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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 ).

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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.

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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.

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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.

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DNA fragmentation of 50 kb is observed in apoptotic human lymphocytes as measured with pulsed field gel electrophoresis (PFGE). Standard PFGE assay involves embedding of cells into agarose blocks followed by lysis in the presence of proteinase K. In this study, we modified the PFGE protocol by omitting the proteinase K. In this study, we modified the PFGE assay by omitting the proteinase K and changing lysis solution according to the method of anomalous viscosity time dependence (AVTD). The conditions of PFGE were adjusted aiming to compress apoptotic fragments, increasing sensitivity and the number of samples that could be loaded on the same gel. Lymphocytes were irradiated with gamma-rays and apoptotic fragmentation of DNA was determined by PFGE using standard lysis with proteinase K and lysis protocol from AVTD method. Both protocols of lysis resulted in the same pattern of DNA fragments. The yield of radiation-induced apoptotic fragmentation was higher with the AVTD protocol of lysis. The novel PFGE protocol is simple and relatively non-expensive, requires only 7 h running time and gives a possibility to analyse simultaneously up to 69 samples in the same gel. The sensitivity of our protocol provides reproducible detection of 50 kb fragmentation after irradiation of human lymphocytes with 5 cGy of gamma-rays. In 2 of 6 donors tested, this DNA fragmentation was detected at dose on 2 cGy. The novel protocol can be used for quantification of 50 kb apoptotic fragments induced by different agents including low dose ionising radiations, chemicals and electromagnetic fields.

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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.

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Extremely low-frequency (ELF) magnetic fields have previously been shown to affect conformation of chromatin, cell proliferation, and calcium metabolism. Possible mutagenic and carcinogenic effects of ELF have also been discussed and tested. In this study, intrachromosomal recombination in the hprt gene after exposure to ELF magnetic field was investigated using the SPD8 recombination assay. SPD8 cells, derived from V79 Chinese hamster cells were exposed to ELF at a specific combination of static and ELF magnetic fields, that has been proven to have effects on chromatin conformation in several cell types. The genotoxic agent camptothecin (CPT) was used either as a positive control or simultaneously with ELF. We also analysed the effect of ELF and CPT on chromatin conformation with the anomalous viscosity time dependence (AVTD) technique, cell growth kinetics, and cell survival with clonogenic assay. DNA fragmentation was analysed by pulsed field gel electrophoresis (PFGE). ELF did not induce recombination alone, neither did ELF modify the recombinogenic effect of CPT. Although, there was no effect on cell survival in response to ELF exposure, inhibition of cell growth was observed. On the other hand, ELF exposure partly counteracted the growth inhibition seen with CPT. The data suggest that ELF exposure may stimulate or inhibit cell growth depending on the state of the cells. Although, ELF did not induce recombination, a weak but statistically significant DNA fragmentation comparable with CPT-induced fragmentation was observed with PFGE 48h after exposure to ELF.

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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.

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PURPOSE: To investigate and compare the propensity of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL), derived from ataxia-telangiectasia (A-T) patients and from unaffected healthy individuals (controls), to undergo apoptosis after exposure to high-linear energy transfer (LET) radiation. MATERIALS AND METHODS: Four A-T (ARO, BMA, CSA and RJO) and two control (JAC and KKB3) LCL were exposed to doses of up to 4Gy of accelerated nitrogen ions (32-45 MeV/u, 8-12Gy/min). For comparative purposes X-ray irradiation (1.36 Gy/min) was also performed. The induction of apoptosis was studied 0-48 h after irradiation with the use of two methods: (1) monitoring of high molecular weight (HMW) DNA fragments by field inversion pulse gel electrophoresis (FIGE); and (2) morphological characterization ofapoptotic cells after fluorescent staining. In parallel, cell-cycle distribution, monitored by DNA flow cytometry, as well as measurements of p53/p21(WAF1) protein levels by Western blots, were investigated in these cells. RESULTS: High-LET radiation-induced apoptosis and G2/M-arrest in both A-T and control LCL. No significant increase in the amount of p53/p21(WAF1) proteins preceded apoptosis in control or in A-T LCL after high-LET irradiation. However, low-LET radiation did induce significant enhanced levels of p53 proteins in control but not in A-T LCL. CONCLUSIONS: LCL from both A-T homozygous and unaffected healthy individuals undergo apoptosis without accumulation of p53/p21(WAF1) proteins after exposure to high-LET radiation. In contrast, low-LET radiation induces apoptosis and significantly increases levels of p53 protein in control but not in A-T LCL.

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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.

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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.

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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.

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The early observations on the possible induction of transmissible genomic instability after exposure to ionising radiation has received a strong support in the literature during the last 10 years. Aided by new research tools in biology, the better understanding of the mechanisms behind genomic instability leads to conclusions which are challenging the existing views on the interaction and response of the genome to radiation or chemicals. It has become commonly accepted that the full revelation of biological pathways leading to the loss of stability of the genome will also be a major step in the understanding of carcinogenesis. In this short review, some aspects of the recent knowledge and their implications are discussed.

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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.

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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.

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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.

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BACKGROUND/AIMS: In order to examine whether iron and alcohol act synergistically during tumor initiation in vivo, we investigated the effects of dietary iron overload and a liquid ethanol-containing diet on the initiation phase of the Solt & Farber model of chemical hepatocarcinogenesis. METHODS: Following dietary supplementation with carbonyl iron for 8 weeks and ethanol pair-feeding according to Lieber deCarli for 5 weeks, animals were subjected to partial hepatectomy in order to induce regenerative cell proliferation and thereby "fix" putative DNA lesions. Levels of malondialdehyde, reduced and oxidized ubiquinone-9, alpha-tocopherol and 8-oxo-2'-deoxyguanosine were analyzed in liver tissue removed at the time of partial hepatectomy, and blood was collected for determination of alanine amino-transferase activities. Following a 2-week recovery period, promotion was achieved with 0.02% dietary 2-acetylaminofluorene and carbon tetrachloride. Two weeks after the completion of promotion, animals were sacrificed and the number of preneoplastic, glutathione S-transferase 7,7-positive lesions counted. Animals initiated with diethylnitrosamine served as a positive control group. RESULTS: Serum aminotransferase activities were significantly increased, and hepatic contents of ubiquinol-9 (reduced ubiquinone-9) were significantly decreased in animals exposed to the combination of iron and ethanol in comparison to the other groups. Livers from iron-treated animals had decreased levels of alpha-tocopherol and increased contents of malondialdehyde, whereas treatment with ethanol did not further enhance these alterations. Levels of 8-oxo-2'-deoxyguanosine were not significantly different in animals treated with iron, ethanol or iron + ethanol as compared with controls. The number of preneoplastic foci at the time of sacrifice was not increased in livers exposed to iron and/or ethanol as compared with those from control animals. As expected, the number of foci was significantly increased in positive controls which were initiated with diethylnitrosamine. CONCLUSIONS: Iron potentiated the cytotoxic effects of ethanol, resulting in increased serum aminotransferase activities and decreased hepatic contents of ubiquinol. However, the combination of iron and ethanol did not exert genotoxic effects detectable as enhanced hepatic levels of 8-oxo-2'-deoxyguanosine, or increased formation of preneoplastic, glutathione S-transferase 7,7-positive lesions in the Solt & Farber model of chemical hepatocarcinogenesis.

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In several cell types, apoptosis is associated with intracellular acidification and activation of a pH-dependent endonuclease. We have examined the effect of acidic pH on the DNA of permeabilized human fibroblasts, and observed cleavage of DNA into high-molecular-mass fragments. This pH-dependent DNA breakage was modulated by temperature, the presence of histones and diethyl pyrocarbonate. Superoxide dismutase and chelators with high affinity for Cu prevented DNA fragmentation, whereas catalase, DMSO and Desferal (desferrioxamine mesylate) offered no protection. Fragmentation of DNA into high-molecular-mass fragments, which is occasionally observed as an early phase of apoptosis, is thought to result from the activation of endonuclease(s). Our results suggest that such fragmentation also occurs through induction of copper-mediated site-specific DNA damage that is enhanced by intracellular acidification.

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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.

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