Toxicology Letters 209 (2012) 129135

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

Inhibitory effect of tungsten carbide nanoparticles on voltage-gated potassium

currents of hippocampal CA1 neurons
Dehong Shan a,b , Yongling Xie a , Guogang Ren c , Zhuo Yang a,
a
b
c

College of Medicine Science, Nankai University, Tianjin 300071, China

Fundamental Medicine College, Liaoning University of Traditional Chinese Medicine, Shenyang 110032, China
Science and Technology Research Institute, University of Hertfordshire, Hateld, Herts AL10 9AB, UK

a r t i c l e

i n f o

Article history:
Received 25 August 2011
Received in revised form 5 December 2011
Accepted 6 December 2011
Available online 13 December 2011
Keywords:
Tungsten carbide nanoparticles
Hippocampus
Transient outward potassium current
Delayed rectier potassium current
Neurotoxicity

a b s t r a c t
The effects of tungsten carbide nanoparticles (nano-WC) on the properties of voltage-dependent potassium currents and evoked action potentials were studied in the hippocampal CA1 pyramidal neurons of
rats at the ages of postnatal days 1014 using the whole-cell patch-clamp technique. The results indicated
that: (1) the amplitudes of transient outward potassium current (IA ) and delayed rectier potassium current (IK ) were signicantly decreased by 107 g/ml nano-WC, while the currentvoltage curves of IA and
IK were signicantly decreased by nano-WC from +10 to +90 mV. (2) Nano-WC produced a depolarizing
shift in the steady-state activation curve of IA and IK with increased slope factors, and delayed the recovery of IA from inactivation, but no signicant effects were found on the inactivation of IA . (3) Nano-WC
prolonged the evoked action potential duration and lowered the ring rate. These results suggest that
107 g/ml nano-WC can decrease the amplitudes of IA and IK currents by reducing the opening number of
voltage-gated potassium channels and delaying the recovery of IA from inactivation, which indicate that
nano-WC has the potential neurotoxicity.
2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
With the development of nanotechnique, more and more
nanomaterials and nanoproductions appear. It is believed that
nanoparticles can enter into the body more easily, and have better
mobility inside the body than bigger ones, so the safety of nanomaterials and nanoproductions should be investigated.
Tungsten carbide (WC) is an inorganic chemical compound containing equal parts of tungsten and carbon atoms. Generally, the
form of WC is a ne gray powder. WC based hard metals display extreme hardness, wear resistance and red hardness, so WC
is widely used in industrial machinery, tools, abrasives, as well as
jewelry. WC can also be used as a potential catalyst replacement for
platinum in electrocatalysis, or as a replacement for the iridium catalyst in hydrazine powered satellite thrusters. Because reduction
of the WC size can give a marked improvement of the mechanical
properties, WC nanoparticles (nano-WC) are produced to replace
WC. And now, a new form of nano-WC, WC nanowires, appears
which can be used to produce the carbide micro-drills or the probes
of the scanning probe microscope.
Occupational exposure to WC is associated with dermatitis,
inammation of the conjunctiva and mucous membranes of the
upper respiratory tract, chronic bronchitis, bronchial asthma, inter-

Corresponding author. Tel.: +86 22 23504364; fax: +86 22 23502554.

E-mail address: zhuoyang@nankai.edu.cn (Z. Yang).
0378-4274/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2011.12.001

stitial brosis, brosing alveolitis, and lung cancer (Lison and

Lauwerys, 1992; Moulin et al., 1998). And WC might also have
the mutagenic and apoptogenic potential in human peripheral
blood mononucleated cells (Lombaert et al., 2004). The toxicological mechanism of WC exposure might be relative to oxidative stress
(Ding et al., 2009). Generally cobalt (Co) is mixed with WC in the
manufacture of hard metals to improve toughness and strength of
the hard metals. The acute toxicity of WCcobalt mixture is much
higher than that of each individual component (Gerard et al., 1992),
and the toxicity of Co is higher than that of WC due to the lower
solubility and bioavailability of WC in the body (Krausa et al., 2001).
However, the toxicity of nano-WC needs to be recognized due to
the obvious changes of physiochemical properties. Nanoparticles
can enter into the brain through the olfactory neuronal pathway
(Oberdrster et al., 2004), so the brain becomes one of the nanoWC exposure organs, and it is necessary to study the neurotoxicity
of nano-WC. An in vitro experiment suggested that nano-WC could
enter into neurons, but there was no acute neuronal loss after nanoWC exposure (Bastian et al., 2009). Though no acute neurotoxicity
was observed, it cannot be concluded that nano-WC is safe because
the structural alteration of neurons caused by nano-WC might be
chronic.
Generally, the neuronal dysfunction often appears prior to
the structure alteration, and the examination of the neuronal
electrophysiological changes might be helpful to nd the acute
neurotoxicities of nano-WC earlier. A survey among former
WC workers with hard metal disease demonstrated decits in

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D. Shan et al. / Toxicology Letters 209 (2012) 129135

Fig. 1. TEM image of nano-WC. The image showed that most of the nanoparticles
were circular or oval shape with diameters of 520 nm.

the short-term verbal memory, allocation of central processing

resources and remote verbal memory (Catherine et al., 1993). This
indicated that WC might inuence the hippocampal function. Neurotoxicity studies of nanoparticles showed that nanomaterials can
inuence electrophysiological properties of hippocampal neurons
(Liu et al., 2009; Zhao et al., 2009). The effect of nano-WC on
the electrophysiological properties of the hippocampal neurons is
known little. Therefore, the aim of the present study is to investigate the possible effect of nano-WC on the voltage-gated potassium
current in hippocampal CA1 neurons.
2. Materials and methods
2.1. Drug application
The nano-WC particles used in the present study was synthesized by plasma
process and provided by QinetiQ Nanomaterials, Farnborough, Hampshire, UK. The
transmission electron microscopy (TEM, Tecnai G2 20 S-TWIN, FEI, USA) images
showed that most of the nanoparticles was circular or oval shape with diameters of
520 nm (Fig. 1). Stock solution (103 g/ml) of nano-WC was prepared in articial
cerebrospinal uid (ACSF) and dispersed by ultrasonic vibration for 20 min. NanoWC suspension (103 g/ml) was characterized by dynamic light scattering using
a Zeta-PALS + BI-90Plus (Brookhaven Instruments Corp., USA) at a wavelength of
659 nm. The scattering angle was xed at 90 C. The particle size distribution had
a wide range from 115.26 nm to 703.70 nm due to the aggregation, and the mean
hydrodynamic diameter was 414.90 nm. The concentrations of nano-WC used in the
experiment were 106 , 107 , 108 g/ml diluted from the stock solution of nano-WC.
The nano-WC suspension was stirred on the vortex agitator before every use.
ACSF contained in mM: 125 NaCl, 25 NaHCO3 , 1.25 KCl, 1.25 KH2 PO4 , 1.5 MgCl2 ,
2.0 CaCl2 , 16 glucose. Tetrodotoxin (TTX) was obtained from the Research Institute
of the Aquatic Products of Hebei (China). 4-Aminopyridine (4-AP), tetraethylammonium chloride (TEA-Cl), CdCl2 , EGTA, HEPES and ATP-Na2 were purchased from
Sigma (USA), and other reagents were of A.R. grade. All other drugs were diluted in
oxygenated ACSF immediately prior to usage, and were applied via the perfusion
system.
2.2. Slice preparation
Male Wistar rats from the Experimental Animal Center of Chinese Academy of
Medical Science were used on postnatal days 1014. The experiments were conducted in accordance with the guidelines of the Medicine Experimental Animal
Administrative Committee of Nation. Horizontal slices that included the entire hippocampus and subiculum (400 m in thickness) were prepared with a vibratome
(VT1000 M/E, Leica, Germany) and incubated with ACSF for at least 1 h before
electrophysiological recording. During recording/data logging, the slices were kept
submerged in a chamber perfused with ACSF. In the experiments, ACSF was saturated with 95% O2 and 5% CO2 .

Fig. 2. Effects of nano-WC (106 , 107 , 108 g/ml) on the amplitudes of IA (A) and IK
(B). The current traces of IA were recorded when the neurons were held at 70 mV
and evoked by +70 mV. And the current traces of IK were recorded when the neurons
were held at 50 mV and evoked by +60 mV. Data are presented as mean S.E.M.,
n = 6, *p < 0.05, **p < 0.01 vs. controls.

2.3. Electrophysiological recordings

The whole cell membrane currents were recorded at room temperature
(2224 C) using the whole-cell patch-clamp technique (Liu et al., 2007). The standard pipette solution for recording voltage-dependent potassium current contained
(in mM): KCl 140, MgCl2 6H2 O2 , HEPES 10, EGTA 10, ATP-Na2 2, buffered to pH
7.4 with KOH. In order to record potassium currents, TTX (0.001 mM) and CdCl2
(0.2 mM) were added into the ACSF to block Na+ and Ca2+ currents. For whole-cell
recording, slices were transferred into a recording chamber (1 ml volume) placed on
the stage of a modied upright infrared DIC microscope equipped with Nomarski
optics. Hippocampal CA1 neurons were visualized on a television monitor connected
to a low light sensitive CCD camera. Signals were ltered at 5 kHz and digitized at a
sampling rate of 2 kHz. The series resistance was compensated at least 60%. Leakage
and capacitive currents were subtracted on-line using a P/4 subtraction procedure.
Data acquisition and analysis were performed with EPC10 patch-clamp amplier
(HEKA, Germany).
After giga-seal formation and membrane rupture, the neurons were allowed
to stabilize for 35 min before starting the pulse protocols. Two types of voltagedependent potassium currents known as transient outward potassium currents (IA )
and delayed rectier potassium currents (IK ) were recorded in the present experiments. IA and IK were distinguished respectively in the presence of 4-AP (3 mM) and
TEA-Cl (25 mM).

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131

Fig. 3. Effects of 107 g/ml nano-WC on the IV curves of IA (B) and IK (D). IV curve of IA was generated by applying 80 ms constant depolarizing pulse from a command
potential of 50 to +90 mV in increments of 10 mV, and the holding potential was 70 mV (A). IV curve of IK was generated by applying 300 ms constant depolarizing pulse
by a similar pulse protocol (C). Data are presented as mean S.E.M., n = 6, *p < 0.05, **p < 0.01 vs. controls.

All data were analyzed by Clampt 9.0, Origin 8.0 and SPSS11.5. The values are
represented as mean S.E.M. and statistical comparisons were made using Students
paired t-test and one-way analysis of variance. p < 0.05 was considered signicant.

and 41.53 7.6% (p < 0.01) (Fig. 2A), respectively. And the amplitudes of IK were reduced approximately 23.93 8.31% (p > 0.05),
34.13 4.26% (p < 0.01) and 35.07 8.9% (p < 0.01) (Fig. 2B), respectively.

3. Results

3.2. Effects of nano-WC on IV curves of IA and IK

3.1. Concentration-dependent effects of nano-WC on IA and IK

107 g/ml nano-WC was used to observe the detailed effect of

nano-WC on voltage-gated K+ current. When recording IA , TEA-Cl
(25 mM) was applied to inhibit IK , and the holding potential was
70 mV, using an 80 ms constant depolarizing pulse from a command potential of 50 to +90 mV in increments of 10 mV (Fig. 3A).
And when recording IK , 4-AP (3 mM) was used to block IA , and
IK was obtained using a 300 ms constant depolarizing pulse by a
similar pulse protocol (Fig. 3C). After nano-WC application, the IA
and IK amplitudes were reduced in a different way at the different
membrane potentials, showing a concentration-dependent manner (Fig. 3B and D). Nano-WC signicantly decreased amplitudes of
IA and IK from +10 to +90 mV and +10 to +90 mV, respectively.

2.4. Data analysis

To determine the minimal concentration used in later experiments, the maximal IA and IK amplitudes obtained before and
after nano-WC application in different concentrations (106 ,
107 , 108 g/ml) were concerned. When recording IA , the neurons were held at 70 mV, current traces were evoked by
+70 mV. And to record IK , the neurons were held at 50 mV,
current traces were evoked by +60 mV. The amplitudes of IA
and IK before and after nano-WC application were normalized in Fig. 2. Results showed that the amplitudes of IA were
decreased about 22.75 10.32% (p > 0.05), 33.18 4.8% (p < 0.05)

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D. Shan et al. / Toxicology Letters 209 (2012) 129135

Fig. 5. Effect of 107 g/ml nano-WC on the steady-state inactivation curve of IA

(n = 6). The currents were obtained with the double-pulse protocols, the neurons
were held at 70 mV and were elicited with an 80 ms test pulse to +50 mV proceeded
by 80 ms prepulse to potentials between 110 and +10 mV.

3.4. Effects of nano-WC on the steady-state inactivation

properties of IA

Fig. 4. Effects of 107 g/ml nano-WC on the steady-state activation curves of IA (A)
and IK (B) (n = 6).

The steady-state inactivation was obtained with the doublepulse protocols as follow. Neurons were held at 70 mV and
currents were elicited with an 80 ms test pulse to +50 mV proceeded by 80 ms prepulse to potentials between 110 and +10 mV.
Peak amplitudes for IA were normalized and plotted vs. prepulse
potentials. The curves were well tted with Boltzmann equation:
I/Imax = 1/{1 + exp[(Vm Vh )/k]}, where I/Imax is normalized current,
Vh the potential for half-maximal inactivation, and k the slope factor. The values of Vh before and after nano-WC application were
70.94 0.91 mV and 70.71 0.77 mV (n = 6, p > 0.05), respectively. And k values before and after nano-WC application were
11.95 0.79 and 10.36 0.67 (n = 6, p > 0.05), respectively. There
was no signicant effect of nano-WC on the inactivation of IA
(Fig. 5).

3.3. Effects of nano-WC on the activation kinetics of IA and IK

Fig. 4A and B shows the steady-state activation curves for IA
and IK before and after nano-WC application. For activation, currents at each test potential were converted to conductance (G)
using the formula G = I/(Vm Vr ), where Vr is reversal potential.
The peak conductance value for each test potential was normalized to Gmax and plotted against the test potential to produce a
voltageconductance relationship curves, which were tted using
Boltzmann equation G/Gmax = 1/{1 + exp[(Vm Vh )/k]}, where Vh is
the voltage at which conductance being half-maximal, and k is a
slope factor. The Vh values of IA before and after nano-WC application were 29.14 1.58 mV and 91.89 23.33 mV (n = 6, p < 0.05),
and k values were 32.25 2.11 and 70.80 22.27 (n = 6, p < 0.05).
Nano-WC produced a depolarizing shift in the activationvoltage
curve with slope factor increase on IA currents. The Vh values of
IK were 46.53 2.47 mV and 62.43 8.38 mV (n = 6, p < 0.05), and
the values of k were 24.21 2.01 and 29.04 4.65 (n = 6, p < 0.05).
Nano-WC produced a depolarizing shift in the activationvoltage
curve with slope factor increase on IK currents.

Fig. 6. Effect of 107 g/ml nano-WC on the recovery of IA (n = 6). Neurons were held
at 70 mV, and an 80 ms conditioning depolarizing pulse of +50 mV was applied to
inactivate the transient outward potassium channels fully, then an 80 ms test pulse
of +50 mV was applied after a series of 80 mV intervals varying from 10 to 265 ms.

D. Shan et al. / Toxicology Letters 209 (2012) 129135

133

3.5. Effects of nano-WC on the recovery from inactivation of IA

4.1. Effect of nano-WC on amplitudes of IA and IK

To observe the kinetics of recovery from the inactivated state,

neurons were held at 70 mV, and an 80 ms conditioning depolarizing pulse of +50 mV was applied to inactivate the transient
outward potassium channels fully, then an 80 ms test pulse of
+50 mV was applied after a series of 80 mV intervals varying
from 10 to 265 ms. The peak value of IA evoked by the conditioning pulse was designated as I1 , while the peak value of the
IA evoked by the test pulse was designated as I2 . The ratio of I2
to I1 represented the recovery of IA from inactivation. The plot of
I2 /I1 vs. the duration of the 80 mV intervals was well tted with
a monoexponential equation: I/Imax = A + B exp (t/), where Imax is
the maximal current amplitude, I is the current after a recovery
period of t,  is the time constant and A is the amplitude coefcient. The values of  before and after nano-WC application were
37.60 9.04 ms and 118.29 14.90 ms (n = 6, p < 0.01), respectively.
Results showed that nano-WC delayed the recovery of IA from inactivation (Fig. 6).

In the nervous system, voltage-gated potassium currents inuence many neuronal properties, such as the resting membrane
potential (RP), the action potential (AP), and the ring rate. There
are two kinds of potassium currents in hippocampal CA1 neurons:
IA and IK . IA , the transient outward potassium current, is important to determine the spike threshold, affects the latency of the
rst spike, and participates in early polarization. IK , the delayed
rectier potassium current, is responsible for the repolarization,
determines the spike width and the postspike hyperpolarization,
and helps shaping the maximal spike frequency of neurons.
In the beginning of this study, 106 , 107 , 108 g/ml nano-WC
were applied to hippocampal slices to detect the effects of nano-WC
on the amplitudes of IA and IK . The results showed that nano-WC
inhibited the amplitudes of IA and IK at a concentration-dependent
manner, and the minimal effective concentration was 107 g/ml.
The changes of IV curves of IA and IK after nano-WC application
indicated that 107 g/ml nano-WC markedly inhibited the IA amplitude from +20 mV, and decreased the IK amplitude from +30 mV.
These results suggested that nano-WC of very low concentration
had the negative effect on IA and IK , and the potential effect of
nano-WC on the neurons should be concerned.

3.6. Effects of nano-WC on action potential duration and action

potential ring rate
The neurons were held at 70 mV and repetitive ring was
evoked with a depolarizing current pulse (500 ms, 50 pA) (Fig. 7A).
The rst evoked APs were used to measure action potential
durations (APDs). The values of APDs before and after nano-WC
application were 0.33 0.05 ms and 0.53 0.02 ms (n = 6, p < 0.01),
respectively. Nano-WC caused a marked prolongation of APDs
(Fig. 7B). The evoked AP number was calculated to assess the ring rate, and the ring rates before and after nano-WC application
were 6.5 0.7 and 4.8 0.7 (n = 6, p < 0.01), respectively. Nano-WC
decreased the ring rates (Fig. 7C).

4. Discussion
Nano-WC and Co are mixed together to achieve extreme hardness and wear resistance in the manufacture of hard metals. WCCo
hard metal is classied by the International Agency for Research on
Cancer (IARC) as the probably carcinogen to human being. Generally, the toxicity of WCCo hard metal is mainly from Co, not from
WC. However, this opinion needs to be reconsidered because when
the materials become nanoscale, their physicochemical properties
are modied, which enable increased uptake and interaction with
biological tissues, consequently, the nano effect occurs. The nano
effect is that nanomaterials are more toxic in regard to reduction
of cell viability or induction of oxidative stress and inammatory
mediators (Worle-Knirsch et al., 2007; Zhang et al., 2000, 2003).
Nanoparticles can enter into the brain by passive diffusion, carriermediated endocytosis and trans-synaptic transport (Hoet et al.,
2004; Oberdrster et al., 2004). So the brain is one of the potentially
nanomaterial-exposed organs. The results from in vitro experiments indicated that neurons were not susceptible to acute toxicity
of nano-WC, and acute neuronal loss was not observed (Bastian
et al., 2009). However, the toxicity of nano-WC might be chronic
(Peters et al., 2006).
Studies about effects of nanoparticles on hippocampal neurons
indicate that nanoparticles can inuence the electrophysiological
properties of hippocampal neurons (Liu et al., 2009; Xu et al., 2009;
Zhao et al., 2009). It is known little that nano-WC has the effect on
hippocampal neurons. In the present study, nano-WC was applied
to the hippocampal slices of rats to observe the effects on voltagegated potassium currents.

4.2. Effect of nano-WC on kinetic properties of voltage-gated

potassium channels in hippocampal CA1 neurons
Ion channels in the cell membrane are major media of intraand extracellular communication, and many ion channels have
their own kinetic properties. The ion channels are the targets of
many toxins and drugs, and their kinetic properties can be modied
by toxins and drugs. IA and IK channels have the different kinetic
properties of ion channels. IA channels can be fast activated and
inactivated, and can be recovered form inactivation. But IK channels
are slowly activated, and are thought as no-inactivation. Therefore,
in the present study the activations of both IA and IK channels were
observed, while the inactivation and the recovery from inactivation
of IA channels were studied.
To the steady-state activation curves for IA and IK , nano-WC
produced a depolarizing shift with slope factor increase, which
suggested that the activations of IA and IK channels became difcult after nano-WC application, and larger membrane potential
changes were needed for their activations to reach the same degree
at normal. To the steady-state inactivation for IA , it was observed
that nano-WC had no marked effect, which indicated that the process of steady-state inactivation for IA channels was not interfered
by nano-WC. But to the curves of recovery from inactivation for IA
channels, it was found that nano-WC application delayed the recovery of IA channels, meaning that IA channels need more time to be
ready to open again after they were inactivated. Based on these
results, it could be inferred that nano-WC application reduced the
opening number of IA and IK channels, which was the reason that
nano-WC inhibited the amplitude of IA and IK .
The inhibitions of nano-WC on the IA and IK can induce many
cellular events. For example, IA inhibition can decrease RP because
IA channels are activated near the resting membrane potential
range. The reduced RP can shorten the distance between the membrane potential and the ring potential; consequently, the neuronal
excitability will be improved. This might involve in the occurrence of epilepsy (Leppert and Singh, 1999; Singh et al., 1998) and
deciency of learning and memory (Ramakers and Storm, 2002;
Watanabe et al., 2002; Scannevin et al., 2004). Because IA and IK
are all responsible for the repolarization of AP, their inhibition
will cause the slow repolarization and a prolonged APD; consequently, decrease the neuronal ring rate. These events occurring
at rest and during AP can cause increased inux of monovalent and

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D. Shan et al. / Toxicology Letters 209 (2012) 129135

Fig. 7. Effect of 107 g/ml nano-WC on the evoked APs. The repetitive rings of neurons in control and after nano-WC application were elicited using a 500 ms, 50 pA
depolarizing current pulse and the neurons were held at 70 mV (A). Bar graphs show the effects of 107 g/ml nano-WC on the APD (B) and ring rate (C), and the data are
presented as mean S.E.M., n = 6, **p < 0.01 vs. controls.

divalent cations, and there will be a tendency of intracellular saturation or crystallization. So the inhibitory effect of
nano-WC on IA and IK might be the fundament mechanism of
neurotoxicity.
4.3. Effect of nano-WC on the APD and ring rate
AP is one of the fundamental properties of neurons, including the
depolarization and repolarization. The depolarization is caused by
rapid Na+ inux, and repolarization is cause by K+ efux. Many toxins and drugs can inuence APs through interfering ion channels.
Because nano-WC could inhibit the opening number of IA and IK
channels, it could be inferred that the AP events might by inuenced
by nano-WC application. However, in order to evoke the APs, the
membrane potential was held at 70 mV, so the neuronal hyperexcitability caused by nano-WC inhibiting IA could not been observed.
Therefore, APDs and the ring rate were concerned when studying
the potential effect of nano-WC on APs.

To detect the effect of nano-WC on APDs, the rst evoked APs

were chosen to measure the APDs. The results showed that APDs
were markedly prolonged after nano-WC application, and the reason was that K+ efux via IA and IK channels in repolarization phase
was inhibited by nano-WC. Because prolonged APDs will lower the
ring rate of neurons, it could be expected that the repetitive ring of hippocampal CA1 neurons after nano-WC application was
decreased. And the results were the same as the expectation.
In conclusion, nano-WC could decrease the amplitudes of IA
and IK currents by reducing the opening number of voltage-gated
potassium channels, consequently, inuenced the AP events of CA1
neurons. The inhibitory effect of nano-WC on CA1 neurons might
cause abnormal change of neuronal ring, which is thought as an
early feature of some neuronal diseases, possibly serving as a mechanism driving neuronal dysfunction (Vucic and Kiernan, 2006). So
the potential neurotoxicity of nano-WC might exist, and the individuals exposed occupationally to nano-WC need the necessary
protection.

D. Shan et al. / Toxicology Letters 209 (2012) 129135

Conict of interest statement

The authors declare that there are no conicts of interest.
Acknowledgement
This work was partly supported by the National Natural Science
Foundation of China (31070890).
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