Kodama, Y., Pawel, D., Nakamura, N., Preston, D., Honda, T., Itoh, M., Nakano, M., Ohtaki, K., Funamoto, S. and Awa, A. A. Stable Chromosome Aberrations in Atomic Bomb Survivors: Results from 25 Years of Investigation. Radiat. Res. 156, 337–346 (2001).

Frequencies of stable chromosome aberrations from more than 3,000 atomic bomb survivors were used to examine the nature of the radiation dose response. The end point was the proportion of cells with at least one translocation or inversion detected in Giemsa-stained cultures of approximately 100 lymphocytes per person. The statistical methods allow for both imprecision of individual dose estimates and extra-binomial variation. A highly significant and nonlinear dose response was seen. The shape of the dose response was concave upward for doses below 1.5 Sv but exhibited some leveling off at higher doses. This curvature was similar for the two cities, with a crossover dose (i.e. the ratio of the linear coefficient to the quadratic coefficient) of 1.7 Sv (95% CI 0.9, 4). The low-dose slopes for the two cities differed significantly: 6.6% per Sv (95% CI 5.5, 8.4) in Hiroshima and 3.7% (95% CI 2.6, 4.9) in Nagasaki. This difference was reduced considerably, but not eliminated, when the comparison was limited to people who were exposed in houses or tenements. Nagasaki survivors exposed in factories, as well as people in either city who were outside with little or no shielding, had a lower dose response than those exposed in houses. This suggests that doses for Nagasaki factory worker survivors may be overestimated by the DS86, apparently by about 60%. Even though factory workers constitute about 20% of Nagasaki survivors with dose estimates in the range of 0.5 to 2 Sv, calculations indicate that the dosimetry problems for these people have little impact on cancer risk estimates for Nagasaki.

Tomilin, N. V., Solovjeva, L. V., Svetlova, M. P., Pleskach, N. M., Zalenskaya, I. A., Yau, P. M. and Bradbury, E. M. Visualization of Focal Nuclear Sites of DNA Repair Synthesis Induced by Bleomycin in Human Cells. Radiat. Res. 156, 347–354 (2001).

In this study, we examined DNA repair synthesis in human cells treated with the radiomimetic drug bleomycin, which efficiently induces double-strand breaks (DSBs). Using tyramide-biotin to amplify fluorescent signals, discrete nuclear foci from the incorporation of 5-iododeoxyuridine (IdU) were detected in proliferating human cells treated with bleomycin. We believe this comes from the repair of DSBs. An increase in the number of foci (>5 per nucleus) was detected in a major fraction (75%) of non-S-phase cells labeled for 30 min with IdU 1 h after the end of bleomycin treatment. The fraction of cells with multiple IdU-containing foci was found to decrease 18 h after treatment. The average number of foci per nucleus detected 1 h after bleomycin treatment was found to decrease twofold between 1 and 3.5 h, indicating that the foci may be associated with the slow component of DSB repair. The presence of DSBs in bleomycin-treated cells was confirmed using antibodies against phosphorylated histone H2AX (γ-H2AX), which is strictly associated with this type of DNA damage. After treatment with bleomycin, non-S-phase cells also displayed heterogeneous nuclear foci containing tightly bound proliferating cell nuclear antigen (PCNA), suggesting an ongoing process of unscheduled DNA synthesis. PCNA is known to be involved in base excision repair, but a fraction of the PCNA foci may also be associated with DNA synthesis occurring during the repair of DSBs.

Belyaev, I. Y., Czene, S. and Harms-Ringdahl, M. Changes in Chromatin Conformation during Radiation-Induced Apoptosis in Human Lymphocytes. Radiat. Res. 156, 355–364 (2001).

Human peripheral lymphocytes in G₀ phase were irradiated with 1–5 Gy of γ rays. The biochemical and morphological changes characteristic of apoptosis were examined for 72 h after irradiation. In parallel, changes in chromatin conformation were studied by the method of anomalous viscosity time dependence (AVTD) and by measurements of nuclear halo size. An immediate and dose-dependent relaxation of chromatin, which became saturated at doses above 2–3 Gy, was revealed by the AVTD method. The state of relaxed chromatin lasted up to 12–24 h after irradiation, a response considerably longer than the time attributable to repair of radiation-induced DNA breaks. Measurements of nuclear halo size also indicated the initial relaxation of chromatin in the irradiated cells and its subsequent condensation. This condensation of chromatin as revealed with AVTD correlated well with nuclear condensation, as measured with dual fluorescence staining, and with DNA fragmentation, as measured by conventional and pulsed-field gel electrophoresis (PFGE). Late apoptotic cells did not contribute significantly to the AVTD signal, showing that the chromatin of these cells was completely condensed and fragmented.

Stewart, R. D. Two-Lesion Kinetic Model of Double-Strand Break Rejoining and Cell Killing. Radiat. Res. 156, 365–378 (2001).

Radiobiological models, such as the lethal and potentially lethal (LPL) model and the repair-misrepair (RMR) model, have been reasonably successful at explaining the cell killing effects of radiation. However, the models have been less successful at relating cell killing to the formation, repair and misrepair of double-strand breaks (DSBs), which are widely accepted as the main type of DNA damage responsible for radiation-induced cell killing. A fully satisfactory model should be capable of predicting cell killing for a wide range of exposure conditions using a single set of model parameters. Moreover, these same parameters should give realistic estimates for the initial DSB yield, the DSB rejoining rate, and the residual number of unrepaired DSBs after all repair is complete. To better link biochemical processing of the DSB to cell killing, a two-lesion kinetic (TLK) model is proposed. In the TLK model, the family of all possible DSBs is subdivided into simple and complex DSBs, and each kind of DSB may have its own repair characteristics. A unique aspect of the TLK model is that break ends associated with both kinds of DSBs are allowed to interact in pairwise fashion to form irreversible lethal and nonlethal damages. To test the performance of the TLK model, nonlinear optimization methods are used to calibrate the model based on data for the survival of CHO cells for an extensive set of single-dose and split-dose exposure conditions. Then some of the postulated mechanisms of action are tested by comparing measured and predicted estimates of the initial DSB yield and the rate of DSB rejoining. The predictions of the TLK model for CHO cell survival and the initial DSB yield and rejoining rate are all shown to be in good agreement with the measured data. Studies suggest a yield of about 25 DSBs Gy⁻¹ cell⁻¹. About 20 DSBs Gy⁻¹ cell⁻¹ are rejoined quickly (15-min repair half-time), and 4 to 6 DSBs Gy⁻¹ cell⁻¹ are rejoined very slowly (10- to 15-h repair half-time). Both the slowly and fast-rejoining DSBs make substantial contributions to the killing of CHO cells by radiation. Although the TLK model provides a much more satisfactory formalism to relate biochemical processing of DSBs to cell killing than did the earlier kinetic models, some small differences among the measured and predicted CHO cell survival and DSB rejoining data suggest that additional factors and processes not considered in the present work may affect biochemical processing of DSBs and hence cell killing.

De Sanctis, V., Bertozzi, C., Costanzo, G., Di Mauro, E. and Negri, R. Cell Cycle Arrest Determines the Intensity of the Global Transcriptional Response of Saccharomyces cerevisiae to Ionizing Radiation. Radiat. Res. 156, 379–387 (2001).

Whole-genome analysis was performed using DNA microarrays to define the changes in the gene expression patterns occurring in Saccharomyces cerevisiae cells exposed to ionizing radiation. The effects of sublethal dose on wild-type, rad53 (enhanced sensitivity to radiation and impaired in a cell cycle damage checkpoint), and rad6 (enhanced sensitivity to radiation and functional cell cycle block by radiation) mutant backgrounds and of a higher dose on the wild-type and G₂-phase-arrested cells were analyzed. Several gene pathways were identified as being implicated in the response to radiation. In particular, the cell cycle blockage that occurred in the wild-type strain after a high radiation dose and in the rad6 mutant after a lower dose entailed modifications of defined gene expression patterns, which are described here and are compared with the gene modulation patterns observed in the rad53 strain in the absence of efficient blockage. Loss of the RAD53 function caused a major increase in the number of genes modulated by radiation. Given that Rad53-Sad1p, the protein encoded by RAD53, has functions other than those directly connected to cell cycle arrest, we determined the gene patterns that were modulated upon irradiation of rad53 cells that had been forced to arrest in G₂ phase by nocodazole treatment. These differential whole-genome analyses shed light on the multiplicity of functions of the pivotal Rad53-Sad1p protein. The results obtained describe how the cells respond to different irradiation conditions by modulating important gene classes, including those associated with stress defense, ribosomal proteins, histones, ergosterol and GCR1-controlled sugar metabolism.

Horan, A. D. and Koch, C. J. The K_m for Radiosensitization of Human Tumor Cells by Oxygen is Much Greater than 3 mmHg and is Further Increased by Elevated Levels of Cysteine. Radiat. Res. 156, 388–398 (2001).

We studied the role of cysteine as an intracellular radiation protector under conditions in which both oxygen and thiols were monitored at 37°C. In HCT-116 human colon cancer cells, the intracellular cysteine content affects the radiation survival dramatically at intermediate oxygen levels, but not at zero or high oxygen levels. Using a spin-through-oil method with a dual radioactive label detection system, we measured intracellular cysteine and glutathione (GSH) levels for cells in suspension culture. A comparison of the cysteine levels of monolayer cells lysed in situ and of trypsinized monolayer cells in suspension (Horan et al., Cytometry 29, 76–82, 1997) revealed that, upon trypsinization from monolayer culture and transfer to a spinner apparatus at 37°C, HCT-116 cells lose most of their intracellular cysteine. Over the 60-min time course of control experiments, these cells do not recover intracellular cysteine despite the availability of cystine (the disulfide of cysteine) in the medium. When cells in spinner culture are provided with exogenous cysteine, they initially concentrate it to 10-fold the extracellular concentration, with the concentration factor decreasing to about 5-fold over the course of an hour. The intracellular GSH concentration changes little throughout this period, regardless of the changes in cysteine levels. The same apparatus was used to assess the survival of HCT-116 cells irradiated at 37°C under conditions of constant pO₂ monitoring. For cells without added cysteine, the oxygen concentration for half-maximal radiation sensitivity was about 7.5 mmHg (intermediate hypoxia), more than twice the commonly accepted value (3 mmHg). At 7.5 mmHg, cells with added cysteine (intracellular concentration 3.5 mM) were almost as radioresistant as severely hypoxic cells (≈0.005% oxygen). Cells in parallel experiments in which the cells were grown in monolayers on glass Petri dishes had intermediate cysteine values and also intermediate radiosensitivity. We conclude that the radiation response of cells at intermediate oxygen levels is controlled predominantly by intracellular cysteine levels and that the cysteine levels commonly found in tumors may increase the K_m for radiosensitivity to values much higher than suggested previously.

Leszczynski, D., Pitsillides, C. M., Pastila, R. K., Anderson, R. R. and Lin, C. P. Laser-Beam-Triggered Microcavitation: A Novel Method for Selective Cell Destruction. Radiat. Res. 156, 399–407 (2001).

We describe a new method of cell destruction that may have potential for use in antitumor therapy. Cells are loaded by phagocytosis with microparticles (<1 μm) and irradiated with short laser pulses. Absorption of laser energy by the microparticles causes localized vaporization of the fluid surrounding the microparticles, leading to the generation of transient vapor bubbles (microcavitation) around the microparticles. Using cultures of bovine aortic endothelial cells, we demonstrate that induction of intralysosomal microcavitation is an efficient, rapid and selective method of cell killing that is dependent on the number of microparticles, the number of laser pulses, and the fluence of the laser pulses. Cell killing by microcavitation is a very selective process that is restricted to cells containing microparticles, leaving other cells unaffected. Intracytoplasmic release of lysosomal hydrolases is, in part, responsible for cell death, because the protease inhibitors E64d and TLCK diminished cell killing. Using the broad-specificity caspase inhibitor Z-VAD-fmk, we determined that lysosomal hydrolases could induce apoptosis in a caspase-independent manner. We also examined the possibility of microcavitation-induced delayed effects in the cells that survived the treatment. Using flow cytometry, we determined that there was no delayed cell death between 1 and 4 days after microcavitation. Moreover, we did not observe changes in the cell cycle, in expression of the proteins BCL2, HSP70 and HSP27, or in PARP degradation. In conclusion, microcavitation induces rapid and specific cells death (limited only to cells containing microparticles), without producing delayed effects among the surviving cells.

Rezvani, M., Birds, D. A., Hodges, H., Hopewell, J. W., Milledew, K. and Wilkinson, J. H. Modification of Radiation Myelopathy by the Transplantation of Neural Stem Cells in the Rat. Radiat. Res. 156, 408–412 (2001).

In a novel approach, neural stem cells were transplanted to ameliorate radiation-induced myelopathy in the spinal cords of rats. A 12-mm section of the cervical spinal cord (T2–C2) of 5-week-old female Sprague-Dawley rats was locally irradiated with a single dose of 22 Gy of ⁶⁰Co γ rays. This dose is known to produce myelopathy in all animals within 6 months of irradiation. After irradiation, the animals were subdivided into three groups, and at 90 days after irradiation, neural stem cells or saline (for controls) were injected into the spinal cord, intramedullary, at two sites positioned 6 mm apart on either side of the center of the irradiated length of spinal cord. The injection volume was 2 μl. Group I received a suspension of MHP36 cells, Group II MHP15 cells, and Group III (controls) two injections of 2 μl saline. All rats received 10 mg/kg cyclosporin (10 mg/ml) daily i.p. to produce immunosuppression. All animals that received saline (Group III) developed paralysis within 167 days of irradiation. The paralysis-free survival rates of rats that received transplanted MHP36 and MHP15 cells (Groups I and II) were 36.4% and 32% at 183 days, respectively. It was concluded that transplantation of neural stem cells 90 days after irradiation significantly (P = 0.03) ameliorated the expression of radiation-induced myelopathy in the spinal cords of rats.

Utteridge, T. D., Charlton, D. E. and Allen, B. J. Monte Carlo Modeling of the Effects of Injected Short-, Medium- and Longer-Range Alpha-Particle Emitters on Human Marrow at Various Ages. Radiat. Res. 156, 413–418 (2001).

The effects of injected short-, medium- and longer-range α-particle emitters (¹⁴⁹Tb, ²¹¹At/²¹¹Po and ²¹³Bi/²¹³Po, respectively) on the total hemopoietic stem cell population of active normal bone marrow in humans of various ages has been estimated using Monte Carlo modeling. The fraction of the normal hemopoietic stem cells that are hit and survive has been calculated as a first step toward estimating the risk of development of therapy-induced leukemia. The fraction was lowest for the shorter-range α-particle emitter (¹⁴⁹Tb) and highest for the longer-range α-particle emitter (²¹³Bi/²¹³Po), with the value for the medium-range α-particle emitter ²¹¹At/²¹¹Po being intermediate between these. There was little variation in the data with the age of the subject within each α-particle emitter. This lack of age dependence provides reassurance that the fraction of cells hit in any subject of any age with normal marrow can be estimated by modeling newborn marrow (which requires little computing time) despite age-related differences in microarchitecture.

Li, W. B., Friedland, W., Pomplun, E., Jacob, P., Paretzke, H. G., Lassmann, M. and Reiners, C. Track Structures and Dose Distributions from Decays of ¹³¹I and ¹²⁵I in and around Water Spheres Simulating Micrometastases of Differentiated Thyroid Cancer. Radiat. Res. 156, 419–429 (2001).

The disintegration of the radionuclides ¹³¹I and ¹²⁵I and the subsequent charged-particle tracks left behind in water (as a model substance for a biological cell) are simulated by the Monte Carlo track structure simulation code PARTRAC, using new inelastic electron scattering cross sections for condensed water. Every photon and electron emitted was followed in detail, event by event, down to 10 eV. From the spatial information on the track structures, absorbed dose distributions per ¹³¹I and ¹²⁵I decay were calculated in and around water spheres simulating micrometastases as well as in the tissue surrounding such metastases. These radionuclides were assumed to be distributed uniformly inside spheres of different diameters (0.01, 0.03, 0.1, 0.3, 1.0 and 3.0 mm). The respective electron degradation spectra, the nearest-neighbor distance distributions between inelastic events, and the distance distributions for all activations for both iodine radionuclides were calculated. The absorbed fractions of the initial electron energies, absorbed doses and energy depositions, and single-event distributions, F₁(ϵ), inside the six water spheres described above and in the surrounding tissue were also calculated. The absorbed doses per decay inside the six water spheres, i.e., the calculated S values (listed from 0.01 to 3.0 mm), were 6.8 × 10^–4, 7.2 × 10^–5, 5.5 × 10^–6, 4.9 × 10^–7, 3.1 × 10^–8 and 1.8 × 10^–9 Gy Bq^–1 s^–1 for ¹³¹I, and 3.4 × 10^–3, 1.7 × 10^–4, 5.1 × 10^–6, 2.0 × 10^–7, 5.6 × 10^–9 and 2.2 × 10^–10 Gy Bq^–1 s^–1 for ¹²⁵I. It is concluded that, in the treatment of thyroid cancer, the geometrical track structure properties of ¹²⁵I might be superior to those of ¹³¹I in micrometastases with diameters less than 0.1 mm; however, in this medical context, many other factors also have to be considered.

Vijayalaxmi, Bisht, K. S., Pickard, W. F., Meltz, M. L., Roti Roti, J. L. and Moros, E. G. Chromosome Damage and Micronucleus Formation in Human Blood Lymphocytes Exposed In Vitro to Radiofrequency Radiation at a Cellular Telephone Frequency (847.74 MHz, CDMA). Radiat. Res. 156, 430–432 (2001).

Peripheral blood samples collected from four healthy nonsmoking human volunteers were diluted with tissue culture medium and exposed in vitro for 24 h to 847.74 MHz radiofrequency (RF) radiation (continuous wave), a frequency employed for cellular telephone communications. A code division multiple access (CDMA) technology was used with a nominal net forward power of 75 W and a nominal power density of 950 W/m² (95 mW/cm²). The mean specific absorption rate (SAR) was 4.9 or 5.5 W/kg. Blood aliquots that were sham-exposed or exposed in vitro to an acute dose of 1.5 Gy of γ radiation were included in the study as controls. The temperatures of the medium during RF-radiation and sham exposures in the Radial Transmission Line facility were controlled at 37 ± 0.3°C. Immediately after the exposures, lymphocytes were cultured at 37 ± 1°C for 48 or 72 h. The extent of genetic damage was assessed from the incidence of chromosome aberrations and micronuclei. The kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation-exposed and sham-exposed lymphocytes with respect to mitotic indices, frequencies of exchange aberrations, excess fragments, binucleate cells, and micronuclei. The response of γ-irradiated lymphocytes was significantly different from that of both RF-radiation-exposed and sham-exposed cells for all of these indices. Thus there was no evidence for induction of chromosome aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 847.74 MHz RF radiation (CDMA) at SARs of 4.9 or 5.5 W/kg.