1. In vitro genotoxicity assessment of graphene quantum dots using a 3D HepG2 model : version v1Irma Durmišević, Anja Haverić, Katja Kološa, Bojana Žegura, 2026, zaključena znanstvena zbirka raziskovalnih podatkov Povzetek: Graphene quantum dots (GQDs) are nano-sized fragments of graphene sheets, with great potential for applications in optics, electrochemistry, and biomedicine, including drug delivery. In this study, two types of GQDs were investigated: green-emitting (G-GQD) and blue-emitting (B-GQD) and their genotoxic potential was examined on the protein level. Flow cytometric analysis of γH2AX, a marker of DNA double-strand breaks, and phosphorylated histone H3 (p-H3), a marker of aneugenic activity, was performed in HepG2 spheroids after 24 hours of exposure to G-GQDs and B-GQDs. HepG2 spheroids were exposed to graded concentrations of B- and G-GQD (12.5, 25, 50, and 100 μg/mL) for 24 hours, respectively. Spheroids were dissociated into single-cell suspensions, washed twice with 1x PBS and fixed in 4% PFA. For flow cytometric analysis, cells were labelled with anti-H2AX pS139 antibody and anti-histone H3 pS128 antibody. REA-APC and REA-PE controls were applied to exclude non-specific antibody binding. For each sample, 10,000 events were acquired using a MACSQuant Analyzer 10 flow cytometer and MACSQuantify™ software (Miltenyi Biotech, Germany). Raw data were exported from the MACSQuantify software and analysed with FlowJo V10 software (Becton Dickinson, New Jersey, USA).
Ključne besede: nanomaterials, graphene quantum dots, HepG2 spheroids, toxicity
Objavljeno v DiRROS: 31.07.2026; Ogledov: 123; Prenosov: 79
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2. Glucose coated FeO@Fe3O4 nanoparticles show tunable catalytic reactivity and safety in a 3D hepatic in vitro modelMarco A. Morales Ovalle, Iza Rozman, Elin L. Winkler, Enio Lima, Alja Štern, Katja Kološa, Bojana Žegura, Gerardo F. Goya, 2026, izvirni znanstveni članek Povzetek: Iron-oxide magnetic nanoparticles (MNPs) have been extensively investigated as magnetically actuated nanocatalysts for diagnostic and therapeutic applications. However, because wüstite/magnetite/maghemite phases can interconvert, coexisting Fe2+/Fe3+ species may redirect Fenton-like chemistry and generate reactive oxygen species (ROS) profiles that differ from the intended biocatalytic pathway. Here, we investigate monodisperse biphasic FeO@Fe3O4 core-shell MNPs with an average particle size ⟨d⟩ = 9.6(5) nm, and their glucose-coated analogue, combining EPR radical analysis with toxicity testing in a 3D HepG2 hepatic spheroid model. Naked particles exhibited conventional Fenton-like behavior dominated by hydroxyl radicals (⋅OH), whereas glucose coating markedly suppressed ⋅OH while increasing hydroperoxyl radicals (⋅OOH; ≈55 pM at 60 min), demonstrating ligand-controlled rerouting of the radical pathway. TEM mapping across spheroid cross-sections showed preferential MNP accumulation in the outer layer, with most observed events confined to the outer ≈10–15 μm, corresponding to an approximately one-cell-thick rim; sparse deeper events were observed up to ≈30–35 μm. MNPs produced dose- and time-dependent cytotoxicity in HepG2 spheroids, with IC50 values of 29.3 (24 h) and 10.8 (96 h) µg·cm− 2, without evidence of lipid peroxidation or genotoxicity. MDA levels remained unchanged, the comet assay showed no increase in DNA damage, and γH2AX and phospho-H3 (p-H3) positive events were not detected. Our results show that glucose functionalization provides a simple route to modulate radical pathways and define operational windows for redox-active FeO@Fe3O4 nano-reactors in oxidative nanomedicine.
Ključne besede: iron-oxide nanoparticles, Fenton-like catalysis, cytotoxicity, genotoxicity, HepG2 spheroids
Objavljeno v DiRROS: 02.06.2026; Ogledov: 239; Prenosov: 232
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3. In vitro toxicity assessment of graphene quantum dots using a 3D HepG2 modelIrma Durmišević, Anja Haverić, Sonja Žabkar, Alja Štern, Katja Kološa, Petra Jenuš, Iza Rozman, Bojana Žegura, 2026, izvirni znanstveni članek Povzetek: In the present study, two types of graphene quantum dots (GQDs) were investigated: green-emitting (G-GQDs) and blue-emitting (B-GQDs). Physicochemical characterisation was performed using transmission electron microscopy (TEM), zeta potential, and hydrodynamic radius measurements to evaluate the morphology, particle size, aggregation behaviour, and colloidal stability of the GQDs in both water and cell culture medium. G-GQDs exhibited superior colloidal stability and more uniform dispersion than B-GQDs, whereas both types showed reduced aggregation and surface charge in cell culture medium due to protein corona formation. Toxicological characterisation was performed using an in vitro human hepatocellular carcinoma (HepG2) 3D spheroid model, with GQDs exposures up to 250 µg/mL (100 µg/cm2). Cytotoxicity was measured using the CellTiter-Glo luminometric assay, while genotoxicity was evaluated by the comet assay and flow cytometric analysis of γH2AX and phosphorylated histone H3 (p-H3) after 24 h of exposure. Both GQDs induced dose-dependent cytotoxic effects in HepG2 spheroids. At non-cytotoxic concentrations, a dose-dependent increase in DNA damage was observed, as determined by the comet assay. However, no evidence of DNA double-strand breaks (γH2AX) or elevated p-H3 levels was detected, suggesting the absence of clastogenic and aneugenic activity. The observed DNA single-strand breaks may be partly attributed to reactive oxygen species induction. These results indicate that, although GQDs induced cytotoxicity and single-strand DNA damage, no clear evidence of more severe genotoxic effects was observed under the tested conditions. Further studies are warranted to elucidate underlying mechanisms and comprehensively assess the safety profile of GQDs for biomedical applications.
Ključne besede: nanomaterials, graphene quantum dots, HepG2 spheroids, toxicity
Objavljeno v DiRROS: 19.03.2026; Ogledov: 520; Prenosov: 192
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4. New mitochondrial ▫$K_V$▫1.3 conjugates are potent and specific inducers of apoptosis in cancer modelsŠpela Gubič, Marzia Fois, Ivan Džajić, Katja Kološa, Alja Štern, Maša Omerzel, Tim Božič, Boštjan Markelc, Tanja Jesenko, Maja Čemažar, Bojana Žegura, Tina Kosjek, Andrej Emanuel Cotman, Tihomir Tomašič, Lucija Peterlin-Mašič, 2026, izvirni znanstveni članek Ključne besede: mitochondrial targeting, mitochondrial KV1.3, apoptosis, cancer
Objavljeno v DiRROS: 12.02.2026; Ogledov: 737; Prenosov: 410
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5. 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, izvirni znanstveni članek Povzetek: 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.
Ključne besede: ZFL cell line, zebrafish embryos, cytotoxicity, genotoxicity, Danube River
Objavljeno v DiRROS: 28.01.2026; Ogledov: 524; Prenosov: 685
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6. 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, izvirni znanstveni članek Povzetek: 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.
Ključne besede: DNA damage, genotoxicity, HepG2 spheroids, magnetic ferrite-based nanoparticles, ROS induction, safety assessment, toxicogenomics
Objavljeno v DiRROS: 27.01.2026; Ogledov: 609; Prenosov: 793
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7. 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, izvirni znanstveni članek Povzetek: 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.
Ključne besede: BPA, BPAF, BPAP, BPPH, cytotoxicity, genotoxicity, ZFL spheroids
Objavljeno v DiRROS: 26.09.2025; Ogledov: 836; Prenosov: 426
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8. Evidence driven indoor air quality improvement : an innovative and interdisciplinary approach to improving indoor air qualityMario Lovrić, Goran Gajski, Jessica Fernández-Agüera, Mira Pöhlker, Bojana Žegura, Matjaž Novak, Alja Štern, Katja Kološa, Martina Štampar, 2024, pregledni znanstveni članek Povzetek: Indoor air pollution is a recognized emerging threat, claiming millions of lives annually. People are constantly exposed to ambient and indoor air pollution. The latest research shows that people in developed countries spend up to 90% of their time indoors and almost 70% at home. Although impaired IAQ represents a significant health risk, it affects people differently, and specific populations are more vulnerable: children, the elderly, and people with respiratory illnesses are more sensitive to these environmental risks. Despite rather extensive research on IAQ, most of the current understanding about the subject, which includes pollution sources, indoor–outdoor relationships, and ventilation/filtration, is still quite limited, mainly because air quality monitoring in the EU is primarily focused on ambient air quality and regulatory requirements are lacking for indoor environments. Therefore, the EDIAQI project aims to improve guidelines and awareness for advancing the IAQ in Europe and beyond by allowing user-friendly access to information about indoor air pollution exposures, sources, and related risk factors. The solution proposed with EDIAQI consists of conducting a characterization of sources and routes of exposure and dispersion of chemical, biological, and emerging indoor air pollution in multiple cities in the EU. The project will deploy cost-effective/user-friendly monitoring solutions to create new knowledge on sources, exposure routes, and indoor multipollutant body burdens. The EDIAQI project brings together 18 organizations from 11 different European countries that provide interdisciplinary skills and expertise in various fields, including environmental science and technology, medicine, and toxicology, as well as policy design and public engagement.
Ključne besede: indoor air pollution, health risk, vulnerable populations, IAQ (Indoor Air Quality), EDIAQI project, monitoring solutions, exposure routes
Objavljeno v DiRROS: 06.11.2024; Ogledov: 1758; Prenosov: 2327
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9. APS8, a polymeric alkylpyridinium salt blocks [alpha]7 nAChR and induces apoptosis in non-small cell lung carcinomaAna Zovko, Kristina Viktorsson, Rolf Lewensohn, Katja Kološa, Metka Filipič, Hong Xing, William R. Kem, Laura Paleari, Tom Turk, 2013, izvirni znanstveni članek Povzetek: Naturally occurring 3-alkylpyridinium polymers (poly-APS) from the marine sponge Reniera sarai, consisting of monomers containing polar pyridinium and nonpolar alkyl chain moieties, have been demonstrated to exert a wide range of biological activities, including a selective cytotoxicity against non-small cell lung cancer (NSCLC) cells. APS8, an analog of poly-APS with defined alkyl chain length and molecular size, non-competitively inhibits α7 nicotinic acetylcholine receptors (nAChRs) at nanomolar concentrations that are too low to be acetylcholinesterase (AChE) inhibitory or generally cytotoxic. In the present study we show that APS8 inhibits NSCLC tumor cell growth and activates apoptotic pathways. APS8 was not toxic for normal lung fibroblasts. Furthermore, in NSCLC cells, APS8 reduced the adverse anti-apoptotic, proliferative effects of nicotine. Our results suggest that APS8 or similar compounds might be considered as lead compounds to develop antitumor therapeutic agents for at least certain types of lung cancer.
Ključne besede: 3-alkylpyridinium polymers, apoptosis, nicotine, nicotinic acetylcholine receptor, non-small cell lung carcinoma
Objavljeno v DiRROS: 02.08.2024; Ogledov: 1197; Prenosov: 1204
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10. Improved protective effect of umbilical cord stem cell transplantation on cisplatin-induced kidney injury in mice pretreated with antithymocyte globulinŽeljka Večerić-Haler, Andreja Erman, Anton Cerar, Helena Motaln, Katja Kološa, Tamara Lah Turnšek, Snežna Sodin-Šemrl, Katja Lakota, Katjuša Mrak Poljšak, Špela Škrajnar, Simona Kranjc Brezar, Miha Arnol, Martina Perše, 2016, izvirni znanstveni članek Povzetek: Mesenchymal stem cells (MSCs) are recognised as a promising tool to improve renal recovery in experimental models of cisplatin-induced acute kidney injury. However, these preclinical studies were performed on severely immunodeficient animals. Here, we investigated whether human umbilical cord derived MSC treatment could equally ameliorate acute kidney injury induced by cisplatin and prolong survival in mice with a normal immune system and those with a suppressed immune system by polyclonal antithymocyte globulin (ATG). We demonstrated that ATG pretreatment, when followed by MSC transplantation, significantly improved injured renal function parameters, as evidenced by decreased blood urea nitrogen and serum creatinine concentration, as well as improved renal morphology. This tissue restoration was also supported by increased survival of mice. The beneficial effects of ATG were associated with reduced level of inflammatory protein serum amyloid A3 and induced antioxidative expression of superoxide dismutase-1 (SOD-1), glutathione peroxidase (GPx), and hem oxygenase-1 (HO-1). Infused MSCs became localised predominantly in peritubular areas and acted to reduce renal cell death. In conclusion, these results show that ATG diminished in situ inflammation and oxidative stress associated with cisplatin-induced acute kidney injury, the effects that may provide more favourable microenvironment for MSC action, with consequential synergistic improvements in renal injury and animal survival as compared to MSC treatment alone.
Ključne besede: mesenchymal stem cells, nephrotoxicity
Objavljeno v DiRROS: 25.07.2024; Ogledov: 1332; Prenosov: 1069
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