Tyburski, J. B., Patterson, A. D., Krausz, K. W., Slavík, J., Fornace, A. J., Jr., Gonzalez, F. J. and Idle, J. R. Radiation Metabolomics. 1. Identification of Minimally Invasive Urine Biomarkers for Gamma-Radiation Exposure in Mice. Radiat. Res. 170, 1–14 (2008).

Gamma-radiation exposure has both short- and long-term adverse health effects. The threat of modern terrorism places human populations at risk for radiological exposures, yet current medical countermeasures to radiation exposure are limited. Here we describe metabolomics for γ-radiation biodosimetry in a mouse model. Mice were γ-irradiated at doses of 0, 3 and 8 Gy (2.57 Gy/min), and urine samples collected over the first 24 h after exposure were analyzed by ultra-performance liquid chromatography–time-of-flight mass spectrometry (UPLC–TOFMS). Multivariate data were analyzed by orthogonal partial least squares (OPLS). Both 3- and 8-Gy exposures yielded distinct urine metabolomic phenotypes. The top 22 ions for 3 and 8 Gy were analyzed further, including tandem mass spectrometric comparison with authentic standards, revealing that N-hexanoylglycine and β-thymidine are urinary biomarkers of exposure to 3 and 8 Gy, 3-hydroxy-2-methylbenzoic acid 3-O-sulfate is elevated in urine of mice exposed to 3 but not 8 Gy, and taurine is elevated after 8 but not 3 Gy. Gene Expression Dynamics Inspector (GEDI) self-organizing maps showed clear dose–response relationships for subsets of the urine metabolome. This approach is useful for identifying mice exposed to γ radiation and for developing metabolomic strategies for noninvasive radiation biodosimetry in humans.

Tsuboi, I., Hirabayashi, Y., Harada, T., Hiramoto, M., Kanno, J., Inoue, T. and Aizawa, S. Predominant Regeneration of B-Cell Lineage, Instead of Myeloid Lineage, of the Bone Marrow after 1 Gy Whole-Body Irradiation in Mice: Role of Differential Cytokine Expression between B-Cell Stimulation by IL10, Flt3 Ligand and IL7 and Myeloid Suppression by GM-CSF and SCF. Radiat. Res. 170, 15–22 (2008).

Irradiation of mice at doses of 1–1.5 Gy induced a predominant regeneration of the B-cell lineage but suppressed the regeneration of the myeloid lineage. The mechanisms underlying such reciprocal regulation of regeneration and the relationship between the two lineages remain unclear. Because the predominant regeneration of the B-cell lineage observed is considered to depend on the stromal cell function, and because the impairment of such stromal function may nullify such reciprocal responses, mouse models of senescent stromal cell impairment (SCI) and the less senescent stage of SCI (non-SCI) were compared to elucidate the mechanisms underlying the reciprocal regulation of both lineages after radiation exposure. In non-SCI mice irradiated with 1 Gy, the numbers of B-lymphocyte progenitor (CFU-preB) and granulocyte-macrophage progenitor (CFU-GM) cells in the bone marrow decreased rapidly during the first 24 h. Then the number of CFU-preB cells in the bone marrow promptly recovered from the nadir and exceeded the pretreatment level, whereas that of CFU-GM cells remained lower than the pretreatment level. The expression of genes encoding positive regulators of the B-lymphoid lineage [interleukin (IL)10, Flt3 ligand and IL7] was up-regulated; in contrast, expression of the positive regulators of the myeloid lineage [granulocyte macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF)] was down-regulated. In SCI mice irradiated with 1 Gy, the oscillatory changes in the numbers of femoral CFU-preB and CFU-GM cells and in the expression levels of cytokine genes were less marked than those in the non-SCI mice. These results thus imply that the reciprocal regeneration depends on the up-regulation of IL10, Flt3 ligand and IL7 expression and the down-regulation of GM-CSF and SCF expression in the bone marrow, possibly depending on the hematopoietic microenvironment.

Sudo, H., Garbe, J., Stampfer, M. R., Barcellos-Hoff, M. H. and Kronenberg, A. Karyotypic Instability and Centrosome Aberrations in the Progeny of Finite Life-Span Human Mammary Epithelial Cells Exposed to Sparsely or Densely Ionizing Radiation. Radiat. Res. 170, 23–32 (2008).

The human breast is sensitive to radiation carcinogenesis, and genomic instability occurs early in breast cancer development. This study tests the hypothesis that ionizing radiation elicits genomic instability in finite life-span human mammary epithelial cells (HMEC) and asks whether densely ionizing radiation is a more potent inducer of instability. HMEC in a non-proliferative state were exposed to X rays or 1 GeV/nucleon iron ions followed by delayed plating. Karyotypic instability and centrosome aberrations were monitored in expanded clonal isolates. Severe karyotypic instability was common in the progeny of cells that survived X-ray or iron-ion exposure. There was a lower dose threshold for severe karyotypic instability after iron-ion exposure. More than 90% of X-irradiated colonies and >60% of iron-ion-irradiated colonies showed supernumerary centrosomes at levels above the 95% upper confidence limit of the mean for unirradiated clones. A dose response was observed for centrosome aberrations for each radiation type. There was a statistically significant association between the incidence of karyotypic instability and supernumerary centrosomes for iron-ion-exposed colonies and a weaker association for X-irradiated colonies. Thus genomic instability occurs frequently in finite life-span HMEC exposed to sparsely or densely ionizing radiation and may contribute to radiation-induced breast cancer.

Takai, K., Sakamoto, S., Sakai, T., Yasunaga, J., Komatsu, K. and Matsuoka, M. A Potential Link between Alternative Splicing of the NBS1 Gene and DNA Damage/Environmental Stress. Radiat. Res. 170, 33–40 (2008).

NBS1 forms a multimetric complex with MRE11/RAD50, which acts as the sensor of DNA double-strand breaks (DSBs). The mechanisms controlling the expression of NBS1 remain largely unknown. Here we show that NBS1 is transcribed as both a wild-type and an alternatively spliced form exhibiting a premature stop codon in an alternative 50-bp exon in intron 2. Although the wild-type transcript predominates in most tissues, the spliced transcript is abundant in resting peripheral blood mononuclear cells (PBMCs). Levels of the spliced form of NBS1 decreased rapidly after irradiation as levels of the wild-type NBS1 transcript increased, resulting in increased levels of NBS1 protein. Both mitogenic stimulation and methyl methanesulfonate treatment also altered the splicing pattern of NBS1. Resting PBMCs, which predominantly express spliced NBS1, were more susceptible to radiation than mitogen-stimulated cells, which showed predominant expression of the wild-type transcript. Since the alternatively spliced NBS1 gene likely did not produce protein, this alternative splicing seems to be associated with the control of NBS1 protein. Thus alternative splicing of the NBS1 gene may be associated with the regulation of NBS1 in response to DSBs, DNA alkylation damage, and mitogenic response.

Bottke, D., Koychev, D., Busse, A., Heufelder, K., Wiegel, T., Thiel, E., Hinkelbein, W. and Keilholz, U. Fractionated Irradiation Can Induce Functionally Relevant Multidrug Resistance Gene and Protein Expression in Human Tumor Cell Lines. Radiat. Res. 170, 41–48 (2008).

The molecular basis of radiotherapy-related multidrug resistance (MDR) is still unclear. Here we report on a study investigating the effect of fractionated irradiation on expression of the MDR-associated proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and lung resistance-related protein (LRP), the respective mRNAs, and the functional consequences. Cells of six colon and five breast cancer cell lines were irradiated with a total dose of 27 Gy, five fractions of 1.8 Gy per week. The mRNA expression was measured by quantitative RT-PCR, protein levels and drug sensitivity to cisplatin, doxorubicin and bendamustine were assessed by flow cytometry. Breast cancer cell lines showed enhancement of the mRNAs encoding for P-gp, MRP1 and LRP in comparison to nonirradiated cells. No up-regulation of the three mRNA species was observed in the colon cancer cell lines. After irradiation, three breast cancer cell lines showed an up-regulation of LRP, one line an up-regulation of MRP1, and four lines a small up-regulation of P-gp. In the colon cancer cell lines, radiation induced significant enhancement of all three proteins. In comparison to controls, the irradiated cells lines showed a significant resistance to cisplatin, doxorubicin and bendamustine. This study confirms the prior reports of enhancement of P-gp and MRP1 after irradiation, which is accompanied by a multidrug resistance phenomenon, but in addition proposes a novel mechanism in the appearance of MDR after radiation-induced enhancement of LRP.

Burri, R. J., Stock, R. G., Cesaretti, J. A., Atencio, D. P., Peters, S., Peters, C. A., Fan, G., Stone, N. N., Ostrer, H. and Rosenstein, B. S. Association of Single Nucleotide Polymorphisms in SOD2, XRCC1 and XRCC3 with Susceptibility for the Development of Adverse Effects Resulting from Radiotherapy for Prostate Cancer. Radiat. Res. 170, 49–59 (2008).

The objective of this study was to determine whether an association exists between certain single nucleotide polymorphisms (SNPs), which have previously been linked with adverse normal tissue effects resulting from radiotherapy, and the development of radiation injury resulting from radiotherapy for prostate cancer. A total of 135 consecutive patients with clinically localized prostate cancer and a minimum of 1 year of follow-up who had been treated with radiation therapy, either brachytherapy alone or in combination with external-beam radiotherapy, with or without hormone therapy, were genotyped for SNPs in SOD2, XRCC1 and XRCC3. Three common late tissue toxicities were investigated: late rectal bleeding, urinary morbidity, and erectile dysfunction. Patients with the XRCC1 rs25489 G/A (Arg280His) genotype were more likely to develop erectile dysfunction after irradiation than patients who had the G/G genotype (67% compared to 24%; P = 0.048). In addition, patients who had the SOD2 rs4880 T/C (Val16Ala) genotype exhibited a significant increase in grade 2 late rectal bleeding compared to patients who had either the C/C or T/T genotype for this SNP (8% compared to 0%; P = 0.02). Finally, patients with the combination of the SOD2 rs4880 C/T genotype and XRCC3 rs861539 T/C (Thr241Met) genotype experienced a significant increase in grade 2 late rectal bleeding compared to patients without this particular genotypic arrangement (14% compared to 1%; P = 0.002). These results suggest that SNPs in the SOD2, XRCC1 and XRCC3 genes are associated with the development of late radiation injury in patients treated with radiation therapy for prostate adenocarcinoma.

Kiger, J. L., Kiger, W. S., III, Riley, K. J., Binns, P. J., Patel, H., Hopewell, J. W., Harling, O. K., Busse, P. M. and Coderre, J. A. Functional and Histological Changes in Rat Lung after Boron Neutron Capture Therapy. Radiat. Res. 170, 60–69 (2008).

The motivation for this work was an unexpected occurrence of lung side effects in two human subjects undergoing cranial boron neutron capture therapy (BNCT). The objectives were to determine experimentally the biological weighting factors in rat lung for the high-LET dose components for a retrospective assessment of the dose to human lung during cranial BNCT. Lung damage after whole-thorax irradiation was assessed by serial measurement of breathing rate and evaluation of terminal lung histology. A positive response was defined as a breathing rate 20% above the control group mean and categorized as occurring either early (<110 days) or late (>110 days). The ED₅₀ values derived from probit analyses of the early breathing rate dose–response data for X rays and neutrons were 11.4 ± 0.4 and 9.2 ± 0.6 Gy, respectively, and were similar for the other end points. The ED₅₀ values for irradiation with neutrons plus p-boronophenylalanine were 8.7 ± 1.0 and 6.7 ± 0.4 for the early and late breathing rate responses, respectively, and 7.0 ± 0.5 Gy for the histological response. The RBEs for thermal neutrons ranged between 2.9 ± 0.7 and 3.1 ± 1.2 for all end points. The weighting factors for the boron component of the dose differed significantly between the early (1.4 ± 0.3) and late (2.3 ± 0.3) breathing rate end points. A reassessment of doses in patients during cranial BNCT confirmed that the maximum weighted doses were well below the threshold for the onset of pneumonitis in healthy human lung.

Balagurumoorthy, P., Chen, K., Adelstein, S. J. and Kassis, A. I. Auger Electron-Induced Double-Strand Breaks Depend on DNA Topology. Radiat. Res. 170, 70–82 (2008).

From a structural perspective, the factors controlling and the mechanisms underlying the toxic effects of ionizing radiation remain elusive. We have studied the consequences of superhelical/torsional stress on the magnitude and mechanism of DSBs induced by low-energy, short-range, high-LET Auger electrons emitted by ¹²⁵I, targeted to plasmid DNA by m-[¹²⁵I]iodo-p-ethoxyHoechst 33342 (¹²⁵IEH). DSB yields per ¹²⁵I decay for torsionally relaxed nicked (relaxed circular) and linear DNA (1.74 ± 0.11 and 1.62 ± 0.07, respectively) are approximately threefold higher than that for torsionally strained supercoiled DNA (0.52 ± 0.02), despite the same affinity of all forms for ¹²⁵IEH. In the presence of DMSO, the DSB yield for the supercoiled form remains unchanged, whereas that for nicked and linear forms decreases to 1.05 ± 0.07 and 0.76 ± 0.03 per ¹²⁵I decay, respectively. DSBs in supercoiled DNA therefore result exclusively from direct mechanisms, and those in nicked and linear DNA, additionally, from hydroxyl radical-mediated indirect effects. Iodine-125 decays produce hydroxyl radicals along the tracks of Auger electrons in small isolated pockets around the decay site. We propose that relaxation of superhelical stress after radical attack could move a single-strand break lesion away from these pockets, thereby preventing further breaks in the complementary strand that could lead to DSBs.

Wiklund, K., Olivera, G. H., Brahme, A. and Lind, B. K. Radial Secondary Electron Dose Profiles and Biological Effects in Light-Ion Beams Based on Analytical and Monte Carlo Calculations using Distorted Wave Cross Sections. Radiat. Res. 170, 83–92 (2008).

To speed up dose calculation, an analytical pencil-beam method has been developed to calculate the mean radial dose distributions due to secondary electrons that are set in motion by light ions in water. For comparison, radial dose profiles calculated using a Monte Carlo technique have also been determined. An accurate comparison of the resulting radial dose profiles of the Bragg peak for ¹H , ⁴He² and ⁶Li³ ions has been performed. The double differential cross sections for secondary electron production were calculated using the continuous distorted wave-eikonal initial state method (CDW-EIS). For the secondary electrons that are generated, the radial dose distribution for the analytical case is based on the generalized Gaussian pencil-beam method and the central axis depth-dose distributions are calculated using the Monte Carlo code PENELOPE. In the Monte Carlo case, the PENELOPE code was used to calculate the whole radial dose profile based on CDW data. The present pencil-beam and Monte Carlo calculations agree well at all radii. A radial dose profile that is shallower at small radii and steeper at large radii than the conventional 1/r² is clearly seen with both the Monte Carlo and pencil-beam methods. As expected, since the projectile velocities are the same, the dose profiles of Bragg-peak ions of 0.5 MeV ¹H , 2 MeV ⁴He² and 3 MeV ⁶Li³ are almost the same, with about 30% more δ electrons in the sub keV range from ⁴He² and ⁶Li³ compared to ¹H . A similar behavior is also seen for 1 MeV ¹H , 4 MeV ⁴He² and 6 MeV ⁶Li³ , all classically expected to have the same secondary electron cross sections. The results are promising and indicate a fast and accurate way of calculating the mean radial dose profile.

Mestres, M., Caballín, M. R., Barrios, L., Ribas, M. and Barquinero, J. F. RBE of X Rays of Different Energies: A Cytogenetic Evaluation by FISH. Radiat. Res. 170, 93–100 (2008).

Mammography using 26–30 kVp X rays is routinely used in breast cancer screening. Discussion about the radiation-related risk associated with this methodology is ongoing. For radioprotection purposes, a quality factor of 1 has been assigned for all photon energies. However, the relative biological effectiveness (RBE) could increase as the photon energy decreases. Analyzing different biological parameters, for 30 kVp X rays, RBE values from 1 to 8 have been estimated. In the present study, a cytogenetic FISH evaluation of the RBE of 30, 80 and 120 kVp X rays has been done. Blood samples were irradiated with 10 doses from 0.05 to 3 Gy for each energy studied. The yields of translocations and dicentrics were determined by fluorescence in situ hybridization (FISH) using whole chromosome probes for chromosomes 1, 4 and 11 together with a pancentromeric probe. The α coefficients of the dose–effect curves for dicentrics, minimum number of breaks needed to produce exchange-type aberrations, and apparently simple translocations were used to estimate the RBE. Using the curves obtained for 120 kVp as a reference, the RBE values for dicentrics were 1.08 ± 0.43 and 1.73 ± 0.59 for 80 and 30 kVp X rays, respectively; for minimum number of breaks these values were 1.38 ± 0.39 and 1.42 ± 0.41, and for apparently simple translocations they were 1.26 ± 0.40 and 1.51 ± 0.47, respectively. Moreover, the induction of complex aberrations by these energies was compared. The percentage of complex aberrations relative to total aberrations showed a significant tendency to increase as X-ray energy decreased: 7.8 ± 1.19, 9.8 ± 1.6 and 14.1 ± 1.9 for 120, 80 and 30 kVp, respectively (P < 0.02).

Sasaki, M. S., Nomura, T., Ejima, Y., Utsumi, H., Endo, S., Saito, I., Itoh, T. and Hoshi, M. Experimental Derivation of Relative Biological Effectiveness of A-Bomb Neutrons in Hiroshima and Nagasaki and Implications for Risk Assessment. Radiat. Res. 170, 101–117 (2008).

Epidemiological data on the health effects of A-bomb radiation in Hiroshima and Nagasaki provide the framework for setting limits for radiation risk and radiological protection. However, uncertainty remains in the equivalent dose, because it is generally believed that direct derivation of the relative biological effectiveness (RBE) of neutrons from the epidemiological data on the survivors is difficult. To solve this problem, an alternative approach has been taken. The RBE of polyenergetic neutrons was determined for chromosome aberration formation in human lymphocytes irradiated in vitro, compared with published data for tumor induction in experimental animals, and validated using epidemiological data from A-bomb survivors. The RBE of fission neutrons was dependent on dose but was independent of the energy spectrum. The same RBE regimen was observed for lymphocyte chromosome aberrations and tumors in mice and rats. Used as a weighting factor for A-bomb survivors, this RBE system was superior in eliminating the city difference in chromosome aberration frequencies and cancer mortality. The revision of the equivalent dose of A-bomb radiation using DS02 weighted by this RBE system reduces the cancer risk by a factor of 0.7 compared with the current estimates using DS86, with neutrons weighted by a constant RBE of 10.

Pierce, D. A., Væth, M. and Cologne, J. B. Allowance for Random Dose Estimation Errors in Atomic Bomb Survivor Studies: A Revision. Radiat. Res. 170, 118–126 (2008).

Allowing for imprecision of radiation dose estimates for A-bomb survivors followed up by the Radiation Effects Research Foundation can be improved through recent statistical methodology. Since the entire RERF dosimetry system has recently been revised, it is timely to reconsider this. We have found that the dosimetry revision itself does not warrant changes in these methods but that the new methodology does. In addition to assumptions regarding the form and magnitude of dose estimation errors, previous and current methods involve the apparent distribution of true doses in the cohort. New formulas give results conveniently and explicitly in terms of these inputs. Further, it is now possible to use assumptions about two components of the dose errors, referred to in the statistical literature as “classical” and “Berkson-type”. There are indirect statistical indications, involving non-cancer biological effects, that errors may be somewhat larger than assumed before, in line with recommendations made here. Inevitably, methods must rely on uncertain assumptions about the magnitude of dose errors, and it is comforting to find that, within the range of plausibility, eventual cancer risk estimates are not very sensitive to these.

Cucinotta, F. A., Kim, M-H. Y., Willingham, V. and George, K. A. Physical and Biological Organ Dosimetry Analysis for International Space Station Astronauts. Radiat. Res. 170, 127–138 (2008).

In this study, we analyzed the biological and physical organ dose equivalents for International Space Station (ISS) astronauts. Individual physical dosimetry is difficult in space due to the complexity of the space radiation environment, which consists of protons, heavy ions and secondary neutrons, and the modification of these radiation types in tissue as well as limitations in dosimeter devices that can be worn for several months in outer space. Astronauts returning from missions to the ISS undergo biodosimetry assessment of chromosomal damage in lymphocyte cells using the multicolor fluorescence in situ hybridization (FISH) technique. Individual-based pre-flight dose responses for lymphocyte exposure in vitro to γ rays were compared to those exposed to space radiation in vivo to determine an equivalent biological dose. We compared the ISS biodosimetry results, NASA's space radiation transport models of organ dose equivalents, and results from ISS and space shuttle phantom torso experiments. Physical and biological doses for 19 ISS astronauts yielded average effective doses and individual or population-based biological doses for the approximately 6-month missions of 72 mSv and 85 or 81 mGy-Eq, respectively. Analyses showed that 80% or more of organ dose equivalents on the ISS are from galactic cosmic rays and only a small contribution is from trapped protons and that GCR doses were decreased by the high level of solar activity in recent years. Comparisons of models to data showed that space radiation effective doses can be predicted to within about a ±10% accuracy by space radiation transport models. Finally, effective dose estimates for all previous NASA missions are summarized.