Senescence-Associated Heterochromatin Foci Suppress γ-H2AX Focus Formation Induced by Radiation Exposure.

DNA damage is induced by both endogenous and exogenous factors. Repair of DNA double-strand break (DSB), a serious damage that threatens genome stability, decreases with senescence. However, the molecular mechanisms underlying the decline in DNA repair capacity during senescence remain unclear. We performed immunofluorescence staining for phosphorylated histone H2AX (γ-H2AX) in normal human fetal lung fibroblasts and human skin fibroblasts of different ages after chronic irradiation (total dose, 1 Gy; dose rate, 1 Gy/day) to investigate the effect of cellular senescence and organismal aging on DSB repair. Accumulation of DSBs was observed with cellular senescence and organismal aging, probably caused by delayed DSB repair. Importantly, the formation of γ-H2AX foci, an early event in DSB repair, is delayed with cellular senescence and organismal aging. These results suggest that the delay in γ-H2AX focus formation might delay the overall DSB repair. Interestingly, immediate γ-H2AX foci formation was suppressed in cells with senescence-associated heterochromatin foci (SAHF). To investigate the relationship between the γ-H2AX focus formation and SAHF, we used LiCl to relax the SAHFs, followed by irradiation. We demonstrated that LiCl rescued the delayed γ-H2AX foci formation associated with cellular senescence. This indicates that SAHF interferes with γ-H2AX focus formation and inhibits DSB repair in radiation-induced DSB. Our results suggest that therapeutic targeting of SAHFs have potential to resolve DSB repair dysfunction associated with cellular senescence.

Identification of Novel Senescent Markers in Small Extracellular Vesicles.

Senescent cells exhibit several typical features, including the senescence-associated secretory phenotype (SASP), promoting the secretion of various inflammatory proteins and small extracellular vesicles (EVs). SASP factors cause chronic inflammation, leading to age-related diseases. Recently, therapeutic strategies targeting senescent cells, known as senolytics, have gained attention; however, noninvasive methods to detect senescent cells in living organisms have not been established. Therefore, the goal of this study was to identify novel senescent markers using small EVs (sEVs). sEVs were isolated from young and senescent fibroblasts using three different methods, including size-exclusion chromatography, affinity column for phosphatidylserine, and immunoprecipitation using antibodies against tetraspanin proteins, followed by mass spectrometry. Principal component analysis revealed that the protein composition of sEVs released from senescent cells was significantly different from that of young cells. Importantly, we identified ATP6V0D1 and RTN4 as novel markers that are frequently upregulated in sEVs from senescent and progeria cells derived from patients with Werner syndrome. Furthermore, these two proteins were significantly enriched in sEVs from the serum of aged mice. This study supports the potential use of senescent markers from sEVs to detect the presence of senescent cells in vivo.

DNA Damage Regulates Senescence-Associated Extracellular Vesicle Release via the Ceramide Pathway to Prevent Excessive Inflammatory Responses.

DNA damage, caused by various oncogenic stresses, can induce cell death or cellular senescence as an important tumor suppressor mechanism. Senescent cells display the features of a senescence-associated secretory phenotype (SASP), secreting inflammatory proteins into surrounding tissues, and contributing to various age-related pathologies. In addition to this inflammatory protein secretion, the release of extracellular vesicles (EVs) is also upregulated in senescent cells. However, the molecular mechanism underlying this phenomenon remains unclear. Here, we show that DNA damage activates the ceramide synthetic pathway, via the downregulation of sphingomyelin synthase 2 (SMS2) and the upregulation of neutral sphingomyelinase 2 (nSMase2), leading to an increase in senescence-associated EV (SA-EV) biogenesis. The EV biogenesis pathway, together with the autophagy-mediated degradation pathway, functions to block apoptosis by removing cytoplasmic DNA fragments derived from chromosomal DNA or bacterial infections. Our data suggest that this SA-EV pathway may play a prominent role in cellular homeostasis, particularly in senescent cells. In summary, DNA damage provokes SA-EV release by activating the ceramide pathway to protect cells from excessive inflammatory responses.

Albumin Hydrogel-Coated Mesoporous Silica Nanoparticle as a Carrier of Cationic Porphyrin and Ratiometric Fluorescence pH Sensor.

Here, we report that a mesoporous silica nanoparticle (MSN) coated with a fluoresceine-labeled bovine serum albumin (F-BSA) hydrogel layer works as a temperature-responsive nanocarrier for tetrakis-N-methylpyridyl porphyrin (TMPyP) and as a fluorescence ratiometric pH probe. F-BSA hydrogel-coated MSN containing TMPyP (F-BSA/MSN/TMPyP) was synthesized by thermal gelation of denatured F-BSA on the external surface of MSN. The F-BSA hydrogel layer was composed of an inner hard corona layer and an outer soft layer and was stable under physiological conditions. F-BSA/MSN/TMPyP exhibited temperature-dependent exponential release of TMPyP. In this release profile, the MSN was found to be a suitable host for stable encapsulation of tetracationic TMPyP by electrostatic interactions, and the F-BSA hydrogel layer mediated the diffusion of TMPyP from the MSN pore interior into the solution phase. Increasing temperature promoted partitioning of TMPyP from the pore interior to the F-BSA hydrogel layer, from where it was spontaneously released into the bulk solution phase by cation exchange. F-BSA/MSN/TMPyP also gave a linear ratiometric fluorescence response (1.3 per pH unit) in the pH range from 6.1 to 8.9.

Tumors induce complex DNA damage in distant proliferative tissues in vivo.

That tumors cause changes in surrounding tissues is well documented, but whether they also affect distant tissues is uncertain. Such knowledge may be important in understanding the relationship between cancer and overall patient health. To address this question, we examined tissues distant to sites of implanted tumors for genomic damage using cohorts of C57BL/6 and BALB/c mice with early-stage subcutaneous syngeneic grafts, specifically, B16 melanoma, MO5076 sarcoma, and COLON26 carcinoma. Here we report that levels of two serious types of DNA damage, double-strand breaks (DSBs) measured by γ-H2AX focus formation and oxidatively induced non-DSB clustered DNA lesions (OCDLs), were elevated in tissues distant from the tumor site in tumor-bearing mice compared with their age- and sex-matched controls. Most affected were crypts in the gastrointestinal tract organs and skin, both highly proliferative tissues. Further investigation revealed that, compared with controls, tumor-bearing mice contained elevated amounts of activated macrophages in the distant gastrointestinal tissues, as well as elevated serum levels of several cytokines. One of these cytokines, CCL2/MCP-1, has been linked to several inflammation-related conditions and macrophage recruitment, and strikingly, CCL2-deficient mice lacked increased levels of DSBs and OCDLs in tissues distant from implanted tumors. Thus, this study is unique in being a direct demonstration that the presence of a tumor may induce a chronic inflammatory response in vivo, leading to increased systemic levels of DNA damage. Importantly, these findings suggest that tumors may have more profound effects on their hosts than heretofore expected.

Prediction of late adverse events in pelvic cancer patients receiving definitive radiotherapy using radiation-induced gamma-H2AX foci assay.

Radiation can induce DNA double-stranded breaks, which are typically detected by the fluorescence of phosphorylated histone H2AX. In this study, we examined the usefulness of the dynamics of radiation-induced gamma-H2AX foci of peripheral blood lymphocytes (PBLs), as a marker of DNA repair ability, in predicting late adverse events from radiotherapy. A total of 46 patients with cervical, vaginal and anal canal cancers treated with radical radiotherapy between 2014 and 2019 were included in this analysis. Concurrent chemotherapy was administered in 36 cases (78.3%). Peripheral blood was obtained before treatment, and then irradiated ex vivo with 1 Gy X-ray. The ratio of radiation-induced gamma-H2AX foci in PBLs measured at 30 min and at 4 h was defined as the foci decay ratio (FDR). With a median follow-up of 54 months, 9 patients (19.6%) were observed to have late genitourinary or gastrointestinal (GU/GI) toxicity. The FDR ranged from 0.51 to 0.74 (median 0.59), with a significantly higher incidence of Grade 1 or higher late adverse events in the FDR ≥ 0.59 group. In multivariate analysis, FDR ≥ 0.59 and hypertension also emerged as significant factors associated with the development of late toxicities. Overall, our results suggest that measurement of radiation-induced gamma-H2AX foci in PBLs may predict the risk of late GU/GI toxicities from chemoradiotherapy, which can enable tailoring the radiation dose to minimize adverse effects.

Beyond visualization of DNA double-strand breaks after radiation exposure.

PURPOSE: Radiation science and radiation biology are fields where milestones have been set by numerous woman researchers, as represented by Marie Curie. This shows that it is a research field that is like a model of research diversity in modern society. In this review, I will describe what kind of research activities I have conducted as a Japanese woman researcher in the field of radiation science research. In addition, as a Japanese woman radiobiologist, I will describe the sense of mission I felt after the Fukushima Nuclear Power Plant accident and the research issues we must challenge in the future. CONCLUSION: As a Japanese woman researcher, I have felt a bias in gender balance in the field of science in Japan. Also, after the Fukushima nuclear Power Plant accident, I sometimes felt that woman researchers would be more suitable when sharing research results and specialized knowledge with the general public. In recent years, the importance of STEAM (Science-Technology-Engineering-Art-Mathematics) education has been highlighted all over the world, and I believe that the field of radiation science falls exactly into the STEAM education category. STEAM education is for people of all gender. I hope that radiation science research will lead to various younger generations, and that the gender balance of Japanese scientific researchers will increase.

Ataxia telangiectasia mutated activation by transcription- and topoisomerase I-induced DNA double-strand breaks.

Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)-which are potent transcription-blocking lesions-also produce transcription-dependent DNA double-strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post-mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, gamma-H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1). The DSB-ATM-DDR pathway was suppressed by inhibiting transcription and gamma-H2AX signals were reduced by RNase H1 transfection, which removes transcription-mediated R-loops. Thus, we propose that Top1cc produce transcription arrests with R-loop formation and generate DSBs that activate ATM in post-mitotic cells.

Hypothermia postpones DNA damage repair in irradiated cells and protects against cell killing.

Hibernation is an established strategy used by some homeothermic organisms to survive cold environments. In true hibernation, the core body temperature of an animal may drop to below 0°C and metabolic activity almost cease. The phenomenon of hibernation in humans is receiving renewed interest since several cases of victims exhibiting core body temperatures as low as 13.7°C have been revived with minimal lasting deficits. In addition, local cooling during radiotherapy has resulted in normal tissue protection. The experiments described in this paper were prompted by the results of a very limited pilot study, which showed a suppressed DNA repair response of mouse lymphocytes collected from animals subjected to 7-Gy total body irradiation under hypothermic (13°C) conditions, compared to normothermic controls. Here we report that human BJ-hTERT cells exhibited a pronounced radioprotective effect on clonogenic survival when cooled to 13°C during and 12h after irradiation. Mild hypothermia at 20 and 30°C also resulted in some radioprotection. The neutral comet assay revealed an apparent lack on double strand break (DSB) rejoining at 13°C. Extension of the mouse lymphocyte study to ex vivo-irradiated human lymphocytes confirmed lower levels of induced phosphorylated H2AX (γ-H2AX) and persistence of the lesions at hypothermia compared to the normal temperature. Parallel studies of radiation-induced oxidatively clustered DNA lesions (OCDLs) revealed partial repair at 13°C compared to the rapid repair at 37°C. For both γ-H2AX foci and OCDLs, the return of lymphocytes to 37°C resulted in the resumption of normal repair kinetics. These results, as well as observations made by others and reviewed in this study, have implications for understanding the radiobiology and protective mechanisms underlying hypothermia and potential opportunities for exploitation in terms of protecting normal tissues against radiation.

The role of DNA damage response pathways in chromosome fragility in Fragile X syndrome.

FRAXA is one of a number of fragile sites in human chromosomes that are induced by agents like fluorodeoxyuridine (FdU) that affect intracellular thymidylate levels. FRAXA coincides with a >200 CGG*CCG repeat tract in the 5' UTR of the FMR1 gene, and alleles prone to fragility are associated with Fragile X (FX) syndrome, one of the leading genetic causes of intellectual disability. Using siRNA depletion, we show that ATR is involved in protecting the genome against FdU-induced chromosome fragility. We also show that FdU increases the number of gamma-H2AX foci seen in both normal and patient cells and increases the frequency with which the FMR1 gene colocalizes with these foci in patient cells. In the presence of FdU and KU55933, an ATM inhibitor, the incidence of chromosome fragility is reduced, suggesting that ATM contributes to FdU-induced chromosome fragility. Since both ATR and ATM are involved in preventing aphidicolin-sensitive fragile sites, our data suggest that the lesions responsible for aphidicolin-induced and FdU-induced fragile sites differ. FRAXA also displays a second form of chromosome fragility in absence of FdU, which our data suggest is normally prevented by an ATM-dependent process.

Q(γ-H2AX), an analysis method for partial-body radiation exposure using γ-H2AX in nonhuman primate lymphocytes.

We previously used the γ-H2AX assay as a biodosimeter for total-body-irradiation (TBI) exposure (γ-rays) in a rhesus macaque (Macaca mulatta) model. Utilizing peripheral blood lymphocytes and plucked hairs, we obtained statistically significant γ-H2AX responses days after total-body exposure to 1-8.5 Gy ((60)Co γ-rays at 55 cGy min(-1)). Here, we introduce a partial-body exposure analysis method, Q(γ-H2AX), which is based on the number of γ-H2AX foci per damaged cells as evident by having one or more γ-H2AX foci per cell. Results from the rhesus monkey - TBI study were used to establish Q(γ-H2AX) dose-response calibration curves to assess acute partial-body exposures. γ-H2AX foci were detected in plucked hairs for several days after in vivo irradiation demonstrating this assay's utility for dose assessment in various body regions. The quantitation of γ-H2AX may provide a robust biodosimeter for analyzing partial body exposures to ionizing radiation in humans.

Health effects triggered by tritium: how do we get public understanding based on scientifically supported evidence?

The Commission for 'Corresponding to Radiation Disaster of the Japanese Radiation Research Society' formulated a description of potential health effects triggered by tritium. This was in response to the issue of discharging water containing tritium filtered by the Advanced Liquid Processing System (ALPS), generated and stored in Fukushima Daiichi Nuclear Power Station after the accident. In this review article, the contents of the description, originally provided in Japanese, which gives clear and detailed explanation about potential health effects triggered by tritium based on reliable scientific evidence in an understandable way for the public, were summarized. Then, additional information about biochemical or environmental behavior of organically bound tritium (OBT) were summarized in order to help scientists who communicate with general public.

An Enriched Environment Alters DNA Repair and Inflammatory Responses After Radiation Exposure.

After the Fukushima Daiichi Nuclear Power Plant accident, there is growing concern about radiation-induced carcinogenesis. In addition, living in a long-term shelter or temporary housing due to disasters might cause unpleasant stress, which adversely affects physical and mental health. It's been experimentally demonstrated that "eustress", which is rich and comfortable, has beneficial effects for health using mouse models. In a previous study, mice raised in the enriched environment (EE) has shown effects such as suppression of tumor growth and enhancement of drug sensitivity during cancer treatment. However, it's not yet been evaluated whether EE affects radiation-induced carcinogenesis. Therefore, to evaluate whether EE suppresses a radiation-induced carcinogenesis after radiation exposure, in this study, we assessed the serum leptin levels, radiation-induced DNA damage response and inflammatory response using the mouse model. In brief, serum and tissues were collected and analyzed over time in irradiated mice after manipulating the raising environment during the juvenile or adult stage. To assess the radiation-induced DNA damage response, we performed immunostaining for phosphorylated H2AX which is a marker of DNA double-strand break. Focusing on the polarization of macrophages in the inflammatory reaction that has an important role in carcinogenesis, we performed analysis using tissue immunofluorescence staining and RT-qPCR. Our data confirmed that EE breeding before radiation exposure improved the responsiveness to radiation-induced DNA damage and basal immunity, further suppressing the chronic inflammatory response, and that might lead to a reduction of the risk of radiation-induced carcinogenesis.

H2AX: functional roles and potential applications.

Upon DNA double-strand break (DSB) induction in mammals, the histone H2A variant, H2AX, becomes rapidly phosphorylated at serine 139. This modified form, termed gamma-H2AX, is easily identified with antibodies and serves as a sensitive indicator of DNA DSB formation. This review focuses on the potential clinical applications of gamma-H2AX detection in cancer and in response to other cellular stresses. In addition, the role of H2AX in homeostasis and disease will be discussed. Recent work indicates that gamma-H2AX detection may become a powerful tool for monitoring genotoxic events associated with cancer development and tumor progression.

Role of oxidatively induced DNA lesions in human pathogenesis.

Genome stability is essential for maintaining cellular and organismal homeostasis, but it is subject to many threats. One ubiquitous threat is from a class of compounds known as reactive oxygen species (ROS), which can indiscriminately react with many cellular biomolecules including proteins, lipids, and DNA to produce a variety of oxidative lesions. These DNA oxidation products are a direct risk to genome stability, and of particular importance are oxidative clustered DNA lesions (OCDLs), defined as two or more oxidative lesions present within 10 bp of each other. ROS can be produced by exposure of cells to exogenous environmental agents including ionizing radiation, light, chemicals, and metals. In addition, they are produced by cellular metabolism including mitochondrial ATP generation. However, ROS also serve a variety of critical cellular functions and optimal ROS levels are maintained by multiple cellular antioxidant defenses. Oxidative DNA lesions can be efficiently repaired by base excision repair or nucleotide excision repair. If ROS levels increase beyond the capacity of its antioxidant defenses, the cell's DNA repair capacity can become overwhelmed, leading to the accumulation of oxidative DNA damage products including OCDLs, which are more difficult to repair than individual isolated DNA damage products. Here we focus on the induction and repair of OCDLs and other oxidatively induced DNA lesions. If unrepaired, these lesions can lead to the formation of mutations, DNA DSBs, and chromosome abnormalities. We discuss the roles of these lesions in human pathologies including aging and cancer, and in bystander effects.

Repair Kinetics of DNA Double Strand Breaks Induced by Simulated Space Radiation.

Radiation is unavoidable in space. Energetic particles in space radiation are reported to induce cluster DNA damage that is difficult to repair. In this study, normal human fibroblasts were irradiated with components of space radiation such as proton, helium, or carbon ion beams. Immunostaining for γ-H2AX and 53BP1 was performed over time to evaluate the kinetics of DNA damage repair. Our data clearly show that the repair kinetics of DNA double strand breaks (DSBs) induced by carbon ion irradiation, which has a high linear energy transfer (LET), are significantly slower than those of proton and helium ion irradiation. Mixed irradiation with carbon ions, followed by helium ions, did not have an additive effect on the DSB repair kinetics. Interestingly, the mean γ-H2AX focus size was shown to increase with LET, suggesting that the delay in repair kinetics was due to damage that is more complex. Further, the 53BP1 focus size also increased in an LET-dependent manner. Repair of DSBs, characterized by large 53BP1 foci, was a slow process within the biphasic kinetics of DSB repair, suggesting non-homologous end joining with error-prone end resection. Our data suggest that the biological effects of space radiation may be significantly influenced by the dose as well as the type of radiation exposure.

Space Radiation Biology for "Living in Space".

Space travel has advanced significantly over the last six decades with astronauts spending up to 6 months at the International Space Station. Nonetheless, the living environment while in outer space is extremely challenging to astronauts. In particular, exposure to space radiation represents a serious potential long-term threat to the health of astronauts because the amount of radiation exposure accumulates during their time in space. Therefore, health risks associated with exposure to space radiation are an important topic in space travel, and characterizing space radiation in detail is essential for improving the safety of space missions. In the first part of this review, we provide an overview of the space radiation environment and briefly present current and future endeavors that monitor different space radiation environments. We then present research evaluating adverse biological effects caused by exposure to various space radiation environments and how these can be reduced. We especially consider the deleterious effects on cellular DNA and how cells activate DNA repair mechanisms. The latest technologies being developed, e.g., a fluorescent ubiquitination-based cell cycle indicator, to measure real-time cell cycle progression and DNA damage caused by exposure to ultraviolet radiation are presented. Progress in examining the combined effects of microgravity and radiation to animals and plants are summarized, and our current understanding of the relationship between psychological stress and radiation is presented. Finally, we provide details about protective agents and the study of organisms that are highly resistant to radiation and how their biological mechanisms may aid developing novel technologies that alleviate biological damage caused by radiation. Future research that furthers our understanding of the effects of space radiation on human health will facilitate risk-mitigating strategies to enable long-term space and planetary exploration.

Pilot clinical study of ascorbic acid treatment in cardiac catheterization.

Clinical radiodiagnosis and radiotherapy sometimes induce tissue damage and/or increase the risk of cancer in patients. However, in radiodiagnosis, a reduction in the exposure dose causes a blockier image that is not acceptable for diagnosis. Approximately 70% of DNA damage is induced via reactive oxygen species and/or radicals created during X-ray irradiation. Therefore, treatment with anti-oxidants and/or radical scavengers is considered to be effective in achieving a good balance between image quality and damage. However, few studies have examined the effect of using radical scavengers to reduce radiation damage in the clinical setting. In this study, we administrated 20 mg/kg ascorbic acid (AA) to patients before cardiac catheterization (CC) for diagnostic purposes. We analyzed changes in the number of phosphorylated H2AX (γH2AX) foci (a marker of DNA double-strand breaks) in lymphocytes, red blood cell glutathione levels, blood cell counts, and biochemical parameters. Unfortunately, we did not find satisfactory evidence to show that AA treatment reduces γH2AX foci formation immediately after CC. AA treatment did, however, cause a higher reduced/oxidized glutathione ratio than in the control arm immediately after CC. This is a preliminary study, but this result suggests that reducing radiation damage in clinical practice can be achieved using a biological approach.

The Causal Relationship between DNA Damage Induction in Bovine Lymphocytes and the Fukushima Nuclear Power Plant Accident.

The Fukushima Daiichi Nuclear Power Plant (FNPP) accident, the largest nuclear incident since the 1986 Chernobyl disaster, occurred when the plant was hit by a tsunami triggered by the Great East Japan Earthquake on March 11, 2011. The subsequent uncontrolled release of radioactive substances resulted in massive evacuations in a 20-km zone. To better understand the biological consequences of the FNPP accident, we have been measuring DNA damage levels in cattle in the evacuation zone. DNA damage was evaluated by assessing the levels of DNA double-strand breaks in peripheral blood lymphocytes by immunocytofluorescence-based quantification of γ-H2AX foci. A greater than two-fold increase in the fraction of damaged lymphocytes was observed in all animal cohorts within the evacuation zone, and the levels of DNA damage decreased slightly over the 700-day sample collection period. While the extent of damage appeared to be independent of the distance from the accident site and the estimated radiation dose from radiocesium, we observed age-dependent accumulation of DNA damage. Thus, this study, which was the first to evaluate the biological impact of the FNPP accident utilizing the γ-H2AX assays, indicated the causal relation between high levels of DNA damage in animals living in the evacuation zone and the FNPP accident.

Evidence for chromosome fragility at the frataxin locus in Friedreich ataxia.

Friedreich ataxia (FRDA) is a member of the Repeat Expansion Diseases, a group of genetic conditions resulting from an increase/expansion in the size of a specific tandem array. FRDA results from expansion of a GAA/TTC-tract in the first intron of the frataxin gene (FXN). The disease-associated tandem repeats all form secondary structures that are thought to contribute to the propensity of the repeat to expand. The subset of these diseases that result from a CGG/CCG-repeat expansion, such as Fragile X syndrome, also express a folate-sensitive fragile site coincident with the repeat on the affected chromosome. This chromosome fragility involves the generation of chromosome/chromatid gaps or breaks, or the high frequency loss of one or both copies of the affected gene when cells are grown under folate stress or as we showed previously, in the presence of an inhibitor of the ATM checkpoint kinase. Whether Repeat Expansion Disease loci containing different repeats form similar fragile sites was not known. We show here that the region of chromosome 9 that contains the FXN locus is intrinsically prone to breakage in vivo even in control cells. However, like FXS alleles, FRDA alleles show significantly elevated levels of chromosome abnormalities in the presence of an ATM inhibitor, consistent with the formation of a fragile site.