Ljungkvist, A. S. E., Bussink, J., Kaanders, J. H. A. M. and van der Kogel, A. J. Dynamics of Tumor Hypoxia Measured with Bioreductive Hypoxic Cell Markers. Radiat. Res. 167, 127–145 (2007).

Hypoxic cells are common in tumors and contribute to malignant progression, distant metastasis and resistance to radiotherapy. It is well known that tumors are heterogeneous with respect to the levels and duration of hypoxia. Several strategies, including high-oxygen-content gas breathing, radiosensitizers and hypoxic cytotoxins, have been developed to overcome hypoxia-mediated radioresistance. However, with these strategies, an increased tumor control rate is often accompanied by more severe side effects. Consequently, development of assays for prediction of tumor response and early monitoring of treatment responses could reduce both over- and undertreatment, thereby avoiding unnecessary side effects. The purpose of this review is to discuss different assays for measurement of hypoxia that can be used to detect changes in oxygen tension. The main focus is on exogenous bioreductive hypoxia markers (2-nitroimidazoles) such as pimonidazole, CCI-103F, EF5 and F-misonidazole. These are specifically reduced and bind to macromolecules in viable hypoxic cells. A number of these bioreductive drugs are approved for clinical use and can be detected with methods ranging from noninvasive PET imaging (low resolution) to microscopic imaging of tumor sections (high resolution). If the latter are stained for multiple markers, hypoxia can be analyzed in relation to different microenvironmental parameters such as vasculature, proliferation and endogenous hypoxia-related markers, for instance HIF1α and CA-IX. In addition, temporal and spatial changes in hypoxia can be analyzed by consecutive injection of two different hypoxia markers. Therefore, bioreductive exogenous hypoxia markers are promising as tools for development of predictive assays or as tools for early treatment monitoring and validation of potential endogenous hypoxia markers.

Greene, K. F., Budzinski, E. E., Iijima, H., Davidzik, J. B., DeFedericis, H-C., Patrzyc, H. B., Evans, M. S., Bailey, D. T., Freund, H. F. and Box, H. C. Assessment of DNA Damage at the Dimer Level: Measurement of the Formamide Lesion. Radiat. Res. 167, 146–151 (2007).

UVC-radiation-induced DNA damage was measured in mouse fibroblast cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conjunction with isotopically labeled internal standards. The thymine glycol and formamide lesions were assayed in the form of modified dinucleoside monophosphates. The 8-oxo-7,8-dihydroguanine lesion was measured as the modified nucleoside. DNA damage in cells treated with tirapazamine was also measured. Tirapazamine is a chemotherapeutic agent that acts via a free radical mechanism. The two agents, UVC radiation and tirapazamine, produce markedly different profiles of DNA damage, reflecting their respective mechanisms of action. Both agents produce significant amounts of thymine glycol and formamide damage, but only the former produced a measurable amount of the 8-oxo-7,8-dihydroguanine lesion. The merits of measuring DNA damage at the dimer level are discussed.

Datta, K., Weinfeld, M., Neumann, R. D. and Winters, T.A. Determination and Analysis of Site-Specific ¹²⁵I Decay-Induced DNA Double-Strand Break End-Group Structures. Radiat. Res. 167, 152–166 (2007).

End groups contribute to the structural complexity of radiation-induced DNA double-strand breaks (DSBs). As such, end-group structures may affect a cell's ability to repair DSBs. The 3′-end groups of strand breaks caused by γ radiation, or oxidative processes, under oxygenated aqueous conditions have been shown to be distributed primarily between 3′-phosphoglycolate and 3′-phosphate, with 5′-phosphate ends in both cases. In this study, end groups of the high-LET-like DSBs caused by ¹²⁵I decay were investigated. Site-specific DNA double-strand breaks were produced in plasmid pTC27 in the presence or absence of 2 M DMSO by ¹²⁵I-labeled triplex-forming oligonucleotide targeting. End-group structure was assessed enzymatically as a function of the DSB end to serve as a substrate for ligation and various forms of end labeling. Using this approach, we have demonstrated 3′-hydroxyl (3′-OH) and 3′-phosphate (3′-P) end groups and 5′-ends (≥42%) terminated by phosphate. A ³²P postlabeling assay failed to detect 3′-phosphoglycolate in a restriction fragment terminated by the ¹²⁵I-induced DNA double-strand break, and this is likely due to restricted oxygen diffusion during irradiation as a frozen aqueous solution. Even so, end-group structure and relative distribution varied as a function of the free radical scavenging capacity of the irradiation buffer.

Yasui, L. S., Chen, K., Wang, K., Jones, T. P., Caldwell, J., Guse, D. and Kassis, A. I. Using Hoechst 33342 to Target Radioactivity to the Cell Nucleus. Radiat. Res. 167, 167–175 (2007).

We have explored the use of Hoechst 33342 (H33342) to carry radioactivity to the cell nucleus. H33342 enters cells and targets DNA at adenine-thymine-rich regions of the minor groove. Considerable membrane blebbing and ruffling occur in CHO cells within minutes after its addition to the culture medium in micromolar quantities. Blue vesicles are apparent in the cell cytoplasm, and by 30 min the nuclei are stained dark blue. Upon its binding to DNA, a visible emission shift of the dye can be observed with fluorescence microscopy. We have radioiodinated (¹²⁵I) H33342 and specifically irradiated nuclear DNA by incubating CHO cells with ¹²⁵I-H33342 at 37°C and accumulating ¹²⁵I decays at −90°C. At various times, the cells are thawed and assayed for survival (clonogenicity) and DSB (γ-H2AX) formation. ¹²⁵I-H33342 decay leads to a monoexponential decrease in cell survival with a D₀ of 122 ¹²⁵I decays per cell and a linear increase in DNA DSB induction (equivalent to 15 γ-H2AX foci/cell). Cell death is not modified by the radioprotective effects of H33342 because we use considerably lower concentrations than those that provide a slight protection against γ radiation. We conclude that cell killing by ¹²⁵I-H33342 and the induction of γ-H2AX foci are highly correlated.

Malone, J. and Ullrich, R. Novel Radiation Response Genes Identified in Gene-Trapped MCF10A Mammary Epithelial Cells. Radiat. Res. 167, 176–184 (2007).

We have used a gene-trapping strategy to screen human mammary epithelial cells for radiation response genes. Relative mRNA expression levels of five candidate genes in MCF10A cells were analyzed, both with and without exposure to radiation. In all five cases, the trapped genes were significantly down-regulated after radiation treatment. Sequence analysis of the fusion transcripts identified the trapped genes: (1) the human androgen receptor, (2) the uncharacterized DREV1 gene, which has known homology to DNA methyltransferases, (3) the human creatine kinase gene, (4) the human eukaryotic translation elongation factor 1 beta 2, and (5) the human ribosomal protein L27. All five genes were down-regulated significantly after treatment with varying doses of ionizing radiation (0.10 to 4.0 Gy) and at varying times (2–30 h after treatment). The genes were also analyzed in human fibroblast and lymphoblastoid cell lines to determine whether the radiation response being observed was cell-type specific. The results verified that the observed radiation response was not a cell-type-specific phenomenon, suggesting that the genes play essential roles in the radiation damage control pathways. This study demonstrates the potential of the gene-trap approach for the identification and functional analysis of novel radiation response genes.

Gaugler, M. H., Neunlist, M., Bonnaud, S., Aubert, P., Benderitter, M. and Paris, F. Intestinal Epithelial Cell Dysfunction is Mediated by an Endothelial-Specific Radiation-Induced Bystander Effect. Radiat. Res. 167, 185–193 (2007).

The response of endothelial cells (EC) to high radiation doses leads to damage of normal tissue or tumor. The precise mechanisms of the endothelial-tissue linkage are still largely unknown. We investigated the possible involvement of a bystander effect, secondary to endothelial damage, in tissue response to radiation. Proliferating human intestinal epithelial T84 cells were grown in a non-contact co-culture with confluent primary human microvascular EC (HMVEC-L). The bystander response in unirradiated T84 cells co-cultured with irradiated EC was studied by evaluating cell growth, cell death and epithelial morphology. Twenty-four hours after exposure of EC to 15 Gy, unirradiated T84 cells showed a decreased cell number (29%) and percentage in mitosis (66%) as well as increased apoptosis (1.5-fold) and cell surface area (1.5-fold), highlighting the involvement of bystander effects on T84 cells after irradiation of EC. Furthermore, the responses of T84 cells were amplified when EC and T84 cells were irradiated together, indicating that the bystander response in T84 cells adds further to direct radiation damage. As opposed to direct irradiation, the T84 cell bystander response did not involve the cell cycle-related protein p21^Waf1 (CDKN1A) and pro-apoptosis protein BAX. The bystander effect was specific to EC since the irradiation of human colon fibroblasts did not induce bystander responses in unirradiated T84 cells. These results strengthen previous in vivo evidence of the role of EC in tissue damage by radiation. In addition, this study provides a suitable and useful model to identify soluble factors involved in bystander effects secondary to endothelial damage. Modulating such factors may have important clinical implications.

Fournier, C., Becker, D., Winter, M., Barberet, P., Heiß, M., Fischer, B., Topsch, J. and Taucher-Scholz, G. Cell Cycle-Related Bystander Responses are not Increased with LET after Heavy-Ion Irradiation. Radiat. Res. 167, 194–206 (2007).

Evidence has accumulated that irradiated cells affect their unirradiated neighbors, so that they in turn display cellular responses typically associated with direct radiation exposure. These responses are generally known as bystander effects. In this study, cell cycle-related bystander responses were investigated in three strains of human fibroblasts after exposure to densely ionizing radiation. Varying the linear energy transfer (LET) from 11 to 15,000 keV μm⁻¹ allowed a study of the impact of the complexity of DNA damage in the inducing cells on the responses of bystander cells. Using both broad-beam and microbeam irradiation, transient bystander responses were obtained for the induction of CDKN1A (p21). The latter was also observed when the transmission of bystander signals was limited to soluble factors. Targeted irradiation of single cells in confluent cell monolayers revealed no correlation between the amount of CDKN1A protein in the bystander cells and the radial distance to the targeted cells. In line with the induction of CDKN1A in bystander cells after irradiation with different LETs, a transient delay in the first G₁ phase after irradiation of G₀/G₁ cells was observed. However, the CDKN1A induction revealed no significant effect on premature terminal differentiation considered to underlie fibrosis in irradiated tissue. Thus the unchanged differentiation pattern in bystander cells does not indicate pronounced, long-lasting effects.

Gollapalle, E., Wang, R., Adetolu, R., Tsao, D., Francisco, D., Sigounas, G. and Georgakilas, A. G. Detection of Oxidative Clustered DNA Lesions in X-Irradiated Mouse Skin Tissues and Human MCF-7 Breast Cancer Cells. Radiat. Res. 167, 207–216 (2007).

Bistranded oxidative clustered DNA lesions are closely spaced lesions (1–10 bp) that challenge the DNA repair mechanisms and are associated with genomic instability. The endogenous levels of oxidative clustered DNA lesions in cells of human cancer cell lines or in animal tissues remain unknown, and these lesions may persist for a long time after irradiation. We measured the different types of DNA clusters in cells of two human cell lines, MCF-7 and MCF-10A, and in skin obtained from mice exposed to either 12.5 Gy or sham X radiation. For the detection and measurement of oxidative clustered DNA lesions, we used adaptations of number average length analysis, constant-field agarose gel electrophoresis, putrescine, and the repair enzymes APE1, OGG1 (human) and Nth1 (E. coli). Increased levels of all cluster types were detected in skin tissue from animals exposed to radiation at 20 weeks postirradiation. The level of endogenous (no radiation treatment) oxidative clustered DNA lesions was higher in MCF-7 cells compared to nonmalignant MCF-10A cells. To the best of our knowledge, this is the first study to demonstrate persistence of oxidative clustered DNA lesions for up to 20 weeks in animal tissues exposed to radiation and to detect these clusters in human breast cancer cells. This may underscore the biological significance of clustered DNA lesions.

Koana, T., Okada, M. O., Ogura, K., Tsujimura, H. and Sakai, K. Reduction of Background Mutations by Low-Dose X Irradiation of Drosophila Spermatocytes at a Low Dose Rate. Radiat. Res. 167, 217–221 (2007).

A sex-linked recessive lethal mutation assay was performed in Drosophila melanogaster using immature spermatocytes and spermatogonia irradiated with X rays at a high or low dose rate. The mutation frequency in the sperm irradiated with a low dose at a low dose rate was significantly lower than that in the sham-irradiated group, whereas irradiation with a high dose resulted in a significant increase in the mutation frequency. It was obvious that the dose–response relationship was not linear, but rather was U-shaped. When mutant germ cells defective in DNA excision repair were used instead of wild-type cells, low-dose irradiation at a low dose rate did not reduce the mutation frequency. These observations suggest that error-free DNA repair functions were activated by low dose of low-dose-rate radiation and that this repaired spontaneous DNA damage rather than the X-ray-induced damage, thus producing a practical threshold.

Schubauer-Berigan, M. K., Daniels, R. D., Fleming, D. A., Markey, A. M., Couch, J. R., Ahrenholz, S. H., Burphy, J. S., Anderson, J. L. and Tseng, C-Y. Risk of Chronic Myeloid and Acute Leukemia Mortality after Exposure to Ionizing Radiation among Workers at Four U.S. Nuclear Weapons Facilities and a Nuclear Naval Shipyard. Radiat. Res. 167, 222–232 (2007).

A nested case-control study was conducted among workers at five U.S. nuclear facilities to evaluate leukemia mortality risk (excluding chronic lymphocytic) from ionizing radiation using worksite doses and adjusting for potential confounding. Conditional logistic regression was used to estimate the relative risk (RR) of exposed workers and the excess relative risk (ERR) per unit of radiation among 206 cases and 823 age-matched controls. Adjusting for sex and benzene, the RR of leukemia for workers receiving more than 10 mSv was higher compared to those receiving lower or no dose; however, the risk increase was attenuated in the highest dose group. The ERR per 10 mSv was 1.44% (95% CI: <−1.03%, 7.59%) but was higher for workers born after 1921 compared to workers born earlier or when excluding leukemias of uncertain type. Excluding the 7% who were high-dose workers (>100 mSv), the sex- and benzene-adjusted ERR per 10 mSv was 6.82% (95% CI: −2.87%, 24.1%). The results suggest that risks among these nuclear workers are comparable to those observed in high-dose populations, although no evidence was observed of a positive quadratic dose–response term in this study. This large study is among the first to jointly evaluate benzene and ionizing radiation risk.

Worgul, B. V., Kundiyev, Y. I., Sergiyenko, N. M., Chumak, V. V., Vitte, P. M., Medvedovsky, C., Bakhanova, E. V., Junk, A. K., Kyrychenko, O. Y., Musijachenko, N. V., Shylo, S. A., Vitte, O. P., Xu, S., Xue, X. and Shore, R. E. Cataracts among Chernobyl Clean-up Workers: Implications Regarding Permissible Eye Exposures. Radiat. Res. 167, 233–243 (2007).

The eyes of a prospective cohort of 8,607 Chernobyl clean-up workers (liquidators) were assessed for cataract at 12 and 14 years after exposure. The prevalence of strictly age-related cataracts was low, as expected (only 3.9% had nuclear cataracts at either examination), since 90% of the cohort was younger than 55 years of age at first examination. However, posterior subcapsular or cortical cataracts characteristic of radiation exposure were present in 25% of the subjects. The data for Stage 1 cataracts, and specifically for posterior subcapsular cataracts, revealed a significant dose response. When various cataract end points were analyzed for dose thresholds, the confidence intervals all excluded values greater than 700 mGy. Linear-quadratic dose–response models yielded mostly linear associations, with weak evidence of upward curvature. The findings do not support the ICRP 60 risk guideline assumption of a 5-Gy threshold for “detectable opacities” from protracted exposures but rather point to a dose–effect threshold of under 1 Gy. Thus, given that cataract is the dose-limiting ocular pathology in current eye risk guidelines, revision of the allowable exposure of the human visual system to ionizing radiation should be considered.

EL-Faramawy, N. A. and Rühm, W. Additional Criteria for EPR Dosimetry using Tooth Enamel. Radiat. Res. 167, 244– 250 (2007).

Currently, EPR measurements are based on the assumption that odontogenesis (the series of events between the bud formation stage until the complete maturation of the tooth) is finished as soon as the tooth erupts. Consequently, it is also assumed that the hydroxyapatite concentration of the enamel (source of free radicals) does not depend on tooth age. However, the present work provides evidence that odontogenesis does not end after tooth eruption but continues for several years after eruption. Fifty-nine molars and pre-molars were analyzed by EPR spectroscopy. Tooth enamel samples were irradiated with different doses of γ radiation from a ⁶⁰Co source. The resulting EPR signals were evaluated in terms of posteruption tooth age and tooth position. It was found that, except for wisdom teeth, the concentration of the dosimetric EPR free radicals increased with tooth age after eruption and became constant after a certain period. A mathematical equation was developed to describe this effect as a function of tooth age, tooth position and applied dose. The results suggest that EPR measurements obtained on young teeth should be interpreted carefully unless data are available that would allow one to describe the effect of posteruptive enamel maturation on the EPR estimated dose quantitatively. Little or no correction is needed for older teeth. Since only a limited number of young teeth were available for the present study, further studies are needed to clarify the situation and quantify this effect.