Bioelectromagnetics 31:270^276 (2010)
Sinusoidal ELF Magnetic Fields Affect Acetylcholinesterase Activity in Cerebellum Synaptosomal Membranes Silvia Ravera,1 Bruno Bianco,2 Carlo Cugnoli,3 Isabella Panfoli,1 Daniela Calzia,1 Alessandro Morelli,1 and Isidoro M. Pepe1* 1
2
Department of Biology, University of Genoa, Genoa, Italy Department of Electronic and Biophysical Engineering, University of Genoa, Genoa, Italy 3 Institute of Cybernetics and Biophysics, C.N.R., Genoa, Italy
The effects of extremely low frequency magnetic fields (ELF-MF) on acetylcholinesterase (AChE) activity of synaptosomal membranes were investigated. Sinusoidal fields with 50 Hz frequency and different amplitudes caused AChE activity to decrease about 27% with a threshold of about 0.74 mT. The decrease in enzymatic activity was independent of the time of permanence in the field and was completely reversible. Identical results were obtained with exposure to static MF of the same amplitudes. Moreover, the inhibitory effects on enzymatic activity are spread over frequency windows with different maximal values at 60, 200, 350, and 475 Hz. When synaptosomal membranes were solubilized with Triton, ELF-MF did not affect AChE activity, suggesting the crucial role of the membrane, as well as the lipid linkage of the enzyme, in determining the conditions for inactivation. The results are discussed in order to give an interpretation at molecular level of the macroscopic effects produced by ELF-MF on biological systems, in particular the alterations of embryo development in many organisms due to acetylcholine accumulation. Bioelectromagnetics 31:270– 276, 2010. 2009 Wiley-Liss, Inc. Key words: ELF magnetic exposure; acetylcholinesterase activity; magnetic field exposure; ELF-MF effects on enzymes
INTRODUCTION The effects of extremely low frequency magnetic fields (ELF-MF) on nerve cells of different organisms have been extensively studied [Seegal et al., 1989; Stuchly and Esselle, 1992; Azanza and Del Moral, 1994; Coelho et al., 1995; Wieraszko, 2004; Marchionni et al., 2006]. Results are highly variable and contradictory due to the different experimental conditions and the intrinsic phenomenological complexity. Moreover, a generally accepted theoretical model able to explain these phenomena is lacking. A contribution to simplify the matter could be that of investigating the molecular level of the macroscopic effects produced by ELF-MF. In fact, studies on the central nervous system showed a significant activity change with various types of neurotransmitters, such as acetylcholine, dopamine, serotonin, and amino acids, in the brain of animals exposed to EMF [Vasquez et al., 1988; Seegal et al., 1989; Lai et al., 1993; Graham et al., 1999; Jelenkovi et al., 2005]. Our attention focused on the enzymatic activity, which is known to be underlying many biological processes. The hypothesis that ELF-MF could affect enzyme activities was tested on a number of soluble 3 2009 Wiley-Liss, Inc.
enzymes [Litovitz et al., 1991; Zhang and Berg, 1992; Dutta et al., 1994; Thumm et al., 1999; Harada et al., 2001] as well as on membrane-associated enzymes [Blank and Soo, 2001]. Among the latter, two lipidlinked enzymes such as alkaline phosphatase and acetylcholinesterase (AChE) lowered dramatically their activities by about 54% and 58%, respectively [Morelli et al., 2005], when exposed to ELF-MF generated by a square wave of 75 Hz. Moreover, the effects of the applied field on these enzymes vanished when they were mildly solubilized by Triton, suggesting that the membranes could have an important role for ————— — Grant sponsor: Italian Ministry of Research MIUR (PRIN 2007 program). *Correspondence to: Isidoro Mario Pepe, DISTBIMO, University of Genoa, 16132 Genoa, Italy. E-mail:
[email protected] Received for review 18 February 2009; Final revision received 22 July 2009 DOI 10.1002/bem.20563 Published online 29 December 2009 in Wiley InterScience (www.interscience.wiley.com).
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mediating the effect of the field on the enzymatic activity [Morelli et al., 2005]. The effects of MF on AChE are important. In particular for the nerve cells, acetylcholine acts as a transmitter for cell–cell communications. A deficiency of acetylcholine seems present in Alzheimer’s disease and an uptake of AChE inhibitors is a common treatment. On the other hand, inhibition of AChE activity leads to alterations of embryo development in many organisms due to acetylcholine accumulation. Experiments on fertilized eggs of sea urchin made by our group showed that exposure to MF of 75 Hz frequency and 2.5 mT amplitude dramatically affected embryo development by altering both cell cycle clock and chromosome pattern [Ravera et al., 2006]. In this paper the effects of ELF-MF on AChE activity were further studied using a sinusoidal field with 50 Hz frequency, or a static MF with amplitudes from 0.5 to 2.0 mT. The results showed that AChE activity is lowered by about 27% when exposed to sinusoidal or static MF with thresholds of about 0.74– 0.80 mT. Moreover, the inhibitory effects on enzymatic activity are spread over frequency windows with different maximal values. MATERIALS AND METHODS Magnetic Field Exposure System A suitable current to generate the MF was carried by a 100-turn coil wound around a plexiglass cylindrical sample holder of 2 cm diameter and 6 cm height. This coil of copper wire (0.5 mm diameter, 0.6 mm with insulator) was supplied with AC voltage produced by a wave generator (sweep/function generator model TG 320, Thurlby Thandar Instruments, Huntingdon, UK) and amplified by an integrated amplifier (Sony TA-1140, Tokyo, Japan, frequency response: 10 Hz–35 kHz 0.2 dB) as shown in Figure 1. At a given point P inside the solenoid, the
Fig. 1. Schematic representation of the experimental exposure system.
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MF B was calculated with the formula [Stratton, 1941, p. 232]: I m i dl ^ r B¼ 0 4p l r 3 where m0 is the permeability constant of the vacuum, i is the electric current, l is the length of the solenoid, and r is the distance between P and dl, a vector element of the coil wire. For instance, with 1 mA of current measured with an amperometer with an error of 5%, the calculated maximal component of the MF at the center of the solenoid was B ¼ 1.98 mT with an error of about 5%. The sample (1 ml) was put in a polycarbonate cuvette of 1 cm 1 cm base and 4 cm height, which was then placed in the center of the sample holder. The amplitude of the MF calculated in the central region filled with the sample (1 cm3) was uniform and differences were less than 1%. The radial component in this region was negligible. The exposure system was kept at a constant temperature of 25 8C by a thermostat, with flowing water in contact with the external surface of the cylindrical sample holder, around which the isolated coils of copper wire were wound. The inside of the sample holder was kept open to the air. The control samples were not exposed to the field but were kept in the sample holder without any current flowing through the wires. Temperature variations of the order of magnitude of experimental error (0.1 8C) were measured with a thermocouple placed inside the sample during the experimental measurements. The MF was calculated with the above formula because no gaussmeter was available with a small head suitable to measure the field inside the polycarbonate cuvette. However, measurements with our gaussmeter (Mag-03SVC, Bartington, Witney, UK, with probe Mag-03MS250, square head of 3 cm 3 cm) outside or inside a polycarbonate beaker exposed to MF showed identical results, suggesting that the polycarbonate cuvette containing the experimental samples was not altering the applied MF. The mean electric component of the electromagnetic field was calculated with the formula Ems ¼ (1/2) ro Brms [Amaroli et al., 2006] where r is the radius of the sample (0.005 m), o ¼ 2pf ¼ 314.16 r/s. Exposure to static MF was done using the same system supplied with a continuous current (ELVI model 32 DC power supply, ELVI Elettrotecnica, Delebio, Italy). In order to avoid exposure to AC generated MF component and to keep the time of the system constant at about 104 s, the samples were put into the cuvette 5 min after the DC was turned on. Bioelectromagnetics
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Synaptosomal Preparation For synaptosomal preparation, the method published by Rawls et al. [1999] was followed, with minor modifications. For each preparation, six dorsal striata were removed from cerebellum of decapitated Mus musculus mouse and pooled in ice-cold 100 mM Tris buffer pH 7.4. The striata were transferred to 0.32 M unbuffered sucrose and manually disrupted in a glass homogenizer with a Teflon pestle. The homogenate was centrifuged at 3000g for 2 min. The supernatant was centrifuged at 14000g for 12 min. The soft pellet was resuspended in 0.32 M sucrose containing 10 mM N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid (TES, pH 4), and 4 ml aliquots were loaded onto discontinuous gradients consisting of three layers of Ficoll (Sigma–Aldrich, St. Louis, MO) in sucrose (w/v; 12%, 4 ml; 9%, 1 ml; 6%, 4 ml). The gradients were centrifuged at 62483g for 35 min. Synaptosomes were harvested from the 9% layer, diluted with 3 volumes of 10 mM TES buffer containing 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.2 mM Na2HPO4, 1 mM MgCl2, and 10 mM glucose (pH 7.4), and centrifuged at 14000g for 12 min. Synaptosomes were resuspended in 0.5 ml of 10 mM TES buffer (pH 7.4). The preparation was divided into aliquots containing 0.8 mg of protein and centrifuged at 10000g for 2 min. Synaptosomes were overlaid with 200 ml of 0.25 M sucrose containing 5 mM TES (pH 7.4) and stored as pellets at 80 8C. Acetylcholinesterase Activity AChE activity was measured by using acetylthiocholine chloride (Sigma–Aldrich) as substrate and monitoring the thiocholine production as described by Bergmeyer and Grassl [1983] Synaptosomal preparations (100 mg of protein) were added to a reaction mixture containing 0.4 mM acetylthiocholine in 50 mM phosphate buffer, pH 7.2. The final volume of 1 ml was kept in a spectrophotometer cuvette of 1 cm 1 cm (width depth) at the middle of the electromagnetic field. At different time intervals, aliquots of 100 ml were withdrawn and added to 50 ml of 25% perchloric acid (PCA). Control samples were run in the same experimental conditions as above, but in the absence of field. Each sample was centrifuged for 3 min at 14000 rpm and then 100 ml of supernatant was withdrawn and neutralized with 50 ml 2 M K2CO3. Centrifugation was repeated to remove potassium perchlorate. Aliquots of 100 ml of each neutralized extract were used for thiocholine assay by adding 0.5 mM 5,50 -Dithiobis(2-nitrobenzoic acid) (DTNB) in phosphate buffer, pH 7.2 (1 ml final volume). Thiocholine reaction with DTNB gave thionitrobenzoate formation (e405 ¼ 1.33 mM/cm) which was Bioelectromagnetics
monitored spectrophotometrically by following the rise in absorbance. RESULTS Effects of Sinusoidal MF on AChE In order to measure the effects of sinusoidal MF of 50 Hz frequency on the AChE activity, a sample containing the synaptosomal preparation was placed at the center of the cylindrical sample holder of the exposure system and kept at a constant temperature of 25 8C by a thermostat. The experiment started by adding the substrate to the sample which was immediately exposed to the MF. Figure 2 shows the time course of the product formation in the absence or presence of the field. It can easily be seen that the enzymatic activity appears reduced during the first 5 min exposure to an alternating field of maximal magnetic component of about 1.0 mT. In fact, the quantity of product calculated on the straight lines (y ¼ 0.86x for the control and y ¼ 0.61x for the exposed sample) resulted in about 0.86 U/mg (mmol thiocholine produced/min/mg protein) for the control sample and about 0.61 U/mg for the sample exposed for 5 min to the field, corresponding to a decrease in AChE activity of about 29%. After 5 min exposure, the field was withdrawn and the enzymatic activity returned to the same slope of the control values indicating that the effect is reversible. In a different experiment, the synaptosomal preparation containing AChE, but not the substrate, was kept in the field (up to 30 min) and then, soon after withdrawal of the field, the substrate acetylcholine was immediately added and the enzymatic activity assayed. The results were identical to those of the controls, indicating that the effect of the field on the enzyme was completely reversible.
Fig. 2. Thiocholine production (mmol/mg protein) by AChE activity in the absence (*, &) or in the presence (&) of a sinusoidal field of 50 Hz frequency and 1.0 mT maximal amplitude. Each point represents the mean of three measurements on the same membrane preparation.
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A more precise measurement of the AChE activity decrease was run with different samples of the same synaptosomal preparation. Two identical aliquots of the same sample were used: one was exposed for 1 min to a field of 1.0 mT, and the other one was kept as control without the field. The results of 10 measurements were the following: the samples exposed to the field gave a mean activity value of 0.67 0.03 U/mg while those in the absence of field gave 0.93 0.06 U/mg with a decrease of 27% (4%) with a statistical significance of P < 0.001. When the same experiments were run on the synaptosomal preparation solubilized by 0.1% Triton, the results showed that the activity of the mildly solubilized AChE was not affected by the field (0.91 0.05 U/mg without the field; 0.95 0.05 U/mg with the field of 50 Hz, 1.0 mT) as already reported for AChE and for other lipid-linked enzymes exposed to a 75 Hz field [Morelli et al., 2005]. The experiments were usually run with AChE activities above the Km ¼ 0.095 mM [Bergmeyer and Grassl, 1983]. The AChE activity measured at the Km resulted in about (0.45 0.05 U/mg). In order to further investigate the effects of the field on AChE, experiments were run with different enzymatic activities. Figure 3 shows that the activity inhibition of about 27% remained unchanged when AChE activity was decreased by lowering the substrate concentrations below the Km. Amplitude Threshold The decrease of the enzymatic activity was studied using the same frequency of 50 Hz with different amplitudes of the applied field. Figure 4 shows
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Fig. 4. Enzymatic activity of AChE measured during 1min exposure to 50 Hz magnetic field of different amplitudes. Each value represents the mean SD of five measurements.
that a field amplitude of about 0.74 0.04 mT was the lowest value that produced a supra-threshold response. Instead, amplitudes of 0.7 or 0.2 mT gave results of enzymatic activities statistically similar to those of the controls. The effect of the field on the AChE activity seems independent of its amplitude, because it appears to maintain the same value for field amplitudes varying from 0.74 to 2.0 mT. Experiments With Different Frequencies AChE activity was measured under exposure of sinusoidal MF of different frequencies and of 1.0 mT maximal amplitude. Figure 5 shows the AChE activity exposed to different field frequencies in the range from 10 to 650 Hz. The first maximal decrease of enzymatic activity (about 30%) is between 50 and 75 Hz. Other frequency windows show maximal effect on AChE activity at 200, 350, and 475 Hz. Effects of Static Magnetic Fields on AChE In order to rule out the effect of the small electric component of the sinusoidal field on the decrease of
Fig. 3. Effect of sinusoidal field of 50 Hz frequency and 1.0 mT amplitude on different AChE activities. E/C, the ratio of enzyme activity with the field/without the field versus the enzyme activity of control samples (without the field).The AChE activity of 0.45 U/mg was measured at the Km (0.095 mM). Values of enzymatic activities below 0.02 U/mg were not significant due to their high experimental errors. Each plotted value represents the mean SD of five measurements.
Fig. 5. Enzymatic activity of AChE measured during 1min exposure to magnetic fields of 1.0 mT amplitude and different frequencies. Each value represents the mean SD of five measurements. Bioelectromagnetics
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Fig. 6. Effects of static magnetic fields of different amplitudes and 50 Hz frequency on AChE activity measured during 1min exposure. Each value represents the mean SD of five measurements.
AChE activity, the samples were exposed to a static MF obtained by supplying the exposure system with a continuous current. The results of AChE activity under exposure to a field of 1.0 mT were identical to those shown in Figure 2 obtained with sinusoidal field with 50 Hz frequency. The decrease of AChE activity was calculated to be 27% (4%) with a statistical significance of P < 0.001. In order to measure a threshold for this effect, the decrease of the enzymatic activity was studied with different amplitudes of the constant MF. Figure 6 shows that a field amplitude of about 0.80 0.04 mT was the lowest value that produced a supra-threshold response. Instead, amplitudes from 0.76 to 0.40 mT gave results of enzymatic activities statistically similar to those of the controls. Also in this case, the effect of the field on the AChE activity seems independent of its amplitude, because it appears to maintain the same value for static field amplitudes varying from 0.80 to 1.50 mT. DISCUSSION The results of this work show that sinusoidal MF of 50 Hz frequency and an amplitude threshold of maximal magnetic component of about 0.74 mT (0.04 mT), decrease AChE activity by about 27%. The effect of the field on enzymatic activity seems independent of the exposure time and was detectable only during the field exposure. No effects were measured immediately after the withdrawal of the field, suggesting a perfect reversibility of the mechanism. Moreover, the decrease of the enzymatic activity shown in Figure 2 was independent of the time of permanence in the field and appeared constant within the experimental errors under exposure lengths of 1–5 min, up to 10 min (data not shown). In other words, the same decrease in the enzymatic activity was measured independently of the exposure time, indicating that Bioelectromagnetics
the effect of the field starts within the first minute of exposure and remains constant during all the field permanence. The action of the field seems to switch the enzyme to a state of a reduced activity which is immediately restored when the field is removed. The effect of the field on enzymatic activities seems also independent of its amplitude above the threshold of 0.74 mT, up to 2.0 mT, as well as for an amplitude of 3.0 mT (data not shown). The decrease of AChE activity of about 27% is lower than that of 58% reported in a previous publication [Morelli et al., 2005] where the AChE activity was exposed to an ELF-MF of 75 Hz. This discrepancy could be due to the exposure system, which, in the latter case, supplied a square wave with a period of 13.3 ms (75 Hz of frequency) and a duty cycle of 10%, to a couple of Helmotz coils aligned on a central axis. The Fourier analysis of this wave showed different harmonic components. As shown in Figure 5, in addition to 75 Hz, other frequencies showed inhibitory effect on AChE activity. The decrease of about 58% obtained with the square wave may be due to additive effects of different frequency components on the enzymatic activity. The static MF seem to have the same effect of that produced by sinusoidal MF of 50 Hz, that is an AChE activity decrease of about 27%. The effect also seems to have the same threshold amplitude, because 0.74 (0.05 mT) for the sinusoidal field and 0.80 (0.05 mT) of the constant field are statistically indistinguishable. This suggests that the inhibitory effect of sinusoidal field on AChE activity is due only to the magnetic component. The electric component, in fact, is very low, being on the order of 0.78 mV/m as calculated from the formula E ¼ (1/2)roB [Amaroli et al., 2006] where r is the radius of the sample (0.005 m), o ¼ 2pf ¼ 314.16 r/s and B ¼ 1 mT. The effect of the sinusoidal field of 50 Hz on AChE activity vanished when the enzyme was mildly solubilized by 0.1% Triton, as already reported for AChE and for other lipid-linked enzymes [Morelli et al., 2005] exposed to a square wave of 75 Hz. These results strongly suggest that the field action is mediated by the membrane organization and structure, which is crucial in determining the conditions of the enzyme inactivation. The fact that the activity of the mildly solubilized AChE was not changed by the field would suggest also that the field does not directly affect the enzyme activity. This is in accordance with the measurements shown in Figure 3, where the activity decrease of about 27% was independent on enzyme activities, being induced by the field at different substrate concentrations above and below the Km. However, the effect of the field on the membrane is not
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sufficient alone to explain the decrease in enzymatic activity. AChE, as well as carbonic anhydrase and alkaline phosphatase, are known to be anchored to the membrane through a glycosylphosphatidylinositol. On the other hand, integral membrane enzymes such as CaATPase and Na/K-ATPase, or peripheral membrane enzymes such as phosphodiesterase (PDE), were not affected by the field [Morelli et al., 2005], suggesting also the importance of the linkage to the membrane. However, some authors reported that Na/K-ATPase or cytochrome oxidase were found to increase its enzymatic activity by approximately 30% and 45%, respectively, under exposure to a pulsed MF of 60 Hz with an amplitude of only 10 mT [Blank and Soo, 2001] with a threshold ever lower for Na/K-ATPase activity [Blank and Soo, 1996; Blank, 2005]. In those papers the enzymatic activities were kept lower than those reported in our experiments where the activities were measured above the Km. On the other hand, when we exposed AChE to similar low fields (from 10 to 500 mT, 50 Hz) we were not able to detect any change either in the maximal enzymatic activity (about 0. 8 mmol thiocholine/min/mg protein) or in much lower activities (down to about 0.05 mmol thiocholine/min/mg protein). In a recent report [Caseli et al., 2005], the enzymatic activity of alkaline phosphatase incorporated into artificial phospholipid monolayers dropped about 40% following a lipid phase transition from liquid-expanded to liquid-condensed state, when surface pressure increased above 18 mN/m. Fluorescence microscopy revealed that above this pressure, proteins aggregated and formed clusters which would affect the substrate accessibility to the catalytic site and, therefore, decrease the enzymatic activity. It has also been reported that a pulsed field of 50 Hz is able to induce a decrease in lipid molecular dynamics of cell membranes [Volpe et al., 1998] and significant clustering of the distribution of the membrane proteins [Bersani et al., 1997]. Therefore, we can speculate that the field of 50 Hz would be able to induce a transition of the lipid bilayer to a more liquid ordered phase, as suggested by the sharp threshold behavior shown in Figure 4, followed by the partition of the lipid-anchored enzymes in clusters with a subsequent activity decrease. The results presented in this paper are important for the interpretation at molecular level of macroscopic effects produced by ELF-EMF on biological systems. There are several studies indicating that ELF-EMF affect cell division timing and embryo development of many organisms [Delgado et al., 1982; Dixey and Rein, 1982; Koch et al., 1993]. In particular, the effects of low-intensity electromagnetic fields on the early development of the sea urchin Paracentrotus lividus [Falugi et al., 1987] could be interpreted as due to AChE
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inhibition. A recent report on exposure of fertilized eggs of the sea urchin P. lividus to an electromagnetic field of 75 Hz frequency shows a dramatic loss of synchronization of the first cell cycle, with formation of anomalous embryos linked to irregular separation of chromatides during the mitotic events [Ravera et al., 2006]. Moreover, AChE activity assayed in embryo homogenates was found to decrease by 48% when exposed to the same pulsed field of 75 Hz, with the same threshold of about 0.75 mT found for the effect on the formation of anomalous embryos [Ravera et al., 2006]. The conclusion was that one of the main causes of the dramatic effects on the early development of the sea urchin by field exposure could be the accumulation of acetylcholine due to AChE inactivation. Moreover, the effects of accumulation of acetylcholine last presumably even after the removal of the field and the consequent reversible restoration of AChE activity. In other words, even though the effects of ELFEMF are likely transient, the consequences of the exposure may not be overlooked because they can be irreversible. On the other hand, some field bioeffect could be positive. It was reported that cholinesterase inhibitors, such as methanesulfonyl fluoride, improve cognitive performance in patients with senile dementia of the Alzheimer type [Moss et al., 1999]. In this case, ELF-MF may be of some help for treatment of the disease as a mild tool without pharmacological side effects. REFERENCES Amaroli A, Trielli F, Bianco B, Giordano S, Moggia E, Corrado MUD. 2006. Effects of a 50 Hz magnetic field on Dictyostelium discoideum (Protista). Bioelectromagnetics 27:528– 534. Azanza MJ, Del Moral A. 1994. Cell membrane biochemistry and neurobiological approach to biomagnetism. Prog Neurobiol 44:517–601. Bergmeyer HU, Grassl MW. 1983. Enzymes. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis, Vol. 2. Weinheim: Verlag Chemie. pp. 126–281. Bersani F, Marinelli F, Ognibene A, Matteucci A, Cecchi S, Squarzoni S, Maraldi NM. 1997. Intramembrane protein distribution in cell cultures is affected by 50 Hz pulsed magnetic fields. Bioelectromagnetics 18:463–469. Blank M. 2005. Do electromagnetic fields interact with electrons in the Na,K-ATPase? Bioelectromagnetics 26:677–683. Blank M, Soo L. 1996. The threshold for Na,K-ATPase stimulation by electromagnetic fields. Bioelectrochem Bioenerg 40: 63–65. Blank M, Soo L. 2001. Optimal frequencies for magnetic acceleration of cytochrome oxidase and Na,K-ATPase reactions. Bioelectrochemistry 53:171–174. Caseli L, Oliveira RG, Masui DC, Furriel RP, Leone FA, Maggio B, Zaniquelli MED. 2005. Effect of molecular surface packing on the enzymatic activity modulation of an anchored protein Bioelectromagnetics
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