DNA damage in cultured skin microvascular endothelial cells exposed to gamma rays and treated by the combination pentoxifylline and α-tocopherol

Purpose: This in vitro study aims at evaluating the effect of the combination of pentoxifylline (PTX) and trolox (Tx), the water-soluble analogue of α-tocopherol, on the oxidative state and DNA damage in dermal microvascular endothelial cells exposed to doses up to 10 Gy of ionizing radiation.

Materials and methods: Confluent primary cultures of dermal endothelial cells were gamma irradiated at 3 and 10 Gy, and 0.5 mM of both drugs, PTX and Tx, was added either before (15 min) or after (30 min or 24 h) irradiation. Reactive oxygen species (ROS), measured by the dichlorodihydrofluorescein diacetate assay, and DNA damage, assessed by the comet and micronucleus assays, were measured at different times after exposure (0 – 21 days).

Results: The PTX/Tx treatment decreased the early and delayed peak of ROS production by a factor of 2.8 in 10 Gy-irradiated cells immediately after irradiation and the basal level by a factor of 2 in non-irradiated control cells. Moreover, the level of DNA strand breaks, as measured by the comet assay, was shown to be reduced by half immediately after irradiation when the PTX/Tx treatment was added 15 min before irradiation. However, unexpectedly, it was decreased to a similar extent when the drugs were added 30 min after radiation exposure. This reduction was accompanied by a 2.2- and 3.6-fold higher yield in the micronuclei (MN) frequency observed on days 10 and 14 post-irradiation, respectively.

Conclusion: These results suggest that oxidative stress and DNA damage induced in dermal microvascular endothelial cells by radiation can be modulated by early PTX/Tx treatment. These drugs acted not only as radical scavengers, but they were also responsible for the increased MN frequency in 10 Gy-irradiated cells. Thus, these drugs may cause a possible interference with DNA repair processes.     

人参皂苷Rh2对小鼠X射线辐射损伤的防护作用

目的 旨在探讨人参皂苷Rh2对X射线辐射损伤所致遗传毒性和免疫损伤的保护作用。方法 小鼠接受4 Gy X射线全身照射后,灌胃给予5、10和20 mg/kg剂量的人参皂苷Rh2,24 h后以碱性彗星电泳方法观察小鼠外周血淋巴细胞DNA损伤程度,以吖啶橙荧光染色法观察小鼠骨髓微核形成情况,以MTT法测定刀豆球蛋白和脂多糖诱导的脾淋巴细胞转化率。结果 人参皂苷Rh2剂量依赖性地抑制了X射线所致小鼠外周血DNA损伤和骨髓微核形成,对辐射所致脾淋巴细胞转化能力下降也有明显的防护作用。结论 人参皂苷Rh2对X射线所致遗传损伤和辐射防护均有明显保护作用,有望成为辐射防护药物。     

Persistence of genetic damage in mice exposed to low dose of X rays

PURPOSE: The aim of this work was to evaluate the persistence of genetic damage in CBA/J mice treated with a single irradiation of 0.1 or 1 Gy of X rays. MATERIALS AND METHODS: Peripheral blood was collected from irradiated and control mice after 30 min, 24 h, 7 days, 1, 3 and 6 months from exposure and analysed by comet assay. To investigate if the whole-body irradiation affect DNA repair, half of the sampled blood cells were in vitro-irradiated with additional 4 Gy and immediately analysed. Six months from exposure haematopoietic organs were sampled for measuring apoptotic index. RESULTS: In mice exposed to 1 Gy genetic damage was initially high and decreased during the experimental-time, while in the 0.1 Gy group damage, at first low, persisted and slightly increased. The 0.1 Gy-irradiated mice showed also a time-dependent increasing sensitivity to the in vitro-irradiation. Six months after whole-body irradiation, the percentage of apoptotic cells observed in haematopoietic compartments from 0.1 Gy-irradiated mice was significantly higher compared to controls and to 1 Gy mice. CONCLUSIONS: Results demonstrated that a single exposure to low-dose might induce long-term damage. Persistence of genetic damage might have relevant implications for estimating risk for low doses.     

Persistence of genetic damage in mice exposed to low dose of X rays

Purpose:?The aim of this work was to evaluate the persistence of genetic damage in CBA/J mice treated with a single irradiation of 0.1 or 1 Gy of X rays.

Materials and methods:?Peripheral blood was collected from irradiated and control mice after 30 min, 24 h, 7 days, 1, 3 and 6 months from exposure and analysed by comet assay. To investigate if the whole-body irradiation affect DNA repair, half of the sampled blood cells were in vitro-irradiated with additional 4 Gy and immediately analysed. Six months from exposure haematopoietic organs were sampled for measuring apoptotic index.

Results:?In mice exposed to 1 Gy genetic damage was initially high and decreased during the experimental-time, while in the 0.1 Gy group damage, at first low, persisted and slightly increased. The 0.1 Gy-irradiated mice showed also a time-dependent increasing sensitivity to the in vitro-irradiation. Six months after whole-body irradiation, the percentage of apoptotic cells observed in haematopoietic compartments from 0.1 Gy-irradiated mice was significantly higher compared to controls and to 1 Gy mice.

Conclusions:?Results demonstrated that a single exposure to low-dose might induce long-term damage. Persistence of genetic damage might have relevant implications for estimating risk for low doses.     

Protection of mice against X-ray injuries by the post-irradiation administration of guanosine and inosine

Purpose:?To examine the radioprotective action of guanosine (Guo) and inosine (Ino) administered to mice after irradiation with X-rays.

Materials and methods:?Survival of mice exposed to lethal and sublethal doses of X-rays was studied. Peripheral blood cells were counted using a light microscope. The damage to bone marrow cells was assessed by micronucleus (MN) test. Damage and repair of DNA in blood leukocytes were estimated using the comet assay.

Results:?Mice injected intraperitoneally (i.p.) with Guo or Ino (～30 μg g^?1, i.e., ～0.6 mg per 20-g mouse) 15 min after acute whole-body irradiation with 7 Gy recovered from X-ray injury. On the 30th day after irradiation, 50 and 40% of mice injected with Guo and Ino, respectively, remained alive. The dose reduction factor (DRF) was 1.23 for Guo and 1.15 for Ino. The protective effect gradually decreased as the time interval between the irradiation and injection was increased to 3, 5, 8 h. Guo and Ino facilitated the restoration of peripheral blood cell counts. These compounds protected bone marrow cells from damage and normalized erythropoiesis. Guo and Ino contributed to a more rapid and complete repair of DNA in mouse leukocytes irradiated both in vitro and in vivo.

Conclusion:?Guo and Ino introduced shortly after irradiation reduce leukopenia and thrombocytopenia and offer promise as therapeutic agents for treatment of radiation injuries.     

Radiation-Induced Comet-Formation in Human Skin Fibroblasts from Radiotherapy Patients with Different Normal Tissue Reactions

BACKGROUND: In clinical radiotherapy most patients tolerate the applied dosage with no or moderate side effects. However, 5 to 10% of all individuals show increased acute and/or late reactions. In-vitro test systems are investigated for their suitability for predictive purposes. This paper attempts a correlation between the induction and repair of DNA damage measured in the comet assay and the clinical observed reaction in order to evaluate the suitability of the comet assay for prediction of radiation sensitivity. PATIENTS AND METHODS: Skin fibroblasts of 30 patients with average tissue reactions or acute and/or late increased side effects and cell lines of 4 individuals carrying the heritable disease ataxia telangiectasia (AT) were irradiated in vitro. The induction and repair of DNA damage was measured at different time points after irradiation in the comet assay (single cell gel electrophoresis). These results were compared to the acute and late clinical reactions classified according to the RTOG grading system. RESULTS: The radiation induced DNA damage decreased over time reflecting DNA repair. Cells of the AT individuals showed an elevated damage induction and a reduced repair capacity compared to patients with average tissue reactions. Fibroblasts of patients with increased acute and late side effects exhibited slower DNA repair. In addition to the known lack of cell cycle control, our results indicate that AT cells show reduced DNA repair capacity. CONCLUSIONS: The comet assay seems to be able to detect some types of increased individual radiation sensitivity. In contrast to other predictive in-vitro tests, the comet assay needs less time and fewer cells, which would be useful in a clinical setting.     

In vitro response of human and porcine vascular cells exposed to high dose-rate γ-irradiation

Aim : The objective of this study was to compare the in vitro response of human and pig endothelial cells, smooth muscle cells and fibroblasts exposed to conventional high dose-rate γ-irradiation. Materials and methods : Clonogenic cell survival and growth responses were obtained after irradiation of plateau-phase cells with a 60 Co source at a dose-rate of 1.5 Gy/min. DNA single-strand breaks were also evaluated using an alkaline filter elution technique. Results : Overall, both the pig and human cell lines showed a similar response to conventional high dose-rate irradiation. Using clonogenic assays, the human aortic smooth muscle cell line was more sensitive than the fibroblast and endothelial cell lines, whereas the pig endothelial cell line was more sensitive than smooth muscle cells and fibroblasts. Shortly after irradiation (10 days) there was a temporary growth arrest, which was similar for endothelial, smooth muscle cells and fibroblasts with doses above 6 Gy. There was also a non-linear, dose-dependent growth delay up to 4 weeks after irradiation. This effect was also consistent between the different cell lines. Using alkaline filter elution, there was no significant difference in relative elution between endothelial cells, smooth muscle cells and fibroblasts, indicating similar DNA damage among the different cell lines. Conclusion : The in vitro response of human and pig endothelial cells, smooth muscle cells and fibroblasts exposed to high doserate irradiation appeared similar. The pig model seems well suited to evaluate the short- and long-term effects of ionizing radiation in the prevention of restenosis after vessel injury.     

Differential response to acute low dose radiation in primary and immortalized endothelial cells

Abstract

Purpose: The low dose radiation response of primary human umbilical vein endothelial cells (HUVEC) and its immortalized derivative, the EA.hy926 cell line, was evaluated and compared.

Material and methods: DNA damage and repair, cell cycle progression, apoptosis and cellular morphology in HUVEC and EA.hy926 were evaluated after exposure to low (0.05–0.5 Gy) and high doses (2 and 5 Gy) of acute X-rays.

Results: Subtle, but significant increases in DNA double-strand breaks (DSB) were observed in HUVEC and EA.hy926 30 min after low dose irradiation (0.05 Gy). Compared to high dose irradiation (2 Gy), relatively more DSB/Gy were formed after low dose irradiation. Also, we observed a dose-dependent increase in apoptotic cells, down to 0.5 Gy in HUVEC and 0.1 Gy in EA.hy926 cells. Furthermore, radiation induced significantly more apoptosis in EA.hy926 compared to HUVEC.

Conclusions: We demonstrated for the first time that acute low doses of X-rays induce DNA damage and apoptosis in endothelial cells. Our results point to a non-linear dose-response relationship for DSB formation in endothelial cells. Furthermore, the observed difference in radiation-induced apoptosis points to a higher radiosensitivity of EA.hy926 compared to HUVEC, which should be taken into account when using these cells as models for studying the endothelium radiation response.     

Effect of low-dose X-rays on the liver of whole-body irradiated rats

Abstract

Purpose: To investigate the effect of whole-body low-dose radiation (10 cGy of 6?MV x-rays) given prior to a challenging dose of 2?Gy x-rays on adaptive response in the liver of rats

Material and methods: Rats were either irradiated with 10 cGy, 2?Gy or 10 cGy 24?h prior to 2?Gy (10 cGy–2?Gy irradiated). Liver samples were analyzed for apoptosis, caspase-3, Bcl-2, by flow cytometry. DNA damage was determined by alkaline comet assay. The protein damage, lipid damage, and expression of endogenous antioxidants, Il-1β, Il-6, and TNF-α were determined by ELISA.

Results: An increase in apoptosis paralleled with increase in DNA damage and expression of Il-1β, Il-6, and TNF-α, protein oxidation, and lipid peroxidation were detected in all irradiated groups. The irradiated rats with 10 cGy–2?Gy and the 2?Gy-irradiated showed the highest effect. The increase in oxidative damage of protein and lipid was accompanied with depletion of endogenous antioxidants. In all cases, the10 cGy–2?Gy irradiated group showed similar or higher effect than the 2?Gy-irradiated group.

Conclusions: Under the present experimental conditions, There is no evidence that 10 cGy-irradiation protected the liver cells against a subsequent higher dose. The significant effect of 10 cGy-irradiation emphasizes the low-dose radiation-associated risk.     

慧星电泳用于肿瘤细胞辐射敏感性检测的研究

目的：探讨彗星电泳方法检测肿瘤细胞对γ射线的辐射敏感性的可行性。方法：以自行设计的图像分析系统，用彗星电泳方法检测4种人肿瘤细胞受γ射线照射后细胞初始DNA损伤，以及细胞径30min修复时DNA损伤残余率；用细胞集落存活法检测2Gy γ射线照射后细胞存活率。结果：4种肿瘤细胞初始DNA损伤与辐射敏感性无关，2Gyγ射线照射后4种细胞存活率（SF2）与细胞经30min修复后的DNA损伤残余率相关明显（r=-0.87)，结论：本实验方法有可能成为一种快速、准确检测肿瘤细胞内在辐射敏感性的方法。     

Analysis of the Action of the Restriction Endonuclease AluI Using Three Different Comet Assay Protocols

BACKGROUND AND PURPOSE: The comet assay offers the opportunity to measure the amount of DNA damage and the effectiveness of DNA repair in single cells. In a first part, experiments are presented comparing three different protocols of the comet assay technique with respect to the analysis of the induction of DNA damage after X-irradiation in isolated human lymphocytes and CHO cells. In a second part, the restriction enzyme AluI, an agent producing DNA double-strand breaks exclusively, was introduced into CHO cells by electroporation and the effects were analyzed using the different comet assay protocols. The experiments were carried out in order to test the assertion that comet assay techniques can measure different types of DNA damages at different pH conditions of lysis and electrophoresis. MATERIAL AND METHODS: Three different comet assay protocols were used for the analysis of DNA damage in lymphocytes and CHO cells. RESULTS: The results clearly indicate that among the three protocols the modified comet assay technique used by the authors showed the highest sensitivity in the radiotherapy-relevant dose range between 0 and 2 Gy. All three protocols were capable of detecting an effect by AluI. This effect, however, was clearly different from radiation effects. Whereas after radiation exposure all cell nuclei show a dose-dependent increase in DNA content in the comet tail, most of the cell nuclei were unaffected by an AluI uptake. Nevertheless, there was an effect by AluI that could be detected in all three assay versions: between 5% and 15% of the nuclei showed clearly abnormal comet morphologies. CONCLUSION: Neither the strictly alkaline nor the strictly neutral comet assay is applicable in the radiation dose range of about 2 Gy. The restriction enzyme results show that other factors than just DNA strand breaks contribute to DNA migration into the tail of the comets.     

Magnitude of radiation-induced DNA damage in peripheral blood leukocytes and its correlation with aggressiveness of thymic lymphoma in Swiss mice

Abstract

Purpose: The present study is aimed to investigate the magnitude and kinetics of DNA damage in peripheral blood leukocytes of mice exposed to whole body gamma irradiation (WBI; 3 Gy) and its correlation with aggressiveness of thymic lymphoma (TL).

Materials and methods: DNA damage was monitored in peripheral blood cells of individual mice by comet assay at different intervals of post-irradiation, which were correlated with weight of TL in respective mice at 120th day. To further study genomic radiosensitivity in TL development, peripheral blood samples collected at the 15th and 90th day of post-irradiation from control and WBI animals were irradiated (0.5 Gy) ex vivo followed by assessment of DNA damage by comet assay.

Results: The maximum DNA damage (tail moment) was observed at 5 min after WBI, which decreased at longer period, and was minimum at the 7th day after WBI. However, residual damage was observed in comparison to control and it persisted up to 90 days of irradiation. Tail moment values observed at an early time (5 min) of post-irradiation was better correlated (correlation coefficient, r = 0.84) with weight of TL than at longer time period (60 days; r = 0.21). Our results showed that in ex vivo irradiated (0.5 Gy) peripheral blood, the magnitude of DNA damage was higher in samples obtained from WBI mice than sham-irradiated controls suggesting enhanced genomic radiosensitivity in WBI mice. Genomic susceptibility to radiation observed in peripheral blood from WBI animals showed better correlation with weight of TL at the 15th day (r = 0.9) post-irradiation period than at the 90th day (r = 0.44).

Conclusion: These results suggest that the magnitude of radiation-induced initial DNA damage in peripheral blood leukocytes and genomic radiosensitivity could be an indicator of TL aggressiveness in mice.     

Effect of low-dose X-ray irradiation on micronucleus formation in human embryo,newborn and child cells

Purpose: It is well known that a high-dose of ionizing radiation is sufficient to break DNA strands, which leads to elevated genotoxic risks; however, the risks associated with low doses of ionizing radiation remain unclear. In addition, there is little data about the effect of low-dose ionizing radiation on human-derived embryo, newborn and child cells. We investigated the frequency of micronucleus (MN) formation in these cells to understand the genotoxic effects of ionizing radiation.

Materials and methods: We irradiated the cells with X-rays from 0.02–2?Gy at a rate of 0.0635?Gy/min. After irradiation, we investigated the effect of low-dose X-ray irradiation on cellular viability and frequency of MN formation.

Results: Increases in MN formation were largely dose-dependent; however, there were no differences between controls and doses lower than 0.2?Gy, except in KMST-6 human transformed embryo cells.

Conclusion: We could not detect an obvious effect of low-dose X-ray irradiation at doses lower than 0.1?Gy. The embryonic cells were more sensitive to X-ray irradiation than newborn and child cells. The threshold for X-ray-induced MN formation appears to be in the range of 0.05–0.1?Gy in cultured human embryo, newborn and child cells.     

Radioprotection of thyroid cells mediated by methimazole

Purpose:?The radioprotective effect of antithyroid drugs on radioiodine treatment is a controversial issue. However, it is of clinical relevance whether antithyroid medication has to be interrupted for therapy and when antithyroid medication can be continued after radioiodine treatment. We investigated DNA damage caused by internal or external radiation using thyroid cells (sodium iodine symporter [NIS] positive).

Materials and methods:?Adherent thyroid cells were irradiated following incubation with the mediators methimazole and perchlorate using either X-ray tube or Re-188-perrhenate. DNA damage was quantified by OTM (Olive's tail moment) of the alkaline comet assay.

Results:?Following external irradiation of 15?Gy OTM was 4.3?±?4.2 compared to 0.5?±?1.4 in controls. DNA damage was reduced by methimazole to 70%. Incubation with Re-188 showed effects depending on presence of the mediators. Non-irradiated controls had a mean OTM?<?1, internal irradiation increased OTM to 25.5?±?9.1 in cells without mediators. OTM decreased to 60% after pre-incubation with methimazole and to 15% with perchlorate. Re-188 uptake was modified by both perchlorate and, to a lesser extent, methimazole.

Conclusions:?Methimazole was shown to have a radioprotective effect not only by its scavenger capacity but also by interaction with NIS. Perchlorate acted by competitive inhibition of NIS mediated Re-188 uptake.     

Optimization and Standardization of the “Comet Assay” for Analyzing the Repair of DNA Damage in Cells

BACKGROUND: The "comet assay" has become an interesting and a very useful tool for the analysis of the induction and amount of DNA damage in single cells thus offering the opportunity to measure the effectiveness of DNA repair. On the basis of the Ostling and Johanson protocol we have developed a modified method with increased sensitivity and high reproducibility. MATERIAL AND METHODS: Human tumor cells or isolated human peripheral blood lymphocytes were analyzed in the experiments. The amount of DNA damage and the effectiveness of DNA repair was measured after X-irradiation using the "comet assay" technique. RESULTS: In this presentation the influences of different methodological factors like agarose concentration, buffer pH, electrophoresis time, electric field strength on the applicability of the "comet assay" are described in detail and optimum conditions for "comet assay" experiments have been evaluated. Additionally the authors will show a comparison of different fluorescent DNA dyes pointing out their advantages or disadvantages for "comet" analysis. The usefulness of this technique and its capabilities are exemplified by showing DNA repair kinetics of human lymphocytes of different healthy or radiosensitive donors after in-vitro irradiation with 2 Gy X-rays. CONCLUSIONS: This paper presents data on the optimization and standardization of the original "comet assay" leading to an extremely fast and practicable protocol in the field of single cell gel electrophoresis. After irradiation with 0.1 Gy an increase in the amount of DNA damage can be measured with high statistical significance and the DNA repair capacity of individual cells after X-ray doses of 2 Gy can be analyzed with high reproducibility. The results comparing DNA repair capacities of different donors point out that the "comet assay" may have the potential for the estimation of individual radiosensitivity.     

High levels of chromosome aberrations correlate with impaired in vitro radiation-induced apoptosis and DNA repair in human B-chronic lymphocytic leukaemia cells

Purpose : To investigate the relationship between the susceptibility of B-chronic lymphoid leukaemia (B-CLL) cells to DNA damage-induced apoptosis, the kinetics of DNA strand-break rejoining, and chromosome damage after exposure to ionizing irradiation. Materials and methods : Lymphocytes from B-CLL patients were γ-irradiated in vitro with 0.2-5 Gy and stimulated by Staphylococcus aureus cowan I (SAC I) for estimation of chromosomal damage. Induction of apoptosis after irradiation was studied in 50 patients by two methods: morphological characterization of apoptotic cells after fluorescent staining (Hoechst), and specific quantification of mono- and oligonucleosomes in cytoplasmic cell fractions (ELISA assay). Morphological chromosome damage was scored in the first cell generation after irradiation (13 patients). In parallel, the kinetics of DNA single-strand break rejoining were investigated by the alkaline comet assay (12 patients). Results : Ionizing irradiation did not induce apoptosis in lymphocytes from a subset of B-CLL patients. The results suggest that B-CLL cells resistant to radiation-induced apoptosis could repair DNA strand-breaks more rapidly and showed a higher level of chromosome aberrations than radiation-sensitive B-CLL cells. Conclusion : Each of three biological effects observed (apoptosis, kinetics of DNA single-strand-break repair, chromosomal damage) might be explained by different modifications occurring in irradiated B-CLL cells. Their convergence strongly suggests that resistance to apoptotic death initiation by DNA damage may be impeded by a rapid engaging of the DNA repair mechanisms. The higher level of chromosome aberrations observed in these cells suggests that the type of DNA repair system involved may generate inaccurate repair.     

Investigations of antioxidant-mediated protection and mitigation of radiation-induced DNA damage and lipid peroxidation in murine skin

Abstract

Purpose: Radioprotection and mitigation effects of the antioxidants, Eukarion (EUK)-207, curcumin, and the curcumin analogs D12 and D68, on radiation-induced DNA damage or lipid peroxidation in murine skin were investigated. These antioxidants were studied because they have been previously reported to protect or mitigate against radiation-induced skin reactions.

Methods: DNA damage was assessed using two different assays. A cytokinesis-blocked micronucleus (MN) assay was performed on primary skin fibroblasts harvested from the skin of C3H/HeJ male mice 1 day, 1 week and 4 weeks after 5 Gy or 10 Gy irradiation. Local skin or whole body irradiation (100 kVp X-rays or caesium (Cs)-137 γ-rays respectively) was performed. DNA damage was further quantified in keratinocytes by immunofluorescence staining of γ-histone 2AX (γ-H2AX) foci in formalin-fixed skin harvested 1 hour or 1 day post-whole body irradiation. Radiation-induced lipid peroxidation in the skin was investigated at the same time points as the MN assay by measuring malondialdehyde (MDA) with a Thiobarbituric acid reactive substances (TBARS) assay.

Results: None of the studied antioxidants showed significant mitigation of skin DNA damage induced by local irradiation. However, when EUK-207 or curcumin were delivered before irradiation they provided some protection against DNA damage. In contrast, all the studied antioxidants demonstrated significant mitigating and protecting effects on radiation-induced lipid peroxidation at one or more of the three time points after local skin irradiation.

Conclusion: Our results show no evidence for mitigation of DNA damage by the antioxidants studied in contrast to mitigation of lipid peroxidation. Since these agents have been reported to mitigate skin reactions following irradiation, the data suggest that changes in lipid peroxidation levels in skin may reflect developing skin reactions better than residual post-irradiation DNA damage in skin cells. Further direct comparison studies are required to confirm this inference from the data.     

DNA damage and repair in individual cells: applications of the comet assay in radiobiology.

The comet assay is a single-cell gel electrophoresis method that can measure a variety of types of DNA damage, and repair of damage, in individual cells. It is now in widespread use in genetic toxicology and oncology. This review describes the history of the development of this method and its applications in radiation biology, with particular emphasis on the use of the comet assay to measure heterogeneity in DNA damage in cells exposed to ionizing radiation.     

High levels of chromosome aberrations correlate with impaired in vitro radiation-induced apoptosis and DNA repair in human B-chronic lymphocytic leukaemia cells

PURPOSE: To investigate the relationship between the susceptibility of B-chronic lymphoid leukaemia (B-CLL) cells to DNA damage-induced apoptosis, the kinetics of DNA strand-break rejoining, and chromosome damage after exposure to ionizing irradiation. MATERIALS AND METHODS: Lymphocytes from B-CLL patients were gamma-irradiated in vitro with 0.2-5 Gy and stimulated by Staphylococcus aureus cowan I (SAC I) for estimation of chromosomal damage. Induction of apoptosis after irradiation was studied in 50 patients by two methods: morphological characterization of apoptotic cells after fluorescent staining (Hoechst), and specific quantification of mono- and oligonucleosomes in cytoplasmic cell fractions (ELISA assay). Morphological chromosome damage was scored in the first cell generation after irradiation (13 patients). In parallel, the kinetics of DNA single-strand break rejoining were investigated by the alkaline comet assay (12 patients). RESULTS: Ionizing irradiation did not induce apoptosis in lymphocytes from a subset of B-CLL patients. The results suggest that B-CLL cells resistant to radiation-induced apoptosis could repair DNA strand-breaks more rapidly and showed a higher level of chromosome aberrations than radiation-sensitive B-CLL cells. CONCLUSION: Each of three biological effects observed (apoptosis, kinetics of DNA single-strand-break repair, chromosomal damage) might be explained by different modifications occurring in irradiated B-CLL cells. Their convergence strongly suggests that resistance to apoptotic death initiation by DNA damage may be impeded by a rapid engaging of the DNA repair mechanisms. The higher level of chromosome aberrations observed in these cells suggests that the type of DNA repair system involved may generate inaccurate repair.     

γ射线诱导人淋巴细胞损伤及磷酸化组蛋白H2AX和ATM表达

目的 探讨人外周血经不同剂量60co γ射线照射后淋巴细胞损伤情况,以及与磷酸化的H2AX、ATM表达的关系.方法 永生化淋巴细胞及人新鲜外周血淋巴细胞经0～8 Gy 60 Coγ射线照射后0.5 h,分别采用Western blot和流式细胞术检测磷酸化ATM和γ-H2AX的表达情况,并通过CB微核法检测受照射后人外周血淋巴细胞微核验证细胞损伤程度.结果 流式细胞检测发现人外周血淋巴细胞照射后0.5 h,γ-H2AX表达的剂量效应关系呈线性平方模式,其拟合曲线为:Y=3.96+11.29D-0.45D2,而相同方法检测磷酸化ATM蛋白的表达未见明显变化.Western blot检测结果表明,照射后0.5 h,各受照射组磷酸化ATM表达在0.1～8 Gy范围内具有呈剂量依赖性增高的趋势.人外周血淋巴细胞微核结果显示,γ射线照射后DNA损伤情况明显加重,随吸收剂量的增加,微核细胞率明显增多.结论 60Co γ射线可诱导DNA双链断裂,随着吸收剂量的增加,微核率明显增加,磷酸化的H2AX、ATM表达水平亦明显增高,本研究为辐射损伤及辐射事故生物剂量估算领域的研究提供了理论依据.

Abstract：

Objective To investigate 60Co γ-ray induced damage in lymphocytes and the relationship between doses of 60Co γ-ray irradiation and the levels of phosphorylated H2AX and ATM.Methods Cells were irradiated with 60Co γ-rays in the range of 0-8 Gy.The levels of phosphorylated H2AX and ATM were detected by Western blot and FACScan,respectively.The micronucleus(MN)was analyzed by CB method to evaluate DNA damage.Results FACScan results showed the dose-effect relationship of γ-H2AX expression were linear.square at 0.5 h post-irradiation to different doses,and the fitting curve was shown as Y=3.96+11.29D-0.45D2.The level of phosphorylated ATM(p-ATM)was not changed significantly by using the same method.Western blot showed that p-ATM protein expression was significandy increased after irradiation compared with sham.irradiated group.The MN assay which represented DNA damage was sensitive to different doses.Conclusions γ-ray irradiation could induce the phosphorylation of H2AX and ATM,which may play an important role in indicating DNA damage.Both of H2AX and ATM have the potential as sensitive biomarker and biodosimeter for radiation damage.