Sgura, A., Cherubini, A. R. and Tanzarella, C. Chromosome Nondisjunction and Loss Induced by Protons and X Rays in Primary Human Fibroblasts: Role of Centromeres in Aneuploidy. Radiat. Res. 156, 225–231 (2001).

To study the origin of micronuclei induced in human primary fibroblasts by low-energy protons (7.7 and 28.5 keV/μm) and X rays, we have developed a combined antikinetochore-antibody (CREST) and FISH staining with pancentromeric probes. This technique allowed us to analyze the integrity of the kinetochore and centromeric DNA structures and to assess their role in induced aneuploidy. The effect of LET on radiation-induced chromosome nondisjunction was studied in binucleated cells with centromeric-specific DNA probes for chromosomes 7 and 11. Our results indicate that, though more than 90% of radiation-induced micronuclei were CREST⁻/FISH⁻, 28.5 keV/μm protons and X rays were also able to induce statistically significant increases in the number of micronuclei that were CREST⁻/FISH and CREST /FISH , respectively. One interpretation of these results could be that the protons induced chromosome loss by kinetochore detachment or by breakage in the centromeric DNA region, whereas X rays induced aneuploidy through a non-DNA damage mechanism. Nondisjunction appears to be a far more important mechanism leading to radiation-induced aneuploidy. Irrespective of the higher frequency of micronuclei induced by 28.5 keV/μm protons, the frequency of chromosome loss was markedly higher for X rays than for 28.5 keV/μm protons, strengthening the hypothesis that non-DNA targets, such as components of the mitotic spindle apparatus, may be involved in aberrations in chromosome segregation after X irradiation.

Oh, C-W., Bump, E. A., Kim, J-S., Janigro, D. and Mayberg, M. R. Induction of a Senescence-Like Phenotype in Bovine Aortic Endothelial Cells by Ionizing Radiation. Radiat. Res. 156, 232–240 (2001).

Treatment of confluent monolayers of bovine aortic endothelial cells (BAEC) with γ rays resulted in the delayed appearance of cells with an enlarged surface area that were morphologically similar to senescent cells. The majority of these cells stained positively for senescence-associated β-galactosidase (SA-β-gal), indicating that these cells are biochemically similar to senescent cells. The incidence of the senescence-like phenotype increased with dose (5–15 Gy) and time after irradiation. Cells with a senescence-like phenotype began to appear in the monolayer several days after irradiation. The onset of the appearance of this phenotype was accelerated by subculturing 24 h after irradiation. This acceleration was not entirely due to stimulation of progression through the cell cycle, since a high percentage of the senescent-like cells that appeared after subculture were not labeled with BrdUrd during the period after subculture. Prolonged up-regulation of expression of CDKN1A (also known as p21^CIP1/WAF1) after irradiation was noted by Western blot analysis, again suggesting a similarity to natural senescence. Phenotypically altered endothelial cells were present in the irradiated monolayers as long as 20 weeks after irradiation, suggesting that a subpopulation of altered endothelial cells that might be functionally deficient could persist in the vasculature of irradiated tissue for a prolonged period after irradiation.

Mothersill, C., Lyng, F., Mulford, A., Seymour, C., Cottell, D., Lyons, M. and Austin, B. Effect of Low Doses of Ionizing Radiation on Cells Cultured from the Hematopoietic Tissue of the Dublin Bay Prawn, Nephrops norvegicus. Radiat. Res. 156, 241–250 (2001).

Explant cultures from the hematopoietic tissue of the Dublin Bay prawn, Nephrops norvegicus, were exposed to low doses of ⁶⁰Co γ radiation. Cells growing from the explants were examined 7 days after irradiation using light and transmission electron microscopy and were also tested for their ability to produce signals indicative of a bystander effect. The exposed cultures displayed pronounced damage and were orders of magnitude more sensitive than the data in the literature would suggest for arthropod cells. The cultures were also more sensitive than mammalian cells that were exposed to similar doses. Cellular abnormalities included damage to cytoplasmic organelles, particularly the cytoskeleton. Abnormal mitochondria were also prominent. At low doses (0.5 Gy), nuclear damage was not apparent in the cultures, but there was evidence of a dose-dependent increase in apoptosis. The irradiated cultures released a factor into the medium that was capable of inducing apoptosis and cell death in unirradiated fish and human cells. This bystander effect was of a similar magnitude to that reported for mammalian cell systems. It is suggested that these crustaceans may be highly sensitive to radiation, unlike terrestrial arthropods and certain other invertebrates, which are generally considered to be radioresistant.

Lewis, D. A., Mayhugh, B. M., Qin, Y., Trott, K. and Mendonca, M. S. Production of Delayed Death and Neoplastic Transformation in CGL1 Cells by Radiation-Induced Bystander Effects. Radiat. Res. 156, 251–258 (2001).

Other investigators have demonstrated by transfer of medium from irradiated cells and by irradiation with low-fluence α particles or microbeams that cells do not have to be directly exposed to ionizing radiation to be detrimentally affected, i.e. bystander effects. In this study, we demonstrate by transfer of medium from X-irradiated human CGL1 hybrid cells that the killing of bystander cells reduces the plating efficiency of the nonirradiated CGL1 cells by 33 ± 6%. In addition, we show that the amount of cell death induced by bystander effects is not dependent on X-ray dose, and that the induction of apoptosis does not appear to be responsible for the cell death. Furthermore, we found that the reduction in plating efficiency in bystander cells is evident for over 18 days, or 22 cell population doublings, after medium transfer, despite repeated refeeding of the cell cultures. Finally, we report the novel observation that bystander effects induced by the transfer of medium from irradiated cells can induce neoplastic transformation. Exposing unirradiated CGL1 cells to medium from cells irradiated with 5 or 7 Gy increased the frequency of neoplastic transformation significantly from 6.3 × 10^–6 in unirradiated controls to 2.3 × 10^–5 (a factor of nearly four). We conclude that the bystander effect induces persistent, long-term, transmissible changes in the progeny of CGL1 cells that result in delayed death and neoplastic transformation. The data suggest that neoplastic transformation in bystander cells may play a significant role in radiation-induced neoplastic transformation at lower doses of X rays.

Evans, H. H., Horng, M. F., Ricanati, M., Diaz-Insua, M., Jordan, R. and Schwartz, J. L. Diverse Delayed Effects in Human Lymphoblastoid Cells Surviving Exposure to High-LET ⁵⁶Fe Particles or Low-LET ¹³⁷Cs Gamma Radiation. Radiat. Res. 156, 259–271 (2001).

To obtain information on the origin of radiation-induced genomic instability, we characterized a total of 166 clones that survived exposure to ⁵⁶Fe particles or ¹³⁷Cs γ radiation, isolated approximately 36 generations after exposure, along with their respective control clones. Cytogenetic aberrations, growth alterations, responses to a second irradiation, and mutant frequencies at the Na /K ATPase and thymidine kinase loci were determined. A greater percentage of clones that survived exposure to ⁵⁶Fe particles exhibited instability (defined as clones showing one or more outlying characteristics) than in the case of those that survived γ irradiation. The phenotypes of the unstable clones that survived exposure to ⁵⁶Fe particles were also qualitatively different from those of the clones that survived γ irradiation. A greater percentage (20%) of the unstable clones that survived γ irradiation than those that survived exposure to ⁵⁶Fe particles (4%) showed an altered response to the second irradiation, while an increase in the percentage of clones that had an outlying frequency of ouabain-resistant and thymidine kinase mutants was more evident in the clones exposed to ⁵⁶Fe particles than in those exposed to γ rays. Growth alterations and increases in dicentric chromosomes were found only in clones with more than one alteration. These results underscore the complex nature of genomic instability and the likelihood that radiation-induced genomic instability arises from different original events.

Turcotte, S. and Averill-Bates, D. A. Sensitization to the Cytotoxicity of Melphalan by Ethacrynic Acid and Hyperthermia in Drug-Sensitive and Multidrug-Resistant Chinese Hamster Ovary Cells. Radiat. Res. 156, 272–282 (2001).

The ability of physical and pharmacological modulators to increase the cytotoxicity of melphalan was investigated in Chinese hamster ovary cells using a clonogenic cell survival assay. Hyperthermia has potential for use in cancer treatment, particularly as an adjuvant to chemotherapy or radiotherapy. Ethacrynic acid is a glutathione S-transferase inhibitor and also undergoes conjugation with glutathione. Interactions between hyperthermia (41–43°C), ethacrynic acid and melphalan were evaluated in multidrug-resistant (CH^RC5) cells with overexpression of P-glycoprotein (33.69-fold), and in drug-sensitive (AuxB1) cells. GST alpha was expressed at a higher level (3.65-fold) in CH^RC5 cells than in sensitive cells, whereas levels of isoforms pi and mu were the same. GST pi was the most highly expressed isoform in the two cell populations. Ethacrynic acid was cytotoxic at elevated temperatures, while it caused little or no cytotoxicity at 37°C. This effect occurred in drug-resistant and drug-sensitive cells, and attributes thermosensitizing properties to ethacrynic acid. Ethacrynic acid (20 μM) alone did not alter the cytotoxicity of melphalan at 37°C. Hyperthermia potentiated drug cytotoxicity in cells, both with and without ethacrynic acid treatment. Ethacrynic acid could be useful in cancer treatment by acting as a thermosensitizer when combined with heat and by enhancing the cytotoxicity of melphalan at elevated temperatures. A major advantage arising from the use of regional hyperthermia is the ability to target drug cytotoxicity to the tumor volume. A useful finding is that ethacrynic acid, heat and/or melphalan are also effective against multidrug-resistant cells with overexpression of P-glycoprotein.

Whitnall, M. H., Inal, C. E., Jackson, W. E., III, Miner, V. L., Villa, V. and Seed, T. M. In Vivo Radioprotection by 5-Androstenediol: Stimulation of the Innate Immune System. Radiat. Res. 156, 283–293 (2001).

We showed previously that 5-androstenediol stimulates myelopoiesis, increases the numbers of circulating neutrophils and platelets, and enhances resistance to infection in γ-irradiated mice. We have extended those studies to include monocytes, natural killer (NK) cells, eosinophils and basophils, and we have measured the activation marker CD11b using flow cytometry. Androstenediol (160 mg/kg) was administered subcutaneously to female B6D2F1 mice 24 h before whole-body γ irradiation. Androstenediol treatments increased the blood levels of neutrophils, monocytes and NK cells in unirradiated animals; decreased the numbers of circulating eosinophils; and ameliorated radiation-induced decreases in neutrophils, monocytes, NK cells, erythrocytes and platelets. The androstenediol treatments had no significant effect on the numbers of circulating B cells or T cells. CD11b labeling intensity on monocytes was decreased slightly after androstenediol treatment. In contrast, radiation or androstenediol alone caused increases in CD11b labeling intensity on NK cells. Androstenediol and radiation combined caused a marked increase in NK cell CD11b. The results indicate that androstenediol increases the numbers of the three major cell types of the innate immune system (neutrophils, monocytes and NK cells), that androstenediol-induced changes in blood elements in irradiated animals persist for at least several weeks, and that there is a significant positive interaction between radiation and administration of androstenediol in the activation of NK cells.

Amorino, G. P., Lee, H., Holburn, G. E., Paschal, C. B., Hercules, S. K., Shyr, Y., Steffen, R. P. and Choy, H. Enhancement of Tumor Oxygenation and Radiation Response by the Allosteric Effector of Hemoglobin, RSR13. Radiat. Res. 156, 294–300 (2001).

Prior studies using pO₂ microelectrodes have shown that RSR13, an allosteric modifier of hemoglobin, increases tissue oxygenation in vivo. Recently, measurements of tissue oxygenation have been performed by many investigators using blood oxygen level-dependent magnetic resonance imaging (BOLD MRI). In this study, we tested the hypothesis that the BOLD MRI signal ratio in tumors will change after administration of RSR13. NCI-H460 human lung carcinoma cells were used as a xenograft in athymic nude mice. Mice with 1-cm³ tumors in the flank were anesthetized and mounted on the MRI apparatus, and various doses of RSR13 were administered intraperitoneally (i.p.). MR images were then acquired at 10-min intervals for up to 60 min after injection. The effect of RSR13 on tumor response was studied using the same mouse xenograft model with tumor growth delay measurements. RSR13 increased the MRI signal ratio [Intensity(t)/Intensity(t = 0)] in a dose-dependent manner, with maximum increases occurring 30 min after RSR13 was administered. An RSR13 dose of 200 mg/kg proved to be optimum. Since the MRI signal ratio has been shown previously to be linearly related to tissue oxygenation, the changes in the MRI signal ratio can be attributed to changes in tumor oxygen levels. Using a 200-mg/kg dose of RSR13, with a 10-Gy dose of radiation administered to tumors 30 min later, enhancement of radiation-induced tumor growth delay by RSR13 was observed (enhancement factor = 2.8). Thus our MRI results support and verify the previously reported RSR13-induced increase in tumor oxygenation obtained using pO₂ microelectrodes. Based upon these results and other previous studies, the mechanism of enhancement of the effect of radiation by RSR13 probably involves an increase in tumor oxygenation.

Huber, P. E. and Debus, J. Tumor Cytotoxicity In Vivo and Radical Formation In Vitro Depend on the Shock Wave-Induced Cavitation Dose. Radiat. Res. 156, 301–309 (2001).

Local tumor therapy using focused ultrasonic waves may become an important treatment option. This technique exploits the ability of mechanical waves to induce thermal and nonthermal effects noninvasively. The cytotoxicity to cultured cells and biological tissues in vivo that results from exposure to ultrasonic shock waves is considered to be a nonthermal effect that is partly a consequence of ultrasound-induced cavitation. Cavitation is defined as the formation of bubbles during the negative wave cycle; their subsequent oscillation and/or violent implosion can affect surrounding structures. To investigate cavitational effects in cells and tissues, defined cavitation doses must be applied while ideally holding all other potential ultrasound parameters constant. The application of independent cavitation doses has been difficult and has yielded little knowledge about quantitative cavitation–tissue interactions. By using a special shock-wave pulse regimen and laser optical calibration in this study, we were able to control the cavitation dose independently of other physical parameters such as the pressure amplitudes, and averaged acoustic intensity. We treated Dunning prostate tumors (subline R3327-AT1) transplanted into Copenhagen rats with shock waves at three cavitation dose levels and then determined the tumor growth delay and the histopathological changes. All of the treated animals exhibited a significant tumor growth delay compared to the controls. Higher cavitation doses were associated with a greater delay in the growth of the tumor and more severe effects on tumor histopathology, such as hemorrhaging, tissue disruption, and necrosis. In vitro, the cavitation dose level correlated with the amount of radical formation. We concluded that the process of acoustic cavitation was responsible; higher cavitation doses caused greater effects in tumors both in vivo and in vitro. These findings may prove important in local tumor therapy and other applications of ultrasound such as ultrasound-mediated drug delivery.

Doke, T., Hayashi, T. and Borak, T. B. Comparisons of LET Distributions Measured in Low-Earth Orbit Using Tissue-Equivalent Proportional Counters and the Position-Sensitive Silicon-Detector Telescope (RRMD-III). Radiat. Res. 156, 310–317 (2001).

Determinations of the LET distribution, ϕ(L), of charged particles within a spacecraft in low-Earth orbit have been made. One method used a cylindrical tissue-equivalent proportional counter (TEPC), with the assumption that for each measured event, lineal energy, y, is equal to LET and thus ϕ(L) = ϕ(y). The other was based on the direct measurement of LETs for individual particles using a charged-particle telescope consisting of position-sensitive silicon detectors called RRMD-III. There were differences of up to a factor of 10 between estimates of ϕ(L) using the two methods on the same mission. This caused estimates of quality factor to vary by a factor of two between the two methods.

Saito, N. and Suzuki, I. H. Photon W Value for Krypton in the M-Shell Transition Region. Radiat. Res. 156, 317–323 (2001).

Absolute W values for krypton have been measured for incident X rays with energies in the range of 85 to 1000 eV, using monochromatic synchrotron radiation and a multiple-electrode ion chamber technique that yields the absolute intensity of the X-ray beam and the photoabsorption cross section. To improve the purity of the incident X rays, the electron storage ring was operated at an energy lower than the normal mode, and thin filters were used. The W values are derived from the measured photon intensity and photoabsorption cross section, using the mean charges of the residual ions obtained in previous work. A considerable oscillation of the W values with the photon energy was found in the region near the krypton 3d electron ionization edge. The results are discussed and compared with data in the literature for low-energy electrons and with the calculations from a model that includes multiple photoionization effects related to inner-shell ionization.

MacCallum, D. E., Hall, P. A. and Wright, E. G. The Trp53 Pathway is Induced In Vivo by Low Doses of Gamma Radiation. Radiat. Res. 156, 324–327 (2001).

The induction of the Trp53 response after very low doses (0.01–1 Gy) of ionizing radiation was studied in the adult mouse using immunochemical and immunohistochemical methods. We found a detectable response at 0.01 Gy and an increased induction of Trp53 with increasing dose in both radiation-resistant and radiation-sensitive tissues. These results suggest that there is no lower threshold for induction. This response was heterogeneous, since cells that received the same dose had different staining intensities, suggesting that the induction of Trp53 is not based simply on dose-dependent responses to DNA damage. These data also demonstrate the exquisite sensitivity of the Trp53 pathway and show that this response is controlled by cell- and tissue-specific factors that have yet to be defined.

Li, L., Bisht, K. S., LaGroye, I., Zhang, P., Straube, W. L., Moros, E. G. and Roti Roti, J. L. Measurement of DNA Damage in Mammalian Cells Exposed In Vitro to Radiofrequency Fields at SARs of 3–5 W/kg. Radiat. Res. 156, 328–332 (2001).

In the present study, we determined whether exposure of mammalian cells to 3.2–5.1 W/kg specific absorption rate (SAR) radiofrequency fields could induce DNA damage in murine C3H 10T½ fibroblasts. Cell cultures were exposed to 847.74 MHz code-division multiple access (CDMA) and 835.62 frequency-division multiple access (FDMA) modulated radiations in radial transmission line (RTL) irradiators in which the temperature was regulated to 37.0 ± 0.3°C. Using the alkaline comet assay to measure DNA damage, we found no statistically significant differences in either comet moment or comet length between sham-exposed cells and those exposed for 2, 4 or 24 h to CDMA or FDMA radiations in either exponentially growing or plateau-phase cells. Further, a 4-h incubation after the 2-h exposure resulted in no significant changes in comet moment or comet length. Our results show that exposure of cultured C3H 10T½ cells at 37°C CDMA or FDMA at SAR values of up to 5.1 W/kg did not induce measurable DNA damage.