Wilson, G. D. and Marples, B. Flow Cytometry in Radiation Research: Past, Present and Future. Radiat. Res. 168, 391–403 (2007).

Flow cytometry is an invaluable technique in research and clinical laboratories. The technique has been applied extensively to many areas of radiation research at both the experimental and clinical level. In the past few years, there has been a significant increase in the capabilities of modern flow cytometers to undertake multicolor analysis in a user-friendly manner. The developments in cytometric technology are being matched by the rapid development of new reagents, new fluorochromes and new platforms such as bead arrays. These developments are facilitating many new applications in both basic and clinical research that have relevance for many fields of biology, including radiation research. This review provides a historical overview of the application of flow cytometry to radiobiology and an update on how technology and reagents have changed and cites examples of new applications relevant to radiation researchers. In addition, some entirely new flow instrumentation is currently under development that has significant potential for applications in radiation research.

Neriishi, K., Nakashima, E., Minamoto, A., Fujiwara, S., Akahoshi, M., Mishima, H. K., Kitaoka, K. and Shore, R. E. Postoperative Cataract Cases among Atomic Bomb Survivors: Radiation Dose Response and Threshold. Radiat. Res. 168, 404–408 (2007).

Recent evidence argues against a high threshold dose for vision-impairing radiation-induced cataractogenesis. We conducted logistic regression analysis to estimate the dose response and used a likelihood profile procedure to determine the best-fitting threshold model among 3761 A-bomb survivors who underwent medical examinations during 2000–2002 for whom radiation dose estimates were available, including 479 postoperative cataract cases. The analyses indicated a statistically significant dose–response increase in the prevalence of postoperative cataracts [odds ratio (OR), 1.39; 95% confidence interval (CI), 1.24–1.55] at 1 Gy, with no indication of upward curvature in the dose response. The dose response was suggestive when the restricted dose range of 0 to 1 Gy was examined. A nonsignificant dose threshold of 0.1 Gy (95% CI, <0–0.8) was found. The prevalence of postoperative cataracts in A-bomb survivors increased significantly with A-bomb radiation dose. The estimate (0.1 Gy) and upper bound (0.8 Gy) of the dose threshold for operative cataract prevalence was much lower than the threshold of 2–5 Gy usually assumed by the radiation protection community and was statistically compatible with no threshold at all.

Lyon, C. M., Klinge, D. M., Liechty, K. C., Gentry, F. D., March, T. H., Kang, T., Gilliland, F. D., Adamova, G., Rusinova, G., Telnov, V. and Belinsky, S. A. Radiation-Induced Lung Adenocarcinoma is Associated with Increased Frequency of Genes Inactivated by Promoter Hypermethylation. Radiat. Res. 168, 409–414 (2007).

Epigenetic inactivation of genes by promoter hypermethylation, a major mechanism in the initiation and progression of tobacco-induced cancer, has also been associated with lung cancer induced through environmental and occupational exposures. Our previous study of gene methylation in workers from the MAYAK nuclear enterprise identified a significantly higher prevalence for methylation of the p16 gene (CDKN2A) in adenocarcinomas from workers compared to tumors from non-worker controls. The purpose of this investigation was to determine whether genes in addition to p16 are “targeted” for silencing and whether overall gene methylation was more common in radiation-induced adenocarcinoma. A significant increase in the prevalence of methylation of GATA5 was seen in tumors from workers compared to tumors from controls. The prevalence for methylation of PAX5 β and H-cadherin did not differ in tumors from workers and controls. Evaluating the frequency for methylation of a five-gene panel revealed that 93% of adenocarcinomas from workers compared to 66% of tumors from controls were methylated for at least one gene. Moreover, a twofold increase was seen in the number of tumors methylated for three or more genes for tumors from workers compared to controls. Increased frequency for inactivation of genes by promoter hypermethylation and targeting of tumor suppressor genes such as GATA5 may be factors that contribute to the increased risk for lung cancer associated with radiation exposure.

Pinar, B., Lara, P. C., Lloret, M., Bordón, E., Núñez, M. I., Villalobos, M., Guerrero, R., Luna, J. D. and Ruiz de Almodóvar, J. M. Radiation-Induced DNA Damage as a Predictor of Long-Term Toxicity in Locally Advanced Breast Cancer Patients Treated with High-Dose Hyperfractionated Radical Radiotherapy. Radiat. Res. 168, 415–422 (2007).

This 14-year-long study makes a novel contribution to the debate on the relationship between the in vitro radiosensitivity of peripheral blood lymphocytes and normal tissue reactions after radiation therapy. The aims were (1) to prospectively assess the degree and time of onset of skin side effects in 40 prospectively recruited consecutive patients with locally advanced breast cancer treated with a hyperfractionated dose-escalation radiotherapy schedule and (2) to assess whether initial radiation-induced DNA damage in peripheral blood lymphocytes of these patients could be used to determine their likelihood of suffering severe late damage to normal tissue. Initial radiation-induced DNA double-strand breaks (DSBs) were assessed in peripheral blood lymphocytes of these patients by pulsed-field electrophoresis. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity score. A wide interindividual variation was observed in toxicity grades and in radiation-induced DNA DSBs in peripheral blood lymphocytes (mean 1.61 ± 0.76 DSBs/Gy per 200 MBp, range 0.63– 4.08), which were not correlated. Multivariate analysis showed a correlation (P < 0.008) between late toxicity and higher prescribed protocol dose (81.6 Gy). Analysis of the 29 patients referred to 81.6 Gy revealed significantly (P < 0.031) more frequent late subcutaneous toxicity in those with intrinsic sensitivity to radiation-induced DNA DSBs of >1.69 DSBs/Gy per DNA unit. Our demonstration of a relationship between the sensitivity of in vitro-irradiated peripheral blood lymphocytes and the risk of developing late toxic effects opens up the possibility of predicting normal tissue response to radiation in individual patients, at least in high-dose non-conventional radiation therapy regimens.

Swancutt, K. L., Mezyk, S. P. and Kiddle, J. J. Free Radical-Induced Redox Chemistry of Platinum-Containing Anti-cancer Drugs. Radiat. Res. 168, 423–427 (2007).

Arrhenius parameters for the reactions of oxidizing hydroxyl radicals and reducing hydrated electrons with cisplatin, transplatin and carboplatin in aqueous solution have been determined using pulsed electron radiolysis and absorption spectroscopy techniques. Under physiological pH and chloride concentration conditions, hydroxyl radical reaction rate constants of (9.99 ± 0.20) × 10⁹, (8.38 ± 0.55) × 10⁹, and (6.03 ± 0.08) × 10⁹ M⁻¹ s⁻¹ at 24.0, 20.7 and 24.0°C, respectively, with corresponding activation energies of 12.79 ± 0.57, 13.88 ± 1.14, and 14.35 ± 0.56 kJ mol⁻¹ for these three reactions, were determined. These oxidations of cisplatin and transplatin to form a Pt^III transient are significantly faster than reported previously at room temperature. The lower rate constant for carboplatin is consistent with hydroxyl radical reaction partitioning between reaction at the platinum center and the cyclobutanedicarboxylate ligand. The equivalent reductive hydrated electron reaction rate constants measured were (1.99 ± 0.04) × 10¹⁰ (24.0°C), (1.77 ± 0.08) × 10¹⁰ (22.0°C), and (8.92 ± 0.06) × 10⁹ M⁻¹ s⁻¹ (24.0°C), with corresponding activation energies of 15.75 ± 1.00, 19.74 ± 1.82, and 19.99 ± 0.34 kJ mol⁻¹. Again, the values determined for cisplatin and transplatin are faster than reported; however, all three values are consistent with direct reduction of the platinum center to form a Pt^I moiety.

Jordan, B. F., Christian, N., Crokart, N., Grégoire, V., Feron, O. and Gallez, B. Thyroid Status is a Key Modulator of Tumor Oxygenation: Implication for Radiation Therapy. Radiat. Res. 168, 428–432 (2007).

In normal tissues, thyroid hormones play a major role in the metabolic activity and oxygen consumption of cells. Because the rate of oxygen consumption is a key factor in the response of tumors to radiation, we hypothesized that thyroid hormones may affect the metabolic activity of tumor cells and hence modulate the response to cytotoxic treatments. We measured the influence of thyroid status on the tumor microenvironment in experimental tumors. Hypothyroidism and hyperthyroidism were generated in mice by chronic treatment with propyl thiouracil and l-thyroxine. Thyroid status significantly modified tumor pO₂ as measured with EPR oximetry. Mechanistically, this was the result of the profound changes in oxygen consumption rates. Thyroid status was associated with a significant change in tumor radiosensitivity since the regrowth delay was increased in hypothyroid mice compared to euthyroid mice, an effect that was abolished when temporarily clamped tumors were irradiated. This study provides unique insights into the impact of modulating tumor oxygen consumption and could have implications in the management of cancer patients with thyroid disorders.

Nakayama, T., Yamazumi, K., Uemura, T., Yoshizaki, A., Yakata, Y., Matsuu-Matsuyama, M., Shichijo, K. and Sekine, I. X Radiation Up-regulates the Occurrence and the Multiplicity of Invasive Carcinomas in the Intestinal Tract of Apc^min/ Mice. Radiat. Res. 168, 433–439 (2007).

X rays are well known to cause genetic damage and to induce many types of carcinomas in humans. The Apc^min/ mouse, an animal model for human familial adenomatous polyposis (FAP), contains a truncating mutation in the APC gene and spontaneously develops intestinal adenomas. To elucidate the role of X rays in the development of intestinal tumors, we examined the promotion of carcinogenesis in X-irradiated Apc^min/ mice. Forty out of 77 (52%) X-irradiated Apc^min/ mice developed adenocarcinomas that invaded the proprial muscle layer of the small intestine; 24 of 44 (55%) were in males, and 16 of 33 (49%) were in females. In contrast, invasive carcinomas were detected in the small intestines of only 13 of 64 (20%) nonirradiated Apc^min/ mice; nine of 32 (28%) were in males and four of 32 (13%) were in females. These differences between X-irradiated and nonirradiated Apc^min/ mice in the occurrence of invasive intestinal carcinomas were statistically significant (P < 0.05 for males, P < 0.005 for females). In wild-type mice, invasive carcinomas were not detected in either X-irradiated or nonirradiated mice. Apc^min/ mice had many polyps in the large intestine with or without X irradiation; there was no difference in the number of polyps between the two groups. Also, invasive carcinomas were not detected in the large intestine with or without irradiation. The occurrence of mammary tumors, which was observed in Apc^min/ mice, was found to be increased in irradiated Apc^min/ mice (P < 0.01). Apc^min/ mice had many polyps in the small and large intestines with or without X irradiation. X-irradiated Apc^min/ mice had highly invasive carcinomas in the small intestine with multiplicities associated with invasiveness. Our results suggest that X radiation may promote the invasive activity of intestinal tumors in Apc^min/ mice.

Kohl, R. R., Kolozsvary, A., Brown, S. L., Zhu, G. and Kim, J. H. Differential Radiation Effect in Tumor and Normal Tissue after Treatment with Ramipril, an Angiotensin-Converting Enzyme Inhibitor. Radiat. Res. 168, 440–445 (2007).

The angiotensin-converting enzyme inhibitor, ramipril, has been shown to mitigate radiation injury in normal tissues. Using A549 cell xenografts grown in athymic mice, we measured the effect of ramipril on radiation damage to tumors. Ramipril did not alter tumor response to radiation despite different times of drug administration with respect to radiation delivery (drug started 2 weeks before or immediately after irradiation). In contrast, using the same dose, ramipril reduced normal tissue radiation injury (30 Gy × 2 or 6 Gy × 10) as assessed by a semi-quantitative scale of skin damage and relative leg contraction. The results indicate that ramipril could offer therapeutic gain due to its different effect on normal tissues and tumors.

Park, H-R., Jung, U. and Jo, S-K. Impairment of Natural Killer (NK) Cells is an Important Factor in a Weak Th1-like Response in Irradiated Mice. Radiat. Res. 168, 446–452 (2007).

In whole-body-irradiated (WBI) mice, levels of the canonical Th1 cytokine IFN-γ (IFNG) have been shown to be markedly reduced, resulting in a Th1/Th2 imbalance. In this study, the influence of natural killer (NK) cells on the balance of this Th1/Th2 immune response was evaluated in WBI mice. Although NK cells are one of the types of cells that secreteIFN-γ, NK cell activity tends to be minimal, even at 7 weeks after irradiation. In NK cell-depleted mice, the levels of Th1-related cytokines were lower than those of the control mice and were correlated with lower IgG2a production and elevated IgE and IgG1 production. These results indicated that NK cells have a crucial role in the final differentiation of Th cells into Th1 cells. The impairment of NK cells in the WBI mice was confirmed by the observation that NK cells from the WBI mice induced a decrease in the generation of IFN-γ by the NK cell-depleted spleen lymphocytes from normal mice. Also, the WBI mice that received NK cells obtained from the normal mice generated more IgG2a, IL12 and IFN-γ. Our results indicate that the impairment of NK cells is an important factor in the reduced Th1-like response in irradiated mice.

Lu, F. and Wong, C. S. Time-Dependent Neurosphere-Forming Ability of Adult Rat Spinal Cord after Irradiation. Radiat. Res. 168, 453–461 (2007).

To determine whether there was evidence for long-term time-dependent changes in neurosphere-forming ability of rat spinal cord after irradiation, a 15-mm length of spinal cord (C2–T2) of 10-week-old female rats was irradiated with a single dose of 2, 5, 10 or 19 Gy. Cells were isolated from the central 10-mm segment of the irradiated spinal cord immediately or at 0.5, 1, 2 or 5 months to form neurospheres. The number and sizes of neurospheres were determined at day 10, 12, 14 and 16 in vitro. The multipotential properties of neurosphere cells were assessed by immunocytochemistry using lineage-specific markers for neurons and glia. In nonirradiated controls, the number and size of the neurospheres decreased with increasing age of the animals. Regardless of the time after irradiation, there was a dose-dependent decrease in the number and size of neurospheres obtained from the irradiated cord compared to age-matched controls. Using three-way ANOVA, the number of neurospheres was dependent on radiation dose (P < 0.0001), time after irradiation (P < 0.0001), and day of counting in vitro (P < 0.0001). Compared to cells cultured immediately after irradiation, there was an increase in the relative plating efficiency of neurospheres cultured 1 month after irradiation. However, no further increase was apparent up to 5 months after irradiation. The multipotential properties of neurosphere cells in vitro remained unchanged with increasing time after irradiation. These results may suggest a time-dependent recovery of radiation damage using neurosphere-forming ability as the end point and agree with data that show time-dependent recovery of radiation damage in spinal cord using histological or functional end points.

Vlkolinský, R., Krucker, T., Smith, A. L., Lamp, T. C., Nelson, G. A. and Obenaus, A. Effects of Lipopolysaccharide on ⁵⁶Fe-Particle Radiation-Induced Impairment of Synaptic Plasticity in the Mouse Hippocampus. Radiat. Res. 168, 462–470 (2007).

Space radiation, including high-mass, high-Z, high-energy particles (HZE; e.g. ⁵⁶Fe), represents a significant health risk for astronauts, and the central nervous system (CNS) may be a vulnerable target. HZE-particle radiation may directly affect neuronal function, or during immunological challenge, it may alter immune system-to-CNS communication. To test these hypotheses, we exposed mice to accelerated iron particles (⁵⁶Fe; 600 MeV/nucleon; 1, 2, 4 Gy; brain only) and 1 month later prepared hippocampal slices to measure the effects of radiation on neurotransmission and synaptic plasticity in CA1 neurons. In a model of immune system-to-CNS communication, these electrophysiological parameters were measured in irradiated mice additionally challenged with the peripheral immunological stressor lipopolysaccharide (LPS) injected intraperitoneally 4 h before the slice preparation. Exposure to ⁵⁶Fe particles alone increased dendritic excitability and inhibited plasticity. In control mice (0 Gy), LPS treatment also inhibited synaptic plasticity. Paradoxically, in mice exposed to 2 Gy, the LPS treatment restored synaptic plasticity to levels similar to those found in controls (0 Gy, no LPS). Our results indicate that HZE-particle radiation alters normal electrophysiological properties of the CNS and the hippocampal response to LPS.

Kumlin, T., Iivonen, H., Miettinen, P., Juvonen, A., van Groen, T., Puranen, L., Pitkäaho, R., Juutilainen, J. and Tanila, H. Mobile Phone Radiation and the Developing Brain: Behavioral and Morphological Effects in Juvenile Rats. Radiat. Res. 168, 471–479 (2007).

The increasing use of mobile phones by children and teenagers has raised concerns about their safety. Addressing such concerns is difficult, because no data are available on possible effects from long-term exposure to radiofrequency (RF) fields during the development of the nervous system. Possible morphological and functional changes were evaluated in the central nervous system of young male Wistar rats exposed to 900 MHz mobile phone signal for 2 h/day on 5 days/week. After 5 weeks of exposure at whole-body average specific energy absorption rates of 0.3 or 3.0 W/kg or sham exposure, six rats per group were examined histologically, and the remaining 18 rats per group were subjected to behavioral tests. No degenerative changes, dying neurons, or effects on the leakage of the blood-brain barrier were detected. No group differences were observed in the open-field test, plus maze test or acoustic startle response tests. In the water maze test, however, significantly improved learning (P = 0.012) and memory (P = 0.01) were detected in rats exposed to RF fields. The results do not indicate a serious threat to the developing brain from mobile phone radiation at intensities relevant to human exposure. However, the interesting finding of improved learning and memory warrants further studies.

Smith, P. A., Kuster, N., Ebert, S. and Chevalier, H-J. GSM and DCS Wireless Communication Signals: Combined Chronic Toxicity/Carcinogenicity Study in the Wistar Rat. Radiat. Res. 168, 480–492 (2007).

A total of 1170 rats comprised of 65 male and 65 female Han Wistar rats per group were exposed for 2 h/day, 5 days/ week for up to 104 weeks to GSM or DCS wireless communication signals at three nominal SARs of 0.44, 1.33 and 4.0 W/kg. A preliminary study confirmed that the highest exposure level was below that which was capable of causing a measurable increase in the core temperature of the rat. Additional groups for each modulation were sham exposed, and there was also an unrestrained, unexposed (cage) control group. Fifteen male and 15 female rats per group were killed after 52 weeks. From the remaining 50 male and 50 female rats per group, surviving animals were killed after 104 weeks. Evaluations during the study included mortality rate, clinical signs, recording of palpable masses, body weight, food consumption, ophthalmoscopic examination, and clinical pathological investigations. Terminal investigations included organ weight measurement and macroscopic and microscopic pathology examinations. There was no adverse response to the wireless communication signals. In particular, there were no significant differences in the incidence of primary neoplasms, the number of rats with more than one primary neoplasm, the multiplicity and latency of neoplasms, the number of rats with metastases, and the number of benign and malignant neoplasms between the rats exposed to wireless communication signals and rats that were sham exposed.

Medvedeva, N. G., Panyutin, I. V., Panyutin, I. G. and Neumann, R. D. Phosphorylation of Histone H2AX in Radiation-Induced Micronuclei. Radiat. Res. 168, 493–498 (2007).

DNA double-strand breaks are thought to precede the formation of most radiation-induced micronuclei. Phosphorylation of the histone H2AX is an early indicator of DNA double-strand breaks. Here we studied the phosphorylation status of the histone H2AX in micronuclei after exposure of cultured cells to ionizing radiation or treatment with colchicine. In human astrocytoma SF268 cells, after exposure to γ radiation, the proportion of γ-H2AX-positive to γ-H2AX-negative micronuclei increases. The majority of the γ-H2AX-positive micronuclei are centromere-negative. The number of γ-H2AX-positive micronuclei continues to increase even 24 h postirradiation when most γ-H2AX foci in the main nucleus have disappeared. In contrast, in normal human fibroblasts (BJ), the proportion of γ-H2AX-positive to γ-H2AX-negative micronuclei remains constant, and the majority of the centromere-negative cells are γ-H2AX-negative. Treatment of both cell lines with colchicine results in mostly centromere-positive, γ-H2AX-negative micronuclei. Immunostaining revealed co-localization of MDC1 and ATM with γ-H2AX foci in both main nuclei and micronuclei; however, other repair proteins, such as Rad50, 53BP1 and Rad17, that co-localized with γ-H2AX foci in the main nuclei were not found in the micronuclei. Combination of the micronucleus assay with γ-H2AX immunostaining provides new insights into the mechanisms of the formation and fate of micronuclei.

Chow, K-H. and Courcelle, J. RecBCD and RecJ/RecQ Initiate DNA Degradation on Distinct Substrates in UV-Irradiated Escherichia coli. Radiat. Res. 168, 499–506 (2007).

After UV irradiation, recA mutants fail to recover replication, and a dramatic and nearly complete degradation of the genomic DNA occurs. Although the RecBCD helicase/nuclease complex is known to mediate this catastrophic DNA degradation, it is not known how or where this degradation is initiated. Previous studies have speculated that RecBCD targets and initiates degradation from the nascent DNA at replication forks arrested by DNA damage. To test this question, we examined which enzymes were responsible for the degradation of genomic DNA and the nascent DNA in UV-irradiated recA cells. We show here that, although RecBCD degrades the genomic DNA after UV irradiation, it does not target the nascent DNA at arrested replication forks. Instead, we observed that the nascent DNA at arrested replication forks in recA cultures is degraded by RecJ/RecQ, similar to what occurs in wild-type cultures. These findings indicate that the genomic DNA degradation and nascent DNA degradation in UV-irradiated recA mutants are mediated separately through RecBCD and RecJ/RecQ, respectively. In addition, they demonstrate that RecBCD initiates degradation at a site(s) other than the arrested replication fork directly.

DeVeaux, L. C., Müller, J. A., Smith, J. R., Petrisko, J., Wells, D. P. and DasSarma, S. Extremely Radiation-Resistant Mutants of a Halophilic Archaeon with Increased Single-Stranded DNA-Binding Protein (RPA) Gene Expression. Radiat. Res. 168, 507–514 (2007).

Extremely halophilic archaea are highly resistant to multiple stressors, including radiation, desiccation and salinity. To study the basis of stress resistance and determine the maximum tolerance to ionizing radiation, we exposed cultures of the model halophile Halobacterium sp. NRC-1 to four cycles of irradiation with high doses of 18–20 MeV electrons. Two independently obtained mutants displayed an LD₅₀ > 11 kGy, which is higher than the LD₅₀ of the extremely radiation-resistant bacterium Deinococcus radiodurans. Whole-genome transcriptome analysis comparing the mutants to the parental wild-type strain revealed up-regulation of an operon containing two single-stranded DNA-binding protein (RPA) genes, VNG2160 (rfa3) and VNG2162, and a third gene of unknown function, VNG2163. The putative transcription start site for the rfa3 operon was mapped ∼40 bp upstream of the ATG start codon, and a classical TATA-box motif was found centered about 25 bp further upstream. We propose that RPA facilitates DNA repair machinery and/or protects repair intermediates to maximize the ionizing radiation resistance of this archaeon.