1. Lethal and sub-lethal effects and modulation of gene expression induced by T kinase inhibitors in zebrafish (Danio Rerio) embryosTina Eleršek, Matjaž Novak, Mateja Mlinar, Igor Virant, Nika Bahor, Karin Leben, Bojana Žegura, Metka Filipič, 2022, original scientific article Abstract: Tyrosine kinase inhibitors (TKIs) are designed for targeted cancer therapy. The consumption of these drugs during the last 20 years has been constantly rising. In the zebrafish (Danio rerio) embryo toxicity test, we assessed the toxicity of six TKIs: imatinib mesylate, erlotinib, nilotinib, dasatinib, sorafenib and regorafenib. Imatinib mesylate and dasatinib induced lethal effects, while regorafenib, sorfenib and dasatinib caused a significant increase of sub-lethal effects, predominantly oedema, no blood circulation and formation of blood aggregates. The analyses of the changes in the expression of selected genes associated with the hormone system after the exposure to imatinib mesylate, dasatinib and regorafenib demonstrated that all three tested TKIs deregulated the expression of oestrogen receptor esr1, cytochrome P450 aromatase (cypa19b) and hydroxysteroid-dehydrogenase (hsd3b), regorafenib, and also thyroglobulin (tg). The expression of genes involved in the DNA damage response (gadd45 and mcm6) and apoptosis (bcl2) was deregulated only by exposure to regorafenib. The data indicate that common mechanisms, namely antiangiogenic activity and interference with steroidogenesis are involved in the TKI induced sub-lethal effects and potential hormone disrupting activity, respectively. The residues of TKIs may represent an environmental hazard; therefore, further ecotoxicological studies focusing also on the effects of their mixtures are warranted.
Keywords: aquatic toxicity, tyrosine kinase inhibitors, zebrafish embryo toxicity test, gene expression, environmental hazard
Published in DiRROS: 16.07.2024; Views: 26; Downloads: 10
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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, original scientific article Abstract: 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.
Keywords: in vitro 3D cell model, HepG2, flow cytometry, cell cycle, proliferation, DNA strand, breaks
Published in DiRROS: 16.07.2024; Views: 26; Downloads: 20
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3. Adverse toxic effects of tyrosine kinase inhibitors on non-target zebrafish liver (ZFL) cellsKatja Kološa, Bojana Žegura, Martina Štampar, Metka Filipič, Matjaž Novak, 2023, original scientific article Keywords: zebrafish liver cells, ZFL, tyrosine kinase inhibitors, cytotoxicity, cell cycle, genotoxicity, environmental hazard
Published in DiRROS: 12.07.2024; Views: 57; Downloads: 30
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4. Exploring the safety of cannabidiol (CBD) : a comprehensive in vitro evaluation of the genotoxic and mutagenic potential of a CBD isolate and extract from Cannabis sativa LAlja Štern, Matjaž Novak, Katja Kološa, Jurij Trontelj, Sonja Žabkar, Tjaša Šentjurc, Metka Filipič, Bojana Žegura, 2024, original scientific article Abstract: Cannabidiol (CBD), a naturally occurring cyclic terpenoid found in Cannabis sativa L., is renowned for its diverse
pharmacological benefits. Marketed as a remedy for various health issues, CBD products are utilized by patients
as a supplementary therapy or post-treatment failure, as well as by healthy individuals seeking promised advantages. Despite its widespread use, information regarding potential adverse effects, especially genotoxic
properties, is limited. The present study is focused on the mutagenic and genotoxic activity of a CBD isolate
(99.4 % CBD content) and CBD-rich Cannabis sativa L extract (63.6 % CBD content) in vitro. Both CBD samples
were non-mutagenic, as determined by the AMES test (OECD 471) but exhibited cytotoxicity for HepG2 cells
(~IC50 (4 h) 26 µg/ml, ~IC50 (24 h) 6–8 µg/ml, MTT assay). Noncytotoxic concentrations induced upregulation of
genes encoding metabolic enzymes involved in CBD metabolism, and CBD oxidative as well as glucuronide
metabolites were found in cell culture media, demonstrating the ability of HepG2 cells to metabolize CBD. In this
study, the CBD samples were found non-genotoxic. No DNA damage was observed with the comet assay, and no
influence on genomic instability was observed with the cytokinesis block micronucleus and the γH2AX and p-H3
assays. Furthermore, no changes in the expression of genes involved in genotoxic stress response were detected in
the toxicogenomic analysis, after 4 and 24 h of exposure. Our comprehensive study contributes valuable insights
into CBD’s safety profile, paving the way for further exploration of CBD’s therapeutic applications and potential
adverse effects.
Keywords: cannabidiol, CBD, metabolism, cytotoxicity, genotoxicity, mutagenicity
Published in DiRROS: 09.07.2024; Views: 57; Downloads: 31
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7. Subchronic exposure of rats to sublethal dose of microcystin-YR induces DNA damage in multiple organsMetka Filipič, Bojana Žegura, Bojan Sedmak, Irena Horvat-Žnidaršič, Aleksandra Milutinović Živin, Dušan Šuput, 2007, original scientific article Abstract: Background. Microcystins (MCs) are cyclic heptapeptides that are considered tobe liver specific toxins. They are potent tumour promoters and recent studies indicate that they are also genotoxic. In this study we measured DNA damage in lymphocytes, liver, kidney (cortex and medulla), lung, spleen and brain cells of male Fisher F344 rats that were exposed to sublethal dose (every second day 10 Ugžkg b.w.č i.p) of microeysrin-YR (MCYR) for one month. Methods. At the end of exposure the animals were sacrificed, the lymphocytes were isolated from blood taken from jugular vein, liver cells were obtained byperfusion with collagenase A and the cells from other organs were isolated by incubating small tissue pieces with eollagenase A. The DNA damage in isolated cells was measured with the single cells gel electrophoresis (SCGE) also called the comet assay. Results. A significant increase of the % tail DNAin MCYR-exposed animals compared to the nonexposed control ones was observed in brain (2.5 fold), liver (2.1 fold), kidney medulla (1.9 fold), kidney cortex (1.8 fold) and lung (1.7 fold) cells, while the DNA from lymphocytes and spleen cells was not affected. Conclusion. This study demonstrated that subehronic exposure to sublethal doses of MCs can induce systemicgenotoxicity in mammals, and it affects not only the liver but also other vital organs.
Keywords: DNA damage, comet assay, cyanobacteria, bacterial toxins, rats, inbred F344
Published in DiRROS: 20.02.2024; Views: 296; Downloads: 71
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