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Multiple sclerosis is a clinically heterogeneous autoimmune disease leading to severe neurological disability. Although during the last years many disease-modifying agents as treatment options for multiple sclerosis have been made available, their mechanisms of action are still not fully determined. In the present study radiosensitivity in lymphocytes of patients with relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis and healthy controls was investigated. Whole blood cultures from multiple sclerosis patients and healthy controls were used to analyze the spontaneous and radiation-induced micronuclei in binucleated lymphocytes. A subgroup of patients with relapsing-remitting multiple sclerosis was treated with immunomodulatory agents, interferon β or glatiramer acetate. The secondary progressive multiple sclerosis patients group was not receiving any treatment. Our results reveal that the basal DNA damage was not different between relapsing-remitting and secondary progressive multiple sclerosis patients, and healthy controls. No differences between gamma-irradiation induced micronuclei frequencies in binucleated cells from relapsing-remitting and secondary progressive multiple sclerosis patients, and healthy controls were found either. Nevertheless, when we compared the radiation induced DNA damage in binucleated cells from healthy individuals with the whole group of patients, a reduction in the frequency of micronuclei was obtained in the patients group. Induced micronuclei yield was significantly lower in the irradiated samples from treated relapsing-remitting multiple sclerosis patients than in healthy controls and relapsing-remitting not treated patients. Intrinsic sensitivity of lymphocytes subpopulations to the apoptotic effect of immunomodulatory treatment could be responsible for this result., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The exposure of tumour cells to high doses of ionizing radiation can induce endopolyploidization as an escape route from cell death. This strategy generally results in mitotic catastrophe during the first few days after irradiation. However, some cells escape mitotic catastrophe, polyploidize and attempt to undergo genome reduction and de-polyploidization in order to create new, viable para-diploid tumour cell sub-clones. In search for the consequences of ionizing radiation induced endopolyploidization, genome and chromosome architecture in nuclei of polyploid tumour cells, and sub-nuclei after division of bi- or multi-nucleated cells were investigated during 7 days following irradiation. Polyploidization was induced in p53-function deficient HeLa cells by exposure to 10Gy of X-irradiation. Chromosome territories #1, #4, #12 and centromeres of chromosomes #6, #10, #X were labelled by FISH and analysed for chromosome numbers, volumes and spatial distribution during 7 days post irradiation. The numbers of interphase chromosome territories or centromeres, respectively, the positions of the most peripherally and centrally located chromosome territories, and the territory volumes were compared to non-irradiated controls over this time course. Nuclei with three copies of several chromosomes (#1, #6, #10, #12, #X) were found in the irradiated as well as non-irradiated specimens. From day 2 to day 5 post irradiation, chromosome territories (#1, #4, #12) shifted towards the nuclear periphery and their volumes increased 16- to 25-fold. Consequently, chromosome territories returned towards the nuclear centre during day 6 and 7 post irradiation. In comparison to non-irradiated cells (∼500μm(3)), the nuclear volume of irradiated cells was increased 8-fold (to ∼4000μm(3)) at day 7 post irradiation. Additionally, smaller cell nuclei with an average volume of about ∼255μm(3) were detected on day 7. The data suggest a radiation-induced generation of large intra-nuclear chromosome territories and their repositioning prior to genome reduction., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The dicentric chromosome assay (DCA), which involves counting the frequency of dicentric chromosomes in mitotic lymphocytes and converting it to a dose-estimation for ionizing radiation exposure, is considered to be the gold standard for radiation biodosimetry. Furthermore, for emergency response, the DCA has been adapted for triage by simplifying the scoring method [1]. With the development of new technologies such as the imaging flow cytometer, it may now be possible to adapt this microscope-based method to an automated cytometry method. This technology allows the sensitivity of microscopy to be maintained while adding the increased throughput of flow cytometry. A new protocol is being developed to adapt the DCA to the imaging cytometer in order to further increase the rapid determination of a biological dose. Peripheral blood mononuclear cells (PBMC) were isolated from ex vivo irradiated whole blood samples using a density gradient separation method and cultured with PHA and Colcemid. After 48h incubation, the chromosomes were isolated, stained for DNA content with propidium iodide (PI) and labelled with a centromere marker. Stained chromosomes were then analyzed on the ImageStream(×) (EMD-Millipore, Billerica, MA). Preliminary results indicate that individual chromosomes can be identified and mono- and dicentric chromosomes can be differentiated by imaging cytometry. A dose response curve was generated using this technology. The details of the method and the dose response curve are presented and compared to traditional microscope scoring. Imaging cytometry is a new technology which enables the rapid, automated analysis of fluorescently labelled chromosomes. Adapting the dicentric assay to this technology has the potential for high throughput analysis for mass casualty events., (Copyright © 2013. Published by Elsevier B.V.)

Mass casualty scenarios of radiation exposure require high throughput biological dosimetry techniques for population triage in order to rapidly identify individuals who require clinical treatment. The manual dicentric assay is a highly suitable technique, but it is also very time consuming and requires well trained scorers. In the framework of the MULTIBIODOSE EU FP7 project, semi-automated dicentric scoring has been established in six European biodosimetry laboratories. Whole blood was irradiated with a Co-60 gamma source resulting in 8 different doses between 0 and 4.5Gy and then shipped to the six participating laboratories. To investigate two different scoring strategies, cell cultures were set up with short term (2-3h) or long term (24h) colcemid treatment. Three classifiers for automatic dicentric detection were applied, two of which were developed specifically for these two different culture techniques. The automation procedure included metaphase finding, capture of cells at high resolution and detection of dicentric candidates. The automatically detected dicentric candidates were then evaluated by a trained human scorer, which led to the term 'semi-automated' being applied to the analysis. The six participating laboratories established at least one semi-automated calibration curve each, using the appropriate classifier for their colcemid treatment time. There was no significant difference between the calibration curves established, regardless of the classifier used. The ratio of false positive to true positive dicentric candidates was dose dependent. The total staff effort required for analysing 150 metaphases using the semi-automated approach was 2 min as opposed to 60 min for manual scoring of 50 metaphases. Semi-automated dicentric scoring is a useful tool in a large scale radiation accident as it enables high throughput screening of samples for fast triage of potentially exposed individuals. Furthermore, the results from the participating laboratories were comparable which supports networking between laboratories for this assay., (Copyright © 2013 Elsevier B.V. All rights reserved.)

It is well established that chromosomes exist in discrete territories (CTs) in interphase and are positioned in a cell-type specific probabilistic manner. The relative localisation of individual CTs within cell nuclei remains poorly understood, yet many cancers are associated with specific chromosome rearrangements and there is good evidence that relative territorial position influences their frequency of exchange. To examine this further, we characterised the complexity of radiation-induced chromosome exchanges in normal human bronchial epithelial (NHBE) cells by M-FISH analysis of PCC spreads and correlated the exchanges induced with their preferred interphase position, as determined by 1/2-colour 2D-FISH analysis, at the time of irradiation. We found that the frequency and complexity of aberrations induced were reduced in ellipsoid NHBE cells in comparison to previous observations in spherical cells, consistent with aberration complexity being dependent upon the number and proximity of damaged CTs, i.e. lesion proximity. To ask if particular chromosome neighbourhoods could be identified we analysed all radiation-induced pair-wise exchanges using SCHIP (statistics for chromosome interphase positioning) and found that exchanges between chromosomes (1;13), (9;17), (9;18), (12;18) and (16;21) all occurred more often than expected assuming randomness. All of these pairs were also found to be either sharing similar preferred positions in interphase and/or sharing neighbouring territory boundaries. We also analysed a human small cell lung cancer cell line, DMS53, by M-FISH observing the genome to be highly rearranged, yet possessing rearrangements also involving chromosomes (1;13) and (9;17). Our findings show evidence for the occurrence of non-random exchanges that may reflect the territorial organisation of chromosomes in interphase at time of damage and highlight the importance of cellular geometry for the induction of aberrations of varying complexity after exposure to both low and high-LET radiation., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

A number of authors have suggested that a Bayesian approach may be most appropriate for analysis of cytogenetic radiation dosimetry data. In the Bayesian framework, probability of an event is described in terms of previous expectations and uncertainty. Previously existing, or prior, information is used in combination with experimental results to infer probabilities or the likelihood that a hypothesis is true. It has been shown that the Bayesian approach increases both the accuracy and quality assurance of radiation dose estimates. New software entitled CytoBayesJ has been developed with the aim of bringing Bayesian analysis to cytogenetic biodosimetry laboratory practice. CytoBayesJ takes a number of Bayesian or 'Bayesian like' methods that have been proposed in the literature and presents them to the user in the form of simple user-friendly tools, including testing for the most appropriate model for distribution of chromosome aberrations and calculations of posterior probability distributions. The individual tools are described in detail and relevant examples of the use of the methods and the corresponding CytoBayesJ software tools are given. In this way, the suitability of the Bayesian approach to biological radiation dosimetry is highlighted and its wider application encouraged by providing a user-friendly software interface and manual in English and Russian., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The conventional dicentric assay does not provide an accurate dose estimate in the case of accidental exposure to ionizing radiation above 6 Gy due to mitotic delay and poor mitotic index. The present study aims to establish a simple and rapid dose assessment technique based on scoring of rings and fragments in PCC spreads of stimulated lymphocytes. Human peripheral blood lymphocytes were gamma irradiated to different doses (6.2-24.5 Gy), cultured for two days with PHA and were forced to condense prematurely using 500 nM Okadaic acid (OA). The chromosome spreads were prepared, stained with Giemsa and observed under a microscope. The PCC index, PCC rings, and PCC fragments were scored for each dose point to arrive at the dose effect curve for various end points such as induction of rings and fragments and dicentrics. The PCC index varied from 12-18% up to 18 Gy and thereafter dropped to 6-8% at higher doses. The dose dependent increase in rings and fragments was found to be linear with a slope of 0.054+/-0.001 Gy(-1) for rings and 0.45+/-0.03 Gy(-1) for PCC fragments. An experiment was carried out to simulate partial-body exposure by mixing 10 Gy in vitro irradiated blood with un-irradiated blood in different proportions. The ratio of frequency of damaged cells among the total number of cells analyzed was found to be a good index of partial-body exposure. The culture duration was extended to 72 h to overcome the cell cycle delay induced by high doses of radiation. The conventional dicentrics rings and fragments also showed a dose response at high doses. The response can be best fitted to a linear model with a slope of 0.28+/-0.0007 Gy(-1) for the induction of dicentrics. However, long culture duration, technical skill and time required to analyse multi-aberrant cells makes the dicentric assay less suitable for high dose exposures requiring a rapid dose estimate. The PCC assay can be performed in 50 h with biodosimetric information about the irradiated fraction in cases of acute radiation exposures. The automated finding of PCC spreads significantly increased the speed of scoring PCC fragments., (Copyright 2010 Elsevier B.V. All rights reserved.)

The mechanism behind chromatid break formation is as yet unclear, although it is known that DNA double-strand breaks (DSBs) are the initiating lesions. Chromatid breaks formed in cells in the G2-phase of the cell-cycle disappear ('rejoin') as a function of time between radiation exposure and cell fixation. However, the kinetics of disappearance of chromatid breaks does not correspond to those of DSB rejoining, leading us to seek alternative models. We have proposed that chromatid breaks could be formed indirectly from DSB and that the mechanism involves topoisomerase IIalpha. In support of this hypothesis we have recently shown that frequencies of radiation-induced chromatid breaks are lower in two variant human promyelocytic leukaemic cell lines with reduced topoisomerase IIalpha expression. Here we report that suppression of topoisomerase IIalpha in human hTERT-RPE1 cells, either by its abrogation using specific siRNA or by inhibition of its catalytic activity with the inhibitor ICRF-193, causes a reduction in frequency of chromatid breaks in radiation-exposed cells. The findings support our hypothesis for the involvement of topoisomerase IIalpha in the formation of radiation-induced chromatid breaks, and could help explain inter-individual variation in human chromosomal radiosensitivity; elevation of which has been linked with cancer susceptibility.

We describe a low level of chromatid-type aberrations which included the relatively rare isochromatid/chromatid triradial in peripheral blood lymphocytes that were irradiated, ostensibly in G0, with accelerated heavy (12)C ions. These were produced only at the energies of 69 MeV/n (34.6 keV/microm), almost absent at the energy of either 58.6 MeV/n (46.07 keV/ microm) or 19.3 MeV/n (97 keV/microm), nor were they found after low-LET X-rays. Mechanisms potentially responsible for their formation are discussed.

We have measured rejoining kinetics of chromosome breaks using a modified cell fusion-based premature chromosome condensation (PCC) technique in confluent cultures of normal human fibroblasts irradiated at low doses of X-rays. In order to enhance the sensitivity of the fusion-based PCC assay, we added a DNA double strand break (DSB) repair inhibitor wortmannin during the incubation period for PCC/fusion process resulting in a significantly higher yield of G1-type chromosome breaks. The initial number of chromosome breaks (without repair) gave a linear dose response with about 10 breaks per cell per Gy which is about two times higher than the value with the conventional G1-type PCC method. The chromosome rejoining kinetics at 0.5 and 2.0 Gy X-rays reveal a bi-phasic curve with both a fast and a slow component. The fast component (0-30 min) is nearly identical for both doses, but the slow component for 2 Gy kinetics is much slower than that for 0.5 Gy, indicating that the process occurring during this period may be crucial for the ultimate fate of irradiated cells. The chromosome rejoining kinetics obtained here is similar to that of other methods of detecting DNA DSB repair such as the gammaH2AX assay. Our chromosome repair assay is useful for evaluating the accuracy of other assays measuring DNA DSB repair at doses equal or less than 0.5 Gy of ionizing radiation.

The frequency, distribution pattern and localisation of gamma radiation-induced break points on the chromosomes of patients with various inherited metabolic disorders were studied to detect: (i) whether the break point distribution following irradiation is random and proportional to the length or the DNA content of the chromosome, or non-proportionally distributed on their length and at times clustering to form hot spots on certain region of the chromosomes; and (ii) to find whether there exists a syndrome-related chromosome-specific pattern of radiation-induced break points. Lymphocyte cultures from patients of haemophilia, ichthyosis, Duchenne muscular dystrophy, retinitis pigmentosa and alpha-thalassemia, whose defective gene loci were located by DNA probe method, were subjected to 3Gy of gamma radiation at G(0). The chromosomal break point analysis was carried out on all the 23 types of chromosomes (excluding Y chromosome) using G banding and FISH painting. The exact location of the break points on G-banded chromosomes was identified using a semi-automated microscope densitometer system (Leitz MPV2). In normal individuals in all the chromosomes except the chromosome 1, a random distribution of break points proportional to their length based on their DNA content was observed. However, in all the syndromes studied a mixture of hypersensitive chromosomes with a non-random distribution pattern of chromosomal break points invariably clustering to form hot spots, and chromosomes with random distribution of break points proportional to their length were observed. The hypersensitive chromosomes and their hot spots were syndrome-specific.

To establish the optimal experimental conditions for the use of the micronuclei (MN) test to determine the level of chromosomal damage induced by ionising radiation (IR) exposure in lymphoblastoid cell lines, a time-course study was performed comparing a normal and an ataxia telangiectasia (AT) cell line, the latter being characterised by an extreme radiation sensitivity. Several parameters were analysed: the use of cytochalasin-B (Cyt-B) to quantify MN, the optimum fixation time to measure radiation-induced MN, the most appropriate treatment dose of IR to distinguish between the normal and the radiosensitive cells and the cell-cycle distribution after irradiation. The results obtained showed that the spontaneous as well as the radiation-induced levels of MN were significantly higher in the AT cell compared to the normal cells (P < 0.001 and P = 0.005, respectively). In both cell types the number of radiation-induced MN were lower in the cultures without Cyt-B than those with Cyt-B (P < 0.001), with the AT cells being distinguished in terms of IR-induced MN from the normal cells only with the addition of Cyt-B. The level of MN formation was independent of the dose of Cyt-B used (3 or 6 microg/ml). The optimum time for radiation-induced MN measured was found to be between 48 and 72 h post-irradiation, with 2 and 4 Gy exposures inducing similar levels of MN. However, as the higher dose caused a greater delay in the cell-cycle, treatment with 2 Gy with MN measurement at 48 h in the presence of 3 microg/ml Cyt-B were chosen as the optimum experimental conditions. This choice was validated using two additional normal and AT cell lines. In conclusion, our results show that the use of Cyt-B increases the sensitivity of the MN test for comparing differences in radiosensitivity between lymphoblastoid cell lines., (Copyright 2002 Elsevier Science B.V.)

Immunodeficiency, centromeric region instability, and facial anomalies (ICF), a rare recessive chromosome instability syndrome, involves the loss of DNA methyltransferase 3B activity and the consequent hypomethylation of a small portion of the genome. We demonstrate for the first time that ICF cells are strongly hypersensitive to a genotoxic agent, namely, ionizing radiation. However, unlike cell lines from patients with ataxia telangiectasia or Nijmegen breakage syndrome, chromosome instability syndromes also associated with unusual sensitivity to ionizing radiation, ICF cells did not show any deficiencies in their cell cycle checkpoints. ICF lymphoblastoid cell lines demonstrated increased apoptosis, long-term cell cycle arrest, and loss of viability in clonogenicity assays after irradiation compared to analogous normal cell lines. Also, the ICF cell lines were subject to high frequencies of rapid non-apoptotic cell death upon irradiation but not to abnormally high levels of radiation-induced, cytogenetically detectable chromosome abnormalities. ICF-associated undermethylation of some regulatory gene(s) might lead to an exaggerated response to radiation-induced breaks in DNA yielding increased rates of cell death and irreversible cell cycle arrest. As a defense against their frequent spontaneous breaks in chromosomes 1 and 16, ICF patients may be abnormally prone to chromosome break-induced apoptosis, non-apoptotic cell death, and permanent cell cycle arrest so as to minimize the number of cycling cells with spontaneous rearrangements. A similarly increased cell death and cycle-arrest response to chromosome breaks due to cancer-linked DNA hypomethylation might occur during carcinogenesis.

We developed a simple and rapid method to study chromosome aberrations involving specific chromosomes using unstimulated human peripheral blood lymphocytes (HPBL). Premature chromosome condensation (PCC) was induced by incubating unstimulated HPBL in the presence of okadaic acid (OA, a phosphatase inhibitor), adenosine triphosphate (ATP), and p34(cdc2)/cyclin B kinase [an essential component of mitosis-promoting factor (MPF)], which eliminated the need for fusion with mitotic cells. OA concentration and duration of incubation for PCC induction was optimized using mitogen-stimulated HPBL; a final concentration of 0.75 microM incubated for 3 h was optimum, resulting in approximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the addition of p34(cdc2)/cyclin B kinase at concentrations as low as 5 units/ml to a cell culture medium containing OA. Increases in the concentration of p34(cdc2)/cyclin B kinase from 5 to 50 units/ml resulted in a concentration-dependent increase in PCC yield (30% to 42%). We demonstrate that this technique of inducing PCC in unstimulated HPBL is suitable for studying radiation-induced aberrations involving a specific chromosome (chromosome 1) after 24 h repair using a whole-chromosome in situ hybridization probe and chromosome painting. Cells with aberrant chromosome number 1 are characterized with more than two chromosome spots. The frequency of cells with aberrant chromosome 1 increased with 60Co gamma-radiation doses in the region 0-7.5 Gy. The observed dose-effect relationship for the percentage of cells with aberrant chromosome 1 (Y) was explained by using both a linear [Y=(2.77+/-0.230)D+0.90+/-0.431, r(2)=0.966] and a nonlinear power [Y=(5.70+/-0.46)D((0.61+/-0.05)), r(2)=0.9901) model. This technique can be applied to biological dosimetry of radiation exposures involving uniform whole-body low linear energy transfer (LET) exposures.

The radiation space environment includes particles such as protons and multiple species of heavy ions, with much of the exposure to these radiations occurring at extremely low average dose-rates. Limitations in databases needed to predict cancer hazards in human beings from such radiations are significant and currently do not provide confidence that such predictions are acceptably precise or accurate. In this article, we outline the need for animal carcinogenesis data based on a more sophisticated understanding of the dose-response relationship for induction of cancer and correlative cellular endpoints by representative space radiations. We stress the need for a model that can interrelate human and animal carcinogenesis data with cellular mechanisms. Using a broad model for dose-response patterns which we term the "subalpha-alpha-omega (SAO) model", we explore examples in the literature for radiation-induced cancer and for radiation-induced cellular events to illustrate the need for data that define the dose-response patterns more precisely over specific dose ranges, with special attention to low dose, low dose-rate exposure. We present data for multiple endpoints in cells, which vary in their radiosensitivity, that also support the proposed model. We have measured induction of complex chromosome aberrations in multiple cell types by two space radiations, Fe-ions and protons, and compared these to photons delivered at high dose-rate or low dose-rate. Our data demonstrate that at least three factors modulate the relative efficacy of Fe-ions compared to photons: (i) intrinsic radiosensitivity of irradiated cells; (ii) dose-rate; and (iii) another unspecified effect perhaps related to reparability of DNA lesions. These factors can produce respectively up to at least 7-, 6- and 3-fold variability. These data demonstrate the need to understand better the role of intrinsic radiosensitivity and dose-rate effects in mammalian cell response to ionizing radiation. Such understanding is critical in extrapolating databases between cellular response, animal carcinogenesis and human carcinogenesis, and we suggest that the SAO model is a useful tool for such extrapolation.

Manned space missions recently increased in number and duration, thus it became important to estimate the biological risks encountered by astronauts. They are exposed to cosmic and galactic rays, a complex mixture of different radiations. In addition to the measurements realized by physical dosimeters, it becomes essential to estimate real biologically effective doses and compare them to physical doses. Biological dosimetry of radiation exposures has been widely performed using cytogenetic analysis of chromosomes. This approach has been used for many years in order to estimate absorbed doses in accidental or chronic overexposures of humans. In addition to conventional techniques (Giemsa or FPG staining, R- or G-banding), faster and accurate means of analysis have been developed (fluorescence in situ hybridization [FISH] painting). As results accumulate, it appears that strong interindividual variability exists in the basal level of aberrations. Moreover, some aberrations such as translocations exhibit a high background level. Radiation exposures seem to induce variability between individual responses. Its extent strongly differs with the mode of exposure, the doses delivered, the kind of radiation, and the cytogenetic method used. This paper aims to review the factors that may influence the reliability of cytogenetic dosimetry. The emphasis is on the exposure to high linear energy transfer (LET) particles in space as recent studies demonstrated interindividual variations in doses estimated from aberration analysis after long-term space missions. In addition to the problem of dose estimates, the heterogeneity of cosmic radiation raises questions relating to the real numbers of damaged cells in an individual, and potential long-term risks. Actually, densely ionizing particles are extremely potent to induce late chromosomal instability, and again, interindividual variability exists in the expression of damage.

Cytogenetic analysis of chromosomal aberrations (CA) in 175,229 cells from 1113 individuals, both unexposed and occupationally or environmentally exposed to heavy metals (mercury and lead), organic (styrene, formaldehyde, phenol and benzo(a)pyrene) and inorganic (sulfur and nitrogen oxides, hydrogen and ammonium fluorides) volatile substances and/or ionizing radiation was performed. In addition, 11,250 cells from 225 individuals were scored for the frequency of sister-chromatid exchanges (SCE). Increased frequencies of CA were found in all occupationally exposed groups. A principal difference between the exposure to heavy metals and organic substances was found: increase in the CA frequency was dependent on duration of exposure to mercury but not dependent on duration of exposure to styrene, formaldehyde and phenol. A higher CA incidence was found in lymphocytes of children living in the vicinity of a plant manufacturing phosphate fertilizers. This indicates that children are a sensitive study group for the assessment of environmental exposure. However, the results of SCE analysis in these children were inconclusive. Exposure to ionizing radiation was found to cause chromosome breaks and chromatid exchanges in Chernobyl clean-up workers and chromatid breaks, chromatid exchanges, dicentric chromosomes and chromosome translocations in workers from the Ignalina Nuclear Power Plant. The increased frequency of chromatid exchanges in individuals exposed to ionizing radiation was quite unexpected. This may be attributed to the action of some unrecognized life-style or occupational factors, or to be a result of radiation-induced genomic instability. Also an increased SCE frequency was found in lymphocytes of Chernobyl clean-up workers.

Telomerase, a ribonucleoprotein enzyme, has been detected in immortalised cells and in majority of human cancers. Numerical and structural chromosome aberrations are commonly observed in tumour cell lines. To study the expression of telomerase and its influence on the formation of chromosomal aberrations, human colon carcinoma cell line (SW480) and human nonpolyposis colorectal carcinoma (HNPCC) cell lines (NA50600, NA59 and NA61) were exposed to 2 or 4 Gy X-rays. Increased telomerase activity was observed in all these cell lines at 24 h postirradiation and a 3 to 7 fold increase was seen at 4 Gy dose as detected by Telomere Repeat Amplification Protocol. Chromosomal rearrangements (dicentrics, translocations and breaks/fragments) analysed by Giemsa staining and chromosome painting were increased significantly following X- Quantitative fluorescence in situ hybridisation using a peptide nucleic acid telomeric probe to measure telomere length at irradiation chromosomal level revealed that all cell lines have very short telomeres in the range of 0.29 to 2.1 kb. Following X-irradiation, an increase in the chromosome end-to-end (telomere) associations was observed. The present results demonstrate that presence or upregulation of telomerase activity did not prevent the formation of chromosome aberrations and/or telomere associations in tumour cell lines after X-irradiation., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Frequencies of intra-chromosomal exchanges (pericentric inversions and centric rings) and inter-chromosomal exchanges (dicentrics and translocations) in X-irradiated (2.5 Gy) human lymphocytes have been estimated. To detect these events we employed FISH (fluorescence in situ hybridization) technique and arm specific painting probes for chromosomes #1 and #3. The ratio between centric rings and pericentric inversions was found to be about 1. For intra-changes to inter-changes, the ratio (F) was between 6 and 9. Based on the total number of colour junctions involving chromosomes #1 and #3 it was found that exchanges between the arms of the same chromosome occur about 8.7 times more than inter-chromosomal exchanges calculated on the basis of the DNA content of the chromosomes and random induction of aberrations in the total genome. Chromosomal organization in interphase nucleus appears to promote the formation of more intra-changes than inter-changes following X-irradiation, most probably due to close proximity of the two arms of a chromosome.

Chromosome aberrations induced by radiation have been used for the purpose of dosimetry for a long time. Translocations are especially useful for retrospective dosimetry, since they are assumed to be stable. The method of chromosome painting (FISH) has facilitated objective scoring of aberrations considerably. Translocation frequencies, obtained by FISH, for retrospective dosimetry rely on the main assumptions of neutral selection value and that the distribution of aberrations over the chromosomes is a known function of the DNA content of the chromosomes. Data scrutinising the two above-mentioned assumptions indicate deviations from both. Other factors potentially causing problems for retrospective dosimetry, such as inter-individual variations in background and induction patterns, are discussed. Finally, a brief analysis of the statistical power of dosimetry studies shows that establishing low doses (approximately 0.25 Gy) with good precision requires a great effort, which is probably unrealistic for individual dose estimates in epidemiological studies.

Frequencies of spontaneous and radiation-induced chromosome aberrations were documented in lymphocytes from patients with basal cell nevus syndrome (BCNS) and from those with epidermodysplasia verruciformis (EV). Cells were irradiated with single or double doses of gamma-rays or UV light. For the double dose irradiation protocol, the two doses were separated from each other by 60 min. After irradiation of cells with 4 or 2 + 2 J/m2 UV light, lymphocytes from seven BCNS patients had a similar number of aberrant cells compared with normal controls, while cells from three EV patients showed a significantly increased number of cells containing aberrations, particularly chromatid-type aberrations, compared to controls (p < 0.03, Student's t-test). Similar results were observed whether the cells were irradiated with single or double doses of UV light. In addition, lymphocytes from EV patients had significantly lower mitotic indices than controls whether the cultures were irradiated with UV light or unirradiated (p < 0.05; Student's t-test). After irradiation with 100, 100 + 100 or 50 + 50 cGy gamma-rays, the mean chromosome aberration frequencies from nine BCNS patients and two EV patients were not significantly different from the controls. The spontaneous chromosome aberration frequencies were similar for the patients and the controls. Our data indicate that BCNS and EV lymphocytes irradiated with gamma-rays have a normal DNA repair response. However, cells from EV patients have an abnormal repair response to UV light induced DNA damage. This abnormality is probably caused by deficiency in DNA repair and it is consistent with the patients' sensitivity to UV light induced cutaneous squamous cell carcinomas.

As part of a regional International Atomic Energy Agency (IAEA) collaborative project within Latin America, five countries participated in an intercomparison in cytogenetic dosimetry. Coded slides for chromosomal aberrations and micronucleus analyses were prepared by the coordinator laboratory which organized the exercise and sent to the other participating laboratories. For estimates of dose, each laboratory scored the frequency of dicentrics in metaphases and the frequency of micronuclei in binucleated cells. The lymphocytes were irradiated with 60Co gamma-rays (0, 0.75, 1.5 and 3.0 Gy). Eleven of the 15 estimates of dose based on dicentrics and nine of the 12 based on micronuclei fell within +/- 30% of the true dose. When considering the uncertainties of the dose estimates, the true dose fell within the 95% confidence limits of the estimates on eight of the 15 occasions for dicentrics and four of the 12 for micronuclei.

The repair of X-ray-induced DNA damage during G2 cell-cycle phase has been examined in lines of skin fibroblasts from three patients with trichothiodystrophy (TTD), one with apparently normal and two with defective nucleotide excision repair (NER). These responses are compared with those of five lines from clinically normal controls, lines from xeroderma pigmentosum (XP), Cockayne syndrome (CS), Down syndrome (DS), and ataxia telangiectasia (AT) patients. Chromosomal DNA repair was measured as the chromatid aberration frequency (CAF) or total number of chromatid breaks and long gaps per 100 metaphase cells, determined 0.5-1.5 h after X-irradiation (53 rad). Chromatid breaks and gaps (as defined herein) represent unrepaired DNA strand breaks. Only one of the TTD lines, TTD 1BR, showed an abnormally high CAF. This line was shown subsequently to be of a different complementation group, representing a new nucleotide excision repair gene. An abnormally high CAF was also observed, as reported previously, in XP-C, AT and DS but not in CS skin fibroblasts. In addition, cell lines were examined for DNA incision activity by an indirect method in which chromatid aberrations were enumerated with or without ara-C, an inhibitor of repair synthesis, added after X-irradiation. All TTD lines had abnormally low incision activity.

A dose-effect curve obtained by analysis of dicentric chromosomes after irradiation of peripheral blood samples, from one donor, at 11 different doses of gamma-rays is presented. For the elaboration of this curve, more than 18,000 first division metaphases have been analyzed. The results fit very well to the linear-quadratic model. To validate the curve, samples from six individuals (three controls and three occupationally exposed persons) were irradiated at 2 Gy. The results obtained, when compared with the curve, showed that in all cases the 95% confidence interval included the 2 Gy dose, with estimated dose ranges from 1.82 to 2.19 Gy.

Cells in human peripheral blood were treated in vitro with increasing concentrations of melatonin (0.5 or 1.0 or 2.0 mM) for 20 min at 37 +/- 1 degrees C and then exposed to 150 cGy gamma-radiation from a 137Cs source. The lymphocytes which were pre-treated with melatonin exhibited a significant and concentration-dependent decrease in the frequency of radiation-induced chromosome damage as compared with the irradiated cells which did not receive the pre-treatment. The extent of the reduction in radiation-induced chromosome damage observed with 2.0 mM melatonin was similar to that found in lymphocytes pre-treated with 1.0 M dimethyl sulfoxide, a known free radical scavenger. Melatonin at 2.0 mM (a 500 x lower concentration) was as effective in decreasing the radiation-induced chromosome damage as dimethyl sulfoxide at 1.0 M. These observations may have implications for human protection against damage due to endogenously produced free radicals and also due to exposure to free radical producing physical and chemical mutagens and carcinogens.

Ataxia telangiectasia (AT) skin fibroblasts in G1 phase and peripheral blood lymphocytes in G0 and G1 phase were studied for their DNA replication response to X-rays. The irradiation of normal cells in G1 but not in G0 phase caused a delay of onset of DNA replication, which was less pronounced in AT cells. However, such radioresistant DNA replication itself cannot be the sole mechanism of the increased sensitivity of AT cells to chromosome aberration formation by X-rays for the following two reasons: (1) due to the intrinsically slow cell cycle progression of AT fibroblasts, the time of traverse to DNA replication of AT cells was comparable with that of normal cells after exposure to 1 Gy while AT cells gave rise to a greatly increased number of chromatid aberrations; (2) in peripheral blood lymphocytes irradiated in G0 phase, the traversal to the DNA replication phase was the same for normal and AT cells in spite of the well documented chromosomal radiosensitivity of G0-irradiated AT cells. The AT factor may be better explained as a key element directly involved in DNA damage processing, which in turn provides messages to suppress replication if recombination and replication are mutually exclusive.

Cytogenetic data on cultured lymphocytes of the in utero exposed A-bomb survivors in the RERF Adult Health Study cohort have been analyzed using the G-banding technique to determine the frequency of aneuploid cells. The data consist of blood samples collected between 1985 and 1987 from 264 Hiroshima individuals for whom DS86 maternal uterine dose estimates are available: 124 proximally exposed (74 males and 50 females) with an estimated dose of 0.005 Sv or more, and 140 distally exposed (76 males and 64 females) with a dose estimate of 0 Sv, assuming the neutron relative biological effectiveness (RBE) of 10. A main feature of aneuploidy was that aneuploid frequency in autosomes depended generally on chromosome length; aneuploidies were significantly more frequent in shorter chromosomes than in longer chromosomes. The frequency of aneuploidies also depended on type, with chromosome loss approximately five times more frequent than chromosome gain. However, chromosome 21, as well as the sex chromosomes, were notable in that aneuploidy was much more frequent for these chromosomes than would be predicted from a simple relationship with length. X chromosome aneuploidies were significantly more frequent in females than in males. There was no dependence of aneuploid frequencies on dose when measured 40 years after the exposure.

From more than 500 tumours reported in human primary immune deficiencies a majority has been observed in two disorders: ataxia telangiectasia (A-T) and common variable immune deficiency (CVID). Since both diseases have an increased risk of lymphomas/leukaemias and gastrointestinal tumours, suggesting a common risk factor, and the cells derived from A-T patients exhibit an increased chromosomal radiosensitivity we analysed chromosome damage in the G2 lymphocytes of 24 CVID patients and 21 controls after X-irradiation in vitro. There was a significant difference in mean aberration yields between patients and controls. Three CVID patients had yields higher than the mean + 3SD of the controls. Six patients but only one control had yields higher than the mean + 2SD of controls. The patient with the highest chromosomal radiosensitivity subsequently developed a lymphoma. Repeat assays on the same blood sample, with a 24-h delay in setting up the second culture, showed as much variability for control donors as the variation between control donors although for CVID patients inter-individual variation was greater than the difference between results of repeat samples. There was a weak positive correlation between radiosensitivity and age of donor. Chromosomal radiosensitivity of five patients with X-linked hypogammaglobulinaemia was not different from healthy donors. The mean mitotic index (MI) for unirradiated samples from CVID patients was significantly lower than for controls and there was an inverse relationship between MI and aberration yields in the patients, but not in controls. We suggest that the defect in CVID patients that reduces response to mitogenic stimuli may have mechanism(s) in common with those involved in cellular repair processes.

Cultures of human skin fibroblasts were exposed to heavy ions: neon (E = 10.74 MeV/u) and argon (E = 10.52 MeV/u) at fluences of 10(6), 2 x 10(6) and 4 x 10(6) and lead (E = 9.5 MeV/u) at a fluence of 2 x 10(6) particles/cm2. Cultures were further prolonged for up to 25 passages and karyotyping was performed at various times. Radiation-induced chromosome anomalies progressively decreased, became quite rare at passages 5-7 and increased at later passages. Around passages 20-25, most anomalies occurring were dicentrics, involving telomeric regions of 13p and q arms principally and to a lesser degree those of 1p, 16p and 16q arms. These non-random rearrangements paralleled the appearance of clones with unbalanced karyotypes. In particular, two independent proliferating clones were characterized by a monosomy 13. It is concluded that most chromosome lesions directly induced by heavy ions are hardly compatible with cell survival and thus disappear after a few cell generations. However, surviving cells acquire a de novo chromosome instability leading to the formation of clones with unbalanced karyotypes at late passages.

Chromosomal aberration (CA) and sister-chromatid exchange (SCE) frequencies have been assessed in 9 patients with von Recklinghausen's neurofibromatosis (NF1) and 8 apparently healthy controls. In separate experiments over a 5-year period, blood lymphocytes, skin fibroblast cell strains, and lymphoblastoid lines from both groups were treated with X-rays or mitomycin C (MMC) to determine whether the NF1 group was more sensitive to these agents than the control group. No difference between cells from NF1 patients and controls was observed with respect to spontaneous or X-ray-induced CA. Spontaneous or X-ray- and MMC-induced SCE frequencies were also similar in NF1 patients and controls.

The premature chromosome condensation (PCC) technique and conventional chromosome analysis were applied to examine the kinetics of radiation-induced primary chromosome breaks, their rejoining and formation of dicentrics in human peripheral blood lymphocytes (PBLs). Numbers of chromosomal elements and dicentrics per cell were analyzed for each dose. Dose-dependent increases were observed for chromosome fragments (linear) and dicentrics (linear/quadratic). For an assessment of repair kinetics, numbers of breaks and dicentrics were estimated immediately and at several recovery periods after irradiation, using the PCC technique. It was found that chromosome fragments restitute with time, whereas the dicentrics are formed very quickly and their frequency remains the same, despite the decline in the number of chromosome breaks at later recovery times. Fractionation experiments were conducted to study the time-dependent interaction of primary breaks in the formation of dicentric chromosomes. PBLs were irradiated with 3 Gy X-rays split into 2 equal fractions separated by different intervals up to 5 h. No marked difference was observed in the yield of dicentrics following the different fractionation protocols, except that the mean levels of dicentrics declined when the fraction interval was 4 h or more. It appears that most of the dicentrics are formed by misrepair of strand breaks, produced directly by radiation and not resulting from rejoining of existing breaks during the slow repair process which follows. We also studied the role of the chromatin configuration at the time of irradiation on the yield of chromosome fragments and dicentrics. Highly condensed chromatin due to pretreatment with 0.3 M NaCl was found to reduce the frequency of radiation-induced chromosomal aberrations.

Porokeratosis is a rare genetic skin disorder known to be associated with a propensity to develop skin cancer. To further elucidate the previously reported cytogenetic and cellular abnormalities, we studied karyotypic changes and the sensitivity to X-ray irradiation of cultured fibroblasts derived from skin lesions and normal-appearing skin of 3 patients with porokeratosis. Cultured fibroblasts from normal-appearing skin of 9 controls were similarly examined. Porokeratosis subjects had a greater number of cells with chromosomal abnormalities than controls. Two porokeratosis strains which were derived from the normal-appearing skin of a patient had a noticeable clone of abnormal cells. Porokeratosis fibroblasts were hypersensitive to the lethal effects of X-radiation. This hypersensitivity was common to both the lesion-derived strains and the ones derived from normal-appearing skin. The 2 strains with clonal abnormal cells were also similarly hypersensitive to X-radiation. These results suggest that chromosomal instability is strongly related to porokeratosis and that X-ray hypersensitivity is an inherent abnormality in cultured fibroblasts of porokeratosis patients.

Two aspects of the relationship between Asymmetrical (A) and Symmetrical (S) radiation-induced chromosomal aberrations are considered in this paper. (1) Are A and S truly alternative modes of lesion interactions? Relative frequencies for chromatid-type and chromosome-type are examined, and new lymphocyte data using banding is used to look at this, and also for parallelism in chromosome participation of the two forms for various aberration categories. All the tests applied suggest that A and S are alternative interaction modes. (2) The long-term survival characteristics of A and S are discussed, and the differences in expected frequencies of derived S per surviving cell from chromosome-type and chromatid-types are stressed. Since many in vivo tissues have varying mixtures of potential chromatid and chromosome aberration-bearing target cells, ultimate cell survival and derived S frequencies may differ between tissues for the same absorbed dose. An Appendix gives Relative Corrected Lengths (RCL) for chromosomes of the human karyotype which should be used when testing the various exchange aberration categories for random chromosome participation.

The yield of X-ray-induced chromosome aberrations in unstimulated human lymphocytes is greatly enhanced when the cells are incubated with cytosine arabinoside (ara-C) after irradiation. There is an increase in aberration yield with increasing time of incubation in ara-C (1, 2 or 3 h). When G2 cells are X-irradiated and incubated with ara-C until fixation, the deletion yields are considerably increased, but no interchanges are observed. In the absence of ara-C, 0.29 interchanges per cell were observed. The G2 results suggest that ara-C inhibits the repair of damage that leads to the formation of exchange aberrations. The increase in yield in unstimulated lymphocytes is interpreted to be due to an accumulation of strand breaks caused by ara-C inhibition of repair; when the inhibition is reversed with deoxycytidine, these breaks can interact to form aberrations. A possible mechanism for the induction of chromosome aberrations by X-rays is offered on the basis of these results.

Frequencies of chromosomal abberations after irradiation with X-rays of peripheral blood lymphocytes in vitro were determined at different times after initiation of cultures. In each culture, the kinetics of cell multiplication was followed by using BrdU labelling and differential staining of chromosomes. The results indicate that the mixing up of first and second cell cycle cells at later sampling times cannot explain the observed variation in the frequencies of chromosomal aberrations but that donor-to-donor variation is a predominant factor influencing yields of aberrations. The condition of a donor seems to be most important because repeats on the same donor also showed marked variability.

Dicentric and total aberration yields induced in human lymphocytes by 15-MeV neutrons under conditions of oxygenation and of anoxia have been fitted to a dose-response curve using the function Y = alpha D + beta D2. An oxygen-enhancement ration (OER) ranging from 3.7 at low yields to 1.6 at high yields was calculated from the coefficients of the dicentric yield curves and evidence is presented which suggests that oxygen does not act as a dose-modifying agent in this system. High dose RBE values of 1.2 and 2.1 with respect to 250 kVp X-rays for oxygenated and anoxic conditions were also obtained. Coefficients for total aberration yield gave similar values to dicentrics for both OER and RBE.

We have compared the frequencies of chromosomal aberrations in skin fibroblasts from persons with Down's syndrome (trisomy 21) with those from normal diploid controls exposed to 60Co gamma-radiation in vitro. No difference between the chromosomal radiosensitivities of the two groups was observed, nor did the two groups differ in the background frequency of spontaneous aberrations. These data support the hypothesis that the increased in vitro chromosomal radiosensitivity of lymphocytes reported to be associated with trisomy 21 is not typical of all tissues.

Human blood from normal donors was irradiated with 200 R during the Go phase, and the X-ray sensitivity of early and late dividing lymphocytes in culture was expressed as percentage of induced dicentrics. Cells in first or subsequent divisions were individualized by BrdU-Giemsa techniques. Lymphocytes in the first division at 40, 44 and 72 h after the start of culture had a lower sensitivity to radiation than lymphocytes making their first division at 48, 52 and 56 h. It was observed that: (a) the combination of radiation followed by BrdU did not increase the clastogenic action of X-rays, (b) X-rays in the dose and duration used in our cultures did not increase the frequency of SCEs, and (c) minor changes in culture conditions probably influenced the frequency of SCEs.

The quantitative analysis of the chromosome rearrangements detected in 2128 R-banded metaphases, obtained from gamma-irradiated human lymphocytes after 48 to 96 h in culture is reported. Depending on the culture time, and possibly on the dose of radiation (from 1 to 3 Gy), the most frequent type of rearrangement was either dicentrics or reciprocal translocations. In first generation mitoses, the frequency of cells without rearrangement ranged from 0.66 to 0.18, and the mean number of rearranged chromosomes per cell from 0.79 to 3.28. The dose-response curve follows a quadratic function for dicentric aberration yields, but not for other rearrangements.

Development of the procedure to stimulate peripheral blood lymphocytes has greatly facilitated the understanding of chromosome aberration formation and repair mechanisms in human cells. Yet, because radiation induces far more initial chromosome breaks than are observed as aberrations in metaphase, it has not been possible to examine the kinetics of primary chromosome breakage and rejoining with this procedure. An improved method to induce premature chromosome condensation in unstimulated lymphocytes has been used to study primary chromosome breakage, rejoining, and ring formation at various times after irradiation with up to 800 rad of X-rays. The dose-response relations for chromosome fragments analyzed immediately or 1, 2, or 24 h after exposure were found to be linear. Rapid rejoining of chromosome fragments, which takes place in the first 3 h after X-ray exposure, was not correlated with a simultaneous increase in the formation of rings. The yield of rings per cell scored 24 h after irradiation, however, increased significantly and fit a linear quadratic equation. Both chromosome fragment rejoining and ring formation were completed about 6 h after irradiation. The frequency distributions of rings among cells followed a Poisson distribution, whereas chromosome fragments were overdispersed.

A comparison has been made of dicentric yields in GO lymphocytes between man and crab-eating monkey, Macaca fascicularis, after acute and chronic gamma-irradiations. With acute irradiation (49.6 rad/min) there was no significant difference between them, but for the chronic irradiation (17.1 rad/h) a significant difference was observed between the species. When the dose-response relations were fitted to the linear-quadratic model (Y = alpha D + beta D2), the species-difference observed for chronic irradiation was almost entirely due to change in the value of beta. In addition, after chronic irradiation the beta-value for monkey was almost negligible, but that for man was significant. Post-irradiation incubation experiment showed that cells with dicentrics were partly eliminated during the course of chronic irradiation, because there were appreciable reductions of dicentric yields (ca. 25% for both man and monkey at 400 rad) together with mitotic indices (ca. 30% and 60% for man and monkey, respectively, at 400 rad). Accordingly, it would be reasonable to postulate that GO repair for dicentrics other than selection mechanism must play a major role in the effects of low dose rate. It can be further suggested that GO-repair capacity for chromosal damages leading to dicentrics may be different among different primate species.

Human lymphocytes from normal and Down syndrome (DS) subjects were examined to determine the effect of 3-aminobenzamide (3AB) on X-ray-induced chromosome aberrations. Lymphocytes were treated with 150 or 300 rad of X-rays in the presence of 3 mM 3AB for various times after irradiation, and then the cells were analyzed for the presence of chromosome aberrations in mitotic cells. 3-Aminobenzamide had no effect on the frequency of chromosome aberrations produced by X-rays in G0 lymphocytes from normal subjects. In contrast, lymphocytes from DS patients displayed an increase in the frequency of chromosome aberrations as a result of treatment with X-rays in the presence of 3AB. These observations indicate that DS lymphocytes are more sensitive to the inhibition of poly(ADP)ribose synthetase than normal lymphocytes.

The induction of chromosome aberrations, micronuclei and sister-chromatid exchanges in human G0 lymphocytes by 24-keV epithermal neutrons has been measured. Positive linear dose responses were obtained for the 3 end points, with a tendency to saturation at higher dose for SCE production. In all cases, the responses to 24-keV neutrons were characteristic of high-LET radiations.

Human peripheral blood lymphocytes cultured in the presence of low concentrations of bleomycin (BLM), 0.01-0.1 microgram/ml, for 48 h and then treated with a high concentration (1.5 microgram/ml) of the same agent or with 1.5 Gy X-rays, became significantly less sensitive to the induction of chromosomal damage than those which did not receive the pre-treatment with BLM. They responded with lower frequencies of chromatid and isochromatid breaks. These results lend further support to the operation of an adaptive repair system in lymphocytes which offers resistance and cross-resistance to the induction of chromosomal damage by the same or similar DNA-damaging agents.