Illegitimate recombination can repair DNA double-strand breaks in one of two ways, either without sequence homology or by using a few base pairs of homology at the junctions. The second process is known as microhomology-mediated recombination. Previous studies showed that ionizing radiation and restriction enzymes increase the frequency of microhomology-mediated recombination in trans during rejoining of unirradiated plasmids or during integration of plasmids into the genome. Here we show that radiation-induced microhomology-mediated recombination is reduced by deletion of RAD52, RAD1 and RAD10 but is not affected by deletion of RAD51 and RAD2. The rad52 mutant did not change the frequency of radiation-induced microhomology-mediated recombination but rather reduced the length of microhomology required to undergo repair during radiation-induced recombination. The rad1 and rad10 mutants exhibited a smaller increase in the frequency of radiation-induced microhomology-mediated recombination, and the radiation-induced integration junctions from these mutants did not show more than 4 bp of microhomology. These results suggest that Rad52 facilitates annealing of short homologous sequences during integration and that Rad1/Rad10 endonuclease mediates removal of the displaced 3′ single-stranded DNA ends after base-pairing of microhomology sequences, when more than 4 bp of microhomology are used. Taken together, these results suggest that radiation-induced microhomology-mediated recombination is under the same genetic control as the single-strand annealing apparatus that requires the RAD52, RAD1 and RAD10 genes.

Double-strand breaks (DSBs) can be generated in the DNA of mammalian cells when heat-labile sites induced by ionizing radiation within a clustered DNA damage site are thermally transformed to single-strand breaks (SSBs) and combine with other SSBs or heat-labile sites in the opposite DNA strand. When this thermal transformation of heat-labile sites to SSBs occurs during DNA preparation using high-temperature lysis, the DSB yield is overestimated by including DSBs not present in the tested cell. Low-temperature lysis avoids this artifact but shows slower repair kinetics for prompt DSBs than high-temperature lysis. The apparent slowdown of DSB repair after low-temperature lysis is particularly pronounced in mutants defective in the DNA-PK-dependent pathway of NHEJ (D-NHEJ) and has led to the postulate that these cells may actually be entirely deficient in DSB repair. Since our work as well as that of others provides strong evidence for DSB repair in D-NHEJ-deficient cells by alternative repair pathways operating as a backup (B-NHEJ), we re-examine here DSB repair in several D-NHEJ-deficient mutants using low-temperature lysis. The results demonstrate extensive but slow repair of DSBs after low-temperature lysis indicative of a robust function of B-NHEJ. At the same time, the results demonstrate a pronounced sensitivity of heat-labile sites to lysis at 37°C, which indicates that DSBs generated by transformation of heat-labile sites may be in part a reality that the irradiated cell faces at all times.

Mammalian POLQ (pol θ) is a specialized DNA polymerase with an unknown function in vivo. Roles have been proposed in chromosome stability, as a backup enzyme in DNA base excision repair, and in somatic hypermutation of immunoglobulin genes. The purified enzyme can bypass AP sites and thymine glycol. Mice defective in POLQ are viable and have been reported to have elevated spontaneous and radiation-induced frequencies of micronuclei in circulating red blood cells. To examine the potential roles of POLQ in hematopoiesis and in responses to oxidative stress responses, including ionizing radiation, bone marrow cultures and marrow stromal cell lines were established from Polq ^/ and Polq^−/− mice. Aging of bone marrow cultures was not altered, but Polq^−/− cells were more sensitive to γ radiation than were Polq ^/ cells. The D₀ was 1.38 ± 0.06 Gy for Polq ^/ cells compared to 1.27 ± 0.16 and 0.98 ± 0.10 Gy (P  =  0.032) for two Polq^−/− clones. Polq^−/− cells were moderately more sensitive to bleomycin than Polq ^/ cells and were not hypersensitive to paraquat or hydrogen peroxide. ATM kinase activation appeared to be normal in γ-irradiated Polq^−/− cells. Inhibition of ATM kinase activity increased the radiosensitivity of Polq ^/ cells slightly but did not affect Polq^−/− cells. Polq^−/− mice had more spontaneous and radiation-induced micronucleated reticulocytes than Polq / and ^/− mice. The sensitivity of POLQ-defective bone marrow stromal cells to ionizing radiation and bleomycin and the increase in micronuclei in red blood cells support a role for this DNA polymerase in cellular tolerance of DNA damage that can lead to double-strand DNA breaks.

Dietary antioxidants have radioprotective effects after γ-radiation exposure that limit hematopoietic cell depletion and improve animal survival. The purpose of this study was to determine whether a dietary supplement consisting of l-selenomethionine, vitamin C, vitamin E succinate, α-lipoic acid and N-acetyl cysteine could improve survival of mice after proton total-body irradiation (TBI). Antioxidants significantly increased 30-day survival of mice only when given after irradiation at a dose less than the calculated LD_50/30; for these data, the dose-modifying factor (DMF) was 1.6. Pretreatment of animals with antioxidants resulted in significantly higher serum total white blood cell, polymorphonuclear cell and lymphocyte cell counts at 4 h after 1 Gy but not 7.2 Gy proton TBI. Antioxidants significantly modulated plasma levels of the hematopoietic cytokines Flt-3L and TGFβ1 and increased bone marrow cell counts and spleen mass after TBI. Maintenance of the antioxidant diet resulted in improved recovery of peripheral leukocytes and platelets after sublethal and potentially lethal TBI. Taken together, oral supplementation with antioxidants appears to be an effective approach for radioprotection of hematopoietic cells and improvement of animal survival after proton TBI.

Mammalian MTH1 protein is an antimutagenic (2′-deoxy)ribonucleoside 5′-triphosphate pyrophosphohydrolase that prevents the incorporation of oxidatively modified nucleotides into nucleic acids. It decomposes most specifically the miscoding products of oxidative damage to purine nucleic acid precursors (e.g. 8-oxo-dGTP, 2-oxo-dATP, 2-oxo-ATP, 8-oxo-GTP) that may cause point mutations or transcription errors when incorporated into DNA and RNA, respectively. The increased expression of MTH1 mRNA and MTH1 protein was previously proposed as a molecular marker of oxidative stress. Therefore, we hypothesized that increased 8-oxo-dGTPase activity of MTH1 protein in mouse organs could serve as a dose-dependent marker of exposure to ionizing radiation, which is known to induce oxidative stress. To test our hypothesis, we measured 8-oxo-dGTPase activity in six organs of male BL6 mice after exposure to 0, 10, 25 and 50 cGy and 1 Gy of ¹³⁷Cs γ radiation given as a single whole-body dose (1 Gy/min). The mice were killed 4, 8 and 24 h after irradiation. A statistically significant induction of 8-oxo-dGTPase was found in brains, testes and kidneys but not in lungs, hearts or livers. Brains, which demonstrated the highest (4.3-fold) increase of 8-oxo-dGTPase activity, were shown to express ∼50% higher levels of MTH1 protein. However, due to the lack of a simple positive correlation between the dose and the observed 8-oxo-dGTPase activity in brain, testes and kidneys, we conclude that measurements of 8-oxo-dGTPase activity in these organs may serve as a rough indicator rather than a quantifiable marker of radiation-induced oxidative stress.

Radiation metabolomics employing mass spectral technologies represents a plausible means of high-throughput minimally invasive radiation biodosimetry. A simplified metabolomics protocol is described that employs ubiquitous gas chromatography-mass spectrometry and open source software including random forests machine learning algorithm to uncover latent biomarkers of 3 Gy γ radiation in rats. Urine was collected from six male Wistar rats and six sham-irradiated controls for 7 days, 4 prior to irradiation and 3 after irradiation. Water and food consumption, urine volume, body weight, and sodium, potassium, calcium, chloride, phosphate and urea excretion showed major effects from exposure to γ radiation. The metabolomics protocol uncovered several urinary metabolites that were significantly up-regulated (glyoxylate, threonate, thymine, uracil, p-cresol) and down-regulated (citrate, 2-oxoglutarate, adipate, pimelate, suberate, azelaate) as a result of radiation exposure. Thymine and uracil were shown to derive largely from thymidine and 2′-deoxyuridine, which are known radiation biomarkers in the mouse. The radiation metabolomic phenotype in rats appeared to derive from oxidative stress and effects on kidney function. Gas chromatography-mass spectrometry is a promising platform on which to develop the field of radiation metabolomics further and to assist in the design of instrumentation for use in detecting biological consequences of environmental radiation release.

Estimates of cancer risks posed to space-flight crews by exposure to high atomic number, high-energy (HZE) ions are subject to considerable uncertainty because epidemiological data do not exist for human populations exposed to similar radiation qualities. We assessed the leukemogenic efficacy of one such HZE species, 1 GeV ⁵⁶Fe ions, a component of space radiation, in a mouse model for radiation-induced acute myeloid leukemia. CBA/CaJ mice were irradiated with 1 GeV/nucleon ⁵⁶Fe ions or ¹³⁷Cs γ rays and followed until they were moribund or to 800 days of age. We found that 1 GeV/nucleon ⁵⁶Fe ions do not appear to be substantially more effective than γ rays for the induction of acute myeloid leukemia (AML). However, ⁵⁶Fe-ion-irradiated mice had a much higher incidence of hepatocellular carcinoma (HCC) than γ-irradiated mice, with an estimated RBE of approximately 50. These data suggest a difference in the effects of HZE iron ions on the induction of leukemia compared to solid tumors, suggesting potentially different mechanisms of tumorigenesis.

The oxygen consumption rate in tumors affects tumor oxygenation and response to therapies. A new EPR method was developed to measure tissue oxygen consumption non-invasively. The protocol was based on the measurement of pO₂ during a carbogen challenge. The following sequence was used: (1) basal value during air breathing; (2) saturation of tissue with oxygen by carbogen breathing; (3) switch back to air breathing. The assumption was that the kinetics of the return to the basal value after oxygen saturation would be governed mainly by tissue oxygen consumption. This challenge was applied in hyperthyroid mice (generated by chronic treatment with l-thyroxine) and control mice because hyperthyroidism is known to dramatically affect the oxygen consumption rate of tumor and muscle cells. Muscle and tumor cells from the hyperthyroid mice consumed oxygen faster than muscle and tumor cells from the control mice, which is consistent with the results of in vitro studies. Tumor perfusion was not affected by the treatment with l-thyroxine. This method gives an index that may reasonably be ascribed to the local oxygen consumption and has the unique advantage of being adaptable to in vivo studies.

X irradiation of sucrose single crystals at room temperature leads to the production of stable radicals, which give rise to the dosimetric electron paramagnetic resonance (EPR) signal. In the first few hours after irradiation, however, the shape of the EPR spectrum changes drastically. Based on two-dimensional field-frequency electron nuclear double resonance (FF-ENDOR) measurements, we demonstrate that, after high-dose (∼5 kGy) and high-dose-rate irradiation, several species with limited stability at room temperature are produced next to the stable radicals. For two of these species, the main characteristics could be determined. Analysis of the time evolution of the FF-ENDOR and room-temperature EPR spectra in the first few hours after irradiation leads to the conclusion that these meta-stable radicals mainly recombine into diamagnetic species, while transformation into stable radicals is at most a marginal process.

Genomic instability has been suggested as a mechanism by which exposure to ionizing radiation can lead to cancer in exposed humans. However, the data from human cells needed to support or refute this idea are limited. In our previous study on clonal lymphocyte populations carrying stable-type aberrations derived from A-bomb survivors, we found no increase in the frequency of sporadic additional aberrations among the clonal cell populations compared with the spontaneous frequency in vivo. That work has been extended by using multicolor FISH (mFISH) to quantify the various kinds of chromosome aberrations known to be indicative of genomic instability in cloned T lymphocytes after they were expanded in culture for 25 population doublings. The blood T cells used were obtained from each of two high-dose-exposed survivors (>1 Gy) and two control subjects, and a total of 66 clonal populations (36 from exposed and 30 from control individuals) were established. For each clone, 100 metaphases were examined. In the case of exposed lymphocytes, a total of 39 additional de novo stable, exchange-type aberrations [translocation (t) derivative chromosome (der)] were found among 3600 cells (1.1%); the corresponding value in the control group was 0.6% (17/3000). Although the ratio (39/3600) obtained from the exposed cases was greater than that of the controls (17/3000), the difference was not statistically significant (P  =  0.101). A similar lack of statistical difference was found for the total of all structural chromosome alterations including t, der, dicentrics, duplications, deletions and fragments (P  =  0.142). Thus there was no clear evidence suggesting the presence of chromosome instabilities among the clonally expanded lymphocytes in vitro from A-bomb survivors.

The European project EMFnEAR was undertaken to assess potential changes in human auditory function after a short-term exposure to radiofrequency (RF) radiation produced by UMTS (Universal Mobile Telecommunication System) mobile phones. Participants were healthy young adults with no hearing or ear disorders. Auditory function was assessed immediately before and after exposure to radiofrequency radiation, and only the exposed ear was tested. Tests for the assessment of auditory function were hearing threshold level (HTL), distortion product otoacoustic emissions (DPOAE), contralateral suppression of transiently evoked otoacoustic emission (CAS effect on TEOAE), and auditory evoked potentials (AEP). The exposure consisted of speech at a typical conversational level delivered via an earphone to one ear, plus genuine or sham RF-radiation exposure produced by a commercial phone controlled by a personal computer. Results from 134 participants did not show any consistent pattern of effects on the auditory system after a 20-min UMTS exposure at the maximum output of the phone with 69 mW/kg SAR in the cochlea region in a double blind comparison of genuine and sham exposure. An isolated effect on the hearing threshold at high frequencies was identified, but this was statistically nonsignificant after correction for multiple comparisons. It is concluded that UMTS short-term exposure at the maximum output of consumer mobile phones does not cause measurable immediate effects on the human auditory system.

Pataky, K., Villanueva, G., Liani, A., Zgheib, O., Jenkins, N., Halazonetis, D. J., Halazonetis, T. D. and Brugger, J. Microcollimator for Micrometer-Wide Stripe Irradiation of Cells Using 20–30 keV X Rays. Radiat. Res. 172, 252–259 (2009).

The exposure of subnuclear compartments of cells to ionizing radiation is currently not trivial. We describe here a collimator for micrometer-wide stripe irradiation designed to work with conventional high-voltage X-ray tubes and cells cultured on standard glass cover slips. The microcollimator was fabricated by high-precision silicon micromachining and consists of X-ray absorbing chips with grooves of highly controlled depths, between 0.5–10 μm, along their surfaces. These grooves form X-ray collimating slits when the chips are stacked against each other. The use of this device for radiation biology was examined by irradiating human cells with X rays having energies between 20–30 keV. After irradiation, p53 binding protein 1 (53BP1), a nuclear protein that is recruited at sites of DNA double-strand breaks, clustered in lines corresponding to the irradiated stripes.

Persistent, chronic oxidative injury may play a mechanistic role in late radiation injury. Thus antioxidants may be useful as mitigators of radiation injury. The antioxidants deferiprone, genistein and apocynin were tested in a rat radiation nephropathy model that uses single-fraction total-body irradiation (TBI) followed by syngeneic bone marrow transplant. Deferiprone was added to the drinking water at 1.0 or 2.5 g/liter, starting 3 days after the TBI. Urinary bleomycin-detectable iron, which could enhance production of oxygen radicals, was reduced in the rats on deferiprone compared to untreated rats, but deferiprone did not mitigate radiation nephropathy. Genistein added to the chow at 750 mg/kg starting immediately after TBI did not mitigate radiation nephropathy. Apocynin added to the drinking water at 250 mg/liter immediately after TBI did not mitigate radiation nephropathy. Thus three different types of antioxidants, when used at doses consistent with an antioxidant effect, had no mitigation efficacy against radiation nephropathy.