Xenobiotics represent a significant health risk due to their ability to induce DNA damage, which can lead to cancer, chronic disease and other pathologies. Conventional 2D cell models as well as in vivo animal testing have several limitations in mimicking the human metabolism. Thus in vitro 3D models represent an innovative alternative for genotoxicity assessment. In this master’s thesis we optimised and validated a dynamic in vitro 3D spheroid model constructed from the HepG2 cell line. This 3D model was later applied to assess the genotoxic effects of polycyclic aromatic hydrocarbons (PAH). In the first experimental part, we monitored the growth, differentiation and metabolic activity during the spheroids’ cultivation. The results demonstrated overall stability of the spheroids during their cultivation, gradual maturation, and peak metabolic activity between days 17 and 22 of the spheroids’ age, confirmed by the expression of hepatic markers (ALB, CK18, HNF4α) and induction of metabolic enzymes (CYP1A1, CYP3A4, NAT2, UGT1A1). Based on these results, 21-day-old spheroids were selected as the optimal model. In the second experimental part, the spheroids were exposed to PAHs: benzo(g)perylene (BGP), benzo(b)fluoranthene (BBF), their mixture, and benzo(a)pyrene (BaP) for 24 and 96 hours. A flow cytometry analysis revealed that BGP increased the P21- and γH2AX-positive cells, whereas BBF and BaP additionally elevated the percentage of GADD45α-positive cells. The mixture did not display synergistic effects. Higher concentrations produced stronger responses, while long-term exposure indicated either activation of repair mechanisms or accumulation of DNA damage, depending on the species of PAH. Based on biomarker expression, the genotoxic potential of PAHs was ranked as following: BGP < BBF < BaP.
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