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

Lungu-Mitea, Sebastian and Lundqvist, Johan (2020). Potentials and pitfalls of transient in vitro reporter bioassays: interference by vector geometry and cytotoxicity in recombinant zebrafish cell lines. Archives of Toxicology. 94 , 2769-2784
[Journal article]

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Abstract

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 mu M and 7.8-250 mu M) 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.

Authors/Creators:Lungu-Mitea, Sebastian and Lundqvist, Johan
Title:Potentials and pitfalls of transient in vitro reporter bioassays: interference by vector geometry and cytotoxicity in recombinant zebrafish cell lines
Year of publishing :2020
Volume:94
Page range:2769-2784
Number of Pages:16
Publisher:Springer
ISSN:0340-5761
Language:English
Publication Type:Journal article
Article category:Scientific peer reviewed
Version:Published version
Copyright:Creative Commons: Attribution 4.0
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Cell Biology
Keywords:In vitro bioassays, Effect-based tools, Transient transfection, Cytotoxicity, Non-specific effects, Oxidative stress
URN:NBN:urn:nbn:se:slu:epsilon-p-105996
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-105996
Additional ID:
Type of IDID
DOI10.1007/s00204-020-02783-6
Web of Science (WoS)000534994500001
ID Code:17386
Faculty:VH - Faculty of Veterinary Medicine and Animal Science
Department:(VH) > Department of Biomedical Science and Veterinary Public Health
Deposited By: SLUpub Connector
Deposited On:02 Sep 2020 09:56
Metadata Last Modified:02 Sep 2020 09:56

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