1. Double strand breaks and cell-cycle arrest induced by the cyanobacterial toxin cylindrospermopsin in HepG2 cellsAlja Štern, Metka Filipič, Matjaž Novak, Bojana Žegura, 2013, izvirni znanstveni članek Povzetek: The newly emerging cyanobacterial cytotoxin cylindrospermopsin (CYN) is increasingly found in surface freshwaters, worldwide. It poses a potential threat to humans after chronic exposure as it was shown to be genotoxic in a range of test systems and is potentially carcinogenic. However, the mechanisms of CYN toxicity and genotoxicity are not well understood. In the present study CYN induced formation of DNA double strand breaks (DSBs), after prolonged exposure (72 h), in human hepatoma cells, HepG2. CYN (0.1–0.5 µg/mL, 24–96 h) induced morphological changes and reduced cell viability in a dose and time dependent manner. No significant increase in lactate dehydrogenase (LDH) leakage could be observed after CYN exposure, indicating that the reduction in cell number was due to decreased cell proliferation and not due to cytotoxicity. This was confirmed by imunocytochemical analysis of the cell-proliferation marker Ki67. Analysis of the cell-cycle using flow-cytometry showed that CYN has an impact on the cell cycle, indicating G0/G1 arrest after 24 h and S-phase arrest after longer exposure (72 and 96 h). Our results provide new evidence that CYN is a direct acting genotoxin, causing DSBs, and these facts need to be considered in the human health risk assessment.
Ključne besede: cylindrospermopsin, cell-cycle, cell-proliferation, double-strand breaks, HepG2 cells
Objavljeno v DiRROS: 02.08.2024; Ogledov: 107; Prenosov: 82
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2. 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: 110; Prenosov: 84
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3. 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: 126; Prenosov: 106
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