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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, Johan


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."


Bioassays; Reporter gene assays; Transient transfection; Fish cell lines; Squelching

Published in

Cell Biology and Toxicology
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

UKÄ Subject classification

Environmental Sciences
Cell Biology

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