Yiin, J. H., Schubauer-Berigan, M. K., Silver, S. R., Daniels, R. D., Kinnes, G. M., Zaebst, D. D., Couch, J. R., Kubale, T. L. and Chen, P-H. Risk of Lung Cancer and Leukemia from Exposure to Ionizing Radiation and Potential Confounders among Workers at the Portsmouth Naval Shipyard. Radiat. Res. 163, 603–613 (2005).

Significantly elevated lung cancer deaths and statistically significantly positive linear trends between leukemia mortality and radiation exposure were reported in a previous analysis of Portsmouth Naval Shipyard workers. The purpose of this study was to conduct a modeling-based analysis that incorporates previously unanalyzed confounders in exploring the exposure–response relationship between cumulative external ionizing radiation exposure and mortality from these cancers among radiation-monitored workers in this cohort. The main analyses were carried out with Poisson regression fitted with maximum likelihood in linear excess relative risk models. Sensitivity analyses varying model components and using other regression models were conducted. The positive association between lung cancer risk and ionizing radiation observed previously was no longer present after adjusting for socioeconomic status (smoking surrogate) and welding fume and asbestos exposures. Excesses of leukemia were found to be positively, though not significantly, associated with external ionizing radiation, with or without including potential confounders. The estimated excess relative risk was 10.88% (95% CI −0.90%, 38.77%) per 10 mSv of radiation exposure, which was within the ranges of risk estimates in previous epidemiological studies (−4.1 to 19.0%). These results are limited by many factors and are subject to uncertainties of the exposure and confounder estimates.

Mendonca, M. S., Mayhugh, B. M., McDowell, B., Chin-Sinex, H., Smith, M. L., Dynlacht, J. R., Spandau, D. F. and Lewis, D. A. A Radiation-Induced Acute Apoptosis Involving TP53 and BAX Precedes the Delayed Apoptosis and Neoplastic Transformation of CGL1 Human Hybrid Cells. Radiat. Res. 163, 614–622 (2005).

Exposing CGL1 (HeLa × fibroblast) hybrid cells to 7 Gy of X rays results in the onset of a delayed apoptosis in the progeny of the cells 10 to 12 cell divisions postirradiation that correlates with the emergence of neoplastically transformed foci. The delayed apoptosis begins around day 8 postirradiation and lasts for 11 days. We now demonstrate that the delayed apoptosis is also characterized by the appearance of ∼50-kb apoptotic DNA fragments and caspase 3 activation postirradiation. In addition, we confirm that stabilization of TP53 and transactivation of pro-apoptosis BAX also occurs during the delayed apoptosis and show that anti-apoptosis BCL-X_L is down-regulated. To test whether the delayed apoptosis was due to a nonfunctional acute TP53 damage response in CGL1 cells, studies of acute apoptosis were completed. After irradiation, CGL1 cells underwent an acute wave of apoptosis that involves TP53 stabilization, transactivation of BAX gene expression, and a rapid caspase activation that ends by 96 h postirradiation. In addition, the acute onset of apoptosis correlates with transactivation of a standard wild-type TP53-responsive reporter (pG13-CAT) in CGL1 cells after radiation exposure. We propose that the onset of the delayed apoptosis is not the result of a nonfunctional acute TP53 damage response pathway but rather is a consequence of X-ray-induced genomic instability arising in the distant progeny of the irradiated cells.

Franco, N., Lamartine, J., Frouin, V., Le Minter, P., Petat, C., Leplat, J.-J., Libert, F., Gidrol, X. and Martin, M. T. Low-Dose Exposure to γ Rays Induces Specific Gene Regulations in Normal Human Keratinocytes. Radiat. Res. 163, 623–635 (2005).

Skin is the organ most exposed to various environmental aggressors, including ionizing radiation. Low-dose and low-dose-rate exposures to γ rays account for most occupational, medical or environmental irradiations. To examine whether this type of exposure triggers specific molecular responses, cultured primary keratinocytes isolated from adult normal skin were irradiated with single acute doses of 1 cGy or 2 Gy. DNA microarrays containing 10,500 probes were used to assess transcriptional changes over a time course between 3 and 72 h postirradiation. Keratinocytes were studied at a differentiated stage to mimic the response of cells from the suprabasal layers of the epidermis. A major finding of this study was the identification of an important number of low-dose-specific genes (140), most of which were modulated at 48 h. Clustering analysis also revealed low-dose-specific profiles. One of these clusters (17 known genes) was further analyzed using Gibbs sampling algorithm, which led to the identification of 7 putative promoter sequences. These results show for the first time that low-dose ionizing radiation is able to induce specific transcriptional responses in human keratinocytes. Our findings support the potential usefulness of microarrays in biological dosimetry studies after low-dose exposures.

Philippo, H., van der Kogel, A. J., Winter, E. A. M. and Huiskamp, R. Recovery Capacity of Glial Progenitors after In Vivo Fission-Neutron or X Irradiation: Age Dependence, Fractionation and Low-Dose-Rate Irradiations. Radiat. Res. 163, 636–643 (2005).

Previous experiments on the radiosensitivity of O-2A glial progenitors determined for single-dose fission-neutron and X irradiation showed log-linear survival curves, suggesting a lack of accumulation of recovery of sublethal damage. In the present study, we addressed this question and further characterized the radiobiological properties of these glial stem cells by investigating the recovery capacity of glial stem cells using either fractionated or protracted whole-body irradiation. Irradiations were performed on newborn, 2-week-old or 12-week-old rats. Fractionated irradiations (four fractions) were performed with 24-h intervals, followed by cell isolations 16– 24 h after the last irradiation. Single-dose irradiations were followed by cell isolation 16–24 h after irradiation or delayed cell isolation (4 days after irradiation) of the O-2A progenitor cells from either spinal cord (newborns) or optic nerve (2- and 12-week-old rats). Results for neonatal progenitor cell survival show effect ratios for both fractionated fission-neutron and X irradiation of the order of 1.8 when compared with single-dose irradiation. A similar ratio was found after single-dose irradiation combined with delayed plating. Comparable results were observed for juvenile and adult optic nerve progenitors, with effect ratios of the order of 1.2. The present investigation clearly shows that fractionated irradiation regimens using X rays or fission neutrons and CNS tissue from rats of various ages results in an increase in O-2A progenitor cell survival while repair is virtually absent. This recovery of the progenitor pool after irradiation can be observed at all ages but is greatest in the neonatal spinal cord and can probably be attributed to repopulation.

Chen, J. K., Hu, L. J., Wang, J., Lamborn, K. R., Kong, E. L. and Deen, D. F. Hypoxia-Induced BAX Overexpression and Radiation Killing of Hypoxic Glioblastoma Cells. Radiat. Res. 163, 644–653 (2005).

One major challenge in treating glioblastoma multiforme (GBM) has been the presence of radiation-resistant hypoxic cells. The pro-apoptosis protein BAX has been reported to be a possible radiation sensitizer of cancer cells; however, to our knowledge, no studies have reported on the effects of BAX on radiation sensitivity under hypoxic conditions. Therefore, in this study, we specifically overexpressed murine Bax in hypoxic cells in an attempt to enhance radiation-induced cell killing. We have previously stably transfected U-251 MG and U-87 MG human GBM cells with constructs containing murine Bax under the regulation of nine copies of hypoxia-responsive elements (HREs). During hypoxia, the transcriptional complex hypoxia-inducible factor 1 (HIF1) forms and binds to HRE; this binding facilitates the transcription of downstream genes. In the experiments reported here, two protocols were used. In the first protocol, parent and clone cells were exposed to graded doses of X rays under hypoxic conditions, left hypoxic for 0, 4, 16 or 24 h, and then assayed for clonogenic cell survival. In the second protocol, cells were incubated under hypoxic conditions for 20 h, irradiated with graded doses under hypoxia, then left in hypoxic conditions for 4 h before being assayed for clonogenic cell survival. Western blots showed that we had successfully increased Bax expression in both U-251 MG and U-87 MG Bax clone cells after 16 h of hypoxic incubation, yet dose–response curves showed no difference in radiation-induced cell killing between control non-Bax-expressing pNeo clone cells and the U-251 MG Bax clone cells using either protocol. In U-87 MG cells, the first protocol showed no difference in radiation response between control pNeo clone cells and the Bax clone, similar to the results obtained in U-251 cells. However, the second protocol revealed that Bax overexpression did render these cells more sensitive to radiation under hypoxic conditions. Therefore, we conclude that whether Bax is a radiation enhancer under hypoxia not only is cell line-dependent but also depends on when the Bax overexpression occurs.

Collins, C., Zhou, X., Wang, R., Barth, M. C., Jiang, T., Coderre, J. A. and Dedon, P. C. Differential Oxidation of Deoxyribose in DNA by γ and α-Particle Radiation. Radiat. Res. 163, 654–662 (2005).

Emerging evidence points to the importance of deoxyribose oxidation in the toxicity of oxidative DNA damage, including the formation of protein-DNA crosslinks and base adducts. With the goal of understanding the differences in deoxyribose oxidation chemistry known to occur with different oxidants, we have compared the formation of one product of 3′-oxidation of deoxyribose in DNA, 3′-phosphoglycolaldehyde (PGA) residues, in isolated DNA and cells exposed to ionizing radiations. A recently developed gas chromatography/negative chemical ionization mass spectrometry method was used to quantify PGA residues in purified DNA and in human TK6 lymphoblastoid cells exposed to γ radiation (⁶⁰Co) and α particles (²⁴¹Am). The level of PGA residues was then correlated with the total quantity of deoxyribose oxidation determined by plasmid topoisomer analysis. Alpha-particle irradiation (0–100 Gy) of purified DNA in 50 mM potassium phosphate (pH 7.4) produced a linear dose response of 0.13 PGA residues per 10⁶ nucleotides per gray. When normalized to an estimate of the total number of deoxyribose oxidation events (2.0 per 10⁶ nucleotides per gray), PGA formation occurred in 7% (±0.5) of deoxyribose oxidation events produced by α-particle radiation. In contrast, the efficiency of PGA formation in γ-irradiated DNA was found to be 1% (±0.02), which indicates a shift in the chemistry of deoxyribose oxidation, possibly as a result of the different track structures of the two types of ionizing radiation. Studies with γ radiation were extended to TK6 cells, in which it was observed that γ radiation produced a linear dose response of 0.0019 PGA residues per 10⁶ nucleotides per gray. This is consistent with an approximately 1000-fold quenching effect in cells, similar to the results of other published studies of oxidative DNA damage in vivo.

Tokdemir, S. and Nelson, W. H. Radiation-Induced Hydroxyl Addition to Purine Molecules: EPR and ENDOR Study of Hypoxanthine Hydrochloride Monohydrate Single Crystals. Radiat. Res. 163, 663–672 (2005).

Three radical species were detected in an EPR/ENDOR study of X-irradiated hypoxanthine·HCl·H₂O single crystals at room temperature: RI was identified as the product of net H addition to C8, RII was identified as the product of net H addition to C2, and RIII was identified as the product of OH addition to C8. The observed set of radicals was the same for room-temperature irradiation as for irradiation at 10 K followed by warming the crystals to room temperature; however, the C2 H-addition and C8 OH-addition radicals were not detectable after storage of the crystals for about 2 months at room temperature. Use of selectively deuterated crystals permitted unique assignment of the observed hyperfine couplings, and results of density functional theory calculations on each of the radical structures were consistent with the experimental results. Comparison of these experimental results with others from previous crystal-based systems and model system computations provides insight into the mechanisms by which the biologically important purine C8 hydroxyl addition products are formed. The evidence from solid systems supports the mechanism of net water addition to one-electron oxidized purine bases and demonstrates the importance of a facial approach between the reactants.

Nelson, W. H. Dose–Response Relationships for Radicals Trapped in Irradiated Solids. Radiat. Res. 163, 673–680 (2005).

This work develops a modified description of the dose–response relationships for radical production by ionizing radiation. The main new feature is incorporation of the concept of the target and the possibility that the population of targets can undergo depletion at increased doses. The resulting mathematical relationships are capable of describing the decrease in product yield at increasing doses as is sometime observed. In addition, the description preserves the general properties of the widely used relationships. Finally, the expressions provide the possibility for estimating the average mass of the target.

Jakob, B., Rudolph, J. H., Gueven, N., Lavin, M. F. and Taucher-Scholz, G. Live Cell Imaging of Heavy-Ion-Induced Radiation Responses by Beamline Microscopy. Radiat. Res. 163, 681–690 (2005).

To study the dynamics of protein recruitment to DNA lesions, ion beams can be used to generate extremely localized DNA damage within restricted regions of the nuclei. This inhomogeneous spatial distribution of lesions can be visualized indirectly and rapidly in the form of radiation-induced foci using immunocytochemical detection or GFP-tagged DNA repair proteins. To analyze faster protein translocations and a possible contribution of radiation-induced chromatin movement in DNA damage recognition in live cells, we developed a remote-controlled system to obtain high-resolution fluorescence images of living cells during ion irradiation with a frame rate of the order of seconds. Using scratch replication labeling, only minor chromatin movement at sites of ion traversal was observed within the first few minutes of impact. Furthermore, time-lapse images of the GFP-coupled DNA repair protein aprataxin revealed accumulations within seconds at sites of ion hits, indicating a very fast recruitment to damaged sites. Repositioning of the irradiated cells after fixation allowed the comparison of live cell observation with immunocytochemical staining and retrospective etching of ion tracks. These results demonstrate that heavy-ion radiation-induced changes in subnuclear structures can be used to determine the kinetics of early protein recruitment in living cells and that the changes are not dependent on large-scale chromatin movement at short times postirradiation.