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

Cell Biology and Toxicology · 3 authors, 1 centre

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Researchers developed a transient reporter gene assay in zebrafish liver cells to study the xenobiotic metabolism pathway and found that viral, constitutive promoters can cause signal silencing, a phenomenon they term 'squelching'.

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This preclinical study developed and characterized a transient reporter gene assay in the zebrafish liver cell line (ZFL) to monitor the AhR xenobiotic metabolism pathway. The researchers identified that synthetic, constitutive promoters can interfere with reporter signal transduction through a 'squelching' phenomenon, leading to artificial signal inhibition. The work also characterized the response of zebrafish cells to the ligand beta-naphthoflavone (BNF), finding it promiscuously activates multiple pathways and can lead to multi-level inhibition termed 'maisonette squelching'. A key limitation is the low responsiveness of the zebrafish system to AhR-mediated effects, which may limit its utility for predicting low-dose effects.

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preclinical research

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ORIGINAL PAPER

PMC10406726

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

Cell Biology and Toxicology

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ABSTRACT

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. Supplementary Information The online version contains supplementary material available at 10.1007/s10565-021-09659-0.

CITATION EXPORT

Sebastian Lungu-Mitea, Yuxin Han, Johan Lundqvist. Development, scrutiny, and modulation of transient reporter gene assays of the xenobiotic metabolism pathway in zebrafish hepatocytes. Cell Biology and Toxicology. (2021). https://doi.org/10.1007/s10565-021-09659-0

AUTHORS

3 · 1 CENTRES

Sebastian Lungu-Mitea, Yuxin Han, Johan Lundqvist

grid.6341.00000 0000 8578 2742Department of Biomedicine and Veterinary Public Health, Swedish University of Agricultural Sciences, Box 7028, 750 07 Uppsala, Sweden