Irradiated Human Lymphocytes Research Articles

Automation of the cytokinesis-block micronucleus cytome assay by laser scanning cytometry and its potential application in radiation biodosimetry.

Here we describe the adaptation of laser scanning cytometry (LSC) to measure micronuclei (MN) automatically in lymphocytes. MN frequencies were determined in irradiated human lymphocytes using the cytokinesis-block technique, and the results from LSC were compared with visual scoring results obtained from slides of cells stained using Fast Green and 4',6-diamidino-2-phenylindole (DAPI). This fluorescent approach allowed clear identification of binucleated cells and detection of MN. The dose responses measured visually and by LSC showed similar trends and correlated positively (r = 0.9689; P < 0.0001). High-content analysis was developed to further automatically score MN within mono-, tri- and tetra-nucleated cells and to determine the nuclear division index and nuclear circularity values. The high-throughput nature of LSC can provide unique advantages in future DNA damage diagnostics in experimental and epidemiological studies. Importantly, it allows for co-detection of other biomarkers of interest within a single lymphocyte, and further development of this capability is anticipated.

Genotoxicity and cytotoxicity of sevoflurane in two human cell lines in vitro with ionizing radiation

To determine the in vitro toxicity of different concentrations of sevoflurane in cells exposed to X-ray. The genotoxic effects of sevofluorane were studied by means of the micronucleus test in cytokinesis-blocked cells of irradiated human lymphocytes. Subsequently, its cytotoxic effects on PNT2 (normal prostate) cells was determined using the cell viability test (MTT) and compared with those induced by different doses of X-rays. A dose- and time-dependent cytotoxic effect of sevofluorane on PNT2 cells was determined (p >0.001) and a dose-dependent genotoxic effect of sevofluorane was established (p >0.001). However, at volumes lower than 30 μL of sevofluorane at 100%, a non-toxic effect on PNT2 cells was shown. Sevofluorane demonstrates a genotoxic capacity as determined in vitro by micronucleus test in cytokinesis-blocked cells of irradiated human lymphocytes.

Cytogenetic characteristics of the radiation-induced bystander effect and its persistence in human blood lymphocytes

Cytogenetic characteristics of the radiation-induced chromosomal instability and its persistence in human peripheral blood lymphocytes due to the bystander effect from exposure to ionizing radiation in vitro and in vivo at low and high doses has been established using a coculturing of irradiated and nonirradiated human peripheral blood lymphocytes from persons of different sexes and G-banding metaphase chromosomes staining.

Enhanced cytotoxic and genotoxic effects of gadolinium following ELF-EMF irradiation in human lymphocytes

Gadolinium (Gd) and its chelated derivatives are widely utilized for various industrial and medical purposes, particularly as a contrast agent for magnetic resonance imaging (MRI). There are many studies of Gd nephrotoxicity and neurotoxicity, whereas research on cyto- and genotoxicity in normal human lymphocytes is scarce. It is important to investigate the effect of extremely low-frequency electromagnetic fields (ELF-EMF) on Gd toxicity, as patients are co-exposed to Gd and ELF-EMF generated by MRI scanners. We investigated the cytotoxicity and genotoixcity of Gd and the possible enhancing effect of ELF-EMF on Gd toxicity in cultured human lymphocytes by performing a micronuclei (MN) assay, trypan blue dye exclusion, single cell gel electrophoresis, and apoptosis analyses using flow cytometry. Isolated lymphocytes were exposed to 0.2–1.2 mM of Gd only or in combination with a 60-Hz ELF-EMF of 0.8-mT field strength. Exposing human lymphocytes to Gd resulted in a concentration- and time-dependent decrease in cell viability and an increase in MN frequency, single strand DNA breakage, apoptotic cell death, and ROS production. ELF-EMF (0.8 mT) exposure also increased cell death, MN frequency, olive tail moment, and apoptosis induced by Gd treatment alone. These results suggest that Gd induces DNA damage and apoptotic cell death in human lymphocytes and that ELF-EMF enhances the cytotoxicity and genotoxicity of Gd.

Simultaneous analysis of p53 protein expression and cell proliferation in irradiated human lymphocytes by flow cytometry.

P53 protein has an intrinsic role in modulating the cellular response against DNA radioinduced damages and has been pointed out as an indirect indicator of individual radiosensitivity. The rate of cell proliferation is also a parameter that has been related to tissue sensitivity to radiation. However, this feature is yet understudied. In this context, the aim of this work was to employ Flow Cytometry (FC) for simultaneously assessing of p53 protein expression levels together with cellular proliferation rate of irradiated human lymphocytes. From in vitro irradiated human blood samples, mononuclear cells were isolated and labeled with Carboxylfluorescein Diacetate Succinimidyl Ester (CFSE) prior to phytohaemagglutinin (PHA) stimulation in culture for 96 hours. Cells were also labeled with anti-p53 monoclonal antibody PE-conjugated in order to analyze either proliferation rate or p53 expression levels by FC. It was verified a reduction in the proliferation rate of irradiated lymphocytes and, in parallel, a rise in the p53 expression levels, similar for quiescent and proliferating lymphocytes. The results emphasize the importance of the use of CFSE-stained lymphocytes in assays associated to proliferation rate and the use of this methodology in several studies, such as for evaluating individual radiosensitivity.

Safety evaluation and nutritional composition of a Fraxinus excelsior seed extract, FraxiPure™

A natural extract obtained from the seeds of Fraxinus excelsior L. (FraxiPure™) has been previously reported to reduce glycemia in animal models and in humans. The objective of this work was to evaluate the safety of FraxiPure™ at in vitro, in vivo and human levels. In addition, nutritional analyses revealed an extract high in carbohydrates, with minor levels of protein, dietary fiber, glucose and sucrose. IC50 and IC90 values of 1.447 and 2.530mg/mL, respectively, after 72h incubation were calculated using the MTT assay. FraxiPure™ conferred a magnitude of protection of 69.2% against the formation of micronuclei in irradiated human lymphocytes as determined by the micronucleus assay. An LD50 of greater than 2500mg/kg was concluded following an acute oral toxicity study in Sprague–Dawley rats. A human safety evaluation in a double-blind, placebo-controlled parallel study of 100 healthy volunteers revealed no significant differences between daily consumption of 1000mg of FraxiPure™ for 90days and placebo (maltodextrin) for any of the biochemical or hematological parameters studied. Numbers of adverse events were similar in both groups, and were deemed mild to moderate. These results demonstrate, for the first time, the safety and tolerability of FraxiPure™ for consumption in healthy subjects.

Study of Radioprotective Effect of Green Tea against Gamma Irradiation Using Micronucleus Assay on Binucleated Human Lymphocytes.

The aim of this study was to investigate the radioprotective effect of green tea against genotoxicity induced by gamma irradiation in cultured blood lymphocytes from 5 human volunteers. Peripheral blood samples were collected from volunteers before and 1, 3 and 5 hr after drinking a decoction 4 g green tea in 280 ml boiling water for 5 constitutive days with the same quantity. At each time point, the whole blood samples were exposed to 200 cGy of (60) Co gamma irradiation and then were cultured with mitogenic stimulation to determine the chromosomal aberration in micronucleus assay on cytokinesis-blocked binucleated cells. As expected, for each volunteer, the results showed a significant increase in the incidence of micronuclei after exposure to gamma irradiation as compared to non-irradiated control samples. Only lymphocytes blood sample collected 3 hr after drinking green tea exhibited a significant decrease in incidence of micronuclei compared to non-treated irradiated samples. These results suggest the radioprotective ability of green tea against ionizing radiation in human lymphocytes, at specified time after consumptior.

Enhanced cytotoxic and genotoxic effect of gadolinium under the ELF-EMF irradiation in human lymphocytes

Enhanced cytotoxic and genotoxic effect of gadolinium under the ELF-EMF irradiation in human lymphocytes

Mitochondrial DNA alterations of peripheral lymphocytes in acute lymphoblastic leukemia patients undergoing total body irradiation therapy

BackgroundMitochondrial DNA (mtDNA) alterations, including mtDNA copy number and mtDNA 4977 bp common deletion (CD), are key indicators of irradiation-induced damage. The relationship between total body irradiation (TBI) treatment and mtDNA alterations in vivo, however, has not been postulated yet. The aim of this study is to analyze mtDNA alterations in irradiated human peripheral lymphocytes from acute lymphoblastic leukemia (ALL) patients as well as to take them as predictors for radiation toxicity.MethodsPeripheral blood lymphocytes were isolated from 26 ALL patients 24 hours after TBI preconditioning (4.5 and 9 Gy, respectively). Extracted DNA was analyzed by real-time PCR method.ResultsAverage 2.31 times mtDNA and 0.53 fold CD levels were observed after 4.5 Gy exposure compared to their basal levels. 9 Gy TBI produced a greater response of both mtDNA and CD levels than 4.5 Gy. Significant inverse correlation was found between mtDNA content and CD level at 4.5 and 9 Gy (P = 0.037 and 0.048). Moreover, mtDNA content of lymphocytes without irradiation was found to be correlated to age.ConclusionsmtDNA and CD content may be considered as predictive factors to radiation toxicity.

Modified C-band technique for the analysis of chromosome abnormalities in irradiated human lymphocytes

Abstract A modified C-band technique was developed in order to analyze more accurately dicentric, tricentric, and ring chromosomes in irradiated human peripheral lymphocytes. Instead of the original method relying on treatment with barium hydroxide Ba(OH) 2 , C-bands were obtained using a modified form of heat treatment in formamide followed with DAPI staining. This method was tentatively applied to the analysis of dicentric chromosomes in irradiated human lymphocytes to examine its availability. The frequency of dicentric chromosome was almost the same with conventional Giemsa staining and the modified C-band technique. In the analysis using Giemsa staining, it is relatively difficult to identify the centromere on the elongated chromosomes, over-condensed chromosomes, fragment, and acentric ring. However, the modified C-band method used in this study makes it easier to identify the centromere on such chromosomes than with the use of Giemsa staining alone. Thus, the modified C-band method may give more information about the location of the centromere. Therefore, this method may be available and more useful for biological dose estimation due to the analysis of the dicentric chromosome in human lymphocytes exposed to the radiation. Furthermore, this method is simpler and faster than the original C-band protocol and fluorescence in situ hybridization (FISH) method with the centromeric DNA probe.

The Radioprotective Effect of Zataria multiflora Against Genotoxicity Induced by γ Irradiation in Human Blood Lymphocytes

The radioprotective effect of hydroalcholic Zataria multiflora (Avishan-e shirazi) extract was investigated against genotoxicity induced by γ irradiation in human lymphocytes. Peripheral blood samples were collected from human volunteers and incubated with Z. multiflora extract at different concentrations (5, 10, and 50 μg/mL) for 1 hour. At each dose point, the whole blood was exposed in vitro to 150 cGy of cobalt-60 γ irradiation, and then the lymphocytes were cultured with mitogenic stimulation to determine number of the micronuclei in cytokinesis-blocked binucleated cells. The treatment of lymphocytes with extract showed a significant decrease in the incidence of micronuclei binucleated cells, compared with similarly irradiated lymphocytes without extract against γ irradiation. The maximum protection and decrease in frequency of micronuclei was observed at 50 μg/mL of Zataria extract by 32% reduction. High-performance liquid chromatography analysis of extract showed that it contains high amounts of thymol. Zataria extract exhibited concentration-dependent radical-scavenging activity on 1,1-diphenyl-2-picryl hydrazyl free radicals. These data have an important application for the protection of human lymphocyte from the genetic damage and side-effects induced by γ irradiation in personnel exposed to radiation.

Modification in the Expression of MRE11/RAD50/NBS1 Complex in Low dose Irradiated Human Lymphocytes

Despite the fact that high doses of radiation are detrimental, low dose radiation (LDR) often protects the organism against a subsequent exposure of lethal doses of radiation. Present study was undertaken to understand the role of Mre11, Rad50 and Nbs1 genes in the low dose radio-adapted human peripheral blood mononuclear cells (PBMCs). Optimum time interval between low dose (0.07 Gy) and high dose (5.0 Gy) of (60)Co-gamma-radiation was observed to be 5.0 hours, at which PBMCs showed maximum LDR induced resistance (RIR). At cytogenetic level, micronuclei frequency was found to be reduced in LDR pre-irradiated PBMCs subsequently exposed to high dose radiation (HDR) as compared to controls. At transcriptional level, with reference to sham-irradiated cells significantly (p< or =0.05) altered expression of Mre11, Rad50 and Nbs1 genes was observed in low dose irradiated cells. At protein level, Mre11, Rad50 and Nbs1 were enhanced significantly (p< or =0.05) in low dose pre-irradiated cells subsequently exposed to high dose of radiation as compared to only high dose irradiated cells. Transcriptional as well as translational modulation in the expression of MRN complex components upon low dose irradiation may confer its participation in repair pathways, resulting in induced resistance.

Caffeic acid modulates ultraviolet radiation-B induced oxidative damage in human blood lymphocytes

Ultraviolet (UV) radiation causes inflammation, gene mutation and immunosuppressin in the human skin cells. These biological changes are responsible for photocarcinogenesis and photoaging. Normal lymphocytes are highly sensitive to the damaging effect of UV-radiation and undergo cell death. In the present study, the photoprotective effect of caffeic acid (3,4-dihydroxy cinnamic acid), a dietary phenolic compound, has been examined in the UVB (280–320) irradiated human blood lymphocytes. Lymphocytes pretreated with increasing concentration of caffeic acid (l, 5 and 10 μg/mL) for 30 min were irradiated and lipid peroxidation, antioxidant defence status, cell viability (by MTT assay) and DNA damage (by comet assay) were examined. UVB-irradiation causes increased levels of lipid peroxidation, DNA damage and decreased antioxidant status, cell viability in human lymphocytes. Caffeic acid pretreatment significantly reduced the levels of lipid peroxidation markers i.e. thiobarbituric acid reactive substance (TBARS), lipid hydroperoxide (LPH), conjugated diene (CD) and decreased DNA damage (tail length and % tail DNA) in UVB-irradiated lymphocytes. Further, caffeic acid pretreatment significantly maintains antioxidant status and decreased UVB-induced cytotoxicity. The maximum dose of caffeic acid (l0 μg/mL) normalized the UVB induced cellular changes indicating the photoprotective effect of caffeic acid in irradiated lymphocytes.

Radioprotective effects of hesperidin against genotoxicity induced by -irradiation in human lymphocytes

The radioprotective effect of hesperidin against genotoxicity induced by gamma-irradiation has been investigated in vivo/in vitro in cultured blood lymphocytes from human volunteers. Peripheral blood samples were collected at 0 (10 min before) and at 1, 2 and 3 h after a single oral ingestion of 250 mg hesperidin. At each time point, the whole blood was exposed in vitro to 150 cGy of (60)Co gamma-irradiation and then the lymphocytes were cultured with mitogenic stimulation to determine the micronuclei in cytokinesis-blocked binucleated cells. For each volunteer, the results showed a significant increase in the incidence of micronuclei after exposure of cells to gamma-irradiation as compared to control samples. The lymphocytes in the blood samples collected at 1 h after hesperidin ingestion and exposed in vitro to gamma-rays exhibited a significant decrease in the incidence of micronuclei, compared with similarly irradiated lymphocytes from blood samples collected at 0 h. The maximum protection and decrease in frequency of micronuclei (33%) was observed at 1 h after ingestion of hesperidin. These data have important application for the protection of human lymphocytes from the genetic damage and side effects induced by gamma-irradiation in patients undergoing radiotherapy.

Adaptive response in different mitotic cycles after irradiation

The frequency of cells with chromosome aberrations and the number of aberrations per cell by metaphase analysis have been studied in the nonirradiated progeny of irradiated human blood lymphocytes. The DNA fragmentation (DNA double strand breaks) have simultaneously been investigated by the DNA comet assay. PHA stimulated lymphocytes have been irradiated in the adaptive dose 0.05 Gy 24 h and in the challenge dose 1 Gy 48 h after stimulation to study the adaptive response (AR). 5-bromodeoxyuridine have been added for the identification the first--the fourth mitoses. It has been discovered that the frequency of chromosome aberrations is increased is all mitotic cycles after challenge irradiation, the level of double strand breaks is increased too. The adaptive response in induced by the adaptive and challenge irradiation in the first and the second mitotic cycles (fixation 48 and 72 h after stimulation) for the most parts of individuals, but it is absent in the third and the fourth mitosis. Only chromatid aberrations are observed in the first mitosis, but chromosome aberrations--in the following mitosis. Investigation by the DNA comet assay have showed the adaptive response is noticed 48-72 h after stimulation but it is insignificant 96 h. The conclusion is that the genomic instability is observed in nonirradiated progeny irradiated lymphocytes; the adaptive response is manifested up to third mitosis and is explained by the decreasing of the number of the chromatid and chromosome aberrations and DNA fragmentation. We can suppose that double strand DNA breaks can be signaling damage for the adaptive response induction.

Characterization of late-arising chromosome aberrations in human B-cell lines established from α-ray– or γ-ray–irradiated lymphocytes

To clarify the characteristics of late-arising (delayed) chromosome aberrations after irradiation in human lymphocytes, 30 B-cell lines were established from the peripheral blood from a healthy adult donor, the lymphocytes of which were exposed to α-rays or γ-rays and then used for experiments. Chromosome aberrations were serially observed at several passages by both conventional cytogenetics and fluorescence in situ hybridization analysis using subtelomere probes. These B-cell lines derived from lymphocytes with a history of radiation exposure had higher percentages of delayed chromosome aberrations, such as dicentrics, rings, endoreduplication, hyperdiploid, hyperploidy, and telomere association. Furthermore, α-ray exposure induced higher chromosome instability than γ-ray exposure, indicating that delayed chromosome aberrations were related with radiation quality. Chromosome instabilities were also observed at the subtelomere. Cell lines showing high chromosome instability had high DNA-PK activity, low expressions of Ku70, p53, and TRF1 proteins after stimulation with radiation. These results indicate that mechanisms underlying delayed chromosome aberrations might be epigenetic, and multiple factors such as defects of DNA-PK, subtelomere, and telomere might be associated.

Residual γH2AX after irradiation of human lymphocytes and monocytes in vitro and its relation to late effects after prostate brachytherapy

Background and purpose Retention of γH2AX foci in irradiated cells can signify a deficiency in DNA double-strand break repair that may be useful as an indicator of individual radiosensitivity. Materials and Methods To examine this possibility, the retention of γH2AX after irradiation was compared using white blood cells from 20 prostate brachytherapy patients who developed late normal tissue toxicity and 20 patients with minimal toxicity. Peripheral blood lymphocytes and monocytes were coded for analysis, exposed in vitro to 4 doses of 0.7 Gy X-rays at 3 hourly intervals, and retention of γH2AX was measured by flow cytometry 18 hours after the final irradiation. Results Excellent reproducibility in duplicate samples and a range in residual γH2AX from 7% above background to 244% above background were observed. Residual γH2AX in lymphocytes showed a positive correlation with patient age. However, no relation was observed between the level of residual γH2AX in peripheral blood mononuclear cells and late normal tissue damage. Conclusions We conclude that the method of detection of residual γH2AX after in vitro irradiation of lymphocytes and monocytes was simple, reproducible, and sensitive. However, it failed to predict for late normal tissue toxicity after brachytherapy. Possible reasons are discussed.

Trifluoperazine stimulates ionizing radiation induced cell killing through inhibition of DNA repair

The effect of trifluoperazine (TFP), a phenothiazine derivative antipsychotic drug, on ionizing radiation (IR) induced cell killing through inhibition of DNA repair was investigated in human cell lines. In clonogenic survival assay, TFP augmented IR induced cell killing. Also, TFP enhanced micronucleus formation in irradiated human lymphocytes. The effect of TFP and other known DNA repair inhibitors like wortmannin and caffeine, on irradiated cells, was compared by MTT assay. On the other hand, TFP failed to increase the toxicity induced by H 2O 2. Repair of DNA double strand breaks induced by IR was markedly inhibited by TFP, as determined by field inversion gel electrophoresis (FIGE). Further, TFP increased radiation induced apoptosis, which was accompanied by enhanced G2/M arrest. Thus, our results strongly suggest that TFP inhibits repair of DNA damage induced by IR, which significantly implicates the possibility of using TFP as an adjuvant to radiotherapy.

Artificial neural network modeling of apoptosis in gammairradiated human lymphocytes

Purpose: To develop an artificial neural network (ANN) model of apoptotic response in gamma irradiated human lymphocytes. To assess the feasibility of training ANN radiobiological models using data collected with flow cytometry.Materials and methods: Irradiated isolated human lymphocytes were labelled with Annexin V-Fluorescein Isothiocyanate (FITC) and 7-Amino-Actinomycin D (7AAD) then analysed using flow cytometry. Twenty-four dose responses per donor from 14 donors were collected from a flow cytometer and used in model development as the training and cross-validation datasets. The general ANN model architecture was a multi-layer perceptron using the mean squared error of a cross validation dataset as the objective function. The ANN model was optimized by varying the number of hidden layers and the number of processing elements per layer. The optimized model constituted of three hidden layers with 80, 40, and 10 hidden layers in the first, second, and third layers respectively.Results: The optimized model was used to simulate dose responses at the training doses of 0, 2, 4 and 8 Gray. A strong agreement between the model and measured dose responses was observed. The model was also used to simulate a dose response at 0.1 Gray and results were compared to the measured dose response from a donor not used in model development. Again, strong agreement between the model and the observed dose response was found.Conclusions: This study shows that artificial neural networks can be trained to provide high resolution, high accuracy models of multivariate radiobiological data collected by flow cytometry.

Photoprotective effect of sesamol on UVB-radiation induced oxidative stress in human blood lymphocytes in vitro

Normal lymphocytes are highly sensitive to the damaging effect of radiation and undergo cell death. In the present study, the photoprotective effect of sesamol, a constituent of sesame oil, has been examined in the UVB-(280–320 nm) irradiated human blood lymphocytes. Lymphocytes pretreated with increasing concentrations of sesamol (1, 5 and 10 μg/ml) for 30 min, were irradiated and lipid peroxidation and antioxidant defense were examined. UVB-irradiated lymphocytes exhibited increased levels of lipid peroxidation and disturbances in antioxidant defense. Sesamol pretreatment resulted in significant reduction in lipid peroxidation marker, thiobarbituric acid reactive substances (TBARS). Further, antioxidants like reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) increased, in a dose-dependent manner, in sesamol pretreated and UVB-irradiated lymphocytes. The maximum dose of sesamol (10 μg/ml) normalized the UVB induced lipid peroxidation, indicating the photoprotective effect of sesamol in irradiated lymphocytes.