Jetrni 3D celični modeli za zaznavanje genotoksičnega delovanja kemikalij

Žabkar, Sonja (author), Žegura, Bojana (mentor)

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.

• Language: Slovenian
• Keywords: in vitro 3D celični modeli, sferoidi, HepG2 celice, prisilna plavajoča metoda, genotoksičnost, citotoksičnost, benzo(a)piren, 2-amino-1-metil-6-fenilimidazo(4, 5-b)piridin, pretočna citometrija, celični cikel, proliferacija, dvoverižni prelomi DNA
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2020
• PID: 20.500.12556/RUL-124063
• COBISS.SI-ID: 44408067
• Publication date in RUL: 24.12.2020

Secondary language

• Language: English
• Title: Liver 3D cell models for detection of genotoxic activity of chemicals

Abstract

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.

• Keywords: in vitro 3D cell models, spheroids, HepG2 cell line, force floating method, genotoxicity, cytotoxicity, benzo(a)pyrene, 2-amino-1-methyl-6-phenylimidazo(4, 5-b)pyridine, flow citometry, cell cycle, cell proliferation, DNA double-strand breaks