Evaluation of oxidative stress and genetic instability among residents near mobile phone base stations in Germany.

Human exposure to radiofrequency electromagnetic fields (RF-EMF) is restricted to prevent thermal effects in the tissue. However, at very low intensity exposure "non-thermal" biological effects, like oxidative stress, DNA or chromosomal aberrations, etc. collectively termed genomic-instability can occur after few hours. Little is known about chronic (years long) exposure with non-thermal RF-EMF. We identified two neighboring housing estates in a rural region with residents exposed to either relatively low (control-group) or relatively high (exposed-group) RF-EMF emitted from nearby mobile phone base stations (MPBS). 24 healthy adults that lived in their homes at least for 5 years volunteered. The homes were surveyed for common types of EMF, blood samples were tested for oxidative status, transient DNA alterations, permanent chromosomal damage, and specific cancer related genetic markers, like MLL gene rearrangements. We documented possible confounders, like age, sex, nutrition, life-exposure to ionizing radiation (X-rays), occupational exposures, etc. The groups matched well, age, sex, lifestyle and occupational risk factors were similar. The years long exposure had no measurable effect on MLL gene rearrangements and c-Abl-gene transcription modification. Associated with higher exposure, we found higher levels of lipid oxidation and oxidative DNA-lesions, though not statistically significant. DNA double strand breaks, micronuclei, ring chromosomes, and acentric chromosomes were not significantly different between the groups. Chromosomal aberrations like dicentric chromosomes (p=0.007), chromatid gaps (p=0.019), chromosomal fragments (p<0.001) and the total of chromosomal aberrations (p<0.001) were significantly higher in the exposed group. No potential confounder interfered with these findings. Increased rates of chromosomal aberrations as linked to excess exposure with ionizing radiation may also occur with non-ionizing radiation exposure. Biological endpoints can be informative for designing exposure limitation strategies. Further research is warranted to investigate the dose-effect-relationship between both, exposure intensity and exposure time, to account for endpoint accumulations after years of exposure. As established for ionizing radiation, chromosomal aberrations could contribute to the definition of protection thresholds, as their rate reflects exposure intensity and exposure time.

Effects of low-dose ionizing radiation on genomic instability in interventional radiology workers.

Interventional radiologists are chronically exposed to low-dose ionizing radiation (IR), which may represent a health risk. The aim of the present study was to evaluate genomic instability by analyzing chromosomal aberrations, micronuclei, and 53BP1 DNA repair foci in peripheral blood lymphocytes of radiologists. Based on the IAEA guidelines on biodosimetry using dicentrics, the average protracted whole-body dose in radiologists were estimated. Since preleukemic fusion genes (PFG) are the primary events leading to leukemia, we also studied their presence by RT-qPCR and FISH. No significant difference in 53BP1 foci and incidence of PFG (MLL-AF4, MLL-AF9, AML1-ETO, BCR-ABL p190) was found in cells of interventional radiologists in comparison to controls. However, our results showed an increased frequency of micronuclei and various types of chromosomal aberrations including dicentrics in interventional radiologists. The average protracted whole body estimated dose was defined at 452.63 mGy. We also found a significantly higher amplification of the MLL gene segment and increased RNA expression in cells of interventional radiologists in comparison to controls. In conclusion, our results showed that long-term low-dose IR induces genomic instability in interventional radiologists.

Assessment of Individual Radiosensitivity in Breast Cancer Patients Using a Combination of Biomolecular Markers.

About 5% of patients undergoing radiotherapy (RT) develop RT-related side effects. To assess individual radiosensitivity, we collected peripheral blood from breast cancer patients before, during and after the RT, and γH2AX/53BP1 foci, apoptosis, chromosomal aberrations (CAs) and micronuclei (MN) were analyzed and correlated with the healthy tissue side effects assessed by the RTOG/EORTC criteria. The results showed a significantly higher level of γH2AX/53BP1 foci before the RT in radiosensitive (RS) patients in comparison to normal responding patients (NOR). Analysis of apoptosis did not reveal any correlation with side effects. CA and MN assays displayed an increase in genomic instability during and after RT and a higher frequency of MN in the lymphocytes of RS patients. We also studied time kinetics of γH2AX/53BP1 foci and apoptosis after in vitro irradiation of lymphocytes. Higher levels of primary 53BP1 and co-localizing γH2AX/53BP1 foci were detected in cells from RS patients as compared to NOR patients, while no difference in the residual foci or apoptotic response was found. The data suggested impaired DNA damage response in cells from RS patients. We suggest γH2AX/53BP1 foci and MN as potential biomarkers of individual radiosensitivity, but they need to be evaluated with a larger cohort of patients for clinics.

Chemotherapy and cryopreservation affects DNA repair foci in lymphocytes of breast cancer patients.

PURPOSE: Although breast cancer (BC) patients benefit from radiotherapy (RT), some radiosensitive (RS) patients suffer from side effects caused by ionizing radiation in healthy tissues. It is thought that RS is underlaid by a deficiency in the repair of DNA double-strand breaks (DSB). DNA repair proteins such as p53-binding protein 1 (53BP1) and phosphorylated histone H2AX (γH2AX), form DNA repair foci at the DSB locations and thus serve as DSB biomarkers. Peripheral blood lymphocytes (PBL) are commonly believed to be an appropriate cell system for RS assessment using DNA repair foci. The amount of DSB may also be influenced by chemotherapy (CHT), which is often chosen as the first treatment modality before RT. As it is not always possible to analyze blood samples immediately after collection, there is a need for cryopreservation of PBL in liquid nitrogen. However, cryopreservation may potentially affect the number of DNA repair foci. In this work, we studied the effect of cryopreservation and CHT on the amount of DNA repair foci in PBL of BC patients undergoing radiotherapy. MATERIALS AND METHODS: The effect of cryopreservation was studied by immunofluorescence analysis of 53BP1 and γH2AX proteins at different time intervals after in vitro irradiation. The effect of chemotherapy was analyzed by fluorescent labelling of 53BP1 and γH2AX proteins in PBL collected before, during, and after RT. RESULTS: Higher number of primary 53BP1/γH2AX foci was observed in frozen cells indicating that cryopreservation affects the formation of DNA repair foci in PBL of BC patients. In CHT-treated patients, a higher number of foci were found before RT, but no differences were observed during and after the RT. CONCLUSIONS: Cryopreservation is the method of choice for analyzing DNA repair residual foci, but only similarly treated and preserved cells should be used for comparison of primary foci. CHT induces DNA repair foci in PBL of BC patients, but this effect disappears during radiotherapy.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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 MLL2-AF4 and BCR-ABL (p190) fusion genes in UCB of two out from four pediatric patients, apparently not detected at diagnosis, while UCB cells of TEL-AML1+ 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.

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

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.

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

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.

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

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.

Recombinant Tyrosinase from Polyporus arcularius: Overproduction in Escherichia coli, Characterization, and Use in a Study of Aurones as Tyrosinase Effectors.

Tyrosinases act in the development of organoleptic properties of tea, raisins, etc., but also cause unwanted browning of fruits, vegetables, and mushrooms. The tyrosinase from Agaricus bisporus has been used as a model to study tyrosinase inhibitors, which are also indispensable in the treatment of skin pigmentation disorders. However, this model has disadvantages such as side enzyme activities and the presence of multiple isoenzymes. Therefore, we aimed to introduce a new tyrosinase model. The pro-tyrosinase from Polyporus arcularius was overproduced in Escherichia coli. Trypsin digestion led to a cleavage after R388 and hence enzyme activation. The tyrosinase was a homodimer and transformed L-DOPA and tert-butylcatechol preferentially. Various aurons were examined as effectors of this enzyme. 2'- and 3'-hydroxyaurones acted as its activators and 2',4'-dihydroxyaurone as an inhibitor, whereas 4'-hydroxyaurones were its substrates. The enzyme is a promising model for tyrosinase effector studies, being a single isoenzyme and void of side enzyme activities.

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

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.

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

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.

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

PURPOSE: In order to evaluate DNA damage induced by protons at low and radiotherapeutic doses at the therapeutic proton complex at Ruzomberok, 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.