Toxicology Letters 270 (2017) 1216

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Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet

Comparison of the acute inhibitory effects of Tetrodotoxin (TTX) in rat

and human neuronal networks for risk assessment purposes
Emma E.J. Kasteel, Remco H.S. Westerink*
Neurotoxicology Research Group, Division Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O.
Box 80.177, NL-3508 TD, Utrecht, The Netherlands

H I G H L I G H T S

 TTX inhibits neuronal electrical activity in rat cortical cultures (IC50 7 nM).
 TTX is equipotent in human iPSC-derived neurons (IC50 10 nM).
 Our and literature data indicate that interspecies differences for TTX are limited.
 Experimental animal data could be used to derive human acute reference dose (ARfD).
 The ARfD amounts to 1.33 mg/kg bw, or 200 mg/kg TTX in shellsh.

A R T I C L E I N F O A B S T R A C T

Article history:
Received 4 January 2017 Tetrodotoxin (TTX) is an extremely toxic marine neurotoxin. TTX inhibits voltage-gated sodium channels,
Received in revised form 7 February 2017 resulting in a potentially lethal inhibition of neurotransmission. Despite numerous intoxications in Asia
Accepted 8 February 2017 and Europe, limited (human) toxicological data are available for TTX. Additionally, the degree of
Available online 10 February 2017 interspecies differences for TTX is not well established, hampering the use of available (animal) data for
human risk assessment and establishing regulatory limits for TTX concentrations in (shell)sh.
Keywords: We therefore used micro-electrode array (MEA) recordings as an integrated measure of
Acute reference dose (ARfD) neurotransmission to demonstrate that TTX inhibits neuronal electrical activity in both primary rat
Human risk assessment
cortical cultures and human-induced pluripotent stem cell (hIPSC)-derived iCell1 neurons in co-culture
Interspecies extrapolation
with hIPSC-derived iCell1 astrocytes, with IC50 values of 7 and 10 nM, respectively.
Marine neurotoxin
Tetrodotoxin (TTX) From these data combined with LD50 values and IC50 concentrations of voltage-gated sodium channels
derived from literature it can be concluded that interspecies differences are limited for TTX.
Consequently, we used experimental animal data to derive a human acute reference dose of 1.33 mg/
kg body weight, which corresponds to maximum concentration of TTX in shellsh of 200 mg/kg.
2017 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).

1. Introduction sh (Tetraodontidae family) is the best known source of TTX (Bane

et al., 2014; Noguchi and Arakawa, 2008). TTX is not produced by
Tetrodotoxin (TTX) is a potent neurotoxin that is naturally puffer sh itself, but most likely originates from a symbiosis of
present in certain marine and terrestrial species. The fugu or puffer bacteria with marine animals (Lago et al., 2015). TTX is a voltage-
gated sodium channel blocker, which binds to the sodium channel
and thereby prevents the ux of sodium ions through the channel.
Abbreviations: ARfD, acute reference dose; DIV, days in vitro; EFSA, European Consequently, depolarization of the nerve cell membrane and
Food Safety Authority; FBS, fetal bovine serum; hIPSC, human-induced pluripotent generation of action potential is prevented, resulting in inhibition
stem cell; IC50, half maximal inhibitory concentration; LD50, median lethal dose; of neurotransmission in the central and peripheral nervous system
LOEAL, lowest observed adverse effect level; (mw)MEA, (multi-well)micro-
electrode array; MLD, minimum lethal dose; MSR, mean spike rate; NBA,
(Bane et al., 2014). In total, 26 naturally occurring analogues of TTX
Neurobasal1-A; NOAEL, no observed adverse effects levels; PEI, polyethyleneimine; are known, which may differ in potency but have comparable
Pen/Strep, penicillin/streptomycin; PND, post-natal day; TTX, tetrodotoxin. toxicological properties (Bane et al., 2014, 2016; Miyazawa and
* Corresponding author. Noguchi, 2001). Intoxicated patients suffer from paresthesia of the
E-mail address: r.westerink@uu.nl (R.H.S. Westerink).

http://dx.doi.org/10.1016/j.toxlet.2017.02.014
0378-4274/ 2017 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
 E.E.J. Kasteel, R.H.S. Westerink / Toxicology Letters 270 (2017) 1216 13

tongue and lips, headache, vomiting, ataxia and in severe cases 2.2.1. Rat primary cortical cells
respiratory- and heart failure (Bane et al., 2014; Lago et al., 2015). Rat primary cortical cells were isolated from the neonatal
While the presence of TTX used to be conned to Asia and cortex from post-natal day (PND) 1 Wistar rat pups as described
especially Japan, TTX has also been found in the last decade in previously (Dingemans et al., 2016; de Groot et al., 2016;
marine organisms, including shellsh, in Europe (Turner et al., Hondebrink et al., 2016). Briey, rat pups were decapitated and
2015; Rodriguez et al., 2008; Vlamis et al., 2015). As such, the cortices were rapidly dissected on ice. Tissues were kept in
appearance of TTX in European shellsh is an emerging problem dissection medium containing NBA medium, supplemented with
and uniform regulatory limits for TTX have not yet been set as TTX 25 g/L sucrose, 450 mM L-glutamine, 30 mM glutamate, 1% Pen/
is currently under review by the European Food Safety Authority Strep and 10% FBS, pH was set to 7.4. Cells were seeded in dissection
(EFSA). medium on poly-L-lysine (50 mg/mL) coated culture materials.
Unfortunately, it is hard to establish regulatory limits for TTX After 1 day in culture (DIV1), 90% of the dissection medium was
since human data is based on case studies. Human no observed replaced with comparable medium, but with 2% B-27 supplement
adverse effects levels (NOAELs) or lethal human levels are instead of FBS. At DIV4, 90% of the medium was replaced with NBA
therefore not reliable. There are reports of LD50 values for mice medium, supplemented with 15 g/L sucrose, 450 mM <***>l-
and cats, but the extrapolation to the human situation is difcult glutamine, 1% Pen/Strep and 10% FBS, pH was set to 7.4
due to limited knowledge regarding interspecies differences for (glutamate-free medium). For MEA experiments, a 50 mL drop of
TTX. It is therefore of great importance to compare animal and cell suspension (1*105 cells/well) was placed on the electrode eld
human models to determine if interspecies difference in sensitivity in each well of the 48-wells of a multi-well MEA plate. All animal
to TTX exist. experiments were performed in accordance with the Dutch law
Since TTX inhibits neurotransmission by blocking voltage-gated and were approved by the Ethical Committee for Animal
sodium channels, measurements of neuronal network activity in Experimentation of Utrecht University (project number
vitro provide a suitable integrated endpoint to assess effects of TTX AVD108002015443). All efforts were made to treat the animals
on neuronal signaling (Nicolas et al., 2014). Using micro-electrode humanely and for alleviation of suffering.
array (MEA) recordings, changes in (spontaneous) neuronal
electrical activity can be measured in vitro (Johnstone et al., 2.2.2. iCell1 neurons/astrocytes
2010). Analogues to the in vivo situation, neuronal networks grown iCell1 neurons (NRC-100-010-001, Cellular Dynamics Interna-
on MEAs develop spontaneous activity (Robinette et al., 2011; tional (CDI), Madison, WI, USA) and iCell1 astrocytes (ASC-100-
Dingemans et al., 2016; de Groot et al., 2016) and are responsive to 020-001-PT, CDI, Madison, WI, USA) were seeded as an iCell1
different neurotransmitters and chemicals (Hondebrink et al., neurons/iCell1 astrocytes co-culture according to CDI protocol
2016). While rat primary cortical cultures are currently the golden with slight modications. Briey, iCell1 neurons and iCell1
standard for MEA recordings, the use of human induced pluripo- astrocytes were seeded as a 10 mL droplet of cell suspension at a
tent stem cell (hIPSC)-derived neuronal models is increasing density of 1.4*105 cells/mL, with 7.0*104 iCell1 neurons and 7.0*104
(Tukker et al., 2016), thereby potentially eliminating the need for astrocytes in Complete iCell1 Neurons Maintenance Medium,
interspecies extrapolation. The aim of the present study is supplemented with 2% iCell1 Neurons Medium Supplement and
therefore to compare the sensitivity of rat primary cortical cultures 10 mg/mL laminin. The cell suspension was placed directly over the
and co-cultures of hIPSC-derived iCell1 neurons and hIPSC- electrode eld of each polyethyleneimine (PEI)-coated well of a 48-
derived iCell1 astrocytes to the inhibitory effects of TTX on well MEA plate (Axion Biosystems Inc., Atlanta, USA) that has been
neuronal electrical activity using MEA recordings, and to calculate pre-dotted with 10 mL of iCell1 Neurons Maintenance medium
an acute reference dose (ARfD) for TTX taking into account this with 80 mg/mL laminin. The cell suspension droplet was allowed to
interspecies extrapolation. attach to the electrode eld for 35 min, after which 300 mL of
Complete iCell1 Maintenance Medium supplemented with lami-
nin was added to each well. Every 23 days, 50% of the medium was
2. Materials and methods refreshed.

2.1. Chemicals 2.3. Multi-well microelectrode array recordings

Tetrodotoxin citrate (TTX, purity >98%) was obtained from Multi-well microelectrode array (mwMEA) plates contained 48
Abcam (Cambridge, United Kingdom). Neurobasal1-A (NBA) wells per plate, with each well containing an electrode array of 16
Medium, L-glutamine, fetal bovine serum (FBS), B-27 supplement, individual embedded nanotextured gold microelectrodes with four
KnockOut Serum Replacement, 50/50 DMEM/F12 medium and integrated ground electrodes, yielding a total of 768 channels
penicillin-streptomycin (Pen/Strep) (10,000 U/mL10,000 mg/mL) (Axion Biosystems Inc.). Spontaneous electrical activity was
were purchased from Life Technologies (Bleiswijk, The recorded as described previously (Dingemans et al., 2016; de
Netherlands). iCell1 Neurons Maintenance Medium (NRM-100- Groot et al., 2016; Hondebrink et al., 2016; Tukker et al., 2016).
121-001) and iCell1 Neurons Medium Supplement (NRM-100- Briey, experiments were performed at DIV9 (rat cortical cultures)
031-001) were purchased from Cellular Dynamics International or at DIV7 (iCell1 neurons/astrocytes) at 37  C, using a Maestro
(Madison, WI, USA). All other chemicals were obtained from 768-channel amplier with integrated heating system, tempera-
Sigma-Aldrich (Zwijndrecht, The Netherlands). ture controller and data acquisition interface (Axion BioSystems
Stock solutions of TTX (1 mM) were prepared in MilliQ1 water Inc., Atlanta, USA).
and stored at 4  C for a maximum of 3 months. Stock solutions were After a 5 min equilibration period, a 30 min baseline recording
diluted in cell culture medium to obtain the desired concentrations of the spontaneous activity was started. Only wells with at least
just prior to the experiments. one visibly active electrode after equilibration were included. After
the baseline recording, 33 mL TTX (nal concentrations 130 nM,
2.2. Cell culture diluted in NBA medium, supplemented with 10% KnockOut Serum
Replacement and 1% Pen/Strep) was added to the wells (iCell1
All cells were cultured in a humidied 5% CO2 atmosphere at neurons/astrocytes). For rat cortical cultures, 5 mL TTX (diluted in
37  C. FBS medium) was added to the wells. Following addition of TTX
14 E.E.J. Kasteel, R.H.S. Westerink / Toxicology Letters 270 (2017) 1216

(nal concentrations 130 nM) activity was recorded for another

30 min to determine the inhibitory effect of TTX on neuronal
activity.

2.4. Data analysis and statistics

Data were analyzed by NeuroExplorer5 software (NEX Tech-

nologies, Madison, USA) and custom-made MS Excel macros as
described previously (Hondebrink et al., 2016). Raw data les were
re-recorded for spike detection using the AxIS spike detector
(Adaptive threshold crossing, Ada BandFit v2) with a threshold
spike detector set at 7*SD (rat cortical culture) or 6*SD (iCell1
neurons/astrocytes co-culture) of the internal noise level on each
electrode. The re-recorded raw data were loaded into Neuro-
Explorer to determine the average mean spike rate (MSR; spikes/s)
of all active electrodes per active well. Electrodes were considered
active when MSR > 0.1 spikes/s and wells were considered active
when 1 active electrode was present. The development of
spontaneous activity (MSR) over time and the effect of TTX on
spontaneous activity was determined by custom-made MS Excel
macros. The MSR during the exposure recording (MSRexposure) was
expressed as a percentage of the MSR during the baseline recording
(MSRbaseline) to derive a treatment ratio. Treatment ratios of
individual electrodes were averaged per well and subsequently per
condition. Outliers (effects 2*SD above or below average) in control
and effect data were excluded for further analysis (<5%). The
treatment ratios in control experiments were set to 100% and
effects of TTX were expressed as percentage of control. For all data
analysis, GraphPad Prism v6.05 (GraphPad Software, San Diego,
California) was used. Groups were compared using an ANOVA
followed by a post-hoc Bonferroni test. Effects were considered
statistically signicant when p < 0.05. Fig. 1. Examples of Spike Raster Plots of iCell1 neurons/astrocytes co-cultures
before (A) and after (B) exposure to 10 nM TTX. Each row depicts one electrode;
every mark represents one spike. Concentration-effect curves (C) of TTX on
3. Results neuronal activity in primary rat cortical cultures (black dotted line, nwells = 2229,
Nplates = 4) and iCell1 neurons/astrocytes co-cultures (grey solid line, nwells = 724,
Effects of acute TTX exposure were investigated in rat cortical Nplates = 13). Results are expressed as treatment ratio  SEM (change in% between
cultures and iCell1 neurons/astrocytes co-cultures. Following a MSRbaseline and MSRexposure). Data were synchronized to the time of exposure to
eliminate the application artifact (Hondebrink et al., 2016). Effects of TTX exposure
baseline recording (Fig. 1A), cultures were exposed for 30 min to are signicantly different from control at 3 nM.
different concentrations of TTX (Fig. 1B). Each well was exposed to
only one concentration to prevent potential effects of cumulative
dosing.
In rat primary cortical cultures, TTX concentration-dependently
inhibits neuronal activity (Fig. 1C). At 3 nM and 10 nM, TTX factors such as bioactivation/metabolisms or exposure routes such
inhibited neuronal activity signicantly to 75% (p < 0.05) and 25% as the gastrointestinal tract. In vitro data, such as our measure-
(p < 0.0001) from control, respectively. At 30 nM, neuronal activity ments of neuronal electrical activity using MEA recordings, thus
of rat primary cortical cultures was almost completely abolished cannot (yet) fully replace experimental animal studies for risk
and the calculated IC50 for inhibition of neuronal activity by TTX assessment purposes. Nevertheless, these in vitro studies can shed
amounted to 7 nM. light on the degree of interspecies variation, which can subse-
In human iPSC-derived iCell1 neurons/astrocytes co-cultures, quently be used to further shape risk assessments based on
TTX induced a comparable concentration-dependent inhibition of (existing) experimental data.
neuronal activity (Fig. 1C). At 3 nM and 10 nM, TTX inhibited Our results demonstrate that TTX potently and concentration-
neuronal activity signicantly to 78% (p < 0.0001) and 41% dependently inhibits neuronal electrical activity in both models.
(p < 0.0001) from control, respectively. Comparable with rat The IC50s for inhibition of rat cortical cultures obtained in the
primary cortical cultures, 30 nM TTX almost completely abolished present study are in line with those obtained earlier [Nicolas et al.,
neuronal activity and the calculated IC50 for inhibition of neuronal 2014, IC50 = 4 nM], demonstrating the reproducibility of this
activity by TTX amounted to 10 nM. approach. Interestingly, neuronal activity completely ceased at
30 nM TTX, which is close to the estimated lethal blood level of
4. Discussion 9 ng/mL TTX (28 nM) (Bane et al., 2014; Islam et al., 2011; Leung
et al., 2011). More importantly, TTX is roughly equipotent in both
We earlier demonstrated the suitability of rat cortical cultures the rat and human in vitro models, indicating that for inhibition of
grown on mwMEAs to investigate the effects of various marine neuronal activity by TTX, rat-human interspecies differences are
neurotoxins, including TTX, on neuronal activity (Nicolas et al., limited.
2014). Here we investigated the acute inhibitory effects of TTX on Notably, interspecies differences appear to be also limited for
neuronal electrical activity in rat primary cortical cultures and TTX-induced inhibition of voltage-gated sodium channel, which
iCell1 neurons/astrocytes co-cultures. It is important to note that are highly conserved in mammalian species (Goldin et al., 2000;
currently most in vitro models do not sufciently take into account Lopreato et al., 2001; Grosson et al., 1996). For example, TTX is
 E.E.J. Kasteel, R.H.S. Westerink / Toxicology Letters 270 (2017) 1216 15

roughly equipotent in different species in inhibiting voltage-gated hydration status could be more prone to TTX poisoning, due to
NaV1.4 sodium channels from skeletal muscle (rat IC50 = 17 nM delayed excretion of TTX. This is in agreement with the suggestion
(Walker et al., 2012), human IC50 = 25 nM (Chahine et al., 1994)), that people who suffer from diabetic neuropathy, uremia and Na-
voltage-gated NaV1.6 sodium channels from the central nervous K-adenosine-triphosphate deciency can have more severe TTX-
system (rat IC50 = 1 nM (Dietrich et al., 1998), mouse IC50 = 6 nM induced adverse effects than healthy patients (Bane et al., 2014;
(Smith et al., 1998)), and voltage-gated NaV1.7 sodium channels Leung et al., 2011). Consequently, a subsequent factor 10 should be
from dorsal root ganglia in the peripheral nervous system (rat applied to take into account any intraspecies differences due to for
IC50 = 4 nM (Sangameswaran et al., 1997), human IC50 = 1925 nM example poor renal clearance and/or poor hydration status. This
(Walker et al., 2012; Klugbauer et al., 1995)). Similarly, when would result in an ARfD for TTX of 1.33 mg/kg bw. Using this ARfD
comparing minimum lethal dose (MLD) levels in mammals and assuming 400 g of shellsh meat as an appropriate estimate of
following subcutaneous injection, little interspecies differences a large portion size consumed in Europe to be used in its risk
are observed with rats and guinea pigs being most sensitive assessments in order to protect high consumers against acute
(estimated MLD of crystalline TTX of 2.7 mg/kg and 4.5 mg/kg, effects of marine biotoxins (EFSA, 2010), a person of 60 kg can
respectively) and mouse, dogs and cats being least sensitive safely consume shellsh with a maximum concentration of TTX of
(estimated MLD of crystalline TTX of 8 mg/kg, 9 mg/kg and 10 mg/ 200 mg/kg.
kg, respectively) (Kao, 1966). Since at least for TTX rat-human
interspecies differences appear limited, experimental animal (rat)
Conict of interest
data for TTX may play a more prominent role in human risk
assessment of TTX.
The authors state that they have no conict of interest.
Current human risk assessment is hampered by the lack of well-
documented human intoxications. While a grading system for the
severity of TTX poisoning was already provided in 1941 (Fukuda Acknowledgements
and Tani, 1941) and numerous reports on TTX intoxication exist,
only a few measured urine and/or blood concentrations of patients Anke Tukker and Fiona Wijnolts are greatly acknowledged for
and none can provide a reliable estimate of the amount of TTX help with cell culture. This work was supported by a grant from The
ingested. Human lethal dose estimations of 14 mg reported Netherlands Food and Consumer Product Safety Authority (NVWA;
earlier (Zimmer, 2010; Klaassen, 2013) should therefore be treated grant number 60010896).
with caution. It thus seems viable to use experimental animal data
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