20.500.12556/RUL-187881 Proučevanje škodljivih učinkov policikličnih aromatskih ogljikovodikov v onesnaženem zraku na in vitro humanih celičnih modelih z uporabo mikroskopskih tehnik Assessment of harmful effects of polycyclic aromatic hydrocarbons in polluted air in human cell models in vitro using microscopy techniques Onesnaženost zraka je eden od vodilnih dejavnikov tveganja za javno zdravje. Med pomembnejša onesnažila spadajo policiklični aromatski ogljikovodiki (PAH), ki pa so kot obsežna družina sorodnih spojin relativno slabo toksikološko opredeljeni. V namen celostne identifikacije njihove škodljivosti smo razvili delovni postopek avtomatizirane visokopretočne fluorescenčne mikroskopije in kvantitativne analize slik. S pristopom smo na celični liniji HepG2 proučevali potencialno genotoksičnost (PARP1, γH2AX, pH3), odziv na poškodbe DNK (Gadd45α, p21, Ki67) in indukcijo oksidativnega stresa (8-OxoG, Hmox1, SRXN1). Metodološki pristop smo potrdili v 2D kulturi z uporabo modelnih kemikalij z znanim mehanizmom delovanja (etopozid, kolhicin, KBrO3) in benzo[a]pirena (BAP) kot referenčnega PAH-a ter potrdili visoko zanesljivost in primerljivost kvantifikacije med komercialno opremo (Agilent BioTek Gen5) in uveljavljenim programom FIJI/ImageJ. Avtomatska kvantifikacija je dosegla visoko korelacijo z ročno analizo brez sistemske pristranskosti, pri čemer je bila pretočnost analize med 2D vzorci in parafinskimi rezinami sferoidov primerljiva. V drugem delu smo z razvitim postopkom ocenili toksikološki profil manj raziskanih PAH-ov – benzo[b]fluorantena (BBF), benzo[ghi]perilena (BGP) in njune binarne mešanice – ter primerjali odzive med standardnim 2D modelom in naprednim 3DD celičnim modelom (zreli sferoidi HepG2, gojeni 21 dni pod dinamičnimi pogoji). Podatke smo analizirali z linearnimi (mešanimi) modeli in multivariatnimi pristopi (PERMANOVA, PCA) v programu RStudio. Rezultati so pokazali, da BBF izkazuje izrazito genotoksičnost primerljivo z BAP, saj povzroča dvoverižne prelome DNK (γH2AX) in aktivira odziv na poškodbe DNK (p21). BGP se je izkazal za manj genotoksičnega, vendar pri višjih koncentracijah primarno sproža oksidativni stres (Hmox1, SRXN1). V 3D celičnem modelu so se celični odzivi na vse testirane ksenobiotike znatno spremenili in okrepili, z izrazitejšim aneugenim delovanjem (pH3), spremembami v proliferaciji (Ki67) in povečanim oksidativnim stresom (8-OxoG, SRXN1). Zaključujemo, da je razviti mikroskopski pristop HTS zanesljiv, objektiven in visokopretočen, kar predstavlja robustno platformo za celostno toksikološko oceno onesnaževal zraka. Testirana PAH-a in njuna mešanica so izrazito biološko aktivni ter škodljivi za humane celične modele, pri čemer delujejo preko različnih prevladujočih mehanizmov (genotoksičnost ali oksidativni stres), kompleksnejši 3DD celični model pa bistveno poveča dovzetnost celic za škodljive učinke. Air pollution is one of the leading risk factors for public health. Polycyclic aromatic hydrocarbons (PAHs) are among the most important pollutants, yet as a large family of related compounds, they remain relatively poorly characterized toxicologically. For the purpose of a comprehensive identification of their harmful effects, we developed a workflow using automated high-throughput fluorescence microscopy and quantitative image analysis. Using this approach, we studied potential genotoxicity (PARP1, γH2AX, pH3), DNA damage response (Gadd45α, p21, Ki67), and induction of oxidative stress (8-OxoG, Hmox1, SRXN1) in the HepG2 cell line. The methodological approach was validated in a 2D culture using model chemicals with known mechanisms of action (etoposide, colchicine, KBrO3) and benzo[a]pyrene (BAP) as a reference PAH, confirming high reliability and comparability of quantification between commercial software (Agilent BioTek Gen5) and the established software FIJI/ImageJ. Automated quantification achieved high correlation with manual analysis without systematic bias, while the throughput of analysis was comparable between 2D samples and paraffin sections of spheroids. In the second part, using the developed workflow, we evaluated the toxicological profile of less studied PAHs – benzo[b]fluoranthene (BBF), benzo[ghi]perylene (BGP), and their binary mixture – and compared responses between the standard 2D model and an advanced 3DD cell model (mature HepG2 spheroids, cultured for 21 days under dynamic conditions). Data were analyzed using linear (mixed) models and multivariate approaches (PERMANOVA, PCA) in RStudio software. The results showed that BBF exhibits pronounced genotoxicity comparable to BAP, as it causes DNA double-strand breaks (γH2AX) and activates the DNA damage response (p21). BGP proved to be less genotoxic, but at higher concentrations primarily triggered oxidative stress (Hmox1, SRXN1). In the 3D cell model, cellular responses to all tested xenobiotics were significantly altered and enhanced, with more pronounced aneugenic action (pH3), changes in proliferation (Ki67), and increased oxidative stress (8-OxoG, SRXN1). We conclude that the developed HTS microscopy approach is reliable, objective, and high-throughput, representing a robust platform for the comprehensive toxicological evaluation of air pollutants. The tested PAHs and their mixture are highly biologically active and harmful to human cell models, acting through different predominant mechanisms (genotoxicity or oxidative stress), while the more complex 3D cell model significantly increases cell susceptibility to harmful effects. HepG2 sferoidi in vitro 3DD celični model ksenobiotiki genotoksičnost oksidativni stres policiklični aromatski ogljikovodiki avtomatska mikroskopija HTS kvantitativna analiza slike multivariatne statistične metode razvoj delovnega postopka HepG2 spheroids in vitro 3DD cell model xenobiotics genotoxicity oxidative stress polycyclic aromatic hydrocarbons automatic HTS microscopy quantitative image analysis multivariate statistics workflow development true false false [B. režonja] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-09-16 07:17:58 2026-09-16 07:18:09 2026-09-25 10:25:49 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2027-09-11 502.3:547.555:614(043.2) 289328 292484355 62203.pdf 62203.pdf 1 ABC8E9B7EDA8518FA46C37A0F59E6083 a09bd169059b52af7b048a827ad67b78532630f86008e0ce914645654a45c593 f90c0937-b18d-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=247705 Biotehniška fakulteta 0 0 0