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Objective. The arrangement of chromosomes in the cell nucleus has implications for cell radiosensitivity. The development of new tools to utilize Hi-C chromosome conformation data in nanoscale radiation track structure simulations allows for in silico investigation of this phenomenon. We have developed a framework employing Hi-C-based cell nucleus models in Monte Carlo radiation simulations, in conjunction with mechanistic models of DNA repair, to predict not only the initial radiation-induced DNA damage, but also the repair outcomes resulting from this damage, allowing us to investigate the role chromosome conformation plays in the biological outcome of radiation exposure. Approach. In this study, we used this framework to generate cell nucleus models based on Hi-C data from fibroblast and lymphoblastoid cells and explore the effects of cell type-specific chromosome structure on radiation response. The models were used to simulate external beam irradiation including DNA damage and subsequent DNA repair. The kinetics of the simulated DNA repair were compared with previous results. Main results. We found that the fibroblast models resulted in a higher rate of inter-chromosome misrepair than the lymphoblastoid model, despite having similar amounts of initial DNA damage and total misrepairs for each irradiation scenario. Significance. This framework represents a step forward in radiobiological modeling and simulation allowing for more realistic investigation of radiosensitivity in different types of cells., (Creative Commons Attribution license.)

In cells that are exposed to terrestrial sunlight, the indole moiety in the side chain of tryptophan (Trp) can suffer photo/oxidative damage (POD) by reactive oxygen species (ROS) and/or ultraviolet light (UV-B). Trp is oxidized to produce N-formylkynurenine (NFK), a UV-A-responsive photosensitizer that further degenerates into photosensitizers capable of generating ROS through exposure to visible light. Thus, Trp-containing proteins function as both victims, and perpetrators, of POD if they are not rapidly replaced through protein turnover. The literature indicates that protein turnover and DNA repair occur poorly in chromosomal interiors. We contend, therefore, that basic chromosomal proteins (BCPs) that are enveloped by DNA should have evolved to lack Trp residues in their amino acid sequences, since these could otherwise function as 'Trojan horse-type' DNA-damaging agents. Our global analyses of protein sequences demonstrates that BCPs consistently lack Trp residues, although DNA-binding proteins in general do not display such a lack. We employ HU-B (a wild-type, Trp-lacking bacterial BCP) and HU-B F47W (a mutant, Trp-containing form of the same bacterial BCP) to demonstrate that the possession of Trp is deleterious to BCPs and associated chromosomal DNA. Basically, we show that UV-B and UV-A (a) cause no POD in HU-B, but cause extensive POD in HU-B F47W (in vitro), as well as (b) only nominal DNA damage in bacteria expressing HU-B, but extensive DNA damage in bacteria expressing F47W HU-B (in vivo). Our results suggest that Trp-lacking BCPs could have evolved to reduce scope for protein-facilitated, sunlight-mediated damage of DNA by UV-A and visible light, within chromosomal interiors that are poorly serviced by protein turnover and DNA repair machinery., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest,, (Copyright © 2023 Elsevier B.V. All rights reserved.)

The quest for the discovery and validation of radiosensitivity biomarkers is ongoing and while conventional bioassays are well established as biomarkers, molecular advances have unveiled new emerging biomarkers. Herein, we present the validation of a new 4-gene signature panel of CDKN1, FDXR, SESN1 and PCNA previously reported to be radiation-responsive genes, using the conventional G2 chromosomal radiosensitivity assay. Radiation-induced G2 chromosomal radiosensitivity at 0.05 Gy and 0.5 Gy IR is presented for a healthy control ( n = 45) and a prostate cancer ( n = 14) donor cohort. For the prostate cancer cohort, data from two sampling time points (baseline and Androgen Deprivation Therapy (ADT)) is provided, and a significant difference ( p > 0.001) between 0.05 Gy and 0.5 Gy was evident for all donor cohorts. Selected donor samples from each cohort also exposed to 0.05 Gy and 0.5 Gy IR were analysed for relative gene expression of the 4-gene signature. In the healthy donor cohort, there was a significant difference in gene expression between IR dose for CDKN1, FXDR and SESN1 but not PCNA and no significant difference found between all prostate cancer donors, unless they were classified as radiation-induced G2 chromosomal radiosensitive. Interestingly, ADT had an effect on radiation response for some donors highlighting intra-individual heterogeneity of prostate cancer donors.

Chromoanagenesis is a genomic catastrophe that results in chromosomal shattering and reassembly. These extreme single chromosome events were first identified in cancer, and have since been observed in other systems, but have so far only been formally documented in plants in the context of haploid induction crosses. The frequency, origins, consequences, and evolutionary impact of such major chromosomal remodeling in other situations remain obscure. Here, we demonstrate the occurrence of chromoanagenesis in poplar (Populus sp.) trees produced from gamma-irradiated pollen. Specifically, in this population of siblings carrying indel mutations, two individuals exhibited highly frequent copy number variation (CNV) clustered on a single chromosome, one of the hallmarks of chromoanagenesis. Using short-read sequencing, we confirmed the presence of clustered segmental rearrangement. Independently, we identified and validated novel DNA junctions and confirmed that they were clustered and corresponded to these rearrangements. Our reconstruction of the novel sequences suggests that the chromosomal segments have reorganized randomly to produce a novel rearranged chromosome but that two different mechanisms might be at play. Our results indicate that gamma irradiation can trigger chromoanagenesis, suggesting that this may also occur when natural or induced mutagens cause DNA breaks. We further demonstrate that such events can be tolerated in poplar, and even replicated clonally, providing an attractive system for more in-depth investigations of their consequences., Competing Interests: The authors have declared that no competing interests exist.

The large majority of chromosome damage produced by ionizing radiations takes the form of exchange aberrations. For simple exchanges between two chromosomes, multi-fluor fluorescence in situ hybridization (mFISH) studies confirm that the dose response to X rays or gamma rays is quasilinear with dose. This result is in seeming conflict with generalized theories of radiation action that depend on the interaction of lesions as the source of curvature in dose-response relationships. A qualitative explanation for such "linearization" had been previously proposed but lacked quantitative support. The essence of this explanation is that during the rejoining of radiogenic chromosome breaks, competition for breaks (CFB) between different aberration types often results in formation of complex exchange aberrations at the expense of simple reciprocal exchange events. This process becomes more likely at high radiation doses, where the number of contemporaneous breaks is high and complex exchanges involving multiple breaks become possible. Here we provide mathematical support for this CFB concept under the assumption that the mean and variance for exchange complexity increase with radiation dose., (©2021 by Radiation Research Society. All rights of reproduction in any form reserved.)

This study aimed to investigate the impact of chronic low-level exposure to chemical carcinogens with different modes of action on the cellular response to ionising radiation. Human lymphoblastoid GM1899A cells were cultured in the presence of 4-nitroquinoline N-oxide (4NQO), N-nitroso-N-methylurea (MNU) and hydrogen peroxide (H 2 O 2 ) for up to 6 months at the highest non-(geno)toxic concentration identified in pilot experiments. Acute challenge doses of 1 Gy X-rays were given and chromosome damage (dicentrics, acentric fragments, micronuclei, chromatid gaps/breaks) was scored. Chronic exposure to 20 ng/ml 4NQO, 0.25 μg/ml MNU or 10 μM H 2 O 2 hardly induced dicentrics and did not significantly alter the yield of X-ray-induced dicentrics. Significant levels of acentric fragments were induced by all chemicals, which did not change during long-term exposure. Fragment data in combined treatment samples compared to single treatments were consistent with an additive effect of chemical and radiation exposure. Low level exposure to 4NQO induced micronuclei, the yields of which did not change throughout the 6 month exposure period. As for fragments, micronuclei yields for combined treatments were consistent with an additive effect of chemical and radiation. These results suggest that cellular radiation responses are not affected by long-term low-level chemical exposure.

The interpretation of radiation dose is an important procedure for both radiological operators and persons who are exposed to background or artificial radiations. Dicentric chromosome assay (DCA) is one of the representative methods of dose estimation that discriminates the aberration in chromosomes modified by radiation. Despite the DCA-based automated radiation dose estimation methods proposed in previous studies, there are still limitations to the accuracy of dose estimation. In this study, a DCA-based automated dose estimation system using deep learning methods is proposed. The system is comprised of three stages. In the first stage, a classifier based on a deep learning technique is used for filtering the chromosome images that are not appropriate for use in distinguishing the chromosome; 99% filtering accuracy was achieved with 2,040 test images. In the second stage, the dicentric rate is evaluated by counting and identifying chromosomes based on the Feature Pyramid Network, which is one of the object detection algorithms based on deep learning architecture. The accuracies of the neural networks for counting and identifying chromosomes were estimated at over 97% and 90%, respectively. In the third stage, dose estimation is conducted using the dicentric rate and the dose-response curve. The accuracies of the system were estimated using two independent samples; absorbed doses ranging from 1- 4 Gy agreed well within a 99% confidential interval showing highest accuracy compared to those in previous studies. The goal of this study was to provide insights towards achieving complete automation of the radiation dose estimation, especially in the event of a large-scale radiation exposure incident., (©2021 by Radiation Research Society. All rights of reproduction in any form reserved.)

Developments in the genome organisation field has resulted in the recent methodology to infer spatial conformations of the genome directly from experimentally measured genome contacts (Hi-C data). This provides a detailed description of both intra- and inter-chromosomal arrangements. Chromosomal intermingling is an important driver for radiation-induced DNA mis-repair. Which is a key biological endpoint of relevance to the fields of cancer therapy (radiotherapy), public health (biodosimetry) and space travel. For the first time, we leverage these methods of inferring genome organisation and couple them to nano-dosimetric radiation track structure modelling to predict quantities and distribution of DNA damage within cell-type specific geometries. These nano-dosimetric simulations are highly dependent on geometry and are benefited from the inclusion of experimentally driven chromosome conformations. We show how the changes in Hi-C contract maps impact the inferred geometries resulting in significant differences in chromosomal intermingling. We demonstrate how these differences propagate through to significant changes in the distribution of DNA damage throughout the cell nucleus, suggesting implications for DNA repair fidelity and subsequent cell fate. We suggest that differences in the geometric clustering for the chromosomes between the cell-types are a plausible factor leading to changes in cellular radiosensitivity. Furthermore, we investigate changes in cell shape, such as flattening, and show that this greatly impacts the distribution of DNA damage. This should be considered when comparing in vitro results to in vivo systems. The effect may be especially important when attempting to translate radiosensitivity measurements at the experimental in vitro level to the patient or human level., Competing Interests: The authors have declared that no competing interests exist.

Background Although the National Lung Screening Trial reported a significant reduction in lung cancer mortality when low-dose (LD) CT chest examinations are used for a diagnosis, their biologic effects from radiation exposure remain unclear. Purpose To compare LD CT and standard-dose (SD) CT for DNA double-strand breaks and chromosome aberrations (CAs) in peripheral blood lymphocytes. Materials and Methods Between March 2016 and June 2018, 209 participants who were referred to a respiratory surgery department for chest CT studies were prospectively enrolled in this study. Individuals were excluded if they had undergone radiography examinations within the last 3 days or had undergone chemotherapy or radiation therapy. Peripheral blood samples were obtained before and 15 minutes after CT. The number of γ-H2AX foci and unstable CAs in lymphocytes was quantified by immunofluorescent staining of γ-H2AX and by fluorescence in situ hybridization by using peptide nucleic acid probes for centromeres and telomeres, respectively. The Wilcoxon signed rank test was used for statistical analysis. Bonferroni correction was applied for multiple comparisons. Results Of the 209 participants (105 women, 104 men; mean age, 67.0 years ± 11.3 [standard deviation]), 107 underwent chest LD CT and 102 underwent chest SD CT. Sex distribution, age, and body size metrics were similar between the two groups. The median effective dose of LD CT and SD CT was 1.5 and 5.0 mSv, respectively. The number of double-strand breaks and CAs increased after a SD CT examination (γ-H2AX, P < .001; CAs, P = .003); the number of double-strand breaks and CAs before and after LD CT was not different (γ-H2AX, P = .45; CAs, P = .69). Conclusion No effect of low-dose CT on human DNA was detected. In the same setting, DNA double-strand breaks and chromosome aberrations increased after standard-dose CT. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Brenner in this issue.

Purpose: The main aim of this study was to comparatively investigate the effects of culturing methods on the response of human peripheral blood lymphocytes to irradiation exposure. Materials and methods: Whole blood and isolated lymphocytes were ex vivo exposed to two radiation sources (60 MeV proton or 250 kV X-ray radiation) with different doses (0.3, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, and 4.0 Gy), and genotoxic markers were subsequently assayed. The observed effects were compared as dose-response relationships using two end points (CBMN and PCC tests) and different biomarkers (NDI, PCC index, MNi frequency and excess PCC fragments). Results and conclusions: The results showed different effects of the culturing techniques on the response of human peripheral blood lymphocytes to radiation. The MNi frequency and excess PCC fragments were significantly higher when lymphocytes were cultured after being isolated. After irradiation, no differences were seen in the NDI between the lymphocytes of the two culturing techniques; however, there were differences in the PPC index. When planning or performing cytogenetic studies, the possibility of such effects and their potential to impact the variability of the results of human biomonitoring studies should be considered important and taken into account.

Purpose: The premature chromosome condensation (PCC) technique is used to study exposure to external radiation through the determination of chromosome fragments observed in interphase cells. The presence of large telomeric signals in CHO cells interferes with the detection of PCC fragments and the identification of dicentric chromosomes. We present an improved method for the fusion of G0-lymphocytes with mitotic Akodon cells (few chromosomes and weakly-staining telomeric sequences) to induce PCC in combination with rapid quantification of dicentric chromosomes and centric rings as an alternative to the classical CHO cell fusion technique. Materials and methods: Whole blood from three healthy volunteers was γ-irradiated with 0, 2, or 4 Gy. Following a 24 h incubation post-exposure at 37 °C, chromosome spreads of isolated lymphocytes were prepared by standard PCC procedures using mitotic Akodon cells. Results: The percentage of scorable fusions, measured by telomere/centromere (T/C) staining, for Akodon -induced PCC was higher than that for CHO-induced PCC, irrespective of radiation exposure. Importantly, both techniques gave the same result for biodosimetry evaluation. Conclusion: The mitotic Akodon cell-induced PCC fusion assay, in combination with the scoring of dicentric chromosomes and rings by T/C staining of G0-lymphocytes is a suitable alternative for fast and reliable dose estimation after accidental radiation exposure.

A fully optimized staining method for detecting sister chromatid exchanges in cultured cells is presented. The method gives reproducibly robust quantitative results. Sister chromatid exchange is a classic toxicology assay for genotoxicity and for detecting alterations to the biochemistry underlying cellular homologous recombination. Growth of cells in the presence of 5'-bromo-deoxyuridine for two rounds of DNA replication followed by collecting metaphase spreads on glass slides, treatment with the UV-sensitive dye Hoechst 33258, long-wave UV light exposure, and Giemsa staining gives a permanent record of the exchanges.

One of the most common usages of radiation in current medical diagnosis is computed tomography (CT) using X-rays. The potential health risk of CT scans has been discussed in various studies to determine whether low-dose radiation from CT could enhance the chromosome aberration yields in pediatric patients and increase their risk of carcinogenesis. For this reason, it is of great interest to study the effects of low-dose radiation. The induction of DNA damage by a CT scan examination has been demonstrated in several reports by the γ-H2AX assay, the micronuclei assay and dicentrics measurements. However, the results of most studies showed limitations. On the other hand, epidemiological studies give contradictory results for post-natal radiation exposure in the low-dose range, so it is still difficult to draw conclusions about the effects of CT examinations and risk of carcinogenesis. This article provides an overview of previously published data and summarizes the current state of knowledge., (© The Author(s) 2019. Published by Oxford University Press.)

Purpose: Based on clinical and molecular data, breast cancer is a heterogeneous disease. Breast cancers that have no expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) are defined as triple negative breast cancers (TNBCs); luminal cancers have different expressions of ER, PR and/or HER2. TNBCs are frequently linked with advanced disease, poor prognosis and occurrence in young African women, and about 15% of the cases are associated with germline BRCA1/2 mutations. Since radiotherapy is utilized as a principle treatment in the management of TNBC, we aimed to investigate the chromosomal instability and radiosensitivity of lymphocytes in TNBC patients compared to luminal breast cancer patients and healthy controls using the micronucleus (MN) assay. The effect of mutations in breast cancer susceptibility genes on chromosomal radiosensitivity was also evaluated. Methods: Chromosomal radiosensitivity was evaluated in the G0 (83 patients and 90 controls) and S/G2 (34 patients and 17 controls) phase of the cell cycle by exposing blood samples from all patients and controls to 2 and 4 Gy ionizing radiation (IR). Results: In the G0 MN assay, the combined cohort of all breast cancer, TNBC and luminal patients' exhibit significantly elevated spontaneous MN values compared to controls indicating chromosomal instability. Chromosomal radiosensitivity is also significantly elevated in the combined cohort of all breast cancer patients compared to controls. The TNBC patients, however, do not exhibit enhanced chromosomal radiosensitivity. Similarly, in the S/G2 phase, 76% of TNBC patients do not show enhanced chromosomal radiosensitivity compared to the controls. In both the G0 and S/G2 phase, luminal breast cancer patients demonstrate a shift toward chromosomal radiosensitivity compared to TNBC patients and controls. Conclusions: The observations of the MN assay suggest increased chromosomal instability and chromosomal radiosensitivity in South African breast cancer patients. However, in TNBC patients, the irradiated MN values are not elevated. Our results suggest that the healthy lymphocytes in TNBC patients could handle higher doses of IR.

18 F-FDG PET/CT imaging is used in the diagnosis of diseases, including cancers. The principal photons used for imaging are 511 ke V gamma photons resulting from positron annihilation. The absorbed dose varies among body organs, depending on administered radioactivity and biological clearance. We have attempted to evaluate DNA double-strand breaks (DSB) and toxicity induced in V79 lung fibroblast cells in vitro by 18 F-FDG, at doses which might result from PET procedures. Cells were irradiated by 18 F-FDG at doses (14.51 and 26.86 mGy), comparable to absorbed doses received by critical organs during PET procedures. The biological endpoints measured were formation of γ-H2AX foci, mitochondrial stress, chromosomal aberrations, and cell cycle perturbation. Irradiation induced DSB (γH2AX assay), mitochondrial depolarization, and both chromosome and chromatid types of aberrations. At higher radiation doses, increased aneuploidy and reduced mitotic activity were also seen. Thus, significant biological effects were observed at the doses delivered by the 18 F-FDG exposure and the effects increased with dose., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Environmental exposure to modern microwave telecommunication electromagnetic fields (EMFs) has increased to unprecedented levels with consequent health complaints and concerns. Many studies have already reported genotoxic effects on a variety of organisms and cell/tissue types. Human peripheral blood lymphocytes from six healthy donors were stimulated for mitosis and exposed to microwave EMF of Universal Mobile Telecommunications System (UMTS) or third generation (3G) mobile telephony (MT) EMF/radiation emitted by a commercially available mobile phone handset. Lymphocytes exposed during the G2 phase of the cell division cycle and observed at metaphase, exhibited chromatid-type aberrations (gaps and breaks) at highly significant percentages - up to 275% - compared to the control (sham-exposed) samples. Each subject exhibited a different sensitivity to the microwave exposure. Moreover, the percentages of aberrations in the control samples among subjects were different due to genetic and environmental factors. The MT EMF exposure induced mainly achromatic lesions (gaps), and secondarily terminal deletions (breaks) in a smaller degree. In conclusion, the present study shows that microwave 3G MT EMF/radiation - within the current exposure limits - has significant genotoxic action on human cells, and human exposure to this EMF/radiation should be kept at levels as low as possible.

Charged-particle microbeams (CPMs) provide a unique opportunity to investigate the effects of ionizing radiation on living biological specimens with a precise control of the delivered dose, i.e. the number of particles per cell. We describe a methodology to manipulate and micro-irradiate early stage C. elegans embryos at a specific phase of the cell division and with a controlled dose using a CPM. To validate this approach, we observe the radiation-induced damage, such as reduced cell mobility, incomplete cell division and the appearance of chromatin bridges during embryo development, in different strains expressing GFP-tagged proteins in situ after irradiation. In addition, as the dosimetry of such experiments cannot be extrapolated from random irradiations of cell populations, realistic three-dimensional models of 2 cell-stage embryo were imported into the Geant4 Monte-Carlo simulation toolkit. Using this method, we investigate the energy deposit in various chromatin condensation states during the cell division phases. The experimental approach coupled to Monte-Carlo simulations provides a way to selectively irradiate a single cell in a rapidly dividing multicellular model with a reproducible dose. This method opens the way to dose-effect investigations following targeted irradiation.

The classical G 2 -assay is widely used to assess cell-radiosensitivity and cancer phenotype: Cells are exposed to low doses of ionizing-radiation (IR) and collected for cytogenetic- analysis ˜1.5 h later. In this way, chromosome-damage is measured in cells irradiated in G 2 -phase, without retrieving information regarding kinetics of chromosome-break-repair. Modification of the assay to include analysis at multiple time-points after IR, has enabled kinetic-analysis of chromatid-break-repair and assessment of damage in a larger proportion of G 2 -phase cells. This modification, however, increases the probability that at later time points not only cells irradiated in G 2 -phase, but also cells irradiated in S-phase will reach metaphase. However, the response of cells irradiated in G 2 -phase can be mechanistically different from that of cells irradiated in S-phase. Therefore, indiscriminate analysis may confound the interpretation of experiments designed to elucidate mechanisms of chromosome-break-repair and the contributions of the different DSB-repair-pathways in this response. Here we report an EdU based modification of the assay that enables S- and G 2 -phase specific analysis of chromatid break repair. Our results show that the majority of metaphases captured during the first 2 h after IR originate from cells irradiated in G 2 -phase (EdU - metaphases) in both rodent and human cells. Metaphases originating from cells irradiated in S-phase (EdU + metaphases) start appearing at 2 h and 4 h after IR in rodent and human cells, respectively. The kinetics of chromatid-break-repair are similar in cells irradiated in G 2 - and S-phase of the cell-cycle, both in rodent and human cells. The protocol is applicable to classical-cytogenetic experiments and allows the cell-cycle specific analysis of chromosomal-aberrations. Finally, the protocol can be applied to the kinetic analysis of chromosome-breaks in prematurely-condensed-chromosomes of G 2 -phase cells. In summary, the developed protocol provides means to enhance the analysis of IR-induced-cytogenetic-damage by providing information on the cell-cycle phase where DNA damage is inflicted., (Copyright © 2019 Elsevier B.V. All rights reserved.)

It has been hypothesized that species with holocentric chromosomes have a selective evolutionary advantage for developmental and reproductive success because holocentric chromosomes are less susceptible to chromosome breakage than monocentric chromosomes. We analyzed data on sterilizing doses of ionizing radiation for more than 250 species of arthropods to test whether the minimal dose for reproductive sterilization is higher for species with holocentric chromosomes than for species with monocentric chromosomes. Using linear mixed models that account for phylogeny, we show that holocentric arthropods are more tolerant of sterilizing radiation than monocentrics. Moreover, higher dose rates correlate with lower sterilizing doses in monocentrics, but not in holocentrics, which is a novel finding that may be of importance for radiosanitation practice. Under the dose rate of 1 Gy/min, holocentric arthropods are sterilized on average with a 2.9 times higher minimal dose than monocentrics. Life stage and sex have significant but considerably weaker effects on sterilizing dose than chromosome type. Adults and males require 1.2 and 1.4 times higher sterilizing doses than juveniles and females, respectively. These results support the hypothesis that holocentric lineages may originate and thrive better in times of increased exposure to chromosome-breaking factors.

Homologous recombination (HR) is the principal mechanism of DNA repair acting during meiosis and is fundamental for the segregation of chromosomes and the increase of genetic diversity. Nevertheless, non-homologous end joining (NHEJ) mechanisms can also act during meiosis, mainly in response to exogenously-induced DNA damage in late stages of first meiotic prophase. In order to better understand the relationship between these two repair pathways, we studied the response to DNA damage during male mouse meiosis after gamma radiation. We clearly discerned two types of responses immediately after treatment. From leptotene to early pachytene, exogenous damage triggered the massive presence of γH2AX throughout the nucleus, which was associated with DNA repair mediated by HR components (DMC1 and RAD51). This early pathway finished with the sequential removal of DMC1 and RAD51 and was no longer inducible at mid pachytene. However, from mid-pachytene to diplotene, γH2AX appeared as large discrete foci. This late repair pattern was mediated initially by NHEJ, involving Ku70 and XRCC4, which were constitutively present, and 53BP1, which appeared at sites of damage soon after irradiation. Nevertheless, 24 hours after irradiation, a HR pathway involving RAD51 but not DMC1 mostly replaced NHEJ. Additionally, we observed the occurrence of synaptonemal complex bridges between bivalents, most likely representing chromosome translocation events that may involve DMC1, RAD51 or 53BP1. Our results reinforce the idea that the early "meiotic" repair pathway that acts by default at the beginning of meiosis is replaced from mid-pachytene onwards by a "somatic-like" repair pattern. This shift might be important to resolve DNA damage (either endogenous or exogenous) that could not be repaired by the early meiotic mechanisms, for instance those in the sex chromosomes, which lack a homologous chromosome to repair with. This transition represents another layer of functional changes that occur in meiotic cells during mid pachytene, in addition to epigenetic reprograming, reactivation of transcription, changes in the gene expression profile and acquisition of competence to proceed to metaphase., Competing Interests: The authors have declared that no competing interests exist.

Premature chromosome condensation (PCC) is a sensitive and unique way to detect interphase chromosome damage and its recovery in mammalian cells irradiated with ionizing radiation. In this chapter, we describe G1 PCC assay with which one can measure immediate chromosome breaks in G1 type chromosomes and their repair/rejoining. In order to induce G1 PCC, one needs to fuse mitotic cells with G1 cells to be tested. There are two methods to fuse cells; one is to use Sendai virus or its equivalent, and another method needs polyethylene glycol (PEG) as a fusing agent. The date obtained with PCC assay can bridge the gap between radiation-induced DNA damage (mainly double strand breaks) and chromosome aberrations observable at metaphase stage.

Here, we report the findings of a 25-year cytogenetic follow-up study on a male patient who received 2 rounds of radioiodine treatment within a span of 26 months (1.78 GBq in 1992 and 14.5 GBq in 1994). The patient was 34 years old with a body mass index of 25 at the time of the first radioiodine treatment. Multicolor FISH and multicolor banding (mBAND) techniques performed on the patient detected inter- and intrachromosomal exchanges. Although the frequency of chromosome translocations remained essentially the same as reported in our earlier study (0.09/cell), the percentage of reciprocal (balanced) translocations increased from 54.38 to 80.30% in the current study. In addition to simple chromosome translocations, complex exchanges (0.29%) involving more than 2 chromosomes were detected for the first time in this patient. Strikingly, a clonal translocation involving chromosomes 14 and 15, t(14p;15q), was found in 7 of the 677 cells examined (1.03%). The presence of complex and clonal translocations indicates the onset of chromosomal instability induced by internal radioiodine exposure. mBAND analysis using probes specific for chromosomes 1, 2, 4, 5, and 10 revealed 5 inversions in a total of 717 cells (0.69%), and this inversion frequency is several-fold higher than the baseline frequency reported in healthy individuals using the classical G-banding technique. Collectively, our study suggests that stable chromosome aberrations such as translocations and inversions can be useful not only for retrospective biodosimetry but also for long-term monitoring of chromosomal instability caused by past radioiodine exposure., (© 2019 S. Karger AG, Basel.)

The G2 chromosomal radiosensitivity assay or, simply G2 assay, measures the number of chromatid type aberrations induced by radiation in G2 phase. Typically, asynchronous growing cells are irradiated with less than 1 Gy and allowed 0.5-1 h for cells in mitosis, at the time of irradiation, to transit into G1. Later, the G2 phase cells, at the time irradiation, are blocked by colcemid for 1-4 h at metaphase. Cells are collected by standard hypotonic solution and Carnoy solution fixation or directly fixed onto the culture vessels. The G2 assay can detect severe radiosensitivity in ATM homozygous mutated cells and relatively small differences among cellular radiosensitivity such as heterozygous mutation carriers of ATM and BRCA1/2 mutation carriers. The G2 assay also has the capability to detect cancer prone individuals. This assay only requires a conventional cell culture facility and the standard microscopic observation.

Photons, such as X- or γ-rays, induce DNA damage (distributed throughout the nucleus) as a result of low-density energy deposition. In contrast, particle irradiation with high linear energy transfer (LET) deposits high-density energy along the particle track. High-LET heavy-ion irradiation generates a greater number and more complex critical chromosomal aberrations, such as dicentrics and translocations, compared with X-ray or γ irradiation. In addition, the formation of >1000 bp deletions, which is rarely observed after X-ray irradiation, has been identified following high-LET heavy-ion irradiation. Previously, these chromosomal aberrations have been thought to be the result of misrepair of complex DNA lesions, defined as DNA damage through DNA double-strand breaks (DSBs) and single-strand breaks as well as base damage within 1-2 helical turns (<3-4 nm). However, because the scale of complex DNA lesions is less than a few nanometers, the large-scale chromosomal aberrations at a micrometer level cannot be simply explained by complex DNA lesions. Recently, we have demonstrated the existence of clustered DSBs along the particle track through the use of super-resolution microscopy. Furthermore, we have visualized high-level and frequent formation of DSBs at the chromosomal boundary following high-LET heavy-ion irradiation. In this review, we summarize the latest findings regarding the hallmarks of DNA damage structure and the repair pathway following heavy-ion irradiation. Furthermore, we discuss the mechanism through which high-LET heavy-ion irradiation may induce dicentrics, translocations and large deletions.

The RTGene study was focused on the development and validation of new transcriptional biomarkers for prediction of individual radiotherapy patient responses to ionizing radiation. In parallel, for validation purposes, this study incorporated conventional biomarkers of radiation exposure, including the dicentric assay. Peripheral blood samples were taken with ethical approval and informed consent from a total of 20 patients undergoing external beam radiotherapy for breast, lung, gastrointestinal or genitourinary tumors. For the dicentric assay, two samples were taken from each patient: prior to radiotherapy and before the final fraction. Blood samples were set up using standard methods for the dicentric assay. All the baseline samples had dicentric frequencies consistent with the expected background for the normal population. For blood taken before the final fraction, all the samples displayed distributions of aberrations, which are indicative of partial-body exposures. Whole-body and partial-body cytogenetic doses were calculated with reference to a 250-kVp X-ray calibration curve and then compared to the dose to blood derived using two newly developed blood dosimetric models. Initial comparisons indicated that the relationship between these measures of dose appear very promising, with a correlation of 0.88 ( P = 0.001). A new Bayesian zero-inflated Poisson finite mixture method was applied to the dicentric data, and partial-body dose estimates showed no significant difference ( P > 0.999) from those calculated by the contaminated Poisson technique. The next step will be further development and validation in a larger patient group.

Purpose: Exposure to ionizing radiation causes damage to the genomic integrity and stability of the cell. Though a large number of molecules have been studied for their radioprotective capability, no single agent is available today that meets all the requirements of a good radiprotector. In this study, we have investigated a combination of Resveratrol (RSV) and 3,3'-Diindolyl methane (DIM) for its efficacy for radioprotection. It is our hypothesis that this combination that possesses less toxicity than synthetic compounds, free radical scavenging potential, and the capacity to interfere with the several of the signaling cascades that trigger damage to cell by ionizing radiation may possess good radioprotective capability., Materials and Methods: Mice were pre-treated with a combination of RSV and DIM and the 30-day mortality assay, endogenous antioxidant levels in intestinal mucosa, metaphase chromosomal aberrations, and micronuclei formation were assessed after exposed to ionizing radiation., Results: The dose modifying factor (DRF) obtained for RSV, DIM, and the combination is 1.15, 1.17, and 1.3, respectively. Pre-treatment of mice with the combination results in significant (***p = .001) protection of the endogenous antioxidant levels, chromosomal aberrations, micronuclei formation, after exposure to ionizing radiation., Conclusions: Our findings suggest that pre-treatment with the combination of RSV and DIM protects effectively from the ionizing radiation-induced damage at the molecular, cellular, and tissue levels by counteracting both the direct and indirect effects.

Purpose: Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage. It was suggested to be due to an effective transformation of DNA damage to chromosomal damage at low temperature. The purpose of the study was to analyze the kinetics of aberration formation during the first hours after exposing human peripheral blood lymphocytes to ionizing radiation at 0.8 °C and 37 °C., Materials and Methods: To this end, we applied the technique of premature chromosome condensation. In addition, DNA damage response was analyzed by measuring the levels of phosphorylated DNA damage responsive proteins ATM, DNA-PK and p53 and mRNA levels of the radiation-responsive genes BBC3, FDXR, GADD45A, XPC, MDM2 and CDKN1A., Results: A consistently lower frequency of chromosomal breaks was observed in cells exposed at 0.8 °C as compared to 37 °C already after 30 minutes postexposure. This effect was accompanied by elevated levels of phosphorylated ATM and DNA-PK proteins and a reduced immediate level of phosphorylated p53 and of the responsive genes., Conclusions: Low temperature at exposure appears to promote DNA repair leading to reduced transformation of DNA damage to chromosomal aberrations.

Exposure to a small number of high-energy heavy charged particles (HZE ions), as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/μm) in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/μm) at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis.

Meiotic recombination occurs as a programmed event that initiates by the formation of DNA double-strand breaks (DSBs) that give rise to the formation of crossovers that are observed as chiasmata. Chiasmata are essential for the accurate chromosome segregation and the generation of new combinations of parental alleles. Some treatments that provoke exogenous DSBs also lead to alterations in the recombination pattern of some species in which full homologous synapsis is achieved at pachytene. We have carried out a similar approach in males of the grasshopper Stethophyma grossum, whose homologues show incomplete synapsis and proximal chiasma localization. After irradiating males with γ rays we have studied the distribution of both the histone variant γ-H2AX and the recombinase RAD51. These proteins are cytological markers of DSBs at early prophase I. We have inferred synaptonemal complex (SC) formation via identification of SMC3 and RAD 21 cohesin subunits. Whereas thick and thin SMC3 filaments would correspond to synapsed and unsynapsed regions, the presence of RAD21 is only restricted to synapsed regions. Results show that irradiated spermatocytes maintain restricted synapsis between homologues. However, the frequency and distribution of chiasmata in metaphase I bivalents is slightly changed and quadrivalents were also observed. These results could be related to the singular nuclear polarization displayed by the spermatocytes of this species., Competing Interests: The authors have declared that no competing interests exist.

The calculated risk of cancer in humans due to radiation exposure is based primarily on long-term follow-up studies, e.g. the life-span study (LSS) on atomic bomb (A-bomb) survivors in Hiroshima and Nagasaki. Since A-bomb radiation consists of a mixture of γ-rays and neutrons, it is essential that the relative biological effectiveness (RBE) of neutrons is adequately evaluated if a study is to serve as a reference for cancer risk. However, the relatively small neutron component hampered the direct estimation of RBE in LSS data. To circumvent this problem, several strategies have been attempted, including dose-independent constant RBE, dose-dependent variable RBE, and dependence on the degrees of dominance of intermingled γ-rays. By surveying the available literature, we tested the chromosomal RBE of neutrons as the biological endpoint for its equivalence to the microdosimetric quantities obtained using a tissue-equivalent proportional counter (TEPC) in various neutron fields. The radiation weighting factor, or quality factor, Q n , of neutrons as expressed in terms of the energy dependence of the maximum RBE, RBE m , was consistent with that predicted by the TEPC data, indicating that the chromosomally measured RBE was independent of the magnitude of coexisting γ-rays. The obtained neutron RBE, which varied with neutron dose, was confirmed to be the most adequate RBE system in terms of agreement with the cancer incidence in A-bomb survivors, using chromosome aberrations as surrogate markers. With this RBE system, the cancer risk in A-bomb survivors as expressed in unit dose of reference radiation is equally compatible with Hiroshima and Nagasaki cities, and may be potentially applicable in other cases of human radiation exposure., (© The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

Many studies have investigated exposure biomarkers for high dose radiation. However, no systematic study on which biomarkers can be used in dose estimation through premature chromosome condensation (PCC) analysis has been conducted. The present study aims to screen the high-dose radiation exposure indicator in calyculin A-induced PCC. The dose response of multiple biological endpoints, including G2/A-PCC (G2/M and M/A-PCC) index, PCC ring (PCC-R), ratio of the longest/shortest length (L/L ratio), and length and width ratio of the longest chromosome (L/B ratio), were investigated in calyculin A-induced G2/A-PCC spreads in human peripheral blood lymphocytes exposed to 0-20Gy (dose-rate of 1Gy/min) cobalt-60 gamma-rays. The G2/A-PCC index was decreased with enhanced absorbed doses of 4-20Gy gamma-rays. The G2/A PCC-R at 0-12Gy gamma-rays conformed to Poisson distribution. Three types of PCC-R were scored according to their shape and their solidity or hollowness. The frequencies of hollow PCC-R and PCC-R including or excluding solid ring in G2/A-PCC spreads were enhanced with increased doses. The length and width of the longest chromosome, as well as the length of the shortest chromosome in each G2/M-PCC or M/A-PCC spread, were measured. All L/L or L/B ratios in G2/M-PCC or M/A-PCC spread increased with enhanced doses. A blind test with two new irradiated doses was conducted to validate which biomarker could be used in dose estimation. Results showed that hollow PCC-R and PCC-R including solid ring can be utilized for accurate dose estimation, and that hollow PCC-R was optimal for practical application., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.

Biodosimetry is a method for measuring the dose of radiation to individuals using biological markers such as chromosome damage. Following mass casualty events, it is important to provide this information rapidly in order to assist with the medical management of potentially exposed casualties. Currently, the gold standard for biodosimetry is the dicentric chromosome assay, which accurately estimates the dose from the number of dicentric chromosomes in lymphocytes. To increase throughput of analysis following a large-scale mass casualty event, this assay has been adapted for use on the imaging flow cytometer. This chapter describes the methods for the identification and quantification of mono- and multicentric chromosomes using the imaging flow cytometer.

This chapter presents a detailed methodology for somatic cell nuclear transfer-cloning of zebrafish. We aim to place the reader in a virtual lab experience to assist acquisition of the technical skills required for reproducing the published protocol. All materials, including catalog numbers for reagents and techniques for their preparation, are provided. Our protocols describe laser inactivation of egg chromosomes, the transfer of a cell through the oocyte micropyle, and spontaneous activation of the reconstructed embryo. High-quality eggs are the key to cloning success, and Chinook salmon ovarian fluid is indispensable for keeping eggs arrested at the metaphase of meiosis II. This protocol continues to be refined by our laboratory. However, naive investigators should be able to apply it in its present form to generate cloned zebrafish., (Copyright © 2016 Elsevier Inc. All rights reserved.)

DNA double strand breaks (DSB) are the most deleterious lesions for the integrity of the genome, as their misrepair can lead to the formation of chromosome translocations. Cells have evolved two main repair pathways to suppress the formation of these genotoxic lesions: homology-dependent, error-free homologous recombination repair (HRR), and potentially error-prone, classical, DNA-PK-dependent non-homologous end-joining (c-NHEJ). The most salient feature of c-NHEJ, speed, will largely suppress chromosome translocation formation, while sequence alterations at the junction remain possible. It is now widely accepted that when c-NHEJ is inactivated, globally or locally, an alternative form of end-joining (alt-EJ) removes DSBs. Alt-EJ operates with speed and fidelity markedly lower than c-NHEJ, causing thus with higher probability chromosome translocations, and generating more extensive sequence alterations at the junction. Our working hypothesis is that alt-EJ operates as a backup to c-NHEJ. Recent results show that alt-EJ can also backup abrogated HRR in G2 phase cells, again at the cost of elevated formation of chromosome translocations. These observations raise alt-EJ to a global rescuing mechanism operating on ends that have lost their chromatin context in ways that compromise processing by HRR or c-NHEJ. While responsible for eliminating from the genome highly cytotoxic DNA ends, alt-EJ provides this function at the price of increased translocation formation. Here, we analyze recent literature on the mechanisms of chromosome translocation formation and propose a functional hierarchy among DSB processing pathways that makes alt-EJ the global backup pathway. We discuss possible ramifications of this model in cellular DSB management and pathway choice, and analyze its implications in radiation carcinogenesis and the design of novel therapeutic approaches., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The timely determination of the fact of radiation impact on the organism is extremely important for preventive and curative interventions. Despite the fact that so far cytogenetic violations are considered to be the best biomarkers to determine the impact of ionizing radiation on the organism, actual problem is to find the optimal combination of different biomarkers. The aim of the work was investigation of the extended set of biomarkers in distant periods of exposure in people previously assigned to the radiation risk group, as well as the identification of genetic disorders in the process of radiotherapy. The object of the study were 37 residents of districts, where at the beginning of this century radioactive sources were discovered, and 6 oncology patients in the course of radiotherapy. Chromosome disorders, the overall level of DNA cells single-stranded damage by comet-assay method and a method of level detection of buccal micronuclei in were investigated. The results showed heterogeneity of different organism response to irradiation. Determination of absorbed dose, identification of various genetic disorders in individuals exposed to identical doses of radiation, offers the opportunity to judge the individual biological effect and is very important for individual preventive activities.

This review describes my personal reflections on the development of whole chromosome painting using fluorescence in situ hybridization and how my laboratory applied the technology in humans and in animal models. The trials and triumphs of the early years are emphasized, along with some of the scientific surprises that were encountered along the way. Scientific issues that my laboratory addressed using chromosome painting technologies are summarized and related to questions in radiation dosimetry, chemical clastogenesis, translocation persistence, and translocation frequencies in unexposed people. A description is provided of scientific controversies that were encountered and how they were resolved. I hope this paper will encourage young scientists to follow their passions and pursue their scientific dreams even if the task seems daunting and the circumstances appear exceedingly difficult. In my case the journey has been challenging, exciting, and richly rewarding on many levels., (Copyright © 2014 Elsevier B.V. All rights reserved.)

High-energy protons found in the space environment can induce mutations and cancer, which are inextricably linked. We hypothesized that some mutants isolated from proton-exposed kidneys arose through a genome-wide incident that causes loss of heterozygosity (LOH)-generating mutations on multiple chromosomes (termed here genomic LOH). To test this hypothesis, we examined 11 pairs of nonselected chromosomes for LOH events in mutant cells isolated from the kidneys of mice exposed to 4 or 5 Gy of 1 GeV protons. The mutant kidney cells were selected for loss of expression of the chromosome 8-encoded Aprt gene. Genomic LOH events were also assessed in Aprt mutants isolated from isogenic cultured kidney epithelial cells exposed to 5 Gy of protons in vitro. Control groups were spontaneous Aprt mutants and clones isolated without selection from the proton-exposed kidneys or cultures. The in vivo results showed significant increases in genomic LOH events in the Aprt mutants from proton-exposed kidneys when compared with spontaneous Aprt mutants and when compared with nonmutant (i.e., nonselected) clones from the proton-exposed kidneys. A bias for LOH events affecting chromosome 14 was observed in the proton-induced Aprt mutants, though LOH for this chromosome did not confer increased radiation resistance. Genomic LOH events were observed in Aprt mutants isolated from proton-exposed cultured kidney cells; however the incidence was fivefold lower than in Aprt mutants isolated from exposed intact kidneys, suggesting a more permissive environment in the intact organ and/or the evolution of kidney clones prior to their isolation from the tissue. We conclude that proton exposure creates a subset of viable cells with LOH events on multiple chromosomes, that these cells form and persist in vivo, and that they can be isolated from an intact tissue by selection for a mutation on a single chromosome.

Radiation sensitivity at low and high dose exposure to X-rays was investigated by means of chromosomal aberration (CA) analysis in heterozygous ATM mutation carrier and A-T patient (biallelic ATM mutation) lymphoblastoid cell lines (LCLs). Targeted and non-targeted responses to acutely delivered irradiation were examined by applying a co-culture system that enables study of both directly irradiated cells and medium-mediated bystander effects in the same experimental setting. No indication of radiation hypersensitivity was observed at doses of 0.01 Gy or 0.1 Gy for the ATM mutation carrier LCL. The A-T patient cells also did not show low-dose response. There was significant increase in unstable CA yields for both ATM mutation carrier and A-T LCLs at 1 and 2 Gy, the A-T cells displaying more distinct dose dependency. Both chromosome and chromatid type aberrations were induced at an increased rate in the irradiated A-T cells, whereas for ATM carrier cells, only unstable chromosomal aberrations were increased above the level observed in the wild type cell line. No bystander effect could be demonstrated in any of the cell lines or doses applied. Characteristics typical for the A-T cell line were detected, i.e., high baseline frequency of CA that increased with dose. In addition, dose-dependent loss of cell viability was observed. In conclusion, CA analysis did not demonstrate low-dose (≤100 mGy) radiosensitivity in ATM mutation carrier cells or A-T patient cells. However, both cell lines showed increased radiosensitivity at high dose exposure.

Purpose: Despite concerns over risks from exposure to low-dose ionizing radiations encountered in the environment and workplace, the molecular consequences of these exposures, particularly at representative doses and dose-rates, remains poorly understood., Materials and Methods: Using a novel flood source construct, we performed a direct comparison of genome-wide gene expression regulations resulting from exposure of primary human prostate fibroblast cultures to acute (10 cGy and 200 cGy) and longer-term chronic (1.0-2.45 cGy cumulative over 24 h) exposures., Results: Expression profiling showed significant differential regulation of 396 genes with no measureable changes in the acute 10 cGy dose. However, there were 106 genes in common between samples given an acute 200 cGy dose compared to those given chronic doses, most of which were decreased and related to cell cycle or chromosomal movement in M-phase. Biological pathway analysis showed decreases in cell cycle, chromosomal movement, cell survival and DNA replication, recombination and repair as well as a predicted activation of transcriptional regulators TP53, RB1 and CDKN2A. In agreement with these results, prostate epithelial cells given 200 cGy or chronic doses displayed functional decreases in proliferation and mitotic cells., Conclusions: In summary, we showed a contrast to the common observation of constant or reduced effect per unit dose as the dose (acute) was diminished, that even very low total doses delivered chronically could rival the perturbing effect of acute doses 100 times as intense. Underscored is the importance of the means of dose delivery, shown to be as important as dose size when considering biologic effect.

The presence of γH2AX foci on apparently intact mitotic chromosomes is controversial because they challenge the assumed relationship between γH2AX foci and DNA double-strand breaks (DSBs). In this work, we show that after irradiation during interphase, a variety of γH2AX foci are scored in mitotic cells. Surprisingly, approximately 80% of the γH2AX foci spread over apparently undamaged chromatin at Terminal or Interstitial positions and they can display variable sizes, thus being classified as Small, Medium and Big foci. Chromosome and chromatid breaks that reach mitosis are spotted with Big (60%) and Medium (30%) Terminal γH2AX foci, but very rarely are they signaled with Small γH2AX foci. To evaluate if Interstitial γH2AX foci might be signatures of misrejoining, an mFISH analysis was performed on the same slides. The results show that Interstitial γH2AX foci lying on apparently intact chromatin do not mark sites of misrejoining, and that misrejoined events were never signaled by a γH2AX foci during mitosis. Finally, when analyzing the presence of other DNA-damage response (DDR) factors we found that all γH2AX foci-regardless their coincidence with a visible break-always colocalized with MRE11, but not with 53BP1. This pattern suggests that these γH2AX foci may be hallmarks of both microscopically visible and invisible DNA damage, in which an active, although incomplete or halted DDR is taking place.

Purpose: In order to evaluate fluorescent in situ hybridization (FISH) as a method for predicting radiosensitivity, this study examined the incidence of translocations, after exposure to in vitro radiation, in both normally responding patients and those exhibiting severe late effects after radiotherapy treatment., Materials and Methods: Patients were selected from a randomized trial for intermediate-risk prostate cancer. Of the patients entered on trial with mature follow-up, 3% developed grade 3 late proctitis. Blood samples were taken from this radiosensitive cohort along with matched control patients with no late proctitis. Whole blood samples were exposed to 0 or 4 Gy and cultured according to the International Atomic Energy Agency (IAEA) recommended methods. Colour junctions were evaluated in the resulting metaphases and scored according to the Protocol for Aberration Identification and Nomenclature Terminology (PAINT) system., Results: Both groups were statistically similar at 0 Gy. After 4 Gy in vitro radiation, the radiosensitive group had significantly higher rates of chromosome damage in the number of colour junctions per cell (p = 0.002), the number of deletions per cell (p = 0.01) and the number of dicentrics per cell (p = 0.005)., Conclusions: These results indicate that the analysis of translocations using FISH after in vitro irradiation correlates with clinical response to radiation. This cytogenetic assay should be considered as a potential predictor of radiosensitivity.

Meiosis generates haploid cells or spores for sexual reproduction. As a prelude to haploidization, homologous chromosomes pair and recombine to undergo segregation during the first meiotic division. During the entire meiotic prophase of the yeast Saccharomyces cerevisiae, chromosomes perform rapid movements that are suspected to contribute to the regulation of recombination. Here, we investigated the impact of ionizing radiation (IR) on movements of GFP-tagged bivalents in live pachytene cells. We find that exposure of sporulating cultures with >40 Gy (4-krad) X-rays stalls pachytene chromosome movements. This identifies a previously undescribed acute radiation response in yeast meiosis, which contrasts with its reported radioresistance of up to 1,000 Gy in survival assays. A modified 3'-end labeling assay disclosed IR-induced dsDNA breaks (DSBs) in pachytene cells at a linear dose relationship of one IR-induced DSB per cell per 5 Gy. Dihydroethidium staining revealed formation of reactive oxygen species (ROS) in irradiated cells. Immobility of fuzzy-appearing irradiated bivalents was rescued by addition of radical scavengers. Hydrogen peroxide-induced ROS did reduce bivalent mobility similar to 40 Gy X IR, while they failed to induce DSBs. IR- and H2O2-induced ROS were found to decompose actin cables that are driving meiotic chromosome mobility, an effect that could be rescued by antioxidant treatment. Hence, it appears that the meiotic actin cytoskeleton is a radical-sensitive system that inhibits bivalent movements in response to IR- and oxidant-induced ROS. This may be important to prevent motility-driven unfavorable chromosome interactions when meiotic recombination has to proceed in genotoxic environments.

A chemicogenetic screen was performed in budding yeast mutants that have a weakened replication stress response. This identified an inhibitor of target of rapamycin (TOR) complexes 1 and 2 that selectively enhances the sensitivity of sgs1Δ cells to hydroxyurea and camptothecin. More importantly, the inhibitor has strong synthetic lethality in combination with either the break-inducing antibiotic Zeocin or ionizing radiation, independent of the strain background. Lethality correlates with a rapid fragmentation of chromosomes that occurs only when TORC2, but not TORC1, is repressed. Genetic inhibition of Tor2 kinase, or its downstream effector kinases Ypk1/Ypk2, conferred similar synergistic effects in the presence of Zeocin. Given that Ypk1/Ypk2 controls the actin cytoskeleton, we tested the effects of actin modulators latrunculin A and jasplakinolide. These phenocopy TORC2 inhibition on Zeocin, although modulation of calcineurin-sensitive transcription does not. These results implicate TORC2-mediated actin filament regulation in the survival of low levels of DNA damage., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Analysis of premature chromosome condensation (PCC) mediated by fusion of G0-lymphocytes with mitotic CHO cells in combination with rapid visualization and quantification of rings (PCC-Rf) is proposed as an alternative technique for dose assessment of radiation-exposed individuals. Isolated lymphocytes or whole blood from six individuals were γ-irradiated with 5, 10, 15 and 20Gy at a dose rate of 0.5Gy/min. Following either 8- or 24-h post-exposure incubation of irradiated samples at 37°C, chromosome spreads were prepared by standard PCC cytogenetic procedures. The protocol for PCC fusion proved to be effective at doses as high as 20Gy, enabling the analysis of ring chromosomes and excess PCC fragments. The ring frequencies remained constant during the 8-24-h repair time; the pooled dose relationship between ring frequency (Y) and dose (D) was linear: Y=(0.088±0.005)×D. During the repair time, excess fragments decreased from 0.91 to 0.59 chromatid pieces per Gy, revealing the importance of information about the exact time of exposure for dose assessment on the basis of fragments. Compared with other cytogenetic assays to estimate radiation dose, the PCC-Rf method has the following benefits: a 48-h culture time is not required, allowing a much faster assessment of dose in comparison with conventional scoring of dicentrics and rings in assays for chemically-induced premature chromosome condensation (PCC-Rch), and it allows the analysis of heavily irradiated lymphocytes that are delayed or never reach mitosis, thus avoiding the problem of saturation at high doses. In conclusion, the use of the PCC fusion assay in conjunction with scoring of rings in G0-lymphocytes offers a suitable alternative for fast dose estimation following accidental exposure to high radiation doses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Assessment of radiation doses through measurement of dicentric chromosomes may be difficult due to the inability of damaged cells to reach mitosis. After high-dose exposures, premature chromosome condensation (PCC) has become an important method in biodosimetry. PCC can be induced upon fusion with mitotic cells, or by treatment with chemicals such as calyculin A or okadaic acid. Several different cytogenetic endpoints have been measured with chemically induced PCC, e.g., via scoring of extra chromosome pieces or ring chromosomes. The dose-effect curves published with chemically induced PCC show differences in their coefficients and in the distribution of rings among cells. Here we present a study with calyculin A to induce PCC in peripheral blood lymphocytes irradiated at nine different doses of γ-rays up to 20Gy. Colcemid was also added in order to observe metaphase cells. During microscopical analysis the chromosome aberrations observed in the different cell-cycle phases (G2/M-PCC, M/A-PCC and M cells) were recorded. The proportion of G2/M-PCC cells was predominant from 3 to 20Gy, M cells decreased above 1Gy and M/A-PCC cells remained constant at all doses and showed the highest frequencies of PCC rings. Depending on the cell-cycle phase there was a difference in the linear coefficients in the dose-effect curves of extra fragments and rings. Poisson distribution among PCC rings was observed after calyculin A+colcemid treatment, facilitating the use of this methodology also for partial body exposures to high doses. This has been tested with two simulated partial exposures to 6 and 12Gy. The estimated doses in the irradiated fraction were very close to the real dose, indicating the usefulness of this methodology., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An atomic force microscope probe can be formed into an ultra-sharp cylindrical shape (a nanoneedle) using micro-fabrication techniques such as focused ion beam etching. This nanoneedle can be effectively inserted through the plasma membrane of a living cell to not only access the cytosol, but also to penetrate through the nuclear membrane. This technique shows great potential as a tool for performing intranuclear measurements and manipulations. Repeated insertions of a nanoneedle into a live cell were previously shown not to affect cell viability. However, the effect of nanoneedle insertion on the nucleus and nuclear components is still unknown. DNA is the most crucial component of the nucleus for proper cell function and may be physically damaged by a nanoneedle. To investigate the integrity of DNA following nanoneedle insertion, the occurrence of DNA double-strand breaks (DSBs) was assessed. The results showed that there was no chromosomal DNA damage due to nanoneedle insertion into the nucleus, as indicated by the expression level of γ-H2AX, a molecular marker of DSBs., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

UVB radiation suppresses photosynthesis and protein biosynthesis in plants, which in turn decreases growth and productivity. Here, an ultraviolet-B (UVB)-tolerant rice mutant, utr319 (UV Tolerant Rice 319), was isolated from a mutagenized population derived from 2500 M1 seeds (of the UVB-resistant cultivar 'Sasanishiki') that were exposed to carbon ions. The utr319 mutant was more tolerant to UVB than the wild type. Neither the levels of UVB-induced cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts], nor the repair of CPDs or (6-4) photoproducts, was altered in the utr319 mutant. Thus, the utr319 mutant may be impaired in the production of a previously unidentified factor that confers UVB tolerance. To identify the mutated region in the utr319 mutant, microarray-based comparative genomic hybridization analysis was performed. Two adjacent genes on chromosome 7, Os07g0264900 and Os07g0265100, were predicted to represent the mutant allele. Sequence analysis of the chromosome region in utr319 revealed a deletion of 45 419 bp. RNAi analysis indicated that Os07g0265100 is most likely the mutated gene. Database analysis indicated that the Os07g0265100 gene, UTR319, encodes a putative protein with unknown characteristics or function. In addition, the homologs of UTR319 are conserved only among land plants. Therefore, utr319 is a novel UVB-tolerant rice mutant and UTR319 may be crucial for the determination of UVB sensitivity in rice, although the function of UTR319 has not yet been determined.