Multidisciplinary European Low Dose Initiative (MELODI): strategic research agenda for low dose radiation risk research.

MELODI (Multidisciplinary European Low Dose Initiative) is a European radiation protection research platform with focus on research on health risks after exposure to low-dose ionising radiation. It was founded in 2010 and currently includes 44 members from 18 countries. A major activity of MELODI is the continuous development of a long-term European Strategic Research Agenda (SRA) on low-dose risk for radiation protection. The SRA is intended to identify priorities for national and European radiation protection research programs as a basis for the preparation of competitive calls at the European level. Among those key priorities is the improvement of health risk estimates for exposures close to the dose limits for workers and to reference levels for the population in emergency situations. Another activity of MELODI is to ensure the availability of European key infrastructures for research activities, and the long-term maintenance of competences in radiation research via an integrated European approach for training and education. The MELODI SRA identifies three key research topics in low dose or low dose-rate radiation risk research: (1) dose and dose rate dependence of cancer risk, (2) radiation-induced non-cancer effects and (3) individual radiation sensitivity. The research required to improve the evidence base for each of the three key topics relates to three research lines: (1) research to improve understanding of the mechanisms contributing to radiogenic diseases, (2) epidemiological research to improve health risk evaluation of radiation exposure and (3) research to address the effects and risks associated with internal exposures, differing radiation qualities and inhomogeneous exposures. The full SRA and associated documents can be downloaded from the MELODI website ( http://www.melodi-online.eu/sra.html ).

Interaction of lipid peroxidation products with nuclear macromolecules.

The interaction of lipid peroxidation products with nuclear macromolecules was investigated in rat liver nuclei labelled with [3H]arachidonic acid. Lipid peroxidation reactions were driven both non-enzymatically and enzymatically by the addition of ascorbate-Fe2+ or NADPH-ADP-Fe3+, respectively, to the incubation mixtures. The extent of peroxidation was evaluated by the formation of thiobarbituric acid chromophore and of radioactive hydrophilic peroxidation products. The results obtained show that: (1) nuclear membrane lipid peroxidation products formed during incubation interact with DNA and total nuclear proteins; (2) non-enzymatic lipid peroxidation processes induced a 40% larger association of peroxidation products to DNA compared to processes driven enzymatically, whereas the corresponding interaction with total nuclear proteins was similar in both peroxidation systems; (3) the radioactivity associated with histones decreased during incubation in the presence of ascorbate-Fe2+ or NADPH-ADP-Fe3+, and increased in control samples (no additions); (4) inhibition of lipid peroxidation by the iron chelator Desferrioxamine B prevented the association of peroxidation products to nuclear macromolecules; (5) the levels of radioactivity found in DNA after 180 min of incubation would represent the formation of 0.6-1.0 adducts per 10(6) DNA bases. The results obtained provide evidence for an interaction between lipid peroxidation products and chromatin in the interior of the cell nucleus.

Effects of zero magnetic field on the conformation of chromatin in human cells.

The effects of zero magnetic field on human VH-10 fibroblasts and lymphocytes were studied by the method of anomalous viscosity time dependencies (AVTD). A decrease of about 20% in the AVTD peaks was observed within 40 to 80 min of exposure of fibroblasts. This decrease was transient and disappeared 120 min after beginning of exposure. Similar kinetics for the effect of zero field was observed when cells were exposed 20 min and then kept at an ambient field. A 20% decrease of the AVTD peaks (p < 0.005 to 0.05) 40 to 70 min after 20 min exposure to zero field was reproduced in four independent experiments (out of four) with human lymphocytes from the same healthy donor. Contrary to the effects of zero field, irradiation of lymphocytes or fibroblasts with gamma-rays resulted in significant increase of the AVTD peaks immediately after irradiation. We concluded that zero field and gamma-rays caused hypercondensation and decondensation of chromatin, correspondingly. The effect of ethidium bromide served as a positive control and supported this conclusion. The effects of zero field on human lymphocytes were more significant in the beginning of G1-phase than in G0-phase. Thus, human fibroblasts and lymphocytes were shown to respond to zero magnetic field.

Osmotic fragility and fluidity of erythrocyte membranes from rats raised on an essential fatty acid deficient diet.

Erythrocyte membranes from rats raised on a diet with low content of essential fatty acids were studied by osmotic sensitivity tests and spin labeling techniques. This diet induced significant modifications in acylglycerophosphocholine fatty acid composition with regard to 16 : 1, 18 : 1, 18 : 2 (n-6), 20 : 3 (n-9), and 20 : 4 (n-6). No changes in membrane fluidity as monitored by spin label motion were found but the diet caused an increased osmotic sensitivity in essential fatty acid deficient erythrocytes. 50% hemolysis was obtained at a 51.0% dilution of saline with H2O as compared to a 57.0% dilution for the control material. Membrane fluidity was unaffected by gamma-irradiation up to 80 krad.

Studies on lipid peroxidation in rat liver nuclei and isolated nuclear membranes.

Non-enzymatic and enzymatically-driven lipid peroxidation processes were studied in rat liver nuclei and isolated nuclear membranes, by evaluating the formation of thiobarbituric acid-chromophore, free malondialdehyde, lipofuscin-like pigments, and the degradation of polyunsaturated fatty acids of the nuclear membrane lipids. The results obtained show that: (1) both non-enzymatic and enzymatically driven lipid peroxidation processes are operative in cell nuclei and isolated nuclear membranes; (2) only for isolated nuclear membranes, a good qualitative and up to a great extent quantitative correlation between malondialdehyde and lipofuscin-like pigment formation was obtained; (3) there is a qualitative but not quantitative correlation between malondialdehyde formation and polyunsaturated fatty acid degradation; (4) lipid peroxidation processes in isolated nuclear membranes and intact nuclei have an essentially identical kinetic behaviour. No statistical differences in the relative increases in the concentrations of malondialdehyde and lipofuscin-like pigments or in the degradation of polyunsaturated fatty acids were obtained, when the two systems were compared, except in the presence of NADPH-ADP-Fe3+, which induced a significantly larger degradation of polyunsaturated fatty acids in isolated nuclear membranes than in intact nuclei, and (5) no malondialdehyde-DNA fluorescent adduct formation was observed in any of the experimental groups studied, as inferred from the characteristics of the fluorescent spectra of lipofuscin-like pigments extracted from incubated nuclear preparations.

Effects of ethidium bromide on DNA loop organisation in human lymphocytes measured by anomalous viscosity time dependence and single cell gel electrophoresis.

The effects of ethidium bromide (EtBr) on human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD) and by the comet assay. EtBr at low concentrations increased the maximum viscosity and time of radial migration as measured with AVTD at neutral conditions of lysis. A pronounced relaxation of DNA loops was observed with the neutral comet assay. The maximal comet length corresponded to 2 Mb DNA loops. At high concentrations of EtBr, 2 mg/ml, significant reduction in AVTD below control level was seen that suggested hypercondensation of chromatin. The hypercondensation was directly observed with the neutral comet assay. EtBr did not induce DNA strand breaks as measured by the alkaline comet assay. The hypercondensed nuclei could be decondensed by irradiation with gamma-rays or exposure to light. The data provide evidence that EtBr at high concentrations resulted in hypercondensation of chromatin below control level. The comet assay confirmed that the increase in AVTD peaks deals with relaxation of loops and AVTD decrease is caused by chromatin condensation. The prediction of the AVTD theory for a correlation between time of radial migration and condensation of chromatin was verified. Further, the data show that the comet assay at neutral conditions of lysis is rather sensitive to DNA loop relaxation in the absence of DNA damage. Finally, donor specificity was found for the hypercondensation.

Studies on radiation-induced apoptosis in G0 human lymphocytes.

PURPOSE: To determine the relationships between the frequencies of radiation-induced chromosomal alterations and the extent of apoptosis in G0 human lymphocytes. MATERIAL AND METHODS: G0 human peripheral blood lymphocytes (HPBL) were X or gamma-irradiated, in the presence or absence of the repair inhibitor cytosine arabinoside (Ara-C). Directly after irradiation, a part of the lymphocytes were stimulated to grow while the rest were stimulated 48 h after irradiation. These lymphocyte cultures were analysed for induction of chromosomal aberrations. A subset of lymphocytes was kept in G0 and analysed for cell viability, apoptosis and p53 expression. RESULTS: The fraction of cells bearing dicentrics was reduced in lymphocytes stimulated to grow 48 h post irradiation as compared to lymphocytes stimulated immediately after irradiation. The decrease in the frequency of dicentrics correlated with the increase in the number of apoptotic cells. The operative apoptotic pathway in irradiated Go lymphocytes was dependent on the expression of p53. CONCLUSIONS: The radiation-induced apoptotic response of G0 lymphocytes is p53 dependent and increases with the time they are held in G0. When mitogen was added 48 h after irradiation, cells with dicentrics were either preferentially eliminated or did not enter mitosis. Thus the radiation-induced damage can be underevaluated depending on the time between radiation exposure and the induction of proliferation. These results may have relevance for biodosimetry studies or for evaluations of the efficacy of radiotherapy which are based on the frequencies of chromosomal aberrations.

Chronic Gamma-Irradiation Induces a Dose-Rate-Dependent Pro-inflammatory Response and Associated Loss of Function in Human Umbilical Vein Endothelial Cells.

A central question in radiation protection research is dose and dose-rate relationship for radiation-induced cardiovascular diseases. The response of endothelial cells to different low dose rates may contribute to help estimate risks for cardiovascular diseases by providing mechanistic understanding. In this study we investigated whether chronic low-dose-rate radiation exposure had an effect on the inflammatory response of endothelial cells and their function. Human umbilical vein endothelial cells (HUVECs) were chronically exposed to radiation at a dose of 1.4 mGy/h or 4.1 mGy/h for 1, 3, 6 or 10 weeks. We determined the pro-inflammatory profile of HUVECs before and during radiation exposure, and investigated the functional consequences of this radiation exposure by measuring their capacity to form vascular networks in matrigel. Expression levels of adhesion molecules such as E-selectin, ICAM-1 and VCAM-1, and the release of pro-inflammatory cytokines such as MCP-1, IL-6 and TNF-α were analyzed. When a total dose of 2 Gy was given at a rate of 4.1 mGy/h, we observed an increase in IL-6 and MCP-1 release into the cell culture media, but this was not observed at 1.4 mGy/h. The increase in the inflammatory profile induced at the dose rate of 4.1 mGy/h was also correlated with a decrease in the capacity of the HUVECs to form a vascular network in matrigel. Our results suggest that dose rate is an important parameter in the alteration of HUVEC inflammatory profile and function.

H2O2-driven reduction of the Fe3+-quin2 chelate and the subsequent formation of oxidizing species.

The objective of this study was to compare effects of quin2 and EDTA in iron-driven Fenton-type reactions. Seven different assays for detection of strong oxidants were used: the DMSO, deoxyribose, benzoate hydroxylation, and plasmid DNA strand breakage assays, detection of 8-oxo-deoxyguanosine in deoxyguanosine mononucleosides and calf thymus DNA, and electron spin resonance with the spin-trap (4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in the presence of ethanol or DMSO. With H2O2 and Fe3+, quin2 generally strongly increased the formation of reactive species in all assays, whereas with EDTA the results varied between the assays from barely detectable to highly significant increases compared to H2O2 and unchelated Fe3+. We found that the species produced in the reaction between Fe3+-quin2 and H2O2 behaved like the hydroxyl radical in all assays, whereas with Fe3+-EDTA no clear conclusion could be drawn about the nature of the oxidant. The effect of quin2 on the formation of oxidants on Fe2+ autoxidation, varied from generally inhibiting to slightly promoting, depending on the assay used. EDTA had a promoting effect on the amount of oxidant detected by all but one assay. None of the autoxidation systems produced DMSO or ethanol radical adducts with 4-POBN. In the presence of either chelator, H2O2, and Fe2+ DMSO and ethanol radical adducts of 4-POBN were produced. Using the Fe2+ indicator ferrozine, evidence for direct reduction of Fe3+-quin2 by H2O2 was found. Superoxide anion radical appeared to be less efficient than H2O2 as reductant of Fe3+-quin2 as addition of superoxide dismutase in the ferrozine experiments only decreased the amount of Fe2+ available for Fenton reaction by 10-20%. The main conclusions from our study are that the reduction of Fe3+-quin2 can be driven by H2O2 and that Fe2+ in the following oxidation step produces a species indistinguishable from free hydroxyl radical.

Can 8-oxo-dG be used as a predictor for individual radiosensitivity?

PURPOSE: To develop predictive tests for individual radiosensitivity of tumor patients. METHODS AND MATERIALS: Acute skin reactions were clinically scored among 40 women after 46 Gy, given with 2 Gy fractions to breast and regional lymph nodes, adjuvant after surgery. The acute skin reactions were compared to the excretion of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in urine, determined by high-performance liquid chromatography (HPLC) with electrochemical detector. Specimens of urine were collected before and during postoperative radiation treatment at given intervals. We compared a group of 9 patients with the most pronounced skin reactions with another group of 8 patients with almost no skin reactions at 46 Gy. RESULTS: The level of 8-oxo-dG excreted in urine during 8 h was measured. After normalizing the excretion to irradiated volumes, dose per volume and excretion before irradiation, the 8-oxo-dG level in urine was significantly (p < 0.001) lower for the patients with pronounced skin reactions as compared to patients with minor skin reactions, at an accumulated dose of 12 Gy. In addition, the background level of 8-oxo-dG excreted before treatment started, was significantly (p = 0.043) lower for patients with minor skin reactions as compared to patients with pronounced skin reactions. The background level of 8-oxo-dG was corrected for body weight and normalized to BMI. CONCLUSION: We suggest that the excretion of 8-oxo-dG into urine of breast cancer patients is a possible marker for acute radiosensitivity.

Induction of homologous recombination in the hprt gene of V79 Chinese hamster cells in response to low- and high-LET irradiation.

Dense ionization tracks from high linear energy transfer (LET) radiations form multiple damaged sites (MDS), which involve several types of DNA lesions in close vicinity. The primary DNA damage triggers sensor proteins that activate repair processes, cell cycle control or eventually apoptosis in subsequent cellular responses. The question how homologous recombination (HR) and non-homologous end joining (NHEJ) interact in the repair of radiation-induced DNA damage of MDS type has been addressed in different model systems but several questions remain to be answered. We have therefore challenged cells with treatments of ionizing radiation of different qualities to investigate whether primary DNA damages of different complexity are reflected in the processes of repair by HR as well as cell survival. We used the V79 derived SPD8 cell line to determine the induction of HR in the hprt exon 7 and clonogenic assay for survival in response to radiation. SPD8 cells were irradiated with gamma-rays (137Cs 0.5 keV/microm), boron ions (40 and 80 keV/microm) and nitrogen ions (140 keV/microm), with doses up to 5 Gy. Analysis of clonogenic survival showed that B-ions (80 keV/microm) and N-ions were more toxic than gamma-rays, 4.1 and 5.0 times respectively, while B-ions at 40 keV/microm were 2.0 times as toxic as gamma-rays. Homologous recombination in the cells exposed to B-ions (80 keV/microm) increased 2.9 times, a significant response as compared to cells exposed to gamma-rays, while for B-ions (40 keV/microm) and N-ions a nonsignificant increase in HR of 1.2 and 1.4, respectively, was observed. We hypothesize that the high-LET generated formation of MDS is responsible for the enhanced cytotoxicity as well as for the mobilization of the HR machinery.

Effects of gamma rays in the 0.5-50-cGy range on the conformation of chromatin in mammalian cells.

The effects of 137Cs gamma-ray quanta in mammalian cells were studied using the method of anomalous viscosity time dependence (AVTD). Several different cell types were exposed: VH-10 human fibroblasts, BALB/c mouse splenocytes and Sprague-Dawley rat thymocytes. The cells were irradiated with doses of 0.1-50 cGy and then lysed for viscosity measurements. It was established for all types of cells that exposure to a dose of 0.5 cGy resulted in a statistically significant reduction in viscosity peaks. This reduction reached a maximum value approximately 40-60 min after irradiation. The reduction of viscosity was revealed at doses up to 4 cGy for human fibroblasts with the maximum effect observed at about 2 cGy. The opposite response, an increase in viscosity, was observed after exposing the cells to 10-50 cGy. From the linear approximation of this dose dependence, the increase in viscosity started at doses above 4 cGy. The effect of increased viscosity disappeared with time after irradiation, with kinetics similar to that of DNA repair. Repair of this effect of AVTD depended strongly on temperature in the 0-37 degrees C range. In contrast, the kinetics of the effect of 0.5 cGy did not depend on temperature. Thus two different responses of chromatin were observed in mammalian cells after low (<4 cGy) and high (>4 cGy) doses of radiation. The specific inhibitor of DNA topoisomerase II, etoposide, was shown to increase the peaks of AVTD significantly. These data provide further evidence that the effects of AVTD correlate with changes in chromatin conformation.

Protection or sensitization by thiols or ascorbate in irradiated solutions of DNA or deoxyguanosine.

The initial aim of this study was to investigate how charge and other chemical properties of some radical scavengers influence the radiation-induced formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in two model systems. The target molecule, deoxyguanosine (dG), was either organized in the DNA-helix form or present as a free nucleoside in an aerated aqueous phosphate buffer. Samples were irradiated with 137Cs gamma rays, alone or in the presence of different thiols, alcohols or ascorbate with net charges from -1 to +1. The formation of 8-oxo-dG was assayed with reverse-phase HPLC coupled to an electrochemical detector. In the absence of radical scavengers, the radiation-induced formation of 8-oxo-dG in DNA was extensive, and the ratio for formation of 8-oxo-dG was 20-fold higher for DNA compared to dG. The yields of 8-oxo-dG in DNA and dG were 7.7 x 10(-3) micromol J(-1) and 3.8 x 10(-4) micromol J(-1), respectively. Yield-dose plots showed that the efficiency of the positively charged thiol cysteamine to counteract the radiation-induced formation of 8-oxo-dG in DNA was significantly (P < 0.001) greater compared to the uncharged or negatively charged thiols. Uncharged thiols were significantly (0.001 < P < 0.05) more effective in protecting DNA compared to negatively charged thiols. In contrast to the protection against oxidative damage provided by thiols and ascorbate when they were present during irradiation of DNA, the formation of 8-oxo-dG was significantly increased when these compounds were present during irradiation of dG in solution. Compared to the irradiated control, the increase was 11- to 116-fold for thiols and ascorbate, respectively. The enhanced oxidative damage of dG observed in the presence of ascorbate or thiols suggests that secondarily formed radicals from thiols or ascorbate may react with dG, or that transformation of different primary sites of damage on dG to 8-oxo-dG is enhanced.

Changes in chromatin conformation during radiation-induced apoptosis in human lymphocytes.

Human peripheral lymphocytes in G(0) phase were irradiated with 1-5 Gy of gamma rays. The biochemical and morphological changes characteristic of apoptosis were examined for 72 h after irradiation. In parallel, changes in chromatin conformation were studied by the method of anomalous viscosity time dependence (AVTD) and by measurements of nuclear halo size. An immediate and dose-dependent relaxation of chromatin, which became saturated at doses above 2-3 Gy, was revealed by the AVTD method. The state of relaxed chromatin lasted up to 12-24 h after irradiation, a response considerably longer than the time attributable to repair of radiation-induced DNA breaks. Measurements of nuclear halo size also indicated the initial relaxation of chromatin in the irradiated cells and its subsequent condensation. This condensation of chromatin as revealed with AVTD correlated well with nuclear condensation, as measured with dual fluorescence staining, and with DNA fragmentation, as measured by conventional and pulsed-field gel electrophoresis (PFGE). Late apoptotic cells did not contribute significantly to the AVTD signal, showing that the chromatin of these cells was completely condensed and fragmented.

Lipid peroxide levels in a murine adenocarcinoma exposed to hyperthermia: the role of glutathione depletion.

Increased lipid peroxide levels were obtained 1 h after a 60-min 43 degrees C hyperthermia treatment of a solid murine C3H mammary adenocarcinoma, grown subcutaneously in the hind paws of mice. Previous work from our group revealed that this heat treatment depletes the intracellular glutathione (GSH) content in this tumor. To investigate GSH depletion as one tentative mechanism behind the increased lipid peroxide levels obtained, we also measured the formation of lipid peroxidation products after extensive DL-buthionine-S,R-sulfoximine (BSO)-induced GSH depletion. The lipid peroxide effect provoked by BSO was less than that of the 60-min hyperthermia treatment. We therefore propose that the increased lipid peroxide levels induced by heat treatment do not correlate primarily with the observed decrease in GSH levels. Furthermore, in thermotolerance-induced tumors, lipid peroxide levels after a second heat treatment were observed to increase concomitantly with the cessation of thermotolerance. Lipid peroxide levels were also studied in liver, lung, and heart. Following BSO treatments, and up to 2-fold increase was observed in these organs in non-tumor-bearing mice. It was also observed that the intrinsic lipid peroxide levels in these organs from tumor-bearing mice were approximately 1.5- to 4-fold higher in comparison with non-tumor-bearing mice, thus indicating a systemic effect of the tumor implant.

Incorporation of [3H]acetate into the membrane lipids of a murine tumour during the development of thermotolerance.

The incorporation of [3H]acetate into the membrane lipids of a C3H mammary adenocarcinoma, grown s.c. in the hind paw of CBA mice, was followed to estimate the effects on the de novo synthesis of membrane lipids after hyperthermic treatments. Thermotolerance developed in response to a heat treatment at 43 degrees C for 20 min, as verified through growth rate studies of tumours exposed to fractionated heat treatments. Our results show that, during the development of thermotolerance, the relative rates of incorporation of [3H]acetate into the major lipid classes of the tumour cell membranes change significantly. The de novo synthesis of phospholipids decreased while that of cholesteryl esters plus triglycerides increased. The incorporation of [3H]acetate into cholesterol remained constant. Consequently, the ratio [3H]cholesterol/[3H]lecithin increased significantly during the development of thermotolerance. When the incorporation of [3H]acetate was followed 72-96 h after the heat treatment, i.e. at the interval at which heat resistance was observed to approach that of control tumours, the incorporation into cholesterol was significantly reduced while incorporation into phospholipids increased to control levels. Thus, the ratio [3H]cholesterol/[3H]lecithin was significantly lower, when compared to that of control tumours. The functional relationship between the heat-induced changes in the de novo synthesis of membrane lipids and the development of thermotolerance is discussed with regard to a mechanism based on homeoviscous adaptation of the membranes.

Chromosomal instability in human lymphocytes after low dose rate gamma-irradiation and delayed mitogen stimulation.

PURPOSE: To study the possibility that radiation induced chromosomal instability in human lymphocytes is promoted by a conflict between mitogen-induced growth stimulation and radiation-induced genotoxic stress. MATERIALS AND METHODS: Peripheral blood lymphocytes were exposed to low LET-irradiation at: (1) low-dose rate (LDR, 1-3 Gy, 0.024 Gy h[-1]) in order to minimize genotoxic stress; (2) high dose rate (HDR, 1-3 Gy, 45 Gy h[-1]) followed by immediate mitogen stimulation; and (3) HDR followed by a recovery period of 5 days before mitogen stimulation. Subsequent analyses included cell viability and clonogenic cell survival, chromosome aberrations at the first post-irradiation mitosis, and karyotype analysis of long term cultured cells, 11-57 days after mitogen stimulation. RESULTS: Dose (1-3 Gy) and dose rate (LDR and HDR) effects on the frequency of dicentric chromosomes at the first post-irradiation mitosis were in agreement with published data, with a pronounced dose rate effect of 2 and 3 Gy exposures. G-handed karyotypes after 11 days of growth in vitro showed increased frequencies of chromosome breaks and rearrangements in all irradiated cell cultures. Clones with complex karyotype abnormalities and increased frequencies of de novo aberrations developed in the irradiated cultures during extended growth for 22-57 days. These results show that: (1) LDR-irradiation induces chromosomal instability in primary human lymphocytes; (2) mitogen stimulation rescues HDR-irradiated cells from death at the expense of an increased level of chromosome aberrations; and (3) HDR-irradiated cells that are allowed 5 days of recovery before mitogen stimulation develop chromosomal instability during subsequent long-term proliferation. CONCLUSIONS: Neither the acute genotoxic stress of HDR-irradiation compared with LDR-irradiation, nor the hypothesized conflict between mitogen-induced growth stimulation and irradiation-induced growth arrest, seem to be critical conditions for the development of chromosomal instability in primary human T lymphocytes. Post-irradiation incubation allowing apoptotic processes to remove damaged cells does not prevent the subsequent development of chromosomal instability during long-term cell proliferation.

RBE for the induction of apoptosis in human peripheral lymphocytes exposed in vitro to high-LET radiation generated by accelerated nitrogen ions.

PURPOSE: To investigate the relative biological effectiveness (RBE) of accelerated nitrogen ions (32-45 MeV/u) compared with 137Cs gamma-rays for the induction of apoptosis in G0 lymphocytes. MATERIALS AND METHODS: Human peripheral G0 lymphocytes were exposed in vitro to doses up to 3 Gy. RBE for the induction of apoptosis was studied at different times after irradiation (0, 24, 48 and 72 h) with the use of three different methods: (1) morphological characterization of apoptotic cells after fluorescence staining; (2) cell size distribution analysis of the cell population to detect apoptotic bodies; and (3) electrophoretic analysis of DNA to detect 'DNA-ladders'. RESULTS: RBE values in the range of 1.3-3.0 were obtained from the linear components of the dose response curves. The variation in RBE was primarily dependent on post-irradiation time, where the highest RBE values were obtained after 48 h. The three different techniques used for analysis of apoptosis gave similar results. The significantly increased RBE was also seen as an earlier appearance of DNA-ladders, as well as a more rapid disappearance of the total number of viable cells. CONCLUSIONS: These results show that nitrogen ions of high linear energy transfer (LET) produce RBE > 1.0 and induce a faster apoptotic response as compared with gamma-photons of low-LET radiation.

Influence of dose-rate, post-irradiation incubation time and growth factors on interphase cell death by apoptosis and clonogenic survival of human peripheral lymphocytes.

PURPOSE: To investigate the effects of dose-rate, post-irradiation incubation time and growth factors on radiation-induced interphase cell death by apoptosis and reproductive cell death in human peripheral lymphocytes. MATERIALS AND METHODS: Lymphocytes in G0-phase were exposed in vitro to 1-3Gy 137Cs gamma-radiation at a high- (HDR, 45Gy/h) or a low dose-rate (LDR, 0.024Gy/h). HDR exposures were performed either on day 1 (HDR1) simultaneously with the start of the 3 Gy LDR exposure, or on day 6 (HDR6) when the LDR exposures ended. Apoptosis was studied at different times after irradiation by measuring (1) cellular membrane integrity, (2) morphological changes and (3) cell size reduction. Clonogenic survival was analysed for cells plated directly after irradiation (LDR, HDR6, HDR1 day 1) or after 5 days post-irradiation incubation (HDR1 day 6). RESULTS: A significant decrease in reproductive cell death was observed after 3 Gy LDR exposure as compared with the HDR1 exposure for cells plated day 6. For the lower doses applied, the dose-rate effect could not be statistically verified. A decrease in apoptosis for all three doses applied (i.e. 1, 2 and 3Gy) was observed when the LDR exposures were compared with the HDRI analysed day 6, although not of statistical significance. Radiation-induced apoptosis was efficiently counteracted by growth factors up to 24-48 h after 3 Gy HDR exposure. The prevention of radiation-induced cell death by growth factors was dependent on dose and post-irradiation time in G0. When the growth factors were added after a prolonged post-irradiation incubation in G0 (HDR 1 cells plated day 6), a significant increase in reproductive cell death was found (3 Gy) as compared with HDR protocols where the growth factors were added directly after irradiation (HDR 1 plated day 1 and HDR6). CONCLUSIONS: A dose-rate effect on radiation-induced apoptosis was indicated but not statistically verified. A significant dose-rate effect on reproductive cell death was observed. This dose-rate effect was, however, inverted when growth factors were added directly after the HDR irradiations.