1. Tiered genotoxicity testing of enriched river water samples using zebrafish in vitro and in vivo models: a joint Danube Survey 4 case studyMargareta Kračun-Kolarević, Bojana Žegura, Katja Kološa, Jovana Jovanović Marić, Andrea Novaković, Peter Oswald, Martina Oswaldova, Jaroslav Slobodnik, Nikiforos Alygizakis, Momir Paunović, 2026, original scientific article Abstract: The increasing complexity of aquatic pollution, dominated by diverse and often uncharacterized chemical mixtures, challenges traditional monitoring approaches. In this study, we assessed the genotoxic potential of surface water samples collected during the Joint Danube Survey 4 (JDS4) using large-volume solid-phase extraction (LVSPE) combined with a comprehensive battery of bioassays. Twenty-three enriched water samples from the Danube River and its major tributaries were evaluated for genotoxicity using a tiered testing strategy comprising the SOS/umuC assay, zebrafish liver (ZFL) cell-based assays (cytotoxicity, comet assay, cell cycle), and zebrafish embryo assays. While no genotoxicity was detected in the prokaryotic SOS/umuC assay, ZFL assays revealed significant DNA damage in 16 out of 23 samples, with notable genotoxicity observed in samples from the middle Danube section. In contrast, no teratogenic effects were observed in zebrafish embryo assays at concentrations up to REF100. These findings demonstrate the superior sensitivity of ZFL cells compared to both prokaryotic and in vivo embryo models. The study also highlights a critical gap in available genotoxicity data for detected substances, emphasizing the need for standardized databases and testing frameworks. Overall, our results support zebrafish-based in vitro assays as effective tools for effect-based monitoring, providing early warnings of genotoxic pollution in complex aquatic environments.
Keywords: ZFL cell line, zebrafish embryos, cytotoxicity, genotoxicity, Danube River
Published in DiRROS: 28.01.2026; Views: 144; Downloads: 122
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2. 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, original scientific article Abstract: 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.
Keywords: TBQ derivatives, HepG2 cell line, comet assay, γH2AX assay, cell cycle analysis, cytotoxicity
Published in DiRROS: 11.07.2024; Views: 1243; Downloads: 764
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