Irradiated Human Peripheral Blood Lymphocytes Research Articles

THE IMPACT OF ASTAXANTHIN ON THE LEVEL OF DNA METHYLATION IN IRRADIATED IN VITRO HUMAN LYMPHOCYTES.

To investigate an effect of astaxanthin on global DNA methylation in human peripheral blood lympho-cytes exposed to γ-radiation in vitro. Samples of whole blood were diluted with RPMI-1640 medium in proportion 1 : 10 and incubated at 37 °Cfor 3 h. Samples were exposed to γ-ray (emitter IBL-237C, dose-rate 2.34 Gy/min) in dose 1.0 Gy. Astaxanthin wasadded into culture medium in final concentrations 20.0 μg/ml before irradiation. The analysis of the global DNAmethylation state was carried out using modified method of neutral single-cell gel electrophoresis (Comet assay)after digestion with the methylation-sensitive endonuclease HpaII. For every experiment the analysis of the frequency distribution of «comets» was carried out. The treatmentof non-irradiated and irradiated human peripheral blood lymphocytes with astaxanthin resulted to a statisticallysignificant (p < 0.05) shift of comet distribution with tendency to increasing level of DNA migration which indicatesincrease in the pool of cells with a reduced level of DNA methylation. It has been found that astaxanthin in concentration of 20.0 μg/ml reduced the levels of global DNAmethylation both in irradiated in vitro and native human lymphocytes. It suggests that astaxanthin has an effect onmechanisms of epigenetic regulation of gene expression.

The radioprotective effect of metformin against cytotoxicity and genotoxicity induced by ionizing radiation in cultured human blood lymphocytes

Metformin is a widely prescribed drug used in the treatment of patients with type 2 diabetes. In this study, the radioprotective effect of metformin was investigated against cytotoxicity and genotoxicity induced by ionizing radiation (IR) in human peripheral blood lymphocytes. Human lymphocytes were treated with metformin at concentrations 10 and 50μM for 2h and irradiated with 6MV X-rays. The radiation antagonistic potential of metformin was assessed by MTT [3-(4,5-dimethyl-2-thiaozolyl)-2,5-diphenyl-2H tetrazolium bromide] assay, chromosomal aberration (CA) analysis, cytokinesis blocked micronucleus (CBMN) assay, and flow cytometry. Observations demonstrated a radiation-dose-dependent decrease in the percentage of cell viability after 24h. It was found that pretreatment with metformin (10 and 50μM) increased the percentage of cell viability. A highly significant dose modifying factor (DMF) 1.35 and 1.42 was observed for 10 and 50μM metformin, respectively. Metformin (10 and 50μM) pretreatment significantly decreased the frequency of dicentrics (DCs), acentric fragments (AFs), rings (RIs), micronuclei (MN), and nucleoplasmic bridges (NPBs) in irradiated human peripheral blood lymphocytes. Also, treatment with metformin (10 and 50μM) without irradiation did not increase the number of MN, NPBs, DCs, AFs, RIs, and did not show a cytostatic effect in the human peripheral blood lymphocytes. On the other hand, metformin treatment (10 and 50μM) 2h prior to irradiation significantly reduced X-radiation-induced apoptotic incidence in human lymphocytes. The present study demonstrates metformin to be an effective radioprotector against DNA damage and apoptosis induced by IR in human lymphocytes. These data have an important application for the protection of lymphocytes from the genetic damage and side-effects induced by radiotherapy in cancer patients.

Biological dosimetry of ionizing radiation: Evaluation of the dose with cytogenetic methodologies by the construction of calibration curves

In case of a radiation accident, it is well known that in the absence of physical dosimetry biological dosimetry based on cytogenetic methods is a unique tool to estimate individual absorbed dose. Moreover, even when physical dosimetry indicates an overexposure, scoring chromosome aberrations (dicentrics and rings) in human peripheral blood lymphocytes (PBLs) at metaphase is presently the most widely used method to confirm dose assessment. The analysis of dicentrics and rings in PBLs after Giemsa staining of metaphase cells is considered the most valid assay for radiation injury. This work shows that applying the fluorescence in situ hybridization (FISH) technique, using telomeric/centromeric peptide nucleic acid (PNA) probes in metaphase chromosomes for radiation dosimetry, could become a fast scoring, reliable and precise method for biological dosimetry after accidental radiation exposures. In both in vitro methods described above, lymphocyte stimulation is needed, and this limits the application in radiation emergency medicine where speed is considered to be a high priority. Using premature chromosome condensation (PCC), irradiated human PBLs (non-stimulated) were fused with mitotic CHO cells, and the yield of excess PCC fragments in Giemsa stained cells was scored. To score dicentrics and rings under PCC conditions, the necessary centromere and telomere detection of the chromosomes was obtained using FISH and specific PNA probes. Of course, a prerequisite for dose assessment in all cases is a dose-effect calibration curve. This work illustrates the various methods used; dose response calibration curves, with 95% confidence limits used to estimate dose uncertainties, have been constructed for conventional metaphase analysis and FISH. We also compare the dose-response curve constructed after scoring of dicentrics and rings using PCC combined with FISH and PNA probes. Also reported are dose response curves showing scored dicentrics and rings per cell, combining PCC of lymphocytes and CHO cells with FISH using PNA probes after 10 h and 24 h after irradiation, and, finally, calibration data of excess PCC fragments (Giemsa) to be used if human blood is available immediately after irradiation or within 24 h.

Jatropha curcas leaf and bark fractions protect against ultraviolet radiation-B induced DNA damage in human peripheral blood lymphocytes

The present study is conducted to investigate the antioxidant potential of Jatropha curcas root bark extract (RB4 fraction) and leaf extract (L1 fraction), and to study their effects on UVB-radiation-induced DNA damage in cultured human blood lymphocytes. In this study, J. curcas showed strong antioxidant property in different free radical scavenging systems. Both the fractions effectively scavenged hydroxyl (OH), superoxide anion (O2–), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical cation (ABTS+) in a concentration-dependent manner. The IC50 (Inhibitory Concentration 50) values of J. curcas fractions were compared to standard ascorbic acid used in this study. The antioxidant potential of a compound was directly proportional to the photoprotective effect. In this study, human peripheral blood lymphocytes (HPBL) were exposed to UVB-radiation and there was an increase in comet attributes (% tail DNA, tail length, tail movement and Olive tail moment). Jatropha curcas RB4 fraction and L1 fraction treatment before UVB-irradiation significantly decreased the % tail DNA, tail length, tail moment and Olive tail moment in irradiated HPBL. These results suggested that J. curcas exhibited strong antioxidant property and RB4 and L1 fractions protected UVB-radiation-induced DNA damage in HPBL.

Apigenin ameliorates gamma radiation-induced cytogenetic alterations in cultured human blood lymphocytes

The aim of the present study was to assess the protective effect of apigenin, a dietary flavone, against cytogenetic alterations in human peripheral blood lymphocytes (HPBL) induced by Cobalt-60 radiation (3Gy). Results of MTT [3-(4, 5-dimethyl-2-thiaozolyl)-2,5-diphenyl-2H tetrazolium bromide] assay revealed that 37.2μM of apigenin was found to be non-toxic in HPBL. At this dose (37.2μM) of apigenin, the LD50 radiation dose of HPBL increased from 2.9Gy to 3.4Gy, which resulted in a DMF of 1.17. Apigenin (37.2μM) treatment 1h before irradiation significantly (p<0.05) reduced DNA damage in irradiated HPBL as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment). Moreover, apigenin treatment significantly decreased the frequencies of dicentric (DC), acentric fragments (AF), and acentric rings (AR) in irradiated HPBL. Apigenin pretreatment also reduced the radiation-induced CBMN (cytokinesis blocked micronuclei) anomalies such as micronuclei (MNi), nucleoplasmic bridges (NPB) and nuclear buds (NBUD) in HPBL. These results also showed that there was a significant correlation between NPB and DC frequencies and MNi and AF+AR. Treatment with apigenin alone had no significant effect on DNA damage and chromosomal aberrations in HPBL. Thus, the current studies indicate that apigenin protects HPBL from radiation-induced cytogenetic alterations.

Apigenin, a dietary antioxidant, modulates gamma radiation-induced oxidative damages in human peripheral blood lymphocytes

The present study was aimed to explore the antioxidant potential of apigenin and its preventive effect on radiation-induced oxidative damage in human peripheral blood lymphocytes (HPBL). Apigenin (4′, 5, 7-trihydroxy flavone) effectively scavenges hydroxyl (OH•), superoxide anion (O2•–), 1, 1-diphenyl- 2-picrylhydrazyl (DPPH•) and 2, 2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical cation (ABTS•+) in a concentration-dependent manner (37.2–186μM). Gamma-radiation (3Gy) exposure increased the levels of reactive oxygen species (ROS), % apoptotic cells and decreased the mitochondrial membrane potential (ΔΨm) in HPBL. On the other hand, apigenin treatment (37.2μM) 60min prior to irradiation significantly decreased ROS generation in HPBL. It has also been observed that apigenin treatment significantly reduced γ-radiation-induced apoptotic incidence and modulates loss of ΔΨm in HPBL. Furthermore, apigenin treatment significantly maintains cellular antioxidant status (superoxide dismutase, catalase, glutathione peroxidase activities and reduced glutathione level) and protects cellular milieu from γ-radiation-induced lipid peroxidation and micronuclei formation in irradiated HPBL. Radiation exposure increased p53, p21, Bax and NF-kB expressions and decreased Bcl-2 expression in HPBL. Conversely, an increase in Bcl-2 along with a decrease in p53, p21, Bax and NF-kB expression was observed in apigenin plus irradiated HPBL. Taken together, the present study indicates that apigenin showed strong antioxidant property in free radical scavenging systems and protects HPBL from radiation-induced oxidative damages at cellular and molecular level.

Growth hormone protects human lymphocytes from irradiation-induced cell death.

1. Undesired effects of cancer radiotherapy mainly affect the hematopoietic system. Growth hormone (GH) participates in both hematopoiesis and modulation of the immune response. We report both r-hGH cell death prevention and restoration of secretory capacities of irradiated human peripheral blood lymphocytes (PBL) in vitro. 2. r-hGH induced cell survival and increased proliferation of irradiated cells. Western blot analysis indicated that these effects of GH were paralleled by increased expression of the antiapoptotic protein Bcl-2. 3. r-hGH restored mitogen-stimulated release of IL-2 by PBL. Preincubation of irradiated lymphocytes with the growth hormone receptor (GHR) antagonists B2036 and G120 K abrogated r-hGH-dependent IL-2 release. 4. These results demonstrate that r-hGH protects irradiated PBL from death in a specific, receptor-mediated manner. Such effect of r-hGH on PBL involves activation of the antiapoptotic gene bcl-2 and prevention of cell death, associated with preserved functional cell capacity. Finally, potential use of GH as an immunopotentiating agent could be envisioned during radiation therapy of cancer.

DNA fragmentation and morphological changes in apoptotic human lymphocytes

Cell suspensions enriched in cells at various stages of apoptosis were obtained by separation of irradiated human peripheral blood lymphocytes on density gradients at different post-irradiation times. The state of DNA fragmentation in the cells was determined by comet assay and pulsed field gel electrophoresis. The morphologically distinguishable features of apoptosis such as chromatin condensation and cell shrinkage correlated with discrete stages of DNA fragmentation. It was found that ⩾50 kbp fragmentation of DNA occurs already in cells of normal density whereas the subsequent DNA fragmentation onto fragments <50 kbp occurs in parallel with cell shrinkage and simultaneous increase in cell density.The observed stages of DNA fragmentation seem to be separated in time that could allow in case of abortive apoptosis formation of chromosomal aberrations.

Comet assay to sense neutron ‘fingerprint’

The suitability of comet assay to identify DNA damage induced by neutrons of varying energy was tested. For this purpose, monoenergetic neutrons from Hiroshima University Radiobiological Research Accelerator (HIRRAC) were used to induce DNA damage in irradiated human peripheral blood lymphocytes. The level of damage was computed as tail moment for different doses (0.125–1 Gy) and compared with the effects resulting from irradiation with 60 Co γ. The neutron-irradiated cells exhibited longer comet tails consisting of tiny pieces of broken DNA in contrast to the streaking tails generated by 60 Co γ. The peak biological effectiveness occurred at 0.37 and 0.57 MeV; a further increase or decrease in neutron energy led to a reduced RBE value. The RBE values, as measured by the comet assay, were 6.3, 5.4, 4.7, 4.3, 2.6, and 1.7 for 0.37, 0.57, 0.79, 0.186, 1, and 2.3 MeV neutrons. The lower RBE value obtained by the comet assay when compared to that for other biological end points is discussed. This study reports the usefulness of the alkaline comet assay for identifying DNA damage induced by neutrons of the same radiation weighting factor. The comet assay is a potential tool for use in neutron therapy, as well as a method for the rapid screening of samples from individuals accidentally exposed to radiation.

Analysis of the alloreactive T cell repertoire in man. I. Differences in precursor frequency for cytotoxic T cell responses against allogeneic MHC molecules in unrelated individuals.

We demonstrate here that in man the frequency of cytotoxic T cells specific for a given set of allo-major histocompatibility complex (MHC) antigens varies among unrelated individuals. This has been established by limiting dilution analysis of human peripheral blood lymphocytes (HPBL) in the presence of irradiated autologous filler cells, T cell growth factors, and irradiated HPBL carrying allo-MHC antigens. Two unrelated individuals were tested against the same panel of allo-MHC antigens. We have been able to identify frequencies of T cells ranging from 1:5,000 to 1:20,000(high), 1:20,000 to 1:60,000(intermediate) and 1:60,000 to 1:100,000(low). In some cases, the precursor frequency of cytotoxic T cells was so low that it was considered to represent nonresponsiveness. The clear differences in precursor frequency suggest that this system is suitable for further analysis of the allo-MHC-specific repertoire in man.