Phillips, T. L. 50 Years of Radiation Research: Medicine. Radiat. Res. 158, 389–417 (2002).

The advances brought about by research in radiation medicine over the past 50 years are presented. The era began with the atomic explosions in Hiroshima and Nagasaki and the establishment of the Atomic Bomb Casualty Commission to understand what damage was caused by exposure of a large population to radiation. A better understanding of the effects of whole-body exposure led to the development of whole-body radiation treatment techniques and to bone marrow transplantation in the treatment of leukemias. The field of diagnostic imaging was revolutionized by a series of inventions that included angiography, mammography, computed tomography, magnetic resonance imaging, magnetic resonance spectroscopy, and ultrasound imaging. The field of nuclear medicine came of age through new man-made radionuclides and the invention of scanning and imaging techniques including positron emission tomography. Radiotherapy, a minor sideline of radiology, developed into radiation oncology, an extremely important component of modern cancer therapy. The advances in clinical radiotherapy were made possible by discoveries and inventions in physics and engineering and by insights and discoveries in radiobiology. The result of the last 50 years of progress is a very powerful set of clinical tools.

Holmberg, E., Wallgren, A., Holm, L-E., Lundell, M. and Karlsson, P. Dose–Response Relationship for Parathyroid Adenoma after Exposure to Ionizing Radiation in Infancy. Radiat. Res. 158, 418–423 (2002).

Several authors have suggested that there is an excess risk of hyperparathyroidism, adenomas or hyperplasia after exposure to ionizing radiation. There is still, however, some uncertainty about this association, because these diseases are often asymptomatic and escape clinical detection if not specially searched for. This study is based on a pooled Swedish cohort of 27,925 persons with skin hemangiomas. The majority received radiation treatment in infancy between 1920 and 1965 in Stockholm and Gothenburg. The mean age at treatment was 6 months and the median thyroid dose was 0.20 Gy (range 0–28.5 Gy). Record linkage with the Swedish Cancer Register for the period 1958–1997 gave 43 cases of parathyroid adenoma in the cohort. Analyses of excess relative risk (ERR) models were performed using Poisson regression methods. Clinical records were scrutinized to determine if the childhood radiation exposure was known (biased cases) at the time of diagnosis. Seven of the cases of parathyroid adenoma were classified as biased cases. The standardized incidence ratio (SIR) was 2.10 (95% confidence interval 1.52–2.82) when all cases were included and 1.76 (95% CI 1.23–2.43) with the biased cases excluded. A linear dose–response model with stratification for sex fitted the data best. The ERR per gray was 3.84 (95% CI 1.56–8.99) with all cases and 1.56 (95% CI 0.36–4.45) with the biased cases excluded. There was a significant difference in the ERR per gray between the two subcohorts, probably because of different diagnostic activity in the regions. Our findings confirm that there is a dose–response relationship for radiation-induced parathyroid adenomas.

Jones, I. M., Galick, H., Kato, P., Langlois, R. G., Mendelsohn, M. L., Murphy, G. A., Pleshanov, P., Ramsey, M. J., Thomas, C. B., Tucker, J. D., Tureva, L., Vorobstova, I. and Nelson, D. O. Three Somatic Genetic Biomarkers and Covariates in Radiation-Exposed Russian Cleanup Workers of the Chernobyl Nuclear Reactor 6–13 Years after Exposure. Radiat. Res. 158, 424–442 (2002).

Three somatic mutation assays were evaluated in men exposed to low-dose, whole-body, ionizing radiation. Blood samples were obtained between 1992 and 1999 from 625 Russian Chernobyl cleanup workers and 182 Russian controls. The assays were chromosome translocations in lymphocytes detected by FISH, hypoxanthine phosphoribosyltransferase (HPRT) mutant frequency in lymphocytes by cloning, and flow cytometic assay for glycophorin A (GPA) variant frequency of both deletion (N/Ø) and recombination (N/N) events detected in erythrocytes. Over 30 exposure and lifestyle covariates were available from questionnaires. Among the covariates evaluated, some increased (e.g. age, smoking) and others decreased (e.g. date of sample) biomarker responses at a magnitude comparable to Chernobyl exposure. When adjusted for covariates, exposure at Chernobyl was a statistically significant factor for translocation frequency (increase of 30%, 95% CI of 10%–53%, P = 0.002) and HPRT mutant frequency (increase of 41%, 95% CI of 19%–66%, P < 0.001), but not for either GPA assay. The estimated average dose for the cleanup workers based on the average increase in translocations was 9.5 cGy. Translocation analysis is the preferred biomarker for low-dose radiation dosimetry given its sensitivity, relatively few covariates, and dose–response data. Based on this estimated dose, the risk of exposure-related cancer is expected to be low.

Boreham, D. R., Dolling, J-A., Misonoh, J. and Mitchel, R. E. J. Teratogenic Effects of Mild Heat Stress during Mouse Embryogenesis: Effect of Trp53. Radiat. Res. 158, 443–448 (2002).

Hyperthermia can be teratogenic in fetal mice exposed during organogenesis, an effect considered to be due to heat-induced apoptosis of cells in the developing organs. We exposed pregnant mice carrying Trp53 ^/ , Trp53 ^/− and Trp53^−/− fetuses to mild whole-body hyperthermia that raised their core temperature to 40.5°C for 60 min on either day 10 or 11 of gestation. On day 18 of gestation, the fetuses were removed from control and hyperthermia-treated mice and genotyped, and tail length was measured. Limb digits were examined for abnormalities. Tail length in unheated control fetuses was influenced by Trp53 status. A complete lack of functional Trp53 (Trp53^−/−) but not partial lack of function (Trp53 ^/−) resulted in shorter tails compared to Trp53 ^/ fetuses, indicating a role for Trp53 in the regulation of tail lengthening in mouse fetuses. In all three genotypes, hyperthermia on gestation day 10 resulted in tails shorter than unheated controls, and hyperthermia on day 11 resulted in tails longer than controls. There was no effect on limb digit abnormalities. The data suggest that Trp53-dependent or independent apoptosis may not be directly involved in heat-induced teratogenesis, but that the primary teratogenic effect of heat results from the disruption of another tail length-regulating process that is independent of Trp53. However, the nature of the teratogenic outcome of that disruption depends on the gestation time. The ability of Trp53 to additionally regulate the tail lengthening process was also sensitive to the effects of heat, but that sensitivity again depended on the time of the heat stress during gestation.

Boreham, D. R., Dolling, J-A., Misonoh, J. and Mitchel, R. E. J. Radiation-Induced Teratogenic Effects in Fetal Mice with Varying Trp53 Function: Influence of Prior Heat Stress. Radiat. Res. 158, 449–457 (2002).

Teratogenesis induced by radiation in fetal mice has been closely linked to Trp53-dependent apoptosis. This study examined teratogenesis in tails and limb digits of fetal mice with varying Trp53 status after a 4-Gy radiation exposure, with and without a prior 40.5°C, 60-min heat stress. Irradiation earlier in gestation (day 11) produced greater effects than later (day 12) exposure, but in both cases the maximum teratogenic effect of radiation occurred in Trp53 normal fetuses, the minimum in Trp53 null fetuses, and intermediate effects in Trp53 heterozygotes, indicating dominance of Trp53-dependent apoptosis. Heat stress 24 h prior to irradiation on day 11 did not alter the teratogenic effects in Trp53 normal or heterozygous fetuses, but it reduced effects in the Trp53 null fetuses. Conversely, heat stress immediately before irradiation on day 11 amplified teratogenesis in Trp53 null fetuses, still with only a small or no effect on fetuses with full or partial Trp53 function, respectively. These results indicate little effect of mild heat on Trp53-dependent apoptosis after irradiation, but they also suggest heat-induced amplification of Trp53-independent processes that led to apoptosis when heat was delivered near the time of radiation exposure, and heat-induced protection of that process when sufficient expression time was allowed. However, Trp53-dependent apoptosis, when functional, acted as the ultimate determinant of radiation-induced teratogenic effects during early organogenesis. On gestation day 12, radiation effects were diminished, but heat stress 24 h prior to radiation exposure had a large amplifying effect in Trp53 normal or heterozygous fetuses. In the absence of functional Trp53, the sensitizing effect of the heat was diminished. The results may suggest that at later times in organ development, DNA repair is more active, allowing some cells to escape radiation-induced Trp53-dependent apoptosis. However, heat may be able to significantly inhibit this active repair and increase the teratogenic effect of radiation. A diminished effect in the absence of functional Trp53 is consistent with an influence of heat on inhibiting DNA repair, but with a diminished probability of apoptosis.

Mitchel, R. E. J., Dolling, J-A., Misonoh, J. and Boreham, D. R. Influence of Prior Exposure to Low-Dose Adapting Radiation on Radiation-Induced Teratogenic Effects in Fetal Mice with Varying Trp53 Function. Radiat. Res. 158, 458–463 (2002).

Teratogenesis in tails and limb digits of fetal mice with varying Trp53 status was examined after exposure of pregnant females to 4 Gy γ radiation with and without a prior 30-cGy exposure. Prior low-dose exposure modified the teratogenic effects of radiation in a manner dependent upon Trp53 status and gestation time. A 4-Gy exposure on gestation day 11 resulted in tail shortening and digit abnormalities. A 30-cGy exposure 24 h prior to a 4-Gy radiation exposure on day 11 reduced the extent of both digit abnormalities and the tail-shortening effects in Trp53 ^/ fetuses and also reduced tail shortening in Trp53 ^/− fetuses, but to a lesser extent. However, the pre-exposure enhanced the tail-shortening effects of 4 Gy in Trp53^−/− fetuses. In contrast, a 30-cGy exposure given 24 h prior to a 4-Gy exposure on gestation day 12 had no effect on the reduced tail length resulting from the 4-Gy exposure of Trp53 ^/ or Trp53 ^/− fetuses, but it partly protected Trp53^−/− fetuses against reduced tail length. A 4-Gy exposure alone on day 12 did not result in any increase in the frequency of digit abnormalities in Trp53^−/− fetuses so any protective effect of the preirradiation could not be detected. However, the preirradiation did result in protection against in digit abnormalities in Trp53 ^/− fetuses. We conclude that radiation-induced teratogenesis reflects both Trp53-dependent and independent processes that lead to apoptosis, and these respond differently to prior adapting doses.

Benderitter, M., Durand, V., Caux, C. and Voisin, P. Clearance of Radiation-Induced Apoptotic Lymphocytes: Ex Vivo Studies and an In Vitro Co-culture Model. Radiat. Res. 158, 464–474 (2002).

Lymphocytes are very sensitive to radiation. Our aim was to test the possibility of detecting apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure. Our in vitro data confirmed the dose–time–effect relationships involved in radiation-induced apoptosis. The detection of in vivo induction of apoptosis in circulating lymphocytes after exposure of animals to radiation appears to depend critically on the technique used to measure apoptosis. Among the different techniques we investigated, mitochondrial modification was the most appropriate; they allowed establishment of dose–time–effect relationships when animals were observed for 72 h. A model of in vitro phagocytosis of apoptotic lymphocytes by macrophages was developed to mimic clearance of apoptotic cells occurring in vivo. Together, our data show that mitochondrial labeling may make it possible to detect ex vivo radiation-induced apoptosis of lymphocytes before macrophage ingestion occurs. We propose the measurement of apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure.

Green, L. M., Tran, D. T., Murray, D. K., Rightnar, S. S., Todd, S. and Nelson, G. A. Response of Thyroid Follicular Cells to Gamma Irradiation Compared to Proton Irradiation: II. The Role of Connexin 32. Radiat. Res. 158, 475–485 (2002).

The objective of this study was to determine whether connexin 32-type gap junctions contribute to the “contact effect” in follicular thyrocytes and whether the response is influenced by radiation quality. Our previous studies demonstrated that early-passage follicular cultures of Fischer rat thyroid cells express functional connexin 32 gap junctions, with later-passage cultures expressing a truncated nonfunctional form of the protein. This model allowed us to assess the role of connexin 32 in radiation responsiveness without relying solely on chemical manipulation of gap junctions. The survival curves generated after γ irradiation revealed that early-passage follicular cultures had significantly lower values of α (0.04 Gy⁻¹) than later-passage cultures (0.11 Gy⁻¹) (P < 0.0001, n = 12). As an additional way to determine whether connexin 32 was contributing to the difference in survival, cultures were treated with heptanol, resulting in higher α values, with early-passage cultures (0.10 Gy⁻¹) nearly equivalent to untreated late-passage cultures (0.11 Gy⁻¹) (P > 0.1, n = 9). This strongly suggests that the presence of functional connexin 32-type gap junctions was contributing to radiation resistance in γ-irradiated thyroid follicles. Survival curves from proton-irradiated cultures had α values that were not significantly different whether cells expressed functional connexin 32 (0.10 Gy⁻¹), did not express connexin 32 (0.09 Gy⁻¹), or were down-regulated (early-passage plus heptanol, 0.09 Gy⁻¹; late-passage plus heptanol, 0.12 Gy⁻¹) (P > 0.1, n = 19). Thus, for proton irradiation, the presence of connexin 32-type gap junctional channels did not influence their radiosensitivity. Collectively, the data support the following conclusions. (1) The lower α values from the γ-ray survival curves of the early-passage cultures suggest greater repair efficiency and/or enhanced resistance to radiation-induced damage, coincident with the expression of connexin 32-type gap junctions. (2) The increased sensitivity of FRTL-5 cells to proton irradiation was independent of their ability to communicate through connexin 32 gap junctions. (3) The fact that the β components of the survival curves from both γ rays and proton beams were similar (average 0.022 ± 0.008 Gy^–2, P > 0.1, n = 39) suggests that at higher doses the loss of viability occurs at a relatively constant rate and is independent of radiation quality and the presence of functional gap junctions.

Sedelnikova, O. A., Rogakou, E. P., Panyutin, I. G. and Bonner, W. M. Quantitative Detection of ¹²⁵IdU-Induced DNA Double-Strand Breaks with γ-H2AX Antibody. Radiat. Res. 158, 486–492 (2002).

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (γ-H2AX) demonstrates that γ-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of ¹²⁵I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of ¹²⁵IdU and processed immunocytochemically to determine the number of γ-H2AX foci. The numbers of ¹²⁵IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of ¹²⁵I decays and the number of foci per cell, consistent with the assumptions that each ¹²⁵I decay yields a DNA DSB and each DNA DSB yields a visible γ-H2AX focus. Based on these findings, we conclude that γ-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.

Harris, R. E. and Pimblott, S. M. On ³H β-Particle and ⁶⁰Co γ Irradiation of Aqueous Systems. Radiat. Res. 158, 493–504 (2002).

The chemistry of water and aqueous solutions is very different after irradiation with ³H β particles and high-energy electrons or ⁶⁰Co γ rays. The greater the linear energy transfer (LET) of the medium for ³H β particles compared to high-energy electrons or ⁶⁰Co γ rays leads to an increased local concentration of reactants. There is an increased amount of intratrack chemistry, which reduces the escape yield of e_aq⁻ and OH by about 50%, but increases the yield of H₂ by about 50% and of H₂O₂ by about 35%. Analysis of stochastic-diffusion kinetic calculations employing simulated track structures reveals that the yield of H₂ produced by diffusion-kinetic processes increases significantly for ³H β particles compared to ⁶⁰Co γ radiation, while production of H₂ by sub-picosecond processes is essentially the same. In both ³H β-particle and ⁶⁰Co γ radiolysis, the reactions (e_aq⁻ e_aq⁻) and (e_aq⁻ H) are equally important in the production of H₂. In the former case, each reaction has a yield of ∼0.18, and in the latter a yield of ∼0.08. In neutral water, the reaction (H H) is negligible. The yield of Fe(III) in ³H β-particle radiolysis of the Fricke dosimeter is much smaller than in radiolysis with more energetic electrons. Simulations show that this change is primarily due to the reduced escape yield of H, formed from the scavenging of e_aq⁻ by the bulk H₃O of the acid. The chemical differences observed in experiments, and in calculations, reflect the underlying structure of the electron tracks: Examination of the track structure simulations demonstrates that primary events are considerably more well-separated in high-energy electron tracks compared to ³H β-particle tracks.

Regulla, D., Schmid, E., Friedland, W., Panzer, W., Heinzmann, U. and Harder, D. Enhanced Values of the RBE and H Ratio for Cytogenetic Effects Induced by Secondary Electrons from an X-Irradiated Gold Surface. Radiat. Res. 158, 505–515 (2002).

The low-energy secondary electrons emerging from the entrance surface of an X-irradiated gold foil increase the dose to cells in contact with or at micrometer distances from this surface (Radiat. Res. 150, 92–100, 1998). We examined the effect of the spectrum of these low-energy electrons on the RBE for cytogenetic effects and showed that this RBE was increased. A monolayer of surface-attached human T lymphocytes was exposed to 60 kV X rays in the absence or presence of a gold foil positioned immediately behind the cell layer or separated from it by a Mylar foil 0.9 or 2 μm thick. The enhancement of dose in the cell nuclei caused by the photoelectrons and Auger electrons emerging from the entrance surface of the gold foil was measured by TSEE dosimetry. Dose enhancement factors of 55.7, 46.6 and 37.5 were obtained with 0, 0.9 and 2 μm of Mylar inserted between the gold surface and the cell layer. This large enhancement results from the photoelectric effect in the gold foil, as shown by the accompanying Monte Carlo calculations of the secondary electron spectra at the gold surface. Auger electrons from the gold foil generally were not able to penetrate into the cell nuclei except for that fraction of the cells that had a very thin (< 0.7 μm) layer of cytoplasm and membranes between gold surface and cell nucleus. The dose–yield curves for dicentric chromosomes plus centric rings and for acentric fragments obtained after exposures without or with the gold foil were linear-quadratic. The coefficient α, the slope of the linear yield component, was increased in the presence of the gold foil and showed RBE values ranging from 1.7 to 2.2 compared to exposures in absence of the gold foil. The ratio of the yield of interstitial deletions and dicentrics (H ratio) was significantly increased from about 0.17 in the absence of the gold foil to about 0.22 in the presence of the gold foil. The increases in the RBE and the H ratio are interpreted in microdosimetric terms: The preferred occurrence of electron track ends in the vicinity of the gold surface causes an increase in the dose-mean restricted linear energy transfer in cell nuclei exposed to the photoelectrons and Auger electrons.

Pompa, P. P., Nassisi, V. and Alifano, P. Scattering Phenomena Effects on Growth of 308-nm Laser-Irradiated Bacteria in Suspension. Radiat. Res. 158, 516–522 (2002).

In this study we analyzed the effect of 308-nm laser exposure on recovery of irradiated Staphylococcus epidermidis held in liquid after irradiation and before plating. Coexistence of bacterial growth inhibition and stimulation phenomena was observed. Under certain conditions, bacterial recovery was about fivefold higher in irradiated samples than in the controls. The available evidence suggests that the growth inhibition was due to the bactericidal activity of the 308-nm wavelength light, whereas the growth stimulation effect was associated with broadband radiation generated by scattering phenomena in the bacterial suspensions. Spectroscopic investigations revealed that the nutrient broth plays a decisive role in the scattering of laser radiation within the suspension.

McNamee, J. P., Bellier, P. V., Gajda, G. B., Miller, S. M., Lemay E. P., Lavallée, B. F., Marro, L. and Thansandote, A. DNA Damage and Micronucleus Induction in Human Leukocytes after Acute In Vitro Exposure to a 1.9 GHz Continuous-Wave Radiofrequency Field. Radiat. Res. 158, 523–533 (2002).

Human blood cultures were exposed to a 1.9 GHz continuous-wave (CW) radiofrequency (RF) field for 2 h using a series of six circularly polarized, cylindrical waveguides. Mean specific absorption rates (SARs) of 0.0, 0.1, 0.26, 0.92, 2.4 and 10 W/kg were achieved, and the temperature within the cultures during a 2-h exposure was maintained at 37.0 ± 0.5°C. Concurrent negative (incubator) and positive (1.5 Gy ¹³⁷Cs γ radiation) control cultures were run for each experiment. DNA damage was quantified immediately after RF-field exposure using the alkaline comet assay, and four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. No evidence of increased primary DNA damage was detected by any parameter for RF-field-exposed cultures at any SAR tested. The formation of micronuclei in the RF-field-exposed blood cell cultures was assessed using the cytokinesis-block micronucleus assay. There was no significant difference in the binucleated cell frequency, incidence of micronucleated binucleated cells, or total incidence of micronuclei between any of the RF-field-exposed cultures and the sham-exposed controls at any SAR tested. These results do not support the hypothesis that acute, nonthermalizing 1.9 GHz CW RF-field exposure causes DNA damage in cultured human leukocytes.

McNamee, J. P., Bellier, P. V., Gajda, G. B., Lavallée, B. F., Lemay, E. P., Marro, L. and Thansandote, A. DNA Damage in Human Leukocytes after Acute In Vitro Exposure to a 1.9 GHz Pulse-Modulated Radiofrequency Field. Radiat. Res. 158, 534–537 (2002).

Blood cultures from human volunteers were exposed to an acute 1.9 GHz pulse-modulated radiofrequency (RF) field for 2 h using a series of six circularly polarized, cylindrical waveguides. Mean specific absorption rates (SARs) ranged from 0 to 10 W/kg, and the temperature within the cultures during the exposure was maintained at 37.0 ± 0.5°C. DNA damage was quantified in leukocytes by the alkaline comet assay and the cytokinesis-block micronucleus assay. When compared to the sham-treated controls, no evidence of increased primary DNA damage was detected by any parameter for any of the RF-field-exposed cultures when evaluated using the alkaline comet assay. Furthermore, no significant differences in the frequency of binucleated cells, incidence of micronucleated binucleated cells, or total incidence of micronuclei were detected between any of the RF-field-exposed cultures and the sham-treated control at any SAR tested. These results do not support the hypothesis that acute, nonthermalizing 1.9 GHz pulse-modulated RF-field exposure causes DNA damage in cultured human leukocytes.

Box, H. C., Patrzyc, H. B., Dawidzik, J. B., Iijima, H., Freund, H. G., Higbee, A. J. and Budzinski, E. E. Detection and Characterization of Formamido Lesions in DNA by Liquid Chromatography-Mass Spectrometry. Radiat. Res. 158, 538–542 (2002).

DNA X-irradiated in oxygenated aqueous solution produces the formamido lesion from the breakdown of pyrimidine nucleosides. This pyrimidine breakdown product inhibits the hydrolysis by nuclease P1 of the phosphoester bond 3′ to the damaged nucleoside. Consequently, the lesion can be obtained from an enzymatic digest of the DNA as a modified dinucleoside monophosphate in which the 5′ nucleoside contains the lesion. In this form, the formamido lesion can be detected with good sensitivity by liquid chromatography-mass spectrometry (LC-MS). Nucleosides that have lost the base moiety also inhibit nuclease P1. Together, the formamido and abasic lesions account for all of the substantial peaks in the LC-MS ion current profile.