Gaugler, M. H., Vereycken-Holler, V., Squiban, C., Vandamme, M., Vozenin-Brotons, M. C. and Benderitter, M. Pravastatin Limits Endothelial Activation after Irradiation and Decreases the Resulting Inflammatory and Thrombotic Responses. Radiat. Res. 163, 479–487 (2005).

Endothelial dysfunction has been implicated in the pathogenesis of atherosclerosis, fibrosis and vascular occlusion after radiation therapy. Statins have been reported to improve endothelial function; however, this beneficial effect on endothelial cells has never been investigated after irradiation. Therefore, using human microvascular endothelial cells from lung that had been irradiated with 5 or 10 Gy, we assessed the effect of pravastatin on endothelial activation by ELISA, cell-ELISA and electrophoretic mobility shift assay and increased blood-endothelial cell interactions by a flow adhesion assay. Pravastatin inhibited the overproduction of monocyte chemoattractant protein 1, IL6 and IL8 and the enhanced expression of intercellular adhesion molecule 1 but had no effect on platelet-endothelial cell adhesion molecule 1 expression. Moreover, pravastatin down-regulated the radiation-induced activation of the transcription factor activator protein 1 but not of nuclear factor-κB. Finally, an inhibition by pravastatin of increased adhesion of leukocytes and platelets to irradiated endothelial cells was observed. The effect of pravastatin was maintained up to 14 days after irradiation and was reversed by mevalonate. Pravastatin exerts persistent anti-inflammatory and anti-thrombotic effects on irradiated endothelial cells. Statins may be considered in therapeutic strategies for the management of patients treated with radiation therapy.

Jung, M., Velena, A., Chen, B., Petukhov, P. A., Kozikowski, A. P. and Dritschilo, A. Novel HDAC Inhibitors with Radiosensitizing Properties. Radiat. Res. 163, 488–493 (2005).

The members of the histone deacetylase (HDAC) family play important roles in various cellular processes, including transcriptional regulation, cell proliferation, differentiation and apoptosis. Inhibitors of histone deacetylases are emerging as an important new class of chemotherapeutic agents. As such, identifying stable and potent chemical HDAC inhibitory compounds is an important focus for translational research. Here we report the results of a rational drug design of novel HDAC inhibitors with potential for sensitizing cancer cells to radiation therapy. Over 60 HDAC inhibitor analogues incorporating a urea backbone and the hydroxamic acid end moiety were designed and screened. Six were found to confer 50% inhibition of HDAC enzyme activity at nanomolar concentrations. These candidate HDAC inhibitors inhibited cell proliferation at the ranges of IC₅₀ 10–50 μM in various cancer cells, including prostate (PC-3), breast (MCF-7) and head and neck squamous carcinoma (SQ-20B). Furthermore, radiation clonogenic survival assays revealed that these compounds possess radiosensitizing properties that are cell type-specific. The data support the further investigation of these HDAC inhibitors for use as sensitizing agents with potential for clinical application.

Tsuruoka, C., Suzuki, M., Kanai, T. and Fujitaka, K. LET and Ion Species Dependence for Cell Killing in Normal Human Skin Fibroblasts. Radiat. Res. 163, 494–500 (2005).

We studied the LET and ion species dependence of the RBE for cell killing to clarify the differences in the biological effects caused by the differences in the track structure that result from the different energy depositions for different ions. Normal human skin fibroblasts were irradiated with heavy-ion beams such as carbon, neon, silicon and iron ions that were generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS) in Japan. Cell killing was measured as reproductive cell death using a colony formation assay. The RBE-LET curves were different for carbon ions and for the other ions. The curve for carbon ions increased steeply up to around 98 keV/μm. The RBE of carbon ions at 98 keV/μm was 4.07. In contrast, the curves for neon, silicon and iron ions had maximum peaks around 180 keV/μm, and the RBEs at the peak position ranged from 3.03 to 3.39. When the RBEs were plotted as a function of Z*²/β² (where Z* is the effective charge and β is the relative velocity of the ion) instead of LET, the discrepancies between the RBE-LET curves for the different ion beams were reduced, but branching of the RBE-Z*²/β² curves still remained. When the inactivation cross section was plotted as a function of either LET or Z*²/β², it increased with increasing LET. However, the inactivation cross section was always smaller than the geometrical cross section. These results suggest that the differences in the energy deposition track structures of the different ion sources have an effect on cell killing.

Nath, R., Bongiorni, P., Chen, Z., Gragnano, J. and Rockwell, S. Relative Biological Effectiveness of ¹⁰³Pd and ¹²⁵I Photons for Continuous Low-Dose-Rate Irradiation of Chinese Hamster Cells. Radiat. Res. 163, 501–509 (2005).

Monolayers of Chinese hamster lung cells (CCL-16) in a polystyrene phantom were irradiated in vitro by ¹⁰³Pd and ¹²⁵I sources at dose rates of 6 to 72 cGy/h. Cell survival curves for acute high-dose-rate irradiation (over 30 Gy/h) were also measured using nearly monoenergetic X-ray beams which were designed to simulate the mean energies of photons emitted by ¹²⁵I and ¹⁰³Pd and also using a clinical 250 kVp X-ray beam. A profound dose-rate effect is observed over the dose-rate range of 6 to 20 cGy/h. An inverse dose-rate effect was observed for both radionuclides, with its onset occurring at a dose rate of about 20–30 cGy/h. The average RBE of ¹⁰³Pd relative to ¹²⁵I was determined to be 1.45 ± 0.07, 1.41 ± 0.07, 0.70 ± 0.07 and 1.49 ± 0.07 at dose rates of 6.9, 12.6, 19.0 and 26.7 cGy/h, respectively. Because ¹⁰³Pd implants are generally prescribed at a higher initial dose rate (21 cGy/h) than the corresponding ¹²⁵I implants (7 cGy/h), the effects of both dose rate and photon energy on biological response must be considered together. For the CCL-16 cells, the RBE of ¹⁰³Pd at 19.0 cGy/h relative to that of ¹²⁵I at 6.9 cGy/h was estimated to be 2.3 ± 0.5.

Gomolka, M., Rössler, U., Hornhardt, S., Walsh, L., Panzer, W. and Schmid, E. Measurement of the Initial Levels of DNA Damage in Human Lymphocytes Induced by 29 kV X Rays (Mammography X Rays) Relative to 220 kV X Rays and γ Rays. 163, 510–519 (2005).

Experiments using the alkaline comet assay, which measures all single-strand breaks regardless of their origin, were performed to evaluate the biological effectiveness of photons with different energies in causing these breaks. The aim was to measure human lymphocytes directly for DNA damage and subsequent repair kinetics induced by mammography 29 kV X rays relative to 220 kV X rays, ¹³⁷Cs γ rays and ⁶⁰Co γ rays. The level of DNA damage, predominantly due to single-strand breaks, was computed as the Olive tail moment or percentage DNA in the tail for different air kerma doses (0.5, 0.75, 1, 1.5, 2 and 3 Gy). Fifty cells were analyzed per slide with a semiautomatic imaging system. Data from five independent experiments were transformed to natural logarithms and fitted using a multiple linear regression analysis. Irradiations with the different photon energies were performed simultaneously for each experiment to minimize interexperimental variation. Blood from only one male and one female was used. The interexperimental variation and the influence of donor gender were negligible. In addition, repair kinetics and residual DNA damage after exposure to a dose of 3 Gy were evaluated in three independent experiments for different repair times (10, 20, 30 and 60 min). Data for the fraction of remaining damage were fitted to the simple function F_d = A/(t A), where F_d is the fraction of remaining damage, t is the time allowed for repair, and A (the only fit parameter) is the repair half-time. It was found that the comet assay data did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiation types, 220 kV X rays and the γ rays of ¹³⁷Cs and ⁶⁰Co, either for the total dose range or in the low-dose range. These results are, with some restrictions, consistent with physical examinations and predictions concerning, for example, the assessment of the possible difference in effectiveness in causing strand breaks between mammography X rays and conventional (150–250 kV) X rays, indicating that differences in biological effects must arise through downstream processing of the damage.

Yokota, Y., Shikazono, N., Tanaka, A., Hase, Y., Funayama, T., Wada, S. and Inoue, M. Comparative Radiation Tolerance Based on the Induction of DNA Double-Strand Breaks in Tobacco BY-2 Cells and CHO-K1 Cells Irradiated with Gamma Rays. Radiat. Res. 163, 520–525 (2005).

Higher plants are generally more tolerant to ionizing radiation than mammals. To explore the radiation tolerance of higher plants, the induction of DNA double-strand breaks (DSBs) by γ rays was investigated in tobacco BY-2 cells and compared with that in Chinese hamster ovary (CHO)-K1 cells as a reference. This is the first examination of radiation-induced DSBs in a higher plant cell. The resulting DNA fragments were separated by pulsed-field gel electrophoresis and stained with SYBR Green I. The initial yield of DSBs was then quantified from the fraction of DNA fragments shorter than 1.6 Mbp based on the assumption of random distribution of DSBs. The DSB yield in tobacco BY-2 cells (2.0 ± 0.1 DSBs Gbp⁻¹ Gy⁻¹) was only one-third of that in CHO-K1 cells. Furthermore, the calculated number of DSBs per diploid cell irradiated with γ rays at the mean lethal dose was five times greater in BY-2 cells (263 ± 13) than in CHO-K1 cells. These results suggest that the radiation tolerance of BY-2 cells appears to be due not only to a lower induction of DNA damage but also to a more efficient repair of the induced DNA damage.

Rydberg, B., Cooper, B., Cooper, P. K., Holley, W. R. and Chatterjee, A. Dose-Dependent Misrejoining of Radiation-Induced DNA Double-Strand Breaks in Human Fibroblasts: Experimental and Theoretical Study for High- and Low-LET Radiation. Radiat. Res. 163, 526–534 (2005).

Misrejoining of DNA double-strand breaks (DSBs) was measured in human primary fibroblasts after exposure to X rays and high-LET particles (helium, nitrogen and iron) in the dose range 10–80 Gy. To measure joining of wrong DNA ends, the integrity of a 3.2-Mbp restriction fragment was analyzed directly after exposure and after 16 h of repair incubation. It was found that the misrejoining frequency for X rays was nonlinearly related to dose, with less probability of misrejoining at low doses than at high doses. The dose dependence for the high-LET particles, on the other hand, was closer to being linear, with misrejoining frequencies higher than for X rays, particularly at the lower doses. These experimental results were simulated with a Monte Carlo approach that includes a cell nucleus model with all 46 chromosomes present, combined with realistic track structure simulations to calculate the geometrical positions of all DSBs induced for each dose. The model assumes that the main determinant for misrejoining probability is the distance between two simultaneously present DSBs. With a Gaussian interaction probability function with distance, it was found that the data for both low- and high-LET radiation could be fitted with an interaction distance (sigma of the Gaussian curve) of 0.25 μm. This is half the distance previously found to best fit chromosomal aberration data in human lymphocytes using the same methods (Holley et al., Radiat. Res. 158, 568–580, 2002). The discrepancy may indicate inadequacies in the chromosome model, for example insufficient chromosomal overlap, but may also be partly due to differences between fibroblasts and lymphocytes.

Gouk, S-S., Kocherginsky, N. M., Kostetski, Y. Y., Moser, H. O., Yang, P., Lim, T-M. and Sun, W. Q. Synchrotron Radiation-Induced Formation and Reactions of Free Radicals in Human Acellular Dermal Matrix. Radiat. Res. 163, 535–543 (2005).

The present work characterizes the formation of free radicals in an implantable human acellular dermal tissue (Alloderm®, LifeCell Corp., Branchburg, NJ) upon irradiation. The tissue was preserved in a vitreous carbohydrate matrix by freeze-drying. Freeze-dried samples were irradiated using a synchrotron light source, and free radicals generated were investigated using the electron paramagnetic resonance (EPR) technique. At least two free radical populations, with g factors of 1.993 (∼43%) and 2.002 (∼57%), respectively, were identified in the irradiated tissue. The transformation (reaction) kinetics of free radicals produced was investigated in the presence of nitrogen, oxygen and moisture. The reaction kinetics of free radicals was extremely slow in the nitrogen environment. The presence of oxygen and moisture greatly accelerated free radical reactions in the tissue matrix. The reaction of free radicals could not be described by traditional reaction kinetics. A dispersive kinetics model and a diffusion model were developed to analyze the reaction kinetics in the present study. The dispersive model took into consideration molecular mobility and dispersivity of free radicals in the heterogeneous tissue material. The diffusion model described the radical reaction kinetics as two parallel and simultaneous processes: a first-order fast kinetics mainly on tissue surface and a diffusion-limited slow kinetics in deeper layers of the tissue matrix. Both models described quantitative experimental data well. Further investigation is needed to verify whether any of these two models or concepts describes the inherent radical reaction kinetics in the solid tissue matrix.

Yuan, H., Goetz, D. J., Gaber, M. W., Issekutz, A. C., Merchant, T. E. and Kiani, M. F. Radiation-Induced Up-regulation of Adhesion Molecules in Brain Microvasculature and their Modulation by Dexamethasone. Radiat. Res. 163, 544– 551 (2005).

Little is known about the time course and magnitude of the up-regulation of endothelial cell adhesion molecules (ECAMs) in irradiated brain vasculature and the mechanisms by which dexamethasone modulates this up-regulation. We used antibody-conjugated microspheres and a rat closed cranial window model to determine the time course of functional up-regulation of radiation (20 Gy)-induced ICAM1, E-selectin and P-selectin in the pial vasculature of the rat brain and to determine the relationship between suppression of inflammation by dexamethasone and the expression of these ECAMs. The results indicate that ICAM1, E-selectin and P-selectin were up-regulated to a functional level in the microvasculature with distinct time-course patterns. The number of adherent anti-E-selectin and anti-P-selectin microspheres was 5– 12 times greater than that of IgG microspheres 3–6 h postirradiation, and their expression returned to normal at 48 h. The number of adherent anti-ICAM1 microspheres was five and nine times greater than that of IgG at 24 and 48 h, respectively, and returned to baseline by 7 days. Dexamethasone significantly reduced the number of adhering leukocytes and the number of adhering anti-ICAM1, anti-E-selectin and anti-P-selectin microspheres to background levels. Our findings partially identify a key sequence in radiation-induced inflammatory response and provide a potential means to limit radiation-induced inflammatory responses and their potential side effects in the brain.

Ellender, M., Harrison, J. D., Edwards, A. A., Bouffler, S. D. and Cox, R. Direct Single Gene Mutational Events Account for Radiation-Induced Intestinal Adenoma Yields in Apc^Min/ Mice. Radiat. Res. 163, 552–556 (2005).

Data on the induction of small intestinal tumors, predominantly adenomas, by X radiation in Apc^Min/ mice are reported. Comparison of these incidences with estimates of radiation-induced direct single gene mutation frequencies taken from the literature support the hypothesis that direct mutational loss of Apc is the sole requirement for initiation of adenoma. Furthermore, estimates of radiation-induced initiation of adenoma per target stem cell in this animal model are similar to or less than radiation-induced direct somatic gene mutation frequencies. Therefore, while the data reported here do not preclude a role for genomic instability in tumor progression, it is not necessary in this model to postulate the involvement of radiation-induced transmissible genomic instability in initiation of intestinal adenoma.

Bertho, J-M., Prat, M., Frick, J., Demarquay, C., Gaugler, M-H., Dudoignon, N., Clairand, I., Chapel, A., Gorin, N-C., Thierry, D. and Gourmelon, P. Application of Autologous Hematopoietic Cell Therapy to a Nonhuman Primate Model of Heterogeneous High-Dose Irradiation. Radiat. Res. 163, 557– 570 (2005).

We developed a model of heterogeneous irradiation in a nonhuman primate to test the feasibility of autologous hematopoietic cell therapy for the treatment of radiation accident victims. Animals were irradiated either with 8 Gy to the body with the right arm shielded to obtain 3.4 Gy irradiation or with 10 Gy total body and 4.4 Gy to the arm. Bone marrow mononuclear cells were harvested either before irradiation or after irradiation from an underexposed area of the arm and were expanded in previously defined culture conditions. We showed that hematopoietic cells harvested after irradiation were able to expand and to engraft when reinjected 7 days after irradiation. Recovery was observed in all 8-Gy-irradiated animals, and evidence for a partial recovery was observed in 10-Gy-irradiated animals. However, in 10-Gy-irradiated animals, digestive disease was observed from day 16 and resulted in the death of two animals. Immunohistological examinations showed damage to the intestine, lungs, liver and kidneys and suggested radiation damage to endothelial cells. Overall, our results provide evidence that such an in vivo model of heterogeneous irradiation may be representative of accidental radiation exposures and may help to define the efficacy of therapeutic interventions such as autologous cell therapy in radiation accident victims.

Lubin, J. H., Wang, Z. Y., Wang, L. D., Boice, J. D., Jr., Cui, H. X., Zhang, S. R., Conrath, S., Xia, Y., Shang, B., Cao, J. S. and Kleinerman, R. A. Adjusting Lung Cancer Risks for Temporal and Spatial Variations in Radon Concentration in Dwellings in Gansu Province, China. Radiat. Res. 163, 571– 579 (2005).

Our recent study in Gansu Province, China reported an increasing risk of lung cancer with increasing residential radon concentration that was consistent with previous pooled analyses and with meta-analyses of other residential studies (Wang et al., Am. J. Epidemiol. 155, 554–564, 2002). Dosimetry used current radon measurements (1-year track-etch detectors) in homes to characterize concentrations for the previous 30 years, resulting in uncertainties in exposure and possibly reduced estimates of disease risk. We conducted a 3-year substudy in 55 houses to model the temporal and spatial variability in radon levels and to adjust estimates of radon risk. Temporal variation represented the single largest source of uncertainty, suggesting the usefulness of multi-year measurements to assess this variation; however, substantial residual variation remained unexplained. The uncertainty adjustment increased estimates of the excess odds ratio by 50–100%, suggesting that residential radon studies using similar dosimetry may also underestimate radon effects. These results have important implications for risk assessment.

Bit-Babik, G., Guy, A. W., Chou, C-K., Faraone, A., Kanda, M., Gessner, A., Wang, J. and Fujiwara, O. Simulation of Exposure and SAR Estimation for Adult and Child Heads Exposed to Radiofrequency Energy from Portable Communication Devices. Radiat. Res. 163, 580–590 (2005).

The level and distribution of radiofrequency energy absorbed in a child's head during the use of a mobile phone compared to those in an adult head has been a controversial issue in recent years. It has been suggested that existing methods that are used to determine specific absorption rate (SAR) and assess compliance with exposure standards using an adult head model may not adequately account for potentially higher levels of exposure in children due to their smaller head size. The present study incorporates FDTD computations of locally averaged SAR in two different anatomically correct adult and child head models using the IEEE standard (Std. C95.3-2002) SAR averaging algorithm. The child head models were obtained by linear scaling of the adult head model to replicate the conditions of previous studies reported in the literature and also by transforming the different adult head models based on data on the external shapes of children's heads. The tissue properties of the adult and corresponding child head models were kept the same. In addition, modeling and experimental measurements were made using three spheres filled with a tissue-equivalent mixture to approximate heads of increasing size. Results show that the peak local average SAR over 1 g and 10 g of tissue and the electromagnetic energy penetration depths are about the same in all of the head models under the same exposure conditions. When making interlaboratory comparisons, the model and the SAR averaging algorithm used must be standardized to minimize controversy.

Shukla, L. I., Pazdro, R., Becker, D. and Sevilla, M. D. Sugar Radicals in DNA: Isolation of Neutral Radicals in Gamma-Irradiated DNA by Hole and Electron Scavenging. Radiat. Res. 163, 591–602 (2005).

In this investigation of the radical formation and the reaction of radicals in γ-irradiated DNA, we report the isolation of putative neutral radicals by the scavenging of holes by Fe(CN)₆⁴⁻ and of electrons by Fe(CN)₆³⁻. Experiments are performed under conditions that emphasize direct and quasi-direct effects (collectively called direct-type effects.) Samples containing Fe(CN)₆⁴⁻ show effective scavenging of holes and the ESR spectra obtained arise principally from DNA anion radicals and neutral radicals. On the other hand, for samples containing Fe(CN)₆³⁻, electron scavenging is highly efficient, and the resulting spectra arise principally from guanine cation radicals and neutral radicals. When both Fe(CN)₆⁴⁻ and Fe(CN)₆³⁻ are present, a near complete scavenging of cation radicals and anion radicals is observed at 77 K, and the ESR spectra that result originate predominantly with neutral radicals which are assigned predominantly to radicals on the sugar phosphate backbone. A notable finding is the presence of spectral components that indicate the formation, through the rupture of the C3′–O bond, of a neutral deoxyribose radical; a concurrent strand break must accompany formation of this radical. This radical was previously reported in argon-ion-irradiated DNA and now, for the first time, is reported in DNA irradiated with low-LET radiation.