Abstract
The evaluation of genotoxicity plays an important role within hazard identification and risk assessment of chemicals and consumer products. For genotoxicity assessment, in vitro hepatic cells are often used as they have retained certain level of xenobiotic metabolic activity. However, current protocols are designed for the use on 2D monolayer models that are associated with several limitations due to the lack of numerous biological functions, which results in the loss of many hepatic properties. In this respect, an attractive alternative are three-dimensional (3D) models. The aim of our study was to develop physiologically more relevant 3D cell model (spheroids) from the human hepatocellular carcinoma (HepG2) cell line for genotoxicity testing. The spheroids were prepared by the forced floating method, which had been optimized for the production of a large number of uniform spheroids. The sensitivity of the spheroids to detect genotoxicity was determined by the comet assay after the exposure of spheroids to non-cytotoxic concentrations of model indirect acting genotoxic compounds, namely polycyclic aromatic hydrocarbon (B(a)P), mycotoxin (AFB1), two heterocyclic aromatic amines (PhIP and IQ) and a direct acting etoposide (ET). All five tested compounds concentration dependently induced DNA damage. Higher sensitivity of 3D cell model compared to 2D monolayer culture was noticed particularly for detection of the genotoxicity of the heterocyclic aromatic amines and BaP. Deregulation of mRNA expression (qPCR) by genotoxic compounds revealed that HepG2 cells in 3D express important genes encoding phase I and II metabolic enzymes, as well as DNA damage responsive genes in an inducible form. The newly developed HepG2 3D model shows improved sensitivity for detecting genotoxic compounds compared to 2D cultures and can provide a suitable experimental model for genotoxicity assessment.
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Acknowledgements
The authors acknowledge the financial support from the Slovenian Research Agency [research core funding P1-0245, J1-6730 and MR-MStampar], and COST Action CA16119 (In vitro 3-D total cell guidance and fitness) and CA15132 (The comet assay as a human biomonitoring tool).
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The Figure showing the results of protein expression. The Table showing the results of planimetry of formed spheroids after 24, 48, 72 and 96 h. (DOCX 160 kb)
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Štampar, M., Tomc, J., Filipič, M. et al. Development of in vitro 3D cell model from hepatocellular carcinoma (HepG2) cell line and its application for genotoxicity testing. Arch Toxicol 93, 3321–3333 (2019). https://doi.org/10.1007/s00204-019-02576-6
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DOI: https://doi.org/10.1007/s00204-019-02576-6