Structural chromosome organisation and radiation-induced interchromosomal aberrations

The quantitative prediction of the biological effects of radiation is one of the actual tasks of radiobiology. The experimental study may be impossible under certain conditions (low doses, complex radiation fields, etc). The development of theoretical tools is required to predict biological and medical consequence of the irradiation of cell and organism. The effect under the consideration in the present paper is chromosome aberrations (CA) induced by low and high LET radiation. One of the most uncertain factors in CA prediction is the impact of chromosomal and nuclear architecture. In the present study the quantitative evaluation of the mechanisms of CA induction are discussed in the framework of the biophysical modelling technique taking into account interphase chromosomes structure in the nucleus of living (human) cell. We show that the surface contacts mechanism of interchromosomal aberrations (interchange) formation does not explain the observed ratio of simple/complex interchanges induced by both low and high LET radiation. The chromatin structure repositioning following irradiation is proposed as a possible mechanism involved in the formation of the complex aberrations.     

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.     

Formation of chromosome aberrations: insights from FISH

Most of the mutagenic and carcinogenic agents induce chromosome aberrations in vivo and in vitro. Conventional solid staining (such as Giemsa) has been employed to evaluate the frequencies and types of spontaneous and induced chromosomal aberrations. Recently, molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) using chromosome specific or chromosome region-specific DNA libraries have become available, which have increased the resolution of the detection of aberrations. This has lead to a better understanding on the mechanisms of formation of chromosome aberrations, especially following treatment with ionizing radiation. The present paper reviews briefly the results obtained using FISH technique both from basic and applied studies.     

Disappearance rates of radiation-induced chromosome aberrations from swine leukocytes

Whole-blood leukocyte cultures were evaluated at intervals up to 20 weeks following the exposure of mature pigs to 150 or 200 R whole-body doses of γ-radiation. Lymphocyte number was depressed to approximately 35% of preirradiation values by 48 h postexposure; chromosome aberration levels also quite drastically during this period. A rapid decrease in aberrations per cell indicated selective removal of cells containing chromosome anomalues during the early postirradiation period. Elevated levels of both one-track and two-track aberrations persisted at 20 weeks although the former had been at preirradiation levels at 12 and 16 weeks.     

Structural analysis of heavy ion radiation-induced chromosome aberrations by atomic force microscopy

Heavy ion radiation (high linear energy transfer, LET, radiation) induces various types of chromosome aberration. In this report, we describe a new method employing an atomic force microscope (AFM) for nanometer-level structural analysis of chromosome damage induced by heavy ion irradiation. Metaphase mouse chromosomes with chromatid gap or chromatid breaks induced by heavy ion irradiation were marked under a light microscope. Then the detailed structure of chromosomes of Giemsa-stained or unstained samples was visualized by the AFM. The height data of chromosomes obtained by AFM provided useful information to distinguish chromatid gaps and breaks. A fibrous structure was observed on the unstained chromosome, the average width of which was about 45.8 nm in the image of AFM. These structures were considered to be 30-nm fibers on the chromosome. The structure of the break point regions induced by neon- or carbon-ion irradiation was imaged by AFM. In some cases, the fibrous structure of break points was detected by AFM imaging after carbon ion irradiation. These observations indicated that AFM is a useful tool for analysis of chromosome aberrations induced by heavy ion radiation.     

Species comparisons concerning radiation-induced dominant lethals and chromosome aberrations

After X-irradiation of post-meiotic stages, male mice, guinea-pigs, rabbits and golden hamsters differed both in general sensitivity to the induction of dominant lethals, and in the relative sensitivity of the various spermatogenic stages. Guinea-pigs were the least sensitive, and hamsters had a different stage sensitivity pattern, with mature sperm the most sensitive stage.     

Interphase chromosome positioning affects the spectrum of radiation-induced chromosomal aberrations

In interphase, chromosomes occupy defined nuclear volumes known as chromosome territories. To probe the biological consequences of the described nonrandom spatial positioning of chromosome territories in human lymphocytes, we performed an extensive FISH-based analysis of ionizing radiation-induced interchanges involving chromosomes 1, 4, 18 and 19. Since the probability of exchange formation depends strongly on the spatial distance between the damage sites in the genome, a preferential formation of exchanges between proximally positioned chromosomes is expected. Here we show that the spectrum of interchanges deviates significantly from one expected based on random chromosome positioning. Moreover, the observed exchange interactions between specific chromosome pairs as well as the interactions between homologous chromosomes are consistent with the proposed gene density-related radial distribution of chromosome territories. The differences between expected and observed exchange frequencies are more pronounced after exposure to densely ionizing neutrons than after exposure to sparsely ionizing X rays. These experiments demonstrate that the spatial positioning of interphase chromosomes affects the spectrum of chromosome rearrangements.     

The influence of maternal age on radiation-induced chromosome aberrations in mouse oocytes

The relative sensitivities of dictyate oocytes from young and old female mice to radiation-induced chromosome damage were examined in 2 separate experiments. Firstly, females were given either 2 or 4 Gy of X-rays and metaphase I stage oocytes collected 16.5 days later. Analysis of these cells showed dose-related increases in chromosome aberrations in both age groups. The response was significantly greater in oocytes of older females. In the second experiment, females were given 4 Gy of X-rays and metaphase I stage oocytes collected 3.5 days later. Again, a significantly larger frequency of aberrations was present in cells from older animals. Overall, these 2 experiments provide unambiguous evidence that the radiosensitivity of mouse dictyate oocytes increases with advancing maternal age.     

Effect of caffeine on the frequency of radiation-induced chromosome aberrations at different stages of the cell cycle

The effect of caffeine on the frequency of chromosome aberrations in human lymphocytes irradiated at different stages of the cell cycle was studied. Caffeine appeared to nearly double the frequency of chromosome aberrations induced by irradiation at S and G2 stages and did not influence the effect of irradiation at G0 and G1 stages.     

Modulation of radiation-induced chromosome aberrations by DMSO, an OH radical scavenger. 1: Dose-response studies in human lymphocytes exposed to 220 kV X-rays

Human G0 lymphocytes were exposed to 220 kV X-radiation in the presence or absence of DMSO, an efficient selective scavenger of OH radicals. Our studies demonstrate that DMSO affects a concentration-dependent modulation of induced asymmetrical aberrations in human lymphocytes exposed to approximately 3.0 Gy, with maximum protectible fractions of approximately 70 percent at DMSO concentrations of greater than or equal to 1 M. The dose dependency for dicentrics in lymphocytes acutely exposed to X-ray doses of 0.51 to 4.98 Gy in the absence of DMSO is adequately described by the linear-quadratic dose-response function Y = alpha D + beta D2. Data from duplicate cultures exposed in the presence of 1 M DMSO produce an excellent fit to the regression function modified as follows: Y(+ DMSO) = alpha(delta D) + beta(delta D)2 where the 'dose modifying' factor delta = 0.501. We interpret these findings as providing evidence that OH radical-mediated lesions in DNA account for approximately 50 percent of the dose dependency for dicentrics resulting from either one-track or two-track events, following exposures of non-cycling cells to moderate-to-high doses of low LET radiation. These data may be used in additional calculations to derive an estimate of approximately 6 x 10(8) s-1 for the rate of reaction of OH radicals with DNA targets involved in aberration formation.     

Recovery kinetics of radiation-induced chromosome aberrations in human G0 lymphocytes

Summary Declining yields of radiation-induced dicentric chromosomes in human G⁰ lymphocytes were observed in split-dose experiments with time intervals varied up to 8 h. In agreement with microdosimetric intratrack-intertrack interaction models, only the dose-squared yield component was reduced and approached an asymptotic value equal to one half of the corresponding single exposure yield. For 150 kV X-rays and 13 MeV electrons, at total doses up to 6 Gy, the time constant of the approximately exponential decline was practically dose- and quality-independent within a range of 100–180 min. For 10 kV X-rays, in the presence of a dominant linear yield component, only a small split-dose effect, but with a consistent-value, was observed for a total dose of 5 Gy. Since can be interpreted as the mean life time of primary lesions in chromatin fibres, its independence from absorbed dose and radiation quality means that radiation damage of the split-dose recovery mechanism can be excluded for doses up to 6 Gy. By correlating the observed split-dose reduction of the acentric fragment yield to the reduction of the dicentric yield, (1.64 ± 0.03) acentrics/dicentric for 150 kV X-rays and (1.51 ± 0.11) acentrics/dicentric for 13 MeV electrons were obtained. Acentrics formed in the course of dicentric formation as well as in other binary interactions of primary lesions are represented in these ratios. Post-irradiation recovery during time intervals between irradiation and cell stimulation up to 24 h did not occur. The relations to comparable results in cell lethality experiments are discussed, and a hypothesis of fast and slow binary interactions of primary lesions is put forward.Dedicated to Prof. Dr. H. Muth on the occasion of his 65th birthdayThis work was supported by the Bundesministerium des Innern, Bonn, FRG     

Numerical relationship between cells with radiation-induced chromosome aberrations and cells lethally injured by radiation

Summary The numerical relationship between radiation-induced chromosome aberrations in humanG ₀ lymphocytes and reproductive lethality of human bone-marrow lymphocytes is analysed within a large LET interval. The comparison is based upon the evaluation of the coefficient of the linear component of the corresponding dose-effect-relation for the frequency of cells without aberrations and for the frequency of surviving cells respectively. The good correlation between these coefficients over a large LET interval supports the hypothesis that structural chromosome aberrations and reproductive cell death both result from the same gross chromatin damage.This work was supported by the Bundesministerium des Innern, Bonn     

Detection of chromosome aberrations by FISH as a function of cell division cycle (harlequin-FISH)

Chromosome aberrations are a sensitive indicator of genetic change, and the measurement of chromosome aberration frequency in peripheral blood lymphocytes is often used as a biological dosimeter of exposure (1,4). The length of time that cells are maintained in culture before cytogenetic analysis is probably the most important in vitro factor that influences both the frequency and types of aberrations that are seen following exposure to mutagens. Therefore, for accurate cytogenetic measurements of genetic damage, cells must be analyzed in their first mitosis following exposure. As cells progress through subsequent mitotic division cycles, cells with unstable types of aberrations, e.g., dicentrics and acentric fragments, are eliminated (1,3,4). Even the use of synchronized populations of cells does not guarantee that all cells analyzed will be in their first division following treatment. Small variations in growth rate after irradiation can lead to large variations in the proportion of cells that are in their first vs. a subsequent mitosis. For example, 48 h after G0 lymphocytes are stimulated to enter the cell cycle (the standard sampling time for cytogenetic analysis), up to 50% of the cells in mitosis can be in their second division cycle (10). While there are methods available to distinguish cells in different division cycles (see Introduction), they are not easily adapted for use with standard fluorescence in situ hybridization (FISH) procedures. The goal of this study was to develop a simple approach to detect aberrations by FISH whereby cells in different division cycles could be distinguished.