Dewhirst, M. W. Relationships between Cycling Hypoxia, HIF-1, Angiogenesis and Oxidative Stress.

This Failla Lecture focused on the inter-relationships between tumor angiogenesis, HIF-1 expression and radiotherapy responses. A common thread that bonds all of these factors together is microenvironmental stress caused by reactive oxygen and nitrogen species formed during tumor growth and angiogenesis or in response to cytotoxic treatment. In this review we focus on one aspect of the crossroad between oxidative stress and angiogenesis, namely cycling hypoxia. Understanding of the relative importance of this feature of the tumor microenvironment has recently expanded; it influences tumor biology in ways that are separate from chronic hypoxia. Cycling hypoxia can influence angiogenesis, treatment responses and metastatic behavior. It represents an important and relatively less well understood feature of tumor biology that requires additional research.

Kunos, C. A., Chiu, S., Pink, J. and Kinsella, T. J. Modulating Radiation Resistance by Inhibiting Ribonucleotide Reductase in Cancers with Virally or Mutationally Silenced p53 Protein.

Therapeutic ionizing radiation damages DNA, increasing p53-regulated ribonucleotide reductase (RNR) activity required for de novo synthesis of the deoxyribonucleotide triphosphates used during DNA repair. This study investigated the pharmacological inhibition of RNR in cells of virally or mutationally silenced p53 cancer cell lines using 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine®, NSC #663249), a chemotherapeutic radiosensitizer that equally inhibits RNR M2 and p53R2 small subunits. The effects of 3-AP on RNR inhibition and resulting radiosensitization were evaluated in cervical (CaSki, HeLa and C33-a) and colon (RKO, RKO-E6) cancer cells. 3-AP treatment significantly enhanced radiation-related cytotoxicity in cervical and colon cancer cells. 3-AP treatment significantly decreased RNR activity, caused prolonged radiation-induced DNA damage, and resulted in an extended G₁/S-phase cell cycle arrest in all cell lines. Similar effects were observed in both RKO and RKO-E6 cells, suggesting a p53-independent mechanism of radiosensitization. We conclude that inhibition of ribonucleotide reductase by 3-AP enhances radiation-mediated cytotoxicity independent of p53 regulation by impairing repair processes that rely on deoxyribonucleotide production, thereby substantially increasing the radiation sensitivity of human cancers.

Kranjc, S., Tevz, G., Kamensek, U., Vidic, S., Cemazar, M. and Sersa, G. Radiosensitizing Effect of Electrochemotherapy in a Fractionated Radiation Regimen in Radiosensitive Murine Sarcoma and Radioresistant Adenocarcinoma Tumor Model.

Electrochemotherapy can potentiate the radiosensitizing effect of bleomycin, as shown in our previous studies. To bring this treatment closer to use in clinical practice, we evaluated the interaction between electrochemotherapy with bleomycin and single-dose or fractionated radiation in two murine tumor models with different histology and radiosensitivity. Radiosensitive sarcoma SA-1 and radioresistant adenocarcinoma CaNT subcutaneous tumors grown in A/J and CBA mice, respectively, were used. The anti-tumor effect and skin damage around the treated tumors were evaluated after electrochemotherapy with bleomycin alone or combined with single-dose radiation or a fractionated radiation regimen. The anti-tumor effectiveness of electrochemotherapy was more pronounced in SA-1 than CaNT tumors. In both tumor models, the tumor response to radiation was not significantly influenced by bleomycin alone or by electroporation alone. However, electrochemotherapy before the first tumor irradiation potentiated the response to a single-dose or fractionated radiation regimen in both tumors. For the fractionated radiation regimen, normal skin around the treated tumors was damaged fourfold less than for the single-dose regimen. Electrochemotherapy prior to single-dose irradiation induced more damage to the skin around the treated tumors and greater loss of body weight compared to other irradiated groups, whereas electrochemotherapy combined with the fractionated radiation regimen did not. Electrochemotherapy with low doses of bleomycin can also be used safely for radiosensitization of different types of tumors in a fractionated radiation regimen, resulting in a good anti-tumor effect and no major potentiating effect on radiation-induced skin damage.

Jiao, W., Kiang, J. G., Cary, L., Elliott, T. B., Pellmar, T. C. and Ledney, G. D. COX-2 Inhibitors Are Contraindicated for Treatment of Combined Injury.

Casualties of radiation dispersal devices, nuclear detonation or major ionizing radiation accidents, in addition to radiation exposure, may sustain physical and/or thermal trauma. Radiation exposure plus additional tissue trauma is known as combined injury. There are no definitive therapeutic agents. Cyclooxygenase-2 (COX-2), an inducible enzyme expressed in pathological disorders and radiation injury, plays an important role in inflammation and the production of cytokines and prostaglandin E₂ (PGE₂) and could therefore affect the outcome for victims of combined injury. The COX-2 inhibitors celecoxib and meloxicam were evaluated for their therapeutic value against combined injury in mice. In survival studies, the COX-2 inhibitors had no beneficial effect on 30-day survival, wound healing or body weight gain after radiation injury alone or after combined injury. Meloxicam accelerated death in both wounded and combined injury mice. These drugs also induced severe hepatic toxicity, exaggerated inflammatory processes, and did not enhance hematopoietic cell regeneration. This study points to potential contraindications for use of COX-2 inhibitors in patients undergoing therapy for radiation injury and combined injury.

Mishra, K., Bhardwaj, R. and Chaudhury, N. K. Netropsin, a Minor Groove Binding Ligand: A Potential Radioprotective Agent.

Minimizing radiation-induced damages in DNA is an important aspect in the development of chemical radioprotectors. The aim of this study was to evaluate the possible radioprotective ability of the DNA minor groove binding ligand netropsin in an aqueous solution of plasmid DNA (pBR322) and to compare its efficacy with that of Hoechst 33258, a known radioprotector. The radiochemical parameters D₀, G_SSB and DMF were calculated in pBR322 DNA. Based on a comparison of the DMFs of netropsin and Hoechst 33258, netropsin appeared to be the better radioprotector. The ligand binding site accessibility of the restriction enzyme EcoRI at the ligand-pBR322 complex was assessed using a restriction-digestion assay in irradiated solutions. A distinct ligand-bound site protection in netropsin-DNA was observed in irradiated solutions. However, no site protection was observed in the presence of Hoechst 33258. The possible role of ligand-induced structural stabilization in irradiated aqueous solutions was also investigated using netropsin-calf thymus DNA melting temperature measurements. The greater radioprotective ability of netropsin in solutions of DNA was suggested to be due to its higher binding affinity and its ability to provide higher structural stabilization. EcoRI digestion revealed that hydroxyl radical (OH^•) generated by ionizing radiation is not able to radiolyse the netropsin-DNA complex. These results will help in developing better radioprotectors.

Jiang, J., Stoyanovsky, D. A., Belikova, N. A., Tyurina, Y. Y., Zhao, Q., Tungekar, M. A., Kapralova, V., Huang, Z., Mintz, A. H., Greenberger, J. S. and Kagan, V. E. A Mitochondria-Targeted Triphenylphosphonium-Conjugated Nitroxide Functions as a Radioprotector/Mitigator.

Removal of excessive mitochondrial reactive oxygen species by electron scavengers and antioxidants is a promising therapeutic strategy to reduce the detrimental effects of radiation exposure. Here we exploited triphenylphosphonium (TPP) cation as a means to target nitroxide radicals to mitochondria. We synthesized a library of TPP-conjugated nitroxides and tested their radioprotective effects in γ-irradiated mouse embryo cells and human epithelial BEAS-2B cells. Cells were incubated with conjugates either before or after irradiation. We found that [2-(1-oxyl-2,2,6,6-tetramethyl-piperidin-4-ylimino)-ethyl]-triphenyl-phosphonium (TPEY-Tempo) significantly blocked radiation-induced apoptosis as revealed by externalization of phosphatidylserine on the cell surface and inhibition of cytochrome c release from mitochondria. Using electron paramagnetic resonance, we showed that TPEY-Tempo was integrated into cells and mitochondria, where it underwent one-electron reduction to hydroxylamine. TPEY-Tempo acted as an electron scavenger that prevented superoxide generation and cardiolipin oxidation in mitochondria. Finally, TPEY-Tempo increased the clonogenic survival rate of irradiated cells. The cellular integration efficiencies of nonradioprotective TPP conjugates, including Mito-Tempo (Alexis, San Diego, CA), were markedly lower, although these homologues were integrated into isolated succinate-energized mitochondria to a similar extent as TPEY-Tempo. We conclude that mitochondrial targeting of TPP-conjugated nitroxides represents a promising approach for the development of novel radioprotectors.

Okamoto, M., Suzuki, Y., Shirai, K., Mizui, T., Yoshida, Y., Noda, S., Al-Jahdari, W. S., Shirao, T. and Nakano, T. Effect of Radiation on the Development of Immature Hippocampal Neurons In Vitro.

Little is known about the direct biological effects of radiation on immature neurons, despite its relevance to the mental retardation caused by irradiation of the brains of fetuses and children. In this study, we investigated the effects of radiation using primary cultured hippocampal neuronal cells with exclusion of glial cells, focusing on cell survival and structural development. Primary neurons were prepared from the hippocampi of fetal rats at embryonic day 18 and cultured according to Banker's methods. After incubation for 7 days, cells were irradiated with X rays and incubated continuously for 7 or 14 days. The number of neurons, their rate of apoptosis, and the patterns of expression of synaptic proteins on the neural dendrites were investigated by immunohistochemical methods. The total numbers of neurons were the same regardless of whether they were irradiated. The number of TUNEL-positive neurons, which can be considered as undergoing apoptosis, increased significantly in a dose-dependent fashion at both 7 and 14 days after irradiation. The mean numbers of clusters of synaptic proteins on neural dendrites, which are considered to represent their developmental level, decreased dose-dependently at both 7 and 14 days after irradiation. These results suggest that radiation not only induces apoptosis but also produces structural defects in the surviving neurons that may directly suppress neural development.

Titushkin, I. A., Rao, V. S., Pickard, W. F., Moros, E. G., Shafirstein, G. and Cho, M. R. Altered Calcium Dynamics Mediates P19-Derived Neuron-Like Cell Responses to Millimeter-Wave Radiation.

Intracellular calcium oscillations have long been recognized as a principal mediator of many vital cellular activities. Furthermore, Ca² dynamics can be modulated by external physical cues, including electromagnetic fields. While cellular responses to low-frequency electric fields have been established, the possible non-thermal effects of millimeter-wave (MMW) radiation are still a subject of discussion and debate. We used mouse embryonic stem cell-derived neuronal cells and a custom-built 94 GHz applicator to examine in real time the altered Ca² oscillations associated with MMW stimulation. MMW irradiation at 18.6 kW/m² nominal power density significantly increased the Ca² spiking frequency in the cells exhibiting Ca² activity. The N-type calcium channels, phospholipase C enzyme, and actin cytoskeleton appear to be involved in mediating increased Ca² spiking. Reorganization of the actin microfilaments by a 94 GHz field seems to play a crucial role in modulating not only Ca² activity but also cell biomechanics. Many but not all observed cellular responses to MMW were similar to thermally induced effects. For example, cell exposure to a 94 GHz field induced nitric oxide production in some morphologically distinct neuronal cells that could not be reproduced by thermal heating of the cells up to 42°C. The highest observed average temperature rise in the MMW exposure chamber was ∼8°C above the room temperature, with possible complex non-uniform microscopic distribution of heating rates at the cell level. Our findings may be useful to establish quantitative molecular benchmarks for elucidation of nociception mechanisms and evaluation of potential adverse bioeffects associated with MMW exposure. Moreover, control of Ca² dynamics by MMW stimulation may offer new tools for regulation of Ca² -dependent cellular and molecular activities, for example, in tissue engineering applications.

Dayal, D., Martin, S. M., Owens, K. M., Aykin-Burns, N., Zhu, Y., Boominathan, A., Pain, D., Limoli, C. L., Goswami, P. C., Domann, F. E. and Spitz, D. R. Mitochondrial Complex II Dysfunction Can Contribute Significantly to Genomic Instability after Exposure to Ionizing Radiation.

Ionizing radiation induces chronic metabolic oxidative stress and a mutator phenotype in hamster fibroblasts that is mediated by H₂O₂, but the intracellular source of H₂O₂ is not well defined. To determine the role of mitochondria in the radiation-induced mutator phenotype, end points of mitochondrial function were determined in unstable (CS-9 and LS-12) and stable (114) hamster fibroblast cell lines derived from GM10115 cells exposed to 10 Gy X rays. Cell lines isolated after irradiation demonstrated a 20–40% loss of mitochondrial membrane potential and an increase in mitochondrial content compared to the parental cell line GM10115. Surprisingly, no differences were observed in steady-state levels of ATP (P > 0.05). Unstable clones demonstrated increased oxygen consumption (two- to threefold; CS-9) and/or increased mitochondrial electron transport chain (ETC) complex II activity (twofold; LS-12). Using Western blot analysis and Blue Native gel electrophoresis, a significant increase in complex II subunit B protein levels was observed in LS-12 cells. Furthermore, immunoprecipitation assays revealed evidence of abnormal complex II assembly in LS-12 cells. Treatment of LS-12 cells with an inhibitor of ETC complex II (thenoyltrifluoroacetone) resulted in significant decreases in the steady-state levels of H₂O₂ and a 50% reduction in mutation frequency as well as a 16% reduction in CAD gene amplification frequency. These data show that radiation-induced genomic instability was accompanied by evidence of mitochondrial dysfunction leading to increased steady-state levels of H₂O₂ that contributed to increased mutation frequency and gene amplification. These results support the hypothesis that mitochondrial dysfunction originating from complex II can contribute to radiation-induced genomic instability by increasing steady-state levels of reactive oxygen species.

Ainsbury, E. A., Livingston, G. K., Abbott, M. G., Moquet, J. E., Hone, P. A., Jenkins, M. S., Christensen, D. M., Lloyd, D. C. and Rothkamm, K. Interlaboratory Variation in Scoring Dicentric Chromosomes in a Case of Partial-Body X-Ray Exposure: Implications for Biodosimetry Networking and Cytogenetic “Triage Mode” Scoring.

The international radiation biodosimetry community has recently been engaged in activities focused on establishing cooperative networks for biodosimetric triage for radiation emergency scenarios involving mass casualties. To this end, there have been several recent publications in the literature regarding the potential for shared scoring in such an accident or incident. We present details from a medical irradiation case where two independently validated laboratories found very different yields of dicentric chromosome aberrations. The potential reasons for this disparity are discussed, and the actual reason is identified as being the partial-body nature of the radiation exposure combined with differing criteria for metaphase selection. In the context of the recent networking activity, this report is intended to highlight the fact that shared scoring may produce inconsistencies and that further validation of the scoring protocols and experimental techniques may be required before the networks are prepared to deal satisfactorily with a radiological or nuclear emergency. Also, the findings presented here clearly demonstrate the limitations of the dicentric assay for estimating radiation doses after partial-body exposures and bring into question the usefulness of rapid “triage mode” scoring in such exposure scenarios.

Lund, A., Sagstuen, E., Sanderud, A. and Maruani, J. Relaxation-Time Determination from Continuous-Microwave Saturation of EPR Spectra.

Based on the theories of Portis and of Castner 50 years ago, different continuous-wave measurement procedures for analyzing the microwave saturation power dependence of inhomogeneously broadened EPR lines were developed. Although these procedures have been refined, they still use only a few selected points on the saturation curve. A non-linear least-squares procedure for analyzing the microwave-power dependence of inhomogeneously broadened lines using all data points on a saturation curve has been developed. This procedure provides a simple alternative method to obtain magnetic relaxation data when the more direct pulse-saturation techniques are not available or are less suitable. The latter includes applications of quantitative EPR such as dosimetry. Then microwave saturation data should be obtained under conditions similar to those used in the quantitative measurements, which are usually made on first derivative spectra recorded using continuous-wave spectrometers. Selected applications to benchmark literature data and within the field of EPR dosimetry are discussed. The results obtained illustrate that relaxation times comparable to those yielded by various pulse-saturation EPR techniques can be obtained. It appears as a systematic feature that, whenever the pulse EPR data are fitted using bi-exponential functions, the shortest relaxation times obtained are those that correspond best to those measured using the current continuous-wave saturation method.

Hawkins, R. B. The Relationship Between the Sensitivity of Cells to High-Energy Photons and the RBE of Particle Radiation used in Radiotherapy.

The relationship between relative biological effectiveness in the limit of zero dose (RBE₁) and the LET of radiation is examined and compared for several cell lines, including cells from patients with ataxia telangiectasia, in the context of a microdosimetric-kinetic (MK) model of cell killing by radiation. Evidence is presented that the sensitivity of a cell to low-LET photon radiation, as measured by the linear parameter of the linear-quadratic cell survival relationship (α), is largely determined by its vulnerability to formation of a lethal lesion from transformation of a single potentially lethal lesion (PLL) in DNA, as opposed to formation by combination of two PLL. As a result, the RBE₁ of cells that are relatively less sensitive to low-LET photon radiation increases more with increasing LET than the RBE₁ of cells that are more sensitive to low-LET radiation. As a consequence, a pair of cells that have clearly different sensitivity to low-LET radiation tend to have more nearly the same sensitivity as the LET increases into the range of 100 to 200 keV µm⁻¹. Cells with the same, or nearly the same, sensitivity to low-LET photon radiation continue to have nearly the same sensitivity as the LET is similarly increased. Thus there may be a radiobiological advantage to treatment with high-LET particle radiation for situations in which the target tumor cells are less radiosensitive than the cells that determine the tolerance of the normal tissue at risk. This may be the case for treatment of many of the common malignancies that occur in adults. This general principle may be helpful in selection of patients for treatment with particle radiation, such as carbon ions, and in the design of clinical trials to determine the optimal dose and fractionation schedules for such treatment.