Morgan, W. Non-targeted and Delayed Effects of Exposure to Ionizing Radiation: I. Radiation-Induced Genomic Instability and Bystander Effects In Vitro. Radiat. Res. 159, 567–580 (2003).

A long-standing dogma in the radiation sciences is that energy from radiation must be deposited in the cell nucleus to elicit a biological effect. A number of non-targeted, delayed effects of ionizing radiation have been described that challenge this dogma and pose new challenges to evaluating potential hazards associated with radiation exposure. These effects include induced genomic instability and non-targeted bystander effects. The in vitro evidence for non-targeted effects in radiation biology will be reviewed, but the question as to how one extrapolates from these in vitro observations to the risk of radiation-induced adverse health effects such as cancer remains open.

Morgan, W. F. Non-targeted and Delayed Effects of Exposure to Ionizing Radiation: II. Radiation-Induced Genomic Instability and Bystander Effects In Vivo, Clastogenic Factors and Transgenerational Effects. Radiat. Res. 159, 581–596 (2003).

The goal of this review is to summarize the evidence for non-targeted and delayed effects of exposure to ionizing radiation in vivo. Currently, human health risks associated with radiation exposures are based primarily on the assumption that the detrimental effects of radiation occur in irradiated cells. Over the years a number of non-targeted effects of radiation exposure in vivo have been described that challenge this concept. These include radiation-induced genomic instability, bystander effects, clastogenic factors produced in plasma from irradiated individuals that can cause chromosomal damage when cultured with nonirradiated cells, and transgenerational effects of parental irradiation that can manifest in the progeny. These effects pose new challenges to evaluating the risk(s) associated with radiation exposure and understanding radiation-induced carcinogenesis.

Kawata, T., Ito, H., George, K., Wu, H., Uno, T., Isobe, K. and Cucinotta, F. A. Radiation-Induced Chromosome Aberrations in Ataxia Telangiectasia Cells: High Frequency of Deletions and Misrejoining Detected by Fluorescence In Situ Hybridization. Radiat. Res. 159, 597–603 (2003).

The mechanisms underlying the hyper-radiosensitivity of AT cells were investigated by analyzing chromosome aberrations in the G₂ and M phases of the cell cycle using a combination of chemically induced premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) with chromosome painting probes. Confluent cultures of normal fibroblast cells (AG1522) and fibroblast cells derived from an individual with AT (GM02052) were exposed to γ rays and allowed to repair at 37°C for 24 h. At doses that resulted in 10% survival, GM02052 cells were approximately five times more sensitive to γ rays than AG1522 cells. For a given dose, GM02052 cells contained a much higher frequency of deletions and misrejoining than AG1522 cells. For both cell types, a good correlation was found between the percentage of aberrant cells and cell survival. The average number of color junctions, which represent the frequency of chromosome misrejoining, was also found to correlate well with survival. However, in a similar surviving population of GM02052 and AG1522 cells, induced by 1 Gy and 6 Gy, respectively, AG1522 cells contained four times more color junctions and half as many deletions as GM02052 cells. These results indicate that both repair deficiency and misrepair may be involved in the hyper-radiosensitivity of AT cells.

Fletcher, L., Yen, T. J. and Muschel, R. J. DNA Damage in HeLa Cells Induced Arrest at a Discrete Point in G₂ Phase as Defined by CENP-F Localization. Radiat. Res. 159, 604–611 (2003).

G₂ is defined as the time in the cell cycle after DNA synthesis is complete but before the initiation of mitosis. However, as the molecular events of the cell cycle are described, G₂ can be seen to be a sequence of events rather than a static phase. For example, CENP-F increases in amount in early G₂, after DNA synthesis is complete, but localizes to the nuclear rim and then to the kinetochores before mitosis commences. After DNA damage cells may arrest in G₂ through TP53-dependent and independent mechanisms, raising the question of the precise position of the arrest within G₂. HeLa cells lack functional TP53; this allowed us to examine the TP53-independent mechanism of G₂ arrest. Here we showed that the DNA damage-induced G₂ arrest in HeLa cells is positioned in early G₂, before redistribution of CENP-F to the nuclear envelope and kinetochores, and before chromosome condensation commences.

Miederer, M., Seidl, C., Beyer, G-J., Charlton, D. E., Vranjes-Duric, S., Comor, J. J., Huber, R., Nikula, T., Apostolidis, C., Schuhmacher, C., Becker, K-F. and Senekowitsch-Schmidtke, R. Comparison of the Radiotoxicity of Two Alpha-Particle- Emitting Immunoconjugates, Terbium-149 and Bismuth-213, Directed against a Tumor-Specific, Exon 9 Deleted (d9) E-Cadherin Adhesion Protein. Radiat. Res. 159, 612–620 (2003).

We investigated the effects of the α-particle emitters ¹⁴⁹Tb and ²¹³Bi coupled to a tumor-specific antibody targeting the mutated delta 9 E-cadherin (d9 E-Cad) on single cells and cell pellets. The d9 mutation of the adhesion molecule E-cadherin is found in 10% of diffuse-type gastric cancers and is not expressed in normal tissue. Human breast cancer cells (MDA-MB-435S) transfected with d9 E-Cad or the wild-type E-cadherin gene were used to study the effects of anti-d9 E-Cad MAb coupled to ¹⁴⁹Tb and ²¹³Bi (¹⁴⁹Tb-d9 MAb and ²¹³Bi-d9 MAb). The density of binding sites determined on transfected MDA tumor cells by Scatchard analysis and flow cytometry varied from 4 × 10⁴ to 6 × 10⁴ antigens per cell. Internalization of radioimmunoconjugates by cells expressing d9 E-Cad was less than 10% of bound antibody within 240 min. The effect of the radioimmunoconjugates on cell suspensions and cell pellets was quantified by [³H]thymidine incorporation, and the dose to the cell nuclei was determined using microdosimetric calculations. ¹⁴⁹Tb and ²¹³Bi immunoconjugates affected cells in suspension similarly. Significant differences in the proliferation capacity of d9 E-cadherin- and wild-type E-cadherin-expressing cells were observed at activity concentrations around 185 kBq/ml, corresponding to antibody concentrations between 200 ng/ml and 1000 ng/ml. Proliferation after incubation with ²¹³Bi-d9 MAb was 50% greater in pelleted wild-type E-Cad-expressing cells compared to wild-type E-Cad cells in suspension. In contrast, the proliferation of pelleted d9 E-Cad cells was similar to that of d9 E-Cad cells in suspension. For ¹⁴⁹Tb-d9 MAb, no significant difference was found between pelleted cells and cells in suspension for low activity concentrations. However, at high activity concentrations, ¹⁴⁹Tb-d9 MAb had only a small effect on pelleted cells. These in vitro studies demonstrate different effects of ¹⁴⁹Tb and ²¹³Bi conjugated to a tumor-specific antibody toward single cells and tumor cell pellets. Microdosimetric simulation of single cell survival after α-particle irradiation modeled the experimental results with reasonable accuracy.

Zhao, D., Constantinescu, A., Chang, C. H., Hahn, E. W. and Mason, R. P. Correlation of Tumor Oxygen Dynamics with Radiation Response of the Dunning Prostate R3327-HI Tumor. Radiat. Res. 159, 621–631 (2003).

Our previous studies have shown that oxygen inhalation significantly reduces tumor hypoxia in the moderately well-differentiated HI subline of the Dunning prostate R3327 rat carcinoma. To test our hypothesis that modifying hypoxia could improve the radiosensitivity of these tumors, we performed experimental radiotherapy to compare the tumor response to ionizing radiation alone or in combination with oxygen inhalation. Tumor pO₂ measurements were performed on size-selected tumors several hours before radiotherapy using ¹⁹F nuclear magnetic resonance echo planar imaging relaxometry (FREDOM) of the reporter molecule hexafluorobenzene. In common with our previous findings, the larger tumors (>3.5 cm³) exhibited greater hypoxia than the smaller tumors (<2 cm³; P < 0.001), and oxygen inhalation reduced the hypoxic fraction (<10 Torr): In the larger tumors, hypoxic fraction dropped significantly from a mean baseline value of 80% to 17% (P < 0.001). The effect of oxygen administered 30 min before and during irradiation on tumor response to a single 30-Gy dose of photons was evaluated by growth delay. For the smaller tumors, no difference in growth delay was found when treatment was given with or without oxygen breathing. By contrast, breathing oxygen before and during irradiation significantly enhanced the growth delay in the larger tumors (additional 51 days). The differential behavior may be attributed to the low baseline hypoxic fraction (<10 Torr) in small tumors (20%) as a target for oxygen inhalation. There was a strong correlation between the estimated initial pO₂ value and the radiation-induced tumor growth delay (R > 0.8). Our histological studies showed a good match between the perfused vessels marked by Hoechst 33342 dye and the total vessels immunostained by anti-CD31 and indicated extensive perfusion in this tumor line. In summary, the present results suggest that the ability to detect modulation of tumor pO₂, in particular, the residual hypoxic fraction, with respect to an intervention, could have prognostic value for predicting the efficacy of radiotherapy.

Dilmanian, F. A., Morris, G. M., Zhong, N., Bacarian, T., Hainfeld, J. F., Kalef-Ezra, J., Brewington, L. J., Tammam, J. and Rosen, E. M. Murine EMT-6 Carcinoma: High Therapeutic Efficacy of Microbeam Radiation Therapy. Radiat. Res. 159, 632–641 (2003).

Microbeam radiation therapy is an experimental modality using parallel arrays of thin (<100 μm) slices of synchrotron-generated X rays (microplanar beams, microbeams). We used EMT-6 murine mammary carcinoma subcutaneously inoculated in the hind legs of mice to compare the therapeutic efficacies of single-fraction, unidirectional (1) “co-planar” microbeams (an array of vertically oriented microplanar beams), (2) “cross-planar” microbeams (two arrays of parallel microbeams propagated in the same direction, one with vertically and the other with horizontally oriented microplanar beams), and (3) seamless (broad) beams from the same synchrotron source. The microbeams were 90 μm wide and were spaced 300 μm on center; the median energy in all beams was 100 or 118 keV. Tumor ablation rates were 4/8, 4/8 and 6/7 for a 410-, 520- and 650-Gy in-slice cross-planar microbeam dose, respectively, and 1/8, 3/8, 3/7 and 6/8 for a 23-, 30-, 38- and 45-Gy broad-beam dose, respectively. When the data were pooled from the three highest doses (same average tumor ablations of 50–60%), the incidences of normal-tissue acute toxicity (moist desquamation and epilation) and delayed toxicity (failure of hair regrowth) were significantly lower for cross-planar microbeams than broad beams (P < 0.025). Furthermore, for the highest doses in these two groups, which also had the same tumor ablation rate (>75%), not only were the above toxicities lower for the cross-planar microbeams than for the broad beams (P < 0.02), but severe leg dysfunction was also lower (P < 0.003). These findings suggest that single-fraction microbeams can ablate tumors at high rates with relatively little normal-tissue toxicity.

Van den Aardweg, G. J. M. J., Olofsen-van Acht, M. J. J., van Hooije, C. M. C. and Levendag, P. C. Radiation-Induced Rectal Complications are not Influenced by Age: A Dose Fractionation Study in the Rat. Radiat. Res. 159, 642–650 (2003).

Radiation-induced complications of the rectum are an important dose-limiting factor in radiotherapy of pelvic malignancies. In general, animal studies demonstrated no differences in acute and late normal tissue toxicity with age, but little is known about rectal complications in relation to age. For this purpose, an extensive histological and dose fractionation study was carried out on the rectum of young (12 weeks) and older (77–80 weeks) rats. In this paper, the results of dose fractionation are presented in relation to age at the time of irradiation. Young and older animals were irradiated with single and fractionated doses. After irradiation, rectal complications could lead to occlusion and stenosis, eventually resulting in the clinical symptoms of a megacolon and a possible fistula. For each dose group, cumulative survival rates were obtained with Kaplan-Meier analysis, from which dose–effect curves and the associated LD₅₀ values for a megacolon/fistula were calculated. The majority of responders died between 8 and 24 weeks after irradiation, irrespective of age. For both age groups, only the fractionation data showed a reduction in the mean latency with increasing dose. In the older age group, 39% of the responders developed a fistula compared to 26% for the younger animals. The LD₅₀ values increased from around 30 Gy after single doses to nearly 65 Gy after 10 fractions. The increases in LD₅₀ values with the number of fractions were independent of the age of the rats. For each of the dose fractionation schedules, log-rank testing indicated no significant differences in cumulative survival rates between younger and older animals (P > 0.10). The high α/β ratios obtained for both the young and older animals strongly suggested that the late rectal complications were a consequence of early epithelial injury. Associated histological findings indicated that blood vessel damage, which was already evident at a high incidence at 4 weeks after irradiation, could also play a significant role in the occurrence of consequential late injuries. In conclusion, data obtained for the latent period of rectal occlusion, for the dose–effect curves, for the log-rank testing of cumulative survival rates, and for the α/β ratios strongly support the hypothesis that the incidence of radiation-induced rectal complications is independent of age. Late rectal complications could be a consequence of radiation-induced acute injury.

Kiuru, A., Auvinen, A., Luokkamäki, M., Makkonen, K., Veidebaum, T., Tekkel, M., Rahu, M., Hakulinen, T., Servomaa, K., Rytömaa, T. and Mustonen, R. Hereditary Minisatellite Mutations among the Offspring of Estonian Chernobyl Cleanup Workers. Radiat. Res. 159, 651–655 (2003).

A single accidental event such as the fallout released from the Chernobyl reactor in 1986 can expose millions of people to non-natural environmental radiation. Ionizing radiation increases the frequency of germline mutations in experimental studies, but the genetic effects of radiation in humans remain largely undefined. To evaluate the hereditary effects of low radiation doses, we compared the minisatellite mutation rates of 155 children born to Estonian Chernobyl cleanup workers after the accident with those of their siblings born prior to it. All together, 94 de novo paternal minisatellite mutations were found at eight tested loci (52 and 42 mutants among children born after and before the accident, respectively). The minisatellite mutation rate was nonsignificantly increased among children born after the accident (0.042 compared to 0.036, OR 1.33, 95% CI 0.80–2.20). Furthermore, there was some indication of an increased mutation rate among offspring born after the accident to workers who had received doses of 20 cSv or above compared with their siblings born before the accident (OR 3.0, 95% CI 0.97–9.30). The mutation rate was not associated with the father's age (OR 1.04, 95% CI 0.94–1.15) or the sex of the child (OR 0.95, 95% CI 0.50–1.79). Our results are consistent with both no effect of radiation on minisatellite mutations and a slight increase at dose levels exceeding 20 cSv.

Heidenreich, W. F., Nyberg, U. and Hall, P. A Biologically Based Model for Liver Cancer Risk in the Swedish Thorotrast Patients. Radiat. Res. 159, 656–662 (2003).

Data on liver tumors among 416 Swedish patients who were exposed to Thorotrast between 1930 and 1950 were analyzed with the biologically based two-step clonal expansion (TSCE) model. For background data, the Swedish Cancer Register for the follow-up period 1958 to 1997 was used. Effects of radiation on the initiating mutation and on the clonal expansion rate explained the observed patterns well. The TSCE model permits the deduction of several kinetic parameters of the postulated tumorigenesis process. Dose rates of 5 mGy/year double the spontaneous initiation rate. The clonal expansion rate is doubled by 80 mGy/year, and for females it reaches a plateau at dose rates beyond 240 mGy/year. For males the plateau is not significant. The magnitude of the estimated promoting effect of radiation can be explained with a moderate increase in the cell replacement probability for the intermediate cells in the liver, which is strikingly similar to the situation in lung tumorigenesis.

Razskazovskiy, Y., Debije, M. G. and Bernhard, W. A. Strand Breaks Produced in X-Irradiated Crystalline DNA: Influence of Base Sequence. Radiat. Res. 159, 663–669 (2003).

This study reports the radiation-chemical yields for DNA single-strand breaks (SSBs) in crystals of CGCACG:CGTGCG (I) and CACGCG:CGCGTG (II) duplexes induced by direct ionization using X rays. The DNA fragmentation products, consisting of 3′- and 5′-phosphate-terminated fragments, were quantified by ion-exchange chromatography using a set of reference compounds. The yields of single-strand breaks in I and II are 0.16 ± 0.03 μmol/J and 0.07 ± 0.02 μmol/J, respectively. The probability of cleavage at a given site is relatively independent of which of the four bases is at that site. For the very small sample of base sequences studied to date, there is no obvious dependence on base sequence. However, there appears to be an increased frequency of strand breaks at the non-phosphorylated termini of the oligodeoxynucleotides. These results show that direct ionization is efficient at producing single-strand breaks in DNA and that its action is relatively indiscriminate with respect to base sequence.

Moritake, T., Tsuboi, K., Anzai, K., Ozawa, T., Ando, K. and Nose, T. ESR Spin Trapping of Hydroxyl Radicals in Aqueous Solution Irradiated with High-LET Carbon-Ion Beams. Radiat. Res. 159, 670–675 (2003).

The aim of this study was to quantify the hydroxyl radicals (^·OH) produced when aqueous solutions are decomposed by high-linear energy transfer (LET) 290 MeV/nucleon carbon-ion beams using an electron spin resonance (ESR) spectrometer. Aerated cell culture medium containing 200 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was irradiated with doses of 0 to 20 Gy with an LET of 20 to 90 keV/μm. We were able to obtain ESR spectra 10 min after irradiation, and the formation of ^·OH and hydrogen atoms was confirmed by radiolysis of deuterium oxide and ethanol containing DMPO. Our results showed that the yield of ^·OH by carbon-ion radiolysis increased in proportion to the absorbed dose over the range of 0 to 20 Gy. Furthermore, we discovered that the yield of ^·OH decreased linearity as LET increased logarithmically from 20 to 90 keV/μm. The generation of ^·OH by carbon-ion radiolysis at LETs of 20, 40, 60, 80 and 90 keV/μm was 64, 58, 52, 49 and 50%, respectively, of that for low-LET X radiolysis. These unique findings provide a further understanding of the indirect effect of high-LET radiation.

Jakob, B., Scholz, M. and Taucher-Scholz, G. Biological Imaging of Heavy Charged-Particle Tracks. Radiat. Res. 159, 676–684 (2003).

The immunocytochemical response to DNA damage induced by low-energy bismuth and carbon ions was investigated in normal human fibroblasts. Inside the nuclei, the traversing charged particles lead to the accumulation of proteins related to DNA lesions and repair along the ion trajectories. Irradiation under a standard geometric setup with the beam direction perpendicular to the cell monolayer generates spots of these proteins as described previously for MRE11B (hMre11), CDKN1A (p21) and PCNA (Jakob et al., Int. J. Radiat. Biol. 78, 75–88, 2002). Here we present data obtained with a new irradiation geometry characterized by a small angle between the beam direction and the monolayer of cells. This new irradiation geometry leads to the formation of protein aggregates in the shape of streaks stretching over several micrometers in the x/y plane, thus facilitating the analysis of the fluorescence distributions along the particle trajectories. Measurements of fluorescence intensity along the ion tracks in double- and triple-stained samples revealed a strict spatial correlation for the occurrence of CDKN1A and MRE11B clusters. In addition, immunostained γ-H2AX is used as a marker of double-strand breaks (DSBs) to visualize the localized induction of these lesions along the particle paths. A clear coincidence of CDKN1A and γ-H2AX signals within the ion-induced streaks is observed. Also for PCNA, which mainly associates with lesions processed by excision repair, a strict colocalization with the MRE11B aggregations was found along the ion trajectories, despite the higher estimated yield of this type of lesions compared to DSBs. Strikingly similar patterns of protein clusters are generated not only for the various proteins studied but also using different ion species from carbon to bismuth, covering LET values ranging from about 300 to 13600 keV/μm and producing estimated DSB densities differing by a factor around 45. The patterns of protein clustering along the very heavy-ion trajectories appear far more heterogeneous than expected based on idealized DSB distributions arising from model calculations. The results suggest that additional factors like compaction or confined movement of chromatin are responsible for the observed clustering of proteins.

Thalau, H. P., Raczek, J., Marx, B., Hombach, V. and Cooper, J. Temperature Changes in Chicken Embryos Exposed to a Continuous-Wave 1.25 GHz Radiofrequency Electromagnetic Field. Radiat. Res. 159, 685–692 (2003).

A total of 550 fertile chicken eggs (White Leghorn) were exposed to a radiofrequency (RF) electromagnetic field of 1.25 GHz (continuous wave) at six different power flux densities in the range of 9.0–0.75 mW/cm². The eggs were exposed either continuously throughout the whole 21 days of incubation (long-term exposure) or in a short-term exposure (1–2 h/day). The temperatures of the embryonic tissue and the amniotic fluid, respectively, were measured with inserted temperature probes. This study was designed to investigate the relationship between exposure and temperature changes in exposed tissues, without considering biological and medical effects. This knowledge is of general interest for studies of nonthermic teratological or embryo-lethal effects of exposure to electromagnetic fields (EMFs). Throughout the entire 21 days of embryonic development, the mean temperature increases in the eggs during the exposure were found to be up to 0.25°C for a power flux density of 1.25 mW/cm² and increased to 2.3°C for 9.0 mW/cm². The corresponding maximum whole-body SARs for the embryos over the 21 days of embryonic development were 1.45 and 10.44 W/kg, respectively. At 0.75 mW/cm² (0.87 W/kg) the extent of the RF-field induced hyperthermia was within the measurement accuracy (±0.1°C) of the temperature probes used in the tests. The field-induced temperature increase was greatest in the first week of incubation and was less pronounced in the last (third) week before hatching. In both the short- and the long-term exposures, the temperature of the exposed tissue and the amniotic fluid, respectively, reached its maximum (asymptotic) approximately 40–50 min after the RF field was switched on. After the field was switched off, the temperature inside the exposed eggs returned to its initial value within 40–50 min.

McNamee, J. P., Bellier, P. V., Gajda, G. B., Lavallée, B. F., Marro, L., Lemay, E. and Thansandote, A. No Evidence for Genotoxic Effects from 24 h Exposure of Human Leukocytes to 1.9 GHz Radiofrequency Fields. Radiat. Res. 159, 693–697 (2003).

The current study extends our previous investigations of 2-h radiofrequency (RF)-field exposures on genotoxicity in human blood cell cultures by examining the effect of 24-h continuous-wave (CW) and pulsed-wave (PW) 1.9 GHz RF-field exposures on both primary DNA damage and micronucleus induction in human leukocyte cultures. Mean specific absorption rates (SARs) ranged from 0 to 10 W/kg, and the temperature within the cultures was maintained at 37.0 ± 1.0°C for the duration of the 24-h exposure period. No significant differences in primary DNA damage were observed between the sham-treated controls and any of the CW or PW 1.9 GHz RF-field-exposed cultures when processed immediately after the exposure period by the alkaline comet assay. Similarly, no significant differences were observed in the incidence of micronuclei, incidence of micronucleated binucleated cells, frequency of binucleated cells, or proliferation index between the sham-treated controls and any of the CW or PW 1.9 GHz RF-field-exposed cultures. In conclusion, the current study found no evidence of 1.9 GHz RF-field-induced genotoxicity in human blood cell cultures after a 24-h exposure period.