TOXICOLOGICAL SCIENCES, 150(2), 2016, 283–291
doi: 10.1093/toxsci/kfv326
Advance Access Publication Date: December 29, 2015
Research Article
Beauvericin Inhibits Neuromuscular Transmission and
Skeletal Muscle Contractility in Mouse Hemidiaphragm
Preparation
z
,† Breda Jakovac Strajn,† and
ek,* Marjana Grandic
Monika Cecilija Zu
*,1
Robert Frangez
eva
*Institute for physiology, pharmacology and toxicology, Veterinary faculty, University of Ljubljana, Gerbic
60, 1000 Ljubljana, Slovenia; and †Institute for hygiene and pathology of animal nutrition, Veterinary faculty,
University of Ljubljana, Cesta v Mestni log 47, 1000 Ljubljana, Slovenia
1
To whom correspondence should be addressed. Fax: þ386 1 2832243. E-mail: [email protected].
ABSTRACT
The effects of Beauvericin (BEA) produced by the fungus Beauveria bassiana and Fusarium sp. on neuromuscular transmission
and contractility were determined in an isolated neuromuscular mouse hemidiaphragm preparation. BEA (5 mM)
significantly inhibits indirectly elicited twitch amplitude. At higher concentrations (7.5 and 10 mM), BEA produces a
significant reduction of directly elicited, or complete block of indirectly evoked, muscle contraction. BEA also appears to be
myotoxic, as indicated by a slowly developing muscle contracture. Development of neuromuscular blockade and
contracture is concentration dependent. BEA acted by presynaptically depressing spontaneous acetylcholine release as
indicated by the reduction in the frequency of spontaneous miniature endplate potentials (MEPPs), while the membrane
potential of muscle fibers remained unchanged. At higher concentrations (7.5 and 10 mM), BEA progressively reduces or
completely blocks MEPPs and EPPs amplitudes. Changes in MEPPs and EPPs are associated with substantial depolarization
of muscle fibers when exposed to 7.5 and 10 mM of BEA. These results indicate that BEA has neurotoxic and myotoxic effects,
which overlap in a narrow range of concentrations.
Key words: beauvericin; neuromuscular transmission; motor endplate; mouse; muscle contractility.
Beauvericin (BEA) and enniatins are a group of structurally
related cyclic hexadepsipeptides consisting of alternately linked
three d-2 hydroxycarboxylic acid and N-methylamino acid residues (Blais et al., 1992; Hamill et al., 1969; Logrieco et al., 1998).
BEA is an emerging mycotoxin that is primarily produced by the
fungus Beauveria bassiana and is one of the most common graincontaminating genuses of the fungi Fusarium spp. (Harvey et al.,
1994; Logrieco et al., 1998). A wide range of biological activities
of BEA have been reported. It has insecticidal, antimicrobial,
antiviral, and cytotoxic activities (Wang and Xu, 2012). Because
of its cytotoxic activity, BEA is a putative candidate for antitumor therapy. The potential cytotoxic effect of BEA has been
demonstrated in the results of in vitro studies using various cell
lines. For example, BEA induces cell death in insect, murine,
and human cell lines (Calo et al., 2003, 2004; Jow et al., 2004).
The mechanism of BEA cytotoxicity is based on extracellular
calcium movement into the cell. On the basis of its chemical
structure, BEA has been considered a potential ionophore
capable of transporting cations such as Kþ, Naþ, and Ca2þ,
across the plasma membrane (Kouri et al., 2003; Prince et al.,
1974). Despite the fact that BEA may activate Ca2þ influx, some
believe that at the same time it induces Ca2þ release from the
endoplasmic reticulum as internal calcium storage (Kim et al.,
2002). Increased intracellular Ca2þ concentration activates an
unknown signal system, which results in the release of Cyt C
molecules from the mitochondria. Caspase that is activated by
Cyt C triggers apoptosis, which results in cell death (Calo et al.,
2003, 2004; Chen et al., 2006).
C The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.
V
All rights reserved. For Permissions, please e-mail: [email protected]
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Fusarium spp. colonize important cereal grains, such as corn,
wheat, and rice (Logrieco et al., 1998, 2002; Plattner and Nelson,
1994) and have an adverse impact on human and animal health.
Despite a limited amount of data regarding the occurrence of BEA
in grains throughout the world, BEA is a common food and feed
contaminant with concentration levels ranging from trace levels
up to 520 mg/kg. The highest concentration of BEA was found in
corn in mid and Northern Europe, while the lowest concentration
of BEA was found in oats, barley, rye, and wheat in Finland (Jestoi,
2008; Jestoi et al., 2004). A recent study revealed the presence of
BEA and other structurally related mycotoxins in various species
of Chinese medicinal herbs, with ginger having the highest BEA
concentration (Hu and Rychlik, 2014). Regarding feed, Streit et al.
(2013) analyzed 83 samples of feed and feed raw materials. BEA
was the most frequently detected mycotoxin and was present in
98% of the samples.
The available data on the toxicokinetics of BEA are limited.
In vitro data indicate that BEA is absorbed and rapidly metabolized
to a range of uncharacterized metabolites. Although oral bioavailability has not been determined in experimental animals, BEA is
found in rat plasma after oral exposure. BEA and enniatins often
act together and were detected in eggs of laying hens, and in turkey and broiler tissues. This finding indicates that residual BEA
and enniatins from poultry may only marginally contribute to the
human exposure (EFSA, 2014).
The combination of the high contamination levels and the
toxic effects of BEA could pose a risk to human and animal
health because the maximum levels of BEA in food and feed
have not yet been established. Additionally, to better define the
role of BEA in the human and animal food chain and to evaluate
its potential use in the field of medicine and pharmacy, the
mechanism of action of BEA on the cellular, tissue, and organ
level should be more precisely determined. On the basis of the
above-described mechanisms of cytotoxicity, we assume that
an increased intracellular Ca2þ level may affect the function of
muscle cells and, consequently, the function of the neuromuscular junction. Therefore, in this work, we studied (1) the effect
of BEA on skeletal muscle contractility and (2) the mechanism(s)
of neuromuscular toxicity. The results of this study suggest, for
the first time, that BEA has a myotoxic as well as a neurotoxic
mode of action.
phrenic nerves was dissected and divided in half to obtain two
hemidiaphragms. Each hemidiaphragm was then tightly pinned
to the Rhodorsil-coated organ bath containing oxygenated standard Krebs-Ringer solution composed of (in mM): 154 NaCl, 2 CaCl2,
5 KCl, 1 MgCl2, 5 HEPES and 11 D-glucose, pH 7.4, at 22–24 C. A silk
thread was attached with a steel hook to the tendinous side of the
hemidiaphragm and to an isometric force displacement transducer FT 03 (Grass instruments, West Warwick, RI) on the other
side. Nerve-evoked single isometric twitches were recorded as follows: the motor nerve of isolated neuromuscular preparation was
stimulated with a square pulse S-48 stimulator (Grass instruments, West Warwick, RI) via a suction electrode with pulses of
0.1 ms duration, 0.1 Hz stimulation rate, and with the supramaximal voltage of 5–10 V. Directly evoked single isometric twitches
were evoked by stimulating the isolated hemidiaphragm preparation with an electric field derived from a platinum electrode
assembly placed along the organ bath and connected to the isolation unit of the S-48 stimulator. Pulses were 0.1 ms in duration,
with a 0.1 Hz stimulation rate and with the supramaximal voltage
of 60–80 V. Directly or nerve-evoked tetanic muscle contraction
recordings were obtained by stimulating the hemidiaphragm with
series of pulses (1000 ms duration at 80 Hz). To achieve maximal
muscle contraction upon nerve-evoked stimulation, each hemidiaphragm preparation was adjusted to the resting tension, typically 1.5–2.5 g. Each hemidiaphragm preparation was then left to
equilibrate for 20 min to achieve stable resting tension before
beginning the experiments.
Muscle twitch tension was measured using a Grass FT03
force transducer. Electrical signals were amplified by a P122
strain gage amplifier (Grass instruments, West Warwick, RI) and
then continuously digitized at a sampling rate of 1 kHz using a
data acquisition system (Digidata 1440 A; Molecular Devices,
Sunnyvale, CA).
The effect of BEA on the neuromuscular hemidiaphragm preparation was measured 60–90 min after the addition of BEA to the
organ bath. Muscle tension was expressed as maximal twitch
response before and after the addition of BEA. Concentration–
response curve of BEA was plotted 60–90 min after drug perfusion.
In one set of experiments, indirect stimulation was established by
abolition of muscle twitches with 10 lM tubocurarine (DTC), a
nondepolarizing antagonist of muscle-type nicotinic acetylcholine
receptors (nAChRs) (Nguyen-Huu et al., 2005).
MATERIALS AND METHODS
Recordings of Membrane Potential, Miniature Endplate Potential, and
Endplate Potential in Mouse Hemidiaphragms. The experiments
were performed at 22 C–24 C on oxygenated mouse hemidiaphragm preparations, pretreated for 30 min with 2 lM l-conotoxin
GIIIB, an inhibitor of muscle Naþ channels, to record full-sized
endplate potentials (EPPs) without contracting the muscle (Cruz
et al., 1985; Hong and Chang, 1989). The resting membrane
potentials, EPPs, and miniature endplate potentials (MEPPs)
were recorded from endplate regions in superficial muscle
fibers using intracellular borosilicate microelectrodes filled with
3 M KCl and pulled with a P-97 Flaming/Brown microelectrode
puller (Sutter Instruments, Novato, CA) to have a resistance
from 10–20 MX. Recordings were performed on each hemidiaphragm preparation before, 30, 60, and 90 min after application of
BEA and 30 min after washing-out the BEA. EPPs were evoked by
stimulating the phrenic nerve via a suction electrode with
supramaximal square pulses of 0.1 ms duration and with a frequency of 1 pulse per second. EPP and MEPP recordings were
digitized using Digidata 1440A and the pClamp 10 software.
Data were analyzed using the pClamp-Clampfit 10 program
(Molecular Devices, Union City, CA). Amplitudes of EPPs and
Materials
Drugs. BEA was purchased from Santa Cruz Biotechnology (USA).
Before use, it was dissolved in ethanol at a stock concentration
of 1.2 mM. Aliquots of 100 ll were stored at 20 C until further
use. Acetylcholine chloride (Sigma Aldrich, USA), D-tubocurarine
(Acros organics, USA), and l-conotoxin GIIIB (Bachem,
Switzerland) were of the highest grade available.
Experimental Animals. Male Balb/C mice, 2–3 months of age, were
obtained from the animal breeding facility in the Veterinary
Faculty, University of Ljubljana. All experiments followed ethical standards and were approved by The Administration of the
Republic of Slovenia for food safety, veterinary, and plant protection (permit no. 34401-12/2012/2).
Methods
Muscle Twitch Recordings from Isolated Mouse Hemidiaphragms.
Adult Balb/C mice were sacrificed by cervical dislocation, followed
by immediate exsanguination. The diaphragm with corresponding
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MEPPs were normalized to a membrane potential of 70 mV
using the formula Vc ¼ V0 x (70)/E, where Vc is the normalized
amplitude of EPPs or MEPPs, V0 is the recorded amplitude, and E
is the resting membrane potential.
Data Analysis and Statistics. Data were statistically analyzed
using SigmaPlot for Windows 11.0 (Systat Software Inc.,
Germany). Parametric data are presented as the mean 6 SEM.
Concentration–response curves were fitted using the four
parameter nonlinear regression model. Data were firstly tested
for normality (Shapiro–Wilk) and equal variance for assignment
to parametric or nonparametric analysis. For nonpaired
comparison between multiple groups, when equal variance
were not met, nonparametric analysis was performed using
Kruskal–Wallis one-way analysis of variance (ANOVA) on ranks
followed by Dunn’s post-hoc test and are presented as box-andwhisker plots using median and the interquartile range (IQR).
P value 0.05 was considered to be statistically significant. Oneway ANOVA followed by Holm–Sidak testing for multiple comparisons was performed to compare the effects of different BEA
concentrations on EPP amplitude.
RESULTS
The Effects of BEA on Isometric Muscle Contraction in Mouse
Hemidiaphragm in vitro
BEA was tested in vitro for its eventual effects on the neuromuscular junction and the skeletal muscle because previously
reported data describe BEA as having cytotoxic activity. The following concentrations of BEA were used: 1, 2.5, 5, 7.5, and 10 lM.
Under our experimental conditions, no change in amplitude of
twitch and tetanic contraction was observed during the 90 min
exposure period of control recordings (data not shown). In control experiments in which neuromuscular preparations were
exposed to the final concentrations of ethanol (used as a BEA
solvent), only a small drop in the twitch amplitude (2–4%) was
observed immediately after ethanol application in nerveevoked muscle twitches (Fig. 1A). The concentration of ethanol
was kept constant in all experiments. BEA blocked nerve evoked
as well as directly evoked single twitch responses and tetanic
contractions in a concentration-dependent manner (Fig. 2D). At
low concentrations of BEA (1 and 2.5 lM), the amplitude of
muscle twitch and tetanic contractions in mouse hemidiaphragm preparations evoked by direct and indirect stimulation
remained unchanged, while concentrations of 5 lM or higher
reduced, in a time-dependent manner, nerve-evoked (Fig. 2B1)
and directly elicited muscle twitches (Fig. 2C1). The same effect
was observed for directly and indirectly elicited tetanic muscle
contraction (Figs. 2B2 and C2).
Nerve-evoked and directly evoked single twitch contractions
and tetanic contractions were poorly reversed 30 min after
extensive washing of the preparations with a drug-free
medium.
These results indicate that BEA affects both directly and
indirectly elicited muscle twitches.
The addition of higher concentrations of BEA (7.5 and 10 lM)
induced a dose dependent contracture. This contracture at a
concentration of 10 lM BEA was not inhibited by the absence of
indirect or direct electrical stimulation (Fig. 3C).
Complete block of indirectly elicited isometric muscle contraction of the mouse phrenic nerve-diaphragm preparation
was achieved at the highest concentration (10 lM) of BEA
studied (Fig. 1C). BEA also blocked the directly elicited muscle
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contraction. At the highest concentration (10 mM), BEA reduced
the amplitude of directly elicited muscle twitch for 80% 6 1.6%.
Effects of BEA on Resting Membrane Potential and Amplitude of
MEPPs and EPPs in Isolated Mouse Neuromuscular Preparations
The median values for resting membrane potentials measured
from 6 to 9 fibers under control conditions were 66.56 mV (IQR:
68.19 to 63.96), 67.69 mV (IQR: 70.71 to 62.58), 67.57 mV
(IQR: 69.95 to 64.78), and 66.52 mV (IQR: 68.42 to 59.37) at
the time points 0, 30, 60, and 90 min, respectively (Fig. 4). As
shown above, BEA at a concentration of 10 mM almost completely blocked nerve-evoked and directly elicited isometric
muscle contraction. BEA at a concentration of 5 lM did not significantly affect the resting membrane potential recorded in the
endplate region of muscle fibers (Fig. 4). At higher concentrations (7.5 and 10 mM), BEA significantly decreased the resting
membrane potential (Fig. 4). BEA significantly reduced the resting membrane potential 60 and 90 min after exposure to 7.5 mM
concentrations. The effect was the most pronounced at the BEA
concentration of 10 mM, at which the median values for resting
membrane potentials measured between 6 and 13 fibers
dropped from 66.15 mV (IQR: 68.16 to 58.09) to 46.72 mV
(IQR: 59.15 to 34.39), 37.34 mV (IQR: 52.57 to 26.62), and
27.24 mV (IQR 49.41 to 16.77) at 30 min, 60 min, and 90 min
after the exposure to the highest concentration of BEA (10 lM),
respectively (Fig. 4).
The effects of BEA on MEPPs frequency and amplitude are
shown in Figure 5. At concentrations of 10 lM, BEA significantly
reduced the amplitude of MEPPs measured in superficial muscle
fibers of the mouse hemidiaphragm (Figs. 5A and B). At lower
concentrations (5 and 7.5 mM), BEA also reduced the amplitude
of MEPPs, as well as the frequency. Similar effects of BEA on
the amplitude of EPPs were observed after nerve stimulation
(Fig. 6A).
Electrophysiological recordings from endplates of the mouse
hemidiaphragm exposed to BEA (from 5 to 10 lM; Fig. 6A) for 30,
60, and 90 min revealed that BEA significantly reduced the
amplitude of EPPs in a concentration-dependent manner (Fig.
6B). No significant changes in the MEPP and EPPs half-decay
time were observed with BEA in the range of the concentrations
studied. The effects of BEA on MEPPs and EPPs were slowly
reversible at the 5 lM BEA concentration and partially reversible
at concentrations higher than 5 mM (not shown).
DISCUSSION
This study shows, for the first time, the effects of mycotoxin
BEA on neuromuscular function. Activity of BEA on neuromuscular transmission was examined in mice neuromuscular hemidiaphragm preparation. BEA inhibits indirectly and directly
evoked isometric muscle twitches and causes a small but sustained contracture of the mouse hemidiaphragm. The data
revealed that BEA in a concentration-dependent manner
blocked nerve-evoked single twitch response and tetanic contraction. The neuromuscular block produced by the BEA in the
mouse hemidiaphragm occurs at concentrations 5, 7.5, and
10 mM. BEA in 5 mM concentration, without affecting MEPPs
amplitude, decreased MEPPs frequency measured in muscle
fibers without a significant decrease of muscle fiber resting
membrane potential, indicating the presynaptic activity of the
compound. Possible involvement of acetylcholinesterase (AChE)
was excluded because there was no prolongation of half-decay
time constant of spontaneous MEPP and EPP in mouse hemidiaphragm preparations as would be expected if the compound
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FIG. 1. Concentration-dependent effects of BEA on isometric muscle contraction and resting muscle tension. A, Representative control tracing. B, BEA significantly
reduces the muscle twitch amplitude. C, At concentrations above 5 mM, BEA spontaneously induces a contracture of the mouse phrenic nerve-diaphragm preparation.
The arrow designated by the letter c on panel (C) indicates the contracture produced by BEA. All other letters have the same meaning as in Figure 2.
were blocking AChE (Katz and Miledi, 1973). Our results show
that BEA at a slightly higher concentrations (7.5 and 10 mM)
inhibits directly elicited twitches and produces contracture in
mouse hemidiaphragm indicating myotoxicity (Harvey et al.,
1994). As determined by analyzing the contractile responses
upon single and tetanic direct muscle stimulation, BEA affects
muscle excitability and excitation-contraction coupling processes in muscle at 7.5 and 10 mM concentrations. This
contracture cannot be abolished by the presence of selective
muscle-type nAChR antagonist DTC (10 mM), indicating a direct
effect of BEA on skeletal muscle contractility. Rise in baseline
resting tension (contracture) is postsynaptic in origin because
no repetitive EPPs were recorded and contracture developed
also without nerve stimulation and in the presence of 10 mM
DTC. BEA can produce cation-selective channels in artificial
lipid membranes and ventricular myocytes at 10 mM
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FIG. 2. Effects of BEA on both nerve-evoked and directly elicited single twitch and tetanic contractions in isolated mouse hemidiaphragm tissue. A, Representative
whole muscle contraction recordings showing the partial block of nerve-evoked muscle twitch and tetanic contraction by BEA at a concentration of 5 lM. N, nerveevoked muscle contraction; D, directly elicited muscle contraction; Tn, nerve-evoked tetanic contraction; Td, directly elicited tetanic contraction; W, wash-out. B1,
Representative superimposed recordings of nerve-evoked single twitch before (N1), and 90 min after application of 5 mM BEA (N2) showing the blocking effects of BEA.
B2, Representative recordings of tetanic contraction (at 70 Hz) before (Tn1) and 90 min after the application of 5 mM BEA (Tn2), data recorded from the same muscle
showing its blocking effects. C1, Representative superimposed recordings showing that BEA has a significant effect on the peak amplitude of single muscle twitch elicited by direct muscle stimulation before (D1) and 90 min after the application of BEA (D2). Tracings are from the same mouse hemidiaphragm. C2, Representative
superimposed recordings showing tetanic contractions elicited by direct muscle stimulation before (Td1) and 90 min after application of BEA (Td2). D, Concentrationdependent inhibition curve for directly elicited and nerve-evoked contraction for BEA in mouse hemidiaphragms, expressed as the percent of the maximal twitch
response. Values are expressed as the mean 6 SEM (n ¼ 4–5 different muscles for each concentration).
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FIG. 3. Effect of 10 mM BEA on muscle contractility and contracture development. A, BEA produces a complete block of indirectly elicited isometric muscle twitch and
contracture. A partial block of directly evoked muscle twitch is also evident. B, The effects of 10 mM BEA on directly elicited muscle twitches and tetanic contraction in a
neuromuscular preparation incubated with 10 mM dTb. Notice the contracture without development and the partial block of directly elicited isometric contractions. C,
Mouse hemidiaphragm exposed to 10 mM BEA and not stimulated. Notice the contracture without stimulation development and complete block of directly stimulated
isometric contractions. All letters have the same meaning as in Figure 2.
concentration (Kouri et al., 2003). These channels allow Ca2þ
influx and increased [Ca2þ]i (Kouri et al., 2003) may be associated
with skeletal muscle contracture observed in our study. Based
on the results obtained from direct muscle stimulation experiments, a direct effect of BEA on the sarcolemma or excitationcontraction coupling leading to the reduction of muscle twitch
and tetanic contraction amplitude is likely. The muscle
response to the direct electrical stimulation was reduced at 5–
10 mM concentration of BEA. We anticipated that in these concentrations, BEA could depolarize and inactivate voltagedependent sodium Nav1.4 type channels in muscle fibers
because it was shown that BEA forms cation-selective channels
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FIG. 4. Concentration- and time-dependent effect of BEA on the resting membrane potential (rVm) of muscle fibers. Recordings were performed at the end-plate region
of muscle fibers in mouse hemidiaphragms. Muscles were exposed to 5, 7.5, and 10 lM BEA, and recordings were taken before (time 0), after 30 min, 60 min and 90 min
of BEA application, and 15 min after a wash-out of the drug from the medium. Box plots indicate the median values of the data obtained from 3 different hemidiaphragms as well as its IQR, with whiskers showing the full range of the data, and all outliers. (30 min) *P < 0.05 versus control, 5 and 7.5 mM BEA; (60 min) *P < 0.05 versus
control, 5 and 7.5 mM BEA; **P < 0.05 versus control and 10 mM BEA; (90 min) *P < 0.05 versus control, 5 and 7.5 mM BEA, **P < 0.05 versus control, 7.5 and 10 mM BEA,
***P < 0.05 versus 7.5 and 10 mM BEA; (wash-out) *P < 0.05 versus control, 5 and 7.5 mM BEA (Kruskal–Wallis one-way ANOVA on ranks with Dunn’s post-hoc test).
FIG. 5. Effects of BEA on the amplitude and frequency of MEPPs. A, Representative traces of MEPPs recorded from the same mouse hemidiaphragm under control conditions and after the indicated concentrations of BEA applied for 90 min. B, MEPPs amplitude recorded from mouse hemidiaphragms exposed to different BEA concentrations, and normalized to the amplitude recorded before BEA application. Box plots indicate the median values of the data obtained from 4 to 6 different nerve-muscle
preparations as well as its IQR, with whiskers showing the full range of the data, and all outliers. *P < 0.05 versus control, 5 mM BEA, **P < 0.05 versus 5 mM BEA; ***P < 0.05
versus 7.5 and 10 mM BEA (Kruskal–Wallis one-way ANOVA on ranks with Dunn’s post-hoc test). C, Effects of BEA on MEPPs frequency in the same preparation. *P < 0.05
versus control, 5 and 7.5 mM BEA; **P < 0.05 versus control, 10 mM BEA; ***P < 0.05 versus control and 10 mM BEA (Kruskal–Wallis one-way ANOVA on ranks with Dunn’s
post-hoc test).
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FIG. 6. Effects of BEA on evoked neurotransmitter release. A, Examples of EPP tracings recorded under control conditions (control) and 90 min after exposure of nervemuscle preparations to BEA (concentrations are indicated in front of the respective tracings). Mouse hemidiaphragm preparations were pretreated for 30 min with 2 lM
l-conotoxin GIIIB, and all experiments were performed in the presence of 2 lM l-conotoxin GIIIB to record full-sized endplate potentials and to prevent muscle
twitches. Note that the decrease of EPP amplitudes occurred without changes in the resting membrane potential of muscle fibers at 5 mM BEA. B, Block of full-size EPPs
as a function of BEA concentration in isolated mouse hemidiaphragm. Each point represents the mean value 6 SEM obtained from 3 to 5 different nerve-muscle preparations. *P < 0.05 versus control, 5 and 7.5 mM BEA, **P < 0.05 versus control, 5 and 10 mM BEA, ***P < 0.05 versus 7.5 and 10 mM BEA (one-way ANOVA followed by Holm–
Sidak).
in the sarcoplasm of ventricular cardiomyocytes and artificial
lipid membranes at 10 mM concentrations (Kouri et al., 2003).
Slow inactivation of sodium channels due to persistent depolarization may affect excitability and contractility (for review see
Ulbricht, 2005). This assumption was confirmed in this study by
the intracellular recordings of membrane potential in the
muscle fibers exposed to different concentrations of BEA and
different times of exposition to the compound. The results
clearly show that BEA substantially reduces the resting membrane potential in a time and concentration-dependent manner
at the higher concentrations used (7.5 and 10 mM). This response
may explain almost complete directly elicited twitch and tetanic contraction block in neuromuscular preparation exposed to
7.5 and 10 mM BEA.
In the following electrophysiological experiments using
microelectrode technique, we showed that BEA at 5–10 mM concentrations significantly reduces the frequency of spontaneous
MEPPs. Furthermore, significant MEPPs amplitude reduction at
the highest BEA concentration used (10 mM) was observed. This
reduction may be due to muscle membrane depolarization that
reduces the driving force acetylcholine (Er-EM), assuming that the
equilibrium potential for acetylcholine (ACh) remained constant
during the action of BEA. Alternatively, results may indicate
reduced spontaneous and evoked quantal ACh release or postsynaptic antagonizing effect of BEA on nAChR in neuromuscular
junction at the highest concentration of BEA used. It has been
described that BEA at concentrations of 0.3–100 mM reversibly
inhibits L-type voltage-dependent Ca2þ current in NG108-15 neuronal cells with an IC50 value of 4 mM (Wu et al., 2002).
Spontaneous secretion of the neurotransmitter ACh in mammalian neuromuscular synapse depends on the resting Ca2þ concentration in nerve terminals. Spontaneous ACh release is
dependent on P/Q-type VDCC in mammalian neuromuscular
junction (Giovannini et al., 2002). However, there are some reports
that L-type VDCC may be involved in both spontaneous (Losavio
and Muchnik, 1997) and nerve- evoked ACh release in mammalian neuromuscular junction (Lin and Lin-Shiau, 1997). The possibility that BEA may act on P/Q-type VDCC cannot be excluded.
Therefore, reduced frequency in spontaneous quantal release
observed in our experiments may be related to the blockade of
L-type voltage-dependent Ca2þ channels (VDCC) caused by BEA.
As shown in this study, muscle fibers substantially depolarize
when exposed to the 7.5 and 10 mM concentrations of BEA.
Depolarization brings the membrane potential toward the reversal potential of the end-plate current, which diminishes the driving force for inward current flow. Significant reduction of muscle
twitches occurred at 5 mM BEA without changes in the resting
membrane potential of muscle fibers and EPPs amplitude. Since
no significant decrease in MEPPs and EPPs were detected, a direct
effect of BEA on nAChR is unlikely. However, the hypothesis that
BEA may exert an antagonist action on muscle-type nAChRs
remains to be verified. Altogether, our results show that BEA at
5 mM concentrations causes reduction in amplitude of muscle
twitches without altering muscle fiber passive membrane properties and postsynaptic potentials. Reduction in the amplitude of
muscle twitches at 5 mM BEA is probably due to its direct effects
on muscle cells, since no difference in the amplitude of directly
and indirectly evoked muscle twitches were detected. At 7.5 and
10 mM concentrations of BEA, a progressive decrease in resting
membrane potential is associated with changes in frequency and
amplitude of MEPPs and EPPs.
In conclusion, BEA at 5 mM concentration acts presynaptically as revealed by reduced frequency of MEPPs. At higher concentrations (7.5 and 10 mM), BEA produces contracture,
indicating a direct effect of BEA on skeletal muscle fibers. The
final maximal concentration of BEA (10 lM), producing a contracture and complete block of nerve evoked muscle twitch and
tetanic contraction in vitro, is roughly six times lower than the
maximal calculated concentration of BEA in the blood and
extracellular fluid in vivo after injection of one i.p. LD50 (60 mM)
(Omura et al., 1991). Therefore, related to the molecular mechanism of BEA action on biological membranes of excitable tissue
and organs (skeletal and heart muscle, neuronal tissue), clinical
signs such as ataxia, weakness, paresis, paralysis, tremors, and
respiratory failure are expected as a result of BEA intoxication.
In light of these findings, it will be interesting to examine
other mycotoxins for similar neuro and myotoxic activity.
FUNDING
Slovenian Research Agency (Grant/Award Number: P4-0053).
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ACKNOWLEDGMENTS
We thank Boštjan Drolc and Katarina Babnik for excellent
technical support as well as statistician Mateja Nagode for
her consultation on data analysis.
REFERENCES
Blais, L. A., ApSimon, J. W., Blackwell, B. A., Greenhalgh, R., and
Miller, J. D. (1992). Isolation and characterization of enniatins
from Fusarium avenaceum DAOM 196490. Can. J. Chem. 70,
1281–1287.
Calo, L., Fornelli, F., Nenna, S., Tursi, A., Caiaffa, M. F., and
Macchia, L. (2003). Beauvericin cytotoxicity to the invertebrate cell line SF-9. J. Appl. Genet. 44, 515–520.
Calo, L., Fornelli, F., Ramires, R., Nenna, S., Tursi, A., Caiaffa, M.
F., and Macchia, L. (2004). Cytotoxic effects of the mycotoxin
beauvericin to human cell lines of myeloid origin. Pharmacol.
Res. 49, 73–77.
Chen, B. F., Tsai, M. C., and Jow, G. M. (2006). Induction of calcium
influx from extracellular fluid by beauvericin in human leukemia cells. Biochem. Biophys. Res. Commun. 340, 134–139.
Cruz, L. J., Gray, W. R., Olivera, B. M., Zeikus, R. D., Kerr, L.,
Yoshikami, D., and Moczydlowski, E. (1985). Conus geographus toxins that discriminate between neuronal and muscle
sodium channels. J. Biol. Chem. 260, 9280–9288.
EFSA (2014). Scientific Opinion on the risks to human and animal
health related to the presence of beauvericin and enniatins
in food and feed. EFSA J. 12(8), 3802.
Giovannini, F., Sher, E., Webster, R., Boot, J., and Lang, B. (2002).
Calcium channel subtypes contributing to acetylcholine release from normal, 4-aminopyridine-treated and myasthenic
syndrome auto-antibodies-affected neuromuscular junctions. Br. J. Pharmacol. 136, 1135–1145.
Hamill, R. L., Higgens, G. E., Boaz, H. E., and Gorman, M. (1969).
The structure of beauvericin, a new depsipeptide antibiotic
toxic to Artemia salina. Tetrahedron. Lett. 49, 4255–4258.
Harvey, A. L., Barfaraz, A., Thomson, E., Faiz, A., Preston, S., and
Harris, J. B. (1994). Screening of snake venoms for neurotoxic
and myotoxic effects using simple in vitro preparations from
rodents and chicks. Toxicon 32, 257–265.
Hong, S. J., and Chang, C. C. (1989). Use of geographutoxin II (muconotoxin) for the study of neuromuscular transmission in
mouse. Br. J. Pharmacol. 97, 934–940.
Hu, L., and Rychlik, M. (2014). Occurrence of enniatins and beauvericin in 60 Chinese medicinal herbs. Food. Addit. Contam. A
Chem. Anal. Control Expo. Risk. Assess. 31, 1240–1245.
Jestoi, M. (2008). Emerging fusarium-mycotoxins fusaproliferin,
beauvericin, enniatins, and moniliformin: A review. Crit. Rev.
Food. Sci. Nutr. 48, 21–49.
Jestoi, M., Rokka, M., Yli-Mattila, T., Parikka, P., Rizzo, A., and
Peltonen, K. (2004). Presence and concentrations of the
Fusarium-related mycotoxins beauvericin, enniatins and
moniliformin in finnish grain samples. Food Addit. Contam.
21, 794–802.
|
291
Jow, G. M., Chou, C. J., Chen, B. F., and Tsai, J. H. (2004).
Beauvericin induces cytotoxic effects in human acute lymphoblastic leukemia cells through cytochrome c release, caspase 3 activation: the causative role of calcium. Cancer Lett.
216, 165–173.
Katz, B. and Miledi, R. (1973). The binding of acetylcholine to receptors and its removal from the synaptic cleft. J. Physiol. 231,
549–574.
Kim, M. K., Seong, S. Y., Seoh, J. Y., Han, T. H., Song, H. J., Lee, J.
E., Shin, J. H., Lim, B. U., and Kang, J. S. (2002). Orientia
tsutsugamushi inhibits apoptosis of macrophages by
retarding intracellular calcium release. Infect. Immun. 70,
4692–4696.
Kouri, K., Lemmens, M., and Lemmens-Gruber, R. (2003).
Beauvericin-induced channels in ventricular myocytes and
liposomes. Biochim. Biophys. Acta 1609, 203–210.
Lin, M. J. and Lin-Shiau, S. Y. (1997). Multiple types of Ca2þ channels in mouse motor nerve terminals. Eur. J. Neurosci. 9,
817–823.
Logrieco, A., Moretti, A., Castella, G., Kostecki, M., Golinski, P.,
Ritieni, A., and Chelkowski, J. (1998). Beauvericin production
by Fusarium species. Appl. Environ. Microbiol. 64, 3084–3088.
Logrieco, A., Rizzo, A., Ferracane, R., and Ritieni, A. (2002).
Occurrence of beauvericin and enniatins in wheat affected
by Fusarium avenaceum head blight. Appl. Environ. Microbiol.
68, 82–85.
Losavio, A. and Muchnik, S. (1997). Spontaneous acetylcholine
release in mammalian neuromuscular junctions. Am. J.
Physiol. 273, C1835–C1841.
Nguyen-Huu, T., Dobbertin, A., Barbier, J., Minic, J., Krejci, E.,
Duvaldestin, P., and Molgo, J. (2005). Cholinesterases and the
resistance of the mouse diaphragm to the effect of tubocurarine. Anesthesiology 103, 788–795.
Omura, S., Koda, H., and Nishida, H. (1991). Hypolipemics containing beauvericin as acylcoenzyme A cholesterol acyltransferase inhibitor. Patent JP 89-16115019890623.
Plattner, R. D. and Nelson, P. E. (1994). Production of beauvericin
by a strain of Fusarium proliferatum isolated from corn fodder for swine. Appl. Environ. Microbiol. 60, 3894–3896.
Prince, R. C., Crofts, A. R., and Steinrauf, L. K. (1974). A comparison of beauvericin, enniatin and valinomycin as calcium
transporting agents in liposomes and chromatophores.
Biochem. Biophys. Res. Commun. 59, 697–703.
Streit, E., Schwab, C., Sulyok, M., Naehrer, K., Krska, R., and
Schatzmayr, G. (2013). Multi-mycotoxin screening reveals
the occurrence of 139 different secondary metabolites in feed
and feed ingredients. Toxins 5, 504–523.
Ulbricht, W. (2005). Sodium channel inactivation: molecular determinants and modulation. Physiol. Rev. 85, 1271–1301.
Wang, Q. and Xu, L. (2012). Beauvericin, a bioactive compound
produced by fungi: A short review. Molecules 17, 2367–2377.
Wu, S. N., Chen, H., Liu, Y. C., and Chiang, H. T. (2002). Block of
L-type Ca2þ current by beauvericin, a toxic cyclopeptide,
in the NG108-15 neuronal cell line. Chem. Res. Toxicol. 15,
854–860.