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1.  Is FISH painting an appropriate biological marker for dose estimates of suspected accidental radiation overexposure? A review of cases investigated in France from 1995 to 1996. 
Environmental Health Perspectives  1997;105(Suppl 6):1427-1432.
From 1995 to 1996 about 15 people suspected of being overexposed to ionizing radiation were referred to the Institute for Nuclear Safety and Protection in Fontenay-aux-Roses, France, for investigation by chromosome aberration analysis. Biological estimates of accidental overexposure were first obtained by scoring radio-induced unstable structural chromosome aberrations (dicentrics, centric rings, and fragments) in peripheral blood lymphocytes. For dose estimates, the yield of these chromosomal aberrations observed in 500 metaphases was compared with the laboratory dose-response relationship established from human blood irradiated in vitro (gamma-rays, 60Co, 0.5 Gy/min). To extend the possibilities of detecting DNA damage from earlier exposures by visualizing stable chromosome aberrations, chromosome painting by fluorescence in situ hybridization (FISH painting) was developed using a cocktail of three composite whole human chromosome-specific DNA probes (numbers 2, 4, and 12). A laboratory calibration curve for scoring terminal and/or reciprocal translocations was established for the same radiation quality and dose rate as those used for conventional cytogenetics (gamma-rays, 60Co, 0.5 Gy/min). For dosimetry purposes, it was also important to verify whether FISH painting could be applied to each human blood sample assessed for conventional expertise. For each individual, 2000 metaphases were scored for the presence or absence of reciprocal and terminal translocations. We present here a comparison between the results obtained by the two technologies for each of the cases studied separately. We describe their similarities or differences and discuss the suitability of using FISH painting for routine expertise analysis.
Images
PMCID: PMC1469948  PMID: 9467056
2.  Biological Dosimetry by the Triage Dicentric Chromosome Assay – Further validation of International Networking 
Radiation measurements  2011;46(9):923-928.
Biological dosimetry is an essential tool for estimating radiation doses received to personnel when physical dosimetry is not available or inadequate. The current preferred biodosimetry method is based on the measurement of radiation-specific dicentric chromosomes in exposed individuals' peripheral blood lymphocytes. However, this method is labour-, time- and expertise-demanding. Consequently, for mass casualty applications, strategies have been developed to increase its throughput. One such strategy is to develop validated cytogenetic biodosimetry laboratory networks, both national and international. In a previous study, the dicentric chromosome assay (DCA) was validated in our cytogenetic biodosimetry network involving five geographically dispersed laboratories. A complementary strategy to further enhance the throughput of the DCA among inter-laboratory networks is to use a triage DCA where dose assessments are made by truncating the labour-demanding and time-consuming metaphase-spread analysis to 20 to 50 metaphase spreads instead of routine 500 to 1000 metaphase spread analysis. Our laboratory network also validated this triage DCA, however, these dose estimates were made using calibration curves generated in each laboratory from the blood samples irradiated in a single laboratory. In an emergency situation, dose estimates made using pre-existing calibration curves which may vary according to radiation type and dose rate and therefore influence the assessed dose. Here, we analyze the effect of using a pre-existing calibration curve on assessed dose among our network laboratories. The dose estimates were made by analyzing 1000 metaphase spreads as well as triage quality scoring and compared to actual physical doses applied to the samples for validation. The dose estimates in the laboratory partners were in good agreement with the applied physical doses and determined to be adequate for guidance in the treatment of acute radiation syndrome.
doi:10.1016/j.radmeas.2011.03.012
PMCID: PMC3176593  PMID: 21949482
3.  mBAND analysis of early and late damages in the chromosome of human lymphocytes after exposures to gamma rays and Fe ions 
Journal of Radiation Research  2014;55(Suppl 1):i87-i88.
Stable-type chromosome aberrations that survive multiple generations of cell division include translocation and inversions. An efficient method to detect an inversion is multi-color banding fluorescent in situ hybridization (mBAND) which allows identification of both inter- and intra-chromosome aberrations simultaneously. Post irradiation, chromosome aberrations may also arise after multiple cell divisions as a result of genomic instability. This study was aimed at investigating stable or late-arising chromosome aberrations in human lymphocytes induced after low- and high-LET radiation exposure.
Human lymphocytes were exposed in vitro to gamma ray doses of 2 or 4 Gy using a 137Cs source at a dose rate of 0.5 Gy/min. For high LET radiation, cells were exposed to Fe ions (600 MeV/nucleon) of 0.05, 0.5 or 1 Gy at NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). Chromosomes were collected at 48 h which represented the first mitosis, and 7 and 14 days after irradiation using a premature chromosome condensation (PCC) technique as described previously [ 1]. Chromosome 3 was painted with the XCyte3 mBAND kit (MetaSystems), and intra- and inter-chromosomal aberrations were analyzed with the mBAND analysis system (MetaSystems).
With gamma irradiation, about half of the damages observed at first mitosis remained after 7- and 14-day culture, suggesting transmissibility of damages to the surviving progeny. With Fe ions irradiation, at the doses that produced similar frequencies of gamma-induced chromosome aberrations as observed at first mitosis, a significantly lower yield of aberrations remained at the same population doublings after Fe ion exposure. At these equitoxic doses, more complex-type aberrations were observed for Fe ions, indicating that Fe ion-induced initial chromosome damages are more severe and may lead to cell death.
Comparison of the number of breaks indicates that Fe ions produced three or more breaks in ∼20% of the damaged chromosome 3, whereas ∼10% or a less fraction of cells with three or more breaks in chromosome 3 were found after gamma ray exposures. Unlike gamma rays, increasing doses of Fe ions did not produce more breaks in a damaged chromosome 3, indicating that damages from Fe ions exposures were mostly due to the single track effect, in agreement with the results of Hada et al. [ 1, 2].
Increasing doses of exposure resulted in less fraction of chromosome break ends involved in intra-chromosomal exchange induced by either gamma rays or Fe ions in the first mitosis.
Interestingly, simple inversions in chromosome 3 were found in only the 7 and 14-day samples after 4 Gy gamma irradiation. It is not clear whether cells containing simple inversions had already progressed through the first cell division at 48 h, or the simple inversions were induced after the first cell division.
Detailed analysis of breaks participating in total chromosome exchanges within the first cell cycle post-irradiation revealed a common hot spot located in the 3p21 region, which is a known fragile site corresponding to Band 6 in the mBand analysis [ 2]. The breakpoint distribution in chromosomes collected at 7 days, but not at 14 days, post-irradiation appeared similar to the distribution in cells collected within the first cell cycle post-irradiation. The breakpoint distribution for human lymphocytes after radiation exposure was different from the previously published distribution for human mammary epithelial cells [ 2], indicating that interphase chromatin folding structures play a role in the distribution of radiation-induced breaks.
doi:10.1093/jrr/rrt182
PMCID: PMC3941537
heavy ion; genomic instability; chromosome aberrations; mBAND
4.  Sample Tracking in an Automated Cytogenetic Biodosimetry Laboratory for Radiation Mass Casualties 
Radiation measurements  2007;42(6-7):1119-1124.
Chromosome aberration-based dicentric assay is expected to be used after mass casualty life-threatening radiation exposures to assess radiation dose to individuals. This will require processing of a large number of samples for individual dose assessment and clinical triage to aid treatment decisions. We have established an automated, high-throughput, cytogenetic biodosimetry laboratory to process a large number of samples for conducting the dicentric assay using peripheral blood from exposed individuals according to internationally accepted laboratory protocols (i.e., within days following radiation exposures). The components of an automated cytogenetic biodosimetry laboratory include blood collection kits for sample shipment, a cell viability analyzer, a robotic liquid handler, an automated metaphase harvester, a metaphase spreader, high-throughput slide stainer and coverslipper, a high-throughput metaphase finder, multiple satellite chromosome-aberration analysis systems, and a computerized sample tracking system. Laboratory automation using commercially available, off-the-shelf technologies, customized technology integration, and implementation of a laboratory information management system (LIMS) for cytogenetic analysis will significantly increase throughput.
This paper focuses on our efforts to eliminate data transcription errors, increase efficiency, and maintain samples’ positive chain-of-custody by sample tracking during sample processing and data analysis. This sample tracking system represents a “beta” version, which can be modeled elsewhere in a cytogenetic biodosimetry laboratory, and includes a customized LIMS with a central server, personal computer workstations, barcode printers, fixed station and wireless hand-held devices to scan barcodes at various critical steps, and data transmission over a private intra-laboratory computer network. Our studies will improve diagnostic biodosimetry response, aid confirmation of clinical triage, and medical management of radiation exposed individuals.
doi:10.1016/j.radmeas.2007.05.021
PMCID: PMC2084199  PMID: 18037985
5.  TRIAGE DOSE ASSESSMENT FOR PARTIAL-BODY EXPOSURE: DICENTRIC ANALYSIS 
Health physics  2010;98(2):244-251.
Partial-body biodosimetry is likely to be required after a radiological or nuclear exposure. Clinical signs and symptoms, distribution of dicentrics in circulating blood cells, organ-specific biomarkers, physical signals in teeth and nails all can provide indications of non-homogeneous exposures. Organ specific biomarkers may provide early warning regarding physiological systems at risk after radiation injury. Use of a combination of markers and symptoms will be needed for clinical insights for therapeutic approaches. Analysis of dicentrics, a marker specific for radiation injury, is the “Gold standard” of biodosimetry and can reveal partial-body exposures. Automation of sample processing for dicentric analysis can increase throughput with customization of off-the-shelf technologies for cytogenetic sample processing and information management. Automated analysis of the metaphase spreads is currently limited but improvements are in development. Our efforts bridge the technological gaps to allow the use of dicentric chromosome assay (DCA) for risk-based stratification of mass casualties. This article summarizes current knowledge on partial-body cytogenetic dose assessment synthesizing information leading to the proposal of an approach to triage dose prediction in radiation mass casualties, based on equivalent whole-body doses under partial-body exposure conditions and assesses the validity of using this model. An initial screening using only 20 metaphase spreads per subject can confirm irradiation above 2-Gy. A subsequent increase to 50 metaphases improves dose determination to allow risk stratification for clinical triage. Metaphases evaluated for inhomogeneous distribution of dicentrics can reveal partial-body exposures. We tested the validity of this approach in an in vitro model that simulates partial-body irradiation by mixing irradiated and un-irradiated lymphocytes in various proportions. Our preliminary results support the notion that this approach will be effective under a range of conditions including some partial-body exposures, but may have limitations with low doses or small proportions of irradiated body. Our studies address an important problem in the diagnosis of partial-body irradiation and dose assessment in mass casualties and propose a solution. However, additional work is needed to fully develop and validate the application of DCA to partial-body exposures.
doi:10.1097/01.HP.0000348020.14969.4
PMCID: PMC2806648  PMID: 20065689
Partial-body biodosimetry; cytogenetics; triage; dicentric analysis
6.  Spectral Imaging in Preconception/Preimplantation Genetic Diagnosis of Aneuploidy: Multicolor, Multichromosome Screening of Single Cells 
Purpose:Our purpose was to evaluate the utility of spectral imaging for multicolor, multichromosome enumeration in human interphase cell nuclei.
Methods:Chromosome-specific probes labeled with different fluorochromes or nonfluorescent haptens were obtained commercially or prepared in-house. Metaphase spreads, interphase lymphocytes, or blastomeres cells were hybridized with either 7 or 11 distinctly different probes. Following 46 hr of hybridization, slides were washed and detected using either a filter-based quantitative image processing system (QUIPS) developed in-house or a commercial spectral imaging system.
Results:The filter-based fluorescence microscope system is preferred for simultaneous detection of up to seven chromosome targets because of its high sensitivity and speed. However, this approach may not be applicable to interphase cells when 11 or more targets need to be discriminated. Interferometer-based spectral imaging with a spectral resolution of approximately 10 nm allows labeling of chromosome-specific DNA probes with fluorochromes having greatly overlapping emission spectra. This leads to increases in the number of fluorochromes or fluorochrome combinations available to score unambiguously chromosomes in interphase nuclei.
Conclusions:Spectral imaging provides a significant improvement over conventional filter-based microscope systems for enumeration of multiple chromosomes in interphase nuclei, although further technical development is necessary in its application to embryonic blastomeres. When applied to preconception/preimplantation genetic diagnosis, presently available probes for spectral imaging are expected to detect abnormalities responsible for 70–80% of spontaneous abortions caused by chromosomal trisomies.
doi:10.1023/A:1022508930762
PMCID: PMC3454763  PMID: 9604769
aneuploidy; diagnosis; interphase cells; preconception; preimplantation genetic diagnosis; fluorescence in situ hybridization; spectral imaging
7.  Current cytogenetic methods for detecting exposure and effects of mutagens and carcinogens. 
Environmental Health Perspectives  1996;104(Suppl 3):445-448.
Most mutagens and genotoxic carcinogens are efficient inducers of chromosomal alterations in exposed cells. Two important classes of aberrations, namely structural and numerical, are recognized and both types of aberrations are associated with congenital abnormalities and neoplasia in humans. These alterations can be easily detected and quantified in human peripheral blood lymphocytes. Conventional staining techniques can be used to detect these aberrations; this technique was used to estimate absorbed dose in the case of a radiation accident in Goiania, Brazil. A recently introduced fluorescent in situ hybridization technique (FISH) using DNA probes has increased the sensitivity and ease of detecting chromosome aberrations, especially stable chromosome aberrations. This technique allows, to some extent, the estimation of absorbed radiation dose from past exposures. Numerical aberrations can be directly estimated in metaphases by counting the number of FISH-painted chromosomes. Micronuclei are formed by lagging chromosome fragments or whole chromosomes during the anaphase stage of cell division. The nature of micronuclei as to whether they possess a centromere can be determined either by CREST staining (calcinosis, Raynoud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) or FISH with centromere-specific DNA probes. In several carcinogen-exposed populations, such as heavy smokers or people exposed to arsenic, aneuploidy appears to be more common than structural aberrations. In victims of radiation accidents, aneuploidy (hyperploidy) has been found to be common in addition to structural aberrations.
PMCID: PMC1469636  PMID: 8781361
8.  Chromosomal aberrations in human lymphocytes and fibroblasts after exposure to very low doses of high-LET radiation 
Journal of Radiation Research  2014;55(Suppl 1):i50-i51.
Purpose: The relationship between biological effects and low doses of radiation is still uncertain, especially for high-LET radiation exposures. Estimates of risk from exposure to low doses and low dose rates are often extrapolated from the Japanese atomic bomb survivor data using either linear or linear-quadratic models fitted to dose–response data. In this study, we determined the dose–response for chromosome damage after exposure to very low doses of high-LET radiation and assessed the radiation qualities of Fe, Si and Oxygen ions.
Materials and methods: Chromosomal aberrations (CA) were measured in human peripheral blood lymphocytes and normal skin fibroblasts after exposure to very low doses (0.01–0.20 Gy) of 77-MeV/u oxygen (LET = 55 keV/µm), 170-MeV/u 28Si (LET = 99 keV/µm), or 56Fe ions with energies of 600- or 450-MeV/u (LET = 180 or 195 keV/µm). These exposures included doses that, on average, produce fewer than one in five direct ion traversals per cell nucleus. Chromosomes were analyzed using the whole-chromosome fluorescence in situ hybridization (FISH) technique during the first cell division after irradiation, and CA were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving more than two breaks in two or more chromosomes). The frequencies of CA in the painted chromosome(s) were evaluated as the ratio between aberrations scored and total cells analyzed. The dose–response for simple exchanges was assessed using a generalized linear model assuming binomial errors per number of chromosomes scored. The model coefficients were extrapolated to whole-genome equivalents. The linear dose–response denoted as the targete effects (TE) model considered the mean number of radiation tracks per cell. Two different non-targeted effect (NTE) models, P = P0 + αT + κ × I (NTE1), and P = P0 + αT (1 − e−T) + κe−T × I (NTE2), were compared with the simple linear model, P = P0 + αT. Akaike information criteria (AIC) and Bayes information criteria (BIC) were used to compare TE and NTE models for fitting chromosome aberrations in low dose range.
Results: Doses that on average produce more than one ion traversal per cell nucleus showed a linear dose–response for CA in both lymphocytes and fibroblasts. However, for doses that produce fewer than one tracks per cell in fibroblasts, O, Si and Fe particles showed a dose-independent response for CA that was significantly elevated relative to background frequencies. For fibroblasts the NTE model 2, P = P0 + αT (1 − e−T) + κe−T × I, showed improved fit to CA in low dose range compared with TE model or NTE1 model. For lymphocytes, tests of the various models were less clear with TE model optimal for Si and Fe while the NTE2 model optimal for O particles. When low-dose exposures were fractionated with 2-h intervals, increased frequencies of both simple and complex exchanges were observed. Nitric oxide scavenger reduced CA induced by low doses of high-LET irradiation. Inhibition of transforming growth factor-β receptor-1 reduced the frequency of simple exchanges.
Conclusions: The results show a non-linear dose–response for CA in fibroblasts after very low doses of high-LET exposure. Possible explanations for this could involve non-targeted effects due to aberrant cell signaling [ 1], perhaps involving nitric oxide and TGF-β, or could be due to delta-ray dose fluctuations [ 2] where CA are induced in cells that receive a significant dose from delta-rays emanating from the multiple ion tracks that do not directly traverse cell nuclei.
doi:10.1093/jrr/rrt212
PMCID: PMC3941494
low dose; heavy ion; chromosome aberrations
9.  Rapid and reliable diagnosis of murine myeloid leukemia (ML) by FISH of peripheral blood smear using probe of PU. 1, a candidate ML tumor suppressor 
Background
Murine myeloid leukemia (ML) provides a good animal model to study the mechanisms of radiation-induced leukemia in humans. This disease has been cytogenetically characterized by a partial deletion of chromosome 2 with G-banding. For the rapid diagnosis of ML, this study reports a FISH method using spleen cells and peripheral blood smears from ML mice exposed to gamma rays and neutrons with PU.1, a candidate ML tumor suppressor, as a probe.
Results
Among mice that were tentatively diagnosed with ML by clinical findings and blood smear examination, 85% carried spleen cells showing the loss of PU.1 although the frequency of these abnormal cells varied among individuals. Mice with very low frequencies of cells showing the loss of one copy of PU.1 (one-PU.1 frequency) were later diagnosed pathologically not with ML but with blastic or eosinophilic leukemia. Some neutron-irradiated mice had cells showing translocated PU.1, although no pathological features differentiated these ML mice from ML mice expressing the simple loss of PU.1.
The one-PU.1 frequency can be detected from spleen metaphase cells, spleen interphase cells, and blood smears. There was a good correlation between the one-PU.1 frequency in spleen metaphase cells and that in spleen interphase cells (r = 0.96) and between one-PU.1 frequency in spleen interphase cells and that in blood cells (r = 0.83).
Conclusion
The FISH method was capable of detecting aberration of copy number of the PU.1 gene on murine chromosome 2, and using a peripheral blood smear is more practical and less invasive than conventional pathological diagnosis or the cytogenetic examination of spleen cells.
doi:10.1186/1755-8166-1-22
PMCID: PMC2572613  PMID: 18922187
10.  Chromosomal assignment of canine THADA gene to CFA 10q25 
Background
Chromosomal translocations affecting the chromosome 2p21 cluster in a 450 kb breakpoint region are frequently observed in human benign thyroid adenomas. THADA (thyroid adenoma associated) was identified as the affected gene within this breakpoint region. In contrast to man tumours of the thyroid gland of dogs (Canis lupus familiaris) constitute mainly as follicular cell carcinomas, with malignant thyroid tumours being more frequent than benign thyroid adenomas. In order to elucidate if the THADA gene is also a target of chromosomal rearrangements in thyroid adenomas of the dog we have physically mapped the canine THADA gene to canine chromosome 10.
A PCR was established to screen a canine genome library for a BAC clone containing the gene sequence of canine THADA. Further PCR reactions were done using the identified BAC clone as a template in order to verify the corresponding PCR product by sequencing.
Canine whole blood was incubated with colcemid in order to arrest the cultured cells in metaphases. The verified BAC DNA was digoxigenin labeled and used as a probe in fluorescence in situ hybridization (FISH). Ten well spread metaphases were examined indicating a signal on canine chromosome 10 on both chromatids. A detailed fine mapping was performed indicating the canine THADA gene locus on the q-arm of chromosome 10.
Results
The canine THADA gene locus was mapped on chromosome 10q25. Our mapping results obtained in this study following the previously described nomenclature for the canine karyotype.
Conclusion
We analysed whether the THADA gene locus is a hotspot of canine chromosomal rearrangements in canine neoplastic lesions of the thyroid and in addition might play a role as a candidate gene for a possible malignant transformation of canine thyroid adenomas. Although the available cytogenetic data of canine thyroid adenomas are still insufficient the chromosomal region to which the canine THADA has been mapped seems to be no hotspot of chromosomal aberrations seen in canine thyroid adenomas.
doi:10.1186/1755-8166-1-11
PMCID: PMC2430699  PMID: 18522714
11.  No Evidence for the In Vivo Induction of Genomic Instability by Low Doses of 137CS Gamma Rays in Bone Marrow Cells of BALB/CJ and C57BL/6J Mice 
Dose-Response  2011;10(1):11-36.
In spite of extensive research, assessment of potential health risks associated with exposure to low-dose (≤ 0.1 Gy) radiation is still challenging. We evaluated the in vivo induction of genomic instability, expressed as late-occurring chromosome aberrations, in bone-marrow cells of two strains of mouse with different genetic background, i.e. the radiosensitive BALB/cJ and the radioresistant C57BL/6J strains following a whole-body exposure to varying doses of 137Cs gamma rays (0, 0.05, 0.1, and 1.0 Gy). A total of five mice per dose per strain were sacrificed at various times post-irradiation up to 6 months for sample collections. Three-color fluorescence in situ hybridization for mouse chromosomes 1, 2, and 3 was used for the analysis of stable-aberrations in metaphase-cells. All other visible gross structural-abnormalities involving non-painted-chromosomes were also evaluated on the same metaphase-cells used for scoring the stable-aberrations of painted-chromosomes. Our new data demonstrated in bone-marrow cells from both strains that low doses of low LET-radiation (as low as 0.05 Gy) are incapable of inducing genomic instability but are capable of reducing specific aberration-types below the spontaneous rate with time post-irradiation. However, the results showed the induction of genomic instability by 1.0 Gy of 137Cs gamma rays in the radiosensitive strain only.
doi:10.2203/dose-response.11-002.Rithidech
PMCID: PMC3299525  PMID: 22423226
low dose; gamma rays; mouse; genomic instability; cytogenetics
12.  Biodosimetry and assessment of radiation dose 
Aim
When investigating radiation accidents, it is very important to determine the exposition dose to the individuals. In the case of exposures over 1 Gy, clinicians may expect deterministic effects arising the following weeks and months, in these cases dose estimation will help physicians in the planning of therapy. Nevertheless, for doses below 1 Gy, biodosimetry data are important due to the risk of developing late stochastic effects. Finally, some accidental overexposures are lack of physical measurements and the only way of quantifying dose is by biological dosimetry.
Background
The analysis of chromosomal aberrations by different techniques is the most developed method of quantifying dose to individuals exposed to ionising radiations.1,2 Furthermore, the analysis of dicentric chromosomes observed in metaphases from peripheral lymphocytes is the routine technique used in case of acute exposures to assess radiation doses.
Materials and methods
Solid stain of chromosomes is used to determine dicentric yields for dose estimation. Fluorescence in situ hybridization (FISH) for translocations analysis is used when delayed sampling or suspected chronically irradiation dose assessment. Recommendations in technical considerations are based mainly in the IAEA Technical Report No. 405.2
Results
Experience in biological dosimetry at Gregorio Marañón General Hospital is described, including own calibration curves used for dose estimation, background studies and real cases of overexposition.
Conclusion
Dose assessment by biological dosimeters requires a large previous standardization work and a continuous update. Individual dose assessment involves high qualification professionals and its long time consuming, therefore requires specific Centres. For large mass casualties cooperation among specialized Institutions is needed.
doi:10.1016/j.rpor.2011.06.003
PMCID: PMC3863315  PMID: 24376970
Biodosimetry; Dicentrics; Translocations; FISH
13.  Comparison of RBE values of high- LET α-particles for the induction of DNA-DSBs, chromosome aberrations and cell reproductive death 
Background
Various types of radiation effects in mammalian cells have been studied with the aim to predict the radiosensitivity of tumours and normal tissues, e.g. DNA double strand breaks (DSB), chromosome aberrations and cell reproductive inactivation. However, variation in correlations with clinical results has reduced general application. An additional type of information is required for the increasing application of high-LET radiation in cancer therapy: the Relative Biological Effectiveness (RBE) for effects in tumours and normal tissues. Relevant information on RBE values might be derived from studies on cells in culture.
Methods
To evaluate relationships between DNA-DSB, chromosome aberrations and the clinically most relevant effect of cell reproductive death, for ionizing radiations of different LET, dose-effect relationships were determined for the induction of these effects in cultured SW-1573 cells irradiated with gamma-rays from a Cs-137 source or with α-particles from an Am-241 source. RBE values were derived for these effects. Ionizing radiation induced foci (IRIF) of DNA repair related proteins, indicative of DSB, were assessed by counting gamma-H2AX foci. Chromosome aberration frequencies were determined by scoring fragments and translocations using premature chromosome condensation. Cell survival was measured by colony formation assay. Analysis of dose-effect relations was based on the linear-quadratic model.
Results
Our results show that, although both investigated radiation types induce similar numbers of IRIF per absorbed dose, only a small fraction of the DSB induced by the low-LET gamma-rays result in chromosome rearrangements and cell reproductive death, while this fraction is considerably enhanced for the high-LET alpha-radiation. Calculated RBE values derived for the linear components of dose-effect relations for gamma-H2AX foci, cell reproductive death, chromosome fragments and colour junctions are 1.0 ± 0.3, 14.7 ± 5.1, 15.3 ± 5.9 and 13.3 ± 6.0 respectively.
Conclusions
These results indicate that RBE values for IRIF (DNA-DSB) induction provide little valid information on other biologically-relevant end points in cells exposed to high-LET radiations. Furthermore, the RBE values for the induction of the two types of chromosome aberrations are similar to those established for cell reproductive death. This suggests that assays of these aberrations might yield relevant information on the biological effectiveness in high-LET radiotherapy.
doi:10.1186/1748-717X-6-64
PMCID: PMC3127784  PMID: 21651780
14.  The application of an in situ karyotyping technique for mesenchymal stromal cells: a validation and comparison study with classical G-banding 
The cytogenetic analysis of mesenchymal stromal cells (MSCs) is essential for verifying the safety and stability of MSCs. An in situ technique, which uses cells grown on coverslips for karyotyping and minimizes cell manipulation, is the standard protocol for the chromosome analysis of amniotic fluids. Therefore, we applied the in situ karyotyping technique in MSCs and compared the quality of metaphases and karyotyping results with classical G-banding and chromosomal abnormalities with fluorescence in situ hybridization (FISH). Human adipose- and umbilical cord-derived MSC cell lines (American Type Culture Collection PCS-500-011, PCS-500-010) were used for evaluation. The quality of metaphases was assessed by analyzing the chromosome numbers in each metaphase, the overlaps of chromosomes and the mean length of chromosome 1. FISH was performed in the interphase nuclei of MSCs for 6q, 7q and 17q abnormalities and for the enumeration of chromosomes via oligo-FISH in adipose-derived MSCs. The number of chromosomes in each metaphase was more variable in classical G-banding. The overlap of chromosomes and the mean length of chromosome 1 as observed via in situ karyotyping were comparable to those of classical G-banding (P=0.218 and 0.674, respectively). Classical G-banding and in situ karyotyping by two personnel showed normal karyotypes for both cell lines in five passages. No numerical or structural chromosomal abnormalities were found by the interphase-FISH. In situ karyotyping showed equivalent karyotype results, and the quality of the metaphases was not inferior to classical G-banding. Thus, in situ karyotyping with minimized cell manipulation and the use of less cells would be useful for karyotyping MSCs.
doi:10.1038/emm.2013.133
PMCID: PMC3880460  PMID: 24357832
fluorescence in situ hybridization; in situ culture; karyotype; mesenchymal stromal cells
15.  The effect of X-rays and C-ions on pluripotent embryonic stem cells 
Journal of Radiation Research  2014;55(Suppl 1):i55-i56.
Embryonic stem cells (ESC) are characterized by both the capacity of infinite self-renewal and the ability to give rise to all the three germ layers emphasizing the need to strictly control the genetic integrity. To date, ESC are a powerful tool in disease modeling, tissue engineering and drug testing. However, in the field of radiation research, their potential has not been exploited.
We used the mouse ESC line D3 as a model to examine the effects of X-rays or C-ions (spread out Bragg peak, energy 106–147 MeV/u, average LET = 75 keV/µm) [ 1]. Doses of 0.5–5 Gy were applied and endpoints such as cell cycle progression (measured by flow cytometry), apoptosis (microscopic analysis of cell nucleus morphology), induction of chromosome aberrations (mFISH analysis), presence of pluripotency markers Oct3/4 and SOX2 (western blotting) and differentiation capacity by means of an embryoid body formation assay were analyzed up to 17 days post-irradiation. The experiments show that cells undergo a transient G2 arrest following exposure. After G2 checkpoint release, an increase in the apoptotic index is observed for both radiation types (3.7-fold increase for 2 Gy X-ray and 2.4-fold increase for 2 Gy C-ions). C-ions induce more structural chromosomal aberrations in first cycle cells than X-rays. During subsequent cell divisions, the frequency of chromosome aberrations declines: After >7 population doublings (8 days after exposure), the aberration frequency in the progeny of X-ray exposed cells returns to the control level (7% aberrant cells), while the progeny of C-ion exposed cells still harbor significantly more aberrations than control cells, which is mainly due to transmissible translocations.
The expression of pluripotency markers is maintained in cells surviving X-ray or C-ion exposure. This finding is supported by examining the differentiation capacity of ESC through the formation of embryoid bodies. Our experiments show that after X-ray or C-ion exposure, cells are able to develop spontaneous beating activity, indicating the differentiation ability into mesodermal cell lineages, i.e. beating cardiomyocytes. However, following C-ion exposure, the formation of beating clusters was delayed compared with control cells.
Moreover, our chromosome studies revealed that unexposed cells carry a high frequency of numerical aberrations. These comprise trisomies of chromosome 8 and 11 with a frequency of 29 ± 8% and 26 ± 6% respectively, as well as nullisomy of chromosome Y with a frequency of 35 ± 3%. Aneuploidy is a typical feature of mouse ESC and has been related to cell culture methods [ 2] and passage number. Because aneuploidy may affect gene expression and influence the properties of a cell population, the relevance of experiments based on mouse ESC is limited.
To overcome this problem, we recently extended our studies to human ESC. Human ESC are known to be cytogenetically more stable than mouse ESC, and represent a model that is closer to human embryonic development. Indeed, first investigations revealed a lower faction of cells with numerical and structural aberrations in the human ESC line H9 [ 3] compared with the mouse ESC line D3 (2% vs. 73% and 3% vs. 7%, respectively).
doi:10.1093/jrr/rrt175
PMCID: PMC3941490
embryonic stem cells; pluripotency; genomic integrity
16.  Studies on cell division in mammalian cells. VII. A temperature- sensitive cell line abnormal in centriole separation and chromosome movement 
The Journal of Cell Biology  1983;96(1):301-306.
A temperature-sensitive Syrian hamster mutant cell line, ts-745, exhibiting novel mitotic events has been isolated. The cells show normal growth and mitosis at 33 degrees C, the permissive temperature. At the nonpermissive temperature of 39 degrees C, mitotic progression becomes aberrant. Metaphase cells and those cells still able to form a metaphase configuration continue through and complete normal cell division. However, cells exposed to 39 degrees C for longer than 15 min can not form a normal metaphase spindle. Instead, the chromosomes are distributed in a spherical shell, with microtubules (MT) radiating to the chromosomes from four closely associated centrioles near the center of the cell. The cells progress from the spherical monopolar state to other monopolar orientations conical in appearance with four centrioles in the apex region. Organized chromosome movement is present, from the spherical shell state to the asymmetrical orientations. Chromosomes remain in the metaphase configuration without chromatid separation. Prometaphase chromosome congression appears normal, as the chromosomes and MT form a stable monopolar spindle, but bipolar spindle formation is apparently blocked in a premetaphase state. When returned from 39 degrees to 33 degrees C, the defective phenotype is readily reversible. At 39 degrees C, the mitotic abnormality lasts 3-5 h, followed by reformation of a single nucleus and cell flattening in an interphase- like state. Subsequent cell cycle events appear to occur, as the cells duplicate chromosomes and initiate a second round of abnormal mitosis. Cell cycle traversion continues for at least 5 d in some cells despite abnormal mitosis resulting in cells accumulating several hundred chromosomes.
PMCID: PMC2112245  PMID: 6826652
17.  Chromosome aberrations in cultured peripheral lymphocytes from persons with elevated skin radiosensitivity. 
Environmental Health Perspectives  1997;105(Suppl 6):1437-1439.
The purpose of this study was to elucidate whether an enhanced skin radiation reaction correlated with an enhanced chromosome radiation response. Twelve patients with late radiation skin ulcers formed after courses of radiation therapy were chosen as a group of individuals with elevated skin radiosensitivity. Half of the venous blood samples from each donor were irradiated with 2 Gy gamma-rays; the other half remained unexposed. Using standard cytogenetic technique, lymphocyte cultures were prepared with all samples. On the metaphase preparations, all chromosome aberrations detectable without karyotype identification were scored. The frequency of various aberrations in each patient were compared with relevant mean values in healthy unexposed donors. In several patients, the frequency of one aberration type or another exceeded the control value significantly. Comparison of aberration patterns in irradiated and nonirradiated cultures and consideration of elapsed time after therapeutic exposures suggested that the observed increased aberration levels reflected individual features of the patients' radiosensitivity, rather than lesions induced by previous in vivo exposures. Therefore, the question of a correlation between skin and chromosome radiosensitivity can be answered positively. Analysis of the peculiarities of cellular distribution of aberrations and of the relative contribution of different aberration types in patients and healthy donors indicates that the investigation of in vitro-induced aberrations is more suitable for the assessment of individual radiosensitivity than the study of aberrations observed in unexposed cultures.
PMCID: PMC1469925  PMID: 9467058
18.  Genome Damage in Oropharyngeal Cancer Patients Treated by Radiotherapy 
Croatian medical journal  2008;49(4):515-527.
Aim
To estimate genome damage in oropharyngeal cancer patients before, during, and after radiotherapy and to measure the persistence of caused genome damage relevant in the evaluation of secondary cancer risk.
Methods
DNA damage was evaluated in peripheral blood lymphocytes of 10 oropharyngeal cancer patients using alkaline comet assay, analysis of structural chromosome aberrations, and micronucleus assay. Blood samples were taken 2 hours before irradiation on day 1 of the first radiotherapy cycle, 2 hours after the application of the first dose, in the middle of the radiotherapy cycle, within 2 hours after the last received radiotherapy dose, and after 6 and 12 months after radiotherapy.
Results
In most participants, the highest level of primary DNA damage was recorded in blood samples collected after the administration of first radiation dose (mean tail length 25.04 ± 6.23 μm). Most patients also had increased frequency of comets with long tail-nucleus (LTN comets) after the administration of the first radiation dose (mean, 10.50 ± 7.71 per 100 comets), which remained increased in the middle of radiotherapy (mean, 18.30 ± 27.62 per 100 comets). Later on, the levels of primary DNA damage as recorded by the comet assay, slightly diminished. The frequency of structural chromosome aberrations in lymphocytes gradually increased during the radiation cycle (26.50 ± 27.72 per 100 metaphases at the end of the therapy), as well as the frequency of micronuclei (mean total number of micronuclei 167.20 ± 35.69; per 1000 binuclear cells).
Conclusion
Oropharyngeal cancer patients had relatively high levels of primary DNA damage in their peripheral blood lymphocytes even before therapy. The frequency of complex structural chromosome aberrations and the frequency of micronuclei increased with the progression of the radiation cycle and the doses delivered. As the frequency of chromosomal aberrations a year after radiotherapy mostly did not return to pre-therapy values, it represents an important risk factor related to the onset of second cancer.
doi:10.3325/cmj.2008.4.515
PMCID: PMC2525833  PMID: 18716999
19.  CBFB and MYH11 in inv(16)(p13q22) of Acute Myeloid Leukemia Display Close Spatial Proximity in Interphase Nuclei of Human Hematopoietic Stem Cells 
Genes, chromosomes & cancer  2011;50(9):746-755.
To gain a better understanding of the mechanism of chromosomal translocations in cancer, we investigated the spatial proximity between CBFB and MYH11 genes involved in inv(16)(p13q22) found in acute myeloid leukemia patients. Previous studies have demonstrated a role for spatial genome organization in the formation of tumorigenic abnormalities. The non-random localization of chromosomes and, more specifically, of genes appears to play a role in the mechanism of chromosomal translocations. Here, two-color fluorescence in situ hybridization and confocal microscopy were used to measure the interphase distance between CBFB and MYH11 in hematopoietic stem cells, where inv(16)(p13q22) is believed to occur, leading to leukemia development. The measured distances in hematopoietic stem cells were compared to mesenchymal stem cells, peripheral blood lymphocytes and fibroblasts, as spatial genome organization is determined to be cell-type specific. Results indicate that CBFB and MYH11 are significantly closer in hematopoietic stem cells compared to all other cell types examined. Furthermore, the CBFB-MYH11 distance is significantly reduced compared to CBFB and a control locus in hematopoietic stem cells, although separation between CBFB and the control is ~70% of that between CBFB and MYH11 on metaphase chromosomes. Hematopoietic stem cells were also treated with fragile site-inducing chemicals since both genes contain translocation breakpoints within these regions. However, treatment with fragile site-inducing chemicals did not significantly affect the interphase distance. Consistent with previous studies, our results suggest that gene proximity may play a role in the formation of cancer-causing rearrangements, providing insight into the mechanism of chromosomal abnormalities in human tumors.
doi:10.1002/gcc.20896
PMCID: PMC3724351  PMID: 21638519
20.  Homologous recombination pathway may play a major role in high-LET radiation-induced DNA double-strand break repair 
Journal of Radiation Research  2014;55(Suppl 1):i83-i84.
Purpose: Particle beams are increasingly applied to various cancer treatments because of their excellent dose localization to tumors while preserving surrounding normal tissues. However, characteristic of DNA damages induced by particle beams and their repair mechanisms are not fully understood. It is known that the majority of DNA double-strand breaks (DSBs) induced by ionizing radiation are repaired either by non-homologous end-joining (NHEJ) or by homologous recombination (HR) pathways. However, it has not been clarified how NHEJ and HR pathways contribute to the repair of DSBs induced by various particle beams [1, 2]. Thus, the purpose of this study is to clarify how these repair pathways contribute to the DSB repair in cells after irradiation with various radiation qualities.
Material and methods: A control Chinese hamster ovary (CHO) cell line (AA8), its mutant cell line deficient of DNA-PKcs (V3), XRCC4 (XR1) and Chinese hamster lung fibroblast cell line deficient of XRCC2 (IRS1) were exposed to gamma rays, protons, carbon ions and iron ions. V3 and XR1 lack NHEJ pathway, while IRS1 lacks HR pathway. After each irradiation, colony survival and gross-chromosome aberration were examined.
Results: It was demonstrated that colony survival was clearly dependent on the presence of NHEJ or HR pathways as well as radiation qualities. Although HR-deficient cells (IRS1) became more sensitive as LET value increased, NHEJ-deficient cells (V3 and XR1) did not further sensitized as LET value increased (Fig.  1). In addition, values of relative biological effectiveness of iron beams were higher in HR-deficient cells than in NHEJ-deficient cells (3.2 in AA8; 2.7 in IRS1; 1.8 in XR1 and V3). These may suggest that HR plays an important role in repairing DNA lesions induced by high-LET radiation. As for the incidence of total chromosomal aberration, we found that its incidence increased as LET values increased in wild-type (AA8) and NHEJ-deficient cells (V3, XR1), but not in HR-deficient cells (IRS1) (Table  1). However, occurrence of chromosome-type aberrations increased as LET values increased in all cell lines analysed here. This may indicate that the chromosomal aberrations occur from not only unrepaired damages but also the repair process of error-prone NHEJ pathway, suggesting that limited capacity of NHEJ to repair DSBs induced by high-LET irradiation may cause increased number of chromosome-type aberrations.
Conclusions: Taken together, although NHEJ pathway is the major pathway to repair DSBs induced by various types of radiation, HR pathway may play more important roles as LET value increases. Fig. 1.Radio sensitivity after gamma ray and iron beam. Clonogenic survival curves of AA8 (closed circle); XR1 (open circle); V3 (closed square) and IRS1 (open square) after irradiation with gamma ray (dashed) and iron beam (dotted). NHEJ-deficient cells are more sensitive to gamma ray, but HR-deficient cells are most sensitive to iron beam. Table 1.Chromatid and chromosome type aberration per chromosomeCell linesTypeControlProtonCarbonIronAA8 (wild-type)Chromatid0.860.240.611.07Chromosome0.051.180.743.44XR1 (NHEJ)Chromatid0.053.181.553.41Chromosome02.592.504.96V3 (NHEJ)Chromatid0.483.372.417.89Chromosome0.165.264.978.01IRS1 (HR)Chromatid0.028.2610.357.59Chromosome0.014.247.438.18
doi:10.1093/jrr/rrt181
PMCID: PMC3941540
high LET particle; NHEJ and HR pathway; chromosome aberration
21.  Effects of Aneuploidy on Genome Structure, Expression, and Interphase Organization in Arabidopsis thaliana 
PLoS Genetics  2008;4(10):e1000226.
Aneuploidy refers to losses and/or gains of individual chromosomes from the normal chromosome set. The resulting gene dosage imbalance has a noticeable affect on the phenotype, as illustrated by aneuploid syndromes, including Down syndrome in humans, and by human solid tumor cells, which are highly aneuploid. Although the phenotypic manifestations of aneuploidy are usually apparent, information about the underlying alterations in structure, expression, and interphase organization of unbalanced chromosome sets is still sparse. Plants generally tolerate aneuploidy better than animals, and, through colchicine treatment and breeding strategies, it is possible to obtain inbred sibling plants with different numbers of chromosomes. This possibility, combined with the genetic and genomics tools available for Arabidopsis thaliana, provides a powerful means to assess systematically the molecular and cytological consequences of aberrant numbers of specific chromosomes. Here, we report on the generation of Arabidopsis plants in which chromosome 5 is present in triplicate. We compare the global transcript profiles of normal diploids and chromosome 5 trisomics, and assess genome integrity using array comparative genome hybridization. We use live cell imaging to determine the interphase 3D arrangement of transgene-encoded fluorescent tags on chromosome 5 in trisomic and triploid plants. The results indicate that trisomy 5 disrupts gene expression throughout the genome and supports the production and/or retention of truncated copies of chromosome 5. Although trisomy 5 does not grossly distort the interphase arrangement of fluorescent-tagged sites on chromosome 5, it may somewhat enhance associations between transgene alleles. Our analysis reveals the complex genomic changes that can occur in aneuploids and underscores the importance of using multiple experimental approaches to investigate how chromosome numerical changes condition abnormal phenotypes and progressive genome instability.
Author Summary
Most plants and animals have two copies of each chromosome in the normal chromosome set. Unbalanced numerical changes resulting from gains or losses of individual chromosomes (aneuploidy) usually have deleterious consequences. For example, Down syndrome in humans is caused by an extra (triplicate) copy of chromosome 21. Human tumor cells usually display numerous alterations in chromosome number and structure. Little is known about how changes in chromosome number influence gene activity and chromosome integrity, thereby perturbing physiology and development. We have used the model plant A. thaliana to study how triplication of chromosome 5 affects gene expression, chromosome structure, and chromosome packaging in the nucleus. The results indicate that the presence of an extra chromosome 5 has multiple effects: (1) substantial changes in gene expression occur, primarily on the triplicated chromosome 5 but also on the four non-triplicated chromosomes; (2) broken derivatives of chromosome 5 can be retained in the presence of two normal copies; and (3) two copies of the triplicated chromosome 5 may show a slightly enhanced tendency to associate with each other, perhaps to spatially compensate for the chromosome imbalance. The detrimental effects of aneuploidy are likely due to concurrent changes in gene expression, chromosome structure, and arrangement.
doi:10.1371/journal.pgen.1000226
PMCID: PMC2562519  PMID: 18927630
22.  Tradescantia micronucleus bioassay and pollen tube chromatid aberration test for in situ monitoring and mutagen screening. 
The meiotic pollen mother cells (PMC) of Tradescantia (spiderwort) are highly synchronized in their prophase I and tetrad stages. Chromosomes of this stage are sensitive to physical or chemical mutagens. Thus high frequency of acentric fragments or sticky chromosomes can be induced with very low level of mutagens. These induced chromosome aberrations become micronuclei (MCN) in the synchronized tetrads and they can be easily identified and scored. Based upon these features, the Tradescantia micronucleus bioassay was established. This bioassay involves the exposure of PMC in the young inflorescences of the plant cuttings to gaseous agents through diffusion, to liquid agent through absorption and dialysis from the stem to flower buds, or to radiation. The exposed samples are fixed in aceto-alcohol (1:3) and prepared into microslides by using the aceto-carmine squash method. Frequencies of MCN in a large population of synchronized tetrads are the indications of genetic damage caused by the agents. Mature pollen grains of Tradescantia are free cells which can be cultured in lactose-agar medium. The generative cells in the cultured pollen tubes can carry out mitosis similar to the in vivo condition. The G2 interphase chromosomes of pollen mitosis are highly sensitive to gaseous or liquid chemicals and radiation. Treatments can be applied to these mitotic generative cells of the mature pollen or the mitotic generative nuclei of the developing pollen tube. The mitotic chromosomes of the generative cells are allowed to proceed through mitosis in the culture medium and slides are prepared for metaphase figures. Frequencies of various types of chromatid aberrations can be scored and used as the indices of genetic damage.
Images
PMCID: PMC1568629  PMID: 7460887
23.  Array-CGH and multipoint FISH to decode complex chromosomal rearrangements 
BMC Genomics  2006;7:330.
Background
Recently, several high-resolution methods of chromosome analysis have been developed. It is important to compare these methods and to select reliable combinations of techniques to analyze complex chromosomal rearrangements in tumours. In this study we have compared array-CGH (comparative genomic hybridization) and multipoint FISH (mpFISH) for their ability to characterize complex rearrangements on human chromosome 3 (chr3) in tumour cell lines. We have used 179 BAC/PAC clones covering chr3 with an approximately 1 Mb resolution to analyze nine carcinoma lines. Chr3 was chosen for analysis, because of its frequent rearrangements in human solid tumours.
Results
The ploidy of the tumour cell lines ranged from near-diploid to near-pentaploid. Chr3 locus copy number was assessed by interphase and metaphase mpFISH. Totally 53 chr3 fragments were identified having copy numbers from 0 to 14. MpFISH results from the BAC/PAC clones and array-CGH gave mainly corresponding results. Each copy number change on the array profile could be related to a specific chromosome aberration detected by metaphase mpFISH. The analysis of the correlation between real copy number from mpFISH and the average normalized inter-locus fluorescence ratio (ANILFR) value detected by array-CGH demonstrated that copy number is a linear function of parameters that include the variable, ANILFR, and two constants, ploidy and background normalized fluorescence ratio.
Conclusion
In most cases, the changes in copy number seen on array-CGH profiles reflected cumulative chromosome rearrangements. Most of them stemmed from unbalanced translocations. Although our chr3 BAC/PAC array could identify single copy number changes even in pentaploid cells, mpFISH provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels.
doi:10.1186/1471-2164-7-330
PMCID: PMC1769374  PMID: 17196103
24.  Dose estimation by chromosome aberration analysis and micronucleus assays in victims accidentally exposed to 60Co radiation 
The British Journal of Radiology  2009;82(984):1027-1032.
The objective of this study was to assess the radiation exposure levels in victims of a 60Co radiation accident using chromosome aberration analysis and the micronucleus assay. Peripheral blood samples were collected from three victims exposed to 60Co 10 days after the accident and were used for the chromosome aberration and micronucleus assays. After in vitro culture of the lymphocytes, the frequencies of dicentric chromosomes and rings (dic+r) and the numbers of cytokinesis blocking micronuclei (CBMN) in the first mitotic division were determined and used to estimate radiation dosimetry. The Poisson distribution of the frequency of dic+r in lymphocytes was used to assess the uniformity of the exposure to 60Co radiation. Based on the frequency of dic+r in lymphocytes, estimates of radiation exposure of the three victims were 5.61 Gy (A), 2.48 Gy (B) and 2.68 Gy (C). The values were estimated based on the frequencies of CBMN, which were 5.45 Gy (A), 2.78 Gy (B) and 2.84 Gy (C). The estimated radiation dosimetry demonstrated a critical role in estimating the radiation dose and facilitating an accurate clinical diagnosis. Furthermore, the frequencies of dir+r in victims A and B deviated significantly from a normal Poisson distribution. Chromosome aberration analysis offers a reliable means for estimating biological exposure to radiation. In the present study, the micronucleus assay demonstrated a high correlation with the chromosome aberration analysis in determining the radiation dosimetry 10 days after radiation exposure.
doi:10.1259/bjr/62484075
PMCID: PMC3473381  PMID: 19366736
25.  CHROMOSOME PULVERIZATION IN MICRONUCLEI INDUCED BY TRITIATED THYMIDINE 
The Journal of Cell Biology  1972;52(1):97-104.
Cultures of a pseudodiploid cell line (Don) of Chinese hamster origin were exposed to varying doses of tritiated thymidine (TdR-3H) for relatively long periods of time. In addition to previously observed chromosomal aberrations) such as breaks and reunions, a substantial number of interphasic cells with micronuclei and of metaphases associated with pulverized chromosomes was found; both phenomena were dependent on exposure time to and concentration of TdR-3H. The former phenomenon appeared to result from the effects of the β-emissions originating in the TdR-3H. A possible interpretation for chromosome pulverization induction is presented, emphasizing the derivation of the pulverized material from micronuclei in a common cytoplasm with a metaphase nucleus. These observations further substantiate our previously advanced hypothesis regarding the essential role played by substances present in a mitotic cell in the induction of chromosome pulverization and nuclear membrane dissolution.
PMCID: PMC2108688  PMID: 5061680

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