Boudaïffa, B., Cloutier, P., Hunting, D., Huels, M. A. and Sanche, L. Cross Sections for Low-Energy (10 – 50 eV) Electron Damage to DNA. Radiat. Res. 157, 227 – 234 (2002).

We report direct measurements of the formation of single-, double- and multiple strand breaks in pure plasmid DNA as a function of exposure to 10 – 50 eV electrons. The effective cross sections to produce these different types of DNA strand breaks were determined and were found to range from approximately 10⁻¹⁷ to 3  × 10⁻¹⁵ cm². The total effective cross section and the effective range for destruction of supercoiled DNA extend from 3.4 to 4.4  × 10⁻¹⁵ cm² and 12 to 14 nm, respectively, over the range 10 – 50 eV. The variation of the effective cross sections with electron energy is discussed in terms of the electron's inelastic mean free path, penetration depth, and dissociation mechanisms, including resonant electron capture; the latter is found to dominate the effective cross sections for single- and double-strand breaks at 10 eV. The most striking observations are that (1) supercoiled DNA is approximately one order of magnitude more sensitive to the formation of double-strand breaks by low-energy electrons than is relaxed circular DNA, and (2) the dependence of the effective cross sections on the incident electron energy is unrelated to the corresponding ionization cross sections. This finding suggests that the traditional notion that radiobiological damage is related to the number of ionization events would not apply at very low energies.

Debije, M. G., Close, D. M. and Bernhard, W. A. Reductive Damage in Directly Ionized DNA: Saturation of the C5 = C6 Bond of Cytosine in d(CGCG)₂ Crystals. Radiat. Res. 157, 235 – 242 (2002).

Electron paramagnetic resonance (EPR) was used to study an oligodeoxynucleotide duplex of d(CGCG)₂ that is known to crystallize in Z-form. After X irradiation at 4 K, EPR data were collected on single crystals and polycrystalline samples as a function of annealing temperature and dose. A radical produced by the net gain of a hydrogen atom at C6 and a proton at N3, Cyt(C6 H, N3 H ) ^·, is identified. This radical had not been positively identified in polymeric DNA previously. The Cyt(C6 H, N3 H ) ^· makes up about 4 % of the total radical population at 4 K, increasing to about 10 – 15 % after the DNA is annealed to 240 K. There appears to be neither an increase nor a decrease in the absolute concentration of Cyt(C6 H, N3 H ) ^· upon annealing from 4 K to 240 K. Additionally, the presence of another radical, one due to the net gain of hydrogen at C5 of cytosine, the Cyt(C5 H)^·, is implicated. Together, these two radicals appear to account for 60 – 80 % of the reduced species in DNA that has been irradiated at 4 K and annealed to 240 K.

Quarmby, S., West, C., Magee, B., Stewart, A., Hunter, R. and Kumar, S. Differential Expression of Cytokine Genes in Fibroblasts Derived from Skin Biopsies of Patients who Developed Minimal or Severe Normal Tissue Damage after Radiotherapy. Radiat. Res. 157, 243 – 248 (2002).

Curative radiotherapy for cancer patients requires the use of radiation doses that are limited by the tolerance of the surrounding normal tissues. Unfortunately, these tolerance doses vary not only between tissues but also between individuals. In a small proportion of sensitive patients, exposure to radiation can lead to severe irreversible morbidity and even death several months to years after treatment. At present these radiosensitive patients can be identified only retrospectively. Here we describe a cytokine microarray technique that was used to identify differentially expressed gene transcripts in fibroblasts obtained from a small group of patients who suffered either negligible or severe normal damage to tissues after radiotherapy. If our preliminary findings can be confirmed, the availability of such markers may eventually allow the prediction of outcome prior to commencement of radiotherapy, and thus allow modification of radiotherapy protocols to minimize adverse late effects, without compromising tumor control.

Gault, N., Vozenin-Brotons, M. C., Calenda, A., Lefaix, J. L. and Martin, M. T. Promoter Sequences Involved in Transforming Growth Factor β1 Gene Induction in HaCaT Keratinocytes after Gamma Irradiation. Radiat. Res. 157, 249 – 255 (2002).

Transforming growth factor beta 1 (TGFB1) is a cytokine involved in the development of both acute and late cutaneous radiation syndromes. We previously demonstrated that ionizing radiation induces TGFB1 expression in vivo in pig skin within a few hours. The purpose of the present study was to develop an in vitro human model to identify the mechanisms of this early activation. Accordingly, human HaCaT keratinocytes were irradiated with a single dose of 20 Gy. First, radiation-induced TGFB1 overexpression was checked at both the transcriptional and transductional levels in HaCaT cells. Then electrophoretic mobility shift assays (EMSA) and transient transfection with various TGFB1 promoter constructs were used to identify the sequences involved in regulating this promoter. EMSA analysis showed the induction of nuclear protein binding activity by γ irradiation to the −365 AP1 sequence (TGTCTCA), suggesting the involvement of AP1 sequences in the regulation of TGFB1 transcription. In gene reporter assays, maximal TGFB1 promoter activation was found for the longest construct, which contains two AP1 sequences. However, assays with constructs including deletions showed that these two AP1 sequences were not sufficient to confer TGFB1 inducibility. These results showed for the first time, to our knowledge, that transcriptional regulation is involved in radiation-induced activation of TGFB1 gene expression.

Johnston, C. J., Williams, J. P., Okunieff, P. and Finkelstein, J. N. Radiation-Induced Pulmonary Fibrosis: Examination of Chemokine and Chemokine Receptor Families. Radiat. Res. 157, 256 – 265 (2002).

Fibrosis is a common outcome of chronic inflammation or injury. Pulmonary fibrosis may be the result of abnormal repair after an acute inflammatory response. The process of repair initiated by a tissue insult is largely a function of the activation of cells to produce important biological mediators such as cytokines, growth factors and chemokines, which orchestrate most aspects of the inflammatory response. Consequently, altered regulation of the production of inflammatory cell cytokines and chemokines after injury and repair likely contributes to the fibrosis. Our hypothesis is that chronic expression of specific chemokine and chemokine receptors during the fibrotic phase induced by thoracic irradiation may perpetuate the recruitment and activation of lymphocytes and macrophages, which may contribute to the development of fibrosis. Fibrosis-sensitive (C57BL/6) and fibrosis-resistant (C3H/HeJ) mice were irradiated with a single dose of 12.5 Gy to the thorax. Total lung RNA was prepared and hybridized using microarray analysis and RNase protection assays. At 26 weeks postirradiation, messages encoding the chemokines BLC (now known as Scyb13), C10 (now known as Scya6), IP-10 (now known as Scyb10), MCP-1 (now known as Scya2), MCP-3 (now known as Scya7), MIP-1γ (now known as Scya9), and RANTES (now known as Scya5) and the chemokine receptors Ccr1, Ccr2, Ccr5 and Ccr6 were elevated in fibrosis-sensitive (C57BL/6) mice. In contrast, only the messages encoding SDF-1α (now known as Sdf1) and Ccr1 were elevated 26 weeks postirradiation in fibrosis-resistant (C3H/HeJ) mice. Our results point to the CC and CCR family members as the predominant chemokine responders during the development of fibrosis. These studies suggest that monocyte/macrophage and lymphocyte recruitment and activation are key components of radiation-induced fibrosis.

François, A., Ksas, B., Aigueperse, J. and Griffiths, N. M. The Recovery of the Neurally Evoked Secretory Response of Rat Colonic Mucosa after Irradiation is Independent of Mast Cells. Radiat. Res. 157, 266 – 274 (2002).

The ability of the enteric submucosal plexus to influence the transport of water and electrolytes in the colon was investigated in rats for 1 week after acute whole-body γ irradiation. The involvement of neuroimmune links in the epithelial responses to nerve stimulation was confirmed by the sensitivity of the tissue to tetrodotoxin, mepyramine and doxantrazole. At 1 and 3 days after irradiation, colon tissues were hyporesponsive to nerve stimulation. This was associated with a drastic diminution of mucosal mast cell numbers, tissue histamine levels, and rat mast cell protease II (RMCP II) levels, and by a decreased maximal epithelial response to exogenously added histamine. The responses to electric-field stimulation were insensitive to both mepyramine and doxantrazole. At 7 days, neurally evoked responses recovered, despite the virtual absence of mast cells, tissue histamine and RMCP II, and the continuing decreased response to histamine. The responses were insensitive to doxantrazole but were decreased by mepyramine. This study showed that the establishment of a normal epithelial response to neural stimulation can occur despite the radiation-induced depletion of mucosal mast cells. The recovery of the epithelial response, which was sensitive to mepyramine, may be ascribed to the reappearance of an unknown histaminergic pathway, which probably has indirect effects on epithelial transport but is independent of nerve – mast cell connections.

Kojima, S., Ishida, H., Takahashi, M. and Yamaoka, K. Elevation of Glutathione Induced by Low-Dose Gamma Rays and its Involvement in Increased Natural Killer Activity. Radiat. Res. 157, 275 – 280 (2002).

We examined the relationship between the induction of an increase in the level of glutathione and the elevation of natural killer (NK) activity in mouse splenocytes by a low dose of γ rays. The glutathione levels in mouse splenocytes increased significantly between 2 h and 6 h after whole-body γ irradiation at 0.5 Gy, peaked at 4 h, and then decreased almost to the level before irradiation by 12 h postirradiation. A significant enhancement of NK activity was found in the splenocytes obtained from whole-body-irradiated mice between 4 and 6 h postirradiation. Reduced glutathione (GSH) added exogenously to splenocytes obtained from normal mice enhanced both the total cellular glutathione content and the NK activity in a dose-dependent manner. Other precursors of de novo GSH synthesis, such as cysteine, N-acetylcysteine and oxidized glutathione, also increased the activity. These enhancements were completely blocked by buthionine sulfoximine, an inhibitor of de novo GSH synthesis. We conclude that the induction of endogenous glutathione in living cells immediately after low-dose γ irradiation is at least partially responsible for the appearance of enhanced NK activity.

Mizutani, N., Fujikura, Y., Wang, Y-H., Tamechika, M., Tokuda, N., Sawada, T. and Fukumoto, T. Inflammatory and Anti-inflammatory Cytokines Regulate the Recovery from Sublethal X Irradiation in Rat Thymus. Radiat. Res. 157, 281 – 289 (2002).

We investigated the regeneration of rat thymus after sublethal X irradiation (6 Gy). The number of thymocytes was much lower on day 3 after irradiation, and many apoptotic cells were observed. However, by day 5, there had been a rapid proliferation of thymocytes. Since cytokines are considered to be important regulatory factors in postirradiation recovery, we performed in vivo cytokine assays using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and found serial changes in the cytokine message. The messenger RNA (mRNA) expression of the pro-inflammatory cytokines interleukin 1 beta (Il1b), Il6 and tumor necrosis factor alpha (Tnf) was higher than normal on day 3, lower on day 5, and higher again on day 7. In particular, Tnf was completely absent on day 5 and was expressed again on day 7. Of the anti-inflammatory cytokines Il4, transforming growth factor beta (Tgfb) and Il10, only the Il10 message changed substantially. Il10 expression was very high on day 5 but was completely absent on day 7. Thus the Tnf and Il10 messages were expressed alternately. The changes in the distribution of macrophages detected by the immunohistochemical analysis may be related to the changes in the cytokines. Analysis of cytokine messages in the regenerating thymus in vivo may provide new insights into potential therapies for radiation-induced damage.

Srivastava, M., Chandra, D. and Kale, R. K. Modulation of Radiation-Induced Changes in the Xanthine Oxidoreductase System in the Livers of Mice by its Inhibitors. Radiat. Res. 157, 290 – 297 (2002).

The xanthine oxidoreductase (XOD) system, which consists of xanthine dehydrogenase (XDH) and xanthine oxidase (XO), is one of the major sources of free radicals in biological systems. The XOD system is present predominantly in the normal tissues as XDH. In damaged tissues, XDH is converted into XO, the form that generates free radicals. Therefore, the XO form of the XOD system is expected to be found mainly in radiolytically damaged tissue. In this case, XO may catalyze the generation of free radicals and potentiate the effect of radiation. Inhibition of the XOD system is likely to attenuate the detrimental effects of ionizing radiation. We have examined this possibility using allopurinol and folic acid, which are known inhibitors of the XOD system. Swiss albino mice (7 – 8 weeks old) were given single doses of allopurinol and folic acid (12.5 – 50 mg/kg) intraperitoneally and irradiated with different doses of γ radiation at a dose rate of 0.023 Gy/s. The XO and XDH activities as well as peroxidative damage and lactate dehydrogenase (LDH) were determined in the liver. An enhancement of the activity of XO and a simultaneous decrease in the activity of XDH were observed at doses above 3 Gy. The decrease in the ratio XDH/XO and the unchanged total activity (XDH   XO) suggested the conversion of XDH into XO. The enhanced activity of XO may potentiate radiation damage. The increased levels of peroxidative damage and the specific activity of LDH in the livers of irradiated mice supported this possibility. Allopurinol and folic acid inhibited the activities of XDH and XO, decreased their ratio (XDH/XO), and lowered the levels of peroxidative damage and the specific activity of LDH. These results suggested that allopurinol and folic acid have the ability to inhibit the radiation-induced changes in the activities of XDH and XO and to attenuate the detrimental effect of this conversion, as is evident from the diminished levels of peroxidative damage and the decreased activity of LDH.

Fujimori, A., Hashimoto, H., Araki, R., Saito, T., Sato, S., Kasama, Y., Tsutsumi, Y., Mori, M., Fukumura, R., Ohhata, T., Tatsumi, K. and Abe, M. Sequence Analysis of 193.4 and 83.9 kbp of Mouse and Chicken Genomic DNAs Containing the Entire Prkdc (DNA-PKcs) Gene. Radiat. Res. 157, 298 – 305 (2002).

The catalytic subunit of DNA-dependent protein kinase plays critical roles in nonhomologous end joining in repair of DNA double-strand breaks and V(D)J recombination. In addition to the SCID phenotype, it has been suggested that the molecule contributes to the polymorphic variations in radiosensitivity and susceptibility to cancer in mouse strains. Here we show the nucleotide sequence of approximately 193-kbp and 84-kbp genomic regions encoding the entire Prkdc gene (also known as DNA-PKcs) in the mouse and chicken, respectively. A large retroposon was found in intron 51 in the mouse but not in the human or chicken. Comparative analyses of the genome strongly suggested that the region contains only two genes for Prkdc and Mcm4; however, several conserved sequences and cis elements were also predicted.

Inamdar, K. V., Yu, Y. and Povirk, L. F. Resistance of 3′-Phosphoglycolate DNA Ends to Digestion by Mammalian DNase III. Radiat. Res. 157, 306 – 311 (2002).

An essential step in the repair of free radical-mediated DNA strand breaks is the removal of sugar fragments such as phosphoglycolate from the 3′ termini. While the abasic endonuclease Ape1 can remove phosphoglycolate from single-strand breaks in double-stranded DNA, an enzyme capable of removing it from 3′ overhangs of double-strand breaks has yet to be identified. We therefore tested DNase III, the predominant 3′ → 5′ exonuclease in mammalian cell extracts, for possible 3′-phosphoglycolate-removing activity. However, all 3′-phosphoglycolate substrates, as well as a 3′-phosphate substrate, were resistant to DNase III under conditions in which the analogous 3′-hydroxyl substrates were extensively degraded. The DNA end-binding protein Ku (an equimolar mixture of Ku70, now known as G22P1, and Ku86, now known as XRCC5) did not alter the resistance of the 3′-phosphoglycolate substrates, but the protein modulated the susceptibility of 3′-hydroxyl substrates, allowing DNase III to remove a 3′ overhang but inhibiting digestion of the double-stranded portion of the substrate.

Neubauer, S., Arutyunyan, R., Stumm, M., Dörk, T., Bendix, R., Bremer, M., Varon, R., Sauer, R. and Gebhart, E. Radiosensitivity of Ataxia Telangiectasia and Nijmegen Breakage Syndrome Homozygotes and Heterozygotes as Determined by Three-Color FISH Chromosome Painting. Radiat. Res. 157, 312 – 321 (2002).

A three-color chromosome painting technique was used to examine the spontaneous and radiation-induced chromosomal damage in peripheral lymphocytes and lymphoblastoid cells from 11 patients with ataxia telangiectasia (AT) and from 14 individuals heterozygous for an AT allele. In addition, cells from two homozygous and six obligate heterozygous carriers of mutations in the Nijmegen breakage syndrome gene (NBS) were investigated. The data were compared to those for chromosome damage in 10 unaffected control individuals and 48 cancer patients who had not yet received therapeutic treatment. Based on the well-documented radiation sensitivity of AT and NBS patients, it was of particular interest to determine whether the FISH painting technique used in these studies allowed the reliable detection of an increased sensitivity to in vitro irradiation of cells from heterozygous carriers. Peripheral blood lymphocytes and lymphoblastoid cells from both the homozygous AT and NBS patients showed the highest cytogenetic response, whereas the cells from control individuals had a low number of chromosomal aberrations. The response of cells from heterozygous carriers was intermediate and could be clearly differentiated from those of the other groups in double-coded studies. AT and NBS heterozygosity could be distinguished from other genotypes by the total number of breakpoints per cell and also by the number of the long-lived stable aberrations in both AT and NBS. Only AT heterozygosity could be distinguished by the fraction of unstable chromosome changes. The slightly but not significantly increased radiosensitivity that was found in cancer patients was apparently due to a higher trend toward rearrangements compared to the controls. Thus the three-color painting technique presented here proved to be well suited as a supplement to conventional cytogenetic techniques for the detection of heterozygous carriers of these diseases, and may be superior method.

Wild-Type TP53 Inhibits G₂-Phase Checkpoint Abrogation and Radiosensitization Induced by PD0166285, a WEE1 Kinase Inhibitor. Radiat. Res. 157, 322 – 330 (2002).

The WEE1 protein kinase carries out the inhibitory phosphorylation of CDC2 on tyrosine 15 (Tyr15), which is required for activation of the G₂-phase checkpoint in response to DNA damage. PD0166285 is a newly identified WEE1 inhibitor and is a potential selective G₂-phase checkpoint abrogator. To determine the role of TP53 in PD0166285-induced G₂-phase checkpoint abrogation, human H1299 lung carcinoma cells expressing a temperature-sensitive TP53 were used. Upon exposure to γ radiation, cells cultured under nonpermissive conditions (TP53 mutant conformation) underwent G₂-phase arrest. However, under permissive conditions (TP53 wild-type conformation), PD0166285 greatly inhibited the accumulation of cells in G₂ phase. This abrogation was accompanied by a nearly complete blockage of Tyr15 phosphorylation of CDC2, an increased activity of CDC2 kinase, and an enhanced sensitivity to radiation. However, under permissive conditions (TP53 wild-type conformation), PD0166285 neither disrupted the G₂-phase arrest nor increased cell death. The compound inhibited Tyr15 phosphorylation only partially and did not activate CDC2 kinase activity. To understand the potential mechanism(s) by which TP53 inhibits PD0166285-induced G₂-phase checkpoint abrogation, two TP53 target proteins, 14-3-3σ and CDKN1A (also known as p21), that are known to be involved in G₂-phase checkpoint control in other cell models were examined. It was found that 14-3-3σ was not expressed in H1299 cells, and that although CDKN1A did associate with CDC2 to form a complex, the level of CDKN1A associated with CDC2 was not increased in response to radiation or to PD0166285. The level of cyclin B1, required for CDC2 activity, was decreased in the presence of functional TP53. Thus inhibition of PD0166285-induced G₂-phase checkpoint abrogation by TP53 was achieved at least in part through partial blockage of CDC2 dephosphorylation of Tyr15 and inhibition of cyclin B1 expression.

Kakinuma, S., Nishimura, M., Sasanuma, S-I., Mita, K., Suzuki, G., Katsura, Y., Sado, T. and Shimada, Y. Spectrum of Znfn1a1 (Ikaros) Inactivation and its Association with Loss of Heterozygosity in Radiogenic T-Cell Lymphomas in Susceptible B6C3F1 Mice. Radiat. Res. 157, 331 – 340 (2002).

Ikaros (now known as Znfn1a1), a Krüppel-type zinc-finger transcription factor that plays a critical role in both lineage commitment and differentiation of lymphoid cells, has recently been shown to function as a tumor suppressor gene. We have previously reported a high frequency of LOH (∼50 %) at the Znfn1a1 locus in radiation-induced T-cell lymphoma in susceptible B6C3F1 mice. The aim of the present study was to delineate the types of Znfn1a1 inactivation, with special reference to the LOH status, and to determine the relative contribution of each type of Znfn1a1 inactivation in radiation-induced T-cell lymphomas in B6C3F1 mice. We demonstrated that Znfn1a1 was frequently altered (in ∼50 % of T-cell lymphomas), and that its inactivation was caused by a variety of mechanisms, which came under one of the following four categories: (1) null expression (14 %); (2) expression of unusual dominant-negative isoforms (11 %); (3) amino acid substitutions in the N-terminal zinc-finger domain for DNA binding caused by point mutations (22 %); (4) lack of the Znfn1a1 isoform 1 due to the creation of a stop codon by insertion of a dinucleotide in exon 3 (3 %). The null expression, amino acid substitutions, and dinucleotide insertion inactivation types were well correlated with LOH at the Znfn1a1 allele (86 %) and were consistent with Knudson's two-hit theory. On the other hand, T-cell lymphomas expressing dominant-negative Znfn1a1 isoforms retained both alleles. These results indicate that Znfn1a1 inactivation takes place by a variety of mechanisms in radiation-induced murine T-cell lymphomas and is frequently associated with LOH, this association depending on the type of inactivation.

Romanyukha, A. A., Mitch, M. G., Lin, Z., Nagy, V. and Coursey, B. M. Mapping the Distribution of ⁹⁰Sr in Teeth with a Photostimulable Phosphor Imaging Detector. Radiat. Res. 157, 341 – 349 (2002).

The present communication describes the technical aspects of the first application of an imaging plate for visualization of ⁹⁰Sr deposited in human teeth. The teeth were obtained from Techa River area residents who were exposed as a result of releases of radioactivity into the Techa River by the first Soviet nuclear plant Mayak in the early 1950s. The investigations form the basis for an experimental procedure for accurate mapping of the distribution of ⁹⁰Sr in teeth with an imaging plate. This new method can be used as an individual indicator of radionuclide intake. Its advantages are its high sensitivity (0.02 Bq/g mm⁻² of ⁹⁰Sr), it ability to examine small detectable cross-sectional areas of dental tissue (dentin) contaminated with ⁹⁰Sr (from 0.01 mm²), the nondestructive method of analysis, and the simplicity of use. The combined application of this method with EPR tooth biodosimetry can provide more accurate dose reconstruction and may lead to more effective radiation risk assessment.

Gersey, B. B., Borak, T. B., Guetersloh, S. B., Zeitlin, C., Miller, J., Heilbronn, L., Murakami, T. and Iwata, Y. The Response of a Spherical Tissue-Equivalent Proportional Counter to Iron Particles from 200 – 1000 MeV/nucleon. Radiat. Res. 157, 350 – 360 (2002).

The radiation environment on board the space shuttle and the International Space Station includes high-Z and high-energy (HZE) particles that are part of the galactic cosmic radiation (GCR) spectrum. Iron-56 particles are considered to be one of the most biologically important parts of the GCR spectrum. Tissue-equivalent proportional counters (TEPCs) are used as active dosimeters on manned space flights. These TEPCs are further used to determine the average quality factor for each space mission. A TEPC simulating a 1-μm-diameter sphere of tissue was exposed as part of a particle spectrometer to ⁵⁶Fe particles at energies from 200 – 1000 MeV/nucleon. The response of TEPCs in terms of mean lineal energy, ȳ_F, and dose mean lineal energy, ȳ_D, as well as the energy deposited at different impact parameters through the detector was determined for six different incident energies of ⁵⁶Fe particles in this energy range. Calculations determined that charged-particle equilibrium was achieved for each of the six experiments. Energy depositions at different impact parameters were calculated using a radial dose distribution model, and the results were compared to experimental data.