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: 125; Prenosov: 97
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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: 135; Prenosov: 100
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3. 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: 130; Prenosov: 92
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