Chromosome Exchange Aberrations Research Articles

Investigation of Spatial Organization of Chromosome Territories in Chromosome Exchange Aberrations After Ionizing Radiation Exposure.

Higher-order organization of the human genome is well established with chromosomes occupying distinct domains or territories in the interphase nucleus. Spatial organization of chromosome territories in the interphase nucleus occurs in a cell-type-specific manner. Since both stable and unstable aberrations induced by ionizing radiation involve the exchange of material between two or more chromosomes, this study investigated the role of spatial organization of chromosome domains in ionizing-radiation-induced chromosome translocation events. Using multicolor fluorescence in situ hybridization, the study characterized the positioning of each human chromosome relative to its neighborhood territories in the interphase nucleus of lymphocytes and B-lymphoblastoid cells before ionizing radiation and compared this interphase positioning with the spectrum of exchanges observed after ionizing radiation in the metaphase chromosomes. In addition to multicolor fluorescence in situ hybridization, the genome-wide chromosome conformation capture technique (Hi-C) was also performed in mock and x-ray-irradiated human B-lymphoblastoid and fibroblast cells to characterize the interactions among chromosomes and to assess the genome reorganization changes, if any, after ionizing radiation exposure. On average, 35-50% of the total translocations induced by x rays and neutrons correlated with proximity of chromosome territories detected by multicolor fluorescence in situ hybridization in both lymphocytes and lymphoblastoid cells. The translocation rate observed in proximally positioned chromosome territories was consistently higher than distally located territories and was found to be statistically significant (p = 0.01) in human lymphoblastoid cells after x rays. The interchromosome interaction frequencies detected by Hi-C correlate fairly well with ionizing-radiation-induced translocations detected by multicolor fluorescence in situ hybridization, suggesting the importance of chromosome proximity effects in ionizing-radiation-induced chromosomal translocation events.

Complex interchanges as a complex function of chromosome organisation

A Monte Carlo technique was developed for biophysical modelling of structural organisation of 46 chromosomes within human lymphocyte interphase nucleus. The technique takes into account: different levels of chromatin organisation, non-random localisation of particular chromosomes and chromosome loci dynamics. All chromosomes in a nucleus were modelled as polymer globules. A dynamic pattern of intra/interchromosomal contacts was simulated to predict radiation-induced chromosomal exchange aberrations (CA). Distance dependence of interaction probability was calculated directly with taking DNA break repair into account. Dose-response for simple and complex CA was calculated to analyse mFISH data for human lymphocytes. Calculated simple CA frequencies fitted the experimental data well. Unexpectedly, complex aberrations were underestimated, despite the dense packaging of chromosome territories within a nucleus. To study sensitivity of dose-response to uncertainty of chromosome organisation, CA were recalculated for the alternative concept of nucleus organisation, the SCD model. The simulation showed the underestimation of complex CA yield as well. The movement of damaged loci from different chromosomes to common repair factories was proposed as an additional mechanism of complex CA formation.

High yields of isochromatid breaks and successive formation of chromosome exchanges may lead to reproductive cell death following high-LET irradiation

Abstract To clarify the relationship between cell death and chromosomal aberrations following exposure to heavy-charged ion particles beams, exponentially growing Human Salivary Gland Tumor cells (HSG cells) were irradiated with various kinds of high energy heavy ions; 13 keV/μm carbon ions as a low-LET charged particle radiation source, 120 keV/μm carbon ions and 440 keV/μm iron ions as high-LET charged particle radiation sources. X-rays (200 kVp) were used as a reference. Reproductive cell death was evaluated by clonogenic assays, and the chromatid aberrations in G2/M phase and their repairing kinetics were analyzed by the calyculin A induced premature chromosome condensation (PCC) method. High-LET heavy-ion beams introduced much more severe and un-repairable chromatid breaks and isochromatid breaks in HSG cells than low-LET irradiation. In addition, the continuous increase of exchange aberrations after irradiation occurred in the high-LET irradiated cells. The cell death, initial production of isochromatid breaks and subsequent formation of chromosome exchange seemed to be depend similarly on LET with a maximum RBE peak around 100–200 keV/μm of LET value. Conversely, un-rejoined isochromatid breaks or chromatid breaks/gaps seemed to be less effective in reproductive cell death. These results suggest that the continuous yield of chromosome exchange aberrations induced by high-LET ionizing particles is a possible reason for the high RBE for cell death following high-LET irradiation, alongside other chromosomal aberrations additively or synergistically.

Radiation-induced chromosome aberrations: insights gained from biophysical modeling.

Enzymatic misrepair of ionizing-radiation-induced DNA damage can produce large-scale rearrangements of the genome, such as translocations and dicentrics. These and other chromosome exchange aberrations can cause major phenotypic alterations, including cell death, mutation and neoplasia. Exchange formation requires that two (or more) genomic loci come together spatially. Consequently, the surprisingly rich aberration spectra uncovered by recently developed techniques, when combined with biophysically based computer modeling, help characterize large-scale chromatin architecture in the interphase nucleus. Most results are consistent with a picture whereby chromosomes are mainly confined to territories, chromatin motion is limited, and interchromosomal interactions involve mainly territory surfaces. Aberration spectra and modeling also help characterize DNA repair/misrepair mechanisms. Quantitative results for mammalian cells are best described by a breakage-and-reunion model, suggesting that the dominant recombinational mechanism during the G(0)/G(1) phase of the cell cycle is non-homologous end-joining of radiogenic DNA double strand breaks. In turn, better mechanistic and quantitative understanding of aberration formation gives new insights into health-related applications.

Impact of radiation quality on the spectrum of induced chromosome exchange aberrations

Purpose: To study the impact of radiation quality on the spectrum of chromosome exchange aberrations in human lymphocytes using chromosome arm-specific and telomeric probes. The analysis is focused on: (1) incomplete exchanges, (2) interstitial fragments, (3) interarm intrachanges and (4) the complexity of the aberration patterns. The present data after neutron exposure are compared with previously obtained data after X-irradiation. Materials and methods: Isolated human lymphocytes from three donors were irradiated with 1 MeV fast neutrons (0.25, 0.5, 1.0, 1.5, 2.0Gy). Analysis was performed on first post-irradiation metaphases with arm-specific probes for chromosome 1 in combination with a pan-centromeric probe, or with telomeric and centromeric PNA probes. Results: In comparison with X-rays, exposure to neutrons leads to: (1) similar frequencies of incomplete exchanges or terminal deletions, (2) a significantly higher induction of both inter- and intraarm intrachanges, (3) a higher proportion of complex aberrations, and (4) aberrations with a higher degree of complexity, i.e. derived from more chromosome breaks which interact more frequently in a non-reciprocal fashion. Essentially no dose dependence was found for the yield ratios between the various types of chromosomal aberrations. Conclusions: Despite the reduced rejoining eyciency of DNA double-strand breaks induced by high-LET radiation, exposure to neutrons does not lead to enhanced levels of unrejoined chromosome breaks that can be observed as incomplete exchanges in cells that have reached mitosis. Proximity effects are more pronounced after densely ionizing radiation than after sparsely ionizing radiation. Clustered damage produced by neutron tracks results in a high proportion of complex aberrations and in non-reciprocal interactions of chromosome breaks. Most of the exchanges occur within one neutron track and little interaction seems to take place between the breaks formed in different tracks.

Kinetics of DSB rejoining and formation of simple chromosome exchange aberrations

Purpose : To investigate the role of kinetics in the processing of DNA double strand breaks (DSB), and the formation of simple chromosome exchange aberrations following X-ray exposures to mammalian cells based on an enzymatic approach. Methods : Using computer simulations based on a biochemical approach, rate-equations that describe the processing of DSB through the formation of a DNA-enzyme complex were formulated. A second model that allows for competition between two processing pathways was also formulated. The formation of simple exchange aberrations was modelled as misrepair during the recombination of single DSB with undamaged DNA. Non-linear coupled differential equations corresponding to biochemical pathways were solved numerically by fitting to experimental data. Results : When mediated by a DSB-repair enzyme complex, the processing of single DSB showed a complex behaviour that gives the appearance of fast and slow components of rejoining. This is due to the time-delay caused by the action time of enzymes in biomolecular reactions. It is shown that the kinetic- and dose- responses of simple chromosome exchange aberrations are well described by a recombination model of DSB interacting with undamaged DNA when aberration formation increases with linear dose-dependence. Competition between two or more recombination processes is shown to lead to the formation of simple exchange aberrations with a dose-dependence similar to that of a linear-quadratic model. Conclusions : Using a minimal number of assumptions, the kinetics and dose-response observed experimentally for DSB rejoining and the formation of simple chromosome exchange aberrations are shown to be consistent with kinetic models based on enzymatic reaction approaches. A non-linear dose-response for simple exchange aberrations is possible in a model of recombination of DNA containing a DSB with undamaged DNA when two or more pathways compete for DSB repair.

Higher-order structure of interphase chromosomes and radiation-induced chromosomal exchange aberrations

Purpose : To discuss the effects of chromosome structure on the inter- and intrachromosomal exchanges in the framework of the model, which argues that the higher-order chromosome structure contains 10-30 Mbp loop clusters (micelles). Results and conclusions : Intermicelle contacts determine exchangetype chromosome aberrations. Ratios of inter- to intrachromosomal exchanges calculated in the model are similar to the experimental data (literature) for human lymphocytes and flat fibroblasts. The frequency of interchanges is affected by nuclear shape; this might explain the greater number of interchanges observed in 3-D spherical lymphocytes vs. that in 2-D flat fibroblasts. Chromosome configuration (linear vs. folded Rabl) affects the pattern of micelle contacts. The model predicts that chromosomes in haploid Tradescantia microspores have the folded Rabl orientation; this explains quantitatively the low value of the ratio of dicentrics to centric rings observed in these cells.

Dose-response curves for X-ray induced interchanges and interarm intrachanges in human lymphocytes using arm-specific probes for chromosome 1

Frequencies of chromosomal aberrations were estimated in X-irradiated (0; 0.5; 1; 2; 3 and 4 Gy) human lymphocytes using arm-specific probes for chromosome 1 in combination with a pancentromeric probe in a triple colour FISH procedure. This allowed the construction of dose-response curves for both interchanges as well as interarm intrachanges. We found that 11.7–18.4% of aberrant chromosomes contained interarm intrachanges. Assuming a free interaction of randomly induced exchange breakpoints throughout the whole genome, interarm intrachanges occur about 8.7 times more frequently than expected. The close proximity of the two arms of a chromosome in the interphase nucleus appears to promote the formation of interarm intrachanges. Convincing evidence was obtained, by comparing the frequency of colour junctions that occur between 1p–1q and 1p–3q (the arms 1q and 3q are of similar size). With this approach, we observed 8.8 time more colour junctions between 1p–1q than between 1p–3q, illustrating the importance of proximity in the formation of chromosomal exchange aberrations. At doses higher than 2 Gy, a saturation of simple reciprocal aberrations, both for interchanges as well as for interarm intrachanges was observed. Furthermore, we found that symmetrical and assymmetrical simple interchanges as well as interarm intrachanges occur with equal frequencies in human chromosome 1.

Insight into sites

Spatial factors conditioning the formation of radiation-induced chromosome exchange aberrations are reviewed, and concepts such as ‘rejoining distance’ and ‘site’ are re-examined in the light of the unexpectedly high frequencies of multi-break (‘Complex’) exchanges being revealed by FISH painting. Given the anticipated densities of dsb within a nucleus, and assuming random 3-D break distribution, nearest-neighbour analysis indicates that the most likely break interaction distance is a well defined shell, several hundred nm from each break. The sharpness with which this shell is defined increases with break density, and therefore with dose. It is argued that random movement and chance meeting over such distances will not account for the Complex frequencies observed, and that other factors, or modes of formation, must be invoked.

Ultrasoft 1.5 keV Aluminum K X Rays Are Efficient Producers of Complex Chromosome Exchange Aberrations as Revealed by Fluorescence In Situ Hybridization

The electron pairs generated by ultrasoft 1.5 keV aluminum K X-ray photons deposit their energy in tracks of length < 70 nm and provide an ideal tool for analyzing the spatial distribution of breaks and misrepair processes. We have undertaken the analysis of changes in chromosome structure produced by aluminum K X rays in untransformed HF12 human fibroblasts in G1 phase using fluorescence in situ hybridization (FISH). Multicolored chromosome-specific DNA probes for chromosomes 1 and 2 and an alpha-satellite pan-centromeric probe were used to examine in vitro radiation-induced chromosome-type exchange aberrations. After mean doses of 0.37-2.93 Gy the relative frequencies of complex exchanges, derived from three or more breaks in two or more chromosomes, ranged from 15-35%. For the classic break-age-and-rejoining theory to hold, very large interaction distances are needed to account for this high frequency of multibreak interactions, unless many sites pre-exist where several different chromosomes come very close together. Alternatively, damaged DNA may be able to interact with adjacent undamaged DNA, obviating the need for large rejoining distances.

On the time course of the interactions between DNA breaks in the production of a radiation-induced chromosome exchange aberration

Quiescent human lymphocytes were X-irradiated and allowed to repair for various times at 37°C before the cells were challenged with the DNA-repair inhibitor ara-C. The observed yield of chromosome exchange aberrations (dicentrics) was about twice the yield induced by X-rays alone, if ara-C was added immediately after irradiation. The yield as a function of the repair time between X-irradiation and ara-C treatment decreased with a half-life of 15–30 min and was almost down to the baseline yield for X-rays alone after 1 h. This shows that an exchange aberration can be formed from a short-lived DNA break. In contrast, previously published results from dose-split experiments demonstrate that the half-life of the interacting DNA breaks is of the order of several hours. A model is proposed which can account for the different estimates of the time course of the interactions involved in the process which leads to an exchange aberration.

Telomere Staining of Human Chromosomes and the Mechanism of Radiation-Induced Dicentric Formation

The majority of models of radiation action developed over the past half century hold that the curvilinear dose responses exhibited by eukaryotic cells to sparsely ionizing radiations result from the interaction of pairs of lesions produced in sensitive targets of the cell. Within this conceptual framework, chromosomal exchange aberrations (e.g., interchanges) are believed to occur through the interaction of damaged sites on both chromosomes participating in the exchange. In contrast, the model proposed by Chadwick and Leenhouts (as well as some other models) suggests that such exchanges arise from initial radiation damage to only one chromosome, which then becomes associated with an undamaged chromosome. A particular aspect of this theory is that asymmetrical exchanges, such as dicentrics, may be formed from the rejoining of a broken end of one chromosome to the telomere of another. By using a DNA probe that specifically hybridizes to the telomeric region of human chromosomes, we were able to test this assertion directly. After scanning more than 200 dicentrics produced in normal human fibroblasts by 6 Gy of 60Co gamma rays, virtually none were found that contained telomeres located between the centromeres of this aberration type. Therefore, since the proposed telomere-break rejoining process, per se, is not necessarily a central element of the Chadwick-Leenhouts model, we suggest the theory be modified to exclude this mechanism.

X-radiation-induced chromosome breakage in retinoblastoma lymphocytes

We have examined the spontaneous and X-radiation-induced chromosomal damage in normal humans and in patients with retinoblastoma using the BudR-Giemsa technique in lymphocytes cultured for 48 h.9 sporadic unilateral non-hereditary cases, 11 hereditary cases (8 bilateral sporadic and 3 unilateral hereditary cases) and 20 healthy individuals were studied simultaneously. No difference in the spontaneous frequency of chromatid and chromosome aberrations was observed between patients and controls. After treatment with 150 rad the frequency of chromosome exchange aberrations was higher in unilateral hereditary cases than the controls (42.0% ± 5.3 and 22.3 ± 2.6 respectively; p = 0.05). In bilateral sporadic retinoblastoma 2 different groups were observed. A hypersensitive group showed a significant increment in radiation-induced chromosomal exchange aberrations over the control group (46.2% ± 5.4 and 24.2% ± 2.1 respectively; p = 0.01). The other group had a chromosomal exchange frequency similar individuals (26/5% ± 2.0 and 24.2% ± 0.4 respectively; p = 0.10). Sporadic unilateral non-hereditary retinoblastoma had an exchange chromosomal aberration frequency similar to control individuals (26.1% ± 2.8 and 24.6% ± 2.7 respectively; p > 0.10). These results suggest that: 1. (a) There is no relationship between spontaneous chromosome fragility and retinoblastoma. 2. (b) Sporadi unilateral non-hereditary retinoblastoma has normal chromosome sensitivity to X-irradiation. 3. (c) Some hereditary cases of retinoblastoma are sensitive to X-rays while other behave like normals. A mutation or a submicroscopic deletion at a DNA repair locus which is independent of the retinoblastoma gene may cause this radiosensitivity.

The induction of chromosome exchange aberrations by carbon ultrasoft X-rays in V79 hamster cells.

V79 hamster cells in plateau (extended G1) phase were irradiated with either 250 kV ('hard') X-rays or carbon K characteristic ultrasoft X-rays under conditions minimizing cell overlap. These cells were killed most effectively by the carbon X-rays, by a factor of about 3 relative to hard X-rays, in agreement with our previous findings with cells in exponential growth. Chromosome-type aberrations were measured at 3 fixation times within the first division cycle after irradiation, and an approximately uniform sensitivity to aberration induction was found for both radiations. The combined aberration data show that carbon X-rays are 2 or more times as effective as hard X-rays, depending on dose and/or data fit. Exchange aberrations require recombination between two separate chromosomes, but they are induced efficiently by carbon X-rays with a substantial linear component to the dose-response despite the very short electron tracks (approximately less than 7 nm) that they produce in the cell. This implies either that the participating DNA helices must be lying extremely close together at the time of radiation damage, so that one track can effectively damage both helices, or that only one radiation-damaged chromosome is needed to promote an exchange event.

In vivo cytogenetic activity of sulphonylurea drugs in man

A cytogenetic investigation of diabetic patients undergoing treatment with sulphonylurea drugs, particularly chlorpropamide, shows that significantly more chromatid aberrations and chromosome exchange aberrations are present in the lymphocytes of these patients compared with controls. This is taken as evidence of possible mutagenic activity by these drugs, although the possibility cannot be ruled out that the diabetic state itself is a contributory factor

Chromosome-exchange aberrations in human lymphocytes.

SummaryThe probability of finding a given chromosome in a dicentric is directly proportional to the length of that chromosome at interphase, but not to the length of the early replicating portion of the interphase chromosome or to its length at metaphase. The number of dicentric chromosomes and centric rings per cell is limited primarily by the number of centromeres. These results are consistent with the assumption that every chromosome break is a site for chromosome-exchange.

The site distribution parameter for radiation induced chromosome exchanges

The distribution between cells of radiation induced chromosome exchange aberrations in a number of species conforms to the terms of a binomial [ p + (1 − p)] n . The current explanation for this observation is that there are only a few places (termed “sites”) within the nucleus, where the participating chromosomal threads come close enough together for exchange to take place. p is taken as representing the probability of obtaining an exchange in a site, and n the number of “sites” in the cell available for exchange. Mathematical consideration is given to the case where not all cells in the population have the same number of “sites”, and it is concluded that n is a distribution parameter indicating the way in which cells with different numbers of sites are distributed in the population, and that it does not indicate directly the mean number of “sites” available. This conclusion is illustrated by calculated exchange distributions for a number of hypothetical populations having cells in the different site categories distributed in a known manner. It follows from this conclusion that: 1. (1) different types of aberration may have different mean numbers of “sites”, but the same distribution parameter; 2. (2) a Poissonian distribution of aberrations does not necessarily indicate that the mean number of “sites” per cell is unlimited or even large.

THE INDUCTION OF CHROMOSOME EXCHANGE ABERRATIONS BY IONIZING RADIATIONS: THE "SITE CONCEPT".

SummaryWolff and his collaborators have proposed that there are only four ‘sites’ in the Tradescantia nucleus, each with a radius of ∼0·3 μ, within which 2-break aberrations can be formed by x-rays or by fast neutrons. In Vicia faba there are two sites. This paper shows that this concept is incompatible with data describing the production of 2-break aberrations by alpha-particles in Vicia faba. The arguments advanced in support of the ‘site’ concept are criticised.

CHROMOSOME-EXCHANGE SITES IN TRADESCANTIA PALUDOSA MICROSPORES.

SummaryThe distribution of chromosome-exchange aberrations between cells, induced by radiation in Tradescantia paludosa microspores four days before pollen-grain mitosis, has been investigated. Gamma-, x- and neutron-radiations given at a variety of dose-rates, and under different environmental gas conditions, are represented in the data.Dicentric plus centric ring types conform to a binomial distribution (p + q)n with n = 2·4, over the whole yield range investigated. If n is equivalent to the average number of ‘sites’ available for exchange, this is strong evidence that these are pre-determined physical entities.When dicentric and centric rings are considered separately, each type conforms to a binomial with n = 2·0. Non-additivity of the n values suggests that differences in probability exist at the two types of site, and a spurious n value arises from pooling.Some possible reasons for this difference between sites are considered.