Development, scrutiny, and modulation of transient reporter gene assays of the xenobiotic metabolism pathway in zebrafish hepatocytes

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.” Graphical abstract A transient reporter gene assay in zebrafish cell lines utilizing endogenous regulatory gene elements shows increased in vitro toxicity testing performance. Synthetic and constitutive promotors interfere with signal transduction (“squelching”) and might increase cellular stress (cytotoxicity). The squelching phenomenon might occur on multiple levels (toxicity pathway crosstalk and normalization vector), leading to a complete silencing of the reporter signal.