It is well established that high-LET radiations efficiently induce chromosome aberrations. However, data on the effect of protons on telomere maintenance, as involved in genomic stability, are scarce and contradictory. Here we demonstrate that high-LET protons induce telomere lengthening in human primary fibroblasts and that this elongation does not involve the telomerase enzyme, supporting the hypothesis that high-LET radiations are able to activate a telomerase-independent mechanism. In tumor cells that lack telomerase, one or more non-telomerase mechanisms for telomere maintenance are present, which are termed alternative lengthening of telomeres (ALT). Since ALT cells are characterized by recombinational events at telomeres, known as telomeric-sister chromatid exchanges (T-SCE), and colocalization of telomeres and premyelocytic leukemia protein (PML), we analyzed both T-SCE and PML. Our results show that high-LET protons induce a 2.5-fold increase of T-SCE and a colocalization of PML protein and telomeric DNA. Furthermore, our data show that the ALT pathway can be activated in human primary cells after induction of severe DNA damage. Thus, since telomeres are known to be involved in chromosome maintenance, the present work may contribute in the elucidation of the mechanism by which ionizing radiation induces genomic instability.

To achieve a more complete understanding of the molecular mechanisms underlying tumor radioresistance, we established a radioresistant cell line from the human larynx squamous cell carcinoma cell line Hep-2 after long-term radiation induction. The biological features of the resulting cell lines were characterized. cDNA microarray technology was used to measure the alterations of gene expression in the radioresistant cells. We found that certain genes associated with DNA repair, cell cycle, apoptosis, etc. were significantly changed. In particular, genes related to telomeres, such as POT1, were significantly altered. Radioresistant cells had higher telomerase activity and longer telomeres than their parental cells. Our research suggests that telomere function is a novel hallmark of cellular radiosensitivity, and the mechanistic link between telomere maintenance and radiosensitivity may involve the genes and pathways we implicated in this study.

The pathway involving Bre1-dependent monoubiquitination of histone H2B lysine 123, which leads to Dot1-dependent methylation of histone H3 lysine 79 (H3K79me2), has been implicated in survival after exposure to ionizing radiation in Saccharomyces cerevisiae. We found that depletion of mammalian homologs of Bre1 compromises the response to ionizing radiation, leading to increased radiosensitivity and a G₂/M checkpoint defect. The deficiency in Bre1a/b function was also associated with increased sensitivity to crosslinking drugs and defective formation of Rad51 foci in mouse cells, suggesting a defect in homologous recombinational repair analogous to that seen in Saccharomyces. In budding yeast, H3K79me2 is important for the recruitment of the checkpoint signaling protein Rad9 to sites of double-strand breaks (DSBs). However, in mammalian cells, 53BP1 (the Rad9 ortholog) in addition to H3K79me2 recognizes a different residue, H4K20me2, and some studies argue that it is H4K20me2 and not H3K79me2 that is the preferred target for 53BP1. We show here that depletion of Bre1b specifically reduced dimethylation of H3K79 without affecting dimethylation of H4K20. Thus our data suggest that the observed defects in the radiation response of Bre1a/b-deficient cells are associated with reduced H3K79me2 and not with H4K20me2.

Potentially lethal damage (PLD) and its repair were studied in confluent human fibroblasts by analyzing the kinetics of chromosome break rejoining and misrejoining in irradiated cells that were either held in noncycling G₀ phase or allowed to enter G₁ phase of the cell cycle immediately after 6 Gy irradiation. Virally mediated premature chromosome condensation (PCC) methods were combined with fluorescence in situ hybridization (FISH) to study chromosomal aberrations in interphase. Flow cytometry revealed that the vast majority of cells had not yet entered S phase 15 h after release from G₀. By this time some 95% of initially produced prematurely condensed chromosome breaks had rejoined, indicating that most repair processes occurred during G₁. The rejoining kinetics of prematurely condensed chromosome breaks was similar for each culture condition. However, under noncycling conditions misrepair peaked at 0.55 exchanges per cell, while under cycling conditions (G₁) it peaked at 1.1 exchanges per cell. At 12 h postirradiation, complex-type exchanges were sevenfold more abundant for cycling cells (G₁) than for noncycling cells (G₀). Since most repair in G₀/G₁ occurs via the non-homologous end-joining (NHEJ) process, increased PLD repair may result from improved cell cycle-specific rejoining fidelity of the NHEJ pathway.

For repair of damaged DNA, cells increase de novo synthesis of deoxyribonucleotide triphosphates through the rate-limiting, p53-regulated ribonucleotide reductase (RNR) enzyme. In this study we investigated whether pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) enhanced chemoradiation sensitivity through a mechanism involving sustained DNA damage. RNR inactivation by 3-AP and resulting chemoradiosensitization were evaluated in human cervical (CaSki, C33-a) cancer cells through study of DNA damage (γ-H2AX signal) by flow cytometry, RNR subunit p53R2 and p21 protein steady-state levels by Western blot analysis and laser scanning imaging cytometry, and cell survival by colony formation assays. 3-AP treatment led to sustained radiation- and cisplatin-induced DNA damage (i.e. increased γ-H2AX signal) in both cell lines through a mechanism of inhibited RNR activity. Radiation, cisplatin and 3-AP exposure resulted in significantly elevated numbers and persistence of γ-H2AX foci that were associated with reduced clonogenic survival. DNA damage was associated with a rise in p53R2 but not p21 protein levels 6 h after treatment with radiation and/or cisplatin plus 3-AP. We conclude that blockage of RNR activity by 3-AP impairs DNA damage responses that rely on deoxyribonucleotide production and thereby may substantially increase chemoradiosensitivity of human cervical cancers.

Skeletal muscles are the organ of movement, and their growth, regeneration and maintenance are dependent in large part on a population of myogenic stem cells known as satellite cells. Skeletal muscles and these resident myogenic stem cells (i.e., satellite cells) are commonly exposed to significant doses of radiation during diagnostic procedures and/or during the radiotherapeutic management of cancer. The main objective of this study was to examine the effects of clinically relevant doses of γ radiation on satellite cell survival and proliferation, cell cycle regulation, apoptosis, DNA double-strand break repair, oxidative stress and NO production. Overall, our findings demonstrate that doses of γ radiation ≥5 Gy reduced satellite cell numbers by at least 70% due in part to elevated apoptosis and the inhibition of cell cycle progression. Radiation was also found to cause a significant and persistent increase in the level of reactive oxygen and nitrogen species. Interestingly, and within this backdrop of elevated oxidative stress, similar doses were found to produce substantial reductions in the levels of nitric oxide (NO). Proliferation of satellite cells has been shown to depend in part on the production of NO, and our findings give rise to the possibility that radiation-induced reductions in NO levels may provide a mechanism for the inhibition of satellite cell proliferation in vitro and possibly the regrowth of skeletal muscle exposed during clinical irradiation procedures.

Alpha-particle irradiation of cells damages not only the irradiated cells but also nontargeted bystander cells. It has been proposed that the bystander effect is caused by oxidants and free radicals generated by the radiation. Recent studies have shown that α₁-microglobulin protects against cell damage caused by oxidants and free radicals. Using a novel experimental system that allows irradiation of 0.02% of a human hepatoma monolayer, leaving 99.98% as bystander cells, we investigated the influence of oxidative stress and the cell-protective effects of α₁-microglobulin during α-particle irradiation. The results showed an increase in cell death in both irradiated cells and bystander cells. A significant increase in apoptosis, oxidation markers and expression of the stress response genes heme oxygenase 1, superoxide dismutase, catalase, glutathione peroxidase 1, p21 and p53 were observed. Addition of α₁-microglobulin reduced the amount of dead cells and inhibited apoptosis, formation of oxidation markers, and up-regulation of stress response genes. The results emphasize the role of oxidative stress in promoting bystander effects. Furthermore, the results suggest that α₁-microglobulin protects nonirradiated cells by eliminating oxidants and free radicals generated by radiation and imply that α₁-microglobulin can be used in radiation therapy of tumors to minimize damage to surrounding tissues.

Activation of p53 has been causally linked to normal tissue damage after irradiation. Pifithrin-α (PFT-α), a specific inhibitor of p53, has been suggested as a combinatory agent in the treatment of p53-deficient tumors in which inhibition of p53 would not compromise therapeutic efficacy but would decrease p53-mediated side effects in normal tissue. We tested this concept for radiotherapy of p53-deficient and -proficient glioma. We observed significant interaction of PFT-α with radiation-induced G₁ checkpoint activation and plating efficiency only in glioma cells expressing at least one wild-type allele of p53. This interaction was correlated with PFT-α-mediated inhibition of radiation-induced expression of the p53 target gene p21^Waf1. Despite inhibition of p53 function we did not observe significant changes in radiosensitivity after treatment with PFT-α in either p53-deficient or p53-proficient tumor cells. We confirmed these results in p53-proficient lung cancer cells. In contrast, PFT-α significantly increased the fraction of normal astrocytes and fibroblasts surviving irradiation; this was accompanied by improved DNA damage repair, speaking against an accumulation of cells with genetic lesions after PFT-α treatment. In conclusion, PFT-α might prove useful in protecting normal tissue from the side effects of radiotherapy without reducing the efficacy of treatment for both p53-proficient and -deficient tumors.

Changes in gene expression profiles in mouse liver induced by long-term low-dose-rate γ irradiation were examined by microarray analysis. Three groups of male C57BL/6J mice were exposed to whole-body radiation at dose rates of 17–20 mGy/day, 0.86–1.0 mGy/day or 0.042–0.050 mGy/day for 401–485 days with cumulative doses of approximately 8 Gy, 0.4 Gy or 0.02 Gy, respectively. The gene expression levels in the livers of six animals from each exposure group were compared individually with that of pooled sham-irradiated animals. Some genes revealed a large variation in expression levels among individuals within each group, and the number of genes showing common changes in individuals from each group was limited: 20 and 11 genes showed more than 1.5-fold modulation with 17–20 mGy/day and 0.86–1.0 mGy/day, respectively. Three genes showed more than 1.5-fold modulation even at the lowest dose-rate of 0.04–0.05 mGy/day. Most of these genes were down-regulated. RT-PCR analysis confirmed the expression profiles of the majority of these genes. The results indicate that a few genes are modulated in response to very low-dose-rate irradiation. The functional analysis suggests that these genes may influence many processes, including obesity and tumorigenesis.

Exposure to galactic cosmic radiation (GCR) is considered to be a potential health risk in long-term space travel, and it represents a significant risk to the central nervous system (CNS). The most harmful component of GCR is the HZE [high-mass, highly charged (Z), high-energy] particles, e.g. ⁵⁶Fe. In ground-based experiments, exposure to HZE-particle radiation induces pronounced deficits in hippocampus-dependent learning and memory in rodents. The mechanisms underlying these impairments are mostly unknown, but some studies suggest that HZE-particle exposure perturbs the regulation of long-term potentiation (LTP) at the CA1 synapse in the hippocampus. In this study, we irradiated rats with 60 cGy of 1 GeV ⁵⁶Fe-particle radiation and established its impact on hippocampal glutamatergic neurotransmissions at 3 and 6 months after exposure. Exposure to 60 cGy ⁵⁶Fe-particle radiation significantly (P < 0.05) reduced hyperosmotic sucrose evoked [³H]-glutamate release from hippocampal synaptosomes, a measure of the readily releasable vesicular pool (RRP). This HZE-particle-induced reduction in the glutamatergic RRP persisted for at least 6 months after exposure. At 90 days postirradiation, there was a significant reduction in the expression of the NR1, NR2A and NR2B subunits of the glutamatergic NMDA receptor. The level of the NR2A protein remained suppressed at 180 days postirradiation, but the level of NR2B and NR1 proteins returned to or exceeded normal levels, respectively. Overall, this study shows that hippocampal glutamatergic transmission is sensitive to relative low doses of ⁵⁶Fe particles. Whether the observed HZE-particle-induced change in glutamate transmission, which plays a critical role in learning and memory, is the cause of HZE-particle-induced neurocognitive impairment requires further investigation.

In a previous cohort study of workers engaged in uranium milling and mining activities near Grants, Cibola County, New Mexico, we found lung cancer mortality to be significantly increased among underground miners. Uranium mining took place from early in the 1950s to 1990, and the Grants Uranium Mill operated from 1958–1990. The present study evaluates cancer mortality during 1950–2004 and cancer incidence during 1982–2004 among county residents. Standardized mortality (SMR) and incidence (SIR) ratios and 95% confidence intervals (CI) were computed, with observed numbers of cancer deaths and cases compared to expected values based on New Mexico cancer rates. The total numbers of cancer deaths and incident cancers were close to that expected (SMR 1.04, 95% CI 1.01–1.07; SIR 0.97, 95% CI 0.92–1.02). Lung cancer mortality and incidence were significantly increased among men (SMR 1.11, 95% CI 1.02–1.21; SIR 1.40, 95% CI 1.18–1.64) but not women (SMR 0.97, 95% CI 0.85–1.10; SIR 1.01, 95% CI 0.78–1.29). Similarly, among the population of the three census tracts near the Grants Uranium Mill, lung cancer mortality was significantly elevated among men (SMR 1.57; 95% CI 1.21–1.99) but not women (SMR 1.12; 95% CI 0.75–1.61). Except for an elevation in mortality for stomach cancer among women (SMR 1.30; 95% CI 1.03–1.63), which declined over the 55-year observation period, no significant increases in SMRs or SIRs for 22 other caners were found. Although etiological inferences cannot be drawn from these ecological data, the excesses of lung cancer among men seem likely to be due to previously reported risks among underground miners from exposure to radon gas and its decay products. Smoking, socioeconomic factors or ethnicity may also have contributed to the lung cancer excesses observed in our study. The stomach cancer increase was highest before the uranium mill began operation and then decreased to normal levels. With the exception of male lung cancer, this study provides no clear or consistent evidence that the operation of uranium mills and mines adversely affected cancer incidence or mortality of county residents.

After inhalation of plutonium oxides containing various percentages of americium in rats, we identified an acellular transient pulmonary compartment, the epithelial lining fluid (ELF), in which a fraction of actinide oxides dissolve prior to absorption and subsequent extrapulmonary deposit. Chelation therapy is usually considered to be poorly efficient after inhalation of actinide oxides. However, in the present study, prompt pulmonary administration of diethylenetraminepentaacetic acid (DTPA) as a dry powder led to a decrease in actinide content in ELF together with a limitation of bone and liver deposits. Because americium is more soluble than plutonium, higher amounts of americium were found in ELF, extrapulmonary tissues and urine. Our results also demonstrated that the higher efficacy of DTPA on americium compared to plutonium in ELF induced a preferential inhibition of extrapulmonary deposit and a greater urinary excretion of americium compared to plutonium. All together, our data justify the use of an early and local DTPA treatment after inhalation of plutonium oxide aerosols in which americium can be in high proportion such as in aged compounds.

This study reports the effects of denaturation and deoxygenation on radiation-induced formation of 2-deoxyribonolactone (2-dL) and 5′-aldehyde (5′-Ald) lesions in highly polymerized DNA. The radiation-chemical yields of 2-dL were determined through quantification of its dephosphorylation product 5-methylenefuranone (5MF). The formation of 5′-Ald was monitored qualitatively through the release of furfural (Fur) under the same conditions. The yields of 2-dL were found to be 7.3 ± 0.3 nmol J⁻¹, or about 18% of the yield of free base release measured in the same samples. Denaturation increased the efficiency of 2-dL formation approximately twofold while deoxygenation resulted in a fourfold decrease. The release of Fur is about twofold lower than that of 5MF in aerated native DNA samples and is further reduced by denaturation of the DNA. Unlike 5MF, the formation of Fur requires the presence of molecular oxygen, which is consistent with peroxyl radical-mediated oxidation of C5′ radicals into 5′-Ald. In contrast, the existence of an oxygen-independent pathway of 2-dL formation suggests that C1′ sugar radicals can also be oxidized by radiation-produced oxidizing intermediates such as electron-loss centers on guanines.

The radiation stability of the ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) was investigated after ⁶⁰Co γ irradiation at doses up to 600 kGy. Thermal and spectroscopic analysis revealed the formation of a small quantity of radiolytic products and an evident change in the physicochemical properties of the ionic liquid. The presence of anionic FeCl₄⁻ (up to 5 mol%), as measured by UV-vis absorption, differential scanning calorimetry, and Raman spectroscopy, significantly improved the radiation resistance of [Bmim]Cl. The increased resistance may be due to the capture of a solvated electron (e_solv⁻) by FeCl₄⁻ to form FeCl₄²⁻ and results in the radiation protection of the organic cation. The radiation yield of the reductive species (Fe(II)) was estimated to be 0.217 ± 0.010 µmol/J (2.09 ± 0.10/100 eV), which is considered close to the radiation yield of the solvated electrons of [Bmim]Cl.

The structural modifications of polymers irradiated with 14 MeV neutrons were studied. Two elastomers, a polypropylene-type polymer and poly(ethylene oxide) were exposed to low doses of fast neutrons in the range of 0.3–14 Gy. The radiation damages were observed at the molecular scale by infrared spectroscopy. The morphological changes were investigated by steric exclusion chromatography, insoluble fraction measurements, differential scanning calorimetry and X-ray diffraction. It was found that neutrons provoked oxidation processes accompanied by modifications in the polymer architecture, including chain scissions, crosslinking reactions and changes in the crystallinity. Moreover, the conventional antioxidants were shown to be inefficient in inhibiting the aging of the polymers. These results also suggest that the radiation damages could be used successfully for dosimetry applications using an easily implementable protocol.