Effect of static magnetic fields on bacteria: Streptococcus mutans, Staphylococcus aureus, and Escherichia coli

Transcription

1 Pathophysiology 7 (2000) Effect of static magnetic fields on bacteria: Streptococcus mutans, Staphylococcus aureus, and Escherichia coli Masahiro Kohno a, *, Muneyo Yamazaki b, Isao Kimura b, Moriyasu Wada b a Application and Research Center, Analytical Instruments Di ision, JEOL LTD., 1-2 Musashino 3-Chome, Akishima, Tokyo , Japan b Nihon Uni ersity School of Dentistry at Matsudo, 870-1, Sakaecho, Nishi-2, Chiba , Japan Abstract Biological effect of static magnetic field was investigated by using ferrite magnets to conduct a magnetic field exposure experiment on three species of bacteria: Streptococcus mutans, Staphylococcus aureus, and Escherichia coli. The effects were evaluated by culturing the bacteria and determining their growth rate, the maximum numbers of bacteria, and [ 3 H]-thymidine incorporation. The results showed that the ferrite magnet caused strength-dependent decreases in the growth rate and growth maximum number of bacteria for S. mutans and S. aureus when cultured under anaerobic conditions, but that their growth was not inhibited under aerobic conditions. In addition, [ 3 H]-thymidine was added after culturing each of the species of bacteria for 18 h. After that, culture was continued until 24 h, and changes in [ 3 H]-thymidine incorporation were investigated. But no effect of the magnetic fields was detected. These findings suggested that oxygen related to growth the cases of S. mutans, S. aureus. However, no growth effects were detected on E. coli cultures Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bacteria; Ferrite magnet; Oxygen; Free radical 1. Introduction * Corresponding author. Tel.: ; fax: A variety of man-made magnetic fields, such as lowfrequency pulse magnetic fields (EMFs), exist in our everyday environment in addition to the Earth s magnetic field. For example, magnetic fields are generated in the environment by high-voltage power lines and electrical appliances, during diagnosis by magnetic resonance imaging (MRI) in clinical medicine, during testing of super-conducting magnetically levitated trains (maglev trains), and during bone fracture therapy. In addition, there are also many devices that use weak magnetic fields, e.g. portable telephones and personal computer displays [1 6]. For these reasons, it can be concluded that the occasions for exposure to even stronger magnetic fields than in the past have increased, and that we have entered an era in which it is impossible to overlook the relation between magnetic fields and biological function. Numerous effects of magnetic fields on the body have already been reported. For example, weak electromagnetic waves that do not cause Escherichia coli mutations or phenotypic transformation of cells, and that have no effect on the growth of chicken thoracic cartilage cells, are said to have no adverse effects on the body. In addition, in studies on bacteria, it has been reported that breakdown of hydrogen peroxide by the enzyme catalase accelerates in an 8T (Tesla) magnetic field, that this action contributes to dissolved oxygen and does not affect catalase directly [7]. In the field of dentistry, on the other hand, a method of treatment has been established that uses magnetic fields to anchor and stabilize partial dentures. In this method, a magnetic attachment is fitted to the few remaining teeth, and it anchors and stabilizes the false teeth. However, induction of an inflammatory response has been reported based on reports that the magnetic fields induce production of IL-1 and IL-6. On the other hand, induction fibroblasts to ossify has also been reported. Nevertheless, the effect of magnetic fields on periodontal tissue has never been examined in detail. In this study we combined square ferrite magnets of different magnetic strength developed by Tokunaga and reported last year, and prepared static magnetic fields of 30, 60, 80, and 120 mt? We then assessed the effect /00/$ - see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S (00)

2 144 M. Kohno et al. / Pathophysiology 7 (2000) Fig. 1. Schematic figure of aerobic culture system on the ferrite magnetic plate. of the magnetic fields on the growth of three species of bacteria, Streptococcus mutans, Staphylococcus aureus, and E. coli, and obtained interesting results reported below. 2. Materials and methods 2.1. Magnetic plates As reported last year, we prepared four types of ferrite magnet plates ( cm 3 ) having magnetic fields of different strength and used them in the experiment. Magnetic strength, which is a distinctive property of magnets, was 30, 60, 80, and 100 mt. The uniformity of the magnetic strength of the static magnetic fields on the surface of the plate was measured by using a magnetic field counter Cell cultures The bacteria used in this experiment were S. mutans, S. aureus, and E. coli. The bacteria were pretreated by culturing them for 18 h at 37 C in brain-heart infusion (BHI) broth by the gas-substitution (95% N 2,5%CO 2 ) anaerobic method, and then centrifuged at 400 g. The pellet was washed twice with 10 mm Tris HCl buffer Fig. 3. Survival CFU of S. aureus (Wood) and S. mutans (SG5) under the static magnetic fields (0, 60, 100 mt). Fig. 2. Schematic figure of aerobic culture system under static magnetic fields in anerobic glove box. (ph 7.4) at 400 g, so that none of the BHI ingredients remained, and it was then suspended in the same buffer. Next, suspensions of the pre-treated bacteria prepared by pouring aliquots into test tubes were smeared on BHI agar or mitis salivarius (MS) agar, and as shown in Fig. 1, after placing them on each of the different strengths of ferrite magnets and culturing them for 24 h at 37 C by the gas-substitution anaerobic method, they were diluted by the ordinary 10-fold serial

3 M. Kohno et al. / Pathophysiology 7 (2000) dilution method, and the viable CFUs of each of the bacteria were estimated Assessment of culture conditions and bactericidal effect The culture conditions of the individual culture media were as follows. Culture in MS agar was performed for 5 7 days at 35 C in an anaerobic glove box (80% N 2, 10% CO 2, 10% H 2 ). Culture in staphylococcus medium was performed aerobically at 37 C for 2 days. The cultures were then centrifuged (400 g, 5 min), and the pellet was washed with 10 mm Tris HCl buffer (ph 7.4) so that none of the culture medium ingredients remained. The bactericidal effect of the magnetic field strengths was assessed by placing ferrite magnets of different magnetic field strengths in the center in an anaerobic jar, as shown in Fig. 2, and then allowing test tubes into which final pellets had been placed (1 ml of 10 nm Tris HCl buffer) to stand on both sides and estimating the viable CFUs. When this was done, distances were created between the test tubes and the ferrite magnets, and the strength of the static magnetic fields decreased Assessment of DNA synthesis After culturing the different species of bacteria for 18 h, [ 3 H]-thymidine (Amersham) , 22.2 kbq/ well, was added and culture was continued, and [ 3 H]- thymidine uptake by the bacteria was measured at the end of 24 h. When incubation had been completed, the cultures were treated with PBS containing 0.25% trypsin and 0.02% EDTA, and [ 3 H]-thymidine incorporation was measured with a direct gas-flow type counter (Matrix TM96 Direct Counter, Packard Inst.). DNAsynthesizing ability was measured by assigning a value of 1.0 to thymidine incorporation by bacteria cultured in PBS buffer as a control and calculating the ratio of [ 3 H]-thymidine incorporation for each sample. 3. Results and discussion Fig. 4. Survival CFU of S. aureus (Cowen) under the static magnetic field (0 100 mt). It has been investigated that how magnetic fields produced by ferrite magnets affect two types of mutans bacteria. The experiments were conducted by preparing ferrite magnet plates having magnetic field strengths of 30, 60, 80, and 100 mt up to 5 mm from the surface of the plates. As shown in Fig. 3, in an experiment that was conducted by placing culture tubes on both sides of the incubator, the number of viable bacterial CFUs after 48 h of culture had decreased according to the strength of the magnetic field. Because magnetic field strength cannot be estimated accurately by the experimental method as is shown in Fig. 2, we performed the cultures on magnetic plates, as shown in Fig. 1. In this experiment the distance from the surface of the magnet plates was fixed. However, based on the number of bacteria after 48 h of culture, it was unclear whether the magnetic field effect was a bactericidal effect or growth inhibition. We then exposed the bacteria to 30, 60, 80, and 100 mt magnetic fields for different times in order to assess the effect of magnetic fields on the growth of the individual species of bacteria, and, as shown in Fig. 3 and Fig. 4, the growth of all of the species was found to be inhibited, compared to the controls, according to the strength of the magnetic field. This finding sug-

4 146 M. Kohno et al. / Pathophysiology 7 (2000) Fig. 5. Survival CFU of S. mutans (Smith) under the static magnetic field (0 100 mt). gested that the magnetic effect on mutans bacteria was growth inhibition, not a bactericidal effect. When these results were converted to the log phase at 18 h of culture, as shown in Fig. 5, a negative correlation was obtained with every species, and the magnetic field strength that kept the viable bacteria count at 1% or less in the current experiment was 120 mt (Fig. 6). Hashizume et al. already reported on a bactericidal effect of active oxygen on mutans, but the bactericidal effect was reported to be chiefly attributable to the hydroxyl radical ( OH). By contrast, there is also a report that magnetic field activity inhibits OH generation [8]. In order to elucidate differences in magnetic effect according to bacterial species, we conducted a similar experiment on E. coli, which are aerobic bacteria, but as shown in Fig. 7, no effect of the magnetic fields on bacterial growth was observed. Similarly, as shown in Fig. 8, no growth inhibition by magnetic fields was observed on mutans bacteria (Cowan) grown under aerobic conditions. This suggests that oxygen contributes to the magnetic effect on bacterial growth. Oxygen is a paramagnetic substance, and it may undergo kinetic inhibition in magnetic fields. The magnetic effect on bacteria is thought to be weak. It is well known that dissolved oxygen in the water existing metal ions such as ferric iron and cupric ion related to OH production. Therefore, it can be thought that radical reactions with these paramagnetic ion and oxygen are effected by magnetic fields. Although participation of active oxygen was not demonstrated by the present experiment, it at least suggested that there are large differences in the effect of magnetic fields on bacteria depending on whether dissolved oxygen is present. Upregulation of gene expression of matrix proteins by magnetic field activity has also been investigated in molecular biology research. We therefore assessed DNA-synthesizing ability during bacterial growth on the basis of [ 3 H]-thymidine incorporation, but found no difference from the control. This suggests that [ 3 H]- thymidine incorporation during growth in this experiment was close to normal, and that magnetic field activity has no effect on the ability of bacteria to synthesize DNA (Fig. 9). Fig. 6. Relationship between survival CFU of S. mutans (Smith) or S. aureus (Cowen) and strength of static magnetic fields.

5 M. Kohno et al. / Pathophysiology 7 (2000) Fig. 7. Growth curve of S. aureus (Cowen) under the static magnetic field (100 mt), ; without magnetic field,. Fig. 8. Growth curve of E. coli under the static magnetic field (100 mt), without magnetic filed control,. 4. Conclusion In any event, if magnetic fields are involved in decreasing dissolved oxygen and to OH synthesis, assuming that the action of the magnetic field is related to the behavior of the oxygen and active oxygen, magnetic fields may induce active oxygen formation. Involvement of nitrogen oxide ( NO) as a substance that controls cell membrane channels is also possible [9]. We are considering carrying out more detailed experiments in the future on the relation between magnetic fields and active oxygen and free radicals.

6 148 M. Kohno et al. / Pathophysiology 7 (2000) Fig. 9. Uptake of [3H] -thmidine in S. aureus (Cowen) (a) and S. mutans (Smith) under static magnetic field (100 mt). References [1] A.H. Frey, Electromagnetic field interactions with biological systems, FASEB J. 7 (1993) [2] Y. Omura, Electro-magnetic fields in the home environment (Color TV, computer monitor, microwave oven, cellular phone, etc) as potential contributing factor for the induction of oncogen C-fos AB1, Oncogen C-fos AB2, Integrin-a5 b1 and development cancer, as well as effects of microwave on amino acid composition of food and living human brain, Acupunct. Electrother. Res. Int. J. 18 (1993) [3] S.K. Dutta, M. Verma, C.F. Blackman, Frequency-dependent alternations in enolasa activity in Escherichia coli caused by exposure to electric and magnetic fields, Bioelectromagnetics 15 (1994) [4] M.A. Darendeliler, A. Darendeliler, M. Mandurino, Clinical application of magnets in orthodontics and biological implication: a review, Eur. J. Orthodon. 19 (1997) [5] G. Nindl, J.A. Swez, J.M. Miller, W.X. Balcavage, Growth stage dependent effects of electromegnetic field on DNA synthesis of Jurket cells, FERS Lett. 414 (1997) [6] K. Kubota, N. Yoshimura, M. Yokota, R.J. Fitzsimmons, U.M.E. Wikersjo, Overview of effects of electrical stimulation on osteogenesis and alverolar bone, J. Periodon. 66 (1995) 2 6. [7] S. Ueno, M. Iwasaka, Catalytic activity of catalase under strong magnetic fields of up to 8T, J. Appl. Phys. 79 (1996) [8] H. Hashizume, K. Kimura, T. Tomita, K. Matsushima, Y. Tujimoto, M. Ymazaki, Effects of sterilization of Streptococcus species by the hydroxyl radicals, J. Conserv. Density 37 (1994) [9] B. Brocklehurst, K.A. Maclauchlan, Free radical mechanism for effects of environmental electromagnetic field on biological systems, Int. J. Radic. Biol. 9 (1996)