Chromatid Breaks In Lymphocytes Research Articles

Late Biochemical and Cytogenetic Changes in Plasma and Peripheral Blood Lymphocytes in People Exposed to Low-dose Radiation as a Result of the Accident at the Chernobyl Nuclear Power Plant

A study was conducted to evaluate the biochemical and cytogenetic parameters in peripheral blood lymphocytes in participants in the Chernobyl accident liquidation who were exposed to external and internal radiation in the dose range (0.1–70 cSv) and children living in territories with different levels of radionuclide contamination (1–20 Ci/km2) as a result of the Chernobyl accident. The effects of low-intensity radiation were studied in a comparative analysis of the relationship between the chromatid break index, the rate of mutagenesis in somatic cells (lymphocytes), and the level of reduced glutathione in blood plasma. The increased frequency of chromatid breaks in lymphocytes and an increased level of reduced glutathione in the blood plasma in children and liquidators exposed to low doses of radiation (0.1–20 cSv) were found to be statistically significant compared to the control. As the radiation dose increased (20–70 cSv), the increase in cytogenetic abnormalities in the peripheral blood lymphocytes of liquidators and children from Chechersk (Gomelsk oblast, Belarus) was accompanied by a decrease in the water-soluble antioxidant level in the plasma. The data on the relationship of the reduced glutathione level with the major indices of the cytogenetic status are important for identification of later pathological health-related conditions in people exposed to chronic ionizing radiation.

15q12 Variants, Sputum Gene Promoter Hypermethylation, and Lung Cancer Risk: A GWAS in Smokers

Lung cancer is the leading cause of cancer-related mortality worldwide. Detection of promoter hypermethylation of tumor suppressor genes in exfoliated cells from the lung provides an assessment of field cancerization that in turn predicts lung cancer. The identification of genetic determinants for this validated cancer biomarker should provide novel insights into mechanisms underlying epigenetic reprogramming during lung carcinogenesis. A genome-wide association study using generalized estimating equations and logistic regression models was conducted in two geographically independent smoker cohorts to identify loci affecting the propensity for cancer-related gene methylation that was assessed by a 12-gene panel interrogated in sputum. All statistical tests were two-sided. Two single nucleotide polymorphisms (SNPs) at 15q12 (rs73371737 and rs7179575) that drove gene methylation were discovered and replicated with rs73371737 reaching genome-wide significance (P = 3.3×10(-8)). A haplotype carrying risk alleles from the two 15q12 SNPs conferred 57% increased risk for gene methylation (P = 2.5×10(-9)). Rs73371737 reduced GABRB3 expression in lung cells and increased risk for smoking-induced chronic mucous hypersecretion. Furthermore, subjects with variant homozygote of rs73371737 had a two-fold increase in risk for lung cancer (P = .0043). Pathway analysis identified DNA double-strand break repair by homologous recombination (DSBR-HR) as a major pathway affecting susceptibility for gene methylation that was validated by measuring chromatid breaks in lymphocytes challenged by bleomycin. A functional 15q12 variant was identified as a risk factor for gene methylation and lung cancer. The associations could be mediated by GABAergic signaling that drives the smoking-induced mucous cell metaplasia. Our findings also substantiate DSBR-HR as a critical pathway driving epigenetic gene silencing.

Genome Instability and Bleomycin Sensitivity Test

Estimation of individual susceptibility to mutagens is often a part of epidemiological studies monitoring the appearance of malignant disease in different populations. Genome exposure to mutagens can lead to DNA damage. The rate of damage depends on individual differences in response, which are usually associated with differences in DNA repair capacity. Cytogenetic studies have shown that the genome of tumour cells is less stable than normal cells and therefore accumulates more damage. Tumour patients show a higher frequency of chromatid and chromosomal aberrations and a predisposition to certain types of tumours. One of the common biomarkers used in monitoring tumour appearance and changed response to DNA damage is the bleomycin test. In its active form, bleomycin (glycopeptid) is a radiomimetic cytostatic that can damage the DNA molecule and cause multiple single and double strands. The bleomycin test is simple and inexpensive, and is based on scoring chromatid breaks in lymphocytes in vitro exposed to bleomycin during the late G2 phase of the cell cycle. This review looks into different factors that may affect test results and discusses its wide implementation in studies of genome instability usually caused by a combination of factors.

Microarray Analysis of Bleomycin-Exposed Lymphoblastoid Cells for Identifying Cancer Susceptibility Genes

The uncovering of genes involved in susceptibility to the sporadic cancer types is a great challenge. It is well established that the way in which an individual deals with DNA damage is related to the chance to develop cancer. Mutagen sensitivity is a phenotype that reflects an individual's susceptibility to the major sporadic cancer types, including colon, lung, and head and neck cancer. A standard test for mutagen sensitivity is measuring the number of chromatid breaks in lymphocytes after exposure to bleomycin. The aim of the present study was to search for the pathways involved in mutagen sensitivity. Lymphoblastoid cell lines of seven individuals with low mutagen sensitivity were compared with seven individuals with a high score. RNA was isolated from cells exposed to bleomycin (4 hours) and from unexposed cells. Microarray analysis (19K) was used to compare gene expression of insensitive and sensitive cells. The profile of most altered genes after bleomycin exposure, analyzed in all 14 cell lines, included relatively many genes involved in biological processes, such as cell growth and/or maintenance, proliferation, and regulation of cell cycle, as well as some genes involved in DNA repair. When comparing the insensitive and sensitive individuals, other differentially expressed genes were found that are involved in signal transduction and cell growth and/or maintenance (e.g., BUB1 and DUSP4). This difference in expression profiles between mutagen-sensitive and mutagen-insensitive individuals justifies further studies aimed at elucidating the genes responsible for the development of sporadic cancers.

Non-random distribution of chromatid breaks in lymphocytes of laryngeal squamous cell carcinoma patients

The frequency and localization of mutagen-induced chromatid breaks in peripheral blood lymphocytes (PBLs) were investigated for association with squamous cell carcinoma (SCC) of the larynx. The case-control study was performed in 52 patients with laryngeal SCC and in 47 cancer-free controls. The analyses were based on the bleomycin sensitivity assay. The differences between cases and controls were estimated using Mann-Whitney U test and unconditional logistic regression. The total number of chromatid breaks in PBLs of patients was significantly higher compared with healthy controls (p<0.0001); the increase was observed in almost all chromosomal arms. In a number of chromosomal regions, the relative frequency of breaks was higher in patients; this increase was statistically significant for 1p22, 5q31, 6q23 and 10q24. The majority of sites with the increased proportion of breaks in patients were identified as regions containing loci involved in DNA repair, cell cycle regulation, suppressor genes and oncogenes. Revealing non-random distribution of chromatid breaks specifically associated with laryngeal SSC may be instrumental for defining regions involved in the etiology of this disease.

Mutagen sensitivity as a susceptibility marker for endometriosis

The mutagen sensitivity assay has been well established and widely used as a good independent risk predictor for developing cancers. Although endometriosis is considered a benign disorder, it exhibits several features similar to malignancy. The objectives of this study were to evaluate whether mutagen sensitivity can predict the risk of endometriosis development. The subjects were women undergoing different surgical procedures due to different stages of endometriosis. Bleomycin was used as a mutagen, and the mutagen sensitivity of peripheral lymphocytes from women with and without endometriosis was determined by measuring chromatid breaks induced by bleomycin in short-term culture using cytogenetic analysis. The mean +/- SD (range) number of chromatid breaks per cell in women with and without endometriosis was 0.68 +/- 0.12 (0.50-0.94) and 0.52 +/- 0.10 (0.35-0.68), respectively. There was a significant difference with regard to mean chromatid breaks per cell between women with and without endometriosis (P < 0.001). On logistic regression analysis, the odds ratio (95% confidence interval) of chromatid breaks per cell was 5.80 (2.19-15.37, P < 0.001) for cases compared with controls. Yet, variables of interest including age, dysmenorrhoea, previous induced abortion and smoking in the home and workplace were not statistically correlated with chromatid breaks per cell. These preliminary data suggest that sensitivity to bleomycin-induced chromatid breaks in lymphocytes is associated with the risk of endometriosis development.

Induction and disappearance of G2 chromatid breaks in lymphocytes after low doses of low-LET γ -rays and high-LET fast neutrons

Purpose : To determine by means of the G2 assay the number of chromatid breaks induced by low-LET γ-rays and high-LET neutrons, and to compare the kinetics of chromatid break rejoining for radiations of different quality. Materials and methods : The G2 assay was performed on blood samples of four healthy donors who were irradiated with low-LET γ-rays and high-LET neutrons. In a first set of experiments a dose-response curve for the formation of chromatid breaks was carried out for γ-rays and neutrons with doses ranging between 0.1 and 0.5 Gy. In a second set of experiments, the kinetics of chromatid break formation and disappearance were investigated after a dose of 0.5 Gy using post-irradiation times ranging between 0.5 and 3.5 h. For the highest dose of 0.5 Gy, the number of isochromatid breaks was also scored. Results : No significant differences in the number of chromatid breaks were observed between low-LET γ-rays and high-LET neutrons for the four donors at any of the doses given. The dose-response curves for the formation of chromatid breaks are linear for both radiation qualities and RBEs = 1 were obtained. Scoring of isochromatid breaks at the highest dose of 0.5 Gy revealed that high-LET neutrons were, however, more effective at inducing isochromatid breaks (RBE = 6.2). The rejoining experiments further showed that the kinetics of disappearance of chromatid breaks following irradiation with low-LET γ-rays or high-LET neutrons were not significantly different. Half-times of 0.92 h for γ-rays and 0.84 h for neutrons were obtained. Conclusions : Applying the G2 assay, the results demonstrate that at low doses of irradiation, the induction as well as the disappearance of chromatid breaks is independent of the LET of the radiation qualities used (0.24 keV μ m -1 60 Co γ-rays and 20 keV μ m -1 fast neutrons). As these radiation qualities produce the same initial number of double-strand breaks, the results support the signal model that proposes that chromatid breaks are the result of an exchange process which is triggered by a single double-strand break.

Cryopreserving whole blood for functional assays using viable lymphocytes in molecular epidemiology studies

There is an increasing need for viable lymphocytes in performing phenotypic assays for biomarker studies. Both fresh and cryopreserved lymphocytes have been used for cell culture-based functional assays. However, fresh lymphocytes do not allow assays to be done in batches and cryopreservation of isolated lymphocytes results in a considerable loss of viable cells. To investigate the feasibility of using cryopreserved whole blood as a source of viable lymphocytes in molecular epidemiology studies, two well-established biomarkers, the host-cell reactivation (HCR) and mutagen sensitivity assays, were used to compare the method of cryopreserving whole blood with the traditional methods. In 25 paired blood samples assayed for DNA repair capacity (DRC) by the HCR assay, the DRC values of frozen whole blood (mean±SD, 11.59±3.07) were similar to those of frozen isolated lymphocytes (11.08±3.50). The correlation between the paired DRC values was 0.77 ( P<0.001). In 31 paired blood samples assayed for the γ-radiation-induced chromatid breaks by the mutagen sensitivity assay, there was no significant difference between the baseline level of chromatid breaks in lymphocytes from frozen blood (0.05±0.03) and fresh blood (0.06±0.03). The blastogenic rate and mitotic index of the cells used for the two assays were compared between the different processing methods. The lymphocytes from frozen whole blood were more sensitive to γ-radiation, with a higher mean level of chromatid breaks (0.68±0.21) than that in fresh blood (0.42±0.12, P<0.01), and the correlation between the numbers of chromatid breaks in the paired samples was statistically significant ( r=0.61, P<0.001). These data suggest that within the limits of the parameters investigated here, cryopreserved whole blood is a good source of viable lymphocytes for biomarker assays in molecular epidemiological studies.

Radiation-induced chromatid breaks as a predictor of breast cancer risk

Purpose: In in vivo models, radiation-induced genomic instability correlates with the risk of breast cancer development. In addition, homozygous mutations in tumor suppressor genes associated with breast cancer development adversely affects the processing and repair of radiation-induced DNA damage. We performed a case-control study to determine whether an assay measuring radiation-induced chromatid breaks correlated with the risk of having bilateral breast cancer. Methods and Materials: Patients were prospectively studied on an institutional review board-approved protocol. We included only women with bilateral breast cancer as cases to obtain patients with a presumed genetic susceptibility for breast cancer. Controls were healthy women without a previous cancer history. A mutagen sensitivity assay using γ-radiation was performed on lymphocytes obtained from 26 cases and 18 controls. One milliliter of whole blood was cultured with 9 mL of blood medium for 91 h and then treated with 125 cGy using a Cs-137 irradiator. Following an additional 4 h in culture, cells were treated with Colcemid for 1 h to arrest cells in metaphase. The number of chromatid breaks per cell was counted using a minimum of 50 metaphase spreads for each sample. Results: Cases had a statistically higher number of γ-radiation-induced chromatid breaks per cell than controls, with mean values of 0.61 ± 0.24 vs. 0.45 ± 0.14, respectively ( p = 0.034, Wilcoxon rank sum test). Using the 75th percentile value in the control group as a definition of radiation sensitivity, the radiation-sensitive individuals had a 2.83-fold increased odds ratio for breast cancer development compared with individuals who were not radiation sensitive (95% confidence intervals of 0.83 and 9.67). Conclusions: These preliminary data suggest that sensitivity to radiation-induced chromatid breaks in lymphocytes correlates with the risk of bilateral breast cancer. Although the differences between cases and controls were statistically significant, the small sample size necessitates that this finding be validated in a larger study. More data are also needed to determine whether this sensitivity is limited to breast cancer patients with a genetic susceptibility for the disease or also applies to the general breast cancer population.

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.

Increased frequency of chromatid breaks in lymphocytes of heterozygotes of ataxia telangiectasia after in vitro treatment with caffeine

The frequencies of caffeine-induced chromosomal aberrations (CA), mainly chromatid (CdB) and chromosome (CB) breaks, were studied in lymphocyte cultures derived from 6 obligatory heterozygotes and 1 homozygote of ataxia telangiectasia (AT), and from 4 control adult healthy persons. Caffeine (CF, 1 mM) was added at the beginning of the cultures exposed to CF the frequency of CB was 1.9% and of CdB 1.3%. In cells of the AT homozygote, the frequency of CdB was 6.8% in the absence and 8.7% in the presence of caffeine, the frequencies of CB being 3.4 and 10.9%, respectively. In AT heterozygous cells treated with CF, CdB increased 13-fold as compared to a less than 3-fold increase in control cells. Comparing the frequencies of CF-induced chromosomal lesions in control and AT heterozygous cells, potentiation factors (Pf) for the effect of 1 AT gene on cell sensitivity of CF (Pf [AT]) were 3.5 for CB, 6.6 for CdB and 5.5 for CA. These data demonstrate that lymphocytes of AT heterozygotes are significantly more sensitive to caffeine treatment in vitro in terms of increased frequency of CdB than normala cells, which may be useful for the diagnosis of carriers of this defective gene.