1. TEM images of HepG2 spheroids exposed to magnetic Ferite-based Nanoparticles MxFe3-xO4 (M=Fe, Zn, Mn) for 24 and 96 hours : version v1Marco A. Morales Ovalle, Iza Rozman, Alja Štern, Gerardo F. Goya, Álvaro Gallo-Cordova, María del Puerto Morales, Bojana Žegura, 2025, zaključena znanstvena zbirka raziskovalnih podatkov Povzetek: Nanosized spinel-type ferrites have gained recognition as a unique class of engineered nanomaterials with promising applications, but their safety profiles remain insufficiently explored. Although iron (Fe), zinc (Zn), and manganese (Mn) are biologically relevant elements, the use of Zn- and Mn-containing ferrite nanoparticles (NPs) in biomedical contexts demands careful (geno)toxicity evaluation. In this study, three ferrite NPs – γFe2O3 (FeNPs), Zn0.7Fe2.3O4 (ZnNPs), and Mn0.4Fe2.6O4 (MnNPs) – synthesised through a microwave-assisted polyol route, functionalized with citric acid to improve colloidal stability, were evaluated for their potential (geno)toxic effects in an advanced in vitro 3D cell model, HepG2 spheroids. To assess the spatial distribution of the tested NPs within the 3D cellular architecture of HepG2 spheroids, TEM analysis was performed after 24 and 96 hours of exposure. HepG2 spheroids were exposed to each NP type at a concentration of 50 µg/mL. Following incubation, spheroids were collected, gently washed with phosphate-buffered saline (PBS, pH 7.4) to remove unbound NP, and fixed in 2 % glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.2) for 2 hours at room temperature, followed by incubation in 1.5 % glutaraldehyde in 0.05 M PB at 4°C overnight. Post-fixation was carried out with 1% osmium tetroxide for 1 hour at room temperature, followed by dehydration through a graded ethanol series (30%, 50%, 70%, 90%, and absolute ethanol) and infiltration with epoxy resin. For cross-sectional analysis, resin-embedded spheroids were polymerised at 60 °C for 48 hours, and ultrathin sections (~70 nm) were obtained using an ultramicrotome. Sections were collected on copper grids and stained with uranyl acetate (2% aqueous) and lead citrate to enhance contrast. Imaging was performed using a Tecnai T20 transmission electron microscope (Thermo Fisher Scientific, USA) operated at 200 kV. For each sample and time point, at least three spheroids were analysed. Additionally, in one selected spheroid, a systematic series of TEM images was acquired along a straight linear trajectory across the section, beginning at one external edge of the spheroid and progressing through consecutive adjacent fields until reaching the opposite border, and then the images were composed into a single frame.
Ključne besede: TEM, ferrite-based nanoparticles, HepG2 spheroids, internalization, bioaccumulation
Objavljeno v DiRROS: 24.03.2026; Ogledov: 226; Prenosov: 181
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2. Transcriptomic responses of oxidative and genotoxic stress responsive genes after exposure to MxFe3-xO4 (M = Fe, Zn, Mn) in an advanced 3D human hepatic in vitro model : version v1Iza Rozman, Alja Štern, Bojana Žegura, Gerardo F. Goya, Álvaro Gallo-Cordova, María del Puerto Morales, 2025, zaključena znanstvena zbirka raziskovalnih podatkov Povzetek: Nanosized spinel-type ferrites have gained recognition as a unique class of engineered nanomaterials with promising applications, but their safety profiles remain insufficiently explored. Although iron (Fe), zinc (Zn), and manganese (Mn) are biologically relevant elements, the use of Zn- and Mn-containing ferrite nanoparticles in biomedical contexts demands careful (geno)toxicity evaluation. In this study, three ferrite nanoparticles – γFe2O3 (FeNPs), Zn0.7Fe2.3O4 (ZnNPs), and Mn0.4Fe2.6O4 (MnNPs) – synthesised through a microwave-assisted polyol route, functionalized with citric acid to improve colloidal stability, were evaluated for their potential (geno)toxic effects in an advanced in vitro 3D cell model, HepG2 spheroids. Cellular stress responses upon exposure to the particle were assessed using toxicogenomic analysis.This approach allows the identification of early molecular events that may precede overt toxicity, supporting a mechanistic understanding of adverse outcomes and facilitating the development of predictive biomarkers for hazard assessment. In the present study, the expression of selected DNA damage-responsive genes (TP53, MDM2, GADD45a, CDKN1A, OGG1, and JUNB), apoptosis-related genes (BCL2 and BAX) and oxidative stress response genes (SOD1, CAT, GPX1, GCLC, and GSR) was evaluated. The expression of the selected genes after exposure to the tested nanoparticles was analysed by qPCR primer assays (Applied Biosystems, USA) and One 48.48 Dynamic Array IFC for Gene Expression (Fluidigm, USA). After 24 and 96 hours of exposure, the spheroids were collected, and total RNA was isolated using the RNeasy Mini Kit from Qiagen (Qiagen, Germany) according to the manufacturer's instructions. 10 µg/mL etoposide served as athe positive control for the toxicogenomic analysis. RNA concentration and purity were assessed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) by measuring absorbance at 260/280 nm and gele efectrophoresis (Figure 1). Reverse transcription of 1 µg total RNA per sample was performed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, MA, USA) on a BIO-RAD T100 thermal cycler under conditions listed in Table 3. For preamplification, 4 µL of each of the 24 selected TaqMan assays (SM2) were pooled into a primer mix. The reaction mixture was prepared using TATAA PreAmp GrandMasterMix (Tataa Biocenter, Sweden), the primer pool, and nuclease-free water, following manufacturer instructions. Negative controls (NTC for preamplification and NTCq for qPCR) were included. Each reaction contained 8 µL of mix and 2 µL of 5× diluted cDNA, processed in a 96-deep well plate, sealed, vortexed, and centrifuged (1000 g, 1 min). Preamplification was carried out on a BIO-RAD T100 thermal cycler under conditions in Table 4. Gene expression analysis used TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (Table 6). Preamplified samples were diluted 10× with nuclease-free water. Assays were prepared by mixing equal volumes (6 µL) of each assay with Fluidigm Assay Loading Reagent Kit – 10IFCS. The reaction premix combined DNA Sample Loading Reagent and Fast Probe Master Mix (Biotium/Roche) and was added to each diluted cDNA sample. qPCR was performed on 48.48 Dynamic Array™ IFC chips using the Fluidigm BioMark™ HD System under conditions in Table 5. Data were analysed with Fluidigm Gene Expression Analysis Software and quantGenious. Fold changes >1.5 or <0.66 were considered biologically relevant. Statistical significance between NP-exposed cells and solvent controls was assessed using ANOVA and Dunnett’s test in GraphPad Prism v9 (GraphPad Software, CA, USA).
Ključne besede: ferrite-based nanoparticles, HepG2 spheroids, toxicogenomics, changes in gene expression
Objavljeno v DiRROS: 24.03.2026; Ogledov: 209; Prenosov: 205
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3. Cytogenotoxic effects of polycyclic aromatic hydrocarbons complex mixture in human peripheral blood, lung A549 and liver HepG2 cells : translation of a real-scenario exposure to in vitroLuka Kazensky, Marija Jelena Lovrić Štefiček, Vilena Kašuba, Matjaž Novak, Karolina Belingar, Katarina Matković, Marko Gerić, Jasmina Rinkovec, Ivana Jakovljević, Bojana Žegura, 2026, izvirni znanstveni članek Ključne besede: cytotoxicity, genomic instability, in silico, indoor air pollution, public health, environmental toxicology
Objavljeno v DiRROS: 20.03.2026; Ogledov: 244; Prenosov: 185
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4. 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: 289; Prenosov: 94
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5. 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: 544; Prenosov: 222
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6. 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: 355; Prenosov: 390
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7. 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: 375; Prenosov: 348
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8. Načrt za ravnanje z raziskovalnimi podatkiMarta Sendra, Martina Štampar, Bojana Žegura, 2025, druge monografije in druga zaključena dela Povzetek: Načrt za ravnanje z raziskovalnimi podatki za projekt "Prekinitev plastične verige: Razkrivanje poti in celičnih odzivov na okoljsko starano nanoplastiko (NanoBreak)".
Ključne besede: NanoBreak, Danio rerio, inducirane pluripotentne matične celice, človeški jetrni organoid, mikroplastika, nanoplastika, projekti
Objavljeno v DiRROS: 22.12.2025; Ogledov: 1059; Prenosov: 227
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9. Hazard identification and characterization of leachable chemicals from plastic products – a new PARC projectHubert Dirven, Nina Franko, Marija Sollner Dolenc, Tim Ravnjak, Martina Štampar, Bojana Žegura, 2025, pregledni znanstveni članek Povzetek: A recent study has suggested that plastics may contain more than 16,000 chemicals, including additives, processing aids, starting substances, intermediates and Non-Intentionally Added Substances. Plastic chemicals are released throughout the plastic life cycle, from production, use, disposal and recycling. Most of these chemicals have not been studied for potential hazardous properties for humans and in the environment. To refine the risk assessment of these leachable chemicals, additional hazard data are needed. The PlasticLeach project within the EU co-funded Partnership for the Assessment of Risks from Chemicals (PARC) aims to address this data gap by screening several plastic products in daily use. Leachates will be prepared from a number of these plastic items, and these chemical mixtures will be further tested using several test guideline compliant assays and New Approach Methodologies covering both human health and environmental endpoints. The most toxic leachates will be characterized using a non-targeted analysis pipeline to identify chemicals in the leachate. When single chemicals of concern are identified, these will be further tested to determine hazardous properties and identify the respective potency factors to better understand their specific hazard profiles. A tiered approach for hazard testing will be followed. The experimental work will be complemented by in silico toxicological profiling, using publicly available toxicity databases and tools, including Artificial Intelligence tools that cover both human and environmental endpoints. A comprehensive array of endpoints, including cytotoxicity, endocrine disruption, genotoxicity, immunotoxicity, reproductive toxicity and effects related to ecotoxicity will be evaluated. In this paper, we outline the plastic products to be tested and the battery of assays that will be used to identify hazards relevant to both human health and the environment. Data generated from in silico, in vitro, and in vivo approaches will be reported using standardized formats, stored within a centralized repository, and harmonized to adhere to the FAIR data principles (Findable, Accessible, Interoperable, and Reusable). This integrated strategy will not only advance our understanding of the risks associated with plastic-derived chemicals but will also provide critical support for regulatory decision-making and facilitate the development of safer, and more ecofriendly plastic materials in the future.
Ključne besede: plastics, chemicals, leachables, PARC, new approach methodologies, hazard assessment, toxicity, risk assessment
Objavljeno v DiRROS: 19.12.2025; Ogledov: 395; Prenosov: 280
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