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Bisfenoli (BP) so skupina industrijskih kemikalij, ki se uporabljajo pri proizvodnji plastike in epoksi smol. Iz njih izdelujejo najrazličnejše izdelke kot so obloge za konzerve, stekleničke za dojenčke, igrače, papir za račune in mnoge druge, ki se uporabljajo v našem vsakdanjem življenju in jih posledično zasledimo v okolju. Zaradi kopičenja podatkov o nevarnih lastnostih in škodljivih vplivih najpogostejšega izmed BP-jev, bisfenola A (BPA), predvsem motenj endokrinega sistema, je bila njegova uporaba v Evropski uniji postopoma omejena oziroma celo prepovedana, da bi zaščitili zdravje ljudi in okolje. Prepoved uporabe BPA je spodbudila razvoj domnevno varnejših analogov, za katere pa v literaturi ni veliko toksikoloških podatkov. Tako smo v magistrski nalogi proučevali citotoksično in genotoksično aktivnost BPA in njegovih analogov: bisfenol AF (BPAF), bisfenol AP (BPAP), bisfenol C (BPC), bisfenol FL (BPFL), bisfenol S (BPS) na jetrnem in vitro 3D celičnem modelu. Sferoide smo pripravili s prisilno plavajočo metodo, vpliv na živost celic sferoidov po izpostavitvi BP-jem smo merili s testom MTS. S pomočjo mikroskopije, smo spremljali površino, obliko in kompaktnost sferoidov, s testom komet pa smo preučevali genotoksičnost v časovnih izpostavitvah 24 in 96 ur. Statistično značilen vpliv na živost celic v sferoidu po 24 urah so imeli BPFL > BPAF = BPAP, po 96 urah pa BPFL > BPC = BPAP > BPA = BPAF. Mikroskopske analize so pokazale, da sta imela največji vpliv na rast in spremembo površine ter kompaktnosti sferoidov BPC in BPFL po 96-urni izpostavitvi. S testom komet smo pokazali, da sta bila po 24 urah izpostavitve najbolj genotoksična BPAF in BPS (⡥ 10 μM), sledil je BPAP ( ⡥ 40 μM). Po 96 urah izpostavitve je BPAP povzročil poškodbe DNA pri koncentraciji ⡥ 0,1 μM, pri vseh ostalih BP-jih smo poškodbe DNA zaznali pri višjih koncentracijah. Povzamemo lahko, da so za analoge BPA potrebne dodatne študije mehanizmov delovanja za pripravo ustrezne ocene tveganja, saj je toksična aktivnost za posamezne analoge, kljub podobni kemijski strukturi, različna. Bisphenols (BPs) are a group of industrial chemicals used in the manufacture of plastics and epoxy resin. They are found in various products, including the inner linings of cans, baby bottles, toys, thermal paper, and others used in our daily life. Therefore, BPs are present in the environment. The most widely used BP is bisphenol A (BPA), on which most studies have been conducted showing its adverse effects on human health, especially its endocrine disrupting activity. Therefore, the European Union began to restrict and eventually ban the use of BPA to protect human health and the environment. The ban has led to the development of supposedly safer BPA analogues, for which few toxicological data are available. In the master thesis, we investigated the cytotoxic and genotoxic activity of BPA and its analogues (BPAF, BPAP, BPC, BPFL, BPS) in a liver in vitro 3D cell model. In this study, we prepared spheroids by the forced floating method and measured cell viability after exposure to BPs using the MTS assay. We measured the surface area and observed the shape and compactness of the spheroids with microscopy and studied the genotoxic effects with the comet assay at 24 and 96-hour exposure. After 24-hour exposure to BPs, BPFL > BPAF = BPAP statistically significantly decreased the viability of HepG2 and after 96 hours, we observed a similar effect on cell viability in BPFL > BPC = BPAP > BPA = BPAF. BPC and BPFL had the greatest effect on morphological changes in growth, surface area, compactness, and shape of the spheroids that were evident at 96-hour exposure. Of the six BPs tested by the comet assay, BPAF and BPS (⡥ 10 μM) and BPAP (⡥ 40 μM) exhibited stronger genotoxic potential than BPA, BPFL, and BPC after 24 hours of exposure. After 96 hours of exposure, BPAP was the most genotoxic causing/inducing DNA damage at ⡥1 µM. All others BPs studied statistically significantly increased DNA damage at higher concentrations. To summarize, despite their similar chemical structure, the toxic activity of the analogues is not the same, and therefore further studies on the BPA analogues and their molecular activity are needed to provide/make accurate risk assessment.

Določitev genotoksične aktivnosti kemikalij je pomemben del ocene tveganja za ljudi. Za testiranje genotoksičnosti se najpogosteje uporabljajo in vitro jetrne celične linije, saj imajo ohranjeno določeno stopnjo presnovne aktivnosti ksenobiotikov. Vendar imajo tradicionalni dvodimenzionalni (2D) in vitro celični modeli številne pomanjkljivosti, zato v zadnjih letih raziskovalci razvijajo številne in vitro tridimenzionalne (3D) celične modele, ki zaradi svoje večje kompleksnosti bolje posnemajo in vivo pogoje. Prednosti 3D celičnih modelov pred 2D celičnimi modeli so povrnitev številnih bioloških funkcij, kot so medcelične povezave in povezave celica ¬– zunajcelični matriks, kar vodi do boljše diferenciacije celic. V magistrskem delu smo želeli ovrednotiti občutljivost in specifičnost novorazvitega jetrnega 3D in vitro celičnega modela. V prvem delu smo okarakterizirali in optimizirali pogoje za razvoj in vitro 3D celičnega modela (sferoidi), ki smo ga pripravili iz celične linije humanega hepatocelularnega karcinoma (HepG2 celice). Sferoidi, ki smo jih pripravili s prisilno plavajočo metodo, so bili kompaktni ter enakomerni v obliki, velikosti in rasti. V drugem delu smo sferoide pri starosti 72 ur izpostavili različnim koncentracijam dveh posredno delujočih genotoksičnih spojin, in sicer benzo(a)pirenu (BaP) in 2-amino-1-metil-6-fenilimidazo(4,5-b)piridinu (PhIP) za 24 in 72 ur. Proliferacijo celic, celični cikel ter nastanek dvoverižnih prelomov DNA smo spremljali s pretočno citometrijo. Rezultati kažejo, da spojina BaP vpliva tako na celični cikel kot na proliferacijo celic, medtem ko pri spojini PhIP nismo zaznali razlik. Z analizo prisotnosti dvoverižnih prelomov DNA smo pokazali, da BaP povzroči poškodbe DNA tako po 24-urni kot po 72-urni izpostavitvi že pri koncentraciji 䁥 1 μM. PhIP po 24-urni izpostavitvi povzroči poškodbe DNA pri koncentraciji 200 μM, po 72-urni izpostavitvi pa že pri koncentraciji 䁥 25 μM. Genotoksične učinke BaP in PhIP (z izjemo kratkotrajne izpostavitve sferoidov spojini PhIP) smo zaznali pri nižjih koncentracijah, kot so bili ti zaznani v študijah na in vitro 2D celičnem modelu. Zaključimo lahko, da jetrni in vitro 3D celični model kaže izboljšano občutljivost za zaznavanje citotoksičnega in genotoksičnega delovanja posredno delujočih genotoksičnih spojin. Z našo raziskavo smo tako korak bližje k premostitvi vrzeli med in vivo raziskavami in testi, ki temeljijo na in vitro 2D celičnih modelih. Determination of genotoxic activity of chemicals is an essential part of human risk assessment. For genotoxicity assessment, in vitro hepatic cell lines are most commonly used as they have retained certain level of xenobiotic metabolic activity. However, the traditional two-dimensional (2D) in vitro cell models have numerous limitations. Therefore scientists have in the last few years developed many in vitro three-dimensional (3D) cell models, which due to their higher complexity better imitate in vivo conditions. The advantage of 3D cell models compared to 2D monolayer models is the recovery of several biological functions such as intercellular interactions and cell – extracellular matrix interactions, which lead to better cell differentiation. The objective of this master's thesis was to evaluate sensitivity and specificity of newly developed 3D in vitro cell model. In the first part we optimized the conditions for development of in vitro 3D cell model (spheroids) which was formed from human hepatocellular carcinoma (HepG2) cell line. The spheroids were formed by the forced floating method. They were compact and of uniform shape, size and growth. In the second part, 72 hours old spheroids were exposed to graded concentrations of two indirect acting genotoxic compounds, benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) for 24 and 72 hours. Cell proliferation, cell cycle and induction of DNA double-strand breaks were asessed by flow citometry. The results showed that BaP compound affected both cell cycle and cell proliferation, although no differences were noticed for PhIP. By analysing the presence of DNA double-strand breaks it was shown that BaP caused DNA damage after 24- and 72-hour exposure at concentration 䁥 1 μM. PhIP caused DNA damage at 200 μM after 24-hour exposure and at 䁥 25 μM after 72-hour exposure. Genotoxic effects of BaP and PhIP were (with the exception of short term exposure of spheroids to PhIP) detected at lower concentrations as we reported for in vitro 2D monolayer model. We can conclude that hepatic 3D cell model showed improved sensitivity for detection of cytotoxic and genotoxic activity of indirect acting genotoxic compounds. This research presents a step forward to bridge the gap between in vivo research and tests that are based on 2D monolayer models.