Sharma, K. K., Purkayastha, S. and Bernhard, W. A. Unaltered Free Base Release from d(CGCGCG)₂ Produced by the Direct Effect of Ionizing Radiation at 4 K and Room Temperature. Radiat. Res. 167, 501–507 (2007).

Unaltered free base release in d(CGCGCG)₂ exposed to X rays at 4 K or room temperature was measured by HPLC. Samples were prepared either as films hydrated to a level of Γ = 2.5 mol water/mol nucleotide or as polycrystalline with Γ ∼ 7.5 mol water/mol nucleotide. X irradiation of films at 4 K, followed by annealing to room temperature, resulted in yields for cytosine and guanine of G(Cyt) = 0.036 ± 0.001 μmol/J and G(Gua) = 0.090 ± 0.002 μmol/J. Irradiation of films at room temperature gave similar yields. The yields for polycrystalline d(CGCGCG)₂ X-irradiated at room temperature were G(Cyt) = 0.035 ± 0.005 μmol/J and G(Gua) = 0.077 ± 0.023 μmol/J. The total free base release yield, G(fbr), was 0.124 ± 0.008 μmol/J for films and 0.112 ± 0.028 μmol/J for polycrystalline samples. G(fbr) is believed to be a good estimate of total strand break yield. The yields of total free radicals trapped [G(Σfr)] by the d(CGCGCG)₂ films at 4 K were measured by EPR. The measured value, G(Σfr) = 0.450 ± 0.005 μmol/J, was used to calculate the yield of trappable sugar radicals, giving G_sugar(fr) = 0.04–0.07 μmol/J. We found that (1) guanine release exceeded cytosine release by more than twofold, (2) G_sugar(fr) cannot account for more than half of the free base release, and (3) G(fbr), G(Cyt) and G(Gua) were independent of the sample temperature during irradiation. Finding (1) suggests that base and or sequence influences sugar damage, and finding (2) is consistent with our working hypothesis that an important pathway to strand break formation entails two one-electron oxidations at the same sugar site.

Qi, M. Y., Wu, G. Z., Chen, S. M. and Liu, Y. D. Gamma Radiolysis of Ionic Liquid 1-Butyl-3-methylimidazoliumHexafluorophosphate. Radiat. Res. 167, 508–514 (2007).

The applications of room-temperature ionic liquids in the nuclear fuel cycle and radiation chemistry depend on a comprehensive knowledge of their stability and chemical properties under radiation conditions. In this work, the effect of γ radiation on pure ionic liquid [bmim][PF₆] was investigated in detail. The radiolysis of [bmim][PF₆] leads to an increase of UV-vis absorbance and a decrease of fluorescence intensity with increasing radiation dose. Raman spectra proved that γ radiation induced significant chemical scission of the n-butyl group (e.g. C-H and C-C scission) and damage to the [PF₆]⁻ anion. When the irradiated [bmim][PF₆] samples were cooled, two crystal structures were found to coexist, and they suffered a continuous destruction under irradiation; their dose dependence, however, was different.

Kreth, G., Pazhanisamy, S. K., Hausmann, M. and Cremer, C., Cell Type-Specific Quantitative Predictions of Radiation-Induced Chromosome Aberrations: A Computer ModelApproach. Radiat. Res. 167, 515–525 (2007).

A quantitative computer model was applied to simulate the three-dimensional (3D) spatial organization of chromatin in human cell nuclei under defined conditions of virtual irradiation to explore the implications of spatial organization on chromosome aberrations. To calibrate the virtual irradiation algorithm, a dose-dependent spectrum of radiation-induced chromosome aberrations such as dicentrics, translocations and centric rings was calculated for low-LET radiation doses ranging from 0.5 to 5 Gy. This was compared with the results from experimental studies. While the dose–response curves calculated from model simulations agree well with experimental dose–response curves for dicentrics and translocations, centric rings are significantly more frequent in the model simulation than in experiments despite taking into account exclusive arm territories in the applied Spherical 1 Mbp Chromatin Domain (SCD) computer model explicitly. Taking into account the non-random positioning of chromosome territories observed in lymphocyte cell nuclei (a so-called gene density-correlated arrangement of chromosome territories), aberration frequencies were calculated with the calibrated irradiation algorithm to investigate the impact of chromosome territory neighborhood effects (proximity effects). The absolute frequencies of pairwise exchanges agree well with those found in an experimental study. In conclusion, the results obtained using the computer model approach presented here based on only a few adjustable parameters correlated well with those of experimental studies of chromosome aberration frequencies. Thus the model may be a useful tool in radiation-induced cancer risk estimates in combination with epidemiological studies.

Noda, A., Kodama, Y., Cullings, H. M. and Nakamura, N. Radiation-Induced Genomic Instability in Tandem Repeat Sequences is not Predictive of Unique Sequence Instability. Radiat. Res. 167, 526–534 (2007).

Tandem repeat sequences, classified as minisatellite sequences or partially duplicated genes, are inherently unstable. Radiation exposure can increase the instability of such repeat sequences, but the biological consequences of this elevated instability are not well characterized. To learn more about the characteristics of the instability at different sequences in the genome, we created mutant HT1080 cells bearing 8.4 kb of partially duplicated allele at the HPRT locus by gene targeting. The cells were then tested to determine whether repeat-sequence instability (assessed by elevated reversion rate caused by loss of one duplicated segment) accompanied increased forward mutation rates at the restored wild-type HPRT allele. After a 4-Gy X irradiation, 32 clones were selected (out of 500 clones, 6%) that showed elevated reversion rates even after many cell generations. These clones also showed general increases in the forward mutation rate, whereas the paired individual mutation rates did not correlate with each other. Furthermore, levels of intracellular reactive oxygen species (ROS) and nuclear γ-H2AX foci, which are hallmarks for DNA damage responses, were also generally elevated, although the levels did not correlate with the individual reversion rates. It was concluded that repeat sequence instability is not predictive of unique sequence instability, probably because the instability is generated by multiple mechanisms after radiation exposure.

Alsbeih, G., Al-Harbi, N., Al-Buhairi, M., Al-Hadyan, K. and Al-Hamed, M. Association between TP53 Codon 72 Single-Nucleotide Polymorphism and Radiation Sensitivity of Human Fibroblasts. Radiat. Res. 167, 535–540 (2007).

Inherent radiosensitivity varies widely between individuals. We hypothesized that amino acid substitution variants in two highly radiation-responsive proteins, TP53 (p53) and CDKN1A (p21, Waf1, Cip1), are associated with and could explain individual variations in radiosensitivity. The two non-synonymous single-nucleotide polymorphisms (SNPs) TP53 codon 72 Arg/Pro G>C and CDKN1A codon 31 Ser/Arg C>A were genotyped in 92 normal fibroblast cell strains of different radiosensitivity. The clonogenic surviving fraction at 2 Gy (SF2) ranged between 0.15 and 0.50 (mean = 0.34, SD = 0.08). The mean SF2 was used to divide the cell strains into radiosensitive (45) and normal groups (47). A significant association was observed between SF2 and the TP53 codon 72 haplotype (C compared to G, P = 0.01). No association was observed between CDKN1A codon 31 haplotype and radiosensitivity (P = 0.86). The variant TP53 Arg72 allele was associated with a decrease in radiosensitivity, presumably due to suboptimal function leading to less stringent control of cell division. We conclude that certain SNPs in susceptible genes can influence cellular radiation response. Such risk alleles could ultimately be used as predictive markers for radiosensitivity to help stratifying individuals during assessment of risk of radiation exposure.

Anderson, R. M, Stevens, D. L., Sumption, N. D., Townsend, K. M. S., Goodhead, D. T. and Hill, M. A. Effect of Linear Energy Transfer (LET) on the Complexity of α-Particle-Induced Chromosome Aberrations in Human CD34 Cells. Radiat. Res. 167, 541–550 (2007).

The aim of this study was to assess the relative influence of the linear energy transfer (LET) of α particles on the complexity of chromosome aberrations in the absence of significant other differences in track structure. To do this, we irradiated human hemopoietic stem cells (CD34 ) with α particles of various incident LETs (110–152 keV/μm, with mean LETs through the cell of 119–182 keV/μm) at an equi-fluence of approximately one particle/cell and assayed for chromosome aberrations by mFISH. Based on a single harvest time to collect early-division mitotic cells, complex aberrations were observed at comparable frequencies irrespective of incident LET; however, when expressed as a proportion of the total exchanges detected, their occurrence was seen to increase with increasing LET. Cycle analysis to predict theoretical DNA double-strand break rejoining cycles was also carried out on all complex chromosome aberrations detected. By doing this we found that the majority of complex aberrations are formed in single non-reducible cycles that involve just two or three different chromosomes and three or four different breaks. Each non-reducible cycle is suggested to represent “an area” of finite size within the nucleus where double-strand break repair occurs. We suggest that the local density of damage induced and the proximity of independent repair areas within the interphase nucleus determine the complexity of aberrations resolved in metaphase. Overall, the most likely outcome of a single nuclear traversal of a single α particle in CD34 cells is a single chromosome aberration per damaged cell. As the incident LET of the α particle increases, the likelihood of this aberration being classed as complex is greater.

Gault, N., Rigaud, O., Poncy, J-L. and Lefaix, J-L. Biochemical Alterations in Human Cells Irradiated with α Particles Delivered by Macro- or Microbeams. Radiat. Res. 167, 551–562 (2007).

Irradiation of individual cell nuclei with charged-particle microbeams requires accurate identification and localization of cells using Hoechst staining and UV illumination before computer-monitored localization of each cell. Using Fourier-transform infrared microspectroscopy (FT-IRM), we investigated whether the experimental conditions used for cell recognition induce cellular changes prior to irradiation and compared biochemical changes and DNA damage after targeted and nontargeted irradiation with α particles delivered by macro- or microbeams, using γ radiation as a reference. Molecular damage in single HaCaT cells was studied by means of FT-IRM and comet assay (Gault et al., Int. J. Radiat. Biol. 81, 767–779, 2005). Hoechst 33342-stained HaCaT cells were exposed to single doses of 2 Gy ²³⁹Pu α particles from a broad-beam irradiator, five impacted α particles from a microbeam irradiator, or 6 Gy γ rays from ¹³⁷Cs, each of which resulted in about 5% clonogenic survival. FT-IRM of control cells indicated that Hoechst binding to nuclear DNA induced subtle changes in DNA conformation, and its excitation under UV illumination induced a dramatic shift of the DNA conformation from A to B as well as major DNA damage as measured by the comet assay. Comparison of the FT-IRM spectra of cells exposed to γ rays or α particles specifically targeted to the nucleus, α particles from a broad-beam irradiator revealed spectral changes corresponding to all changes in constitutive bases in nucleic acids, suggesting oxidative damage in these bases, as well as structural damage in the deoxyribose-phosphate backbone of DNA and the osidic structure of nucleic acids. Concomitantly, spectral changes specific to protein suggested structural modifications. Striking differences in IR spectra between targeted microbeam- and nontargeted macrobeam-irradiated cells indicated greater residual unrepaired or misrepaired damage after microbeam irradiation. This was confirmed by the comet assay data. These results show that FT-IRM, together with the comet assay, is useful for assessing direct radiation-induced damage to nucleic acids and proteins in single cells and for investigating the effects of radiation quality. Significantly, FT-IRM revealed that Hoechst 33342 binding to DNA and exposure to UV light induce a dramatic change in DNA conformation as well as DNA damage. These findings suggest that fluorochrome staining should be avoided in studies of ionizing radiation-induced bystander effects based on charged-particle microbeam irradiation. An alternative cell nucleus recognition system that avoids nuclear matrix damage and its possible contribution to propagation of biological effects from irradiated cells to neighboring nontargeted cells needs to be developed.

Iizuka, D., Inanami, O., Kashiwakura, I. and Kuwabara, M. Purvalanol A Enhances Cell Killing by Inhibiting Up-Regulation of CDC2 Kinase Activity in Tumor Cells Irradiated with High Doses of X Rays. Radiat. Res. 167, 563–571 (2007).

To clarify the relationship between CDC2 kinase activity and radiation-induced apoptosis, we examined whether the cyclin-dependent kinase (CDK) inhibitor purvalanol A enhanced radiation-induced apoptosis in gastric tumor cells. MKN45 cells exposed to 20 Gy of X rays increased the CDC2 kinase activity and the expression of regulatory proteins (phospho-CDC2 and cyclin B1) of the G₂/M phase, followed by activation of the G₂/M checkpoint, whereas the treatment of X-irradiated MKN45 cells with 20 μM purvalanol A suppressed the increase in the CDC2 kinase activity and expression of the G₂/M-phase regulatory proteins and reduced the fraction of the cells in the G₂/M phase in the cell cycle. Furthermore, this treatment resulted in not only a significant increase in radiation-induced apoptosis but also the loss of clonogenicity in both MKN45 (p53-wild) and MKN28 (p53-mutated) cells. The expression of anti-apoptosis proteins, inhibitor of apoptosis protein (IAP) family members (survivin and XIAP) and BCL2 family members (Bcl-X_L and Bcl-2), in purvalanol A-treated cells with and without X rays was significantly lower than for cells exposed to X rays alone. These results suggest that the inhibition of radiation-induced CDC2 kinase activity by purvalanol A induces apoptosis through the enhancement of active fragments of caspase 3.

Sanchez, S., Haro, E., Ruffié, G., Veyret, B. and Lagroye, I. In Vitro Study of the Stress Response of Human Skin Cells to GSM-1800 Mobile Phone Signals Compared to UVB Radiation and Heat Shock. Radiat. Res. 167, 572–580 (2007).

The evolution of mobile phone technology is toward an increase of the carrier frequency up to 2.45 GHz. Absorption of radiofrequency (RF) radiation becomes more superficial as the frequency increases. This increasingly superficial absorption of RF radiation by the skin, which is the first organ exposed to RF radiation, may lead to stress responses in skin cells. We thus investigated the expression of three heat-shock proteins (HSP70, HSC70, HSP27) using immunohistochemistry and induction of apoptosis by flow cytometry on human primary keratinocytes and fibroblasts. A well-characterized exposure system, SXC 1800, built by the IT'IS foundation was used at 1800 MHz, with a 217 Hz modulation. We tested a 48-h exposure at an SAR of 2 W/kg (ICNIRP local exposure limit). Skin cells were also irradiated with a 600 mJ/cm² single dose of UVB radiation and subjected to heat shock (45°C, 20 min) as positive controls for apoptosis and HSP expression, respectively. The results showed no effect of a 48-h GSM-1800 exposure at 2 W/kg on either keratinocytes or fibroblasts, in contrast to UVB-radiation or heat-shock treatments, which injured cells. We thus conclude that the GSM-1800 signal does not act as a stress factor on human primary skin cells in vitro.

Zhao, W., Iskandar, S., Kooshki, M., Sharpe, J. G., Payne, V. and Robbins, M. E. Knocking Out Peroxisome Proliferator-Activated Receptor (PPAR) α Inhibits Radiation-Induced Apoptosis in the Mouse Kidney through Activation of NF-κB and Increased Expression of IAPs. Radiat. Res. 167, 581–591 (2007).

Peroxisome proliferator-activated receptor (PPAR) α, a member of the ligand-activated nuclear receptor superfamily, plays an important role in lipid metabolism and glucose homeostasis and is highly expressed in the kidney. The present studies were aimed at testing the hypothesis that PPARα knockout mice would exhibit decreased radiation-induced apoptosis due to exacerbated activation of NF-κB (NFKB) and expression of pro-survival factors. Thirty wild-type mice (29S1/SvImJ) and 30 PPARα knockout mice were irradiated with a single total-body dose 10 Gy of ¹³⁷Cs γ rays; controls were sham-irradiated. Tissue samples were collected at 3, 6, 12, 24 and 48 h postirradiation. Apoptosis was quantified using immunohistochemical staining for apoptotic bodies and cleaved caspase 3. Radiation-induced apoptosis was observed in both mouse strains in a time-dependent manner. However, the level of apoptosis was significantly suppressed in PPARα knockout mice compared with wild-type mice at 6 h postirradiation (P < 0.05). This inhibition of radiation-induced apoptosis was associated with time-dependent increases in NF-κB DNA-binding activity, IκBα phosphorylation, and expression of other antiapoptosis factors in the PPARα knockout mouse kidneys but not in wild-type animals. These data support the hypothesis that the loss of PPARα expression leads to the suppression of radiation-induced apoptosis in the mouse kidney, mediated through activation of NF-κB and up-regulation of anti-apoptosis factors.

Tsuruga, M., Taki, K., Ishii, G., Sasaki, Y., Furukawa, C., Sugihara, T., Nomura, T., Ochiai, A., and Magae, J. Amelioration of Type II Diabetes in db/db Mice by Continuous Low-Dose-Rate γ Irradiation. Radiat. Res. 167, 592–599 (2007).

Low-dose-rate radiation modulates various biological responses including carcinogenesis, immunological responses and diabetes. We found that continuous irradiation with low-dose-rate γ rays ameliorated type II diabetes in db/db mice, diabetic mice that lack leptin receptors. Whole-body exposure of db/db mice to low dose-rate γ radiation improved glucose clearance without affecting the response to insulin. Histological studies suggested that degeneration of pancreatic islets was significantly suppressed by the radiation. Insulin secretion in response to glucose loading was increased significantly in the irradiated mice. These results suggest that low-dose-rate γ radiation ameliorates type II diabetes by maintaining insulin secretion, which gradually decreases during the progression of diabetes due to degeneration of pancreatic islets. We also inferred that protection from oxidative damage is involved in the anti-diabetic effect of low-dose-rate γ rays because expression and activity of pancreatic superoxide dismutase were significantly elevated by low-dose-rate γ radiation.

Parazzini, M., Galloni, P., Piscitelli, M., Pinto, R., Lovisolo, G. A., Tognola, G., Ravazzani, P. and Marino, C. Possible Combined Effects of 900 MHZ Continuous-Wave Electromagnetic Fields and Gentamicin on the Auditory System of Rats. Radiat. Res. 167, 600–605 (2007).

The aim of this study was to evaluate the cochlear functionality of Sprague-Dawley rats exposed to electromagnetic fields at 900 MHz and to gentamicin by distortion product otoacoustic emissions, which are a well-known indicator of the status of the cochlea's outer hair cells. A population of 32 rats was divided into four groups: group 1 was treated with daily intramuscular injections of 150 mg/kg body weight gentamicin for 15 days; group 2 was treated with daily intramuscular injections of 150 mg/kg body weight gentamicin for 15 days and exposed to electromagnetic fields; group 3 was exposed to electromagnetic fields; group 4 was sham-exposed. Rats were exposed 2 h/day, 5 days/week for 4 weeks at a local SAR of 4 W/kg in the ear (continuous wave at 900 MHz). Distortion product otoacoustic emissions tests were carried out before, during and after the combined exposure. The analysis of the data showed no subchronic exposure to electromagnetic fields on the inner auditory system of rats in either normal ears or ears exposed to a well-recognized pathological agent.

Chumak, V. V., Worgul, B. V., Kundiyev, Y. I., Sergiyenko, N. M., Vitte, P. M., Medvedovsky, C., Bakhanova, E. V., Junk, A. K., Kyrychenko, O. Y., Musijachenko, N. V., Sholom, S. V., Shylo, S. A., Vitte, O. P., Xu, S., Xue, X. and Shore, R. E. Dosimetry for a Study of Low-Dose Radiation Cataracts among Chernobyl Clean-up Workers. Radiat. Res. 167, 606– 614 (2007).

A cohort of 8,607 Ukrainian Chernobyl clean-up workers during 1986–1987 was formed to study cataract formation after ionizing radiation exposure. Study eligibility required the availability of sufficient exposure information to permit the reconstruction of doses to the lens of the eye. Eligible groups included civilian workers, such as those who built the “sarcophagus” over the reactor, Chernobyl Nuclear Power Plant Workers, and military reservists who were conscripted for clean-up work. Many of the official doses for workers were estimates, because only a minority wore radiation badges. For 106 military workers, electron paramagnetic resonance (EPR) measurements of extracted teeth were compared with the recorded doses as the basis to adjust the recorded γ-ray doses and provide estimates of uncertainties. Beta-particle doses to the lens were estimated with an algorithm devised to take into account the nature and location of Chernobyl work, time since the accident, and protective measures taken. A Monte Carlo routine generated 500 random estimates for each individual from the uncertainty distributions of the γ-ray dose and of the ratio of β-particle to γ-ray doses. The geometric mean of the 500 combined β-particle and γ-ray dose estimates for each individual was used in the data analyses. The median estimated lens dose for the cohort was 123 mGy, while 4.4% received >500 mGy.

Scherthan, H., Abend, M., Müller, K., Beinke, C., Braselmann, H., Zitzelsberger, H., Köhn, F. M., Pillekamp, H., Schiener, R., Das, O., Peter, R. U., Herzog, G., Tzschach, A., Dörr, H. D., Fliedner, T. M. and Meineke, V. Radiation-Induced Late Effects in Two Affected Individuals of the Lilo Radiation Accident. Radiat. Res. 167, 615–623 (2007).

Radiation exposure leads to a risk for long-term deterministic and stochastic late effects. Two individuals exposed to protracted photon radiation in the radiological accident at the Lilo Military site in Georgia in 1997 received follow-up treatment and resection of several chronic radiation ulcers in the Bundeswehr Hospital Ulm, Germany, in 2003. Multi-parameter analysis revealed that spermatogenetic arrest and serum hormone levels in both patients had recovered compared to the status in 1997. However, we observed a persistence of altered T-cell ratios, increased ICAM1 and β1-integrin expression, and aberrant bone marrow cells and lymphocytes with significantly increased translocations 6 years after the accident. This investigation thus identified altered end points still detectable years after the accident that suggest persistent genomic damage as well as epigenetic effects in these individuals, which may be associated with an elevated risk for the development of further late effects. Our observations further suggest the development of a chronic radiation syndrome and indicate follow-up parameters in radiation victims.