Hertel-Aas, T., Oughton, D. H., Jaworska, A., Bjerke, H., Salbu, B. and Brunborg, G. Effects of Chronic Gamma Irradiation on Reproduction in the Earthworm Eisenia fetida (Oligochaeta). Radiat. Res. 168, 515–526 (2007).

Eisenia fetida were exposed continuously to ⁶⁰Co γ radiation during two generations (F₀ and F₁). Adult F₀ reproduction capacity (i.e., number of cocoons produced, hatchability and number of F₁ hatchlings) in controls and at five dose rates (0.18, 1.7, 4, 11 and 43 mGy/h) was measured over a 13-week exposure period. Survival, growth and sexual maturation of F₁ hatchlings were observed for 11 weeks. F₁ adults were exposed for a further 13 weeks to determine their reproduction capacity. There was no radiation-induced effect on the cocoon production rate in either F₀ or F₁. For F₀, hatchability of cocoons produced during the first 4 weeks was reduced to 60% at 43 mGy/h (98% in controls), and none of the cocoons produced at 5–13 weeks hatched. At 11 mGy/h the cocoon hatchability was reduced to 25% at 9–13 weeks. In addition, the number of hatchlings per hatched cocoon was reduced at 11 and 43 mGy/h. Correspondingly, at these dose rates, the total number of F₁ hatchlings per adult F₀ was significantly lower than in the control. This number was also reduced at 4 mGy/h, but the effect was of borderline significance. For adult F₁, the hatchability of cocoons at 11 mGy/h was reduced to 45–69% during the 13-week exposure period. The number of hatchlings (F₂) per cocoon and the total number of F₂ individuals produced was also reduced. However, and in contrast to the results observed for F₀, hatchability increased with time, suggesting a possible acclimatization or adaptation of the F₁ individuals. In conclusion, chronic irradiation reduced the reproduction capacity of E. fetida, but extensive exposure periods (13 weeks) were needed for these effects to be expressed. The lowest dose rates at which an effect was observed were 4 mGy/h in F₀ and 11 mGy/h in F₁.

Holt, S. M. and Georgakilas, A. G. Detection of Complex DNA Damage in γ-Irradiated Acute Lymphoblastic Leukemia Pre-B NALM-6 Cells. Radiat. Res. 168, 527–534 (2007).

Bistranded complex DNA damage, i.e., double-strand breaks (DSBs) and non-DSB oxidative clustered DNA lesions, is hypothesized to challenge the repair mechanisms of the cell and consequently the genomic integrity. The oxidative clustered DNA lesions may be persistent and may accumulate in human cancer cells for long times after irradiation. To evaluate the detection and possible accumulation of oxidative clustered DNA lesions in leukemia cells exposed to doses equivalent to those used in radiotherapy, we measured the induction of DSBs and three different types of oxidative clustered DNA lesions in NALM-6 cells, a human acute lymphoblastic leukemia (ALL) pre-B cell line, after exposure to ¹³⁷Cs γ rays. For the detection and measurement of DSBs and oxidative clustered DNA lesions, we used an adaptation of the neutral comet assay (single-cell gel electrophoresis) using E. coli repair enzymes (Endo IV, Fpg and Endo III) as enzymatic probes. We found a linear dose response for the induction of DSBs and oxidative clustered DNA lesions. Clustered DNA lesions were more prevalent than prompt DSBs. For each DSB induced by radiation, ∼2.5 oxidative clustered DNA lesions were detected. To our knowledge, this is the first study to demonstrate the detection and linear induction of oxidative clustered DNA lesions with radiation dose in an ALL cell line. These results point to the biological significance of clustered DNA lesions.

Warters, R. L., Williams, D. L., Zhuplatov, S. B., Pond, C. D. and Leachman, S. A. Protein Phosphorylation in Irradiated Human Melanoma Cells. Radiat. Res. 168, 535–544 (2007).

In the present study, we examined the response of confluent, primary human fibroblasts and cells of a melanoma (YUSAC2) cell line to ionizing radiation mediated through post-translational protein phosphorylation. Since the purpose of our study was to identify novel radiation-induced phosphoproteins in the DNA damage stress response of melanoma cells, we were primarily interested in changes in protein phosphoserine expression at early times after irradiation. Our rationale was that by examining the overall protein phosphorylation profile (the phosphoproteome) in irradiated cells, we might discover novel radiation-induced phosphoproteins that distinguish fibroblasts from melanoma cells. Cell proteins were separated by gel electrophoresis and phosphoproteins were identified by Western blot analysis using nonspecific anti-phosphoamino acid antibodies. This approach was not pursued previously since adequate antibodies for examining global protein phosphoserine expression were unavailable. While some radiation-induced phosphoprotein changes in high-abundance proteins were identified, in general the sensitivity of this approach was not sufficient to detect changes in low-abundance, regulatory proteins. Characterization of these phosphoproteins will require greater enrichment of low-abundance proteins.

Takahashi, K., Monzen, S., Eguchi-Kasai, K., Abe, Y. and Kashiwakura, I. Severe Damage of Human Megakaryocytopoiesis and Thrombopoiesis by Heavy-Ion Beam Radiation. Radiat. Res. 168, 545–551 (2007).

Heavy ions have a unique efficacy for tumor control in radiotherapy. To clarify the effects of heavy-ion beams on hematopoietic stem/progenitor cells, the effects of carbon-ion beams on megakaryocytopoiesis and thrombopoiesis in CD34 cells derived from human placental and umbilical cord blood were investigated. The cells were exposed to carbon-ion beams (LET = 50 keV/μm) and then were treated with thrombopoietin (TPO) alone or TPO plus other cytokines. Megakaryocytic progenitor cells, such as megakaryocyte colony-forming units (CFU-Meg), were far more sensitive to carbon-ion beams than to X rays, and no restoration of carbon-ion beam-irradiated CFU-Meg by treatment with any cytokine combination was observed. However, total cell expansion in liquid culture was not different after either carbon-ion beam or X irradiation of CD34 cells. The activation of γ-H2AX, a marker of DNA double strand-breaks (DSBs), was promoted by the cytokine treatment in X-irradiated CD34 cells but not in carbon-ion-irradiated cells. These results showed that carbon-ion beams inflicted severe damage on megakaryocytopoiesis and thrombopoiesis and that a better combination of cytokines and other agents may be needed to stimulate the recovery of hematopoietic cells and repair this damage.

Epperly, M., Jin, S., Nie, S., Cao, S., Zhang, X., Franicola, D., Wang, H., Fink, M. P. and Greenberger, J. S. Ethyl Pyruvate, a Potentially Effective Mitigator of Damage after Total-Body Irradiation. Radiat. Res. 168, 552–559 (2007).

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to improve survival and ameliorate organ damage in animal models of sepsis, ischemia/reperfusion injury and hemorrhagic shock. Incubating IL3-dependent mouse hematopoietic progenitor cell 32Dcl3 cells before or after irradiation with 10 mM EP increased resistance to radiation as assessed by clonogenic radiation survival curves, decreased release of mitochondrial cytochrome C into the cytoplasm, and decreased apoptosis. EP inhibited radiation-induced caspase 3 activation and poly(ADP-ribose) polymerase (PARP) cleavage in 32Dcl3 cells in a concentration-dependent fashion. EP was given i.p. to C57BL/6NHsd mice irradiated with 9.75 Gy total-body irradiation (TBI). This treatment significantly improved survival. The survival benefit was apparent irrespective of whether treatment with EP was started 1 h before TBI and continued for 5 consecutive days after TBI or the compound was injected only 1 h before or only for 5 days after TBI. In all of the in vitro and in vivo experiments, ethyl lactate, an inactive analogue of EP, had no detectable radioprotective or mitigating effects. EP may be an effective radioprotector and mitigator of the hematopoietic syndrome induced by TBI.

Epperly, M. W., Epperly, L. D., Niu, Y., Wang, H., Zhang, X., Franicola, D. and Greenberger, J. S. Overexpression of the MnSOD Transgene Product Protects Cryopreserved Bone Marrow Hematopoietic Progenitor Cells from Ionizing Radiation. Radiat Res. 168, 560–566 (2007).

We determined whether manganese superoxide dismutase (MnSOD)-plasmid liposome (PL) transfection of C57BL/ 6NHsd mouse bone marrow protected cells irradiated at room temperature (24°C) or in the cryopreserved state. MnSOD-overexpressing hematopoietic progenitor 2C6 cells were radioresistant compared to the parent 32D cl 3 cells when irradiated frozen or at 24°C. Fresh whole marrow from mice injected intravenously with MnSOD-PL prior to explant as well as explanted marrow single cell suspensions transfected in vitro were irradiated at 24°C or −80°C. In vivo or in vitro transfection of marrow with MnSOD-PL produced significant radiation protection of irradiated marrow progenitor cells compared to controls at 24°C or −80°C. (in vivo transfection D₀ 2.19 ± 0.21 at 24°C, D₀ 2.10 ± 0.07 at −80°C compared to control D₀ 1.56 ± 0.06 or 1.66 ± 0.04, P = 0.047 and 0.017 respectively; in vitro transfection D₀ 2.35 ± 0.11 at 24°C, D₀ 3.42 ± 0.13 at −80°C compared to D₀ 1.81 ± 0.01 or 2.53 ± 0.05, P = 0.0087 and 0.0026, respectively). Thus the MnSOD transgene product protects frozen marrow cells as well as marrow cells irradiated at 24°C.

Kleiman, N. J., David, J., Elliston, C. D., Hopkins, K. M., Smilenov, L. B., Brenner, D. J., Worgul, B. V., Hall, E. J. and Lieberman, H. B. Mrad9 and Atm Haploinsufficiency Enhance Spontaneous and X-Ray-Induced Cataractogenesis in Mice. Radiat. Res. 168, 567–573 (2007).

Rad9 and Atm regulate multiple cellular responses to DNA damage, including cell cycle checkpoints, DNA repair and apoptosis. However, the impact of dual heterozygosity for Atm and Rad9 is unknown. Using 50 cGy of X rays as an environmental insult and cataractogenesis as an end point, this study examined the effect of heterozygosity for one or both genes in mice. Posterior subcapsular cataracts, characteristic of radiation exposure, developed earlier in X-irradiated double heterozygotes than in single heterozygotes, which were more prone to cataractogenesis than wild-type controls. Cataract onset time and progression in single or double heterozygotes were accelerated even in unirradiated eyes. These findings indicate that the cataractogenic effect of combined heterozygosity is greater than for each gene alone and are the first to demonstrate the impact of multiple haploinsufficiency on radiation effects in an intact mammal. These observations may help explain observed interindividual differential radiosensitivity in human populations and have important implications for those undergoing radiotherapy or exposed to elevated levels of cosmic radiation, such as the astronaut corps. These findings demonstrate that Mrad9 and Atm are important determinants of lens opacification and, given the roles of Atm and Rad9 in maintaining genomic stability, are consistent with a genotoxic basis for radiation cataractogenesis.

Atwood, T., Payne, V. S., Zhao, W., Brown, W. R., Wheeler, K. T., Zhu, J-M. and Robbins, M. E. Quantitative Magnetic Resonance Spectroscopy Reveals a Potential Relationship between Radiation-Induced Changes in Rat Brain Metabolites and Cognitive Impairment. Radiat. Res. 168, 574–581 (2007).

To test the efficacy of magnetic resonance spectroscopy (MRS) in identifying radiation-induced brain injury, adult male Fischer 344 rats received fractionated whole-brain irradiation (40 or 45 Gy given in 5-Gy fractions twice a week for 4 or 4.5 weeks, respectively); control rats received sham irradiation. Twelve and 52 weeks after whole-brain irradiation, rats were subjected to high-resolution MRI and proton MRS. No apparent lesions or changes in T₁- or T₂-weighted images were noted at either time. This is in agreement with no gross changes being found in histological sections from rats 50 weeks postirradiation. Analysis of the MR spectra obtained 12 weeks after fractionated whole-brain irradiation also failed to show any significant differences (P > 0.1) in the concentration of brain metabolites between the whole-brain-irradiated and sham-irradiated rats. In contrast, analysis of the MR spectra obtained 52 weeks postirradiation revealed significant differences between the irradiated and sham-irradiated rats in the concentrations of several brain metabolites, including increases in the NAA/tCr (P < 0.005) and Glx/tCr (P < 0.001) ratios and a decrease in the mI/tCr ratio (P < 0.01). Although the cognitive function of these rats measured by the object recognition test was not significantly different (P > 0.1) between the irradiated and sham-irradiated rats at 14 weeks postirradiation, it was significantly different (P < 0.02) at 54 weeks postirradiation. These findings suggest that MRS may be a sensitive, noninvasive tool to detect changes in radiation-induced brain metabolites that may be associated with the radiation-induced cognitive impairments observed after prolonged fractionated whole-brain irradiation.

Bijwaard, H. and Dekkers, F. Bone Cancer Risk of ²³⁹Pu in Humans Derived from Animal Models. Radiat. Res. 168, 582– 592 (2007).

Two-mutation model fits to bone cancer mortality data from mice, rats and beagle dogs injected with ²³⁹Pu or ²²⁶Ra show that (1) it is possible to fit the radiation-related parameters for animals from different strains of the same species together; (2) for every species the same significant parameters are found in the models for ²³⁹Pu and in the models for ²²⁶Ra, and the only difference is in the value of the linear mutation coefficient; and (3) the toxicity ratio, when defined as the ratio of the linear mutation coefficients for ²³⁹Pu over ²²⁶Ra, has a relatively uniform value of approximately 8 for the species considered. This relatively constant ratio enables the development of a ²³⁹Pu model for humans that is based on the radium dial painters and the toxicity ratio for beagles. The model predictions agree well with published risk estimates based on other data and derived using alternative approaches. This has two important implications: (1) The two-mutation model appears to be a useful tool in translating from animal models to humans in a meaningful way; and (2) once a two-mutation model for humans has been derived, radiation risks can be calculated that depend on doses, dose rates and ages at exposure. Such a model therefore supplements published risk estimates that often lack such dependences.

Nakashima, E., Akahoshi, M., Neriishi, K. and Fujiwara, S. Systolic Blood Pressure and Systolic Hypertension in Adolescence of Atomic Bomb Survivors Exposed In Utero. Radiat. Res. 168, 593–599 (2007).

Annual medical examinations were conducted during adolescence for the in utero clinical study sample subjects exposed prenatally to the atomic bombs in Hiroshima and Nagasaki. Systolic blood pressure and several anthropometric measurements were recorded during these examinations. For 1014 persons exposed in utero, two types of longitudinal analyses were performed, for a total of 7029 observations (6.93 observations per subject) of systolic blood pressure (continuous data) and systolic hypertension (binary data) for persons aged 9 to 19 years. Body mass index (BMI) and/or body weight were considered in the analyses as potential confounders. For the measurements of systolic blood pressure, the common dose effect was 2.09 mmHg per Gy and was significant (P = 0.017). The dose by trimester interaction was suggestive (P = 0.060). A significant radiation dose effect was found in the second trimester (P = 0.001), with an estimated 4.17 mmHg per Gy, but in the first and third trimesters, radiation dose effects were not significant (P > 0.50). For prevalence of systolic hypertension, the radiation dose effect was significant (P = 0.009); the odds ratio at 1 Gy was 2.23 [95% confidence interval (CI): 1.23, 4.04], and the dose by trimester interaction was not significant (P = 0.778). The dose response of systolic hypertension had no dose threshold, with a threshold point estimate of 0 Gy (95% CI: <0.0, 1.1 Gy). The dose response for systolic blood pressure was most pronounced in the second trimester, the most active organogenesis period for the organs relevant to blood pressure.

Mudie, N. Y., Gusev, B. I., Pivina, L. M., Schoemaker, M. J., Rijinkova, O. N, Apsalikov, K. N. and Swerdlow, A. J. Sex Ratio in the Offspring of Parents with Chronic Radiation Exposure from Nuclear Testing in Kazakhstan. Radiat. Res. 168, 600–607 (2007).

The former Soviet Union conducted a nuclear test program in the Semipalatinsk region of northeastern Kazakhstan in 1949–1989. The population in the vicinity of the test site was chronically exposed to radiation fallout, especially from above-ground tests during 1949–1956. Male:female sex ratio has been proposed as a measure of reproductive health, with some reports suggesting an alteration in the sex ratio of offspring of parents exposed to radiation. We investigated the impact of radiation exposure and other factors on the sex ratio in the population inhabiting the exposed region. A total of 11,464 singleton births of 3,992 mothers exposed to radiation during 1949–1956 were analyzed. The overall sex ratio was 1.07, similar to the current sex ratio in Kazakhstan (1.06). The sex ratio increased from 1.04 where mothers received <20.0 cSv to 1.12 where mothers received ≥60.0 cSv. However, the linear trend across exposures was not significant (P = 0.42). No consistent association was found between the sex ratio and the time since parental radiation exposure, parental age at exposure, or year of birth. Sex ratio was significantly associated with maternal age, birth order and possibly ethnicity but not with paternal age, parental educational level or season. In conclusion, no significant association was found between radiation exposure level and sex ratio, but some previously suggested demographic factors were positively associated with sex ratio.

Parazzini, M., Brazzale, A. R., Paglialonga, A., Tognola, G., Collet, L., Moulin, A., Lutman, M. E., Bell, S. L., Thomas, N. A., Uloziene, I., Uloza, V., Thuroczy, G., Tavartkiladze, G., Tsalighopoulos, M., Kyriafinis, G. and Ravazzani, P. Effects of GSM Cellular Phones on Human Hearing: The European Project “GUARD”. Radiat. Res. 168, 608–613 (2007).

The European multicenter project named GUARD involved nine centers and aimed to assess potential changes in auditory function as a consequence of exposure to low-intensity electromagnetic fields (EMFs) produced by GSM cellular phones. Participants were healthy young adults without any evidence of hearing or ear disorders. Auditory function was assessed immediately before and after exposure to EMFs, and only the exposed ear was tested. The procedure was conducted twice in a double blinded design, once with a genuine EMF exposure and once with a sham exposure (at least 24 h apart). Tests for assessment of auditory function were hearing threshold level (HTL), transient otoacoustic emissions (TEOAE), distortion product otoacoustic emissions (DPOAE), and auditory brainstem response (ABR). The exposure consisted of speech at a typical conversational level delivered via an earphone to one ear, plus genuine or sham EMF exposure. The EMF exposure used the output of a software-controlled consumer cellular phone at full power for 10 min. A system of phone positioning that allowed participants to freely move their heads without affecting exposure was used. Analysis of the data showed there were no effects of exposure to GSM mobile phone signals on the main measures of the status of the auditory system.

Schöllnberger, H., Mitchel, R. E. J., Redpath, J. L., Crawford-Brown, D. J. and Hofmann, W. Detrimental and Protective Bystander Effects: A Model Approach. Radiat. Res. 168, 614–626 (2007).

This work integrates two important cellular responses to low doses, detrimental bystander effects and apoptosis-mediated protective bystander effects, into a multistage model for chromosome aberrations and in vitro neoplastic transformation: the State-Vector Model. The new models were tested on representative data sets that show supralinear or U-shaped dose responses. The original model without the new low-dose features was also tested for consistency with LNT-shaped dose responses. Reductions of in vitro neoplastic transformation frequencies below the spontaneous level have been reported after exposure of cells to low doses of low-LET radiation. In the current study, this protective effect is explained with bystander-induced apoptosis. An important data set that shows a low-dose detrimental bystander effect for chromosome aberrations was successfully fitted by additional terms within the cell initiation stage. It was found that this approach is equivalent to bystander-induced clonal expansion of initiated cells. This study is an important step toward a comprehensive model that contains all essential biological mechanisms that can influence dose–response curves at low doses.

Liu, Z., Prestwich, W. V., Stewart, R. D., Byun, S. H., Mothersill, C. E., McNeill, F. E. and Seymour, C. B. Effective Target Size for the Induction of Bystander Effects in Medium Transfer Experiments. Radiat. Res. 168, 627–630 (2007).

Although radiation-induced bystander effects are frequently observed biological phenomena, the mechanism for these effects has not been fully determined. The target-hit theory and related concepts from microdosimetry provide a convenient formalism to help identify the nature of the targets responsible for initiating the emission of diffusible factors in medium transfer experiments. We used the microdosimetric models proposed by Stewart et al. (Radiat. Res. 165, 460–469, 2006) to analyze the results of published medium transfer experiments for γ-ray doses in the range of 0.04 mGy to 5 Gy. The analysis suggests that the effective size of the target responsible for initiating signal emission in HPV-G human keratinocyte donor cells is approximately 2 μm.

Słonina, D., Biesaga, B., Urbański, K. and Kojs, Z. The Response of Primary Keratinocytes and Fibroblasts from Cancer Patients to Multiple Low-Dose Irradiations. Radiat. Res. 168, 631–636 (2007).

In our previous study, using the micronucleus (MN) assay, a hyper-radiosensitivity (HRS)-like phenomenon was observed after single low doses for fibroblasts from two and keratinocytes from four of the 40 patients studied. In this paper, we report the response of primary keratinocytes from 23 and fibroblasts from 21 of these cancer patients to multiple low-dose irradiations and answer the question regarding whether the patients with an HRS-like response after single low doses also demonstrate chromosomal hypersensitivity after multiple low doses. The cells were irradiated with three doses of 0.25 Gy separated by 4-h intervals, and MN induction was compared with that after the same total dose given as a single fraction of 0.75 Gy. Similarly, the effect of three doses of 0.5 Gy was compared with that of a single dose of 1.5 Gy. For fibroblasts from two and keratinocytes from four patients who demonstrated a single-dose HRS-like response, a significant inverse effect of fractionation (greater MN induction after three doses of 0.25 Gy than after a single dose of 0.75 Gy) was observed, which suggests a repeated hypersensitive response after each dose of 0.25 Gy. Such an effect was not seen for the cells from 19 patients who were single-dose HRS-like negative. In conclusion, an inverse fractionation effect for MN induction that was observed in fibroblasts from two and keratinocytes from four patients after three doses of 0.25 Gy (but not 3 × 0.5 Gy) reflects the chromosomal hyper-radiosensitivity seen in the same patients in response to single low doses.