Details zur Publikation

Referenztyp Zeitschriften
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Titel (primär) Cellular metabolism in high-throughput in vitro reporter gene assays and implications for the quantitative in vitro-in vivo extrapolation
Autor Fischer, F.C.; Abele, C.; Henneberger, L.; Klüver, N.; König, M.; Mühlenbrink, M.; Schlichting, R.; Escher, B.I.;
Journal / Serie Chemical Research in Toxicology
Erscheinungsjahr 2020
Department OEC; BIOTOX; ZELLTOX;
Band/Volume 33
Heft 7
Sprache englisch;
POF III (gesamt) T42;
Supplements https://pubs.acs.org/doi/suppl/10.1021/acs.chemrestox.0c00037/suppl_file/tx0c00037_si_001.pdf
Abstract High-throughput in vitro reporter gene assays are increasingly applied to assess the potency of chemicals to alter specific cellular signaling pathways. Genetically modified reporter gene cell lines provide stable readouts of the activation of cellular receptors or transcription factors of interest, but such reporter gene assays have been criticized for not capturing cellular metabolism. We characterized the metabolic activity of the widely applied AREc32 (human breast cancer MCF-7), ARE-bla (human liver cancer HepG2), and GR-bla (human embryonic kidney HEK293) reporter gene cells in the absence and in the presence of benzo[a]pyrene (BaP), an AhR ligand known to upregulate cytochrome P450 in vitro and in vivo. We combined fluorescence microscopy with chemical analysis, real-time PCR, and ethoxyresorufin-O-deethylase activity measurements to track temporal changes in BaP and its metabolites in the cells and surrounding medium over time in relation to the expression and activity of metabolic enzymes. Decreasing BaP concentrations and formation of metabolites agreed with the high basal CYP1 activity of ARE-bla and the strong CYP1A1 mRNA induction in AREc32, whereas BaP concentrations were constant in GR-bla, in which neither metabolites nor CYP1 induction was detected. The study emphasizes that differences in sensitivity between reporter gene assays may be caused not only by different reporter constructs but also by a varying biotransformation rate of the evaluated parent chemical. The basal metabolic capacity of reporter gene cells in the absence of chemicals is not a clear indication because we demonstrated that the metabolic activity can be upregulated by AhR ligands during the assay. The combination of methods presented here is suitable to characterize the metabolic activity of cells in vitro and can improve the interpretation of in vitro reporter gene effect data and extrapolation to in vivo human exposure.
ID 23412
dauerhafte UFZ-Verlinkung https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=23412
Fischer, F.C., Abele, C., Henneberger, L., Klüver, N., König, M., Mühlenbrink, M., Schlichting, R., Escher, B.I. (2020):
Cellular metabolism in high-throughput in vitro reporter gene assays and implications for the quantitative in vitro-in vivo extrapolation
Chem. Res. Toxicol. 33 (7), 1770 - 1779