Preston, D. L., Ron, E., Tokuoka, S., Funamoto, S., Nishi, N., Soda, M., Mabuchi, K. and Kodama, K. Solid Cancer Incidence in Atomic Bomb Survivors: 1958–1998. Radiat. Res. 168, 1–64 (2007).

This is the second general report on radiation effects on the incidence of solid cancers (cancers other than malignancies of the blood or blood-forming organs) among members of the Life Span Study (LSS) cohort of Hiroshima and Nagasaki atomic bomb survivors. The analyses were based on 17,448 first primary cancers (including non-melanoma skin cancer) diagnosed from 1958 through 1998 among 105,427 cohort members with individual dose estimates who were alive and not known to have had cancer prior to 1958. Radiation-associated relative risks and excess rates were considered for all solid cancers as a group, for 19 specific cancer sites or groups of sites, and for five histology groups. Poisson regression methods were used to investigate the magnitude of the radiation-associated risks, the shape of the dose response, how these risks vary with gender, age at exposure, and attained age, and the evidence for inter-site variation in the levels and patterns of the excess risk. For all solid cancers as a group, it was estimated that about 850 (about 11%) of the cases among cohort members with colon doses in excess of 0.005 Gy were associated with atomic bomb radiation exposure. The data were consistent with a linear dose response over the 0- to 2-Gy range, while there was some flattening of the dose response at higher doses. Furthermore, there is a statistically significant dose response when analyses were limited to cohort members with doses of 0.15 Gy or less. The excess risks for all solid cancers as a group and many individual sites exhibit significant variation with gender, attained age, and age at exposure. It was estimated that, at age 70 after exposure at age 30, solid cancer rates increase by about 35% per Gy (90% CI 28%; 43%) for men and 58% per Gy (43%; 69%) for women. For all solid cancers as a group, the excess relative risk (ERR per Gy) decreases by about 17% per decade increase in age at exposure (90% CI 7%; 25%) after allowing for attained-age effects, while the ERR decreased in proportion to attained age to the power 1.65 (90% CI 2.1; 1.2) after allowing for age at exposure. Despite the decline in the ERR with attained age, excess absolute rates appeared to increase throughout the study period, providing further evidence that radiation-associated increases in cancer rates persist throughout life regardless of age at exposure. For all solid cancers as a group, women had somewhat higher excess absolute rates than men (F:M ratio 1.4; 90% CI 1.1; 1.8), but this difference disappears when the analysis was restricted to non-gender-specific cancers. Significant radiation-associated increases in risk were seen for most sites, including oral cavity, esophagus, stomach, colon, liver, lung, non-melanoma skin, breast, ovary, bladder, nervous system and thyroid. Although there was no indication of a statistically significant dose response for cancers of the pancreas, prostate and kidney, the excess relative risks for these sites were also consistent with that for all solid cancers as a group. Dose–response estimates for cancers of the rectum, gallbladder and uterus were not statistically significant, and there were suggestions that the risks for these sites may be lower than those for all solid cancers combined. However, there was emerging evidence from the present data that exposure as a child may increase risks of cancer of the body of the uterus. Elevated risks were seen for all of the five broadly classified histological groups considered, including squamous cell carcinoma, adenocarcinoma, other epithelial cancers, sarcomas and other non-epithelial cancers. Although the data were limited, there was a significant radiation-associated increase in the risk of cancer occurring in adolescence and

Papadopoulou, M. V., Bloomer, W. D., Taylor, A. P., Hernandez, M., Blumenthal, R. D. and Hollingshead, M. G. Advantage of Combining NLCQ-1 (NSC 709257) with Radiation in Treatment of Human Head and Neck Xenografts. Radiat. Res. 168, 65–71 (2007).

NLCQ-1 (NSC 709257), a hypoxia-selective cytotoxin that targets DNA through weak intercalation, was investigated for efficacy in combination with single or fractionated radiotherapy of human head and neck xenografts. A staged tumor experiment was performed in tumor-bearing female athymic nude mice that were locally irradiated with or without NLCQ-1. Tumor hypoxia was assessed by immunohistochemistry for pimonidazole adducts in tumors of varying weight. Fractionated radiation, depending on the dose, was administered either once daily for 4 days or once daily for 4 days followed by a 7-day rest and repeat. NLCQ-1 was administered i.p. at 15 mg/kg alone or 45 min before each radiation dose. Hypoxia (1–52%) was detected in all tumors and was positively correlated with tumor size. NLCQ-1 alone resulted in about 10 days of tumor growth delay, measured at sixfold the tumor's original size, without causing toxicity. All combination treatments with NLCQ-1 were more effective than treatments with radiation alone. Radiation at 1 Gy given once daily for 4 days on days 20 and 30 caused 3.5 days of tumor growth delay, whereas in combination with NLCQ-1 it caused 14.5 days of growth delay. Radiation at 5 Gy given in two doses 10 days apart resulted in 3.5 days of tumor growth delay, whereas more than 20 additional days of delay were observed in combination with NLCQ-1. Radiation given as a single dose of 10 Gy resulted in about 7 days of tumor growth delay, whereas in combination with NLCQ-1 about 30 additional days of delay were seen. These results suggest a significant advantage in combining radiation with NLCQ-1 in treatment of human head and neck tumors, which are known to have hypoxic areas.

Sommer, A. M., Bitz, A. K., Streckert, J., Hansen, V. W. and Lerchl, A. Lymphoma Development in Mice Chronically Exposed to UMTS-Modulated Radiofrequency Electromagnetic Fields. Radiat. Res. 168, 72–80 (2007).

There are public concerns regarding possible carcinogenic or cancer-promoting effects of electromagnetic fields (EMFs) from mobile phones and base stations. The objective of the present study was to investigate whether chronic exposure to EMFs of the UMTS (Universal Mobile Telecommunication System) influences the development of lymphoma in a lymphoma animal model, the AKR/J mouse. Unrestrained mice were chronically sham-exposed (n = 160) or exposed (n = 160) in identical exposure systems (radial waveguides) to a generic UMTS test signal (24 h per day, 7 days per week, 0.4 W/kg SAR). Additionally, 30 animals were kept as cage controls. Animals were checked visually each day and were weighed and palpated weekly to detect swollen lymph nodes. Starting at the age of 6 months, blood samples were taken from the tail every 2 weeks to perform differential leukocyte counts and to measure the hematocrit. Visibly diseased animals or those older than 43 weeks were killed humanely, and tissue slices were examined for metastatic infiltrations and lymphoma type. The study was performed in a blinded way. Cage control animals had a significantly lower growth rate than those kept in the radial waveguides. The number of ill animals, the mean survival time, and the severity code of the disease did not differ between the experimental groups. Therefore, the data show no negative effects from exposure and corroborate earlier findings in AKR/J mice exposed to GSM EMF (Sommer et al., BMC Cancer 4, 77–90, 2004).

Igari, Y., Igari, K., Kunugita, N., Ootsuyama, A. and Norimura, T. Prolonged Increase in T-Cell Receptor (TCR) Variant Fractions of Spleen T Lymphocytes in Pregnant Mice after γ Irradiation. Radiat. Res. 168, 81–86 (2007).

To investigate the relationship between the radiation-induced increase of T-cell receptor (TCR) defective variant fractions and physiological status such as pregnancy, C57BL/ 6N mice were irradiated with 3 Gy of γ rays at various days of gestation, just before and just after pregnancy. While the highest level of variant fractions in spleen T lymphocytes appeared at 9 days postirradiation and resolved fairly rapidly for nonpregnant mice, the increased variant fractions for pregnant mice irradiated at 16.5 days of gestation reached a plateau at 14 days postirradiation and remained at high levels until 28 days after irradiation. Therefore, variant fractions 28 days postirradiation were measured to determine the overall effect of radiation on the kinetics of TCR variant fractions during gestation. There was no significant difference in the baseline TCR variant fraction between unirradiated nonpregnant and pregnant mice. TCR variant fractions after irradiation were about twofold higher in pregnant mice (from 10.5 days of gestation until delivery) than those in nonpregnant mice. Both γ radiation and pregnancy caused a decrease in the proportion of naïve T-cell subsets and an increase in TCR variant fractions of naïve T cells. In addition, the prolonged postirradiation increase in the TCR variant fractions of pregnant mice was associated with an increase in serum progesterone level. Differences between pregnant and nonpregnant mice in the kinetics of postirradiation restoration of T-cell systems may be involved in producing the differences in residual TCR variant fractions of these mice.

Tsao, D., Kalogerinis, P., Tabrizi, I., Dingfelder, M., Stewart, R. D. and Georgakilas, A. G. Induction and Processing of Oxidative Clustered DNA Lesions in ⁵⁶Fe-Ion-Irradiated Human Monocytes. Radiat. Res. 168, 87–97 (2007).

Space and cosmic radiation is characterized by energetic heavy ions of high linear energy transfer (LET). Although both low- and high-LET radiations can create oxidative clustered DNA lesions and double-strand breaks (DSBs), the local complexity of oxidative clustered DNA lesions tends to increase with increasing LET. We irradiated 28SC human monocytes with doses from 0–10 Gy of ⁵⁶Fe ions (1.046 GeV/ nucleon, LET = 148 keV/μm) and determined the induction and processing of prompt DSBs and oxidative clustered DNA lesions using pulsed-field gel electrophoresis (PFGE) and Number Average Length Analysis (NALA). The ⁵⁶Fe ions produced decreased yields of DSBs (10.9 DSB Gy⁻¹ Gbp⁻¹) and clusters (1 DSB:∼0.8 Fpg clusters:∼0.7 Endo III clusters: ∼0.5 Endo IV clusters) compared to previous results with ¹³⁷Cs γ rays. The difference in the relative biological effectiveness (RBE) of the measured and predicted DSB yields may be due to the formation of spatially correlated DSBs (regionally multiply damaged sites) which result in small DNA fragments that are difficult to detect with the PFGE assay. The processing data suggest enhanced difficulty compared with γ rays in the processing of DSBs but not clusters. At the same time, apoptosis is increased compared to that seen with γ rays. The enhanced levels of apoptosis observed after exposure to ⁵⁶Fe ions may be due to the elimination of cells carrying high levels of persistent DNA clusters that are removed only by cell death and/or “splitting” during DNA replication.

Hada, M., Cucinotta, F. A., Gonda, S. R. and Wu, H. mBAND Analysis of Chromosomal Aberrations in Human Epithelial Cells Exposed to Low- and High-LET Radiation. Radiat. Res. 168, 98–105 (2007).

Energetic heavy ions pose a potential health risk to astronauts who have participated in extended space missions. High-LET radiation is much more effective than low-LET radiation in the induction of biological effects, including cell inactivation, genetic mutations, cataracts and cancer. Most of these biological end points are closely correlated with chromosomal damage, which can be used as a biomarker for radiation damage. Multicolor banding in situ hybridization (mBAND) has proven to be highly useful for the study of intrachromosomal aberrations, which have been suggested as a biomarker of exposure to high-LET radiation. To investigate biological signatures of radiation quality and the complexity of intrachromosomal aberrations, we exposed human epithelial cells in vitro to ¹³⁷Cs γ rays or iron ions (600 MeV/nucleon) and collected chromosomes using a premature chromosome condensation technique. Aberrations in chromosome 3 were analyzed using mBAND probes. The results of our study confirmed the observation of a higher incidence of inversions for high-LET radiation. However, detailed analysis of the inversion type revealed that both iron ions and γ rays induced a low incidence of simple inversions. Half of the inversions observed in the low-LET-irradiated samples were accompanied by other types of intrachromosome aberrations, but few inversions were accompanied by interchromosome aberrations. In contrast, iron ions induced a significant fraction of inversions that involved complex rearrangements of both inter- and intrachromosome exchanges.

Kataoka, Y., Murley, J. S., Baker, K. L. and Grdina, D. J. Relationship between Phosphorylated Histone H2AX Formation and Cell Survival in Human Microvascular Endothelial Cells (HMEC) as a Function of Ionizing Radiation Exposure in the Presence or Absence of Thiol-Containing Drugs. Radiat. Res. 168, 106–114 (2007).

Human microvascular endothelial cells (HMEC) were exposed to ionizing radiation at doses ranging from 0 to 16 Gy in either the presence or absence of the active thiol forms of amifostine (WR1065), phosphonol (WR255591), N-acetyl-l-cysteine (NAC), captopril or mesna. Each of these clinically relevant thiols, administered to HMEC at a dose of 4 mM for 30 min prior to irradiation, is known to exhibit antioxidant properties. The purpose of this investigation was to determine the relationship(s), if any, between the frequency of radiation-induced histone H2AX phosphorylation at serine 139 (γ-H2AX) in cells and subsequent survival, as assessed by colony-forming ability, in exposed cell populations as a function of the presence or absence of each of the five thiol compounds during irradiation. γ-H2AX formation in irradiated cells, as a function of relative DNA content, was quantified by bivariant flow cytometry analysis with FITC-conjugated γ-H2AX antibody and nuclear DAPI staining. γ-H2AX formation in cells was measured as the relative fold increase as a function of the treatment conditions. The frequency of γ-H2AX-positive cells increased with increasing dose of radiation followed by a dose- and time-dependent decay. The most robust response for γ-H2AX formation occurred 1 h after irradiation with their relative frequencies decreasing as a function of time 4 and 24 h later. To assess the effects of the various thiols on γ-H2AX formation, all measurements were made 1 h after irradiation. WR1065 was not only effective in protecting HMEC against γ-H2AX formation across the entire dose range of radiation exposures used, but it was also significantly more cytoprotective than either its prodrug (WR2721) or disulfide (WR33278) analogue. WR1065 had no significant effect on γ-H2AX formation when administered immediately or up to 30 min after radiation exposure. An inhibitory effect against γ-H2AX formation induced by 8 Gy of radiation was expressed by each of the thiols tested. NAC, captopril and mesna were equally effective in reducing the frequency of γ-H2AX formation, with both WR1065 and WR255591 exhibiting a slightly more robust protective effect. Each of the five thiols was effective in reducing the frequency of γ-H2AX-positive cells across all phases of the cell cycle. In contrast to the relative ability of each of these thiols to inhibit γ-H2AX formation after irradiation, NAC, captopril and mesna afforded no protection to HMEC as determined using a colony-forming survival assay. Only WR1065 and WR255591 were effective in reducing the frequencies of radiation-induced γ-H2AX-positive cells as well as protecting against cell death. These results suggest that the use of γ-H2AX as a biomarker for screening the efficacy of novel antioxidant radioprotective compounds is highly problematic since their formation and disappearance may be linked to processes beyond simply the formation and repair of radiation-induced DSBs.

Zhang, Y., Carr, T., Dimtchev, A., Zaer, N., Dritschilo, A. and Jung, M. Attenuated DNA Damage Repair by Trichostatin A through BRCA1 Suppression. Radiat. Res. 168, 115–124 (2007).

Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and γ-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D₀ = 1.63 Gy), to the control level (D₀ = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.

Ning, S., Chen, Z., Dirks, A., Husbeck, B., Hsu, M., Bedogni, B., O'Neill, M., Powell, M. B. and Knox, S. J. Targeting Integrins and PI3K/Akt-Mediated Signal Transduction Pathways Enhances Radiation-Induced Anti-angiogenesis. Radiat. Res. 168, 125–133 (2007).

The integrins and PI3K/Akt are important mediators of the signal transduction pathways involved in tumor angiogenesis and cell survival after exposure to ionizing radiation. Selective targeting of either integrins or PI3K/Akt can radiosensitize tumors. In this study, we tested the hypothesis that the combined inhibition of integrin ανβ3 by cRGD and PI3K/Akt by LY294002 would significantly enhance radiation-induced inhibition of angiogenesis by vascular endothelial cells. Treatment with cRGD inhibited the adhesion and tube formation of human umbilical vein endothelial cells (HUVECs). The inhibitory effect was further increased when cRGD and LY294002 were applied simultaneously. Both radiation and cRGD induced Akt phosphorylation, up-regulated COX2 expression, and increased PGE2 production in HUVECs. Treatment with LY294002 effectively inhibited radiation- and cRGD-induced Akt phosphorylation and up-regulation of COX2 and increased apoptosis of HUVECs. The combined use of cRGD and LY294002 enhanced radiation-induced cell killing. The clonogenic survival of HUVECs was decreased from 34% with 2 Gy radiation to 4% with these agents combined. These results demonstrate that combined use of ionizing radiation, cRGD and LY294002 inhibited multiple signaling transduction pathways involved in tumor angiogenesis and enhanced radiation-induced effects on vascular endothelial cells.

Nugent, S. M. E., Mothersill, C. E., Seymour, C., McClean, B., Lyng, F. M. and Murphy, J. E. J. Increased Mitochondrial Mass in Cells with Functionally Compromised Mitochondria after Exposure to both Direct γ Radiation and Bystander Factors. Radiat. Res. 168, 134–142 (2007).

The bystander effect describes radiation-like damage in unirradiated cells either in the vicinity of irradiated cells or exposed to medium from irradiated cells. This study aimed to further characterize the poorly understood mitochondrial response to both direct irradiation and bystander factor(s) in human keratinocytes (HPV-G) and Chinese hamster ovarian cells (CHO-K1). Oxygen consumption rates were determined during periods of state 4, state 3 and uncoupled respiration. Mitochondrial mass was determined using MitoTracker FM. CHO-K1 cells showed significantly reduced oxygen consumption rates 4 h after exposure to 5 Gy direct radiation and irradiated cell conditioned medium (ICCM) and an apparent recovery 12–24 h later. The apparent recovery was likely due to the substantial increase in mitochondrial mass observed in these cells as soon as 4 h after exposure. HPV-G cells, on the other hand, showed a sustained increase in oxygen consumption rates after ICCM exposure and a transient increase 4 h after exposure to 5 Gy direct radiation. A significant increase in mitochondrial mass per HPV-G cell was observed after exposure to both direct radiation and ICCM. These findings are indicative of a stress response to mitochondrial dysfunction that increases the number of mitochondria per cell.