TY - JOUR A1 - Hadi, Naji Said Aboud A1 - Bankoglu, Ezgi Eyluel A1 - Stopper, Helga T1 - Genotoxicity of pyrrolizidine alkaloids in metabolically inactive human cervical cancer HeLa cells co-cultured with human hepatoma HepG2 cells JF - Archives of Toxicology N2 - Pyrrolizidine alkaloids (PAs) are secondary plant metabolites, which can be found as contaminant in various foods and herbal products. Several PAs can cause hepatotoxicity and liver cancer via damaging hepatic sinusoidal endothelial cells (HSECs) after hepatic metabolization. HSECs themselves do not express the required metabolic enzymes for activation of PAs. Here we applied a co-culture model to mimic the in vivo hepatic environment and to study PA-induced effects on not metabolically active neighbour cells. In this co-culture model, bioactivation of PA was enabled by metabolically capable human hepatoma cells HepG2, which excrete the toxic and mutagenic pyrrole metabolites. The human cervical epithelial HeLa cells tagged with H2B-GFP were utilized as non-metabolically active neighbours because they can be identified easily based on their green fluorescence in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronuclei in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of cytochrome P450 enzymes with ketoconazole abrogated micronucleus formation. The efflux transporter inhibitors verapamil and benzbromarone reduced micronucleus formation in the co-culture model. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured HeLa cells. KW - co-culture KW - micronuclei KW - mitotic disturbance KW - cytochrome P450s KW - membrane transporters KW - pyrrolizidine alkaloids Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324708 VL - 97 IS - 1 ER - TY - JOUR A1 - Djelić, Ninoslav A1 - Borozan, Sunčica A1 - Dimitrijević-Srećković, Vesna A1 - Pajović, Nevena A1 - Mirilović, Milorad A1 - Stopper, Helga A1 - Stanimirović, Zoran T1 - Oxidative stress and DNA damage in peripheral blood mononuclear cells from normal, obese, prediabetic and diabetic persons exposed to thyroid hormone in vitro JF - International Journal of Molecular Sciences N2 - Diabetes, a chronic group of medical disorders characterized byhyperglycemia, has become a global pandemic. Some hormones may influence the course and outcome of diabetes, especially if they potentiate the formation of reactive oxygen species (ROS). There is a close relationship between thyroid disorders and diabetes. The main objective of this investigation was to find out whether peripheral blood mononuclear cells (PBMCs) are more prone to DNA damage by triiodothyronine (T\(_3\)) (0.1, 1 and 10 μM) at various stages of progression through diabetes (obese, prediabetics, and type 2 diabetes mellitus—T2DM persons). In addition, some biochemical parameters of oxidative stress (catalase-CAT, thiobarbituric acid reactive substances—TBARS) and lactate dehydrogenase (LDH) were evaluated. PBMCs from prediabetic and diabetic patients exhibited increased sensitivity for T\(_3\) regarding elevated level of DNA damage, inhibition of catalase, and increase of TBARS and LDH. PBMCs from obese patients reacted in the same manner, except for DNA damage. The results of this study should contribute to a better understanding of the role of thyroid hormones in the progression of T2DM. KW - diabetes KW - oxidative stress KW - DNA damage KW - lymphocytes KW - thyroid hormone Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285988 SN - 1422-0067 VL - 23 IS - 16 ER - TY - JOUR A1 - Winkelbeiner, Nicola A1 - Wandt, Viktoria K. A1 - Ebert, Franziska A1 - Lossow, Kristina A1 - Bankoglu, Ezgi E. A1 - Martin, Maximilian A1 - Mangerich, Aswin A1 - Stopper, Helga A1 - Bornhorst, Julia A1 - Kipp, Anna P. A1 - Schwerdtle, Tanja T1 - A multi-endpoint approach to base excision repair incision activity augmented by PARylation and DNA damage levels in mice: impact of sex and age JF - International Journal of Molecular Sciences N2 - Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2'-deoxyguanosine (8-oxodG), 5-hydroxy-2'-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery. KW - maintenance of genomic integrity KW - ageing KW - sex KW - DNA damage KW - base excision repair (incision activity) KW - DNA damage response KW - poly(ADP-ribosyl)ation KW - liver Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285706 SN - 1422-0067 VL - 21 IS - 18 ER - TY - JOUR A1 - Bankoglu, Ezgi Eyluel A1 - Schuele, Carolin A1 - Stopper, Helga T1 - Cell survival after DNA damage in the comet assay JF - Archives of Toxicology N2 - The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H\(_{2}\)O\(_{2}\)) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20-30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than H\(_{2}\)O\(_{2}\) or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided. KW - Cell death and comet assay KW - DNA damage KW - DNA repair Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265339 VL - 95 IS - 12 ER - TY - JOUR A1 - Bankoglu, Ezgi Eyluel A1 - Stipp, Franzisca A1 - Gerber, Johanna A1 - Seyfried, Florian A1 - Heidland, August A1 - Bahner, Udo A1 - Stopper, Helga T1 - Effect of cryopreservation on DNA damage and DNA repair activity in human blood samples in the comet assay JF - Archives of Toxicology N2 - The comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction. KW - human biomonitoring KW - DNA damage KW - DNA repair KW - comet assay KW - blood samples Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265326 VL - 95 IS - 5 ER - TY - JOUR A1 - Reimann, Hauke A1 - Stopper, Helga A1 - Hintzsche, Henning T1 - Long-term fate of etoposide-induced micronuclei and micronucleated cells in Hela-H2B-GFP cells JF - Archives of Toxicology N2 - Micronuclei are small nuclear cellular structures containing whole chromosomes or chromosomal fragments. While there is a lot of information available about the origin and formation of micronuclei, less is known about the fate of micronuclei and micronucleated cells. Possible fates include extrusion, degradation, reincorporation and persistence. Live cell imaging was performed to quantitatively analyse the fates of micronuclei and micronucleated cells occurring in vitro. Imaging was conducted for up to 96 h in HeLa-H2B-GFP cells treated with 0.5, 1 and 2 µg/ml etoposide. While a minority of micronuclei was reincorporated into the main nucleus during mitosis, the majority of micronuclei persisted without any alterations. Degradation and extrusion were observed rarely or never. The presence of micronuclei affected the proliferation of the daughter cells and also had an influence on cell death rates. Mitotic errors were found to be clearly increased in micronucleus-containing cells. The results show that micronuclei and micronucleated cells can, although delayed in cell cycle, sustain for multiple divisions. KW - micronuclei KW - cell fate KW - etoposide KW - live imaging KW - DNA damage Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235039 SN - 0340-5761 VL - 94 ER - TY - JOUR A1 - Reimann, Hauke A1 - Stopper, Helga A1 - Polak, Thomas A1 - Lauer, Martin A1 - Herrmann, Martin J. A1 - Deckert, Jürgen A1 - Hintzsche, Henning T1 - Micronucleus frequency in buccal mucosa cells of patients with neurodegenerative diseases JF - Scientific Reports N2 - Neurodegenerative diseases show an increase in prevalence and incidence, with the most prominent example being Alzheimer's disease. DNA damage has been suggested to play a role in the pathogenesis, but the exact mechanisms remain elusive. We enrolled 425 participants with and without neurodegenerative diseases and analyzed DNA damage in the form of micronuclei in buccal mucosa samples. In addition, other parameters such as binucleated cells, karyolytic cells, and karyorrhectic cells were quantified. No relevant differences in DNA damage and cytotoxicity markers were observed in patients compared to healthy participants. Furthermore, other parameters such as lifestyle factors and diseases were also investigated. Overall, this study could not identify a direct link between changes in buccal cells and neurogenerative diseases, but highlights the influence of lifestyle factors and diseases on the human buccal cytome. KW - peripheral-blood lymphocytes KW - Alzheimers disease KW - DNA damage KW - cognitive impairment KW - cytome biomarkers KW - diagnosis KW - association KW - assay KW - life Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231430 VL - 10 ER - TY - JOUR A1 - Naseem, Muhammad A1 - Othman, Eman M. A1 - Fathy, Moustafa A1 - Iqbal, Jibran A1 - Howari, Fares M. A1 - AlRemeithi, Fatima A. A1 - Kodandaraman, Geema A1 - Stopper, Helga A1 - Bencurova, Elena A1 - Vlachakis, Dimitrios A1 - Dandekar, Thomas T1 - Integrated structural and functional analysis of the protective effects of kinetin against oxidative stress in mammalian cellular systems JF - Scientific Reports N2 - Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions. KW - cytokinins KW - 6-benzylaminopurine Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231317 VL - 10 ER - TY - JOUR A1 - Cheng, Cheng A1 - Othman, Eman M. A1 - Stopper, Helga A1 - Edrada-Ebel, RuAngelie A1 - Hentschel, Ute A1 - Abdelmohsen, Usama Ramadan T1 - Isolation of petrocidin A, a new cytotoxic cyclic dipeptide from the marine sponge-derived bacterium \(Streptomyces\) sp. SBT348 JF - Marine Drugs N2 - A new cyclic dipeptide, petrocidin A (\(\textbf{1}\)), along with three known compounds—2,3-dihydroxybenzoic acid (\(\textbf{2}\)), 2,3-dihydroxybenzamide (\(\textbf{3}\)), and maltol (\(\textbf{4}\))—were isolated from the solid culture of \(Streptomyces\) sp. SBT348. The strain \(Streptomyces\) sp. SBT348 had been prioritized in a strain collection of 64 sponge-associated actinomycetes based on its distinct metabolomic profile using liquid chromatography/high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR). The absolute configuration of all α-amino acids was determined by HPLC analysis after derivatization with Marfey’s reagent and comparison with commercially available reference amino acids. Structure elucidation was pursued in the presented study by mass spectrometry and NMR spectral data. Petrocidin A (\(\textbf{1}\)) and 2,3-dihydroxybenzamide (\(\textbf{3}\)) exhibited significant cytotoxicity towards the human promyelocytic HL-60 and the human colon adenocarcinoma HT-29 cell lines. These results demonstrated the potential of sponge-associated actinomycetes for the discovery of novel and pharmacologically active natural products. KW - biology KW - sponges KW - actinomycetes KW - streptomyces KW - cyclic dipeptide KW - cytotoxic Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172644 VL - 15 IS - 12 ER - TY - JOUR A1 - Balasubramanian, Srikkanth A1 - Othman, Eman M. A1 - Kampik, Daniel A1 - Stopper, Helga A1 - Hentschel, Ute A1 - Ziebuhr, Wilma A1 - Oelschlaeger, Tobias A. A1 - Abdelmohsen, Usama R. T1 - Marine sponge-derived Streptomyces sp SBT343 extract inhibits staphylococcal biofilm formation JF - Frontiers in Microbiology N2 - Staphylococcus epidermidis and Staphylococcus aureus are opportunistic pathogens that cause nosocomial and chronic biofilm-associated infections. Indwelling medical devices and contact lenses are ideal ecological niches for formation of staphylococcal biofilms. Bacteria within biofilms are known to display reduced susceptibilities to antimicrobials and are protected from the host immune system. High rates of acquired antibiotic resistances in staphylococci and other biofilm-forming bacteria further hamper treatment options and highlight the need for new anti-biofilm strategies. Here, we aimed to evaluate the potential of marine sponge-derived actinomycetes in inhibiting biofilm formation of several strains of S. epidermidis, S. aureus, and Pseudomonas aeruginosa. Results from in vitro biofilm-formation assays, as well as scanning electron and confocal microscopy, revealed that an organic extract derived from the marine sponge-associated bacterium Streptomyces sp. SBT343 significantly inhibited staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces, without affecting bacterial growth. The extract also displayed similar antagonistic effects towards the biofilm formation of other S. epidermidis and S. aureus strains tested but had no inhibitory effects towards Pseudomonas biofilms. Interestingly the extract, at lower effective concentrations, did not exhibit cytotoxic effects on mouse fibroblast, macrophage and human corneal epithelial cell lines. Chemical analysis by High Resolution Fourier Transform Mass Spectrometry (HRMS) of the Streptomyces sp. SBT343 extract proportion revealed its chemical richness and complexity. Preliminary physico-chemical characterization of the extract highlighted the heat-stable and non-proteinaceous nature of the active component(s). The combined data suggest that the Streptomyces sp. SBT343 extract selectively inhibits staphylococcal biofilm formation without interfering with bacterial cell viability. Due to absence of cell toxicity, the extract might represent a good starting material to develop a future remedy to block staphylococcal biofilm formation on contact lenses and thereby to prevent intractable contact lens-mediated ocular infections. KW - medicine KW - marine sponges KW - actinomycetes KW - Streptomyces KW - staphilococci KW - biofilms KW - contact lens Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171844 VL - 8 ER -