Tofilon, P.J. and Fike, J.R. The Radioresponse of the Central Nervous System: A Dynamic Process.

Radiation continues to be a major treatment modality for tumors located within and close to the central nervous system (CNS). Consequently, alleviating or protecting against radiation-induced CNS injury would be of benefit in cancer treatment. However, the rational development of such interventional strategies will depend on a more complete understanding of the mechanisms responsible for the development of this form of normal tissue injury. Whereas the vasculature and the oligodendrocyte lineage have traditionally been considered the primary radiation targets in the CNS, in this review we suggest that other phenotypes as well as critical cellular interactions may also be involved in determining the radiore[chsponse of the CNS. Furthermore, based on the assumption that the CNS has a limited repertoire of responses to injury, the reaction of the CNS to other types of insults is used as a framework for modeling the pathogenesis of radiation-induced damage. Evidence is then provided suggesting that, in addition to acute cell death, radiation induces an intrinsic recovery/repair response in the form of specific cytokines and may initiate secondary reactive processes that result in the generation of a persistent oxidative stress.

Hagan, M., Wang, L., Hanley, J. R., Park, J. S. and Dent, P. Ionizing Radiation-Induced Mitogen-Activated Protein (MAP) Kinase Activation in DU145 Prostate Carcinoma Cells: MAP Kinase Inhibition Enhances Radiation-Induced Cell Killing and G₂/M-Phase Arrest.

These studies examine the role(s) played by the mitogen-activated protein kinase (MAPK) pathway after exposure of DU145 prostate carcinoma cells to radiation. Radiation (2 Gy) was found to cause both immediate primary (0–30 min) and prolonged secondary activations (90–1440 min) of the MAPK pathway. These activations of the MAPK pathway were abolished by inhibition of epidermal growth factor receptor (EGFR) function. The secondary activation was also abolished by addition of a neutralizing monoclonal antibody against transforming growth factor α (TGFA). Activation of the MAPK pathway could be induced in nonirradiated cells by the transfer of medium from irradiated cultures. Neutralizing antibody to TGFA blocked this effect, indicating that radiation causes secondary activation of the MAPK pathway by release of TGFA in DU145 cells. Radiation induced a transient G₂/M-phase growth arrest that was prolonged for up to 24 h by inhibition of the MAPK pathway. Inhibition of the MAPK pathway significantly increased the ability of radiation to cause apoptosis 24 h after exposure. The ability of DU145 cells to proliferate after irradiation became dependent on MAPK signaling. When cells were subjected to single doses or fractionated radiation exposure, continuous inhibition of the MAPK pathway significantly decreased clonogenic survival. Only a small fraction of this cell killing could be accounted for by apoptosis within the first 96 h. Thus inhibition of the MAPK pathway increased radiation-induced cell killing likely by both apoptotic and nonapoptotic mechanisms. Collectively, our findings indicate that disruption of the TGFA/EGFR/MAPK pathway may represent a strategy that could be exploited to manipulate prostate carcinoma growth and cell survival after irradiation.

Amorino, G. P., Freeman, M. L. and Choy, H. Enhancement of Radiation Effects In Vitro by the Estrogen Metabolite 2-Methoxyestradiol.

2-Methoxyestradiol (2-ME) is an endogenous estradiol metabolite that disrupts microtubule function, suppresses murine tumors, and inhibits angiogenesis. Since some microtubule inhibitors have been shown to alter radiosensitivity, we have evaluated 2-ME as a radiation enhancer in vitro. H460 human lung cancer cells were plated, treated with 2-ME for 24 h, and irradiated; then colony-forming ability was assessed. The radiation dose enhancement ratios (DERs) using this protocol were 1.3, 1.8 and 2.1 for 1, 1.5 and 2 μM 2-ME, respectively. Using a single-cell plating protocol, the respective DERs were 1.2, 1.5 and 1.8. The parent compound of 2-ME, β-estradiol, did not enhance radiation effects at equally cytotoxic doses. Isobologram analysis showed that 1 μM 2-ME was additive with radiation, but that 1.5 and 2 μM were synergistic. Cell cycle analysis showed a dose-dependent increase in the percentage of cells in the radiosensitive G₂/M phase after a 24-h treatment with 2-ME; a threefold increase in the percentage of cells in G₂/M phase was observed using 2 μM 2-ME. Treatment with 2 μM 2-ME almost completely inhibited repair of sublethal damage (SLD) as shown using split-dose recovery. Radiosensitive, repair-deficient murine SCID (severe combined immunodeficient) cells did not show enhancement of radiation effects with 2 μM 2-ME, but enhancement was observed in the wild-type parental cells (CB-17). SCID cells complemented with human DNA-dependent protein kinase restored radioenhancement by 2-ME. In addition, MCF-7 breast cancer cells were also radiosensitized by 2 μM 2-ME (DER = 2.1). These data suggest that 2-ME is a potential radiation sensitizer, in addition to its previously reported antitumor and antiangiogenic properties. We have verified the antiangiogenic activity of 2-ME in vitro using human endothelial cells. Based on these results, we hypothesize that the mechanism of radiation enhancement may involve redistribution of cells into G₂/M phase by 2-ME, and that the resulting population of cells is repair-deficient and thus radiosensitive.

Ishihara, H., Tanaka, I., Furuse, M. and Tsuneoka, K. Increased Expression of Intracisternal A-Particle RNA in Regenerated Myeloid Cells after X Irradiation in C3H/He Inbred Mice.

Myeloid leukemia cells were derived from regenerated hematopoietic cells damaged by sublethal doses of X radiation in C3H/He inbred mice. We previously found that within the genome of the myeloid leukemia cells, a retrotransposon, the intracisternal A-particle (IAP) element, is integrated. Levels of IAP RNA, the source of cDNA for the integration, were analyzed quantitatively in C3H mice. Higher levels of IAP transcripts were observed in normal cells, particularly in hematopoietic cells, from C3H/He mice, than in those from C57BL/6J and STS/A mice. In the C3H/He mice, an approximately twofold increase in IAP RNA was found in the regenerated spleen and bone marrow cells at 5 days and from 12 to 90 days after whole-body X irradiation. In addition, an increased level of IAP RNA was observed in all the myeloid leukemia cells derived from C3H/He mice. This suggests that the elevated levels of IAP RNA in the regenerated hematopoietic cells after irradiation contribute to the increase in retrotransposition of IAP found in myeloid leukemia cells from C3H/He mice.

Bussink, J., Kaanders, J. H. A. M., Rijken, P. F. J. W., Raleigh, J. A. and Van der Kogel, A. J. Changes in Blood Perfusion and Hypoxia after Irradiation of a Human Squamous Cell Carcinoma Xenograft Tumor Line.

The effect of irradiation depends on the oxygenation status of the tissue, while irradiation itself also changes the oxygenation and perfusion status of tissues. A better understanding of the changes in tumor oxygenation and perfusion over time after irradiation will allow a better planning of fractionated radiotherapy in combination with modifiers of blood flow and oxygenation. Vascular architecture (endothelial marker), perfusion (Hoechst 33342) and oxygenation (pimonidazole) were studied in a human laryngeal squamous cell carcinoma tumor line grown as xenografts in nude mice. The effect of a single dose of 10 Gy X rays on these parameters was evaluated from 2 h to 11 days after irradiation. Shortly after irradiation, there was an 8% increase in perfused blood vessels (from 57% to 65%) followed by a significant decrease, with a minimum value of 42% at 26 h after irradiation, and a subsequent increase to control levels at 7 to 11 days after irradiation. The hypoxic fraction showed a decrease at 7 h after treatment from 13% to 5% with an increase to 19% at 11 days after irradiation. These experiments show that irradiation causes rapid changes in oxygenation and perfusion which may have consequences for the optimal timing of radiotherapy schedules employing multiple fractions per day and the introduction of oxygenation- and perfusion-modifying drugs.

Wang, L. W., Fu, X-L., Clough, R., Sibley, G., Fan, M., Bentel, G. C., Marks, L. B. and Anscher, M. S. Can Angiotensin-Converting Enzyme Inhibitors Protect against Symptomatic Radiation Pneumonitis?

This study was designed to determine whether patients taking angiotensin-converting enzyme (ACE) inhibitors while receiving radiation therapy for lung cancer are protected from developing symptomatic radiation pneumonitis. The records of 213 eligible patients receiving thoracic irradiation for lung cancer with curative intent at Duke University Medical Center from 1994–1997 were reviewed. Of the 213 patients, 26 (12.2%) were on ACE inhibitors (usually for the management of hypertension) during radiotherapy (group 1); the remaining 187 patients (group 2) were not. Patients were irradiated, with fields shaped to protect normal tissues, with total doses of 50–80 Gy. After treatment, patients were generally followed every 3 months for 2 years, then every 6 months thereafter. Symptomatic radiation pneumonitis was scored according to modified National Cancer Institute Common Toxicity Criteria (i.e., radiographic changes alone were not sufficient for the diagnosis of pneumonitis). There was no difference in the incidence of pneumonitis between the two groups (P = 0.75). Fifteen percent of the patients on ACE inhibitors (group 1) developed symptomatic radiation-induced lung injury compared to 12% of the patients not receiving these drugs (group 2). Although patients in group 1 tended to develop pneumonitis slightly sooner than did patients in group 2, this difference also was not significant (P = 0.8). Within the dose range prescribed for treating hypertension, ACE inhibitors do not appear to either decrease the incidence or delay the onset of symptomatic radiation pneumonitis among lung cancer patients receiving thoracic irradiation.

Murali Krishna, C., Bose, B. and Gupta, P. K. N₂ Laser-Induced Oxidation of Hemoproteins in Red Blood Cell Lysate.

Irradiation of red blood cell lysate with a N₂ laser (337 nm) was observed to induce oxidation of hemoproteins. This process showed a strong dependence on the concentration of red blood cell lysate and the dose of radiation. Studies of mechanisms and experiments with deoxygenated red blood cell lysate rule out involvement of any reactive oxygen species and suggest that the process is not a photodynamic reaction.

Bishayee, A., Rao, D. V., Bouchet, L. G., Bolch, W. E. and Howell, R. W. Protection by DMSO against Cell Death Caused by Intracellularly Localized Iodine-125, Iodine-131 and Polonium-210.

The mechanisms by which DNA-incorporated radionuclides impart lethal damage to mammalian cells were investigated by examining the capacity of dimethyl sulfoxide (DMSO) to protect against lethal damage to Chinese hamster V79 cells caused by unbound tritium (³H₂O), DNA-incorporated ¹²⁵I- and ¹³¹I-iododeoxyuridine (¹²⁵IdU, ¹³¹IdU), and cytoplasmically localized ²¹⁰Po citrate. The radionuclides ³H and ¹³¹I emit low- and medium-energy β particles, respectively, ¹²⁵I is a prolific Auger electron emitter, and ²¹⁰Po emits 5.3 MeV α particles. Cells were radiolabeled and maintained at 10.5°C for 72 h in the presence of different concentrations of DMSO (5–12.5% v/v), and the surviving fraction compared to that of unlabeled controls was determined. DMSO afforded no protection against the lethal effects of the high-LET α particles emitted by ²¹⁰Po. Protection against lethal damage caused by unbound ³H, ¹³¹IdU and ¹²⁵IdU depended on the concentration of DMSO in the culture medium. Ten percent DMSO provided maximum protection in all cases. The dose modification factors obtained at 10% DMSO for ³H₂O, ¹³¹IdU, ¹²⁵IdU and ²¹⁰Po citrate were 2.9 ± 0.01, 2.3 ± 0.5, 2.6 ± 0.2 and 0.95 ± 0.07, respectively. These results indicate that the toxicity of Auger electron and β-particle emitters incorporated into the DNA of mammalian cells is largely radical-mediated and is therefore indirect in nature. This is also the case for the low-energy β particles emitted by ³H₂O. In contrast, α particles impart lethal damage largely by direct effects. Finally, calculations of cellular absorbed doses indicate that β-particle emitters are substantially more toxic when incorporated into the DNA of mammalian cells than when they are localized extracellularly.

Hofer, K. G., Lin, X. and Schneiderman, M. H. Paradoxical Effects of Iodine-125 Decays in Parent and Daughter DNA: A New Target Model for Radiation Damage.

Chinese hamster ovary cells were synchronized at the G₁/S-phase boundary of the cell cycle and were pulse-labeled with ¹²⁵I-iododeoxyuridine 30 min after they entered the S phase. Cell samples were harvested and frozen for accumulation of ¹²⁵I decays during the first and second G₂ phase after labeling. Cell aliquots that had accumulated the desired number of decays were thawed and plated for evaluation micronucleus formation and cell death. Cells subjected to ¹²⁵I decays during the first G₂ phase after labeling exhibited single-hit kinetics of cell killing (n = 1, D₀ 41 decays/cell). In contrast, decays accumulated during the second G₂ phase killed cells with dual-hit kinetics (n = 1.9, D₀ 81 decays/cell). A similar divergence in the action of ¹²⁵I was noted for micronucleus formation. These findings indicate that the effects of ¹²⁵I varied depending on whether the decays occurred in daughter DNA (first G₂ phase) or parent DNA (second G₂ phase). Control studies with external X rays showed no such divergence of the action of radiation. To account for this paradox, a model is proposed that invokes higher-order chromatin structures as radiation targets. This model implies differential spatial arrangements for parent and daughter DNA in the genome, with DNA strands organized such that a single ¹²⁵I decay originating in daughter DNA damages two targets during the first G₂ phase, but identical decays occurring during the second G₂ phase damage only one of the targets.

Razskazovskiy, Y., Debije, M. G. and Bernhard, W. A. Direct Radiation Damage to Crystalline DNA: What is the Source of Unaltered Base Release?

The radiation chemical yields of unaltered base release have been measured in three crystalline double-stranded DNA oligomers after X irradiation at 4 K. The yields of released bases are between 10 and 20% of the total free radical yields measured at 4 K. Using these numbers, we estimate that the yield of DNA strand breaks due to the direct effect is about 0.1 μmol J^–1. The damage responsible for base release is independent of the base type (C, G, A or T) and is not scavenged by anthracycline drugs intercalated in the DNA. For these reasons, reactions initiated by the hydroxyl radical have been ruled out as the source of base release. Since the intercalated anthracycline scavenges electrons and holes completely but does not inhibit base release, the possibility for damage transfer from the bases to the sugars can also be ruled out. The results are consistent with a model in which primary radical cations formed directly on the sugar-phosphate backbone react by two competing pathways: deprotonation, which localizes the damage on the sugar, and hole tunneling, which transfers the damage to the base stack. Quantitative estimates indicate that these two processes are approximately equally efficient.

Box, H. C., Patrzyc, H. B., Dawidzik, J. B., Wallace, J. C., Freund, H. G., Iijima, H. and Budzinski, E. E. Double Base Lesions in DNA X-Irradiated in the Presence or Absence of Oxygen.

Previously, double lesions in which two adjacent bases are modified were identified in DNA oligomers exposed in solution to ionizing radiation. However, the formation of such lesions in polymer DNA had not been demonstrated. Using reference oligomer containing a specific double lesion and employing liquid chromatography-mass spectrometry (LC-MS), it was possible to show directly that double lesions are formed in irradiated calf thymus DNA. The double lesion in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized to 8-oxoguanine was detected in DNA X-irradiated in oxygenated aqueous solution. The double lesion in which the methyl carbon atom of a thymine base is covalently linked to carbon at the 8-position of an adjacent guanine base was detected in DNA irradiated in a deoxygenated environment.

Taniguchi, H. and Madden, K. P. DMPO-Alkyl Radical Spin Trapping: An In Situ Radiolysis Steady-State ESR Study.

Short-lived free radicals formed in the reaction of 11 substrates and radiolytically produced hydroxyl radicals were trapped successfully with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) in dilute aqueous solution. The in situ radiolysis steady-state ESR spectra of the spin adducts were analyzed to determine accurate ESR parameters for these spin adducts in a uniform environment. Parent alkyl radicals include methyl, ethyl, 1-propyl and 2-propyl (1-methylethyl). Hydroxyalkyl parent radicals were hydroxymethyl, hydroxyethyl, 2-hydroxy-2-propyl (1-methyl-1-hydroxyethyl), 1-hydroxypropyl and 2-hydroxy-2-methylpropyl. Carboxyl radical (carbon dioxide anion, formate radical) and sulfite anion radical were the sigma radicals studied. The DMPO spin adduct of 1-propyl was identified for the first time. For most spin adducts, g factors were also determined for the first time. In DMPO spin adducts of hydroxyalkyl radicals, nitrogen and C₂-proton hyperfine coupling constants are smaller than those of alkyl radical adducts; the hydroxyalkyl spin adducts possess larger g values than their unsubstituted counterparts. These changes are ascribed to the spread of π conjugation to include the hydroxyl group. Strong evidence of spin addend–aminoxyl group interaction can be seen in the asymmetrical line shapes in the hydroxyethyl and the hydroxypropyl spin adducts.

Le Sech, C., Takakura, K., Saint-Marc, C., Frohlich, H., Charlier, M., Usami, N. and Kobayashi, K. Strand Break Induction by Photoabsorption in DNA-Bound Molecules.

Dried samples of a DNA–chloroterpyridine platinum complex were irradiated with monochromatic X rays tuned to the photoabsorption resonance of the L_III inner shell of the platinum atom. The number of single- and double-strand breaks (SSBs and DSBs) triggered by the Auger effect in supercoiled DNA plasmids was measured by the production of circular nicked and linear forms. To probe the specific contribution of the L_III inner-shell excitation of the platinum atom, photon wavelengths were tuned on the resonance energy (on peak) and below (off peak). The quantum yields of the resonance radiation were typically found to be 11 for the SSBs and 1 for the DSBs. The DSB-to-SSB ratio increased by 20% when switching from off-resonance to on-resonance irradiation.

Cucinotta, F. A., Nikjoo, H. and Goodhead, D. T. Model for Radial Dependence of Frequency Distributions for Energy Imparted in Nanometer Volumes from HZE Particles.

This paper develops a deterministic model of frequency distributions for energy imparted (total energy deposition) in small volumes similar to DNA molecules from high-energy ions of interest for space radiation protection and cancer therapy. Frequency distributions for energy imparted are useful for considering radiation quality and for modeling biological damage produced by ionizing radiation. For high-energy ions, secondary electron (δ-ray) tracks originating from a primary ion track make dominant contributions to energy deposition events in small volumes. Our method uses the distribution of electrons produced about an ion's path and incorporates results from Monte Carlo simulation of electron tracks to predict frequency distributions for ions, including their dependence on radial distance. The contribution from primary ion events is treated using an impact parameter formalism of spatially restricted linear energy transfer (LET) and energy-transfer straggling. We validate our model by comparing it directly to results from Monte Carlo simulations for proton and α-particle tracks. We show for the first time frequency distributions of energy imparted in DNA structures by several high-energy ions such as cosmic-ray iron ions. Our comparison with results from Monte Carlo simulations at low energies indicates the accuracy of the method.

Schmollack, J. U., Klaumuenzer, S. L. and Kiefer, J. Stochastic Radial Dose Distributions and Track Structure Theory.

Measured single-event distributions of the specific energy deposited in cylindrical volumes with simulated diameters down to 150 nm for ⁴He and ¹²C ions with energies of 25 MeV/nucleon and ¹⁶O ions with 21 MeV/nucleon and radial distances up to 12 μm are presented. The mean specific energy per ion ⟨z(r)⟩, the mean specific energy per target hit z₁(r), and the relative frequency of target hits ν(r) as a function of radial distance are evaluated and compared with the corresponding quantities of the track structure model of Kiefer and Straaten (Phys. Med. Biol. 31, 1201–1209, 1986). Though there are some discrepancies in the absolute values, the radial dependence of ⟨z(r)⟩, z₁(r) and v(r) for ¹²C and ¹⁶O ions is reproduced satisfactorily. The model fails to describe the data for ⁴He ions. A more detailed comparison of the radial shape of the mean specific energies calculated from the experimental data from the present work and data from the literature reveals a significant projectile charge dependence which is not included in track structure models.

Vijayalaxmi, Leal, B. Z., Szilagyi, M., Prihoda, T. J. and Meltz, M. L. Primary DNA Damage in Human Blood Lymphocytes Exposed In Vitro to 2450 MHz Radiofrequency Radiation.

Human peripheral blood samples collected from three healthy human volunteers were exposed in vitro to pulsed-wave 2450 MHz radiofrequency (RF) radiation for 2 h. The RF radiation was generated with a net forward power of 21 W and transmitted from a standard gain rectangular antenna horn in a vertically downward direction. The average power density at the position of the cells in the flask was 5 mW/cm². The mean specific absorption rate, calculated by finite difference time domain analysis, was 2.135 (±0.005 SE) W/kg. Aliquots of whole blood that were sham-exposed or exposed in vitro to 50 cGy of ionizing radiation from a ¹³⁷Cs γ-ray source were used as controls. The lymphocytes were examined to determine the extent of primary DNA damage (single-strand breaks and alkali-labile lesions) using the alkaline comet assay with three different slide-processing schedules. The assay was performed on the cells immediately after the exposures and at 4 h after incubation of the exposed blood at 37 ± 1°C to allow time for rejoining of any strand breaks present immediately after exposure, i.e. to assess the capacity of the lymphocytes to repair this type of DNA damage. At either time, the data indicated no significant differences between RF-radiation- and sham-exposed lymphocytes with respect to the comet tail length, fluorescence intensity of the migrated DNA in the tail, and tail moment. The conclusions were similar for each of the three different comet assay slide-processing schedules examined. In contrast, the response of lymphocytes exposed to ionizing radiation was significantly different from RF-radiation- and sham-exposed cells. Thus, under the experimental conditions tested, there is no evidence for induction of DNA single-strand breaks and alkali-labile lesions in human blood lymphocytes exposed in vitro to pulsed-wave 2450 MHz radiofrequency radiation, either immediately or at 4 h after exposure.