Potentials and pitfalls of transient in vitro reporter bioassays: interference by vector geometry and cytotoxicity in recombinant zebrafish cell lines

Autor: Johan Lundqvist, Sebastian Lungu-Mitea
Rok vydání: 2020
Předmět:
Zdroj: Archives of Toxicology
ISSN: 1432-0738
0340-5761
DOI: 10.1007/s00204-020-02783-6
Popis: The water framework directive re-evaluation proposes the integration of effect-based tools, increasing the need for alternative methods. Especially within aquatic toxicology, coverage of specific toxicity pathways is scarce, and most applications are based on mammalian or bacterial models, not reflecting realistic exposure scenarios. The use of transient reporter gene assays in cells from organisms of interest could be a quick and inexpensive solution. However, interference with cellular homeostasis may impact the system beyond the function of the manipulated gene and thus lead to non-specific results. We describe how varying vector geometry and different regulatory gene elements on plasmids used for transfection in zebrafish hepatocytes and embryonic fibroblasts may lead up to a tenfold difference in potency. Cells were transiently co-transfected with an Nrf2-responsive Firefly luciferase reporter plasmid and eight different Renilla luciferase normalization plasmids. Transfected cells were exposed to two different regimes (0.1–100 µM and 7.8–250 µM) of the oxidative stress-inducing compounds, sulforaphane, tertbutylhydroquinone, and metazachlor. Nrf2 activity was measured in dual-luciferase assays. In parallel, cytotoxicity was assessed for different endpoints (energy metabolism, protein amount, membrane stability, and cell proliferation) in non-transfected cells and cells co-transfected with constructs of increasing size, to be used for normalization. Transfected cells were more susceptible to cytotoxicity in a vector size-dependent manner. Conclusively, we report that vector geometries (size, backbones, gene-regulatory units), cell line (tissue origin), applied transfection methods, and signal normalization may alter the sensitivity of reporter bioassays in a synergistic manner. Further, we propose that thorough bioassay design is needed to ensure reliability and regulatory acceptance. Electronic supplementary material The online version of this article (10.1007/s00204-020-02783-6) contains supplementary material, which is available to authorized users.
Databáze: OpenAIRE
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