X Chromosome-Specific Repeats in Non-Domestic Bovidae.

Repetitive sequences form a substantial and still enigmatic part of the mammalian genome. We isolated repetitive DNA blocks of the X chromosomes of three species of the family Bovidae: Kobus defassa (KDEXr sequence), Bos taurus (BTAXr sequence) and Antilope cervicapra (ACEXr sequence). The copy numbers of the isolated sequences were assessed using qPCR, and their chromosomal localisations were analysed using FISH in ten bovid tribes and in outgroup species. Besides their localisation on the X chromosome, their presence was also revealed on the Y chromosome and autosomes in several species. The KDEXr sequence abundant in most Bovidae species also occurs in distant taxa (Perissodactyla and Carnivora) and seems to be evolutionarily older than BTAXr and ACEXr. The ACEXr sequence, visible only in several Antilopini species using FISH, is probably the youngest, and arised in an ancestor common to Bovidae and Cervidae. All three repetitive sequences analysed in this study are interspersed among gene-rich regions on the X chromosomes, apparently preventing the crossing-over in their close vicinity. This study demonstrates that repetitive sequences on the X chromosomes have undergone a fast evolution, and their variation among related species can be beneficial for evolutionary studies.

Incorporation of Low Concentrations of Gold Nanoparticles: Complex Effects on Radiation Response and Fate of Cancer Cells.

(1) Background: In oncology research, a long-standing discussion exists about pros and cons of metal nanoparticle-enhanced radiotherapy and real mechanisms behind the tumor cell response to irradiation (IR) in presence of gold nanoparticles (GNPs). A better understanding of this response is, however, necessary to develop more efficient and safety nanoparticle (NP) types designed to disturb specific processes in tumor cells. (2) Aims and Methods: We combined 3D confocal microscopy and super-resolution single molecule localization microscopy (SMLM) to analyze, at the multiscale, the early and late effects of 10 nm-GNPs on DNA double strand break (DSB) induction and repair in tumor cells exposed to different doses of photonic low-LET (linear energy transfer) radiation. The results were correlated to different aspects of short and long-term cell viability. SkBr3 breast cancer cells (selected for the highest incidence of this cancer type among all cancers in women, and because most breast tumors are treated with IR) were incubated with low concentrations of GNPs and irradiated with 60Co γ-rays or 6 MV X-rays. In numerous post-irradiation (PI) times, ranging from 0.5 to 24 h PI, the cells were spatially (3D) fixed and labeled with specific antibodies against γH2AX, 53BP1 and H3K9me3. The extent of DSB induction, multi-parametric micro- and nano-morphology of γH2AX and 53BP1 repair foci, DSB repair kinetics, persistence of unrepaired DSBs, nanoscale clustering of γH2AX and nanoscale (hetero)chromatin re-organization were measured by means of the mentioned microscopy techniques in dependence of radiation dose and GNP concentration. (3) Results: The number of γH2AX/53BP1 signals increased after IR and an additional increase was observed in GNP-treated (GNP(+)) cells compared to untreated controls. However, this phenomenon reflected slight expansion of the G2-phase cell subpopulation in irradiated GNP(+) specimens instead of enhanced DNA damage induction by GNPs. This statement is further supported by some micro- and nano-morphological parameters of γH2AX/53BP1 foci, which slightly differed for cells irradiated in absence or presence of GNPs. At the nanoscale, Ripley's distance frequency analysis of SMLM signal coordinate matrices also revealed relaxation of heterochromatin (H3K9me3) clusters upon IR. These changes were more prominent in presence of GNPs. The slight expansion of radiosensitive G2 cells correlated with mostly insignificant but systematic decrease in post-irradiation survival of GNP(+) cells. Interestingly, low GNP concentrations accelerated DSB repair kinetics; however, the numbers of persistent γH2AX/53BP1 repair foci were slightly increased in GNP(+) cells. (4) Conclusions: Low concentrations of 10-nm GNPs enhanced the G2/M cell cycle arrest and the proportion of radiosensitive G2 cells, but not the extent of DNA damage induction. GNPs also accelerated DSB repair kinetics and slightly increased presence of unrepaired γH2AX/53BP1 foci at 24 h PI. GNP-mediated cell effects correlated with slight radiosensitization of GNP(+) specimens, significant only for the highest radiation dose tested (4 Gy).

Topological Analysis of γH2AX and MRE11 Clusters Detected by Localization Microscopy during X-ray-Induced DNA Double-Strand Break Repair.

DNA double-strand breaks (DSBs), known as the most severe damage in chromatin, were induced in breast cancer cells and normal skin fibroblasts by 2 Gy ionizing photon radiation. In response to DSB induction, phosphorylation of the histone variant H2AX to γH2AX was observed in the form of foci visualized by specific antibodies. By means of super-resolution single-molecule localization microscopy (SMLM), it has been recently shown in a first article about these data that these foci can be separated into clusters of about the same size (diameter ~400 nm). The number of clusters increased with the dose applied and decreased with the repair time. It has also been shown that during the repair period, antibody-labeled MRE11 clusters of about half of the γH2AX cluster diameter were formed inside several γH2AX clusters. MRE11 is part of the MRE11-RAD50-NBS1 (MRN) complex, which is known as a DNA strand resection and broken-end bridging component in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). This article is a follow-up of the former ones applying novel procedures of mathematics (topology) and similarity measurements on the data set: to obtain a measure for cluster shape and shape similarities, topological quantifications employing persistent homology were calculated and compared. In addition, based on our findings that γH2AX clusters associated with heterochromatin show a high degree of similarity independently of dose and repair time, these earlier published topological analyses and similarity calculations comparing repair foci within individual cells were extended by topological data averaging (2nd-generation heatmaps) over all cells analyzed at a given repair time point; thereby, the two dimensions (0 and 1) expressed by components and holes were studied separately. Finally, these mean value heatmaps were averaged, in addition. For γH2AX clusters, in both normal fibroblast and MCF-7 cancer cell lines, an increased similarity was found at early time points (up to 60 min) after irradiation for both components and holes of clusters. In contrast, for MRE11, the peak in similarity was found at later time points (2 h up to 48 h) after irradiation. In general, the normal fibroblasts showed quicker phosphorylation of H2AX and recruitment of MRE11 to γH2AX clusters compared to breast cancer cells and a shorter time interval of increased similarity for γH2AX clusters. γH2AX foci and randomly distributed MRE11 molecules naturally occurring in non-irradiated control cells did not show any significant topological similarity.

DeepFoci: Deep learning-based algorithm for fast automatic analysis of DNA double-strand break ionizing radiation-induced foci.

DNA double-strand breaks (DSBs), marked by ionizing radiation-induced (repair) foci (IRIFs), are the most serious DNA lesions and are dangerous to human health. IRIF quantification based on confocal microscopy represents the most sensitive and gold-standard method in radiation biodosimetry and allows research on DSB induction and repair at the molecular and single-cell levels. In this study, we introduce DeepFoci - a deep learning-based fully automatic method for IRIF counting and morphometric analysis. DeepFoci is designed to work with 3D multichannel data (trained for 53BP1 and γH2AX) and uses U-Net for nucleus segmentation and IRIF detection, together with maximally stable extremal region-based IRIF segmentation. The proposed method was trained and tested on challenging datasets consisting of mixtures of nonirradiated and irradiated cells of different types and IRIF characteristics - permanent cell lines (NHDFs, U-87) and primary cell cultures prepared from tumors and adjacent normal tissues of head and neck cancer patients. The cells were dosed with 0.5-8 Gy γ-rays and fixed at multiple (0-24 h) postirradiation times. Under all circumstances, DeepFoci quantified the number of IRIFs with the highest accuracy among current advanced algorithms. Moreover, while the detection error of DeepFoci remained comparable to the variability between two experienced experts, the software maintained its sensitivity and fidelity across dramatically different IRIF counts per nucleus. In addition, information was extracted on IRIF 3D morphometric features and repair protein colocalization within IRIFs. This approach allowed multiparameter IRIF categorization of single- or multichannel data, thereby refining the analysis of DSB repair processes and classification of patient tumors, with the potential to identify specific cell subclones. The developed software improves IRIF quantification for various practical applications (radiotherapy monitoring, biodosimetry, etc.) and opens the door to advanced DSB focus analysis and, in turn, a better understanding of (radiation-induced) DNA damage and repair.

DNA repair in head and neck tumor cells and possibilities of its monitoring to estimate individual tumor radioresistance and selection of optimal primary treatment.

In order to maximize post-therapeutic quality of life, radio(chemo)therapy becomes preferred over surgery in head-and-neck tumor (HNT) treatment. However, the therapy selection is only based on the clinical experience and patient's preferences as the radiosensitivity markers remain unknown. New possibilities of deciding on the best primary therapy, moving us towards personalized medicine based on quantifiable biomarkers, have been opened by studies on DNA radiation damage and repair in individual patients tumors. Together with the importance of radiotherapy in HNT oncology, we discuss here our preliminary results revealing the existence of several HNT groups with respect to genome stability and repair ability of tumor cells after irradiation. Monitoring of the formation and disappearance of γH2AX/53BP1 foci in tumor cell primo-cultures derived from individual patients suggests that DNA repair capacity of the identified groups correlates with the tumor cell radiosensitivity. Our findings thus improve understanding of HNT biology; nevertheless, the relationship between the repair groups and in vivo response of tumors to radiotherapy must be further studied. Since most HNTs do not suffer from repair defects, although their viability varies after irradiation, pre-therapeutic tests covering the full spectrum of HNT radiosensitivity causes will require the use of a combination of multiple, still undiscovered biomarkers.

Challenges and Contradictions of Metal Nano-Particle Applications for Radio-Sensitivity Enhancement in Cancer Therapy.

From the very beginnings of radiotherapy, a crucial question persists with how to target the radiation effectiveness into the tumor while preserving surrounding tissues as undamaged as possible. One promising approach is to selectively pre-sensitize tumor cells by metallic nanoparticles. However, though the "physics" behind nanoparticle-mediated radio-interaction has been well elaborated, practical applications in medicine remain challenging and often disappointing because of limited knowledge on biological mechanisms leading to cell damage enhancement and eventually cell death. In the present study, we analyzed the influence of different nanoparticle materials (platinum (Pt), and gold (Au)), cancer cell types (HeLa, U87, and SKBr3), and doses (up to 4 Gy) of low-Linear Energy Transfer (LET) ionizing radiation (γ- and X-rays) on the extent, complexity and reparability of radiation-induced γH2AX + 53BP1 foci, the markers of double stand breaks (DSBs). Firstly, we sensitively compared the focus presence in nuclei during a long period of time post-irradiation (24 h) in spatially (three-dimensionally, 3D) fixed cells incubated and non-incubated with Pt nanoparticles by means of high-resolution immunofluorescence confocal microscopy. The data were compared with our preliminary results obtained for Au nanoparticles and recently published results for gadolinium (Gd) nanoparticles of approximately the same size (2⁻3 nm). Next, we introduced a novel super-resolution approach-single molecule localization microscopy (SMLM)-to study the internal structure of the repair foci. In these experiments, 10 nm Au nanoparticles were used that could be also visualized by SMLM. Altogether, the data show that different nanoparticles may or may not enhance radiation damage to DNA, so multi-parameter effects have to be considered to better interpret the radiosensitization. Based on these findings, we discussed on conclusions and contradictions related to the effectiveness and presumptive mechanisms of the cell radiosensitization by nanoparticles. We also demonstrate that SMLM offers new perspectives to study internal structures of repair foci with the goal to better evaluate potential differences in DNA damage patterns.

Changes in Cryopreserved Cell Nuclei Serve as Indicators of Processes during Freezing and Thawing.

The mechanisms underlying cell protection from cryoinjury are not yet fully understood. Recent biological studies have addressed cryopreserved cell survival but have not correlated the cryoprotection effectiveness with the impact of cryoprotectants on the most important cell structure, the nucleus, and the freeze/thaw process. We identified changes of cell nuclei states caused by different types of cryoprotectants and associate them with alterations of the freeze/thaw process in cells. Namely, we investigated both higher-order chromatin structure and nuclear envelope integrity as possible markers of freezing and thawing processes. Moreover, we analyzed in detail the relationship between nuclear envelope integrity, chromatin condensation, freeze/thaw processes in cells, and cryopreservation efficiency for dimethyl sulfoxide, glycerol, trehalose, and antifreeze protein. Our interdisciplinary study reveals how changes in cell nuclei induced by cryoprotectants affect the ability of cells to withstand freezing and thawing and how nuclei changes correlate with processes during freezing and thawing. Our results contribute to the deeper fundamental understanding of the freezing processes, notably in the cell nucleus, which will expand the applications and lead to the rational design of cryoprotective materials and protocols.

Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy.

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10⁻20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.

Chromatin architecture changes and DNA replication fork collapse are critical features in cryopreserved cells that are differentially controlled by cryoprotectants.

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

Nanger, Eudorcas, Gazella, and Antilope form a well-supported chromosomal clade within Antilopini (Bovidae, Cetartiodactyla).

The evolutionary clade comprising Nanger, Eudorcas, Gazella, and Antilope, defined by an X;BTA5 translocation, is noteworthy for the many autosomal Robertsonian fusions that have driven the chromosome number variation from 2n = 30 observed in Antilope cervicapra, to the 2n = 58 in present Eudorcas thomsoni and Eudorcas rufifrons. This work reports the phylogenetic relationships within the Antilopini using comprehensive cytogenetic data from A. cervicapra, Gazella leptoceros, Nanger dama ruficollis, and E. thomsoni together with corrected karyotypic data from an additional nine species previously reported in the literature. Fluorescence in situ hybridization using BAC and microdissected cattle painting probes, in conjunction with differential staining techniques, provide the following: (i) a detailed analysis of the E. thomsoni chromosomes, (ii) the identification and fine-scale analysis the BTA3 orthologue in species of Antilopini, and (iii) the location of the pseudoautosomal regions on sex chromosomes of the four species. Our phylogenetic analysis of the chromosomal data supports monophyly of Nanger and Eudorcas and suggests an affiliation between A. cervicapra and some of the Gazella species. This renders Gazella paraphyletic and emphasizes a closer relationship between Antilope and Gazella than what has previously been considered.

Tribe-specific satellite DNA in non-domestic Bovidae.

Satellite sequences present in the centromeric and pericentric regions of chromosomes represent useful source of information. Changes in satellite DNA composition may coincide with the speciation and serve as valuable markers of phylogenetic relationships. Here, we examined satellite DNA clones isolated by laser microdissection of centromeric regions of 38 bovid species and categorized them into three types. Sat I sequences from members of Bovini/Tragelaphini/Boselaphini are similar to the well-documented 1.715 sat I DNA family. Sat I DNA from Caprini/Alcelaphini/Hippotragini/Reduncini/Aepycerotini/Cephalophini/Antilopini/Neotragini/Oreotragini form the second group homologous to the common 1.714 sat I DNA. The analysis of sat II DNAs isolated in our study confirmed conservativeness of these sequences within Bovidae. Newly described centromeric clones from Madoqua kirkii and Strepsiceros strepsiceros were similar in length and repetitive tandem arrangement but showed no similarity to any other satellite DNA in the GenBank database. Phylogenetic analysis of sat I sequences isolated in our study from 38 bovid species enabled the description of relationships at the subfamily and tribal levels. The maximum likelihood and Bayesian inference analyses showed a basal position of sequences from Oreotragini in the subfamily Antilopinae. According to the Bayesian inference analysis based on the indels in a partitioned mixed model, Antilopinae satellite DNA split into two groups with those from Neotragini as a basal tribe, followed by a stepwise, successive branching of Cephalophini, Aepycerotini and Antilopini sequences. In the second group, Reduncini sequences were basal followed by Caprini, Alcelaphini and Hippotragini.

Molecular cytogenetic insights to the phylogenetic affinities of the giraffe (Giraffa camelopardalis) and pronghorn (Antilocapra americana).

Five families are traditionally recognized within higher ruminants (Pecora): Bovidae, Moschidae, Cervidae, Giraffidae and Antilocapridae. The phylogenetic relationships of Antilocapridae and Giraffidae within Pecora are, however, uncertain. While numerous fusions (mostly Robertsonian) have accumulated in the giraffe's karyotype (Giraffa camelopardalis, Giraffidae, 2n = 30), that of the pronghorn (Antilocapra americana, Antilocapridae, 2n = 58) is very similar to the hypothesised pecoran ancestral state (2n = 58). We examined the chromosomal rearrangements of two species, the giraffe and pronghorn, using a combination of fluorescence in situ hybridization painting probes and BAC clones derived from cattle (Bos taurus, Bovidae). Our data place Moschus (Moschidae) closer to Bovidae than Cervidae. Although the alternative (i.e., Moschidae + Cervidae as sister groups) could not be discounted in recent sequence-based analyses, cytogenetics bolsters conclusions that the former is more likely. Additionally, DNA sequences were isolated from the centromeric regions of both species and compared. Analysis of cenDNA show that unlike the pronghorn, the centromeres of the giraffe are probably organized in a more complex fashion comprising different repetitive sequences specific to single chromosomal pairs or groups of chromosomes. The distribution of nucleolar organiser region (NOR) sites, often an effective phylogenetic marker, were also examined in the two species. In the giraffe, the position of NORs seems to be autapomorphic since similar localizations have not been found in other species within Pecora.

Isolation and comparison of tribe-specific centromeric repeats within Bovidae.

A taxonomic division of the family Bovidae (Artiodactyla) is difficult and the evolutionary relationships among most bovid subfamilies remain uncertain. In this study, we isolated the cattle satellite I clone BTREP15 (1.715 satellite DNA family) and autosomal centromeric DNAs of members of ten bovid tribes. We wished to determine whether the analysis of fluorescence in situ hybridization patterns of the cattle satellite I clone (BTREP15) and tribe-specific centromeric repeats isolated by laser microdissection would help to reveal some of the ambiguities occurring in the systematic classification of the family Bovidae. The FISH study of the presence and distribution of the cattle satellite I clone BTREP15 (1.715 satellite DNA family) within members of ten bovid tribes was not informative. FISH analysis of autosomal centromeric DNA probes in several species within one tribe revealed similar hybridization patterns in autosomes confirming tribal homogeneity of these probes. Sex chromosomes showed considerable variation in sequence composition and arrangement not only between tribes but also between species of one tribe. According to our findings it seems that Oreotragus oreotragus developed its own specific satellite DNA which does not hybridize to any other bovid species analysed. Our results suggest O. oreotragus as well as Aepyceros melampus may be unique species not particularly closely related to any of the recognized bovid tribes. This study indicates the isolation of tribe-specific centromeric DNAs by laser microdissection and cloning the sequence representing the main motif of these repetitive DNAs could offer the perspectives for comparative phylogenetic studies.

Sperm and embryo analysis in a carrier of supernumerary inv dup(15) marker chromosome.

We identified a small, paternally inherited, supernumerary marker chromosome, inv dup(15), in a phenotypically normal and normozoospermic male from a couple with reproductive problems. Sperm analysis by fluorescence in situ hybridization (FISH) showed that the marker was present in 26% of sperm nuclei. The disomy 15 was 10 times higher than in normal control donors. FISH analysis for aneuploidies of the other chromosomes showed an increase in nondisjunction of chromosome 21. We also examined 24 embryos by preimplantation genetic diagnosis, and 10 embryos (41.7%) contained the marker. This report provides information about inheritance of inv dup(15) from a male carrier.

Possible genetic damage in the Czech nuclear power plant workers.

The aim of our study was to identify occupational risk of irradiation exposure in the Czech nuclear power plant workers. We analyzed levels of chromosomal aberrations, a well-known biomarker of early biological effects and a predictor of cancer risk. We applied the conventional method of cytogenetic analysis and fluorescence in situ hybridization (FISH, whole chromosome painting for chromosomes 1 and 4, combined with a pancentromeric probe) to three groups: 123 subjects in the Temelin nuclear power plant (2 years in use), 114 subjects in the Dukovany nuclear power plant (20 years in use), and 53 matched controls from Ceske Budejovice. Nuclear power plant workers were divided into two groups: subjects with admittance into the monitored zone, and others. Following factors were also analyzed: GSTM1, GSTT1, GSTP1, XPD, XRCC1, hOGG1, p53, MTHFR, and MS gene polymorphisms, levels of vitamins A, C, E, and folate in plasma, and level of cotinine in urine. Long-term exposure to ionizing radiation in the monitored zone was 0.47+/-1.50 mSv (miliSievert) in the Temelin nuclear power plant and 5.74+/-9.57 mSv in the Dukovany nuclear power plant. Using the conventional cytogenetic analysis, we observed 1.90+/-0.95 and 1.82+/-1.19% AB.C. (percent of aberrant cells) in the Temelin nuclear power plant, and 2.39+/-1.01 and 2.33+/-1.04% AB.C. in the Dukovany nuclear power plant, for monitored zone workers and others, respectively. In the control group, we found 2.25+/-0.82% AB.C. Genomic frequency of translocations F(G)/100 measured by FISH was 1.89+/-1.40 and 2.01+/-1.68 in the Temelin nuclear power plant, and 2.48+/-1.93 and 2.14+/-1.62 in the Dukovany nuclear power plant for monitored zone workers and others, respectively. In the control group, F(G)/100 was 1.83+/-1.19. Following factors were identified as potential confounders by the conventional cytogenetic analysis: XPD-6, by the FISH: age, GSTP1 and p53Bst genotypes, long-term use of medication, alcohol consumption, and smoking. No association between the dose of irradiation and the level of chromosomal aberrations in any nuclear power plant was detected either by the conventional cytogenetic analysis or by FISH.