Leiden University Scholarly Publications

Persistent URL of this record https://hdl.handle.net/1887/3216873

Documents

In Collections

This item can be found in the following collections:

Braak, S.J. ter; Niemeijer, M.C.; Wolters, L.; Le Dévédec, S.E.; Bouwman, R.J.P.; Water, B. van de (2021)

Towards an advanced testing strategy for genotoxicity using image-based 2D and 3D HepG2 DNA damage response fluorescent protein reporters

Article / Letter to editor
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 (GFP) 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...Show moreIn 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 (GFP) 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 DMEM medium) give superior sensitivity when genotoxic compounds are tested that do not require metabolization 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.Show less
DNA Damage, Mutagenicity
Test Method, HepG2 BAC GFP, 3D Model
Drug Metabolism
High Content Imaging
Next Generation Risk Assessment (NGRA)
New Approach Methods (NAMs)
All authors
Braak, S.J. ter; Niemeijer, M.C.; Wolters, L.; Le Dévédec, S.E.; Bouwman, R.J.P.; Water, B. van de
Date
2021-08-27
Journal
Mutagenesis
Volume
37
Issue
2
Pages
130 - 142
DOI
doi:10.1093/mutage/geab031