1. 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: 91; Downloads: 93
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2. Synthesis, purification, and cell-toxicity of a choline betainateLucija Jurko, Gregor Hostnik, Tobias Alexander Steindorfer, Alja Štern, Perica Bošković, Matej Bračič, Bojana Žegura, Rupert Kargl, 2024, original scientific article Abstract: In this work, choline chloride and betaine hydrochloride were condensed into a - to our knowledge - unreported choline betainate (N,N,N-trimethyl-2-oxo-2-(2-(trimethylammonio)ethoxy)ethanaminium chloride) using 1,1′-carbonyldiimidazole (CDI) activation of betaine hydrochloride in dimethylsulfoxide. The product and reaction intermediates were isolated, purified by preparative HPLC and analyzed in detail by infrared and nuclear magnetic resonance spectroscopy. The final product has a high cytotoxicity for L929 mouse fibroblasts, and low antibacterial activity against P. Aeruginosa and S. Aureus at concentrations of up to 20 mg/ml. It could potentially further be investigated for similar uses as suxamethonium chloride, a muscle relaxant drug. Keywords: choline chloride, betaine hydrochloride, carbonyldiimidazole, HPLC, antimicrobial, cytotoxicity Published in DiRROS: 07.08.2024; Views: 87; Downloads: 55
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3. New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens : a PARC projectMarc Audebert, Ann-Sophie Assmann, Amaya Azqueta, Pavel Babica, Emilio Benfenati, Martina Štampar, Bojana Žegura, 2023, review article Abstract: Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens. Keywords: non-genotoxic carcinogens, NGTxC, new approach methodologies, NAM, PARC Published in DiRROS: 05.08.2024; Views: 94; Downloads: 48
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4. Double strand breaks and cell-cycle arrest induced by the cyanobacterial toxin cylindrospermopsin in HepG2 cellsAlja Štern, Metka Filipič, Matjaž Novak, Bojana Žegura, 2013, original scientific article Abstract: 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. Keywords: cylindrospermopsin, cell-cycle, cell-proliferation, double-strand breaks, HepG2 cells Published in DiRROS: 02.08.2024; Views: 129; Downloads: 103
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5. DNAqua-Net : developing new genetic tools for bioassessment and monitoring of aquatic ecosystems in EuropeFlorian Leese, Tina Eleršek, Cene Fišer, Ana Rotter, Bojana Žegura, Irena Maček, 2016, original scientific article Abstract: The protection, preservation and restoration of aquatic ecosystems and their functions are of global importance. For European states it became legally binding mainly through the EU-Water Framework Directive (WFD). In order to assess the ecological status of a given water body, aquatic biodiversity data are obtained and compared to a reference water body. The quantified mismatch obtained determines the extent of potential management actions. The current approach to biodiversity assessment is based on morpho-taxonomy. This approach has many drawbacks such as being time consuming, limited in temporal and spatial resolution, and error-prone due to the varying individual taxonomic expertise of the analysts. Novel genomic tools can overcome many of the aforementioned problems and could complement or even replace traditional bioassessment. Yet, a plethora of approaches are independently developed in different institutions, thereby hampering any concerted routine application. The goal of this Action is to nucleate a group of researchers across disciplines with the task to identify gold-standard genomic tools and novel eco-genomic indices for routine application in biodiversity assessments of European fresh- and marine water bodies. Furthermore, DNAqua-Net will provide a platform for training of the next generation of European researchers preparing them for the new technologies. Jointly with water managers, politicians, and other stakeholders, the group will develop a conceptual framework for the standard application of eco-genomic tools as part of legally binding assessments. Keywords: aquatic ecosystems, biodiversity, monitoring, genomic tools Published in DiRROS: 25.07.2024; Views: 140; Downloads: 69
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6. 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: 119; Downloads: 78
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7. Genotoxic effects of cylindrospermopsin, microcystin-LR and their binary mixture in human hepatocellular carcinoma (HepG2) cell lineLeticia Díez-Quijada, Klara Hercog, Martina Štampar, Metka Filipič, Ana M. Cameán, Angeles Jos, Bojana Žegura, 2020, original scientific article Abstract: Simultaneous occurrence of cylindrospermopsin (CYN) and microcystin-LR (MCLR) has been reported in the aquatic environment and thus human exposure to such mixtures is possible. As data on the combined effects of CYN/MCLR are scarce, we aimed to investigate the adverse effects related to genotoxic activities induced by CYN (0.125, 0.25 and 0.5 µg/mL) and MCLR (1 µg/mL) as single compounds and their combinations in HepG2 cells after 24 and 72 h exposure. CYN and CYN/MCLR induced DNA double-strand breaks after 72 h exposure, while cell cycle analysis revealed that CYN and CYN/MCLR arrested HepG2 cells in G0/G1 phase. Moreover, CYN and the combination with MCLR upregulated CYP1A1 and target genes involved in DNA-damage response (CDKN1A, GADD45A). Altogether, the results showed that after 72 h exposure genotoxic activity of CYN/MCLR mixture was comparable to the one of pure CYN. On the contrary, MCLR (1 µg/mL) had no effect on the viability of cells and had no influence on cell division. It did not induce DNA damage and did not deregulate studied genes after prolonged exposure. The outcomes of the study confirm the importance of investigating the combined effects of several toxins as the effects can differ from those induced by single compounds. Published in DiRROS: 23.07.2024; Views: 113; Downloads: 123
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8. 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: 125; Downloads: 134
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9. Characterization of In vitro 3D cell model developed from human hepatocellular carcinoma (HepG2) cell lineMartina Štampar, Barbara Breznik, Metka Filipič, Bojana Žegura, 2020, original scientific article Abstract: In genetic toxicology, there is a trend against the increased use of in vivo models as highlighted by the 3R strategy, thus encouraging the development and implementation of alternative models. Two-dimensional (2D) hepatic cell models, which are generally used for studying the adverse effects of chemicals and consumer products, are prone to giving misleading results. On the other hand, newly developed hepatic three-dimensional (3D) cell models provide an attractive alternative, which, due to improved cell interactions and a higher level of liver-specific functions, including metabolic enzymes, reflect in vivo conditions more accurately. We developed an in vitro 3D cell model from the human hepatocellular carcinoma (HepG2) cell line. The spheroids were cultured under static conditions and characterised by monitoring their growth, morphology, and cell viability during the time of cultivation. A time-dependent suppression of cell division was observed. Cell cycle analysis showed time-dependent accumulation of cells in the G0/G1 phase. Moreover, time-dependent downregulation of proliferation markers was shown at the mRNA level. Genes encoding hepatic markers, metabolic phase I/II enzymes, were time-dependently deregulated compared to monolayers. New knowledge on the characteristics of the 3D cell model is of great importance for its further development and application in the safety assessment of chemicals, food products, and complex mixtures. Published in DiRROS: 22.07.2024; Views: 118; Downloads: 117
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10. Minimum Information for Reporting on the Comet Assay (MIRCA) : recommendations for describing comet assay procedures and resultsPeter Møller, Amaya Azqueta, Elisa Boutet-Robinet, Gudrun Koppen, Stefano Bonassi, Mirta Milić, Goran Gajski, Solange Costa, Bojana Žegura, Matjaž Novak, 2020, original scientific article Abstract: The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between ‘desirable’ and ‘essential’ information: ‘essential’ information refers to the precise details that are necessary to assess the quality of the experimental work, whereas ‘desirable’ information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers. Published in DiRROS: 22.07.2024; Views: 130; Downloads: 88
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