Increased Chromosome Aberrations in Cells Exposed Simultaneously to Simulated Microgravity and Radiation.

Space radiation and microgravity (µG) are two major environmental stressors for humans in space travel. One of the fundamental questions in space biology research is whether the combined effects of µG and exposure to cosmic radiation are interactive. While studies addressing this question have been carried out for half a century in space or using simulated µG on the ground, the reported results are ambiguous. For the assessment and management of human health risks in future Moon and Mars missions, it is necessary to obtain more basic data on the molecular and cellular responses to the combined effects of radiation and µG. Recently we incorporated a µG⁻irradiation system consisting of a 3D clinostat synchronized to a carbon-ion or X-ray irradiation system. Our new experimental setup allows us to avoid stopping clinostat rotation during irradiation, which was required in all other previous experiments. Using this system, human fibroblasts were exposed to X-rays or carbon ions under the simulated µG condition, and chromosomes were collected with the premature chromosome condensation method in the first mitosis. Chromosome aberrations (CA) were quantified by the 3-color fluorescent in situ hybridization (FISH) method. Cells exposed to irradiation under the simulated µG condition showed a higher frequency of both simple and complex types of CA compared to cells irradiated under the static condition by either X-rays or carbon ions.

A three-stage genome-wide association study identifies a susceptibility locus for late radiotherapy toxicity at 2q24.1.

There is increasing evidence supporting the role of genetic variants in the development of radiation-induced toxicity. However, previous candidate gene association studies failed to elucidate the common genetic variation underlying this phenotype, which could emerge years after the completion of treatment. We performed a genome-wide association study on a Spanish cohort of 741 individuals with prostate cancer treated with external beam radiotherapy (EBRT). The replication cohorts consisted of 633 cases from the UK and 368 cases from North America. One locus comprising TANC1 (lowest unadjusted P value for overall late toxicity=6.85×10(-9), odds ratio (OR)=6.61, 95% confidence interval (CI)=2.23-19.63) was replicated in the second stage (lowest unadjusted P value for overall late toxicity=2.08×10(-4), OR=6.17, 95% CI=2.25-16.95; Pcombined=4.16×10(-10)). The inclusion of the third cohort gave unadjusted Pcombined=4.64×10(-11). These results, together with the role of TANC1 in regenerating damaged muscle, suggest that the TANC1 locus influences the development of late radiation-induced damage.

Chromosomal radiosensitivity analyzed by FISH in lymphocytes of prostate cancer patients and healthy donors.

It is known that about 5-10% of cancer patients show severe clinical side effects during and after radiotherapy due to enhanced sensitivity to ionizing radiation. Identification of those radiosensitive individuals by a reliable in vitro assay before onset of treatment would have a great impact on successful radiotherapy. We compared the radiosensitivity of the chromosomes 2, 11 and 17 in prostate cancer patients with and without severe side effects after radiotherapy and in age-matched healthy donors. Each cohort consisted of at least 10 donors. Peripheral blood lymphocytes were irradiated ex vivo with 0.5, 1 und 2 Gy ((137)Cs γ rays). We investigated the radiosensitivity of the chromosomes 2, 11 and 17 by scoring of 100 FISH painted metaphases for each dose point and donor group. Statistical analyses were performed by nonparametric tests as Mann-Whitney test and Kruskal-Wallis ANOVA, paired Wilcoxon rank test, χ(2) goodness-of-fit test and Spearman rank-order correlation at a significance level of P < 0.05. Analysis of the overall aberration yield revealed no significant differences between any donor groups. The translocation frequencies of the chromosomes 2, 11 and 17 coincided with their relative size. Thus, none of the chromosomes analyzed were more or less radiosensitive with respect to the genomic translocation frequency. Additionally, neither of the chromosomes showed enhanced or diminished radiosensitivity in one of the donor groups. Furthermore, variance analyses revealed that the distribution pattern of the aberrations per donor did not differ in each donor group even after exposure to 2 Gy. Prostate cancer patients with and without side effects cannot be distinguished from healthy donors based on aberration yield after irradiation with γ rays.

Gadolinium enhances the sensitivity of SW-1573 cells for thermal neutron irradiation.

Gadolinium neutron capture therapy (Gd-NCT) is an experimental cancer treatment based on the physical principal that neutron capture by gadolinium-157 ensures the release of focal high-dose radiation, such as gamma-rays and electrons. Survival and induction of chromosomal aberrations of human SW-1573 cells was studied after thermal neutron irradiation without and with gadolinium. After neutron irradiation with Gd-DTPA, an alpha-enhancement factor of 2.3 was obtained compared to thermal neutron irradiation alone. Gd-DTPA could not radioenhance the cells for gamma-ray irradiation. Induction of colour junctions and chromosome fragments by thermal neutron irradiation and Gd-NCT were studied using PCC-FISH. Correlations (r2-value) between survival and chromosome aberrations ranged from 0.81 to 0.94 for colour junctions and from 0.78 to 0.98 for chromosome fragments of chromosomes 18 and 2 respectively. Thermal neutron irradiation with or without gadolinium induced more chromosome aberrations than gamma-ray irradiation. After correction for chromosome length it appeared that both chromosomes were equally sensitive to radiation. It is concluded that Gd-NCT at a non-toxic concentration of gadolinium is effective in inducing cell death and chromosome aberrations in in vitro cell cultures.

[Comparative investigation of structural and gene somatic mutations in workers of nuclear chemical plants. I. A study of stable and unstable chromosome aberrations].

A study of frequency of unstable chromosome aberrations in 50 workers of nuclear chemical plants in remote period after beginning or finishing professional contact with ionizing radiation was carried out. 14 persons from this cohort were mainly whole-body exposed to external gamma-rays and 36 were exposed to combined external and internal radiation from incorporated Pu nuclides. In results of this irradiating practically every subject had a chronical radiation sickness. In the 1-st group the frequency of unstable aberrations varied from 0.2 to 3.6 per 100 cells and exceeded reliably control level in 5 persons. In the 2-nd group the frequency of unstable aberrations varied from 0 to 11.6 per 100 cells and exceeded reliably control level in 20 examined workers. The FISH study of frequency of stable aberrations was performed in 13 subjects who were exposed to combined external and internal radiation. Total frequency of complete and incomplete translocations varied from 0.6 to 18.5 aberrations per genome per 100 cells and reliable exceeded control level in 9 subjects. Non-random participation in exchange rearrangements (translocations) was revealed for used set of chromosomes (2, 3 and 8).

The radiation sensitivity of human chromosomes 2, 8 and 14 in peripheral blood lymphocytes of seven donors.

PURPOSE: To investigate if deviations from DNA-proportional distribution of radiation-induced chromosomal aberrations are individually variable. MATERIALS AND METHODS: Peripheral blood lymphocytes were collected from seven healthy donors and exposed to different doses of gamma rays. Chromosomes 2, 8 and 14 were painted in different colors and aberrations scored with the help of an image-analysis system. RESULTS: Chromosome 2 was generally less sensitive than expected on the basis of DNA-proportional distribution and the extent of inter-donor variability was minimal. A higher than expected frequency of aberrations was found in chromosome 14 of five donors, while a higher than expected frequency of aberrations was found in chromosome 8 of two donors. CONCLUSIONS: Inter-donor variability may explain some of the controversies regarding the inter-chromosomal distribution of radiation-induced aberrations.

Interstitial telomeric repeats are not preferentially involved in radiation-induced chromosome aberrations in human cells.

Telomeric repeat sequences, located at the end of eukaryotic chromosomes, have been detected at intrachromosomal locations in many species. Large blocks of telomeric sequences are located near the centromeres in hamster cells, and have been reported to break spontaneously or after exposure to ionizing radiation, leading to chromosome aberrations. In human cells, interstitial telomeric sequences (ITS) can be composed of short tracts of telomeric repeats (less than twenty), or of longer stretches of exact and degenerated hexanucleotides, mainly localized at subtelomeres. In this paper, we analyzed the radiation sensitivity of a naturally occurring short ITS localized in 2q31 and we found that this region is not a hot spot of radiation-induced chromosome breaks. We then selected a human cell line in which approximately 800 bp of telomeric DNA had been introduced by transfection into an internal euchromatic chromosomal region in chromosome 4q. In parallel, a cell line containing the plasmid without telomeric sequences was also analyzed. Both regions containing the transfected plasmids showed a higher frequency of radiation-induced breaks than expected, indicating that the instability of the regions containing the transfected sequences is not due to the presence of telomeric sequences. Taken together, our data show that ITS themselves do not enhance the formation of radiation-induced chromosome rearrangements in these human cell lines.

Dose dependency of FISH-detected translocations in stable and unstable cells after Cs gamma irradiation of human lymphocytes in vitro.

Human peripheral lymphocytes were exposed to 137Cs gamma-rays (0-4.3 Gy) in order to check the impact of unstable cells on the dose-response curve for translocations. Chromosomes 2, 4 and 8 were FISH-painted. 17,720 first dividing cells were analysed. For the discrimination between stable and unstable cells the painted and the counter-stained chromosomes were analysed at doses of 1 Gy and higher. The cell distribution of translocations follows a Poisson distribution. The data were fitted to the linear-quadratic function, y = c + alphaD + betaD2. As expected, the alpha coefficients of the dose-response curves for translocations in stable cells or in total cells do not differ. However, at doses >1 Gy, the frequency of all translocations in stable cells seems to be lower than the frequency in total cells. For the establishment of calibration curves for past dose assessment purposes, only complete translocations should be scored, in order to estimate reliable doses.

Lymphocyte chromosomal aberrations and their complexity induced in vitro by plutonium-239 alpha-particles and detected by FISH.

G0 human lymphocytes were exposed in vitro to plutonium-239 alpha-particles, with doses ranging from 0 to 1.62 Gy, to provide a dose response curve and to compare complex rearrangements produced by high LET radiation with low LET data from previous work. Metaphase chromosomes 1 and 2 were painted using fluorescence in situ hybridization (FISH) whole chromosome probes. All unstable and stable aberrations involving the painted chromosomes were scored. The whole genome corrected alpha-coefficient for dicentrics was 0.244 +/- 0.023 and for total translocations 0.346 +/- 0.032, when considering simple and complex exchanges. The ratio of bicoloured total translocations to bicoloured dicentrics was 1.21 +/- 0.15 and the ratio of 2-way to 1-way translocations was 1.73 +/- 0.27 for apparently simple exchanges only. A correlation was noted between the distributions of dicentrics and translocations and this applied even when the complex rearrangements were removed. 20% of the observed rearrangements were complex and this observation was independent of dose. Qualitatively, following irradiation with alpha-particles the complex rearrangements observed were of a greater complexity than seen after X- or gamma-rays. Using the Savage and Simpson system to classify the complex rearrangements, the higher order complexes were found to be the most common type observed. However the insertion type increased while the 2F + 2G types decreased when complex rearrangements induced by alpha-particles were compared to those formed after X- or gamma-irradiation.

Translocation frequencies measured in patients one year after radioactive iodine therapy for thyrotoxicosis.

PURPOSE: To investigate the incidence of translocations induced by iodine-131 therapy in thyrotoxicosis patients 1 year after the administration of the radiolabelled compound. MATERIALS AND METHODS: Tricolour FISH with whole-chromosome-specific probes for chromosomes 2, 4 and 8 was used for scoring translocations. From the genomic translocation frequencies, derived using the Lucas formula, equivalent whole-body doses were calculated, based on the in vitro (60)Co gamma-ray dose-response curve. RESULTS: A total of 101 translocations were observed in 4864 metaphases, 63% being of the two-way type. In the control group used for obtaining dose-response data, nine translocations were observed in 5278 metaphases, 55% being two-way translocations. No correlation was found between the observed frequency of translocations and administered radioactivity. Using the in vitro dose-response, an estimated average dose for the group of nine patients of 0.79 +/- 0.22Gy was obtained. Compared with frequencies following the assumption that the involvement of a particular chromosome in a two-break exchange-type aberration is proportional to its DNA content, chromosome 4 was more frequently involved and chromosomes 2 and 8 less frequently involved in chromosomal rearrangements. CONCLUSION: This study shows that (131)I therapy for thyrotoxicosis patients induced translocations, especially in chromosome 4, which could be detected 1 year after the administration of the radiolabelled compound.

Biological dosimetry of chernobyl cleanup workers: inclusion of data on age and smoking provides improved radiation dose estimates.

We report the results of a study of chromosome translocations in 126 Russian subjects who participated in the cleanup activities at Chernobyl and another 53 subjects, from other places in Russia, who were not exposed at Chernobyl. In agreement with our earlier study, we find increased translocation frequencies among the exposed compared to Russian controls. We describe statistical methods for estimating the dose of ionizing radiation determined by scoring chromosome translocations found in circulating lymphocytes sampled several years after exposure. Two statistical models were fitted to the data. One model assumed that translocation frequencies followed an overdispersed Poisson distribution. The second model assumed that translocation frequencies followed a negative binomial distribution. In addition, the effects of radiation exposure were modeled as additive or as multiplicative to the effects of age and smoking history. We found that the negative binomial model fit the data better than the overdispersed Poisson model. We could not distinguish between the additive and the multiplicative model with our data. Individual dose estimates ranged from 0 (for 43 subjects) to 0.56 Gy (mean 0.14 Gy) under the multiplicative model and from 0 to 0.95 Gy (mean 0.15 Gy) under the additive model. Dose estimates were similar under the two models when the number of translocations was less than 4 per 100 cells. The additive model tended to estimate larger doses when the number of translocations was greater than 4 per 100 cells. We also describe a method for estimating upper 95% tolerance bounds for numbers of translocations in unexposed individuals. We found that inclusion of data on age and smoking history was important for dose estimation. Ignoring these factors could result in gross overestimation of exposures, particularly in older subjects who smoke.

Correlation between cell reproductive death and chromosome aberrations assessed by FISH for low and high doses of radiation and sensitization by iodo-deoxyuridine in human SW-1573 cells.

PURPOSE: To study the relationship between cell reproductive death and exchange frequency in SW-1573 human lung tumour cells with and without incorporated iodo-deoxyuridine (IdUrd) following irradiation of plateau-phase cultures with y-rays. METHOD: Linear-quadratic (LQ) analysis was performed for the data on clonogenic survival and on the frequency of chromosomal exchanges studied with fluorescence in situ hybridization in chromosomes X and 2. RESULTS: Differences in the LQ parameters alpha and beta of both non-sensitized and sensitized chromosomes were found. In both chromosomes an increase in the number of chromosomal exchanges in IdUrd-radiosensitized cells compared with non-sensitized cells was observed. The alpha-enhancement factors of 1.7 and 1.9 for the X-chromosome and for chromosome 2, respectively, are similar. For the X-chromosome, the beta coefficient increased by a factor of 3.9 and for chromosome 2 by a factor of 1.4. After correction to a full genome equivalence, no significant difference in alpha was found between chromosomes X and 2 for both control and sensitized cells. In contrast, an almost 2.8 times higher beta was found for the sensitized X-chromosome compared to this value for chromosome 2. CONCLUSIONS: It can be concluded that the linear-quadratic analysis of dose-response relationships offers insights into the correlation between cell survival and induction of exchanges in non-sensitized and radiosensitized cells.

Inter-laboratory comparison of cytogenetic endpoints for the biomonitoring of radiological workers.

PURPOSE: The evaluation of different cytogenetic endpoints of radiation damage for the biomonitoring of contract workers temporarily employed at nuclear power plants. MATERIALS AND METHODS: Blood samples from six donors were irradiated in vitro with doses ranging from 0.1 to 2Gy 60Co gamma-rays. Compared were a conventional analysis for dicentrics, the conventional micronucleus (MN) assay, the centromere micronucleus assay using p82H and an alphaAllCen pancentromeric probe, and tricolour FISH with chromosome 2, 4 and 8 DNA probes for the scoring of translocations. RESULTS: Agreement in the number of MN between Giemsa-and propidium iodine fluorescence-stained preparations was obtained. The control samples showed higher centromere positivity for the MN after FISH with the p82H probe compared with the alphaAllCen probe. The MN results with both probes showed a slight but systematic increase in the number of centromere-positive MN with dose, indicating that radiation, although principally clastogenic, also has aneuploidogenic properties. The values of the genomic translocation frequency (FG) derived from the observed translocation frequencies were systematically higher than the dicentric yields. Comparing the sensitivity of the different methods with restriction of the scoring time to 1 day for biomonitoring purposes, the centromere micronucleus assay had the lowest dose detection limit (0.1 to 0.2 Gy). CONCLUSION: This study shows that at present only the centromere micronucleus assay can combine high sensitivity with a reasonable scoring time for the biomonitoring of relatively large populations.

The distribution of chromosome damage, non-reciprocal translocations and clonal aberrations in lymphocytes from Chernobyl clean-up workers.

In this paper we determined whether the frequencies of translocations and insertions are proportional to chromosome size in peripheral blood lymphocytes from Chernobyl nuclear accident clean-up workers and healthy unexposed control subjects. The frequency of aberrations among chromosomes 1, 2 and 4 in both groups was found to be significantly different from the distribution expected on the basis of chromosome size, although the difference was only marginally significant in controls. We also determined whether differences exist in aberration frequencies measured by two scoring systems: the classical method, where reciprocal exchanges are scored as one event, and PAINT, where each break junction is scored as a single event. The two scoring systems gave highly correlated results which yielded an interpretable arithmetic relationship between frequency measurements using the two systems. Approximately 34% of all translocations were observed to be non-reciprocal, and cells bearing clones of abnormal cells were observed in 6 of 198 subjects (3.0%). Our results demonstrate that clones of abnormal cells and the presence of non-reciprocal translocations contribute to the non-proportional distribution of radiation-induced and spontaneous cytogenetic damage.

Increased chromosome exchange frequencies in iodo-deoxyuridine-sensitized human SW-1573 cells after gamma-irradiation.

The induction of chromosome exchanges was investigated in SW-1573 human lung tumour cells radiosensitized with iododeoxyuridine (IdUrd) and irradiated with gamma-rays. Following treatment chromosome 2 and X were analyzed using fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries. The yield of chromosome exchanges involving chromosome 2 was higher than those involving chromosome-X. On the basis of the DNA content the relative involvement of the X-chromosome in exchange frequencies after 2 Gy was much higher than of chromosome 2. After 4 Gy the relative involvement of both chromosomes in exchanges is approximately equal. After radiosensitization, increased chromosome exchange frequencies are observed in both studied chromosomes. For the total chromosome exchange frequencies the sensitizer enhancement ratio (SER) at 2 Gy is 1.8 and 1.3 for chromosome 2 and X respectively. The SER at 4 Gy for total exchange frequencies is 1.6 and 1.9 chromosome 2 and X respectively. For reciprocal exchanges at 2 Gy higher SER values and at 4 Gy lower SER values were observed for both chromosomes.

Distribution of radiation-induced exchange aberrations in human chromosomes 1, 2 and 4.

PURPOSE: To examine the distribution of radiation-induced breakpoints in chromosomes 1, 2 and 4 both in relation to their DNA content and by localization of the breaks along each chromosome. MATERIAL AND METHODS: The work consisted of two studies, one with chromosomal aberrations found in persons after accidental exposure in Estonia in 1994 and another involving aberrations seen in in vitro-irradiated lymphocytes. Localization of breakpoints in painted chromosomes involved in complete exchange-type aberrations was conducted by applying a computerized measuring system on stored image-files. RESULTS AND CONCLUSIONS: The yield of exchanges in chromosomes 1, 2 and 4 in both studies was equal to that expected from their DNA content. In contrast, the breakpoint location of complete exchanges within these chromosomes was not random. Chromosomes 1 and 4 contained more breaks in the middle parts of the p and q arms, whereas breaks were observed more uniformly along chromosome 2. Complete exchanges, however, were very rare in the terminal regions of all three chromosomes, most probably resulting from limitations in the resolution of small painted pieces.

Comparison of radiation-induced aberration frequencies in chromosomes 1 and 2 of two human donors.

PURPOSE: The purpose of this study was to analyse donor and time dependent variations in the frequencies of radiation-induced aberrations in chromosomes 1 and 2. MATERIALS AND METHODS: Human lymphocytes from two donors were irradiated with 1 and 2 Gy of X-rays. Chromosomal aberrations were scored in chromosomes 1 and 2 painted with different fluorochromes and in Giemsa stained cells. Two time displaced experiments were performed with lymphocytes of each donor. RESULTS: In cells of both donors chromosome 1 was generally more frequently involved in translocations than chromosome 2. This result was not always reproducible. Chromosome 2 showed a higher frequency of acentric fragments, especially following a dose of 1 Gy. Again interexperimental variations were observed. No differences between the two chromosomes were seen with regard to other aberration types. Both chromosomes showed less dicentrics and more acentric fragments than proportional to their DNA content. CONCLUSIONS: Chromosomes 1 and 2 show a different sensitivity to ionizing radiation. The difference is dependent on the aberration type and is not always reproducible. This variability could contribute to the difficulty in reaching a consensus regarding the radiosensitivity of individual chromosomes.

Effectiveness of 0.28 keV carbon K ultrasoft X-rays at producing simple and complex chromosome exchanges in human fibroblasts in vitro detected using FISH.

PURPOSE: To study the effects of carbon K ultrasoft X-rays, which produce a single photoelectron with a track length of < 7 nm, on the production of structural chromosome-type changes. MATERIALS AND METHODS: Untransformed human fibroblasts (HF12) were irradiated in G1 phase. Aberrations were analysed using fluorescence in situ hybridization using multi-coloured chromosome specific DNA probes for chromosomes 1 and 2 and an alpha-satellite pan-centromeric probe. RESULTS: CK X-rays have a high efficiency per unit absorbed dose for producing simple and complex exchanges. Mean absorbed doses of 0.33-1.31 Gy produce simple exchanges with a predominantly linear dose dependency, and visibly complex exchanges increased by more than the power 2 of the dose, with no evidence of a linear component. The proportion of exchanges that are visibly complex ranged from 9% to 46%. CONCLUSIONS: The linear response for simple exchanges provides further support to the hypothesis that damaged DNA may be able to interact with undamaged DNA. The high proportion of complex exchanges may be due to the increased efficiency of double-strand break induction and to the high density of tracks per unit absorbed dose targeting pre-existing sites, some of which may be close to the incident nuclear membrane.

Cytogenetic effects of energetic ions with shielding.

In order to understand the effects of shielding on the induction of biological damages by charged particles, we conducted experiments with accelerated protons (250 MeV) and iron particles (1 GeV/u). Human lymphocytes in vitro were exposed to particle beams through polyethylene with various thickness, and chromosomal aberrations were determined using FISH technique. Dose response curves for chromosome aberrations were obtained and compared for various particle types. Experimental results indicated that for a given absorbed dose at the cell, the effectiveness of protons and iron particles in the induction of chromosomal aberrations was not significantly altered by polyethylene with thickness up to 30-cm and 15-cm respectively. Comparing with gamma rays, charged particles were very effective in producing complex chromosomal damages, which may be an important mechanism in alterating functions in non dividing tissues, such as nervous systems.

[Registration of stable aberrations in peripheral blood lymphocytes using G-differential chromosome staining and fluorescence in situ hybridization methods]. / Registratsiia stabil'nykh aberratsii v limfotsitakh perifericheskoi krovi metodami G-differentsial'nogo okrashivaniia khromosom i fliuorestsentnoi in situ gibridizatsii.

G-banding analysis and fluorescence in situ hybridization (FISH) for whole chromosomes 1, 2 and 4 were applied in comparative assay for frequency of stable chromosome aberrations in 37 individuals with previous exposure to radiation (15 clean-up workers/liquidators of Chernobyl nuclear power plant accident and 22 residents of radiocontaminated areas) and in 18 individuals of a reference group. In G-banding analysis of stable aberrations, we used classification of Ohtaki K. et al., 1992, in compliance with ISCN, 1985. FISH-assay for translocations is performed in accordance with classification of Tucker J.D. et al., 1995. Comparison of the results reveals statistical trustworthy correlation between the two assays. The results point out that FISH for translocations in as few as three chromosomes, when combined with screening of numerous metaphases, provides sensitivity comparable with that provided by G-banding which covers the whole genome.