Towards an advanced testing strategy for genotoxicity using image-based 2D and 3D HepG2 DNA damage response fluorescent protein reporters
| Autor: | Bob van de Water, Liesanne Wolters, Sylvia E. Le Dévédec, Bas ter Braak, Peter Bouwman, Marije Niemeijer |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Test Method
HepG2 BAC GFP 3D Model DNA damage Health Toxicology and Mutagenesis Next Generation Risk Assessment (NGRA) Toxicology medicine.disease_cause Green fluorescent protein 03 medical and health sciences 0302 clinical medicine Drug Metabolism Mutagenicity New Approach Methods (NAMs) Genetics medicine Humans Viability assay Mode of action Genetics (clinical) 030304 developmental biology 0303 health sciences BTG2 3D Model Mutagenicity Tests Chemistry DNA Damage Mutagenicity In vitro toxicology High Content Imaging Hep G2 Cells HepG2 BAC GFP In vitro Biochemistry Test Method 030220 oncology & carcinogenesis Tumor Suppressor Protein p53 Genotoxicity Mutagens DNA Damage |
| Zdroj: | Mutagenesis, 37(2), 130-142 Mutagenesis |
| Popis: | In vitro assessment of mutagenicity is an essential component in the chemical risk assessment. Given the diverse modes of action by which chemicals can induce DNA damage, it is essential that these in vitro assays are carefully evaluated for their possibilities and limitations. In this study, we used a fluorescent protein HepG2 reporter test system in combination with high content imaging. To measure induction of the DNA damage response (DDR), we used three different green fluorescent protein reporters for p53 pathway activation. These allowed for accurate quantification of p53, p21 and BTG2 (BTG anti-proliferation factor 2) protein expression and cell viability parameters at a single cell or spheroid resolution. The reporter lines were cultured as 2D monolayers and as 3D spheroids. Furthermore, liver maturity and cytochrome P450 enzyme expression were increased by culturing in an amino acid-rich (AAGLY) medium. We found that culture conditions that support a sustained proliferative state (2D culturing with normal DMEM medium) give superior sensitivity when genotoxic compounds are tested that do not require metabolisation and of which the mutagenic mode of action is dependent on replication. For compounds, which are metabolically converted to mutagenic metabolites, more differentiated HepG2 DDR reporters (e.g. 3D cultures) showed a higher sensitivity. This study stratifies how different culture methods of HepG2 DDR reporter cells can influence the sensitivity towards diverse genotoxicants and how this provides opportunities for a tiered genotoxicity testing strategy. |
| Databáze: | OpenAIRE |
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