Reddy, M. C. and Vasquez, K. M. Repair of Genome Destabilizing Lesions. Radiat. Res. 164, 345–356 (2005).

Living organisms are constantly exposed to detrimental agents both from the environment (e.g. ionizing radiation, ultraviolet light, natural and synthetic chemicals) and from endogenous metabolic processes (e.g. oxidative and hydrolytic reactions), resulting in modifications of proteins, lipids and DNA. Proteins and lipids are degraded and resynthesized, but the DNA is replicated only during cell division, when DNA damage may result in mutation fixation. Thus the DNA damage generated has the potential to lead to carcinogenesis, cell death, or other genetic disorders in the absence of efficient error-free repair. Because modifications in DNA sequence or structure may be incompatible with its essential role in preservation and transmission of genetic information from generation to generation, exquisitely sensitive DNA repair pathways have evolved to maintain genomic stability and cell viability. This review focuses on the repair and processing of genome destabilizing lesions and helical distortions that differ significantly from the canonical B-form DNA in mammalian cells. In particular, we discuss the introduction and processing of site-specific lesions in mammalian cells with an emphasis on psoralen interstrand crosslinks.

Moore, S. R., Ritter, L. E., Gibbons, C. F. and Grosovsky, A. J. Spontaneous and Radiation-Induced Genomic Instability in Human Cell Lines Differing in Cellular TP53 Status. Radiat. Res. 164, 357–368 (2005).

Structural chromosomal rearrangements are commonly observed in tumor karyotypes and in radiation-induced genomic instability. Here we report the effects of TP53 deficiency on karyotypic stability before and after irradiation using related cells and clones differing in cellular TP53 status. The parental cell line, TK6, is a TP53 wild-type human B-lymphoblastoid line with a highly stable karyotype. In the two TK6 derivatives used here, TP53 has been inactivated by biochemical means (expression of HPV16 E6; TK6-5E) or genetic means (allelic inactivation; NH32). Biochemical inactivation of TP53 (TK6-5E) had little effect on the spontaneous karyotype, whereas allelic inactivation of TP53 (NH32) resulted in a modest increase in spontaneous karyotypic instability. After 2 Gy γ irradiation, the number of unstable clones derived from TP53-deficient cells was significantly elevated compared to the TP53 wild-type counterpart. Extensively destabilized clones were common after irradiation in the set of clones derived from NH32 cells, and one was observed in the set of TK6-5E clones; however, they were never observed in TK6-derived clones. In two of the irradiated NH32 clones, whole chromosomes or chromosome bands were preferentially involved in alterations. These results suggest that genomic instability may differ both quantitatively and qualitatively as a consequence of altered TP53 expression. Some of the results showing repeated and preferential chromosome involvement in aberrations support a model in which instability may be driven by cis mechanisms.

Coleman, M. A., Yin, E., Peterson, L. E., Nelson, D., Sorensen, K., Tucker, J. D. and Wyrobek, A. J. Low-Dose Irradiation Alters the Transcript Profiles of Human Lymphoblastoid Cells Including Genes Associated with Cytogenetic Radioadaptive Response. Radiat. Res. 164, 369–382 (2005).

Low-dose ionizing radiation alters the gene expression profiles of mammalian cells, yet there is little understanding of the underlying cellular mechanisms responsible for these changes or of their consequences for genomic stability. We investigated the cytogenetic adaptive response of human lymphoblastoid cell lines exposed to 5 cGy (priming dose) followed by 2 Gy (challenge dose) compared to cells that received a single 2-Gy dose to (a) determine how the priming dose influences subsequent gene transcript expression in reproducibly adapting and non-adapting cell lines, and (b) identify gene transcripts that are associated with reductions in the magnitude of chromosomal damage after the challenge dose. The transcript profiles were evaluated using oligonucleotide arrays and RNA obtained 4 h after the challenge dose. A set of 145 genes (false discovery rate = 5%) with transcripts that were affected by the 5-cGy priming dose fell into two categories: (a) a set of common genes that were similarly modulated by the 5-cGy priming dose irrespective of whether the cells subsequently adapted or not and (b) genes with differential transcription in accordance with the cell lines that showed either adaptive or non-adaptive outcomes. The common priming-dose response genes showed up-regulation for protein synthesis genes and down-regulation of metabolic and signal transduction genes (>10-fold differences). The genes associated with subsequent adaptive and non-adaptive outcomes involved DNA repair, stress response, cell cycle control and apoptosis. Our findings support the importance of TP53-related functions in the control of the low-dose cytogenetic radioadaptive response and suggest that certain low-dose-induced alterations in cellular functions are predictive for the risk of subsequent genomic damage.

Cramers, P., Atanasova, P., Vrolijk, H., Darroudi, F., van Zeeland, A. A., Huiskamp, R., Mullenders, L. H. F. and Kleinjans, J. C. S. Pre-exposure to Low Doses: Modulation of X-Ray-Induced DNA Damage and Repair? Radiat. Res. 164, 383–390 (2005).

The adaptive response to ionizing radiation may be mediated by the induction of antioxidant defense mechanisms, accelerated repair or altered cell cycle progression after the conditioning dose. To gain new insight into the mechanism of the adaptive response, nondividing lymphocytes and fibroblasts were used to eliminate possible contributions of cell cycle effects. The effect of conditioning doses of 0.05 or 0.1 Gy followed by challenging doses up to 8 Gy (with a 4-h interval between exposures) on induction and repair of DNA damage was determined by single-cell gel electrophoresis (comet assay), premature chromosome condensation, and immunofluorescence labeling for γ-H2AX. The conditioning dose reduced the induction of DNA strand breaks, but the kinetics of strand break rejoining was not influenced by the conditioning dose in nondividing cells of either cell type. We conclude that adaptation in nondividing cells is not mediated by enhanced strand break rejoining and that protection against the induction of DNA damage is rather small. Therefore, the adaptive response is most likely a reflection of perturbation of cell cycle progression.

Edelmann, M., Gamarra, F., Kemp da Silva, A., Hornung, V., Castro, M., Passlick, B., Huber, R. M. and Bergner, A. Cell Cycle Effects of Radiation on Human Bronchial Epithelium and Lung Carcinoma Cells in Monolayer Cultures and a Three-Dimensional Co-culture System. Radiat. Res. 164, 391–399 (2005).

The aim of this study was to investigate whether the three-dimensional structure of the bronchial tissue and the contact of non-malignant with malignant cells influence the effectiveness of radiotherapy. Monolayer cultures of cells of the human bronchial epithelial cell line BEAS 2B, monolayer co-cultures of BEAS 2B cells and cells of the GFP-transfected lung carcinoma cell line EPLC 32M1, organ cultures of human bronchial epithelium, and organ co-cultures with EPLC 32M1 cells were irradiated with 10 Gy, and the DNA content was analyzed using flow cytometry. In non-malignant epithelial cells, BEAS 2B monolayer cultures without tumor cells were highly radiosensitive. However, contact with tumor cells in monolayer co-cultures markedly reduced radiosensitivity. Non-malignant cells in three-dimensional organ cultures and organ co-cultures with tumor cells showed moderate radiosensitivity. In EPLC 32M1 tumor cells, proliferation was increased without irradiation when the cells were in contact with epithelial cells in both organ and monolayer co-cultures. Radiosensitivity was higher in organ co-cultures than in monolayer cultures and monolayer co-cultures. These data indicate that organ co-cultures in combination with flow cytometry allow investigation of the effects of radiation in an in vivo-like environment and that both the spatial organization and the interaction of non-malignant and tumor cells are crucial for the effectiveness of radiotherapy.

Goblirsch, M., Lynch, C., Mathews, W., Manivel, J. C., Mantyh, P. W. and Clohisy, D. R. Radiation Treatment Decreases Bone Cancer Pain through Direct Effect on Tumor Cells. Radiat. Res. 164, 400–408 (2005).

The most used treatment for bone cancer pain is radiation; however, the mechanism responsible for analgesia after irradiation is unknown. The mechanistic influence of a single, localized 10-, 20- or 30-Gy dose of radiation on painful behaviors, osteolysis, histopathology and osteoclast number was evaluated in mice with painful femoral sarcomas. Dramatic reductions in pain behaviors (P < 0.05) and osteolysis (P < 0.0001) were seen in mice irradiated with 20 and 30 Gy. Irradiation reduced the tumor area by more than 75% (P < 0.05) but did not affect osteoclast frequency per mm² tumor. Treatment with 20 Gy prior to tumor injection had no effect on tumor growth or pain behaviors, suggesting that radiation reduces osteolysis and pain through direct tumor effects. To demonstrate that tumor elimination was responsible for reduction in osteolysis and pain, sarcoma cells containing the suicide gene cytosine deaminase (CD) were inoculated into femora. After onset of bone cancer pain, mice were treated with the prodrug 5-fluorocytosine (5-FC). 5-FC treatment significantly reduced both osteolysis (P < 0.0005) and bone cancer pain (P < 0.05). The findings in this study demonstrate that one mechanism through which radiation decreases bone cancer pain is by direct effects on tumor cells.

Bauer, S., Gusev, B. I., Pivina, L. M., Apsalikov, K. N. and Grosche, B. Radiation Exposure due to Local Fallout from Soviet Atmospheric Nuclear Weapons Testing in Kazakhstan: Solid Cancer Mortality in the Semipalatinsk Historical Cohort, 1960–1999. Radiat. Res. 164, 409–419 (2005).

Little information is available on the health effects of exposures to fallout from Soviet nuclear weapons testing and on the combined external and internal environmental exposures that have resulted from these tests. This paper reports the first analysis of the Semipalatinsk historical cohort exposed in the vicinity of the Semipalatinsk nuclear test site, Kazakhstan. The cohort study, which includes 19,545 inhabitants of exposed and comparison villages of the Semipalatinsk region, was set up in the 1960s and comprises 582,750 person-years of follow-up between 1960 and 1999. Cumulative effective radiation dose estimates in this cohort range from 20 mSv to ∼4 Sv. Rates of mortality and cancer mortality in the exposed group substantially exceeded those of the comparison group. Dose–response analyses within the exposed group confirmed a significant trend with dose for all solid cancers (P < 0.0001) and for digestive and respiratory cancers (P = 0.0255 and P < 0.0001), whereas no consistent dose–response trend was found for all causes of death (P = 0.4296). Regarding specific cancer sites, a significant trend with dose was observed for lung cancer (P = 0.0001), stomach cancer (P = 0.0050), and female breast cancer (P = 0.0040) as well as for esophagus cancer in women (P = 0.0030). The excess relative risk per sievert for all solid cancers combined was 1.77 (1.35; 2.27) based on the total cohort data, yet a selection bias regarding the comparison group could not be entirely ruled out. The excess relative risk per sievert based on the cohort's exposed group was 0.81 (0.46; 1.33) for all solid cancers combined and thus still exceeds current risk estimates from the Life Span Study. Future epidemiological assessments based on this cohort will benefit from extension of follow-up and ongoing validation of dosimetric data.

Whitehead, T. D., Brownstein, B. H., Parry, J. J., Thompson, D., Cha, B. A., Moros, E. G., Rogers, B. E. and Roti Roti, J. L. Expression of the Proto-oncogene Fos after Exposure to Radiofrequency Radiation Relevant to Wireless Communications. Radiat. Res. 164, 420–430 (2005).

In this study the expression levels of the proto-oncogene Fos were measured after exposure to radiofrequency (RF) radiation at two relatively high specific absorption rates (SARs) of 5 and 10 W/kg for three types of modulated signals: 847.74 MHz code division multiple access (CDMA), 835.62 MHz frequency division multiple access (FDMA), and 836.55 MHz time division multiple access (TDMA). This work was undertaken to confirm a previous report by Goswami et al. (Radiat. Res. 151, 300–309, 1999) that CDMA and FDMA radiation caused small but statistically significant increases in Fos levels as cells entered plateau phase during exposure. No effects on Myc or Jun levels were observed in that study. Therefore, in the present study, analyses were restricted to Fos expression during the transition from exponential growth to plateau phase. Fos expression was measured using the real-time polymerase chain reaction (RT-PCR) technique. Serum-stimulated C3H 10T½ cells were used as a positive control for Fos expression. Possible influences of final cell number or pH variability on Fos expression were evaluated. Expression of Fos mRNA in C3H 10T½ cells was not significantly different from that found after sham exposure at either SAR level for any signal modulation. Therefore, the results of Goswami et al. could not be confirmed.

Görlitz, B. D., Müller, M., Ebert, S., Hecker, H., Kuster, N. and Dasenbrock, C. Effects of 1-Week and 6-Week Exposure to GSM/DCS Radiofrequency Radiation on Micronucleus Formation in B6C3F1 Mice. Radiat. Res. 164, 431– 439 (2005).

The aim of this study was to examine the possible induction of micronuclei in erythrocytes of the peripheral blood and bone marrow and in keratinocytes and spleen lymphocytes of mice exposed to radiofrequency (RF) radiation for 2 h per day over periods of 1 and 6 weeks, respectively. The applied signal simulated the exposure from GSM900 and DCS1800 handsets, including the low-frequency amplitude-modulation components as they occur during speaking (GSM Basic), listening (DTX) and moving within the environment (handovers, power control). The carrier frequency was set to the center of the system's uplink band, i.e., 902 MHz for GSM and 1747 MHz for DCS. Uniform whole-body exposure was achieved by restraining the mice in tubes at fixed positions in the exposure setup. Mice were exposed to slot-averaged whole-body SARs of 33.2, 11.0, 3.7 and 0 mW/g during the 1-week study and 24.9, 8.3, 2.8 and 0 mW/g during the 6-week study. Exposure levels for the 1- and 6-week studies were determined in a pretest to confirm that no thermal effect was present that could influence the genotoxic end points. During both experiments and for both frequencies, no clinical abnormalities were detected in the animals. Cells of the bone marrow from the femur (1-week study), erythrocytes of the peripheral blood (6-week study), keratinocytes from the tail root, and lymphocytes from the spleen (both studies) were isolated on slides and stained for micronucleus analysis. Two thousand cells per animal were scored in erythrocyte and keratinocyte samples. In spleen lymphocytes, 1000 binucleated lymphocytes were scored for each animal. The RF-field exposure had no influence on the formation of red blood cells. After 1 week of exposure, the ratio of polychromatic to normochromatic erythrocytes was unchanged in the treated groups compared to the sham-exposed groups. Furthermore, the RF-field exposure of mice did not induce an increase in the number of micronuclei in erythrocytes of the bone marrow or peripheral blood, in keratinocytes, or in spleen lymphocytes compared to the sham-treated control.

Kappes, U. P. and Rünger, T. M. No Major Role for 7,8-Dihydro-8-oxoguanine in Ultraviolet Light-Induced Mutagenesis. Radiat. Res. 164, 440–445 (2005).

Oxidative DNA damage, in particular 7,8-dihydro-8-oxoguanine (8-oxoG), has been suggested to mediate mutation formation and malignant transformation after exposure of the skin to long-wave ultraviolet (UVA) light. It is processed primarily by the base excision repair (BER) pathway. The initial step of BER is the removal of the damaged base by a damage-specific DNA-glycosylase, which is 8-oxoG DNA glycosylase (OGG1) for 8-oxoG. To study the contribution of 8-oxoG to UVA-light mutagenesis, we compared UVA- and UVB-light-induced mutation frequencies in mouse embryonal fibroblasts from OGG1 knockout mice and their OGG1-intact littermates using the ouabain mutagenesis assay. After irradiation with various doses of UVA or UVB radiation, mutations in the Na,K-ATPase gene of single cells were detected by testing for colony-forming ability in a selective medium. OGG1^−/− cells did not exhibit an increased frequency of UV-light-induced mutations compared to OGG1 ^/ cells after exposure to either UVA or UVB radiation. This indicates that 8-oxoG, which is processed by OGG1, does not contribute significantly to either UVA- or UVB-light-induced mutagenesis.

Alegria, A. E., Sanchez-Cruz, P. and Lopez-Colon, D. Sonochemically Induced Covalent Binding of Calf Thymus DNA by Aziridinylquinones. Radiat. Res. 164, 446–452 (2005).

Sonolysis of argon- or oxygen-containing samples in the presence of calf thymus DNA and the diaziridinylquinones 2,5-bis-aziridin-1-yl-3,6-dichloro-1,4-benzoquinone (AZClQ) and 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone (AZQ) produced quinone-DNA covalent adducts at pH 5.5 and to a much lesser extent at pH 7.4. The corresponding semiquinone derivatives are detected using EPR spectroscopy after sonolysis of argon-saturated solutions at pH 7.4. The amount of covalent adducts decreases with addition of SOD, indicating a role of superoxide in this process. Addition of oxygen to the purging gas decreased but did not eliminate this covalent adduct. Thus this work suggests a possible synergism between bioreductive quinones and ultrasound in antitumor therapies based on alkylating quinone–DNA adduct formation with potential applications to both hypoxic and normally oxygenated conditions.

Brooks, A. L. Paradigm Shifts in Radiation Biology: Their Impact on Intervention for Radiation-Induced Disease. Radiat. Res. 164, 454–461 (2005).

New mechanistic cell and molecular studies on the effects of very low doses of radiation have resulted in three major paradigm shifts. First, the observation of bystander effects demonstrated that non-hit cells may respond as well as cells in which energy is deposited. Second, it was thought that gene mutations and chromosome aberrations were the most important early changes that represented the initiation phase of radiation-induced cancer. Now genomic instability that leads to the loss of genetic control appears to play a major role in the development of cancer. Finally, recent studies have demonstrated that radiation-induced changes in gene expression can be demonstrated at very low radiation doses. These changes can result in alterations in response pathways, many of which appear to be involved in protective or adaptive responses. The demonstration that unique genes are up- and down-regulated depending on the radiation type, dose and dose rate suggests that different molecular mechanisms are involved in responses to high and low radiation doses. The ability to alter radiation response by physical and chemical treatments suggests that it may be possible to intervene in the progression of radiation-induced diseases. Such intervention may decrease the cancer risk from radiation exposure. This new research also demonstrates that many nonlinear biological processes have an impact on the induction of cancer and the shape of dose–response functions. Thus, for low-LET radiation delivered at low dose rates, the linear, no-threshold hypothesis is not well supported, but it is adequately conservative in protecting against low-dose radiation risks.

Demant, P. The Genetic Factors in Cancer Development and their Implications for Cancer Prevention and Detection. Radiat. Res. 164, 462–466 (2005).

Experimental data from laboratory animals indicate that the same extent of DNA damage or the same mutations in oncogenes and tumor suppressor genes in different hosts result in widely differing cancer development because of numerous polymorphic tumor susceptibility genes. Similarly, recent epidemiological data indicate that susceptibility to common, “sporadic” cancers in humans is influenced considerably by multiple polymorphic host genes with relatively weak effects. This indicates that in addition to hereditary familial cancer syndromes, the sporadic cancer is also under strong genetic control. The multiplicity of genes involved, variation in exposure to environmental carcinogens, and small sizes of cancer families prevent efficient searches for the responsible genes in humans. Therefore, an alternative strategy based on the definition of susceptibility genes in experimental animals and the subsequent study of their human homologues has been successfully employed by several groups. This strategy also helped reveal several important features of cancer susceptibility, including mutual interactions of cancer susceptibility genes, their functional heterogeneity, and the existence of stage-specific control of cancer development. This latter phenomenon is especially important, because the susceptibility to early stages of cancer development may be quite different from that of late stages of cancer development. This needs to be taken into account when introducing preventive testing of biomarkers of early preneoplastic lesions or early cancers, because their predictive value is greatly influenced by the genetically determined individual tendency to proceed toward a more advanced form of neoplasia. Therefore, genetic testing of persons in danger of being exposed to carcinogenic factors should be an important part of the personnel selection.

Durante, M. Biomarkers of Space Radiation Risk. Radiat. Res. 164, 467–473 (2005).

Radiation risk estimates are based on epidemiological data obtained on Earth for cohorts exposed predominantly to acute doses of γ rays, and the extrapolation to the space environment is highly problematic and error-prone. The uncertainty can be reduced if risk estimates are compared directly to space radiation-induced biological alterations, i.e. by detecting biomarkers in astronauts. Chromosomal aberrations in peripheral blood lymphocytes are the only biomarker that can provide simultaneous information on dose, dose equivalent and risk, and they have been measured extensively in astronauts during the past 10 years. Individual relative risks calculated from chromosomal aberration measurements in crew members after single space missions in low-Earth orbit fall in the same range as the estimates derived from physical dosimetry, suggesting that the current system for radiogenic risk evaluation is essentially sound. However, the output of the biomarker test is dependent upon the sampling time. Recent results show a fast time-dependent decay of chromosomal aberrations in blood lymphocytes after space flight and a lack of correlation between translocations and cumulative dose in astronauts involved in two to five space missions. This “time factor” may reflect individual variability and time dependence in the risk produced by exposure to cosmic radiation during the flight. Biomarkers may be superior to dose in predicting space radiation risk, pending technical improvements in sensitivity, and validation by epidemiological studies.

George, K., Willingham, V. and Cucinotta, F. A. Stability of Chromosome Aberrations in the Blood Lymphocytes of Astronauts Measured after Space Flight by FISH Chromosome Painting. Radiat. Res. 164, 474–480 (2005).

Follow-up measurements of chromosome aberrations in the blood lymphocytes of astronauts were performed by FISH chromosome painting at various intervals from 5 months to more than 5 years after space flight and compared to preflight baseline measurements. For five of the six astronauts studied, the analysis of individual time courses for translocations revealed a temporal decline of yields with half-lives ranging from 10 to 58 months. The yield of exchanges remained unchanged for the sixth astronaut during an observation period of 5 months after flight. These results may indicate complications with the use of stable aberrations for retrospective dose reconstruction, and the differences in the decay time may reflect individual variability in risk from space radiation exposure.

Chang, P. Y., Bakke, J., Orduna, J., Lin, S. and Rupa, D. Proton-Induced Genetic Damage in lacZ Transgenic Mice. Radiat. Res. 164, 481–486 (2005).

The plasmid-based lacZ transgenic mouse model system was used to evaluate the mutagenic and genotoxic potential of 250 MeV/nucleon proton radiation by evaluating the frequency of micronucleated polychromatic reticulocytes in peripheral blood and bone marrow and the mutant frequencies of the lacZ reporter transgene in spleen and brain, respectively. Doses of 0.1–2 Gy produced dose- and time-dependent changes in the frequency of micronucleated polychromatic reticulocytes within 48 h, with peak induction up to sixfold above control levels. The frequency of micronucleated polychromatic reticulocytes returned to control levels within 1 week after exposure. With doses of 4 Gy, the elevation in the frequency of micronucleated polychromatic reticulocytes was delayed up to 1 week after exposure, but complete recovery to control levels was observed at 16 weeks postirradiation. Significant increase in mutant frequencies in brain tissue was observed at 8 week after proton exposure at doses as low of 0.1 Gy. Mutant frequencies in spleen increased up to twofold above spontaneous mutant frequencies at 8 weeks after exposure to 0.5–1 Gy. These effects appeared saturated at doses >1 Gy for both tissues, possibly due to elimination of damaged cells from the tissue systems. These in vivo results highlight the importance of considering tissue specificity, dose and temporal dependence when assessing radiation effects.

Ford, J. F., Maslowski, A. J., Redd, R. A. and Braby, L. A. Radiation Responses of Perfused Tracheal Tissue. Radiat. Res. 164, 487–492 (2005).

We are using a novel perfusion system to examine the effects of radiation on a model respiratory tissue. Tracheas taken from young adult male Fischer 344 rats are embedded in a growth factor-enriched agarose matrix that is mounted in a special apparatus designed to allow growth medium to periodically wash the epithelial surface of the lumen. A comparison of the microarray expression profiles of freshly harvested tracheas and tracheas maintained in perfusion culture for 24 h shows no significant difference except for an increase in expression of a few metabolism- and surfactant-related genes. Perfusion culture samples exposed to 4 Gy of X rays show a lower than expected increase in expression for some cell cycle- and repair-related genes.

Sutherland, B. M., Cuomo, N. C. and Bennett, P. V. Induction of Anchorage-Independent Growth in Primary Human Cells Exposed to Protons or HZE Ions Separately or in Dual Exposures. Radiat. Res. 164, 493–496 (2005).

Travelers on space missions will be exposed to a complex radiation environment that includes protons and heavy charged particles. Since protons are present at much higher levels than are heavy ions, the most likely scenario for cellular radiation exposure will be proton exposure followed by a hit by a heavy ion. Although the effects of individual ion species on human cells are being investigated extensively, little is known about the effects of exposure to both radiation types. One useful measure of mammalian cell damage is induction of the ability to grow in a semi-solid agar medium highly inhibitory to the growth of normal human cells, termed neoplastic transformation. Using primary human cells, we evaluated induction of soft-agar growth and survival of cells exposed to protons only or to heavy charged particles (600 MeV/ nucleon silicon) only as well as of cells exposed to protons followed after a 4-day interval by silicon ions. Both ions alone efficiently transformed the human cells to anchorage-independent growth. Initial experiments indicate that the dose responses for neoplastic transformation of cells exposed to protons and then after 4 days to silicon ions appear similar to that of cells exposed to silicon ions alone.

Zhang, Q., Williams, E. S., Askin, K. F., Peng, Y., Bedford, J. S., Liber, H. L. and Bailey, S. M. Suppression of DNA-PK by RNAi has Different Quantitative Effects on Telomere Dysfunction and Mutagenesis in Human Lymphoblasts Treated with γ Rays or HZE Particles. Radiat. Res. 164, 497–504 (2005).

Basic to virtually all relevant biological effects of ionizing radiation is the underlying damage produced in DNA and the subsequent cellular processing of such damage. The damage can be qualitatively different for different kinds of radiations, and the genetics of the biological systems exposed can greatly affect damage processing and ultimate outcome—the biological effect of concern. The accurate repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and function. Incorrect repair of such lesions results in chromosomal rearrangements and mutations that can lead to cancer and heritable defects in the progeny of irradiated parents. We have focused on the consequent phenotypic effects of faulty repair by examining connections between cellular radiosensitivity phenotypes relevant for carcinogenesis after exposure to ionizing radiation, and deficiencies in various components of the non-homologous end-joining (NHEJ) system. Here we produced deficiencies of individual components of the DNA-dependent protein kinase (DNA-PK) holoenzyme (Ku86 and the catalytic subunit, DNA-PKcs), both singly and in combination, using RNA interference (RNAi) in human lymphoblastoid cell lines. Exposure of cells exhibiting reduced protein expression to either γ rays or 1 GeV/nucleon iron particles demonstrated differential effects on telomere dysfunction and mutation frequency as well as differential effects between radiation qualities.

Suzuki, M., Tsuruoka, C., Uchihori, Y., Ebisawa, S., Yasuda, H. and Fujitaka, K. Reduction in Life Span of Normal Human Fibroblasts Exposed to Very Low-Dose-Rate Charged Particles. Radiat. Res. 164, 505–508 (2005).

We studied the effect of chronic low-dose irradiation with heavy ions on the life span of normal human fibroblasts in vitro. Cells were cultured in a CO₂ incubator that was placed in the irradiation room for biological studies of heavy ions in the Heavy Ion Medical Accelerator in Chiba (HIMAC) at National Institute of Radiological Sciences (NIRS) and were exposed to scattered radiations produced by heavy-ion beams for the life span of the cell population. The absorbed dose, which was measured using a thermoluminescence dosimeter (TLD) and a silicon semiconductor detector, was 1.4 mGy per day when the HIMAC was operated for biological experiments. The total number of population doublings of the exposed cells as reduced to 79–93% of that of nonexposed control cells. However, the life span of cells exposed to low-dose ¹³⁷Cs γ rays (∼1 mGy/day) in the CO₂ incubator in the γ-irradiation room in NIRS was prolonged to 104–106% of that of nonexposed control cells. Thus there is evidence that exposure to chronic low-dose heavy-ion radiation reduces the life span of cells.

Kawata, T., Ito, H., Saito, M., Uno, T., Okayasu, R., Liu, C., Kan'o, M., George, K. and Cucinotta, F. A. Caffeine Sensitizes Nondividing Human Fibroblasts to X Rays by Inducing a High Frequency of Misrepair. Radiat. Res. 164, 509–513 (2005).

Caffeine sensitizes cells to ionizing radiation, and this effect is believed to be associated with the disruption of DNA damage-responsive cell cycle checkpoints, which is controlled by ATM. Recent studies suggest that misrejoining of DSBs is one of the underlying mechanisms of AT cell hyper-radiosensitivity. In this study, we investigated the effects of caffeine and radiation on nongrowing G₀ normal human fibroblast cells by determining cell survival and scoring aberrations in calyculin A-induced G₂ chromosomes. Results from the cell survival study indicate that after X-ray exposure G₀ cells were sensitized by 24 h treatment with caffeine. Analysis of chromosome aberrations using FISH (fluorescence in situ hybridization) revealed a high frequency of aberrant cells and color junctions in the caffeine-treated cells. Since most DNA repair in nongrowing G₀ cells is believed to result from nonhomologous end joining (NHEJ), caffeine may influence the fidelity of the NHEJ pathway in irradiated G₀ cells.

Antonelli, F., Belli, M., Cuttone, G., Dini, V., Esposito, G., Simone, G., Sorrentino, E. and Tabocchini, M. A. Induction and Repair of DNA Double-Strand Breaks in Human Cells: Dephosphorylation of Histone H2AX and its Inhibition by Calyculin A. Radiat. Res. 164, 514–517 (2005).

Phosphorylation of histone H2AX at serine 139 (γ-H2AX) represents one of the earliest steps in DNA DSB signaling and repair, but the mechanisms of coupling this histone modification to DSB processing remain to be established. In this work, H2AX phosphorylation-dephosphorylation kinetics induced by low doses of γ rays in MRC-5 human fibroblasts was studied. The number of γ-H2AX foci increased rapidly, with the maximum reached 20 min after irradiation. Using calyculin A, a protein phosphatase inhibitor, no significant dephosphorylation was found in this time. At longer times, no further induction of γ-H2AX foci occurred. This indicates that the number of γ-H2AX foci scored at 20 min can be used as representative of the initial number of DSBs. Pulsed-field gel electrophoresis (PFGE) was also used to determine whether calyculin A-mediated inhibition of γ-H2AX dephosphorylation and DSB rejoining are independent phenomena. We found that the maintenance of the phosphate group at Ser 139 in γ-H2AX does not represent an obstacle for DSB rejoining. Preliminary experiments performed with 62 MeV/nucleon carbon ions have shown a longer persistence of γ-H2AX foci with respect to γ rays, consistent with the induction of damage that is more severe and difficult to repair.

Desai, N., Davis, E., O'Neill, P., Durante, M., Cucinotta, F. A. and Wu, H. Immunofluorescence Detection of Clustered γ-H2AX Foci Induced by HZE-Particle Radiation. Radiat. Res. 164, 518–522 (2005).

We studied the spatial and temporal distributions of foci of the phosphorylated form of the histone protein H2AX (γ-H2AX), which is known to be activated by double-strand breaks after irradiation of human fibroblast cells with high-energy silicon (54 keV/μm) and iron (176 keV/μm) ions. Here we present data obtained with the ion path parallel to a monolayer of human fibroblast cells that leads to γ-H2AX aggregates in the shape of streaks stretching over several micrometers in an x/y plane, thus enabling the analysis of the fluorescence distributions along the ion trajectories. Qualitative analyses of these distributions provide insights into DNA damage processing kinetics for high charge and energy (HZE) ions, including evidence of increased clustering of DNA damage and slower processing with increasing LET.

Ding, L-H., Shingyoji, M., Chen, F., Chatterjee, A., Kasai, K-E. and Chen, D. J. Gene Expression Changes in Normal Human Skin Fibroblasts Induced by HZE-Particle Radiation. Radiat. Res. 164, 523–526 (2005).

Studies have shown that radiation exposure affects global gene expression in mammalian cells. However, little is known about the effects of HZE particles on gene expression. To study these effects, human skin fibroblasts were irradiated with HZE particles of different energies and LETs. The data obtained from these experiments indicate that changes in gene expression are dependent on the energy of the radiation source. Particles with the highest energy, i.e. iron, induced the biggest expression changes in terms of numbers of genes and magnitudes of changes. Many genes were found to undergo significant expression changes after HZE-particle irradiation, including CDKN1A/p21, MDM2, TNFRSF6/fas, PCNA and RAD52. Unlike X rays, HZE particles expose cells to two types of radiation: primary ions and δ rays. We hypothesized that the biological effects of δ rays, which are secondary electron emissions, should resemble the effects of X rays. To explore this idea, gene expression changes between cells that had been irradiated with HZE particles and X rays were compared. The results support our hypothesis since the number of genes that commonly changed after exposure to both radiations increased as a function of particle energy.

Baumstark-Khan, C., Hellweg, C. E., Arenz, A. and Meier, M. M. Cellular Monitoring of the Nuclear Factor κB Pathway for Assessment of Space Environmental Radiation. Radiat. Res. 164, 527–530 (2005).

A screening assay for the detection of NF-κB-dependent gene induction using the destabilized variant of the reporter protein enhanced green fluorescent protein (d2EGFP) is used for assessing the biological effects of accelerated heavy ions as a model of space environmental radiation conditions. The time course of d2EGFP expression and therefore of activation of NF-κB-dependent gene expression was measured after treatment with TNFA or after heavy-ion exposure using flow cytometry. The reported experiments clearly show that accelerated argon ions (95 MeV/nucleon, LET 230 keV/μm) induce the NF-κB pathway at low particle densities (1–2 particle hits per nucleus), which result in as few as 5–50 induced DSBs per cell.

Chang, P. Y., Bjornstad, K. A., Rosen, C. J., McNamara, M. P., Mancini, R., Goldstein, L. E., Chylack, L. T. and Blakely, E. A. Effects of Iron Ions, Protons and X Rays on Human Lens Cell Differentiation. Radiat. Res. 164, 531–539 (2005).

We have investigated molecular changes in cultured differentiating human lens epithelial cells exposed to high-energy accelerated iron-ion beams as well as to protons and X rays. In this paper, we present results on the effects of radiation on gene families that include or are related to DNA damage, cell cycle regulators, cell adhesion molecules, and cell cytoskeletal function. A limited microarray survey with a panel of cell cycle-regulated genes illustrates that irradiation with protons altered the gene expression pattern of human lens epithelial cells. A focus of our work is CDKN1A (p21^CIP1/WAF1), a protein that we demonstrate here has a role in several pathways functionally related to LET-responsive radiation damage. We quantitatively assessed RNA and protein expression in a time course before and after single 4-Gy radiation doses and demonstrated that transcription and translation of CDKN1A are both temporally regulated after exposure. Furthermore, we show qualitative differences in the distribution of CDKN1A immunofluorescence signals after exposure to X rays, protons or iron ions, suggesting that LET effects likely play a role in the misregulation of gene function in these cells. A model of molecular and cellular events is proposed to account for precataractous changes in the human lens after exposure to low- or high-LET radiations.

Giedzinski, E., Rola, R., Fike, J. R. and Limoli, C. L. Efficient Production of Reactive Oxygen Species in Neural Precursor Cells after Exposure to 250 MeV Protons. Radiat. Res. 164, 540–544 (2005).

The space radiation environment is composed of highly energetic ions, dominated by protons, that pose a range of potential health risks to astronauts. Traversals of these particles through certain tissues may compromise the viability and/or function of sensitive cells, including neural precursors found within the dentate subgranular zone of the hippocampus. Irradiation has been shown to deplete these cells in vivo, and reductions of these critical cells are believed to impair neurogenesis and cognition. To more fully understand the mechanisms underlying the behavior of these precursor cells after irradiation, we have developed an in vitro neural precursor cell system and used it to assess acute (0–48 h) changes in ROS and mitochondrial end points after exposure to Bragg-peak protons of 250 MeV. Relative ROS levels were increased at nearly all doses (1–10 Gy) and postirradiation times (6–24 h) compared to unirradiated controls. The increase in ROS after proton irradiation was more rapid than that observed with X rays and showed a well-defined dose response at 6 and 24 h, increasing approximately 10% and 3% per gray, respectively. However, by 48 h postirradiation, ROS levels fell below controls and coincided with minor reductions in mitochondrial content. Use of the antioxidant α-lipoic acid (before or after irradiation) was shown to eliminate the radiation-induced rise in ROS levels. Our results corroborate earlier studies using X rays and provide further evidence that elevated ROS are integral to the radioresponse of neural precursor cells.

Guida, P., Vazquez, M. E. and Otto, S. Cytotoxic Effects of Low- and High-LET Radiation on Human Neuronal Progenitor Cells: Induction of Apoptosis and TP53 Gene Expression. Radiat. Res. 164, 545–551 (2005).

The induction of apoptosis, TP53 expression, caspase activation and cell toxicity were investigated after exposure of cells of the human neuronal progenitor cell line Ntera2 (NT2) to low-LET radiation (γ and X rays). The data indicates that irradiation of NT2 cells quickly induced TP53 expression, which was followed in time by an increase in caspase activity, and ultimately resulted in the induction of apoptosis. Induction of apoptosis was dependent on dose, and the highest levels were measured 48 h after exposure. For comparison, the level of apoptosis induced by high-LET particle radiation (1 GeV/ nucleon iron ions) was also determined and was found to be dependent on dose. The relative biological effectiveness (RBE) was estimated from the slopes of the dose–response curves for the induction of apoptosis. The RBE_max for apoptosis 48 h after exposure was at least 3.4. In short, exposure to high-LET radiation results in a more efficient and greater induction of apoptosis in human neuronal progenitor cells than low-LET radiation.

Rabin, B. M., Joseph, J. A. and Shukitt-Hale, B. A Longitudinal Study of Operant Responding in Rats Irradiated when 2 Months Old. Radiat. Res. 164, 552–555 (2005).

Two-month-old rats were exposed to ⁵⁶Fe particles (1, 1. 5, 2 Gy; 1 GeV/nucleon). They were tested on the performance of an ascending fixed-ratio operant task (bar pressing for food reward) at 7, 11 and 15 months after irradiation. Previous research had shown that for the same rats tested at 3 months after exposure, only the rats exposed to 2 Gy of ⁵⁶Fe particles showed a significant disruption of performance compared to control (0 Gy) rats. When these rats were tested 7, 11 and 15 months after exposure, all irradiated groups showed significantly poorer performance than the controls. These results suggest that there is an interaction between irradiation and age such that ⁵⁶Fe-particle-induced performance deficits can develop several months after exposure.

Rola, R., Sarkissian, V., Obenaus, A., Nelson, G. A., Otsuka, S., Limoli, C. L. and Fike, J. R. High-LET Radiation Induces Inflammation and Persistent Changes in Markers of Hippocampal Neurogenesis. Radiat. Res. 164, 556–560 (2005).

Exposure to heavy-ion radiation is considered a potential health risk in long-term space travel. It may result in the loss of critical cellular components in complex systems like the central nervous system (CNS), which could lead to performance decrements that ultimately could compromise mission goals and long-term quality of life. Specific hippocampal-dependent cognitive impairment occurs after whole-body ⁵⁶Fe-particle irradiation, and while the pathogenesis of this effect is not yet clear, it may involve damage to neural precursor cells in the hippocampal dentate gyrus. We irradiated mice with 1–3 Gy of ¹²C or ⁵⁶Fe ions and 9 months later quantified proliferating cells and immature neurons in the dentate subgranular zone (SGZ). Our results showed that reductions in these cells were dependent on the dose and LET. When compared with data for mice that were studied 3 months after ⁵⁶Fe-particle irradiation, our current data suggest that these changes are not only persistent but may worsen with time. Loss of precursor cells was also associated with altered neurogenesis and a robust inflammatory response. These results indicate that high-LET radiation has a significant and long-lasting effect on the neurogenic population in the hippocampus that involves cell loss and changes in the microenvironment.

Wang, B., Murakami, M., Eguchi-Kasai, K., Nojima, K., Shang, Y., Tanaka K., Fujita, K., Coffigny, H. and Hayata, I. Effects of Prenatal Irradiation with an Accelerated Heavy-Ion Beam on Postnatal Development in Rats: I. Neurophysiological Alterations. Radiat. Res. 164, 561–566 (2005).

Effects on postnatal neurophysiological development in offspring were studied after exposure of pregnant Wistar rats to accelerated carbon-ion beams with an LET of about 13 keV/ μm at doses ranging from 0.1 Gy to 2.5 Gy on the 15th day of gestation. The age at which four physiological markers appeared and five reflexes were acquired was examined prior to weaning. Gain in body weight was monitored until the offspring were 3 months old. Male offspring were evaluated as young adults using two behavioral tests. The effects of X rays estimated for the same biological end points were studied for comparison. For most of the end points at early age, no significant alterations were observed in offspring that received prenatal irradiation with 0.1 Gy of either accelerated carbon ions or X rays compared to the offspring of sham-irradiated dams. However, all offspring whose dams received 2.5 Gy died prior to weaning. Offspring from dams irradiated with accelerated carbon ions generally showed higher incidences of prenatal death and preweaning mortality, markedly delayed accomplishment in their physiological markers and reflexes, and gain in body weight compared to those exposed to X rays at doses of 0.5 to 2 Gy. Significantly reduced ratios of main organ weight to body weight at the postnatal ages of 30, 60 and 90 days were also observed within this dose range. The results indicate that irradiation with 0.5 to 2 Gy on day 15 of gestation caused permanent alterations in offspring that were dependent on dose. The alterations include permanent growth retardation, morphological malformations in main organs, including microcephaly, diminished reflex attainment, delayed appearance of physiological markers, and changes in adult behavior. Exposure to 1 to 2 Gy of radiation resulted in growth retardation and behavioral alterations that persisted throughout life. Accelerated carbon ions generally induced more detrimental effects than X rays.

Ballarini, F. and Ottolenghi, A. A Model of Chromosome Aberration Induction: Applications to Space Research. Radiat. Res. 164, 567–570 (2005).

A mechanistic model and Monte Carlo code simulating chromosome aberration induction in human lymphocytes is presented. The model is based on the assumption that aberrations arise from clustered DNA lesions and that only the free ends of clustered lesions created in neighboring chromosome territories or in the same territory can join and produce exchanges. The lesions are distributed in the cell nucleus according to the radiation track structure. Interphase chromosome territories are modeled as compact intranuclear regions with volumes proportional to the chromosome DNA contents. Both Giemsa staining and FISH painting can be simulated, and background aberrations can be taken into account. The good agreement with in vitro data provides validation of the model in terms of both the assumptions adopted and the simulation techniques. As an application in the field of space research, the model predictions were compared with aberration yields measured among crew members of long-term missions on board Mir and ISS, assuming an average radiation quality factor of 2.4. The agreement obtained also validated the model for in vivo exposure scenarios and suggested possible applications to the prediction of other relevant aberrations, typically translocations.

Durante, M., George, K., Gialanella, G., Grossi, G., La Tessa, C., Manti, L., Miller, J., Pugliese, M., Scampoli, P. and Cucinotta, F. A. Cytogenetic Effects of High-Energy Iron Ions: Dependence on Shielding Thickness and Material. Radiat. Res. 164, 571–576 (2005).

We report results for chromosomal aberrations in human peripheral blood lymphocytes after they were exposed to high-energy iron ions with or without shielding at the HIMAC, AGS and NSRL accelerators. Isolated lymphocytes were exposed to iron ions with energies between 200 and 5000 MeV/ nucleon in the 0.1–1-Gy dose range. Shielding materials consisted of polyethylene, lucite (PMMA), carbon, aluminum and lead, with mass thickness ranging from 2 to 30 g/cm². After exposure, lymphocytes were stimulated to grow in vitro, and chromosomes were prematurely condensed using a phosphatase inhibitor (calyculin A). Aberrations were scored using FISH painting. The yield of total interchromosomal exchanges (including dicentrics, translocations and complex rearrangements) increased linearly with dose or fluence in the range studied. Shielding decreased the effectiveness per unit dose of iron ions. The highest RBE value was measured with the 1 GeV/nucleon iron-ion beam at NSRL. However, the RBE for the induction of aberrations apparently is not well correlated with the mean LET. When shielding thickness was increased, the frequency of aberrations per particle incident on the shield increased for the 500 MeV/nucleon ions and decreased for the 1 GeV/nucleon ions. Maximum variation at equal mass thickness was obtained with light materials (polyethylene, carbon or PMMA). Variations in the yield of chromosomal aberrations per iron particle incident on the shield follow variations in the dose per incident particle behind the shield but can be modified by the different RBE of the mixed radiation field produced by nuclear fragmentation. The results suggest that shielding design models should be benchmarked using both physics and biological data.

Dini, V., Antonelli, F., Belli, M., Campa, A., Esposito, G., Simone, G., Sorrentino, E. and Tabocchini, M. A. Influence of PMMA Shielding on DNA Fragmentation Induced in Human Fibroblasts by Iron and Titanium Ions. Radiat. Res. 164, 577–581 (2005).

In the framework of a collaborative project on the influence of the shielding on the biological effectiveness of space radiation, we studied DNA fragmentation induced by 1 GeV/nucleon iron ions and titanium ions with and without a 197-mm-thick polymethylmethacrylate (PMMA) shield in AG1522 human fibroblasts. Pulsed- and constant-field gel electrophoresis were used to analyze DNA fragmentation in the size range 1– 5700 kbp. The results show that, mainly owing to a higher production of small fragments (1–23 kbp), titanium ions are more effective than iron ions at inducing DNA double-strand breaks (DSBs), their RBE being 2.4 and 1.5, respectively. The insertion of a PMMA shield decreases DNA breakage, with shielding protection factors (ratio of the unshielded/shielded cross sections for DSB production) of about 1.6 for iron ions and 2.1 for titanium ions. However, the DSB yield (no. of DSBs per unit mass per unit dose) is almost unaffected by the presence of the shield, and the relative contributions of the fragments in the different size ranges are almost the same with or without shielding. This indicates that, under our conditions, the effect of shielding is mainly to reduce the dose per unit incident fluence, leaving radiation quality practically unaffected.

Miller, J. H., Aceves-Gaona, A., Ernst, M. B., Haranczyk, M., Gutowski, M., Vorpagel, E. R. and Dupuis, M. Structure and Energetics of Clustered Damage Sites. Radiat. Res. 164, 582–585 (2005).

Quantum calculations on duplex DNA trimers were used to model the changes in structure, hydrogen bonding, stacking properties, and electrostatic potential induced by oxidized purine bases and abasic (AP) sites. Results for oxidized purine bases were consistent with experimental data that show small structural and energetic perturbations induced by isolated 8-oxoguanine (8oG). Watson-Crick base pairing was preserved, and no major distortions of the backbone were induced. The thermal destabilization of DNA induced by 8oG was comparable to the energy of a single hydrogen bond. In contrast, AP sites caused substantial distortions of the DNA backbone that were accompanied by relocation of counterions. The loss of Watson-Crick hydrogen bonds in AP sites had the potential to destabilize DNA by 10–20 kcal/mol (0.4–0.8 eV); however, new inter- and intrastrand hydrogen bonds formed after removal of a nucleic acid base that significantly affected the energy of AP sites and introduced a strong dependence on sequence context. Quantum calculations on small DNA fragments provided starting conformations and force-field parameters for classical molecular dynamics simulations of radiation-induced single-strand breaks that most often combine hydrolysis of a phosphate-oxygen (P-O) bond with an AP site and fully or partially degraded sugar ring. P-O bond hydrolysis increased the freedom in backbone torsion angles, which allowed the broken strand to compress and partially fill the hole in the DNA created by the AP site. Results for strand breaks with a 3′phosphoglycolate were similar to those with phosphate end groups.

Gerardi, S., Galeazzi, G. and Cherubini, R. A Microcollimated Ion Beam Facility for Investigations of the Effects of Low-Dose Radiation. Radiat. Res. 164, 586–590 (2005).

Charged-particle microbeams are unique tools to mimic low-dose exposure in vitro by delivering a defined number of particles to single mammalian cells down to only one particle per cell or group of cells. A horizontal single-ion microbeam facility has been built at the INFN-Laboratori Nazionali di Legnaro 7 MV Van de Graaff accelerator. Different light ions (¹H , ²H , ³He² , ⁴He² ) are available covering a wide range of LET from 7 to 150 keV/μm. Collimators of different geometries and materials have been tested, and beam spots 2–3 μm in diameter have been obtained using a tantalum disc. Cell visualization and recognition are performed with a phase-contrast optical microscope coupled with dedicated software. One unique characteristic of such a system is that neither cell staining nor UV light is used. Cells are automatically positioned on the beam spot through remotely controlled precision XY translation stages. A particle detector is positioned downstream of a specially designed petri dish to perform energy measurements and count particles crossing the cell. A particle counting rate of less than 1 ion/s can be reached. This feature, combined with a fast beam deflection system, ensures high reproducibility in administering a preset number of particles per cell.