Qutob, S. S., Proulx, D., Mesak, F. M. and Ng, C. E. Effects of N¹, N¹³-Diethylnorspermine (DENSPM) and X-Radiation Treatment on Human Colorectal Tumor Clones with Varying X-Radiation and Drug Responses. Radiat. Res. 163, 357–363 (2005).

This study was designed to examine the effects of treatment with N¹, N¹³-diethylnorspermine (DENSPM), a spermine analog, and X radiation on survival and on the polyamine and spermidine/spermine N¹-acetyltransferase (SSAT) levels in closely related human colorectal tumor (HCT116) clones exhibiting a wide range of X-radiation and drug responses. After treatment with DENSPM and X radiation, clonogenic cell survival was measured. SSAT protein levels were measured by Western blot analysis and SSAT enzymatic activities by the conversion of [1-¹⁴C]acetyl-CoA into [1-¹⁴C]acetylspermidine. Polyamine [i.e. putrescine (PUT), spermine (SPM) and spermidine (SPD)] levels were measured with high-performance liquid chromatography. DENSPM enhanced the efficacy of radiation treatment in HCT116, HCT116-Clone2 (a radiation-resistant clone) and HCT116-Clone10 (a clone with similar X-radiation response as the parental HCT116 cells) but not in HCT116-CloneK (an X-radiation-sensitive but relatively drug-resistant clone). Treatment with DENSPM without X radiation caused the most significant increase in SSAT activity (∼22-fold) and an almost complete depletion of SPD levels in HCT116-CloneK. Our results suggest that (a) the lack of sensitization of X-radiation treatment by DENSPM in HCT116-CloneK was likely due to the prior depletion of SPD levels by DENSPM alone, (b) natural polyamine contents and/or inducibility of SSAT may be important factors influencing cellular response to combined X-radiation and DENSPM treatments, and (c) more importantly, there may be a potentially novel role for combining polyamine analogs such as DENSPM with X rays.

Wan, X. S., Bloch, P., Ware, J. H., Zhou, Z., Donahue, J. J., Guan, J., Stewart, J. and Kennedy, A. R. Detection of Oxidative Stress Induced by Low- and High-Linear Energy Transfer Radiation in Cultured Human Epithelial Cells. Radiat. Res. 163, 364–368 (2005).

A standardized dichlorofluorescin (DCF) fluorometric assay capable of measuring radiation-induced oxidative stress was used to determine the effectiveness of protons and high-mass, high-atomic number (Z) and high-energy (HZE) particles to produce oxidative stress in vitro. Protons were found to be about equally as effective as X rays in the generation of oxidative stress in cultured cells. However, ⁵⁶Fe-ion beams with energies of 1 GeV/nucleon and 5 GeV/nucleon were less effective than X rays or γ rays in inducing dichlorofluorescin (DCFH) oxidation. The relatively lower slope values for the dose responses of HZE-particle radiation-induced DCFH oxidation indicate that the sensitivity of the DCF fluorometric assay is probably dependent on the linear energy transfer (LET) of the radiation beam.

Elmroth, K. and Stenerlöw, B. DNA-Incorporated ¹²⁵I Induces more than one Double-Strand Break per Decay in Mammalian Cells. Radiat. Res. 163, 369–373 (2005).

The Auger-electron emitter ¹²⁵I releases cascades of 20 electrons per decay that deposit a great amount of local energy, and for DNA-incorporated ¹²⁵I, approximately one DNA double-strand break (DSB) is produced close to the decay site. To investigate the potential of ¹²⁵I to induce additional DSBs within adjacent chromatin structures in mammalian cells, we applied DNA fragment-size analysis based on pulsed-field gel electrophoresis (PFGE) of hamster V79-379A cells exposed to DNA-incorporated ¹²⁵IdU. After accumulation of decays at −70°C in the presence of 10% DMSO, there was a non-random distribution of DNA fragments with an excess of fragments <0.5 Mbp and the measured yield was 1.6 DSBs/decay. However, since these experiments were performed under high scavenging conditions (DMSO) that reduce indirect effects, the yield in cells exposed to ¹²⁵IdU under physiological conditions would most likely be even higher. In contrast, using a conventional low-resolution assay without measurement of smaller DNA fragments, the yield was close to one DSB/decay. We conclude that a large fraction of the DSBs induced by DNA-incorporated ¹²⁵I are nonrandomly distributed and that significantly more than one DSB/decay is induced in an intact cell. Thus, in addition to DSBs produced close to the decay site, DSBs may also be induced within neighboring chromatin fibers, releasing smaller DNA fragments that are not detected by conventional DSB assays.

Koshikawa, T., Uematsu, N., Iijima, A., Katagiri, T. and Uchida, K. Alterations of DNA Copy Number and Expression in Genes Involved in Cell Cycle Regulation and Apoptosis Signal Pathways in γ-Radiation-Sensitive SX9 Cells and -Resistant SR-1 Cells. Radiat. Res. 163, 374–383 (2005).

In the present study, genomic differences related to sensitivity to radiation were examined by comparative genomic hybridization and GeneChip 45K microarray in SX9 cells (radiation-sensitive) and their parental line, SR-1 (radiation-resistant). SX9 cells have defective DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity. DNA-PKcs is a DNA double-strand break repair protein that maintains chromosomal stability through nonhomologous end joining. However, the molecular basis of the radiation sensitivity of SX9 cells is unclear. Flow cytometry analysis showed that SR-1 and SX9 cells had a larger G₂/M-phase population at 12 h after 4 Gy γ irradiation, while only SR-1 cells progressed to G₁/S at 24–36 h. SX9 and SR-1 cells had similar patterns of DNA copy number alteration, but the gains were observed on chromosome 9 (cent-E2), 11 (cent-A3), and 12 (C1-E) only in SX9 cells. Expression of genes located on those regions is higher in SX-9 cells than in SR1 cells, and the regions include genes associated with apoptosis and cell cycle regulation. Time-course data for gene expression at 0, 1, 3, 6 and 12 h after 4 Gy γ irradiation revealed that the genes whose expression was altered in SX9 cells but not in SR-1 cells are in 16 clusters. Three of these clusters included genes for cell cycle regulation: JNK, PKC (PRKC) and ceramide cascade protein. These results suggest that amplification and altered expression of genes associated with cell cycle and apoptosis regulators in DNA-PK-deficient SX9 cells affect the differences in response to γ radiation between SX9 and SR-1 cells.

Maguire, P., Mothersill, C., Seymour, C. and Lyng, F. M. Medium from Irradiated Cells Induces Dose-Dependent Mitochondrial Changes and BCL2 Responses in Unirradiated Human Keratinocytes. Radiat. Res. 163, 384–390 (2005).

Exposure of unirradiated human keratinocytes to irradiated cell conditioned medium (ICCM) is known to cause a cascade of events that leads to reproductive death and apoptosis. This study investigates the effect of ICCM on clonogenic survival, mitochondrial mass and BCL2 expression in unirradiated keratinocytes. Exposure to 5 mGy, 0.5 Gy and 5 Gy ICCM resulted in a significant decrease in clonogenic survival. Human keratinocytes incubated with ICCM containing an antioxidant, N-acetylcysteine, showed no significant decrease in clonogenic survival. HPV-G cells incubated with ICCM containing a caspase 9 inhibitor showed no significant decrease in clonogenic survival when the ICCM dose was ≤0.5 Gy. A significant increase in mitochondrial mass per cell was observed after exposure to 5 mGy and 0.5 Gy ICCM. A change in the distribution of the mitochondria from a diffuse cytoplasmic distribution to a more densely concentrated perinuclear distribution was also observed at these doses. No significant increase in mitochondrial mass or change in distribution of the mitochondria was found for 5 Gy ICCM. Low BCL2 expression was observed in HPV-G cells exposed to 5 mGy or 0.5 Gy ICCM, whereas a large significant increase in BCL2 expression was observed in cells exposed to 5 Gy ICCM. This study has shown that low-dose irradiation can cause cells to produce medium-borne signals that can cause mitochondrial changes and the induction of BCL2 expression in unirradiated HPV-G cells. The dose dependence of the mitochondrial changes and BCL2 expression suggests that the mechanisms may be aimed at control of response to radiation at the population level through signaling pathways.

Mothersill, C., Lyng, F., Seymour, C., Maguire, P., Lorimore, S. and Wright, E. Genetic Factors Influencing Bystander Signaling in Murine Bladder Epithelium after Low-Dose Irradiation In Vivo. Radiat. Res. 163, 391–399 (2005).

Radiation-induced bystander effects occur in cells that are not directly hit by radiation tracks but that receive signals from hit cells. They are well-documented in vitro consequences of low-dose exposure, but their relevance to in vivo radiobiology is not established. To investigate the in vivo production of bystander signals, bladder explants were established from two strains of mice known to differ significantly in both short-term and long-term radiation responses. These were investigated for the ability of 0.5 Gy total-body irradiation in vivo to induce production of bystander signals in bladder epithelium. The studies demonstrate that irradiated C57BL/6 mice, but not CBA/Ca mice, produce bystander signals that induce apoptosis and reduce clonogenic survival in reporter HPV-G-transfected keratinocytes. Transfer of medium from explants established from irradiated animals to explants established from unirradiated animals confirmed these differences in bladder epithelium. The responses to the in vivo-generated bystander signal exhibit genotypic differences in calcium signaling and also in signaling pathways indicative of a major role for the balance of pro-apoptosis and anti-apoptosis proteins in determining the overall response. The results clearly demonstrate the in vivo induction of bystander signals that are strongly influenced by genetic factors and have implications for radiation protection, medical imaging, and radiotherapy.

Adachi, Y., Tokuda, N., Sawada, T. and Fukumoto, T. Semiquantitative Detection of Cytokine Messages in X-Irradiated and Regenerating Rat Thymus. Radiat. Res. 163, 400– 407 (2005).

We investigated the expression of cytokine mRNA derived from thymocytes or thymic epithelial cells in X-irradiated (8 Gy) and recovering rat thymuses, according to our previous observation (Mizutani et al., Radiat. Res. 157, 281–289, 2002). The changes in mRNA expression level of interleukin 2 (Il2), Il4, tumor necrosis factor α (Tnf), interferon γ (Ifng), and transforming growth factor β (Tgfb) were examined. The mRNA expression of Il2 and Il4 decreased from day 5 to day 14 after irradiation. Thereafter, the expression level of Il2 mRNA recovered to normal control levels; however, the expression of Il4 mRNA tended toward significantly low levels. Tnf mRNA expression decreased on day 5 after irradiation and then showed a gradual increase back to normal control levels. Tgfb mRNA expression did not change significantly. Ifng mRNA expression was transiently enhanced from day 11 to day 14. The mRNA expression levels of Il10 increased significantly from day 3 to day 7 after irradiation. In addition, the mRNA expression of thymic epithelial cell-derived Il7 showed a transient decrease on day 3; however, then it showed a continuous increase from day 5 to day 21, finally reaching twice the normal control levels after X irradiation. These observations suggest that the expression of cytokine messages in the irradiated thymus changed significantly and did not return to normal for a long time after 8 Gy irradiation.

Prat, M., Demarquay, C., Frick, J., Thierry, D., Gorin, N. C. and Bertho, J. M. Radiation-Induced Increase in Plasma Flt3 Ligand Concentration in Mice: Evidence for the Implication of Several Cell Types. Radiat. Res. 163, 408–417 (2005).

Circulating T lymphocytes were proposed as the main producer of Flt3 ligand. However, during aplasia, there is a drastic reduction in the number of T lymphocytes, while plasma Flt3 ligand concentration is increased. This contradiction prompted us to compare variations in plasma Flt3 ligand during radiation-induced aplasia in BALB/c mice and in T-lymphocyte-deficient NOD-SCID mice to delineate the role of T lymphocytes in the increase in Flt3 ligand concentration. The results showed that plasma Flt3 ligand concentration was increased similarly in the two strains of mice, and that Flt3 ligand concentration was negatively correlated to the number of residual hematopoietic progenitors. Moreover, the Flt3 ligand mRNA expression and Flt3 ligand protein concentration were similar in the two strains of mice in all organs tested, i.e. thymus, spleen, bone marrow, liver, brain and blood cells. These results confirm that Flt3 ligand concentration in the blood is a reflection of bone marrow function and that T lymphocytes are not the main regulator of Flt3 ligand variations during aplasia.

Ina, Y. and Sakai, K. Further Study of Prolongation of Life Span Associated with Immunological Modification by Chronic Low-Dose-Rate Irradiation in MRL-lpr/lpr Mice: Effects of Whole-Life Irradiation. Radiat. Res. 163, 418–423 (2005).

MRL-lpr/lpr mice carry a deletion in the apoptosis-regulating Fas gene that markedly shortens life due to multiple severe diseases. In our previous study (Radiat. Res. 161, 168– 173, 2004), chronic low-dose-rate γ irradiation of mice at 0.35 or 1.2 mGy/h for 5 weeks markedly prolonged the life span, accompanied by immunological activation. This report shows that extension of the irradiation period to the entire life of the mice at the same dose rates improved survival further. The 50% survival time for untreated mice, 134 days, was prolonged to 502 days by 1.2 mGy/h life-long irradiation. Also obtained were a time course and a radiation dose-rate response for the activation of the immune system as indicated by a significant increase in CD4 CD8 T cells in the thymus and CD8 T cells in the spleen and also by a significant decrease in CD3 CD45R/B220 cells and CD45R/B220 CD40 cells in the spleen. Drastic ameliorations of multiple severe diseases, i.e. total-body lymphadenopathy, splenomegaly and serious autoimmune diseases including proteinuria, and kidney and brain-central nervous system syndromes, were found in parallel with these immunological activations, with lifelong low-dose-rate irradiation being more effective than 5-week irradiation at low dose rates.

Sadetzki, S., Chetrit, A., Freedman, L., Stovall, M., Modan, B. and Novikov, I. Long-Term Follow-up for Brain Tumor Development after Childhood Exposure to Ionizing Radiation for Tinea Capitis. Radiat. Res. 163, 424–432 (2005).

Ionizing radiation is an established risk factor for brain tumors, yet quantitative information on the long-term risk of different types of brain tumors is sparse. Our aims were to assess the risk of radiation-induced malignant brain tumors and benign meningiomas after childhood exposure and to investigate the role of potential modifiers of that risk. The study population included 10,834 individuals who were treated for tinea capitis with X rays in the 1950s and two matched nonirradiated groups, comprising population and sibling comparison groups. The mean estimated radiation dose to the brain was 1.5 Gy. Survival analysis using Poisson regression was performed to estimate the excess relative and absolute risks (ERR, EAR) for brain tumors. After a median follow-up of 40 years, an ERR/Gy of 4.63 and 1.98 (95% CI = 2.43–9.12 and 0.73–4.69) and an EAR/Gy per 10⁴ PY of 0.48 and 0.31 (95% CI = 0.28–0.73 and 0.12–0.53) were observed for benign meningiomas and malignant brain tumors, respectively. The risk of both types of tumors was positively associated with dose. The estimated ERR/Gy for malignant brain tumors decreased with increasing age at irradiation from 3.56 to 0.47 (P = 0.037), while no trend with age was seen for benign meningiomas. The ERR for both types of tumor remains elevated at 30-plus years after exposure.

Gillard, N., Spotheim-Maurizot, M. and Charlier, M. Radiation Abolishes Inducer Binding to Lactose Repressor. Radiat. Res. 163, 433–446 (2005).

The lactose operon functions under the control of the repressor-operator system. Binding of the repressor to the operator prevents the expression of the structural genes. This interaction can be destroyed by the binding of an inducer to the repressor. If ionizing radiations damage the partners, a dramatic dysfunction of the regulation system may be expected. We showed previously that γ irradiation hinders repressor-operator binding through protein damage. Here we show that irradiation of the repressor abolishes the binding of the gratuitous inducer isopropyl-1-β-d-thiogalactoside (IPTG) to the repressor. The observed lack of release of the repressor from the complex results from the loss of the ability of the inducer to bind to the repressor due to the destruction of the IPTG binding site. Fluorescence measurements show that both tryptophan residues located in or near the IPTG binding site are damaged. Since tryptophan damage is strongly correlated with the loss of IPTG binding ability, we conclude that it plays a critical role in the effect. A model was built that takes into account the kinetic analysis of damage production and the observed protection of its binding site by IPTG. This model satisfactorily accounts for the experimental results and allows us to understand the radiation-induced effects.

Bowman, M. K., Becker, D., Sevilla, M. D. and Zimbrick, J. D. Track Structure in DNA Irradiated with Heavy Ions. Radiat. Res. 163, 447–454 (2005).

The spatial properties of trapped radicals produced in heavy-ion-irradiated solid DNA at 77 K have been probed using pulsed electron paramagnetic double resonance (PELDOR or DEER) techniques. Salmon testes DNA hydrated to 12 water molecules per nucleotide was irradiated with ⁴⁰Ar ions of energy 100 MeV/nucleon and LET ranging from 300 to 400 keV/μm. Irradiated samples were maintained at cryogenic temperature at all times. PELDOR measurements were made using a refocused echo detection sequence that allows dipolar interaction between trapped radicals to be observed. The EPR spectrum is attributed to electron loss/gain DNA base radicals and neutral carbon-centered radicals that likely arise from sugar damage. We find a radical concentration of 13.5 × 10¹⁸ cm⁻³ in the tracks and a track radius of 6.79 nm. The cross section of these tracks is 144 nm², yielding a lineal radical density of 2.6 radicals/nm. Based on the yields determined previously for particles having calculated LET values of 300–400 keV/μm and our measured lineal density, we obtain an LET of 270 keV/μm, which is in good agreement with the calculated range of values. These measurements of radical density and spatial extent provide the first direct experimental determination of track characteristics in irradiated DNA.

Taguchi, M. and Kojima, T. Yield of OH Radicals in Water under High-Density Energy Deposition by Heavy-Ion Irradiation. Radiat. Res. 163, 455–461 (2005).

The purpose of the present study was to evaluate the dependence of the OH radical yield on the atomic number and the energy of the heavy ions to understand chemical reactions of aqueous solutions. The total yields of oxidized products from phenol in water increased superlinearly as the incident energy increased from 5 MeV/nucleon to 18 MeV/nucleon for carbon and neon ions. The radiolytic yields of OH radicals produced by the ions were determined by analyzing the relationships of the oxidation yields of phenol to the incident energies up to 18 MeV/nucleon for ions in the range of LET from 110 eV/nm to 550 eV/nm and from 320 eV/nm to 1100 eV/nm for carbon and neon ions, respectively. The yields of the OH radicals increased with the specific energy for the same kind of ion and decreased with the atomic number for different ions used at the same specific energy.

Shishkina, E. A., Göksu, H. Y., El-Faramawy, N. A. and Semiochkina, N. Assessment of ⁹⁰Sr Concentration in Dental Tissue using Thin-Layer Beta-Particle Detectors and Verification with Numerical Calculations. Radiat. Res. 163, 462–468 (2005).

Electron paramagnetic resonance (EPR) measurements of tooth enamel can be used as an individual biological dosimeter for external dose assessment. However, the presence of ⁹⁰Sr in the tooth tissues makes the task of interpreting EPR tooth dosimetry more complicated. The determination of the dose contribution of incorporated ⁹⁰Sr in calcified tissue to the total dose measured by EPR is one of the main aspects of correct interpretation of EPR tooth dosimetry. In this work, experimental and numerical calculations were performed to convert the measured β-particle dose rate to ⁹⁰Sr concentration in calcified tissue. The cumulative β-particle dose was measured by exposing artificially contaminated dentin and enamel to thin-layer α-Al₂O₃:C detectors in two different exposure geometries. Numerical calculations were performed for experimental exposure conditions using calculations of electron transport and secondary photons [Monte Carlo n-Particle Transport code version 4C2 (MCNP)^TM]. Numerical calculations were performed to optimize the sample size and exposure geometry. The applicability of two different exposure conditions to be used in routine analysis was tested. Comparison of the computational and experimental results demonstrated very good agreement.

Lynch, D. J., Wilson, W. E., Batdorf, M. T., Sowa Resat, M. B., Kimmel, G. A. and Miller, J. H. Monte Carlo Simulation of the Spatial Distribution of Energy Deposition for an Electron Microbeam. Radiat. Res. 163, 468–472 (2005).

Dosimetry calculations characterizing the spatial variation of the energy deposited by the slowing and stopping of energetic electrons are reported and compared with experimental measurements from an electron microbeam facility. The computations involve event-by-event, detailed-histories Monte Carlo simulations of low-energy electrons interacting in water vapor. Simulations of electron tracks with starting energies from 30 to 80 keV are used to determine energy deposition distributions in thin cylindrical rings as a function of penetration and radial distance from a beam source. Experimental measurements of the spatial distribution of an electron microbeam in air show general agreement with the density-scaled simulation results for water vapor at these energies, yielding increased confidence in the predictions of Monte Carlo track-structure simulations for applications of the microbeam as a single-cell irradiator.