Stieber, V. W., Robbins, M., Balamucki, C., DeGuzman, A., Tatter, S. B., Ekstrand, K. E., McMullen, K. P., Branch, C., Shaw, E. G., Bourland, J. D., Lovato, J., Munley, M. T. and Ellis, T. L. Determination of a Clinical Value for the Repair Half-Time (T_1/2) of the Trigeminal Nerve Based on Outcome Data from Gamma Knife Radiosurgery for Facial Pain. Radiat. Res. 168, 143–148 (2007).

Stereotactic radiosurgery (GKRS) using the Leksell Gamma Knife is a treatment option for patients with trigeminal pain. We analyzed a database of 326 GKRS procedures performed over 4.6 years at three discrete dose levels commonly described in the published literature. Logistic regression was used to model the logit of response as a function of treatment time. The resulting coefficient was converted to an estimated probability of response for the shortest and longest treatment times in clinical practice. The two estimated probabilities were then compared to yield the estimated difference in the biologically effective dose (BED) between the two doses, using a modified linear-quadratic model for stereotactic radiosurgery. This difference was used to back-calculate a clinical value for T_1/2, resulting in a range of 1.28–1.77 h for T_1/2. The biological model appeared to accurately predict that, given the doses and treatment times used in general clinical practice, there would be no significant difference in clinical outcome.

Bladen, C. L., Flowers, M., Miyake, K., Podolsky, R. H., Barrett, J., Kozlowski, D. J. and Dynan, W. S. Quantification of Ionizing Radiation-Induced Cell Death In Situ in a Vertebrate Embryo. Radiat. Res. 168, 149–157 (2007).

Quantitative studies of radiation cytotoxicity have been performed mostly in cells in culture. For a variety of reasons, however, the response of cells in culture may not reflect the response for cells in situ in a whole organism. We describe here an approach for quantification of radiation-induced cell death in vivo using the transparent embryo of the zebrafish, Danio rerio, as a model vertebrate system. Using this system, we show that the number of TUNEL-positive cells within a defined region increases approximately linearly with radiation dose up to 1 Gy. The results are consistent with predictions of a linear-quadratic model. The use of alternative models, accommodating a response threshold or low-dose hypersensitivity, did not significantly improve the fit to the observed data. Attenuation of the expression of the 80-kDa subunit of Ku, an essential protein for the nonhomologous end-joining pathway of repair, led to a dose reduction of 30- to 34-fold, possibly approaching the limit where each double-strand break causes a lethal hit. In both the Ku80-attenuated and the control embryos, apoptotic cells were distributed uniformly, consistent with a cell-autonomous mechanism of cell death. Together, these results illustrate the potential of the zebrafish for quantitative studies of radiation-induced cell death during embryogenesis and in vivo.

Asakawa, J., Nakamura, N., Katayama, H. and Cullings, H. M. Estimation of Mutation Induction Rates in AT-Rich Sequences Using a Genome Scanning Approach after X Irradiation of Mouse Spermatogonia. Radiat. Res. 168, 158–167 (2007).

We have previously used NotI as the marker enzyme (recognizing GCGGCCGC) in a genome scanning approach for detection of mutations induced in mouse spermatogonia and estimated the mutation induction rate as about 0.7 × 10⁻⁵ per locus per Gy. To see whether different parts of the genome have different sensitivities for mutation induction, we used AflII (recognizing CTTAAG) as the marker enzyme in the present study. After the screening of 1,120 spots in each mouse offspring, we found five mutations among 92,655 spots from the unirradiated paternal genome, five mutations among 218,411 spots from the unirradiated maternal genome, and 13 mutations among 92,789 spots from 5 Gy-exposed paternal genome. Among the 23 mutations, 11 involved mouse satellite DNA sequences (AT-rich), and the remaining 12 mutations also involved AT-rich but non-satellite sequences. Both types of sequences were found as multiple, similar-sequence blocks in the genome. Counting each member of cluster mutations separately and excluding results on one hypermutable spot, the spontaneous mutation rates were estimated as 3.2 (± 1.9) × 10⁻⁵ and 2.3 (± 1.0) × 10⁻⁵ per locus per generation in the male and female genomes, respectively, and the mutation induction rate as 1.1 (± 1.2) × 10⁻⁵ per locus per Gy. The induction rate would be reduced to 0.9 × 10⁻⁵ per locus per Gy if satellite sequence mutations were excluded from this analysis. The results indicate that mutation induction rates do not largely differ between GC-rich and AT-rich regions: 1 × 10⁻⁵ per locus per Gy or less, which is close to 1.08 × 10⁻⁵ per locus per Gy, the current estimate for the mean mutation induction rate in mice.

Hafer, K., Iwamoto, K., Scuric, Z. and Schiestl, R. H. Adaptive Response to Gamma Radiation in Mammalian Cells Proficient and Deficient in Components of Nucleotide Excision Repair. Radiat. Res. 168, 168–174 (2007).

Cells preconditioned with low doses of low-linear energy transfer (LET) ionizing radiation become more resistant to later challenges of radiation. The mechanism(s) by which cells adaptively respond to radiation remains unclear, although it has been suggested that DNA repair induced by low doses of radiation increases cellular radioresistance. Recent gene expression profiles have consistently indicated that proteins involved in the nucleotide excision repair pathway are up-regulated after exposure to ionizing radiation. Here we test the role of the nucleotide excision repair pathway for adaptive response to γ radiation in vitro. Wild-type CHO cells exhibited both greater survival and fewer HPRT mutations when preconditioned with a low dose of γ rays before exposure to a later challenging dose. Cells mutated for ERCC1, ERCC3, ERCC4 or ERCC5 did not express either adaptive response to radiation; cells mutated for ERCC2 expressed a survival adaptive response but no mutation adaptive response. These results suggest that some components of the nucleotide excision repair pathway are required for phenotypic low-dose induction of resistance to γ radiation in mammalian cells.

Elmroth, K. and Stenerlöw, B. Influence of Chromatin Structure on Induction of Double-Strand Breaks in Mammalian Cells Irradiated with DNA-Incorporated ¹²⁵I. Radiat. Res. 168, 175–182 (2007).

In this study the induction of double-strand breaks (DSBs) was investigated in Chinese hamster V79-379A cells irradiated with the Auger-electron emitter ¹²⁵I incorporated into DNA. The role of chromatin organization was studied by pulse-labeling synchronized cells with ¹²⁵IdU before decay accumulation in early or late S phase. Pulsed-field gel electrophoresis and fragment-size analysis were used to quantify the distribution of DNA fragments in irradiated intact cells and naked DNA as well as in DNA from asynchronously labeled cultures in a different scavenging environment. The results show that in intact cells, after accumulation of decays at −70°C in the presence of 10% DMSO, almost four times more DSBs were induced in late S phase compared with early S phase and the fragment distribution was clearly non-random with an excess of fragments <0.2 Mbp. The DSB yield was 0.6 DSB/cell and decay for cells irradiated in early S phase and 2.3 DSBs/cell and decay for cells irradiated in late S phase. When similar experiments were performed on naked genomic DNA or intact cells irradiated with γ rays, the difference in yield was not as prominent. These data imply a role of chromatin organization in the induction of DSBs by DNA-incorporated ¹²⁵I. In summary, the results presented here suggest that the yield of DSBs as well as the fragment distribution induced by ¹²⁵IdU decay may vary significantly depending on the chromatin organization during S phase and the labeling procedure used.

Ianzini, F., Domann, F. E., Kosmacek, E. A., Phillips, S. L. and Mackey, M. A. Human Glioblastoma U87MG Cells Transduced with a Dominant Negative p53 (TP53) Adenovirus Construct Undergo Radiation-Induced Mitotic Catastrophe. Radiat. Res. 168, 183–192 (2007).

Human gliomas are among the most aggressive tumors, and they respond poorly to treatment. The efficacy of surgical, radiation and chemotherapy treatment of these tumors is limited by the development of resistance. Interventions aimed at altering the response of these tumors to radiation or chemotherapy treatments are needed to improve survival rate and prognosis. Glioblastomas are generally p53 (TP53) functional tumors; however, DNA repair pathways are activated in these tumors instead of the pathways to apoptosis. Thus resistance to treatment is seen in the ability of these tumors to overcome cell death. We present data that demonstrate that U87MG glioblastoma cells transduced with a dominant-negative p53 adenovirus construct become sensitized to radiation-induced mitotic catastrophe through abrogation of G₂/M checkpoint control and overaccumulation of cyclin B1. These findings suggest that interventions abrogating the G₂/M checkpoint sensitize these cells to radiation-induced mitotic catastrophe and may represent a novel mechanism to increase the efficacy of radiation in wild-type p53 gliomas that are resistant to apoptosis.

Lanza, V., Fadda, P., Iannone, C. and Negri, R. Low-Dose Ionizing Radiation Stimulates Transcription and Production of Endothelin by Human Vein Endothelial Cells. Radiat. Res. 168, 193–198 (2007).

A transient increase of EDN1 mRNA accumulation is observed in human vein endothelial cells (HUVECs) after a low dose of ionizing radiation. The kinetics of this mRNA accumulation parallels that of other AP1-regulated transcripts, showing a sharp peak 2 h after irradiation. This accumulation is followed by a net increase of endothelin 1 and big endothelin 1 in the cytoplasm that reaches a peak 4 h after irradiation. We followed the kinetics of endothelin 1 secretion in cell culture medium and did not find a detectable increase in the rate of secretion by the irradiated cells compared to sham-irradiated cells. We conclude that in HUVEC monolayers, an increase in endothelin production does not automatically correspond to an increase in secretion. These findings suggest that endothelin is an important component in the response of endothelial cells to ionizing radiation and that it could be used as a biomarker for low-dose irradiation of endothelial tissues.

Short, S. C., Buffa, F. M., Bourne, S., Koritzinsky, M., Wouters, B. G. and Bentzen, S. M. Dose- and Time-Dependent Changes in Gene Expression in Human Glioma Cells after Low Radiation Doses. Radiat. Res. 168, 199–208 (2007).

We have used DNA microarrays to identify changes in gene expression in cells of the radioresistant human glioma cell lines T98G and U373 after low radiation doses (0.2–2 Gy). Using Bayesian linear models, we have identified a set of genes that respond to low doses of radiation; furthermore, a hypothesis-driven approach to data analysis has allowed us to identify groups of genes with defined non-linear dose responses. Specifically, one of the cell lines we have examined (T98G) shows increased radiosensitivity at low doses (low-dose hyper-radiosensitivity, HRS); thus we have also assessed sets of genes whose dose response mirrors this survival pattern. We have also investigated a time course for induction of genes over the period when the DNA damage response is expected to occur. We have validated these data using quantitative PCR and also compared genes up-regulated in array data to genes present in the polysomal RNA fraction after irradiation. Several of the radioresponsive genes that we describe code for proteins that may have an impact on the outcome of irradiation in these cells, including RAS homologues and kinases involved in checkpoint signaling, so understanding their differential regulation may suggest new ways of altering radioresistance. From a clinical perspective these data may also suggest novel targets that are specifically up-regulated in gliomas during radiotherapy treatments.

Sasnouski, S., Pic, E., Dumas, D., Zorin, V., D'Hallewin, M-A., Guillemin, F. and Bezdetnaya, L. Influence of Incubation Time and Sensitizer Localization on Meta-tetra(hydroxyphenyl)chlorin (mTHPC)-Induced Photoinactivation of Cells. Radiat. Res. 168, 209–217 (2007).

The present study addresses the impact of different aggregation states of meta-tetra(hydroxyphenyl)chlorin (mTHPC) on the photoinactivation of cells. Measurements of the photophysical properties of mTHPC in MCF-7 cells showed progressive sensitizer aggregation with increasing incubation time. Reconstructed absorption spectra of intracellular mTHPC showed a significant decrease in the molar extinction coefficient and broadening of the Soret band at 24 h incubation compared to 3 h. Intracellular photobleaching of mTHPC slowed down, and the profile changed from mono- to bi-exponential upon incubation. Fluorescence lifetime imaging (FLIM) measurements revealed a substantial decrease in the lifetime of mTHPC fluorescence at 24 h compared to 3 h. In addition, the intracellular localization of mTHPC as observed by fluorescence microscopy changed from a diffuse homogeneous fluorescence pattern at short incubation times to a punctiform pattern at 24 h. The efficiency of photodynamic therapy (PDT) assessed by a clonogenic assay was three times greater at 24 h. However, when the survival curves were replotted as a function of the number of absorbed photons, the efficiency was 1.8 times greater at 3 h than at 24 h. The loss of photosensitizing efficiency at higher mTHPC concentrations was attributed to self-quenching of the triplet states of the sensitizers.

Hou, H., Khan, N., Grinberg, O. Y., Yu, H., Grinberg, S. A., Lu, S., Demidenko, E., Steffen, R. P. and Swartz, H. M. The Effects of Efaproxyn™ (Efaproxiral) on Subcutaneous RIF-1 Tumor Oxygenation and Enhancement of Radiotherapy-Mediated Inhibition of Tumor Growth in Mice. Radiat. Res. 168, 218–225 (2007).

Efaproxiral, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity, facilitating oxygen release from hemoglobin, which is likely to increase tissue pO₂. The purpose of this study was to determine the effect of efaproxiral on tumor oxygenation and growth inhibition of RIF-1 tumors that received X radiation (4 Gy) plus oxygen breathing compared to radiation plus oxygen plus efaproxiral daily for 5 days. Two lithium phthalocyanine (LiPc) deposits were implanted in RIF-1 tumors in C3H mice for tumor pO₂ measurements using EPR oximetry. Efaproxiral significantly increased tumor oxygenation by 8.4 to 43.4 mmHg within 5 days, with maximum increases at 22–31 min after treatment. Oxygen breathing alone did not affect tumor pO₂. Radiation plus oxygen plus efaproxiral produced tumor growth inhibition throughout the treatment duration, and inhibition was significantly different from radiation plus oxygen from day 3 to day 5. The results of this study provide unambiguous quantitative information on the effectiveness of efaproxiral to consistently and reproducibly increase tumor oxygenation over the course of 5 days of treatment, modeling the clinical use of efaproxiral. Also, based on the tumor growth inhibition, the study shows the efaproxiral-enhanced tumor oxygenation was radiobiologically significant. This is the first study to demonstrate the ability of efaproxiral to increase tumor oxygenation and to increase the tumor growth inhibition of radiotherapy over 5 days of treatment.

Korystov, Y. N., Shaposhnikova, V. V., Korystova, A. F. and Emel'yanov, M. O. Detection of Reactive Oxygen Species Induced by Radiation in Cells Using the Dichlorofluorescein Assay. Radiat. Res. 168, 226–232 (2007).

The goal of this study was to determine the amount of reactive oxygen species (ROS) that arises inside cells irradiated in medium containing blood serum using the 2′7′-dichlorofluorescein (DCF) assay. DCF fluorescence in cells and medium was recorded on an MF44 Perkin Elmer fluorimeter, and fluorescence in cells only was recorded on a Partec flow-through cytometer. Human larynx tumor HEp-2 cells and lympholeukosis P388 cells were irradiated with X rays at a dose rate of 1.12 Gy/min. The factors (temperature, pH, serum concentration) affecting the oxidation of 2′7′-dichlorofluorescin (DCFH) to DCF were studied, and errors in the dichlorofluorescein assay of ROS were minimized. The amount of ROS registered by the DCF assay in cells was found to depend on the concentration of serum in the medium during irradiation. In the presence of 10% serum, radiation had no effect on the amount of detectable ROS. The effect of radiation on the formation of intracellular ROS was almost completely abolished if the irradiated medium was removed immediately after radiation exposure. The increase in the formation of ROS in cells irradiated in medium with a low serum content is due mainly to the radiolytic products of water that arise in medium and oxidize DCFH located in cells.

Singh, A., Chen, K., Adelstein, S. J. and Kassis, A. I. Synthesis of Coumarin–Polyamine-Based Molecular Probe for the Detection of Hydroxyl Radicals Generated by Gamma Radiation. Radiat. Res. 168, 233–242 (2007).

To develop a molecular probe for detection of hydroxyl radicals in the vicinity of DNA, the coumarin–polyamine complexes, N¹,N¹²-bis[2-oxo-2H-chromene-3-carbonyl]-1,12-diamine-4,9-diazadodecane (5) and tris[2-(2-oxo-2H-chromene-3-carboxamido)ethyl]amine (7), and their hydroxylated derivatives, N¹,N¹²-bis[7-hydroxy-2-oxo-2H-chromene-3-carbonyl]-1,12-diamine-4,9-diazadodecane (6) and tris[2-(7-hydroxy-2-oxo-2H-chromene-3-carboxamido)ethyl]amine (8), have been synthesized. Using computer-generated molecular modeling, the derivatives have been docked onto DNA dodecamer d(CGCGAATTCGCG)₂, the ligand–DNA complexes have been minimized, and the free binding energies (ΔG_binding) and inhibition constants (K_i) have been calculated. Compound 7 is not water-soluble at the concentrations required for the project. When aqueous solutions of 5 are irradiated with γ rays, the relationship between induced fluorescence and dose is linear in the range of 0 to 10 Gy. The fluorescence emission spectrum of irradiated 5 is similar to that of its dihydroxy derivative 6, indicating conversion of 5 to 6, and induction of fluorescence records formation of hydroxyl radicals in aqueous solution. The dicoumarin–polyamine 5, a novel compound for the detection of hydroxyl radicals close to DNA, is a sensitive and quantitative probe with potential for applications in biological systems.

Kozlowski, D., Marsal, P., Steel, M., Mokrini, R., Duroux, J-L., Lazzaroni, R. and Trouillas, P. Theoretical Investigation of the Formation of a New Series of Antioxidant Depsides from the Radiolysis of Flavonoid Compounds. Radiat. Res. 168, 243–252 (2007).

This paper deals with the formation of a series of antioxidant depsides obtained from flavonoid solutions irradiated with γ rays. These reactions take place in radiolyzed alcohol solutions, a medium that is very rich in many different highly reactive species and that hosts specific reactions. We focus on the first step of those reactions, i.e., reactivity of the solute (flavonoid) with the alkoxy radicals CH₃O^· and CH₃CH₂O^· formed in methanol and ethanol, respectively, and their carbon-centered isomers: the 1-hydroxy-methyl (^·CH₂OH) and the 1-hydroxy-ethyl (CH₃^·CHOH) radicals. Among the different flavonoid groups of molecules, only flavonols are transformed. To establish the structure–reactivity relationship that explains why the radiolytic transformation occurs only for those compounds, the process is rationalized theoretically, with Density Functional Theory calculations, taking into account the solvent effects by a Polarizable Continuum Model and a microhydrated environment (one or two water molecules surrounding the active center). The first redox reaction, occurring between the flavonol and the reactive species formed upon irradiation of the solvent, is studied in terms of (1) the O-H bond dissociation enthalpy of each OH group of the flavonoids and (2) electron abstraction from the molecule. We conclude that the reaction, initiated preferentially by the alkoxy radicals, first occurs at the 3-OH group of the flavonol. It is then followed by the formation of a peroxyl radical (after molecular oxygen or superoxide addition). The different cascades of reactions, which lead to the formation of depsides via C-ring opening, are discussed on the basis of the corresponding calculated energetic schemes.

Erdreich, L. S., Van Kerkhove, M. D., Scrafford, C. G., Barraj, L., McNeely, M., Shum, M., Sheppard, A. R. and Kelsh, M. Factors that Influence the Radiofrequency Power Output of GSM Mobile Phones. Radiat. Res. 168, 253–261 (2007).

Epidemiological studies of mobile phone use and risk of brain cancer have relied on self-reported use, years as a subscriber, and billing records as exposure surrogates without addressing the level of radiofrequency (RF) power output. The objective of this study was to measure environmental, behavioral and engineering factors affecting the RF power output of GSM mobile phones during operation. We estimated the RF-field exposure of volunteer subjects who made mobile phone calls using software-modified phones (SMPs) that recorded output power settings. Subjects recruited from three geographic areas in the U.S. were instructed to log information (place, time, etc.) for each call made and received during a 5-day period. The largest factor affecting energy output was study area, followed by user movement and location (inside or outside), use of a hands-free device, and urbanicity, although the two latter factors accounted for trivial parts of overall variance. Although some highly statistically significant differences were identified, the effects on average energy output rate were usually less than 50% and were generally comparable to the standard deviation. These results provide information applicable to improving the precision of exposure metrics for epidemiological studies of GSM mobile phones and may have broader application for other mobile phone systems and geographic locations.

Dynlacht, J. R. and Zeman, E. M. Recent Initiatives for Radiation Oncology Resident Education in Radiation and Cancer Biology. Radiat. Res. 168, 262–265 (2007).

Nearly all residents from accredited radiation oncology residency programs in the United States are required to take the American College of Radiology (ACR) In-Training examination each year. The test is comprised of three sections: Clinical Radiation Oncology, Radiological Physics, and Radiation (and Cancer) Biology. Here we provide an update on changes to the biology portion of the ACR exam. We also discuss the availability and use of the ACR and biology practice exams as assessment and teaching tools for both the instructors of radiation and cancer biology and the residents they teach.