Meesungnoen, J., Benrahmoune, M., Filali-Mouhim, A., Mankhetkorn, S. and Jay-Gerin, J-P. Monte Carlo Calculation of the Primary Radical and Molecular Yields of Liquid Water Radiolysis in the Linear Energy Transfer Range 0.3–6.5 keV/μm: Application to ¹³⁷Cs Gamma Rays.

Monte Carlo simulations of the radiolysis of neutral liquid water and 0.4 M H₂SO₄ aqueous solutions at ambient temperature are used to calculate the variations of the primary radical and molecular yields (at 10^–6 s) as a function of linear energy transfer (LET) in the range ∼0.3 to 6.5 keV/μm. The early energy deposition is approximated by considering short (∼20–100 μm) high-energy (∼300–6.6 MeV) proton track segments, over which the LET remains essentially constant. The subsequent nonhomogeneous chemical evolution of the reactive species formed in these tracks is simulated by using the independent reaction times approximation, which has previously been used successfully to model the radiolysis of water under various conditions. The results obtained are in good general agreement with available experimental data over the whole LET range studied. After normalization of our computed yields relative to the standard radical and molecular yields for ⁶⁰Co γ radiation (average LET ∼0.3 keV/μm), we obtain empirical relationships of the primary radiolytic yields as a function of LET over the LET range studied. Such relationships are of practical interest since they allow us to predict a priori values of the radical and molecular yields for any radiation from the knowledge of the average LET of this radiation only. As an application, we determine the corresponding yields for the case of ¹³⁷Cs γ radiation. For this purpose, we use the value of ∼0.91 keV/μm for the average LET of ¹³⁷Cs γ rays, chosen so that our calculated yield G(Fe³ ) for ferrous-ion oxidation in air-saturated 0.4 M sulfuric acid reproduces the value of 15.3 molecules/100 eV for this radiation recommended by the International Commission on Radiation Units and Measurements. The uncertainty range on those primary radical and molecular yields are also determined knowing the experimental error (∼2%) for the measured G(Fe³ ) value. The following values (expressed in molecules/100 eV) are obtained: (1) for neutral water: G_e^–aq = 2.50 ± 0.16, G_H^· = 0.621 ± 0.019, G_H2 = 0.474 ± 0.025, G_^·OH = 2.67 ± 0.14, G_H2O2 = 0.713 ± 0.031, and G_–H2O = 4.08 ± 0.22; and (2) for 0.4 M H₂SO₄ aqueous solutions: G_H^· = 3.61 ± 0.09, G_H2 = 0.420 ± 0.019, G_^·OH = 2.78 ± 0.12, G_H2O2 = 0.839 ± 0.037, and G_–H2O = 4.46 ± 0.16. These computed values are found to differ from the standard yields for ⁶⁰Co γ rays by up to ∼6%.

Bonnefont-Rousselot, D., Rouscilles, A., Bizard, C., Delattre, J., Jore, D. and Gardès-Albert, M. Antioxidant Effect of Ethanol toward In Vitro Peroxidation of Human Low-Density Lipoproteins Initiated by Oxygen Free Radicals.

This study was designed to evaluate the effect of ethanol on the peroxidation of human low-density lipoprotein (LDL) initiated by oxygen free radicals (O₂^·– and ^·OH in the absence of ethanol; O₂^·– and ethanol-derived peroxyl radicals, RO₂^·, in the presence of ethanol) generated by γ radiolysis. Initial radiolytic yields as determined by several markers of lipid peroxidation [i.e. decrease in endogenous antioxidants α-tocopherol and β-carotene, formation of conjugated dienes and of thiobarbituric acid-reactive substances (TBARS)] were determined in 3 g liter⁻¹ LDLs (expressed as total LDL concentration) in the absence of ethanol or its presence at six different concentrations (0.42–17 × 10⁻² mol liter⁻¹). Ethanol acted as an antioxidant by decreasing the rate of consumption of LDL endogenous antioxidants and the yields of formation of lipid peroxidation products, and by delaying the onset of the propagation phase for conjugated dienes and TBARS. With regard to the different markers studied, except for α-tocopherol and β-carotene consumption, the effect of ethanol did not appear to be dependent on its concentration. Indeed, ^·OH were scavenged by ethanol at the lowest ethanol concentration (0.42 × 10⁻² mol liter⁻¹), leading to RO₂^·. These RO₂^· resulted in lower radiation-induced yields related to endogenous antioxidant consumption or to formation of lipid peroxidation products (for example, approximately 10% of RO₂^· oxidized LDLs from TBARS). Thus, under our in vitro conditions, ethanol behaved as an antioxidant when added to the LDL solutions. This should be taken into account in the reported antioxidant activity of wine. This is also of interest when lipophilic compounds have to be added as ethanolic solutions to LDLs to evaluate in vitro their antioxidant activity toward LDL peroxidation.

Kvinnsland, Y., Stokke, T. and Aurlien, E. Radioimmunotherapy with Alpha-Particle Emitters: Microdosimetry of Cells with a Heterogeneous Antigen Expression and with Various Diameters of Cells and Nuclei.

Intercellular variations in the level of antigen expression and in cellular and nuclear radii were taken into account in a model used to estimate cell survival for an in vitro experiment with antibodies containing α-particle emitters that target the cell surface. Using measured variations in these characteristics for cells of two human cancer cell lines, the model gave results for cell survival and the fundamental parameter of radiation sensitivity, z₀, that differ substantially from those obtained using only mean values. The cell survival may be underestimated by a factor of 100 if only mean values of these cellular parameters are used, and calculated values of z₀ may be overestimated by a factor of 2. Most of this effect stems from the variation in antigen expression. The magnitudes of the differences were found to be a function of the fractions of mean specific energy delivered by surrounding activity and by activity bound to the cells.

Niedbala, M., Alsbeih, G., Ng, C. E. and Raaphorst, G. P. Equivalence of Pulsed-Dose-Rate to Low-Dose-Rate Irradiation in Tumor and Normal Cell Lines.

To determine whether different fractionation schemes could simulate low-dose-rate irradiation, ovarian cells of the carcinoma cell lines A2780s (radiosensitive) and A2780cp (radioresistant) and AG1522 normal human fibroblasts were irradiated in vitro using different fraction sizes and intervals between fractions with an overall average dose rate of 0.53 Gy/h. For the resistant cell line, the three fractionation schemes, 0.53 Gy given every hour, 1.1 Gy every 2 h, and 1.6 Gy every 3 h, were equivalent to low dose rate (0.53 Gy/h). Two larger fraction sizes, 2.1 Gy every 4 h and 3.2 Gy every 6 h, resulted in lower survival than that after low-dose-rate irradiation for the resistant cell line, suggesting incomplete repair of radiation damage due to the larger fraction sizes. The survival for the sensitive cell line was lower at small doses, but then it increased until it was equivalent to that after low-dose-rate irradiation for some fractionation schemes. The sensitive cell line showed equivalence only with the 1.6-Gy fraction every 3 h, although 0.53 Gy every 1 h and 1.1 Gy every 2 h showed equivalence at lower doses. This cell line also showed an adaptive response. The normal cell line showed a sensitization to the pulsed-dose-rate schemes compared to low-dose-rate irradiation. These data indicate that the response to pulsed-dose-rate irradiation is dependent on the cell line and that compared to the response to low-dose-rate irradiation, it shows some equivalence with the resistant carcinoma cell line, an adaptive response with the parental carcinoma cell line, and sensitization with the normal cells. Therefore, further evaluation is required before implementing pulsed-dose-rate irradiation in the clinic.

Samuni, Y., DeGraff, W., Chevion, M., Mitchell, J. B. and Cook, J. A. Radiation Sensitization of Mammalian Cells by Metal Chelators.

The cell cycle effects, alteration in radiation response, and inherent cytotoxicity of the metal chelators mimosine, desferrioxamine (DFO), N,N′-bis(o-hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid (HBED), and deferiprone (L1) were studied in exponentially growing Chinese hamster V79 cells. Incubation of cells with 200–1000 μM mimosine for 12 h reduced clonogenic survival to 50–60%, while incubation for 24 h reduced survival further to 0.5%. Mimosine treatment resulted in cell cycle blocks at the G₁/S-phase border and in S phase. Pulse labeling with 5-bromodeoxyuridine indicated that the S-phase cells ceased to actively replicate DNA after only 2 h of mimosine treatment and were unable to replicate DNA for extended periods. Treatment of V79 cells with 600 μM mimosine for 12 h resulted in radiosensitization, yielding a sensitizer enhancement ratio (SER) of 2.7 ± 0.3 at the 10% survival level. To study the kinetics of the sensitization, V79 cells were incubated with mimosine for various times up to 12 h and irradiated with a single 10-Gy dose of X rays. It was found that the radiosensitization increased continually up to 8 h (from a 3- to a 100-fold difference in survival) and then reached a plateau after 8 h. Mimosine also equally radiosensitized human lung cancer cells having either a normal or mutated TP53 gene, suggesting a TP53-independent mechanism. To test whether iron binding by mimosine was responsible for the observed radiosensitization, additional experiments were performed using the iron chelators DFO, HBED and L1. V79 cells treated with 500 μM of these agents for 8 h followed by various doses of X rays gave SERs similar to that for mimosine (2.0–2.7). These studies indicate that metal chelators are potent radiosensitizers in V79 and human cells. Importantly, when the DFO was preloaded together with Fe³ [Fe(III)-DFO], the radiosensitizing effect was lost. These preliminary findings warrant further studies for the possible application of metal chelators as radiation sensitizers in radiation oncology.

Jirsová, P., Kozubek, S., Bártová, E., Kozubek, M., Lukášová, E., Cafourková, A., Koutná, I. and Skalníková, M. Spatial Distribution of Selected Genetic Loci in Nuclei of Human Leukemia Cells after Irradiation.

Fluorescence in situ hybridization (FISH) combined with high-resolution cytometry was used to determine the topographic characteristics of the centromeric heterochromatin (of the chromosomes 6, 8, 9, 17) and the tumor suppressor gene TP53 (which is located on chromosome 17) in cells of the human leukemia cell lines ML-1 and U937. Analysis was performed on cells that were either untreated or irradiated with γ rays and incubated for different intervals after exposure. Compared to untreated cells, homologous centromeres and the TP53 genes were found closer to each other and also closer to the nuclear center 2 h after irradiation. The spatial relationship between genetic elements returned to that of the unirradiated controls during the next 2–3 h. Statistical evaluation of our experimental results shows that homologous centromeres and the homologous genes are positioned closer to each other 2 h after irradiation because they are localized closer to the center of the nucleus (probably due to more pronounced decondensation of the chromatin related to repair). This radial movement of genetic loci, however, is not connected with repair of DSBs by processes involving homologous recombination, because the angular distribution of homologous sequences remains random after irradiation.

Endoh, D., Okui, T., Kon, Y. and Hayashi, M. Hypertonic Treatment Inhibits Radiation-Induced Nuclear Translocation of the Ku Proteins G22p1 (Ku70) and Xrcc5 (Ku80) in Rat Fibroblasts.

The effects of X irradiation and hypertonic treatment with 0.5 M NaCl on the subcellular localization of the Ku proteins G22p1 (also known as Ku70) and Xrcc5 (also known as Ku80) in rat fibroblasts with normal radiosensitivity were examined using confocal laser microscopy and immunoblotting. Although these proteins were observed mainly in the nuclei of human fibroblasts, approximately 80% of the intensities of immunofluorescence from both G22p1 and Xrcc5 was observed in the cytoplasm of rat fibroblasts. When the rat cells were X-irradiated with 4 Gy, the intensities of the fluorescence derived from G22p1 and Xrcc5 in the nuclei increased from 20% to 50% of the total cellular fluorescence intensity at 20 min postirradiation. No significant differences were observed between the total intensities of the cellular fluorescence from the proteins in unirradiated and irradiated rat fibroblasts. The results showed that the proteins were translocated from the cytoplasm to the nucleus in the rat cells after X irradiation. The nuclear translocation of the proteins from the cytoplasm was inhibited by hypertonic treatment of the cells with 0.5 M NaCl for 20 min, which inhibits the fast repair process of potentially lethal damage (PLD). When the rat cells were treated with 0.5 M NaCl immediately after X irradiation, the repair of DNA DSBs was inhibited. The surviving fraction was approximately 60% of that of irradiated cells that were not treated with 0.5 M NaCl. The surviving fraction increased with incubation time in the growth medium before treatment with NaCl. The proportions of the intensities of fluorescence from G22p1 in the nuclei of X-irradiated cells also increased from 20% to 50% with increasing interval between X irradiation and treatment with NaCl. These results suggest that nuclear translocation of G22p1 and Xrcc5 is important for the fast repair process of PLD in rat cells.

Yasui, L. S., Hughes, A. and DeSombre, E. R. Relative Biological Effectiveness of Accumulated ¹²⁵IdU and ¹²⁵I-Estrogen Decays in Estrogen Receptor-Expressing MCF-7 Human Breast Cancer Cells.

The therapeutic potential for delivering a cytotoxic dose of radiation (using the decay of Auger-electron emitters) to the cell nucleus of cancer cells that express estrogen receptors (ERs) by radiolabeled estrogen was investigated in the ER-expressing human breast cancer cell line, MCF-7. The radiolabeled estrogen/ER complex irradiates the cell nucleus by binding specific DNA sequences called estrogen response elements (EREs). Cell clonogenicity and induction of DNA double-strand breaks (DSBs) by γ radiation or accumulation of ¹²⁵I-iododeoxyuridine (¹²⁵IdU) or E-17α[¹²⁵I]iodovinyl-11βmethoxyestradiol (¹²⁵IVME2) decays were determined. MCF-7 cells were efficiently killed by accumulation of ¹²⁵IdU (D₀ = 30 decays per cell) and ¹²⁵IVME2 decays (D₀ = 28 decays per cell). DNA DSBs were induced by the accumulation of ¹²⁵IdU (approximately 3750 decays per cell required to reduce the mean value of the elution profile to 50%) or ¹²⁵IVME2 decays (approximately 465 decays per cell required to reduce the mean value to 50%). For survival of MCF-7 cells after γ irradiation, the D₀ was 1 Gy, and approximately 65 Gy was required to reduce the mean value to 50% for induction of DSBs. The RBE values for cell killing and induction of DSBs by ¹²⁵IVME2 relative to γ radiation were 4.8 and 18.8, respectively. The RBE values for cell killing and induction of DSBs by ¹²⁵IdU relative to γ radiation were 4.5 and 2.3, respectively. Cell killing in a manner similar to that induced by high-LET radiation and the high RBE for induction of DSBs by ¹²⁵IVME2 in the ER-expressing MCF-7 cells provide a biological rationale for the use of Auger electron-emitting radionuclides covalently bound to estrogen to deliver a cytotoxic dose of radiation to ER-positive cancers.

Bishayee, A., Hill, H. Z., Stein, D., Rao, D. V. and Howell, R. W. Free Radical-Initiated and Gap Junction-Mediated Bystander Effect due to Nonuniform Distribution of Incorporated Radioactivity in a Three-Dimensional Tissue Culture Model.

To investigate the biological effects of nonuniform distribution of radioactivity in mammalian cells, we have developed a novel three-dimensional tissue culture model. Chinese hamster V79 cells were labeled with tritiated thymidine and mixed with unlabeled cells, and multicellular clusters (∼1.6 mm in diameter) were formed by gentle centrifugation. The short-range β particles emitted by ³H impart only self-irradiation of labeled cells without significant cross-irradiation of unlabeled bystander cells. The clusters were assembled in the absence or presence of 10% dimethyl sulfoxide (DMSO) and/or 100 μM lindane. DMSO is a hydroxyl radical scavenger, whereas lindane is an inhibitor of gap junctional intercellular communication. The clusters were maintained at 10.5°C for 72 h to allow ³H decays to accumulate and then dismantled, and the cells were plated for colony formation. When 100% of the cells were labeled, the surviving fraction was exponentially dependent on the mean level of radioactivity per labeled cell. A two-component exponential response was observed when either 50 or 10% of the cells were labeled. Though both DMSO and lindane significantly protected the unlabeled or bystander cells when 50 or 10% of the cells were labeled, the effect of lindane was greater than that of DMSO. In both cases, the combined treatment (DMSO lindane) elicited maximum protection of the bystander cells. These results suggest that the bystander effects caused by nonuniform distributions of radioactivity are affected by the fraction of cells that are labeled. Furthermore, at least a part of these bystander effects are initiated by free radicals and are likely to be mediated by gap junctional intercellular communication.

Bouffler, S. D., Haines, J. W., Edwards, A. A., Harrison, J. D. and Cox, R. Lack of Detectable Transmissible Chromosomal Instability after In Vivo or In Vitro Exposure of Mouse Bone Marrow Cells to ²²⁴Ra Alpha Particles.

Several studies over recent years have highlighted the possibility that radiation can induce transmissible genomic instability. Most of these involve in vitro irradiation and usually in vitro culture. Here it is reported that the short-half-life bone-seeking α-particle emitter ²²⁴Ra did not induce excess transmissible chromosomal instability in CBA/H mouse bone marrow cells in a 100-day period after in vivo or in vitro exposure. Similarly, no excess transmissible chromosomal instability could be detected after in vivo whole-body X irradiation. It was noted, however, that short-term culture of murine bone marrow cells elevated yields of aberrations, as did transplantation of untreated marrow into radiation-ablated hosts. These findings emphasize the sensitivity of murine hemopoietic tissue to experimental manipulation and reinforce the importance of appropriate concurrent control experiments in any investigation of transmissible genomic instability.

Brooks, A. L., Bao, S., Rithidech, K., Couch, L. A. and Braby, L. A. Relative Effectiveness of HZE Iron-56 Particles for the Induction of Cytogenetic Damage In Vivo.

One of the risks of prolonged manned space flight is the exposure of astronauts to radiation from galactic cosmic rays, which contain heavy ions such as ⁵⁶Fe. To study the effects of such exposures, experiments were conducted at the Brookhaven National Laboratory by exposing Wistar rats to high-mass, high-Z, high-energy (HZE) particles using the Alternating Gradient Synchrotron (AGS). The biological effectiveness of ⁵⁶Fe ions (1000 MeV/nucleon) relative to low-LET γ rays and high-LET α particles for the induction of chromosome damage and micronuclei was determined. The mitotic index and the frequency of chromosome aberrations were evaluated in bone marrow cells, and the frequency of micronuclei was measured in cells isolated from the trachea and the deep lung. A marked delay in the entry of cells into mitosis was induced in the bone marrow cells that decreased as a function of time after the exposure. The frequencies of chromatid aberrations and micronuclei increased as linear functions of dose. The frequency of chromosome aberrations induced by HZE particles was about 3.2 times higher than that observed after exposure to ⁶⁰Co γ rays. The frequency of micronuclei in rat lung fibroblasts, lung epithelial cells, and tracheal epithelial cells increased linearly, with slopes of 7 × 10⁻⁴, 12 × 10⁻⁴, and 11 × 10⁻⁴ micronuclei/binucleated cell cGy⁻¹, respectively. When genetic damage induced by radiation from ⁵⁶Fe ions was compared to that from exposure to ⁶⁰Co γ rays, ⁵⁶Fe-ion radiation was between 0.9 and 3.3 times more effective than ⁶⁰Co γ rays. However, the HZE-particle exposures were only 10–20% as effective as radon in producing micronuclei in either deep lung or tracheal epithelial cells. Using microdosimetric techniques, we estimated that 32 cells were hit by δ rays for each cell that was traversed by the primary HZE ⁵⁶Fe particle. These calculations and the observed low relative effectiveness of the exposure to HZE particles suggest that at least part of the cytogenetic damage measured was caused by the δ rays. Much of the energy deposited by the primary HZE particles may result in cell killing and may therefore be “wasted” as far as production of detectable micronuclei is concerned. The role of wasted energy in studies of cancer induction may be important in risk estimates for exposure to HZE particles.

Fenton, B. M., Lord, E. M. and Paoni, S. F. Effects of Radiation on Tumor Intravascular Oxygenation, Vascular Configuration, Development of Hypoxia, and Clonogenic Survival.

The underlying physiological mechanisms leading to tumor reoxygenation after irradiation have elicited considerable interest, but they remain somewhat unclear. The current study was undertaken to determine the effects of a single dose of 10 Gy γ radiation on both tumor pathophysiology and radiobiologically hypoxic fraction. Immunohistochemical staining and perfusion markers were used to quantify tumor vasculature, uptake of the hypoxia marker EF5 to assess the distribution of hypoxia, and intravascular HbO₂ measurements to determine oxygen availability. Tumor radiosensitivity was measured by a clonogenic assay. At 24 h postirradiation, oxygen availability increased, perfused vessel numbers decreased, EF5 uptake decreased, and the radiobiologically hypoxic fraction was unchanged. Together, these results demonstrate that tumor hypoxia develops at an increased distance from perfused blood vessels after irradiation, suggesting a decrease in oxygen consumption at 24 h. By 72 h postirradiation, all physiological parameters had returned to the levels in volume-matched, nonirradiated controls. These studies clearly show that single measures of either tumor oxygenation or vascular structure are inadequate for assessing the effects of radiation on tumor clonogenicity. Although such direct measurements have previously proven valuable in predicting tumor response to therapy or oxygen manipulation, a combination of parameters is required to adequately describe the mechanisms underlying these changes after irradiation.

Jauchem, J. R., Ryan, K. L., Frei, M. R., Dusch, S. J., Lehnert, H. M. and Kovatch, R. M. Repeated Exposure of C3H/HeJ Mice to Ultra-wideband Electromagnetic Pulses: Lack of Effects on Mammary Tumors.

It has been suggested that chronic, low-level exposure to radiofrequency (RF) radiation may promote the formation of tumors. Previous studies, however, showed that low-level, long-term exposure of mammary tumor-prone mice to 435 MHz or 2450 MHz RF radiation did not affect the incidence of mammary tumors. In this study, we investigated the effects of exposure to a unique type of electromagnetic energy: pulses composed of an ultra-wideband (UWB) of frequencies, including those in the RF range. One hundred C3H/HeJ mice were exposed to UWB pulses (rise time 176 ps, fall time 3.5 ns, pulse width 1.9 ns, peak E-field 40 kV/m, repetition rate 1 kHz). Each animal was exposed for 2 min once a week for 12 weeks. One hundred mice were used as sham controls. There were no significant differences between groups with respect to incidence of palpated mammary tumors, latency to tumor onset, rate of tumor growth, or animal survival. Histopathological evaluations revealed no significant differences between the two groups in numbers of neoplasms in all tissues studied (lymphoreticular tissue, thymus, respiratory, digestive and urinary tracts, reproductive, mammary and endocrine systems, and skin). Our major finding was the lack of effects of UWB-pulse exposure on promotion of mammary tumors in a well-established animal model of mammary cancer.