Avoidance of the use of tryptophan in buried chromosomal proteins as a mechanism for reducing photo/oxidative damage to genomes.

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.

A 4-Gene Signature of CDKN1, FDXR, SESN1 and PCNA Radiation Biomarkers for Prediction of Patient Radiosensitivity.

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 from radiation-induced genome damage in Populus.

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.

Investigating the impact of long term exposure to chemical agents on the chromosomal radiosensitivity using human lymphoblastoid GM1899A cells.

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 (H2O2) 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 H2O2 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.

Robbing Peter to Pay Paul: Competition for Radiogenic Breaks During Rejoining Diminishes Curvature in the Dose Response for Simple Chromosome Exchanges.

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.

Feasibility Study on Automatic Interpretation of Radiation Dose Using Deep Learning Technique for Dicentric Chromosome Assay.

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.

Hi-C implementation of genome structure for in silico models of radiation-induced DNA damage.

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.

Biological Effects of Low-Dose Chest CT on Chromosomal DNA.

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.

The Sister Chromatid Exchange (SCE) Assay.

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.

Effects of culturing technique on human peripheral blood lymphocytes response to proton and X-ray radiation.

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.

An alternative approach for the induction of premature chromosome condensation in human peripheral blood lymphocytes using mitotic Akodon cells.

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.

Detection of Simple, Complex, and Clonal Chromosome Translocations Induced by Internal Radioiodine Exposure: A Cytogenetic Follow-Up Case Study after 25 Years.

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.

18F-FDG-induced DNA damage, chromosomal aberrations, and toxicity in V79 lung fibroblast cells.

18F-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 18F-FDG, at doses which might result from PET procedures. Cells were irradiated by 18F-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 18F-FDG exposure and the effects increased with dose.

RISK EVALUATION IN THE LOW-DOSE RANGE CT FOR RADIATION-EXPOSED CHILDREN, BASED ON DNA DAMAGE.

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.

Chromosome damage in human cells induced by UMTS mobile telephony radiation.

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.

Monte-Carlo dosimetry and real-time imaging of targeted irradiation consequences in 2-cell stage Caenorhabditis elegans embryo.

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.

Chromosomal radiosensitivity of triple negative breast cancer patients.

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.

G1 Premature Chromosome Condensation (PCC) Assay.

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.

G2 Chromosomal Radiosensitivity Assay for Testing Individual Radiation Sensitivity.

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.

A method for the cell-cycle-specific analysis of radiation-induced chromosome aberrations and breaks.

The classical G2-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 G2-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 G2-phase cells. This modification, however, increases the probability that at later time points not only cells irradiated in G2-phase, but also cells irradiated in S-phase will reach metaphase. However, the response of cells irradiated in G2-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 G2-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 G2-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 G2- 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 G2-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.