<p>The bottleneck in data acquisition during biological dosimetry based on a dicentric assay is the need to score dicentrics in a large number of lymphocytes. One way to increase the capacity of a given laboratory is to use the ability of skilled operators from other laboratories. This can be done using image analysis systems and distributing images all around the world. Two exercises were conducted to test the efficiency of such an approach involving 10 laboratories. During the first exercise (E1), the participant laboratories analysed the same images derived from cells exposed to 0.5 and 3 Gy; 100 images were sent to all participants for both doses. Whatever the dose, only about half of the cells were complete with well-spread metaphases suitable for analysis. A coefficient of variation (CV) on the standard deviation of ?15 \% was obtained for both doses. The trueness was better for 3 Gy (0.6 %) than for 0.5 Gy (37.8 %). The number of estimated doses classified as satisfactory according to the z-score was 3 at 0.5 Gy and 8 at 3 Gy for 10 dose estimations. In the second exercise, an emergency situation was tested, each laboratory was required to score a different set of 50 images in 2 d extracted from 500 downloaded images derived from cells exposed to 0.5 Gy. Then the remaining 450 images had to be scored within a week. Using 50 different images, the CV on the estimated doses (79.2 %) was not as good as in E1, probably associated to a lower number of cells analysed (50 vs. 100) or from the fact that laboratories analysed a different set of images. The trueness for the dose was better after scoring 500 cells (22.5 %) than after 50 cells (26.8 %). For the 10 dose estimations, the number of doses classified as satisfactory according to the z-score was 9, for both 50 and 500 cells. Overall, the results obtained support the feasibility of networking using electronically transmitted images. However, before its implementation some issues should be elucidated, such as the number and resolution of the images to be sent, and the harmonisation of the scoring criteria. Additionally, a global website able to be used for the different regional networks, like Share Points, will be desirable to facilitate worldwide communication.</p>

The zebra mussel Dreissena polymorpha is widely used as sentinel organism for the assessment of environmental contamination in freshwater environments. However, in the River Rhine (Germany), the D. polymorpha population is declining, whereas the closely related quagga mussel D. bugensis is found in high numbers at some sites. In the present laboratory study, D. polymorpha and D. bugensis were exposed to resuspended native sediments for ≤2 weeks. Wet sediments (<63 μm, 100 mg l−1 dry weight) were used as surrogate suspended particulate matter to mimic one of the mussels’ main uptake route for chemicals. The sediments were sampled in (1) the River Elbe in Dessau, a site known to be highly polluted with, e.g., organochlorine (OC) pesticides and (2) at a relatively unpolluted site in Havelberg in the River Havel, one of the Elbe’s tributaries. Chemical analysis of persistent OC compounds (seven polychlorinated biphenyls [PCBs], DDT and its metabolites (DDX), hexachlorocylohexanes [HCHs], and hexachlorobenzene [HCB]) in soft tissue of mussels showed significantly greater values of PCBs 101, 118, 153, 138, 180, the sum of seven PCBs, and p,p′-DDD in D. bugensis compared with D. polymorpha. Fourteen days of exposure to Dessau sediment increased the concentration of p,p′-DDE and p,p′-DDD, as well as the sum of DDX, in both species compared with Havelberg sediment. Interspecific differences were less pronounced when regarding chemical concentrations with lipid content instead of dry-weight of tissue because D. bugensis had greater levels of total lipid than D. polymorpha. DNA damage in gills, as measured with the comet assay, was greater in D. bugensis compared with D. polymorpha. Simultaneously, the content of heat-shock protein (hsp70) in gills was greater in D. polymorpha than in D. bugensis. DNA damage and hsp70 were not induced by exposure time or sediment type. This study shows that D. bugensis and D. polymorpha may differ in their bioaccumulation potential of OC pesticides as well as their levels of DNA damage and hsp70. Therefore, more investigations are needed before quagga mussel can be used as alternative test organism for the zebra mussel.

<p>Hypoxic cells have been linked to genetic instability and tumor progression. However, little is known about the exact relationship between DNA repair and genetic instability in hypoxic cells. We therefore tested whether the sensing and repair of DNA double-strand breaks (DNA-dsbs) is altered in irradiated cells kept under continual oxic, hypoxic or anoxic conditions. Synchronized G0-G1 human fibroblasts were irradiated (0-10 Gy) after initial gassing with 0% O₂ (anoxia), 0.2% O₂ (hypoxia) or 21% O₂ (oxia) for 16 hours. The response of phosphorylated histone H2AX (γ-H2AX), phosphorylated ataxia telangiectasia mutated [ATM(Ser1981)], and the p53 binding protein 1 (53BP1) was quantified by intranuclear DNA repair foci and western blotting. At 24 hours following DNA damage, residual γ-H2AX, ATM(Ser1981) and 53BP1 foci were observed in hypoxic cells. This increase in residual DNA-dsbs under hypoxic conditions was confirmed using neutral comet assays. Clonogenic survival was also reduced in chronically hypoxic cells, which is consistent with the observation of elevated G1-associated residual DNA-dsbs. We also observed an increase in the frequency of chromosomal aberrations in chronically hypoxic cells. We conclude that DNA repair under continued hypoxia leads to decreased repair of G1-associated DNA-dsbs, resulting in increased chromosomal instability. Our findings suggest that aberrant DNA-dsb repair under hypoxia is a potential factor in hypoxia-mediated genetic instability.</p>

<p>Exposure to high doses of ionizing radiation (IR) can lead to localized radiation injury of the skin and exposed cells suffer dsDNA breaks that may elicit cell death or stochastic changes. Little is known about the DNA damage response after high-dose exposure of the skin. Here, we investigate the cellular and DNA damage response in acutely irradiated minipig skin.IR-induced DNA damage, repair and cellular survival were studied in 15 cm(2) of minipig skin exposed in vivo to ~50 Co-60 γ rays. Skin biopsies of control and 4 h up to 96 days post exposure were investigated for radiation-induced foci (RIF) formation using γ-H2AX, 53BP1, and active ATM-p immunofluorescence. High-dose IR induced massive γ-H2AX phosphorylation and high 53BP1 RIF numbers 4 h, 20 h after IR. As time progressed RIF numbers dropped to a low of 3-fold elevated at all subsequent time points. Replicating basal cells (Ki67+) were reduced 3 days post IR followed by increased proliferation and recovery of epidermal cellularity after 28 days.Acute high dose irradiation of minipig epidermis impaired stem cell replication and induced elevated apoptosis from 3 days onward. DNA repair cleared the high numbers of DBSs in skin cells, while RIFs that persisted in &lt;1% cells marked complex and potentially lethal DNA damage up to several weeks after exposure. An elevated frequency of keratinocytes with persistent RIFs may thus serve as indicator of previous acute radiation exposure, which may be useful in the follow up of nuclear or radiological accident scenarios.</p>

Ionizing radiation is a known human carcinogen that can induce a variety of biological effects depending on the physical nature, duration, doses and dose-rates of exposure. However, the magnitude of health risks at low doses and dose-rates (below 100mSv and/or 0.1mSvmin(-1)) remains controversial due to a lack of direct human evidence. It is anticipated that significant insights will emerge from the integration of epidemiological and biological research, made possible by molecular epidemiology studies incorporating biomarkers and bioassays. A number of these have been used to investigate exposure, effects and susceptibility to ionizing radiation, albeit often at higher doses and dose rates, with each reflecting time-limited cellular or physiological alterations. This review summarises the multidisciplinary work undertaken in the framework of the European project DoReMi (Low Dose Research towards Multidisciplinary Integration) to identify the most appropriate biomarkers for use in population studies. In addition to logistical and ethical considerations for conducting large-scale epidemiological studies, we discuss the relevance of their use for assessing the effects of low dose ionizing radiation exposure at the cellular and physiological level. We also propose a temporal classification of biomarkers that may be relevant for molecular epidemiology studies which need to take into account the time elapsed since exposure. Finally, the integration of biology with epidemiology requires careful planning and enhanced discussions between the epidemiology, biology and dosimetry communities in order to determine the most important questions to be addressed in light of pragmatic considerations including the appropriate population to be investigated (occupationally, environmentally or medically exposed), and study design. The consideration of the logistics of biological sample collection, processing and storing and the choice of biomarker or bioassay, as well as awareness of potential confounding factors, are also essential.

Chromatin modifications/remodeling are important mechanisms by which cells regulate various functions through providing accessibility to chromatin DNA. Recent studies implicated INO80, a conserved chromatin-remodeling complex, in the process of DNA repair. However, the precise underlying mechanism by which this complex mediates repair in mammalian cells remains enigmatic. Here, we studied the effect of silencing of the Ino80 subunit of the complex on double-strand break repair in mammalian cells. Comet assay and homologous recombination repair reporter system analyses indicated that Ino80 is required for efficient double-strand break repair. Ino80 association with chromatin surrounding double-strand breaks suggested the direct involvement of INO80 in the repair process. Ino80 depletion impaired focal recruitment of 53BP1 but did not impede Rad51 focus formation, suggesting that Ino80 is required for the early steps of repair. Further analysis by using bromodeoxyuridine (BrdU)-labeled single-stranded DNA and replication protein A (RPA) immunofluorescent staining showed that INO80 mediates 5'-3' resection of double-strand break ends.

A semi-automated scoring system has been developed to provide rapid, accurate assessment of micronuclei in preparations of mononuclear mouse lymphoma L5178Y cells. Following exposure to a range of test agents, flat, single-cell preparations were produced from exponentially growing cultures by cytocentrifugation. Following staining with 4'-6-diamidino-2-phenylindole (DAPI), cells were scanned by use of the MicroNuc module of Metafer 4 v 3.4.102, after modifying the classifier developed for selecting micronuclei in binucleate cells to increase its sensitivity. The image gallery of all cells was then sorted to bring aberrant cells to the top of the gallery to assess visually the numbers of cells with micronuclei, as distinct from other debris. Slide quality was shown to be paramount in obtaining accurate results from an automated scan and the data obtained compared very well with the incidence of micronuclei scored conventionally by microscopy. Compared with manual scoring the time saving is considerable, as more than 2000 images are captured in approximately 2min, with subsequent visual assessment of aberrant cells in the image gallery taking about 1-2min/slide. By scanning all aberrant cells, the system also captures additional information on necrotic, apoptotic and fragmented cells. Although optimised for mouse lymphoma cells, it should be simple to adapt the method for any cell type growing in suspension.

Evidence suggests that de novo, therapy-related and benzene-induced acute myeloid leukemias (AML) occur via similar cytogenetic and genetic pathways, several of which involve aneuploidy, the loss or gain of chromosomes. Aneuploidy of specific chromosomes has been detected in benzene-related leukemia patients as well as in healthy benzene-exposed workers, suggesting that aneuploidy precedes and may be a potential mechanism underlying benzene-induced leukemia. Here, we analyzed the peripheral blood lymphocytes of 47 exposed workers and 27 unexposed controls using a novel OctoChrome fluorescence in situ hybridization (FISH) technique that simultaneously detects aneuploidy in all 24 chromosomes. Through this chromosome-wide aneuploidy study (CWAS) approach, we found heterogeneity in the monosomy and trisomy rates of the 22 autosomes when plotted against continuous benzene exposure. In addition, statistically significant, chromosome-specific increases in the rates of monosomy [5, 6, 7, 10, 16 and 19] and trisomy [5, 6, 7, 8, 10, 14, 16, 21 and 22] were found to be dose dependently associated with benzene exposure. Furthermore, significantly higher rates of monosomy and trisomy were observed in a priori defined 'susceptible' chromosome sets compared with all other chromosomes. Together, these findings confirm that benzene exposure is associated with specific chromosomal aneuploidies in hematopoietic cells, which suggests that such aneuploidies may play roles in benzene-induced leukemogenesis.

<p>The COMET assay is recognized as a rapid and sensitive method in quantifying radiation induced DNA damage. We investigated the distorting influence of endogenous, assay-inherent factors onto base (single cell level) and primary outcome measures (experimental/slide level), such as olive tail moment (OTM) and percentage DNA in the tail (\%tail-DNA). From 2003 to 2008, we performed the assay on lymphocytes isolated from the blood samples of 355 lung cancer patients, 170 controls, and 610 relatives, as well as one single reference individual, repeated 170 times. In total, the data from 10,016 single experiments containing around 1,750,000 cells have been included in this study. This is the first time that the endogenous variability of the COMET assay has been validated systematically on such a huge data set over a 5 year period. Assuming that the reference sample reflects assay specific white noise, we estimated a proportion of 7-95% of the variability of the outcome measures due to assay variation (white noise) depending on parameter, exposure level, and study group. The proportion of white noise was largest for the initial radiation damage. The specific endogenous factors considered attribute to 14.8% of the total variability in the primary outcome measurements of the OTM and 6.9% of the %tail-DNA. OTM turns out to be a sensitive parameter to detect variation, but is also more susceptible to disturbance caused by endogenous factors than %tail-DNA. To reduce the experimental variability in COMET assays, we recommend a highly standardized operation protocol as well as inspecting and/or adjusting the primary outcome measures according to endogenous factors before calculating secondary outcome measures, e.g. DNA repair capacity (DRC) or testing statistical inference. A human reference (HR) sample is also useful to inspect homogeneity in the temporal progression of long lasting investigations, but not for calibrating primary outcome measurements.</p>

Automated image analysis scoring of micronuclei (MN) in cells can facilitate the objective and rapid measurement of genetic damage in mammalian and human cells. This approach was repeatedly developed and tested over the past two decades but none of the systems were sufficiently robust for routine analysis of MN until recently. New methodological, hardware and software developments have now allowed more advanced systems to become available. This mini-review presents the current stage of development and validation of the Metasystems Metafer MNScore system for automated image analysis scoring of MN in cytokinesis-blocked binucleated lymphocytes, which is the best-established method for studying MN formation in humans. The results and experience of users of this system from 2004 until today are reviewed in this paper. Significant achievements in the application of this method in research related to mutagen sensitivity phenotype in cancer risk, radiation biodosimetry and biomonitoring studies of air pollution (enriched by new data) are described. Advantages as well as limitations of automated image analysis in comparison with traditional visual analysis are discussed. The current increased use of the Metasystems Metafer MNScore system in various studies and the growing number of publications based on automated image analysis scoring of MN is promising for the ongoing and future application of this approach.

The scoring of micronuclei (MN) is widely used in biomonitoring and mutagenicity testing as a surrogate marker of chromosomal damage inflicted by clastogenic agents or by aneugens. Individual differences in the response to a mutagenic challenge are known from studies on cancer patients and carriers of mutations in DNA repair genes. However, it has not been studied to which extent genetic factors contribute to the observed variability of individual MN frequencies. Our aim was to quantify this heritable genetic component of both baseline and radiation-induced MN frequencies. We performed a twin study comprising 39 monozygotic (MZ) and 10 dizygotic (DZ) twin pairs. Due to the small number of DZ pairs, we had to recruit controls from which 38 age- and gender-matched random control pairs (CPs) were generated. For heritability estimates, we used biometrical modelling of additive genetic, common environmental, and unique environmental components (ACE model) of variance and direct comparison of variance between the sample groups. While heritability estimates from MZ to DZ comparisons produced inconclusive results, both estimation methods revealed a high degree of heritability (h(2)) for baseline MN frequency (h(2) = 0.68 and h(2) = 0.72) as well as for the induced frequency (h(2) = 0.68 and h(2) = 0.57) when MZ were compared to CP. The result was supported by the different intraclass correlation coefficients of MZ, DZ and CP for baseline (r = 0.63, r = 0.31 and r = 0.0, respectively) as well as for induced MN frequencies (r = 0.79, r = 0.74 and r = 0.0, respectively). This study clearly demonstrates that MN frequencies are determined by genetic factors to a major part. The strong reflection of the genetic background supports the idea that MN frequencies represent an intermediate phenotype between molecular DNA repair mechanisms and the cancer phenotype and affirms the approaches that are made to utilise them as predictors of, for example, cancer risk.

Biological dosimetry, based on the analysis of micronuclei (MN) in the cytokinesis-block micronucleus (CBMN) assay can be used as an alternative method for scoring dicentric chromosomes in the field of radiation protection. Biological dosimetry or Biodosimetry, is mainly performed, in addition to physical dosimetry, with the aim of individual dose assessment. Many studies have shown that the number of radiation-induced MN is strongly correlated with dose and quality of radiation. The CBMN assay has become, in the last years, a thoroughly validated and standardised technique to evaluate in vivo radiation exposure of occupational, medical and accidentally exposed individuals. Compared to the gold standard, the dicentric assay, the CBMN assay has the important advantage of allowing economical, easy and quick analysis. The main disadvantage of the CBMN assay is related to the variable micronucleus (MN) background frequency, by which only in vivo exposures in excess of 0.2-0.3 Gy X-rays can be detected. In the last years, several improvements have been achieved, with the ultimate goals (i) of further increasing the sensitivity of the CBMN assay for low-dose detection by combining the assay with a fluorescence in situ hybridisation centromere staining technique, (ii) of increasing the specificity of the test for radiation by scoring nucleoplasmic bridges in binucleated cells and (iii) of making the assay optimally suitable for rapid automated analysis of a large number of samples, viz. in case of a large-scale radiation accident. The development of a combined automated MN-centromere scoring procedure remains a challenge for the future, as it will allow systematic biomonitoring of radiation workers exposed to low-dose radiation.

<p>In the case of a large-scale nuclear or radiological incidents a reliable estimate of dose is an essential tool for providing timely assessment of radiation exposure and for making life-saving medical decisions. Cytogenetics is considered as the #gold##standard# for biodosimetry. The dicentric analysis (DA) represents the most specific cytogenetic bioassay. The micronucleus test (MN) applied in interphase in peripheral lymphocytes is an alternative and simpler approach. A dose-effect calibration curve for the MN frequency in peripheral lymphocytes from 27 adult donors was established after in vitro irradiation at a dose range 0.15-8 Gy of 137Cs gamma rays (dose rate 6 Gy min-1). Dose prediction by visual scoring in a dose-blinded study (0.15-4.0 Gy) revealed a high level of accuracy (R = 0.89). The scoring of MN is time consuming and requires adequate skills and expertise. Automated image analysis is a feasible approach allowing to reduce the time and to increase the accuracy of the dose estimation decreasing the variability due to subjective evaluation. A good correlation (R = 0.705) between visual and automated scoring with visual correction was observed over the dose range 0-2 Gy. Almost perfect discrimination power for exposure to 1-2 Gy, and a satisfactory power for 0.6 Gy were detected. This threshold level can be considered sufficient for identification of sub lethally exposed individuals by automated CBMN assay.</p>

<p>Structural genomic rearrangements are frequent findings in human cancers. Therefore, papillary thyroid carcinomas (PTCs) were investigated for chromosomal aberrations and rearrangements of the RET proto-oncogene. For this purpose, primary cultures from 23 PTC have been established and metaphase preparations were analysed by spectral karyotyping (SKY). In addition, interphase cell preparations of the same cases were investigated by fluorescence in situ hybridisation (FISH) for the presence of RET/PTC rearrangements using RET-specific DNA probes. SKY analysis of PTC revealed structural aberrations of chromosome 11 and several numerical aberrations with frequent loss of chromosomes 20, 21, and 22. FISH analysis for RET/PTC rearrangements showed prevalence of this rearrangement in 72% (16 out of 22) of cases. However, only subpopulations of tumour cells exhibited this rearrangement indicating genetic heterogeneity. The comparison of visual and automated scoring of FISH signals revealed concordant results in 19 out of 22 cases (87%) indicating reliable scoring results using the optimised scoring parameter for RET/PTC with the automated Metafer4 system. It can be concluded from this study that genomic rearrangements are frequent in PTC and therefore important events in thyroid carcinogenesis.</p>

Multicolour fluorescence in situ hybridisation (M-FISH) and multicolour banding (M-BAND) are advanced chromosome painting techniques combining multiple chromosome- or region-specific paints in one step. M-FISH identifies all chromosomes or chromosome arms at once, whereas M-BAND identifies the different regions of a single chromosome. The use of either or both can improve the accuracy of karyotyping and help identify cryptic chromosome rearrangements. These probes are prepared by pooling multiple chromosome- or chromosome region-specific DNA libraries, each labelled with a unique combination of fluorochromes. Commercial probes are available, avoiding the need for probe preparation. In the protocol described here, a commercial probe is used. Well-spread metaphases are prepared according to standard techniques, followed by alkaline denaturation and application of the denatured probe. After an incubation period, the slides are washed. A fluorescence microscope with filter sets specific to the fluorescent labels is used for analysis, together with specialised image analysis software. The software interprets the combination of fluorochromes to identify each chromosome and produce a false colour image specific for each chromosome or region. The single colour galleries - which show the hybridisation patterns of the individual fluorochromes - are useful to help interpret and confirm the false colour images produced by the software, including ambiguous signals.

<p>Adaptive response (AR) is a term describing resistance to ionizing radiation-induced killing or formation of aberrant chromosomes that is mediated by pre-exposure to low ionizing radiation doses. The mechanism of AR remains elusive. Because cell killing and chromosome aberration formation derive from erroneous processing of DNA double-strand breaks (DSB), AR may reflect a modulation of DSB processing by nonhomologous end joining (NHEJ) or homologous recombination repair. Here, we use plasmid end-joining assays to quantify modulations induced by low ionizing radiation doses to NHEJ, the dominant pathway of DSB repair in higher eukaryotes, and investigate propagation of this response through medium transfer to nonirradiated bystander cells. Mouse embryo fibroblasts were conditioned with 10 to 1000 mGy and NHEJ quantified at different times thereafter by challenging with reporter plasmids containing a DSB. We show robust increases in NHEJ efficiency in mouse embryo fibroblasts exposed to ionizing radiation &gt;100 mGy, irrespective of reporter plasmid used. Human tumor cells also show AR of similar magnitude that is compromised by caffeine, an inhibitor of DNA damage signaling acting by inhibiting ATM, ATR, and DNA-PKcs. Growth medium from pre-irradiated cells induces a caffeine-sensitive AR in nonirradiated cells, similar in magnitude to that seen in irradiated cells. In bystander cells, γH2AX foci are specifically detected in late S-G(2) phase and are associated with Rad51 foci that signify the function of homologous recombination repair, possibly on DNA replication-mediated DSBs. The results point to enhanced NHEJ as a mechanism of AR and suggest that AR may be transmitted to bystander cells through factors generating replication-mediated DSBs. Cancer Res; 70(21); 8498-506. ©2010 AACR.</p>

PURPOSE: The backup pathway of nonhomologous end joining (B-NHEJ) enables cells to process DNA double-strand breaks (DSBs) when the DNA-PK-dependent pathway of NHEJ (D-NHEJ) is compromised. Our previous results show marked reduction in the activity of B-NHEJ when LIG4(-/-) mouse embryo fibroblasts (MEFs) cease to grow and enter a plateau phase. The dependence of B-NHEJ on growth state is substantially stronger than that of D-NHEJ and points to regulatory mechanisms or processing determinants that require elucidation. Because the different D-NHEJ mutants show phenotypes distinct in their details, it is necessary to characterize the dependence of their DSB repair capacity on growth state and to explore species-specific responses. METHODS AND MATERIALS: DSB repair was measured in cells of different genetic background from various species using pulsed-field gel electrophoresis, or the formation of ã-H2AX foci, at different stages of growth. RESULTS: Using pulsed-field gel electrophoresis, we report a marked reduction of B-NHEJ during the plateau phase of growth in KU and XRCC4, mouse or Chinese hamster, mutants. Notably, this reduction is only marginal in DNA-PKcs-deficient cells. However, reduced B-NHEJ is also observed in repair proficient, plateau-phase cells after treatment with DNA-PK inhibitors. The reduction of B-NHEJ activity in the plateau phase of growth does not derive from the reduced expression of participating proteins, is detectable by ã-H2AX foci analysis, and leads to enhanced cell killing. CONCLUSIONS: These results further document the marked dependence on growth state of an essential DSB repair pathway and show the general nature of the effect. Molecular characterization of the mechanism underlying this response will help to optimize the administration of DNA repair inhibitors as adjuvants in radiation therapy.

<p>Ionizing radiation (IR) induces a variety of DNA lesions among which DNA double-strand breaks (DSBs) are the biologically most significant. It is currently unclear if DSB repair is equally efficient after low and high doses. Here, we use gamma-H2AX, phospho-ATM (pATM), and 53BP1 foci analysis to monitor DSB repair. We show, consistent with a previous study, that the kinetics of gamma-H2AX and pATM foci loss in confluent primary human fibroblasts are substantially compromised after doses of 10 mGy and lower. Following 2.5 mGy, cells fail to show any foci loss. Strikingly, cells pretreated with 10 microM H(2)O(2) efficiently remove all gamma-H2AX foci induced by 10 mGy. At the concentration used, H(2)O(2) produces single-strand breaks and base damages via the generation of oxygen radicals but no DSBs. Moreover, 10 microM H(2)O(2) up-regulates a set of genes that is also up-regulated after high (200 mGy) but not after low (10 mGy) radiation doses. This suggests that low radical levels induce a response that is required for the repair of radiation-induced DSBs when the radiation damage is too low to cause the induction itself. To address the in vivo significance of this finding, we established gamma-H2AX and 53BP1 foci analysis in various mouse tissues. Although mice irradiated with 100 mGy or 1 Gy show efficient gamma-H2AX and 53BP1 foci removal during 24 h post-IR, barely any foci loss was observed after 10 mGy. Our data suggest that the cellular response to DSBs is substantially different for low vs. high radiation doses.</p>

DNA double-strand breaks (DSBs) are critical cellular lesions that can result from ionizing radiation exposure. A marker for DSB formation is the phosphorylated form of the histone H2 variant H2AX (gamma-H2AX). DSBs also attract the damage sensor p53-binding protein 1 (53BP1) to the DSB-containing chromatin, because 53BP1 associates with the DSB-surrounding chromatin. We studied the induction, persistence, and disappearance of radiation-induced gamma-H2AX and 53BP1 foci after the first (131)I therapy of patients with differentiated thyroid carcinoma, a model for protracted, continuous, internal whole-body irradiation. METHODS: Twenty-six patients (7 men, 19 women; mean age +/- SD, 42 +/- 13 y) underwent posttherapeutic blood dosimetry according to the standard operating procedure of the European Association of Nuclear Medicine, including peripheral blood sampling and external dose rate measurements at 2-144 h after administration of (131)I for thyroid remnant ablation. The mean time curves of dose accumulation and dose rate to the blood were compared with the mean gamma-H2AX and 53BP1 foci counts over the same period in samples of mononuclear peripheral blood leukocytes. RESULTS: The mean absorbed dose to the blood in 24 patients evaluable for physical dosimetry was 0.31 +/- 0.10 Gy (minimum, 0.17 Gy; maximum, 0.57 Gy). After 24 h, the mean daily dose increment was less than 0.05 Gy. The excess focus counts per nucleus–that is, nuclear foci in excess of the low background count–peaked at 2 h after radioiodine administration (median excess foci for gamma-H2AX [n = 21 patients], 0.227, and for 53BP1 [n = 19 patients], 0.235) and progressively declined thereafter. Significantly elevated numbers of excess focus counts per nucleus (median excess foci for gamma-H2AX [n = 8 patients], 0.054, and for 53BP1 [n = 6 patients], 0.046) still were present at 120-144 h after therapy. Because the rate of occurrence of radiation-induced focus counts per nucleus per absorbed dose varied considerably among patients, a dose-response relationship could not be established for this series as a whole. The number of excess radiation-induced focus counts per nucleus per absorbed dose rate increased with time, potentially indicating a slower rate of DNA repair or, alternatively, a higher de novo rate of focus formation. The values over time of both radiation-induced DSB markers correlated closely (r(2) = 0.973). CONCLUSION: Radiation-induced gamma-H2AX and 53BP1 nuclear foci are useful markers for detecting radiation exposure after radionuclide incorporation, even for absorbed doses to the blood below 20 mGy.

<p>The purpose of the present study was to investigate as to what extent differences in the linear energy transfer (LET) are reflected at the chromosomal level. For this study human lymphocytes were exposed to 9.5 MeV/u C-ions (1 or 2 Gy, LET=175 keV/microm) or X-rays (1-6 Gy), harvested at 48, 72 or 96 h post-irradiation and aberrations were scored in first cycle metaphases using 24 color fluorescence in situ hybridization (mFISH). Additionally, in selected samples aberrations were measured in prematurely condensed G2-phase cells. Analysis of the time-course of aberrations in first cycle metaphases showed a stable yield of simple and complex exchanges after X-ray irradiation. In contrast, after C-ion exposure the yields profoundly increased with harvesting time complicating the estimation of the frequency of aberrations produced by high LET particles within the entire cell population. This is especially true for the yield of complex exchanges. Complex aberrations dominate the aberration spectrum produced by C-ions. Their fraction was about 50\% for the two measured doses. In contrast, isodoses of X-rays induced smaller proportions of complex aberrations (i.e. 5% and 15%, respectively). For both radiation qualities the fraction of complexes did not change with harvesting time. As expected from the different dose deposition of high and low LET radiation, complex exchanges produced by high LET C-ions involved more breaks and more chromosomes than those induced by isodoses of X-rays. Noteworthy, C-ions but not X-rays induced a small number of complex chromatid-isochromatid exchanges that are not expected for cells exposed in the G0-phase. The results obtained so far for cells arrested in G2-phase confirm these patterns. Altogether our data show that the increased effectiveness of C-ions for the induction of aberrations in first cycle cells is determined by complex exchanges, whereas for simple exchanges the relative biological effectiveness is about one.</p>