Analysis by FISH of the spectrum of chromosome aberrations induced by X-rays in G0 human lymphocytes and their fate through mitotic divisions in culture

The induction, distribution, and persistence of chromosome aberrations in human lymphocytes exposed to X-rays in G0 were analyzed in 48-, 70-, and 94-hr cultures by conventional metaphase analysis and painting of chromosomes 1, 2, and 4 by FISH. All cells that had been scored by FISH were relocated to determine by differential staining of chromatids whether they had passed through 1, 2, or > or =3 divisions. FISH revealed a dose-dependent induction of stable and unstable aberrations, while chromatid labeling showed mitotic lag caused by irradiation in G0. Relative to their DNA contents, there was a small but significant overrepresentation of chromosome 4 and underrepresentation of chromosome 2 among the aberrations involving chromosomes 1, 2, and 4. FISH slightly underestimated the genomic frequency of unstable aberrations measured by conventional metaphase analysis. There was a slight excess of translocations relative to dicentrics, but the data are compatible with the 1:1 ratio expected from cytogenetic theory. Many of the translocations were apparently incomplete (i.e., nonreciprocal). Incomplete translocations were more frequent at higher X-ray dose and in first division, suggesting that they may be associated with complex damage and are more apt to be lost in mitosis than complete translocations. Among the incomplete translocations, t(Ab) outnumbered t(Ba) -- a difference ascribable to the FISH technique. Aberration frequencies declined as the cells divided in culture. The overall decline in the frequency of aberrant cells (approximately 29% per cell generation) reflects a rapid decline in dicentrics and fragments (approximately 60% per cell generation) and the relative stability of translocations. The frequency of translocation-bearing cells underwent a modest decline in culture (approximately 13% per cell generation).     

Chromosome painting analysis of X-ray-induced aberrations in human lymphocytes in vitro

Chromosomal rearrangements in human lymphocytes induced by X-rays(0, 0.5, 1.0 and 2.0 Gray) were analyzed using chromosome painting.DNA probes for human chromosomes 1, 3 or 4 alone, and a combinationof 1 and 4, were used for analysis. The frequency of cells withrearrangements, i.e. reciprocal translocations, dicentrics,insertions, tricentrics and fragments, involving chromosome4 increased with dose in both 48 and 72 h cultures. The numberof translocations per cell also increased with dose at 48 and72 h. Dicentrics increased with dose in 48 h but not in 72 hcultures. The estimated genomic frequency of aberrations percell was comparable with results in banded cells. No differencewas shown on the detection efficiency of chromosome rearrangementsamong the various DNA probes used. Since this technique doesnot necessarily require well-spread metaphases for analysis,it is possible to increase the number of analyzable metaphasescompared with the banding technique. Chromosome painting isa simpler, more objective and more practical method for detectingchromosome rearrangements than conventional banding analyses.     

p53 deficiency and defective mitotic checkpoint in proliferating T lymphocytes increase chromosomal instability through aberrant exit from mitotic arrest

During the proliferation of T cells for successful immune responses against pathogens, the fine regulation of cell cycle is important to the maintenance of T cell homeostasis and the prevention of lymphoproliferative disorders. However, it remains to be elucidated how the cell cycle is controlled at the mitotic phase in proliferating T cells. Here, we show that during the proliferation of primary T cells, the disruption of the mitotic spindle leads to cell-cycle arrest at mitosis and that prolonged mitotic arrest results in not only apoptosis but also the form of chromosomal instability observed in human cancers. It is interesting that in response to spindle damage, the phosphorylation of BubR1, a mitotic checkpoint kinase, was significantly induced in proliferating T cells, and the expression of the dominant-negative mutant of BubR1 compromised mitotic arrest and subsequent apoptosis and thus led to the augmentation of polyploidy formation. We also show that in response to prolonged spindle damage, the expression of p53 but not of p73 was significantly induced. In addition, following sustained mitotic arrest, p53-deficient T cells were found to be more susceptible to polyploidy formation than the wild type. These results suggest that during flourishing immune response, mitotic checkpoint and p53 play important roles in the prevention of chromosomal instability and in the maintenance of the genomic integrity of proliferating T cells.     

Variabilities in chromosomal aberrations in human somatic cells

Variabilities in the frequency of spontaneous chromosomal aberrations depend on subjective and objective factors, the stochastic nature of occurrence and recording of the aberrations. Due to the low frequency of spontaneous chromosomal aberrations, it is impossible to exactly assess their frequency on an individual basis. The low accuracy of estimating the individual frequency of spontaneous chromosomal aberrations while analyzing 100-1000 cells virtually allows the use of the individual average frequencies of chromosomal aberrations without taking into account the size of a sample to have an average group estimation of the frequency of chromosomal aberrations. The average frequencies of spontaneous chromosomal aberrations can be obtained to a rather high accuracy for individual groups when a sample comprises 50 to several hundreds of individuals. It is shown that the frequency of spontaneous chromosomal aberrations does not depend on sex and age. There is a significant variability in their frequency in relation to a season and to the year of a follow-up. Regular changes are found in the frequency of spontaneous chromosomal aberrations depending on the number of a month and on the year of a follow-up.     

Chromosomal aberrations induced by in vitro irradiation: comparisons between human sperm and lymphocytes

Types and frequencies of structural aberrations in human sperm and lymphocyte chromosomes from one donor were compared after in vitro irradiation with 100, 200, and 400 rad in order to determine if cells with dramatically different chromatin configurations are similarly affected and to investigate the feasibility of using lymphocytes as surrogates for germ cells in risk estimation. Sperm chromosomes were analyzed after fusion with eggs from the golden hamster. Total frequencies of induced aberrations were similar in the two cell types. However, the relative frequencies of rejoined lesions (dicentrics), compared with unrejoined lesions (chromosome breaks and acentric fragments), were different. At the three doses tested, a constant ratio of 5 dicentrics in lymphocytes for every dicentric in sperm was induced. Conversely, for every chromosome break or acentric fragment induced in lymphocytes, 1.7 such events were induced in sperm at the three doses tested.     

Adriamycin-induced sister chromatid exchange and chromosomal aberrations in Down's syndrome lymphocytes.

Blood samples from six Down's syndrome (DS) and six age- and sex-matched controls were cultured for 72 h in the presence of BrdUrd. Lymphocytes were then analysed at their second mitosis for sister chromatid exchange (SCE) and at their first mitosis for chromosome aberrations. Treatment with adriamycin (30 and 60 ng) showed a significant increase in frequency of SCE and chromosome aberrations in DS lymphocytes compared to normal lymphocytes at initiation of culture. Cells treated with adriamycin (ADR) for the last 24 h also showed a significant increase in SCE in DS lymphocytes compared to normal lymphocytes. A significant increase in chromatid-type aberrations was also recorded in DS lymphocytes after both treatments cultured for the last 24 h.     

In vitro induction of sister chromatid exchanges and chromosomal aberrations in peripheral lymphocytes of the oyster toadfish and american eel

A series of experiments was conducted to characterize the proliferation of oyster toadfish lymphocytes in medium containing 5-bromodeoxyuridine (BrdUrd) and to determine the effectiveness of cytogenetic endpoints for assessing the genotoxic effects of in vitro exposure of toadfish and eel lymphocytes to known mammalian clastogens. Although the rate of proliferation of toadfish lymphocytes was low compared to that of mammalian lymphocytes, the effects of increasing BrdUrd concentrations were similar, in that proliferation exhibited a concentration-dependent inhibition for concentrations above 10 μM BrdUrd, and sister chromatid exchange (SCE) frequencies exhibited a concentration-dependent increase for concentrations above 100 μM BrdUrd. Mitomycin C (MMC) and ethylene dibromide (EDB) induced concentration-dependent increases in chromatid-type exchange and SCE frequencies with least effective concentrations (control SCE frequency divided by the slope of the least-squares line) for SCE induction by MMC (6.8 × 10^?9 M) and EDB (2.6 × 10^?4 M) that were comparable to or slightly lower than those that have been obtained with mammalian in vitro systems. In vitro exposure of toadfish lymphocytes to dimethoate (DIM) induced a concentration-dependent increase in SCE frequency with a least effective concentration of 2.8 × 10^?3 M that was much higher than that observed with mammalian in vitro systems. In vitro exposure of American eel lymphocytes to MMC also induced a concentration-dependent increase in the frequency of chromosomal aberrations and SCEs with a least effective concentration for SCE induction of 2.0 × 10^?9 M. These results indicate that cytogenetic endpoints can be effectively scored with cultured lymphocytes from these and perhaps other fish species with comparable karyotypes that contain an average of at least 0.07 pg DNA/chromosome.     

Modulating factors of individual sensitivity to diepoxybutane: chromosome aberrations induced in vitro in human lymphocytes

Peripheral blood lymphocytes from a sample of 62 randomly selecteddonors were analysed for spontaneous and diepoxybutane (DEB)-inducedchromosomal aberrations (CA). These individuals were part ofa larger sample of 122 subjects whose DEB responsiveness wasevaluated by means of sister chromatid exchange (SCE) analysis.Confounding factors (such as smoking, wine and coffee consumption,occupation and haematological factors) were analysed for theireffect on individual DEB-responsiveness, but no statisticallysignificant associations were observed. Interestingly, a bimodaldistribution of aberrant cell frequencies was clearly detectable,showing the existence of DEB-sensitive subjects belonging tothe second mode (CA frequencies >19%). When responsivenessevaluated by means of CA induction was compared with SCE responsiveness,it was noted that all SCE-inducible subjects (>110.9 SCEs/cell)belonged to the second mode of CA frequency distribution. Onthe other hand, highly CA inducible individuals did not necessarilyshow a higher SCE-response, although their DEB-induced SCE frequencieswere above average (92 SCEs/cell). DEB-induced CA frequencycorrelated with baseline levels, indicating that DEB-sensitiveindividuals also showed higher spontaneous chromosome damage(3.6 versus DEB-resistant 2%, P < 0.05). Finally, when simpleand multiple regression analyses were carried out, DEB-sensitivityappeared negatively related to haematic concentrations of proteinsand uric acid (intercept 0.131 ± 0.011, slope –0.029± 0.0116, r = –0.39; P < 0.01), probably dueto its antioxidant activity. This finding confirmed previousobservations on the scavenger activity of plasma factors onDEB mutagenicity. ¹To whom correspondence should be addressed     

Inhibitory effect of ascorbic acid post-treatment on radiation-induced chromosomal damage in human lymphocytes in vitro

In the present study, the effect of exposure to ascorbic acid (vitamin C) after gamma-ray-induced chromosomal damage in cultured human lymphocytes was examined to explore the mechanism by which this antioxidant vitamin protects irradiated cells Non-irradiated lymphocytes were exposed to increasing concentrations of ascorbic acid (1-100 micro g/ml) and DNA damage was estimated using chromosomal aberration analysis and the comet assay. The results showed that ascorbic acid did not influence the frequency of chromosomal aberrations in non-irradiated cells, except at the highest concentration (20 micro g/ml), which induced breakage-type chromosomal aberrations. Vitamin C at the concentration of 50 micro g/ml caused DNA damage detected by the comet assay. A significant (34%) decrease in the frequency of chromosomal aberrations was observed in lymphocytes exposed to gamma-radiation and then cultured in the presence of ascorbic acid (1 micro g/ml). The removal of DNA breaks in cells exposed to 2 Gy of gamma-radiation was accelerated in the presence of ascorbic acid as determined by the comet assay, suggesting that it may stimulate DNA repair processes.     

Effect of vitamin E on chromosomal aberrations in lymphocytes from patients with Down's syndrome

A possible protective effect of vitamin E (DL-alpha-tocopherol) on chromosomal damage was evaluated in lymphocytes from patients with Down's syndrome (DS) and from controls. This included the analysis of the basal and G2 chromosomal aberration frequencies in lymphocytes cultured with and without 100 microM vitamin E. The chromosomal damage in G2 was determined by scoring the number of chromosomal aberrations in lymphocyte cultures treated with 5 mM caffeine, 2 h before harvesting. Vitamin E treatment decreased the basal and G2 chromosomal aberrations both in control and DS lymphocytes. In DS cells, this protective effect, expressed as a decrease in the chromosomal damage, was greater (50%) than in controls (30%). These results suggest that the increment in basal and G2 aberrations yield in DS lymphocytes may be related to the increase in oxidative damage reported in these patients.     

Cytogenetic effect of colistin on human lymphocytes in vitro: chromosome aberrations, sister chromatid exchanges, mitotic index, cell cycle kinetics, and acrocentric associations

Colistin, a peptide antibiotic, was tested at three different concentrations--71, 142, and 214 units/ml. One hundred forty-two units per milliliter corresponds to the plasma level after receiving therapeutic dose. There was a dose-dependent increase in the frequency of chromosome aberrations irrespective of the duration of treatment (T0, T24, T48). This antibiotic decreased the mitotic index and delayed the cell turn over rate indicating inhibition of DNA synthesis by it. Inhibition of DNA synthesis probably results in increase in the frequency of chromosome aberrations. Frequency of satellite associations of acrocentric chromosomes was increased with increasing concentration of the drug, but the differences at three concentrations were not significant compared to controls. There was no increase in the frequency of SCEs at any concentration or duration of treatment compared to controls. It appears that colistin induces the type of lesions that lead to chromosome aberrations and not to SCEs.     

Specificity of asbestos-induced chromosomal aberrations in short-term cultured human mesothelial cells

Short-term cultured normal human mesothelial cells were exposed for 48 hours to three different asbestos compounds, crocidiolite, chrysotile, and amosite. In the concentration used (0.01 mg/ml) all three asbestiform minerals caused, within a few days, a significant increase of cells showing numerical and/or structural abnormalities. The abnormalities were analyzed in detail using banding techniques. The results were compared with the cytogenetic observations in 52 published cases of mesotheliomas. This comparison revealed only a few similarities as regards numerical deviations. The structural rearrangements in asbestos-exposed cultures, however, in many instances involved chromosome types and chromosome regions preferentially affected in mesotheliomas.     

Induction of chromosome aberrations and mitotic arrest by cytomegalovirus in human cells

Human cytomegalovirus (CMV) is potentially an effective but often overlooked genotoxic agent in humans. We report here evidence that indicates that infection by CMV can induce chromosome alterations and mitotic inhibition. The frequency of chromosome aberrations induced was dependent on the input multiplicity of infection (m.o.i.) for human lung fibroblasts (LU), but not for human peripheral blood lymphocytes (PBLs) when both cell types were infected at the GO phase of the cell cycle. The aberrations induced by CMV were mostly chromatid breaks and chromosome pulverizations that resembled prematurely condensed S-phase chromatin. Pulverized chromosomes were not observed in LU cells infected with virus stocks that had been rendered nonlytic by UV-irradiation at 24,000 ergs/mm2 or from infection of human lymphocytes. In LU cells infected with UV-irradiated CMV, the frequency of aberrations induced was inversely dependent on the extent of the exposure of the CMV stock to the UV-light. In permissive CMV infection of proliferating LU cells at 24 hr after subculture, a high percentage (greater than 40%) of the metaphase cells were arrested at their first metaphase and displayed severely condensed chromosomes when harvested 48 hr later. A significant increase (p less than 0.05) in the chromosome aberration frequency was also observed. Our study shows that CMV infection is genotoxic to host cells. The types and extent of damage are dependent on the viral genome expression and on the cell cycle stage of the cells at the time of infection. The possible mechanisms for induction of chromosome damage by CMV are discussed.     

Induction and prevention of micronuclei and chromosomal aberrations in cultured human lymphocytes exposed to the light of halogen tungsten lamps.

Previous studies have shown that the light emitted by halogen tungsten lamps contains UV radiation in the UV-A, UV-B and UV-C regions, induces mutations and irreparable DNA damage in bacteria, enhances the frequency of micronuclei in cultured human lymphocytes and is potently carcinogenic to the skin of hairless mice. The present study showed that the light emitted by an uncovered, traditional halogen lamp induces a significant, dose-related and time-related increase not only in micronuclei but also in chromosome-type aberrations, such as breaks, and even more in chromatid-type aberrations, such as isochromatid breaks, exchanges and isochromatid/chromatid interchanges, all including gaps or not, in cultured human lymphocytes. All these genotoxic effects were completely prevented by shielding the same lamp with a silica glass cover, blocking UV radiation. A new model of halogen lamp, having the quartz bulb treated in order to reduce the output of UV radiation, was considerably less genotoxic than the uncovered halogen lamp, yet induction of chromosomal alterations was observed at high illuminance levels.     

Sensitivity to cisplatin-induced mutations and elevated chromosomal aberrations in lymphocytes from sickle cell disease patients

Sickle cell disease (SCD) is an inherited disorder caused by a single nucleotide substitution in the β-globin gene. The clinical heterogeneity observed in SCD patients has been attributed to environmental and genetic factors. The patients are subjected to increased oxidative stress, particularly during vaso-occlusive crises and acute chest pain. Another possible cause of oxidative stress in SCD is the high concentration of iron in the patients’ plasma. The increase in oxidative stress could be a relevant risk factor for mutagenesis and carcinogenesis. Studies on the frequency of basal chromosomal aberrations in cultured lymphocytes from SCD patients have not been reported so far. In order to contribute to the understanding of the role of the different biomarkers and their relationship with the extremely variable clinical manifestation of SCD, we investigated the frequency of chromosome damage in peripheral lymphocytes from sickle cells patients and healthy controls. We found an increased frequency of chromosome damage and percentage of aberrant metaphases in these patients when compared with control subjects, even at basal values (p < 0.05). In the cytogenetic sensitivity assay, the results showed that these patients presented a marked decrease in the mitotic index values compared with healthy controls. Cisplatin-induced chromosomal damage in lymphocytes from these patients was significantly higher than the frequency measured in healthy controls. The results obtained in the present study showed that more investigations are needed in order to elucidate the susceptibility to genomic instability of SCD patients.     

Utilization of rat lymphocytes for the in vitro chromosomal aberration assay

In vitro cytogenetic assays are widely conducted to assess the mutagenic potential of chemicals. Chinese hamster ovary (CHO) cells or human lymphocytes are often used for these assays; however, these cell types have certain limitations. In order to evaluate an alternate cell system, cultured rat lymphocytes were treated for 4 h at 48 h of incubation with a variety of direct- and indirect-acting clastogens in the presence or absence of an exogenous mammalian activation system. Cytogenetic effects of in vitro physiological alterations such as medium hypertonicity or pH changes were also evaluated. The background aberration rate of rat lymphocytes is approximately 2%, and they respond positively to both direct- and indirect-acting clastogens. In contrast to CHO cells, however, neither the hyperosmolality nor pH changes in the treatment media have significant effect on background aberrations. Unlike samples of human blood, rat blood can be collected under well-controlled environmental conditions. Because of the easy access to rat blood samples, the simplicity of culture, the reproducible nature of its in vitro growth, the positive response to known clastogens and negative response to media pH changes or hyperosmolalities, the rat lymphocyte in vitro chromosomal assay presented is an optimal system to assess the mutagenic potential of chemicals.     

Role of p53 codon 72 polymorphism in chromosomal aberrations and mitotic index in patients with chronic hepatitis B

Polymorphisms of the p53 gene, which participates in DNA repair, can affect the functioning of the p53 protein. The Arg and Pro variants in p53 codon 72 were shown to have different regulation properties of p53-dependent DNA repair target genes that can affect various levels of cytogenetic aberrations in chronic hepatitis B patients. The present study aimed to examine the frequency of chromosomal aberrations and the mitotic index in patients with chronic hepatitis B and their possible association with p53 gene exon 4 codon 72 Arg72Pro (Ex4+119 G>C; rs1042522) polymorphism. Fifty-eight patients with chronic hepatitis B and 30 healthy individuals were genotyped in terms of the p53 gene codon 72 Arg72Pro polymorphism by PCR-RFLP. A 72-h cell culture was performed on the same individuals and evaluated in terms of chromosomal aberrations and mitotic index. A high frequency of chromosomal aberrations and low mitotic index were detected in the patient group compared to the control group. A higher frequency of chromosomal aberrations was detected in both the patient and the control groups with a homozygous proline genotype (13 patients, 3 control subjects) compared to patients and controls with other genotypes [Arg/Pro (38 patients, 20 control subjects) and Arg/Arg (7 patients, 7 control subjects)]. We observed an increased frequency of cytogenetic aberrations in patients with chronic hepatitis B. In addition, a higher frequency of cytogenetic aberrations was observed in p53 variants having the homozygous proline genotype compared to variants having other genotypes both in patients and healthy individuals.     

Characterization of chromosomal aberrations in human gastric carcinoma cell lines using chromosome painting

Using chromosome painting, a study of chromosomal abnormalities was performed in six gastric carcinoma cell lines (SNU-484, 601, 620, 638, 668, 719) from Korean patients. Each carcinoma cell line had unique modal karyotypic characteristics and showed a variable number of numerical and structural clonal cytogenetic aberrations. SNU-484, SNU-620, and SNU-668 had near-triploidy; SNU-601, SNU-638, and SNU-719 had near-diploidy. The origins of the marker chromosomes of these cell lines were identified by fluorescence in situ hybridization with constructed painting probes. In all of six cell lines, rearrangement of chromosome 17 resulting in partial deletion of 17p (and/or partial duplication of 17q) was found. The most frequent marker was a partial gain of chromosome 7 with the breakpoints on 7q22 and 7q31. The nonrandom rearrangements of chromosomes were also determined on 1q32, 5q11-q22, 8q, 14q22, 14q34, and 15q15; suggesting that they may be the candidate regions for the isolation of the genes related to gastric cancer.