Ohtaki, K., Kodama, Y., Nakano, M., Itoh, M., Awa, A. A., Cologne, J. and Nakamura, N. Human Fetuses do not Register Chromosome Damage Inflicted by Radiation Exposure in Lymphoid Precursor Cells except for a Small but Significant Effect at Low Doses. Radiat. Res. 161, 373–379 (2004).

Human fetuses are thought to be highly sensitive to radiation exposure because diagnostic low-dose X rays have been suggested to increase the risk of childhood leukemia. However, animal studies generally have not demonstrated a high radiosensitivity of fetuses, and the underlying causes for the discrepancy remain unidentified. We examined atomic bomb survivors exposed in utero for translocation frequencies in blood lymphocytes at 40 years of age. Contrary to our expectation of a greater radiosensitivity in fetuses than in adults, the frequency did not increase with dose except for a small increase (less than 1%) at doses below 0.1 Sv, which was statistically significant. We interpret the results as indicating that fetal lymphoid precursor cells comprise two subpopulations. One is small in number, sensitive to the induction of both translocations and cell killing, but rapidly diminishing above 50 mSv. The other is the major fraction but is insensitive to registering damage expressed as chromosome aberrations. Our results provide a biological basis for resolving the long-standing controversy that a substantial risk of childhood leukemia is implicated in human fetuses exposed to low-dose X rays whereas animal studies involving mainly high-dose exposures generally do not confirm it.

Asakawa, J., Kuick, R., Kodaira, M., Nakamura, N., Katayama, H., Pierce, D., Funamoto, S., Preston, D., Satoh, C., Neel, J. V. and Hanash, S. A Genome Scanning Approach to Assess the Genetic Effects of Radiation in Mice and Humans. Radiat. Res. 161, 380–390 (2004).

We used Restriction Landmark Genome Scanning (RLGS) to assess, on a genome-wide basis, the mutation induction rate in mouse germ cells after radiation exposure. Analyses of 1,115 autosomal NotI DNA fragments per mouse for reduced spot intensity, indicative of loss of one copy, in 506 progeny derived from X-irradiated spermatogonia (190, 237 and 79 mice in 0-, 3-, and 5-Gy groups, respectively), permitted us to identify 16 mutations affecting 23 fragments in 20 mice. The 16 mutations were composed of eight small changes (1–9 bp) at microsatellite sequences, five large deletions (more than 25 kb), and three insertions of SINE B2 or LINE1 transposable elements. The maximum induction rate of deletion mutations was estimated as (0.17 ± 0.09) × 10−5/locus Gy–1. The estimate is considerably lower than 1 × 10−5/locus Gy–1, the mean induction rate of deletion mutations at Russell's 7 loci, which assumed that deletion mutations comprise 50% of all mutations. We interpret the results as indicating that the mean induction rate of mutations in the whole genome may be substantially lower than that at the 7 loci. We also demonstrate the applicability of RLGS for detection of human mutations, which allows direct comparisons between the two species.

Koana, T., Takashima, Y., Okada, M. O., Ikehata, M., Miyakoshi, J. and Sakai, K. A Threshold Exists in the Dose– Response Relationship for Somatic Mutation Frequency Induced by X Irradiation of Drosophila. Radiat. Res. 161, 391– 396 (2004).

The dose–response relationship of ionizing radiation and its stochastic effects has been thought to be linear without any thresholds. The basic data for this model were obtained from mutational assays in the male germ cells of the fruit fly Drosophila melanogaster. However, it is more appropriate to examine carcinogenic activity in somatic cells than in germ cells. Here the dose–response relationship of X irradiation and somatic mutation was examined in Drosophila. A threshold at approximately 1 Gy was observed in DNA repair-proficient flies. In the repair-deficient siblings, the threshold was smaller and the inclination of the dose–response curve was much steeper. These results suggest that the dose–response relationship between X irradiation and somatic mutation has a threshold and that the DNA repair function contributes to its formation.

Mitchell, S. A., Randers-Pehrson, G., Brenner, D. J. and Hall, E. J. The Bystander Response in C3H 10T½ Cells: The Influence of Cell-to-Cell Contact. Radiat. Res. 161, 397–401 (2004).

Although radiation-induced heritable damage in mammalian cells was thought to result from the direct interaction of radiation with DNA, it is now accepted that biological effects may occur in cells that were not themselves traversed by ionizing radiation but are close to those that were. However, little is known about the mechanism underlying such a bystander effect, although cell-to-cell communication is thought to be of importance. Previous work using the Columbia microbeam demonstrated a significant bystander effect for clonogenic survival and oncogenic transformation in C3H 10T½ cells. The present study was undertaken to assess the importance of the degree of cell-to-cell contact at the time of irradiation on the magnitude of this bystander effect by varying the cell density. When 10% of cells were exposed to a range of 2–12 α particles, a significantly greater number of cells (P < 0.0001) were inactivated when cells were irradiated at high density (>90% in contact with neighbors) than at low density (<10% in contact). In addition, the oncogenic transformation frequency was significantly higher in high-density cultures (P < 0.0004). These results suggest that when a cell is hit by radiation, the transmission of the bystander signal through cell-to-cell contact is an important mediator of the effect, implicating the involvement of intracellular communication through gap junctions.

Reitsema, T. J., Banáth, J. P., MacPhail, S. H. and Olive,P. L. Hypertonic Saline Enhances Expression of Phosphorylated Histone H2AX after Irradiation. Radiat. Res. 161, 402– 408 (2004).

Phosphorylation of histone H2AX at serine 139 occurs at sites surrounding DNA double-strand breaks, producing discrete spots called “foci” that are visible with a microscope after antibody staining. This modification is believed to create changes in chromatin structure and assemble various repair proteins at sites of DNA damage. To examine the role of chromatin structure, human SiHa cells were exposed to hypertonic salt solutions that are known to condense chromatin and sensitize cells to chromosome damage and killing by ionizing radiation. Postirradiation incubation in 0.5 M Na increased γH2AX expression about fourfold as measured by flow cytometry and immunoblotting, and loss of γH2AX was inhibited in the presence of high salt. Focus size rather than the number of radiation-induced γH2AX foci was also increased about fourfold. When high-salt treatment was delayed for 1 h after irradiation, effects on focus size and retention were reduced. The increase in focus size was associated with a decrease in the rate of rejoining of double-strand breaks as measured using the neutral comet assay. We conclude that γH2AX expression after irradiation is sensitive to salt-induced changes in chromatin structure during focus formation, and that a large focus size may be an indication of a reduced ability to repair DNA damage.

Inoue, M., Shen, G-P., Chaudhry, M. A., Galick, H., Blaisdell, J. O. and Wallace, S. S. Expression of the Oxidative Base Excision Repair Enzymes is not Induced in TK6 Human Lymphoblastoid Cells after Low Doses of Ionizing Radiation. Radiat. Res. 161, 409–417 (2004).

Most of the DNA damage produced by ionizing radiation is repaired by the base excision repair (BER) pathway. To determine whether the BER genes were up-regulated by low doses of ionizing radiation, we investigated their expression in TK6 human lymphoblastoid cells by measuring mRNA levels using real-time quantitative PCR. No induction at the transcriptional level of any of the base excision repair genes, NTH1 (NTHL1), OGG1, NEIL1, NEIL2, NEIL3, APE1, POLB, or accessory protein genes, LIG3, XRCC1 or XPG, was found at γ-radiation doses ranging from 1 cGy to 2 Gy in a 24-h period. As has been measured in other cell lines, a dose-dependent induction of CDKN1A (WAF1) mRNA levels was observed in TK6 cells in the dose range of 0.5 to 2.0 Gy. We also examined BER enzyme activity on 8-oxoguanine-, dihydrouracil- and furan-containing oligonucleotide substrates and found no increase in extracts of TK6 cells after γ-ray doses of 0.5–2.0 Gy. These data were corroborated by Western blot analysis of APE1 and NTH1, suggesting that the BER enzymes are also not up-regulated at the post-transcriptional level after ionizing radiation exposure.

Zhao, W., Goswami, P. C. and Robbins, M. E. C. Radiation- Induced Up-regulation of Mmp2 Involves Increased mRNA Stability, Redox Modulation, and MAPK Activation. Radiat. Res. 161, 418–429 (2004).

We have previously observed time- and dose-dependent increases in matrix metalloproteinase 2 (Mmp2) protein levels in rat tubule epithelial cells (NRK52E) after irradiation. However, the mechanism(s) involved remains unclear. In the present study, irradiating NRK52E cells with 0–20 Gy γ rays was associated with time- and dose-dependent increases in Mmp2 mRNA. Studies using the transcription inhibitor actinomycin D (ActD) added 24 h after irradiation revealed the t_1/2 of Mmp2 mRNA to be approximately 8 h in control cells. In contrast, the increase in Mmp2 mRNA levels in irradiated cells was essentially unchanged after incubation with ActD for up to 12 h. Incubating cells with the antioxidants N-acetylcysteine or ebselen or the MEK pathway inhibitors PD98059 and U0126 prior to irradiation abolished the radiation-induced up-regulation of Mmp2. Irradiating NRK52E cells led to reactive oxygen species-mediated Erk1/2 activation; preincubation with NAC prevented the radiation-induced increase in phosphorylated Erk1/2. Transfecting cells with a dominant-negative ERK mutant completely inhibited radiation-induced Erk phosphorylation and abolished the radiation-induced up-regulation of Mmp2 protein. Thus the radiation-induced up-regulation of Mmp2 mRNA is due in part to increased mRNA stability and is mediated by redox; the ERK MAPK signaling pathway may be involved.

Coulton, L. A., Harris, P. A., Barker, A. T. and Pockley, A. G. Effect of 50 Hz Electromagnetic Fields on the Induction of Heat-Shock Protein Gene Expression in Human Leukocytes. Radiat. Res. 161, 430–434 (2004).

Although evidence is controversial, exposure to environmental power-frequency magnetic fields is of public concern. Cells respond to some abnormal physiological conditions by producing cytoprotective heat-shock (or stress) proteins. In this study, we determined whether exposure to power-frequency magnetic fields in the range 0–100 μT rms either alone or concomitant with mild heating induced heat-shock protein gene expression in human leukocytes, and we compared this response to that induced by heat alone. Samples of human peripheral blood were simultaneously exposed to a range of magnetic-field amplitudes using a regimen that was designed to allow field effects to be distinguished from possible artifacts due to the position of the samples in the exposure system. Power-frequency magnetic-field exposure for 4 h at 37°C had no detectable effect on expression of the genes encoding HSP27, HSP70A or HSP70B, as determined using reverse transcriptase-PCR, whereas 2 h at 42°C elicited 10-, 5- and 12-fold increases, respectively, in the expression of these genes. Gene expression in cells exposed to power-frequency magnetic fields at 40°C was not increased compared to cells incubated at 40°C without field exposure. These findings and the extant literature suggest that power-frequency electromagnetic fields are not a universal stressor, in contrast to physical agents such as heat.

Fujii, K., Akamatsu, K. and Yokoya, A. Ion Desorption from DNA Components by Ultrasoft X-Ray Photons. Radiat. Res. 161, 435–441 (2004).

Positive ion desorption from thin films of DNA components, 2-deoxy-d-ribose, thymine, thymidine (dThd), and thymidine 5′-monophosphate (dTMP) was investigated in the oxygen K- shell edge excitation region using synchrotron ultrasoft X rays (538 eV). A large number of molecular fragments, H , CH_x , C₂H_x , CO , CH_xO , C₃H_x , C₂H_xO and C₃H_xO (x = 1, 2 and 3), were observed as desorbed ions from 2-deoxy-d-ribose. Some of these ions are related to simultaneous bond scission at particular C–C and C–O (or C–C) bonds in the furanose ring structure in the 2-deoxy-d-ribose molecule, indicating that the impact of photons on the oxygen atom and the impact of ejected secondary electrons (e.g. Auger electrons) cause an intense destruction of the furanose ring structure. In thymine thin films, H , CH_x , CO , CH_xO , C₂H_xN and CH_xNO (x = 1, 2 and 3) fragments were observed. The yields of these ions were smaller than the yields from 2-deoxy-d-ribose. The desorption of CH₃ from thymine might induce a molecular conversion from thymine to uracil. The mass patterns of dThd and dTMP, and especially that of dTMP, were similar to that of 2-deoxy-d-ribose, indicating that a number of ions were generated at the sugar site, even in the nucleotide molecule. It is therefore predicted that the sugar moiety is more fragile than the thymine base.

Akamatsu, K., Fujii, K. and Yokoya, A. Qualitative and Quantitative Analyses of the Decomposition Products that Arise from the Exposure of Thymine to Monochromatic Ultrasoft X Rays and ⁶⁰Co Gamma Rays in the Solid State. Radiat. Res. 161, 442–450 (2004).

HPLC analyses of condensed thymine irradiated with monochromatic synchrotron ultrasoft X rays in the energy region around nitrogen and oxygen K-shell edges were performed. Cobalt-60 γ rays were used as a reference radiation. The radiation chemical dose response of each separated thymine decomposition product was also determined. Uracil (U), 5-(hydroxymethyl)uracil (HMU), 5,6-dihydrothymine (DHT), 5-formyluracil (foU) and four main unknown products were found in the HPLC chromatogram of the sample irradiated with ultrasoft X rays in vacuo. Similar spectra of the products were also found in the γ-ray experiment; however, some unknown products that appeared after elution of the thymine peak were significantly larger than those in the ultrasoft X- ray experiment. This result indicates the difference in radiation quality. The G value of DHT produced by γ radiation was 10 times larger than those produced by the ultrasoft X- ray photons with energies of 395 and 407 eV corresponding to below and on the nitrogen K-shell edge, respectively. This result suggests that the differences in the photon energy and/ or in the energy spectra of the secondary electron between ultrasoft X rays and γ rays are causing differences in the process of the radiation chemistry. Moreover, the yields of all the thymine decomposition products induced by 538 eV photons (oxygen K-shell edge) were significantly smaller than those induced by photons around the nitrogen K-shell edge. The K-shell excitation of oxygen in thymine may efficiently promote the production of small thymine fragments susceptible to desorption from the sample.

Semenenko, V. A. and Stewart, R. D. A Fast Monte Carlo Algorithm to Simulate the Spectrum of DNA Damages Formed by Ionizing Radiation. Radiat. Res. 161, 451–457 (2004).

Ionizing radiation produces both singly and multiply damaged DNA sites. Multiply damaged sites (MDS) have been implicated in radiation-induced cell killing and mutagenesis. The spatial distribution of elementary damages (strand breaks and base damages) that constitute MDS is of special interest, since the complexity of MDS has an impact on damage repair. A fast and easy-to-implement algorithm to simulate the local clustering of elementary damages produced by ionizing radiation is proposed. This algorithm captures the major trends in the DNA damage spectrum predicted using detailed track- structure simulations. An attractive feature of the proposed algorithm is that only four adjustable parameters need to be identified to simulate the formation of DNA damage. A convenient recipe to determine the parameters used in the fast Monte Carlo damage simulation algorithm is provided for selected low- and high-LET radiations. The good agreement among the damage yields predicted by the fast and detailed damage formation algorithms suggests that the small-scale spatial distribution of damage sites is determined primarily by independent and purely stochastic events and processes.

Bardet, M., Maron, S., Foray, M. F., Berger, M. and Guillermo, A. Investigation of γ-Irradiated Vegetable Seeds with High-Resolution Solid-State ¹³C NMR. Radiat. Res. 161, 458– 463 (2004).

¹³C solid-state NMR was used to investigate the effects of γ radiation on vegetable seeds, Pisum sativum and Latuca sativa, at absorbed doses that inhibit their germination. By combining single-pulse excitation and cross-polarization experiments under magic angle spinning, both liquid and solid domains of seeds can be characterized. We showed that the liquid domains, mostly made of triacylglycerols (TAG), of vegetable seeds are not sensitive to radiation. The main structural changes have been observed in the embryonic axes of seeds when the seeds are water-imbibed before irradiation. These results rule out a starting hypothesis concerning the potential role of TAG contained in oil bodies as a potential source of aldehydes that could further react with DNA moiety.

Gustafsson, H., Olsson, S., Lund, A. and Lund, E. Ammonium Formate, a Compound for Sensitive EPR Dosimetry. Radiat. Res. 161, 464–470 (2004).

Alanine EPR dosimetry has been applied successfully when measuring intermediate and high radiation doses. Although the performance of alanine dosimetry is being improved, the sensitivity of the material is too low for a fast and simple low- dose determination. Here we present the results using ammonium formate as an EPR dosimeter material. Ammonium formate is seven times more sensitive than alanine, using spectrometer settings optimized for the latter. Deuterated ammonium formate is found to be more than eight times more sensitive than alanine. Analysis of signal stability with time shows that the ammonium formate signal is stable by 5 min after irradiation and that no change in signal intensity is found during 8 days. The atomic composition of ammonium formate is closer to that of tissue than alanine, and thus the energy dependence is smaller than that of alanine at photon energies below 200 keV. Power saturation studies indicate that the energy transfer between the spins and the lattice is fast in ammonium formate, which gives the possibility of using high microwave power without saturation to further increase the sensitivity. These results suggest that ammonium formate has some important properties required of an EPR dosimeter for applications in dosimetry in the dose range typical for radiation therapy.

Yoksan, R., Akashi, M., Miyata, M. and Chirachanchai, S. Optimal γ-Ray Dose and Irradiation Conditions for Producing Low-Molecular-Weight Chitosan that Retains its Chemical Structure. Radiat. Res. 161, 471–480 (2004).

This study focuses on the optimal conditions for γ irradiation to reduce the molecular weight of chitosan but still retain its chemical structure. Chitosan was irradiated under various conditions, i.e. flake solid state (condition 1), flake dispersed in water (condition 2), flake dispersed in 0.05, 0.1, 1 and 2% aqueous K₂S₂O₈ solution (conditions 3a, 3b, 3c and 3d, respectively), flake dispersed in 0.5, 1 and 2% aqueous H₂O₂ solution (conditions 4a, 4b and 4c, respectively), and chitosan acetic acid solution (condition 5). Comparative studies were done using three types of chitosans with molecular weights of the order of 10⁵ Da with degrees of deacetylation of 0.80, 0.85 and 0.90%. For all conditions, after irradiation, there were two regions of molecular weight reduction. A severe degradation occurred in the first region with decreases in the molecular weight of 80% for radiation doses up to 50 kGy for conditions 1, 2 and 3 (3a–3c) and 20 kGy for condition 4. In the second region, a slow degradation occurred, which resembled a plateau stage. The results for conditions 3d and 5 were the most dramatic, since the primary structure of chitosan was changed after the irradiation. The degradation of chitosan by γ rays was found to be most effective for the amorphous structure. The retention of the structure of chitosan after γ irradiation makes it possible to produce a low-molecular-weight chitosan that retains its functionality, as demonstrated by its activity in the coupling reaction with N,N′-carbonyldiimidazole.

Chornobyl Thyroid Diseases Study Group of Belarus, Ukraine, and the USA. A Cohort Study of Thyroid Cancer and Other Thyroid Diseases after the Chornobyl Accident: Objectives, Design and Methods. Radiat. Res. 161, 481–492 (2004).

The thyroid gland in children is one of the organs that is most sensitive to external exposure to X and γ rays. However, data on the risk of thyroid cancer in children after exposure to radioactive iodines are sparse. The Chornobyl accident in Ukraine in 1986 led to the exposure of large populations to radioactive iodines, particularly ¹³¹I. This paper describes an ongoing cohort study being conducted in Belarus and Ukraine that includes 25,161 subjects under the age of 18 years in 1986 who are being screened for thyroid diseases every 2 years. Individual thyroid doses are being estimated for all study subjects based on measurement of the radioactivity of the thyroid gland made in 1986 together with a radioecological model and interview data. Approximately 100 histologically confirmed thyroid cancers were detected as a consequence of the first round of screening. The data will enable fitting appropriate dose–response models, which are important in both radiation epidemiology and public health for prediction of risks from exposure to radioactive iodines from medical sources and any future nuclear accidents. Plans are to continue to follow-up the cohort for at least three screening cycles, which will lead to more precise estimates of risk.