Research article - Peer-reviewed, 2021
Development, scrutiny, and modulation of transient reporter gene assays of the xenobiotic metabolism pathway in zebrafish hepatocytes
Lungu-Mitea, Sebastian; Han, Yuxin; Lundqvist, JohanAbstract
The "toxicology in the twenty-first century" paradigm shift demands the development of alternative in vitro test systems. Especially in the field of ecotoxicology, coverage of aquatic species-specific assays is relatively scarce. Transient reporter gene assays could be a quick, economical, and reliable bridging technology. However, the user should be aware of potential pitfalls that are influenced by reporter vector geometry. Here, we report the development of an AhR-responsive transient reporter-gene assay in the permanent zebrafish hepatocytes cell line (ZFL). Additionally, we disclose how viral, constitutive promoters within reporter-gene assay cassettes induce squelching of the primary signal. To counter this, we designed a novel normalization vector, bearing an endogenous zebrafish-derived genomic promoter (zfEF1aPro), which rescues the squelching-delimited system, thus, giving new insights into the modulation of transient reporter systems under xenobiotic stress. Finally, we uncovered how the ubiquitously used ligand BNF promiscuously activates multiple toxicity pathways of the xenobiotic metabolism and cellular stress response in an orchestral manner, presumably leading to a concentration-related inhibition of the AhR/ARNT/XRE-toxicity pathway and non-monotonous concentration-response curves. We named such a multi-level inhibitory mechanism that might mask effects as "maisonette squelching."Keywords
Bioassays; Reporter gene assays; Transient transfection; Fish cell lines; SquelchingPublished in
Cell Biology and Toxicology2021,
Publisher: SPRINGER
Authors' information
Lungu-Mitea, Sebastian (Lungu Mitea, Sebastian)
Swedish University of Agricultural Sciences, Department of Biomedical Science and Veterinary Public Health
Han, Yuxin
Swedish University of Agricultural Sciences, Department of Biomedical Science and Veterinary Public Health
Swedish University of Agricultural Sciences, Department of Biomedical Science and Veterinary Public Health
UKÄ Subject classification
Environmental Sciences
Cell Biology
Publication Identifiers
DOI: https://doi.org/10.1007/s10565-021-09659-0
URI (permanent link to this page)
https://res.slu.se/id/publ/114171