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Attenuated effect of tungsten carbide nanoparticles on voltage-gated sodium current of hippocampal CA1 pyramidal neurons
Toxicology in Vitro 27 (2013) 299–304
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Attenuated effect of tungsten carbide nanoparticles on voltage-gated sodium current of hippocampal CA1 pyramidal neurons Dehong Shan a,b, Yongling Xie a, Guogang Ren c, Zhuo Yang a,⇑ a
College of Medicine Science, Nankai University, Tianjin 300071, China Fundamental Medicine College, Liaoning University of Traditional Chinese Medicine, Shenyang 110032, China c Science and Technology Research Institute, University of Hertfordshire, Hatfield, Herts AL10 9AB, UK b
a r t i c l e
i n f o
Article history: Received 21 April 2012 Accepted 22 August 2012 Available online 29 August 2012 Keywords: Tungsten carbide nanoparticles Hippocampus Voltage-gated sodium current Action potential Neurotoxicity
a b s t r a c t Nanomaterials and relevant products are now being widely used in the world, and their safety becomes a great concern for the general public. Tungsten carbide nanoparticles (nano-WC) are widely used in metallurgy, aeronautics and astronautics, however our knowledge regarding the influence of nano-WC on neurons is still lacking. The aim of this study was to investigate the impact of nano-WC on tetrodotoxin (TTX)-sensitive voltage-activated sodium current (INa) of hippocampal CA1 pyramidal neurons. Results showed that acute exposure of nano-WC attenuated the peak amplitudes of INa in a concentration-dependent manner. The minimal effective concentration was 10 5 g/ml. The exposure of nano-WC significantly decreased current amplitudes of the current–voltage curves of INa from 50 to +50 mV, shifted the steady-state activation and inactivation curves of INa negatively and delayed the recovery of INa from inactivation state. After exposure to nano-WC, the peak amplitudes, overshoots and the V-thresholds of action potentials (APs) were markedly reduced. These results suggested that exposure of nano-WC could influence some characteristics of APs evoked from the hippocampal CA1 neurons by modifying the kinetics of voltage-gated sodium channels (VGSCs). Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Nanoscale materials and relevant products are now being widely used in a variety of areas, ranging from environmental remediation to engineering and medicine. Nanoparticles possess unique physicochemical properties, such as ultra small size, very large surface area to mass ratio and high reactivity. As nanoparticles can easily enter into the body through inhalation, ingestion and dermal penetration, in some sense, everyone might be at the risk of nanoparticle exposure. It has been found that nanoparticles can impair cells through the oxidative stress mechanism (Zhang et al., 1998, 2003; Brooking et al., 2001; Colvin, 2003; Oberdörster et al., 2005; Nel et al., 2006; Choi and Hu, 2008; Hsin et al., 2008). Therefore, the biosafety of nanomaterials becomes a great concern for the public, and the toxicological assessment is very necessary. Tungsten carbide (WC) is an inorganic chemical compound containing equal parts of tungsten and carbon atoms, and its hardness is close to that of the diamond. WC also has properties of high thermal and electrical conductivity, high resistance to corrosion, and high modulus of elasticity. For these excellent properties, WC is widely used in the military, aeronautics/astronautics and metal⇑ Corresponding author. Tel.: +86 22 23504364; fax: +86 22 23502554. E-mail address: [email protected] (Z. Yang). 0887-2333/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2012.08.025
lurgy. With the development of nanotechnique, WC nanoparticles (nano-WC) are produced to replace WC to improve the mechanical properties markedly. The demand of nano-WC is persistently growing owing to the rapid development in the national defense, civil and war industries throughout the globe. The basic form of WC is a fine powder, and levels of airborne particles are very high in the workplace, particularly close to production facilities and deposition. Occupational exposure to WC could cause dermatitis, bronchial asthma, fibrosing alveolitis, and lung cancer (Lison and Lauwerys, 1992; Moulin et al., 1998). WC exposure also had the mutagenic and apoptogenic potentials in human peripheral blood mononucleated cells (Lombaert et al., 2004). It is presumed that the toxicity of nano-WC might be more severe than that of WC because nanoscale particles have better permeability than that of bigger ones inside the body. However, studies of possible toxic effects for nano-WC are few, and much more work should be done to uncover the toxicity of nano-WC. What attracts our attention is the potential neurotoxicity of nano-WC because nanoparticles could enter into the brain through the olfactory neuronal pathway (Oberdörster et al., 2004; Elder et al., 2006). Studies found that, nanoparticles of Ag and ZnO could influence the electrophysiological characteristics of hippocampal CA1 neurons (Liu et al., 2009; Zhao et al., 2009). It is not clear whether nano-WC exposure could induce such effects. Therefore,
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the whole-cell patch-clamp technique was used in the present study to explore the impact of nano-WC on the electrophysiological characteristics of CA1 pyramidal neurons in hippocampal slices. 2. Materials and methods
hippocampus and subiculum (400 lm in thickness) were cut using a vibratome (VT1000 M/E, Leica, Germany) and incubated in ACSF for at least 1 h before transferring into the recording chamber. 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.
2.1. Nano-WC solution 2.4. Electrophysiological recordings WC nanoparticles used in the present study were synthesized and provided by QinetiQ Nanomaterials, Farnborough, Hampshire, UK. The particles were circular or oval shapes with the diameters of 5–20 nm. The stock solution (10 3 g/ml) of nano-WC in artificial cerebrospinal fluid (ACSF) was mixed by an ultrasonic (750 W) mixer (from Sonicator, US), with pulse 1 s on and 1 s off for 60 s, 50% of power input, for 20 min. The nano-WC suspension (10 3g/ml) was characterized by dynamic light scattering (DLS) using a ZetaPALS + BI-90Plus (Brookhaven Instruments Corp., USA) at a wavelength of 659 nm. The scattering angle was fixed at 90 °C. The particle size distribution had a wide range from 115.26 to 703.70 nm due to the aggregation, and the mean hydrodynamic diameter was 414.90 nm (Fig.1). The concentrations of nano-WC used in the experiment were 10 4, 10 5 and 10 6 g/ml diluted from the stock solution of nano-WC. However, the particles do settle after 1 h to the bottom of the container. By the time the tests were set up, the nano-WC suspensions were stirred on the vortex agitator for 20 min to recreate the uniform suspensions.
ACSF contains (in mM): 125 NaCl, 25 NaHCO3, 1.25 KCl, 1.25 KH2PO4, 1.5 MgCl2, 2.0 CaCl2 and 16 glucose. Tetrodotoxin (TTX) was purchased from the Research Institute of the Aquatic Products of Hebei (China). 4-Aminopyridine (4-AP), tetraethylammonium chloride (TEA-Cl), CsOH, CdCl, EGTA, Hepes and Mg-ATP were purchased from Sigma (USA), and other reagents were analytical research (AR) grade. All other drugs were diluted in oxygenated ACSF immediately prior to usage, and were applied via the perfusion system.
Electrophysiological recordings were performed at room temperature (22–24 °C). The standard pipette solution for recording sodium current contained (in mM): CsCl 140, MgCl2 2, Hepes 10, EGTA 10, TEA-Cl 20 and Mg-ATP 2, buffered to pH 7.2 with CsOH. The standard pipette solution for current-clamp experiments was (in mM): KCl 130, CaCl2 1, MgCl2 2, EGTA 10, Hepes 10 and MgATP 2, buffered to pH 7.2 with KOH. 4-AP (10 mM) and CdCl2 (200 lM) were added extracellularly to record voltage-gated sodium current (INa). For the whole-cell recording, slices were transferred into a recording chamber (1 ml volume) placed on the stage of a modified 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 filtered at 5 kHz and digitized at a sampling rate of 2 kHz. The series resistance was compensated at least 60%. The leakage and capacitive currents were subtracted on-line using a P/4 subtraction procedure. Data acquisition and analysis were performed with EPC10 patch-clamp amplifier (HEKA, Germany). The whole cell mode was established after giga-seal formation and membrane rupture. The neurons were allowed to stabilize for 3–5 min before starting the pulse protocols. A total of 42 neurons from 21 rats were analyzed in the present study. These neurons were identified as CA1 pyramidal neurons based on their location in CA1 pyramidal cell layer and the pyramidal shape of cell bodies. TTX-sensitive sodium current was obtained when cells were held at 100 mV, and applied with depolarizing pulses from 90 to +80 mV at 10 mV steps for 20 ms. The activated inward current was completely and reversibly blocked by bath application of 0.5 lM TTX (figures were not shown), indicating that the sodium channels expressed in hippocampal CA1 pyramidal neurons were TTX-sensitive.
2.3. Slice preparation
2.5. Data analysis
Male Wistar rats from the Experimental Animal Center of Chinese Academy of Medical Science were used on postnatal days 10–14. The experiments were conducted in accordance with the guidelines of the Medicine Experimental Animal Administrative Committee of Nation. The animals were anesthetized with Ketamine (0.1 mg/kg). The brain was quickly removed and immersed in the ice-cold ACSF. Horizontal slices that included the entire
All data were analyzed by Clampfit 9.0, Origin 8.0 and SPSS11.5. The values were represented as mean ± SEM and statistical comparisons were made using the Student’s paired t-test and oneway analysis of variance (ANOVA). P < 0.05 was considered significant.
2.2. Drug application
3. Results 3.1. Effects of nano-WC on the peak amplitude of INa
Fig. 1. Dispersion and characterization of nano-WC in ASCF. Nano-WC suspension (10 3 g/ml) was characterized by dynamic light scattering. The particle size distribution had a wide range from 115.26 to 703.70 nm due to the aggregation. The mean hydrodynamic diameter was 414.90 nm.
Effects of 10 4, 10 5 and 10 6 g/ml nano-WC solutions on the peak amplitudes of INa were tested. INa was evoked by a 40-ms single depolarizing pulse from 70 to 30 mV when neurons were held at 70 mV (Fig. 2A). The peak amplitudes of INa were normalized before and after nano-WC application (Fig. 2B). After the application of 10 4, 10 5 and 10 6 g/ml nano-WC on hippocampal slices, the amplitudes of INa were reduced to 59.22 ± 12.51 % (n = 6, P < 0.01), 70.53 ± 4.52 % (n = 6, P < 0.05) and 92.78 ± 7.06 % (n = 6, P > 0.05), respectively. Results showed that nano-WC reduced the peak amplitudes of INa in a dose-dependent manner. Consequently, the nano-WC suspension of 10 5 g/ml was chosen to observe its impact on INa.
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Fig. 2. Effects of 10 4, 10 5 and 10 6 g/ml nano-WC on peak amplitudes of INa. A, original traces of INa before and after application of different concentrations of nano-WC. INa was evoked by a 40-ms single depolarizing pulse from 70 to 30 mV when the neurons were held at 70 mV. B, effects of nano-WC of different concentrations on the peak amplitudes of INa obtained from 6 neurons. Data were presented as mean ± SEM (n = 6, ⁄P < 0.05, ⁄⁄P < 0.01 vs. controls).
3.2. Effect of nano-WC on the current–voltage curve of INa In the whole-cell patch-clamp model, INa was generated by applying pulses from 90 to +80 mV at 10 mV steps for 20 ms when neurons were held at 100 mV. Fig. 3A showed the original traces of INa before and after nano-WC application. The current– voltage (I–V) curves of INa in Fig. 3B showed that application of 10 5g/ml nano-WC markedly decreased the amplitude of INa from 50 to +50 mV (n = 6, P < 0.05). Moreover, the activation threshold of INa was 50 mV in the control condition, while changed to 60 mV in the presence of nano-WC. The voltage evoking the maximal peak INa was 30 mV in the control, while 40 mV after application of nano-WC. 3.3. Effect of nano-WC on steady-state activation curves of INa For steady-state activation curves of INa, currents at each test potential were converted to conductance (G) using the following formula G = I/(V Vk), where Vk is reversal potential. The peak conductance value for each test potential was normalized to Gmax and plotted against the test potential to produce a voltage-conductance
relationship curves, which was fitted using Boltzmann equation G/ Gmax = 1/{1 + exp[(V Vh)/k]}, where Vh is the voltage at which the conductance is half-maximal, and k is slope factor. The steadystate activation curves of INa before and after nano-WC application were shown in Fig. 4. The values of Vh for activation of INa were 50.49 ± 1.30 and 59.41 ± 0.90 mV before and after application of nano-WC (n = 6, P < 0.05), with a slope factor k of 4.05 ± 1.00 and 2.81 ± 1.36 (n = 6, P > 0.05), respectively. Results suggested that nano-WC produced a negative shift in the steady-state activation curves on INa with no effect on the slope factor. 3.4. Effect of nano-WC on the inactivation kinetics of INa To examine the impact of nano-WC on the inactivation kinetics of INa, currents were elicited with a 100 ms conditioning prepulse between 80 and +20 mV in 10 mV increments, followed by a 20 ms pulse of 30 mV, and holding potential at 70 mV. The inactivation curves were obtained through normalizing the test current amplitudes by taking the maximum value under each condition as unity. Steady-state inactivation curves were fitted with the Boltzmann equations G/Gmax = 1/{1 + exp[(V Vh)/k]}, where Vh is the
Fig. 3. The effect of nano-WC on INa. A, original traces of INa before and after application of 10 5 nano-WC. INa was generated by applying pulses from 90 to +80 mV at 10 mV steps for 20 ms when neurons were held at 100 mV. B, the effect of 10 5 nano-WC on I–V curves obtained from 6 neurons. Each point represents mean ± SEM (n = 6, ⁄P < 0.05 vs. controls).
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Fig. 4. The effect of nano-WC on the steady-state activation curve of INa. Peak amplitudes of INa obtained from 6 neurons were converted into conductance. The normalized conductance of VGSCs was plotted against the voltages of command pulses, and fitted with a Boltzmann function. Each point represents mean ± SEM (n = 6).
membrane potential of half-inactivation, and k is the slope factor. The Vh values were 34.10 ± 0.35 and 44.70 ± 2.38 mV before and after nano-WC application (n = 6, P < 0.05), and k values were 4.76 ± 0.69 and 7.69 ± 2.10 (n = 6, P > 0.06), respectively (Fig. 5). Results suggested that nano-WC produced a negative shift in the inactivation–voltage curve on INa with no effect on the slope factor. 3.5. Effect of nano-WC on the recovery of INa To test the impact of nano-WC on the recovery time course of INa from inactivation, the currents were obtained as the following protocol: neurons were held at 70 mV, and a 12 ms conditioning depolarizing pulse to 30 mV was applied to inactivate the sodium channels fully, then a 60 ms test pulse of 30 mV was applied after a series of 70 mV intervals varying from 2 to 24 ms. The peak value of INa evoked by the conditioning pulse was designated as I1, while the peak value of INa evoked by the test pulse was designated
Fig. 6. The effect of nano-WC on recovery from inactivation of INa. The currents were obtained when neurons were held at 70 mV, a 12 ms conditioning depolarizing pulse to 30 mV was applied to inactivate the sodium channels fully, and then a 60 ms test pulse of 30 mV was applied after a series of 70 mV intervals varying from 2 to 24 ms. The peak value of INa evoked from 6 neurons by the conditioning pulse was designated as I1, while the peak value of INa evoked by the test pulse was designated as I2. The ratio of I2 to I1 represents the recovery of INa from inactivation. Each point represents mean ± SEM (n = 6).
as I2. The ratio of I2 to I1 represents the recovery of INa from inactivation. The plot of I2/I1 vs. the duration of the 80 mV intervals was fitted with the monoexponential equation: I/Imax = A + B exp( t/s), where Imax is the maximal current amplitude, I is the current after a recovery period of Dt, s is the time constant and A is the amplitude coefficient. The s values were 0.80 ± 0.12 ms and 1.41 ± 0.16 ms before and after nano-WC application (n = 6, P < 0.05), respectively (Fig. 6). These results indicated that nanoWC delayed the recovery of INa from inactivation. 3.6. Effects of nano-WC on the action potential Effects of nano-WC on the action potential properties were examined in the mode of whole-cell current-clamp recordings. The action potentials (APs) were evoked using a 10 ms depolarizing current pulse when neurons were held at 70 mV. Fig. 7 showed that APs were evoked before and after nano-WC application. Because Na+ influx through voltage-gated sodium channels (VGSCs) determines the depolarization properties of the AP, the evidences occurring during the depolarization were tested before and after nano-WC applications. The results showed that, V-thresholds, peak amplitudes and overshoots were decreased (n = 6, P < 0.05) in the presence of nano-WC, but the spike half-widths had no changes (Table 1). 4. Discussion
Fig. 5. The effect of nano-WC on inactivation kinetics of INa. Currents were elicited with a 100 ms conditioning prepulse to potentials between 80 and +20 mV in 10 mV increments, followed by a 20 ms pulse of 30 mV, and holding potential at 70 mV. Peak amplitudes of INa obtained from 6 neurons were normalized and plotted vs. command potentials, and the data were fitted with Boltzmann function. Each point represents mean ± SEM (n = 6).
Studies found that nanomaterials of Cu, Ag, Al, ferric oxide and titanium dioxide could produce neurotoxicity (Sharma and Sharma, 2007; Hu et al., 2010, 2011; Wang et al., 2011). However, the acute neurotoxicity of nano-WC was not observed (Bastian et al., 2009). In order to find the neurotoxicity of nano-WC much earlier, the whole-cell patch-clamp technique was used to study the effects of nano-WC on electrophysiological characteristics of hippocampal CA1 neurons. In the present study, we observed that nano-WC attenuated the peak amplitudes of INa in a concentration-dependent manner, and the minimal effective concentration was 10 5g/ml. This suggested that nano-WC could influence INa of hippocampal CA1 neurons at a
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Fig. 7. The effect of nano-WC on the APs. The APs were evoked by brief depolarizing current pulses (10 ms, 100 pA) from a holding potential of application of nano-WC (B); C, the overlapped traces of A and B.
70 mV before (A) and after
Table 1 Effects of nano-WC on the APs. Group (n = 6) Control Nano-WC
V-threshold (mV) 40.12 ± 2.12 46.83 ± 4.04⁄
Peak amplitude (mV)
Overshoot (mV)
Spike half-with (ms)
92.08 ± 1.33 83.33 ± 1.28⁄
22.61 ± 0.92 15.76 ± 1.28⁄
3.90 ± 0.25 4.12 ± 0.33
Note: Values are mean ± SEM (⁄P < 0.05 vs. controls).
very low concentration. Consequently, it was observed that the pattern of I-V curve of INa was markedly changed after application of 10 5g/ml nano-WC suspension. This indicated that the kinetics of VGSCs on hippocampal CA1 neurons might be modified by nano-WC. VGSCs on neurons have the characteristics of fast activation and inactivation. Many neurotoxins can target VGSCs to cause neuronal impairment (Catterall 1980; Wang and Wang, 2003). The ultra small size and the particle shape enable nano-WC to reach the brain, so nano-WC could be a potential neurotoxin. By now it is not clear whether nano-WC could affect VGSCs. In our study, it was found that, both steady-state activation curves and inactivation curves of VGSCs were shifted negatively by nano-WC exposure. This suggested that nano-WC might bind with S4 segments of VGSCs to make the activation gates open earlier (Catterall, 2000; Horn, 2000; del Camino and Yellen, 2001), and bind to the fast inactivation gates of the VGSC, leading to the premature closing of VGSCs (McPhee et al., 1998; Strichartz et al., 1987; West et al., 1992). The premature opening and closing of VGSCs might contribute to the attenuated effect of nano-WC on the amplitudes of INa. Moreover, the recovery time of VGSCs was increased by nano-WC exposure, which indicated that nano-WC exposure delayed the conformational changes of the VGSCs from inactivation state to activation state. Based on the above analysis, we surmised that nano-WC could bind with VGSCs and modify their kinetics. VGSCs are responsible for the generation of APs, therefore, it could be speculated that APs of hippocampal CA1 neurons might be affected by nano-WC exposure. The results showed that after nano-WC application, V-thresholds, peak amplitudes and overshoots were reduced significantly. These results indicated that the excitability of hippocampal CA1 neurons was markedly promoted by nano-WC; however, the amplitudes of APs were reduced. Such changes of neurons in electrophysiological characteristics might disturb the transmembrane
ionic distribution, induce excess firing, influence signal transduction, inhibit the neurotransmitter release (Balser, 2001; Meisler and Kearney, 2005; Koopmann et al., 2006). As hippocampal neurons play important roles in cognitive functions, emotion, stress and regulations of visceral function, it could be predicted that nano-WC exposure might induce disorders in learning and memory, emotion and visceral activities. Actually, these disorders were the common problems caused by nanoparticle exposure (Warheit et al., 2004; Shvedova et al., 2005; Sharma and Sharma, 2007; Yokota et al., 2011). Therefore, we postulated that the abnormal changes of neurons in electrophysiological characteristics might be one of the toxic effects of nanoparticle exposure, consequently, these abnormal changes might served as one of the toxicological mechanisms leading to neural damage as well as the oxidative stress mechanism. In conclusion, it was found in this study that nano-WC exposure could influence some characteristics of APs evoked from the hippocampal CA1 neurons by modifying the kinetics of VGSCs, which was one of the toxic effects of nano-WC exposure. Acknowledgements This work was partly supported by the National Natural Science Foundation of China (31070890) and Tianjin Research Program of Application Foundation and Advanced Technology (10JCZDJC19100).
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Contents lists available at SciVerse ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Attenuated effect of tungsten carbide nanoparticles on voltage-gated sodium current of hippocampal CA1 pyramidal neurons Dehong Shan a,b, Yongling Xie a, Guogang Ren c, Zhuo Yang a,⇑ a
College of Medicine Science, Nankai University, Tianjin 300071, China Fundamental Medicine College, Liaoning University of Traditional Chinese Medicine, Shenyang 110032, China c Science and Technology Research Institute, University of Hertfordshire, Hatfield, Herts AL10 9AB, UK b
a r t i c l e
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Article history: Received 21 April 2012 Accepted 22 August 2012 Available online 29 August 2012 Keywords: Tungsten carbide nanoparticles Hippocampus Voltage-gated sodium current Action potential Neurotoxicity
a b s t r a c t Nanomaterials and relevant products are now being widely used in the world, and their safety becomes a great concern for the general public. Tungsten carbide nanoparticles (nano-WC) are widely used in metallurgy, aeronautics and astronautics, however our knowledge regarding the influence of nano-WC on neurons is still lacking. The aim of this study was to investigate the impact of nano-WC on tetrodotoxin (TTX)-sensitive voltage-activated sodium current (INa) of hippocampal CA1 pyramidal neurons. Results showed that acute exposure of nano-WC attenuated the peak amplitudes of INa in a concentration-dependent manner. The minimal effective concentration was 10 5 g/ml. The exposure of nano-WC significantly decreased current amplitudes of the current–voltage curves of INa from 50 to +50 mV, shifted the steady-state activation and inactivation curves of INa negatively and delayed the recovery of INa from inactivation state. After exposure to nano-WC, the peak amplitudes, overshoots and the V-thresholds of action potentials (APs) were markedly reduced. These results suggested that exposure of nano-WC could influence some characteristics of APs evoked from the hippocampal CA1 neurons by modifying the kinetics of voltage-gated sodium channels (VGSCs). Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Nanoscale materials and relevant products are now being widely used in a variety of areas, ranging from environmental remediation to engineering and medicine. Nanoparticles possess unique physicochemical properties, such as ultra small size, very large surface area to mass ratio and high reactivity. As nanoparticles can easily enter into the body through inhalation, ingestion and dermal penetration, in some sense, everyone might be at the risk of nanoparticle exposure. It has been found that nanoparticles can impair cells through the oxidative stress mechanism (Zhang et al., 1998, 2003; Brooking et al., 2001; Colvin, 2003; Oberdörster et al., 2005; Nel et al., 2006; Choi and Hu, 2008; Hsin et al., 2008). Therefore, the biosafety of nanomaterials becomes a great concern for the public, and the toxicological assessment is very necessary. Tungsten carbide (WC) is an inorganic chemical compound containing equal parts of tungsten and carbon atoms, and its hardness is close to that of the diamond. WC also has properties of high thermal and electrical conductivity, high resistance to corrosion, and high modulus of elasticity. For these excellent properties, WC is widely used in the military, aeronautics/astronautics and metal⇑ Corresponding author. Tel.: +86 22 23504364; fax: +86 22 23502554. E-mail address: [email protected] (Z. Yang). 0887-2333/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2012.08.025
lurgy. With the development of nanotechnique, WC nanoparticles (nano-WC) are produced to replace WC to improve the mechanical properties markedly. The demand of nano-WC is persistently growing owing to the rapid development in the national defense, civil and war industries throughout the globe. The basic form of WC is a fine powder, and levels of airborne particles are very high in the workplace, particularly close to production facilities and deposition. Occupational exposure to WC could cause dermatitis, bronchial asthma, fibrosing alveolitis, and lung cancer (Lison and Lauwerys, 1992; Moulin et al., 1998). WC exposure also had the mutagenic and apoptogenic potentials in human peripheral blood mononucleated cells (Lombaert et al., 2004). It is presumed that the toxicity of nano-WC might be more severe than that of WC because nanoscale particles have better permeability than that of bigger ones inside the body. However, studies of possible toxic effects for nano-WC are few, and much more work should be done to uncover the toxicity of nano-WC. What attracts our attention is the potential neurotoxicity of nano-WC because nanoparticles could enter into the brain through the olfactory neuronal pathway (Oberdörster et al., 2004; Elder et al., 2006). Studies found that, nanoparticles of Ag and ZnO could influence the electrophysiological characteristics of hippocampal CA1 neurons (Liu et al., 2009; Zhao et al., 2009). It is not clear whether nano-WC exposure could induce such effects. Therefore,
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the whole-cell patch-clamp technique was used in the present study to explore the impact of nano-WC on the electrophysiological characteristics of CA1 pyramidal neurons in hippocampal slices. 2. Materials and methods
hippocampus and subiculum (400 lm in thickness) were cut using a vibratome (VT1000 M/E, Leica, Germany) and incubated in ACSF for at least 1 h before transferring into the recording chamber. 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.
2.1. Nano-WC solution 2.4. Electrophysiological recordings WC nanoparticles used in the present study were synthesized and provided by QinetiQ Nanomaterials, Farnborough, Hampshire, UK. The particles were circular or oval shapes with the diameters of 5–20 nm. The stock solution (10 3 g/ml) of nano-WC in artificial cerebrospinal fluid (ACSF) was mixed by an ultrasonic (750 W) mixer (from Sonicator, US), with pulse 1 s on and 1 s off for 60 s, 50% of power input, for 20 min. The nano-WC suspension (10 3g/ml) was characterized by dynamic light scattering (DLS) using a ZetaPALS + BI-90Plus (Brookhaven Instruments Corp., USA) at a wavelength of 659 nm. The scattering angle was fixed at 90 °C. The particle size distribution had a wide range from 115.26 to 703.70 nm due to the aggregation, and the mean hydrodynamic diameter was 414.90 nm (Fig.1). The concentrations of nano-WC used in the experiment were 10 4, 10 5 and 10 6 g/ml diluted from the stock solution of nano-WC. However, the particles do settle after 1 h to the bottom of the container. By the time the tests were set up, the nano-WC suspensions were stirred on the vortex agitator for 20 min to recreate the uniform suspensions.
ACSF contains (in mM): 125 NaCl, 25 NaHCO3, 1.25 KCl, 1.25 KH2PO4, 1.5 MgCl2, 2.0 CaCl2 and 16 glucose. Tetrodotoxin (TTX) was purchased from the Research Institute of the Aquatic Products of Hebei (China). 4-Aminopyridine (4-AP), tetraethylammonium chloride (TEA-Cl), CsOH, CdCl, EGTA, Hepes and Mg-ATP were purchased from Sigma (USA), and other reagents were analytical research (AR) grade. All other drugs were diluted in oxygenated ACSF immediately prior to usage, and were applied via the perfusion system.
Electrophysiological recordings were performed at room temperature (22–24 °C). The standard pipette solution for recording sodium current contained (in mM): CsCl 140, MgCl2 2, Hepes 10, EGTA 10, TEA-Cl 20 and Mg-ATP 2, buffered to pH 7.2 with CsOH. The standard pipette solution for current-clamp experiments was (in mM): KCl 130, CaCl2 1, MgCl2 2, EGTA 10, Hepes 10 and MgATP 2, buffered to pH 7.2 with KOH. 4-AP (10 mM) and CdCl2 (200 lM) were added extracellularly to record voltage-gated sodium current (INa). For the whole-cell recording, slices were transferred into a recording chamber (1 ml volume) placed on the stage of a modified 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 filtered at 5 kHz and digitized at a sampling rate of 2 kHz. The series resistance was compensated at least 60%. The leakage and capacitive currents were subtracted on-line using a P/4 subtraction procedure. Data acquisition and analysis were performed with EPC10 patch-clamp amplifier (HEKA, Germany). The whole cell mode was established after giga-seal formation and membrane rupture. The neurons were allowed to stabilize for 3–5 min before starting the pulse protocols. A total of 42 neurons from 21 rats were analyzed in the present study. These neurons were identified as CA1 pyramidal neurons based on their location in CA1 pyramidal cell layer and the pyramidal shape of cell bodies. TTX-sensitive sodium current was obtained when cells were held at 100 mV, and applied with depolarizing pulses from 90 to +80 mV at 10 mV steps for 20 ms. The activated inward current was completely and reversibly blocked by bath application of 0.5 lM TTX (figures were not shown), indicating that the sodium channels expressed in hippocampal CA1 pyramidal neurons were TTX-sensitive.
2.3. Slice preparation
2.5. Data analysis
Male Wistar rats from the Experimental Animal Center of Chinese Academy of Medical Science were used on postnatal days 10–14. The experiments were conducted in accordance with the guidelines of the Medicine Experimental Animal Administrative Committee of Nation. The animals were anesthetized with Ketamine (0.1 mg/kg). The brain was quickly removed and immersed in the ice-cold ACSF. Horizontal slices that included the entire
All data were analyzed by Clampfit 9.0, Origin 8.0 and SPSS11.5. The values were represented as mean ± SEM and statistical comparisons were made using the Student’s paired t-test and oneway analysis of variance (ANOVA). P < 0.05 was considered significant.
2.2. Drug application
3. Results 3.1. Effects of nano-WC on the peak amplitude of INa
Fig. 1. Dispersion and characterization of nano-WC in ASCF. Nano-WC suspension (10 3 g/ml) was characterized by dynamic light scattering. The particle size distribution had a wide range from 115.26 to 703.70 nm due to the aggregation. The mean hydrodynamic diameter was 414.90 nm.
Effects of 10 4, 10 5 and 10 6 g/ml nano-WC solutions on the peak amplitudes of INa were tested. INa was evoked by a 40-ms single depolarizing pulse from 70 to 30 mV when neurons were held at 70 mV (Fig. 2A). The peak amplitudes of INa were normalized before and after nano-WC application (Fig. 2B). After the application of 10 4, 10 5 and 10 6 g/ml nano-WC on hippocampal slices, the amplitudes of INa were reduced to 59.22 ± 12.51 % (n = 6, P < 0.01), 70.53 ± 4.52 % (n = 6, P < 0.05) and 92.78 ± 7.06 % (n = 6, P > 0.05), respectively. Results showed that nano-WC reduced the peak amplitudes of INa in a dose-dependent manner. Consequently, the nano-WC suspension of 10 5 g/ml was chosen to observe its impact on INa.
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Fig. 2. Effects of 10 4, 10 5 and 10 6 g/ml nano-WC on peak amplitudes of INa. A, original traces of INa before and after application of different concentrations of nano-WC. INa was evoked by a 40-ms single depolarizing pulse from 70 to 30 mV when the neurons were held at 70 mV. B, effects of nano-WC of different concentrations on the peak amplitudes of INa obtained from 6 neurons. Data were presented as mean ± SEM (n = 6, ⁄P < 0.05, ⁄⁄P < 0.01 vs. controls).
3.2. Effect of nano-WC on the current–voltage curve of INa In the whole-cell patch-clamp model, INa was generated by applying pulses from 90 to +80 mV at 10 mV steps for 20 ms when neurons were held at 100 mV. Fig. 3A showed the original traces of INa before and after nano-WC application. The current– voltage (I–V) curves of INa in Fig. 3B showed that application of 10 5g/ml nano-WC markedly decreased the amplitude of INa from 50 to +50 mV (n = 6, P < 0.05). Moreover, the activation threshold of INa was 50 mV in the control condition, while changed to 60 mV in the presence of nano-WC. The voltage evoking the maximal peak INa was 30 mV in the control, while 40 mV after application of nano-WC. 3.3. Effect of nano-WC on steady-state activation curves of INa For steady-state activation curves of INa, currents at each test potential were converted to conductance (G) using the following formula G = I/(V Vk), where Vk is reversal potential. The peak conductance value for each test potential was normalized to Gmax and plotted against the test potential to produce a voltage-conductance
relationship curves, which was fitted using Boltzmann equation G/ Gmax = 1/{1 + exp[(V Vh)/k]}, where Vh is the voltage at which the conductance is half-maximal, and k is slope factor. The steadystate activation curves of INa before and after nano-WC application were shown in Fig. 4. The values of Vh for activation of INa were 50.49 ± 1.30 and 59.41 ± 0.90 mV before and after application of nano-WC (n = 6, P < 0.05), with a slope factor k of 4.05 ± 1.00 and 2.81 ± 1.36 (n = 6, P > 0.05), respectively. Results suggested that nano-WC produced a negative shift in the steady-state activation curves on INa with no effect on the slope factor. 3.4. Effect of nano-WC on the inactivation kinetics of INa To examine the impact of nano-WC on the inactivation kinetics of INa, currents were elicited with a 100 ms conditioning prepulse between 80 and +20 mV in 10 mV increments, followed by a 20 ms pulse of 30 mV, and holding potential at 70 mV. The inactivation curves were obtained through normalizing the test current amplitudes by taking the maximum value under each condition as unity. Steady-state inactivation curves were fitted with the Boltzmann equations G/Gmax = 1/{1 + exp[(V Vh)/k]}, where Vh is the
Fig. 3. The effect of nano-WC on INa. A, original traces of INa before and after application of 10 5 nano-WC. INa was generated by applying pulses from 90 to +80 mV at 10 mV steps for 20 ms when neurons were held at 100 mV. B, the effect of 10 5 nano-WC on I–V curves obtained from 6 neurons. Each point represents mean ± SEM (n = 6, ⁄P < 0.05 vs. controls).
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Fig. 4. The effect of nano-WC on the steady-state activation curve of INa. Peak amplitudes of INa obtained from 6 neurons were converted into conductance. The normalized conductance of VGSCs was plotted against the voltages of command pulses, and fitted with a Boltzmann function. Each point represents mean ± SEM (n = 6).
membrane potential of half-inactivation, and k is the slope factor. The Vh values were 34.10 ± 0.35 and 44.70 ± 2.38 mV before and after nano-WC application (n = 6, P < 0.05), and k values were 4.76 ± 0.69 and 7.69 ± 2.10 (n = 6, P > 0.06), respectively (Fig. 5). Results suggested that nano-WC produced a negative shift in the inactivation–voltage curve on INa with no effect on the slope factor. 3.5. Effect of nano-WC on the recovery of INa To test the impact of nano-WC on the recovery time course of INa from inactivation, the currents were obtained as the following protocol: neurons were held at 70 mV, and a 12 ms conditioning depolarizing pulse to 30 mV was applied to inactivate the sodium channels fully, then a 60 ms test pulse of 30 mV was applied after a series of 70 mV intervals varying from 2 to 24 ms. The peak value of INa evoked by the conditioning pulse was designated as I1, while the peak value of INa evoked by the test pulse was designated
Fig. 6. The effect of nano-WC on recovery from inactivation of INa. The currents were obtained when neurons were held at 70 mV, a 12 ms conditioning depolarizing pulse to 30 mV was applied to inactivate the sodium channels fully, and then a 60 ms test pulse of 30 mV was applied after a series of 70 mV intervals varying from 2 to 24 ms. The peak value of INa evoked from 6 neurons by the conditioning pulse was designated as I1, while the peak value of INa evoked by the test pulse was designated as I2. The ratio of I2 to I1 represents the recovery of INa from inactivation. Each point represents mean ± SEM (n = 6).
as I2. The ratio of I2 to I1 represents the recovery of INa from inactivation. The plot of I2/I1 vs. the duration of the 80 mV intervals was fitted with the monoexponential equation: I/Imax = A + B exp( t/s), where Imax is the maximal current amplitude, I is the current after a recovery period of Dt, s is the time constant and A is the amplitude coefficient. The s values were 0.80 ± 0.12 ms and 1.41 ± 0.16 ms before and after nano-WC application (n = 6, P < 0.05), respectively (Fig. 6). These results indicated that nanoWC delayed the recovery of INa from inactivation. 3.6. Effects of nano-WC on the action potential Effects of nano-WC on the action potential properties were examined in the mode of whole-cell current-clamp recordings. The action potentials (APs) were evoked using a 10 ms depolarizing current pulse when neurons were held at 70 mV. Fig. 7 showed that APs were evoked before and after nano-WC application. Because Na+ influx through voltage-gated sodium channels (VGSCs) determines the depolarization properties of the AP, the evidences occurring during the depolarization were tested before and after nano-WC applications. The results showed that, V-thresholds, peak amplitudes and overshoots were decreased (n = 6, P < 0.05) in the presence of nano-WC, but the spike half-widths had no changes (Table 1). 4. Discussion
Fig. 5. The effect of nano-WC on inactivation kinetics of INa. Currents were elicited with a 100 ms conditioning prepulse to potentials between 80 and +20 mV in 10 mV increments, followed by a 20 ms pulse of 30 mV, and holding potential at 70 mV. Peak amplitudes of INa obtained from 6 neurons were normalized and plotted vs. command potentials, and the data were fitted with Boltzmann function. Each point represents mean ± SEM (n = 6).
Studies found that nanomaterials of Cu, Ag, Al, ferric oxide and titanium dioxide could produce neurotoxicity (Sharma and Sharma, 2007; Hu et al., 2010, 2011; Wang et al., 2011). However, the acute neurotoxicity of nano-WC was not observed (Bastian et al., 2009). In order to find the neurotoxicity of nano-WC much earlier, the whole-cell patch-clamp technique was used to study the effects of nano-WC on electrophysiological characteristics of hippocampal CA1 neurons. In the present study, we observed that nano-WC attenuated the peak amplitudes of INa in a concentration-dependent manner, and the minimal effective concentration was 10 5g/ml. This suggested that nano-WC could influence INa of hippocampal CA1 neurons at a
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Fig. 7. The effect of nano-WC on the APs. The APs were evoked by brief depolarizing current pulses (10 ms, 100 pA) from a holding potential of application of nano-WC (B); C, the overlapped traces of A and B.
70 mV before (A) and after
Table 1 Effects of nano-WC on the APs. Group (n = 6) Control Nano-WC
V-threshold (mV) 40.12 ± 2.12 46.83 ± 4.04⁄
Peak amplitude (mV)
Overshoot (mV)
Spike half-with (ms)
92.08 ± 1.33 83.33 ± 1.28⁄
22.61 ± 0.92 15.76 ± 1.28⁄
3.90 ± 0.25 4.12 ± 0.33
Note: Values are mean ± SEM (⁄P < 0.05 vs. controls).
very low concentration. Consequently, it was observed that the pattern of I-V curve of INa was markedly changed after application of 10 5g/ml nano-WC suspension. This indicated that the kinetics of VGSCs on hippocampal CA1 neurons might be modified by nano-WC. VGSCs on neurons have the characteristics of fast activation and inactivation. Many neurotoxins can target VGSCs to cause neuronal impairment (Catterall 1980; Wang and Wang, 2003). The ultra small size and the particle shape enable nano-WC to reach the brain, so nano-WC could be a potential neurotoxin. By now it is not clear whether nano-WC could affect VGSCs. In our study, it was found that, both steady-state activation curves and inactivation curves of VGSCs were shifted negatively by nano-WC exposure. This suggested that nano-WC might bind with S4 segments of VGSCs to make the activation gates open earlier (Catterall, 2000; Horn, 2000; del Camino and Yellen, 2001), and bind to the fast inactivation gates of the VGSC, leading to the premature closing of VGSCs (McPhee et al., 1998; Strichartz et al., 1987; West et al., 1992). The premature opening and closing of VGSCs might contribute to the attenuated effect of nano-WC on the amplitudes of INa. Moreover, the recovery time of VGSCs was increased by nano-WC exposure, which indicated that nano-WC exposure delayed the conformational changes of the VGSCs from inactivation state to activation state. Based on the above analysis, we surmised that nano-WC could bind with VGSCs and modify their kinetics. VGSCs are responsible for the generation of APs, therefore, it could be speculated that APs of hippocampal CA1 neurons might be affected by nano-WC exposure. The results showed that after nano-WC application, V-thresholds, peak amplitudes and overshoots were reduced significantly. These results indicated that the excitability of hippocampal CA1 neurons was markedly promoted by nano-WC; however, the amplitudes of APs were reduced. Such changes of neurons in electrophysiological characteristics might disturb the transmembrane
ionic distribution, induce excess firing, influence signal transduction, inhibit the neurotransmitter release (Balser, 2001; Meisler and Kearney, 2005; Koopmann et al., 2006). As hippocampal neurons play important roles in cognitive functions, emotion, stress and regulations of visceral function, it could be predicted that nano-WC exposure might induce disorders in learning and memory, emotion and visceral activities. Actually, these disorders were the common problems caused by nanoparticle exposure (Warheit et al., 2004; Shvedova et al., 2005; Sharma and Sharma, 2007; Yokota et al., 2011). Therefore, we postulated that the abnormal changes of neurons in electrophysiological characteristics might be one of the toxic effects of nanoparticle exposure, consequently, these abnormal changes might served as one of the toxicological mechanisms leading to neural damage as well as the oxidative stress mechanism. In conclusion, it was found in this study that nano-WC exposure could influence some characteristics of APs evoked from the hippocampal CA1 neurons by modifying the kinetics of VGSCs, which was one of the toxic effects of nano-WC exposure. Acknowledgements This work was partly supported by the National Natural Science Foundation of China (31070890) and Tianjin Research Program of Application Foundation and Advanced Technology (10JCZDJC19100).
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