Guan, J., Stewart, J., Ware, J. H., Zhou, Z., Donahue, J. J. and Kennedy, A. R. Effects of Dietary Supplements on the Space Radiation-Induced Reduction in Total Antioxidant Status in CBA Mice. Radiat. Res. 165, 373–378 (2006).

In the present study, the total antioxidant status was used as a biomarker to evaluate oxidative stress induced by proton, HZE-particle and γ radiation in CBA mice. The results demonstrated that the plasma level of TAS was significantly decreased (P < 0.05) in CBA mice after exposure to a 50-cGy dose of radiation from HZE particles or a 3-Gy dose of radiation from protons or γ rays. Diet supplementation with Bowman-Birk Inhibitor Concentrate (BBIC), l-selenomethionine (l-SeM), or a combination of N-acetyl cysteine, sodium ascorbate, co-enzyme Q10 (CoQ10), α-lipoic acid, l-SeM and vitamin E succinate could partially or completely prevent the reduction in the plasma level of TAS in CBA mice exposed to proton or HZE-particle radiation. The selected antioxidant combination with or without CoQ10 has a comparable protective effect on the γ-radiation-induced drop in TAS in CBA mice. These results indicate that BBIC, l-SeM and the selected antioxidant combinations may serve as countermeasures for space radiation-induced adverse biological effects.

Liu, G. W., Gong, P. S., Zhao, H. G., Wang, Z. C., Gong, S. L. and Cai, L. Effect of Low-Level Radiation on the Death of Male Germ Cells. Radiat. Res. 165, 379–389 (2006).

Hormetic and adaptive responses induced by low-level radiation in hematopoietic and immune systems have been observed, as shown by stimulatory effects on cell growth and resistance to subsequent radiation-induced cytogenetic damage. However, in terms of cell death by apoptosis, the effects of low-level radiation are controversial: Some studies showed decreased apoptosis in response to low-level radiation while others showed increased apoptosis. This controversy may be related to the radiation doses or dose rates and also, more importantly, to the cell types. Testes are one of the most radiosensitive organs. The loss of male germ cells after exposure to ionizing radiation has been attributed to apoptosis. In the present study, the effects of low-level radiation at doses up to 200 mGy on mouse male germ cells in terms of apoptosis and the expression of apoptosis-related proteins were examined at different times after whole-body exposure of mice to low-level radiation. In addition, the effect of pre-exposure to low-level radiation on subsequent cell death induced by high doses of radiation was examined to explore the possibility of low-level radiation-induced adaptive response. The results showed that low-level radiation in the dose range of 25–200 mGy induced significant increases in apoptosis in both spermatogonia and spermatocytes, with the maximal effect at 75 mGy. The increased apoptosis is most likely associated with Trp53 protein expression. Furthermore, 75 mGy low-level radiation given pre-irradiation led to an adaptive response of seminiferous germ cells to subsequent high-level radiation-induced apoptosis. These results suggest that low-level radiation induces increased apoptosis in male germ cells but also induces a significant adaptive response that decreases cell death after a subsequent high-dose irradiation.

Ho, S-Y., Huang, P-C., Guo, H-R., Chang, W-H., Chen, R-J., Wei, B-L., Wu, W-J., Tai, C. and Wang, Y-J. Mechanisms of Apoptosis Induction and Cell Cycle Regulation in Irradiated Leukemia U937 Cells and Enhancement by Arsenic Trioxide. Radiat. Res. 165, 390–399 (2006).

Apoptosis is a common mode of cell death after exposure of tumor cells to radiation and/or chemotherapy. The factors that determine the rate of induction of apoptosis are generally related to the functioning of cell cycle checkpoints. In the present study, we investigated the involvement of several genes in cell cycle redistribution and induction of apoptosis in U937 cells after low and high doses of radiation. Activation of CDC2 was observed after both low and high doses of radiation in U937 cells that underwent apoptosis. Expression of CDK2, CDC2 and cyclin A was induced rapidly in the process of radiation-induced apoptosis. In addition, we investigated the use of a clinically relevant dose of radiation to promote As₂O₃-induced apoptosis in U937 cells. We found that combining radiation and As₂O₃ may be a new and more effective means of cancer treatment.

Lyng, F. M., Maguire, P., McClean, B., Seymour, C. and Mothersill, C. The Involvement of Calcium and MAP Kinase Signaling Pathways in the Production of Radiation-Induced Bystander Effects. Radiat. Res. 165, 400–409 (2006).

Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005–5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min–24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of bystander responses. Further investigations of these signaling pathways may aid in the identification of novel therapeutic targets.

Kabakov, A. E., Malyutina, Y. V. and Latchman, D. S. Hsf1-Mediated Stress Response can Transiently Enhance Cellular Radioresistance. Radiat. Res. 165, 410–423 (2006).

To elucidate how the heat-shock transcription factor 1 (Hsf1)-mediated stress response affects cellular radioresistance, mouse embryo fibroblasts with Hsf1-gene knockout (Hsf1^−/− cells) or with normal wild-type Hsf1 expression (Hsf1 wild-type cells) were preconditioned by heating (43°C, 30 min) without or with quercetin (an inhibitor of Hsf1) and then exposed to γ radiation (4 or 6 Gy). Some cell samples were infected with virus-based vectors to overexpress the constitutively active (mutant) form of Hsf1 or individual heat-shock proteins (Hsps). The heat preconditioning transiently up-regulated the Hsp levels in Hsf1 wild-type cells and significantly improved their postirradiation survival; these effects could be abolished by quercetin or simulated (without preheating) by the Hsf1 overexpression. In contrast, no enhanced radioresistance was found in heat-preconditioned Hsf1^−/− cells that were unable to trigger Hsf1-mediated Hsp induction after heating. However, when the constitutively active Hsf1 was overexpressed in Hsf1^−/− cells, the latter accumulated stress-inducible Hsps and became more radioresistant like heat-preconditioned Hsf1 wild-type cells. The overexpression of Hsp70 or/ and Hsp27 also enhanced radioresistance of both cell cultures. Thus the preirradiation stress response resulting in the intracellular Hsp accumulation can improve survival of severely irradiated mammalian cells.

Chauhan, V., Mariampillai, A., Bellier, P. V., Qutob, S. S., Gajda, G. B., Lemay, E., Thansandote, A. and McNamee, J. P. Gene Expression Analysis of a Human Lymphoblastoma Cell Line Exposed In Vitro to an Intermittent 1.9 GHz Pulse-Modulated Radiofrequency Field. Radiat. Res. 165, 424–429 (2006).

This study was designed to determine whether radiofrequency (RF) fields of the type used for wireless communications could elicit a cellular stress response. As general indicators of a cellular stress response, we monitored changes in proto-oncogene and heat-shock protein expression. Exponentially growing human lymphoblastoma cells (TK6) were exposed to 1.9 GHz pulse-modulated RF fields at average specific absorption rates (SARs) of 1 and 10 W/kg. Perturbations in the expression levels of the proto-oncogenes FOS, JUN and MYC after exposure to sham and RF fields were assessed by real-time RT-PCR. In addition, the transcript levels of the cellular stress proteins HSP27 and inducible HSP70 were also monitored. We demonstrated that transcript levels of these genes in RF-field-exposed cells showed no significant difference in relation to the sham treatment group. However, concurrent positive (heat-shock) control samples displayed a significant elevation in the expression of HSP27, HSP70, FOS and JUN. Conversely, the levels of MYC mRNA were found to decline in the positive (heat-shock) control. In conclusion, our study found no evidence that the 1.9 GHz RF-field exposure caused a general stress response in TK6 cells under our experimental conditions.

Lim, S., Yoon, H., Ryu, S., Jung, J., Lee, M. and Kim, D. A Comparative Evaluation of Radiation-Induced DNA Damage using Real-Time PCR: Influence of Base Composition. Radiat. Res. 165, 430–437 (2006).

To study the radiosensitivity of DNA segments at the open reading frame (gene) level, real-time PCR was used to analyze DNA damages induced by ionizing radiation. After irradiation (1, 3 and 5 kGy) of genomic DNA purified from Salmonella typhimurium, real-time PCR based on SYBR Green fluorescence and melting temperature was performed using various primer sets targeting the rfbJ, rfaJ, rfaB, hilD, ssrB, pipB, sopD, pduQ, eutG, oadB, ccmB and ccmA genes. The ccmA and ccmB genes, which existed as two copies on the chromosome and had a high GC content (∼70%), showed much lower radiosensitivities than the other genes tested, particularly at 5 kGy; this distinctive feature was seen only when the genes were located on the chromosome, regardless of copy number. Our results reinforce the concept that gene sensitivity to ionizing radiation depends on the base composition and/or the spatial localization of the gene on the chromosome.

Bailey, D. T., DeFedericis, H-C. C., Greene, K. F., Iijima, H., Budzinski, E. E., Patrzyc, H. B., Dawidzik, J. B. and Box, H. C. A Novel Approach to DNA Damage Assessments: Measurement of the Thymine Glycol Lesion. Radiat. Res. 165, 438– 444 (2006).

A different approach to the measurement of DNA damage has been developed based on the fact that many lesions can be excised from DNA in the form of modified dinucleoside monophosphates. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used in conjunction with isotopically labeled internal standards to quantify the lesion. The method has several advantages, including high sensitivity for the detection of dinucleoside monophosphates. The method was applied to the measurement of the 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol) lesion in the DNA of mouse fibroblast cells exposed in culture to various treatments including ionizing radiation, UVC light and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. The application of the method to the measurement of other DNA lesions is discussed.

DeFedericis, H-C., Patrzyc, H. B., Rajecki, M. J., Budzinski, E. E., Iijima, H., Dawidzik, J. B., Evans, M. S., Greene, K. F. and Box, H. C. Singlet Oxygen-Induced DNA Damage. Radiat. Res. 165, 445–451 (2006).

Singlet oxygen, hydrogen peroxide, hydroxyl radical and hydrogen peroxide are the reactive oxygen species (ROS) considered most responsible for producing oxidative stress in cells and organisms. Singlet oxygen interacts preferentially with guanine to produce 8-oxo-7,8-dihydroguanine and spiroiminodihydantoin. DNA damage due to the latter lesion has not been detected directly in the DNA of cells exposed to singlet oxygen. In this study, the singlet oxygen-induced lesion was isolated from a short synthetic oligomer after exposure to UVA radiation in the presence of methylene blue. The lesion could be enzymatically excised from the oligomer in the form of a modified dinucleoside monophosphate. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), the singlet oxygen lesion was detected in the form of modified dinucleoside monophosphates in double-stranded DNA and in the DNA of HeLa cells exposed to singlet oxygen. Pentamer containing the singlet oxygen-induced lesion and an isotopic label was synthesized as an internal standard for quantifying the lesion and served as well as for correcting for losses of product during sample preparation.

Panajotovic, R., Martin, F., Cloutier, P., Hunting, D. and Sanche, L. Effective Cross Sections for Production of Single-Strand Breaks in Plasmid DNA by 0.1 to 4.7 eV Electrons. Radiat. Res. 165, 452–459 (2006).

We determined effective cross sections for production of single-strand breaks (SSBs) in plasmid DNA [pGEM 3Zf(-)] by electrons of 10 eV and energies between 0.1 and 4.7 eV. After purification and lyophilization on a chemically clean tantalum foil, dry plasmid DNA samples were transferred into a high-vacuum chamber and bombarded by a monoenergetic electron beam. The amount of the circular relaxed DNA in the samples was separated from undamaged molecules and quantified using agarose gel electrophoresis. The effective cross sections were derived from the slope of the yield as a function of exposure and had values in the range of 10⁻¹⁵– 10⁻¹⁴ cm², giving an effective cross section of the order of 10⁻¹⁸ cm² per nucleotide. Their strong variation with incident electron energy and the resonant enhancement at 1 eV suggest that considerable damage is inflicted by very low-energy electrons to DNA, and it indicates the important role of π* shape resonances in the bond-breaking process. Furthermore, the fact that the energy threshold for SSB production is practically zero implies that the sensitivity of DNA to electron impact is universal and is not limited to any particular energy range.

Stewart, R. D., Ratnayake, R. K. and Jennings, K. Microdosimetric Model for the Induction of Cell Killing through Medium-Borne Signals. Radiat. Res. 165, 460–469 (2006).

Microbeam, medium-transfer and low-dose experiments have demonstrated that intercellular signals can initiate many of the same biological events and processes as direct exposure to ionizing radiation. These phenomena cast doubt on cell-autonomous modes of action and the linear, no-threshold carcinogenesis paradigm. To account for the effects of intercellular signals, new approaches are needed to relate dosimetric quantities to the emission and processing of signals by irradiated and unirradiated cells. In this paper, microdosimetric principles are used to develop a stochastic model to relate absorbed dose to the emission and processing of cell death signals by unirradiated cells. Our analyses of published results of medium transfer experiments performed using HPV-G human keratinocytes suggest that the emission of death signals is a bi-exponential function of dose with a distinct plateau in the 5- to 100-mGy range. However, the emission of death signals by HPV-G cells may not become fully saturated until the absorbed dose becomes larger than 0.6 Gy. Similar saturation effects have been observed in microbeam and medium-transfer experiments with other mammalian cell lines. The model predicts that the cell-killing effect of medium-borne death signals decreases exponentially as the absorbed dose becomes small compared to the frequency-mean specific energy per radiation event.

Swanson, J. and Kheifets, L. Biophysical Mechanisms: A Component in the Weight of Evidence for Health Effects of Power-Frequency Electric and Magnetic Fields. Radiat. Res. 165, 470–478 (2006).

Comparatively high exposures to power-frequency electric and magnetic fields produce established biological effects that are explained by accepted mechanisms and that form the basis of exposure guidelines. Lower exposures to magnetic fields (<1 μT average in the home) are classified as “possibly carcinogenic” on the basis of epidemiological studies of childhood leukemia. This classification takes into consideration largely negative laboratory data. Lack of biophysical mechanisms operating at such low levels also argues against causality. We survey around 20 biophysical mechanisms that have been proposed to explain effects at such low levels, with particular emphasis on plausibility: the principle that to produce biological effects, a mechanism must produce a “signal” larger than the “noise” that exists naturally. Some of the mechanisms are impossible, and some require specific conditions for which there is limited or no evidence as to their existence in a way that would make them relevant to human exposure. Others are predicted to become plausible above some level of field. We conclude that effects below 5 μT are implausible. At about 50 μT, no specific mechanism has been identified, but the basic problem of implausibility is removed. Above about 500 μT, there are established or likely effects from accepted mechanisms. The absence of a plausible biophysical mechanism at lower fields cannot be taken as proof that health effects of environmental electric and magnetic fields are impossible. Nevertheless, it is a relevant consideration in assessing the overall evidence on these fields.

Adhikary, A., Kumar, A. and Sevilla, M. D. Photo-induced Hole Transfer from Base to Sugar in DNA: Relationship to Primary Radiation Damage. Radiat. Res. 165, 479–484 (2006).

This work presents the hypothesis that photo-excitation of G^· in DNA and model systems results in the same electronic states expected from direct ionization of the sugar phosphate backbone and that these states lead to specific sugar radicals on the DNA sugar phosphate backbone. As evidence we show that visible photo-excitation of guanine cation radicals (G^· ) in the dinucleoside phosphate TpdG results in high yields (about 85%) of deoxyribose sugar radicals at the C1′ and C3′ sites. Further, we have calculated transition energies of hole transfer from G^· in TpdG using time-dependent density functional theory (TD-DFT) at the B3LYP/6–31G(d) level in gas phase as well as in a solvated environment. These calculations clearly predict that visible excitation of G^· in TpdG causes transitions from only inner-shell filled molecular orbitals (MOs) to the singly occupied molecular orbital (SOMO) that effectively result in hole transfer from guanine either to the sugar phosphate backbone or to the adjacent base, thymine. The hole transfer is followed by rapid deprotonation from the sugar to form C1′ and C3′ radicals. These experimental and theoretical results are in agreement with our previously published experimental and theoretical results that photo-excitation of G^· results in high yields of deoxyribose sugar radicals in DNA, guanine deoxyribonucleosides and deoxyribonucleotides. Photo-excitation of G^· therefore provides a convenient method to produce and study sugar radicals that are expected to be formed in γ-irradiated DNA systems unencumbered by the many other pathways involved in direct ionization.

Muroya, Y., Plante, I., Azzam, E. I., Meesungnoen, J., Katsumura, Y. and Jay-Gerin, J-P. High-LET Ion Radiolysis of Water: Visualization of the Formation and Evolution of Ion Tracks and Relevance to the Radiation-Induced Bystander Effect. Radiat. Res. 165, 485–491 (2006).

Ionizing radiation-induced bystander effects, commonly observed in cell populations exposed to high-linear energy transfer (LET) radiations, are initiated by damage to a cellular molecule which then gives rise to a toxic signal exported to neighboring cells not directly hit by radiation. A major goal in studies of this phenomenon is the identification of this initial radiation-induced lesion. Liquid water being the main constituent of biological matter, reactive species produced by water radiolysis in the cellular environment are likely to be major contributors to the induction of this lesion. In this context, the radiation track structure is of crucial importance in specifying the precise location and identity of all the radiolytic species and their subsequent signaling or damaging effects. We report here Monte Carlo track structure simulations of the radiolysis of liquid water by four different impacting ions ¹H , ⁴He² , ¹²C⁶ and ²⁰Ne¹⁰ , with the same LET (∼70 keV/ μm). The initial radial distribution profiles of the various water decomposition products (e_aq⁻, ^·OH, H^·, H₂ and H₂O₂) for the different ions considered are presented and discussed briefly in the context of track structure theory. As an example, the formation and temporal evolution of simulated 24 MeV ⁴He² ion tracks (LET ∼26 keV/μm) are reported for each radiolytic species from 1 ps to 10 μs. The calculations reveal that the ion track structure is completely lost by ∼1 μs.