Unirradiated Lymphocytes Research Articles

Oxidative Stress−Mediated Apoptotic Death in Human Peripheral Blood Lymphocytes Treated with Plasma from Gamma-Irradiated Blood

The present study is aimed to investigate the effect of plasma from irradiated human blood on the induction of apoptotic death in human peripheral blood lymphocytes. Our results showed that plasma obtained from γ-irradiated human blood showed a significant decrease in viability and an increase in apoptotic death in unirradiated lymphocytes, which is correlated with increase in intracellular reactive oxygen species level. Moreover, a decrease in the activity of superoxide dismutase in lymphocytes further seems to be associated with oxidative stress-mediated apoptotic death. The diffusible factors from irradiated blood were found to be generated from the cellular components of blood. Based on our results, we suggest induction of oxidative stress-mediated apoptotic death in unirradiated lymphocytes by diffusible factors from irradiated blood.

Gamma-H2AX-Based Dose Estimation for Whole and Partial Body Radiation Exposure

Most human exposures to ionising radiation are partial body exposures. However, to date only limited tools are available for rapid and accurate estimation of the dose distribution and the extent of the body spared from the exposure. These parameters are of great importance for emergency triage and clinical management of exposed individuals. Here, measurements of γ-H2AX immunofluorescence by microscopy and flow cytometry were compared as rapid biodosimetric tools for whole and partial body exposures. Ex vivo uniformly X-irradiated blood lymphocytes from one donor were used to generate a universal biexponential calibration function for γ-H2AX foci/intensity yields per unit dose for time points up to 96 hours post exposure. Foci – but not intensity – levels remained significantly above background for 96 hours for doses of 0.5 Gy or more. Foci-based dose estimates for ex vivo X-irradiated blood samples from 13 volunteers were in excellent agreement with the actual dose delivered to the targeted samples. Flow cytometric dose estimates for X-irradiated blood samples from 8 volunteers were in excellent agreement with the actual dose delivered at 1 hour post exposure but less so at 24 hours post exposure. In partial body exposures, simulated by mixing ex vivo irradiated and unirradiated lymphocytes, foci/intensity distributions were significantly over-dispersed compared to uniformly irradiated lymphocytes. For both methods and in all cases the estimated fraction of irradiated lymphocytes and dose to that fraction, calculated using the zero contaminated Poisson test and γ-H2AX calibration function, were in good agreement with the actual mixing ratios and doses delivered to the samples. In conclusion, γ-H2AX analysis of irradiated lymphocytes enables rapid and accurate assessment of whole body doses while dispersion analysis of foci or intensity distributions helps determine partial body doses and the irradiated fraction size in cases of partial body exposures.

Radiation-induced bystander effects and adaptive response in murine lymphocytes

Purpose: To study the bystander effects of γ-radiation in murine lymphocytes using irradiated conditioned medium (ICM) generated from irradiated lymphocytes.Methods: Proliferation response of unirradiated lymphocytes to mitogen concanavalin A (con A) in presence of ICM, collected from γ-irradiated lymphocytes (60Co source; 0.35 Gy/min; 0.1 – 1 Gy), was studied by 3H-thymidine incorporation and also by dye dilution using carboxyfluorescein succinimidyl ester (CFSE). Expression of proliferation markers, interleukin 2 receptor α chain (CD25) and cyclin D in ICM treated lymphocytes was analyzed by labeling with specific antibodies. Intracellular reactive oxygen species (ROS) and apoptosis were estimated by flow cytometry using dichlorodihydrofluorescein diacetate (H2DCFDA) and propidium iodide, respectively. Nitric oxide (NO) was measured using Griess reagent.Results: Proliferation response to con A in unirradiated lymphocytes was enhanced in the presence of ICM with maximum enhancement observed in the presence of 0.5 Gy ICM. Augmentation of proliferation in the presence of ICM was accompanied by an increase in CD25 and cyclin D expression, enhanced ROS and NO generation. ICM pretreated lymphocytes showed adaptive response to radiation which was not abrogated by wortmannin, a phosphatidyl inositol 3-kinase (PI3K) inhibitor.Conclusion: Soluble factors released from irradiated lymphocytes initiate a signaling cascade in unirradiated lymphocytes resulting in increased response to mitogen and radioresistance which may have an important role in radiation-induced immunomodulation.

Influence of Polymorphisms in DNA Repair GenesXPD, XRCC1andMGMTon DNA Damage Induced by Gamma Radiation and its Repair in LymphocytesIn Vitro

DNA single-strand breaks (SSBs) were quantified by single-cell gel electrophoresis and micronucleated and apoptotic cells were quantified by microscopic assays in peripheral blood lymphocytes after irradiation on ice with 2 Gy of 60Co gamma radiation, and their association with polymorphisms of genes that encode proteins of different DNA repair pathways and influence cancer risk (XPD codon 312Asp --> Asn and 751Lys --> Gln, XRCC1 399Arg --> Gln, and MGMT 84Leu --> Phe) was studied. In unirradiated lymphocytes, SSBs were significantly more frequent in individuals older than the median age (52 years) (P = 0.015; n = 81), and the frequency of apoptotic or micronucleated cells was higher in individuals with alleles coding for Asn at XPD 312 or Gln at 751 (P = 0.030 or 0.023 ANOVA, respectively; n = 54). The only polymorphism associated with the background SSB level was MGMT 84Phe (P = 0.04, ANOVA; n = 66). After irradiation, SSB levels and repair parameters did not differ significantly with age or smoking habit. The SSB level varied more than twofold and the repair rate and level of unrepaired SSBs more than 10-fold between individuals. The presence of variant alleles coding for Asn at XPD 312 was associated with more radiation-induced SSBs (P = 0.014) and fewer unrepaired SSBs (P = 0.008), and the phenotype (> median induced SSBs/< median unrepaired SSBs) was seen in the majority of XPD 312Asn/Asn homozygotes; the odds ratio for variant homozygotes to show this phenotype was 5.2 (95% confidence interval 1.4-19.9). The hypothesis is discussed that XPD could participate in repair of ionizing radiation-induced DNA damage. While it cannot be excluded that the effects observed are due to cosegregating polymorphisms or that the responses of lymphocytes are not typical of other cell types, the results suggest that polymorphism of DNA repair genes, particularly XPD, is one factor implicated in the variability of responses to ionizing radiation between different individuals.

Time-resolved fluorimetric probing of DNA structure in irradiated human lymphocytes

An in situ technique has been developed that detects genomic conformational changes in irradiated human cells. Cells are treated on ice with detergent, mild alkali and ethidium bromide (EB) and the resulting intact nuclei are examined using kinetic spectrofluorimetry. In the nuclei of unirradiated lymphocytes the fluorescence decay profile is tri-exponential with a long-lived component (∼23 ns) attributable to EB intercalated within double-stranded DNA, an intermediate life-time component (∼6 ns) indicative of a loosely bound DNA biomolecular-EB complex, and a short-lived component (∼2 ns) corresponding to unbound EB. Irradiated fresh human lymphocytes show three similar components but their relative contributions are changed. Results from a typical donor, show that after 1 Gy the intermediate component decreased with a concomitant increase in the long-lived component while the short-lived component remained essentially unchanged. Fresh whole blood from healthy donors was irradiated at doses of 0.1–1 Gy, and the samples analyzed with or without post-irradiation incubation at 37 °C for 24 h prior to lymphocyte extraction. For doses of 1.0 Gy in the absence of incubation there is good agreement between multiple samples of the same individual, or among the six donors, as compared with the results from irradiated isolated lymphocytes. Whole blood incubation was unreliable but results from one individual at 0.1 and 1.0 Gy were similar to those observed without incubation. Fluorescence lifetime analysis can detect DNA structural/topological damage in irradiated human lymphoid cells, and it may have potential application to in vivo bio-dosimetry and bio-monitoring.

Effect of ex vivo hyperthermia on radiation-induced micronuclei in lymphocytes of cancer patients before and during radiotherapy

To investigate the effect of ex vivo hyperthermia (HT) and 137 Cs -irradiation on micronucleus (MN) production in cytokinesis-blocked lymphocytes, we obtained the peripheral blood samples from the same cancer patients ( n=6) before and during fractionated partial-body radiotherapy (xRT). The whole blood cultures were heated at 43.5°C for 60 min, followed by 137 Cs irradiation (0–4 Gy). The control cultures from the same patients were incubated at 37°C after being exposed to radiation. The lymphocytes were then stimulated with PHA. Cytochalasin B was applied at 44 h, and lymphocytes were harvested at 72 h. MN frequency was determined on Giemsa-stained slides. We found that in patients before xRT, HT (43.5°C) significantly increased the MN yield (mean±SEM) in unirradiated lymphocytes from 15.6±2.8 (37°C) to 39.7±10.9. Further, in patients either before or during xRT, when the lymphocytes were treated with HT (43.5°C) and combined with ex vivo irradiation, the MN yield ( Y) could be estimated by a linear equation Y= C+ αD. Our findings indicate that as measured by the MN production in cytokinesis-blocked lymphocytes, HT alone at 43.5°C induced DNA damage. Moreover, it enhanced the radiation-induced cytogenetic damage. Therefore, the application of HT may impair the T-cell function in cancer patients who are receiving radiotherapy.

Cytogenetic investigations in three cell types of a Saudi family with ataxia telangiectasia

Chromosomal analyses were performed on lymphocytes, fibroblasts and lymphoblastoid cell lines derived from a Saudi family with ataxia telangiectasia (AT). The three siblings of a consanguineous marriage were all affected. The lymphocytes of the AT homozygotes (probands) showed an increase of 2- to 6-fold and 4- to 8-fold respectively, in the frequency of spontaneous and X-ray-induced chromosomal aberrations compared with controls, while the parents (obligate heterozygotes) of the patients showed no notable difference. The unirradiated lymphocytes from the oldest AT sibling, an 11-year-old boy (AT1), showed specific rearrangements involving chromosomes 7 and 14 [t(7;14)(q35;q12)] and 12 and 14 [t(12;14)(q23;q12)] in two different clones. The most severely affected sibling was a 9-year-old girl (AT2) who presented with a clone showing a novel rearrangement involving chromosomes 14 and 17, namely: del(14) (q31q32) and dup(17)(q21-q24). The lymphocytes from the third sibling, a 2-year-old boy (AT3), showed a t(2;14)(p24;q12). In addition, an inv(14)(q12q32) was observed in all three AT patients, while inv(7)(p14q35) was found only in patients 2 and 3. The lymphocytes from the AT parents and controls showed normal karyotypes. The breakpoints involving chromosomes 2, 12 and 17, observed in our studies, have rarely been reported in other series of AT patients. No non-random chromosomal rearrangements were observed either in the skin fibroblasts or in the lymphoblastoid cell lines derived from the AT patients, although all cell lines showed an increase in both spontaneous and radiation-induced chromosomal breaks per cell. The present study constitutes the first report on a cytogenetic analysis of a Saudi family with three AT siblings.

Interaction of Lymphocytes and High Endothelial Venules in Irradiated Lymph Nodes

After total-body exposure to various doses of ionizing radiation, the ability of lymphocytes to interact specifically with high endothelial venules of rat cervical and mesenteric lymph nodes was analyzed in frozen sections. Following a radiation dose of 1.5 Gy, high endothelial venules remained intact and the binding of unirradiated lymphocytes to the venules was enhanced relative to unirradiated controls. At radiation doses above 5.0 Gy, damage to high endothelial venules was observed histologically as well as assessed functionally. There was a significant decrease in specific lymphocyte-venule binding and a significant increase in nonspecific binding. These findings suggest that radiation-induced damage to high endothelial venules might play a role in radiation-induced immunosuppression by interfering with the normal passage of lymphocytes from the blood into lymph nodes via a specific interaction between lymphocytes and high endothelial venules.

Sensitivity of lymphocytes from patients with cancer to x- and ultraviolet radiation.

It has been suggested that radiation sensitivity may be increased in certain patients with cancer. Using a high-efficiency limiting dilution cloning technique, the sensitivity to x- and ultraviolet (UV)-radiation of lymphocytes from untreated patients with cancer to that of age-matched controls was compared. Sensitivity to x-radiation in 19 patients with carcinoma and 10 with lymphoma, and sensitivity to UV-radiation in 8 patients with carcinoma and 4 with lymphoma were studied. The proportion of unirradiated lymphocytes forming clones was significantly lower in patients with cancer, particularly lymphoma, but there was no suggestion of increased sensitivity to either x- or UV-radiation in any of the patients studied.

Mutations in human lymphocytes: Effect of X- and UV-irradiation

The mutagenic effects of X- and UV-irradiation on human lymphocytes were studied using a highly efficient cloning technique. The hypoxanthine-guanine phosphoribosyl-transferase enzyme locus was used to study mutation induction, with mutant cells being selected by their ability to form a clone in the presence of the purine analogue 6-thioguanine. Mutation dose-response curves for X- and UV-irradiation were established by studying lymphocytes from 11 individuals on day 10 after irradiation. The mean mutation frequency of unirradiated lymphocytes was 2.9 × 10 −6 and there were dose-dependent increase to 9.5 × 10 −5 after 400 rad of X-irradiation, and to 5.6 × 10 −5 after 125 erg/mm 2 of UV. The expression time of X-ray-induced mutations was 3−7 days. Dose-responses were obtained for mutation frequency and survival following X-irradiation of proliferating and non-proliferating lymphocytes from 8 individuals. Compared with non-proliferating lymphocytes, the proliferating lymphocytes developed fewer mutations but had a greater mortality after irradiation.

Age-related variation in the DNA-repair synthesis after UV-C irradiation in unstimulated lymphocytes of healthy blood donors

UV-C light-induced DNA-repair synthesis was studied in unstimulated lymphocytes of 51 healthy blood donors aged between 17 and 74 years. Repair synthesis was measured by the incorporation of [ 3H]thymidine after a 2-h incubation period in the presence of 2 mM hydroxyurea. The evaluation included (1)the spontaneous DNA-synthesis in unirradiated lymphocytes with and without hydroxyurea, (2) the DNA-repair synthesis in lymphocytes irradiated with UV-light. The interindividual variation was significantly higher than the methodological variation ascertained in 24 persons in whom 2 determinations were carried out. In blood donors aged between 17 and 39 years, the spontaneous DNA synthesis, both with and without hydroxyurea, was significantly lower than in older individuals The DNA-repair synthesis was dependent on the dose of UV-C light between 2 and 16 J/M 2. There were no significant differences in DNA-repair synthesis in the age range 17–74 years. The variations in rate of DNA-repair synthesis (greater or lesser degree of [ 3H]thymidine incorporation beyond the 99% confidence range) were wider in older (44–74 years), than in younger individuals.

The effect of x-ray induced mitotic delay on chromosome aberration yields in human lymphocytes

The extent to which X-ray induced mitotic delay at 150 and 400 rad influences chromosome aberration yields was examined in human peripheral blood lymphocytes. The dicentric was used as a marker and aberrations yields were obtained for mixed cultures prepared from equal numbers of normal and irradiated cells. The cultures were terminated following incubation times of 36-120 h. Greater mitotic delay of the order of a few hours was observed at the higher dose. However most reduction in the number of lymphocytes arriving at metaphase by 48 h may be ascribed to interphase death or failure to transform. Analysis of the dicentric distributions which were expected to follow Poisson statistics indicated that cells containing dicentrics were delayed relative to irradiated but aberration-free cells. Cells with one dicentric moved more easily through the first cell cycle than cells containing two dicentrics. Following accidental partial body irradiation, selection in culture favouring the unirradiated lymphocytes does not distort the aberration yield sufficiently to warrant incubation times in excess of the standard 48-52 h.