Inter-chromosomal Aberrations Research Articles

Use of human lymphocyte G0 PCCs to detect intra- and inter-chromosomal aberrations for early radiation biodosimetry and retrospective assessment of radiation-induced effects.

A sensitive biodosimetry tool is required for rapid individualized dose estimation and risk assessment in the case of radiological or nuclear mass casualty scenarios to prioritize exposed humans for immediate medical countermeasures to reduce radiation related injuries or morbidity risks. Unlike the conventional Dicentric Chromosome Assay (DCA), which takes about 3–4 days for radiation dose estimation, cell fusion mediated Premature Chromosome Condensation (PCC) technique in G0 lymphocytes can be rapidly performed for radiation dose assessment within 6–8 hrs of sample receipt by alleviating the need for ex vivo lymphocyte proliferation for 48 hrs. Despite this advantage, the PCC technique has not yet been fully exploited for radiation biodosimetry. Realizing the advantage of G0 PCC technique that can be instantaneously applied to unstimulated lymphocytes, we evaluated the utility of G0 PCC technique in detecting ionizing radiation (IR) induced stable and unstable chromosomal aberrations for biodosimetry purposes. Our study demonstrates that PCC coupled with mFISH and mBAND techniques can efficiently detect both numerical and structural chromosome aberrations at the intra- and inter-chromosomal levels in unstimulated T- and B-lymphocytes. Collectively, we demonstrate that the G0 PCC technique has the potential for development as a biodosimetry tool for detecting unstable chromosome aberrations (chromosome fragments and dicentric chromosomes) for early radiation dose estimation and stable chromosome exchange events (translocations) for retrospective monitoring of individualized health risks in unstimulated lymphocytes.

MBAND analysis of early and late damages in the chromosome of human lymphocytes after exposures to gamma rays and Fe ions

Stable-type chromosome aberrations that survive multiple generations of cell division include translocation and inversions. An efficient method to detect an inversion is multi-color banding fluorescent in situ hybridization (mBAND) which allows identification of both inter- and intra-chromosome aberrations simultaneously. Post irradiation, chromosome aberrations may also arise after multiple cell divisions as a result of genomic instability. This study was aimed at investigating stable or late-arising chromosome aberrations in human lymphocytes induced after low- and high-LET radiation exposure.Human lymphocytes were exposed in vitro to gamma ray doses of 2 or 4 Gy using a 137Cs source at a dose rate of 0.5 Gy/min. For high LET radiation, cells were exposed to Fe ions (600 MeV/nucleon) of 0.05, 0.5 or 1 Gy at NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). Chromosomes were collected at 48 h which represented the first mitosis, and 7 and 14 days after irradiation using a premature chromosome condensation (PCC) technique as described previously [ 1]. Chromosome 3 was painted with the XCyte3 mBAND kit (MetaSystems), and intra- and inter-chromosomal aberrations were analyzed with the mBAND analysis system (MetaSystems).With gamma irradiation, about half of the damages observed at first mitosis remained after 7- and 14-day culture, suggesting transmissibility of damages to the surviving progeny. With Fe ions irradiation, at the doses that produced similar frequencies of gamma-induced chromosome aberrations as observed at first mitosis, a significantly lower yield of aberrations remained at the same population doublings after Fe ion exposure. At these equitoxic doses, more complex-type aberrations were observed for Fe ions, indicating that Fe ion-induced initial chromosome damages are more severe and may lead to cell death.Comparison of the number of breaks indicates that Fe ions produced three or more breaks in ∼20% of the damaged chromosome 3, whereas ∼10% or a less fraction of cells with three or more breaks in chromosome 3 were found after gamma ray exposures. Unlike gamma rays, increasing doses of Fe ions did not produce more breaks in a damaged chromosome 3, indicating that damages from Fe ions exposures were mostly due to the single track effect, in agreement with the results of Hada et al. [ 1, 2].Increasing doses of exposure resulted in less fraction of chromosome break ends involved in intra-chromosomal exchange induced by either gamma rays or Fe ions in the first mitosis.Interestingly, simple inversions in chromosome 3 were found in only the 7 and 14-day samples after 4 Gy gamma irradiation. It is not clear whether cells containing simple inversions had already progressed through the first cell division at 48 h, or the simple inversions were induced after the first cell division.Detailed analysis of breaks participating in total chromosome exchanges within the first cell cycle post-irradiation revealed a common hot spot located in the 3p21 region, which is a known fragile site corresponding to Band 6 in the mBand analysis [ 2]. The breakpoint distribution in chromosomes collected at 7 days, but not at 14 days, post-irradiation appeared similar to the distribution in cells collected within the first cell cycle post-irradiation. The breakpoint distribution for human lymphocytes after radiation exposure was different from the previously published distribution for human mammary epithelial cells [ 2], indicating that interphase chromatin folding structures play a role in the distribution of radiation-induced breaks.

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.

No significant level of inheritable interchromosomal aberrations in the progeny of bystander primary human fibroblasts after alpha particle irradiation

A major concern for bystander effects is the probability that normal healthy cells adjacent to the irradiated cells become genomically unstable and undergo further carcinogenesis after therapeutic irradiation or space mission where astronauts are exposed to low dose of heavy ions. Genomic instability is a hallmark of cancer cells. In the present study, two irradiation protocols were performed in order to ensure pure populations of bystander cells and the genomic instability in their progeny were investigated. After irradiation, chromosomal aberrations of cells were analyzed at designated time points using G2 phase premature chromosome condensation (G2-PCC) coupled with Giemsa staining and with multiplex fluorescent in situ hybridization (mFISH). Our Giemsa staining assay demonstrated that elevated yields of chromatid breaks were induced in the progeny of pure bystander primary fibroblasts up to 20days after irradiation. mFISH assay showed no significant level of inheritable interchromosomal aberrations were induced in the progeny of the bystander cell groups, while the fractions of gross aberrations (chromatid breaks or chromosomal breaks) significantly increased in some bystander cell groups. These results suggest that genomic instability occurred in the progeny of the irradiation associated bystander normal fibroblasts exclude the inheritable interchromosomal aberration.

Chromosome aberrations induced by dual exposure of protons and iron ions

During space travel, astronauts will be exposed to protons and heavy charged particles. Since the proton flux is high compared to HZE particles, on average, it is assumed that a cell will be hit by a proton before it is hit by an HZE ion. Although the effects of individual ion species on human cells have been investigated extensively, little is known about the effects of exposure to mixed beam irradiation. To address this, we exposed human epithelial cells to protons followed by HZE particles and analyzed chromosomal damage using the multicolor banding in situ hybridization (mBAND) procedure. With this technique, individually painted chromosomal bands on one chromosome allowed the identification of intra-chromosomal aberrations (inversions and deletions within a single painted chromosome) as well as inter-chromosomal aberrations (translocation to unpainted chromosomes). Our results indicated that chromosome aberration frequencies from exposures to protons followed by Fe ions did not simply decrease as the interval between the two exposures increased, but peak when the interval was 30 min.

Biomarkers specific to densely-ionising (high LET) radiations.

There have been several suggestions of biomarkers that are specific to high LET radiation. Such a biomarker could significantly increase the power of epidemiological studies of individuals exposed to densely-ionising radiations such as alpha particles (e.g. radon, plutonium workers, individuals exposed to depleted uranium) or neutrons (e.g. radiation workers, airline personnel. We discuss here a potentially powerful high LET biomarker (the H value) which is the ratio of induced inter-chromosomal aberrations to intra-arm aberrations. Both theoretical and experimental studies have suggested that this ratio should differ by a factor of about three between high LET radiation and any other likely clastogen, and will yield more discrimination than the previously suggested F value (ratio of inter-chromosomal aberrations to intra-chromosomal inter-arm aberrations). Evidence of the long-term stability of such chromosomal biomarkers has also been generated. Because these stable intra-arm anld inter-chromosomal aberrations are (1) frequent and (2) measurable at long times after exposure, this H value appears to be a practical biomarker of high LET exposure, and several in vitro studies have confirmed the approach for unstable aberrations. The approach is currently being tested in a population of Russian radiation workers exposed several decades ago to high- or low LET radiation.

An Optimized Probe Set for the Detection of Small Interchromosomal Aberrations by Use of 24-Color FISH

The rapid spread of the use of new 24-color karyotyping techniques has preceded their standardization. This is best documented by the fact that the exact resolution limits have not yet been defined. Indeed, it is shown here that a substantial proportion of interchromosomal aberrations will be missed by all multicolor karyotyping systems currently in use. We demonstrate that both the sensitivity and the specificity of 24-color karyotyping critically depend on the fluorochrome composition of chromosomes involved in an interchromosomal rearrangement. As a solution, we introduce a conceptual change in probe labeling. Seven-fluorochrome sets that overcome many of the current limitations are described, and examples of their applications are shown. The criteria presented here for an optimized probe-set design and for the estimation of resolution limits should have important consequences for pre- and postnatal diagnostics and for research applications.

LET dependence of yield ratios of radiation-induced intra- and interchromosomal aberrations in human lymphocytes

Purpose: To investigate the LET dependence of various yield ratios of intra- and interchromosomal aberrations in order to quantify proximity effects for illegitimate rejoining of chromosome breaks. Materials and methods: Calculations are based on 13 human lymphocyte data sets obtained from in vitro irradiation experiments with various radiation qualities covering an LET range of 0.5 to 150 keV/ mu m. A total of 93 000 first-division metaphases was analysed for all categories of chromosome-type aberrations. Results: No dose dependence was found for the various yield ratios of chromosomal aberrations. No LET dependence became apparent for the yield ratio of inter- to intrachromosomal exchange-type aberrations (dicentrics/centric rings F value). However, a clear LET dependence was found for the yield ratio of intra-arm intrachanges to inter-arm intrachanges (interstitial deletions/centric rings G ratio) and for the yield ratio of intraarm intrachanges to interchromosomal exchanges (interstitial deletions/dicentrics H ratio). Conclusion: Measurements of intrachanges are informative for interpretation and quantification of proximity effects in the formation of radiation-induced chromosome aberrations. In particular, yield ratios for intra-arm intrachanges are likely to have a high potential as an indicator of high-LET radiations.