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

Sebastian Lungu-Mitea,Johan Lundqvist

doi:10.1007/s00204-020-02783-6

Sebastian Lungu-Mitea, Johan Lundqvist

Open Access

https://doi.org/10.1007/s00204-020-02783-6

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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 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.

Highlights

Legislation on the manufacturing of chemicals to protect human health and the environment from adverse effects, such as the REACH (Registration, Evaluation, Authorization, and Restriction of Chemicals) directive or TSCA (ToxicElectronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Substances Control Act) reauthorization, have increased the demand in animal toxicity testing (Goldberg 2010; Hartung 2011)
As a step towards this direction, we previously developed a transient reporter gene assay for analysis of oxidative stress in D. rerio fibroblast (ZF4) and liver (ZFL) cell lines (Lungu-Mitea et al 2018), by measuring induction of the nuclear factor erythroid 2-related factor 2 (Nrf2), a major upstream regulator of ROS detoxification and metabolization (Itoh et al 2004)
We monitored various endpoints of cytotoxicity to investigate how luciferase induction and reporter activity are impacted by the plasmid geometry, and how cytotoxicity further affects the outcome

Summary

Introduction

Development of in vitro assays using fish cells has been proposed as a promising strategy to reduce and replace the use of fish in aquatic toxicity testing (Ankley et al 2010; Halder et al 2014; Worth et al 2014). Such assays would allow highthroughput screening, application of omics technologies, and modeling approaches to risk assessment, such as conducted in ToxCast using mammalian cells (Judson et al 2014). Some fish cell cultures and fish embryo tests have emerged as useful alternative approaches in environmental toxicology (Garcia et al 2016)

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