Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants
Archives of Toxicology
https://doi.org/10.1007/s00204-019-02473-y
IN VITRO SYSTEMS
Development of a neurotoxicity assay that is tuned to detect
mitochondrial toxicants
Johannes Delp1,2 · Melina Funke1 · Franziska Rudolf1 · Andrea Cediel3 · Susanne Hougaard Bennekou4 ·
Wanda van der Stel5 · Giada Carta6 · Paul Jennings6 · Cosimo Toma7 · Iain Gardner8 · Bob van de Water5 ·
Anna Forsby3,9 · Marcel Leist1
Received: 5 March 2019 / Accepted: 7 May 2019
© The Author(s) 2019
Abstract
Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito- and
neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into
mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based
on culturing the cells in either glucose or galactose (Glc–Gal–NeuriTox) as the main carbohydrate source during toxicity
testing. Using this Glc–Gal–NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel
of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and
2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more
dependent on mitochondrial function, which made them 2–3 orders of magnitude more sensitive to various mitotoxicants.
Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma
membrane lysis) of the toxicants. The Glc–Gal–NeuriTox assay worked particularly well for inhibitors of cI and cIII, while
the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose ↔ galactose exchange.
As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/
complexes in LUHMES cells. The combination of the Glc–Gal–NeuriTox neurotoxicity screening assay with the mechanistic
follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of
the mode of action of mitochondrial toxicants.
Keywords Neurotoxicity · Mitotoxicity · Metabolic reprogramming · High-throughput toxicity screening · High content
imaging · Mechanistic safety assessment
Abbreviations
ADP Adenosine triphosphate
AOP Adverse outcome pathway
Asp
l-Aspartate
ATP Adenosine diphosphate
cAMP N6,2′-O-Dibutyryladenosine 3′,5′-cyclic
monophosphate
cI–V MRC complex I–V
CNS Central nervous system
Cyt c Cytochrome c
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00204-019-02473-y) contains
supplementary material, which is available to authorized users.
* Marcel Leist
marcel.leist@uni‑konstanz.de
Extended author information available on the last page of the article
FAD(H2) Flavin adenine dinucleotide (FAD: oxidized, FADH2: reduced)
FCCP Carbonyl cyanide-4-(trifluoromethoxy)
phenylhydrazone
G6P Glucose-6-phosphate
Gal1P Galactose-1-phosphate
GALK Galactokinase
GALT Galactose-1-phosphate uridylyltransferase
GDNF Glial derived neurotrophic factor
Glc1P Glucose-1-phosphate
Hex Hexose, in this study either glucose or
galactose
HK Hexokinase
Lac Lactate
Mal Malate
MoA Mode of action
MPP 1-Methyl-4-phenylpyridinium
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Archives of Toxicology
MRC Mitochondrial respiratory chain
NA Neurite area
NAD(H) Nicotinamide adenine dinucleotide (NAD:
oxidized, NADH: reduced)
NPA 3-Nitropropionic acid
O2 Oxygen
OA Oxaloacetate
OCR Oxygen consumption rate
PC Pyruvate carboxylase
PGM Phosphoglucomutase
Pyr Pyruvate
Q Ubiquinone or coenzyme Q
ROS Reactive oxygen species
Ser
l-Serine
SSA 5′-Sulfosalicylic acid
TCA Citric acid cycle or Krebs cycle or tricarbonic acid cycle
UDP Uridine diphosphate
UDP-GALE UDP-glucose 4-epimerase
V Viability
Introduction
Specific identification of neurotoxicants still remains
a problem to be solved, and assay conditions need to
be optimized to increase the sensitivity of the available
in vitro tests (Schmidt et al. 2017; van Thriel et al. 2017).
The metabolic situation of a given cell type is one of the
parameters that may be tuned as it is known to be one
of the key determinants affecting the type and extent of
toxicity triggered by a chemical (Delp et al. 2018a; Latta
et al. 2000; Leist et al. 1997b, 1999; Volbracht et al. 1999).
Replacement of glucose for galactose in cell culture media
was reported to tune cellular metabolism from a primary
glycolytic to a predominantly mitochondrial phenotype
without impairing ATP production (Reitzer et al. 1979;
Robinson et al. 1992).
Mitochondria are key organelles of eukaryotic cells, best
known for their central role in energy homeostasis (Zhang
and Avalos 2017). However, their failure also affects other
cell functions, such as calcium signaling (Huang et al.
2017; Leist and Nicotera 1998), (phospho)lipid metabolism
(Wajner and Amaral 2015), neurotransmitter turnover (Leist
et al. 1998; Nicotera et al. 1999), amino acid metabolism
including urea generation (Porporato et al. 2016) and steroid
metabolism (Martin et al. 2016). Mitochondrial dysfunctions
may therefore have largely different manifestations in different tissues and metabolic situations. Moreover, such defects
may escape detection in commonly used toxicological models due to the compensatory capacities that are often seen in
animal models (Blomme and Will 2016). Also, most commonly used cell cultures lack sensitivity, as culture media
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contain frequently supra-physiological amounts of glucose,
and thereby facilitate aerobic glycolysis instead of mitochondrial activity for energy production (Jones and Bianchi 2015;
Lunt and Vander Heiden 2011; Reitzer et al. 1979).
Considering the difficulties of detecting mitochondrial
toxicity (mitotoxicity) in rodents, it is not surprising that
many drugs that had to be withdrawn from the market (e.g.,
troglitazone and cerivastatin), had later been linked to mitotoxicity (Blomme and Will 2016; Tirmenstein et al. 2002;
Westwood et al. 2005). Indeed, a sizable fraction of compounds causing, e.g., drug-induced liver injury are mitotoxicants (Aleo et al. 2014; Begriche et al. 2011; Pessayre et al.
2012; Rana et al. 2018; Tilmant et al. 2018).
The need to obtain data on mitochondrial toxicity has
been realized in several large European research projects
(Desprez et al. 2018; Dragovic et al. 2016; Jennings et al.
2014; Kinsner-Ovaskainen et al. 2013; Kohonen et al.
2017; Wolters et al. 2018) as well as by the Tox21 program
(Attene-Ramos et al. 2015; Xia et al. 2018).
During the past 2 decades, technologies have become
available that allow the investigation of cellular oxygen consumption for large numbers of samples. Examples are the
Agilent Seahorse devices, with sensors fixed to dedicated
plates (Nadanaciva et al. 2012) or various methods using
solu (...truncated)
