Ionizing radiation responses appear incidental to desiccation responses in the bdelloid rotifer Adineta vaga.

BACKGROUND: The remarkable resistance to ionizing radiation found in anhydrobiotic organisms, such as some bacteria, tardigrades, and bdelloid rotifers has been hypothesized to be incidental to their desiccation resistance. Both stresses produce reactive oxygen species and cause damage to DNA and other macromolecules. However, this hypothesis has only been investigated in a few species. RESULTS: In this study, we analyzed the transcriptomic response of the bdelloid rotifer Adineta vaga to desiccation and to low- (X-rays) and high- (Fe) LET radiation to highlight the molecular and genetic mechanisms triggered by both stresses. We identified numerous genes encoding antioxidants, but also chaperones, that are constitutively highly expressed, which may contribute to the protection of proteins against oxidative stress during desiccation and ionizing radiation. We also detected a transcriptomic response common to desiccation and ionizing radiation with the over-expression of genes mainly involved in DNA repair and protein modifications but also genes with unknown functions that were bdelloid-specific. A distinct transcriptomic response specific to rehydration was also found, with the over-expression of genes mainly encoding Late Embryogenesis Abundant proteins, specific heat shock proteins, and glucose repressive proteins. CONCLUSIONS: These results suggest that the extreme resistance of bdelloid rotifers to radiation might indeed be a consequence of their capacity to resist complete desiccation. This study paves the way to functional genetic experiments on A. vaga targeting promising candidate proteins playing central roles in radiation and desiccation resistance.

Duplicated chromosomal fragments stabilize shortened telomeres in normal human IMR-90 cells before transition to senescence.

To assess why during in vitro aging of fibroblasts the maintenance of chromosomal stability is effective or occasionally fails, a detailed cytogenetic analysis was performed in normal human IMR-90 fetal lung fibroblasts. The onset of senescence was inferred from proliferation activity, expression pattern of cell cycle regulating proteins, activity of ß-galactosidase, and morphological features. Over the period of proliferation, a moderate increase of non-transmissible structural chromosomal aberrations was observed. In addition, using fluorescence in situ hybridization (mFISH and mBAND) techniques, we detected clonally expanding translocations in up to 70% of the analyzed metaphases, all involving one homolog of chromosome 9 as an acceptor. Notably, chromosomes are randomly involved as donor-chromosomes of the translocated terminal acentric fragments. These fragments result from duplication because the donor chromosomes are apparently unchanged. Interstitial telomeric signals were detectable at fusion sites, most likely belonging to chromosome 9. Quantitative fluorescence in situ hybridization (QFISH) detecting telomere sequences, followed by mFISH technique revealed that already in young cells the respective telomeres of one chromosome 9 were particularly short. For the first time, we have observed dysfunctional telomeres of one specific chromosome in normal human cells that have been stabilized by duplicated terminal sequences.

Chromosome aberration measurements in mitotic and G2-PCC lymphocytes at the standard sampling time of 48 h underestimate the effectiveness of high-LET particles.

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 ß 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.

Chromosome Aberrations in Lymphocytes of Patients Undergoing Radon Spa Therapy: An Explorative mFISH Study.

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.

A Human 3D Cardiomyocyte Risk Model to Study the Cardiotoxic Influence of X-rays and Other Noxae in Adults.

The heart tissue is a potential target of various noxae contributing to the onset of cardiovascular diseases. However, underlying pathophysiological mechanisms are largely unknown. Human stem cell-derived models are promising, but a major concern is cell immaturity when estimating risks for adults. In this study, 3D aggregates of human embryonic stem cell-derived cardiomyocytes were cultivated for 300 days and characterized regarding degree of maturity, structure, and cell composition. Furthermore, effects of ionizing radiation (X-rays, 0.1-2 Gy) on matured aggregates were investigated, representing one of the noxae that are challenging to assess. Video-based functional analyses were correlated to changes in the proteome after irradiation. Cardiomyocytes reached maximum maturity after 100 days in cultivation, judged by α-actinin lengths, and displayed typical multinucleation and branching. At this time, aggregates contained all major cardiac cell types, proven by the patch-clamp technique. Matured and X-ray-irradiated aggregates revealed a subtle increase in beat rates and a more arrhythmic sequence of cellular depolarisation and repolarisation compared to non-irradiated sham controls. The proteome analysis provides first insights into signaling mechanisms contributing to cardiotoxicity. Here, we propose an in vitro model suitable to screen various noxae to target adult cardiotoxicity by preserving all the benefits of a 3D tissue culture.

Heavy-ion induced chromosomal aberrations: a review.

Heavy-ion radiobiology is attracting increasing interest for its implications in radiation oncology and space radiation protection. The analysis of chromosome aberrations induced by heavy-ions started already in the 1960s, but the new FISH-painting methodologies are revealing unique features of the action of the heavy charged particles. Heavy-ions induce a high fraction of complex-type exchanges, and possibly unique chromosome rearrangements. The relative biological effectiveness for the induction of cytogenetic damage is strongly dependent on the time between irradiation and chromosome harvest, due to cell-cycle delays and loss of heavily damaged cells. In this review we will concentrate on recent data obtained with multicolor FISH methods in mammalian chromosomes exposed to heavy-ions, and the open questions that remain to be addressed.

The number and not the molecular structure of DNA double-strand breaks is more important for the formation of chromosomal aberrations: a hypothesis.

DNA double-strand breaks (DSB) are known to cause chromosomal aberrations (CA). The ends of DSB have different molecular structures depending on the inducing agent. Restriction endonucleases, DNase I and benzon nuclease produce DSB with 3'-OH and 5'-phosphate ends which should be easily rejoinable ("clean" DSB). Other DSB-inducing agents, such as neocarzinostatin, bleomycin and ionizing radiation induce DSB with chemically modified ends ("dirty" DSB) which are not religatable before enzymatic pruning to make them "clean". Both "clean" and "dirty" DSB lead to CA whose quantities and distributions are quite similar. We conclude that the number and not the molecular structures of DSB are essential for the production of CA. All DSB-inducing agents discussed in this review induce exchange events in the G1 phase of the cell cycle which look like sister chromatid exchanges (SCE) and are therefore called "false" SCE. "False" SCE seem to be mainly intrachanges and would therefore outweigh the frequencies of interchanges. Consequently, the factor F of inter- to intrachanges would then be <1, indicating that the majority of DSB would lead to CA inside the chromosome domains in which they were induced and not between different chromosomes.

Complex exchanges are responsible for the increased effectiveness of C-ions compared to X-rays at the first post-irradiation mitosis.

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.

Radiation-induced premature senescence is associated with specific cytogenetic changes.

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) beta-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.

Novel in vitro assay to investigate radiation induced changes in the functionality of human embryonic stem cell-derived neurospheres.

Ionizing radiation (IR) is increasingly used for diagnostics and therapy of severe brain diseases. However, IR also has adverse effects on the healthy brain tissue, particularly on the neuronal network. This is true for adults but even more pronounced in the developing brain of unborn and pediatric patients. Epidemiological studies on children receiving radiotherapy showed an increased risk for cognitive decline ranging from mild deficits in academic functioning to severe late effects in intellectual ability and language as a consequence of altered neuronal development and connectivity. To provide a comprehensive approach for the analysis of radiation-induced alterations in human neuronal functionality, we developed an in vitro assay by combining microelectrode array (MEA) analyses and human embryonic stem cell (hESC) derived three-dimensional neurospheres (NS). In our proof of principle study, we irradiated hESC with 1 Gy X-rays and let them spontaneously differentiate into neurons within NS. After the onset of neuronal activity, we recorded and analyzed the activity pattern of the developing neuronal networks. The network activity in NS derived from irradiated hESC was significantly reduced, whereas no differences in molecular endpoints such as cell proliferation and transcript or protein expression analyses were found. Thus, the combination of MEA analysis with a 3D model for neuronal functionality revealed radiation sequela that otherwise would not have been detected. We therefore strongly suggest combining traditional biomolecular methods with the new functional assay presented in this work to improve the risk assessment for IR-induced effects on the developing brain.

Spontaneous and Radiation-Induced Chromosome Aberrations in Primary Fibroblasts of Patients With Pediatric First and Second Neoplasms.

The purpose of the present study was to investigate whether former childhood cancer patients who developed a subsequent secondary primary neoplasm (SPN) are characterized by elevated spontaneous chromosomal instability or cellular and chromosomal radiation sensitivity as surrogate markers of compromised DNA repair compared to childhood cancer patients with a first primary neoplasm (FPN) only or tumor-free controls. Primary skin fibroblasts were obtained in a nested case-control study including 23 patients with a pediatric FPN, 22 matched patients with a pediatric FPN and an SPN, and 22 matched tumor-free donors. Clonogenic cell survival and cytogenetic aberrations in Giemsa-stained first metaphases were assessed after X-irradiation in G1 or on prematurely condensed chromosomes of cells irradiated and analyzed in G2. Fluorescence in situ hybridization was applied to investigate spontaneous transmissible aberrations in selected donors. No significant difference in clonogenic survival or the average yield of spontaneous or radiation-induced aberrations was found between the study populations. However, two donors with an SPN showed striking spontaneous chromosomal instability occurring as high rates of numerical and structural aberrations or non-clonal and clonal translocations. No correlation was found between radiation sensitivity and a susceptibility to a pediatric FPN or a treatment-associated SPN. Together, the results of this unique case-control study show genomic stability and normal radiation sensitivity in normal somatic cells of donors with an early and high intrinsic or therapy-associated tumor risk. These findings provide valuable information for future studies on the etiology of sporadic childhood cancer and therapy-related SPN as well as for the establishment of predictive biomarkers based on altered DNA repair processes.

Differential effects of irradiation with carbon ions and x-rays on macrophage function.

Macrophages are potent elicitors of inflammatory reactions that can play both positive and negative roles in radiotherapy. While several studies have investigated the effects of X-rays or gamma-rays on macrophages, virtually no work has been done on the responses of these cells to irradiation with carbon ions. Investigations into the effects of carbon ion irradiation are of particular interest in light of the fact that this type of radiation is being used increasingly for cancer therapy. In the present investigation we compared the effects of 250 kV X-rays with those of 9.8 MeV/u carbon ions on RAW 264.7 macrophages over a wide range of radiation doses. Macrophage functions including vitality, phagocytic activity, production of the proinflammatory cytokines IL-1beta and TNFalpha and production of nitric oxide (NO) were measured. In comparison to lymphocytes and fibroblasts, macrophages showed only a small decrease in vitality after irradiation with either X-rays or carbon ions. Proinflammatory cytokines and NO were induced in macrophages by LPS but not by irradiation alone. X-rays or carbon ions had little modulating effect on LPS-induced TNFalpha production. However, LPS-induced NO increased in a dose dependent manner up to 6-fold after carbon ion irradiation, while X-ray irradiation did not have this effect. Carbon ion irradiation mediated a concomitant decrease in IL-1beta production. Carbon ions also had a greater effect than X-rays in enhancing the phagocytic activity of macrophages. These results underscore the greater potential of carbon ion irradiation with regard to radiobiological effectiveness.

Modeling radiation-induced cell cycle delays.

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 <20%.

Patterns of host use by brood parasitic Maculinea butterflies across Europe.

The range of hosts exploited by a parasite is determined by several factors, including host availability, infectivity and exploitability. Each of these can be the target of natural selection on both host and parasite, which will determine the local outcome of interactions, and potentially lead to coevolution. However, geographical variation in host use and specificity has rarely been investigated. Maculinea (= Phengaris) butterflies are brood parasites of Myrmica ants that are patchily distributed across the Palæarctic and have been studied extensively in Europe. Here, we review the published records of ant host use by the European Maculinea species, as well as providing new host ant records for more than 100 sites across Europe. This comprehensive survey demonstrates that while all but one of the Myrmica species found on Maculinea sites have been recorded as hosts, the most common is often disproportionately highly exploited. Host sharing and host switching are both relatively common, but there is evidence of specialization at many sites, which varies among Maculinea species. We show that most Maculinea display the features expected for coevolution to occur in a geographic mosaic, which has probably allowed these rare butterflies to persist in Europe. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Electrophysiological investigation of human embryonic stem cell derived neurospheres using a novel spike detection algorithm.

Microelectrode array (MEA) technology in combination with three-dimensional (3D) neuronal cell models derived from human embryonic stem cells (hESC) provide an excellent tool for neurotoxicity screening. Yet, there are significant challenges in terms of data processing and analysis, since neuronal signals have very small amplitudes and the 3D structure enhances the level of background noise. Thus, neuronal signal analysis requires the application of highly sophisticated algorithms. In this study, we present a new approach optimized for the detection of spikes recorded from 3D neurospheres (NS) with a very low signal-to-noise ratio. This was achieved by extending simple threshold-based spike detection utilizing a highly sensitive algorithm named SWTTEO. This analysis procedure was applied to data obtained from hESC-derived NS grown on MEA chips. Specifically, we examined changes in the activity pattern occurring within the first ten days of electrical activity. We further analyzed the response of NS to the GABA receptor antagonist bicuculline. With this new algorithm method we obtained more reliable results compared to the simple threshold-based spike detection.

Functional video-based analysis of 3D cardiac structures generated from human embryonic stem cells.

Human embryonic stem cells (hESCs) differentiated into cardiomyocytes (CM) often develop into complex 3D structures that are composed of various cardiac cell types. Conventional methods to study the electrophysiology of cardiac cells are patch clamp and microelectrode array (MEAs) analyses. However, these methods are not suitable to investigate the contractile features of 3D cardiac clusters that detach from the surface of the culture dishes during differentiation. To overcome this problem, we developed a video-based motion detection software relying on the optical flow by Farnebäck that we call cBRA (cardiac beat rate analyzer). The beating characteristics of the differentiated cardiac clusters were calculated based on the local displacement between two subsequent images. Two differentiation protocols, which profoundly differ in the morphology of cardiac clusters generated and in the expression of cardiac markers, were used and the resulting CM were characterized. Despite these differences, beat rates and beating variabilities could be reliably determined using cBRA. Likewise, stimulation of ß-adrenoreceptors by isoproterenol could easily be identified in the hESC-derived CM. Since even subtle changes in the beating features are detectable, this method is suitable for high throughput cardiotoxicity screenings.

Persistence of radiation-induced aberrations in patients after radiotherapy with C-ions and IMRT.

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 the treatment. MATERIALS AND METHODS: Cytogenetic damage was analyzed in lymphocytes from 14 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 multiplex fluorescence in situ hybridization 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.

High LET radiation shows no major cellular and functional effects on primary cardiomyocytes in vitro.

It is well known that ionizing radiation causes adverse effects on various mammalian tissues. However, there is little information on the biological effects of heavy ion radiation on the heart. In order to fill this gap, we systematically examined DNA-damage induction and repair, as well as proliferation and apoptosis in avian cardiomyocyte cultures irradiated with heavy ions such as titanium and iron, relevant for manned space-flight, and carbon ions, as used for radiotherapy. Further, and to our knowledge for the first time, we analyzed the effect of heavy ion radiation on the electrophysiology of primary cardiomyocytes derived from chicken embryos using the non-invasive microelectrode array (MEA) technology. As electrophysiological endpoints beat rate and field action potential duration were analyzed. The cultures clearly exhibited the capacity to repair induced DNA damage almost completely within 24 h, even at doses of 7 Gy, and almost completely recovered from radiation-induced changes in proliferative behavior. Interestingly, no significant effects on apoptosis could be detected. Especially the functionality of primary cardiac cells exhibited a surprisingly high robustness against heavy ion radiation, even at doses of up to 7 Gy. In contrast to our previous study with X-rays the beat rate remained more or less unaffected after heavy ion radiation, independently of beam quality. The only change we could observe was an increase of the field action potential duration of up to 30% after titanium irradiation, diminishing within the following three days. This potentially pathological observation may be an indication that heavy ion irradiation at high doses could bear a long-term risk for cardiovascular disease induction.

Interrelation amongst differentiation, senescence and genetic instability in long-term cultures of fibroblasts exposed to different radiation qualities.

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 5months 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.