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Povzetek: 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.
Ključne besede: genotoxicity, advanced 3D in vitro models, hepatic cells, spheroids, comet assay, micronucleus assay, genotoxicology, toxicological studies
Objavljeno v DiRROS: 14.11.2024; Ogledov: 120; Prenosov: 59
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Povzetek: 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.
Ključne besede: hepatic cell lines, p53, comet assay, genotoxicity
Objavljeno v DiRROS: 24.07.2024; Ogledov: 298; Prenosov: 195
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Povzetek: 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.
Ključne besede: cyanobacteria, algae, extracts, complex mixtures, genotoxicity, cytotoxicity, retinoic acids, microcystins, cyanotoxins, chemical analysis
Objavljeno v DiRROS: 19.07.2024; Ogledov: 315; Prenosov: 219
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Ključne besede: zebrafish liver cells, ZFL, tyrosine kinase inhibitors, cytotoxicity, cell cycle, genotoxicity, environmental hazard
Objavljeno v DiRROS: 12.07.2024; Ogledov: 576; Prenosov: 232
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Povzetek: The applications of magnetic nanoparticles (MNPs) as biocatalysts in different biomedical areas have been evolved very recently. One of the main challenges in this field is to design affective MNPs surfaces with catalytically active atomic centres, while producing minimal toxicological side effects on the hosting cell or tissues. MNPs of vanadium spinel ferrite (VFe2O4) are a promising material for mimicking the action of natural enzymes in degrading harmful substrates due to the presence of active V5+ centres. However, the toxicity of this material has not been yet studied in detail enough to grant biomedical safety. In this work, we have extensively measured the structural, compositional, and magnetic properties of a series of VxFe3-xO4 spinel ferrite MNPs to assess the surface composition and oxidation state of V atoms, and also performed systematic and extensive in vitro cytotoxicity and genotoxicity testing required to assess their safety in potential clinical applications. We could establish the presence of V5+ at the particle surface even in water-based colloidal samples at pH 7, as well as different amounts of V2+ and V3+ substitution at the A and B sites of the spinel structure. All samples showed large heating efficiency with Specific Loss Power values up to 400 W/g (H0 = 30 kA/m; f = 700 kHz). Samples analysed for safety in human hepatocellular carcinoma (HepG2) cell line with up to 24h of exposure showed that these MNPs did not induce major genomic abnormalities such as micronuclei, nuclear buds, or nucleoplasmic bridges (MNIs, NBUDs, and NPBs), nor did they cause DNA double-strand breaks (DSBs) or aneugenic effects—types of damage considered most harmful to cellular genetic material. The present study is an essential step towards the use of these type of nanomaterials in any biomedical or clinical application.
Ključne besede: magnetic nanoparticles, vanadium ferrite, cytotoxicity, genotoxicity, specific power absorption, cell viability
Objavljeno v DiRROS: 23.05.2024; Ogledov: 437; Prenosov: 394
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