1. 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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2. 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: 56; Downloads: 31
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3. Nature-inspired substituted 3-(imidazol-2-yl) morpholines targeting human topoisomerase IIα : dynophore-derived discoveryBarbara Herlah, Matej Janežič, Iza Ogris, Simona Golič Grdadolnik, Katja Kološa, Sonja Žabkar, Bojana Žegura, Andrej Perdih, 2024, original scientific article Abstract: The molecular nanomachine, human DNA topoisomerase IIα, plays a crucial role in replication, transcription, and recombination by catalyzing topological changes in the DNA, rendering it an optimal target for cancer chemotherapy. Current clinical topoisomerase II poisons often cause secondary tumors as side effects due to the accumulation of double-strand breaks in the DNA, spurring the development of catalytic inhibitors. Here, we used a dynamic pharmacophore approach to develop catalytic inhibitors targeting the ATP binding site of human DNA topoisomerase IIα. Our screening of a library of nature-inspired compounds led to the discovery of a class of 3-(imidazol-2-yl) morpholines as potent catalytic inhibitors that bind to the ATPase domain. Further experimental and computational studies identified hit compound 17, which exhibited selectivity against the human DNA topoisomerase IIα versus human protein kinases, cytotoxicity against several human cancer cells, and did not induce DNA double-strand breaks, making it distinct from clinical topoisomerase II poisons. This study integrates an innovative natural product-inspired chemistry and successful implementation of a molecular design strategy that incorporates a dynamic component of ligand-target molecular recognition, with comprehensive experimental characterization leading to hit compounds with potential impact on the development of more efficient chemotherapies.
Keywords: topoisomerase II, catalytic inhibitors, chemotherapy, DNA damage, cancer
Published in DiRROS: 03.06.2024; Views: 264; Downloads: 124
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