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Giemsa stained premature chromosome condensation s in human lymphocytes: (a) Demonstrating 46 single chromatid chromosomes in a nonirradiated lymphocyte; (b) 50 premature chromosome condensation fragments can be visualized in an irradiated lymphocyte a b
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Cytogenetic dosimetry plays an important role in the triage and medical management of affected people in radiological incidents/accidents. Cytogenetic biodosimetry uses different methods to estimate the absorbed dose in the exposed individuals, and each approach has its advantages and disadvantages. Premature chromosome condensation (PCC) assay pre...
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The cytokinesis-block micronucleus assay in peripheral blood lymphocytes is an established technique for biodosimetry. The aim of this project was to generate a X-ray induced micronuclei (MN) curve for peripheral blood lymphocytes taken from five healthy donors. The blood samples were irradiated with X-rays of 122 KeV at a dose rate of 0.652 Gy/min...
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... This method shows the G0 PCC technique's potential as a biodosimetry tool for estimating radiation doses early on by locating unstable chromosomal abnormalities such as fragmented and dicentric chromosomes. Furthermore, by identifying stable chromosomal exchange events like translocations in resting cells, it demonstrates its potency for retroactively monitoring individual health risks (Neronova, 2016). Premature chromosomal condensation (PCC) spread analysis and automated image collection were used to evaluate the efficacy of a quick biodosimetry technique. ...
... There are published reports from few laboratories about the dose response calibration curves for low LET radiation using PCC assay (Lamadrid et al., 2007;Balakrishnan et al., 2010;Lindholm et al., 2010;Puig et al., 2013;Neronova 2016). Table 5 shows the dose response coefficients of RC from different studies including the present study. ...
Quantification of chromosomal aberrations in the exposed personnel blood samples is considered as a ‘gold standard’ and sensitive biomarker in biological dosimetry. Despite technological developments, culture of cells for 48–52 hrs remains an unmet need in case of triage biodosimetry. Moreover, it is difficult to get sufficient number of metaphase spreads for scoring after high doses of exposures. The technique which causes condensation of chromatin before mitosis using biological or chemical agent is named as Premature Chromosome Condensation (PCC) assay. This assay is considered as an alternative to chromosome aberration assay, particularly at high acute doses of low and high LET radiation. To establish the PCC assay, blood samples were collected from healthy non-smoking individuals (n = 3) and exposed to various doses (0–20 Gy) of 6 MV X-rays at a dose rate of 5.6 Gy/min, using a high energy Linear accelerator (LINAC). Irradiated blood samples were subjected to Calyculin-A induced PCC. About 500 cells or more than 100 Ring Chromosomes (RC) were scored at each dose. Dicentric chromosomes (DC) and acentric fragments were also scored at each dose; the number of chromosomal aberrations in G1, M, G2/M and M/A phase of cell cycle were recorded and the frequency was used to construct the dose response curve. A dose dependent increase in RC and DC frequency were observed with a slope of 0.049 ± 0.002 and 0.30 ± 0.02 respectively. This study is first of its kind to construct a dose response curve for LINAC X-rays using a PCC assay.
... Despite the distinct advantages of G0-PCC over the conventional methods, studies using G0-PCC are highly restricted in the literature, as this technique requires a great deal of expertise and optimizations. Recently, there has been a rejuvenated interest and focus on G0-PCC to overcome challenges in biodosimetry (Bezrookove et al. 2003;Hatzi et al. 2006;Lamadrid Boada et al. 2013;Suto et al. 2013;Karachristou et al. 2015;Neronova 2016;Terzoudi et al. 2017;Ryan et al. 2019). A recent study demonstrated utility of the method in large scale accident with small volume of 100 ml of blood in 96 well plate (Pantelias and Terzoudi 2018). ...
... The dose response curve generated 24 h post irradiation allowing complete repair and offered convenience better than that of 8 h for performing experiments. Our results are in agreement with earlier reports by Neronova 2016). ...
... Background frequency of excess fragments was found to be 0.19 ± 0.10 per cell (seven donors, $50 cells each), the data is shown in the Table 1. These were in agreement with earlier reports (Pantelias and Maillie 1984;Lamadrid Boada et al. 2013;Karachristou et al. 2015;Neronova 2016). The background did not differ significantly among male and female donors. ...
Biodosimetry with persistent cytogenetic indicators in peripheral blood lymphocytes (PBLs) plays crucial role in regulatory/medical management of individuals overexposed to radiation. Conventional methods require ∼48h culture and have limited dose range (0.1-5Gy) applications due to checkpoint arrest/poor stimulation. G0-Phase Premature chromosome condensation (G0-PCC) allows chromosome aberration analysis within hours after blood collection. Due to high skill demand, applications of G0-PCC were not very well explored and being re-visited worldwide. Among all aberrations, analysis of excess chromosomal fragments is quickest. Radiation dose response curve for the fragments has been reported.
Purpose: In present study, excess fragment analysis has been addressed in detail, in addition to validation of radiation dose response curve, gender variation in the response, dose dependent repair kinetics, minimum detection limit (MDL), duration and accuracy of final dose estimation with 5blindfolded, ex-vivo irradiated samples have been studied. In extension, feasibility of multiparametric dosimetry with Fluorescent in situ hybridization (FISH) based endpoints were qualitatively explored.
Material and Methods: PBLs were exposed to Gamma-Radiation and G0-PCC was performed at different time points. Decay kinetics and dose response curve were established. Gender Variation of the frequency of the fragments were assessed at 0, 2 and 4Gy. FISH was performed with G0-PCC applying centromere probe, whole chromosome paints, multi-color FISH and multi-color banding probes.
Results: Radiation response curve for fragments was found to be linear (Slope 1.09 ± 0.031 Gy-1). Background frequency as well as dose response did not show significant gender bias. Based on variation in background frequency of fragments MDL was calculated to be ∼0.3Gy. Kinetics of fragment tested at 0, 4, 8, 16 & 24h showed exponential decay pattern from 0 to 8h and without further decay. Final dose estimation of 5 samples was completed within 13 man-hours. Dicentric chromosomes, translocations, insertions and breaks were identifiable in combination with centromere FISH and WCP. Advanced methods employing multicolor FISH and multi-color banding were also demonstrated with PCC spreads.
Conclusion: G0-PCC, can be useful tool for high dose biodosimetry with quick assessment of fragment frequency. Further, it holds potential for multi-parametric dosimetry in combination of FISH.
... Therefore, we decided to investigate the potential detrimental effects of the process of lymphocyte sample preparation on DNA damage and proliferation ability using the cytokinesis-block micronucleus (CBMN) assay and the premature chromosome condensation (PCC) test. These methods have been widely approved as biodosimetry tests to accurately detect the effect of exposure to low and high linear energy transfer (LET) ionizing radiation (Gotoh and Asakawa 1996;Rothkamm et al. 2013;Neronova 2016). These methods are now used for population monitoring, evaluating genetic damage, predicting the radiosensitivity of tumors and determining inter-individual variation in radiosensitivity (Gotoh et al. 1995;Gotoh 2015;Hayashi 2016). ...
... The CBMN and PCC assays are considered to be suitable and valuable methods for cell response studies for analysing the amount of DNA damage, cell cycle stage, and proliferation index after exposure to ionising radiation (Vian et al. 1993;Rothkamm et al. 2013;Gotoh 2015;Neronova 2016;Hayashi 2016). In human biomonitoring or radiobiological studies, the effects of culturing technique (whole blood or isolated HPBLs) are often omitted. ...
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.
... In our study, the fusion between mitotic CHO cells and human G 0 lymphocytes at a ratio of 1:5 with PEG resulted in a PCC index of about 3 %. A comparable index of 3.6 % was reported by Neronova [32], following 1:5 ratio for CHO-lymphocyte fusion. Scoring of PCC induced by cell fusion may be demanding [33] as the CHO: lymphocyte hybrids represent only a small fraction of the CHO metaphase spreads. ...
The inherent capacity of individuals to efficiently repair ionizing radiation induced DNA double strand breaks (DSBs) may be inherited, however, it is influenced by several epigenetic and environmental factors. A pilot study tested whether chronic low dose natural radiation exposure influences the rejoining of initial DNA DSBs induced by a 2 Gy γ-irradiation in 22 individuals from high (>1.5 mGy/year) and normal (≤1.5 mGy/year) level natural radiation areas (H&NLNRA) of Kerala. Rejoining of DSBs (during 1 h at 37 °C, immediately after irradiation) was evaluated at the chromosome level in the presence and absence of wortmannin (a potent inhibitor of DSB repair in normal human cells) using a cell fusion-induced premature chromosome condensation (PCC) assay. The PCC assay quantitates DSBs in the form of excess chromosome fragments in human G0 lymphocytes without the requirement for cell division. A quantitative difference was observed in the early rejoining of DNA DSBs between individuals from HLNRA and NLNRA, with HLNRA individuals showing a higher (P = 0.05) mean initial repair ratio. The results indicate an influence of chronic low dose natural radiation on initial DNA DSB repair in inhabitants of HLNRA of the Kerala coast.
... The book published by Academic Press in 1982 gives details on the applications of PCC for basic research, clinical research and in the areas of mutation research [7]. Recently, a calibration curve was reported for PCC for assessment of γ radiation exposure and a comparative analysis between dicentrics and PCC fragments was performed for validation of PCC for biodosimetric applications [8]. Similar dose-response curves have been constructed for emergency preparedness [9]. ...
Premature chromosome condensation (PCC) involves induction of near-chromosome-like morphology to interphase chromatin. Experimental induction of PCC was achieved by somatic cell hybridization (SCH), an approach which evolved into a chemical-induction process. PCC presents most probably the only way in which cytogenetic assessment of damages can be analyzed in special situations such as availability of limited numbers of sample cells and for cells which have lost their ability to divide. Initial experiments on PCC were reported in late 1960s and the technique has evolved into one with wide range of applications owing to its increased efficiency in detecting primary DNA damages. Biodosimetry remains as the primary area which utilizes PCC technique to the maximum efficiency with several multiple-groups participating in collaborative exercises for biodosimetric applications. However, in spite of the advantages that the technique offers, it is yet to reach its full potential. This is due to the inherent limitations of the manner in which PCC is induced currently; by the somatic cell hybridization and chemical-induction processes. An approach which combines these two would sure help in taking PCC to its highest potential as the preferred technique for assessment of primary DNA damages. We present the chronological events of evolution of the PCC technique along with its applications. Also, the limitations of the technique along with the suggestions for further refinement of the PCC technique are discussed.
... Furthermore, it has been documented experimentally that PCC also has the potential to discriminate accurately between total-and partial-body exposures. [63,64] The fluorescence in situ hybridization chromosome painting has been used to measure the frequency of stable translocations in peripheral lymphocytes of Mayak nuclear-industrial workers. [65] The frequency of such "stable chromosomal translocations" is thought to reflect the degree of radiation genetic injury; in turn, the relative injurious radiation dose received. ...
The possible detonation of a radiological dispersal device or improvised nuclear device in a metropolitan city, or the accidental exposures to a radiation source, nuclear accidents, or the all-to-often threats of radiological/nuclear terrorism have led to the urgent need to develop essential analytic tools to assess such radiation exposures, especially radiation doses to exposed individuals. This exposure-assessing work using biological samples, and discipline, is known as biodosimetry. As of late, this field has progressed significantly as it has made use of the advances within newer areas of biologic analytics, namely omics (genomics, proteomics, metabolomics, and transcriptomics), lymphocyte kinetics, optically stimulated luminescence, and electron paramagnetic resonance technology in addition to conventional cytogenetic techniques. The use of automated high throughput platforms and the planning for laboratory surge capacity during the time of need are the latest developments in the field of biomarkers for biodosimetry. Such biomarkers are also needed for radiation exposure/dose conversion estimates that are essential for the development and application of radiation countermeasures, from animals to humans and that are currently being developed following the US Food and Drug Administration Animal Rule. Here, we present and discuss the current status of various biomarkers for assessing radiation dose after radiation exposure. It is anticipated that with the advent of improved biomarkers and associated biomarker platforms for the acute radiation syndrome, exposed victims can be more efficiently triaged and appropriately treated than is currently allowable. The latest advances in the field, and identify the areas where improvement is needed are also listed and discussed.
Mitotic cell fusion induced Premature Chromosome Condensation (G0-PCC) assay in human lymphocytes allows rapid detection of cytogenetic damage in interphase stage, within few hours after blood collection. Hence, it is the most suitable method for rapid and high dose biodosimetry. Mitotic cells, used for G0-PCC could be either freshly isolated or previously cryo-preserved. However, under emergency scenarios, only cryo-preserved cells can be relied upon, fresh isolation will only delay the process by 18–24 h. Impact of cryopreservation on mitotic cells and their efficacy to induce PCC are not reported. In the present study, we investigated effect of cryopreservation on mitotic cells and refined the parameters for G0-PCC. More than 95% of the cells were recoverable after 4 months of cryopreservation, within 20 min recovery at 37 °C, without significant change in the mitotic index or viability. Recovered mitotic cells have shown mitotic index of 89 ± 4% and viability of 90 ± 4%, similar to that of freshly isolated cells. Decrease in metaphases was observed within 40 min after recovery as the mitotic cells progressed through cell cycle and reduced to 21% at 1.5 h. Nevertheless, in presence of Colcemid, the cells progressed slowly and considerably high metaphase index (60%) persisted up to ~ 2 h. The recovered cells efficiently fused with lymphocytes and induced PCC. Average PCC index varied from 10 to 20%, which did not change with cryopreservation duration. Post fusion incubation duration of 2 h was found to be optimum for proper chromosome condensation. In conclusion, use of cryo-preserved mitotic cells is the most practical approach for rapid biodosimetry. The cells can be recovered quickly and efficiently without alteration in viability or mitotic index. Recovered cells are fully competent to induce G0-PCC.
Ionizing radiation is ubiquitous in the environment. Its source can be natural, such as radioactive materials present in soil and cosmic rays, or artificial, such as the fuel for nuclear power plants. Overexposure to ionizing radiation may damage living tissue and could cause severe health problems (i.e., mutations, radiation sickness, cancer, and death). Cytogenetic bio-dosimetry has the great advantage to take into account the inter-individual variation, and it is informative even when physical dosimetry is not applicable; moreover, it is the definitive method to assess exposure to ionizing radiation recommended by the World Health Organization (WHO). Such a procedure involves counting the frequency of dicentric chromosomes (DCs), which are the most studied chromosomal aberrations used as absorbed radiation biomarkers, during the metaphase of cells. A set of algorithms, tested on different programming languages to automatically identify DCs, is analyzed by the authors together with an Automated Dicentric Chromosome Identifying software (ADCI) mostly based on OpenCV programming libraries. The purpose of this work is to review the main results regarding the correlation between ionizing radiation and dicentric chromosomes in cytogenetic bio-dosimetry.
