In Vitro Toxicology Sciences
Vesna Munic Kos, PhD, senior research fellow
Accumulating chemicals may pose a chronic threat for an organism, when present in high concentrations. As our knowledge about biological systems expands it is necessary to constantly revalidate our conclusions about the safety of such chemicals because of their prolonged presence.
Therefore, the main focus of my research are chemicals (pharmaceuticals and environmental toxins) which accumulate in organisms. During 13 years of my career in the research of pharmaceutical industry large part of my research was dealing with highly accumulating pharmaceuticals, especially of macrolide class.
Predicting the effects of highly accumulating compounds
In this research project we use in vitro techniques to analyze the consequences of compound’s accumulation in cells and their binding to biological membranes. We are looking at how these compounds affect various processes in cells, physiological functions of membranes and extracellular membrane vesicles. In collaboration we use structure-property relationship studies to improve detection and prediction of accumulating chemicals.
Developing cell based methods
Additionally, we are developing cell based methods for various types of screenings of environmental toxicants and pharmaceuticals. We mostly use cell function assays, high content quantitative imaging techniques and gene expression analyses on human and murine primary cells and cell lines.
Previous team members
Sam Ulrich – guest student – University of Tuebingen, Germany
Beril Inci – guest student – Anadolu University, Turkey
Arrabi Easwaranathan – student – KTH Royal Institute of Technology
Inna Sitnik – student – Uppsala University
Diana Sanchez Garcia – student – KTH Royal Institute of Technology
Erica Selin – student – Karolinska Institutet
Issra Dawi – trainee
Patrik Kagebeck – student – Uppsala University
Eric Wassen – student – Stockholm University
Predicting the effects of highly accumulating and lipid binding compounds
The aim of this interdisciplinary project is to improve the detection and prediction of highly accumulating and lipid binding chemicals and to analyze in detail the toxicological consequences of their binding to biological membranes. To achieve this, we use:
- extensive in vitro measurements and comparisons of different toxicological outputs together with quantification of compounds in cells,
- in silico modelling with structure-property relationship studies, and
- physico-chemical methods to assess the molecular properties of accumulating compounds.
Mechanisms of chemical accumulation and adverse effects are studied together with the functionality of biological membranes in the cell and in the extracellular nano- and microvesicles, especially in terms of compounds immunotoxic, neurotoxic and obesogenic effects. Cell-differentiation processes, intercellular communication via extracellular vesicles, as well as protein-lipid interactions within cells is being assessed in prolonged exposure to accumulating compounds. Looking at these processes is relatively new in toxicology, but it is necessary to estimate the risk associated with accumulating and lipid-binding compounds.
The project started in 2015 and it focuses on pharmaceuticals and environmental toxicants (persistent organic pollutants) with accumulating properties, aiming to find common points as well as the differences between subclasses of these compounds.
EU-ToxRisk – An Integrated European ‘Flagship’ Programme Driving Mechanism-based Toxicity Testing and Risk Assessment for the 21st century
For this project we have introduced a quantitative high content imaging assay that we now use for screening of various chemicals on their potential effects on neuron differentiation and degradation. Parameters describing neurite length, number, branching points and straightness, together with cell body area and cell number are quantified using our ImageXpress Micro high content analysis system.
Intracellular exposure: a new parameter for prediction of toxicological outcome after exposure to endocrine disrupting chemicals? – Swedish Research Council Formas 2018-2021
This project is a collaboration with Prof. Per Artursson from Uppsala University (Department of Pharmacy), and Prof. Ian Cotgreave and Dr Daniel Mucs from Swetox (Karolinska Institutet, Unit for Toxicology Sciences). The aim of the project is to determine the intracellular unbound fraction of selected endocrine disrupting chemicals (EDCs), and assess the impact of this understudied kinetic parameter on the assessment of adverse effects of EDCs in various cell based test systems.
The majority of toxicological targets are found inside cells but intracellular exposure is not routinely determined in toxicological outcomes predictions due to technical challenges. These challenges have recently been addressed in the pharmaceutical area by our collaborators with a method for determination of intracellular free concentration (FFi) and showed that FFi of drugs correlates with their binding to pharmacological targets and subsequent cellular response. There have been very few attempts to study the FFi of foreign compounds and reconcile these with toxicological outcomes. Therefore, in various established in vitro test systems we will apply the developed methods to systematic study of the relationships between extracellular concentration of selected EDCs, their achieved intracellular free concentration (FFi), and their observed adverse effect. Using computational methods we aim to build quantitative in silico models to improve the risk assessment of endocrine disruptors (EDCs).
Cell culture – primary cells and cell lines
Isolation of human and rodent cells
High content cell imaging and quantitative image analysis
Isolation and function of extracellular vesicles (exosomes)
Fluorimetry/spectrofotometry/luminometry plate based assays
qRT-PCR – gene expression analysis
- Karolinska Institutet
- Swedish Research Council Formas
- European Union’s Horizon 2020 research and innovation programme
Feel free to contact me for possibilities of collaboration at firstname.lastname@example.org .
Kagebeck P, Nikiforova V, Brunken L, Easwaranathan A, Ruegg J, Cotgreave I, Munic Kos V.
Lysosomotropic cationic amphiphilic drugs inhibit adipocyte differentiation in 3T3-L1K cells via accumulation in cells and phospholipid membranes, and inhibition of autophagy. Eur J Pharmacol. 2018; 829:44-53. https://doi.org/10.1016/j.ejphar.2018.04.004
Sanchez Garcia D, Sjödin M, Hellstrandh M, Norinder U, Nikiforova V, Lindberg J, Wincent E, Bergman Å, Cotgreave I, Munic Kos V. Cellular accumulation and lipid binding of perfluorinated alkylated substances (PFASs) – A comparison with lysosomotropic drugs. Chem Biol Interact. 2018; 281:1-10. https://www.ncbi.nlm.nih.gov/pubmed/29248446
Sanchez Garcia D, Sjödin M, Hellstrandh M, Norinder U, Nikiforova V, Lindberg J, Wincent E, Bergman Å, Cotgreave I, Munic Kos V. Cellular accumulation and lipid binding of perfluorinated alkylated substances (PFASs) – A comparison with lysosomotropic drugs. Chem Biol Interact. 2017 Dec 14; 281:1-10. doi: 10.1016/j.cbi.2017.12.021. [Epub ahead of print]
Koštrun S, Munic Kos V, Matanović Škugor M, Palej Jakopović I, Malnar I, Dragojević S, Ralić J, Alihodžić S. Around the macrolide – Impact of 3D structure of macrocycles on lipophilicity and cellular accumulation, Eur J Med Chem. 2017; 133:351-364
Munić Kos V, Koštrun S, Fajdetić A, Bosnar M, Kelnerić Ž, Stepanić V, Eraković Haber V Structure-property relationship for cellular accumulation of macrolones in human polymorphonuclear leukocytes (PMNs). Eur J Pharm Sci. 2013; 49(2):206-19,
Matijašić M, Munić Kos V, Nujić K, Cužić S, Padovan J, Kragol G, Alihodžić S, Mildner B, Verbanac D, Eraković Haber V. Fluorescently labeled macrolides as a tool for monitoring cellular and tissue distribution of azithromycin. Pharmacol Res. 2012; 66(4):332-42.
Polancec DS, Munic Kos V, Banjanac M, Vrancic M, Cuzic S, Belamaric D, Parnham MJ, Polancec D, Erakovic Haber V. Azithromycin drives in vitro GM-CSF/IL-4-induced differentiation of human blood monocytes toward dendritic-like cells with regulatory properties. J Leukoc Biol. 2012; 91(2):229-43.
Banjanac M, Munić Kos V, Nujić K, Vrančić M, Belamarić D, Crnković S, Hlevnjak M, Eraković Haber V. Anti-inflammatory mechanism of action of azithromycin in LPS-stimulated J774A.1 cells. Pharmacol Res. 2012; 66(4):357-62.
Nujić K, Banjanac M, Munić V, Polančec D, Eraković Haber V. Impairment of lysosomal functions by azithromycin and chloroquine contributes to anti-inflammatory phenotype. Cell Immunol. 2012; 279(1):78-86.
Vrančić M, Banjanac M, Nujić K, Bosnar M, Murati T, Munić V, Stupin Polančec D, Belamarić D, Parnham MJ, Eraković Haber V. Azithromycin distinctively modulates classical activation of human monocytes in vitro. Br J Pharmacol. 2012; 165(5):1348-60.
Munić V, Banjanac M, Koštrun S, Nujić K, Bosnar M, Marjanović N, Ralić J, Matijašić M, Hlevnjak M, Eraković Haber V. Intensity of macrolide anti-inflammatory activity in J774A.1 cells positively correlates with cellular accumulation and phospholipidosis. Pharmacol Res. 2011; 64(3):298-307.
Stepanić V, Koštrun S, Malnar I, Hlevnjak M, Butković K, Ćaleta I, Dukši M, Kragol G, Makaruha-Stegić O, Mikac L, Ralić J, Tatić I, Tavčar B, Valko K, Zulfikari S, Munić V. Modeling cellular pharmacokinetics of 14- and 15-membered macrolides with physicochemical properties. J Med Chem. 2011; 54(3):719-33.
Munić V, Kelnerić Z, Mikac L, Eraković Haber V. Differences in assessment of macrolide interaction with human MDR1 (ABCB1, P-gp) using rhodamine-123 efflux, ATPase activity and cellular accumulation assays. Eur J Pharm Sci. 2010; 41(1):86-95.
Vesna Munic Kos (University of Zagreb) – PhD thesis: Interaction of macrolides and mammalian ABC transporters, 2007
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