Yamada, M., Wong, F. L., Fujiwara, S., Akahoshi, M. and Suzuki, G. Noncancer Disease Incidence in the Atomic Bomb Survivors, 1958–1998. Radiat. Res. 161, 622–632 (2004).

We examined the relationships between the incidence of noncancer diseases and atomic bomb radiation dose using the longitudinal data for about 10,000 Adult Health Study (AHS) participants during 1958–1998. The current report updates the analysis we presented in 1993 with 12 additional years of follow-up. In addition to the statistically significant positive linear dose–response relationships detected previously for the incidence of thyroid disease (P < 0.0001), chronic liver disease and cirrhosis (P = 0.001), and uterine myoma (P < 0.00001), we also found a significant positive dose response for cataract (P = 0.026), a negative linear dose–response relationship for glaucoma (P = 0.025), and significant quadratic dose–response relationships for hypertension (P = 0.028) and for myocardial infarction among survivors exposed at less than 40 years of age (P = 0.049). Significant radiation effects for calculus of the kidney and ureter were evident for men but not for women (test of heterogeneity by sex: P = 0.007). Accounting for smoking and drinking did not alter the results. Radiation effects for cataract, glaucoma, hypertension, and calculus of the kidney and ureter in men are new findings. These results attest to the need for continued follow-up of the aging A-bomb survivors to fully elucidate the effects of radiation exposure on the occurrence of noncancer diseases.

Zablotska, L. B., Ashmore, J. P. and Howe, G. R. Analysis of Mortality among Canadian Nuclear Power Industry Workers after Chronic Low-Dose Exposure to Ionizing Radiation. Radiat. Res. 161, 633–641 (2004).

Studies of radiation-associated risks among workers chronically exposed to low doses of radiation are important, both to estimate risks directly and to assess the adequacy of extrapolations of risk estimates from high-dose studies. This paper presents results based on a cohort of 45,468 nuclear power industry workers from the Canadian National Dose Registry monitored for more than 1 year for chronic low-dose whole-body ionizing radiation exposures sometime between 1957 and 1994 (mean duration of monitoring = 7.4 years, mean cumulative equivalent dose = 13.5 mSv). The excess relative risks for leukemia [excluding chronic lymphocytic leukemia (CLL)] and for all solid cancers were 52.5 [95% confidence interval (CI): 0.205, 291] and 2.80 (95% CI: −0.038, 7.13) per sievert, respectively, both associations having P values close to 0.05. Relative risks by dose categories increased monotonically for leukemia excluding CLL but were less consistent for all solid cancers combined. Although the point estimates are higher than those found in other studies of whole-body irradiation, the difference could well be due to chance. Further follow-up of this cohort or the combination of results from multiple worker studies will produce more stable estimates and thus complement the risk estimates from higher-dose studies.

Suzuki, G., Shimada, Y., Hayashi, T., Akashi, M., Hirama, T. and Kusunoki, Y. An Association between Oxidative Stress and Radiation-Induced Lymphomagenesis. Radiat. Res. 161, 642–647 (2004).

It is generally thought that reactive oxygen species (ROS) play an important role in carcinogenesis. However, direct evidence supporting this idea is still lacking. In the present study, we measured ROS in thymocytes at the thymic prelymphoma stage in C57BL/6 mice. Mice (n = 20) were irradiated at 1.6 Gy/week for 4 consecutive weeks and the levels of ROS were measured 8 to 11 weeks later by dehydrorhodamine 123, which accumulated in mitochondria and became fluorescent dye upon oxidation. Unirradiated littermates (n = 17) served as controls. Thymic prelymphoma cells were diagnosed by the aberrant CD4/CD8 staining profile and monoclonal or oligoclonal T-cell receptor gene rearrangement. A significant fraction of mice (11/13) bearing thymic prelymphoma cells exhibited elevated levels of ROS in thymocytes (P < 0.001). The result is consistent with the hypothesis that ROS may play an important role in radiation carcinogenesis.

Tucker, J. D., Marples, B., Ramsey, M. J. and Lutze-Mann, L. H. Persistence of Chromosome Aberrations in Mice Acutely Exposed to ⁵⁶Fe ²⁶ Ions. Radiat. Res. 161, 648–655 (2004).

Space exploration has the potential to yield exciting and significant discoveries, but it also brings with it many risks for flight crews. Among the less well studied of these are health effects from space radiation, which includes the highly charged, energetic particles of elements with high atomic numbers that constitute the galactic cosmic rays. In this study, we demonstrated that 1 Gy iron ions acutely administered to mice in vivo resulted in highly complex chromosome damage. We found that all types of aberrations, including dicentrics as well as translocations, insertions and acentric fragments, disappear rapidly with time after exposure, probably as a result of the death of heavily damaged cells, i.e. cells with multiple and/or complex aberrations. In addition, numerous cells have apparently simple exchanges as their only aberrations, and these cells appear to survive longer than heavily damaged cells. Eight weeks after exposure, the frequency of cells showing cytogenetic damage was reduced to less than 20% of the levels evident at 1 week, with little further decline apparent over an additional 8 weeks. These results indicate that exposure to 1 Gy iron ions produces heavily damaged cells, a small fraction of which appear to be capable of surviving for relatively long periods. The health effects of exposure to high-LET radiation in humans on prolonged space flights should remain a matter of concern.

Hicks, K. O., Siim, B. G., Pruijn, F. B. and Wilson, W. R. Oxygen Dependence of the Metabolic Activation and Cytotoxicity of Tirapazamine: Implications for Extravascular Transport and Activity in Tumors. Radiat. Res. 161, 656–666 (2004).

The hypoxic cytotoxin tirapazamine (TPZ) is currently in phase III clinical trial and appears to have clinical activity. One hypothesis as to why TPZ has been used more successfully in the clinic than most other bioreductive drugs is that its unusual O₂ dependence allows killing of radioresistant cells at “intermediate” O₂ concentrations. We have determined the O₂ dependence of the metabolism of TPZ to its reduction product SR 4317, and its cytotoxicity, in stirred suspensions of HT29 colon carcinoma cells while monitoring O₂ in solution with an Oxylite™ probe. The O₂ dependence of the cytotoxicity of TPZ is entirely accounted for by its inhibition of the metabolism of TPZ, with a K_O2 value (O₂ concentration for 50% inhibition) of 1.21 ± 0.09 (SEM) μM. We used this experimental O₂ dependence to extend a recent (Hicks et al., Cancer Res. 63, 5970–5977, 2003) pharmacokinetic/pharmacodynamic model for the cytotoxicity of TPZ in anoxic HT29 multicellular layers to model cell killing in tumors. The model indicates that the O₂ dependence of killing by TPZ complements that of radiation well during fractionated radiotherapy. It predicts that lowering K_O2 would decrease killing in radioresistant cells at intermediate O₂ concentrations, while higher K_O2 values would exacerbate metabolic consumption of TPZ and thus further impede its penetration into hypoxic regions. Raising K_O2 would also increase metabolic activation at physiological O₂ concentrations, thereby compromising hypoxic selectivity. We conclude that the K_O2 value of TPZ is indeed close to the optimum for a bioreductive drug of this class (i.e. one that kills only cells in which it is reduced).

Zhang, Y., Jung, M., Dritschilo, A. and Jung, M. Enhancement of Radiation Sensitivity of Human Squamous Carcinoma Cells by Histone Deacetylase Inhibitor. Radiat. Res. 161, 667–674 (2004).

Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents with potential for disruption of critical cellular processes in cancer cells. Transcriptional regulation, differentiation, cell cycle arrest, radiation sensitization, and apoptosis have been observed in response to exposure to HDAC inhibitors. In the present study, we observed that several potent HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, M344 (an analogue of hydroxamic acid), and the cyclic tetrapeptide, depsipeptide (FR90228), modulate cellular responses to ionizing radiation in cells of two human squamous carcinoma lines (SQ-20B and SCC-35), previously characterized as intrinsically resistant to radiation. Also exposure to IC₅₀ concentrations of these inhibitors, radiation sensitivities were enhanced in both cell lines. Depsipeptide exhibited the greatest effect on SQ-20B cells, decreasing D₀ values from 2.62 Gy to 1.64 Gy. M344 was the most active drug in sensitizing SCC-35 cells, decreasing D₀ values from 1.91 Gy to 1.21 Gy. The mechanisms underlying HDAC inhibitor-induced radiosensitization were further investigated by extending trichostatin A studies to assess cell cycle distributions and levels of apoptosis. Treatment of SQ-20B cells with radiosensitizing concentrations of trichostatin A resulted in cell cycle arrest in G₁ phase (>70%) and inhibition of DNA synthesis. Contrary to previous reports, induction of apoptosis was very low and caspase 3 and 9 were not activated. Taken together, these results implicate G₁ arrest and inhibition of DNA synthesis in the mechanisms underlying radiation sensitization by trichostatin A and support the use of HDAC inhibitors for targeting radioresistant cancers.

Fournier, C., Wiese, C. and Taucher-Scholz, G. Accumulation of the Cell Cycle Regulators TP53 and CDKN1A (p21) in Human Fibroblasts after Exposure to Low- and High-LET Radiation. Radiat. Res. 161, 675–684 (2004).

The accumulation of the cell cycle regulators TP53 and CDKN1A (p21/CIP1/WAF1) was investigated after exposure to X rays and carbon ions (170 keV μm⁻¹) and xenon, bismuth and uranium ions (8900–15,000 keV μm⁻¹) in normal human fibroblasts. The influence of the overall dose and the LET of these radiation types was studied systematically and the kinetics of the cell response was followed up to 24 h after exposure. The accumulation of TP53 protein was dependent on the dose and the LET, and TP53 levels declined to lower levels for all radiation types within 24 h after exposure. CDKN1A levels increased and peaked at 3 to 6 h after exposure. The persisting level of this protein at 24 h was strongly dependent on the dose and the LET for X rays and carbon ions. The exposure to very high-LET ions (8900–15,000 keV μm⁻¹) did not lead to a further increase in CDKN1A, suggesting a saturation effect for the induction of this protein. The cellular effects of elevated CDKN1A after particle irradiation are discussed.

Oksvold, M. P., Thien, C. B. F, Widerberg, J., Chantry, A., Huitfeldt, H. S. and Langdon, W. Y. UV-Radiation-Induced Internalization of the Epidermal Growth Factor Receptor Requires Distinct Serine and Tyrosine Residues in the Cytoplasmic Carboxy-Terminal Domain. Radiat. Res. 161, 685–691 (2004).

The mechanism of UV-radiation-induced EGF receptor (EGFR) internalization remains to be established. In the present study, we found UV-radiation-mediated internalization of the EGFR to be dependent on the cytoplasmic carboxy-terminal region. UV radiation was unable to induce internalization of EGFR carboxy-terminal truncation mutants where all or four of the five major autophosphorylation sites were missing (963- and 1028-EGFR, respectively). Mutational removal of serine residues 1046, 1047, 1057 and 1142 within the carboxy-terminal receptor region was also sufficient to abolish UV-radiation-induced internalization of the EGFR. Furthermore, the UV-radiation-induced internalization was abrogated for an EGFR mutated in tyrosine 1045 (Y1045F), the major c-Cbl binding site. However, UV radiation did not induce phosphorylation at tyrosine 1045, in contrast to the prominent phosphorylation induced by EGF. Our results suggest a mechanism for UV-radiation-induced internalization of EGFR involving a conformational change that is dependent on structural elements formed by specific serine and tyrosine residues in the carboxy-terminal domain.

Gu, Q., Wang, D., Wang, X., Peng, R., Liu, J., Jiang, T., Wang, Z., Wang, S. and Deng, H. Basic Fibroblast Growth Factor Inhibits Radiation-Induced Apoptosis of HUVECs. I. The PI3K/AKT Pathway and Induction of Phosphorylation of BAD. Radiat. Res. 161, 692–702 (2004).

Radiation-induced endothelial cell apoptosis is involved in the development of many radiation injuries, including radiation-induced skin ulcers. The proangiogenic growth factors basic fibroblast growth factor (bFGF, NUDT6) and VEGF enhance endothelial cell survival. In the present study, we used primary cultured human umbilical vein endothelial cells (HUVECs) irradiated with ⁶⁰Co γ rays to explore the effects of bFGF on radiation-induced apoptosis of HUVECs and its signaling pathways. We found that bFGF inhibited radiation-induced apoptosis of HUVECs, and that the effect was mediated by the PI3K/AKT pathway. This pathway was activated by exposure of irradiated HUVECs to bFGF, involving phosphorylation of FGFR, PI3K and AKT. The survival-enhancing effect of bFGF was abrogated by wortmannin and LY294002. Transfection of a dominant-negative mutant of AKT completely blocked the anti-apoptosis effect of bFGF in irradiated HUVECs. We also found evidence for the first time that bFGF induced BAD phosphorylation in the γ-irradiated HUVECs. These results showed that the PI3K/AKT pathway participated in the bFGF-induced modulation of the survival of irradiated HUVECs. Activation of the PI3K/AKT pathway plays an important role in bFGF-induced endothelial cell survival in the treatment of radiation-induced skin ulcers.

Gu, Q., Wang, D., Wang, X., Peng, R., Liu, J., Deng, H., Wang, Z. and Jiang, T. Basic Fibroblast Growth Factor Inhibits Radiation-Induced Apoptosis of HUVECs. II. The RAS/ MAPK Pathway and Phosphorylation of BAD at Serine 112. Radiat. Res. 161, 703–711 (2004).

Radiation-induced endothelial cell apoptosis is involved in the development of many radiation injuries, including radiation-induced skin ulcers. The proangiogenic growth factor basic fibroblast growth factor (bFGF, NUDT6) enhances endothelial cell survival. In the present study, we set up a model of apoptosis in which primary cultured human umbilical vein endothelial cells (HUVECs) were irradiated with ⁶⁰Co γ rays to explore the effects of bFGF on radiation-induced apoptosis of HUVECs and the signaling pathways involved. We found that bFGF inhibited radiation-induced apoptosis of HUVECs, and that the effect was mediated in part by the RAS/MEK/ MAPK/RSK (p90 ribosomal S6 kinase)/BAD pathway. This pathway was activated by exposure of irradiated HUVECs to bFGF, involving phosphorylation of FGFR, MEK and p44/42 MAPK. The survival-enhancing effect of bFGF was partly inhibited by U0126 and PD98059. The fact that the anti-apoptosis effect of bFGF on irradiated HUVECs was not completely abrogated by U0126 and PD98059 suggests that other survival signaling pathways may exist. Transfection of a dominant-negative form of RSK2 (DN RSK2) partly blocked the anti-apoptosis effect of bFGF in irradiated HUVECs. Moreover, we provide evidence for the first time that bFGF induced BAD phosphorylation (at serine 112) and CREB (cAMP response element-binding protein) activation (phosphorylation at serine 133) in γ-irradiated HUVECs. In our model, inhibition of MAPK signaling-dependent phosphorylation of BAD at serine 112 promoted increased association with BCL-X_L, suggesting that MAPK pathway-dependent serine 112 phosphorylation of BAD is critical for the effect of bFGF on cell survival. These results showed that RAS/MAPK/BAD pathway participated in the bFGF-induced effect on survival of HUVECs exposed to radiation. It is suggested that RAS/ MAPK pathway in tumor vascular endothelium could be a potential therapeutic target to enhance the efficacy of ionizing radiation.

Lebaron-Jacobs, L., Wysocki, J. and Griffiths, N. M. Differential Qualitative and Temporal Changes in the Response of the Hypothalamus-Pituitary-Adrenal Axis in Rats after Localized or Total-Body Irradiation. Radiat. Res. 161, 712–722 (2004).

Stress such as exposure to ionizing radiation is able to activate the hypothalamus-pituitary-adrenal axis. The present study sought to examine the effects of different configurations of a 10-Gy γ irradiation in rats on the hypothalamus-pituitary-adrenal axis to understand the mechanism of negative feedback by glucocorticoids induced by ionizing radiation. Specifically, we determined adrenocorticotropin and corticosterone levels in plasma as well as corticotrophin-releasing factor expression in the paraventricular nucleus of the hypothalamus by in situ hybridization from 6 h to 4 days after total-body, abdominal or head irradiation. In this study, we found an activation of the hypothalamus-pituitary-adrenal axis after radiation exposure. Plasma adrenocorticotropin and corticosterone levels were significantly increased after total-body and abdominal irradiation 3 days after exposure, in parallel with decreased labeling of corticotrophin-releasing factor mRNA in the parvocellular region of the paraventricular nucleus of the hypothalamus. Our results suggest that ionizing radiation activates the neuroendocrine system to protect the organism from the occurrence of radiation-induced inflammation.

Huang, Z., Chen, Q., Trncic, N., LaRue, S. M., Brun, P., Wilson, B. C., Shapiro, H. and Hetzel, F. W. Effects of Pd-bacteriopheophorbide (TOOKAD)-Mediated Photodynamic Therapy on Canine Prostate Pretreated with Ionizing Radiation. Radiat. Res. 161, 723–731 (2004).

The aim of this study was to evaluate the effects of photodynamic therapy (PDT) using a novel palladium bacteriopherophorbide photosensitizer TOOKAD (WST09) on canine prostate that had been pretreated with ionizing radiation. To produce a physiological and anatomical environment in canine prostate similar to that in patients for whom radiotherapy has failed, canine prostates (n = 4) were exposed to ionizing radiation (54 Gy) 5 to 6 months prior to interstitial TOOKAD-mediated PDT. Light irradiation (763 nm, 50–200 J/cm at 150 mW/cm from a 1-cm cylindrical diffusing fiber) was delivered during intravenous infusion of TOOKAD at 2 mg/kg over 10 min. Interstitial measurements of tissue oxygen profile (pO₂) and of local light fluence rate were also measured. The prostates were harvested for histological examination 1 week after PDT. The baseline pO₂ of preirradiated prostate was in the range 10–44 mmHg. The changes in relative light fluence rate during PDT ranged from 12 to 43%. The acute lesions were characterized by hemorrhagic necrosis, clearly distinguishable from the radiotherapy-induced pre-existing fibrosis. The lesion size was correlated with light fluence and comparable to that in unirradiated prostate treated with a similar TOOKAD-PDT protocol. There was no noticeable damage to the urethra, bladder or adjacent colon. The preliminary results obtained from a small number of animals indicate that TOOKAD-PDT can effectively ablate prostate pretreated with ionizing radiation, and so it may provide an alternative modality for those prostate cancer patients for whom radiotherapy has failed.

Neti, P. V. S. V., de Toledo, S. M., Perumal, V., Azzam, E. I. and Howell, R. W. A Multi-port Low-Fluence Alpha-Particle Irradiator: Fabrication, Testing and Benchmark Radiobiological Studies. Radiat. Res. 161, 732–738 (2004).

A new multi-port irradiator, designed to facilitate the study of the effects of low fluences of α particles on monolayer cultures, has been developed. The irradiator consists of four individual planar ²⁴¹Am α-particle sources that are housed inside a helium-filled Lucite chamber. Three of the radioactive sources consist of 20 MBq of ²⁴¹Am dioxide foil. The fourth source, used to produce higher dose rates, has an activity of 500 MBq. The four sources are mounted on rotating turntables parallel to their respective 1.5-μm-thick Mylar exit windows. A stainless steel honeycomb collimator is placed between the four sources and their exit windows by a cantilever attachment to the platform of an orbital shaker that moves its table in an orbit of 2 cm. Each exit window is equipped with a beam delimiter to optimize the uniformity of the beam and with a high-precision electronic shutter. Opening and closing of the shutters is controlled with a high-precision timer. Custom-designed stainless steel Mylar-bottomed culture dishes are placed on an adapter on the shutter. The α particles that strike the cells have a mean energy of 2.9 MeV. The corresponding LET distribution of the particles has a mean value of 132 keV/μm. Clonogenic cell survival experiments with AG1522 human fibroblasts indicate that the RBE of the α particles compared to ¹³⁷Cs γ rays is about 7.6 for this biological end point.

Pond, C. D., Leachman, S. A. and Warters, R. L. Accumulation, Activation and Interindividual Variation of the Epidermal TP53 Protein in Response to Ionizing Radiation in Organ Cultured Human Skin. Radiat. Res. 161, 739–745 (2004).

In this study, we examined effects of low-dose ionizing radiation on organ cultured human foreskin and, in particular, on the epidermis. Diagnostic, therapeutic, natural environmental and incidental exposures to moderate to low doses of radiation are inevitable and, although information on cultured cells continues to accumulate, little is known about the effects of low-dose radiation on human tissues. Our hypothesis is that ex vivo organ cultured foreskin is a simple and reliable model to study the biochemical effects of low-dose radiation exposure on skin. A model such as this will aid in the identification and quantification of low-dose radiation-induced changes in proteins in human skin and may be useful in the development of a precise, non-invasive, and reliable assay of exposure. In this work, several aspects of skin responses to culture conditions and radiation were examined. The responses of epidermal TP53 from organ cultured skin irradiated in medium with and without serum were found to be similar. TP53 levels in organ cultured neonatal foreskin epidermis were then examined for baseline TP53 expression. After an initial increase at 4 h, the TP53 D01 signal returned to low steady-state levels for at least 72 h. Irradiated skin samples from different individuals revealed variations in the TP53 D01 signal. The dose and temporal response of dermis and epidermis to radiation were examined by Western blotting from 0 to 24 h after exposure. After irradiation and incubation, the epidermis was removed and assayed by Western blotting and was found to have increases in the TP53 D01 epitope and the TP53 phosphoserine 15 (TP53-S15p) epitope that reached a maximum at about 3 h. In the epidermis, doses of 1–5 cGy of radiation were detectable with the TP53 D01, and CDKN1A antibodies and doses greater than 10 cGy were detectable with the TP53-S15p antibody. When the dermis was compared to epidermis, it was found that dermis had a smaller response to radiation and more phosphorylated TP53.