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research-article |
Department of Biology, University of Genoa, 16132 Genova, Italy
ABSTRACT
Exposure of fertilized eggs of the sea urchin Paracentrotus lividus to an electromagnetic field of 75-Hz frequency and low amplitudes (from 0.75 to 2.20 mT of magnetic component) leads to a dramatic loss of synchronization of the first cell cycle, with formation of anomalous embryos linked to irregular separation of chromatids during the mitotic events. Because acetylcholinesterase (ACHE) is thought to regulate the embryonic first developmental events of the sea urchin, its enzymatic activity was assayed in embryo homogenates and decreased by 48% when the homogenates were exposed to the same pulsed field. This enzymatic inactivation had a threshold of about 0.75 ± 0.01 mT. The same field threshold was found for the effect on the formation of anomalous embryos of P. lividus. Moreover, ACHE inhibitors seem to induce the same teratological effects as those caused by the field, while blockers of acetylcholine (ACh) receptors are able to antagonize those effects. We conclude that one of the main causes of these dramatic effects on the early development of the sea urchin by field exposure could be the accumulation of ACh due to ACHE inactivation. The crucial role of the membrane in determining the conditions for enzyme inactivation is discussed.
acetylcholinesterase inactivation, anomalous embryos, early development, embryo, sea urchin, fertilization, LF-EMF effects, neurotransmitters, Paracentrotus lividus
The effects of extremely low-frequency electromagnetic fields (ELF-EMFs) on biological systems have been widely investigated. Several studies indicate that ELF-EMFs affect cell division timing and embryo development of many organisms [1–4], although a sound explanation of such effects is not available so far. A suitable experimental model for this kind of study is the well-known early development of the sea urchin Paracentrotus lividus, because embryos can be obtained in large numbers, are transparent, and display synchronous development [5, 6]. Exposure to ELF-EMFs can induce alterations in the timing of the cell cycle of sea urchins by advancing the first and second cell divisions [7]. A similar effect of acceleration of the cell cleavages was observed when sea urchin embryos were exposed to fields with a frequency of 5 kHz, well above the ELF range [8]. An opposite effect, although in different experimental conditions, was found after continuous exposure of sea urchin embryos to a cyclic 60-Hz magnetic field, which caused a significant developmental delay during the subsequent 23 h [9].
Herein, we report an investigation on the effect of an ELF-EMF of 75-Hz frequency on the early developmental events of the sea urchin P. lividus, from zygote to the eight-blastomere stage. A dramatic loss of synchronization of the first cell cycle was observed, with formation of anomalous embryos in the successive cleavages, linked to irregular separation of chromatids during the mitotic events.
To give an interpretation of the macroscopic effects produced by ELF-EMFs at the molecular level, our attention was focused on the enzymatic activity of acetylcholinesterase (ACHE). In fact, cholinergic molecules of the neurotransmitter system were found to play a role during fertilization and the early cell cycles of a large number of invertebrate and vertebrate organisms [10–13]. Addition of acetylcholine (ACh) to sea urchin eggs before fertilization can cause developmental anomalies [14, 15]. Moreover, the involvement of ACh binding to muscarinic receptors was proposed in the modulation of the nuclear status during the first cell cycle [14]. The enzyme ACHE, which was found in ovarian eggs of sea urchin using immunofluorescence [15] and whose activity was revealed after fertilization in cortical granules [16], is thought to regulate the embryonic first developmental events [17]. Therefore, ACHE activity of embryos was assayed and was found to be decreased when exposed to the same field, suggesting a strict correlation between the enzymatic inactivation and the formation of sea urchin anomalous embryos.
ACh chloride and 2,6-diamidine-4-phenylindole (DAPI) were from Sigma-Aldrich Co. (St. Louis, MO). All other reagents were of analytical grade.
Gamete Handling and Fertilization
Gametes from the sea urchin P. lividus were obtained in the laboratory, according to the usual methods: eggs were obtained by intracoelomic injection of 0.5 M KCl and were collected in ultrafiltered seawater (SW); sperm were collected "dry" and were stored at 4°C for no more than 2 days. For fertilization, 5 µl of the sea urchin sperm was added to 1 ml of SW, and 10 µl of this diluted sperm was added to six bowls (three for test and three for control), each containing about 2000 eggs in 20 ml of SW. Immediately after fertilization, samples were exposed to the ELF-EMF for the duration of the experiment as described herein. The extent of fertilization was checked at 30 sec to 1 min after addition of sperm; experiments were carried out only when more than 85% of eggs raised the fertilization layer within 5 min. Fertilization and development were carried out at 20°C in a temperature-controlled room. Experiments were repeated five times, in different seasonal periods. For each experiment, eggs and sperm of five adult specimens were used. At the end of exposures, embryos were stained, and three slides for each test were prepared. For each slide, three random microscope fields were analyzed, and anomalous embryos were counted vs. normal ones. The count was stopped when 110 embryos per microscope field (20x) were counted, analyzing 330 embryos for each slide. In total, about 1000 embryos per bowl were counted. Statistical elaboration of data was performed, including percentage of embryos, SD, and Student-Neuman-Keuls test.
Embryo Homogenate Preparations
Eggs were fertilized, and embryos were frozen at –80°C after 70 min (corresponding to the first division). Thawed embryos (1 ml) were homogenized in a small glass-glass potter on ice in ultrafiltered SW. The sample was centrifuged at 3500 rpm for 15 min at 4°C, and the pellet (2 mg/ml of protein) was resuspended in ultrafiltered SW and used for the assay of ACHE activity. Protein content was determined by the method of Bradford [18].
Electromagnetic Field Production
EMFs were produced using equipment (Biostim Igea, Modena, Italy) mainly used for clinical application and already described [19, 20]. The generator system supplies a square wave with a maximal applied tension of 180 V, a period of 13.3 milliseconds (75-Hz frequency), a duty cycle of 10%, to a couple of identical coils aligned on a central axis. Each coil, made up of 1000 turns of copper wire with a diameter of 0.2 mm, had internal and external diameters of 72.5 mm and 82.5 mm, respectively. Because the current supplied by the equipment to the coils was not adjustable, different flux density magnetic fields were obtained by separating the two coils with different distances. Measurements of the magnetic field B maximal intensity with a gaussmeter showed that it was fairly constant midway between the coils, giving values of about 2.20 ± 0.08 mT when the distance between the coils was 12 cm and about 0.45 ± 0.02 mT at 29.6-cm distance. An error of 0.5 cm in determining the distance between the coils gave an error of 4% in the field measurements. The values of the field near the threshold were determined more accurately (1% SD, n = 10). Because of an erroneous calibration of the gaussmeter, the values of magnetic fields that appear in previous publications [20, 21] in which the same exposure system was used should be multiplied by a factor of 9. The time course of the magnetic field and that of the induced electric field were measured as already described [19, 20] at the center of the distance between the two coils where the samples were placed. Temperature variations of the order of magnitude of experimental error (± 0.1°C) were measured with a thermocouple inside the sample in the presence of the applied field.
About 2000 eggs were fertilized in 20 ml of ultrafiltered SW in a glass cuvette, and immediately after fertilization, an aliquot of 10 ml was exposed for 150 min to the ELF-EMF in a glass Petri dish (5-cm diameter) put at the center of the exposure system as already described. The remaining 10 ml of the sample was considered a control not exposed to the field. The control and the sample were maintained in the same conditions of temperature, light, and evaporation for the duration of the experiment. After 150 min, 10 ml of paraformaldehyde (4%) was added to the control or to the sample to stop the development of sea urchins. These rates were used for morphological and histochemical analyses.
Experiments were repeated eight times, using gametes obtained from different adult specimens collected from the wild (Portofino bay) in different periods.
The morphology of the embryos was analyzed on a Wild stereomicroscope. Photographs were taken with a Leitz microscope, equipped with a photographic apparatus (Leica, I).
Embryos stored in methanol were rehydrated with 0.1 M citrate buffer, pH 7. The buffer was replaced twice and then was substituted with a solution of DAPI 1:1000 in citrate buffer and incubated for 5 min in the dark. The DAPI causes fluorescence of DNA, binding to A-T clusters in the small DNA furrow. After rinsing with citrate buffer, samples were mounted on a slide supplied with antifading Gelvatol [22] and observed on a Zeiss microscope equipped with UV (wavelength, 
= 360–400 nm).
ACHE activity of embryo homogenate preparations was measured using ACh chloride (Sigma) as a substrate by monitoring the choline production as already described [23]. Briefly, embryo homogenate preparations (30 µg of total protein) were added to a reaction mixture (240 µl) containing 100 mM phosphate buffer, pH 7.2, without the substrate and were exposed to the field. The enzymatic activity was measured during the exposure to the field of 75 Hz by adding 10 µl of 0.25 mM acetylcholine. The reaction was blocked by withdrawal of 100 µl of reaction mixture, to which was added 50 µl of 25% PCA (perchloric acid). Samples clarified by centrifugation were neutralized with 50 µl of 2 M K2CO3. Centrifugation was repeated to remove salt. Control samples were run in the same experimental conditions but in the absence of field. Neutralized extracts were used for choline assay by adding 0.5 mM DTNB (5,5'-Dithiobis(2-nitrobenzoic acid)) in phosphate buffer, pH 7.2 (1-ml final volume), following the formation of nitrobenzoate by spectrophotometrically monitoring the rise in absorbance at 405 nm. Optical densities were converted to rates of nitrobenzoate production by using the molar extinction coefficient of 1.33 mmol–1 mm–1.
An ELF-EMF (75 Hz) of 2.20 ± 0.08 mT magnetic amplitude was applied soon after fertilization of sea urchin eggs for the duration of the experiments. At about 150 min after fertilization, while the controls showed the usual synchronous development corresponding to embryos at eight-blastomere stage (Fig. 1A), about 80% of the exposed samples showed shape anomalies, such as membrane wrinkles and a loss of synchronization: in Figure 1, some embryos showed a deceleration of development to four blastomeres (Fig. 1B), while others were able to develop to eight blastomeres (Fig. 1C) but with a wrinkled and opaque surface. The DAPI fluorescent staining of blastomeres revealed irregular chromatin distribution and size in about 80% of the exposed samples (Fig. 1, B1 and C1) compared with the usual distribution of the control (Fig. 1A1). Panels D, E, and F of Figure 2 show a sort of polarization of chromatin, with the spindles oriented along the same direction. Furthermore, the nuclear material seems to be irregularly distributed (Fig. 2B), fragmented (Fig. 2G), or loosely diffused in the cytoplasm (Fig. 2, C, H, and I).
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Similar results were obtained using different amplitudes of the 75-Hz field. Figure 3A shows that the effects of a field amplitude of 1.02 ± 0.04 mT already appeared at the first 10 min of exposure with a percentage of anomalous embryos of about 80%, which remained almost constant during the experiment. Figure 3B shows a typical example of cleavage deregulation: at 10 min of field exposure, three DAPI spots appeared in a cell with a nucleus fragmentation; at 45 min, the cell showed an acceleration with a sort of quadruple cleavage, while at 60 min, a cell appeared with two nuclei and a nuclear fragment. At 150 min, the cleavage seemed decelerated, with irregular chromatin distribution, as already shown in the preceding figures. When the field amplitude was further decreased to 0.45 ± 0.02 mT, the exposed samples showed no effects and were indistinguishable from the controls.
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To connect the observed development anomalies of sea urchin eggs to the cholinergic system, the effects of the same ELF-EMF on ACHE activity were measured. Exposure of embryo homogenates to the same field of 75 Hz, 2.2 mT caused a 48% drop in the activity of ACHE, from 9.2 units/mg (±0.4 SD of five measurements) of the control samples to 4.8±0.5 units/mg of the exposed samples. This effect was all or none, independent of the exposure time (range, 5–150 min) and of the field amplitude (range, 2.2–0.80 mT). As shown in Figure 4, an amplitude of about 0.80 ± 0.01 mT was the lowest value that produced a suprathreshold response, while amplitudes of 0.45 mT or 0.75 ± 0.01 mT gave results of enzymatic activities that were statistically indistinguishable from those of the controls (P < 0.001). Enzymatic inhibition was fully reversible on removal of the field and was independent of the exposure time.
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Figure 4 shows the macroscopic effects of different field amplitudes on sea urchin development. At 0.45 mT or 0.75 ± 0.01 mT, the exposed samples showed the same low percentage of anomalous embryos of the controls, while at 0.80 ± 0.01 mT or 1.80 ± 0.07 mT, the percentage of anomalous embryos was about 80%, as for the exposure to 2.2 mT and 1.02 mT amplitudes shown in Figure 1 and in Figure 3. The amplitude of 0.80 ± 0.01 mT was the lowest value that produced a suprathreshold response. This result shows that the effects of field exposure on the sea urchin development have the same amplitude threshold found for the effect on ACHE activity.
To test whether this enzyme indeed functionally mediates between the field and the observed effects on the cells, BW284c51 (1,5-bis [4-allyldimethylammoniumphenyl] pentan-3–1 dibromide) or physostigmine, which are well known ACHE inhibitors, was added to sea urchin eggs soon after fertilization. Such inhibitors affected the DNA structure of 80% of embryos compared with controls (P < 0.005) (Fig. 5). This result may suggest a similarity of action between the treatments with the ACHE inhibitors and the electromagnetic field. However, analysis of the transduction pathways is needed to demonstrate the mechanism of the phenomenon. Conversely, the field effects could be prevented by adding blockers of muscarinic and nicotinic ACh receptors such as atropine and tubocurarine chloride, respectively. Figure 6 shows the development of embryos sampled at 180 min after fertilization at the stage of 32 blastomeres; 100% of embryos exposed to the field in the presence of these blockers are identical to the controls.
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The reversibility of the field effects on embryo development was also investigated. Figure 7 shows that the first 5 min of exposure to the 75-Hz field are sufficient to trigger the anomalies in chromatin distribution sampled at the stage of 150 min after fertilization. Moreover, different times of field exposure seem to have the same effects. After several hours (up to 36 h) from the withdrawal of the field, the embryos did not resume their usual prism shape of normal development. They assumed instead anomalous "spheroid" forms similar to those already described in experiments run in the presence of organophosphorous compounds that are ACHE inhibitors [24].
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The results reported herein show that ELF-EMFs of 75-Hz frequency and low intensities are able to produce strong anomalies in the early development of the sea urchin P. lividus. The field effects were detected morphologically and using DAPI fluorescent staining, which revealed irregular chromatin distribution and size. Alterations in the early embryonic development of the sea urchin due to exposure to magnetic fields have been reported, and differences in the observed effects could be due to difference in the experimental conditions used by various authors. For instance, the choice of field frequency is crucial as the interaction of the electromagnetic wave with molecules can only operate through frequency windows. Therefore, different frequencies can produce different effects. Field frequencies of 5 kHz [8] and 450 MHz [25], which are well above the ELF range, produced an acceleration of the cleavage of embryo cells and an increase in the number of zygotes with abnormal fertilization envelopes, respectively. Fields of 60-Hz frequency, close to the 75 Hz used in our experiments, caused opposite alterations in the timing of the cell cycle of sea urchin embryos, namely, a significant developmental delay [9] or an advance of first and second cell divisions [7], a discrepancy that can be explained by the different experimental conditions set up by the two groups. However, the alterations in the early embryonic development of sea urchin were never shown at the chromosome level by means of DAPI staining. Moreover, we tried to correlate the teratological effects with ACHE inactivation by the field exposure. In fact, ACHE activity was measured in embryo homogenate and was found to be decreased by the field exposure. Moreover, the formation of anomalous embryos had a field threshold of about 0.75 mT, the same threshold found for the inhibitory effect on ACHE activity. This suggests a close correlation between the enzymatic and morphological effects of the applied field. Accumulation of ACh due to ACHE inactivation by the field exposure could be the trigger for the formation of anomalous embryos. The hypothesis that ACHE could functionally mediate between the field and the observed effects on embryos is strengthened by the results shown in Figure 5, in which ACHE inhibitors seem to induce the same teratological effects as those caused by the field. Conversely, blockers of ACh receptors are able to antagonize the effects of the field, giving further support to the biochemical model.
The interactions between molecules related to the cholinergic signal system and the first phases of embryonic development have been investigated [10, 26]. Addition of ACh to zygotes of P. lividus after fertilization led to embryos with anomalous nuclear and chromatin structure [14], similar to those shown in Figure 1. The hypothesis was proposed that muscarinic receptors may be involved in the (presumably Ca2+ dependent) modulation of the nuclear status during the first cell cycles [14]. The correlation between ionic and cytoskeletal dynamics is crucial for the correct embryonic development, particularly for leading the nuclear dysjunction during cleavage [27], ultimately depending on ACh availability. A link between [Ca2+]i variations and the regulation of cytoskeletal elements and events during the first cleavages of sea urchin zygotes and early embryos has been demonstrated [28–30]. The inhibition of ACHE is known to cause an overflow of ACh at the receptor sites, affecting the nicotinic and muscarinic receptor state. In the early stages of development of sea urchins, these receptors are known to be involved in sodium [31, 32] and calcium [14] intracellular dynamics. The intracellular concentration of calcium spike, for instance, is in turn responsible for the chromosome metaphasic plate formation by acting on the aster microtubular structure [28–30].
With regard to the mechanisms underlying the effects of ELF-EMFs on membrane-associated enzymes, a unified physical-chemical explanation has not been provided yet, despite the large number of reports available [33–40]. It was previously found that a field of 75 Hz decreased only the activities of some lipid-linked enzymes such as alkaline phosphatase or ACHE, while it had no effects on the activities of integral membrane enzymes (such as calcium, Na/K, adenosine triphosphatase, and succinic dehydrogenase) or of peripheral membrane enzymes (such as photoreceptor phosphodiesterase) [21]. The decrease in enzymatic activity of the lipid-linked enzymes was independent of the time of permanence in the field and was completely reversible. When these enzymes were solubilized with Triton X-100, no effect of the field was obtained on the enzymatic activity, suggesting the crucial roles of membrane and lipid linkage in determining the conditions for enzyme inactivation.
ACHE and alkaline phosphatase are anchored to the membrane through a glycosylphosphatidylinositol, which allows the protein to move along the membrane surface to meet the substrate molecules. Changing the conditions of the lipid matrix where the enzyme is inserted through its anchor may have a profound effect on the protein flexibility, a requisite for its functional activity. A recent report [41] on alkaline phosphatase incorporated into artificial phospholipid monolayers showed that the formation of aggregated protein clusters, due to a surface pressure increase, caused a drop in enzymatic activity of about 40%. Considering that a significant clustering of the distribution of membrane proteins was induced by a pulsed field of 50 Hz [42], we can speculate that the 75-Hz field used in our experiments would be able to induce a partition of the lipid-anchored enzymes in clusters, with a subsequent activity decrease.
Finally, this article should attract attention on the risks that biological organisms could receive from exposure to electromagnetic fields with frequencies usually generated by high-voltage and low-voltage transmission lines, display terminals, laboratory instrumentations, and home appliances. In the case of ACHE, the effects of accumulation of ACh persist presumably even after the removal of the field and the consequent reversible restoration of ACHE activity. In other words, although the effects of ELF-EMFs are likely transient, the consequences of the exposure can be irreversible.
Correspondence: 1 Isidoro M. Pepe, Department of Biology, University of Genoa, Viale Benedetto XV 3.5, Genova 16132, Italy. FAX: 39 010 353 8153; e-mail: mario.pepe{at}unige.it
Received: 31 January 2006.
First decision: 6 March 2006.
Accepted: 28 August 2006.
REFERENCES
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