Age dependence of T-lymphocyte apoptosis induced by high-energy proton exposure

Peripheral blood samples from three donors of different ages were exposed to 300 kVp x-rays or 138 MeV protons (0, 2, and 9 Gy dose). After 48 h incubation, CD4 and CD8 T-lymphocytes were labelled with specific monoclonal antibodies and cellular DNA was stained by propidium iodine. Radiation-induced apoptosis was followed by flow cytometry and the data were processed by LYSIS II software. The data analysis revealed an age-dependent sensitivity to radiation-induced apoptosis by 300 kVp x-rays and 138 MeV protons, for both CD4 and CD8 T-lymphocytes. Radiation-induced apoptosis was about 4 times greater in CD4 lymphocytes from the youngest donor than the oldest donor and was about 2 times greater in CD8 T-lymphocytes, both after x-ray and proton exposures. RBE values for CD4 T-lymphocytes ranged from 0.9 to 1.4 and for CD8-positive cells from 0.7 to 0.9. It is concluded that radiation-induced apoptosis of CD4 and CD8 T-lymphocytes, which is already exploited to predict patient response in conventional radiotherapy, may also be used to predict response in proton treatment planning. Received: 23 June 2000 / Accepted: 29 January 2001     

射线诱导在体造血细胞凋亡的量时效关系的研究

以4—10Gy射线在体损伤的小鼠为模型,应用DNA电泳和流式细胞术等方法证实细胞凋亡是射线损伤在体骨髓造血细胞的途径之一,发现射线诱导的造血细胞凋亡有明显的量效和时效关系。4、6、8和10Gy照射后,细胞凋亡发生率均表现为升高-降低过程,各剂量诱导的凋亡发生率峰值分别出现在照后12、8、4和4h,6和8Gy诱导的凋亡发生率已达最高水平,约为30％,10Gy诱导的凋亡反而要低。上述结果表明,诱导细胞凋亡是射线损伤骨髓造血细胞的一个重要途径,而且凋亡的发生率受照射剂量和照后时间的影响。因此,深入研究射线诱导的细胞凋亡,将有助于揭示射线损伤造血功能的机理。     

The importance of abrogation of G2-phase arrest in combined effect of TRAIL and ionizing radiation

BACKGROUND: In this work we studied the relationship between the enhanced expression of DR5 receptor and the effect of combination of TRAIL and ionizing radiation on cell cycle arrest and apoptosis induction in human leukemia cell line HL-60. MATERIAL AND METHODS: DR5, APO2.7 and cell cycle were analyzed by flow cytometry. Proteins Bid and Mcl-1 were analyzed by Western-blotting. For clonogenic survival, colony assay on methylcellulose was used. RESULTS: Ionizing radiation caused significantly enhanced positivity of DR5 receptors 24 h after irradiation with high doses (6 and 8 Gy). An increase of DR5 receptor positivity after a dose of 2 Gy was not statistically significant and application of TRAIL 48 h after irradiation did not increase the apoptosis induction. However, a decrease of radiation-induced G(2) phase arrest and an increase of apoptosis were observed when TRAIL was applied 16 h before irradiation with the dose of 2 Gy. Incubation with 6 microg/l TRAIL for 16 h reduced D(0) value from 2.9 Gy to 1.5 Gy. The induction of apoptosis by TRAIL was accompanied by Bid cleavage and a decrease of antiapoptotic Mcl-1 16 h after incubation with TRAIL. CONCLUSION: TRAIL in concentration of 6 microg/l applied 16 h before irradiation by the dose of 1.5 Gy caused the death of 63% of clonogenic tumor cells, similarly as the dose of 2.9 Gy alone, which is in good correlation with the enhanced apoptosis induction.     

Radiation-induced DNA double-stranded breaks and the dynamics of apoptotic death of human peripheral lymphocytes

DNA double-stranded breaks and their association with the development of radiation-induced peripheral lymphocyte apoptosis were studied in healthy donors exposed to in vitro gamma-irradiation in a dose of 1 Gy. It was shown that irradiation in 1-Gy dose caused a significant (p < 0.05) increase in the frequency of cells in late apoptosis 4 hours after irradiation and a rise in their frequency in early apoptosis 24 hours following this procedure. A significant correlation (r = 0.52, p < 0.05) was recorded between the primary level of radiation-induced DNA double-stranded breaks and the frequency of cells in late apoptosis following 4 hours, which suggests that DNA double-stranded breaks as a signal to trigger cell apoptotic death are of great importance.     

Radiation-induced apoptosis: a new approach using infrared microspectroscopy

PURPOSE: to characterize radiation-induced apoptosis in human cells using Fourier transform infrared microspectroscopy (FT-IRM) as a new analytical tool. MATERIAL AND METHODS: Normal human circulating lymphocytes were given a gamma ray dose of 6 Gy, or treated with t-butyl hydroperoxide (t-BuOH). HaCaT keratinocytes were given a dose of 20 Gy. Cells were deposited on ZnS windows for infrared spectral acquisition 2 days and 2 h after irradiation and 2 h after t-BuOH treatment. Apoptosis was simultaneously assessed by flow cytometry analysis of cells displaying annexin-V-positive staining. RESULTS: The flow cytometry study showed that about 90% of sham and irradiated cells were annexin-V negative 2 h after irradiation. Two days after irradiation, 68% of lymphocytes and 76% of HaCaT cells were apoptotic, as well as 43% of lymphocytes treated with t-BuOH. In infrared spectra of these apoptotic cells, qualitative and quantitative changes were observed. In the 960-1245 and 1690-1720 cm-1 ranges, mainly attributed to nucleic acids, changes corresponding to conformational changes in DNA were associated with a decrease in the amount of detectable DNA. Conformational changes were also observed in secondary protein structure, in particular an increase in the amount of beta structures. These DNA and protein changes were associated with an increase in the detectable amount of lipids in apoptotic HaCaT cells only. Two hours after irradiation, depending on the dose and (or) the cell type, qualitative and quantitative changes were observed in the IR spectra in the amide I and amide II bands, mainly attributed to proteins. These changes were associated with a significant decrease in the 1700-1750 cm-1 range, mainly attributed to the -C=O ester groups of DNA and phospholipids, in the irradiated HaCaT cells only. CONCLUSION: Our results are in agreement with biochemical published data on radiation-induced apoptosis, and show that DNA is the first cellular target of radiation-induced apoptosis, which, however, also requires conformational changes and synthesis of cell proteins. They also demonstrate that FT-IRM may be useful for assessing the early radiation damage at the molecular level in human cells.     

Changes in phosphorylation of histone H2A.X and p53 in response of peripheral blood lymphocytes to gamma irradiation

The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to gamma-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as gammaH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G(0) phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5-5 Gy) induction of foci containing gammaH2A.X (1 h after gamma-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3(+) lymphocytes were A(+). Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A(+)PI(-)) in comparison to non-stimulated PBMCs (38% A(+) resp. 13.4%). After PHA-stimulation also the amount of gammaH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to gamma-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2-7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3(+) T lymphocytes by the dose of 4 Gy 65% of cells were A(+).     

Nuclear scintigraphic assessment of radiation-induced intestinal dysfunction

Radiation-induced damage to the intestine can be measured by abnormalities in the absorption of various nutrients. Changes in intestinal absorption occur after irradiation because of loss of the intestinal absorptive surface and a consequent decrease in active transport. In our study, the jejunal absorption of (99m)Tc-pertechnetate, an actively transported gamma-ray emitter, was assessed in C3H/Kam mice given total-body irradiation with doses of 4, 6, 8 and 12.5 Gy and correlated with morphological changes in the intestinal epithelium. The absorption of (99m)Tc-pertechnetate from the intestinal lumen into the circulation was studied with a dynamic gamma-ray-scintigraphy assay combined with a multichannel analyzer to record the radiometry data automatically in a time-dependent regimen. The resulting radioactivity-time curves obtained for irradiated animals were compared to those for control animals. A dose-dependent decrease in absorptive function was observed 3.5 days after irradiation. The mean absorption rate was reduced to 78.8 +/- 9.3% of control levels in response to 4 Gy total-body irradiation (mean +/- SEM tracer absorption lifetime was 237 +/- 23 s compared to 187 +/- 12 s in nonirradiated controls) and to 28.3 +/- 3.7% in response to 12.5 Gy (660 +/- 76 s). The decrease in absorption of (99m)Tc-pertechnetate at 3.5 days after irradiation correlated strongly (P < 0.001) with TBI dose, with the number of cells per villus, and with the percentage of cells in the crypt compartment that were apoptotic or mitotic. A jejunal microcolony assay showed no loss of crypts and hence no measured dose-response effects after 4, 6 or 8 Gy TBI. These results show that dynamic enteroscintigraphy with sodium (99m)Tc-pertechnetate is a sensitive functional assay for rapid evaluation of radiation-induced intestinal damage in the clinically relevant dose range and has a cellular basis.     

Radioprotective potential of histamine on rat small intestine and uterus

The aim of this study was to improve knowledge about histamine radioprotective potential investigating its effect on reducing ionising radiation-induced injury and genotoxic damage on the rat small intestine and uterus. Forty 10-week-old male and 40 female Sprague-Dawley rats were divided into 4 groups. Histamine and histamine-5Gy groups received a daily subcutaneous histamine injection (0.1 mg/kg) starting 24 h before irradiation. Histamine-5Gy and untreated-5Gy groups were irradiated with a dose of whole-body Cesium-137 irradiation. Three days after irradiation animals were sacrificed and tissues were removed, fixed, and stained with haematoxylin and eosin, and histological characteristics were evaluated. Proliferation, apoptosis and oxidative DNA markers were studied by immunohistochemistry, while micronucleus assay was performed to evaluate chromosomal damage. Histamine treatment reduced radiation-induced mucosal atrophy, oedema and vascular damage produced by ionising radiation, increasing the number of crypts per circumference (239±12 vs 160±10; P<0.01). This effect was associated with a reduction of radiation-induced intestinal crypts apoptosis. Additionally, histamine decreased the frequency of micronuclei formation and also significantly attenuated 8-OHdG immunoreactivity, a marker of DNA oxidative damage. Furthermore, radiation induced flattening of the endometrial surface, depletion of deep glands and reduced mitosis, effects that were completely blocked by histamine treatment. The expression of a proliferation marker in uterine luminal and glandular cells was markedly stimulated in histamine treated and irradiated rats.The obtained evidences indicate that histamine is a potential candidate as a safe radio-protective agent that might increase the therapeutic index of radiotherapy for intra-abdominal and pelvic cancers. However, its efficacy needs to be carefully investigated in prospective clinical trials.Key words: histamine, ionising radiation, radio-protectors, small intestine, uterus.     

The radioenhancement of two human head and neck squamous cell carcinomas by 2'-2' difluorodeoxycytidine (gemcitabine; dFdC) is mediated by an increase in radiation-induced residual chromosome aberrations but not residual DNA DSBs

PURPOSE: The present study aimed at investigating if 2'-2' difluorodeoxycytidine (dFdC) radioenhancement was mediated by an effect on induction and/or repair of radiation-induced DNA DSBs and chromosome aberrations in cells with different intrinsic radiosensitivity. METHODS: Confluent human head and neck squamous cell carcinoma cell lines designated SCC61 and SQD9 were treated with 5 microM dFdC for 3 or 24 h prior to irradiation. DNA DSBs induction and repair were analyzed by PFGE. Radiation-induced chromosome aberrations were examined with a FISH technique. RESULTS: In both cell lines, dFdC did not modify radiation-induced DNA DSBs in a dose range between 0 and 40 Gy. After a single dose of 40 Gy, dFdC affected neither the kinetic of repair nor the residual amount of DNA DSBs up to 4 h after irradiation. Whereas dFdC did not increase the induction of chromosome aberrations, after a single dose of 5 Gy, the percentage of aberrant cells and the number of aberrations per aberrant cells were significantly higher in combination with dFdC. CONCLUSION: Our data suggest that under experimental conditions yielding substantial radioenhancement, dFdC decreases the repair of genomic lesions inducing secondary chromosome breaks but has no effect on DNA DSBs repair as measured by PFGE.     

巴西莓和诺尼果粉对C57BL/6J小鼠抗8Gy 剂量辐射的初步研究

目的:比较研究巴西莓果粉Herbal Clean Energy和诺尼果粉Noni GIA通过消除自由基、降低造血细胞凋亡等作用对8Gy大剂量γ线照射后小鼠活存的影响。方法:将C57BL/6J小鼠随机分组、每组雌雄各半,单一果粉在相同灌胃剂量下采用照前灌胃10天、照后灌胃10天以及照前照后灌胃10天三种灌胃方式,首先观察了小鼠在用钴60γ线8Gy致死剂量照后40天活存率;其次在上述相同条件处理下,照后10天对小鼠外周血白细胞计数、CD4+和CD8+淋巴细胞类型、活性氧、骨髓造血细胞凋亡率等分析。结果:生理盐水灌胃组C57BL/6J小鼠受8Gy照射在第18天全部死亡(n=20,下同),死亡率100%,而照前10天灌胃后照射8Gy试验组:巴西莓和诺尼果粉组照后40天活存10/20只,诺尼果粉组第40天活存9/20,巴西莓果粉组第40天活存8/20。在照后灌胃的组别中,诺尼果粉组第40天活存7/20,巴西莓和诺尼果粉组第40天活存4/20只,巴西莓果粉组第40天活存2/20。照射前后单独或联合灌胃两种果粉组外周血白细胞均有升高,而骨髓造血细胞凋亡降低,而且果粉灌胃小鼠外周血Th/Tc比率同对照组相比明显保持于正常值范围。红细胞和血小板数据无明显变化,活性氧含量则呈现无规律表现。结论:巴西莓和诺尼果粉对小鼠抗辐射有预防作用,其细胞学表现为保持造血增殖能力、降低造血细胞凋亡,并维持Th/Tc免疫平衡;显示果粉对保持造血和免疫能力是提高抵抗辐射损伤、提高活存的基础。同时表明,服用巴西莓果粉和诺尼果粉,对人体防止辐射损伤有预防作用。     

The effects of ionizing radiation on stem cells and hematopoietic progenitors: the place of apoptosis and the therapeutic potential of anti-apoptosis treatments

Abstract: Bone marrow aplasia observed following ionizing radiation exposure (Total Body Irradiation; gamma dose range: 2-10 Gy) is a result, in particular, of the radiation-induced (RI) apoptosis in hematopoietic stem and progenitor cells (HSPC). We have previously shown in a baboon model of mobilized peripheral blood CD34+ cell irradiation in vitro that RI apoptosis in HSPC was an early event, mostly occurring within the first 24 hours, which involves the CD95 Fas pathway. Apoptosis may be significantly reduced with a combination of 4 cytokines (4F): Stem Cell Factor (SCF), FLT-3 Ligand (FL), thrombopoietin (TPO), and interleukin-3 (IL-3), each at 50 ng x mL(-1) (15% survival versus <3% untreated cells, 24 h post-irradiation at 2.5 Gy). In this study we show that addition of TNF-alpha(800 IU/ml) induces an increase in 4F efficacy in terms of cell survival 24 h after incubation (26% survival after 24 h irradiation exposure at 2.5 Gy) and amplification (k) of CD34+ cells after 6 days in a serum free culture medium (SFM) (kCD34+ = 4.3 and 6.3 respectively for 4F and successive 4F + TNF-a/ 4F treatments). In addition, the 4F combination allows culture on pre-established allogenic irradiated stromal cells in vitro at 4 Gy (kCD34+ = 4.5). Overall this study suggests (i) the potential therapeutic interest for an early administration of anti-apoptotic cytokines with or without hematopoiesis inhibitors (emergency cytokine therapy) and (ii) the feasibility in the accidentally irradiated individual, of autologous cell therapy based on ex vivo expansion in order to perform autograft of residual HSPC collected after the accident.     

Suppression of thymic lymphoma induction by life-long low-dose-rate irradiation accompanied by immune activation in C57BL/6 mice

The induction of thymic lymphomas by whole-body X irradiation with four doses of 1.8 Gy (total dose: 7.2 Gy) in C57BL/6 mice was suppressed from a high frequency (90%) to 63% by preirradiation with 0.075 Gy X rays given 6 h before each 1.8-Gy irradiation. This level was further suppressed to 43% by continuous whole-body irradiation with 137Cs gamma rays at a low dose rate of 1.2 mGy/h for 450 days, starting 35 days before the challenging irradiation. Continuous irradiation at 1.2 mGy/h resulting in a total dose of 7.2 Gy over 258 days yielded no thymic lymphomas, indicating that this low-dose-rate radiation does not induce these tumors. Further continuous irradiation up to 450 days (total dose: 12.6 Gy) produced no tumors. Continuously irradiated mice showed no loss of hair and a greater body weight than unirradiated controls. Immune activities of the mice, as measured by the numbers of CD4+ T cells, CD40+ B cells, and antibody-producing cells in the spleen after immunization with sheep red blood cells, were significantly increased by continuous 1.2-mGy/h irradiation alone. These results indicate the presence of an adaptive response in tumor induction, the involvement of radiation-induced immune activation in tumor suppression, and a large dose and dose-rate effectiveness factor (DDREF) for tumor induction with extremely low-dose-rate radiation.     

Infrared microspectroscopic characteristics of radiation-induced apoptosis in human lymphocytes

Infrared microspectroscopic characterization of radiation-induced apoptosis was used as a new analytical tool to study the kinetics of apoptosis in human peripheral blood lymphocytes at the molecular level. This vibrational technique, which has already been used to investigate biomolecules in normal and tumor cells, allows the simultaneous detection of the biochemical changes in the various subcellular compartments. Normal circulating lymphocytes from five healthy human donors were given a single dose of 6 Gy ((60)Co) and deposited on ZnS windows for infrared spectral acquisition 1, 2 and 4 days after irradiation. Apoptosis was assessed simultaneously by flow cytometry analysis of lymphocytes displaying annexin V-positive staining, and by detection of the DNA laddering that is characteristic of apoptosis. The flow cytometry study showed that about 80% of sham-irradiated lymphocytes were annexin V(neg)/PI(neg) at 1, 2 and 4 days. One day after irradiation, 46% of irradiated lymphocytes were annexin V(neg)/PI(neg), 48% were annexin V(pos)/PI(neg), 5% were annexin V(pos)/PI(pos), and 1% were annexin V(neg)/PI(pos). These mean percentages were respectively 31, 59, 9 and 1 at day 2 and 23, 36, 30, and 11 at day 4. Irradiated lymphocytes presented a DNA laddering pattern characteristic of apoptosis from day 1 after irradiation. In the infrared spectra of irradiated lymphocytes, qualitative and quantitative changes were observed from days 1 and 2, respectively. In the range of 960-1245 cm(-1) mainly attributed to nucleic acids, changes corresponding to conformational changes in DNA were associated with a decrease in the amount of detectable DNA. Conformational changes were also observed in secondary protein structures, in particular an increase in the amount of beta structures. These DNA and protein changes were associated with an increase in the detectable amount of lipids at day 4 after irradiation. These results showed that DNA is probably the first cellular target of radiation-induced apoptosis, which, however, also requires conformational changes and synthesis of cell proteins. Our results are in agreement with biochemical and morphological data on radiation-induced apoptosis of normal human circulating lymphocytes, and they demonstrate that infrared microspectroscopy may be useful for assessing the process of apoptosis at the molecular level.     

Degradation of the nuclear matrix is a common element during radiation-induced apoptosis and necrosis

Human promyelocytic leukemia (HL60) cells were irradiated with 10 or 50 Gy of X rays and studied for up to 72 h postirradiation to determine the mode of death and assess changes in the nuclear matrix. After 50 Gy irradiation, cells were found to die early, primarily by apoptosis, while cells irradiated with 10 Gy died predominantly by necrosis. Disassembly of the nuclear lamina and degradation of the nuclear matrix protein lamin B occurred in cells undergoing radiation-induced apoptosis or necrosis. However, using Western blotting and a recently developed flow cytometry assay to detect changes in nuclear matrix protein content, we found that the kinetics and mechanisms of disassembly of the nuclear lamina are different for each mode of cell death. During radiation-induced apoptosis, cleavage and degradation of lamin B to a approximately 28-kDa fragment was detected in most cells within 4-12 h after irradiation. Measurements of dual-labeled apoptotic cells revealed that nonrandom DNA fragmentation was evident prior to or concomitant with breakdown of the nuclear lamina. Disassembly of the nuclear lamina during radiation-induced necrosis occurred much later (between 30-60 h after irradiation), and a different cleavage pattern of lamin B was observed. Degradation of the nuclear lamina was also inhibited in apoptosis-resistant BCL2-overexpressing HL60 cells exposed to 50 Gy until approximately 48 h after irradiation. These data indicate that breakdown of the nuclear matrix may be a common element in radiation-induced apoptosis and necrosis, but that the mechanisms and temporal patterns of breakdown of the nuclear lamina during apoptosis are distinct from those of necrosis.     

Protection of Radiation-Induced Damage to the Hematopoietic System,Small Intestine and Salivary Glands in Rats by JNJ7777120 Compound,a Histamine H4 Ligand

Based on previous data on the histamine radioprotective effect on highly radiosensitive tissues, in the present work we aimed at investigating the radioprotective potential of the H₄R ligand, JNJ7777120, on ionizing radiation-induced injury and genotoxic damage in small intestine, salivary glands and hematopoietic tissue. For that purpose, rats were divided into 4 groups. JNJ7777120 and JNJ7777120-irradiated groups received a daily subcutaneous JNJ7777120 injection (10 mg/kg) starting 24 h before irradiation. Irradiated groups received a single dose of 5 Gy on whole-body using Cesium-137 source and were sacrificed 3 or 30 days after irradiation. Tissues were removed, fixed, stained with hematoxylin and eosin or PAS staining and histological characteristics were evaluated. Proliferative and apoptotic markers were studied by immunohistochemistry, while micronucleus assay was performed to evaluate DNA damage. Submandibular gland (SMG) function was evaluated by methacholine-induced salivation. Results indicate that JNJ7777120 treatment diminished mucosal atrophy and preserved villi and the number of crypts after radiation exposure (240±8 vs. 165±10, P<0.01). This effect was associated to a reduced apoptosis and DNA damage in intestinal crypts. JNJ7777120 reduced radiation-induced aplasia, preserving medullar components and reducing formation of micronucleus and also it accelerated bone marrow repopulation. Furthermore, it reduced micronucleus frequency in peripheral blood (27±8 vs. 149±22, in 1,000 erythrocytes, P<0.01). JNJ7777120 completely reversed radiation-induced reduced salivation, conserving glandular mass with normal histological appearance and reducing apoptosis and atrophy of SMG. JNJ7777120 exhibits radioprotective effects against radiation-induced cytotoxic and genotoxic damages in small intestine, SMG and hematopoietic tissues and, thus, could be of clinical value for patients undergoing radiotherapy.     

Membrane oxidative damage induced by ionizing radiation detected by fluorescence polarization

Effects of ionizing radiation on biological membranes include alterations in membrane proteins, peroxidation of unsaturated lipids accompanied by perturbations of the lipid bilayer polarity. We have measured radiation-induced membrane modifications using two fluorescent lipophilic membrane probes (TMA-DPH and DPH) by the technique of fluorescence polarization on two different cell lines (Chinese hamster ovary CHO-K1 and lymphoblastic RPMI 1788 cell lines). γ-Irradiation was performed using a ⁶⁰Co source with dose rates of 0.1 and 1 Gy/min for final doses of 4 and 8 Gy. Irradiation induced a decrease of fluorescence intensity and anisotropy of DPH and TMA-DPH in both cell lines, which was dose-dependent but varied inversely with the dose rate. Moreover, the fluorescence anisotropy measured in lymphoblastic cells using TMA-DPH was found to decrease as early as 1 h after irradiation, and remained significantly lower 24 h after irradiation. This study indicates that some alterations of membrane fluidity are observed after low irradiation doses and for some time thereafter. The changes in membrane fluidity might reflect oxidative damage, thus confirming a radiation-induced fluidization of biological membranes. The use of membrane fluidity changes as a potential biological indicator of radiation injury is discussed. Received: 14 May 1996 / Accepted in revised form: 30 September 1996     

Modification of gene expression by dietary antioxidants in radiation-induced apoptosis of mice splenocytes

The modification of radiation-induced apoptosis in splenocytes by a vitamin-containing dietary supplement was studied. For 45 days prior to irradiation at a lethal dose of 6 Gy, mice received a dietary supplement containing vitamins with antioxidant properties and microelements. The expression of TRPM-2 (a marker for programmed cell death), bcl-2 (the product of which has been shown to prevent apoptosis), superoxide dismutase, and catalase genes was studied at different time intervals after irradiation. Radiation-induced alterations in gene expression were different in the control and the antioxidant mixture-fed mice. The antioxidant mixture administration resulted in an inhibition of TRPM-2 expression both before and after irradiation. The bcl-2 mRNA content steadily increased after irradiation in splenocytes from antioxidant mixture-fed mice, while in the control group 2-h after irradiation only trace amount of bcl-2 mRNA was detected. In splenocytes from control mice, the expression of superoxide dismutase and catalase genes significantly decreased within 2-h after irradiation; whereas in mice receiving the antioxidant mixture, inhibition of catalase gene expression was not as prominent. The expression of superoxide dismutase gene was still high 24-h after irradiation. The antioxidant administration decreased the radiation-induced apoptosis and delayed internucleosomal fragmentation of DNA. Our data suggest that radiation-induced alteration of gene expression is, at least in part, determined by reactive oxygen species.     

Evaluation of delayed apoptotic response in lethally irradiated human melanoma cell lines

To assess the lethal doses of gamma radiation and corresponding apoptotic response in new established human melanoma cell lines we exposed exponentially growing cultures to 8-100 Gy gamma radiation. The apoptosis and cell survival were determined by trypan blue exclusion, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction, agarose gel electrophoresis, colony forming assay, and long-term survival assay. The maximal DNA fragmentation 3 days after irradiation was observed in cultures irradiated with 20 Gy (36.9% TUNEL positive cells). The cultures irradiated with 50 and 100 Gy contained 18.7% and 16.4% TUNEL positive cells, respectively. Cultures exposed to 8 and 20 Gy gamma radiation recovered by week 3-4. Lethally irradiated (50 and 100 Gy) cultures which contained less apoptotic cells by day 3 died by week 5. A detectable increase in melanoma cell pigmentation after irradiation was also observed. The survival of human melanoma cell cultures after exposure to gamma radiation does not correlate with the level of apoptotic cells by day 3. At high radiation doses (> 50 Gy) when the radiation induced cell pigmentation is not inhibited the processes of apoptotic DNA fragmentation might be preferentially inactivated.     

Analysis of radiation-induced DNA double-strand breaks misrepair is not compromized by broken DNA in human fibroblasts

It has been suggested that the technique for measuring repair fidelity of radiation-induced DNA double-strand breaks (DSBs) using Southern blotting and hybridization to defined regions of the genome could be compromised by broken or poorly-digested DNA. Since misrepair of DNA DSBs is an important aspect of radiation-induced chromosome aberrations, mutations, and cell killing, we checked for such a supposition in non-transformed human fibroblasts. DSB misrepair was assessed in a NotI-cleavable DNA fragment of 3.2 Mbp located on the long arm of chromosome 21 and detected by D21S1 probe. We hypothesized that the suggested DNA degradation, whether spurious in nature or the results of irradiation-induced phenomena such as apoptosis and/or necrosis, should be detectable with or without NotI restriction enzyme treatment. When the DNA embedded in agarose plugs was separated by electrophoresis without prior NotI restriction, no significant difference was observed in the relative amount of migrating DNA between the control (no irradiation) and 24 h of repair following 80 Gy irradiation. Furthermore, only about 10% of the total signal was located below the 3.2 Mbp band. This suggests that the amount of DNA fragmentation due to biological (apoptosis or necrosis) or technical processes was negligible. The Tunel assay supported these results, as there was little to no apoptosis detectable in these fibroblasts up to 24 h after irradiation. We conclude that in primary human fibroblasts, the NotI method for measuring radiation-induced misrepair is not compromised by DNA degradation.     

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.