Zhong, N., Morris, G. M., Bacarian, T., Rosen, E. M. and Dilmanian F. A. Response of Rat Skin to High-Dose Unidirectional X-Ray Microbeams: A Histological Study. Radiat. Res. 160, 133–142 (2003).

There is growing interest in evaluating microbeam radiation therapy as a potential clinical modality. Microbeam radiation therapy uses arrays of parallel, microscopically thin (<100 μm) planes of synchrotron-generated X rays (microplanar beams, or microbeams). Due to the relatively low beam energies involved in microbeam radiation therapy (a median beam energy of 120 keV was used in the present study), the dose penetration of microbeams in tissue is lower than that used in conventional radiotherapy. This lower energy necessitates using a significantly elevated dose to the skin's surface during clinical microbeam therapy to ensure an adequate dose distribution in the target tumor. The findings of the present study, using a rat skin model, indicated that the skin had an extremely high tolerance to microbeam radiation at doses considerably in excess of those that were therapeutically effective in preclinical studies. A histological study was undertaken to evaluate the biological mechanisms underlying this high tolerance. The irradiation configuration employed single-exposure, unidirectional microbeams 90 μm wide, with 300 μm beam spacing on-center. The in-beam skin-surface absorbed doses were in the range 835–1335 Gy. Monte Carlo simulations of the dose distribution indicated that the “valley” dose, i.e. the radiation leakage between adjacent microbeams, was about 2.5% of the in-beam dose. The high tolerance of the rats' skin to microbeams and the rapid regeneration of the damaged segments of skin were attributed to the surviving clonogenic cells situated between the adjacent microplanar beams. In the epidermis, clonogenic cells in the hair follicular epithelium appeared to play a key role in the regeneration process.

La Regina, M., Moros, E. G., Pickard, W. F., Straube, W. L., Baty, J. and Roti Roti, J. L. The Effect of Chronic Exposure to 835.62 MHz FDMA or 847.74 MHz CDMA Radiofrequency Radiation on the Incidence of Spontaneous Tumors in Rats. Radiat. Res. 160, 143–151 (2003).

This study was designed to determine whether chronic exposure to radiofrequency (RF) radiation from cellular phones increased the incidence of spontaneous tumors in F344 rats. Eighty male and 80 female rats were randomly placed in each of three irradiation groups. The sham group received no irradiation; the Frequency Division Multiple Access (FDMA) group was exposed to 835.62 MHz FDMA RF radiation; and the Code Division Multiple Access (CDMA) group was exposed to 847.74 MHz CDMA RF radiation. Rats were irradiated 4 h per day, 5 days per week over 2 years. The nominal time-averaged specific absorption rate (SAR) in the brain for the irradiated animals was 0.85 ± 0.34 W/kg (mean ± SD) per time-averaged watt of antenna power. Antennas were driven with a time-averaged power of 1.50 ± 0.25 W (range). That is, the nominal time-averaged brain SAR was 1.3 ± 0.5 W/kg (mean ± SD). This number was an average from several measurement locations inside the brain, and it takes into account changes in animal weight and head position during irradiation. All major organs were evaluated grossly and histologically. The number of tumors, tumor types and incidence of hyperplasia for each organ were recorded. There were no significant differences among final body weights or survival days for either males or females in any group. No significant differences were found between treated and sham-exposed animals for any tumor in any organ. We conclude that chronic exposure to 835.62 MHz FDMA or 847.74 MHz CDMA RF radiation had no significant effect on the incidence of spontaneous tumors in F344 rats.

Zeni, O., Schiavoni, A. S., Sannino, A., Antolini, A., Forigo, D., Bersani, F. and Scarfì, M. R. Lack of Genotoxic Effects (Micronucleus Induction) in Human Lymphocytes Exposed In Vitro to 900 MHz Electromagnetic Fields. Radiat. Res. 160, 152–158 (2003).

In the present study, we investigated the induction of genotoxic effects in human peripheral blood lymphocytes after exposure to electromagnetic fields used in mobile communication systems (frequency 900 MHz). For this purpose, the incidence of micronuclei was evaluated by applying the cytokinesis-block micronucleus assay. Cytotoxicity was also investigated using the cytokinesis-block proliferation index. The experiments were performed on peripheral blood from 20 healthy donors, and several conditions were tested by varying the duration of exposure, the specific absorption rate (SAR), and the signal [continuous-wave (CW) or GSM (Global System of Mobile Communication) modulated signal]. The following exposures were carried out: (1) CW intermittent exposure (SAR = 1.6 W/kg) for 6 min followed by a 3-h pause (14 on/off cycles); (2) GSM signal, intermittent exposure as described in (1); (3) GSM signal, intermittent exposure as described in (1) 24 h before stimulation with phytohemagglutinin (8 on/off cycles); (4) GSM signal, intermittent exposure (SAR = 0.2 W/kg) 1 h per day for 3 days. The SARs were estimated numerically. No statistically significant differences were detected in any case in terms of either micronucleus frequency or cell cycle kinetics.

Carnes, B. A., Grahn, D. and Hoel, D. Mortality of Atomic Bomb Survivors Predicted from Laboratory Animals. Radiat. Res. 160, 159–167 (2003).

Exposure, pathology and mortality data for mice, dogs and humans were examined to determine whether accurate interspecies predictions of radiation-induced mortality could be achieved. The analyses revealed that (1) days of life lost per unit dose can be estimated for a species even without information on radiation effects in that species, and (2) accurate predictions of age-specific radiation-induced mortality in beagles and the atomic bomb survivors can be obtained from a dose–response model for comparably exposed mice. These findings illustrate the value of comparative mortality analyses and the relevance of animal data to the study of human health effects.

Zdravkova, M., Crokart, N., Trompier, F., Asselineau, B., Gallez, B., Gaillard-Lecanu, E. and Debuyst, R. Retrospective Dosimetry after Criticality Accidents Using Low-Frequency EPR: A Study of Whole Human Teeth Irradiated in a Mixed Neutron and Gamma-Radiation Field. Radiat. Res. 160, 168–173 (2003).

In the context of accidental or intentional radiation exposures (nuclear terrorism), it is essential to separate rapidly those individuals with substantial exposures from those with exposures that do not constitute an immediate threat to health. Low-frequency electron paramagnetic resonance (EPR) spectroscopy provides the potential advantage of making accurate and sensitive measurements of absorbed radiation dose in teeth without removing the teeth from the potential victims. Up to now, most studies focused on the dose–response curves obtained for γ radiation. In radiation accidents, however, the contribution of neutrons to the total radiation dose should not be neglected. To determine how neutrons contribute to the apparent dose estimated by EPR dosimetry, extracted whole human teeth were irradiated at the SILENE reactor in a mixed neutron and γ-radiation field simulating criticality accidents. The teeth were irradiated in free air as well as in a paraffin head phantom. Lead screens were also used to eliminate to a large extent the contribution of the γ radiation to the dose received by the teeth. The EPR signals, obtained with a low-frequency (1.2 GHz) spectrometer, were compared to dosimetry measurements at the same location. The contribution of neutrons to the EPR dosimetric signal was negligible in the range of 0 to 10 Gy and was rather small (neutron/γ-ray sensitivity in the range 0–0.2) at higher doses. This indicates that the method essentially provides information on the dose received from the γ-ray component of the radiation.

Becker, D., Bryant-Friedrich, A., Trzasko, C. and Sevilla, M. D. Electron Spin Resonance Study of DNA Irradiated with an Argon-Ion Beam: Evidence for Formation of Sugar Phosphate Backbone Radicals. Radiat. Res. 160, 174–185 (2003).

In this study, the effects of high-LET radiation on DNA were investigated and compared with the effects of γ radiation. Hydrated DNA samples at 77 K were irradiated with argon-ion beams (³⁶Ar or ⁴⁰Ar beam at energies between 60 and 100 MeV/nucleon). The individual free radicals formed were identified and their yields were investigated by electron spin resonance spectroscopy. Argon-ion irradiation resulted in lower yields of base ion radicals and higher yields of neutral radicals than γ irradiation. A hitherto unknown species was assigned to the radical formed by C–O bond rupture at the deoxyribose C3′, resulting in a sugar carbon-centered radical. A previously characterized phosphorus-centered radical was also found. The formation of each of these species was accompanied by an immediate strand break. G values, k values, and analyses for the individual yields of neutral radicals and ion radical composition for argon-ion-irradiated hydrated DNA are reported and compared to those found previously for γ-irradiated DNA. The lower G values and k values for ion radicals and the higher fraction of neutral radicals found for argon-ion-irradiated DNA are attributed to differences in track structure inherent in the two radiations.

Malinen, E. and Sagstuen, E. Radical Formation in Pyrimidine Bases after X, Proton and α-Particle Irradiation. Radiat. Res. 160, 186–197 (2003).

Single crystals of anhydrous thymine (Ta) and cytosine monohydrate (Cm) were irradiated at room temperature using X rays, 20 MeV protons and 35 MeV α particles, and the relative distributions of the various radiation-induced stable radicals in the crystals were investigated. These two crystal systems were chosen because of systematic differences in their molecular packing and hydrogen-bonding network. The radicals stabilized in these systems have previously been identified and analyzed by several authors. Experimental EPR spectra could thus be reconstructed based on simulated benchmark spectra using a fitting procedure, yielding the relative amounts of the different radical species. It was found that the relative amounts of a given radical species varied with the type of radiation used, with differences being most prominent between α particles and protons or X rays. In Ta, an increased production of hydrogen addition or abstraction radicals was found after exposure to α particles. These radicals are believed to be formed predominantly from superexcited states, resulting from the higher density of ionizations along the track of the α particle. A corresponding reduction in radicals derived from proton transfer reactions was observed in comparison with exposure to protons or X rays. In Cm, the differences were smaller than for Ta. This is probably due to differences in the arrangement of the crystal lattice between the two systems, with Cm having an extended hydrogen-bonding network promoting fast proton transfers after ionization. Most interesting, however, was the observation that more than 50% of the radical population in Cm not could be accounted for by known radical species. The population with an unknown origin was represented with a broad singlet, having a line width of 1.5 mT and a g value of 2.0045, in the spectral reconstruction procedure.

Zohar-Perez, C., Chernin, L., Chet, I. and Nussinovitch, A. Structure of Dried Cellular Alginate Matrix Containing Fillers Provides Extra Protection for Microorganisms against UVC Radiation. Radiat. Res. 160, 198–204 (2003).

Soil microorganisms in general and biocontrol agents in particular are very sensitive to UV light. The packaging of biocontrol microorganisms into cellular solids has been developed as a means of reducing loss caused by exposure to environmental UV radiation. The bacterial and fungal biocontrol agents Pantoea agglomerans and Trichoderma harzianum were immobilized in freeze-dried alginate beads containing fillers and subjected to 254 nm UV radiation (UVC). Immobilization of cells in freeze-dried alginate-glycerol beads resulted in greater survival after UV irradiation than for a free cell suspension. Adding chitin, bentonite or kaolin as fillers to the alginate-glycerol formulation significantly increased bacterial survival. Immobilization in alginate-glycerol-kaolin beads resulted in the highest levels of survival. The transmissive properties of the dried hydrocolloid cellular solid had a major influence on the amount of protection by the cell carrier. Dried alginate matrix (control) transmitted an average of 7.2% of the radiation. Filler incorporation into the matrix significantly reduced UV transmission: Alginate with kaolin, bentonite and chitin transmitted an average of 0.15, 0.38 and 3.4% of the radiation, respectively. In addition, the filler inclusion had a considerable effect on the bead's average wall thickness, resulting in a ∼1.5- to threefold increase relative to beads based solely on alginate. These results suggest that the degree of protection of entrapped microorganisms against UVC radiation is determined by the UV-transmission properties of the dried matrix and the cellular solid's structure. It is concluded that for maximum protection against UV-radiation-induced cell loss, biocontrol microorganisms should be immobilized in alginate-glycerol beads containing kaolin.

Larsson, C. L. and Ng, C. E. p21 ^/ (CDKN1A ^/ ) and p21^–/– (CDKN1A^–/–) Human Colorectal Carcinoma Cells Display Equivalent Amounts of Thermal Radiosensitization. Radiat. Res. 160, 205–209 (2003).

The mechanism of thermal radiosensitization is related to the inhibition of repair of radiation-induced DNA damage by heat. Due to the interaction of the gene p21/WAF1/CIP1 (now known as CDKN1A) with a variety of DNA repair proteins, its involvement in thermal radiosensitization was investigated. Two isogenetic human colorectal cancer cell lines with wild-type TP53 status were used. The 80S4 cell line was deficient in CDKN1A and the HCT116 cells were CDKN1A proficient. Both cell lines were significantly more sensitive to 44°C than 42°C heating (P < 0.01), and both cell lines expressed thermotolerance for heating times longer than about 2 h at the lower temperature. There were no significant differences in the X-radiation response of the two cell lines. Further, the two cell lines displayed similar cell survival levels after hyperthermia given before or after X radiation for both hyperthermia temperatures. Comparison of thermal enhancement ratios confirmed that there was no difference in the amount of thermal radiosensitization induced in the two cell lines. The induction and subsequent repair of DNA double-strand breaks, as measured by clamped homogeneous gel electrophoresis, was also the same in both cell lines. These findings strongly suggest that the gene CDKN1A does not play an important role in the expression of thermal radiosensitization.

Kashiwakura, I., Inanami, O., Takahashi, K., Takahashi, T. A., Kuwabara, M. and Takagi, Y. Protective Effects of Thrombopoietin and Stem Cell Factor on X-Irradiated CD34 Megakaryocytic Progenitor Cells from Human Placental and Umbilical Cord Blood. Radiat. Res. 160, 210–216 (2003).

In previous studies we characterized the radiosensitivity of CFU-megakaryocytes from human placental and umbilical cord blood and the effects of various early-acting cytokines. We found that the maximal clonal growth of CFU-megakaryocytes in vitro and maximal protection against X-ray damage were supported by a combination of thrombopoietin and stem cell factor. However, the mechanism by which the two cytokines exert a synergistic effect remained unclear, so we extended these studies to investigate the radioprotective action of synergistic thrombopoietin and stem cell factor on the survival of X-irradiated CD34 CFU-megakaryocytes. A combination of thrombopoietin and stem cell factor led to activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and to suppression of caspase 3 in X-irradiated CD34 cells. When PD98059 and various synthetic substrates—specific inhibitors of these proteins—were used, the combination had less effect on the clonal growth of X-irradiated CD34 CFU-megakaryocytes. However, the addition of wortmannin, a specific inhibitor of the phosphatidylinositol-3 kinase pathway, did not alter the synergistic action of thrombopoietin plus stem cell factor. We suggest that part of this synergistic effect can be explained by activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and by suppression of the caspase cascade.

Qi, W. and Martinez J. D. Reduction of 14-3-3 Proteins Correlates with Increased Sensitivity to Killing of Human Lung Cancer Cells by Ionizing Radiation. Radiat. Res. 160, 217–223 (2003).

The 14-3-3 proteins have a wide range of ligands and are involved in a variety of biological pathways. Importantly, 14-3-3 proteins are known to be overexpressed in some human lung cancers, suggesting that they may play a role in tumorigenesis. Here we examined 14-3-3 expression in several lung cancer-derived cell lines and found that four of the seven 14-3-3 isoforms, β, ϵ, θ and ζ, were highly expressed in both lung cancer cell lines and normal lung fibroblasts. Two isoforms, σ and γ, were present only at very low levels. Immunoprecipitation data showed 14-3-3ζ could bind to CDC25C in irradiated A549 cells, and suppression of 14-3-3ζ in A549 cells with antisense resulted in a decrease in CDC25C localization in cytoplasm and CDC2 phosphorylation on Tyr15. As a consequence, CDC2 activity remained elevated which resulted in release from radiation-induced G₂/M-phase arrest. Moreover, 16% 14-3-3ζ antisense-transfected cells underwent apoptosis when exposed to 10 Gy ionizing radiation. These data indicate that 14-3-3ζ is involved in G₂ checkpoint activation and that inhibition of 14-3-3 may be a useful approach to sensitize human lung cancers to ionizing radiation.

Kim, K-U., Xiao, J., Ni, H-T., Cho, K. H., Spellman, S. R., Low, W. C. and Hall, W. A. Changes in Expression of Transferrin, Insulin-like Growth Factor 1, and Interleukin 4 Receptors after Irradiation of Cells of Primary Malignant Brain Tumor Cell Lines. Radiat. Res. 160, 224–231 (2003).

Various immunotoxins have been developed for the treatment of cancer. The toxin is internalized by target cells through cell-surface receptors, and it is essential for these receptors to be expressed for the immunotoxin to have specific anti-tumor activity. Radiation therapy is one of the main treatment modalities for primary malignant brain tumors. The purpose of this study was to determine whether radiation influences the expression of cell-surface receptors. Cells of one human medulloblastoma (Daoy) and two glioblastoma (U373-MG and T98-G) cell lines were tested by exposing the cells to a single dose of 5 Gy γ rays. Expression of transferrin receptors, type-1 insulin-like growth factor receptors (IGF1R), and interleukin 4 receptors (IL4R) was measured by flow cytometry analysis on unirradiated cells and on cells 3 to 120 h after irradiation. In Daoy cells, the absolute expression index of transferrin receptors increased during the 24 h after irradiation with the greatest change of 26% above control at 9 h. The absolute expression index of IGF1R increased 26.5% above control at 12 h. The absolute expression index of IL4R decreased 9 h after irradiation. In U373-MG cells the absolute expression index of transferrin receptors increased during the 24 h after irradiation, and the greatest increase was 45% above control at 9 h. The absolute expression index of IGF1R increased during the 12 h after irradiation with a maximum increase of 33% above control at 6 h. The absolute expression index of IL4R decreased with time after irradiation. In T98-G cells, the absolute expression index of both transferrin receptors and IL4R decreased after irradiation. The results suggest that the expression of growth factor receptors on brain tumor cells may be influenced by radiation. The effect of ionizing radiation on receptor expression should be considered when administration of targeted toxin is combined with radiation. Similar studies with other growth factor receptors used in targeted toxin therapy are recommended.

Ding, G-R., Honda, N., Nakahara, T., Tian, F., Yoshida, M., Hirose, H. and Miyakoshi, J. Radiosensitization by Inhibition of IκB-α Phosphorylation in Human Glioma Cells. Radiat. Res. 160, 232–237 (2003).

To assess the role of nuclear factor κB (NFKB) in cellular radiosensitivity, three different IκB-α (also known as NFKBIA) expression plasmids, i.e., S-IκB (mutations at ^{32, 36}Ser), Y-IκB (a mutation at ⁴²Tyr), and SY-IκB, were constructed and introduced into human brain tumor M054 cells. The clones were named as M054-S8, M054-Y2 and M054-SY4, respectively. Compared to the parental cell line, M054-S8 and M054-Y2 cells were more sensitive to X rays while M054-SY4 cells exhibited the greatest sensitivity. After treatment with N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor, the X-ray sensitivity of M054-S8 and M054-SY4 cells did not change, while that of M054-Y2 cells and the parental cells was enhanced. An increase in X-ray sensitivity accompanied by a decrease in translocation of NFKB to the nucleus in parental cells was observed after treatment with pervanadate, an inhibitor of tyrosine phosphatase, as well as in M054-S8 and M054-SY4 cells. Repair of potentially lethal damage (PLD) was observed in the parental cells but not in the clones. Four hours after irradiation (8 Gy), the expression of TP53 and phospho-p53 (¹⁵Ser) was induced in the parental cells but not in M054-S8, M054-Y2 or M054-SY4 cells. Our data suggest that inhibition of IκB-α phosphorylation at serine or tyrosine acts independently in sensitizing cells to X rays. NFKB may play a role in determining radiosensitivity and PLD repair in malignant glioma cells; TP53 may also be involved.

Gault, N. and Lefaix, J-L. Infrared Microspectroscopic Characteristics of Radiation-Induced Apoptosis in Human Lymphocytes. Radiat. Res. 160, 238–250 (2003).

Infrared microspectroscopic characterization of radiation-induced apoptosis was used as a new analytical tool to study the kinetics of apoptosis in human peripheral blood lymphocytes at the molecular level. This vibrational technique, which has already been used to investigate biomolecules in normal and tumor cells, allows the simultaneous detection of the biochemical changes in the various subcellular compartments. Normal circulating lymphocytes from five healthy human donors were given a single dose of 6 Gy (⁶⁰Co) and deposited on ZnS windows for infrared spectral acquisition 1, 2 and 4 days after irradiation. Apoptosis was assessed simultaneously by flow cytometry analysis of lymphocytes displaying annexin V-positive staining, and by detection of the DNA laddering that is characteristic of apoptosis. The flow cytometry study showed that about 80% of sham-irradiated lymphocytes were annexin V^neg/PI^neg at 1, 2 and 4 days. One day after irradiation, 46% of irradiated lymphocytes were annexin V^neg/PI^neg, 48% were annexin V^pos/PI^neg, 5% were annexin V^pos/PI^pos, and 1% were annexin V^neg/PI^pos. These mean percentages were respectively 31, 59, 9 and 1 at day 2 and 23, 36, 30, and 11 at day 4. Irradiated lymphocytes presented a DNA laddering pattern characteristic of apoptosis from day 1 after irradiation. In the infrared spectra of irradiated lymphocytes, qualitative and quantitative changes were observed from days 1 and 2, respectively. In the range of 960–1245 cm⁻¹ mainly attributed to nucleic acids, changes corresponding to conformational changes in DNA were associated with a decrease in the amount of detectable DNA. Conformational changes were also observed in secondary protein structures, in particular an increase in the amount of β structures. These DNA and protein changes were associated with an increase in the detectable amount of lipids at day 4 after irradiation. These results showed that DNA is probably the first cellular target of radiation-induced apoptosis, which, however, also requires conformational changes and synthesis of cell proteins. Our results are in agreement with biochemical and morphological data on radiation-induced apoptosis of normal human circulating lymphocytes, and they demonstrate that infrared microspectroscopy may be useful for assessing the process of apoptosis at the molecular level.

Koc, M., Taysi, S., Buyukokuroglu, M. E. and Bakan, N. The Effect of Melatonin against Oxidative Damage during Total-Body Irradiation in Rats. Radiat. Res. 160, 251–255 (2003).

Melatonin has been reported to participate in the regulation of a number of important physiological and pathological processes. Melatonin, which is a powerful endogenous antioxidant, may play a role in the prevention of oxidative damage. The aim of this study was to investigate the effect of pretreatment with melatonin (5 mg kg^–1 and 10 mg kg^–1) on γ-radiation-induced oxidative damage in plasma and erythrocytes after total-body irradiation with a single dose of 5 Gy. Total-body irradiation resulted in a significant increase in plasma and erythrocyte MDA levels. Melatonin alone increased the levels of SOD and GSH-Px. Erythrocyte and plasma MDA levels in irradiated rats that were pretreated with melatonin (5 or 10 mg kg^–1) were significantly lower than those in rats that were not pretreated. There was no significant difference between the effects of 5 and 10 mg kg^–1 on plasma MDA activities and CAT activities. However, erythrocyte MDA levels showed a dose-dependent decrease, while GSH-Px activities increased with dose. Our study suggests that melatonin administered prior to irradiation may protect against the damage produced by radiation by the up-regulation of antioxidant enzymes and by scavenging free radicals generated by ionizing radiation.