Lessons on harmonization of scoring criteria for dicentric chromosome assay in South Korea.

PURPOSE: Networking with other biodosimetry laboratories is necessary to assess the radiation exposure of many individuals in large-scale radiological accidents. The Korea biodosimetry network, K-BioDos, prepared harmonized scoring guidelines for dicentric chromosome assay to obtain homogeneous results within the network and investigated the efficiency of the guidelines. MATERIALS AND METHODS: Three laboratories in K-BioDos harmonized the scoring guidelines for dicentric chromosome assay. The results of scoring dicentric chromosomes using the harmonized scoring guidelines were compared with the laboratories' results using their own methods. Feedback was collected from the scorers following the three intercomparison exercises in 3 consecutive years. RESULTS: K-BioDos members showed comparable capacity to score dicentrics in the three exercises. However, the results of the K-BioDos guidelines showed no significant improvement over those of the scorers' own methods. According to the scorers, our harmonized guidelines led to more rejected metaphases and ultimately decreased the number of scorable metaphases compared with their own methods. Moreover, the scoring time was sometimes longer with the K-BioDos protocol because some scorers were not yet familiar with the guidelines, though most scorers reported that the time decreased or was unchanged. These challenges may cause low adherence to the guidelines. Most scorers expressed willingness to use the guidelines to select scorable metaphases or identify dicentrics for other biodosimetry works, whereas one did not want to use it due to the difference from their calibration curves. CONCLUSIONS: We identified potential resistance to following the harmonized guidelines and received requests for more detailed methods. Our findings suggest that the harmonized criteria should be continually updated, and education and training should be provided for all scorers. These changes could allow members within the biodosimetry network to successfully collaborate and support each other in large-scale radiological accidents.

Improving radiation dosimetry with an automated micronucleus scoring system: correction of automated scoring errors.

Radiation dose estimations performed by automated counting of micronuclei (MN) have been studied for their utility for triage following large-scale radiological incidents; although speed is essential, it also is essential to estimate radiation doses as accurately as possible for long-term epidemiological follow-up. Our goal in this study was to evaluate and improve the performance of automated MN counting for biodosimetry using the cytokinesis-block micronucleus (CBMN) assay. We measured false detection rates and used them to improve the accuracy of dosimetry. The average false-positive rate for binucleated cells was 1.14%; average false-positive and -negative MN rates were 1.03% and 3.50%, respectively. Detection errors seemed to be correlated with radiation dose. Correction of errors by visual inspection of images used for automated counting, called the semi-automated and manual scoring method, increased accuracy of dose estimation. Our findings suggest that dose assessment of the automated MN scoring system can be improved by subsequent error correction, which could be useful for performing biodosimetry on large numbers of people rapidly, accurately, and efficiently.

The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis.

People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure.

High-Throughput γ-H2AX Assay Using Imaging Flow Cytometry.

The γ-H2AX assay is a sensitive and reliable method to evaluate radiation-induced DNA double-strand breaks. The conventional γ-H2AX assay detects individual nuclear foci manually, but is labor-intensive and time-consuming, and hence unsuitable for high-throughput screening in cases of large-scale radiation accidents. We have developed a high-throughput γ-H2AX assay using imaging flow cytometry. This method comprises (1) sample preparation from small volumes of blood in the Matrix™ 96-tube format, (2) automated image acquisition of cells stained with immunofluorescence-labeled γ-H2AX using ImageStream®X, and (3) quantification of γ-H2AX levels and batch processing using the Image Data Exploration and Analysis Software (IDEAS®). This enables the rapid analysis of γ-H2AX levels in several thousand of cells from a small volume of blood with accurate and reliable quantitative measurements for γ-H2AX foci and mean fluorescence levels. This high-throughput γ-H2AX assay could be a useful tool not only for radiation biodosimetry in mass casualty events, but also for large-scale molecular epidemiological studies and individualized radiotherapy.

LDR-adapted liver-derived cytokines have potential to induce atherosclerosis.

PURPOSE: Atherosclerosis is a lipid-driven chronic inflammatory disease that causes cardiovascular diseases (CVD). The association between radiation and atherosclerosis has already been demonstrated; however, the effects of low-dose radiation (LDR) exposure on atherosclerosis have not been reported. Our study aims to propose that LDR may cause atherosclerosis phenotypes by the upregulation of plasminogen activator inhibitor-1 (PAI-1) and downregulation of androgen receptor (AR), which are cytokines secreted from the liver. METHODS: Low-density lipoprotein (LDL) receptor deficient (Ldlr-/-) mice were irradiated at 50 mGy, 100 mGy, and 1000 mGy. LDR irradiated Ldlr-/- mice serum was analyzed by cytokine array and proteomics with silver staining. Oil Red O staining and BODIPY staining were performed to determine lipid accumulation in Human umbilical vein endothelial cells (HUVECs). Foam cell formation and monocyte recruitment were assessed through co-culture system with HUVECs and THP-1 cells. RESULTS: After irradiation with LDR (100 mGy) the mice showed atherosclerotic phenotypes and through analysis results, we selected regulated cytokines, PAI-1 and AR, and found that these were changed in the liver. LDR-regulated cytokines have the potential to be transported to endothelial cells and induce lipid accumulation, inflammation of monocytes, increased oxidized low-density lipoprotein (oxLDL) and foam cells formation, that were series of phenotypes lead to plaque formation in endothelial cells and induces atherosclerosis. As a further aspect of this study, testosterone undecanoate (TU) was found to pharmacologically inhibit a series of atherosclerotic phenotypes exhibited by LDR. This study suggests a role for PAI-1 and AR in regulating the development of atherosclerosis after LDR exposure. Targeting PAI-1 and AR could serve as an attractive strategy for the management of atherosclerosis following LDR exposure.

A subset of macrophages and monocytes in the mouse bone marrow express atypical chemokine receptor 1.

Duffy antigen receptor for chemokines (DARC)/CD234, also known as atypical chemokine receptor 1 (ACKR1), is a seven-transmembrane domain protein expressed on erythrocytes, vascular endothelium, and a subset of epithelial cells (Peiper et al., 1995). Previously, we reported that ACKR1 was expressed in bone marrow macrophages. ACKR1 interacts with CD82 on long-term repopulating hematopoietic stem cells (LT-HSCs) to maintain the dormancy of LT-HSCs during homeostasis (Hur et al., 2016). We also demonstrated that ACKR1 interacts with CD82 in HSCs from human umbilical cord blood (hUCB). These findings demonstrated that CD82 is a functional surface marker of LT-HSCs and this molecule maintains LT-HSC quiescence by interactions with ACKR1-expressing macrophages in mice and humans.

The Current Safety Regulation for Radiation Emergency Medicine in Korea.

Radiation emergency medicine (REM) systems are operated around the world to provide specialized care for injured individuals who require immediate medical attention in accidents. This manuscript describes the current status of REM safety regulation in Korea and summarizes an assessment of the effects of this regulation. Responding to the requests of people for stronger safety regulations related to radiation exposure, a unique REM safety regulation for nuclear licensees, which is enforceable by laws, has been established and implemented. It is not found in other countries. It can provide a good example in practice for sustainable REM management including document reviews on medical response procedures and inspections of equipment and facilities. REM preparedness of nuclear or radiologic facilities has been improved with systematic implementation of processes contained in the regulation. In particular, the medical care system of licensees has become firmly coordinated in the REM network at the national level, which has enhanced their abilities by providing adequate medical personnel and facilities. This legal regulation service has contributed to preparing the actual medical emergency response for unexpected accidents and should ultimately secure the occupational safety for workers in radiation facilities.

Chromosome aberration dynamics in breast cancer patients treated with radiotherapy: Implications for radiation biodosimetry.

Although radiological accidents often result in partial-body radiation exposure, most biodosimetry studies focus on estimating whole-body exposure doses. We have evaluated time-dependent changes in chromosomal aberrations before, during, and after localized fractionated radiotherapy. Twelve patients with carcinoma in situ of the breast who underwent identical adjuvant radiation therapy (50 Gy in 25 fractions) were included in the study. Lymphocytes were collected from patients before, during, and after radiotherapy, to measure chromosome aberrations, such as dicentric chromosomes and translocations. Chromosome aberrations were then used to calculate whole- and partial-body biological absorbed doses of radiation. Dicentric chromosome frequencies in all study participants increased during radiotherapy (p < 0.05 in Kruskal-Wallis test). Increases of translocation frequencies during radiotherapy were observed in seven of the twelve patients. The increased levels of dicentric chromosomes and translocations persisted throughout our 1-year follow-up, and evidence of partial-body exposure (such as Papworth's U-value > 1.96) was observed more than 1 year after radiotherapy. We found that cytogenetic biomarkers reflected partial-body fractionated radiation exposure more than 1 year post-exposure. Our findings suggest that chromosome aberrations can be used to estimate biological absorbed radiation doses and can inform medical intervention for individuals suspected of fractionated or partial-body radiation exposure.

Expansion of monocytic myeloid-derived suppressor cells ameliorated intestinal inflammatory response by radiation through SOCS3 expression.

Radiation-induced colitis is a common clinical problem after radiation therapy and accidental radiation exposure. Myeloid-derived suppressor cells (MDSCs) have immunosuppressive functions that use a variety of mechanisms to alter both the innate and the adaptive immune systems. Here, we demonstrated that radiation exposure in mice promoted the expansion of splenic and intestinal MDSCs and caused intestinal inflammation due to the increased secretion of cytokines. Depletion of monocytic MDSCs using anti-Ly6C exacerbated radiation-induced colitis and altered the expression of inflammatory cytokine IL10. Adoptive transfers of 0.5 Gy-derived MDSCs ameliorated this radiation-induced colitis through the production IL10 and activation of both STAT3 and SOCS3 signaling. Intestinal-inflammation recovery using 0.5 Gy-induced MDSCs was assessed using histological grading of colitis, colon length, body weight, and survival rate. Using in vitro co-cultures, we found that 0.5 Gy-induced MDSCs had higher expression levels of IL10 and SOCS3 compared with 5 Gy-induced MDSCs. In addition, IL10 expression was not enhanced in SOCS3-depleted cells, even in the presence of 0.5 Gy-induced monocytic MDSCs. Collectively, the results indicate that 0.5 Gy-induced MDSCs play an important immunoregulatory role in this radiation-induced colitis mouse model by releasing anti-inflammatory cytokines and suggest that IL10-overexpressing mMDSCs may be potential immune-therapy targets for treating colitis.

An intercomparison exercise to compare scoring criteria and develop image databank for biodosimetry in South Korea.

PURPOSE: Mutual cooperation of biodosimetry laboratories is required for dose assessments of large numbers of people with potential radiation exposure, as in mass casualty accidents. We launched an intercomparison exercise to validate the performance of biodosimetry laboratories in South Korea. MATERIALS AND METHODS: Participating laboratories shared metaphase images from dicentric chromosome assays (DCAs) and fluorescence in situ hybridization (FISH)-based translocation assays, which were evaluated based on their own scoring protocols. RESULTS: Overall, the coefficient of variation among three laboratories was less than 10% for counting scorable metaphases and chromosomal aberrations. However, there was variation in the interpretation of the International Atomic Energy Agency guidelines for selecting scorable metaphases and identifying chromosomal aberrations. In a technical workshop, scoring discrepancies were extensively discussed in order to harmonize biodosimetry protocols in Korea. In addition, metaphase images with agreement among all participating laboratories were compiled into an image databank, which can be used for education and training of scorers. CONCLUSIONS: These findings and exercises may improve the accuracy of dose assessment, as well as increase the capacity for biodosimetry in South Korea.

Chronic radiation exposure aggravates atherosclerosis by stimulating neutrophil infiltration.

BACKGROUND: Radiation exposure is known to increase the risk of chronic inflammatory diseases, such as atherosclerosis, by modulating inflammation. METHODS: To investigate the infiltration of leukocytes in radiation-aggravated atherosclerosis, we examined low-density lipoprotein receptor-deficient (Ldlr-/-) mice and C57BL/6j mice after exposure to 0.5 or 1 Gy radiation over 16 weeks. RESULTS: We found that radiation exposure induced atherosclerosis development in Ldlr-/- mice, as demonstrated by increased lipid-laden plaque size, reactive oxygen species levels, and levels of the pro-inflammatory cytokines, IL-1ß and TNF-α, in the aortas and spleens. Total plasma cholesterol, triglyceride, and LDL cholesterol levels were also increased by radiation exposure, along with cardiovascular risk. We also showed dose-dependent increases in neutrophils and monocytes that coincided with a reduction in lymphocytes in the spleens of Ldlr-/- mice. The correlation between the infiltration of leukocytes and cytokine production was also confirmed in the hearts and spleens of these mice. CONCLUSIONS: We concluded that chronic radiation exposure increased the production of pro-inflammatory mediators, which was associated with the migration of neutrophils and inflammatory monocytes into sites of atherosclerosis. Thus, our data suggest that the accumulation of neutrophils and inflammatory monocytes, together with the reduction of lymphocytes, contribute to aggravated atherosclerosis in Ldlr-/- mice under prolonged exposure to radiation.

Assessment of an Emergency Medicine System for Radiation Accidents in Korea: A State Survey of the Workers Involved the Medical Response to Radiation Accidents.

Radiation emergency medicine systems are operated around the world to provide special care for the injured that require immediate medical attention in accidents. The objective of this survey was to evaluate people's perception of those who design the emergency medical plan for radiation accidents and those who supervise it in Korea. A questionnaire survey was conducted on the people involved in a regulatory system for medical response in a radiation emergency. Of 150 survey recipients, 133 (88.7%) completed the survey, including 92 workers and 41 inspectors. The respondents expressed the view that the national emergency medical plan is prepared above the average level using a Likert-style scale of 1 to 5 (mean = 3.55, SD = 0.74). Interestingly, using the Mann-Whitney U test, it could be shown that inspectors evaluated the emergency medical system for radiation accidents more strictly in all of the questions than the licensee workers, especially on radiation medical emergency preparedness (p = 0.004) and the governmental regulatory policy for radiation safety (p = 0.007). For a more efficient system of radiation emergency medicine, licensee workers prioritized the workforce, whereas inspectors favored laws and regulations for safety. The survey results show different perspectives between inspectors and licensee workers, which stem from the actual properties of each occupational role in the regulatory system for radiation medical emergency. These data could be utilized for communication and interaction with relevant people to improve the medical response preparedness against radiation accidents.

Autophagic Organelles in DNA Damage Response.

Autophagy is an important subcellular event engaged in the maintenance of cellular homeostasis via the degradation of cargo proteins and malfunctioning organelles. In response to cellular stresses, like nutrient deprivation, infection, and DNA damaging agents, autophagy is activated to reduce the damage and restore cellular homeostasis. One of the responses to cellular stresses is the DNA damage response (DDR), the intracellular pathway that senses and repairs damaged DNA. Proper regulation of these pathways is crucial for preventing diseases. The involvement of autophagy in the repair and elimination of DNA aberrations is essential for cell survival and recovery to normal conditions, highlighting the importance of autophagy in the resolution of cell fate. In this review, we summarized the latest information about autophagic recycling of mitochondria, endoplasmic reticulum (ER), and ribosomes (called mitophagy, ER-phagy, and ribophagy, respectively) in response to DNA damage. In addition, we have described the key events necessary for a comprehensive understanding of autophagy signaling networks. Finally, we have highlighted the importance of the autophagy activated by DDR and appropriate regulation of autophagic organelles, suggesting insights for future studies. Especially, DDR from DNA damaging agents including ionizing radiation (IR) or anti-cancer drugs, induces damage to subcellular organelles and autophagy is the key mechanism for removing impaired organelles.

Risk perception of radiation emergency medical staff on low-dose radiation exposure: Knowledge is a critical factor.

Radiation emergency medical (REM) staff respond to many types of disasters such as radiological and nuclear accidents as well as environmental radioactivity exposure. The objective of this study was to evaluate the risk perception of REM staff on radiation exposure in various situations and to analyze the factors that affect their risk perception. A questionnaire was given to 284 REM staff affiliated with various organizations, including nuclear power generation, nuclear fuel manufacturing, large-scale irradiation, and radiation-waste disposal facilities, as well as research and development institutions. To determine the substantially influential factors for risk perception, we analyzed the questionnaire responses using ordinal logistic regression, Kruskal-Wallis, and Spearman correlation analyses. It was generally perceived by REM staff that low-dose radiation exposure in daily life and work environments does not pose any health risks. A higher level of radiation knowledge was tightly associated with a lower risk perception of REM staff on extremely low-dose (several mSv) radiation exposure, thus exhibiting an inverse correlation. In contrast to radiation researchers, the work experience of REM staff was not a contributing factor to their risk perception. In our study, REM staff with a high level of radiation knowledge did not have any health concerns in their work environments. Efforts to enhance the radiation knowledge of REM staff through proper education and training would result in analytical risk evaluation, which may also improve their willingness to help meet surge capacity needs in large-scale radiological events.

Role of Commensal Microbes in the γ-Ray Irradiation-Induced Physiological Changes in Drosophila melanogaster.

Ionizing radiation induces biological/physiological changes and affects commensal microbes, but few studies have examined the relationship between the physiological changes induced by irradiation and commensal microbes. This study investigated the role of commensal microbes in the γ-ray irradiation-induced physiological changes in Drosophila melanogaster. The bacterial load was increased in 5 Gy irradiated flies, but irradiation decreased the number of operational taxonomic units. The mean lifespan of conventional flies showed no significant change by irradiation, whereas that of axenic flies was negatively correlated with the radiation dose. γ-Ray irradiation did not change the average number of eggs in both conventional and axenic flies. Locomotion of conventional flies was decreased after 5 Gy radiation exposure, whereas no significant change in locomotion activity was detected in axenic flies after irradiation. γ-Ray irradiation increased the generation of reactive oxygen species in both conventional and axenic flies, but the increase was higher in axenic flies. Similarly, the amounts of mitochondria were increased in irradiated axenic flies but not in conventional flies. These results suggest that axenic flies are more sensitive in their mitochondrial responses to radiation than conventional flies, and increased sensitivity leads to a reduced lifespan and other physiological changes in axenic flies.

Inhibition of EphA2 by Dasatinib Suppresses Radiation-Induced Intestinal Injury.

Radiation-induced multiorgan dysfunction is thought to result primarily from damage to the endothelial system, leading to a systemic inflammatory response that is mediated by the recruitment of leukocytes. The Eph-ephrin signaling pathway in the vascular system participates in various disease developmental processes, including cancer and inflammation. In this study, we demonstrate that radiation exposure increased intestinal inflammation via endothelial dysfunction, caused by the radiation-induced activation of EphA2, an Eph receptor tyrosine kinase, and its ligand ephrinA1. Barrier dysfunction in endothelial and epithelial cells was aggravated by vascular endothelial-cadherin disruption and leukocyte adhesion in radiation-induced inflammation both in vitro and in vivo. Among all Eph receptors and their ligands, EphA2 and ephrinA1 were required for barrier destabilization and leukocyte adhesion. Knockdown of EphA2 in endothelial cells reduced radiation-induced endothelial dysfunction. Furthermore, pharmacological inhibition of EphA2-ephrinA1 by the tyrosine kinase inhibitor dasatinib attenuated the loss of vascular integrity and leukocyte adhesion in vitro. Mice administered dasatinib exhibited resistance to radiation injury characterized by reduced barrier leakage and decreased leukocyte infiltration into the intestine. Taken together, these data suggest that dasatinib therapy represents a potential approach for the protection of radiation-mediated intestinal damage by targeting the EphA2-ephrinA1 complex.

The Emerging Roles of Exosomes as EMT Regulators in Cancer.

Epithelial-mesenchymal transition (EMT) causes epithelial cells to lose their polarity and adhesion property, and endows them with migratory and invasive properties to enable them to become mesenchymal stem cells. EMT occurs throughout embryonic development, during wound healing, and in various pathological processes, including tumor progression. Considerable research in the last few decades has revealed that EMT is invariably related to tumor aggressiveness and metastasis. Apart from the interactions between numerous intracellular signaling pathways known to regulate EMT, extracellular modulators in the tumor microenvironment also influence tumor cells to undergo EMT, with extracellular vesicles (EVs) receiving increasing attention as EMT inducers. EVs comprise exosomes and microvesicles that carry proteins, nucleic acids, lipids, and other small molecules to stimulate EMT in cells. Among EVs, exosomes have been investigated in many studies, and their role has been found to be significant with respect to regulating intercellular communications. In this review, we summarize recent studies on exosomes and their cargoes that induce cancer-associated EMT. Furthermore, we describe the possible applications of exosomes as promising therapeutic strategies.