Exposures to low- and high-linear energy transfer (LET) radiation induce clustered damage in DNA that is difficult to repair. These lesions are manifested as DNA-associated foci positive for DNA repair proteins and have been shown to persist in vitro and in vivo for days in several cell types and tissues in response to low-LET radiation. Although in some experimental conditions these residual foci have been linked with genomic instability and chromosomal aberrations, it remains poorly understood what type of damage they represent. Because high-LET radiation induces complex DNA lesions more efficiently than low-LET radiation, we compared the efficacy of several heavy ions (oxygen, silicon and iron) in a range (17 , 70 and 175 keV/μm, respectively) of LET and X rays at a 1 Gy dose. Persistent genomic damage was measured by γ-H2AX-53BP1-positive residual foci and micronucleus levels during the first three days and up to a week after in vitro and in vivo irradiation in lung cells and tissue. We demonstrate that in an in vitro irradiated mouse bronchial epithelial cell line, the expression of residual foci is readily detectable at 24 h with levels declining in the following 72 h postirradiation, but still persisting elevated over background at day 7. At this time, foci numbers are low but significant and proportional to the dose and quality of the radiation. The expression of residual foci in vitro was mirrored by increased micronuclei generation measured in cytokinesis-blocked cells, indicating long-term, persistent effects of genomic damage in this cell type. We also tested the expression of residual foci in lung tissue of C57BL/6 mice that received whole-body X-ray or heavy-ion irradiation. We found that at day 7 postirradiation, Clara/Club cells, but not pro-SPC-positive pneumocytes, contained a subpopulation of cells expressing γ-H2AX-53BP1-positive foci in a radiation quality-dependent manner. These findings suggest that in vivo persistent DNA repair foci reflect the initial genotoxic damage induced by radiation and a differential vulnerability among cells in the lung.

In preclinical studies, several tumor cell lines have demonstrated an epithelial-to-mesenchymal (EMT)-dependent enhancement in migration when exposed to ionizing radiation at doses of 10 Gy or higher. The goal of this study was to determine whether a lower dose (2.3 Gy) of radiation enhances breast tumor cell migration, and to elucidate the potential contribution of EMT and pro-migratory secreted factors in radiation-induced tumor cell migration. Three human breast cancer cell lines were irradiated and imaged in real-time over 72 h to quantify changes in single cell migration, chemotactic migration and invasion. EMT markers were assessed and conditioned media from irradiated cells was used to determine whether cellular migration was influenced by secreted factors. We observed that a 2.3 Gy dose of radiation did not induce EMT in epithelial-like MCF-7 cells and did not increase the ability of MCF-7 cells or highly motile MDA-MB-231 LM2-4 cells to migrate. In addition, a 2.3 Gy dose significantly increased MDA-MB-231 migration, as detected by single cell tracking and transwell migration assays, but did not increase invasion of MDA-MB-231 cells through reconstituted basement membrane. Cells from all three cell lines migrated further from their point of origin after irradiation, suggesting the cells may be responding to soluble factors produced by other irradiated cells. Consistently, conditioned media derived from 2.3 Gy irradiated MDA-MB-231 cells contained increased levels of several pro-migratory chemokines, and conditioned media from irradiated cells enhanced the migration of nonirradiated MDA-MB-231 cells. These findings indicate that 2.3 Gy dose of radiation is sufficient to increase migration of MDA-MB-231 cells and to alter the single cell migration behavior of three human breast cancer cell lines. Our data suggest the involvement of soluble factors released by 2.3 Gy irradiated cells, and support further in vitro and in vivo studies to identify potential therapeutic targets to prevent tumor cell migration after irradiation.

The purpose of this study was to determine whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived stress can account for unloading- and radiation-induced endothelial damage and neurovascular remodeling in a mouse model. Wild-type (WT, Nox2 ^/ ) C57BL/6 mice or Nox2^–/– (B6.129S6-CYBBM) knockout (KO) mice were placed into one of the following groups: age-matched control; hindlimb unloading (HLU); low-dose/low-dose-rate radiation (LDR); or HLU with LDR simultaneously for 21 days. The mice were then sacrificed one month later. Anti-orthostatic tail suspension was used to model the unloading, fluid shift and physiological stress aspects of microgravity. The LDR was delivered using ⁵⁷Co plates (0.04 Gy at 0.01 cGy/h) to the simulate whole-body irradiation, similar to that experienced while in space. Brains were isolated for characterization of various oxidative stress markers and vascular topology. The level of 4-hydroxynonenal (4-HNE) protein, a specific marker for lipid peroxidation, was measured. Expression of aquaporin-4 (AQP4), a water channel protein expressed in astrocyte end-feet, was quantified. Thirty days after simulated spaceflight, KO mice showed decreased apoptosis (P < 0.05) in the brain compared to WT counterparts. The HLU-dependent increase in apoptosis in WT mice was not observed in KO mice. The level of 4-HNE protein was significantly elevated in the hippocampus of the LDR with HLU treatment group compared to WT controls (P < 0.05). However, there were no significant differences among groups of Nox2-KO mice at the one-month time point. In contrast to findings in the WT animals, superoxide dismutase (SOD) level and expression of AQP4 were similar among all KO groups. In summary, for most of the parameters, the oxidative response to HLU and LDR was suppressed in Nox2-KO mice. This suggests that Nox2-containing NADPH oxidase may contribute to spaceflight environment-induced oxidative stress.

Survivors of the atomic bombings of Hiroshima and Nagasaki are monitored for health effects within the Life Span Study (LSS). The LSS results represent the most important source of data about cancer effects from ionizing radiation exposure, which forms the foundation for the radiation protection system. One uncertainty connected to deriving universal risk factors from these results is related to the problem of mixed radiation qualities. The A-bomb explosions generated a mixed beam of the sparsely ionizing gamma radiation and densely ionizing neutrons. However, until now the possible interaction of the two radiation types of inducing biological effects has not been taken into consideration. The existence of such interaction would suggest that the application of risk factors derived from the LSS to predict cancer effects after pure gamma-ray irradiation (such as in the Fukushima prefecture) leads to an overestimation of risk. To analyze the possible interaction of radiation types, a mixed-beam exposure facility was constructed where cells can be exposed to sparsely ionizing X rays and densely ionizing alpha particles. U2OS cells were used, which are stably transfected with a plasmid coding for the DNA repair gene 53BP1 coupled to a gene coding for the green fluorescent protein (GFP). The induction and repair of DNA damage, which are known to be related to cancer induction, were analyzed. The results suggest that alpha particles and X rays interact, leading to cellular and possibly cancer effects, which cannot be accurately predicted based on assuming simple additivity of the individual mixed-beam components.

Retrospective estimation of the doses received by atomic bomb (A-bomb) survivors by cytogenetic methods has been hindered by two factors: One is that the photon energies released from the bomb were widely distributed, and since the aberration yield varies depending on the energy, the use of monoenergetic ⁶⁰Co gamma radiation to construct a calibration curve may bias the estimate. The second problem is the increasing proportion of newly formed lymphocytes entering into the lymphocyte pool with increasing time intervals since the exposures. These new cells are derived from irradiated precursor/stem cells whose radiosensitivity may differ from that of blood lymphocytes. To overcome these problems, radiation doses to tooth enamel were estimated using the electron spin resonance (ESR; or EPR, electron paramagnetic resonance) method and compared with the cytogenetically estimated doses from the same survivors. The ESR method is only weakly dependent on the photon energy and independent of the years elapsed since an exposure. Both ESR and cytogenetic doses were estimated from 107 survivors. The latter estimates were made by assuming that although a part of the cells examined could be lymphoid stem or precursor cells at the time of exposure, all the cells had the same radiosensitivity as blood lymphocytes, and that the A-bomb gamma-ray spectrum was the same as that of the ⁶⁰Co gamma rays. Subsequently, ESR and cytogenetic endpoints were used to estimate the kerma doses using individual DS02R1 information on shielding conditions. The results showed that the two sets of kerma doses were in close agreement, indicating that perhaps no correction is needed in estimating atomic bomb gamma-ray doses from the cytogenetically estimated ⁶⁰Co gamma-ray equivalent doses. The present results will make it possible to directly compare cytogenetic doses with the physically estimated doses of the survivors, which would pave the way for testing whether or not there are any systematic trends or factors affecting physically estimated doses.

The relative biological effectiveness (RBE) of neutrons depends on their physical nature (e.g., energy) and the biological context (e.g., end points, materials). From the perspective of radiological protection, age is an important biological context that influences radiation-related cancer risk, but very few studies have addressed its potential impact on neutron effects. We therefore investigated the influence of age on the effect of accelerator-generated fast neutrons (mean energy, ∼2 MeV) in an animal model of breast carcinogenesis. Female Sprague-Dawley rats at 1, 3 and 7 weeks of age were irradiated with fast neutrons at absorbed doses of 0.0485–0.97 Gy. All animals were kept under specific pathogen-free conditions and screened weekly for mammary tumors by palpation until they were 90 weeks old. Tumors were diagnosed based on histology. Mathematical modeling was used to analyze mammary cancer incidence, collectively using data from this study and a previously reported experiment on ¹³⁷Cs gamma rays. The results indicate that neutron irradiation elevated the risk of palpable mammary carcinoma with a linear dose response, the slope of which depended on age at time of irradiation. The RBE of neutron radiation was 7.5 ± 3.4, 9.3 ± 3.5 and 26.1 ± 8.9 (mean ± SE) for animals exposed at 1, 3 and 7 weeks of age, respectively. Our results indicate that age of the animal is an important factor influencing the effect of fast neutrons on breast cancer risk.

The goal of this study was to determine the critical ion-radiation conditions under which heavy ion beams can induce DNA double-strand breaks. Helium, nitrogen and argon-ion beams in the energy range of 20 eV to 2 keV were used to irradiate naked DNA plasmid pGFP to fluences of 1, 2 and 4 × 10¹⁵ ions/cm². The topological forms of DNA were subsequently analyzed using gel electrophoresis. The DNA forms were changed from the original supercoiled to damaged relaxed and linear forms, depending on the ion mass, energy, fluence and inertia. We found ion energy and fluence thresholds above which direct double-strand breaks can occur. The threshold is discussed in terms of the areal ion-energy density and the cross-section.

Exposure to high-dose ionizing radiation during medical treatment exerts well-documented deleterious effects on bone health, reducing bone density and contributing to bone growth retardation in young patients and spontaneous fracture in postmenopausal women. However, the majority of human radiation exposures occur in a much lower dose range than that used in the radiation oncology clinic. Furthermore, very few studies have examined the effects of low-dose ionizing radiation on bone integrity and results have been inconsistent. In this study, mice were irradiated with a total-body dose of 0.17, 0.5 or 1 Gy to quantify the early (day 3 postirradiation) and delayed (day 21 postirradiation) effects of radiation on bone microarchitecture and bone marrow stromal cells (BMSCs). Female BALBc mice (4 months old) were divided into four groups: irradiated (0.17, 0.5 and 1 Gy) and sham-irradiated controls (0 Gy). Micro-computed tomography analysis of distal femur trabecular bone from animals at day 21 after exposure to 1 Gy of X-ray radiation revealed a 21% smaller bone volume (BV/TV), 22% decrease in trabecular numbers (Tb.N) and 9% greater trabecular separation (Tb.Sp) compared to sham-irradiated controls (P < 0.05). We evaluated the differentiation capacity of bone marrow stromal cells harvested at days 3 and 21 postirradiation into osteoblast and adipocyte cells. Osteoblast and adipocyte differentiation was decreased when cells were harvested at day 3 postirradiation but enhanced in cells isolated at day 21 postirradiation, suggesting a compensatory recovery process. Osteoclast differentiation was increased in 1 Gy irradiated BMSCs harvested at day 3 postirradiation, but not in those harvested at day 21 postirradiation, compared to controls. This study provides evidence of an early, radiation-induced decrease in osteoblast activity and numbers, as well as a later recovery effect after exposure to 1 Gy of X-rays, whereas osteoclastogenesis was enhanced. A better understanding of the effects of radiation on osteoprogenitor cell populations could lead to more effective therapeutic interventions that protect bone integrity for individuals exposed to low-dose ionizing radiation.

Cranial radiation severely affects brain health and function, including glial cell production and myelination. Recent studies indicate that voluntary exercise has beneficial effects on oligodendrogenesis and myelination. Here, we hypothesized that voluntary running would increase oligodendrocyte numbers in the corpus callosum after irradiation of the juvenile mouse brain. The brains of C57Bl/6J male mice were 6 Gy irradiated on postnatal day 9 during the main gliogenic developmental phase, resulting in a loss of oligodendrocyte precursor cells. Upon adulthood, the mice were injected with bromodeoxyuridine and allowed to exercise on a running wheel for four weeks. Cell proliferation and survival, Ascl1 oligodendrocyte precursor and Olig2 oligodendrocyte cell numbers as well as CC1 mature oligodendrocytes were quantified using immunohistology. Radiation induced a reduction in the number of Olig2 oligodendrocytes by nearly 50% without affecting production or survival of new Olig2 cells. Ascl1 cells earlier in the oligodendroglial cell lineage were also profoundly affected, with numbers reduced by half. By three weeks of age, Olig2 cell numbers had not recovered, and this was paralleled by a volumetric loss in the corpus callosum. The deficiency of Olig2 oligodendrocytes persisted into adulthood. Additionally, the depletion of Ascl1 progenitor cells was irreversible, and was even more pronounced at 12 weeks postirradiation compared to day 2 postirradiation. Furthermore, the overall number of CC1 mature oligodendrocytes decreased by 28%. The depletion of Olig2 cells in irradiated animals was reversed by 4 weeks of voluntary exercise. Moreover, voluntary exercise also increased the number of Ascl1 progenitor cells in irradiated animals. Taken together, these results demonstrate that exercise in adulthood significantly ameliorates the profound and long-lasting effects of moderate exposure to immature oligodendrocytes during postnatal development.

Low-dose radiation has been used in clinical and experimental models for the prevention of scarring and for fracture healing. There is evidence that low-dose radiation improves the hormesis of various cell types but little is known about its effects on peripheral nerve tissue. In this study, we investigated the beneficial effects of low-dose radiation on the regeneration of transectional peripheral nerve injury in an experimental rat model. Seventy-two male Sprague-Dawley rats received transection injury to the left sciatic nerves, and the nerves were subsequently sutured by epineurium end-to-end anastomosis to restore continuity. Animals were randomly assigned to one of two treatment groups (n = 36/group): 1 Gy X-ray irradiation or control (sham irradiation). Gait analysis, electrophysiological examination and morphological investigations were performed. In addition, Western blot and qRT-PCR were performed to determine the level of vascular endothelial growth factor (VEGF) and growth-associated protein-43 (GAP-43). Content of VEGF and GAP-43 in the regenerated sciatic nerve of the irradiated group was higher than the control group. At 4 to 12 weeks after surgery, the irradiated animals exhibited a significantly improved functional recovery relative to controls. At 12 weeks after surgery, amplitude and conduction velocity of the irradiated group were higher than the control group (P < 0.05). The number of nerve fibers, diameter of axons and morphological structure of the myelin sheath in the irradiated group were superior to those of the control group. These results suggest that low-dose radiation contributed to regeneration and functional recovery after transverse peripheral nerve injury by inducing increased production of VEGF and GAP-43, which promote the axonal regeneration and myelination.