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Breast carcinomas (BC) are among the most frequent cancers in women. Studies on radiosensitivity and ionizing radiation response of BC cells are scarce and mainly focused on intrinsic molecular mechanisms but do not include clinically relevant features as chromosomal rearrangements important for radiotherapy. The main purpose of this study was to compare the ionizing radiation response and efficiency of repair mechanisms of human breast carcinoma cells (Cal 51) and peripheral blood lymphocytes (PBL) for different doses and radiation qualities (60Co γ-rays, 150 MeV and spread-out Bragg peak (SOBP) proton beams). The radiation response functions obtained using the conventional metaphase assay and premature chromosome condensation (PCC) technique enabled us to determine the number of chromosomal breaks at different time after irradiation. Both cytogenetic assays used confirmed the higher biological radiosensitivity for proton beams in tumor cells compared to PBL, corresponding to higher values of the linear LQ parameter α. additionally, the ratio of the LQ parameters β/α describing efficiency of the repair mechanisms, obtained for chromosome aberrations, showed higher numbers for PBL than for Cal 51 for all exposures. Similar results were observed for the ratio of PCC breaks determined directly after irradiation to that obtained 12 h later. This parameter (t0/t12) showed faster decrease of the repair efficiency with increasing LET value for Cal 51 cells. This finding supports the use of the proton therapy for breast cancer patients.

The mechanism underlying the carcinogenic potential of α radiation is not fully understood, considering that cell inactivation (e.g., mitotic cell death) as a main consequence of exposure efficiently counteracts the spreading of heritable DNA damage. The aim of this study is to improve our understanding of the effectiveness of α particles in inducing different types of chromosomal aberrations, to determine the respective values of the relative biological effectiveness (RBE) and to interpret the results with respect to exposure risk. Human peripheral blood lymphocytes (PBLs) from a single donor were exposed ex vivo to doses of 0-6 Gy X rays or 0-2 Gy α particles. Cells were harvested at two different times after irradiation to account for the mitotic delay of heavily damaged cells, which is known to occur after exposure to high-LET radiation (including α particles). Analysis of the kinetics of cells reaching first or second (and higher) mitosis after irradiation and aberration data obtained by the multiplex fluorescence in situ hybridization (mFISH) technique are used to determine of the cytogenetic risk, i.e., the probability for transmissible aberrations in surviving lymphocytes. The analysis shows that the cytogenetic risk after α exposure is lower than after X rays. This indicates that the actually observed higher carcinogenic effect of α radiation is likely to stem from small scale mutations that are induced effectively by high-LET radiation but cannot be resolved by mFISH analysis.

Clinical and translational radiation oncology 13, 57 - 63 (2018). doi:10.1016/j.ctro.2018.10.002, Background and purpose:Chromosomal aberrations in peripheral blood lymphocytes are a biomarker for radiation exposure and are associated with an increased risk for malignancies. To determine the long-term cytogenetic effect of radiotherapy, we analyzed the persistence of different aberration types up to 2.5 years after C-ion boost + IMRT treatment.Materials and Methods:Cytogenetic damage was analyzed in lymphocytes from 13 patients that had undergone C-ion boost + IMRT treatment for prostate cancer. Samples were taken immediately, 1 year and 2.5 years after therapy. Aberrations were scored using the mFISH technique and grouped according to their transmissibility to daughter cells.Results:Dicentric chromosomes (non-transmissible) and translocations (transmissible) were induced with equal frequencies. In the follow-up period, the translocation yield remained unchanged while the yield of dicentrics decreased to ≈40% of the initial value (p=0.011 and p=0.001 for 1 and 2.5 years after compared to end of therapy). In 2 patients clonal aberrations were observed; however they were also found in samples taken before therapy and thus were not radiotherapy induced.Conclusion:The shift in the aberrations spectrum towards a higher fraction of translocations indicates the exposure of hematopoietic stem and progenitor cells underlining the importance of a careful sparing of bone marrow during radiotherapy to minimize the risk for secondary cancers., Published by Elsevier, Amsterdam

In the present exploratory study, we aim to elucidate the action of radon in vivo and to assess the possible health risks. Chromosome aberrations were analyzed in lymphocytes of two patients (P1, P2) undergoing radon spa therapy in Bad Steben (Germany). Both patients, suffering from painful chronic degenerative disorders of the spine and joints, received nine baths (1.2 kBq/L at 34 °C) over a 3-week period. Chromosome aberrations were analyzed before and 6, 12 and 30 weeks after the start of therapy using the high-resolution multiplex fluorescence in situ hybridization (mFISH) technique. For comparison, the lymphocytes from two healthy donors (HD1, HD2) were examined. P1 had a higher baseline aberration frequency than P2 and both healthy donors (5.3 ± 1.3 vs. 2.0 ± 0.8, 1.4 ± 0.3 and 1.1 ± 0.1 aberrations/100 analyzed metaphases, respectively). Complex aberrations, biomarkers of densely ionizing radiation, were found in P1, P2 and HD1. Neither the aberration frequency nor the fraction of complex aberrations increased after radon spa treatment, i.e., based on biological dosimetry, no increased health risk was found. It is worth noting that a detailed breakpoint analysis revealed potentially clonal aberrations in both patients. Altogether, our data show pronounced inter-individual differences with respect to the number and types of aberrations, complicating the risk analysis of low doses such as those received during radon therapy.

International audience; Purpose: Our study aimed at evaluating: 1) whether well-established variability in radioadaptive response (AR) in various donor blood lymphocytes might be attributed to inter-individual differences in radiosensitivity to different low dose levels; 2) whether AR is reproducibly present over time in the lymphocytes of AR-positive individuals.Experimental procedure: Whole blood samples of three donors were exposed to low doses (2–30 cGy) of γ-radiation alone (G0 phase) or followed by a 1 Gy challenge dose (late S/early G2 phase), and chromosome aberration were scored to assess the dose-response relationship and adaptive response, correspondingly. Three experiments were performed on blood samples of the same donors at six month intervals.Results: Significant differences in dose response relationship for blood lymphocytes were found among individuals. In most cases, the donors exhibited initial low-dose hypersensitivity (HRS) followed by an increase in radioresistance (IRR). AR could be successfully induced by some particular priming doses in the lymphocytes of each donor; however, the doses resulting in a protective response were quite different for all three donors. These protective doses could equally belong to either HRS or IRR region on the individual dose-response curves. In most cases, no clear AR outcome dependence on the priming dose was found at all. Moreover, pre-exposure to the same low dose could result in opposite effects in the lymphocytes of the same donor in different experiments.Conclusions: AR variability in human lymphocytes is not attributed to variation in radiosensitivity among individuals and is more drastic than was believed. It seems doubtful that AR is a universal phenomenon which has a consistent impact on the effects of radiation exposure on humans.

This study is based on our already published experimental data (Kowalska et al. in Radiat Environ Biophys 58:99–108, 2019) and is devoted to modeling of chromosome aberrations in human lymphocytes induced by 22.1 MeV/u 11B ions, 199 MeV/u 12C ions, 150 MeV and spread-out Bragg peak (SOBP) proton beams as well as by 60Co γ rays. The curvature of the dose–effect curves determined by the linear-quadratic model was considered in the frame of a simple analytical approach taking into account increase in the irradiation dose due to overlapping interaction regions of ion tracks. The model enabled to estimate effective interaction radius which could be compared with the physical expectations. The results were also compared to the Amorphous Track Structure Model of Katz which allows to get some additional information about the ion track structure. The analysis showed that the curvature of the experimental dose–effect curves mainly results from highly efficient repair processes of the DNA damage.

International audience; The biological phenomena observed at low doses of ionizing radiation (adaptive response, bystander effects, genomic instability, etc.) are still not well understood. While at high irradiation doses, cellular death may be directly linked to DNA damage, at low doses, other cellular structures may be involved in what are known as non-(DNA)-targeted effects. Mitochondria, in particular, may play a crucial role through their participation in a signaling network involving oxygen/nitrogen radical species. According to the size of the implicated organelles, the fluctuations in the energy deposited into these target structures may impact considerably the response of cells to low doses of ionizing irradiation. Based on a recent simulation of these fluctuations, a theoretical framework was established to have further insight into cell responses to low doses of photon irradiation, namely the triggering of radioresistance mechanisms by energy deposition into specific targets. Three versions of a model are considered depending on the target size and on the number of targets that need to be activated by energy deposition to trigger radioresistance mechanisms. These model versions are applied to the fraction of radiation-induced chromosomal aberrations measured at low doses in human carcinoma cells (CAL51). For this cell line, it was found in the present study that the mechanisms of radioresistance could not be triggered by the activation of a single small target (nanometric size, 100 nm), but could instead be triggered by the activation of a large target (micrometric, 10μm) or by the activation of a great number of small targets. The mitochondria network, viewed either as a large target or as a set of small units, might be concerned by these low-dose effects.

Chromosome aberrations in human lymphocytes exposed to different doses of particle radiation: 150 MeV and spread out Bragg peak proton beams, 22 MeV/u boron beam and 199 V/u carbon beam were studied. For comparison, an experiment with 60Co γ-rays was also performed. We investigated distributions of aberration frequency and the shape of dose–response curves for the total aberration yield as well as for exchange and non-exchange aberrations, separately. Applying the linear-quadratic model, we could derive a relation between the fitted parameters and the ion track radius which could explain experimentally observed curvature of the dose–response curves. The results compared with physical expectations clearly show that the biological effects of cell repair are much more important than the ion track overlapping.

Hematopoietic stem and progenitor cells (HSPC), that is, the cell population giving rise not only to all mature hematopoietic lineages but also the presumed target for leukemic transformation, can transmit (adverse) genetic events, such as are acquired from chemotherapy or ionizing radiation. Data on the repair of DNA double-strand-breaks (DSB) and its accuracy in HSPC are scarce, in part contradictory, and mostly obtained in murine models. We explored the activity, quality and molecular components of DSB repair in human HSPC as compared with mature peripheral blood lymphocytes (PBL). To consider chemotherapy/radiation-induced compensatory proliferation, we established cycling HSPC cultures. Comparison of pathway-specific repair activities using reporter systems revealed that HSPC were severely compromised in non-homologous end joining and homologous recombination but not microhomology-mediated end joining. We observed a more pronounced radiation-induced accumulation of nuclear 53BP1 in HSPC relative to PBL, despite evidence for comparable DSB formation from cytogenetic analysis and γH2AX signal quantification, supporting differential pathway usage. Functional screening excluded a major influence of phosphatidylinositol-3-OH-kinase (ATM/ATR/DNA-PK)- and p53-signaling as well as chromatin remodeling. We identified diminished NF-κB signaling as the molecular component underlying the observed differences between HSPC and PBL, limiting the expression of DSB repair genes and bearing the risk of an inaccurate repair.

Background and purpose To investigate the cytogenetic damage in blood lymphocytes of patients treated for prostate cancer with different radiation qualities and target volumes. Materials and methods Twenty patients receiving carbon-ion boost irradiation followed by IMRT or IMRT alone for the treatment of prostate cancer entered the study. Cytogenetic damage induced in peripheral blood lymphocytes of these patients was investigated at different times during the radiotherapy course using Giemsa staining and mFISH. A blood sample from each patient was taken before initiation of radiation therapy and irradiated in vitro to test for individual radiosensitivity. In addition, in vitro dose–effect curves for the induction of chromosomal exchanges by X-rays and carbon ions of different energies were measured. Results The yield of chromosome aberrations increased during the therapy course, and the frequency was lower in patients irradiated with carbon ions as compared to patients treated with IMRT with similar target volumes. A higher frequency of aberrations was measured by increasing the target volume. In vitro , high-LET carbon ions were more effective than X-rays in inducing aberrations and yielded a higher fraction of complex exchanges. The yield of complex aberrations observed in vivo was very low. Conclusion The investigation showed no higher aberration yield induced by treatment with a carbon-ion boost. In contrast, the reduced integral dose to the normal tissue is reflected in a lower chromosomal aberration yield when a carbon-ion boost is used instead of IMRT alone. No cytogenetic "signature" of exposure to densely ionizing carbon ions could be detected in vivo .

Exposure of the embryo to ionizing radiation (IR) is detrimental as it can cause genotoxic stress leading to immediate and latent consequences such as functional defects, malformations, or cancer. Human embryonic stem (hES) cells can mimic the preimplantation embryo and help to assess the biological effects of IR during early development. In this study, we describe the alterations H9 hES cells exhibit after X-ray irradiation in respect to cell cycle progression, apoptosis, genomic stability, stem cell signaling, and their capacity to differentiate into definitive endoderm. Early postirradiation, hES cells responded with an arrest in G2/M phase, elevated apoptosis, and increased chromosomal aberrations. Significant downregulation of stem cell signaling markers of the TGF beta-, Wnt-, and Hedgehog pathways was observed. Most prominent were alterations in the expression of activin receptors. However, hES cells responded differently depending on the culture conditions chosen for maintenance. Enzymatically passaged cells were less sensitive to IR than mechanically passaged ones showing fewer apoptotic cells and fewer changes in the stem cell signaling 24 h after irradiation, but displayed higher levels of chromosomal aberrations. Even though many of the observed changes were transient, surviving hES cells, which were differentiated 4 days postirradiation, showed a lower efficiency to form definitive endoderm than their mock-irradiated counterparts. This was demonstrated by lower expression levels of SOX17 and microRNA miR-375. In conclusion, hES cells are a suitable tool for the IR risk assessment during early human development. However, careful choice of the culture methods and a vigorous monitoring of the stem cell quality are mandatory for the use of these cells. Exposure to IR influences the stem cell properties of hES cells even when immediate radiation effects are overcome. This warrants consideration in the risk assessment of radiation effects during the earliest stages of human development.

In tomographic optoacoustic imaging, multiple parameters related to both light and ultrasound propagation characteristics of the medium need to be adequately selected in order to accurately recover maps of local optical absorbance. Speed of sound in the imaged object and surrounding medium is a key parameter conventionally assumed to be uniform. Mismatch between the actual and predicted speed of sound values may lead to image distortions but can be mitigated by manual or automatic optimization based on metrics of image sharpness. Although some simple approaches based on metrics of image sharpness may readily mitigate distortions in the presence of highly contrasting and sharp image features, they may not provide an adequate performance for smooth signal variations as commonly present in realistic whole-body optoacoustic images from small animals. Thus, three new hybrid methods are suggested in this work, which are shown to outperform well-established autofocusing algorithms in mouse experiments in vivo.

We investigated induction of chromosome aberrations (CA) in human lymphocytes when exposed to 150 MeV and spread out Bragg peak (SOBP) proton beams, and 199 MeV/u carbon beam which are currently widely used for cancer treatment and simultaneously are important components of cosmic radiation. For a comparison, the boron ions of much lower energy 22 MeV/u and a 60Co γ rays were used. Dose–effect curves as well as the distributions of CA were studied using Poisson and Neyman type A statistics. Systematics of experimentally determined parameters, their dependence on applied doses and irradiation quality are presented.

The relationship between heavy-ion-induced cell cycle delay and the time-course of aberrations in first-cycle metaphases or prematurely condensed G(2)-cells (G(2)-PCC) was investigated. Lymphocytes of the same donor were irradiated with X-rays or various charged particles (carbon, iron, xenon, and chromium) covering an LET range of 2-3,160 keV/μm. Chromosome aberrations were measured in samples collected at 48, 60, 72, and 84 h postirradiation. Linear-quadratic functions were fitted to the data, and the fit parameters α and β were determined. At any sampling time, α values derived from G(2)-cells were higher than those from metaphases. The α value derived from metaphase analysis at 48 h increased with LET, reached a maximum around 155 keV/μm, and decreased with a further rise in LET. At the later time-points, higher α values were estimated for particles with LET > 30 keV/μm. Estimates of α values from G(2)-cells showed a similar LET dependence, yet the time-dependent increase was less pronounced. Altogether, our data demonstrate that heavily damaged lymphocytes suffer a prolonged G(2)-arrest that is clearly LET dependent. For this very reason, the standard analysis of aberrations in metaphase cells 48 h postirradiation will considerably underestimate the effectiveness of high-LET radiation. Scoring of aberrations in G(2)-PCC at 48 h as suggested by several authors will result in higher aberration yields. However, when particles with a very high-LET value (LET > 150 keV/μm) are applied, still a fraction of multiple damaged cells escape detection by G(2)-analysis 48 h postirradiation.

The biological response to high linear energy transfer (LET) radiation differs considerably from that to low LET radiation and this has been attributed to differences in the spatial energy deposition of both radiation qualities. In the case of X-rays the energy is deposited uniformly within the cell nucleus and produces damages in a purely stochastic manner. In contrast, for particles the energy is deposited inhomogeneously along the ion trajectory and the local dose decays with the square radial distance from the center of the track. This nonuniformity affects the yield and the distribution of aberrations among cells. Moreover, after high LET exposure a relationship between the aberration yield and cell cycle delay was observed. In this study, we present a detailed analysis of the distribution of aberrations in human lymphocytes reaching mitosis at early and later times after low and high LET exposure. Aberration data were fit to stochastic distributions demonstrating that the delay is related to the number of particle traversals per cell nucleus. To further elucidate this relationship, we introduce a Monte Carlo phenomenological model which incorporates the number of particle hits per nucleus. This value was derived by fitting theoretical distributions to the experimental data. Additionally, the probability that a cell traversed by a particle reaches mitosis at a given time was calculated. The analysis of biological data and numerical simulations clearly show the impact of the track structure on the formation of chromosome aberrations and their distribution among cells.

To investigate further the relationship between high linear energy transfer (LET) induced cell cycle arrests and the yield of chromosome aberrations observable in normal human fibroblasts at the first post-irradiation mitosis.Normal human fibroblasts (AG01,522C) were exposed in G0/G1 to either 11 MeV u(-1) C ions (LET = 153.5 keV microm(-1)) or 9.9 MeV u(-1) Ni ions (LET = 2,455 keV microm(-1)), subcultured in medium containing 5-Bromo-2'-deoxyuridine (BrdU) and at multiple time-points post-irradiation the yield of chromosomal damage, the mitotic index and the cumulative BrdU-labelling index were determined. Furthermore, a mathematical approach was used to analyse the entire cell population.Following high LET exposure normal fibroblasts suffer a transient delay into S-phase and into mitosis as well as a prolonged, probably permanent cell cycle arrest in the initial G0/G1-phase. Cells that reach the first mitosis at early times carried less aberrations than those collected at later times indicating a relationship between cell cycle delay and the number of aberrations. However, with respect to the whole cell population, only a few aberrant fibroblasts are able to progress to the first mitosis. For all endpoints studied the relative biological effectiveness (RBE) of C ions is in the range of 2 - 4, while for Ni ions RBE1 is estimated. In contrast, when compared on a per particle basis Ni ions with the higher ionization density were found to be more effective.Detailed analysis of the data demonstrates that the number of fibroblasts at risk for neoplastic transformation is significantly reduced by a chronic cell cycle arrest in the initial G0/G1-phase and, for the first time, the LET-dependence of this effect has been shown.

Chromosome aberration data obtained for various types of mammalian cells after exposure to low and high LET radiation clearly demonstrate differences in the energy deposition pattern of both radiation qualities. In the present study we focus on the distributions of chromosome aberrations induced in human peripheral blood lymphocytes after exposure to 990 MeV/u Fe ions (LET = 155 keV/μm) or X-rays. For the analysis three different types of distributions were applied, namely a Poisson distribution, a compound Poisson–Poisson (Neyman type A) distribution and a convoluted Poisson–Neyman distribution. The analysis showed that after low LET radiation the distribution of aberrations can be well described by Poisson statistics, reflecting a simple random distribution of damages as expected according to the homogeneous pattern of energy depositions. In contrast, for particles the energy is deposited spatially very inhomogeneous and concentrated along the ion trajectories. After exposure to high energy, high LET particles where the track radius is much larger than the cell nucleus, best fits to the data were achieved by a convoluted Poisson–Neyman statistics. The analysis indicates that, under this exposure condition, the distribution of aberrations is determined by two independent components. The first component is determined by the damage induced by a center of the tracks and follows the Neyman distribution. The second component is determined by the overlapping part of tracks which in the case of very high energetic particles leads to a “photon-like” background dose and is thus characterized by a Poisson distribution.

The speed of sound (SoS) in the imaged sample and in the coupling medium is an important parameter in optoacoustic tomography that must be specified in order to accurately restore maps of local optical absorbance. In this work, several hybrid focusing functions are described that successfully determine the most suitable SoS based on post-reconstruction images. The SoS in the coupling medium (water) can be determined from temperature readings. Thereby, this value is suggested to be used as an initial guess for faster SoS calibration in the reconstruction of tissues having a different SoS than water.

Summary The premature chromosome condensation (PCC) technique has been used to compare chromatin breakage and repair in non-cycling CHO-K1 cells following high LET (C ions) and low LET (X-rays) irradiation. For both radiation qualities the average initial number of excess PCC fragments increases linearly with dose. However, the frequency of chromatin breaks follows the pattern of energy deposition and at higher LET values reveals clustering due to the large number of ionizing events being concentrated in a small volume of the cell nucleus. In consequence, the distribution of PCC chromosomes plus excess fragments among cells has followed Poisson statistics after X-ray irradiation while the overdispersion of the frequencies has been observed after C-irradiation indicating that a single particle traversal through a cell nucleus can produce multiple chromatin lesions.

To examine the relationship between cell proliferation and the expression of chromosomal damage in normal human skin fibroblasts after X-ray and particle irradiation.Confluent G0/G1 AG1522B cells were exposed to X-rays or 195MeV u(-1) C ions with a linear energy transfer of 16.6 keV microm(-1) in the dose range 1-4 Gy. Directly after irradiation, cells were reseeded at a low density in medium containing 5-bromo-2'-deoxyuridine. At multiple time points post-irradiation, the cumulative BrdU-labelling index, mitotic index and aberration frequency were measured. Based on these data, the total amount of damage induced within the entire cell population was estimated by means of mathematical analysis.Both types of radiation exposure exert a pronounced effect on the cell cycle progression of fibroblasts. They result in delayed entry of cells into S-phase and into the first mitosis, and cause a dramatic reduction in mitotic activity. Measurement of chromosomal damage in first-cycle cells at multiple time points post-irradiation shows that the frequencies of aberrant cells and aberrations increase with time up to twofold for the lower doses. However, for the higher doses, this effect is less pronounced or even disappears. When the data for the whole cell population are analysed, it becomes evident that only a few damaged fibroblasts can progress to the first mitosis, a response attributable at least in part to a long-term arrest of injured cells in the initial G0/G1-phase. As observed in other investigations, the effectiveness of 195 MeV u(-1) C ions was similar or slightly higher than X-rays for all endpoints studied leading to a relative biological effectiveness in the range 1.0-1.4.Cell cycle arrests affect the aberration yield observable in normal human fibroblasts at mitosis. The data obtained for the cell population as a whole reveal that injured cells are rapidly removed from the mitotically active population through a chronic cell cycle arrest, which is consistent with other studies that indicate that this response is a specific strategy of fibroblasts to minimize the fixation and propagation of genetic alterations.

In the present study, we set out to investigate cytogenetic changes in the progeny of two normal human fibroblast cell strains after exposure to sparsely or densely ionizing irradiation (X-rays or 9.8 MeV u −1 carbon ions). The cells were regularly subcultured up to senescence. The transition to senescence was determined by measurement of population doubling numbers and senescence associated (SA) β-galactosidase activity. Chromosomal changes (structural aberrations, tetraploidy) were investigated by solid staining. In temporal proximity to senescence, we observed for all populations of the two fibroblasts cell strains an increase in the fraction of cells with structural and numerical aberrations. The observed changes in the yield of structural chromosomal aberrations were similar for the progeny of controls and irradiated cells, except that a previous irradiation with a high, fractionated X-ray dose resulted in a stronger increase. Noteworthy, delayed tetraploidy in the descendants of irradiated cells exceeded the level in control cells. In addition, tetraploidy and the time of onset of senescence were significantly correlated for all populations, regardless of a preceding radiation exposure. However, the time of the onset of senescence depends on previous exposure to radiation. We conclude that the occurrence of tetraploidy is associated with senescence independently of exposure to radiation.

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.

human lymphocytes, heavy ions, time-course of aberrations, calyculin A, apoptosis The time-course of Fe-ion (200 MeV/u, 440 keV/µm) and X-ray induced chromosomal damage was investigated in human lymphocytes. After cells were exposed in G0 and stimulated to grow, aberrations were measured in first-cycle metaphases harvested 48, 60 and 72h post-irradiation. Additionally, lesions were analysed in G2 and mitotic (M) cells collected at 48h using calyculin A-induced premature chromosome condensation (PCC). Following X-irradiation, similar aberration yields were found in all of the samples scored. In contrast, after Fe-ion exposure a drastic increase in the aberration frequency with sampling time was observed, i.e. cells arriving late at the first mitosis carried more aberrations than those arriving at earlier times. The PCC data indicate that the delayed entry of heavily damaged cells into mitosis observed after Fe-ion irradiation resulted from a prolonged arrest in G2. Altogether these experiments provide further evidence that in the case of high-LET exposure cell-cycle delays of severely damaged cells have to be taken into account for any meaningful quantification of chromosomal damage and, consequently, for an accurate estimate of the RBE.

The premature chromosome condensation technique has been used to compare chromatin breakage and repair in noncycling V79 cells following high and low LET radiation.Plateau-phase V79 cells were exposed to graded doses of low energy Ar ions (LET 1233 keV/microm) and X-rays. Cells were fused to mitotic V79 cells immediately after exposure to examine initial chromatin breakage or after various time intervals of post-irradiation incubation to investigate the kinetics of chromatin break rejoining as well as the fraction of unrejoined fragments.For both radiation qualities an average initial number of about 2.4 excess PCC fragments per cell per Gy was found increasing linearly with dose. The distributions of PCC chromosomes plus excess fragments among cells followed Poisson statistics after X-ray irradiation, while an overdispersion of the frequencies was observed after Ar-irradiation indicating that a single particle traversal through a cell nucleus can produce multiple chromatin lesions. Moreover, for both radiation types the rejoining of excess fragments has been examined. Both data sets could be fitted well to first-order kinetics with a single component. Despite similar rates of rejoining cellular repair was noticeably less effective for Ar ions than for X-rays. While after 10 h of post-irradiation incubation 60% of Ar ion induced excess fragments remained unrejoined, only 14% of X-ray-induced lesions were not rejoined. Furthermore, comparison of the residual number of excess PCC fragments with recently published data on the yield of chromosome aberrations in first post-irradiation metaphases shows that for both radiation types more aberrations are detected in interphase than in metaphase cells. Yet, for comparable doses this difference is more pronounced for Ar ions indicating that scoring of high LET induced aberrations in metaphase cells might result in a significant underestimation of the produced damage.

The risk of radiation exposure during embryonic development is still a major problem in radiotoxicology. In this study we investigated the response of the murine embryonic stem cell (mESC) line D3 to two radiation qualities: sparsely ionizing X-rays and densely ionizing carbon ions. We analyzed clonogenic cell survival, proliferation, induction of chromosome aberrations as well as the capability of cells to differentiate to beating cardiomyocytes up to 3 days after exposure. Our results show that, for all endpoints investigated, carbon ions are more effective than X-rays at the same radiation dose. Additionally, in long term studies (≥8 days post-irradiation) chromosomal damage and the pluripotency state were investigated. These studies reveal that pluripotency markers are present in the progeny of cells surviving the exposure to both radiation types. However, only in the progeny of X-ray exposed cells the aberration frequency was comparable to that of the control population, while the progeny of carbon ion irradiated cells harbored significantly more aberrations than the control, generally translocations. We conclude that cells surviving the radiation exposure maintain pluripotency but may carry stable chromosomal rearrangements after densely ionizing radiation.

In the present study we examined the cytogenetic effects of 177 MeV/u Fe-ions (LET = 335 keV/μm) and 4.1 MeV/u Cr-ions (LET = 3160 keV/μm) in human lymphocytes under exposure conditions that result on average in one particle hit per cell nucleus. In non-cycling (G0-phase) lymphocytes the induction and the repair of excess fragments was measured by means of the premature chromosome condensation (PCC) technique and the distribution of breaks among cells was analysed. The PCC-data were further compared with those reported recently for stimulated lymphocytes at the first post-irradiation mitosis. Our experiments show that a single nuclear traversal by a Fe-ion produced more initial chromatin breakage than one Cr-ion, but after 24 h of repair the number of excess fragments/cell was similar for both ion species. All distributions of aberrations were overdispersed. For low energy Cr-ions, where the track radius is smaller than the radius of the cell nucleus, the data could be well described by a Neyman type A distribution. In contrast, the data obtained for high energy Fe-ions were fitted with a convoluted Poisson–Neyman distribution to account for the fact that the dose is deposited not only in the cell actually traversed but also in neighbouring cells. By applying metaphase analysis a different picture emerged with respect to the aberration yield, i.e. more aberrations were detected in cells exposed to Fe-ions than in those irradiated with Cr-ions. Yet, as observed for non-cycling lymphocytes all aberration distributions generated for metaphase cells were overdispersed. The obtained results are discussed with respect to differences in particle track structure. Additionally, the impact of confounding factors such as apoptosis that affect the number of aberrations expressed in a cell population is addressed.

The amount of chromosomal damage induced in synchronous V79 cells by either 250 kV X-rays or 4.6 MeV/u Ar ions (LET: 1850 keV/microns) was determined at five successive sampling times. The experiments show that the time course of the appearance of damaged cells is strongly influenced by radiation-induced cell cycle perturbations and mitotic delay and depends on radiation quality and dose. The yield of chromosomal damage was found to increase with sampling time, but this increase was more pronounced for Ar ions. Because of the observed differences in the yield time profiles induced by sparsely and densely ionizing radiation the contribution of each sample to the overall damage was considered, i.e. the total (time integrated) amount of damage was determined. The obtained data are interpreted in terms of differences in the spatial energy deposition by sparsely and densely ionizing radiation.

Embryonic stem cells (ESC) are characterized by both the capacity of infinite self-renewal and the ability to give rise to all the three germ layers emphasizing the need to strictly control the genetic integrity. To date, ESC are a powerful tool in disease modeling, tissue engineering and drug testing. However, in the field of radiation research, their potential has not been exploited. We used the mouse ESC line D3 as a model to examine the effects of X-rays or C-ions (spread out Bragg peak, energy 106–147 MeV/u, average LET = 75 keV/µm) [ 1]. Doses of 0.5–5 Gy were applied and endpoints such as cell cycle progression (measured by flow cytometry), apoptosis (microscopic analysis of cell nucleus morphology), induction of chromosome aberrations (mFISH analysis), presence of pluripotency markers Oct3/4 and SOX2 (western blotting) and differentiation capacity by means of an embryoid body formation assay were analyzed up to 17 days post-irradiation. The experiments show that cells undergo a transient G2 arrest following exposure. After G2 checkpoint release, an increase in the apoptotic index is observed for both radiation types (3.7-fold increase for 2 Gy X-ray and 2.4-fold increase for 2 Gy C-ions). C-ions induce more structural chromosomal aberrations in first cycle cells than X-rays. During subsequent cell divisions, the frequency of chromosome aberrations declines: After >7 population doublings (8 days after exposure), the aberration frequency in the progeny of X-ray exposed cells returns to the control level (7% aberrant cells), while the progeny of C-ion exposed cells still harbor significantly more aberrations than control cells, which is mainly due to transmissible translocations. The expression of pluripotency markers is maintained in cells surviving X-ray or C-ion exposure. This finding is supported by examining the differentiation capacity of ESC through the formation of embryoid bodies. Our experiments show that after X-ray or C-ion exposure, cells are able to develop spontaneous beating activity, indicating the differentiation ability into mesodermal cell lineages, i.e. beating cardiomyocytes. However, following C-ion exposure, the formation of beating clusters was delayed compared with control cells. Moreover, our chromosome studies revealed that unexposed cells carry a high frequency of numerical aberrations. These comprise trisomies of chromosome 8 and 11 with a frequency of 29 ± 8% and 26 ± 6% respectively, as well as nullisomy of chromosome Y with a frequency of 35 ± 3%. Aneuploidy is a typical feature of mouse ESC and has been related to cell culture methods [ 2] and passage number. Because aneuploidy may affect gene expression and influence the properties of a cell population, the relevance of experiments based on mouse ESC is limited. To overcome this problem, we recently extended our studies to human ESC. Human ESC are known to be cytogenetically more stable than mouse ESC, and represent a model that is closer to human embryonic development. Indeed, first investigations revealed a lower faction of cells with numerical and structural aberrations in the human ESC line H9 [ 3] compared with the mouse ESC line D3 (2% vs. 73% and 3% vs. 7%, respectively).

Ionizing radiation is known to delay the cell cycle progression. In particular after particle exposure significant delays have been observed and it has been shown that the extent of delay affects the expression of damage such as chromosome aberrations. Thus, to predict how cells respond to ionizing radiation and to derive reliable estimates of radiation risks, information about radiation-induced cell cycle perturbations is required. In the present study we describe and apply a method for retrieval of information about the time-course of all cell cycle phases from experimental data on the mitotic index only. We study the progression of mammalian cells through the cell cycle after exposure. The analysis reveals a prolonged block of damaged cells in the G2 phase. Furthermore, by performing an error analysis on simulated data valuable information for the design of experimental studies has been obtained. The analysis showed that the number of cells analyzed in an experimental sample should be at least 100 to obtain a relative error less than 20%., Comment: 19 pages, 11 figures, accepted for publication in Radiation and Environmental Biophysics

Carbon ions are successfully used in tumour treatment for more than 10 years in Chiba and Hyogo (Japan) and at GSI Darmstadt (Germany). Carbon ions are beneficial for the treatment of deep seated tumours, as they deliver the highest dose at the end of their range, and in addition this dose shows an enhanced radiobiological effectiveness. In a clinical study at GSI and at the Clinical Radiology Heidelberg carbon ion therapy is combined with conventional IMRT to treat prostate cancer patients (intermediate risk). In parallel, a study investigating chromosome aberrations in blood lymphocytes of prostate cancer patients is performed. Chromosome aberrations in blood lymphocytes are widely used in biological dosimetry as they represent a sensitive marker for ionizing radiation. Since they are unavoidably exposed to radiation during tumour treatment, this study makes it possible to investigate the cytogenetic effects of carbon ion irradiation in vivo and to compare with conventional tumour treatment. The results may be helpful regarding biological dosimetry, as only very little is known about the effects of partial body irradiation with heavy ions. In this clinical trial, prostate cancer patients are irradiated with a Carbon ion boost (6x3GyE) followed by IMRT (30x2Gy) or solely with IMRT (38x2Gy). Blood samples are drawn from each patient before, during, at the end of and one year after therapy. Lymphocytes are isolated and cultured according to standard techniques, chromosome spreads are prepared and slides are stained using Fluorescence-Plus-Giemsa staining (FPG) and multiplex fluorescence in situ hybridization (mFISH), respectively. In addition, in vitro irradiation experiments with lymphocytes are performed. The number of aberrations found in lymphocytes of prostate cancer patients increases during therapy course, and decreases slightly during the first year after treatment. There is up to now no significant difference visible between patients treated with combined therapy of carbon ions plus IMRT (n=9) and patients treated solely with IMRT (n=2). In addition, prostate cancer patients treated with a larger irradiation field IMRT were included in the study (n=3). The lymphocytes of these patients showed a significantly higher amount of aberrations during therapy course as well as one year after treatment. At the present status of this study, we can conclude that an important influence of irradiation field size on the induced damage in surrounding tissue exists, but that under the given treatment conditions differences in the radiation quality are not observable at chromosomal level.

Background and purpose The goal of the present study was to investigate aging and genetic instability in the progeny of human fibroblasts exposed to X-rays and carbon ions. Materials and methods Following irradiation, cells were regularly subcultured until senescence. At selected time-points BrdU-labelling index, expression of cell cycle related proteins, cell differentiation pattern and chromosome aberrations were assessed. Results After exposure, an immediate cell cycle arrest occurred followed by a period of a few weeks where premature differentiation and senescence were observed. In all cultures cycling cells expressing low levels of cell cycle inhibiting proteins were present and finally dominated the populations. About 5 months after exposure, the cellular and molecular changes attributed to differentiation and senescence reappeared and persisted. Concurrently, genetic instability was observed, but the aberration yields and types differed between repeated experiments. The descendants of cells exposed to carbon ions did not senesce earlier and displayed a similar rate of genetic instability as the X-ray progeny. For high doses an impaired cell cycle regulation and extended life span was observed, but finally cell proliferation ceased in all populations. Conclusions The descendants of irradiated fibroblasts undergo stepwise senescence and differentiation. Genetic instability is frequent and an extension of the life span may occur.

Chromosome damage and the spectrum of aberrations induced by low doses of gamma-irradiation, X-rays and accelerated carbon ions (195 MeV/u, LET 16.6 keV/microm) in peripheral blood lymphocytes of four donors were studied. G0-lymphocytes were exposed to 1-100 cGy, stimulated by PHA, and analyzed for chromosome aberrations at 48 h post-irradiation by the metaphase method. A complex nonlinear dose-effect dependence was observed over the range of 1 to 50 cGy. At 1-7 cGy, the cells showed the highest radiosensitivity per unit dose (hypersensitivity, HRS), which was mainly due to chromatid-type aberration. According to the classical theory of aberration formation, chromatid-type aberrations should not be induced by irradiation of unstimulated lymphocytes. With increasing dose, the frequency of aberrations decreased significantly, and in some cases it even reached the control level. At above 50 cGy the dose-effect curves became linear. In this dose range, the frequency of chromatid aberrations remained at a low constant level, while the chromosome-type aberrations increased linearly with dose. The high yield of chromatid-type aberrations observed in our experiments at low doses confirms the idea that the molecular mechanisms which underlie the HRS phenotype may differ from the classical mechanisms of radiation-induced aberration formation. The data presented, as well as recent literature data on bystander effects and genetic instability expressed as chromatid-type aberrations on a chromosomal level, are discussed with respect to possible common mechanisms underlying all low-dose phenomena.

The aim was to investigate further the relationship between radiation-induced mitotic delay and the expression of chromosome damage in V79 cells. Recently published data on the time-course of chromosome aberrations in V79 first-cycle metaphases after exposure to 10.4 MeV u(-1) Ar ions (LET = 1226 keV microm(-1)) were supplemented and reanalysed. A statistical analysis of the distribution of aberrations among cells was performed. Furthermore, cells were grouped into subpopulations carrying 0, 1 -2, 3-4, 5- 6 and 7 or more aberrations. Then, based on the mitotic index, the flux of each subgroup through the first mitosis was determined and the average entrance time to mitosis was estimated. For comparison, the flux of aberrant V79 cells generated by X-irradiation was analysed. Analysis of the Ar ion data revealed that the flux of each subpopulation through the first mitosis is strongly affected by its aberration burden, i.e. a positive correlation between the mitotic delay and the number of aberrations carried by a cell was observed. The distribution of aberrations among cells could be well described by Neyman-type A statistics; the corresponding fit parameters also reflect the damage-dependent mitotic delay. Interestingly, comparison of the flux of Ar ion and X-ray-irradiated V79 cells through mitosis revealed (1) that a direct correlation exists between the number of aberrations carried by a cell and its average entrance time to mitosis, and (2) that this effect is independent of the linear energy transfer. The role of these observations for radiation cytogenetics is discussed.

In the present paper the relationship between cell cycle delays induced by Fe-ions of differing LET and the aberration yield observable in human lymphocytes at mitosis was examined. Cells of the same donor were irradiated with 990 MeV/n Fe-ions (LET=155 keV/micrometers), 200 MeV/n Fe-ions (LET=440 keV/micrometers) and X-rays and aberrations were measured in first cycle mitoses harvested at different times after 48-84 h in culture and in prematurely condensed G2-cells (PCCs) collected at 48 h using calyculin A. Analysis of the time-course of chromosomal damage in first cycle metaphases revealed that the aberration frequency was similar after X-ray irradiation, but increased two and seven fold after exposure to 990 and 200 MeV/n Fe-ions, respectively. Consequently, RBEs derived from late sampling times were significantly higher than those obtained at early times. The PCC-data suggest that the delayed entry of heavily damaged cells into mitosis results especially from a prolonged arrest in G2. Preliminary data obtained for 4.1 MeV/n Cr-ions (LET=3160 keV/micrometers) revealed, that these delays are even more pronounced for low energy Fe-like particles. Additionally, for the different radiation qualities, BrdU-labeling indices and apoptotic indices were determined at several time-points. Only the exposure to low energy Fe-like particles affected the entry of lymphocytes into S-phase and generated a significant apoptotic response indicating that under this particular exposure condition a large proportion of heavily damaged cells is rapidly eliminated from the cell population. The significance of this observation for the estimation of the health risk associated with space radiation remains to be elucidated.

To investigate further the effect of linear energy transfer (LET) on the yield and quality of aberrations at different post-irradiation sampling times.V79 G(1)-cells were exposed to either 10.6 MeV u-1 Ne ions (360 keV microm-1) or 11.1 MeV u-1 Kr ions (3980 keV microm-1) and chromosomal damage was measured in metaphase cells at several 2-h sampling intervals up to 30 h post-irradiation. To differentiate between cells in the first and second post-irradiation cycle, the fluorescence-plus-Giemsa technique was applied.In both experiments, an increase in the yield of aberrant cells as well as the number of aberrations per cell was observed in first- and second-cycle metaphases. Yet, the increase in the number of aberrations per cells was more pronounced for Kr ions and at comparable fluences Kr ions produced more aberrations than Ne ions. Because no sampling time was representative for the whole cell population, the total amount of Ne and Kr ion-induced chromosomal damage was determined by means of a mathematical approach and used for the comparison of data. Furthermore, in accordance with previous studies, LET-dependent changes in the spectrum of aberration types were detected, i.e. with increasing LET a higher fraction of chromatid-type aberrations was observed, although cells had been exposed in G1. In addition, more chromosomal breaks and less exchange-type aberrations were found.The observation that cell-cycle progression is related to the amount of aberrations harboured by a cell demonstrates that the routinely applied method to measure aberration frequencies in metaphase cells at only one post-irradiation sampling time will unavoidably result in an under- or overestimation of the cytogenetic effects of particles. Consequently, for a meaningful quantification of chromosomal damage, multiple fixation regimes should be used so that the complete time-course of aberrations can be taken into account. Moreover, to avoid bias, all aberration types should be recorded and included in the analysis since the aberration spectrum changes with LET.

The cytogenetic effects of X-rays and Au ions were investigated in repair-proficient CHO-K1 cells and their radiosensitive mutant strain xrs5, which shows a defect in the rejoining of DNA double-strand breaks. Both cell lines were synchronized by mitotic shake off, irradiated in G1-phase with either 250 kV X-rays or 780 MeV/u Au ions (LET: 1150 keV/micrometer) and chromosome aberrations were analyzed in first post-irradiation metaphases. Isoeffective doses of X-rays for the induction of aberrant cells and aberrations per cell were about 14 times lower for xrs5 than for CHO-K1 cells. After high LET radiation the difference in the cytogenetic response of both cell lines was drastically diminished. Furthermore, the analysis of the aberration types induced by sparsely and densely ionizing radiation showed for both cell lines specific changes in the spectrum of aberration types as LET increases. The experimental results are discussed with respect to the different types of lesions induced by sparsely and densely ionizing radiation.

As an extension of previous studies, the time-course of high-LET-induced chromosomal damage was investigated in first- and second-cycle V79 Chinese hamster cells.Cells were exposed in G1 to 10.4 MeV/u Ar ions (LET = 1226 keV/microm) and chromosomal damage was measured at 2h sampling intervals between 10 h and 34 h after irradiation. To distinguish between cells in different post-irradiation cycles, the fluorescence-plus-Giemsa technique was applied.For first- and second-generation cells, the number of aberrant metaphases and aberrations per metaphase were found to increase markedly with sampling time, demonstrating that cell cycle progression was delayed according to the number of lesions carried by the cell. To account for the time-dependent expression of chromosomal damage a mathematical approach was used based on the integrated flux of aberrant cells entering mitosis. Moreover, the analysis of Ar ion-induced chromosome lesions confirmed that high-LET radiation results in specific changes in the spectrum of aberration types. In particular, an increased rate of chromatid-type aberrations as well as a high frequency of chromosomal breaks was found, although the cells were exposed in G1.Due to the fact that cells collected at one sampling time are not representative of the entire population, the complete time-course of chromosomal damage has to be taken into account for the determination of a meaningful RBE value. Otherwise, the analysis of chromosomal damage can result in a pronounced over- or underestimation of the RBE depending on the subpopulation of cells entering mitosis at that particular sampling time.

Synchronous V79 Chinese hamster cells were exposed in G1 to either X-rays or 4.6 MeV/u Ar-ions (LET = 1840 keV/micrometer) and the induction of chromosomal damage was measured at five sampling times ranging from 14 to 30 h after treatment. To distinguish between cells in the first and second post-irradiation cycle the fluorescence-plus-Giemsa technique was applied. The experiment showed that the time-course of the appearance of damaged cells was markedly influenced by radiation-induced cell cycle delays and depended on both radiation quality and dose. The yield of aberrant metaphases and the number of aberrations per metaphase was found to increase with sampling time, but this increase was more pronounced for Ar ions. These differences in yield-time profiles of X-ray and Ar ion induced chromosomal damage are particularly important for an accurate determination of the RBE for particles. Our data clearly indicate that meaningful RBEs can only be obtained if chromosomal damage is analysed at several post-irradiation sampling times and the complete time-course of the expression of chromosomal damage is taken into account. Besides these quantitative differences, differences in the spectrum of chromosomal lesions were observed for X-rays and Ar ions. Following particle exposure more breaks and less exchange-type aberrations were formed compared with X-irradiation and, despite irradiation in G(1), a significant number of chromatid-type aberrations occurred in Ar-irradiated samples. The experimental results are interpreted on the basis of the different pattern of energy deposition by sparsely and densely ionizing radiation. In addition, a statistical analysis based on the Neyman type A distribution is performed, which takes into account the specific stochastic properties of particle irradiation.