Cytogenetic effect in peripheral blood lymphocytes of children living in villages in the Ovruch District, Zhitomir Region of the Ukraine contaminated by radionuclides]

For the purpose of genetic indication of low level of chronic radiation exposure the cytogenetic monitoring of some critical children groups living in two contaminated Ukrainian villages was carried out. In all the groups the mean frequency of aberrant cells and chromosome type aberrations (including dicentrics, centric rings, chromosome translocations as well as polyploid cells) significantly exceeded control level. During the repeated examination of children from Vistupovichi (in 13 months after the first one) the striking increase of cytogenetic effect was revealed. Dicentrics and rings were registered in 79% of persons with the individual rate 0.5-1.5 and mean group frequency 0.57 per 100 cells. The authors tried to evaluate the average cumulative doses of radiation for examined groups using G. Littlefield equation for dicentric outcome under the low dose rate Cs-137 source and taking into account the assumption about the reducing of 50% dicentrics per year. According to this rough calculation, the revealed cytogenetic effect can correspond to the mean total dose of 33 cSv for Vistupovichi children.     

Chromosomal aberrations and sister-chromatid exchanges in Lithuanian populations: effects of occupational and environmental exposures

Cytogenetic analysis of chromosomal aberrations (CA) in 175,229 cells from 1113 individuals, both unexposed and occupationally or environmentally exposed to heavy metals (mercury and lead), organic (styrene, formaldehyde, phenol and benzo(a)pyrene) and inorganic (sulfur and nitrogen oxides, hydrogen and ammonium fluorides) volatile substances and/or ionizing radiation was performed. In addition, 11,250 cells from 225 individuals were scored for the frequency of sister-chromatid exchanges (SCE). Increased frequencies of CA were found in all occupationally exposed groups. A principal difference between the exposure to heavy metals and organic substances was found: increase in the CA frequency was dependent on duration of exposure to mercury but not dependent on duration of exposure to styrene, formaldehyde and phenol. A higher CA incidence was found in lymphocytes of children living in the vicinity of a plant manufacturing phosphate fertilizers. This indicates that children are a sensitive study group for the assessment of environmental exposure. However, the results of SCE analysis in these children were inconclusive. Exposure to ionizing radiation was found to cause chromosome breaks and chromatid exchanges in Chernobyl clean-up workers and chromatid breaks, chromatid exchanges, dicentric chromosomes and chromosome translocations in workers from the Ignalina Nuclear Power Plant. The increased frequency of chromatid exchanges in individuals exposed to ionizing radiation was quite unexpected. This may be attributed to the action of some unrecognized life-style or occupational factors, or to be a result of radiation-induced genomic instability. Also an increased SCE frequency was found in lymphocytes of Chernobyl clean-up workers.     

Instability of chromosomes in human nerve cells (Normal and with Neuromental Diseases)

It is assumed that the genetic mechanism of pathogenesis of such widely spread neural and mental diseases as schizophrenia (SZ), autism, ataxia-telangiectasia (AT), and Alzheimer’s disease (AD) is associated with structural and functional genomi biological markers of genomic instability. The currently available methods of molecular cytogenetics (I-MFISH, QFISH, and ICS-MCB) facilitate the solution of numerous fundamental biological problems, including analysis of genomic variations in brain cells. Using these methods, we have studied for the first time aneuploidy in human embryo and adult brain cells (normal and with AT, AD, and SZ) as well as in blood cells of children with autism. The level of aneuploidy was increased two- to threefold in the embryo brain with a subsequent reduction of the number of abnormal cells in the adult brain. In the case of SZ, mosaic aneuploidy for chromosomes 1, 18, and X was found. The study of blood cells from children with autism showed chromosomal mosaicism for chromosomes X, 9, and 15. In the case of AT, we observed a global expression of aneuploidy in up to 20–50% of cortex and cerebellum neurons. In addition, a local instability of chromosome 14 was revealed in the degenerating cerebellum in the form of breaks in the 14q12 region. In the case of AD, a tenfold increase was observed in the level of aneuploidy for chromosome 21 in brain sections subjected to neurodegeneration. These data indicate that mosaic genomic instability in nerve cells is one of the mechanism of neurodegenerative and mental diseases.     

The effect of low-dose irradiation and of age on the in vitro radiosensitivity of human lymphocytes

The effect of low-dose irradiation and of age on the radiosensitivity of human lymphocytes was studies in two groups: control (67 people) and exposed to uncontrolled low-dose irradiation in past (165 people). Radiosensitivity of lymphocytes was estimated by the level of chromosome aberrations induced in vitro by gamma-radiation Cs137 at the dose 1.5 Gy. In exposed children the frequency of induced chromosome aberrations was higher and in the exposed adults--lower in comparison to the coresponding controls. To investigate an age response of the number of chromosome aberrations three statistical approaches were used: the correlation analysis of individual data, the correlation analysis of means for 10-years intervals, the comparison of 3 age groups. In control group no significant alteration in the level of induced chromosome aberrations with age was found. However the significant negative correlation between these two parameters was revealed in exposed group, which likely is due to the opposite direction of differences in radiosensitivity of exposed children and adults from the corresponding controls.     

Complex cytogenetic characteristic of people suffered from Chernobyl accident

A cytogenetic study was performed on Chernobyl cleanup workers, on their children, on persons evacuated from contaminated aeria (adult and children), on so named "veterans of particular risk" irradiated due to the accidents on the nuclear plant, testing of nuclear weapons etc. and on control donors. The yield of stable (FISH analysis) and of unstable chromosome aberrations, micronuclei in both lymphocytes and erythrocytes, HPRT mutations was found to be increased in exposed groups as compared to control ones. In children of liquidators and in evacuated children we observed genomic instability and increased in vitro chromosomal radiosensitivity. Acceleration of age accumulation of translocations characterized the exposed population in comparison with control group. People with the highest level of routine chromosome aberrations had cardiovascular and digestive diseases more often likely than those with the lowest level. In frame of International Project ECP-6--"Biological dosimetry" the dose-responses for dicentrics and translocations were constructed in dose range 0-100 cGy of gamma-irradiation on the base of data of 8 laboratories. On cancer patients undergone whole-body gamma-irradiation (every day at the dose 11.5 cGy to a total of dose 57.5 cGy) we constructed the dose-responses for the dicentrics and translocations and compared them with the dose-responses for these aberrations after the in vitro irradiation of lymphocytes of the same patients. For the dicentrics the effectiveness of the in vivo irradiation was less than of the in vitro one. No differences were found for translocations.     

Cytogenetic changes in human lymphoma U-937 induced to apoptosis by tumor necrosis factor

The frequency of cytogenetic abnormalities in cell populations of U-937 line in control group and after their exposure to tumor necrosis factor (TNF) was assayed. It has been found that the maximum effect of TNF is observed after 48-h exposure. It was exhibited as apoptosis induction, accumulation of cells with micronuclei and binuclear cells. An increased number of cells with premature chromosome condensation is an early marker of TNF action. Changes in distribution of the cells with various chromosome numbers may lead to the appearance of sublines with properties different from those of the original cell population.     

DDIAS在川崎病患儿血液中的表达及意义

目的:研究DNA损伤诱导凋亡抑制因子(DDIAS)在川崎病(KD)患儿血液中的表达及意义。方法:通过彩色多普勒超声心动图检查96例KD患儿的冠状动脉损伤(CAL)情况,根据CAL存在情况将患儿分为CAL组和非CAL组。选取我院同期体检的30例健康儿童作为对照组。通过ELISA试剂盒检测受试者外周血中DDIAS的水平。将靶向DDIAS的小干扰RNA(siRNA)(DDIAS-siRNA)和阴性对照siRNA(NC-siRNA)转染到人冠状动脉内皮细胞(HCAEC)中,并用10 ng/mL TNF-α处理细胞。通过RT-PCR分析细胞中的TNF-α、IL-6和HO-1 mRNA水平,通过Western blot分析DDIAS、NF-κB p65和I-κBα的蛋白水平。此外,评估了各组HCAEC细胞中的超氧化物和谷胱甘肽(GSH)含量及其与单核细胞的粘附。结果:与对照组比较,KD组的DDIAS水平显著升高(P<0.001)。与非CAL组比较,CAL组的DDIAS水平显著升高(P<0.01)。外周血DDIAS诊断KD的AUC、敏感性和特异性依次为0.747、65.62%和80.65%,外周血DDIAS诊断CAL的AUC、敏感性和特异性依次为0.733、63.83%和75.51%。与NC-siRNA+TNF-α组相比,DDIAS-siRNA+TNF-α组HCAEC细胞中的TNF-αmRNA表达水平降低了51.45%,IL-6 mRNA表达水平降低了59.46%,细胞核NF-κB p65的蛋白表达水平降低了26.40%,细胞质I-κBα的蛋白水平升高了91.30%(P<0.05)。与NC-siRNA+TNF-α组相比,DDIAS-siRNA+TNF-α组粘附实验的相对荧光强度降低了53.42%(P<0.05)。与NC-siRNA+TNF-α组相比,DDIAS-siRNA+TNF-α组HCAEC细胞中的超氧化物相对荧光强度降低了35.38%,HO-1 mRNA水平升高了1.35倍,GSH水平升高了94.59%(P<0.05)。结论:DDIAS对KD及CAL均有较高的诊断价值,下调DDIAS减轻TNF-α诱导的HCAEC细胞损伤。     

Decrease of CD4+ T-lymphocytes in children exposed to environmental lead

The effects of environmental lead on the immune system of young children were assessed by determining the peripheral blood lymphocytes CD3⁺, CD4⁺, CD8⁺, B(CD19⁺) counts, and natural killer (CD16⁺ CD56⁺) cells in 35 preschool children whose mean blood lead level was 140.6 μg/L. The results were compared to an age- and sex-matched control group with a mean blood lead level of 64.3 μg/L. Compared to the controls, a significant reduction in the percentage of CD4⁺ cells and a significant increase of CD8⁺ cells were seen in the high-lead group. The negative correlation between the percentage of CD4⁺ cells and blood lead levels was found to be significant (p<0.01). These results suggest that exposure to environmental lead might result in alterations in the immune function of young children.     

DNA damage and repair in children with Down's syndrome

Down's syndrome (DS) is associated with the presence of a third 21 chromosome and is generally considered as a non-cancer-prone genetic disease. However, leukaemias occur more frequently in children with the syndrome than in general population and there is an open question, whether the presence of an additional chromosome may contribute to genomic instability, which, in turn, may play a role in a higher susceptibility to cancer and leukaemias in particular. In order to assess genomic instability associated with the presence of a third 21 chromosome, we determined the level of endogenous DNA damage and susceptibility to a genotoxic stress-inducing factor, hydrogen peroxide and N-methyl-N'-nitro-N-nitrosoguanidyne (MNNG) as well as the ability to remove DNA damage in the peripheral blood lymphocytes of children with DS and healthy kids. The level of DNA damage and the kinetics of DNA repair were evaluated by alkaline comet assay. Oxidative DNA damage was assayed with DNA repair enzymes: endonuclease III-like NTH1 and formamidopyrimidine-DNA glycosylase. The cells taken from children with DS did not display an effective DNA repair after treatment with 10 mM hydrogen peroxide. No difference in the sensitivity to DNA-damaging agents and the efficacy of DNA repair due to age and gender in DS children was observed. These results suggest that children with DS may be characterized by the increased sensitivity to the DNA-damaging agents impaired cellular reaction to DNA damage, which, in turn, may increase the probability of cancers in these children. Therefore, a special care to avoid exposure to potential mutagenic factor my be considered in these children.     

Evaluation of micronucleated lymphocytes, constitutional karyotypes and anti-p53 antibodies in 21 children with various malignancies

The implication of environmental carcinogens in childhood cancer is still unknown. To assess a possible link between DNA damage and alterations of the tumor suppressor gene p53, blood samples of 21 children with malignancies were examined for the presence of micronuclei in lymphocytes using the cytokinesis blocked micronucleus assay (CBMA). The constitutional karyotypes were analyzed for chromosome abnormalities and the presence of anti-p53 antibodies in blood sera was evaluated by an enzyme-linked immuno sorbent assay (ELISA). A control group of 20 children was also included. The rates of micronucleated cells were 5.1 per thousand+/-3.9 and 2.4 per thousand+/-2.3 for the cancer and control groups, respectively. The difference between the groups were statistically significant (P<0.05 by the Mann-Withney rank sum test). Two children in the cancer group showed extensive chromosome breakage in lymphocytes. The sera of two other children from the cancer group and of one child from the control group contained anti-p53 antibodies. Chromosome breakage and anti-p53 antibodies from the five children were associated with increased micronucleated cell rates. The results of the present study suggest that genotoxic events can occur in the lymphocytes of children with a cancerous state.     

Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli.

The DnaA protein concentration in Escherichia coli was increased above the wild-type level by inducing a lacP-controlled dnaA gene located on a plasmid. In these cells with different DnaA protein levels, we measured several parameters: dnaA gene expression; cell size, amount of DNA per cell, and number of origins per cell by flow cytometry; and origin-to-terminus ratio and the frequencies of five other markers on the chromosome by Southern hybridization. The response of the cells to higher levels of DnaA protein could be divided into three states. From the normal level to a level 1.5-fold higher, DnaA protein had little effect on dnaA gene expression and the rate of DNA replication but led to nearly proportional increases in DNA and origin concentrations. Between 1.5- and 3-fold, the normal DnaA protein concentration, dnaA gene expression was gradually decreased. In this interval, the origin concentration increased significantly; however, the replication rate was severely affected, becoming slower--especially near the origin--the higher the DnaA protein concentration, and as a result, the DNA concentration was constant. Further increases in the DnaA protein concentration did not lead to an increased origin concentration. Thus, the initiation mass was set by the DnaA protein from the normal level to an at least twofold-increased level, but the increased initiation did not lead to a large increase in the amount of DNA per unit of mass because of the inhibition of replication fork velocity.     

急性臭氧暴露对大鼠肺部细胞遗传毒性的影响*

目的: 探讨不同浓度臭氧急性暴露对大鼠肺部细胞的遗传毒性的影响。方法: 36只wistar大鼠随机分为对照组(过滤空气暴露)、臭氧暴露组(0.12 ppm、0.5 ppm、1.0 ppm、2.0 ppm、4.0 ppm)共6组,每组6只。以不同浓度的臭氧对大鼠进行动态染毒4 h后,取肺组织并分离单细胞,采用酶联免疫吸附法检测8-羟基脱氧鸟苷(8-OHdG),利用彗星实验、微核试验和DNA-蛋白质交联实验进行DNA和染色体损伤分析。结果: 与对照组相比,肺组织中8-OHdG含量从臭氧暴露浓度为0.12 ppm起即显著增加,在0.5 ppm时达到最高值。随着臭氧暴露浓度升高,彗星拖尾率逐渐上升,且存在明显的剂量-效应关系;DNA-蛋白质交联率有先升高后下降的趋势,且在2.0 ppm时达到最大值;而肺部细胞微核率尽管呈现出上升趋势,但与对照组相比无显著性差异。结论: 急性臭氧暴露在较低浓度(0.12 ppm)时即可导致大鼠肺部细胞的DNA损伤;而在较高浓度(4 ppm)时却未见显著的染色体损伤。     

Low level chromosome instability in embryonic cells of primary aneuploid mice

Karyotypic analyses of Down syndrome patients have identified a low level of chromosome mosaicism, suggesting that the primary aneuploid status of the cells promotes further chromosomal segregation errors. Sycp3-null female mice produce aneuploid oocytes, which after fusion with normal haploid sperm, result in offspring with systemic whole chromosome, aneuploid embryo cells. Using the Sycp3-null female as a model, we observe an increase in the number of embryonic cells at E7.0 that exhibit abnormal chromosomal bridges at the anaphas estage of mitosis. This result suggests that global changes in gene expression patterns resulting from primary aneuploidy can affect mitotic chromosome segregation, resulting in a low level of chromosomal instability. The increased level of chromosomal instability could in the absence of mitotic checkpoints, lead to chromosomal mosaicism within the adult organism, as seen in Down syndrome patients.     

Long-term cytogenetic effects in children prenatally-exposed to radiation as a result of the accident at the Chernobyl Atomic Energy Station

Forty-two children exposed to ionizing radiation in prenatal period and 15 children of control group were examined in the remote terms after the accident using the method of differential G-staining of chromosomes in lymphocytes of peripheral blood. It was found that the average group rate of aberrant cells and chromosome aberrations was reliably higher in the children exposed in utero compared to control. Long-term cytogenetic consequences of the pre-natal exposure were characterized by prevalence of aberrations of a chromosome type, mainly stable chromosome lesions. At chronic exposure to low doses of ionizing radiation the increase in the rate both stable and unstable chromosome aberrations.     

Cell selection in vivo

Summary A cytogenetic follow-up has been made of nine mixoploid children found among 11 148 consecutive newborn children.The frequency of the cell line with normal chromosomes increased in all but two, and the increase was statistically significant, being from 20% to 39% in four cases, and from 1% to 17% in three, while in one case there was no difference from the first to the last examination. The possibility that children with mixoploid chromosome abnormalities at birth will reveal no cell line with a chromosome abnormality in lymphocyte cultures as adults, despite having clinical signs of the chromosome aberration found in one cell line at birth is discussed, as is the question of cell selection in vivo.The mixoploid children had fewer clinical symptoms and fewer signs of the chromosome abnormalities found in some of their cells than children with the same chromosome abnormalities in all cells.     

Proliferative advantage of specific aneuploid cells drives evolution of tumor karyotypes

Most tumors have abnormal karyotypes, which arise from mistakes during mitotic division of healthy euploid cells and evolve through numerous complex mechanisms. In a recent mouse model with increased chromosome missegregation, chromosome gains dominate over losses both in pretumor and tumor tissues, whereas T-cell lymphomas are characterized by gains of chromosomes 14 and 15. However, the quantitative understanding of clonal selection leading to tumor karyotype evolution remains unknown. Here we show, by introducing a mathematical model based on a concept of a macro-karyotype, that tumor karyotypes can be explained by proliferation-driven evolution of aneuploid cells. In pretumor cells, increased apoptosis and slower proliferation of cells with monosomies lead to predominant chromosome gains over losses. Tumor karyotypes with gain of one chromosome can be explained by karyotype-dependent proliferation, whereas, for those with two chromosomes, an interplay with karyotype-dependent apoptosis is an additional possible pathway. Thus, evolution of tumor-specific karyotypes requires proliferative advantage of specific aneuploid karyotypes.     

Mental retardation in heterozygotes for the fragile-X mutation: evidence in favor of an X inactivation-dependent effect.

The still debated question of whether the expression of mental retardation in heterozygous carriers of the Martin-Bell syndrome is influenced by X inactivation has been investigated in a group of phase-known double heterozygotes for the FRA-X mutant and the G6PD Mediterranean variant. In these individuals, the number of somatic cells (fibroblasts or red cells) with an active FRA-X chromosome could be assessed through the G6PD phenotype at the single-cell level. The data reported indicate a significant inverse correlation between the IQ level (as measured by the Wechsler-Bellevue test) and the percentage of fibroblast cells with an FRA-X active chromosome. In contrast, no significant correlation was found when the IQ level and red cell data were compared, thus suggesting the occurrence of somatic selection against hematopoietic stem cells with an active FRA-X chromosome.     

Positive and negative roles of homologous recombination in the maintenance of genome stability in Saccharomyces cerevisiae

In previous studies of the loss of heterozygosity (LOH), we analyzed a hemizygous URA3 marker on chromosome III in S. cerevisiae and showed that homologous recombination is involved in processes that lead to LOH in multiple ways, including allelic recombination, chromosome size alterations, and chromosome loss. To investigate the role of homologous recombination more precisely, we examined LOH events in rad50 Delta, rad51 Delta, rad52 Delta, rad50 Delta rad52 Delta, and rad51 Delta rad52 Delta mutants. As compared to Rad(+) cells, the frequency of LOH was significantly increased in all mutants, and most events were chromosome loss. Other LOH events were differentially affected in each mutant: the frequencies of all types of recombination were decreased in rad52 mutants and enhanced in rad50 mutants. The rad51 mutation increased the frequency of ectopic but not allelic recombination. Both the rad52 and rad51 mutations increased the frequency of intragenic point mutations approximately 25-fold, suggesting that alternative mutagenic pathways partially substitute for homologous recombination. Overall, these results indicate that all of the genes are required for chromosome maintenance and that they most likely function in homologous recombination between sister chromatids. In contrast, other recombination pathways can occur at a substantial level even in the absence of one of the genes and contribute to generating various chromosome rearrangements.     

The increase in aneuploidy of embryos Is associated with pathological morphology of the sperm

Chromosomes of embryos of couples with reduced number of morphologically normal sperm cells (less than 4%) and couples with a normal level of morphologically normal sperm cells (over 4%) has revealed significant differences. In the group with a low level of normal spermatozoa, the frequency of embryos with normal chromosomes is significantly reduced and incidence of sex chromosome trisomies and autosomal monosomies and trisomies is increased; a tendency to decrease has been found for the frequency of male embryos. The obtained data may be useful to establish additional criteria for preimplantation genetic screening in the case of male infertility.     

Chromosome abnormalities in newborn children. Physical aspects

Summary A chromosome examination was made on 11,148 consecutively live-born children: 93 had a chromosome abnormality and 192 a chromosome variant. The physical aspects of the children with chromosome abnormalities and variants were compared with those of the children with normal karyotypes. Children with aneuploid or unbalanced chromosome abnormalities were more frequently immature or not fully developed at birth than those with normal karyotypes. Birth weight was lower in children with all types of chromosome abnormalities, including reciprocal translocations and chromosome variants. The low birth weight in children with chromosome variants was mainly due to the low birth weight of children with G variants. These children were also subject to a higher frequency of special delivery treatment. Heart disorders were increased in children with aneuploid or unbalanced chromosome abnormalities. The frequency of foetal erythroblastosis was increased in children with short Y as well as in children with acentric fragments. Neonatal mortality was higher in children with aneuploid or unbalanced chromosome abnormalities than in children with normal karyotypes.