Interphase cytogenetics reveals somatic pairing of chromosome 17 centromeres in normal human brain tissue, but no trisomy 7 or sex-chromosome loss

Nuclei isolated from normal human brain tissue, collected from six autopsies, were hybridized with a panel of nine satellite DNA probes specific for the centromeric regions of chromosomes 1, 6, 7, 10, 11, 17, 18, and the X and Y chromosomes. The results did not confirm the recently reported trisomy 7 and loss of sex chromosomes observed in metaphases obtained from normal brain tissue after short-term cultures; however, cells of all six brains displayed somatic pairing of the chromosome 17 centromeres in approximately 50% of the nuclei.     

Seventeen-year follow-up study on chromosomal aberrations in five victims accidentally exposed to several Gy of <Superscript>60</Superscript>Co γ-rays

On 25 June 1990, a radiation accident occurred in a ⁶⁰Co source radiation unit in Shanghai, due to violations in operation regulations. This accident resulted in the exposure of seven individuals to acute high-dose and dose-rate whole-body external irradiation. Conventional chromosomal aberration analysis, G-banding automatic karyotype analysis and/or fluorescent in situ hybridization (FISH) painting methods were used to analyze chromosomal aberrations in peripheral blood lymphocytes from five of the victims 24 h to 17 years after accidental exposure to 1.9–5.1 Gy of ⁶⁰Co γ-rays. The frequency of unstable chromosomal aberrations (dicentrics and rings) remained at constant levels 1 month after exposure. Three months after exposure, the frequency was reduced by 20–40% in three victims, while no reduction was seen in the other two victims. Twelve years after exposure, the number of dicentrics and rings decreased by more than 90%, and did not reveal a dose-dependent relationship. However, even at 12–17 years after exposure, stable chromosome aberrations, dominated by translocations, remained at a high level in a dose-dependent manner. The frequency of stable chromosomal aberrations detected by FISH showed a similar dose-dependent relationship as that detected by karyotype analysis of G-banding chromosomes. The G-banding analysis also suggested that the pattern of chromosome breakpoints is random. The FISH data showed a decreasing tendency with time for chromosome translocation frequency in the peripheral lymphocytes, and the rate of reduction varied among different individuals. It is likely that the higher dose the victim received, the lesser the translocation frequency decreased with time. The G-banding data also showed that the rate of reduction of translocations is different among individuals. From 5 to 17 years after accidental irradiation, a very small reduction (~10%) of translocation frequency was observed in victims C and D, while there was about a 35% reduction (the highest among the victims) for victim G who received the smallest dose (1.9 Gy). These observations can be used to validate the existence of chromosomal aberrations in peripheral blood lymphocytes as a biological dosimeter for radiation exposures.     

MAMMALIAN CHROMOSOMES IN VITRO : XVIII. DNA Replication Sequence in the Chinese Hamster

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H³-thymidine labeling and the method of 5-fluorodeoxyuridine block with H³-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.     

Increased frequency of sister-chromatid exchange and chromatid breaks in lymphocytes after treatment of human volunteers with therapeutic doses of paracetamol

Paracetamol was given to 10 healthy human volunteers in 3 doses of 1 g each during a period of 8 h. Blood samples for lymphocyte cultures were taken before and 24 h after paracetamol administration. A small but significant increase was found in the frequency of sister-chromatid exchanges (SCE) after intake of paracetamol (0.187 +/- 0.030 per chromosome before and 0.208 +/- 0.024 per chromosome after). After exposure the mean frequency of chromatid breaks per 100 cells was significantly increased (2.16 +/- 1.33 versus 0.33 +/- 0.50 before exposure). Exposure of human lymphocytes in vitro showed that concentrations of paracetamol above 0.1 mM induced inhibition of replicative DNA synthesis. Increased SCE was found in lymphocytes exposed to 1-10 mM paracetamol for 2 h. Furthermore, 0.75-1.5 mM paracetamol exposure for 24 h increased the frequency of chromatid and chromosome breaks in the lymphocytes. The paracetamol-induced SCE and chromosome aberrations may be secondary effects of paracetamol-induced inhibition of DNA synthesis or due to covalent binding of paracetamol metabolite(s) to DNA.     

Investigation of the repair of single-strand breaks in human DNA using alkaline gel electrophoresis

Unstimulated lymphocytes from eight healthy persons were exposed to 10-, 30-, and 100-Gy doses of 60Co gamma radiation. The repair of damaged DNA was measured by (1) alkaline gel electrophoresis (extracted DNA loaded on 0.25% agarose gel, run at 1 V/cm for 39-44 h) at 0, 1, and 2 h after exposure and (2) incorporation of [3H]thymidine into unstimulated lymphocytes in the presence of 2 mM hydroxyurea 1 and 2 h after exposure. Both methods--alkaline gel electrophoresis and thymidine incorporation--showed that repair was completed within 2 h.     

Increased levels of numerical chromosome aberrations after in vitro exposure of human peripheral blood lymphocytes to radiofrequency electromagnetic fields for 72 hours

Mazor, R., Korenstein-Ilan, A., Barbul, A., Eshet, Y., Shahadi, A., Jerby, E. and Korenstein, R. Increased Levels of Numerical Chromosome Aberrations after In Vitro Exposure of Human Peripheral Blood Lymphocytes to Radiofrequency Electromagnetic Fields for 72 Hours. Radiat. Res. 169, 28-37 (2008). We investigated the effects of 72 h in vitro exposure of 10 human lymphocyte samples to radiofrequency electromagnetic fields (800 MHz, continuous wave) on genomic instability. The lymphyocytes were exposed in a specially designed waveguide resonator at specific absorption rates (SARs) of 2.9 and 4.1 W/kg in a temperature range of 36-37 degrees C. The induced aneuploidy of chromosomes 1, 10, 11 and 17 was determined by interphase FISH using semi-automated image analysis. We observed increased levels of aneuploidy depending on the chromosome studied as well as on the level of exposure. In chromosomes 1 and 10, there was increased aneuploidy at the higher SAR, while for chromosomes 11 and 17, the increases were observed only for the lower SAR. Multisomy (chromosomal gains) appeared to be the primary contributor to the increased aneuploidy. The effect of temperature on the level of aneuploidy was examined over the range of 33.5-40 degrees C for 72 h with no statistically significant difference in the level of aneuploidy compared to 37 degrees C. These findings suggest the possible existence of an athermal effect of RF radiation that causes increased levels of aneuploidy. These results contribute to the assessment of potential health risks after continuous chronic exposure to RF radiation at SARs close to the current levels set by ICNIRP guidelines.     

Spatial distribution of selected genetic loci in nuclei of human leukemia cells after irradiation

Fluorescence in situ hybridization (FISH) combined with high-resolution cytometry was used to determine the topographic characteristics of the centromeric heterochromatin (of the chromosomes 6, 8, 9, 17) and the tumor suppressor gene TP53 (which is located on chromosome 17) in cells of the human leukemia cell lines ML-1 and U937. Analysis was performed on cells that were either untreated or irradiated with gamma rays and incubated for different intervals after exposure. Compared to untreated cells, homologous centromeres and the TP53 genes were found closer to each other and also closer to the nuclear center 2 h after irradiation. The spatial relationship between genetic elements returned to that of the unirradiated controls during the next 2-3 h. Statistical evaluation of our experimental results shows that homologous centromeres and the homologous genes are positioned closer to each other 2 h after irradiation because they are localized closer to the center of the nucleus (probably due to more pronounced decondensation of the chromatin related to repair). This radial movement of genetic loci, however, is not connected with repair of DSBs by processes involving homologous recombination, because the angular distribution of homologous sequences remains random after irradiation.     

Temporal Characterization of Homology-Independent Centromere Coupling in Meiotic Prophase

Background

Over the past thirty years several reports of the pairing or association of non-homologous centromeres during meiotic prophase have appeared in the literature. Recently, the homology-independent pairwise association of centromeres, termed centromere coupling, was also reported in budding yeast. It seems paradoxical that centromeres would pair with non-homologous partners during a process intended to align homologous chromosomes, yet the conservation of this phenomenon across a wide range of species suggests it may play an important role in meiosis.

Principal Findings

To better define the role of this phenomenon in budding yeast, experiments were preformed to place centromere coupling within the context of landmark meiotic events. Soon after the initiation of the meiotic program, centromeres were found to re-organize from a single cluster into non-homologous couples. Centromere coupling is detected as soon as chromosome replication is finished and persists while the recombination protein Dmc1 is loaded onto the chromosomes, suggesting that centromere coupling persists through the time of double strand break formation. In the absence of the synaptonemal complex component, Zip1, centromere coupling was undetectable, at all times examined, confirming the essential role of this protein on this process. Finally, the timely release of centromere coupling depends on the recombination-initiating enzyme, Spo11, suggesting a connection between events in homologous pairing/recombination and the regulation of centromere coupling.

Conclusions

Based on our results we propose a role for centromere coupling in blocking interactions between homologous centromeres as recombination initiation is taking place.     

Biomarkers of genotoxicity and genomic instability in a non-human primate, Cebus libidinosus (Cebidae, Platyrrhini), exposed to nitroimidazole derivatives

The genotoxicity of two nitroimidazole derivatives, ornidazole (ONZ) and metronidazole (MTZ) in the peripheral blood lymphocytes of Cebus libidinosus (CLI) (Primates, Cebidae) was assessed. Endpoints measured included sister chromatid exchange (SCE) frequency, cell proliferation kinetics (CPK), replication index (RI), mitotic index (MI), and damage incidence in or near CLI heterochromatin regions. MI and SCE values following ONZ or MTZ treatments were significantly different (p<0.001) from control. SCE frequency per chromosome was not proportional to chromosome length. The chromosomes most affected for SCE were 1, 2, 4, 6, 11-13, 17, and 18, many of which possess interstitial or terminal heterochromatin. In the CLI genome, chromosomes 11 and 17 showed higher susceptibility to damage RI was the only biomarker that did not show statistically significant differences between control and treated cultures. C. libidinosus bands 11q1.4 and 11q1.5 may be hot-spots in the context of nitroimidazole exposure.     

Terahertz radiation induces spindle disturbances in human-hamster hybrid cells

The aim of this study was to investigate and quantify the production of spindle disturbances in A(L) cells, a human-hamster hybrid cell line, by 0.106 THz radiation (continuous wave). Monolayer cultures in petri dishes were exposed for 0.5 h to 0.106 THz radiation with power densities ranging from 0.043 mW/cm(2) to 4.3 mW/cm(2) or were kept under sham conditions (negative control) for the same period. As a positive control, 100 μg/ml of the insecticide trichlorfon, which is an aneuploidy-inducing agent, was used for an exposure period of 6 h. During exposure, the sample containers were kept at defined environmental conditions in a modified incubator as required by the cells. Based on a total of 6,365 analyzed mitotic cells, the results of two replicate experiments suggest that 0.106 THz radiation is a spindle-acting agent as predominately indicated by the appearance of spindle disturbances at the anaphase and telophase (especially lagging and non-disjunction of single chromosomes) of cell divisions. The findings in the present study do not necessarily imply disease or injury but may be important for evaluating possible underlying mechanisms.     

Satellite 2 demethylation induced by 5-azacytidine is associated with missegregation of chromosomes 1 and 16 in human somatic cells

Satellite sequences are an important part of the pericentromeric regions in mammalian genomes; they play a relevant role in chromosome stability and DNA hypomethylation of these sequences has been reported in ICF syndrome and in some cancers that are closely associated with chromosomal abnormalities. Epigenetic modifications of satellite sequences and their consequences have not been extensively studied in human cells. In the present work, we evaluated satellite 2 methylation patterns in human lymphocytes exposed to 5-azacytidine (5-azaC) and assessed the relationship between these patterns and chromosome missegregation. Human lymphocytes were exposed to 10μM 5-azaC for 24, 48, and 72h. Segregation errors were evaluated in binucleate cells using FISH against pericentromeric regions of chromosomes 1, 9, and 16. DNA methylation patterns were evaluated by immunodetection, and by bisulfite plus urea conversion and sequencing. We have identified that 5-azaC induced missegregation of chromosomes 1 and 16, which have highly methylated satellite 2, after 72h of exposure. Chromosome methylation patterns showed a notable decrease in pericentromeric methylation. Bisulfite conversion and sequencing analysis demonstrated demethylation of satellite 2 associated to 5-azaC exposure, principally after 72h of treatment. This change occurred in a non-specific pattern. Our study demonstrates an association between loss of satellite 2 DNA methylation and chromosome loss in human lymphocytes.     

Replication patterns of the fragile X in heterozygous carriers: analysis by a BrdUrd antibody method.

The replication status of the fragile X chromosomes was studied in short-term cultures of lymphocytes from six female heterozygous carriers. The fragile X was induced by adding 0.1 microM fluorodeoxyuridine during the last 24 h of culturing. The replication status of the X chromosomes was studied using a bromodeoxyuridine (BrdUrd) antibody method. BrdUrd was added (1) at a final concentration of 0.2 micrograms/ml during the early S phase of chromosome replication (16-10 h before harvest), (2) at 0.2 microgram/ml during the late S phase (the last 6 h of culturing), (3) at 20 micrograms/ml during the early S phase, and (4) at 20 micrograms/ml during the late S phase. BrdUrd that was incorporated into replicating chromosomes was detected by using a nuclease and BrdUrd monoclonal antibody. The frequency of the fragile X was reduced by BrdUrd treatment. The degree of reduction was more severe in the 20 micrograms/ml than in the 0.2 microgram/ml series and was more severe with late S than with early S treatment. Of the early- and late-replicating fragile X chromosomes, those which were actively replicating during a BrdUrd treatment were more reduced than the others. Thus, the average rate of early and late S treatment with 0.2 microgram BrdUrd/ml was assumed to be the closest reflection of the situation in vivo. There was no correlation between the average rate of the early replicating, active fragile X and the intelligence of the heterozygous carriers studied.     

Induction of SCEs and DNA fragmentation in bovine peripheral lymphocytes by in vitro exposure to tolylfluanid-based fungicide

The potential for genotoxic and cytotoxic effects of tolylfluanid-based fungicide (50% active agent) was evaluated using sister chromatid exchange (SCE) and proliferation indices (PI) in cultured bovine peripheral lymphocytes. For the detection of possible genetic damage, DNA fragmentation assay was also applied. Bovine lymphocytes cultured for 72 h were treated with the fungicide at the final concentrations of 1.75, 3.5, 8.75, and 17.5 μg/mL for the last 24 and 48 h of culture without S9 metabolic activation, and during the last 2 h of culture with S9 metabolic activation. In the SCE assays no evidence for genotoxic activity of the fungicide was found in treatments of 24 h without and 2 h with S9. After the 24 h exposure to tolylfluanid, a weak decrease in the PI was observed. With the prolonged exposure time (48 h), dose dependence in the increase of SCE frequencies was observed. Moreover, after 48 h exposure slight fragmentation of DNA at the concentrations of 3.5 and 8.75 μg/mL was demonstrated. SCE quantification is the most widely used approach for the assessment of genotoxic/cytogenetic effects of chemical compounds. Positive results in the assay at 48 h exposure indicated a potential of the fungicide to increase frequency of chromosomal damage (replication injuries) that is the confirmation of early effect of exposure.     

Simple method for culture of peripheral blood lymphocytes of Testudinidae

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.     

Radioprotective effect of melatonin assessed by measuring chromosomal damage in mitotic and meiotic cells.

This study was taken to evaluate the radioprotective effects of melatonin. Male adult albino mice were treated (intraperitoneal, i.p.) with 10 mg/kg melatonin either 1 h before or 1/2 h after exposure to 1.5 Gy of gamma-irradiation. Control, melatonin, irradiated and melatonin plus irradiation groups were sacrificed 24 h following treatment. The incidence of micronuclei (MN) in bone marrow cells was determined in all groups. The results show that melatonin caused a significant reduction in micronuclei polychromatic erythrocytes (MNPCE) when animals were treated with melatonin before and not after exposure to radiation. Mitotic and meiotic metaphases were prepared from spermatogonial and primary spermatocytes, respectively. Examination and analysis of metaphases showed no mutagenic effect of melatonin on chromosomal aberration (CA) frequency in spermatogonial chromosomes. Administration of one single dose of melatonin to animals before irradiation lowered total CA from 46 to 32%. However, no significant effect was observed when melatonin was given after irradiation. Similarly, the frequency of CA in meiotic metaphases decreased from 43.5% in the irradiated group to 31.5% in the irradiated group treated with melatonin 1 h before irradiation, but no change was observed when melatonin was administered after irradiation. The data obtained in this study suggest that melatonin administration confers protection against damage inflicted by radiation when given prior to exposure to irradiation and not after, and support the contention that melatonin radioprotection is achieved by its ability as a scavenger for free radicals generated by ionizing radiation.     

A cereal centromeric sequence

We report the identification of a family of sequences located by in situ hybridisation to the centromeres of all the Triticeae chromosomes studied, including the supernumerary and midget chromosomes, the centromeres of all maize chromosomes and the heterochromatic regions of rice chromosomes. This family of sequences (CCS1), together with the cereal genome alignments, will allow the evolution of the cereal centromeres and their sites to be studied. The family of sequences also shows homology to the CENP-B box. The centromeres of the cereal species and the proteins that interact with them can now be characterised. Received: 11 July 1996; in revised form: 19 September 1996 / Accepted: 24 September 1996     

Retrospective dose estimation in remote period after exposure using different biological methods

Investigation of application of chromosome aberrations of lymphocytes in peripheral blood for biological dosimetry purposes in remote (up to 40 years) period after acute exposure to doses of 1 Gy and more was carried out. The comparative analysis of frequency of unstable and stable (using FISH and G-banding methods) aberrations was performed for 24 subjects accidentally exposed to radiation on nuclear submarines during 1961-1985. Statistically significant increasing of frequency of dicentrics and centric rings was determined in the exposed subjects in remote period after exposure to compare with controls. Their sum frequency in the exposed group varied depending on ARS heaviness from 0.1 to 1.0 aberrations per 100 cells. In control group it was from 0 to 0.2 correspondingly. Translocation frequency (complete + incomplete) fixed by FISH method (2, 4, and 12 chromosomes) varied within the limits of 0.2-16.0 for exposed subjects and 0.3-1.26 translocations per genome per 100 cells for controls. Some examined persons (5 subjects) exposed to accident in 1985 had results of analysis of unstable chromosome aberration in acute period after exposure that allow to estimate obtained doses by dicentrics frequency which having good correlation with ARS heaviness. Individual dosed using traslocation frequency were defined retrospectively in 11 from 21 exposed persons. They correlate with calculated physics doses and doses estimated by haematolotical parameters in acute period and also doses obtained by ESR spectroscopy of tooth enamel in remote period.     

Structural changes in the nuclei of human lymphocytes under the effects of ionizing radiation within the range of doses that cause the adaptive response

Low-level radiation either from external, or from incorporated sources is shown to cause a non-monotonous change in some intranuclear parameters of human peripheral blood lymphocytes. For instance, radiation induces changes in the parameters that characterize the location of perecentromeric regions within the interphase nucleus and a non-monotonous increase in nuclear sizes. The exposure to doses exceeding 5 cGy impairs the relationship between the nuclear sizes and location therein of the perecentromeric regions of interphase chromosomes which exist in the control and after exposure to 2.5 cGy. Differences between the dose ranges of 1.5-3.5 and 17-25 cGy are manifested by the kinetics of restoration of the pattern of distribution of lymphocyte nuclear sizes.     

Genomic imbalances in 61 renal cancers from the proximal tubulus detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been applied to characterize 61 primary renal cell carcinomas derived histogenetically from the proximal tubulus. The tumor samples comprised 46 clear-cell renal cell carcinomas (ccRCCs) and 15 papillary renal cell carcinomas (pRCCs). Changes in the copy number of entire chromosomes or subregions were detected in 56 tumors (92%). In ccRCCs, losses of chromosome 3 or 3p (63%); 14q (30%); 9 (26%); 1 and 6 or 6q (17% each); 4 and 8 or 8p (15% each); 22 (11%); 2 or 2q and 19 (9% each); 7q, 10, 16, 17p, 18, and Y (7% each); and 5, 11, 13, 15, and 21 (4% each) were detected. Most frequent genomic gains in ccRCC were found on chromosome 5 (63%); 7 (35%); 1 or 1q (33%); 2q (24%); 8 or 8q, 12, and 20 (20% each); 3q (17%); 16 (15%); 19 (13%); 6 and 17 or 17q (11% each); and 4, 10, 11, 21, and Y (9% each). In pRCCs, gains in the copy number of chromosomes 7 and 17 (7/15, each) and 16 and 20 (6/15, each) were frequent. One pRCC showed amplification of subchromosome regions 2q22-->q33, 16q, 17q and the entire X chromosome. In pRCC, losses were less frequently seen than gains. Losses of chromosomes 1, 14, 15, and Y (3/15 each) and 2, 4, 6, and 13 (2/15 each) were observed. In ccRCCs, statistical evaluation revealed significant correlations of chromosomal imbalances with tumor stage and grade, i.e., a gain in copy number of chromosome 5 correlated positively with low tumor grade, whereas a gain of chromosomes 10 and 17 correlated positively with high tumor grade. Furthermore, loss of chromosome 4 correlated positively with high tumor stage.