Wang, R. and Coderre, J. A. A Bystander Effect in Alpha-Particle Irradiations of Human Prostate Tumor Cells. Radiat. Res. 164, 711–722 (2005).

Alpha-particle exposures were used to determine whether cells of the human prostate carcinoma cell line DU-145 can produce and respond to a bystander effect signal. An apparatus for α-particle irradiation of cells growing as a monolayer on a 1.4-μm-thick Mylar membrane directly above an ²⁴¹Am α-particle source was constructed and calibrated. At the cell irradiation position, the α-particle fluence was 998 counts/mm² s⁻¹, the average α-particle energy was 3.14 MeV, and the average linear energy transfer was 128 keV/μm. The average dose rate to the cells growing on the Mylar surface was 1.2 Gy/min. A co-culture system was used to examine bystander effects transmitted through the medium from the directly targeted cells to tumor cells growing on an insert well beyond the range of the α particles. Alpha-particle doses from 0.1 to 6.0 Gy to the targeted cells on the Mylar membrane, followed by a 2-h co-incubation of the cells on the insert in the irradiated medium above the irradiated cells, all caused an ∼50% increase in micronucleus formation in the nontargeted co-cultured cells. Addition of the radical scavenger DMSO to the medium during the irradiation and the 2-h postirradiation incubation period completely blocked the bystander effect, whereas addition of a nitric oxide scavenger had no effect. Irradiation of medium containing serum, followed by a 2-h incubation, caused no bystander effect in the co-cultured cells. When the co-cultured cells on the insert were placed into the irradiated medium above the directly targeted cells immediately (∼1 min) after the irradiation and co-incubated for 2 h, there was no bystander effect. These data indicate that the observed bystander effect requires that the co-cultured cells be present in the medium during the irradiation of the directly targeted cells and suggest the involvement of a short-lived radical species.

Tokuzumi, S., Hori, M., Monobe, M., Hosoi, Y. and Kojima, S. Effect of Nitric Oxide on γ-Ray-Induced Micronucleus Frequency in RAW264.7 Cells. Radiat. Res. 164, 723–732 (2005).

The effect of low-dose nitric oxide (NO) on γ-ray-induced micronucleus (MN) frequency was investigated in RAW264.7 cells. Treatment of RAW264.7 cells with 0.25 mM sodium nitroprusside (SNP), a chemical NO donor, reduced the frequency of micronuclei induced by 5 Gy γ rays by 43 to 45% between 3 and 12 h post-treatment. This effect was blocked by carboxy-PTIO, suggesting that NO may play a role in the reduction of radiation-induced MN frequency. To examine possible mechanisms underlying this effect, we first looked at changes in the antioxidant system after SNP treatment. A significant increase in intracellular glutathione (GSH) was seen in SNP-treated cells between 3 and 12 h post-treatment. Depletion of GSH with buthionine sulfoximine (BSO) increased the γ-ray-induced increase in MN frequency. Detailed studies using various inducers of intracellular GSH suggested that GSH induction has a partial role in the reducing effect of NO on the γ-ray-induced MN frequency. Next, the effect of NO on DNA repair and replication systems was examined. Wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK), dose-dependently inhibited the reducing effect of NO, while caffeine, an inhibitor of ATM kinase and ATR kinase, did not. DNA-PK activity was increased by NO treatment. Etoposide, a topoisomerase II inhibitor, dose-dependently blocked the effect of NO in reducing the γ-ray-induced MN frequency. These results suggest that the mechanisms of the effect of NO on the γ-ray-induced MN frequency include elevation of GSH and up-regulation of DNA-PK activity for repairing double-strand breaks. NO may act as a signal for repair systems, e.g. for nonhomologous recombination and for the replication system in S phase, to reduce the MN frequency.

Banáth, J. P., Sinnott, L., Larrivée, B., MacPhail, S. H. and Olive, P. L. Growth of V79 Cells as Xenograft Tumors Promotes Multicellular Resistance but does not Increase Spontaneous or Radiation-Induced Mutant Frequency. Radiat. Res. 164, 733–744 (2005).

A Chinese hamster V79 xenograft model was developed to determine whether cells subjected to a hypoxic tumor microenvironment would be more likely to undergo mutation at the HPRT locus. V79-171b cells stably transfected with VEGF and EGFP were grown subcutaneously in immunodeficient NOD/ SCID mice. V79-VE tumors were characterized for host cell infiltration, doubling time, hypoxic fraction, vascular perfusion, and response to ionizing radiation. When irradiated in vitro, the mutant frequency for a given surviving fraction did not differ for cells grown in vivo or in vitro. Similar results were obtained using HCT116 human colorectal carcinoma cells grown as xenografts. However, V79-VE cells grown as xenografts were significantly more resistant to killing than monolayers. The background mutant frequency and the radiation-induced mutant frequency did not differ for tumor cells close to or distant from blood vessels. Similarly, tumor cells from well-perfused regions showed the same rate of strand break rejoining and the same rate of loss of phosphorylated histone H2AX as cells sorted from poorly perfused regions. Therefore, deleterious effects of the tumor microenvironment on DNA repair efficiency or mutation induction could not be demonstrated in these tumors. Rather, development of multicellular resistance in V79-VE tumors acted to reduce mutant frequency for a given dose of radiation.

Tyrsina, E. G., Slanina, S. V., Kakpakova, E. S., Kalendo, G. S., Kan, N. G., Tyrsin, O. Y. and Ryskov, A. P. Isolation and Characterization of Highly Radioresistant Malignant Hamster Fibroblasts that Survive Acute γ Irradiation with 20 Gy. Radiat. Res. 164, 745–754 (2005).

To study the acquired radioresistance of tumor cells, a model system of two cell lines, Djungarian hamster fibroblasts (DH-TK^–) and their radioresistant progeny, was established. The progeny of irradiated cells were isolated by treating the parental cell monolayer with a single dose of 20 Gy (PIC-20). The genetic and morphological features, clonogenic ability, radiosensitivity, cell growth kinetics, ability to grow in methylcellulose, and tumorigenicity of these cell lines were compared. The plating efficiency of PIC-20 cells exceeded that of DH-TK^– cells. The progeny of irradiated cells were more radioresistant than parental cells. The average D₀ for PIC-20 cells was 7.4 ± 0.2 Gy, which is three times higher than that for parental cells (2.5 ± 0.1 Gy). Progeny cell survival in methylcellulose after irradiation with a dose of 10 Gy was 15 times higher than that of DH-TK^– cells. In contrast to parental cells, the progeny of irradiated cells showed fast and effective repopulation after irradiation with doses of 12.5 and 15 Gy. The tumor formation ability of irradiated progeny cells was higher than that of parental cells; after 15 Gy irradiation, PIC-20 cells produced tumors as large as unirradiated progeny of irradiated cells, whereas the tumor development of DH-TK^– cells diminished by 70%. High radioresistance of progeny of irradiated cells was reproduced during the long period of cultivation (more than 80 passages). The stability of the radioresistant phenotype of PIC-20 cells allows us to investigate the possible mechanisms of acquired tumor radioresistance.

Pang, D., Rodgers, J. E., Berman, B. L., Chasovskikh, S. and Dritschilo, A. Spatial Distribution of Radiation-Induced Double-Strand Breaks in Plasmid DNA as Resolved by Atomic Force Microscopy. Radiat. Res. 164, 755–765 (2005).

Atomic force microscopy (AFM) has been used to directly visualize, size and compare the DNA fragments resulting from exposure to low- and high-LET radiation. Double-stranded pUC-19 plasmid (“naked”) DNA samples were irradiated by electron-beam or reactor neutron fluxes with doses ranging from 0.9 to 10 kGy. AFM scanning in the tapping mode was used to image and measure the DNA fragment lengths (ranging from a few bp up to 2864 bp long). Double-strand break (DSB) distributions resulting from high-LET neutron and lower-LET electron irradiation revealed a distinct difference between the effects of these two types of radiation: Low-LET radiation-induced DSBs are distributed more uniformly along the DNA, whereas a much larger proportion of neutron-induced DSBs are distributed locally and densely. Furthermore, comparisons with predictions of a random DSB model of radiation damage show that neutron-induced DSBs deviate more from the model than do electron-induced DSBs. In summary, our high-resolution AFM measurements of radiation-induced DNA fragment-length distributions reveal an increased number of very short fragments and hence clustering of DSBs induced by the high-LET neutron radiation compared with low-LET electron radiation and a random DSB model prediction.

Lobachevsky, P. N. and Martin, R. F. DNA Breakage by Decay of Auger Electron Emitters: Experiments with ¹²³I-iodoHoechst 33258 and Plasmid DNA. Radiat. Res. 164, 766– 773 (2005).

The Auger electron-emitting isotope ¹²³I is of interest in the context of potential exploitation of Auger electron emitters in radioimmunotherapy. The efficiency of induction of cytotoxic lesions by decay of DNA-associated ¹²⁵I, the prototype Auger electron emitter, is well established, but its long half-life (60 days) is a limitation. However, the advantage of the much shorter half-life of ¹²³I (13.2 h) might be outweighed by its “weaker” Auger electron cascade with an average of 8–11 Auger electrons, compared to about 15–21 electrons for ¹²⁵I. Accordingly, the efficiency of DNA breakage for DNA-associated ¹²³I was investigated by incubation of ¹²³I-iodoHoechst 33258 with plasmid DNA. The efficiency of double-strand break induction by decay of ¹²³I was 0.62 compared to 0.82 per decay of ¹²⁵I in the same experimental system. In the presence of dimethylsulfoxide, the values were 0.54 and 0.65 for decay of ¹²³I and ¹²⁵I, respectively. The results also showed that at a very low ligand/plasmid molar ratio (<1), the majority of cleavage seemed to occur at a particular site on the plasmid molecule, indicating preferential binding of the ¹²³I-ligand to a unique site or a cluster of neighboring sites.

Culard, F., Bouffard, S. and Charlier, M. High-LET Irradiation of a DNA-Binding Protein: Protein-Protein and DNA-Protein Crosslinks. Radiat. Res. 164, 774–780 (2005).

The chromosomal protein MC1 is a monomeric protein of 93 amino acids that is able to bind any DNA but has a slight preferential affinity for some sequences and structures, like cruciform and minicircles. The protein has been irradiated with ³⁶Ar¹⁸ ions of 95 MeV/nucleon. The LET of these particles in water is close to 270 keV/μm. We tested the activity of the protein by measuring its ability to form complexes with DNA. We tested the integrity of the protein by measuring the molecular weight of the species formed. Compared with γ radiation, we observed for the same dose a less efficient inactivation of the protein, a greater protection of the protein by the bound DNA, a lower induction of chain breakage, and a greater production of protein-protein and DNA-protein crosslinks. The results are discussed in terms of the quantitative and the qualitative differences between the two types of radiation: The global radical yield is slightly higher with γ rays, whereas the density of radicals produced along the particle track is considerably higher with argon ions.

Singh, R. K. and Krishna, M. DNA Strand Breaks Signal the Induction of DNA Double-Strand Break Repair in Saccharomyces cerevisiae. Radiat. Res. 164, 781–790 (2005).

Genotoxic stress induces a checkpoint signaling cascade to generate a stress response. Saccharomyces cerevisiae shows an altered radiation response under different type of stress. Although the induction of repair has been implicated in enhanced survival after exposure to the challenging stress, the nature of the signal remains poorly understood. This study demonstrates that low doses of γ radiation and bleomycin induce RAD52-dependent recombination repair pathway in the wild-type strain D-261. Prior exposure of cells to DNA-damaging agents (γ radiation or bleomycin) equips them better for the subsequent damage caused by challenging doses. However, exposure to UV light, which does not cause strand breaks, was ineffective. This was confirmed by PFGE studies. This indicates that the strand breaks probably serve as the signal for induction of the recombination repair pathway while pyrimidine dimers do not. The nature of the induced repair was investigated by mutation scoring in special strain D-7, which showed that the induced repair is essentially error free.

McNamee, J. P., Bellier, P. V., Chauhan, V., Gajda, G. B., Lemay, E. and Thansandote, A. Evaluating DNA Damage in Rodent Brain after Acute 60 Hz Magnetic-Field Exposure. Radiat. Res. 164, 791–797 (2005).

In recent years, numerous studies have reported a weak association between 60 Hz magnetic-field exposure and the incidence of certain cancers. To date, no mechanism to explain these findings has been identified. The objective of the current study was to investigate whether acute magnetic-field exposure could elicit DNA damage within brain cells from both whole brain and cerebellar homogenates from adult rats, adult mice and immature mice. Rodents were exposed to a 60 Hz magnetic field (0, 0.1, 1 or 2 mT) for 2 h. Then, at 0, 2 and 4 h after exposure, animals were killed humanely, their brains were rapidly removed and homogenized, and cells were cast into agarose gels for processing by the alkaline comet assay. Four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. For each species, a significant increase in DNA damage was detected by each of the four parameters in the positive control (2 Gy X rays) relative to the concurrent nonirradiated negative and sham controls. However, none of the four parameters detected a significant increase in DNA damage in brain cell homogenates from any magnetic-field exposure (0– 2 mT) at any time after exposure. The dose–response and time-course data from the multiple animal groups tested in this study provide no evidence of magnetic-field-induced DNA damage.

Galloni, P., Parazzini, M., Piscitelli, M., Pinto, R., Lovisolo, G. A., Tognola, G., Marino, C. and Ravazzani, P. Electromagnetic Fields from Mobile Phones do not Affect the Inner Auditory System of Sprague-Dawley Rats. Radiat. Res. 164, 798–804 (2005).

The auditory system is the first biological structure facing the electromagnetic fields emitted by mobile phones. The aim of this study was to evaluate the cochlear functionality of Sprague-Dawley rats exposed to electromagnetic fields at the typical frequencies of GSM mobile phones (900 and 1800 MHz) by distortion product otoacoustic emissions, which are a well-known indicator of the status of the cochlea's outer hair cells. A population of 48 rats was divided into exposed and sham-exposed groups. Three sets of four loop antennas, one for sham-exposed animals and two for exposed animals, were used for the local exposures. Rats were exposed 2 h/day, 5 days/week for 4 weeks at a local SAR of 2 W/kg in the ear. Distortion product otoacoustic emissions tests were carried out before, during and after the exposure. The analysis of the data shows no statistically significant differences between the audiological signals recorded for the different groups.

Gueulette, J., Binns, P. J., De Coster, B. M., Lu, X-Q., Roberts, S. A. and Riley, K. J. RBE of the MIT Epithermal Neutron Beam for Crypt Cell Regeneration in Mice. Radiat. Res. 164, 805–809 (2005).

The RBE of the new MIT fission converter epithermal neutron capture therapy (NCT) beam has been determined using intestinal crypt regeneration in mice as the reference biological system. Female BALB/c mice were positioned separately at depths of 2.5 and 9.7 cm in a Lucite phantom where the measured total absorbed dose rates were 0.45 and 0.17 Gy/ min, respectively, and irradiated to the whole body with no boron present. The γ-ray (low-LET) contributions to the total absorbed dose (low- high-LET dose components) were 77% (2.5 cm) and 90% (9.7 cm), respectively. Control irradiations were performed with the same batch of animals using 6 MV photons at a dose rate of 0.83 Gy/min as the reference radiation. The data were consistent with there being a single RBE for each NCT beam relative to the reference 6 MV photon beam. Fitting the data according to the LQ model, the RBEs of the NCT beams were estimated as 1.50 ± 0.04 and 1.03 ± 0.03 at depths of 2.5 and 9.7 cm, respectively. An alternative parameterization of the LQ model considering the proportion of the high- and low-LET dose components yielded RBE values at a survival level corresponding to 20 crypts (16.7%) of 5.2 ± 0.6 and 4.0 ± 0.7 for the high-LET component (neutrons) at 2.5 and 9.7 cm, respectively. The two estimates are significantly different (P = 0.016). There was also some evidence to suggest that the shapes of the curves do differ somewhat for the different radiation sources. These discrepancies could be ascribed to differences in the mechanism of action, to dose-rate effects, or, more likely, to differential sampling of a more complex dose–response relationship.

Kubale, T. L., Daniels, R. D., Yiin, J. H., Couch, J., Schubauer-Berigan, M. K., Kinnes, G. H., Silver, S. R., Nowlin, S. J. and Chen, P. A Nested Case-Control Study of Leukemia Mortality and Ionizing Radiation at the Portsmouth Naval Shipyard. Radiat. Res. 164, 810–819 (2005).

A nested case-control study using conditional logistic regression was conducted to evaluate the exposure–response relationship between external ionizing radiation exposure and leukemia mortality among civilian workers at the Portsmouth Naval Shipyard (PNS), Kittery, Maine. The PNS civilian workers received occupational radiation exposure while performing construction, overhaul, repair and refueling activities on nuclear-powered submarines. The study age-matched 115 leukemia deaths with 460 controls selected from a cohort of 37,853 civilian workers employed at PNS between 1952 and 1992. In addition to radiation doses received in the workplace, a secondary analysis incorporating doses from work-related medical X rays and other occupational radiation exposures was conducted. A significant positive association was found between leukemia mortality and external radiation exposure, adjusting for gender, radiation worker status, and solvent exposure duration (OR = 1.08 at 10 mSv of exposure; 95% CI = 1.01, 1.16). Solvent exposure (including benzene and carbon tetrachloride) was also significantly associated with leukemia mortality adjusting for radiation dose, radiation worker status, and gender. Incorporating doses from work-related medical X rays did not change the estimated leukemia risk per unit of dose.

Wong, F. L., Yamada, M., Tominaga, T., Fujiwara, S. and Suzuki, G. Effects of Radiation on the Longitudinal Trends of Hemoglobin Levels in the Japanese Atomic Bomb Survivors. Radiat. Res. 164, 820–827 (2005).

The late effects of radiation on the hematopoietic system have not been fully evaluated. We examined the long-term effects of radiation exposure on hemoglobin levels in the Japanese atomic bomb survivors over a 40-year period from 1958 to 1998. Compared to the unexposed survivors, the mean hemoglobin levels for those exposed to a bone marrow dose of 1 Gy were significantly reduced by 0.10 g/dl (95% CI: 0.04 to 0.16) or 0.67% at 40 years of age (P < 0.0001) and by 0.24 g/dl (95% CI: 0.08 to 0.40) or 1.8% at 80 years of age. Radiation effects are greater for smokers than for nonsmokers at age less than 35 years (P < 0.01), although cigarette smoking was associated with increased hemoglobin levels. Sex and birth cohort differences in radiation effects were not found after adjusting for smoking. The radiation-induced reduction in hemoglobin levels could not be explained by the presence of certain anemia-associated diseases.