1. Do cytotoxicity and cell death cause false positive results in the in vitro comet assay?Amaya Azqueta, Helga Stopper, Bojana Žegura, Maria Dusinska, Peter Møller, 2022, izvirni znanstveni članek Povzetek: The comet assay is used to measure DNA damage induced by chemical and physical agents. High concentrations of test agents may cause cytotoxicity or cell death, which may give rise to false positive results in the comet assay. Systematic studies on genotoxins and cytotoxins (i.e. non-genotoxic poisons) have attempted to establish a threshold of cytotoxicity or cell death by which DNA damage results measured by the comet assay could be regarded as a false positive result. Thresholds of cytotoxicity/cell death range from 20% to 50% in various publications. Curiously, a survey of the latest literature on comet assay results from cell culture studies suggests that one-third of publications did not assess cytotoxicity or cell death. We recommend that it should be mandatory to include results from at least one type of assay on cytotoxicity, cell death or cell proliferation in publications on comet assay results. A combination of cytotoxicity (or cell death) and proliferation (or colony forming efficiency assay) is preferable in actively proliferating cells because it covers more mechanisms of action. Applying a general threshold of cytotoxicity/cell death to all types of agents may not be applicable; however, 25% compared to the concurrent negative control seems to be a good starting value to avoid false positive comet assay results. Further research is needed to establish a threshold value to distinguish between true and potentially false positive genotoxic effects detected by the comet assay.
Ključne besede: comet assay, cytotoxicity, genotoxicity, DNA damage, cell death
Objavljeno v DiRROS: 17.07.2024; Ogledov: 6; Prenosov: 3
 Celotno besedilo (1,37 MB)
Gradivo ima več datotek! Več... |
2. Lethal and sub-lethal effects and modulation of gene expression induced by T kinase inhibitors in zebrafish (Danio Rerio) embryosTina Eleršek, Matjaž Novak, Mateja Mlinar, Igor Virant, Nika Bahor, Karin Leben, Bojana Žegura, Metka Filipič, 2022, izvirni znanstveni članek Povzetek: Tyrosine kinase inhibitors (TKIs) are designed for targeted cancer therapy. The consumption of these drugs during the last 20 years has been constantly rising. In the zebrafish (Danio rerio) embryo toxicity test, we assessed the toxicity of six TKIs: imatinib mesylate, erlotinib, nilotinib, dasatinib, sorafenib and regorafenib. Imatinib mesylate and dasatinib induced lethal effects, while regorafenib, sorfenib and dasatinib caused a significant increase of sub-lethal effects, predominantly oedema, no blood circulation and formation of blood aggregates. The analyses of the changes in the expression of selected genes associated with the hormone system after the exposure to imatinib mesylate, dasatinib and regorafenib demonstrated that all three tested TKIs deregulated the expression of oestrogen receptor esr1, cytochrome P450 aromatase (cypa19b) and hydroxysteroid-dehydrogenase (hsd3b), regorafenib, and also thyroglobulin (tg). The expression of genes involved in the DNA damage response (gadd45 and mcm6) and apoptosis (bcl2) was deregulated only by exposure to regorafenib. The data indicate that common mechanisms, namely antiangiogenic activity and interference with steroidogenesis are involved in the TKI induced sub-lethal effects and potential hormone disrupting activity, respectively. The residues of TKIs may represent an environmental hazard; therefore, further ecotoxicological studies focusing also on the effects of their mixtures are warranted.
Ključne besede: aquatic toxicity, tyrosine kinase inhibitors, zebrafish embryo toxicity test, gene expression, environmental hazard
Objavljeno v DiRROS: 16.07.2024; Ogledov: 26; Prenosov: 10
Celotno besedilo (9,13 MB)
Gradivo ima več datotek! Več... |
3. HepG2 spheroids as a biosensor-like cell-based system for (geno)toxicity assessmentMartina Štampar, Sonja Žabkar, Metka Filipič, Bojana Žegura, 2022, izvirni znanstveni članek Povzetek: 3D spheroids developed from HepG2 cells were used as a biosensor-like system for the detection of (geno)toxic effects induced by chemicals. Benzo(a)pyrene (B(a)P) and amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) with well-known mechanisms of action were used for system validation. HepG2 spheroids grown for 3 days were exposed to BaP and PhIP for 24 and 72 h. The growth and viability of spheroids were monitored by planimetry and Live/Dead staining of cells. Multi-parametric flow cytometric analysis was applied for simultaneous detection of specific end-effects including cell cycle analysis (Hoechst staining), cell proliferation (KI67 marker), and DNA double-strand breaks (ℽH2AX) induced by genotoxic compounds. Depending on the exposure concentration/time, BaP reduced spheroid growth, affected cell proliferation by arresting cells in S and G2 phase and induced DNA double-strand breaks (DSB). Simultaneous staining of ℽH2AX formation and cell cycle analysis revealed that after BaP (10 μM; 24 h) exposure 60% of cells in G0/G1 phase had DNA DSB, while after 72 h only 20% of cells contained DSB indicating efficient repair of DNA lesions. PhIP did not influence the spheroid size whereas accumulation of cells in the G2 phase occurred after both treatment times. The evaluation of DNA damage revealed that at 200 μM PhIP 50% of cells in G0/G1 phase had DNA DSB, which after 72-h exposure dropped to 40%, showing lower repair capacity of PhIP-induced DSB compared to BaP-induced. The developed approach using simultaneous detection of several parameters provides mechanistic data and thus contributes to more reliable genotoxicity assessment of chemicals as a high-content screening tool.
Ključne besede: in vitro 3D cell model, HepG2, flow cytometry, cell cycle, proliferation, DNA strand, breaks
Objavljeno v DiRROS: 16.07.2024; Ogledov: 26; Prenosov: 20
Celotno besedilo (6,21 MB)
Gradivo ima več datotek! Več... |
4. Cytotoxicity and antibacterial efficacy of betaine- and choline-substituted polymersLucija Jurko, Damjan Makuc, Alja Štern, Janez Plavec, Bojana Žegura, Perica Bošković, Rupert Kargl, 2023, izvirni znanstveni članek Povzetek: Cationic charge has been widely used to increase polymer adsorption and flocculation of dispersions or to provide antimicrobial activity. In this work, cationization of hydroxyethyl cellulose (HEC) and polyvinyl alcohol (PVA) was achieved by covalently coupling betaine hydrochloride and choline chloride to the polymer backbones through carbonyl diimidazole (CDI) activation. Two approaches for activation were investigated. CDI in excess was used to activate the polymers’ hydroxyls followed by carbonate formation with choline chloride, or CDI was used to activate betaine hydrochloride, followed by ester formation with the polymers’ hydroxyls. The first approach led to a more significant cross-linking of PVA, but not of HEC, and the second approach successfully formed ester bonds. Cationic, nitrogen-bearing materials with varying degrees of substitution were obtained in moderate to high yields. These materials were analyzed by Fourier transform infrared spectroscopy, nuclear magnetic resonance, polyelectrolyte titration, and kaolin flocculation. Their dose-dependent effect on the growth of Staphylococcus aureus and Pseudomonas aeruginosa, and L929 mouse fibroblasts, was investigated. Significant differences were found between the choline- and betaine-containing polymers, and especially, the choline carbonate esters of HEC strongly inhibited the growth of S. aureus in vitro but were also cytotoxic to fibroblasts. Fibroblast cytotoxicity was also observed for betaine esters of PVA but not for those of HEC. The materials could potentially be used as antimicrobial agents for instance by coating surfaces, but more investigations into the interaction between cells and polysaccharides are necessary to clarify why and how bacterial and human cells are inhibited or killed by these derivatives, especially those containing choline.
Ključne besede: hydroxyethyl cellulose, polyvinyl alcohol, antimicrobial, S. aureus, P. aeruginosa, L929 mouse fibroblast, cationic polymer
Objavljeno v DiRROS: 12.07.2024; Ogledov: 60; Prenosov: 35
Celotno besedilo (3,21 MB)
Gradivo ima več datotek! Več... |
5. Combined toxic effects of BPA and its two analogues BPAP and BPC in a 3D HepG2 cell modelMartina Štampar, Tim Ravnjak, Ana-Marija Domijan, Bojana Žegura, 2023, izvirni znanstveni članek Povzetek: Bisphenol A (BPA) is one of the most commonly used substances in the manufacture ofvarious everyday products. Growing concerns about its hazardous properties, including endocrinedisruption and genotoxicity, have led to its gradual replacement by presumably safer analogues inmanufacturing plastics. The widespread use of BPA and, more recently, its analogues has increasedtheir residues in the environment. However, our knowledge of their toxicological profiles is limitedand their combined effects are unknown. In the present study, we investigated the toxic effectscaused by single bisphenols and by the combined exposure of BPA and its two analogues, BPAP andBPC, after short (24-h) and prolonged (96-h) exposure in HepG2 spheroids. The results showed thatBPA did not reduce cell viability in HepG2 spheroids after 24-h exposure. In contrast, BPAP andBPC affected cell viability in HepG2 spheroids. Both binary mixtures (BPA/BPAP and BPA/BPC)decreased cell viability in a dose-dependent manner, but the significant difference was only observedfor the combination of BPA/BPC (both at 40μM). After 96-h exposure, none of the BPs studiedaffected cell viability in HepG2 spheroids. Only the combination of BPA/BPAP decreased cellviability in a dose-dependent manner that was significant for the combination of 4μM BPA and 4μMBPAP. None of the BPs and their binary mixtures studied affected the surface area and growth ofspheroids as measured by planimetry. In addition, all BPs and their binary mixtures studied triggeredoxidative stress, as measured by the production of reactive oxygen species and malondialdehyde,at both exposure times. Overall, the results suggest that it is important to study the effects of BPsas single compounds. It is even more important to study the effects of combined exposures, as thecombined effects may differ from those induced by single compounds.
Ključne besede: BP analogues, hepatic in vitro 3D cell model, combined exposure, viability, oxidative stress, toxicology
Objavljeno v DiRROS: 12.07.2024; Ogledov: 40; Prenosov: 17
Celotno besedilo (2,18 MB)
Gradivo ima več datotek! Več... |
6. Impact of deoxynivalenol and zearalenone as single and combined treatment on DNA, cell cycle and cell proliferation in HepG2 cellsAna-Marija Domijan, Klara Hercog, Martina Štampar, Goran Gajski, Marko Gerić, Marijana Sokolović, Bojana Žegura, 2023, izvirni znanstveni članek Povzetek: The study aimed to investigate toxicity and the mechanism of toxicity of two Fusarium mycotoxins, deoxynivalenol (DON) and zearalenone (ZEA). DON and ZEA were applied to HepG2 cells as single compounds and in combination at low environmentally relevant concentrations. HepG2 cells were exposed to DON (0.5, 1, and 2 µM), ZEA (5, 10, and 20 µM) or their combinations (1 µM DON + 5 µM ZEA, 1 µM DON + 10 µM ZEA and 1 µM DON + 20 µM ZEA) for 24 h and cell viability, DNA damage, cell cycle and proliferation were assessed. Both mycotoxins reduced cell viability, however, combined treatment with DON and ZEA resulted in higher reduction of cell viability. DON (1 µM) induced primary DNA damage, while DON (1 µM) in combination with higher ZEA concentrations showed antagonistic effects compared to DON alone at 1 µM. DON arrested HepG2 cells in G2 phase and significantly inhibited cell proliferation, while ZEA had no significant effect on cell cycle. The combined treatment with DON and ZEA arrested cells in G2 phase to a higher extend compared to treatment with single mycotoxins. Potentiating effect observed after DON and ZEA co-exposure at environmentally relevant concentrations indicates that in risk assessment and setting governments’ regulations, mixtures of mycotoxins should be considered.
Ključne besede: mycotoxins, comet assay, flow cytometry, co-exposure, food monitoring
Objavljeno v DiRROS: 12.07.2024; Ogledov: 52; Prenosov: 17
Celotno besedilo (1,60 MB)
Gradivo ima več datotek! Več... |
7. Adverse toxic effects of tyrosine kinase inhibitors on non-target zebrafish liver (ZFL) cellsKatja Kološa, Bojana Žegura, Martina Štampar, Metka Filipič, Matjaž Novak, 2023, izvirni znanstveni članek Ključne besede: zebrafish liver cells, ZFL, tyrosine kinase inhibitors, cytotoxicity, cell cycle, genotoxicity, environmental hazard
Objavljeno v DiRROS: 12.07.2024; Ogledov: 57; Prenosov: 30
Celotno besedilo (3,30 MB)
Gradivo ima več datotek! Več... |
8. Adverse (geno)toxic effects of bisphenol A and its analogues in hepatic 3D cell modelMarta Sendra, Martina Štampar, Katarina Fras, Beatriz Novoa, Antonio Figueras, Bojana Žegura, 2023, izvirni znanstveni članek Ključne besede: BPA analogues, in vitro 3D cell model, genotoxic, DNA strand breaks, cell proliferation, cell death
Objavljeno v DiRROS: 12.07.2024; Ogledov: 47; Prenosov: 21
Celotno besedilo (6,52 MB)
Gradivo ima več datotek! Več... |
9. Influence of alkylthio and arylthio derivatives of tert-butylquinone on the induction of DNA damage in a human hepatocellular carcinoma cell line (HepG2)Jelena Djordjević, Stoimir Kolarević, Jovana Jovanović Marić, Margareta Kračun-Kolarević, Bojana Žegura, Alja Štern, Dušan M. Sladić, Irena Novaković, Branka Vuković-Gačić, 2024, izvirni znanstveni članek Povzetek: The aim of this study was to investigate the effects of tert-butylquinone (TBQ) and its alkylthio and arylthio
derivatives on DNA in vitro, using acellular and cellular test systems. Direct interaction with DNA was studied
using the plasmid pUC19. Cytotoxic (MTS assay) and genotoxic (comet assay and γH2AX focus assays) effects,
and their influence on the cell cycle were studied in the HepG2 cell line. Our results show that TBQ and its
derivatives did not directly interact with DNA. The strongest cytotoxic effect on the HepG2 cells was observed for
the derivative 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone (IC50 64.68 and 55.64 μM at 24-h and 48-h
treatment, respectively). The tested derivatives did not significantly influence the cell cycle distribution in the
exposed cellular populations. However, all derivatives showed a genotoxic activity stronger than that of TBQ in
the comet assay, with 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone producing the strongest effect. The
same derivative also induced DNA double-strand breaks in the γH2AX focus assay.
Ključne besede: TBQ derivatives, HepG2 cell line, comet assay, γH2AX assay, cell cycle analysis, cytotoxicity
Objavljeno v DiRROS: 11.07.2024; Ogledov: 64; Prenosov: 41
 Celotno besedilo (7,34 MB)
Gradivo ima več datotek! Več... |
10. Exploring the safety of cannabidiol (CBD) : a comprehensive in vitro evaluation of the genotoxic and mutagenic potential of a CBD isolate and extract from Cannabis sativa LAlja Štern, Matjaž Novak, Katja Kološa, Jurij Trontelj, Sonja Žabkar, Tjaša Šentjurc, Metka Filipič, Bojana Žegura, 2024, izvirni znanstveni članek Povzetek: Cannabidiol (CBD), a naturally occurring cyclic terpenoid found in Cannabis sativa L., is renowned for its diverse
pharmacological benefits. Marketed as a remedy for various health issues, CBD products are utilized by patients
as a supplementary therapy or post-treatment failure, as well as by healthy individuals seeking promised advantages. Despite its widespread use, information regarding potential adverse effects, especially genotoxic
properties, is limited. The present study is focused on the mutagenic and genotoxic activity of a CBD isolate
(99.4 % CBD content) and CBD-rich Cannabis sativa L extract (63.6 % CBD content) in vitro. Both CBD samples
were non-mutagenic, as determined by the AMES test (OECD 471) but exhibited cytotoxicity for HepG2 cells
(~IC50 (4 h) 26 µg/ml, ~IC50 (24 h) 6–8 µg/ml, MTT assay). Noncytotoxic concentrations induced upregulation of
genes encoding metabolic enzymes involved in CBD metabolism, and CBD oxidative as well as glucuronide
metabolites were found in cell culture media, demonstrating the ability of HepG2 cells to metabolize CBD. In this
study, the CBD samples were found non-genotoxic. No DNA damage was observed with the comet assay, and no
influence on genomic instability was observed with the cytokinesis block micronucleus and the γH2AX and p-H3
assays. Furthermore, no changes in the expression of genes involved in genotoxic stress response were detected in
the toxicogenomic analysis, after 4 and 24 h of exposure. Our comprehensive study contributes valuable insights
into CBD’s safety profile, paving the way for further exploration of CBD’s therapeutic applications and potential
adverse effects.
Ključne besede: cannabidiol, CBD, metabolism, cytotoxicity, genotoxicity, mutagenicity
Objavljeno v DiRROS: 09.07.2024; Ogledov: 57; Prenosov: 31
Celotno besedilo (4,31 MB)
Gradivo ima več datotek! Več... |
