Olsson, S., Sagstuen, E., Bonora, M. and Lund, A. EPR Dosimetric Properties of 2-Methylalanine: EPR, ENDOR and FT-EPR Investigations. Radiat. Res. 157, 113–121 (2002).

To find an EPR dosimeter material that is sensitive enough for clinical use, the substance 2-methylalanine (2MA) with the chemical structure (CH₃)₂C(NH₃ )COO⁻ was tested for its sensitivity to ionizing radiation, dose response, and radical stability over time. At equal and moderate settings of microwave power and modulation amplitude, 2MA was found to be 70% more sensitive than l-α-alanine, which is the most common EPR dosimeter material today. The dose response is linear, at least in the dose range of interest (0.5–100 Gy), and the time-dependent variations in signal intensity are very small and may be corrected for easily. The energy dependence of the stopping power and energy absorption was calculated and was found to be similar to that of alanine. The dependence of the signal intensity on microwave power and modulation amplitude was investigated, and the optimal settings were found to be 25 mW (Bruker ER 4102ST) and 12 gauss, respectively. Single crystals of 2MA were analyzed using ENDOR and ENDOR-induced EPR to identify the radiation-induced radicals that formed. Only one radical, in which the amino group is detached from the original molecule, was identified. This radical is obviously dominating and is apparently the only one relevant for dosimetry purposes. The complete set of coupling parameters for three hyperfine couplings is reported. The power saturation properties and spectral line width are ruled by the relaxation times T₁ and T₂. To determine the relaxation times of 2MA, pulsed EPR experiments were performed on single crystals. Two different values of T₁ were obtained, one in the range 1–3 , shown to be of importance for the dosimetry properties, and another that is strongly anisotropic with a value between 10 and 35 that does not seem to affect the saturation behavior. T₂ was estimated to be of the order of 200–300 ns.

Berovic, N., Pratontep, S., Bryant, A., Montouris, A. and Green, R. G. The Kinetics of Radiation Damage to the Protein Luciferase and Recovery of Enzyme Activity after Irradiation. Radiat. Res. 157, 122–127 (2002).

Experimental observations are reported which follow the bioluminescence intensity of luciferase during irradiation by a 5 MeV proton beam. Bioluminescence is a measure of the protein enzyme activity and provides an assay of the enzyme rate of reaction in real time. Transient responses after a pulse of protons show recovery of the reaction rate with two time constants of 0.3 s⁻¹ and 0.01 s⁻¹. Changes in the reaction rate are due to radiation damage to the active form of the protein luciferase. Quantitative analysis of the radiation damage and recovery of the protein shows that products of the radiolysis of water play major part in the process of enzyme damage at room temperature. A few minutes after the pulse of protons, most of the enzyme activity has recovered. We attribute the fast recovery to the removal of charged ions, while the slow recovery involves refolding of denatured protein.

Fayard, B., Touati, A., Abel, F., Hervé du Penhoat, M. A., Despiney-Bailly, I., Gobert, F., Ricoul, M., L'Hoir, A., Politis, M. F., Hill, M. A., Stevens, D. L., Sabatier, L., Sage, E., Goodhead, D. T. and Chetioui, A. Cell Inactivation and Double-Strand Breaks: The Role of Core Ionizations, as Probed by Ultrasoft X Rays. Radiat. Res. 157, 128–140 (2002).

The large RBE (∼7) measured for the killing of Chinese hamster V79 cells by 340 eV ultrasoft X rays, which preferentially ionize the K shell of carbon atoms (Hervé du Penhoat et al., Radiat. Res. 151, 649–658, 1999), was used to investigate the location of sensitive sites for cell inactivation and the physical modes of action of radiation. The enhancement of the RBE above the carbon K-shell edge either may indicate a high intrinsic efficiency of carbon K-shell ionizations (due, for example, to a specific physical or chemical effect) or may be related to the preferential localization of these ionizations on the DNA. The second interpretation would indicate a strong local (within 3 nm) action of K-shell ionizations and consequently the importance of a direct mechanism for radiation lethality (without excluding an action in conjunction with an indirect component). To distinguish between these two hypotheses, the efficiencies of core ionizations in DNA atoms (phosphorus L-shell, carbon K-shell, and oxygen K-shell ionizations) to induce damages were investigated by measuring their capacities to produce DNA double-strand breaks (DSBs). The effect of photoionizations in isolated DNA was studied using pBS plasmids in a partially hydrated state. No enhancement of the efficiency of DSB induction by carbon K-shell ionizations compared to oxygen K-shell ionizations was found, supporting the hypothesis that it is the localization of these carbon K-shell events on DNA which gives to the 340 eV photons their high killing efficiency. In agreement with this interpretation, cell inactivation and DSB induction, which do not appear to be correlated when expressed in terms of yields per unit dose in the sample, exhibit a rather good correlation when expressed in terms of efficiencies per core event in the DNA. These results suggest that core ionizations in DNA, through core-hole relaxation in conjunction with localized effects of spatially correlated secondary and Auger electrons, may be the major critical events for cell inactivation, and that the resulting DSBs (or a constant fraction of these DSBs) may be a major class of unrepairable lesions.

Kiefer, J., Egenolf, R. and Ikpeme, S. Heavy Ion-Induced DNA Double-Strand Breaks in Yeast. Radiat. Res. 157, 141–148 (2002).

Induction of DSBs in the diploid yeast, Saccharomyces cerevisiae, was measured by pulsed-field gel electrophoresis (PFGE) after the cells had been exposed on membrane filters to a variety of energetic heavy ions with values of linear energy transfer (LET) ranging from about 2 to 11,500 keV/μm, ²⁴¹Am α particles, and 80 keV X rays. After irradiation, the cells were lysed, and the chromosomes were separated by PFGE. The gels were stained with ethidium bromide, placed on a UV transilluminator, and analyzed using a computer-coupled camera. The fluorescence intensities of the larger bands were found to decrease exponentially with dose or particle fluence. The slope of this line corresponds to the cross section for at least one double-strand break (DSB), but closely spaced multiple breaks cannot be discriminated. Based on the known size of the native DNA molecules, breakage cross sections per base pair were calculated. They increased with LET until they reached a transient plateau value of about 6 × 10^–7 μm² at about 300–2000 keV/μm; they then rose for the higher LETs, probably reflecting the influence of δ electrons. The relative biological effectiveness for DNA breakage displays a maximum of about 2.5 around 100–200 keV/μm and falls below unity for LET values above 10³ keV/μm. For these yeast cells, comparison of the derived breakage cross sections with the corresponding cross section for inactivation derived from the terminal slope of the survival curves shows a strong linear relationship between these cross sections, extending over several orders of magnitude.

Owen, D. G., McNamee, J. P., Raaphorst, G. P. and Ng, C. E. Potentiation of Cell Killing by Low-Dose-Rate Radiation by Camptothecin is Related to an Increase in the Level of DNA Double-Strand Breaks. Radiat. Res. 157, 149–157 (2002).

We investigated the ability of camptothecin to potentiate cell killing by low-dose-rate irradiation and whether this potentiation was associated with an increase in the level of residual DNA double-strand breaks (DSBs). Human melanoma (Sk-Mel-3) cells, grown to the confluent phase, were treated with low-dose-rate radiation (0.88 cGy/min) alone, camptothecin alone, or concurrent camptothecin and low-dose-rate radiation. Cell survival was determined using a clonogenic assay. The interactions between camptothecin and low-dose-rate radiation were analyzed further using isobolograms. DNA DSBs were determined using the neutral comet assay. We found that 10 and 25 μM camptothecin, but not 1 μM, camptothecin potentiated cell killing significantly relative to that seen with low-dose-rate radiation alone. Unexpectedly, the potentiation of the effects of low-dose-rate radiation by camptothecin was accompanied by large increases in the α parameter of the linear-quadratic fit rather than in the β parameter. This suggests a modification of intrinsic radiosensitivity rather than of repair of sublethal damage. From isobologram analysis, low-dose-rate radiation interacted either additively or supra-additively with 25 or 10 μM camptothecin. Conversely, the interaction of low-dose-rate radiation with 1 μM camptothecin was subadditive. Finally, there were strong correlations (correlation coefficients >0.9) between surviving fraction and either comet tail length or comet tail moment after concurrent treatment with 25 μM camptothecin and low-dose-rate radiation. This suggests that the level of residual DNA DSBs was a good indicator of cell killing after treatment with low-dose-rate radiation plus 25 μM camptothecin.

Boyle, J. M., Spreadborough, A. R., Greaves, M. J., Birch, J. M., Varley, J. M. and Scott, D. Delayed Chromosome Changes in Gamma-Irradiated Normal and Li-Fraumeni Fibroblasts. Radiat. Res. 157, 158–165 (2002).

Knockout mice with only one Trp53 allele ( /– genotype) are highly susceptible to radiation-induced cancers, possibly through numerical chromosome changes. Patients with the Li-Fraumeni syndrome, having heterozygous TP53 germline mutations ( /mut genotype), are also susceptible to spontaneous and radiogenic cancers. We have investigated the susceptibility of six Li-Fraumeni syndrome /mut and six normal fibroblast strains to induced numerical and unstable structural aberrations at six population doublings after exposure to 3 or 6 Gy γ rays. Four of the irradiated Li-Fraumeni syndrome strains showed small increases in both aberration types, similar to those seen in the normal strains. In two irradiated Li-Fraumeni syndrome strains, there were high levels of induced structural changes, and one of these showed a modest increase in hyperploidy. We suggest that enhanced sensitivity to delayed radiation-induced chromosome changes in Li-Fraumeni syndrome cells requires other genetic alterations in addition to TP53 heterozygosity, apparently in contrast to the situation in Trp53 heterozygous null mice. If such additional alterations occur in vivo in Li-Fraumeni syndrome patients, they may predispose them to radiogenic cancers, mainly through enhanced structural rather than numerical chromosome changes. Our findings raise questions about the validity of quantitative extrapolation of cytogenetic data from Trp53-defective mice to radiogenic cancer risk in humans.

Qi, W., Qiao, D. H. and Martinez, J. D. Caffeine Induces TP53-Independent G₁-Phase Arrest and Apoptosis in Human Lung Tumor Cells in a Dose-Dependent Manner. Radiat. Res. 157, 166–174 (2002).

Caffeine is a model radiosensitizing agent that is thought to work by abrogating the radiation-induced G₂-phase checkpoint. In this study, we examined the effect that various concentrations of caffeine had on cell cycle checkpoints and apoptosis in cells of a human lung carcinoma cell line and found that a concentration of 0.5 mM caffeine could abrogate the G₂-phase arrest normally seen after exposure to ionizing radiation. Surprisingly, at a concentration of 5 mM, caffeine not only induced apoptosis by itself and acted synergistically to enhance radiation-induced apoptosis, but also induced a TP53-independent G₁-phase arrest. Examination of the molecular mechanisms by which caffeine produced these effects revealed that caffeine had opposing effects on different cyclin-dependent kinases. CDK2 activity was suppressed by caffeine, whereas activity of CDC2 was enhanced by suppressing phosphorylation on Tyr15 and by interfering with 14-3-3 binding to CDC25C. These data indicate that the effect of caffeine on cell cycle checkpoints and apoptosis is dependent on dose and that caffeine acts through differential regulation of cyclin-dependent kinase activity.

Jyothi Lakshmi, R., Kartha, V. B., Murali Krishna, C., Solomon, J. G. R., Ullas, G. and Uma Devi, P. Tissue Raman Spectroscopy for the Study of Radiation Damage: Brain Irradiation of Mice. Radiat. Res. 157, 175–182 (2002).

Radiotherapy is routinely employed in the treatment of head and neck cancers. Acute cell death, radiation-activated chemical cascades, and the induction of genes coding for protective factors like cytokines are considered to be the major processes involved in radiation damage and repair. It should be possible to follow these processes by monitoring the biochemical interactions initiated by radiation. We have carried out Raman spectroscopy studies on tissue from mice subjected to brain irradiation to identify the biochemical changes occurring in tissue and brain as a result of radiation injury. These studies show that brain irradiation produces drastic spectral changes even in tissue far removed from the irradiation site. The changes are very similar to those produced by the stress of inoculation and restraint and the administration of an anesthetic drug. While the changes produced by stress or anesthetics last for only a short time (a few hours to 1 or 2 days), radiation-induced changes persist even after 1 week. The spectral changes can be interpreted in terms of the observation of new spectra that are dominated by bands due to proteins. The results thus support the hypothesis that various protective factors are released throughout the body when the central nervous system (CNS) is exposed to radiation.

Bartsch, H., Bartsch, C., Seebald, E., Deerberg, F., Dietz, K., Vollrath, L. and Mecke, D. Chronic Exposure to a GSM-like Signal (Mobile Phone) does not Stimulate the Development of DMBA-Induced Mammary Tumors in Rats: Results of Three Consecutive Studies. Radiat. Res. 157, 183–190 (2002).

Certain epidemiological and experimental studies raised concerns about the safety of radiofrequency (RF) electromagnetic fields because of a possible increased risk of leukemia and lymphoma. In this study, an RF field used in mobile telecommunication was tested using 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in female Sprague-Dawley rats as a model for human breast cancer. Three experiments were carried out under strictly standardized conditions and were started on the same day of three consecutive years. The field consisted of a GSM-like signal (900 MHz pulsed at 217 Hz, pulse width 577 μs) of relatively low power flux density (100 μW/cm² ± 3 dB) and was applied continuously throughout each experiment to freely moving animals. The specific absorption rates averaged over the whole body were 17.5–70 mW/kg. The highest values in young animals were at or around the exposure limit permissible for the general public (i.e. 80 mW/kg). The animals were palpated weekly for the presence of mammary tumors and were killed humanely when tumors reached a diameter of 1–2 cm to allow a reliable histopathological classification and a distinction between malignant and benign subtypes. The overall results of the three studies are that there was no statistically significant effect of RF-field exposure on tumor latency and that the cumulative tumor incidence at the end of the experiment was unaffected as well. The risk ratios were 1.08 (95% CI: 0.91–1.29) and 0.96 (95% CI: 0.85–1.07) for benign and malignant tumors, respectively. These observations are in agreement with other published findings. In the first experiment, however, the median latency for the development of the first malignant tumor in each animal was statistically significantly extended for RF-field-exposed animals compared to controls (278 days compared to 145 days, P = 0.009). No such differences were detected in the two subsequent experiments. These results show that low-level RF radiation does not appear to possess carcinogenic or cancer-promoting effects on DMBA-induced mammary tumors. To explain the mechanisms underlying the different results obtained in the three experiments, a hypothesis is presented which is based upon the neuroendocrine control mechanisms involved in the promotion of DMBA-induced mammary tumors. Despite the apparent absence of stimulatory effects of low-level RF-field exposure on the development and growth of solid tumors, it will be necessary to verify these results for leukemias and lymphomas, which may have completely different biological control mechanisms.

Huang, L., Tani, K., Ogushi, F., Ogawa, H., Shimizu, T., Motoki, Y., Moriguchi, H. and Sone, S. Role of CD13/Aminopeptidase N in Rat Lymphocytic Alveolitis Caused by Thoracic Irradiation. Radiat. Res. 157, 191–198 (2002).

CD13/aminopeptidase N is a cell surface glycoprotein that is widely distributed in a variety of mammalian cells. It was recently shown to have chemotactic activity for T lymphocytes. This study examined the role of CD13/aminopeptidase N in lymphocytic alveolitis in radiation-induced lung injury caused by a single-dose thoracic irradiation (15 Gy) in rats. Significantly increased aminopeptidase activity was detected in bronchoalveolar lavage fluid obtained from irradiated rats at 4 weeks after irradiation compared to the activity in unirradiated rats. Significantly higher aminopeptidase activity was detected on alveolar macrophages from irradiated rats at 2 and 4 weeks than on those from unirradiated rats. Western blot analysis showed an increased expression of CD13/aminopeptidase N protein in alveolar macrophages from irradiated rats at 4 weeks. Chemotactic activity for normal rat lymphocytes was detected in bronchoalveolar lavage fluid from irradiated rats at 4 weeks, and approximately 60% of the activity was inhibited by pretreatment of bronchoalveolar lavage fluid with bestatin, a specific aminopeptidase inhibitor. This study suggests that CD13/aminopeptidase N may play an important role as a lymphocyte chemoattractant in lymphocyte-mediated alveolitis in experimental radiation-induced lung injury.

Moulder, J. E., Fish, B. L., Regner, K. R., Cohen, E. P. and Raife, T. J. Retinoic Acid Exacerbates Experimental Radiation Nephropathy. Radiat. Res. 157, 199–203 (2002).

Studies have shown that angiotensin-converting enzyme inhibitors and an angiotensin II receptor blocker can delay, but cannot reverse, the progression of experimentally induced radiation nephropathy. In an effort to find a method for reversing injury, three agents were tested in a rat model of radiation nephropathy. Pirfenidone (a phenyl-pyridone antifibrotic) and thiaproline (an inhibitor of collagen deposition) were not capable of retarding the development of radiation nephropathy. However, all-trans retinoic acid (an anti-inflammatory agent) exacerbated radiation nephropathy. We speculated that the detrimental effects of retinoic acid might be the result of stimulation of renal cell proliferation. However, retinoic acid had no effect on tubular or glomerular cell proliferation in normal animals and did not enhance radiation-induced proliferation. A recent report that retinoic acids inhibit nitric oxide production suggested an alternative mechanism, since inhibition of production of nitric oxide is known to exacerbate radiation nephropathy. Experiments demonstrated that retinoic acid exacerbated the radiation-induced drop in renal production of nitric oxide, suggesting that the detrimental effect of all-trans retinoic acid might be explained by inhibition of renal nitric oxide activity. Particularly in view of the recent clinical report of enhancement of radiation nephropathy by retinoic acid in patients receiving bone marrow transplantation, the combination of retinoic acid and renal irradiation should be carried out with great caution.

Stone, H. B., McBride, W. H. and Coleman, C. N. Modifying Normal Tissue Damage Postirradiation. Report of a Workshop Sponsored by the Radiation Research Program, National Cancer Institute. Radiat. Res. 157, 204–223 (2002).

Late effects that develop in normal tissues adjacent to the tumor site in the months to years after radiotherapy can reduce the quality of life of cancer survivors. They can be dose-limiting and debilitating or life-threatening. There is now evidence that some late effects may be preventable or partially reversible. A workshop, “Modifying Normal Tissue Damage Postirradiation”, was sponsored by the Radiation Research Program of the National Cancer Institute to identify the current status of and research needs and opportunities in this area. Mechanistic, genetic and physiological studies of the development of late effects are needed and will provide a rational basis for development of treatments. Interdisciplinary teams will be needed to carry out this research, including pathologists, physiologists, geneticists, molecular biologists, experts in functional imaging, wound healing, burn injury, molecular biology, and medical oncology, in addition to radiation biologists, physicists and oncologists. The participants emphasized the need for developing and choosing appropriate models, and for radiation dose–response studies to determine whether interventions remain effective at the radiation doses used clinically. Both preclinical and clinical studies require long-term follow-up, and easier-to-use, more objective clinical scoring systems must be developed and standardized. New developments in biomedical imaging should provide useful tools in all these endeavors. The ultimate goals are to improve the quality of life and efficacy of treatment for cancer patients treated with radiotherapy.