Suzuki, K., Okada, H., Yamauchi, M., Oka, Y., Kodama, S. and Watanabe, M. Qualitative and Quantitative Analysis of Phosphorylated ATM Foci Induced by Low-Dose Ionizing Radiation. Radiat. Res. 165, 499–504 (2006).

We examined the formation of phosphorylated ataxia telangiectasia mutated (ATM) foci in exponentially growing normal human diploid cells exposed to low doses of X rays. Phosphorylated ATM foci were detected immediately after irradiation, and the number of foci decreased as the time after irradiation increased. The kinetics of phosphorylated ATM foci was comparable to that of phosphorylated histone H2AX. We found that there were fewer spontaneous phosphorylated ATM foci than that phosphorylated histone H2AX foci. Notably, significant numbers of phosphorylated histone H2AX foci, but not phosphorylated ATM foci, were detected in the S-phase cells. The induction of foci showed a linear dose–response relationship with doses ranging for 10 mGy to 1 Gy, and the average number of phosphorylated ATM foci per gray was approximately 50. The average size of the foci was comparable for the cells irradiated with 20 mGy and 1 Gy, and there was no significant difference in the kinetics of disappearance of foci, indicating that DNA double-strand breaks are similarly recognized by DNA damage checkpoints and are repaired irrespective of the dose.

Costes, S. V., Boissière, A., Ravani, S., Romano, R., Parvin, B. and Barcellos-Hoff, M. H. Imaging Features that Discriminate between Foci Induced by High- and Low-LET Radiation in Human Fibroblasts. Radiat. Res. 165, 505–515 (2006).

In this study, we investigated the formation of radiation-induced foci in normal human fibroblasts exposed to X rays or 130 keV/μm nitrogen ions using antibodies to phosphorylated protein kinase ataxia telangiectasia mutated (ATMp) and histone H2AX (γ-H2AX). High-content automatic image analysis was used to quantify the immunofluorescence of radiation-induced foci. The size of radiation-induced foci increased for both proteins over a 2-h period after nitrogen-ion irradiation, while the size of radiation-induced foci did not change after exposure to low-LET radiation. The number of radiation-induced ATMp foci showed a more rapid rise and greater frequency after X-ray exposure and was resolved more rapidly such that the frequency of radiation-induced foci decreased by 90% compared to 60% after exposure to high-LET radiation 2 h after 30 cGy. In contrast, the kinetics of radiation-induced γ-H2AX focus formation was similar for high- and low-LET radiation in that it reached a plateau early and remained constant for up to 2 h. High-resolution 3D images of radiation-induced γ-H2AX foci and dosimetry computation suggest that multiple double-strand breaks from nitrogen ions are encompassed within large nuclear domains of 4.4 Mbp. Our work shows that the size and frequency of radiation-induced foci vary as a function of radiation quality, dose, time and protein target. Thus, even though double-strand breaks and radiation-induced foci are correlated, the dynamic nature of both contradicts their accepted equivalence for low doses of different radiation qualities.

Wykes, S. M., Piasentin, E., Joiner, M. C., Wilson, G. D. and Marples, B. Low-Dose Hyper-radiosensitivity is not Caused by a Failure to Recognize DNA Double-Strand Breaks. Radiat. Res. 165, 516–524 (2006).

One of the earliest cellular responses to radiation-induced DNA damage is the phosphorylation of the histone variant H2AX (γ-H2AX). γ-H2AX facilitates the local concentration and focus formation of numerous repair-related proteins within the vicinity of DNA DSBs. Previously, we have shown that low-dose hyper-radiosensitivity (HRS), the excessive sensitivity of mammalian cells to very low doses of ionizing radiation, is a response specific to G₂-phase cells and is attributed to evasion of an ATM-dependent G₂-phase cell cycle checkpoint. To further define the mechanism of low-dose hyper-radiosensitivity, we investigated the relationship between the recognition of radiation-induced DNA double-strand breaks as defined by γ-H2AX staining and the incidence of HRS in three pairs of isogenic cell lines with known differences in radiosensitivity and DNA repair functionality (disparate RAS, ATM or DNA-PKcs status). Marked differences between the six cell lines in cell survival were observed after high-dose exposures (>1 Gy) reflective of the DNA repair capabilities of the individual six cell lines. In contrast, the absence of functional ATM or DNA-PK activity did not affect cell survival outcome below 0.2 Gy, supporting the concept that HRS is a measure of radiation sensitivity in the absence of fully functional repair. No relationship was evident between the initial numbers of DNA DSBs scored immediately after either low- or high-dose radiation exposure with cell survival for any of the cell lines, indicating that the prevalence of HRS is not related to recognition of DNA DSBs. However, residual DNA DSB damage as indicated by the persistence of γ-H2AX foci 4 h after exposure was significantly correlated with cell survival after exposure to 2 Gy. This observation suggests that the persistence of γ-H2AX foci could be adopted as a surrogate assay of cellular radiosensitivity to predict clinical radiation responsiveness.

Suzuki, M., Amano, M., Choi, J. H., Park, H. J., Williams, B. W., Ono, K. and Song, C. W. Synergistic Effects of Radiation and β-Lapachone in DU-145 Human Prostate Cancer Cells In Vitro. Radiat. Res. 165, 525–531 (2006).

It has been reported that β-lapachone (β-lap), a bioreductive anti-cancer drug, synergistically interacts with ionizing radiation and that the sensitivity of cells to β-lap is closely related to the activity of NAD(P)H:quinone oxidoreductase 1 (NQO1). Here we report the results of our studies of mechanisms underlying the synergistic interaction of β-lap and radiation in killing cancer cells using the DU-145 human prostate cancer cell line. The clonogenic cell death caused by the combination of radiation and β-lap was synergistic when β-lap was administered 0–10 h after irradiation but not when it was given before irradiation. The expression and activity of NQO1 increased significantly and remained elevated for longer than 12 h after 4 Gy irradiation, suggesting that the long-lasting elevation of NQO1 sensitized the cells to β-lap. Studies with split-dose irradiation demonstrated that β-lap given immediately after irradiation effectively inhibited sublethal radiation damage (SLD) repair. Taken together, these results lead us to conclude that the synergistic interaction between β-lap and radiation in killing cells is the result of two distinct mechanisms: First, radiation sensitizes cells to β-lap by up-regulating NQO1, and second, β-lap sensitizes cells to radiation by inhibiting SLD repair. The combination of β-lap and radiotherapy is potentially promising modality for the treatment of cancer in humans.

Talà, A., Belloni, F., Monaco, C., Lorusso, A., Nassisi, V. and Alifano, P. Effects of XeCl UV_{308 nm} Laser Radiation on Survival and Mutability of recA-Proficient and recA-Defective Escherichia coli Strains. Radiat. Res. 165, 532–537 (2006).

recA1, recA13 and recA56 are considered null alleles of the Escherichia coli recA gene because they were shown to have essentially no activity in vivo. In this study, we used strains harboring the recA null alleles and their recA-proficient congenic counterpart to assess the lethal and the mutagenic effects elicited by near-UV_{308 nm} coherent radiation generated by a XeCl excimer laser. We compared these effects with those produced by a conventional far-UV_{254 nm} germicidal lamp. Compared to the germicidal lamp, the excimer laser was able to better discriminate the different recA-defective strains on the basis of their UV-radiation sensitivity, which was progressively higher in the strains with the alleles in the order recA1, recA56 and recA13. This finding was consistent with previous data on residual biochemical activities of the respective mutated RecA proteins in vitro. The discrepancy between the results obtained with the lamp and laser irradiation suggested that the biological response to the two radiations involves distinct mechanisms. This hypothesis was supported by the evidence that exposure to near-UV_{308 nm} radiation induced mutagenesis in recA-defective strains at an extent considerably greater than in recA-proficient strains. In contrast, far-UV_{254 nm}-radiation-induced mutagenesis was reported to be largely dependent on a functional recA allele.

Gudkov, S. V., Shtarkman, I. N., Smirnova, V. S., Chernikov, A. V. and Bruskov, V. I. Guanosine and Inosine Display Antioxidant Activity, Protect DNA In Vitro from Oxidative Damage Induced by Reactive Oxygen Species, and Serve as Radioprotectors in Mice. Radiat. Res. 165, 538–545 (2006).

The effect of ribonucleosides on 8-oxoguanine formation in salmon sperm DNA dissolved in 1 mM phosphate buffer, pH 6.8, upon exposure to γ rays was examined by ELISA using monoclonal antibodies against 8-oxoguanine. Nucleosides (1 mM) decreased the radiation-induced yield of 8-oxoguanine in the order Guo > Ino > Ado > Thd > Urd > Cyd. Guanosine and inosine considerably reduced deamination of cytosine in the DNA solutions upon heating for 24 h at 80°C. The action of nucleosides on the heat-induced generation of reactive oxygen species in the phosphate buffer was studied. The concentration of hydrogen peroxide was measured by enhanced chemiluminescence in a peroxidase–luminol–p-iodophenol system; the hydroxyl radical formation was measured fluorometrically by the use of coumarin-3-carboxylic acid. Guanosine and inosine considerably decreased the heat-induced production of both hydrogen peroxide and OH radicals. Guanosine and inosine increased survival of mice after a lethal dose of radiation. They especially enhanced the survival of animals when were administered shortly after irradiation. The results indicate that guanosine and inosine, natural antioxidants, prevent oxidative damage to DNA, decrease the generation of ROS, and protect mice against γ-radiation-induced death.

Han, G., Zhou, Y. F., Zhang, M. S., Cao, Z., Xie, C. H., Zhou, F. X., Peng, M. and Zhang, W. J. Angelica sinensis Down-regulates Hydroxyproline and Tgfb1 and Provides Protection in Mice with Radiation-Induced Pulmonary Fibrosis. Radiat. Res. 165, 546–552 (2006).

Pulmonary fibrosis is a common delayed side effect of radiation therapy, and it has a poor prognosis. Tgfb1 is a potent chemoattractant for fibroblasts and stimulates the production of collagen, the protein that contains hydroxyproline. Since collagen is by far the most abundant protein in the lung, comprising 60–70% of the tissue mass, analysis of the hydroxyproline content in lung tissues provides a reliable quantitative index for pulmonary fibrosis. Thus hydroxyproline and Tgfb1 may be involved in the development of fibrosis. In this study, we investigated radiation-induced pulmonary fibrosis in a mouse model. C57BL/6 mice were assigned into four groups: no treatment, treated with Angelica sinensis treated only, X-irradiated only (a single fraction of 12 Gy to the thorax), and Angelica sinensis treatment plus radiation. We assayed expression of hydroxyproline and the mRNA and protein of Tgfb1 in the four groups. We found that Angelica sinensis down-regulated the production of Tgfb1 and hydroxyproline in mice with radiation-induced pulmonary fibrosis. This study has demonstrated for the first time that Angelica sinensis inhibits the progress of radiation-induced pulmonary fibrosis, possibly by down-regulating the expression of the proinflammatory cytokine Tgfb1. These data suggest that Angelica sinensis may be useful in preventing and/or treating radiation-induced pulmonary fibrosis in the clinic.

Amendola, R., Basso, E., Pacifici, P. G., Piras, E., Giovanetti, A., Volpato, C. and Romeo, G. Ret, Abl1 (cAbl) and Trp53 Gene Fragmentations in Comet-FISH Assay Act as In Vivo Biomarkers of Radiation Exposure in C57BL/6 and CBA/J Mice. Radiat. Res. 165, 553–561 (2006).

The International Commission on Radiation Protection (ICRP) has lowered the dose limits for workers and for the general public exposed to ionizing radiation. Consequently, a reliable dosimetric method for monitoring possible radiation-induced damage is of great importance in radioprotection. The counting of dicentric chromosomal aberrations and of micronuclei in peripheral blood lymphocytes is unreliable when it is applied to in vivo biopsies and for low-dose exposures. Single-cell gel electrophoresis (SCGE or comet assay), although sensitive and rapid, shows high variability when applied in vivo, probably due to prompt repair of the DNA breaks and confounding environmental factors. In this paper, we describe specific in situ hybridization of Ret, Abl1 (cAbl), and Trp53 gene fragmentations on SCGE slides (comet-FISH assay) in peripheral blood cells from C57BL/6 and CBA/J mice as an indicator of radiation-induced DNA damage. The results obtained from four mice for each experimental point (0, 1, 2 and 4 Gy of X rays) discriminated in a statistically significant way the effects of all doses when fragmentations were analyzed for the Ret, Ab1 and Trp53 genes. SCGE alone, when applied to the same specimens, produced no significant results because of interindividual and experimental variability.

Lee, W-J., Majumder, Z. R., Jeoung, D-I., Lee, H-J., Kim, S-H., Bae, S. and Lee, Y-S. Organ-Specific Gene Expressions in C57BL/6 Mice after Exposure to Low-Dose Radiation. Radiat. Res. 165, 562–569 (2006).

The possibility that radiation-induced alterations in gene expression are tissue specific and are related to apoptosis was examined using samples from brain, heart, lung, spleen and intestine from female C57BL6 mice after exposure to 0.2 Gy radiation. Apoptosis was the highest in spleen and intestine, moderate in lung, and absent in brain and heart. However, the mRNA expression of Trp53 and Cdkn1a (p21) after irradiation was not different among the organ types, and immunohistochemistry revealed that all the organs expressed these two proteins after irradiation. When expression patterns of 23 genes in the organs were examined by RT-PCR, neogenine, Apo1, nuclease sensitive element binding protein 1, syntaxin, cyclin G1, hNOP56, paraoxonase and glutathione peroxidase were overexpressed after irradiation in all the organs sampled, suggesting them as universal exposure markers for low-dose radiation. Sialyltransferase may be a candidate for radiation detection in spleen and intestine, which are radiosensitive organs. Because Sod1 (Cu/ZnSOD) and αB crystalline were expressed only in spleen, and protein tyrosine kinase and platelet membrane glycoprotein lib were expressed in both spleen and lung, these genes may also be potential markers for detection of radiation exposure, especially low-dose radiation, in these tissues. These data suggested possible tissue-specific markers of low-dose radiation exposure and suggested potential novel genetic modifiers of radiation response.

Zhang, R., Burns, F. J., Chen, H., Chen, S. and Wu, F. Alterations in Gene Expression in Rat Skin Exposed to ⁵⁶Fe Ions and Dietary Vitamin A Acetate. Radiat. Res. 165, 570– 581 (2006).

The purpose of the present work was to examine gene expression patterns in rat skin exposed to a beam of ⁵⁶Fe ions, a surrogate for the high-energy, heavy-ion galactic radiation background, as a basis for obtaining a better understanding of the possible mechanism(s) behind the radioprotective activity of vitamin A. A 2 × 4-cm rectangle of dorsal rat skin was exposed to 1.01 GeV/nucleon ⁵⁶Fe ions generated by the Alternating Gradient Synchrotron at Brookhaven National Laboratory. Gene expression patterns were monitored in either the presence or absence of a 250-ppm dietary supplement of vitamin A acetate in powdered lab chow. Although vitamin A and other retinoids show anti-carcinogenic activity in several animal models, the underlying changes in gene expression have not been examined extensively. At either 1 or 7 day after irradiation, a 1-cm square of irradiated and control rat skin was excised and analyzed using the Affymetrix rat microarray (RG_U34A) system. Microarray responses were displayed and processed by GeneSpring 7.0 and GOTree software. At 1 day after 3 Gy of ⁵⁶Fe-ion irradiation, the expression of 110 genes was significantly up-regulated (P < = 0.05) in comparison to levels in control rat skin, while no genes were altered by the vitamin A acetate supplement alone. Combined with ⁵⁶Fe-ion radiation, the vitamin A acetate supplement blocked the expression of 88 (80%) of the 110 genes and eliminated 16 of 18 gene categories that were significantly altered (all increased) by the ⁵⁶Fe-ion radiation. Categories with large numbers of genes eliminated by the retinoid included response to stress, 33 genes; response to biotic stimulus, 38 genes; signal transduction, 35 genes; and regulation of cellular/physiological process, 40 genes. Even for immune response and response to biotic stimulus, the only two categories that remained significantly altered in the presence of the vitamin, the combined number of altered genes was reduced from 74 to 13. No significant alterations in gene expression were found at 7 days relative to the numbers in controls. The results indicate that at 1 day dietary vitamin A acetate strongly interfered with ⁵⁶Fe-ion-induced gene expression within the broad categories of stimulus- and stress-related genes, implying that the latter gene categories likely play a role in the radioprotective action of the vitamin.

Otsuka, S., Coderre, J. A., Micca, P. L., Morris, G. M., Hopewell, J. W., Rola, R. and Fike, J. R. Depletion of Neural Precursor Cells after Local Brain Irradiation is due to Radiation Dose to the Parenchyma, not the Vasculature. Radiat. Res. 165, 582–591 (2006).

The underlying mechanisms associated with radiation-induced cognitive impairments remain elusive but may involve changes in hippocampal neural precursor cells. Proliferating neural precursor cells have been shown to be extremely sensitive to X rays, either from damage to the cells themselves and/or through microenvironmental factors, including the anatomical relationship with the microvasculature, which is altered by radiation. The neutron capture reaction in boron was used to determine whether the sensitivity of neural precursor cells was dominated by direct radiation effects or was mediated through changes in the microvasculature. Young adult rats were irradiated with X rays, neutrons only, or neutrons plus either mercapto-undecahydro-dodecaborane (BSH) or p-dihydroxyboryl-phenylalanine (BPA). BSH remains inside cerebral vessels, thereby limiting the neutron capture intravascularly; BPA readily passes into the parenchyma. One month after irradiation, cell proliferation and numbers of immature neurons were determined using immunohistochemistry. Results showed that (1) neural precursor cells and their progeny were decreased in a dose-dependent manner by mixed high- and low-LET radiation, and (2) selective irradiation of the microvasculature resulted in less loss of neural precursor cells than when the radiation dose was delivered uniformly to the parenchyma. This information, and in particular the approach of selectively irradiating the vasculature, may be useful in developing radioprotective compounds for use during therapeutic irradiation.

Tawn, E. J., Whitehouse, C. A. and Riddell, A. E. FISH Chromosome Analysis of Plutonium Workers from the Sellafield Nuclear Facility. Radiat. Res. 165, 592–597 (2006).

Chromosome analysis using a single-color FISH technique to paint three pairs of chromosomes was undertaken on a group of 46 retired plutonium workers with assessed bone marrow doses >60 mSv, 34 of whom were categorized as having robust dosimetry and 12 for whom internal doses were considered less reliable. Comparisons were made with a group of 34 workers with negligible radiation exposure and a group of 34 workers with similar recorded external γ-ray doses but negligible internal dose. The simple translocation frequency of 17.65 ± 1.96 × 10⁻³ per genome equivalent for the 34 plutonium workers with robust dosimetry was significantly increased in comparison with that of 10.06 ± 1.16 × 10⁻³ per genome equivalent for the unirradiated control group (P = <0.001) and that of 13.55 ± 1.43 × 10⁻³ per genome equivalent for the group with similar external γ-ray exposure (P = 0.012). Thus, although in vitro studies have indicated that the majority of α-particle-irradiated cells suffer complex non-transmissible chromosome damage, in vivo a significant proportion survive with simple exchanges that can be passed on to descendant cells. In contrast, the three groups demonstrated no significant differences in stable complex aberrations. No evidence of an increase in dicentrics or unstable complex aberrations associated with plutonium exposure was observed, and it can therefore be assumed that there is little, if any, ongoing irradiation of mature lymphocytes. The translocation frequency of 12.08 ± 1.92 × 10⁻³ per genome equivalent for the group of 12 plutonium workers with less reliable internal dosimetry could adequately be accounted for by age and external dose and indicates that the internal bone marrow doses are likely to have been overestimated. Cytogenetic analysis can therefore make a valuable contribution to the validation of internal doses from plutonium deposition.

Verschaeve, L., Heikkinen, P., Verheyen, G., Van Gorp, U., Boonen, F., Vander Plaetse, F., Maes, A., Kumlin, T., Mäki-Paakkanen, J., Puranen, L. and Juutilainen, J. Investigation of Co-genotoxic Effects of Radiofrequency Electromagnetic Fields In Vivo. Radiat. Res. 165, 598–607 (2006).

We investigated the possible combined genotoxic effects of radiofrequency (RF) electromagnetic fields (900 MHz, amplitude modulated at 217 Hz, mobile phone signal) with the drinking water mutagen and carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Female rats were exposed to RF fields for a period of 2 years for 2 h per day, 5 days per week at average whole-body specific absorption rates of 0.3 or 0.9 W/kg. MX was given in the drinking water at a concentration of 19 μg/ml. Blood samples were taken at 3, 6 and 24 months of exposure and brain and liver samples were taken at the end of the study (24 months). DNA damage was assessed in all samples using the alkaline comet assay, and micronuclei were determined in erythrocytes. We did not find significant genotoxic activity of MX in blood and liver cells. However, MX induced DNA damage in rat brain. Co-exposures to MX and RF radiation did not significantly increase the response of blood, liver and brain cells compared to MX exposure only. In conclusion, this 2-year animal study involving long-term exposures to RF radiation and MX did not provide any evidence for enhanced genotoxicity in rats exposed to RF radiation.

Zook, B. C. and Simmens, S. J. The Effects of Pulsed 860 MHz Radiofrequency Radiation on the Promotion of Neurogenic Tumors in Rats. Radiat. Res. 165, 608–615 (2006).

In a previous study, this laboratory reported a statistically nonsignificant trend for shortened latency of ethylnitrosourea (ENU)-induced brain tumors in Sprague-Dawley rats exposed to an 860 MHz pulsed radiofrequency (RF) signal. The present study was designed to investigate further any promoting effect of the pulsed RF signal on latency and other characteristics of neurogenic tumors in the progeny of pregnant rats treated with 6.25 or 10 mg/kg ENU. The resulting 1080 offspring were randomized equally by number, sex and ENU dose into pulsed RF, sham and cage control groups. The rats were exposed to the pulsed RF signal 6 h per day 5 days per week; the sham-exposed group was similarly confined for the same periods, and the cage controls were housed in standard cages. An essentially equal number of rats from each group were killed humanely every 30 days between the ages of 171 and 325 days; 32 rats died and 225 rats were killed when they were moribund. Postmortem examinations on the 1080 rats revealed 38 spinal cord tumors, 191 spinal nerve tumors, 232 cranial nerve tumors, and 823 brain tumors. A methodical study of the tumor characteristics disclosed no evidence that exposure to the pulsed RF signal affected the incidence, malignancy, volume, multiplicity, latency or fatality associated with any kind of neurogenic tumor.

Hafer, K., Scuric, Z., Iwamoto, K. S. and Schiestl, R. H. Point-Source Irradiation with Eccentric Rotation Causes Inhomogeneous Dose Distribution. Radiat. Res. 165, 616–620 (2006).

Radioactive point sources are regularly used for irradiating cell culture and other biological materials. Eccentric rotation is often used to minimize dose disparities that arise from irradiating samples that span a distance from the point source. Rotation provides a great improvement in dose homogeneity compared to inert irradiation yet still presents an obvious shortcoming for exposures in which the sample completes only partial rotation or fractional rotation. In such cases, certain areas of the sample have a closer average distance to the radiation source than other areas within the same sample. This obstacle can be partially overcome by adjusting rotation speed so the sample traverses a full rotation (or multiple thereof) throughout the total irradiation time. Here we investigate the effects of irradiation with eccentric rotation on dose homogeneity. We show that due to the inverse square law that governs dose, even exposures with full rotation result in inhomogeneous dose distributions. This dose inhomogeneity can be substantial, especially for large samples and small source– sample distances. We observed a 33% difference in survival across 100-mm dishes and a 400% difference for 150-mm dishes. The dose inhomogeneity inherent to eccentric rotation increases the actual average dose delivered across the sample compared to that delivered at sample center. We offer a table of correction factors that account for this dose increase and correct the dose delivered at center to the actual average dose delivered across the entire sample.