examination and gene expression of epithelial and inflammatory markers. Viral clearance was assessed 7 days post-influenza infection. Following influenza infection, irradiated animals that were infected at 26 and 46 weeks postirradiation lost significantly more weight and demonstrated reduced survival compared with those infected at 12 weeks postirradiation, with the greatest deleterious effect seen at the late time point. The results of these experiments suggest that radiation injury during early life may affect the lung's response to a subsequent pathogenic aerial challenge, possibly through a chronic and progressive defect in the immune system. This finding may have implications for the development of countermeasures in the context of systemic radiation exposure.

Radiation leads to a rapid burst of reactive oxygen species (ROS), which is considered to be one of the major causes of radiation-induced injury. ROS have previously been shown to induce changes in cytosolic Ca² ([Ca² ]_i) including [Ca² ]_i oscillation. However, the role of radiation in [Ca² ]_i oscillation is poorly understood. The purpose of this study was to identify the effect of ROS and X ray on [Ca² ]_i oscillation, as well as their role in radiation-induced lung injury. Alveolar macrophages were cultured in the absence and presence of different doses of hydrogen peroxide (H₂O₂) or exposed to X-ray irradiation with or without pretreatment of diphenyleneiodonium chloride (DPI, an inhibitor of NADPH oxidases) or tetrandrine (TET, a calcium entry blocker) and cytosolic Ca² concentration was detected by fluorescent Ca² indicator Fura-2. Rat radiation lung injury was induced in vivo by using 40 Gy X ray and DPI or TET was used to prevent radiation-induced lung injury. The results showed that there was spontaneous [Ca² ]_i oscillation in alveolar macrophages under normal conditions, and treatment of H₂O₂ (100–500 μM) or 2 Gy X ray inhibited the spontaneous [Ca² ]_i oscillation and induced [Ca² ]_i rise. TET abolished H₂O₂ or X ray induced [Ca² ]_i rise in alveolar macrophages, and attenuated X ray- induced rat alveolitis in vivo. DPI prevented X-ray-induced inhibition of [Ca² ]_i oscillation in alveolar macrophages and prevented X-ray-induced rat alveolitis. Taken together, the data suggest that the disruption of [Ca² ]_i oscillation and induction of [Ca² ]_i rise through ROS is involved in the mechanism of radiation-induced lung injury.

Apolipoprotein E (ApoE) plays an important role in lipid metabolism and neuronal repair. In humans, there are three major apoE isoforms: apoE2, apoE3 and apoE4. Compared to apoE3, apoE4 increases the risk to develop Alzheimer's disease, particularly in women, and of developing cognitive impairments after specific environmental challenges. ApoE isoform might also be a determinant of cognitive injury after cranial ⁵⁶Fe irradiation. To assess this possibility, in this study female apoE2, apoE3 and apoE4 mice were cranially irradiated with ⁵⁶Fe particles (600 MeV, 0, 1 or 2 Gy) and behaviorally tested 3 months later. Exploratory activity and measures of anxiety were also assessed as they can affect performance on cognitive tests. There were no effects of irradiation on exploratory activity or measures of anxiety in the open field or elevated zero maze. However, there were dose- and apoE isoform-dependent effects of irradiation on novel object recognition and spatial memory retention in the water maze. Compared to apoE2 and apoE3 mice, apoE4 mice were more sensitive to 2 Gy induced impairments in hippocampus-dependent spatial memory retention in the water maze after training to locate the first hidden platform location, but less sensitive to 2 Gy induced cortical impairments in novel object recognition. Conversely, of the irradiated mice, apoE4 mice irradiated with 1 Gy were the only group of mice that showed spatial memory retention for the second platform location after reversal learning in the water maze. Together, these data show that cranial ⁵⁶Fe irradiation causes dose- and apoE isoform-dependent cognitive impairments in female mice and that anatomical specificity might contribute to the relative sensitivity of apoE4 mice to develop space radiation-induced cognitive impairments.