Search

High-LET-type cell survival curves have been observed in cells that were allowed to incorporate 125I-UdR into their DNA. Incorporation of tritiated thymidine into the DNA of cells has also been shown to result in an increase in relative biological effectiveness in cell survival experiments, but the increase is smaller than observed after incorporation of 125I-UdR. These findings are explained in the literature by the overall complexity of the induced DNA damage resulting from energies of the ejected electron(s) during the decay of 3H and 125I. Chromosomal aberrations (CA) are defined as morphological or structural changes of one or more chromosomes, and can be induced by ionizing radiation. Whether the number of CA is associated with the linear energy transfer (LET) of the radiation and/or the actual complexity of the induced DNA double-strand breaks (DSB) remains elusive. In this study, we investigated whether DNA lesions induced at different cell cycle stages and by different radiation types [Auger-electrons (125I), β– particles (3H), or γ radiation (137Cs)] have an impact on the number of CA induced after induction of the same number of DSB as determined by the γ-H2AX foci assay. Cells were synchronized and pulse-labeled in S phase with low activities of 125I-UdR or tritiated thymidine. For decay accumulation, cells were cryopreserved either after pulse-labeling in S phase or after progression to G2/M or G1 phase. Experiments with γ irradiation (137Cs) were performed with synchronized and cryopreserved cells in S, G2/M or G1 phase. After thawing, a CA assay was performed. All experiments were performed after a similar number of DSB were induced. CA induction after 125I-UdR was incorporated was 2.9-fold and 1.7-fold greater compared to exposure to γ radiation and radiation from incorporated tritiated thymidine, respectively, when measured in G2/M cells. In addition, measurement of CA in G2/M cells after incorporation of 125I-UdR was 2.5-fold greater when compared to cells in G1 phase. In contrast, no differences were observed between the three radiation qualities with respect to exposure after cryopreservation in S or G1 phase. The data indicate that the 3D organization of replicated DNA in G2/M cells seems to be more sensitive to induction of more complex DNA lesions compared to the DNA architecture in S or G1 cells. Whether this is due to the DNA organization itself or differences in DNA repair capability remains unclear. [ABSTRACT FROM AUTHOR]

Objective: To study the biological effectiveness of Auger electrons emitted by 99mTc on cell survival, induction of apoptosis and micronucleus (MN) formation in the human squamous cell carcinoma cell line SCL-II and compare the effects observed to those observed after exposure to external 60Co gamma radiation. Material and methods: Cells were either gamma(60Co)-irradiated 0.67 Gy/min) or exposed to 99mTc-pertechnetate (0.95-14.3 MBq/ml) for 24 h under cell culture conditions and assayed for cell survival (colony-forming assay), micronucleus formation (cytochalasin B assay) and the frequency of apoptotic cells (fluorescence microscopy). Monte Carlo based dosimetry has been applied to derive the absorbed dose corresponding to the accumulated decays of 99mTc under the given geometry. Results: Absorbed doses up to 0.5 Gy could be achieved after 99mTc-exposure leading to no substantial cell killing in this dose range except at one dose point (0.1 Gy) resulting in an relative biological effectiveness (RBE)SP = 0.9 of 0.64 when compared to the 60Co reference radiation. MN formation was described best by a linear dose response and was consistently lower after 99mTc exposure when compared to 60Co irradiated cells resulting in an RBE of 0.37. Apoptosis induction was significantly increased after 99mTc exposure at much lower doses (0.1 Gy) when compared to the reference radiation. The 99mTc uptake experiments revealed an activity concentration ratio cells vs. medium of 0.07 after 24 h of exposure. Conclusion: No overall increased biological effectiveness due to the emitted Auger electrons of 99mTc, applied as sodium-pertechnetate, could be observed in the investigated cell line when compared to acute external gamma radiation. The RBEs in the range of 0.37-0.64 might be well explained by dose rate effects. The significantly increased apoptotic response after 99mTc-exposure at very low doses has to be further investigated. [ABSTRACT FROM AUTHOR]

Background: In order to clarify the relationship between delayed reproductive death and radiation-induced genomic instability, the colony-forming efficiency of surviving, irradiated human squamous carcinoma cells and centromere positive as well as centromere negative micronuclei in surviving progeny were examined. Materials and methods: Colony-forming ability and micronucleus (MN) frequency in binucleated cells 24h after the addition of cytochalasin B during 2 weeks of post-irradiation growth were determined in a squamous cell carcinoma cell line (SCL-II) of human origin. In addition, centromeres in micronuclei were detected using FISH. Results: In the human epithelial cell line used for these experiments, delayed reproductive death was pronounced and persisted for at least 2 weeks after irradiation. Although there is evidence for an increased rate of centromere positive micronuclei, but not of centromere negative micronuclei, arising during the first week of post-irradiation proliferation, this decreases later while the rate of delayed reproductive death remains elevated. Conclusion: In the studied cell line, the observed delayed reproductive death is not closely related to the investigated criteria of radiation-induced genomic instability. This casts doubt on the common assumption that delayed reproductive death is a direct manifestation of radiation-induced genomic instability. [ABSTRACT FROM AUTHOR]

High-LET-type cell survival curves have been observed in cells that were allowed to incorporate 125I-UdR into their DNA. Incorporation of tritiated thymidine into the DNA of cells has also been shown to result in an increase in relative biological effectiveness in cell survival experiments, but the increase is smaller than observed after incorporation of 125I-UdR. These findings are explained in the literature by the overall complexity of the induced DNA damage resulting from energies of the ejected electron(s) during the decay of 3H and 125I. Chromosomal aberrations (CA) are defined as morphological or structural changes of one or more chromosomes, and can be induced by ionizing radiation. Whether the number of CA is associated with the linear energy transfer (LET) of the radiation and/or the actual complexity of the induced DNA double-strand breaks (DSB) remains elusive. In this study, we investigated whether DNA lesions induced at different cell cycle stages and by different radiation types [Auger-electrons (125I), β- particles (3H), or γ radiation (137Cs)] have an impact on the number of CA induced after induction of the same number of DSB as determined by the γ-H2AX foci assay. Cells were synchronized and pulse-labeled in S phase with low activities of 125I-UdR or tritiated thymidine. For decay accumulation, cells were cryopreserved either after pulse-labeling in S phase or after progression to G2/M or G1 phase. Experiments with γ irradiation (137Cs) were performed with synchronized and cryopreserved cells in S, G2/M or G1 phase. After thawing, a CA assay was performed. All experiments were performed after a similar number of DSB were induced. CA induction after 125I-UdR was incorporated was 2.9-fold and 1.7-fold greater compared to exposure to γ radiation and radiation from incorporated tritiated thymidine, respectively, when measured in G2/M cells. In addition, measurement of CA in G2/M cells after incorporation of 125I-UdR was 2.5-fold greater when compared to cells in G1 phase. In contrast, no differences were observed between the three radiation qualities with respect to exposure after cryopreservation in S or G1 phase. The data indicate that the 3D organization of replicated DNA in G2/M cells seems to be more sensitive to induction of more complex DNA lesions compared to the DNA architecture in S or G1 cells. Whether this is due to the DNA organization itself or differences in DNA repair capability remains unclear., (©2024 by Radiation Research Society. All rights of reproduction in any form reserved.)

Purpose: Ionizing radiation causes various types of DNA damage e.g. single strand breaks (SSB) and double strand breaks (DSB), whereby the SSB/DSB ratio is shifted toward the DSB with increasing LET. For the DNA-incorporated Auger electron emitter Iodine-125 a SSB/DSB ratio of 5.4:1 is calculated based on computer simulations. In the presented work the SSB/DSB ratio of DNA-incorporated Iodine-125 was experimentally determined and compared to external homogenous γ-irradiation., Materials and Methods: Iodine-125-iododeoxyuridine (I-125-UdR) was incorporated into the DNA of SCL-II cells and cells were subsequently frozen for decay accumulation. Accordingly, external γ-irradiation (Cs-137) experiments were performed in frozen cells. After exposure the neutral or alkaline Comet Assay was performed to quantify DSB or DSB and SSB, respectively. Automated quantification of the comets was performed using the Olive Tail Moment (Metafer CometScan; MetaSystems). Calculation of absorbed dose for Auger electrons on cellular level is extremely biased due to the exclusive DNA localization of I-125-UdR. To avoid dose calculation the γ-H2AX assay was used in order to allow the comparison of the Comet Assay data between both investigated radiation qualities., Results: For low-LET γ-radiation, a SSB/DSB ratio of 10:1 was determined. In contrast, a lower SSB/DSB ratio of 6:1 was induced by DNA-incorporated Iodine-125 which compares very well to the calculated values of Pomplun and co-authors., Conclusion: DNA-incorporated Iodine-125 induces a high-LET type DNA damage pattern in respect to SSB/DSB ratio.

Despite its extensive use in clinical studies, the molecular mechanisms underlying the effects of transcranial direct current stimulation (tDCS) remain to be elucidated. We previously described subacute effects of tDCS on immune- and stem cells in the rat brain. To investigate the more immediate effects of tDCS regulating those cellular responses, we treated rats with a single session of either anodal or cathodal tDCS, and analyzed the gene expression by microarray; sham-stimulated rats served as control. Anodal tDCS increased expression of several genes coding for the major histocompatibility complex I (MHC I), while cathodal tDCS increased the expression of the immunoregulatory protein osteopontin (OPN). We confirmed the effects of gene upregulation by immunohistochemistry at the protein level. Thus, our data show a novel mechanism for the actions of tDCS on immune- and inflammatory processes, providing a target for future therapeutic studies., (Copyright © 2019 Rabenstein, Unverricht-Yeboah, Keuters, Pikhovych, Hucklenbroich, Vay, Blaschke, Ladwig, Walter, Beiderbeck, Fink, Schroeter, Kriehuber and Rueger.)

Gene expression analysis was carried out in Jurkat cells in order to identify candidate genes showing significant gene expression alterations allowing robust discrimination of the Auger emitter 123I, incorporated into the DNA as 123I-iododeoxyuridine (123IUdR), from α- and γ-radiation. The γ-H2AX foci assay was used to determine equi-effect doses or activity, and gene expression analysis was carried out at similar levels of foci induction. Comparative gene expression analysis was performed employing whole human genome DNA microarrays. Candidate genes had to show significant expression changes and no altered gene regulation or opposite regulation after exposure to the radiation quality to be compared. The gene expression of all candidate genes was validated by quantitative real-time PCR. The functional categorization of significantly deregulated genes revealed that chromatin organization and apoptosis were generally affected. After exposure to 123IUdR, α-particles and γ-rays, at equi-effect doses/activity, 155, 316 and 982 genes were exclusively regulated, respectively. Applying the stringent requirements for candidate genes, four (PPP1R14C, TNFAIP8L1, DNAJC1 and PRTFDC1), one (KLF10) and one (TNFAIP8L1) gene(s) were identified, respectively allowing reliable discrimination between γ- and 123IUdR exposure, γ- and α-radiation, and α- and 123IUdR exposure, respectively. The Auger emitter 123I induced specific gene expression patterns in Jurkat cells when compared with γ- and α-irradiation, suggesting a unique cellular response after 123IUdR exposure. Gene expression analysis might be an effective tool for identifying biomarkers for discriminating different radiation qualities and, furthermore, might help to explain the varying biological effectiveness at the mechanistic level.

Metal toxicity is a global environmental challenge. Fish are particularly prone to metal exposure, which can be lethal or cause sublethal physiological impairments. The objective of this study was to investigate how adverse effects of chronic exposure to non-toxic levels of essential and non-essential metals in early life stage zebrafish may be explained by changes in the transcriptome. We therefore studied the effects of three different metals at low concentrations in zebrafish embryos by transcriptomics analysis. The study design compared exposure effects caused by different metals at different developmental stages (pre-hatch and post-hatch). Wild-type embryos were exposed to solutions of low concentrations of copper (CuSO 4 ), cadmium (CdCl 2 ) and cobalt (CoSO 4 ) until 96h post-fertilization (hpf) and microarray experiments were carried out to determine transcriptome profiles at 48 and 96hpf. We found that the toxic metal cadmium affected the expression of more genes at 96hpf than 48hpf. The opposite effect was observed for the essential metals cobalt and copper, which also showed enrichment of different GO terms. Genes involved in neuromast and motor neuron development were significantly enriched, agreeing with our previous results showing motor neuron and neuromast damage in the embryos. Our data provide evidence that the response of the transcriptome of fish embryos to metal exposure differs for essential and non-essential metals., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Environmental metals are known to cause harmful effects to fish of which many molecular mechanisms still require elucidation. Particularly concentration dependence of gene expression effects is unclear. Focusing on this matter, zebrafish embryo toxicity tests were used in combination with transcriptomics. Embryos were exposed to three concentrations of copper (CuSO 4 ), cadmium (CdCl 2 ) and cobalt (CoSO 4 ) from just after fertilization until the end of the 48hpf pre- and 96hpf post-hatch stage. The RNA was then analyzed on Agilent's Zebrafish (V3, 4×44K) arrays. Enrichment for GO terms of biological processes illustrated for cadmium that most affected GO terms were represented in all three concentrations, while for cobalt and copper most GO terms were represented in the lowest test concentration only. This suggested a different response to the non-essential cadmium than cobalt and copper. In cobalt and copper treated embryos, many developmental and cellular processes as well as the Wnt and Notch signaling pathways, were found significantly enriched. Also, different exposure concentrations affected varied functional networks. In contrast, the largest clusters of enriched GO terms for all three concentrations of cadmium included responses to cadmium ion, metal ion, xenobiotic stimulus, stress and chemicals. However, concentration dependence of mRNA levels was evident for several genes in all metal exposures. Some of these genes may be indicative of the mechanisms of action of the individual metals in zebrafish embryos. Real-time quantitative RT-PCR (qRT-PCR) verified the microarray data for mmp9, mt2, cldnb and nkx2.2a., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Triplex-Forming oligonucleotides (TFO) bind sequence-specific to the DNA double helix in-vitro and in-vivo and are a promising tool to manipulate genes or gene regulatory elements. TFO as a carrier molecule for short-range particle emitter such as Auger-Electron-Emitters (AEE) bear the potential to introduce radiation-induced site-specific complex DNA lesions, which are known to induce chromosomal translocations. We studied gene expression, translocation frequency and protein expression in SCL-II cells after transfection with the AEE Iodine-125 (I-125) labeled TFO-BCL2 targeting the human BCL2 gene. The TFO-BCL2 binds to the BCL2 gene in close proximity to a known major-breakage-region (mbr). SCL-II cells were transfected with I-125 labeled TFO and stored for decay accumulation. Monitoring of BCL2 translocations was done with the Fluorescence-In-Situ-Hybridization (FISH) method. The utilized FISH probes were designed to detect a t(14;18) translocation of the BCL2 gene, which is a common translocation leading to an overexpression of BCL2 protein. Analysis of BCL2 gene expression levels was done via quantitative Real-Time PCR. Verification of gene expression on the protein level was analyzed by Western blotting. The relative gene expression of BCL2 in I-125-TFO-BCL2 transfected cells showed a significant up-regulation when compared to controls. Analysis of the BCL2 t(14;18) translocation frequency revealed a significant 1.8- to 2-fold increase when compared to control cells. This 2-fold increase was not reflected on the protein level. We conclude that I-125 decays within the BCL2 gene facilitate the t(14;18) chromosomal translocation in the SCL-II cells and that the increased frequency contributes to the observed overall enhanced BCL2 gene expression., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose: To uncover the role of EZH2 and its opponent ASHL2, a polycomb and trithorax group protein, respectively, on the radioresponsiveness of glioma cell lines., Materials and Methods: Expression of EZH2 and ASHL2 was inhibited by siRNA in glioma cell lines. The effect on histone methylation, gene expression, DNA damage repair signaling, cell cycle checkpoints, apoptosis and tumor control were evaluated., Results: Inhibition of EZH2 (EZH2i) led to a transcriptional dysregulation with upregulation of 544 and downregulation of 445 genes. In comparison, ASH2L inhibition (ASH2Li) had an opposed effect with upregulation of 289 and downregulation of 970 genes. EZH2i and ASH2Li significantly reduced methylation of H3K27 and increased methylation of H3K9, respectively. EZH2i and ASH2Li significantly increased and decreased the number of residual γH2AX foci at 24 h after IR, respectively. The former significantly increased radiation-induced cell cycle arrest in G2/M and apoptotic cell death, while ASH2Li decreased both. In addition, a significant shift of the radioresponse curve by -1.22 + 0.23 Gy (p < 0.0001) in the plaque monolayer assay was found after EZH2i in A7 but not in M059K., Conclusion: Overall, epigenetic modulation is a promising approach to evaluate the role of chromatin structure for the radioresponsiveness of glioma cell lines.

Purpose: This collaboration of five established European gene expression labs investigated the potential impact of culture conditions on the transcriptional response of peripheral blood to radiation exposure., Materials and Methods: Blood from one healthy donor was exposed ex vivo to a Cobalt 60 source to produce a calibration curve in addition to four unknown doses. After exposure, the blood samples were either diluted with RPMI medium or left untouched. After 24-h incubation at 37 °C the diluted blood samples were lysed, while the undiluted samples were mixed with the preservative RNALater and all samples were shipped frozen to the participating labs. Samples were processed by each lab using microarray (one lab) and QRT-PCR (four labs)., Results: We show that although culture conditions affect the total amount of RNA recovered (p < .0001) and its integrity (p < .0001), it does not significantly affect dose estimates (except for the true dose at 1.1 Gy). Most importantly, the different analysis approaches provide comparable mean absolute difference of estimated doses relative to the true doses (p = .9) and number of out of range (>0.5 Gy) measurements (p = .6)., Conclusion: This study confirms the robustness of gene expression as a method for biological dosimetry.

Purpose: A European network was initiated in 2012 by 23 partners from 16 European countries with the aim to significantly increase individualized dose reconstruction in case of large-scale radiological emergency scenarios., Results: The network was built on three complementary pillars: (1) an operational basis with seven biological and physical dosimetric assays in ready-to-use mode, (2) a basis for education, training and quality assurance, and (3) a basis for further network development regarding new techniques and members. Techniques for individual dose estimation based on biological samples and/or inert personalized devices as mobile phones or smart phones were optimized to support rapid categorization of many potential victims according to the received dose to the blood or personal devices. Communication and cross-border collaboration were also standardized. To assure long-term sustainability of the network, cooperation with national and international emergency preparedness organizations was initiated and links to radiation protection and research platforms have been developed. A legal framework, based on a Memorandum of Understanding, was established and signed by 27 organizations by the end of 2015., Conclusions: RENEB is a European Network of biological and physical-retrospective dosimetry, with the capacity and capability to perform large-scale rapid individualized dose estimation. Specialized to handle large numbers of samples, RENEB is able to contribute to radiological emergency preparedness and wider large-scale research projects.

Purpose: RENEB, 'Realising the European Network of Biodosimetry and Physical Retrospective Dosimetry,' is a network for research and emergency response mutual assistance in biodosimetry within the EU. Within this extremely active network, a number of new dosimetry methods have recently been proposed or developed. There is a requirement to test and/or validate these candidate techniques and inter-comparison exercises are a well-established method for such validation., Materials and Methods: The authors present details of inter-comparisons of four such new methods: dicentric chromosome analysis including telomere and centromere staining; the gene expression assay carried out in whole blood; Raman spectroscopy on blood lymphocytes, and detection of radiation-induced thermoluminescent signals in glass screens taken from mobile phones., Results: In general the results show good agreement between the laboratories and methods within the expected levels of uncertainty, and thus demonstrate that there is a lot of potential for each of the candidate techniques., Conclusions: Further work is required before the new methods can be included within the suite of reliable dosimetry methods for use by RENEB partners and others in routine and emergency response scenarios.

Purpose: The efficacy of DNA-targeting radionuclide therapies might be strongly enhanced by employing short range particle-emitters. However, the gain of effectiveness is not yet well substantiated. We compared the Auger electron emitter I-125 to the ß - -emitter P-32 in terms of biological effectiveness per decay and radiation dose when located in the close proximity to DNA using DNA Triplex-forming oligonucleotides (TFO). The clonogenicity and the induction of DNA double-strand breaks (DSB) were investigated in SCL-II cells after exposure to P-32- or I-125-labeled TFO targeting the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene and after external homogeneous exposure to gamma-rays as reference radiation., Materials and Methods: TFO were labeled with P-32 or I-125 using the primer extension method. Cell survival was analyzed by colony-forming assay and DNA damage was assessed by microscopic quantification of protein 53 binding protein 1 (53BP1) foci in SCL-II cells., Results: I-125-TFO induced a pronounced decrease of cell survival (D 37 at ∼360 accumulated decays per cell, equivalent to 1.22 Gy cell nucleus dose) and a significant increase of 53BP1 foci with increasing decays. The P-32-labeled TFO induced neither a strong decrease of cell survival nor an increase of 53BP1 foci up to ∼4000 accumulated decays per cell, equivalent to ∼1 Gy cell nucleus dose. The RBE for I-125-TFO was in the range of 3-4 for both biological endpoints., Conclusions: I-125-TFO proved to be much more radiotoxic than P-32-TFO per decay and per unit dose although targeting the same sequence in the GAPDH gene. This might be well explained by the high number of low energy Auger electrons emitted by I-125 per decay, leading to a high ionization density in the immediate vicinity of the decay site, probably producing highly complex DNA lesions overcharging DNA repair mechanisms.

Aim: To identify predictive assays for radiation-induced toxicity in prostate cancer patients., Patients & Methods: Patients have been surveyed prospectively before and up to 16 months after radiotherapy using a validated questionnaire. Subgroups of 25 patients with minor and larger score changes, respectively, were selected for γ-H2AX, G2 and Annexin V assays., Results: A significantly higher spontaneous chromatid aberration yield (HR: 1.46 [95% CI: 1.02-2.09]; p = 0.04), higher levels of early apoptotic (HR: 1.12 [95% CI: 1.01-1.24]; p = 0.04) and late apoptotic and necrotic (HR: 1.10 [95% CI: 0.99-1.23]; p = 0.08) lymphocytes 24 h post-irradiation were found in patients with a bowel bother score decrease greater than 20 points more than 1 year after treatment., Conclusion: Chromatid aberration and apoptosis/necrosis assays appear to be suitable for the prediction of radiation-induced toxicity.

BCL-2 is a multifunctional protein involved in the regulation of apoptosis, cell cycle progression and neural developmental processes. Its function in the latter process is not well understood and needs further elucidation. Therefore, we characterized the protein expression kinetics of BCL-2 and associated regulatory proteins of the intrinsic apoptosis pathway during the process of neuronal differentiation in ReNcell VM cells with and without functional inhibition of BCL-2 by its competitive ligand HA14-1. Inhibition of BCL-2 caused a diminished BCL-2 expression and higher levels of cleaved BAX, activated Caspase-3 and cleaved PARP, all pro-apoptotic markers, when compared with untreated differentiating cells. In parallel, flow cytometric analysis of HA14-1-treated cells revealed a delayed differentiation into HuC/D+ neuronal cells when compared to untreated differentiating cells. In conclusion, BCL-2 possess a protective function in fully differentiated ReNcell VM cells. We propose that the pro-survival signaling of BCL-2 is closely connected with its stimulatory effects on neurogenesis of human neural progenitor cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Apoptosis is an essential physiological process accompanying the development of the central nervous system and human neurogenesis. However, the time scale and the underlying molecular mechanisms are yet poorly understood. Due to this fact, we investigated the functionality and general inducibility of apoptosis in the human neural ReNcell VM progenitor cell line during differentiation and also after exposure to staurosporine (STS) and ultraviolet B (UVB) irradiation. Transmission light microscopy, flow cytometry, and Western-/Immunoblot analysis were performed to compare proliferating and differentiating, in addition to STS- and UVB-treated cells. In particular, from 24 to 72 h post-initiation of differentiation, G0/G1 cell cycle arrest, increased loss of apoptotic cells, activation of pro-apoptotic BAX, Caspase-3, and cleavage of its substrate PARP were observed during cell differentiation and, to a higher extent, after treatment with STS and UVB. We conclude that redundant or defective cells are eliminated by apoptosis, while otherwise fully differentiated cells were less responsive to apoptosis induction by STS than proliferating cells, likely as a result of reduced APAF-1 expression, and increased levels of BCL-2. These data provide the evidence that apoptotic mechanisms in the neural ReNcell VM progenitor cell line are not only functional, but also inducible by external stimuli like growth factor withdrawal or treatment with STS and UVB, which marks this cell line as a suitable model to investigate apoptosis signaling pathways in respect to the differentiation processes of human neural progenitor cells in vitro.

DNA-associated Auger electron emitters (AEE) cause cellular damage leading to high-LET type cell survival curves indicating an enhanced relative biological effectiveness. Double strand breaks (DSBs) induced by Iodine-125-deoxyuridine ((125)I-UdR) decays are claimed to be very complex. To elucidate the assumed genotoxic potential of (125)I-UdR, chromatid aberrations were analysed in exposed human peripheral blood lymphocytes (PBL). PBL were stimulated with medium containing phytohaemagglutinin (PHA). After 24h, cultures were labelled with (125)I-UdR for 18h (activity concentration 1-45 kBq) during the S-phase. Following standard cytogenetic procedure, at least 100 metaphases were analysed microscopically for each activity concentration. Cell death was measured by apoptosis assay using flow cytometry. Radiation doses were determined by using point kernel calculations. After 18h labelling with (125)I-UdR the cell cycle distribution is severely disturbed. About 40% of PBL are fully labelled and 20% show a moderate labelling of (125)I-UdR, whereas 40% of cells remain un-labelled. The dose-response relationship fits to a polynomial curve in the low dose range, whereas a linear fit supplies a better estimation in the high dose range. Even the lowest dose of 0.2Gy leads to a 13-fold increase of aberrations compared to the controls. On average every fifth (125)I-decay produces a single chromatid aberration in PBL. Additionally, a dose-dependent increase of cell death is observed. (125)I-UdR has a very strong genotoxic capacity in human PBL, even at 0.2Gy. Efficiently labelled cells displaying a prolonged cell cycle compared to moderately labelled cells and cell death contribute substantially to the desynchronisation of the cell cycle. Our data, showing for the first time, that one (125)I-decay induces ∼ 0.2 chromatid aberrations, are in very good accordance to DSB data, stating that ∼0.26 DSB are induced per decay, indicating that it takes on average 250 decays to induce one chromosome aberration (CA). [Corrected], (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Exposure to environmental chemicals known as endocrine disruptors (EDs) is in many cases associated with an unpredictable hazard for wildlife and human health. The identification of endocrine disruptive properties of chemicals certain to enter the aquatic environment relies on toxicity tests with fish, assessing adverse effects on reproduction and sexual development. The demand for quick, reliable ED assays favored the use of fish embryos as alternative test organisms. We investigated the application of a transcriptomics-based assay for estrogenic and anti-androgenic chemicals with zebrafish embryos. Two reference compounds, 17α-ethinylestradiol and flutamide, were tested to evaluate the effects on development and the transcriptome after 48h-exposures. Comparison of the transcriptome response with other estrogenic and anti-androgenic compounds (genistein, bisphenol A, methylparaben, linuron, prochloraz, propanil) showed commonalities and differences in regulated pathways, enabling us to classify the estrogenic and anti-androgenic potencies. This demonstrates that different mechanism of ED can be assessed already in fish embryos., (Copyright © 2013 Elsevier Inc. All rights reserved.)

It is known that about 5-10% of cancer patients show severe clinical side effects during and after radiotherapy due to enhanced sensitivity to ionizing radiation. Identification of those radiosensitive individuals by a reliable in vitro assay before onset of treatment would have a great impact on successful radiotherapy. We compared the radiosensitivity of the chromosomes 2, 11 and 17 in prostate cancer patients with and without severe side effects after radiotherapy and in age-matched healthy donors. Each cohort consisted of at least 10 donors. Peripheral blood lymphocytes were irradiated ex vivo with 0.5, 1 und 2 Gy ((137)Cs γ rays). We investigated the radiosensitivity of the chromosomes 2, 11 and 17 by scoring of 100 FISH painted metaphases for each dose point and donor group. Statistical analyses were performed by nonparametric tests as Mann-Whitney test and Kruskal-Wallis ANOVA, paired Wilcoxon rank test, χ(2) goodness-of-fit test and Spearman rank-order correlation at a significance level of P < 0.05. Analysis of the overall aberration yield revealed no significant differences between any donor groups. The translocation frequencies of the chromosomes 2, 11 and 17 coincided with their relative size. Thus, none of the chromosomes analyzed were more or less radiosensitive with respect to the genomic translocation frequency. Additionally, neither of the chromosomes showed enhanced or diminished radiosensitivity in one of the donor groups. Furthermore, variance analyses revealed that the distribution pattern of the aberrations per donor did not differ in each donor group even after exposure to 2 Gy. Prostate cancer patients with and without side effects cannot be distinguished from healthy donors based on aberration yield after irradiation with γ rays.

We report the case of an 84 years old prostate cancer patient with severe side effects after radiotherapy in 2006. He was cytogenetically analysed in 2009 and in 2012 in a comparative study for individual radiosensitivity of prostate cancer patients. No other patient had clonal aberrations, but this patient showed ring chromosomes in the range of 21-25% of lymphocytes. He received 5 cycles of 5-fluorouracil/folic acid for chemotherapy of sigmoid colon carcinoma in 2003, three years before radiotherapy of prostate cancer. Blood samples were irradiated ex vivo with Cs-137 γ-rays (0.7Gy/min) in the G0-phase of the cell cycle. 100 FISH painted metaphases were analysed for the control and the irradiated samples each. Multicolour in situ hybridisation techniques like mFISH and mBand as well as MYC locus, telomere and centromere painting probes were used to characterise ring metaphases. Metaphase search and autocapture was performed with a Zeiss Axioplan 2 imaging microscope followed by scoring and image analysis using Metafer 4/ISIS software (MetaSystems). In 2009 chromosome 8 rings were found in about 25% of lymphocytes. Rings were stable over time and increased to about 30% until 2012. The ring chromosome 8 always lacked telomere signals and a small amount of rings displayed up to four centromere signals. In aberrant metaphases 8pter and 8qter were either translocated or deleted. Further analyses revealed that the breakpoint at the p arm is localised at 8p21.2-22. The breakpoint at the q arm turned out to be distal from the MYC locus at 8q23-24. We hypothesise that the ring chromosome 8 has been developed during the 5 FU/folic acid treatments in 2003. The long term persistence might be due to clonal expansion of a damaged but viable hematopoietic stem cell giving rise to cycling progenitor cells that permit cell survival and proliferation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Glycogen synthase kinase-3beta is a multifunctional key regulator enzyme in neural developmental processes and a main component of the canonical Wnt signaling pathway. It is already known that the Wnt-driven differentiation of neural progenitor cells is accompanied by an increase of apoptosis at which the pro-apoptotic function of GSK-3beta is still discussed. The aim of the present study was to investigate whether the phosphorylation level of GSK-3beta at serine 9 is the primary regulatory mechanism of differentiation-induced apoptosis. Differentiating human neural ReNcell VM progenitor cells were treated with the specific GSK-3beta inhibitor SB216763 (10 μM) and analyzed in respect to the intrinsic apoptosis pathway regulation using microscopy and protein expression analysis. Differentiation of ReNcell VM cells was accompanied by cell morphological changes, cytoskeleton rearrangement and apoptosis increase. Treatment of differentiating cells with SB216763 induced a significant dephosphorylation of GSK-3beta at serine 9 accompanied by a significant decrease of apoptosis of about 0.7±0.03% and reduced activation of caspase-3 as well as BAX and PARP cleavage during the first 12h of differentiation compared to untreated, differentiating cells. Dephosphorylation of GSK-3beta at serine 9 appears not solely to be responsible for its pro-apoptotic function, because we observed a decrease of intrinsic apoptosis after treatment of the cells with the specific GSK-3beta inhibitor SB216763. We assume that GSK-3beta drives neural progenitor cell apoptosis by direct interaction with pro-apoptotic BAX or by indirect influence on the canonical Wnt/beta-catenin target gene transcription., (Copyright © 2012 ISDN. Published by Elsevier Ltd. All rights reserved.)

Assessment of endocrine disruption currently relies on testing strategies involving adult vertebrates. In order to minimize the use of animal tests according to the 3Rs principle of replacement, reduction and refinement, we propose a transcriptomics and fish embryo based approach as an alternative to identify and analyze an estrogenic activity of environmental chemicals. For this purpose, the suitability of 48 h and 7 days post-fertilization zebrafish and medaka embryos to test for estrogenic disruption was evaluated. The embryos were exposed to the phytoestrogen genistein and subsequently analyzed by microarrays and quantitative real-time PCR. The functional analysis showed that the genes affected related to multiple metabolic and signaling pathways in the early fish embryo, which reflect the known components of genistein's mode of actions, like apoptosis, estrogenic response, hox gene expression and steroid hormone synthesis. Moreover, the transcriptomic data also suggested a thyroidal mode of action and disruption of the nervous system development. The parallel testing of two fish species provided complementary data on the effects of genistein at gene expression level and facilitated the separation of common from species-dependent effects. Overall, the study demonstrated that combining fish embryo testing with transcriptomics can deliver abundant information about the mechanistic effects of endocrine disrupting chemicals, rendering this strategy a promising alternative approach to test for endocrine disruption in a whole organism in-vitro scale system., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: We studied the response of human embryonic stem cells (hESC) to the β-emitter (131)I, which affects the entire cell and to the Auger electron emitter (125)I-deoxyuridine ((125)I-dU), primarily affecting the deoxyribonuleic acid (DNA). The effects were also studied in keratinocytes as a prototype for somatic cells., Methods: HESC (H1) and human keratinocytes (HaCaT, human) were exposed to (125)I-dU (5 × 10(-5) - 5 MBq/ml) and (131)I-iodide (5 × 10(-5) - 12.5 MBq/ml) and apoptosis was measured by DNA-fragmentation. Cell morphology was studied by light microscopy and electron microscopy. Transcriptional profiling was done on the Agilent oligonucleotide microarray platform., Results: Auger-process induced no apoptosis but a strong transcriptional response in hESC. In contrast, HaCaT cells showed a pronounced induction of apoptosis but only a moderate transcriptional response. Transcriptional response of hESC was similar after (125)I-dU and (131)I treatments, whereas HaCaT cells expressed a much more pronounced response to (125)I-dU than to (131)I. A striking radiation-induced down-regulation of pluripotency genes was observed in hESC whereas in keratinocytes the enriched gene annotations were related primarily to apoptosis, cell division and proliferation., Conclusions: Human embryonic stem cells respond to ionizing radiation by (125)I-dU and (131)I in a different way compared to keratinocytes. Transcriptional response and gene expression appear to facilitate an escape from programmed cell death by striking a new path which probably leads to cell differentiation.

Purpose: Triplex-forming oligonucleotides (TFO) bind to the DNA double helix in a sequence-specific manner. Therefore, TFO seem to be a suitable carrier for Auger electron emitters to damage exclusively targeted DNA sequences, e.g., in tumor cells. We studied the influence of I-125 labeled TFO with regard to cell survival and induction of DNA double-strand breaks (DSB) using TFO with different genomic targets and target numbers. Furthermore, the ability of TFO to alter the gene expression of targeted genes was examined., Materials and Methods: TFO were labeled with I-125 using the primer extension method. DNA triplex formation and sequence-specific DSB were demonstrated in vitro. Cell survival was analyzed by colony-forming assay and DNA damage was assessed by microscopic quantification of protein 53 binding protein 1 (53BP1) foci in the human squamous carcinoma cell line II (SCL-II). Quantitative real-time polymerase-chain-reaction (qRT-PCR) was performed to analyze gene expression alterations., Results: The sequence-specific induction of a single DSB in a 1695 bp long DNA double stranded fragment was demonstrated in vitro. I-125-labeled TFO binding to single and multiple targets were shown to induce a pronounced decrease in cell survival and an increase of DSB. TFO targeting multiple sites differing in the total target number showed a significant different cell killing per decay that is also in good accordance with the observed induction of DSB. Single gene targeting I-125-labeled TFO significantly decreased cell survival and altered gene expression in the targeted gene., Conclusions: I-125-labeled TFO enable specific targeting of DNA in vitro as well as in a cellular environment and thus induce sequence-specific complex DNA lesions. Therefore I-125-labeled TFO might be a very useful tool for basic DNA repair research.

To overcome the limitations of existing biodosimetry methods, we examined dose- and time-dependent gene expression changes in human peripheral blood lymphocytes after exposure to low-, medium- and high-dose ionizing radiation and searched for genes suitable for predicting radiation doses in the low-dose range. Additionally, the experiments are intended to provide new insights into the biological effects of exposures to low-, medium- and high-dose radiation. Gene expression analysis using whole human genome DNA microarrays was performed in human blood from six healthy donors irradiated ex vivo with 0, 0.02, 0.1, 0.5, 1, 2 and 4 Gy (γ rays, (137)Cs) at 6, 24 and 48 h after high-dose exposure (0.5-4 Gy), and at 24 and 48 h after low-dose exposures of 0.02 or 0.1 Gy. DNA microarray-based alterations in gene expression were found in a wide dose range in vitro and allowed us to identify nine genes with which low radiation doses could be accurately predicted with a sensitivity of 95.6%. In the low-, medium- and high-dose range, expression alterations increased with increasing dose and time after exposure, and were assigned to different biological processes such as nucleosome assembly, apoptosis and DNA repair response. We conclude from our results that gene expression profiles are suitable for predicting low-dose radiation exposure in a rapid and reliable manner and that acute low-dose exposure, as low as 20 mGy, leads to well-defined physiological responses in human peripheral blood lymphocytes.

Since nano-sized particles (NPs) are increasingly used in various fields of innovative biomedicine and industrial technologies, it is of importance to identify their potential human health risk. We investigated whether ROS-induced mitochondrial DNA damage is the mode of action of titanium dioxide-NPs (TiO2-NPs; ≤20 nm) to induce cytotoxic and genotoxic effects in human HaCaT keratinocytes in vitro. We showed that TiO2-NPs accumulate at the cell surface and are taken up by endocytosis. Micronucleus (MN) formation was found to be significantly maximal increased 24 h after treatment with 10 μg/ml and 48 h after treatment with 5 μg/ml TiO2-NPs about 1.8-fold respectively 2.2-fold of control. Mitochondrial DNA damage measured as "common deletion" was observed to be significantly 14-fold increased 72 h after treatment with 10 μg/ml TiO2-NPs when compared to control. Four hours after treatment with 5 and 50 μg/ml TiO2-NPs the level of ROS in HaCaT cells was found to be significantly increased about 7.5-fold respectively 16.7-fold of control. In conclusion, for the first time we demonstrate the induction of the mitochondrial "common deletion" in HaCaT cells following exposure to TiO2-NPs, which strongly suggests a ROS-mediated cytotoxic and genotoxic potential of NPs. However, the effects of the modification of TiO2-NPs, such as agglomeration, size distribution pattern and exposure time have to be further critically examined., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: A high cellular radiosensitivity may be connected with a risk for development of severe side effects after radiotherapy and indicate cancer susceptibility. Hence, a fast and robust in vitro test is desirable to identify radiosensitive individuals., Materials and Methods: The study included 25 prostate cancer patients with severe side effects (S) and 25 patients without severe side effects (0) after radiotherapy as well as 23 male healthy age-matched donors. Blood samples were exposed to 0.5 Gy or 1 Gy of γ-rays. The initial level of double-strand breaks (dsb) and repair kinetics measured by phosphorylation of histone H2A (γ-H2AX-assay), apoptosis (Annexin V-assay) and the induction of chromatid aberrations after irradiation in the G2-phase of the cell cycle (G2-assay) were analysed., Results: A significant higher chromatid aberration yield was found in lymphocytes from prostate cancer patients when compared to healthy donors. We found no significant differences between patients S and patients 0., Conclusions: There is no obvious correlation between clinical and cellular radiosensitivity in lymphocytes of prostate cancer patients when all chosen in vitro assays are considered. Although 25% of the patients showed both severe side effects and increased radiation-induced chromosomal sensitivity, predictive value of G2-assay is doubtful.

Purpose: A fast, radiation-specific and highly accurate prediction of the radiation dose of accidentally exposed individuals is essential for medical decision-making. The aim of the present study is to identify small gene signatures allowing the discrimination between low and medium dose exposure of low linear energy transfer (LET)-radiation., Material and Methods: We developed a framework for dose prediction using a frequency-based gene selection approach, based on a p-value and fold-change criterion applied to microarray expression data. A repeated cross-validated classification guarantees unbiased performance results. Human blood from six healthy donors was irradiated ex vivo with 0.5, 1, 2 and 4 Gy (Cs-137 γ-rays). Expression levels of isolated blood lymphocytes were measured at 6, 24 and 48 h after irradiation., Results: We identified radiation-responsive genes, most of them functionally linked to apoptosis, DNA-damage or cell-cycle regulation. We extracted small subsets of genes, with which 95.7% of all samples can be correctly predicted, regardless of the time post irradiation. Seven of these genes were used for validation by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)., Conclusion: The genes identified are potential robust biomarkers, which are particularly suitable for dose level discrimination at a window of time that would be appropriate for life-saving medical triage.

Cell survival, induction of apoptosis, and micronucleus formation have been examined in non-transformed human amnion fluid fibroblast-like (AFFL) cells and in a human squameous cell carcinoma (SCL-II) cell line after exposure to the Auger electron emitter (65)Zn and after external low-LET radiation. Cellular uptake and subcellular distribution of (65)Zn(2+) were studied in vitro and the absorbed radiation dose was calculated applying analytical dosimetry models. Auger electrons generated during decay of (65)Zn induced a prominent decrease in cell survival and increased the levels of apoptotic as well as micronucleated cells when compared to external low-LET irradiation. Relative biological effectiveness has been determined for cell survival (RBE approximately 4), micronucleus formation (RBE approximately 2) and apoptosis induction (RBE approximately 5-8) in SCL-II cells and for micronucleus formation (RBE approximately 4-5) and apoptosis induction (RBE approximately 6-10) in AFFL cells, respectively. This demonstrates a general enhanced biological effectiveness of (65)Zn in both investigated cell lines when compared to external low-LET radiation. The distribution pattern of intracellular Zn(2+) was found to be non-uniform, showing enhanced amounts of Zn(2+) in the perinuclear region and low amounts inside the cell nucleus, suggesting a major energy deposition close to the nuclear envelope.

The effect of exposure to 50 Hz, 1 mT magnetic fields (MF) on the cell cycle in general, on the DNA synthesis in S-phase, and on the G1-phase regulating proteins Cdk4, cyclin D1, p16INK4a, and p21CIP1 was investigated in human amniotic fluid cells. The BrdU-incorporation assay revealed a significant diminution of S-phase cells in MF-exposed cultures. The protein level of Cdk4 did not change, but MF induced a decreased expression of cyclin D1 after 24 h and 30 h exposures. The level of p16INK4a increased at 1 h and 12 h after exposure, whereas the expression of p21CIP1 was enhanced at 6 h and 12 h after exposure. Reduced levels of both Cdk inhibitors were observed at longer exposure times (24 h, 30 h). Our results suggest an inhibitory effect of MF on the G1-phase induced by altered expression of p16INK4a and p21CIP1.

It still is an unsolved issue whether exposure to power-line frequency electromagnetic fields (EMF) may promote carcinogenesis and if so whether it does so by influencing the proliferation, the survival, and the differentiation of cells. Since the family of protein kinases C (PKC) takes part in these processes by interacting with signal transduction pathways at several levels including the activation of transcription factors, we evaluated in the present study the effects of exposure of human amniotic fluid cells (AFC) to 50 Hz, 1 mT electromagnetic fields (EMF) alone and in combination with the tumour promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on the subcellular localization of PKC protein, on PKC enzyme activity, and on the cell cycle distribution. Quantitative analyses of the PKC expression pattern demonstrated the translocation of PKC from the cytosolic to the membrane fraction after exposure to 10, 50, 100 nM, and 1 microM TPA. EMF exposure alone showed no effect on PKC translocation. Co-exposure to 10, 50, and 100 nM TPA and I mT EMF revealed a significant additive effect (25 +/- 50, 66 +/- 29, 22 +/- 50%, respectively) with the most prominent increase at the concentration of 50 nM TPA. At the highest concentration of TPA used (1 microM) no additive effect of EMF could be observed. Data on enzymatic activity indicate that EMF modulate the PKC activity, showing a significant increase of 10 +/- 16% in total PKC activity after co-exposure to 50 nM TPA and 1 mT EMF when compared to 50 nM TPA alone. Flow cytometric analyses showed a transient cell cycle arrest in G0/G1-phase followed by a delayed transit through S-phase in response to TPA, which was, however, not enhanced by co-exposure with EMF. We conclude that in AFC cells TPA at lower concentrations (< or = 100 nM) induces a less than maximum effect on the PKC pathway, which can be enhanced by the applied EMF.

Electromagnetic fields (EMFs) have been associated with increased incidence of cancer suggested by epidemiological studies. To test the carcinogenic potency of EMF, the in vitro micronucleus assay with SHE cells has been used as a screening method for genotoxicity. A 50Hz magnetic field (MF) of 1mT field strength was applied either alone or with the tumour initiator benzo(a)pyrene (BP) or the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). All three treatments were applied in single, double or triple treatment regimes. MF or TPA (1nM) alone did not affect the number of micronuclei (MN) in initiated and non-initiated SHE cells. Changing the schedule of the typical initiation protocol, namely applying the initiator (BP) during exposure to MF, results in an 1.8-fold increased MN formation compared to BP treatment alone. Combined experiment with BP, TPA and MF did not cause further MN formation. Since initiation during MF exposure caused a significant increased MN formation, our findings suggest that MFs enhance the initiation process of BP. We think that this MF-enhanced co-carcinogenic effect is caused by an indirect "cell activation" process. The resulting genomic instability is proposed to be due to free radicals and/or to the unscheduled "switching-on" of signal transduction pathways.

Effects of 50 Hz electromagnetic fields on phagocytosis and free radical production were examined in mouse bone marrow-derived macrophages. Macrophages were in vitro exposed to electromagnetic fields using different magnetic field densities (0.5-1.5 mT). Short-time exposure (45 min) to electromagnetic fields resulted in significantly increased phagocytic uptake (36.3% +/- 15.1%) as quantified by measuring the internalization rate of latex beads. Stimulation with 1 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) showed the same increased phagocytic activity as 1 mT electromagnetic fields. However, co-exposure to electromagnetic fields and TPA showed no further increase of bead uptake, and therefore we concluded that because of the absence of additive effects, the electromagnetic fields-induced stimulation of mouse bone marrow-derived macrophages does not involve the protein kinase C signal transduction pathway. Furthermore, a significant increased superoxide production after exposure to electromagnetic fields was detected.