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issn:0301-634
1.  Microbeam Irradiation of C. elegans Nematode in Microfluidic Channels 
Radiation and environmental biophysics  2013;52(4):10.1007/s00411-013-0485-6.
To perform high-throughput studies on the biological effects of ionizing radiation in vivo, we have implemented a microfluidic tool for microbeam irradiation of Caenorhabditis elegans. The device allows the immobilization of worms with minimal stress for a rapid and controlled microbeam irradiation of multiple samples in parallel. Adapted from an established design, our microfluidic clamp consists of 16 tapered channels with 10-μm thin bottoms to ensure charged particle traversal. Worms are introduced into the microfluidic device through liquid flow between an inlet and an outlet and the size of each microchannel guarantees that young adult worms are immobilized within minutes without the use of anesthesia. After site-specific irradiation with the microbeam, the worms can be released by reversing the flow direction in the clamp and collected for analysis of biological endpoints such as repair of radiation-induced DNA damage. For such studies, minimal sample manipulation and reduced use of drugs such as anesthetics that might interfere with normal physiological processes are preferable.
By using our microfluidic device that allows simultaneous immobilization and imaging for irradiation of several whole living samples on a single clamp, here we show that 4.5 MeV proton microbeam irradiation induced DNA damage in wild type C.elegans, as assessed by the formation of Rad-51 foci that are essential for homologous repair of radiation-induced DNA damage.
doi:10.1007/s00411-013-0485-6
PMCID: PMC3809145  PMID: 23942865
Microbeam irradiation with microfluidic devices; C. elegans microbeam irradiation; Small animal microbeam irradiation; Rad-51 foci in C. elegans
2.  Relative Biological Effectiveness of 12C and 28Si radiation in C57BL/6J mice 
Study of heavy ion radiation–induced effects on mice could provide insight into the human health risks of space radiation exposure. The purpose of the present study is to assess the relative biological effectiveness (RBE) of 12C and 28Si ion radiation, which has not been reported previously in the literature. Female C57BL/6J mice (n=15) were irradiated using 4 to 8 Gy of 28Si (300MeV/nucleon energy; LET 70 keV/μm) and 5 to 8 Gy of 12C (290MeV/nucleon energy; LET 13 keV/μm) ions. Post-exposure, mice were monitored regularly and their survival observed for 30 days. The LD50/30 dose (the dose at which 50% lethality occurred by 30-days post-exposure) was calculated from the survival curve and was used to determine the RBE of 28Si and 12C in relation to γ radiation. The LD50/30 for 28Si and 12C ion is 5.17 Gy and 7.34 Gy respectively and the RBE in relation to γ radiation (LD50/30 – 7.25 Gy) is 1.4 for 28Si and 0.99 for 12C. Determination of RBE of 28Si and 12C for survival in mice is not only important for space radiation risk estimate studies, but also has implications for HZE radiation in cancer therapy.
doi:10.1007/s00411-012-0418-9
PMCID: PMC4208103  PMID: 22562428
Relative biological effectiveness; RBE; Heavy ion charged particles; Space radiation; linear energy transfer; 12C-ion; 28Si-ion
3.  NUNDO: a numerical model of a human torso phantom and its application to effective dose equivalent calculations for astronauts at the ISS 
The health effects of cosmic radiation on astronauts need to be precisely quantified and controlled. This task is important not only in perspective of the increasing human presence at the International Space Station (ISS), but also for the preparation of safe human missions beyond low earth orbit. From a radiation protection point of view, the baseline quantity for radiation risk assessment in space is the effective dose equivalent. The present work reports the first successful attempt of the experimental determination of the effective dose equivalent in space, both for extra-vehicular activity (EVA) and intra-vehicular activity (IVA). This was achieved using the anthropomorphic torso phantom RANDO® equipped with more than 6,000 passive thermoluminescent detectors and plastic nuclear track detectors, which have been exposed to cosmic radiation inside the European Space Agency MATROSHKA facility both outside and inside the ISS. In order to calculate the effective dose equivalent, a numerical model of the RANDO® phantom, based on computer tomography scans of the actual phantom, was developed. It was found that the effective dose equivalent rate during an EVA approaches 700 μSv/d, while during an IVA about 20 % lower values were observed. It is shown that the individual dose based on a personal dosimeter reading for an astronaut during IVA results in an overestimate of the effective dose equivalent of about 15 %, whereas under an EVA conditions the overestimate is more than 200 %. A personal dosemeter can therefore deliver quite good exposure records during IVA, but may overestimate the effective dose equivalent received during an EVA considerably.
doi:10.1007/s00411-014-0560-7
PMCID: PMC4206298  PMID: 25119442
Effective dose equivalent; International Space Station; Space radiation environment; Space dosimetry; Phantom experiments
4.  UV Microspot Irradiator at Columbia University 
The Radiological Research Accelerator Facility (RARAF) at Columbia University has recently added a UV microspot irradiator to a microbeam irradiation platform. This UV microspot irradiator applies multiphoton excitation at the focal point of an incident laser as the source for cell damage, and with this approach, a single cell within a 3D sample can be targeted and exposed to damaging UV. The UV microspot’s ability to impart cellular damage within 3D is an advantage over all other microbeam techniques, which instead impart damage to numerous cells along microbeam tracks. This short communication is an overview and a description of the UV microspot including the following applications and demonstrations of selective damage to live single cell targets: DNA damage foci formation, patterned irradiation, photoactivation, targeting of mitochondria, and targeting of individual cardiomyocytes in the live zebrafish embryo.
doi:10.1007/s00411-013-0474-9
PMCID: PMC3723145  PMID: 23708525
DNA damage; Microbeams; UV
5.  Relative biological effectiveness of the 60-MeV therapeutic proton beam at the Institute of Nuclear Physics (IFJ PAN) in Kraków, Poland 
Abstract
The aim of the study was to determine the relative biological effectiveness (RBE) of a 60-MeV proton radiotherapy beam at the Institute of Nuclear Physics, Polish Academy of Sciences (IFJ PAN) in Kraków, the first one to operate in Poland. RBE was assessed at the surviving fractions (SFs) of 0.01, 0.1, and 0.37, for normal human fibroblasts from three cancer patients. The cells were irradiated near the Bragg peak of the pristine beam and at three depths within a 28.4-mm spread-out Bragg peak (SOBP). Reference radiation was provided by 6-MV X-rays. The mean RBE value at SF = 0.01 for fibroblasts irradiated near the Bragg peak of pristine beam ranged between 1.06 and 1.15. The mean RBE values at SF = 0.01 for these cells exposed at depths of 2, 15, and 27 mm of the SOBP ranged between 0.95–1.00, 0.97–1.02, and 1.05–1.11, respectively. A trend was observed for RBE values to increase with survival level and with depth in the SOBP: at SF = 0.37 and at the depth of 27 mm, RBE values attained their maximum (1.19–1.24). The RBE values estimated at SF = 0.01 using normal human fibroblasts for the 60-MeV proton radiotherapy beam at the IFJ PAN in Kraków are close to values of 1.0 and 1.1, used in clinical practice.
doi:10.1007/s00411-014-0559-0
PMCID: PMC4220900  PMID: 25037857
Proton beam; Spread-out Bragg peak; RBE; Human fibroblasts
6.  A Review of Non-Cancer Effects, Especially Circulatory and Ocular Diseases1 
There is a well-established association between high doses (> 5 Gy) of ionizing radiation exposure and damage to the heart and coronary arteries, although only recently have studies with high quality individual dosimetry been conducted that would enable quantification of this risk adjusting for concomitant chemotherapy. The association between lower dose exposures and late occurring circulatory disease has only recently begun to emerge in the Japanese atomic bomb survivors and in various occupationally-exposed cohorts, and is still controversial. Excess relative risks per unit dose in moderate and low dose epidemiological studies are somewhat variable, possibly a result of confounding and effect modification by well known (but unobserved) risk factors.
Radiation doses of 1 Gy or more are associated with increased risk of posterior subcapsular cataract. Accumulating evidence from the Japanese atomic bomb survivors, Chernobyl liquidators, US astronauts and various other exposed groups suggest that cortical cataracts may also be associated with ionizing radiation, although there is little evidence that nuclear cataracts are radiogenic. The dose response appears to be linear, although modest thresholds (of no more than about 0.6 Gy) cannot be ruled out.
A variety of other non-malignant effects have been observed after moderate/low dose exposure in various groups, in particular respiratory and digestive disease and central nervous system (and in particular neuro-cognitive) damage. However, because these are generally only observed in isolated groups, or because the evidence is excessively heterogeneous, these associations must be treated with caution.
doi:10.1007/s00411-013-0484-7
PMCID: PMC4074546  PMID: 23903347
circulatory disease; radiation; heart disease; stroke; cataract; central nervous system; review
7.  Cancer incidence in northern Sweden before and after the Chernobyl nuclear power plant accident 
Sweden received about 5 % of the total release of 137Cs from the Chernobyl nuclear power plant accident in 1986. The distribution of the fallout mainly affected northern Sweden, where some parts of the population could have received an estimated annual effective dose of 1–2 mSv per year. It is disputed whether an increased incidence of cancer can be detected in epidemiological studies after the Chernobyl nuclear power plant accident outside the former Union of Soviet Socialist Republics. In the present paper, a possible exposure–response pattern between deposition of 137Cs and cancer incidence after the Chernobyl nuclear power plant accident was investigated in the nine northernmost counties of Sweden (2.2 million inhabitants in 1986). The activity of 137Cs from the fallout maps at 1986 was used as a proxy for the received dose of ionizing radiation. Diagnoses of cancer (ICD-7 code 140-209) from 1980 to 2009 were received from the Swedish Cancer Registry (273,222 cases). Age-adjusted incidence rate ratios, stratified by gender, were calculated with Poisson regression in two closed cohorts of the population in the nine counties 1980 and 1986, respectively. The follow-up periods were 1980–1985 and 1986–2009, respectively. The average surface-weighted deposition of 137Cs at three geographical levels; county (n = 9), municipality (n = 95) and parish level (n = 612) was applied for the two cohorts to study the pre- and the post-Chernobyl periods separately. To analyze time trends, the age-standardized total cancer incidence was calculated for the general Swedish population and the population in the nine counties. Joinpoint regression was used to compare the average annual percent change in the general population and the study population within each gender. No obvious exposure–response pattern was seen in the age-adjusted total cancer incidence rate ratios. A spurious association between fallout and cancer incidence was present, where areas with the lowest incidence of cancer before the accident coincidentally had the lowest fallout of 137Cs. Increasing the geographical resolution of exposure from nine county averages to 612 parish averages resulted in a two to three times higher value of variance in the regression model. There was a secular trend with an increase in age-standardized incidence of cancer in both genders from 1980 to 2009, but significant only in females. This trend was stronger and statistically significant for both genders in the general Swedish population compared to the nine counties. In conclusion, using both high quality cancer registry data and high resolution exposure maps of 137Cs deposition, it was not possible to distinguish an effect of 137Cs on cancer incidence after the Chernobyl nuclear power plant accident in Sweden.
doi:10.1007/s00411-014-0545-6
PMCID: PMC4102770  PMID: 24811728
Cancer; Cesium-137; Chernobyl; Ecological study; Environment; Epidemiology; Ionizing radiation; Nuclear accident; Radiation; Sweden
8.  Electron paramagnetic resonance measurements of absorbed dose in teeth from citizens of Ozyorsk 
In 1945, within the frame of the Uranium Project for the production of nuclear weapons, the Mayak nuclear facilities were constructed at the Lake Irtyash in the Southern Urals, Russia. The nuclear workers of the Mayak Production Association (MPA), who lived in the city of Ozyorsk, are the focus of epidemiological studies for the assessment of health risks due to protracted exposure to ionising radiation. Electron paramagnetic resonance measurements of absorbed dose in tooth enamel have already been used in the past, in an effort to validate occupational external doses that were evaluated in the Mayak Worker Dosimetry System. In the present study, 229 teeth of Ozyorsk citizens not employed at MPA were investigated for the assessment of external background exposure in Ozyorsk. The annually absorbed dose in tooth enamel from natural background radiation was estimated to be (0.7 ± 0.3) mGy. For citizens living in Ozyorsk during the time of routine noble gas releases of the MPA, which peaked in 1953, the average excess absorbed dose in enamel above natural background was (36 ± 29) mGy, which is consistent with the gamma dose obtained by model calculations. In addition, there were indications of possible accidental gaseous MPA releases that affected the population of Ozyorsk, during the early and late MPA operation periods, before 1951 and after 1960.
doi:10.1007/s00411-014-0527-8
PMCID: PMC3996272  PMID: 24604722
Dose reconstruction; Mayak Production Association; Background radiation; Radioactive noble gas isotopes; EPR; Tooth enamel
9.  State of the art in nail dosimetry: free radicals identification and reaction mechanisms 
Until very recently, analysis of bone biopsies by means of the method of electron paramagnetic resonance (EPR) collected after surgery or amputation has been considered as the sole reliable method for radiation dose assessment in hands and feet. EPR measurements in finger- and toenail have been considered for accident dosimetry for a long time. Human nails are very attractive biophysical materials because they are easy to collect and pertinent to whole body irradiation. Information on the existence of a radiation-induced signal in human nails has been reported almost 25 years ago. However, no practical application of EPR dosimetry on nails is known to date because, from an EPR perspective, nails represent a very complex material. In addition to the radiation-induced signal (RIS), parasitic and intense signals are induced by the mechanical stress caused when collecting nail samples (mechanically induced signals—MIS). Moreover, it has been demonstrated that the RIS stability is strongly influenced not only by temperature but also by humidity. Most studies of human nails were carried out using conventional X-band microwave band (9 GHz). Higher frequency Q-band (37 GHz) provides higher spectral resolution which allows obtaining more detailed information on the nature of different radicals in human nails. Here, we present for the first time a complete description of the different EPR signals identified in nails including parasitic, intrinsic and RIS. EPR in both X- and Q-bands was used. Four different MIS signals and five different signals specific to irradiation with ionizing radiation have been identified. The most important outcome of this work is the identification of a stable RIS component. In contrast with other identified (unstable) RIS components, this component is thermally and time stable and not affected by the physical contact of fingernails with water. A detailed description of this signal is provided here. The discovery of stable radiation-induced radical(s) associated with the RIS component mentioned opens a way for broad application of EPR dosimetry in human nails. Consequently, several recent dosimetry assessments of real accident cases have been performed based on the described measurements and analyses of this component.
doi:10.1007/s00411-014-0512-2
PMCID: PMC3996284  PMID: 24469226
EPR spectroscopy; Dosimetry; Human nails; Radiological accident; Q- and X-bands
10.  Risk of second bone sarcoma following childhood cancer: role of radiation therapy treatment 
Bone sarcoma as a second malignancy is rare but highly fatal. The present knowledge about radiation-absorbed organ dose–response is insufficient to predict the risks induced by radiation therapy techniques. The objective of the present study was to assess the treatment-induced risk for bone sarcoma following a childhood cancer and particularly the related risk of radiotherapy. Therefore, a retrospective cohort of 4,171 survivors of a solid childhood cancer treated between 1942 and 1986 in France and Britain has been followed prospectively. We collected detailed information on treatments received during childhood cancer. Additionally, an innovative methodology has been developed to evaluate the dose–response relationship between bone sarcoma and radiation dose throughout this cohort. The median follow-up was 26 years, and 39 patients had developed bone sarcoma. It was found that the overall incidence was 45-fold higher [standardized incidence ratio 44.8, 95 % confidence interval (CI) 31.0–59.8] than expected from the general population, and the absolute excess risk was 35.1 per 100,000 person-years (95 % CI 24.0–47.1). The risk of bone sarcoma increased slowly up to a cumulative radiation organ absorbed dose of 15 Gy [hazard ratio (HR) = 8.2, 95 % CI 1.6–42.9] and then strongly increased for higher radiation doses (HR for 30 Gy or more 117.9, 95 % CI 36.5–380.6), compared with patients not treated with radiotherapy. A linear model with an excess relative risk per Gy of 1.77 (95 % CI 0.6213–5.935) provided a close fit to the data. These findings have important therapeutic implications: Lowering the radiation dose to the bones should reduce the incidence of secondary bone sarcomas. Other therapeutic solutions should be preferred to radiotherapy in bone sarcoma-sensitive areas.
doi:10.1007/s00411-013-0510-9
PMCID: PMC3996275  PMID: 24419490
Bone sarcoma; Childhood cancer; Iatrogenous effects; Radiation therapy; Secondary tumor
11.  Ultrasonography survey and thyroid cancer in the Fukushima Prefecture 
Thyroid cancer is one of the major health concerns after the accident in the Fukushima Dai-ichi nuclear power station (NPS). Currently, ultrasonography surveys are being performed for persons residing in the Fukushima Prefecture at the time of the accident with an age of up to 18 years. Here, the expected thyroid cancer prevalence in the Fukushima Prefecture is assessed based on an ultrasonography survey of Ukrainians, who were exposed at an age of up to 18 years to 131I released during the Chernobyl NPS accident, and on differences in equipment and study protocol in the two surveys. Radiation risk of thyroid cancer incidence among survivors of the atomic bombings of Hiroshima and Nagasaki and preliminary estimates of thyroid dose due to the Fukushima accident were used for the prediction of baseline and radiation-related thyroid cancer risks. We estimate a prevalence of thyroid cancer of 0.027 % (95 % CI 0.010 %; 0.050 %) for the first screening campaign in the Fukushima Prefecture. Compared with the incidence rate in Japan in 2007, the ultrasonography survey is predicted to increase baseline thyroid cancer incidence by a factor of 7.4 (95 % CI 0.95; 17.3). Under the condition of continued screening, thyroid cancer during the first fifty years after the accident is predicted to be detected for about 2 % of the screened population. The prediction of radiation-related thyroid cancer in the most exposed fraction (a few ten thousand persons) of the screened population of the Fukushima Prefecture has a large uncertainty with the best estimates of the average risk of 0.1–0.3 %, depending on average dose.
Electronic supplementary material
The online version of this article (doi:10.1007/s00411-013-0508-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s00411-013-0508-3
PMCID: PMC3996282  PMID: 24398917
Fukushima; Radiation; Thyroid cancer; Ultrasonography
12.  Karyopathological Traits of Thyrocytes and Exposure to Radioiodines in Belarusian Children and Adolescents following the Accident at the Chernobyl Nuclear Power Plant 
Radiation and environmental biophysics  2012;51(2):10.1007/s00411-012-0407-z.
The Belarus-American (BelAm) Thyroid Study cohort consists of persons 0–18 years of age at the time of exposure to radioiodines from the 1986 Chernobyl nuclear power plant accident who have undergone serial thyroid screenings with referral for fine-needle aspiration biopsy (FNAB) using standardized criteria. We investigated thyrocyte nuclear abnormalities in cytological samples from FNABs in 50 BelAm subjects with thyroid nodules and 43 control patients from Leningrad, Russia, unexposed to Chernobyl fallout. Nuclear abnormalities such as internuclear chromosome bridges and derivative nuclei with broken bridges (i.e., “tailed” nuclei), formed from dicentric and ring chromosomes, may be cellular markers of radiation exposure. In the exposed BelAm cohort, thyrocytes with bridges were present in 80% of subjects with single-nodular goiters compared with 27% of unexposed controls. The average frequency of thyrocytes with bridges was also significantly higher in the BelAm subjects than in controls as was the mean frequency of thyrocytes with tailed nuclei. In the case of multi-nodular goiters, thyrocytes with bridges were present in 75.0% of exposed BelAm patients compared with 16.7% of unexposed controls. Thyrocytes with tailed nuclei were observed in all of the BelAm subjects but in only 35% of controls, and the mean frequency of tailed nuclei was significantly higher. Unusually long bridges were detected in 29% of BelAm patients with single-nodular goiters and 35% of cases with multi-nodular goiters, while no such abnormalities were observed in the follicular thyroid epithelium of patients from the Leningrad region. Further study is needed to understand whether these phenomena represent irradiation consequences in the human organism.
doi:10.1007/s00411-012-0407-z
PMCID: PMC3857023  PMID: 22382464
thyroid gland; thyrocytes; nuclear abnormalities; Chernobyl accident
13.  Behavior and food consumption pattern of the population exposed in 1949–1962 to fallout from Semipalatinsk nuclear test site in Kazakhstan 
Radiation and environmental biophysics  2010;50(1):10.1007/s00411-010-0334-9.
The relationship between radiation exposure from nuclear weapons testing fallout and thyroid disease in a group of 2,994 subjects has been the subject of study by the U.S. National Cancer Institute. In that study, radiation doses to the thyroid were estimated for residents of villages in Kazakhstan possibly exposed to deposition of radioactive fallout from nuclear testing conducted by the Soviet Union at the Semipalatinsk Nuclear Test Site in Kazakhstan between 1949 and 1962. The study subjects included individuals of both Kazakh and Russian origin who were exposed during childhood and adolescence. An initial dose reconstruction used for the risk analysis of Land et al. (2008) was based on individual information collected from basic questionnaires administered to the study population in 1998. However, because data on several key questions for accurately estimating doses was not obtained from the 1998 questionnaires it was decided to conduct a second data collection campaign in 2007. Due to the many years elapsed since exposure, a well developed strategy was necessary to encourage accurate memory recall. In our recent study, a focus group interview data collection methodology was used to collect historical behavioral and food consumption data. The data collection in 2007 involved interviews conducted within four eight-person focus groups (three groups of women and one group of men) in each of four exposed villages where thyroid disease screening was conducted in 1998. Population-based data on relevant childhood behaviors, including time spent in- and outdoors and consumption rates of milk and other dairy products were collected from women’s groups. The data were collected for five age groups of children and adolescents ranging from less than 1 year of age to 21 years of age. Dairy products considered included fresh milk and other products from cows, goats, mares, and sheep. Men’s focus group interviews pertained to construction materials of houses and schools, and animal grazing patterns and feeding practices. The response data collected are useful for improving estimates of thyroid radiation dose estimates for the subjects of an ongoing epidemiological study.
doi:10.1007/s00411-010-0334-9
PMCID: PMC3853382  PMID: 20938673
14.  Modeling hematopoietic system response caused by chronic exposure to ionizing radiation 
Radiation and environmental biophysics  2011;50(2):10.1007/s00411-011-0351-3.
A new model of the hematopoietic system response in humans chronically exposed to ionizing radiation describes the dynamics of the hematopoietic stem cell compartment as well as the dynamics of each of the four blood cell types (lymphocytes, neutrophiles, erythrocytes, and platelets). The required model parameters were estimated based on available results of human and experimental animal studies. They include the steady-state number of hematopoietic stem cells and peripheral blood cell lines in an unexposed organism, amplification parameters for each blood line, parameters describing proliferation and apoptosis, parameters of feedback functions regulating the steady-state numbers, and characteristics of radiosensitivity related to cell death and non-lethal cell damage. The model predictions were tested using data on hematological measurements (e.g., blood counts) performed in 1950–1956 in the Techa River residents chronically exposed to ionizing radiation since 1949. The suggested model of hematopoiesis is capable of describing experimental findings in the Techa River Cohort, including: i) slopes of the dose-effect curves reflecting the inhibition of hematopoiesis due to chronic ionizing radiation, ii) delay in effect of chronic exposure and accumulated character of the effect, and iii) dose-rate patterns for different cytopenic states (e.g., leukopenia, thrombocytopenia).
doi:10.1007/s00411-011-0351-3
PMCID: PMC3830531  PMID: 21259022
15.  Free radicals properties of gamma-irradiated penicillin-derived antibiotics: piperacillin, ampicillin, and crystalline penicillin 
The aim of this work was to determine the concentrations and properties of free radicals in piperacillin, ampicillin, and crystalline penicillin after gamma irradiation. The radicals were studied by electron paramagnetic resonance (EPR) spectroscopy using an X-band spectrometer (9.3 GHz). Gamma irradiation was performed at a dose of 25 kGy. One- and two-exponential functions were fitted to the experimental data, in order to assess the influence of the antibiotics’ storage time on the measured EPR lines. After gamma irradiation, complex EPR lines were recorded confirming the presence of a large number of free radicals formed during the irradiation. For all tested antibiotics, concentrations of free radicals and parameters of EPR spectra changed with storage time. The results obtained demonstrate that concentration of free radicals and other spectroscopic parameters can be used to select the optimal parameters of radiation sterilization of β-lactam antibiotics. The most important parameters are the constants τ (τ1(A),(I) and τ2(A),(I)) and K (K0(A),(I), K1(A),(I), K2(A),(I)) of the exponential functions that describe free radicals decay during samples storage.
doi:10.1007/s00411-013-0498-1
PMCID: PMC3935104  PMID: 24213588
Radiosterilization; Paramagnetic centers; Free radicals; EPR spectroscopy; Antibiotics; β-Lactam antibiotics
16.  Single-cell responses to ionizing radiation 
Radiation and environmental biophysics  2013;52(4):10.1007/s00411-013-0488-3.
While gene expression studies have proved extremely important in understanding cellular processes, it is becoming more apparent that there may be differences in individual cells that are missed by studying the population as a whole. We have developed a qRT-PCR protocol that allows us to assay multiple gene products in small samples, starting at 100 cells and going down to a single cell, and have used it to study radiation responses at the single-cell level. Since the accuracy of qRT-PCR depends greatly on the choice of “housekeeping” genes used for normalization, initial studies concentrated on determining the optimal panel of such genes. Using an endogenous control array, it was found that for IMR90 cells, common housekeeping genes tend to fall into one of two categories—those that are relatively stably expressed regardless of the number of cells in the sample, e.g., B2M, PPIA, and GAPDH, and those that are more variable (again regardless of the size of the population), e.g., YWHAZ, 18S, TBP, and HPRT1. Further, expression levels in commonly studied radiation-response genes, such as ATF3, CDKN1A, GADD45A, and MDM2, were assayed in 100, 10, and single-cell samples. It is here that the value of single-cell analyses becomes apparent. It was observed that the expression of some genes such as FGF2 and MDM2 was relatively constant over all irradiated cells, while that of others such as FAS was considerably more variable. It was clear that almost all cells respond to ionizing radiation but the individual responses were considerably varied. The analyses of single cells indicate that responses in individual cells are not uniform and suggest that responses observed in populations are not indicative of identical patterns in all cells. This in turn points to the value of single-cell analyses.
doi:10.1007/s00411-013-0488-3
PMCID: PMC3812812  PMID: 23995963
Ionizing radiation; Single-cell analyses; qRT-PCR; Endogenous controls
17.  Current concepts in clinical radiation oncology 
Based on its potent capacity to induce tumor cell death and to abrogate clonogenic survival, radiotherapy is a key part of multimodal cancer treatment approaches. Numerous clinical trials have documented the clear correlation between improved local control and increased overall survival. However, despite all progress, the efficacy of radiation-based treatment approaches is still limited by different technological, biological, and clinical constraints. In principle, the following major issues can be distinguished: (1) The intrinsic radiation resistance of several tumors is higher than that of the surrounding normal tissue, (2) the true patho-anatomical borders of tumors or areas at risk are not perfectly identifiable, (3) the treatment volume cannot be adjusted properly during a given treatment series, and (4) the individual heterogeneity in terms of tumor and normal tissue responses toward irradiation is immense. At present, research efforts in radiation oncology follow three major tracks, in order to address these limitations: (1) implementation of molecularly targeted agents and ‘omics’-based screening and stratification procedures, (2) improvement of treatment planning, imaging, and accuracy of dose application, and (3) clinical implementation of other types of radiation, including protons and heavy ions. Several of these strategies have already revealed promising improvements with regard to clinical outcome. Nevertheless, many open questions remain with individualization of treatment approaches being a key problem. In the present review, the current status of radiation-based cancer treatment with particular focus on novel aspects and developments that will influence the field of radiation oncology in the near future is summarized and discussed.
doi:10.1007/s00411-013-0497-2
PMCID: PMC3935099  PMID: 24141602
Radiotherapy; IMRT/IGRT; Particle therapy; Targeted therapy; Biomarkers; Personalized medicine
18.  Single-cell responses to ionizing radiation 
While gene expression studies have proved extremely important in understanding cellular processes, it is becoming more apparent that there may be differences in individual cells that are missed by studying the population as a whole. We have developed a qRT-PCR protocol that allows us to assay multiple gene products in small samples, starting at 100 cells and going down to a single cell, and have used it to study radiation responses at the single-cell level. Since the accuracy of qRT-PCR depends greatly on the choice of “housekeeping” genes used for normalization, initial studies concentrated on determining the optimal panel of such genes. Using an endogenous control array, it was found that for IMR90 cells, common housekeeping genes tend to fall into one of two categories—those that are relatively stably expressed regardless of the number of cells in the sample, e.g., B2M, PPIA, and GAPDH, and those that are more variable (again regardless of the size of the population), e.g., YWHAZ, 18S, TBP, and HPRT1. Further, expression levels in commonly studied radiation-response genes, such as ATF3, CDKN1A, GADD45A, and MDM2, were assayed in 100, 10, and single-cell samples. It is here that the value of single-cell analyses becomes apparent. It was observed that the expression of some genes such as FGF2 and MDM2 was relatively constant over all irradiated cells, while that of others such as FAS was considerably more variable. It was clear that almost all cells respond to ionizing radiation but the individual responses were considerably varied. The analyses of single cells indicate that responses in individual cells are not uniform and suggest that responses observed in populations are not indicative of identical patterns in all cells. This in turn points to the value of single-cell analyses.
doi:10.1007/s00411-013-0488-3
PMCID: PMC3812812  PMID: 23995963
Ionizing radiation; Single-cell analyses; qRT-PCR; Endogenous controls
19.  Damage clusters after gamma irradiation of a nanoparticulate plasmid DNA peptide condensate 
We have gamma irradiated plasmid DNA in aqueous solution in the presence of sub millimolar concentrations of the ligand tetra-arginine. Depending upon the ionic strength, under these conditions the plasmid can adopt a highly compacted and aggregated form which attenuates by some two orders of magnitude the yield of damage produced by the indirect effect. The yields of DNA single and double strand breaks (SSB and DSB) which result are closely comparable with those produced in living cells. The radical lifetimes, diffusion distances, and track structure are expected to be similarly well reproduced. After irradiation, the aggregation was reversed by adjusting the ionic conditions. The approximate spatial distribution of the resulting DNA damage was then assayed using by comparing the increases in the SSB and DSB yields produced by a subsequent incubation with limiting concentrations of the eukaryotic base excision repair enzymes formamidopyrimidine-DNA N-glycosylase (the FPG protein) and endonuclease III. Smaller increases in DSB yields were observed in the plasmid target that was irradiated in the condensed form. By modeling the spatial distribution of DNA damage, this result can be interpreted in terms of a greater extent of damage clustering.
doi:10.1007/s00411-011-0388-3
PMCID: PMC3288216  PMID: 21964719
Gamma radiation; DNA damage; DNA condensation; Clustered damage
20.  Background stratified Poisson regression analysis of cohort data 
Background stratified Poisson regression is an approach that has been used in the analysis of data derived from a variety of epidemiologically important studies of radiation-exposed populations, including uranium miners, nuclear industry workers, and atomic bomb survivors. We describe a novel approach to fit Poisson regression models that adjust for a set of covariates through background stratification while directly estimating the radiation-disease association of primary interest. The approach makes use of an expression for the Poisson likelihood that treats the coefficients for stratum-specific indicator variables as ‘nuisance’ variables and avoids the need to explicitly estimate the coefficients for these stratum-specific parameters. Log-linear models, as well as other general relative rate models, are accommodated. This approach is illustrated using data from the Life Span Study of Japanese atomic bomb survivors and data from a study of underground uranium miners. The point estimate and confidence interval obtained from this ‘conditional’ regression approach are identical to the values obtained using unconditional Poisson regression with model terms for each background stratum. Moreover, it is shown that the proposed approach allows estimation of background stratified Poisson regression models of non-standard form, such as models that parameterize latency effects, as well as regression models in which the number of strata is large, thereby overcoming the limitations of previously available statistical software for fitting background stratified Poisson regression models.
doi:10.1007/s00411-011-0394-5
PMCID: PMC3288408  PMID: 22193911
Cohort studies; Poisson regression; Ionizing radiation; Survival analysis
21.  Enhanced Intestinal Tumor Multiplicity and Grade in vivo after HZE Exposure: Mouse Models for Space Radiation Risk Estimates 
Carcinogenesis induced by space radiation is considered a major risk factor in manned interplanetary and other extended missions. The models presently used to estimate the risk for cancer induction following deep space radiation exposure are based on data from A-bomb survivor cohorts and do not account for important biological differences existing between high-linear energy transfer (LET) and low-LET-induced DNA damage. High-energy and charge (HZE) radiation, the main component of galactic cosmic rays (CGR), causes highly complex DNA damage compared to low-LET radiation, which may lead to increased frequency of chromosomal rearrangements, and contribute to carcinogenic risk in astronauts. Gastrointestinal (GI) tumors are frequent in the United States, and colorectal cancer (CRC) is the third most common cancer accounting for 10% of all cancer deaths. On the basis of the aforementioned epidemiological observations and the frequency of spontaneous precancerous GI lesions in the general population, even a modest increase in incidence by space radiation exposure could have a significant effect on health risk estimates for future manned space flights. Ground-based research is necessary to reduce the uncertainties associated with projected cancer risk estimates and to gain insights into molecular mechanisms involved in space radiation-induced carcinogenesis. We investigated in vivo differential effects of γ-rays and HZE ions on intestinal tumorigenesis using two different murine models, ApcMin/+ and Apc1638 N/+. We showed that γ- and/or HZE exposure significantly enhances development and progression of intestinal tumors in a mutant-line-specific manner, and identified suitable models for in vivo studies of space radiation–induced intestinal tumorigenesis.
doi:10.1007/s00411-010-0292-2
PMCID: PMC3580182  PMID: 20490531
Apc; intestinal tumorigenesis; space radiation; risk estimates
22.  Using electron beam radiation to simulate the dose distribution for whole body solar particle event proton exposure 
As a part of the near solar system exploration program, astronauts may receive significant total body proton radiation exposures during a solar particle event (SPE). In the Center for Acute Radiation Research (CARR), symptoms of the acute radiation sickness syndrome induced by conventional radiation are being compared to those induced by SPE-like proton radiation, to determine the relative biological effectiveness (RBE) of SPE protons. In an SPE, the astronaut’s whole body will be exposed to radiation consisting mainly of protons with energies below 50 MeV. In addition to providing for a potentially higher RBE than conventional radiation, the energy distribution for an SPE will produce a relatively inhomogeneous total body dose distribution, with a significantly higher dose delivered to the skin and subcutaneous tissues than to the internal organs. These factors make it difficult to use a 60Co standard for RBE comparisons in our experiments. Here, the novel concept of using megavoltage electron beam radiation to more accurately reproduce both the total dose and the dose distribution of SPE protons and make meaningful RBE comparisons between protons and conventional radiation is described. In these studies, Monte Carlo simulation was used to determine the dose distribution of electron beam radiation in small mammals such as mice and ferrets as well as large mammals such as pigs. These studies will help to better define the topography of the time-dose-fractionation versus biological response landscape for astronaut exposure to an SPE.
doi:10.1007/s00411-010-0315-z
PMCID: PMC3575686  PMID: 20725839
23.  Bomb-pulse 14C analysis combined with 13C and 15N measurements in blood serum from residents of Malmö, Sweden 
The 14C content of 60 human blood serum samples from residents of Malmö (Sweden) in 1978, obtained from a biobank, has been measured to estimate the accuracy of 14C bomb-pulse dating. The difference between the date estimated using the Calibomb software and sampling date varied between −3 ± 0.4 and +0.2 ± 0.5 years. The average age deviation of all samples was −1.5 ± 0.7 years, with the delay between production and consumption of foodstuffs being probably the dominating cause. The potential influence of food habits on the 14C date has been evaluated using stable isotope δ13C and δ15N analysis and information about the dietary habits of the investigated individuals. Although the group consisting of lacto-ovo vegetarians and vegans (pooled group) was not completely separated from the omnivores in a stable isotopic trophic level diagram, this analysis proved to add valuable information on probable dietary habits. The age deviation of the sampling date from the respective Calibomb date was found strongly correlated with the δ13C values, probably due to influence from marine diet components. For the omnivore individuals, there were indications of seasonal effects on δ13C and the age deviation. No significant correlation was found between the age deviation and the δ15N values of any dietary group. No influence of sex or year of birth was found on neither the 14C nor the δ13C and δ15N values of the serum samples. The data were also divided into two groups (omnivores and pooled group), based on the level of δ15N in the samples. The consumption of high δ15N-valued fish and birds can be responsible for this clustering.
doi:10.1007/s00411-013-0458-9
PMCID: PMC3677238  PMID: 23358598
Serum; Bomb-pulse dating; Stable isotopes
24.  Are mouse lens epithelial cells more sensitive to γ-irradiation than lymphocytes? 
In this pilot study we compared for the first time the radiation sensitivity of mouse lens epithelial cells (LECs) and mouse lymphocytes. We freshly prepared LECs and lymphocytes and irradiated them with γ-rays (137Cs; doses ranging from 0.25 to 2 Gy). DNA damage and repair were evaluated by alkaline comet assay and γH2AX foci assay. Using the comet assay, we observed a dose-dependent increase in DNA damage in both cell types. The faster formation of single- and double-strand breaks in LECs of C57BL/6 mice at doses below 1 Gy needs to be confirmed in other mouse strains. Immunofluorescence for γH2AX foci showed a higher degree of lesions in LECs from C57BL/6J mice compared to those of JF1 mice and to lymphocytes of both strains. Correspondingly, repair of DNA damage proceeded faster in LECs of C57BL/6J mice compared to LECs of JF1 mice and lymphocytes of both strains. It is obvious that the lymphocytes of both strains repaired DNA lesions more slowly than the corresponding LECs. In conclusion, our results demonstrate that LECs of C57Bl/6 mice show a steeper dose–response than lymphocytes in both types of experiments. It shows that both test systems are able to be used also at doses below 0.25 Gy. The observed difference in DNA repair between the LECs from C57BL/6J mice compared to the LECs from JF1 mice and to the lymphocytes of both strains warrants further experiments to identify the underlying molecular mechanisms.
doi:10.1007/s00411-012-0451-8
PMCID: PMC3677237  PMID: 23321930
Lens epithelial cells; Lymphocytes; Gamma-irradiation; Comet assay; Gamma-H2AX assay; Radiation sensitivity
25.  Independent analysis of the radiation risk for leukaemia in children and adults with mortality data (1950–2003) of Japanese A-bomb survivors 
A recent analysis of leukaemia mortality in Japanese A-bomb survivors has applied descriptive models, collected together from previous studies, to derive a joint excess relative risk estimate (ERR) by multi-model inference (MMI) (Walsh and Kaiser in Radiat Environ Biophys 50:21–35, 2011). The models use a linear-quadratic dose response with differing dose effect modifiers. In the present study, a set of more than 40 models has been submitted to a rigorous statistical selection procedure which fosters the parsimonious deployment of model parameters based on pairwise likelihood ratio tests. Nested models were consequently excluded from risk assessment. The set comprises models of the excess absolute risk (EAR) and two types of non-standard ERR models with sigmoidal responses or two line spline functions with a changing slope at a break point. Due to clearly higher values of the Akaike Information Criterion, none of the EAR models has been selected, but two non-standard ERR models qualified for MMI. The preferred ERR model applies a purely quadratic dose response which is slightly damped by an exponential factor at high doses and modified by a power function for attained age. Compared to the previous analysis, the present study reports similar point estimates and confidence intervals (CI) of the ERR from MMI for doses between 0.5 and 2.5 Sv. However, at lower doses, the point estimates are markedly reduced by factors between two and five, although the reduction was not statistically significant. The 2.5 % percentiles of the ERR from the preferred quadratic-exponential model did not fall below zero risk in exposure scenarios for children, adolescents and adults at very low doses down to 10 mSv. Yet, MMI produced risk estimates with a positive 2.5 % percentile only above doses of some 300 mSv. Compared to CI from a single model of choice, CI from MMI are broadened in cohort strata with low statistical power by a combination of risk extrapolations from several models. Reverting to MMI can relieve the dilemma of needing to choose between models with largely different consequences for risk assessment in public health.
Electronic supplementary material
The online version of this article (doi:10.1007/s00411-012-0437-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s00411-012-0437-6
PMCID: PMC3579470  PMID: 23124826
Leukaemia mortality; Radiation risk; A-bomb survivors; Nonlinear dose response; Multi-model inference

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