Michaud, M., Wen, A. and Sanche, L. Cross Sections for Low-Energy (1–100 eV) Electron Elastic and Inelastic Scattering in Amorphous Ice. Radiat. Res. 159, 3–22 (2003).

We report the integral cross sections per scatterer (i.e. elastic collision, phonon excitations, vibrational excitations, electronic excitations and ionization) for 1–100 eV electron scattering in an amorphous film of ice condensed at a temperature of 14 K. The integral cross sections are determined relative to the total from a two-stream multiple-scattering analysis of the electron energy distribution backscattered from the film. Their energy dependence is obtained from both the analysis of the elastic electron reflectivity as a function of the film thickness and the vibrational electron energy-loss spectra measured for several incident energies and large film thickness. The magnitude and various features found in the energy dependence of the cross sections are discussed, whenever possible, by comparison with data and with scattering mechanisms available in the gas phase. Microcospic effects, which are implicitly included in cross sections determined in this way, are discussed in terms of interference and coherent multiple-scattering contributions among the scattering sites as well as interactions of the scattering sites with their neighbors in the condensed phase.

Malinen, E., Heydari, M. Z., Sagstuen, E. and Hole, E. O. Alanine Radicals, Part 3: Properties of the Components Contributing to the EPR Spectrum of X-Irradiated Alanine Dosimeters. Radiat. Res. 159, 23–32 (2003).

The amino acid l-α-alanine has attracted considerable interest for use in radiation dosimetry and has been formally accepted as a secondary standard for high-dose and transfer dosimetry. Recent results have shown that the alanine EPR spectrum consists of contributions from three different radicals. A set of benchmark spectra describing the essential spectral features of these three radical components was used for reconstructions of the experimental spectra. In the present work, these basis spectra have been used to investigate the differential effects of variations in radiation doses and microwave power, as well as the dependence upon temperature annealing and UV illumination. The results presented here, based solely on relatively low-energy (60–80 keV) X rays, indicate that the three components behave very similarly with respect to radiation dose at room temperature. However, with respect to the thermal annealing/fading behavior and microwave power saturation properties, the three species behave significantly differently. It is concluded that even if it is now realized that three different radicals contribute to the composite EPR alanine spectrum, this has a minor impact on the established protocols for present-day applications (high-dose) of EPR/alanine dosimetry. However, some care should be exercised when e.g. constructing calibration curves, since fading and power saturation behavior may vary over the dose range in question. New results from UV-illumination experiments suggest a possible procedure for experimental spectral separation of the EPR signals due to the three radicals.

Tsoulou, E., Kalfas, C. A. and Sideris, E. G. Changes in DNA Flexibility after Irradiation with γ Rays and Neutrons Studied with the Perturbed Angular Correlation Method. Radiat. Res. 159, 33–39 (2003).

Neutron and γ irradiation of buffered solutions of calf thymus DNA resulted in changes in the dynamics of the macromolecule. In the low-dose region (0.8–10 cGy of ²³⁹Pu-Be neutrons and 0.34–3 Gy of ⁶⁰Co γ rays), the flexibility of DNA decreased as indicated by slower rotation of the molecules. Neutrons appeared to be approximately 35 times more effective than ⁶⁰Co γ rays. The rotational correlation time, τ_C, was measured using the perturbed angular correlation (PAC) method. Its variation appears to follow a linear-exponential behavior. An attempt is made to formulate this behavior as a function of the energy deposited on the macromolecule (radiation dose), the average threshold energy (dose) required to form new lesions, and the available population of intact DNA sites.

Anderson, R. M., Marsden, S. J., Paice, S. J., Bristow, A.;thE., Kadhim, M. A., Griffin, C. S. and Goodhead, D. T. Transmissible and Nontransmissible Complex Chromosome Aberrations Characterized by Three-Color and mFISH Define a Biomarker of Exposure to High-LET α Particles. Radiat. Res. 159, 40–48 (2003).

Insertions have been proposed as potential stable biomarkers of chronic high-LET radiation exposure. To examine this in vitro, we irradiated human peripheral blood lymphocytes in G₀ with either 50 cGy ²³⁸Pu α particles (LET 121.4 keV/μm) or 3 Gy 250 kV X rays and stimulated their long-term culture up to ∼22 population doublings postirradiation. Mitotic cells were harvested at regular intervals throughout this culture period and were assayed for chromosome aberrations using the techniques of three-color and 24-color mFISH. We observed the stable persistence of transmissible-type complex rearrangements, all involving at least one insertion. This supports the hypothesis that insertions are relevant indicators of exposure to high-LET radiation. However, one practical caveat of insertions being effective biomarkers is that their frequency is low due to the complexity and cell lethality of the majority of α-particle-induced complexes. Therefore, we propose a “profile of damage” that relies on the presence of insertions, a low frequency of stable simple reciprocal translocations (2B), and, significantly, the complexity of the damage initially induced. We suggest that the complexity of first- and second-division α-particle-induced nontransmissible complex aberrations reflects the structure of the α-particle track and as a consequence adds radiation-quality specificity to the biomarker, increasing the signal:noise ratio of the characteristic 2B:insertion ratio.

McKenna, D. J., Rajab, N. F., McKeown, S. R., McKerr, G. and McKelvey-Martin, V. J. Use of the Comet-FISH Assay to Demonstrate Repair of the TP53 Gene Region in Two Human Bladder Carcinoma Cell Lines. Radiat. Res. 159, 49–56 (2003).

The alkaline single-cell gel electrophoresis (comet) assay can be combined with fluorescence in situ hybridization (FISH) methodology to investigate the localization of specific gene domains within an individual cell. The position of the fluorescent hybridization spots in the comet head or tail indicates whether the sequence of interest lies within or in the vicinity of a damaged region of DNA. In this study, we used the comet-FISH assay to examine initial DNA damage and subsequent repair in the TP53 gene region of RT4 and RT112 bladder carcinoma cells after 5 Gy γ irradiation. In addition to standard comet parameter measurements, the number and location of TP53 hybridization spots within each comet was recorded at each repair time. The results indicate that the rate of repair of the TP53 gene region was fastest during the first 15 min after damage in both cell lines. When compared to overall genomic repair, the repair of the TP53 gene region was observed to be significantly faster during the first 15 min and thereafter followed a rate similar to that for the overall genome. The data indicate that the TP53 domain in RT4 and RT112 cells is repaired rapidly after γ irradiation. Furthermore, this repair may be preferential compared to the repair of overall genomic DNA, which gives a measure of the average DNA repair response of the whole genome. We suggest that the comet-FISH assay has considerable potential in the study of gene-specific repair after DNA damage.

Redpath, J. L., Bengtsson, U., DeSimone, J., Lao, X., Wang, X. and Stanbridge, E. J. Sticky Anaphase Aberrations after G₂-Phase Arrest of Gamma-Irradiated Human Skin Fibroblasts: TP53 Independence of Formation and TP53 Dependence of Consequences. Radiat. Res. 159, 57–71 (2003).

We have studied the impact of TP53 status on the extent and nature of chromosome damage seen in human skin fibroblasts after γ irradiation beyond the G₁-phase checkpoint but prior to the G₂-phase checkpoint. Mitotic cells were examined in the absence and presence of treatment with nocodazole and the yield of aberrations was scored as a function of time postirradiation. The results revealed substantially greater damage in the absence of nocodazole, indicating that damage was being masked in its presence. While metaphase aberrations were seen exclusively in the presence of nocodazole, anaphase aberrations were seen principally in its absence. Furthermore, these were mostly of an unseparated, or “sticky”, type that showed separation of the chromatids in the centromeric region, indicating normal degradation of cohesin, with retention of adhesion further out on the chromatid arms. Using postirradiation BrdU labeling and the absence of nocodazole, we were able to identify mitotic figures up to the third postirradiation mitosis. Analysis of the data revealed that in cells wild-type for TP53 the aberrant anaphases were lost after the first postirradiation mitosis, although they were still found in gradually decreasing amounts into the second and third postirradiation mitoses in E6-expressing cells. The data indicate that the formation of these sticky anaphases is independent of TP53 status, an observation that is consistent with the TP53 independence of transient G₂-phase arrest. However, the consequences of the formation of these lesions appear to be very different. In the case of cells wild-type for TP53 this is chronic G₁-phase arrest, while in E6 cells it is anaphase catastrophe.

DeSimone, J. N., Bengtsson, U., Wang, X. Q., Lao, X. Y., Redpath, J. L, and Stanbridge, E. J. Complexity of the Mechanisms of Initiation and Maintenance of DNA Damage-Induced G₂-Phase Arrest and Subsequent G₁-Phase Arrest: TP53-Dependent and TP53-Independent Roles. Radiat. Res. 159, 72–85 (2003).

Through a detailed study of cell cycle progression, protein expression, and kinase activity in γ-irradiated synchronized cultures of human skin fibroblasts, distinct mechanisms of initiation and maintenance of G₂-phase and subsequent G₁-phase arrests have been elucidated. Normal and E6-expressing fibroblasts were used to examine the role of TP53 in these processes. While G₂ arrest is correlated with decreased cyclin B1/CDC2 kinase activity, the mechanisms associated with initiation and maintenance of the arrest are quite different. Initiation of the transient arrest is TP53-independent and is due to inhibitory phosphorylation of CDC2 at Tyr15. Maintenance of the G₂ arrest is dependent on TP53 and is due to decreased levels of cyclin B1 mRNA and a corresponding decline in cyclin B1 protein level. After transiently arresting in G₂ phase, normal cells chronically arrest in the subsequent G₁ phase while E6-expressing cells continue to cycle. The initiation of this TP53-dependent G₁-phase arrest occurs despite the presence of substantial levels of cyclin D1/CDK4 and cyclin E/CDK2 kinase activities, hyperphosphoryated RB, and active E2F1. CDKN1A (also known as p21^WAF1/CIP1) levels remain elevated during this period. Furthermore, CDKN1A-dependent inhibition of PCNA activity does not appear to be the mechanism for this early G₁ arrest. Thus the inhibition of entry of irradiated cells into S phase does not appear to be related to DNA-bound PCNA complexed to CDKN1A. The mechanism of chronic G₁ arrest involves the down-regulation of specific proteins with a resultant loss of cyclin E/CDK2 kinase activity.

Park, H. J., Lee, S. H., Chung, H., Rhee, Y. H., Lim, B. U., Ha, S. W., Griffin, R. J., Lee, H. S., Song, C. W. and Choi, E.;thK. Influence of Environmental pH on G₂-Phase Arrest Caused by Ionizing Radiation. Radiat. Res. 159, 86–93 (2003).

We investigated the effects of an acidic environment on the G₂/M-phase arrest, apoptosis, clonogenic death, and changes in cyclin B1-CDC2 kinase activity caused by a 4-Gy irradiation in RKO.C human colorectal cancer cells in vitro. The time to reach peak G₂/M-phase arrest after irradiation was delayed in pH 6.6 medium compared to that in pH 7.5 medium. Furthermore, the radiation-induced G₂/M-phase arrest decayed more slowly in pH 6.6 medium than in pH 7.5 medium. Finally, there was less radiation-induced apoptosis and clonogenic cell death in pH 6.6 medium than in pH 7.5 medium. It appeared that the prolongation of G₂-phase arrest after irradiation in the acidic environment allowed for greater repair of radiation-induced DNA damage, thereby decreasing the radiation-induced cell death. The prolongation of G₂-phase arrest after irradiation in the acidic pH environment appeared to be related at least in part to a prolongation of the phosphorylation of CDC2, which inhibited cyclin B1-CDC2 kinase activity.

Vordermark, D., Menke, D. R. and Brown, J. M. Similar Radiation Sensitivities of Acutely and Chronically Hypoxic Cells in HT 1080 Fibrosarcoma Xenografts. Radiat. Res. 159, 94–101 (2003).

It has been suggested that chronically hypoxic tumor cells may be more radiosensitive than acutely hypoxic or even aerobic cells. In the present study we have used the fact that chronically, but not acutely, hypoxic cells that are transformed with a vector containing an enhanced green fluorescent protein (EGFP) driven by a hypoxia-responsive promoter become green (high EGFP) at low oxygen concentrations and can be viably sorted from transplanted tumors in vitro. We showed that the fluorescence of HT 1080 human fibrosarcoma cells stably transfected with this vector increases constantly with decreasing O₂ concentrations (<2%, longer than 1 h, half maximum ∼0.2% for longer than 8 h), and that cells subjected to repeated cycles of hypoxia/reoxygenation (simulating acutely hypoxic cells) showed only background fluorescence. To test the radiosensitivity of acutely and chronically hypoxic cells in tumors, we isolated high-EGFP (“chronically hypoxic”) and low-EGFP cells (containing both acutely hypoxic and aerobic cells) from HT 1080 xenograft tumors by fluorescence-activated cell sorting (FACS), immediately after in situ treatment with 20 Gy (ambient or clamped), and plated the cells to determine clonogenic survival in vitro. We found that the survival of high-EGFP cells after irradiation was not affected by clamping, suggesting that all, or almost all, of these cells were fully (chronically) hypoxic. Also, the survival of the low-EGFP cells irradiated under clamped conditions (acutely hypoxic cells) was not significantly different from that of the high-EGFR cells (chronically hypoxic) cells irradiated under nonclamped (or clamped) conditions. We therefore conclude that, at least in this tumor model, the radiation sensitivity of chronically hypoxic cells is similar to that of the acutely hypoxic cells.

Di Majo, V., Rebessi, S., Pazzaglia, S., Saran, A. and Covelli, V. Carcinogenesis in Laboratory Mice after Low Doses of Ionizing Radiation. Radiat. Res. 159, 102–108 (2003).

Experimental data on the incidence of solid tumors from various long-term mouse studies performed at the Casaccia laboratories over several years were reconsidered, limiting the analysis to the results available for doses equal to or less than 17 cGy of neutrons and 32 cGy of X rays since these dose limits are reasonably close to the generally accepted low-dose levels for high- and low-LET radiation (i.e. D_high-LET < 5 cGy and D_low-LET < 20 cGy, respectively). The following long-term experiments with BC3F1 mice were reviewed: (a) females treated with single doses of 1.5 MeV neutrons or 250 kVp X rays, (b) males treated with fractionated doses of fission neutrons, and (c) mice of both sexes irradiated in utero 17.5 days post coitus with single doses of fission neutrons or X rays. An experiment with CBA mice of both sexes treated with single doses of fission neutrons was also included in this study. Analysis was done on animals at risk; thus all incidences of tumor-bearing animals were expressed as the percentage excess incidence with respect to the controls. Ovarian tumors and other solid neoplasms were considered. The percentage frequencies and mean survival times of tumor-free mice were also recalculated. The results indicate the existence of a region at low doses where the final incidence of solid neoplasms is indistinguishable from the background incidence. These data reinforce the idea that at low doses the effectiveness of ionizing radiation in inducing solid neoplasms in laboratory mice is very low.

Kuzmenok, O., Potapnev, M., Potapova, S., Smolnikova, V., Rzheutsky, V., Yarilin, A. A., Savino, W. and Belyakov, I. M. Late Effects of the Chernobyl Radiation Accident on T Cell-Mediated Immunity in Cleanup Workers. Radiat. Res. 159, 109–116 (2003).

The main goal of this investigation was to evaluate the abnormal T-cell immunity in cleanup workers who took part in the cleanup after the Chernobyl accident in 1986. Peripheral blood mononuclear cells (MNCs) of apparently healthy cleanup workers (n = 134) were used to analyze the phenotype and proliferative response to mitogens in vitro. Evaluation of the MNC phenotype of cleanup workers did not reveal a significant disturbance in the T-cell subpopulation content except for an increase in CD3 CD16 56 (NKT) cells. Immunophenotyping of phytohemagglutinin (PHA)-activated MNCs demonstrated suppression of CD4 T-cell propagation and augmentation of CD8 T-cell propagation in vitro compared to control individuals. DNA synthesis in the MNCs of cleanup workers was markedly inhibited after activation for 3 days with suboptimal concentrations of PHA, pokeweed mitogen and PMA. In contrast to control individuals, the monocytes of cleanup workers were able to stimulate the proliferation of T cells from healthy individuals but inhibited the proliferation of T cells from cleanup workers. This study affords a better understanding of the response of MNCs to stimulation with suboptimal concentrations of PHA and provides an approach to a more accurate analysis of the immunological disorders found after exposure to radiation from Chernobyl-related activities.

Tawn, E. J., Whitehouse, C. A., Daniel, C. P., Tarone, R.;thE., Bothwell, A. M. and Fisher, A. Somatic Cell Mutations at the Glycophorin A Locus in Erythrocytes of Radiation Workers from the Sellafield Nuclear Facility. Radiat. Res. 159, 117–122 (2003).

The glycophorin A (GPA) somatic mutation assay for N0 and NN mutant erythrocytes was performed on 245 current and 48 retired workers who had been occupationally exposed to radiation at the British Nuclear Fuels plc facility at Sellafield. A positive association with increasing age was found for current workers for both N0 and NN frequencies of 0.14 ± 0.05 × 10^–6 (P = 0.012) and 0.25 ± 0.07 × 10^–6 (P = 0.0003) per year, respectively. No association with age was found for the retired workers. In a comparison of ever-smokers with never-smokers, no difference was observed for N0 frequencies for current workers, but a significantly higher frequency was found for ever-smokers in the retired group (P = 0.001). NN mutant frequencies were slightly higher in ever-smokers than in never-smokers for both current and retired workers, but in neither case was the increase significant. In age-adjusted analyses for N0 mutant frequencies, a slight positive radiation dose response was found for current workers (1.6 ± 3.8 × 10^–6 per Sv), for retired workers (2.9 ± 2.5 × 10^–6 per Sv), and in the combined analysis (2.6 ± 2.2 × 10^–6 per Sv), but in no case did this reach significance. Similar analyses for NN mutant frequencies revealed a positive dose response for current workers (4.7 ± 4.6 × 10^–6 per Sv) and a negative response for retired workers (–2.4 ± 3.6 × 10^–6 per Sv) that was maintained in the combined analysis (–1.4 ± 2.8 × 10^–6 per Sv), but none of these slopes was significantly different from zero. The results suggest that the GPA mutation assay is insufficiently sensitive to be used as a biological marker of low-dose chronic exposure and provide further evidence that, in contrast to high acute radiation exposure, protracted exposure is much less effective at inducing somatic mutations in vivo.

Ying, H., Serhir, L., Mahy, P., Reniers, B. and Gueulette, J. Design of a Cylindrical Brachytherapy Implant Applicator for the Irradiation of an Intestinal Segment in Mice. Radiat. Res. 159, 123–127 (2003).

Experimental determination of the RBE of new isotopes for brachytherapy implants (e.g. iodine-125 and palladium-103) remains a very difficult problem, especially in small animals, where the seeds cannot be implanted easily in the planned geometry in a reproducible way. This technical note describes an original device that makes it possible to irradiate a segment of the intestine in mice for the purpose of determining the RBE for crypt regeneration. The device is a length of tube (3.4 mm and 7 mm internal and external diameter, respectively) whose external surface has been longitudinally grooved and into which the seeds can be squeezed (each groove holds either one or two seeds). The tube is composed of two sections. This seed container can be surgically positioned around an intestinal ansa while the mice are anesthetized. The mean dose rates in the intestine (for eight seeds) were found to be 86.3 ± 5.9 and 79.0 ± 5.4 cGy/h for 29.2 MBq (1 U) iodine and 28.6 MBq (1 U) palladium seeds, respectively. So far, more than 100 mice have been irradiated successfully. Full dose–effect relationships can be obtained using the same seeds and applying them successively in different groups of animals (which ensured the accuracy of the relative doses).

Adair, R. K. Environmental Objections to the PAVE PAWS Radar System: A Scientific Review. Radiat. Res. 159, 128–134 (2003).

As part of our continental defense system, the United States Air Force has operated a radar system, known generally by the label PAVE PAWS, off of Cape Cod, MA since 1978. Some populated areas in the vicinity of the system are subject to a low level of background radiofrequency radiation from the system, and local citizens' groups have expressed concern that this radiofrequency radiation may affect their health. These concerns have been fueled by presentations and letters by Dr. R. A. Albanese, an applied mathematician at the Air Force Research Laboratory, who has proposed standards by which that PAVE PAWS radiofrequency radiation which is incident on populations should be judged. I discuss those standards that are sufficiently well defined to be subject to analysis and show that they are not based on sound quantitative reasoning.