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: 195; Downloads: 158
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2. Safety of ferrite nanoparticles for biomedical applications: cyto- and genotoxic effects of MxFe3-xO4 (M = Fe, Zn, Mn) in an advanced 3D human hepatic in vitro modelIza Rozman, Álvaro Gallo-Cordova, María del Puerto Morales, Marco A. Morales Ovalle, Gerardo F. Goya, Katja Kološa, Domen Hočevar, Bojana Žegura, Alja Štern, 2026, original scientific article Abstract: Given the growing interest in nanosized spinel-type ferrite nanoparticles for biomedical applications and the limited information on their safety, this study aimed to assess their cellular and genotoxic effects in an in vitro 3D human hepatic cell model (HepG2 spheroids). Ferrite nanoparticles – γFe2O3 (FeNPs; 14 ± 4 nm), Zn0.7Fe2.3O4 (ZnNPs; 14 ± 5 nm), and Mn0.4Fe2.6O4 (MnNPs; 7 ± 2 nm) – were synthesised through a microwave-assisted polyol route, functionalized with citric acid, and characterised using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD), and Fourier Transform Infrared Spectroscopy (FTIR). Nanoparticle uptake was analysed using TEM, cytotoxicity was measured with CellTiter-Glo®, and oxidative stress induction was assessed using the 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) and malondialdehyde (MDA) assay. Genotoxic effects were evaluated using the comet, γH2AX and p-H3 assays. Cellular stress responses were assessed using toxicogenomic analysis. Significant cytotoxicity of the tested nanoparticles (0.1–250 µg/mL) was observed; however, TEM analysis revealed limited penetration to the outermost cell layers of spheroids. Notably, only FeNPs induced ROS generation, while MDA levels remained unchanged in all tested samples. Low DNA damage was detected at 24 h, but a significant increase was observed at 96 h (5–50 µg/mL). No increase in γH2AX or p-H3 was found. No substantial alterations in DNA damage or oxidative stress-response gene expression were detected. Altogether, our findings suggest that the effects of ferrite nanoparticles are time- and composition-dependent, underlining the importance of further mechanistic and chronic exposure evaluations in 3D cell models.
Keywords: DNA damage, genotoxicity, HepG2 spheroids, magnetic ferrite-based nanoparticles, ROS induction, safety assessment, toxicogenomics
Published in DiRROS: 27.01.2026; Views: 188; Downloads: 150
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3. Deregulations of DNA damage-responsive genes, genes involved in the endocrine system, in an advanced in vitro 3D zebrafish hepatic cell model after exposure to Bisphenol A (BPA) and its emerging alternatives BPAF, BPAP and BPPH : version v1Alja Štern, Špela Rozman, Bojana Žegura, 2025, complete scientific database of research data Abstract: Bisphenol AF (BPAF), Bisphenol AP (BPAP), and Bisphenol PH (BPPH) are being introduced into consumer products to replace BPA and are subsequently detected in ecosystems. This study investigates the genotoxic and endocrine-disruptive potential of these emerging bisphenols using a 3D in vitro liver spheroid model derived from Danio rerio (ZFL cell line), on the transcriptional level. The selected genes targeted DNA damage response pathways (TP53, NER, BER) and endocrine-related signalling (Table 1). ZFL spheroids were prepared by a force floating method as described by Štampar et al. (2019)1. Four-day-old ZFL spheroids were exposed to BPA (50 and 200 µM), BPAF (25 and 100 µM), BPAP (25 and 100 µM), and BPPH (10 and 50 µM) for 24 (Table 2) and 96 (Table 3) hours. Following exposure, total RNA was extracted using the RNeasy® Mini Kit (Qiagen, Germany). RNA quality and quantity were assessed prior to reverse transcription (Applied Biosystems, USA), followed by gene-specific preamplification (TATAA PreAmp GrandMasterMix, Tataa Biocenter, Sweden). Gene expression analysis was performed using TaqMan Gene Expression Assays (Applied Biosystems, USA) on the Fluidigm One 48.48 Dynamic Array IFC microfluidic system as described by Štern et al. (2024)2. The generated data was analysed using the Fluidigm Gene Expression Analysis Software and with a free-accessible web program, quantGenious3. The difference in gene expression greater than 1.5-fold was considered a biologically important up/downregulation (relative expression >1.5 or <0.66, respectively). Statistically significant differences were analysed using ANOVA and Dunnett’s multiple comparison test in GraphPad Prism v9 (GraphPad Software, San Diego, CA, USA).
Keywords: bisphenols, genotoxicity, endocrine disruption, ZFL spheroids, gene expression, data
Published in DiRROS: 30.09.2025; Views: 454; Downloads: 308
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4. Exploring the genotoxic potential of Bisphenol A and its emerging alternatives in an advanced in vitro 3D zebrafish hepatic cell modelAlja Štern, Katja Kološa, Špela Rozman, Bojana Žegura, 2025, original scientific article Abstract: With global restrictions on Bisphenol A (BPA), various BPA alternatives are increasingly found in ecosystems, raising concerns. This study focuses on the genotoxic potential of three emerging BPA alternatives — Bisphenol AF (BPAF), Bisphenol AP (BPAP), and Bisphenol PH (BPPH) — using an advanced in vitro 3D model system, spheroids, prepared from a Zebrafish (Danio rerio) liver cell line (ZFL). Their cytotoxicity was evaluated using the CellTiter-Glo® 2.0 assay, while their genotoxic potential was assessed using the comet assay, γH2AX assay, and toxicogenomic analysis. The BPA alternatives were more cytotoxic to ZFL spheroids than BPA. Non-cytotoxic concentrations caused transient DNA damage without a significant increase in DNA double-strand breaks (DSBs). The toxicogenomic analysis confirmed these findings, indicating activation of the TP53 DNA damage response pathway, the nucleotide excision repair (NER) and base excision repair (BER) mechanisms, likely in response to bulky DNA lesions and oxidative DNA damage. In addition, the gene expression analysis indicated the influence of the tested BPs on the endocrine system. Our results indicate that BPAF, BPAP and BPPH have considerable genotoxic potential and pose a significant ecotoxicological risk, underscoring the need for further investigation and careful consideration of these chemicals as BPA replacements.
Keywords: BPA, BPAF, BPAP, BPPH, cytotoxicity, genotoxicity, ZFL spheroids
Published in DiRROS: 26.09.2025; Views: 450; Downloads: 195
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5. Hazard characterization of the mycotoxins enniatins and beauvericin to identify data gaps and improve risk assessment for human healthAnne-Cathrin Behr, Christiane Kruse Fæste, Amaya Azqueta, Ana P. M. Tavares, Anastasia Spyropoulou, Anita Solhaug, Ann-Karin Olsen, Ariane Vettorazzi, Bojana Žegura, Matjaž Novak, 2025, review article Abstract: Enniatins (ENNs) and beauvericin (BEA) are cyclic hexadepsipeptide fungal metabolites which have demonstrated antibiotic, antimycotic, and insecticidal activities. The substantial toxic potentials of these mycotoxins are associated with their ionophoric molecular properties and relatively high lipophilicities. ENNs occur extensively in grain and grain-derived products and are considered a food safety issue by the European Food Safety Authority (EFSA). The tolerable daily intake and maximum levels for ENNs in humans and animals remain unestablished due to key toxicological and toxicokinetic data gaps, preventing full risk assessment. Aiming to find critical data gaps impeding hazard characterization and risk evaluation, this review presents a comprehensive summary of the existing information from in vitro and in vivo studies on toxicokinetic characteristics and cytotoxic, genotoxic, immunotoxic, endocrine, reproductive and developmental effects of the most prevalent ENN analogues (ENN A, A1, B, B1) and BEA. The missing information identified showed that additional studies on ENNs and BEA have to be performed before sufficient data for an in-depth hazard characterisation of these mycotoxins become available.
Keywords: enniatins, beauvericin, genotoxicity, endocrine effects, immunotoxicology, toxicokinetics
Published in DiRROS: 14.04.2025; Views: 769; Downloads: 370
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6. In vitro hepatic 3D cell models and their application in genetic toxicology : a systematic reviewMartina Štampar, Bojana Žegura, 2024, review article Abstract: The rapid development of new chemicals and consumer products has raised concerns about their potential genotoxic effects on human health, including DNA damage leading to serious diseases. For such new chemicals and pharmaceutical products, international regulations require genotoxicity data, initially obtained through in vitro tests, followed by in vivo experiments, if needed. Traditionally, laboratory animals have been used for this purpose, however, they are costly, ethically problematic, and often unreliable due to species differences. Therefore, innovative more accurate in vitro testing approaches are rapidly being developed to replace, refine and reduce (3R) the use of animals for experimental purposes and to improve the relevance for humans in toxicology studies. One of such innovative approaches are in vitro three-dimensional (3D) cell models, which are already being highlighted as superior alternatives to the two-dimensional (2D) cell cultures that are traditionally used as in vitro models for the safety testing of chemicals and pharmaceuticals. 3D cell models provide physiologically relevant information and more predictive data for in vivo conditions. In the review article, we provide a comprehensive overview of 3D hepatic cell models, including HepG2, HepG2/C3A, HepaRG, human primary hepatocytes, and iPSC-derived hepatocytes, and their application in the field of genotoxicology. Through a detailed literature analysis, we identified 31 studies conducted between 2007 and April 2024 that used a variety of standard methods, such as the comet assay, the micronucleus assay, and the γH2AX assay, as well as new methodological approaches, including toxicogenomics, to assess the cytotoxic and genotoxic activity of chemicals, nanoparticles and natural toxins. Based on our search, we can conclude that the use of in vitro 3D cell models for genotoxicity testing has been increasing over the years and that 3D cell models have an even greater potential for future implementation and further refinement in genetic toxicology and risk assessment.
Keywords: genotoxicity, advanced 3D in vitro models, hepatic cells, spheroids, comet assay, micronucleus assay, genotoxicology, toxicological studies
Published in DiRROS: 14.11.2024; Views: 1310; Downloads: 518
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7. Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human healthHenriqueta Louro, Ariane Vettorazzi, Adela López de Cerain, Bojana Žegura, Matjaž Novak, 2024, review article Abstract: Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.
Keywords: mycotoxin, exposure routes, genotoxicity, endocrine disruption, immunosuppression, biotransformation, toxicokinetics, tenuazonic acid, alternariol, altenuene, tentoxin, altertoxin
Published in DiRROS: 07.08.2024; Views: 1094; Downloads: 683
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8. Use of HuH6 and other human-derived hepatoma lines for the detection of genotoxins : a new hope for laboratory animals?Monika Waldherr, Miroslav Mišík, Franziska Ferk, Jana Tomc, Bojana Žegura, Metka Filipič, Wolfgang Mikulits, Sören Mai, Oskar Haas, Wolfgang W. Huber, Elisabeth Haslinger, Siegfried Knasmüller, 2018, original scientific article Abstract: Cell lines which are currently used in genotoxicity tests lack enzymes which activate/detoxify mutagens. Therefore, rodent-derived liver preparations are used which reflect their metabolism in humans only partly; as a consequence misleading results are often obtained. Previous findings suggest that certain liver cell lines express phase I/II enzymes and detect promutagens without activation; however, their use is hampered by different shortcomings. The aim of this study was the identification of a suitable cell line. The sensitivity of twelve hepatic cell lines was investigated in single cell gel electrophoresis assays. Furthermore, characteristics of these lines were studied which are relevant for their use in genotoxicity assays (mitotic activity, p53 status, chromosome number, and stability). Three lines (HuH6, HCC1.2, and HepG2) detected representatives of five classes of promutagens, namely, IQ and PhIP (HAAs), B(a)P (PAH), NDMA (nitrosamine), and AFB1 (aflatoxin), and were sensitive towards reactive oxygen species (ROS). In contrast, the commercially available line HepaRG, postulated to be a surrogate for hepatocytes and an ideal tool for mutagenicity tests, did not detect IQ and was relatively insensitive towards ROS. All other lines failed to detect two or more compounds. HCC1.2 cells have a high and unstable chromosome number and mutated p53, these features distract from its use in routine screening. HepG2 was frequently employed in earlier studies, but pronounced inter-laboratory variations were observed. HuH6 was never used in genotoxicity experiments and is highly promising, it has a stable karyotype and we demonstrated that the results of genotoxicity experiments are reproducible.
Keywords: hepatic cell lines, p53, comet assay, genotoxicity
Published in DiRROS: 24.07.2024; Views: 1057; Downloads: 614
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9. Plastics in cyanobacterial blooms - genotoxic effects of binary mixtures of cylindrospermopsin and bisphenols in HepG2 cellsKlara Hercog, Alja Štern, Sara Maisanaba Hernández, Metka Filipič, Bojana Žegura, 2020, original scientific article Abstract: Ever-expanding environmental pollution is causing a rise in cyanobacterial blooms and the accumulation of plastics in water bodies. Consequently, exposure to mixtures of cyanotoxins and plastic-related contaminants such as bisphenols (BPs) is of increasing concern. The present study describes genotoxic effects induced by co-exposure to one of the emerging cyanotoxins—cylindrospermopsin (CYN)—(0.5 µg/mL) and BPs (bisphenol A (BPA), S (BPS), and F (BPF); (10 µg/mL)) in HepG2 cells after 24 and 72 h of exposure. The cytotoxicity was evaluated with an MTS assay and genotoxicity was assessed through the measurement of the induction of DNA double strand breaks (DSB) with the γH2AX assay. The deregulation of selected genes (xenobiotic metabolic enzyme genes, DNA damage, and oxidative response genes) was assessed using qPCR. The results showed a moderate reduction of cell viability and induction of DSBs after 72 h of exposure to the CYN/BPs mixtures and CYN alone. None of the BPs alone reduced cell viability or induced DSBs. No significant difference was observed between CYN and CYN/BPs exposed cells, except with CYN/BPA, where the antagonistic activity of BPA against CYN was indicated. The deregulation of some of the tested genes (CYP1A1, CDKN1A, GADD45A, and GCLC) was more pronounced after exposure to the CYN/BPs mixtures compared to single compounds, suggesting additive or synergistic action. The present study confirms the importance of co-exposure studies, as our results show pollutant mixtures to induce effects different from those confirmed for single compounds.
Keywords: cylindrospermopsin, CYN, bisphenols, BPA, BPS, BPF, BPAF, co-exposure, genotoxicity, cytotoxicity
Published in DiRROS: 23.07.2024; Views: 1162; Downloads: 799
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10. Cytotoxic and genotoxic effects of cyanobacterial and algal extracts-microcystin and retinoic acid contentMichal Bittner, Alja Štern, Marie Smutna, Klara Hilscherova, Bojana Žegura, 2021, original scientific article Abstract: In the last decade, it has become evident that complex mixtures of cyanobacterial bioactive substances, simultaneously present in blooms, often exert adverse effects that are different from those of pure cyanotoxins, and awareness has been raised on the importance of studying complex mixtures and chemical interactions. We aimed to investigate cytotoxic and genotoxic effects of complex extracts from laboratory cultures of cyanobacterial species from different orders (Cylindrospermopsis raciborskii, Aphanizomenon gracile, Microcystis aeruginosa, M. viridis, M. ichtyoblabe, Planktothrix agardhii, Limnothrix redekei) and algae (Desmodesmus quadricauda), and examine possible relationships between the observed effects and toxin and retinoic acid (RA) content in the extracts. The cytotoxic and genotoxic effects of the extracts were studied in the human hepatocellular carcinoma HepG2 cell line, using the MTT assay, and the comet and cytokinesis-block micronucleus (cytome) assays, respectively. Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) was used to detect toxins (microcystins (MC-LR, MC-RR, MC-YR) and cylindrospermopsin) and RAs (ATRA and 9cis-RA) in the extracts. Six out of eight extracts were cytotoxic (0.04–2 mgDM/mL), and five induced DNA strand breaks at non-cytotoxic concentrations (0.2–2 mgDM/mL). The extracts with genotoxic activity also had the highest content of RAs and there was a linear association between RA content and genotoxicity, indicating their possible involvement; however further research is needed to identify and confirm the compounds involved and to elucidate possible genotoxic effects of RAs.
Keywords: cyanobacteria, algae, extracts, complex mixtures, genotoxicity, cytotoxicity, retinoic acids, microcystins, cyanotoxins, chemical analysis
Published in DiRROS: 19.07.2024; Views: 1027; Downloads: 683
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