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Environmental science and pollution research international
2018

Toxicological evaluation of nail polish waste discarded in the environment.

Felzenszwalb, Israel, Fernandes, Andreia da Silva, Brito, Lara Barroso, Oliveira, Gisele Augusto Rodrigues, Silva, Paula Aquino Soeiro, Arcanjo, Maria Elena, Marques, Monica Regina da Costa, Vicari, Taynah, Leme, Daniela Morais, Cestari, Marta Margarete, Ferraz, Elisa Raquel Anastacio

Nail polish has been widely used around the world. However, the hazards of nail polishes discarded in the environment are still poorly investigated. Thus, the toxicogenetic effects of solubilized (SE) and leached (LE) extracts from nail polishes were investigated, simulating their disposal on water and landfill, respectively, and identifying their physicochemical properties and chemical constituents. Organic compounds and metals were detected in both extracts. SE and LE only induced mutagenic effects in TA98 Salmonella strain in the presence and absence of exogenous metabolic activation. Although both extracts did not significantly increase the frequency of micronucleated HepG2 cells, the cell viability was affected by 24-h exposure. No DNA damage was observed in gonad fish cells (RTG-2) exposed to both extracts; however, the highest SE and LE concentrations induced significant lethal and sublethal effects on zebrafish early-life stages during 96-h exposure. Based on our findings, it can be concluded that if nail polishes enter aquatic systems, it may cause negative impacts to the environment.

Digital object identifier (DOI): 10.1007/s11356-018-1880-y

Mutation research, 826, 47--52
2018

Folate modulates guanine-quadruplex frequency and DNA damage in Werner syndrome.

Tavakoli Shirazi, Paniz, Leifert, Wayne Richard, Fenech, Michael Felix, François, Maxime

Guanine-quadruplexes (G4) are stable tetra-stranded DNA structures that may cause DNA replication stress and inhibit gene expression. Defects in unwinding these structures by DNA helicases may result in telomere shortening and DNA damage. Furthermore, due to mutations in WRN helicase genes in Werner syndrome, G4 motifs are likely to be key elements in the expression of premature aging phenotypes. The methylation of DNA plays a significant role in the stability and occurrence of G4. Thus, G4 frequency and DNA methylation mechanisms may be affected by excesses or deficiencies in methyl donors such as folate. B-Lymphocytes from Werner patients (n?=?5) and healthy individuals (n?=?5) were cultured in RPMI medium under condition of folate deficiency (20?nM) or sufficiency (200?nM) for 14 days. Cells were fixed on microscope slides for immunofluorescent staining to measure G4 frequency and ?H2AX (a marker of DNA strand breaks) intensity, using automated quantitative imaging fluorescent microscopy. There was a significant increase (p?<?0.05) in G4 levels in Werner syndrome patients compared to healthy controls. Werner and control cells grown in 20?nM folate media also showed significant increases in G4 (p?<?0.001) and ?H2AX (p?<?0.01) signals compared with the same cells grown in 200?nM folate. Control cells grown in 20?nM folate also showed a significant reduction in DNA methylation levels (P?<?0.05). The results of this study suggest that the occurrence of DNA G4 structures can be modulated in vitro via nutrients with important roles in methylation.

Digital object identifier (DOI): 10.1016/j.mrgentox.2017.12.002

Radiology, 288, 529--535
2018

Abdominopelvic 1.5-T and 3.0-T MR Imaging in Healthy Volunteers: Relationship to Formation of DNA Double-Strand Breaks.

Suntharalingam, Saravanabavaan, Mladenov, Emil, Sarabhai, Theresia, Wetter, Axel, Kraff, Oliver, Quick, Harald H, Forsting, Michael, Iliakis, Georg, Nassenstein, Kai

Purpose To investigate the relationship between abdominopelvic magnetic resonance (MR) imaging and formation of DNA double-strand breaks (DSBs) in peripheral blood lymphocytes among a cohort of healthy volunteers. Materials and Methods Blood samples were obtained from 40 healthy volunteers (23 women and 17 men; mean age, 27.2 years [range, 21-37 years]) directly before and 5 and 30 minutes after abdominopelvic MR imaging performed at 1.5 T (n = 20) or 3.0 T (n = 20). The number of DNA DSBs in isolated blood lymphocytes was quantified after indirect immunofluorescent staining of a generally accepted DSB marker, ?-H2AX, by means of high-throughput automated microscopy. As a positive control of DSB induction, blood lymphocytes from six volunteers were irradiated in vitro with x-rays at a dose of 1 Gy (70-90 keV). Statistical analysis was performed by using a Friedman test. Results No significant alteration in the frequency of DNA DSB induction was observed after MR imaging (before imaging: 0.22 foci per cell, interquartile range [IQR] = 0.54 foci per cell; 5 minutes after MR imaging: 0.08 foci per cell, IQR = 0.39 foci per cell; 30 minutes after MR imaging: 0.09 foci per cell, IQR = 0.63 foci per cell; P = .057). In vitro radiation of lymphocytes with 1 Gy led to a significant increase in DSBs (0.22 vs 3.43 foci per cell; P = .0312). The frequency of DSBs did not differ between imaging at 1.5 T and at 3.0 T (5 minutes after MR imaging: 0.23 vs 0.06 foci per cell, respectively [P = .57]; 30 minutes after MR imaging: 0.12 vs 0.08 foci per cell [P = .76]). Conclusion Abdominopelvic MR imaging performed at 1.5 T or 3.0 T does not affect the formation of DNA DSBs in peripheral blood lymphocytes.

Digital object identifier (DOI): 10.1148/radiol.2018172453

Nanoscale, 10, 4320--4331
2018

Super-resolution localization microscopy of radiation-induced histone H2AX-phosphorylation in relation to H3K9-trimethylation in HeLa cells.

Hausmann, Michael, Wagner, Emma, Lee, Jin-Ho, Schrock, Gerrit, Schaufler, Wladimir, Krufczik, Matthias, Papenfuß, Franziska, Port, Matthias, Bestvater, Felix, Scherthan, Harry

Ionizing radiation (IR)-induced damage confers functional and conformational changes to nuclear chromatin associated with DNA single and double strand breaks. This leads to the activation of complex DNA repair machineries that aim to preserve the integrity of the DNA molecule. Since hetero- and euchromatin are differentially accessible to DNA repair pathways, local chromatin re-arrangements and structural changes are among the consequences of an activated DNA damage response. Using super-resolution localization microscopy (SRLM), we investigated the X-ray-induced repositioning of γ-H2AX and histone H3K9me3 heterochromatin marks in the nuclei of HeLa cells. Aliquots of cells exposed to different IR doses (0.5, 1 and 2 Gy) were fixed at certain repair times for SRLM imaging. The number and size of nano-scale γ-H2AX molecule signal clusters detected increased with rising irradiation doses, with the number and size being the highest 0.5 h after irradiation. With growing repair time both the number and size of γ-H2AX nano-clusters decreased. Eight hours after irradiation, the number of clusters reached control levels, in agreement with the disappearance of most IR-induced foci seen by conventional microscopy. SRLM investigation of heterochromatin marks in spatial relation to γ-H2AX clusters showed that on average the heterochromatin density was high in the vicinity of γ-H2AX, which is in agreement with the observation that DSBs seem to relocate to the surface of heterochromatin clusters for DNA repair. The data demonstrate the potential of pointillist images obtained by SRLM for quantitative investigations of chromatin conformation changes and repair-protein recruitment on the nanoscale as measures for a radiation response.

Digital object identifier (DOI): 10.1039/c7nr08145f

International journal of radiation biology, 93, 58--64
2017

The second gamma-H2AX assay inter-comparison exercise carried out in the framework of the European biodosimetry network (RENEB).

Moquet, Jayne, Barnard, Stephen, Staynova, Albena, Lindholm, Carita, Monteiro Gil, Octávia, Martins, Vanda, Rößler, Ute, Vral, Anne, Vandevoorde, Charlot, Wojewódzka, Maria, Rothkamm, Kai

Within the EU RENEB project, seven laboratories have taken part in training and harmonisation activities to strengthen triage gamma-H2AX-based radiation exposure assessment. This has culminated in a second triage biodosimetry exercise. Whole blood and separated lymphocyte samples were homogenously irradiated with (60)Co gamma rays at 0.5, 2.5 (blind samples), 0 and 2 Gy (reference samples). Following post-exposure incubations of 4 and 24 h, 16 samples were shipped on ice packs to each partner. The samples were stained and scored for gamma-H2AX foci, using manual and/or automated fluorescence microscope scoring strategies. Dose estimates were obtained and used to assign triage categories to the samples. Average dose estimates across all the laboratories correlated well with true doses. The most accurate assignment of triage category was achieved by manual scoring of the 4-h blood and lymphocyte samples. Only three samples out of a total of 46 were miscategorized in a way that could have adversely effected the clinical management of a radiation casualty. This inter-comparison exercise has demonstrated that following a recent acute radiation exposure, the gamma-H2AX assay could be a useful triage tool that can be successfully applied across a network of laboratories.

Digital object identifier (DOI): 10.1080/09553002.2016.1207822

Archives of toxicology
2017

Dose-response relationship of temozolomide, determined by the Pig-a, comet, and micronucleus assay.

Guérard, M, Johnson, G, Dertinger, S, Duran-Pacheco, G, Funk, J, Zeller, A

Temozolomide (TMZ), a monofunctional alkylating agent, was selected as a model compound to determine its quantitative genotoxic dose-response relationship in different tissues (blood, liver, and jejunum) and endpoints [Pig-a-, comet-, and micronucleus assay (MNT)] in male rats. TMZ was administered p.o. over 5 consecutive days (day 1-5), followed by a treatment-free period of 50 days (day 6-56) and a final administration prior to necropsy (day 57-59). TMZ showed a dose-dependent increase in DNA damage in all interrogated endpoints. A statistically significant increase in Pig-a mutant phenotypes was observed on day 44 starting at 7.5 mg/kg/day for mutant reticulocytes (for RET(CD59-)) and at 3.75 mg/kg/day for mutant red blood cells (RBC(CD59-)), respectively. In addition, a statistically significant increase in cytogenetic damage, as measured by micronucleated reticulocytes, was observed starting at 3.75 mg/kg/day on day 3 and 1.5 mg/kg/day on day 59. DNA strand breaks, as detected by the comet assay, showed a dose-dependent and statistically significant increase in liver, blood, and jejunum starting at doses of 3.75, 3.75, and 7.5 mg/kg/day, respectively. The dose-response relationships of the Pig-a, MNT, and comet data were analyzed for possible points of departure (PoD) using the benchmark-dose (BMD) software PROAST with different critical effect sizes (CES) (BMD0.1, BMD0.5, BMD1, and BMD1SD). Overall, PoD values show a high concordance between different tissues and endpoints, underlining the suitability of this experimental design to explore quantitative dose-response relationships in a variety of different tissues and endpoints, while minimizing animal use.

Digital object identifier (DOI): 10.1007/s00204-016-1923-4

Leukemia
2017

RECQ1 helicase is involved in replication stress survival and drug resistance in multiple myeloma.

Viziteu, E, Klein, B, Basbous, J, Lin, Y-L, Hirtz, C, Gourzones, C, Tiers, L, Bruyer, A, Vincent, L, Grandmougin, C, Seckinger, A, Goldschmidt, H, Constantinou, A, Pasero, P, Hose, D, Moreaux, J

Multiple myeloma (MM) is a plasma cell cancer with poor survival, characterized by the expansion of multiple myeloma cells (MMCs) in the bone marrow. Using a microarray-based genome-wide screen for genes responding to DNA methyltransferases (DNMT) inhibition in MM cells, we identified RECQ1 among the most downregulated genes. RecQ helicases are DNA unwinding enzymes involved in the maintenance of chromosome stability. Here we show that RECQ1 is significantly overexpressed in MMCs compared to normal plasma cells and that increased RECQ1 expression is associated with poor prognosis in three independent cohorts of patients. Interestingly, RECQ1 knockdown inhibits cells growth and induces apoptosis in MMCs. Moreover, RECQ1 depletion promotes the development of DNA double-strand breaks, as evidenced by the formation of 53BP1 foci and the phosphorylation of ataxia-telangiectasia mutated (ATM) and histone variant H2A.X (H2AX). In contrast, RECQ1 overexpression protects MMCs from melphalan and bortezomib cytotoxicity. RECQ1 interacts with PARP1 in MMCs exposed to treatment and RECQ1 depletion sensitizes MMCs to poly(ADP-ribose) polymerase (PARP) inhibitor. DNMT inhibitor treatment results in RECQ1 downregulation through miR-203 deregulation in MMC. Altogether, these data suggest that association of DNA damaging agents and/or PARP inhibitors with DNMT inhibitors may represent a therapeutic approach in patients with high RECQ1 expression associated with a poor prognosis.Leukemia advance online publication, 10 March 2017; doi:10.1038/leu.2017.54.

Digital object identifier (DOI): 10.1038/leu.2017.54

Oncotarget
2017

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

Kosik, Pavol, Skorvaga, Milan, Durdik, Matus, Jakl, Lukas, Nikitina, Ekaterina, Markova, Eva, Kozics, Katarina, Horvathova, Eva, Belyaev, Igor

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.

Digital object identifier (DOI): 10.18632/oncotarget.16211

Environmental and molecular mutagenesis
2017

Sesamol ameliorates radiation induced DNA damage in hematopoietic system of whole body γ-irradiated mice.

Kumar, Arun, Choudhary, Sandeep, Adhikari, Jawahar S, Chaudhury, Nabo K

Ionizing radiation exposure is harmful and at high doses can lead to acute hematopoietic radiation syndrome. Therefore, agents that can protect hematopoietic system are important for development of radioprotector. Sesamol is a potential molecule for development of radioprotector due to its strong free radical scavenging and antioxidant properties. In the present study, sesamol was evaluated for its role in DNA damage and repair in hematopoietic system of γ-irradiated CB57BL/6 mice and compared with amifostine. C57BL/6 male mice were administered with sesamol 20 mg/kg (i.p.) followed by 2 Gy whole body irradiation (WBI) at 30 min. Mice were sacrificed at 0.5, 3, 24 h postirradiation; bone marrow, splenocytes, and peripheral blood lymphocytes were isolated to measure DNA damages and repair using alkaline comet,γ-H2AXand micronucleus assays. An increase in % of tail DNA was observed in all organs of WBI mice. Whereas in pre-administered sesamol reduced %DNA in tail (P ≤ 0.05). Sesamol has also reduced formation of radiation induced γ-H2AX foci after 0.5 h in these organs and further lowered to respective control values at 24 h of WBI. Similar reduction of % DNA in tail and γ-H2AX foci were observed with amifostine (P ≤ 0.05). Analysis of mnPCE frequency at 24 h has revealed similar extent of protection by sesamol and amifostine. Interestingly, both sesamol and amifostine, alone and with radiation, also increased the granulocytes count significantly compared to the control (P ≤ 0.05). These findings suggest that sesamol has strong potential to protect hematopoietic system by lowering radiation induced DNA damages and can prevent acute hematopoietic syndrome in mice. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.

Digital object identifier (DOI): 10.1002/em.22118

Oncotarget, 7, 75996--76005
2016

The prognostic value of DNA damage level in peripheral blood lymphocytes of chemotherapy-naïve patients with germ cell cancer.

Sestakova, Zuzana, Kalavska, Katarina, Hurbanova, Lenka, Jurkovicova, Dana, Gursky, Jan, Chovanec, Michal, Svetlovska, Daniela, Miskovska, Vera, Obertova, Jana, Palacka, Patrik, Rejlekova, Katarina, Sycova-Mila, Zuzana, Cingelova, Silvia, Spanik, Stanislav, Mardiak, Jozef, Chovanec, Miroslav, Mego, Michal

Germ cell tumors (GCTs) are extraordinarily sensitive to cisplatin (CDDP)-based chemotherapy. DNA damage represents one of the most important factors contributing to toxic effects of CDDP-based chemotherapy. This study was aimed to evaluate the prognostic value of DNA damage level in peripheral blood lymphocytes (PBLs) from chemo-naïve GCT patients. PBLs isolated from 59 chemotherapy-naïve GCT patients were included into this prospective study. DNA damage levels in PBLs were evaluated by the Comet assay and scored as percentage tail DNA by the Metafer-MetaCyte analyzing software. The mean ± SEM (standard error of the mean) of endogenous DNA damage level was 5.25 ± 0.64. Patients with DNA damage levels lower than mean had significantly better progression free survival (hazard ratio [HR] = 0.19, 95% CI (0.04 - 0.96), P = 0.01) and overall survival (HR = 0.00, 95% CI (0.00 - 0.0), P < 0.001) compared to patients with DNA damage levels higher than mean. Moreover, there was significant correlation between the DNA damage level and presence of mediastinal lymph nodes metastases, IGCCCG (International Germ Cell Cancer Collaborative Group) risk group, and serum tumor markers level. These data suggest that DNA damage levels in PBLs of GCT patients may serve as an important prognostic marker early identifying patients with poor outcome.

Digital object identifier (DOI): 10.18632/oncotarget.12515

Mutagenesis
April, 2015

Sesamol attenuates genotoxicity in bone marrow cells of whole-body γ-irradiated mice.

Arun Kumar, Tamizh G. Selvan, Akanchha M. Tripathi, Sandeep Choudhary, Shahanshah Khan, Jawahar S. Adhikari, Nabo K. Chaudhury

Ionising radiation causes free radical-mediated damage in cellular DNA. This damage is manifested as chromosomal aberrations and micronuclei (MN) in proliferating cells. Sesamol, present in sesame seeds, has the potential to scavenge free radicals; therefore, it can reduce radiation-induced cytogenetic damage in cells. The aim of this study was to investigate the radioprotective potential of sesamol in bone marrow cells of mice and related haematopoietic system against radiation-induced genotoxicity. A comparative study with melatonin was designed for assessing the radioprotective potential of sesamol. C57BL/6 mice were administered intraperitoneally with either sesamol or melatonin (10 and 20mg/kg body weight) 30min prior to 2-Gy whole-body irradiation (WBI) and sacrificed after 24h. Total chromosomal aberrations (TCA), MN and cell cycle analyses were performed using bone marrow cells. The comet assay was performed on bone marrow cells, splenocytes and lymphocytes. Blood was drawn to study haematological parameters. Prophylactic doses of sesamol (10 and 20mg/kg) in irradiated mice reduced TCA and micronucleated polychromatic erythrocyte frequency in bone marrow cells by 57\% and 50\%, respectively, in comparison with radiation-only groups. Sesamol-reduced radiation-induced apoptosis and facilitated cell proliferation. In the comet assay, sesamol (20mg/kg) treatment reduced radiation-induced comets (\% DNA in tail) compared with radiation only (P

Dental materials : official publication of the Academy of Dental Materials, 31, 1335--1344
2015

Dental composite components induce DNA-damage and altered nuclear morphology in gingiva fibroblasts.

Styllou, Marianthi, Reichl, Franz-Xaver, Styllou, Panorea, Urcan, Ebru, Rothmund, Lena, Hickel, Reinhard, Högg, Christof, Scherthan, Harry

Released dental composite components can damage human gingival fibroblasts (HGFs) and their DNA. The cytotoxicity, chromatin condensation and the induction of DNA double strand breaks (DSBs) by different compounds of dental composites was investigated using an improved γ-H2AX focus assay. HGFs were incubated with the monomers: bisphenol-A-ethoxylate-dimethacrylate (Bis-DMA), bisphenol-A-glycerolate-dimethacrylate (BisGMA), ethyltriethylen glycol methacrylate (ETEGMA), glycidyl methacrylate (GMA), 1,6-hexandiol-dimethycrylate (HDDMA), trimethylolpropane ethoxylate triacrylate (TMPTA), and acrylamide (ACR). DSBs were determined by enumerating γ-H2AX and 53BP1 foci colocalized at DSBs. A concentration-dependent induction of DSBs was found in the order: GMA>BisGMA>ACR>Bis-DMA>HDDMA>TMPTA>ETEGMA. HGFs exposure to GMA (0.3mM) and to BisGMA (0.09mM) induced the highest rate of DSB foci, i.e. 12-fold and 8-fold, respectively, relative to control (0.33 DSB foci/cell). At the highest concentrations (EC50) prominent changes in the chromatin morphology of HGF cell nuclei, i.e. compaction of nuclear chromatin and reduction of the area covered by the ovoid fibroblast nuclei, were observed. Nuclear condensation was significantly induced by GMA (1.7-fold at 0.3mM) and BisGMA (1.6-fold at 0.09mM), which correlated with the highest numbers of induced DSB foci (GMA, BisGMA, 3.9 and 2.6 foci/cell, respectively). The improved γ-H2AX/53BP1 focus assay revealed a concentration-dependent increase in DSBs for all tested substances. Furthermore, concentration-dependent changes in HGF cell nucleus morphology was noted, demonstrating genotoxic effects of the substances tested.

Digital object identifier (DOI): 10.1016/j.dental.2015.08.156

Toxicol Sci, 140(1), 103--117
July, 2014

The gamma-H2AX Assay for Genotoxic and Nongenotoxic Agents: Comparisonof H2AX Phosphorylation with Cell Death Response.

Teodora Nikolova, Mirek Dvorak, Fabian Jung, Isabell Adam, Elisabeth Kr{\"a}mer, Aslihan Gerhold-Ay, Bernd Kaina

DNA double-strand breaks (DSBs) and blocked replication forks resulting from bulky adducts and inhibitors of replication activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phosphorylated form, gamma-H2AX, accumulates at the site of the damage and can be visualized as foci by immunocytochemistry. The objective of this study was to assess if gamma-H2AX is a reliable biomarker for genotoxic exposures. To this end, we selected 14 well-known genotoxic compounds and compared them with 10 nongenotoxic chemicals, using CHO-9 cells because they are well characterized as to DNA repair and DDR. We quantified gamma-H2AX foci manually and automatically. In addition, total gamma-H2AX activation was determined by flow cytometry. For all chemicals the cytotoxic dose response was assayed by a metabolic cytotoxicity assay. We show that (1) all genotoxic agents induced ?H2AX dose-dependently whereas nongenotoxic agents do not; (2) gamma-H2AX was observed for genotoxicants in the cytotoxic dose range, revealing a correlation between cytotoxicity and gamma-H2AX for genotoxic agents; for nongenotoxic agents cytotoxicity was not related to gamma-H2AX; (3) manual scoring of gamma-H2AX and automated scoring provided comparable results, the automated scoring was faster and investigator independent; (4) data obtained by foci counting and flow cytometry showed a high correlation, suggesting that gamma-H2AX scoring by flow cytometry has the potential for high-throughput analysis. However, the microscopic evaluation can provide additional information as to foci size, distribution, colocalization and background staining; (5) gamma-H2AX foci were colocalized with 53BP1 and Rad51, supporting the notion that they represent true DSBs. Collectively, the automated analysis of gamma-H2AX foci allows for rapid determination of genetic damage in mammalian cells. The data revealed that the induction of gamma-H2AX by genotoxicants is related to loss of viability and support gamma-H2AX as a reliable bio-indicator for pretoxic DNA damage.

Environ Mol Mutagen, 55(2), 114--121
March, 2014

Influence of experimental conditions on data variability in the liver comet assay.

M. Gu{\'e}rard, C. Marchand, U. Plappert-Helbig

The in vivo comet assay has increasingly been used for regulatory genotoxicity testing in recent years. While it has been demonstrated that the experimental execution of the assay, for example, electrophoresis or scoring, can have a strong impact on the results; little is known on how initial steps, that is, from tissue sampling during necropsy up to slide preparation, can influence the comet assay results. Therefore, we investigated which of the multitude of steps in processing the liver for the comet assay are most critical. All together eight parameters were assessed by using liver samples of untreated animals. In addition, two of those parameters (temperature and storage time of liver before embedding into agarose) were further investigated in animals given a single oral dose of ethyl methanesulfonate at dose levels of 50, 100, and 200 mg/kg, 3 hr prior to necropsy. The results showed that sample cooling emerged as the predominant influence factor, whereas variations in other elements of the procedure (e.g., size of the liver piece sampled, time needed to process the liver tissue post-mortem, agarose temperature, or time of lysis) seem to be of little relevance. Storing of liver samples of up to 6 hr under cooled conditions did not cause an increase in tail intensity. In contrast, storing the tissue at room temperature, resulted in a considerable time-dependent increase in comet parameters. Environ. Mol. Mutagen. 55:114-121, 2014. © 2013 Wiley Periodicals, Inc.

Int J Radiat Biol, 90(2), 149--158
February, 2014

Induction and disappearance of gammaH2AX foci and formation of micronuclei after exposure of human lymphocytes to (60)Co gamma-rays and p(66)+ Be(40) neutrons.

Veerle Vandersickel, Philip Beukes, Bram {Van Bockstaele}, Julie Depuydt, Anne Vral, Jacobus Slabbert

To investigate both the formation of micronuclei (MN) and the induction and subsequent loss of phosphorylated histone H2AX foci (gammaH2AX foci) after in vitro exposure of human lymphocytes to either (60)Co gamma-rays or p(66)+ Be(40) neutrons.MN dose response (DR) curves were obtained by exposing isolated lymphocytes of 10 different donors to doses ranging from 0-4 Gy gamma-rays or 0-2 Gy neutrons. Also, gammaH2AX foci DR curves were obtained following exposure to doses ranging from 0-0.5 Gy of either gamma-rays or neutrons. Foci kinetics for lymphocytes for a single donor exposed to 0.5 Gy gamma-rays or neutrons were studied up to 24 hours post-irradiation.Micronuclei yields following neutron exposure were consistently higher compared to that from (60)Co gamma-rays. All MN yields were over-dispersed compared to a Poisson distribution. Over-dispersion was higher after neutron irradiation for all doses > 0.1 Gy. Up to 4 hours post-irradiation lower yields of neutron-induced gammaH2AX foci were observed. Between 4 and 24 hours the numbers of foci from neutrons were consistently higher than that from gamma-rays. The half-live of foci disappearance is only marginally longer for neutrons compared to that from gamma-rays. Foci formations were more likely to be over-dispersed for neutron irradiations.Although neutrons are more effective to induce MN, the absolute number of induced gammaH2AX foci are less at first compared to gamma-rays. With time neutron-induced foci are more persistent. These findings are helpful for using £^H2AX foci in biodosimetry and to understand the repair of neutron-induced cellular damage.

J Dermatol Sci, 72(3), 304--310
December, 2013

A purified Feverfew extract protects from oxidative damage by inducing DNA repair in skin cells via a PI3-kinase-dependent Nrf2/ARE pathway.

Karien J. Rodriguez, Heng-Kuan Wong, Thierry Oddos, Michael Southall, Balz Frei, Simarna Kaur

Environmental factors such as solar ultraviolet (UV) radiation and other external aggressors provide an oxidative challenge that is detrimental to skin health. The levels of endogenous antioxidants decrease with age, thus resulting in less protection and a greater potential for skin damage. The NF-E2-related factor-2 (Nrf2) - antioxidant response element (ARE) pathway is a primary defense mechanism against oxidative stress, and induces the expression of antioxidant, detoxification and repair genes. Activation of ARE-Nrf2 can help restore oxidative homeostasis of the skin and play a role in inflammatory response and DNA repair mechanisms.To evaluate the role of a purified parthenolide-depleted Feverfew (PD-Feverfew) extract on the ARE-Nrf2 pathway and DNA repair in skin cells.These studies were undertaken in primary human keratinocytes or KB cells using Luciferase Promoter assay, siRNA transfection studies, Western blot analyses, Immunofluorescence microscopy, comet assay and quantitative real-time PCR.PD-Feverfew was found to induce Nrf2 nuclear translocation and to increase ARE activity in a dose dependent manner. Furthermore, knockdown of Nrf2 resulted in suppression of PD-Feverfew-induced ARE activity. PD-Feverfew was also found to induce phosphorylation of Akt, a kinase downstream of PI3K. Inhibition of PI3K via pre-treatment with the selective pharmacological inhibitor, LY294002, abolished PD-Feverfew-induced Nrf2/ARE activation. PD-Feverfew also reduced UV-induced DNA damage in a PI3K and Nrf2-dependent manner.Therefore, by increasing endogenous defense mechanisms and aid in DNA repair of damaged skin cells via activation of a PI3K-dependent Nrf2/ARE pathway, PD-Feverfew may help protect the skin from numerous environmental aggressors.

Digital object identifier (DOI): 10.1016/j.jdermsci.2013.08.004

Int J Radiat Biol, 89(9), 716--723
September, 2013

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

Svetlana Sorokina, Eva Markova, Jan Gursky, Jozef Dobrovodsky, Igor Belyaev

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).DNA repair γH2AX/53BP1 foci were analyzed by fluorescent microscopy and flow cytometry.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.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.

Digital object identifier (DOI): 10.3109/09553002.2013.797619

Toxicol Sci
July, 2013

Genotoxicity Profile of Azidothymidine In Vitro.

Andreas Zeller, Julie Koenig, Georg Schmitt, Thomas Singer, Melanie Gu{\'e}rard

Azidothymidine (Zidovudine, AZT) is part of the standard care of treatment for acquired immunodeficiency syndrome since many years. A great number of studies on the genotoxic potential of AZT have been published, but no comprehensive hypothesis yet explains all observations. We investigated a multitude of genotoxic endpoints, both in vitro and in vivo, with the goal to complete the picture. The mutagenic potential of AZT in bacteria was found to be restricted to strains with an #ochre# target sequence and could be abrogated both by thymidine supplementation and rat liver S9 mix. Single-strand breaks in mammalian cells were detected in the comet assay after short-term treatment (3h) with AZT, which did not induce micronuclei. The latter were mainly seen after prolonged exposure (24 and 48h) and are probably not directly related to AZT incorporation into DNA. Our data demonstrate that short-term exposure to low AZT concentrations does not induce biologically relevant micronucleation. Only treatment with high concentrations of AZT for prolonged time periods manifests in substantial micronucleus induction. Furthermore, we found that high concentrations of thymidine have no effect in the comet assay but increase micronucleus frequency in a manner very similar to AZT. These results lead us to the following hypothesis: AZT is triphosphorylated and then incorporated into DNA strands, leading to mutations and cytotoxicity. Cellular attempts to repair these DNA lesions as well as stalled replication forks due to chain termination are detectable with the comet assay. Increased micronucleus frequency is likely related to nucleotide pool imbalance.

Radiother Oncol, 107(3), 377--381
June, 2013

Early biomarkers related to secondary primary cancer risk in radiotherapytreated prostate cancer patients: IMRT versus IMAT.

Joke Werbrouck, Piet Ost, Valerie Fonteyne, Gert {De Meerleer}, Wilfried {De Neve}, Evelien Bogaert, Laurence Beels, Klaus Bacher, Anne Vral, Hubert Thierens

To investigate whether rotational techniques (Volumetric Modulated Arc Therapy - VMAT) are associated with a higher risk for secondary primary malignancies compared to step-and-shoot Intensity Modulated Radiation Therapy (ss-IMRT). To this end, radiation therapy (RT) induced DNA double-strand-breaks and the resulting chromosomal damage were assessed in peripheral blood T-lymphocytes of prostate cancer (PCa) patients applying {\~a}H2AX foci and G0 micronucleus (MN) assays.The study comprised 33PCa patients. A blood sample was taken before start of therapy and after the 1st and 3rd RT fraction to determine respectively the RT-induced {\~a}H2AX foci and MN. The equivalent total body dose (D(ETB)) was calculated based on treatment planning data.A linear dose response was obtained for {\~a}H2AX foci yields versus D(ETB) while MN showed a linear-quadratic dose response. Patients treated with large volume (LV) VMAT show a significantly higher level of induced {\~a}H2AX foci and MN compared to IMRT and small volume (SV) VMAT (p