Publications by authors named "Igor Belyaev"

39 Publications

Imaging flow cytometry and fluorescence microscopy in assessing radiation response in lymphocytes from umbilical cord blood and cancer patients.

Cytometry A 2021 Jun 4. Epub 2021 Jun 4.

Department of Radiobiology, Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, Bratislava, Slovakia.

DNA double strand breaks (DSB) induced by ionizing radiation (IR) are usually measured using γH2AX/53BP1 DNA repair foci, that is considered to be the most sensitive assay for DSB analysis. While fluorescence microscopy (FM) is the gold standard for this analysis, imaging flow cytometry (IFC) may offer number of advantages such as lack of the fluorescence background, higher number of cells analyzed, and higher sensitivity in detection of DNA damage induced by IR at low doses. Along with appearance of γH2AX foci, the variable fraction of the cells exhibits homogeneously stained γH2AX signal resulting in so-called γH2AX pan-staining, which is believed to appear at early stages of apoptosis. Here, we investigated incidence of γH2AX pan-staining at different time points after irradiation with γ-rays using IFC and compared the obtained data with the data from FM. Appearance of γH2AX pan-staining during the apoptotic process was further analyzed by fluorescence-activated cell sorting (FACS) of cells at different stages of apoptosis and subsequent immunofluorescence analysis. Our results show that IFC was able to reveal dose dependence of pan-staining, while FM failed to detect all pan-staining cells. Moreover, we found that γH2AX pan-staining could be induced by therapeutic, but not low doses of γ-rays and correlate well with percentage of apoptotic cells was analyzed using flow cytometric Annexin-V/7-AAD assay. Further investigations showed that γH2AX pan-staining is formed in the early phases of apoptosis and remains until later stages of apoptotic process. Apoptotic DNA fragmentation as detected with comet assay using FM correlated with the percentage of live and late apoptotic/necrotic cells as analyzed by flow cytometry. Lastly, we successfully tested IFC for detection of γH2AX pan-staining and γH2AX/53BP1 DNA repair foci in lymphocyte of breast cancer patients after radiotherapy, which may be useful for assessing individual radiosensitivity in a clinically relevant cohort of patients.
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http://dx.doi.org/10.1002/cyto.a.24468DOI Listing
June 2021

Evaluation of Calyculin A Effect on γH2AX/53BP1 Focus Formation and Apoptosis in Human Umbilical Cord Blood Lymphocytes.

Int J Mol Sci 2021 May 22;22(11). Epub 2021 May 22.

Department of Radiobiology, Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, University Science Park for Biomedicine, Dúbravská Cesta 9, 845 05 Bratislava, Slovakia.

Dephosphorylation inhibitor calyculin A (cal A) has been reported to inhibit the disappearance of radiation-induced γH2AX DNA repair foci in human lymphocytes. However, other studies reported no change in the kinetics of γH2AX focus induction and loss in irradiated cells. While apoptosis might interplay with the kinetics of focus formation, it was not followed in irradiated cells along with DNA repair foci. Thus, to validate plausible explanations for significant variability in outputs of these studies, we evaluated the effect of cal A (1 and 10 nM) on γH2AX/53BP1 DNA repair foci and apoptosis in irradiated (1, 5, 10, and 100 cGy) human umbilical cord blood lymphocytes (UCBL) using automated fluorescence microscopy and annexin V-FITC/propidium iodide assay/γH2AX pan-staining, respectively. No effect of cal A on γH2AX and colocalized γH2AX/53BP1 foci induced by low doses (≤10 cGy) of γ-rays was observed. Moreover, 10 nM cal A treatment decreased the number of all types of DNA repair foci induced by 100 cGy irradiation. 10 nM cal A treatment induced apoptosis already at 2 h of treatment, independently from the delivered dose. Apoptosis was also detected in UCBL treated with lower cal A concentration, 1 nM, at longer cell incubation, 20 and 44 h. Our data suggest that apoptosis triggered by cal A in UCBL may underlie the failure of cal A to maintain radiation-induced γH2AX foci. All DSB molecular markers used in this study responded linearly to low-dose irradiation. Therefore, their combination may represent a strong biodosimetry tool for estimation of radiation response to low doses. Assessment of colocalized γH2AX/53BP1 improved the threshold of low dose detection.
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http://dx.doi.org/10.3390/ijms22115470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196852PMC
May 2021

Induction of AML Preleukemic Fusion Genes in HSPCs and DNA Damage Response in Preleukemic Fusion Gene Positive Samples.

Antioxidants (Basel) 2021 Mar 18;10(3). Epub 2021 Mar 18.

Department of Radiobiology, Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, 845 05 Bratislava, Slovakia.

Preleukemic fusion genes (PFGs) occurring after DNA damage in hematopoietic stem progenitor cells (HSPCs) in utero often represent the initial event in the development of childhood leukemia. While the incidence of PFGs characteristic for acute lymphoblastic leukemia (ALL) was relatively well examined by several research groups and estimated to be 1-5% in umbilical cord blood (UCB) of healthy newborns, PFGs that are relevant to acute myeloid leukemia (AML) were poorly investigated. Therefore, this study is focused on the estimation of the incidence of the most frequent AML PFGs in newborns. For the first time, this study considered the inducibility of AML PFGs in different subsets of UCB HSPCs by low-dose γ-rays and also compared endogenous DNA damage, apoptosis, and reactive oxygen species (ROS) level between UCB samples containing or lacking AML PFGs. We found that: (i) the incidence of AML PFGs in UCB was 3.19% for RUNX1-RUNX1T1, 3.19% for PML-RARα, and 1.17% for KMT2A-MLLT3, (ii) 50 cGy of γ-rays did not induce RUNX1-RUNX1T1, PML-RARα, or KMT2A-MLLT3 PFGs in different subsets of sorted and expanded HSPCs, and (iii) the AML PFG samples accumulated the same level of endogenous DNA damage, as measured by the γH2AX/53BP1 focus formation, and also the same ROS level, and apoptosis as compared to PFG controls. Our study provides critical insights into the prevalence of AML PFGs in UCB of newborns, without the evidence of a specific HSPC population more susceptible for PFG formation after irradiation to low-dose γ-rays or increased amount of ROS, apoptosis and DNA damage.
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http://dx.doi.org/10.3390/antiox10030481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003332PMC
March 2021

DNA damage response and apoptosis induced by hyperthermia in human umbilical cord blood lymphocytes.

Toxicol In Vitro 2021 Jun 27;73:105127. Epub 2021 Feb 27.

Department of Radiobiology, Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, Slovakia.

While hyperthermia (HT) is a promising modality for cancer treatment, the knowledge on mechanisms of its effect on cells is still limited. We have investigated DNA double-strand break (DSB) and apoptosis induced by HT. Umbilical cord blood lymphocytes (UCBL) were subjected to HT at 43 °C. We have treated cells for 1 h (1 h HT), 2 h (2 h HT) and by combined HT and ice treatment (both lasting 1 h). Enumeration of DSB by 53BP1/γH2AX DNA repair focus formation and early apoptosis by γH2AX pan-staining was conducted by automated fluorescent microscopy. Apoptotic stages and viability were assessed by the annexin/propidium iodide (PI) assay using flow cytometry 0, 18, and 42 h post-treatment. HT induced either immediate (2 h HT) or postponed (1 h HT) DNA damage. The levels of 53BP1 and γH2AX foci differed under the same treatment conditions, suggesting that the ratio of co-localized γH2AX/53BP1 foci to all γH2AX and also to all 53BP1 foci could be a valuable marker. The ratio of co-localized foci increased immediately after 2 h HT regardless the way of assessment. For the first time we show, by both annexin/PI and γH2AX pan-staining assay that apoptosis can be induced during or immediately after the 2 h HT treatment. Our results suggest that HT may induce DSB in dependence on treatment duration and post-treatment time due to inhibition of DNA repair pathways and that HT-induced apoptosis might be dependent or associated with DSB formation in human lymphocytes. Assessment of γH2AX pan-staining in lymphocytes affected by HT may represent a valuable marker of HT treatment side effects.
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http://dx.doi.org/10.1016/j.tiv.2021.105127DOI Listing
June 2021

Effects of different mobile phone UMTS signals on DNA, apoptosis and oxidative stress in human lymphocytes.

Environ Pollut 2020 Dec 11;267:115632. Epub 2020 Sep 11.

Department of Radiobiology, Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, 845 05, Slovak Republic. Electronic address:

Different scientific reports suggested link between exposure to radiofrequency radiation (RF) from mobile communications and induction of reactive oxygen species (ROS) and DNA damage while other studies have not found such a link. However, the available studies are not directly comparable because they were performed at different parameters of exposure, including carrier frequency of RF signal, which was shown to be a critical for appearance of the RF effects. For the first time, we comparatively analyzed genotoxic effects of UMTS signals at different frequency channels used by 3G mobile phones (1923, 1947.47, and 1977 MHz). Genotoxicity was examined in human lymphocytes exposed to RF for 1 h and 3 h using complimentary endpoints such as induction of ROS by imaging flow cytometry, DNA damage by alkaline comet assay, mutations in TP53 gene by RSM assay, preleukemic fusion genes (PFG) by RT-qPCR, and apoptosis by flow cytometry. No effects of RF exposure on ROS, apoptosis, PFG, and mutations in TP53 gene were revealed regardless the UMTS frequency while inhibition of a bulk RNA expression was found. On the other hand, we found relatively small but statistically significant induction of DNA damage in dependence on UMTS frequency channel with maximal effect at 1977.0 MHz. Our data support a notion that each specific signal used in mobile communication should be tested in specially designed experiments to rule out that prolonged exposure to RF from mobile communication would induce genotoxic effects and affect the health of human population.
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http://dx.doi.org/10.1016/j.envpol.2020.115632DOI Listing
December 2020

Effect of Intermittent ELF MF on Umbilical Cord Blood Lymphocytes.

Bioelectromagnetics 2020 Dec 15;41(8):649-655. Epub 2020 Nov 15.

Department of Radiobiology, Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, Bratislava, Slovakia.

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http://dx.doi.org/10.1002/bem.22302DOI Listing
December 2020

DNA damage response and preleukemic fusion genes induced by ionizing radiation in umbilical cord blood hematopoietic stem cells.

Sci Rep 2020 08 24;10(1):13722. Epub 2020 Aug 24.

Deparment of Radiobiology, Biomedical Research Center, Cancer Research Institute, Slovak Academy of Sciences, Dubravska cesta 9, 845 05, Bratislava, Slovakia.

There is clear evidence that ionizing radiation (IR) causes leukemia. For many types of leukemia, the preleukemic fusion genes (PFG), as consequences of DNA damage and chromosomal translocations, occur in hematopoietic stem and progenitor cells (HSPC) in utero and could be detected in umbilical cord blood (UCB) of newborns. However, relatively limited information is available about radiation-induced apoptosis, DNA damage and PFG formation in human HSPC. In this study we revealed that CD34+ HSPC compared to lymphocytes: (i) are extremely radio-resistant showing delayed time kinetics of apoptosis, (ii) accumulate lower level of endogenous DNA damage/early apoptotic γH2AX pan-stained cells, (iii) have higher level of radiation-induced 53BP1 and γH2AX/53BP1 co-localized DNA double stranded breaks, and (iv) after low dose of IR may form very low level of BCR-ABL PFG. Within CD34+ HSPC we identified CD34+CD38+ progenitor cells as a highly apoptosis-resistant population, while CD34+CD38- hematopoietic stem/multipotent progenitor cells (HSC/MPP) as a population very sensitive to radiation-induced apoptosis. Our study provides critical insights into how human HSPC respond to IR in the context of DNA damage, apoptosis and PFG.
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http://dx.doi.org/10.1038/s41598-020-70657-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445283PMC
August 2020

Biodosimetry of Low Dose Ionizing Radiation Using DNA Repair Foci in Human Lymphocytes.

Genes (Basel) 2020 01 4;11(1). Epub 2020 Jan 4.

Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, Dúbravska cesta 9, 845 05 Bratislava, Slovakia.

Purpose: Ionizing radiation induced foci (IRIF) known also as DNA repair foci represent most sensitive endpoint for assessing DNA double strand breaks (DSB). IRIF are usually visualized and enumerated with the aid of fluorescence microscopy using antibodies to γH2AX and 53BP1. This study analyzed effect of low dose ionizing radiation on residual IRIF in human lymphocytes to the aim of potential biodosimetry and possible extrapolation of high-dose γH2AX/53BP1 effects to low doses and compared kinetics of DSB and IRIF. We also analyzed whether DNaseI, which is used for reducing of clumps, affects the IRIF level.

Materials And Methods: The cryopreserved human lymphocytes from umbilical cord blood (UCB) were thawed with/without DNaseI, γ-irradiated at doses of 0, 5, 10, and 50 cGy and γH2AX/53BP1 foci were analyzed 30 min, 2 h, and 22 h post-irradiation using appropriate antibodies. We also analyzed kinetics of DSB using PFGE.

Results: No significant difference was observed between data obtained by γH2AX foci evaluation in cells that were irradiated by low doses and data obtained by extrapolation from higher doses. Residual 53BP1 foci induced by low doses significantly outreached the data extrapolated from irradiation by higher doses. 53BP1 foci induced by low dose-radiation remain longer at DSB loci than foci induced by higher doses. There was no significant effect of DNaseI on DNA repair foci.

Conclusions: Primary γH2AX, 53BP1 foci and their co-localization represent valuable markers for biodosimetry of low doses, but their usefulness is limited by short time window. Residual γH2AX and 53BP1 foci are more useful markers for biodosimetry in vitro. Effects of low doses can be extrapolated from high dose using γH2AX residual foci while γH2AX/53BP1 foci are valuable markers for evaluation of initial DSB induced by ionizing radiation. Residual IRIF induced by low doses persist longer time than those induced by higher doses.
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http://dx.doi.org/10.3390/genes11010058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016656PMC
January 2020

Microwaves from mobile phone induce reactive oxygen species but not DNA damage, preleukemic fusion genes and apoptosis in hematopoietic stem/progenitor cells.

Sci Rep 2019 11 7;9(1):16182. Epub 2019 Nov 7.

Deparment of Radiobiology, Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, Bratislava, Slovak Republic.

Exposure to electromagnetic fields (EMF) has been associated with the increased risk of childhood leukemia, which arises from mutations induced within hematopoietic stem cells often through preleukemic fusion genes (PFG). In this study we investigated whether exposure to microwaves (MW) emitted by mobile phones could induce various biochemical markers of cellular damage including reactive oxygen species (ROS), DNA single and double strand breaks, PFG, and apoptosis in umbilical cord blood (UCB) cells including CD34+ hematopoietic stem/progenitor cells. UCB cells were exposed to MW pulsed signals from GSM900/UMTS test-mobile phone and ROS, apoptosis, DNA damage, and PFG were analyzed using flow cytometry, automated fluorescent microscopy, imaging flow cytometry, comet assay, and RT-qPCR. In general, no persisting difference in DNA damage, PFG and apoptosis between exposed and sham-exposed samples was detected. However, we found increased ROS level after 1 h of UMTS exposure that was not evident 3 h post-exposure. We also found that the level of ROS rise with the higher degree of cellular differentiation. Our data show that UCB cells exposed to pulsed MW developed transient increase in ROS that did not result in sustained DNA damage and apoptosis.
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http://dx.doi.org/10.1038/s41598-019-52389-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838175PMC
November 2019

A high throughput screening system of coils for ELF magnetic fields experiments: proof of concept on the proliferation of cancer cell lines.

BMC Cancer 2019 Feb 28;19(1):188. Epub 2019 Feb 28.

Department of Radiobiology, Cancer Research Institute, Biomedical Center, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05, Bratislava, Slovakia.

Background: It has been demonstrated that relatively small variations of the parameters of exposure to extremely low frequency magnetic fields (ELF-MF) can change significantly the outcome of experiments. Hence, either in trying to elucidate if these fields are carcinogenic, or in exploring their possible therapeutic use, it is desirable to screen through as many different exposures as possible. The purpose of this work is to provide a proof of concept of how a recently reported system of coils allows testing different field exposures, in a single experiment.

Methods: Using a novel exposure system, we subjected a glioblastoma cancer cell line (U251) to three different time modulations of an ELF-MF at 60 different combinations of the alternated current (AC) and direct current (DC) components of the field. One of those three time modulations was also tested on another cell line, MDA-MB-231 (breast cancer). After exposure, proliferation was assessed by colorimetric assays.

Results: For the U251 cells, a total of 180 different exposures were tested in three different experiments. Depending on exposure modulation and AC field intensity (but, remarkably, not on DC intensity), we found the three possible outcomes: increase (14.3% above control, p < 0.01), decrease (16.6% below control, p < 0.001), and also no-effect on proliferation with respect to control. Only the time modulation that inhibited proliferation of U251 was also tested on MDA-MB-231 cells which, in contrast, showed no alteration of their proliferation on any of the 60 AC/DC field combinations tested.

Conclusions: We demonstrated, for the first time, the use of a novel system of coils for magnetobiology research, which allowed us to find that differences of only a few μT resulted in statistically different results. Not only does our study demonstrate the relevance of the time modulation and the importance of finely sweeping through the AC and DC amplitudes, but also, and most importantly, provides a proof of concept of a system that sensibly reduces the time and costs of screening.
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http://dx.doi.org/10.1186/s12885-019-5376-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396543PMC
February 2019

Some recommendations for experimental work in magnetobiology, revisited.

Bioelectromagnetics 2018 10 10;39(7):556-564. Epub 2018 Oct 10.

Department of Radiobiology, Cancer Research Institute, Biomedical Research Center, Slovak Academy of Science, Bratislava, Slovakia.

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http://dx.doi.org/10.1002/bem.22144DOI Listing
October 2018

Thermal and non-thermal health effects of low intensity non-ionizing radiation: An international perspective.

Environ Pollut 2018 Nov 6;242(Pt A):643-658. Epub 2018 Jul 6.

European Cancer Environment Research Institute, Brussels, Belgium; Institute for Health and the Environment, University at Albany, Albany, NY, USA; Child Health Research Centre, The University of Queensland, Faculty of Medicine, Brisbane, Australia. Electronic address:

Exposure to low frequency and radiofrequency electromagnetic fields at low intensities poses a significant health hazard that has not been adequately addressed by national and international organizations such as the World Health Organization. There is strong evidence that excessive exposure to mobile phone-frequencies over long periods of time increases the risk of brain cancer both in humans and animals. The mechanism(s) responsible include induction of reactive oxygen species, gene expression alteration and DNA damage through both epigenetic and genetic processes. In vivo and in vitro studies demonstrate adverse effects on male and female reproduction, almost certainly due to generation of reactive oxygen species. There is increasing evidence the exposures can result in neurobehavioral decrements and that some individuals develop a syndrome of "electro-hypersensitivity" or "microwave illness", which is one of several syndromes commonly categorized as "idiopathic environmental intolerance". While the symptoms are non-specific, new biochemical indicators and imaging techniques allow diagnosis that excludes the symptoms as being only psychosomatic. Unfortunately standards set by most national and international bodies are not protective of human health. This is a particular concern in children, given the rapid expansion of use of wireless technologies, the greater susceptibility of the developing nervous system, the hyperconductivity of their brain tissue, the greater penetration of radiofrequency radiation relative to head size and their potential for a longer lifetime exposure.
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http://dx.doi.org/10.1016/j.envpol.2018.07.019DOI Listing
November 2018

Backtracked analysis of preleukemic fusion genes and DNA repair foci in umbilical cord blood of children with acute leukemia.

Oncotarget 2018 Apr 10;9(27):19233-19244. Epub 2018 Apr 10.

Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovak Republic.

The first event in origination of many childhood leukemias is a specific preleukemic fusion gene (PFG) that arises, often in utero, in hematopoietic stem/progenitor cells (HSPC) from misrepaired DNA double strand break (DSB). An immanently elevated level of DSB and impaired apoptosis may contribute to origination and persistence of PFG and donor cell-derived leukemia in recipients of allogeneic transplantation of umbilical cord blood (UCB). We investigated DSB, apoptosis and PFG in the backtracked UCB cells of leukemic patients. RNA from UCB of three patients with acute lymphoblastic leukemia, patient with acute megakaryoblastic leukemia and Down syndrome, and four healthy children was screened for common PFG by RT-qPCR. Presence of PFG was validated by sequencing. Endogenous γH2AX and 53BP1 DNA repair foci, cell populations, and apoptosis were analyzed in UCB CD34+/- cells with imaging and standard flow cytometry. We found and fusion genes in UCB of two out from four pediatric patients, apparently not detected at diagnosis, while UCB cells of ALL patient were tested negative for this PFG and no PFG were detected in UCB cells of healthy children. No significant difference in DNA damage and apoptosis between UCB CD34+/- cells from healthy children and leukemic patients was observed, while Down syndrome trisomy increased DNA damage and resulted in distribution of cell populations resembling transient abnormal myelopoiesis. Our findings indicate increased genetic instability in UCB HSPC of leukemic patients and may be potentially used for diagnostics and exclusion of possibly affected UCB from transplantation.
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http://dx.doi.org/10.18632/oncotarget.24976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922391PMC
April 2018

Heart rate variability affected by radiofrequency electromagnetic field in adolescent students.

Bioelectromagnetics 2018 May 22;39(4):277-288. Epub 2018 Feb 22.

Jessenius Faculty of Medicine in Martin, Department of Medical Biophysics, Comenius University in Bratislava, Martin, Slovakia.

This study examines the possible effect of radiofrequency (RF) electromagnetic fields (EMF) on the autonomic nervous system (ANS). The effect of RF EMF on ANS activity was studied by measuring heart rate variability (HRV) during ortho-clinostatic test (i.e., transition from lying to standing and back) in 46 healthy grammar school students. A 1788 MHz pulsed wave with intensity of 54 ± 1.6 V/m was applied intermittently for 18 min in each trial. Maximum specific absorption rate (SAR ) value was determined to 0.405 W/kg. We also measured the respiration rate and estimated a subjective perception of EMF exposure. RF exposure decreased heart rate of subjects in a lying position, while no such change was seen in standing students. After exposure while lying, a rise in high frequency band of HRV and root Mean Square of the Successive Differences was observed, which indicated an increase in parasympathetic nerve activity. Tympanic temperature and skin temperature were measured showing no heating under RF exposure. No RF effect on respiration rate was observed. None of the tested subjects were able to distinguish real exposure from sham exposure when queried at the end of the trial. In conclusion, short-term RF EMF exposure of students in a lying position during the ortho-clinostatic test affected ANS with significant increase in parasympathetic nerve activity compared to sham exposed group. Bioelectromagnetics. 39:277-288, 2018. © 2018 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bem.22115DOI Listing
May 2018

Low numbers of pre-leukemic fusion genes are frequently present in umbilical cord blood without affecting DNA damage response.

Oncotarget 2017 May;8(22):35824-35834

Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia.

Despite widely accepted notion that many childhood leukemias are likely developed from hematopoietic stem/progenitor cells (HSPC) with pre-leukemic fusion genes (PFG) formed in embryonic/fetal development, the data on PFG incidence in newborns are contradictive. To provide a better understanding of a prenatal origin of leukemia, umbilical cord blood from 500 newborns was screened for the presence of the most frequent PFG associated with pediatric B-cell acute lymphoblastic leukemia. This screening revealed relatively high incidence of ETV6-RUNX1, BCR-ABL1 (p190) and MLL-AF4 at very low frequencies, averaging ~14 copies per 100,000 cells. We assume that most of these PFG might originate relatively late in embryonic/fetal development and will be eliminated later during postnatal development. The obtained results suggested that higher PFG copy numbers originating in specific time windows of the hematopoietic stem cell hierarchy may define a better prognostic tool for the assessment of leukemogenic potential. We have observed no significant effect of low-copy PFG on radiation-induced DNA damage response, accumulation of endogenous DNA double-stranded breaks, and apoptosis in either lymphocytes or HSPC. Imaging flow cytometry showed lower level of γH2AX foci in HSPC in comparison to lymphocytes suggesting better protection of HSPC from DNA damage.
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http://dx.doi.org/10.18632/oncotarget.16211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482620PMC
May 2017

Hematopoietic stem/progenitor cells are less prone to undergo apoptosis than lymphocytes despite similar DNA damage response.

Oncotarget 2017 Jul;8(30):48846-48853

Laboratory of Radiobiology, Cancer Research Institute, Biomedical Center, Slovak Academy of Sciences, Bratislava, Slovakia.

Hematopoietic stem/progenitor CD34+ cells (HSPC) give rise to all types of blood cells and represent a key cellular target for origination of leukemia. Apoptosis and repair of DNA double strand breaks (DSB) are vital processes in leukemogenesis. High doses of ionizing radiation are the best known agent that induces leukemia, but less is known about the leukemogenic potential of low doses. While umbilical cord blood (UCB) serves as a valuable source of the HSPC for both research and clinics, the data on DNA damage response and apoptosis in UCB HSPC are very limited. We have studied apoptosis and DSB in the UCB-derived CD34+HSPC and CD34- lymphocytes at different time points post-irradiation with low and therapeutic doses of γ-rays. DSB were enumerated with γH2AX foci using imaging flow cytometry. Different stages of apoptosis were analyzed using Annexin/7-AAD assay and γH2AX pan-staining by flow cytometry and imaging flow cytometry, respectively. Our results have consistently shown significantly higher resistance of CD34+ stem/progenitor cells to endogenous and radiation induced apoptosis as compared to CD34- lymphocytes. At the same time, no statistically significant difference was found in DSB repair between HSPC and lymphocytes as enumerated by the γH2AX foci. To conclude, we show for the first time that hematopoietic stem/progenitor cells are less prone to undergo apoptosis than lymphocytes what may be accounted for higher expression of anti-apoptotic proteins in CD34+ cells but was unlikely dealt with DSB repair.
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http://dx.doi.org/10.18632/oncotarget.16455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5564729PMC
July 2017

Validation of JCountPro software for efficient assessment of ionizing radiation-induced foci in human lymphocytes.

Int J Radiat Biol 2016 12 20;92(12):766-773. Epub 2016 Sep 20.

a Laboratory of Radiobiology , Cancer Research Institute, Biomedical Research Centre SAS, Slovak Academy of Sciences , Bratislava , Slovakia.

Purpose: Ionizing radiation-induced foci (IRIF) known also as DNA repair foci represent the most sensitive and specific assay for assessing DNA double-strand break (DSB). IRIF are usually visualized and enumerated with the aid of fluorescence microscopy using antibodies to phosphorylated γH2AX and 53BP1. Although several approaches and software packages were developed for quantification of IRIF, not one of them was commonly accepted and inter-laboratory variability in the outputs was reported. In this study, JCountPro software was validated for IRIF enumeration in two independent laboratories.

Materials And Methods: Human lymphocytes were γ-irradiated at doses of 0, 2, 5, 10 and 50 cGy. The cells were fixed, permeabilized and IRIF were immunostained using appropriate antibodies. Cell images were acquired with automatic Metafer system. Endogenous and radiation-induced γH2AX and 53BP1 foci were enumerated using JCountPro. This analysis was performed from the same cell galleries by the researchers from two laboratories. Yield of foci was analyzed by either arithmetic mean (AM) value (foci/cell) or principal average (PA) derived from the approximation of foci distribution with Poisson statistics. Statistical analysis was performed using factorial ANOVA.

Results: Enumeration of 53BP1, γH2AX and co-localized 53BP1/γH2AX foci by JCountPro was essentially the same between laboratories. IRIF were detected at all doses and linear dose response was obtained in the studied dose range. PA values from Poisson distribution fitted the data better as compared to AM values and were more powerful and sensitive for IRIF analysis than the AM values. All JCountPro data were confirmed by visual focus enumeration.

Conclusions: We concluded that the JCountPro software was efficient in objectively enumerating IRIF regardless of an individual researcher's bias and has a potential for usage in clinics and molecular epidemiology.
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http://dx.doi.org/10.1080/09553002.2016.1222093DOI Listing
December 2016

EUROPAEM EMF Guideline 2016 for the prevention, diagnosis and treatment of EMF-related health problems and illnesses.

Rev Environ Health 2016 Sep;31(3):363-97

Chronic diseases and illnesses associated with non-specific symptoms are on the rise. In addition to chronic stress in social and work environments, physical and chemical exposures at home, at work, and during leisure activities are causal or contributing environmental stressors that deserve attention by the general practitioner as well as by all other members of the health care community. It seems necessary now to take "new exposures" like electromagnetic fields (EMF) into account. Physicians are increasingly confronted with health problems from unidentified causes. Studies, empirical observations, and patient reports clearly indicate interactions between EMF exposure and health problems. Individual susceptibility and environmental factors are frequently neglected. New wireless technologies and applications have been introduced without any certainty about their health effects, raising new challenges for medicine and society. For instance, the issue of so-called non-thermal effects and potential long-term effects of low-dose exposure were scarcely investigated prior to the introduction of these technologies. Common electromagnetic field or EMF sources: Radio-frequency radiation (RF) (3 MHz to 300 GHz) is emitted from radio and TV broadcast antennas, Wi-Fi access points, routers, and clients (e.g. smartphones, tablets), cordless and mobile phones including their base stations, and Bluetooth devices. Extremely low frequency electric (ELF EF) and magnetic fields (ELF MF) (3 Hz to 3 kHz) are emitted from electrical wiring, lamps, and appliances. Very low frequency electric (VLF EF) and magnetic fields (VLF MF) (3 kHz to 3 MHz) are emitted, due to harmonic voltage and current distortions, from electrical wiring, lamps (e.g. compact fluorescent lamps), and electronic devices. On the one hand, there is strong evidence that long-term exposure to certain EMFs is a risk factor for diseases such as certain cancers, Alzheimer's disease, and male infertility. On the other hand, the emerging electromagnetic hypersensitivity (EHS) is more and more recognized by health authorities, disability administrators and case workers, politicians, as well as courts of law. We recommend treating EHS clinically as part of the group of chronic multisystem illnesses (CMI), but still recognizing that the underlying cause remains the environment. In the beginning, EHS symptoms occur only occasionally, but over time they may increase in frequency and severity. Common EHS symptoms include headaches, concentration difficulties, sleep problems, depression, a lack of energy, fatigue, and flu-like symptoms. A comprehensive medical history, which should include all symptoms and their occurrences in spatial and temporal terms and in the context of EMF exposures, is the key to making the diagnosis. The EMF exposure is usually assessed by EMF measurements at home and at work. Certain types of EMF exposure can be assessed by asking about common EMF sources. It is very important to take the individual susceptibility into account. The primary method of treatment should mainly focus on the prevention or reduction of EMF exposure, that is, reducing or eliminating all sources of high EMF exposure at home and at the workplace. The reduction of EMF exposure should also be extended to public spaces such as schools, hospitals, public transport, and libraries to enable persons with EHS an unhindered use (accessibility measure). If a detrimental EMF exposure is reduced sufficiently, the body has a chance to recover and EHS symptoms will be reduced or even disappear. Many examples have shown that such measures can prove effective. To increase the effectiveness of the treatment, the broad range of other environmental factors that contribute to the total body burden should also be addressed. Anything that supports homeostasis will increase a person's resilience against disease and thus against the adverse effects of EMF exposure. There is increasing evidence that EMF exposure has a major impact on the oxidative and nitrosative regulation capacity in affected individuals. This concept also may explain why the level of susceptibility to EMF can change and why the range of symptoms reported in the context of EMF exposures is so large. Based on our current understanding, a treatment approach that minimizes the adverse effects of peroxynitrite - as has been increasingly used in the treatment of multisystem illnesses - works best. This EMF Guideline gives an overview of the current knowledge regarding EMF-related health risks and provides recommendations for the diagnosis, treatment and accessibility measures of EHS to improve and restore individual health outcomes as well as for the development of strategies for prevention.
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http://dx.doi.org/10.1515/reveh-2016-0011DOI Listing
September 2016

EUROPAEM EMF Guideline 2015 for the prevention, diagnosis and treatment of EMF-related health problems and illnesses.

Rev Environ Health 2015 ;30(4):337-71

Chronic diseases and illnesses associated with unspecific symptoms are on the rise. In addition to chronic stress in social and work environments, physical and chemical exposures at home, at work, and during leisure activities are causal or contributing environmental stressors that deserve attention by the general practitioner as well as by all other members of the health care community. It seems certainly necessary now to take "new exposures" like electromagnetic field (EMF) into account. Physicians are increasingly confronted with health problems from unidentified causes. Studies, empirical observations, and patient reports clearly indicate interactions between EMF exposure and health problems. Individual susceptibility and environmental factors are frequently neglected. New wireless technologies and applications have been introduced without any certainty about their health effects, raising new challenges for medicine and society. For instance, the issue of so-called non-thermal effects and potential long-term effects of low-dose exposure were scarcely investigated prior to the introduction of these technologies. Common EMF sources include Wi-Fi access points, routers and clients, cordless and mobile phones including their base stations, Bluetooth devices, ELF magnetic fields from net currents, ELF electric fields from electric lamps and wiring close to the bed and office desk. On the one hand, there is strong evidence that long-term-exposure to certain EMF exposures is a risk factor for diseases such as certain cancers, Alzheimer's disease and male infertility. On the other hand, the emerging electromagnetic hypersensitivity (EHS) is more and more recognized by health authorities, disability administrators and case workers, politicians, as well as courts of law. We recommend treating EHS clinically as part of the group of chronic multisystem illnesses (CMI) leading to a functional impairment (EHS), but still recognizing that the underlying cause remains the environment. In the beginning, EHS symptoms often occur only occasionally, but over time they may increase in frequency and severity. Common EHS symptoms include headaches, concentration difficulties, sleeping problems, depression, lack of energy, fatigue and flu-like symptoms. A comprehensive medical history, which should include all symptoms and their occurrences in spatial and temporal terms and in the context of EMF exposures, is the key to the diagnosis. The EMF exposure can be assessed by asking for typical sources like Wi-Fi access points, routers and clients, cordless and mobile phones and measurements at home and at work. It is very important to take the individual susceptibility into account. The primary method of treatment should mainly focus on the prevention or reduction of EMF exposure, that is, reducing or eliminating all sources of EMF at home and in the workplace. The reduction of EMF exposure should also be extended to public spaces such as schools, hospitals, public transport, and libraries to enable persons with EHS an unhindered use (accessibility measure). If a detrimental EMF exposure is reduced sufficiently, the body has a chance to recover and EHS symptoms will be reduced or even disappear. Many examples have shown that such measures can prove effective. Also the survival rate of children with leukemia depends on ELF magnetic field exposure at home. To increase the effectiveness of the treatment, the broad range of other environmental factors that contribute to the total body burden should also be addressed. Anything that supports a balanced homeostasis will increase a person's resilience against disease and thus against the adverse effects of EMF exposure. There is increasing evidence that EMF exposure has a major impact on the oxidative and nitrosative regulation capacity in affected individuals. This concept also may explain why the level of susceptibility to EMF can change and why the number of symptoms reported in the context of EMF exposures is so large. Based on our current understanding, a treatment approach that minimizes the adverse effects of peroxynitrite - as has been increasingly used in the treatment of multisystem disorders - works best. This EMF Guideline gives an overview of the current knowledge regarding EMF-related health risks and provides concepts for the diagnosis and treatment and accessibility measures of EHS to improve and restore individual health outcomes as well as for the development of strategies for prevention.
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http://dx.doi.org/10.1515/reveh-2015-0033DOI Listing
March 2016

DNA repair foci and late apoptosis/necrosis in peripheral blood lymphocytes of breast cancer patients undergoing radiotherapy.

Int J Radiat Biol 2015 4;91(12):934-45. Epub 2015 Nov 4.

a Laboratory of Radiobiology , Cancer Research Institute, Slovak Academy of Sciences , Bratislava , Slovakia.

Purpose: Double-strand breaks (DSB) repair and apoptosis are assumed to be key factors in the determination of individual variability in response to radiation treatment. In this study we investigated tumor protein p53 (TP53) binding protein 1 (53BP1) and phosphorylated histone 2A family member X (γH2AX) foci, γH2AX pan-staining and late apoptosis/necrosis (LAN) in lymphocytes from breast cancer (BC) patients undergoing radiotherapy.

Materials And Methods: BC patients were subjected to local radiotherapy with fractionated doses using linear accelerator. Adverse reactions of patients were classified according to the Radiation Therapy Oncology Group (RTOG)/European Organization for Research and Treatment of Cancer (EORTC) criteria. Blood samples were collected before treatment, at various time-points during and after radiotherapy. Residual 53BP1 and γH2AX foci, γH2AX pan-staining were analyzed in peripheral blood lymphocytes (PBL) using the Metafer system and confocal laser scanning microscopy. LAN cells were counted by the trypan blue (TB) exclusion assay. Statistical analysis was performed using Mann-Whitney test, Spearman rank correlation test and analysis of covariance (ANCOVA).

Results: No statistically significant changes were observed in the levels of γH2AX foci during radiotherapy. In contrast, radiation-induced residual 53BP1 were detected already after the first fraction. Increased individual variability in the 53BP1 focus formation was observed during treatment. The background level of DNA repair foci and its individual variability in response to radiotherapy decreased after the end of radiotherapy indicating successful removal of DNA-damaging effects. A correlation between stage of cancer and 53BP1 focus formation was established which suggests the prognostic value of this test. We show that the fraction of LAN cells negatively correlates with the level of 53BP1 and positively correlates with individual radiosensitivity. Only weak correlation was observed between γH2AX pan-staining and LAN cells. Due to large interindividual variability, both in vivo assays, LAN and focus formation, have shown relatively low predictive power at the individual level.

Conclusions: It is likely that radiosensitive patients have less efficient mechanisms of elimination of apoptotic cells with DNA damage resulting in accumulation of LAN cells and facilitating adverse reactions. Our data suggested that the grade of adverse reaction may positively correlate with LAN cells in PBL before and during radiotherapy.
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http://dx.doi.org/10.3109/09553002.2015.1101498DOI Listing
May 2016

Imaging flow cytometry as a sensitive tool to detect low-dose-induced DNA damage by analyzing 53BP1 and γH2AX foci in human lymphocytes.

Cytometry A 2015 Dec 4;87(12):1070-8. Epub 2015 Aug 4.

Laboratory of Radiobiology, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia.

Ionizing radiation induced foci (IRIF) are considered the most sensitive indicator for DNA double-strand break (DSB) detection. Monitoring DSB induction by low doses of ionizing radiation is important due to the increasing exposure in the general population. γH2AX and 53BP1 are commonly used molecular markers for in situ IRIF assessment. Imaging flow cytometry (IFC) via ImageStream system provides a new opportunity in this field. We analyzed the formation of 53BP1, γH2AX foci and their co-localization induced by γ-rays (2, 5, 10, 50, 200 cGy) in human lymphocytes using ImageStream and the automated microscopic system Metafer. We observed very similar sensitivity of both systems for the detection of endogenous and low-dose-induced IRIF. Statistically significant induction of γH2AX foci was found at doses of 2 and 10 cGy using ImageStream and Metafer, respectively. Statistically significant induction of 53BP1 foci was evident at doses ≥ 5 cGy when analyzed by IFC. Analysis of the co-localizing foci by ImageStream and Metafer showed statistical significance at doses ≥ 2 cGy, suggesting that foci co-localization is a sensitive parameter for DSB quantification. Assessment of γH2AX, 53BP1 foci and their co-localization by Metafer and ImageStream showed similar linear dose responses in the low-dose range up to 10 cGy, although IFC showed slightly better resolution for IRIF in this dose range. At higher doses, IFC underestimated IRIF numbers. Using the imaging ability of ImageStream, we introduced an optimized assay by gating γH2AX foci positive (with 1 or more γH2AX foci) and negative (cells without foci) cells. This assay resulted in statistically significant IRIF induction at doses ≥ 5cGy and a linear dose response up to 50 cGy. In conclusion, we provide evidence for the use of IFC as an accurate high throughput assay for the prompt detection and enumeration of endogenous and low-dose induced IRIF.
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http://dx.doi.org/10.1002/cyto.a.22731DOI Listing
December 2015

Combined multiplex and monoplex RT-PCR as a reliable and cost-effective method for molecular diagnostics of pediatric acute lymphoblastic leukemia.

Neoplasma 2014 ;61(6):758-65

The precise diagnosis of acute lymphoblastic leukemia is essential for correct prognosis assessment and therapy regimen selection. At present, immunophenotyping, cytogenetics and molecular screening are major and complementary methods utilized in a routine leukemia diagnostics. The aim of this study was to validate the application of multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay for molecular diagnosis of the most common pediatric acute lymphoblastic leukemia-associated fusion transcripts. Our data show that screening of bone marrow and/or peripheral blood by RT-PCR, consisting of multiplex and monoplex PCR, confirmed results of real-time quantitative PCR (RT qPCR). This screening may provide a reliable, specific and sensitive method amenable to standard laboratory practice and a cost-effective alternative to more complex and expensive RT qPCR techniques.
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http://dx.doi.org/10.4149/neo_2014_092DOI Listing
June 2015

Incidence of common preleukemic gene fusions in umbilical cord blood in Slovak population.

PLoS One 2014 12;9(3):e91116. Epub 2014 Mar 12.

Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic.

The first event in origination of many childhood leukemias is likely the presence of preleukemic clone (transformed hematopoietic stem/progenitor cells with preleukemic gene fusions (PGF)) in newborn. Thus, the screening of umbilical cord blood (UCB) for PGF may be of high importance for developing strategies for childhood leukemia prevention and treatment. However, the data on incidence of PGF in UCB are contradictive. We have compared multiplex polymerase chain reaction (PCR) and real-time quantitative PCR (RT qPCR) in neonates from Slovak National Birth Cohort. According to multiplex PCR, all 135 screened samples were negative for the most frequent PGF of B-lineage acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). To explore the prevalence of prognostically important TEL-AML1, MLL-AF4 and BCR-ABL (p190), 200 UCB were screened using RT qPCR. The initial screening showed an unexpectedly high incidence of studied PGF. The validation of selected samples in two laboratories confirmed approximately ¼ of UCB positive, resulting in ∼4% incidence of TEL-AML1, ∼6.25% incidence of BCR-ABL1 p190, and ∼0.75% frequency of MLL-AF4. In most cases, the PGF presented at very low level, about 1-5 copies per 105 cells. We hypothesize that low PGF numbers reflect their relatively late origin and are likely to be eliminated in further development while higher number of PGF reflects earlier origination and may represent higher risk for leukemia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091116PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951330PMC
May 2015

Relative biological efficiency of protons at low and therapeutic doses in induction of 53BP1/γH2AX foci in lymphocytes from umbilical cord blood.

Int J Radiat Biol 2013 Sep 22;89(9):716-23. Epub 2013 May 22.

Cancer Research Institute, Slovak Academy of Science, Bratislava, Slovak Republic.

Purpose: In order to evaluate DNA damage induced by protons at low and radiotherapeutic doses at the therapeutic proton complex at Ružomberok, Slovak Republic, lymphocytes from umbilical cord blood (UCB) of the same four probands were irradiated in the dose range of 1-200 cGy with γ-rays and protons (200 MeV, irradiation in the Bragg peak).

Materials And Methods: DNA repair γH2AX/53BP1 foci were analyzed by fluorescent microscopy and flow cytometry.

Results: Statistically significant effects of radiations were detected by fluorescent microscopy at all doses higher 1 cGy. Almost all distributions of foci in irradiated cells fitted to the Poisson distribution. In general, there was no difference in the levels of γH2AX and 53BP1 foci in irradiated cells. Flow cytometry was less sensitive and detected radiation induced effects at doses of 50 cGy and higher. Factorial analysis of variance in the whole studied dose range has shown no significant effect of radiation quality on number of γH2AX and 53BP1 foci. The ratio of proton-induced foci to γ-ray-induced foci was 0.86 ± 0.16 (53BP1) and 0.99 ± 0.34 (γH2AX) as measured by fluorescent microscopy and 0.99 ± 0.16 (γH2AX) as measured by flow cytometry at the radiotherapeutic dose of 2 Gy.

Conclusions: Both flow cytometry and fluorescent microscopy indicated that the average value of relative biological efficiency (RBE) at radiation doses ≥ 20 cGy was about 1.0. Our data that RBE increased at low doses ≤ 20 cGy are relevant both to the development of treatment modalities and exposures that take place during space exploration and should be verified by further studies.
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http://dx.doi.org/10.3109/09553002.2013.797619DOI Listing
September 2013

DNA damage response in CD133 + stem/progenitor cells from umbilical cord blood: low level of endogenous foci and high recruitment of 53BP1.

Int J Radiat Biol 2013 Apr 8;89(4):301-9. Epub 2013 Jan 8.

Laboratory of Molecular Genetics, Cancer Research Institute, Bratislava, Slovak Republic.

Unlabelled: Abstract Purpose: Human hematopoietic stem cells (HSC) are thought to be a major target of radiation-induced leukemogenesis and also provide a relevant cellular model for assessing cancer risk. Cluster of designation 133+ (CD133+) is a marker found in human progenitor and hematopoietic stem cells. Our study examined the repair of radiation-induced DNA double-strand breaks (DSB) in CD133 + umbilical cord blood cells (UCBC).

Materials And Methods: After γ-irradiation, endogenous and induced DSB were evaluated in CD133 + UCBC, CD133 - UCBC and peripheral blood lymphocytes (PBL) in terms of phosphorylated histone 2A family member X (γH2AX) and tumor suppressor p53 binding protein 1 (53BP1) foci.

Results: We found that repair signaling in CD133 + UCBC is different from CD133 - UCBC and PBL. These differences include lower endogenous DSB levels and higher 53BP1 recruitment.

Conclusions: Our data, together with a recent report on radiation-induced γH2AX and 53BP1 foci in CD34 + cells, indicate enhanced DNA repair capacity in HSC as compared to mature lymphocytes.
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http://dx.doi.org/10.3109/09553002.2013.754555DOI Listing
April 2013

Long time persistence of residual 53BP1/γ-H2AX foci in human lymphocytes in relationship to apoptosis, chromatin condensation and biological dosimetry.

Int J Radiat Biol 2011 Jul;87(7):736-45

Department of Genetics, Microbiology and Toxicology, Stockholm University, Sweden.

Purpose: Novel assay for radiosensitivity is based on measurements of residual DNA repair foci produced by several proteins including phosphorylated H2AX (γ-H2AX), recombinase Rad51 (Rad51) and tumour suppressor p53 binding protein 1 (53BP1), which co-localise with radiation-induced DNA double-strand breaks (DSB). Here, we studied dose-response for residual 53BP1, Rad51, and γ-H2AX foci in relationship to apoptosis and chromatin condensation in human G(0)-lymphocytes.

Materials And Methods: Residual foci, apoptosis and condensation of chromatin were studied following irradiation with γ-rays at doses of 0.5-10 Gy.

Results: No clear dose response for residual Rad51 was seen. Residual 53BP1/γ-H2AX foci remained in human lymphocytes up to four weeks after irradiation. No foci formed during radiation-induced apoptosis. We provide evidence that irreversible apoptotic condensation of chromatin is responsible for arrest of residual foci and preventing de novo focus formation. Similar linear dose dependences up to 2 Gy were observed for the 53BP1/γ-H2AX foci at all studied time points. At higher doses, saturation and decline were caused by preferential elimination of apoptotic lymphocytes with residual foci. While primary 53BP1 and γ-H2AX foci almost completely co-localised, co-localisation of residual foci did not exceed 17%, indicating that 53BP1 and γ-H2AX proteins may remain for different times at the locations of DSB repair.

Conclusions: Prolonged persistence of residual 53BP1/γ-H2AX foci may be used for biological dosimetry within the dose range up to 2 Gy. While foci are not formed during radiation-induced apoptosis in human lymphocytes, elimination of apoptotic cells with residual foci may affect the dose response.
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http://dx.doi.org/10.3109/09553002.2011.577504DOI Listing
July 2011

Fifty hertz magnetic fields individually affect chromatin conformation in human lymphocytes: dependence on amplitude, temperature, and initial chromatin state.

Bioelectromagnetics 2011 Oct 15;32(7):570-9. Epub 2011 Apr 15.

Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden.

Effects of magnetic field (MF) at 50 Hz on chromatin conformation were studied by the method of anomalous viscosity time dependence (AVTD) in human lymphocytes from two healthy donors. MF within the peak amplitude range of 5-20 µT affected chromatin conformation. These MF effects differed significantly between studied donors, and depended on magnetic flux density and initial condensation of chromatin. While the initial state of chromatin was rather stable in one donor during one calendar year of measurements, the initial condensation varied significantly in cells from another donor. Both this variation and the MF effect depended on temperature during exposure. Despite these variations, the general rule was that MF condensed the relaxed chromatin and relaxed the condensed chromatin. Thus, in this study we show that individual effects of 50 Hz MF exposure at peak amplitudes within the range of 5-20 µT may be observed in human lymphocytes in dependence on the initial state of chromatin and temperature.
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http://dx.doi.org/10.1002/bem.20674DOI Listing
October 2011

Toxicity and SOS-response to ELF magnetic fields and nalidixic acid in E. coli cells.

Authors:
Igor Belyaev

Mutat Res 2011 May 8;722(1):56-61. Epub 2011 Apr 8.

Laboratory of Molecular Genetics, Cancer Research Institute, Bratislava, Slovak Republic.

Extremely low-frequency magnetic fields (ELF-MF) have previously been shown to affect conformation of chromatin and cell proliferation. Possible genotoxic and carcinogenic effects of ELF-MF have also been discussed and tested. In this study, we analysed the effect of ELF-MF on chromatin conformation in E. coli GE499 cells by the anomalous viscosity time-dependence (AVTD) technique. Possible genotoxic effects of the specific combination of static and ELF-MF, which has been proven to affect chromatin conformation, were investigated by a clonogenic assay, by assessing cell-growth kinetics, and by analysis of the SOS-response by means of inducible recA-lacZ fusion-gene products and the β-galactosidase assay. The genotoxic agent nalidixic acid (NAL) was used as a positive control and in combination with ELF-MF. Nalidixic acid at 3-30μg/ml decreased the AVTD peaks and induced a cytotoxic effect. In contrast to NAL, ELF-MF fields increased AVTD, stimulated cell growth, and increased cloning efficiency. These effects depended on the frequency within the range of 7-11Hz. While NAL induced an SOS-response, exposure to ELF-MF did not induce the recA-lacZ fusion-gene product. Exposure to ELF-MF did not modify the genotoxic effects of NAL either. All together, the data show that ELF-MF, under specific conditions of exposure, acted as a non-toxic but cell-growth stimulating agent.
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http://dx.doi.org/10.1016/j.mrgentox.2011.03.012DOI Listing
May 2011

Toxicity and SOS response to ELF magnetic field and nalidixic acid in E. coli cells.

Authors:
Igor Belyaev

Mutat Res 2011 May 29;722(1):84-8. Epub 2011 Mar 29.

Department of Genetics, Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden.

Extremely low frequency (ELF) magnetic fields have previously been shown to affect conformation of chromatin and cell proliferation. Possible genotoxic and carcinogenic effects of ELF have also been discussed and tested. In this study, we analyzed the effect of ELF on chromatin conformation in E. coli GE499 cells by the anomalous viscosity time dependence (AVTD) technique. Possible genotoxic ELF effects at the specific combination of static and ELF magnetic fields, that has been proven to have effects on chromatin conformation, were investigated by clonogenic assay, cell growth kinetics, and analysis of SOS-response using inducible recA-lacZ fusion and the β-galactosidase assay. Genotoxic agent nalidixic acid (NAL) was used as positive control and in combination with ELF. Nalidixic acid at 3-30μg/ml decreased the AVTD peaks and induced cytotoxic effect. In contrast to NAL, ELF increased AVTD, stimulated cell growth, and increased cloning efficiency. These effects depended on frequency within the frequency range of 7-11Hz. While NAL induced SOS response, ELF exposure did not induce the recA-lacZ fusion. Exposure to ELF did not modify the genotoxic effects of NAL either. All together, the data show that ELF, under specific conditions of exposure, acted as nontoxic but cell growth stimulating agent.
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http://dx.doi.org/10.1016/j.mrgentox.2011.03.011DOI Listing
May 2011