Mechanistic modelling allows to assess pathways of DNA lesion interactions underlying chromosome aberration formation

The knowledge of radiation-induced chromosomal aberration (CA) mechanisms is required in many fields of radiation genetics, radiation biology, biodosimetry, etc. However, these mechanisms are yet to be quantitatively characterised. One of the reasons is that the relationships between primary lesions of DNA/chromatin/chromosomes and dose-response curves for CA are unknown because the pathways of lesion interactions in an interphase nucleus are currently inaccessible for direct experimental observation. This article aims for the comparative analysis of two principally different scenarios of formation of simple and complex interchromosomal exchange aberrations: by lesion interactions at chromosome territories?? surface vs. in the whole space of the nucleus. The analysis was based on quantitative mechanistic modelling of different levels of structures and processes involved in CA formation: chromosome structure in an interphase nucleus, induction, repair and interactions of DNA lesions. It was shown that the restricted diffusion of chromosomal loci, predicted by computational modelling of chromosome organization, results in lesion interactions in the whole space of the nucleus being impossible. At the same time, predicted features of subchromosomal dynamics agrees well with in vivo observations and does not contradict the mechanism of CA formation at the surface of chromosome territories. On the other hand, the ??surface mechanism?? of CA formation, despite having certain qualities, proved to be insufficient to explain high frequency of complex exchange aberrations observed by mFISH technique. The alternative mechanism, CA formation on nuclear centres is expected to be sufficient to explain frequent complex exchanges.     

Comparing DNA damage-processing pathways by computer analysis of chromosome painting data.

Chromosome aberrations are large-scale illegitimate rearrangements of the genome. They are indicative of DNA damage and informative about damage processing pathways. Despite extensive investigations over many years, the mechanisms underlying aberration formation remain controversial. New experimental assays such as multiplex fluorescent in situ hybridyzation (mFISH) allow combinatorial "painting" of chromosomes and are promising for elucidating aberration formation mechanisms. Recently observed mFISH aberration patterns are so complex that computer and graph-theoretical methods are needed for their full analysis. An important part of the analysis is decomposing a chromosome rearrangement process into "cycles." A cycle of order n, characterized formally by the cyclic graph with 2n vertices, indicates that n chromatin breaks take part in a single irreducible reaction. We here describe algorithms for computing cycle structures from experimentally observed or computer-simulated mFISH aberration patterns. We show that analyzing cycles quantitatively can distinguish between different aberration formation mechanisms. In particular, we show that homology-based mechanisms do not generate the large number of complex aberrations, involving higher-order cycles, observed in irradiated human lymphocytes.     

Effect of linear energy transfer (LET) on the complexity of alpha-particle-induced chromosome aberrations in human CD34+ cells

The aim of this study was to assess the relative influence of the linear energy transfer (LET) of alpha particles on the complexity of chromosome aberrations in the absence of significant other differences in track structure. To do this, we irradiated human hemopoietic stem cells (CD34+) with alpha particles of various incident LETs (110-152 keV/microm, with mean LETs through the cell of 119-182 keV/microm) at an equi-fluence of approximately one particle/cell and assayed for chromosome aberrations by mFISH. Based on a single harvest time to collect early-division mitotic cells, complex aberrations were observed at comparable frequencies irrespective of incident LET; however, when expressed as a proportion of the total exchanges detected, their occurrence was seen to increase with increasing LET. Cycle analysis to predict theoretical DNA double-strand break rejoining cycles was also carried out on all complex chromosome aberrations detected. By doing this we found that the majority of complex aberrations are formed in single non-reducible cycles that involve just two or three different chromosomes and three or four different breaks. Each non-reducible cycle is suggested to represent "an area" of finite size within the nucleus where double-strand break repair occurs. We suggest that the local density of damage induced and the proximity of independent repair areas within the interphase nucleus determine the complexity of aberrations resolved in metaphase. Overall, the most likely outcome of a single nuclear traversal of a single alpha particle in CD34+ cells is a single chromosome aberration per damaged cell. As the incident LET of the alpha particle increases, the likelihood of this aberration being classed as complex is greater.     

SKY and FISH analysis of radiation-induced chromosome aberrations: a comparison of whole and partial genome analysis

For a retrospective dose estimation of human exposure to ionising radiation, a partial genome analysis is routinely used to quantify radiation-induced chromosome aberrations. For this purpose, fluorescence in situ hybridisation (FISH) with whole chromosome painting probes for selected chromosomes is usually applied covering about 20% of the whole genome. Since genome-wide screening techniques like spectral karyotyping (SKY) and multiplex FISH (mFISH) have been developed the detection of radiation-induced aberrations within the whole genome has now become feasible. To determine the correspondence between partial and whole genome analysis of radiation-induced chromosome aberrations, they were measured comprehensively in this study using in vitro irradiated blood samples from three donors. We were able to demonstrate that comparable results can be detected with both approaches. However, complex aberrations might be misinterpreted by partial genome analysis. We therefore conclude that whole genome analysis by SKY is useful especially in the high dose range to correct aberration data for complex exchange aberrations.     

Directional genomic hybridization: inversions as a potential biodosimeter for retrospective radiation exposure

Chromosome aberrations in blood lymphocytes provide a useful measure of past exposure to ionizing radiation. Despite the widespread and successful use of the dicentric assay for retrospective biodosimetry, the approach suffers substantial drawbacks, including the fact that dicentrics in circulating blood have a rather short half-life (roughly 1–2 years by most estimates). So-called symmetrical aberrations such as translocations are far more stable in that regard, but their high background frequency, which increases with age, also makes them less than ideal for biodosimetry. We developed a cytogenetic assay for potential use in retrospective biodosimetry that is based on the detection of chromosomal inversions, another symmetrical aberration whose transmissibility (stability) is also ostensibly high. Many of the well-known difficulties associated with inversion detection were circumvented through the use of directional genomic hybridization, a method of molecular cytogenetics that is less labor intensive and better able to detect small chromosomal inversions than other currently available approaches. Here, we report the dose-dependent induction of inversions following exposure to radiations with vastly different ionization densities [i.e., linear energy transfer (LET)]. Our results show a dramatic dose-dependent difference in the yields of inversions induced by low-LET gamma rays, as compared to more damaging high-LET charged particles similar to those encountered in deep space.     

mFISH analysis reveals complexity of chromosome aberrations in individuals occupationally exposed to internal plutonium: a pilot study to assess the relevance of complex aberrations as biomarkers of exposure to high-LET alpha particles

We recently demonstrated that a significant proportion of apparently stable insertions induced after exposure to a mean of one alpha particle/cell, detected using three-color FISH, were part of larger unstable complexes when visualized by 24-color FISH. Interestingly, regardless of the long-term persistence capability of the cell, the complexity of each alpha-particle-induced complex appeared to be specific to the nuclear traversal of a single alpha particle. To assess whether aberrations of a similar complexity are observed in vivo and also to examine the usefulness of detecting such aberrations as a biomarker of chronic exposure to alpha particles, we have carried out a limited pilot study of Russian workers with large body burdens of alpha-particle-emitting plutonium. We found unstable cells containing non-transmissible complex aberrations in all of the plutonium-exposed subjects analyzed by mFISH. In addition, all of the complexes seen were consistent with those previously observed in vitro. Non-transmissible complex aberrations were more common than transmissible-type complexes, consistent with ongoing/chronic exposure, and insertions were dominant features of both types of complex. Accordingly, this preliminary study supports the proposal that aberration complexity and non-transmissibility are the major cytogenetic features of alpha-particle exposure that could potentially be exploited as a specific indicator of chronic exposures to high-LET alpha particles.     

Radiation-produced chromosome aberrations: colourful clues

Ionizing radiation produces many chromosome aberrations. A rich variety of aberration types can now be seen with the technique of chromosome painting. Apart from being important in medicine and public health, radiation-produced aberrations act as colorful molecular clues to damage-processing mechanisms and, because juxtaposition of different parts of the genome is involved, to interphase nuclear organization. Recent studies using chromosome painting have helped to identify DNA double-strand-break repair and misrepair pathways, to determine the extent of chromosome territories and motions, and to characterize different aberration patterns left behind by different kinds of radiation.     

On time dependency of frequency and distribution of chromatid aberrations induced by mutagens with delayed effects. An interpretation

This paper provides a theoretical analysis of pecularities of both the frequency and intrachromosomal distribution of chromatid aberrations observed in the first post-treatment mitosis and induced by clastogenic agents showing delayed effects (S-phase dependent clastogens), as functions of recovery time. The theoretical deductions are based on the following facts: (1) DNA is the target of clastogen action. Lesions induced by clastogens showing delayed effects (e.g. mono- and polyfunctional alkylating agents, ultraviolet light) give rise to aberrations only after interference with the process(es) associated with DNA replication. (2) DNA replication occurs asynchronously with respect to the local involvement in replication of different chromatin regions and according to a highly ordered pattern. (3) Lesions may be removed from DNA (or otherwise modified) by repair processes prior to replication. The removal of lesions from DNA is a time-dependent function.Several possibilities are analysed (i.e. random or non-random distribution of DNA lesions, uniform or locally differing capacities of pre-replicative repair of lesions, uniform or locally differing rates of DNA synthesis) and the frequencies and distribution patterns of chromosome structural changes, as expressed in form of aberration yield-time curves, have been discussed. The theory presented in this paper offers a simple interpretation both of variations of aberration frequency and aberration distribution in dependence on the cell's position within the cell cycle during induction of lesions.It is shown that the intrachromosomal aberration distribution is non-random even if random distribution of lesions and uniform repair rates between chromosome regions replicating at different time periods during S are assumed. Non-random aberration distributions are a necessary consequence of at least two factors: (a) the temporal replication pattern, and (b) the repair activities acting prior to replication. Random distribution of aberrations is only to be expected for the most simplified situation (random distribution of lesions along the DNA and equal transformation probabilities of a given kind of lesion into aberrations) when no loss of lesions prior to replication takes place (no pre-replicative repair) and cells treated with the mutagen during G1 are analysed.     

The role of short-lived DNA lesions in the production of chromosome-exchange aberrations

Human lymphocytes were treated with combined UVC radiation and X-rays or they were X-irradiated and incubated for 60–90 min in the presence of DNA-repair inhibitor ara-C. The X-ray induced chromosome exchange aberration yield was enhanced both by UVC and ara-C by approximately a factor of two in the linear (low dose) portion of the dose-response curve. The enhancement was small in the dose squared (high dose) portion where previous dose-fractionation experiments have shown that X-ray-induced lesions leading to aberrations exist for several hours. The yield of aberrations in lymphocytes incubated after irradiation in the presence of ara-C reaches a saturation level almost immediately after irradiation (5–15 min). These cytogenetic observations together with a previous finding (Holmberg and Strausmanis, 1983) give direct and indirect evidence that the enhanced aberration yield is due to short-lived DNA breaks formed immediately after X-irradiation.

Measurements on the repair kinetics of the DNA breaks induced by X-irradiation show that ara-C strongly impairs the repair of short-lived X-ray-induced DNA breaks. It was also observed that the DNA breaks generated after UVC irradiation occur almost immediately after irradiation and the level of these transient DNA breaks reaches saturation even for short incubation times. Thus, the repair of these breaks can compete with the repair of short-lived X-ray-induced DNA-breaks in combined irradiation with UVC and X-rays.

The experimental results can be explained on the assumption that X-ray-induced aberrations originate from exchange complexes formed in interactions between both short-lived DNA breaks. The short-lived DNA breaks give rise to exchange complexes mainly within single ionization tracks where the DNA breaks are close together. The time between irradiation and exchange complex formation is of the order of 5–15 min within such a track, and short-lived breaks might be repaired before complexes have been formed. If the DNA repair of these breaks is delayed by UVC or ara-C treatment this results in a higher probability of exchange-complex formation. In contrast, interactions between breaks in different tracks originate from long-lived DNA breaks and the probability for complex formation from these breaks is not markedly affected by UVC or ara-C.     

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 degrees 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.     

No increase in radiation-induced chromosome aberration complexity detected by m-FISH after culture in the presence of 5'-bromodeoxyuridine

The thymidine analogue, 5'-bromodeoxyuridine (BrdU), is a known mutagen that is routinely introduced into culture media for subsequent Harlequin stain analysis and determination of cell cycle status. Previously, we examined the induction of chromosome aberrations in human peripheral blood lymphocytes (PBL) known to be in their 1st cell division following exposure to a low dose (0.5 Gy, average one alpha-particle per cell) of high-LET alpha-particles. We found complex chromosome aberrations to be characteristic of exposure to high-LET radiation and suggested the features of complex exchange to reflect qualitatively the spatial deposition of this densely ionising radiation. To exclude the possibility that BrdU addition post-irradiation influenced the complexity of chromosomal damage observed by m-FISH, the effect of increasing BrdU concentration on aberration complexity was investigated. Comparisons between BrdU concentration (0, 10 and 40 microM) and between sham- and alpha-particle-irradiated PBL, were made both independently and in combination to enable discrimination between BrdU and high-LET radiation effects. Aberration type, size, complexity and completeness were assessed by m-FISH, and the relative progression through cell division was evaluated. We found no evidence of any qualitative difference in the complexity of damage as visualised by m-FISH but did observe an increase in the frequency of complex exchanges with increasing BrdU concentration indicative of altered cell cycle kinetics. The parameters measured here are consistent with findings from previous in vitro and in vivo work, indicating that each complex aberration visualised by m-FISH is characteristic of the structure of the high-LET alpha-particle track and the geometry of cell irradiated.     

Karyotypes of human lymphocytes exposed to high-energy iron ions

Chromosomal aberrations were analyzed using multicolor fluorescence in situ hybridization (mFISH) in human peripheral blood lymphocytes after in vitro exposure to gamma rays or accelerated (56)Fe ions (1 GeV/nucleon, 145 keV/microm) at Brookhaven National Laboratory (Upton, NY). Doses of 0.3 and 3 Gy were used for both radiation types. Chromosomes were prematurely condensed by a phosphatase inhibitor (calyculin A) to avoid the population selection bias observed at metaphase as a result of the severe cell cycle delays induced by heavy ions. A total of 1053 karyotypes (G(2) and M phases) were analyzed in irradiated lymphocytes. Results revealed different distribution patterns for chromosomal aberrations after low- and high-LET radiation exposures: Heavy ions induced a much higher fraction of cells with multiple aberrations, while the majority of the aberrant cells induced by low doses of gamma rays contained a single aberration. The high fraction of complex-type exchanges after heavy ions leads to an overestimation of simple-type asymmetrical interchanges (dicentrics) from analysis of Giemsa-stained samples. However, even after a dose of 3 Gy iron ions, about 30% of the cells presented no complex-type exchanges. The involvement of individual chromosomes in exchanges was similar for densely and sparsely ionizing radiation, and no statistically significant evidence of a nonrandom involvement of specific chromosomes was detected.     

The RABiT: high-throughput technology for assessing global DSB repair

At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a rapid automated biodosimetry tool (RABiT); this is a completely automated, ultra-high-throughput robotically based biodosimetry workstation designed for use following a large-scale radiological event, to perform radiation biodosimetry measurements based on a fingerstick blood sample. High throughput is achieved through purpose built robotics, sample handling in filter-bottomed multi-well plates and innovations in high-speed imaging and analysis. Currently, we are adapting the RABiT technologies for use in laboratory settings, for applications in epidemiological and clinical studies. Our overall goal is to extend the RABiT system to directly measure the kinetics of DNA repair proteins. The design of the kinetic/time-dependent studies is based on repeated, automated sampling of lymphocytes from a central reservoir of cells housed in the RABiT incubator as a function of time after the irradiation challenge. In the present study, we have characterized the DNA repair kinetics of the following repair proteins: γ-H2AX, 53-BP1, ATM kinase, MDC1 at multiple times (0.5, 2, 4, 7 and 24 h) after irradiation with 4 Gy γ rays. In order to provide a consistent dose exposure at time zero, we have developed an automated capillary irradiator to introduce DNA DSBs into fingerstick-size blood samples within the RABiT. To demonstrate the scalability of the laboratory-based RABiT system, we have initiated a population study using γ-H2AX as a biomarker.     

Increased complexity of radiation-induced chromosome aberrations consistent with a mechanism of sequential formation

Complex chromosome aberrations (any exchange involving three or more breaks in two or more chromosomes) are effectively induced in peripheral blood lymphocytes (PBL) after exposure to low doses (mostly single particles) of densely ionising high-linear energy transfer (LET) alpha-particle radiation. The complexity, when observed by multiplex fluorescence in situ hybridisation (m-FISH), shows that commonly four but up to eight different chromosomes can be involved in each rearrangement. Given the territorial organisation of chromosomes in interphase and that only a very small fraction of the nucleus is irradiated by each alpha-particle traversal, the aim of this study is to address how aberrations of such complexity can be formed. To do this, we applied theoretical "cycle" analyses using m-FISH paint detail of PBL in their first cell division after exposure to high-LET alpha-particles. In brief, "cycle" analysis deconstructs the aberration "observed" by m-FISH to make predictions as to how it could have been formed in interphase. We propose from this that individual high-LET alpha-particle-induced complex aberrations may be formed by the misrepair of damaged chromatin in single physical "sites" within the nucleus, where each "site" is consistent with an "area" corresponding to the interface of two to three different chromosome territories. Limited migration of damaged chromatin is "allowed" within this "area". Complex aberrations of increased size, reflecting the path of alpha-particle nuclear intersection, are formed through the sequential linking of these individual sites by the involvement of common chromosomes.     

Rapid detection of radiation-induced chromosomal aberrations in lymphocytes and hematopoietic progenitor cells by mFISH

Structural chromosome aberrations (SCAs) are sensitive indicators of a preceding exposure of the hematopoietic system to ionizing radiation. Cytogenetic investigations have therefore become routine tools for an assessment of absorbed radiation doses and their biological effects after occupational exposure or radiation accidents.Due to its speed and ease of use, fluorescence in situ hybridization (FISH) with whole chromosome painting (WCP) probes has become a method of choice to visualize SCAs. Until recently, this technique was limited to a rather small number of chromosomes, which could be tested simultaneously. As a result, only a fraction of the structural aberrations present in a sample could be detected and the overall dose effect had to be calculated by extrapolation. The recent introduction of two genome-wide screening techniques in tumor research, i.e., Spectral Karyotyping (SKY) and multicolor FISH (mFISH) now allows the detection of translocations involving any two non-homologous chromosomes.The present study was prompted by our desire to bring the power of mFISH to bear for the rapid identification of radiation-induced SCAs. We chose two model systems to investigate the utility of mFISH: lymphocytes that were exposed in vitro to 3 Gy photons and single hematopoietic progenitor cell colonies isolated from a Chernobyl victim 9 years after in vivo exposure to 5.4 Sv.In lymphocytes, we found up to 15 different chromosomes involved in rearrangements indicating complex radiation effects. Stable aberrations detected in hematopoietic cell colonies, on the other hand, showed involvement of up to three different chromosomes. These results demonstrated that mFISH is a rapid and powerful approach to detect and characterize radiation-induced SCAs in the hemopoietic system. The application of mFISH is expected to result in a more detailed and, thus, more informative picture of radiation effects. Eventually, this technique will allow researchers to rapidly delineate chromosomal breakpoints and facilitate the identification of the genes involved in radiation tumorigenesis.     

Current status of biodosimetry based on standard cytogenetic methods

Knowledge about dose levels in radiation protection is an important step for risk assessment. However, in most cases of real or suspected accidental exposures to ionizing radiation (IR), physical dosimetry cannot be performed for retrospective estimates. In such situations, biological dosimetry has been proposed as an alternative for investigation. Briefly, biodosimetry can be defined as individual dose evaluation based on biological endpoints induced by IR (so-called biomarkers). The relationship between biological endpoints and absorbed dose is not always straightforward: nausea, vomiting and diarrhoea, for example, are the most well-known biological effects of individual irradiation, but a precise correlation between those symptoms and absorbed dose is hardly achieved. The scoring of unstable chromosomal-type aberrations (such as dicentrics and rings) and micronuclei in mitogen-stimulated peripheral blood, up till today, has been the most extensively biodosimetry assay employed for such purposes. Dicentric assay is the gold standard in biodosimetry, since its presence is generally considered to be specific to radiation exposure; scoring of micronuclei (a kind of by-product of chromosomal damages) is easier and faster than that of dicentrics for dose assessment. In this context, the aim of this work is to present an overview on biodosimetry based on standard cytogenetic methods, highlighting its advantages and limitations as tool in monitoring of radiation workers’ doses or investigation into accidental exposures. Recent advances and perspectives are also briefly presented.     

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.     

Technical report: application of the Metafer2 fluorescence scanning system for the analysis of radiation-induced chromosome aberrations measured by FISH-chromosome painting

The Metafer2 fluorescence scanning system was used for routine analysis of radiation-induced exchange aberrations measured by fluorescence in situ hybridisation (FISH) chromosome painting in human peripheral lymphocytes. The system enables a rapid and unbiased fully-automated finding and image acquisition of fluorescently stained metaphase spreads. The chromosome aberration analysis is performed interactively from stored digitised processed gallery images, presented on a screen. Appropriate software image filters are available to further improve these pictures by background correction, noise reduction and fluorescence signal enhancement.Data sets generated by computer-assisted and manual scoring of radiation-induced reciprocal translocations (2B) and total 2B (2B+related 'one-way' types) or complete dicentrics (2A) and total 2A (2A+related 'one-ways') involving painted target chromosomes 2, 3 or 4 were compared and no significant differences were found.A linear-quadratic dose-response curve for total translocations (2B+'one-ways'+complex-derived types) based on computer-assisted analysis of 27,741 metaphases with chromosome 4 painting was compared to a curve obtained earlier for manually scored translocations in a set of target chromosomes 1, 4 and 12. After extrapolation to the whole genome, no significant difference between both curves was found.From our results it can be derived that computer-assisted aberration analysis using the Metafer2 system is a reliable alternative to manual analysis. Since time saving for computer-assisted translocation analysis is about 50% compared to manual scoring, this system is highly promising for a practical application in retrospective biodosimetry of human radiation exposure.