A 50 Hz, 14 mT magnetic field is not mutagenic or co-mutagenic in bacterial mutation assays

A 50 Hz, 14 mT magnetic field is not mutagenic or co-mutagenic in bacterial...

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A 50 Hz, 14 mT magnetic field is not mutagenic or co-mutagenic in bacterial mutation assays a Bio-Science Department, Abiko Research Laboratory, Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko-city, Chiba 270-1194, Japan b Environmental Biotechnology Laboratory, Railway Technical Research Institute, 2-8-38 Hikari, Kokubunji-city, Tokyo 185-8540, Japan

Abstract

We used bacterial mutation assays to assess the mutagenic and co-mutagenic effects of power frequency magnetic fields

(MF). For the former, we exposed four strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and two strains of Escherichia coli (WP2 uvrA,W P2 uvrA/pKM101) to 50Hz, 14mT circularly polarized MF for 48h. All results were negative.Forthelatter,wetreatedS.typhimurium(TA98,TA100)andE.coli(WP2uvrA,WP2uvrA/pKM101)cellswitheight model mutagens (N-ethyl-N0-nitro-N-nitrosoguanidine, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, 4-nitroquinoline-N-oxide, 2-aminoanthracene, N4-aminocytidine, t-butyl hydroperoxide, cumen hydroperoxide, and acridine orange) with and without the MF. The MF induced no significant, reproducible enhancement of mutagenicity. We also investigated the effect of MF on mutagenicity and co-mutagenicity of fluorescent light (ca. 900lx for 30min) with and without acridine orange on the most sensitive tester strain, E. coli WP2 uvrA/pKM101. Again, we observed no significant difference between the mutation rates induced with and without MF. Thus, a 50Hz, 14mT circularly polarized MF had no detectable mutagenic or co-mutagenic potential in bacterial tester strains under our experimental conditions. Nevertheless, some evidence supporting a mutagenic effect for power frequency MFs does exist; we discuss the potential mechanisms of such an effect in light of the present study and studies done by others. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Magnetic fields; Power frequency; Bacterial mutation assay; Mutagenesis; Co-mutagenesis

1. Introduction

Public concern regarding the health effects of power frequency electric and magnetic fields (EMFs) has been increasing since an epidemiological study in 1979 reported an increased cancer risk in children

Corresponding author. Tel.: C81-471-82-1181/ext. 8628; fax: C81-471-83-3347. E-mail addresses: nakasono@criepi.denken.or.jp (S. Nakasono), ikehata@rtri.or.jp (M. Ikehata). 1 Co-corresponding author. Tel.: C81-42-573-7316; fax: C81-42-573-7349.

living near power lines [1]. Since then, many studies have been conducted in an effort to explain the findings, and some recent research suggests that weak power frequency fields (50/60Hz) may exert a biological effect. The NIEHS working group report [2] of the EMF Research and Public Information Dissemination Program stated that extremely low frequency (ELF) EMFs are possibly carcinogenic to humans. A mechanism for EMF carcinogenesis, however, has not been forthcoming, and some laboratory results are in disagreement [2–4].

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McCann et al. reviewed the literature on the genotoxic potential of EMFs in 1993 and again in 1998 [3,4]. Most of the reports showed that ELF EMFs ranging from 0.15 to 5mT were not genotoxic. Nevertheless, some reports were positive. A 50Hz, 400mT MF increased the mutation frequency in MeWo cells [5], and a 60Hz, 0.1–0.5mT MF inhibited DNA repair in rat brain cells [6,7] in a reaction that could be blocked by free radical scavengers [7], suggesting involvement of oxygen radicals. In third-instar larvae of Drosophila melanogaster,a strong static MF (5T, 24h exposure) increased the frequency of somatic recombination, a finding that could be suppressed by Vitamin E, again suggesting involvement of oxygen radicals [8]. Although those studies suggest a biologically plausible mechanism for an MF effect, i.e. lengthening or shortening of radical lifetime, no mechanism has yet been clearly demonstrated. More mechanism studies are needed.

Bacterial mutation assays [9] are simple standard tests commonly used to screen chemicals and environmental contaminants for mutagenic activity. Assays in Salmonella typhimurium showed that a 100Hz, 0.2mT MF and a 0.3Hz triangular wave MF (0.08mT) do not affect mutagenic frequency (tester strain TA100) [10,1], nor do 0.3mT MF of 60, 600, or 6000Hz (TA97a, TA98, TA100, and TA102) [12]. We recently reported the co-mutagenic potential of a strong static MF (5T, 48h exposure) with the tryptophane auxotroph Escherichia coli WP2 uvrA [13]. Exposure doubled the mutagenicity of some compounds, such as N-ethyl-N0-nitro-N-nitrosoguanidine, probably due to an effect on radical behavior, repair enzymes and the membrane permeability of the chemicals. Here we report on the co-mutagenic potential of high density power frequency MFs in bacterial mutation assays.

On the assumption that in bacterial mutation assays, a MF effect on the mutation rate or related chemical reactions would be more apparent at a higher density, we evaluated the mutagenicity and co-mutagenicity of a 50Hz, 14mT circularly polarized MF. This MF density is several hundred times higher than densities found in homes, and it is tens of thousand times higher than the density that caused the considerable health effects reported in the 1979 epidemiological study [1].

2. Materials and methods 2.1. Exposure system

For exposures, it is possible to generate a high density ELF-MF of up to 10mT (root mean square) for both the vertical and horizontal axes [14,15]. We determined the exact MF density from the output current of the power source. This system generates a large and uniform MF in the exposure space (40cm3, field variation below 5%). All assay exposures were to a 50Hz, 14mT circularly polarized MF for 48h. An incubator with a water jacket was located in the center of the system, and the temperature was maintained at 37 0:2 C. In a sham exposure system, the stray field from the exposure coils was less than 0.01mT.

2.2. Chemicals

N-ethyl-N0-nitro-N-nitrosoguanidine (ENNG, CAS no. 4245-7-6), t-butyl hydroperoxide (BH, CAS no. 75-91-2), cumen hydroperoxide (CH, CAS no. 80-15-10), and 4-nitroquinoline-N-oxide (4-NQO, CAS no. 56-57-5) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2-(2-Furyl)-3-(5-nitro- 2-furyl) acrylamide (AF-2, CAS no. 3688-53-7), acridine orange (AO, CAS no. 10127-02-03), and 2-aminoanthracene (2AA, CAS no. 613-13-8) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). N4-aminocytidine (AC, CAS no. 57294-74-3) was purchased from Funakoshi Ltd. (Tokyo, Japan). The chemicals were selected to represent various classes of mutagens: ENNG, AF-2, and 4-NQO are DNA binding agents, 2AA is a promutagen that binds to DNA following activation by liver S9 enzymes, AC is a base analog, BH and CH are hydroxyl radical precursors, and AO is a photosensitizer for generating active oxygen upon exposure to light.

Nutrient broth (Oxoid, nutrient broth no. 2) was purchased from Unipath Ltd. (Hampshire, UK). Bacto-agar was purchased Difco Laboratories (Detroit, MI, USA). The S9 mix, prepared from the livers of phenobarbital- and 5,6-benzoflavone-treated male Sprague-Dawley rats, was purchased from Kikkoman Co. (Chiba, Japan). Other reagents were laboratory-grade materials purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). All reagents

Table 1 Genotypes of the tester strains used in this study

Strain Amino acid marker Other relevant mutations Plasmid

Mutation Type of mutation Main DNA target DNA repair Cell wall

S. typhimurium

TA98 his D 3052 Frameshift GC uvrB rfa pKM101 TA100 hisG4 6 Base pair substitution GC uvrB rfa pKM101 TA1535 hisG4 6 Base pair substitution GC uvrB rfa – TA1537 his C 3076 Frameshift GC uvrB rfa –

E. coli

WP2 uvrA trp E 56 Base pair substitution AT uvrA C – WP2 uvrA/pKM101 trpE5 6 Base pair substitution AT uvrA C pKM101 were of the highest grade and were used as they were received.

2.3. Bacterial strains

The four S. typhimurium strains (TA98, TA100,

TA1535, TA1537) were provided by Dr. H. Shimizu of the Jikei University School of Medicine, Tokyo, who had received them from Dr. B.N. Ames of the University of California, Berkeley. The two strains of E. coli (WP2 uvrA and WP2 uvrA/pKM101) were provided by Dr. T. Matsushima of the University of Tokyo. Table 1 shows the strain genotypes.

2.4. Mutagenicity assays

The plate incorporation method [9,16] was used with four strains of S. typhimurium and two of E. coli. The cell suspension (0.1ml of preculture containing (1–3/ 108 cells) was mixed with phosphate buffer (0.5ml) and top agar (2ml). The mixture was plated onto minimal glucose agar plates (90mm diameter). The plates were incubated for 48h at 37 C with MF exposure (except for the controls), then revertant colonies were scored. Each experiment was repeated at least three times.

2.5. Co-mutagenicity assays

The plate incorporation method [9,17,18] was used, with a slight modification. Mutagen doses were those that, in preliminary experiments, induced from a doubling of the background revertant rate to up to 500 revertants per plate (Table 3). The compounds were dissolved in DMSO (ENNG, 4-NQO, AF-2, 2AA, and CH) or distilled water (AC, AO, and BH). Cell suspensions (0.1ml of preculture (1–3/ 108 cells), mutagen solution (0.1ml), and phosphate buffer (0.5ml) were diluted with top agar (2ml), and the mixture was plated onto minimal glucose agar plates (90mm diameter). When 2AA was the mutagen, S9 mixture (0.5ml) was added in place of phosphate buffer. In the control experiment, solvent (0.1ml) was used in place of mutagen solution. Six plates were prepared and randomly divided into two groups. One group was incubated at the center of the MF exposure system, and one was placed in a control incubator. Revertant colonies were scored after 48h. Each experiment was repeated four times.

2.6. Effect of MF on mutagenicity of light irradiation with and without AO

The effect of MF on mutation induction by light irradiation was investigated with and without AO (25mg per plate). Plating procedures were carried out under dark-red light. Bacteria were exposed to fluorescent light for 0, 5, 15, 30, or 60 min, then to the MF for 48h, then scored. The fluorescent light intensity was 8 11lx (mean S.D.) (Lux-Meter; Model ANA-9, Tokyo Photo-Electric Co. Ltd.), and it included UV-A at 0.9mW/cm2 (UV-A Light Meter; Model UVA-365, Kastam Co. Ltd., Tokyo) and UV-C at below 0.1mW/cm2 (UV-C Light Meter; Model UV-C 254, Kastam Co. Ltd., Tokyo). The intensity of UV-B was not measured. Each experiment was repeated three times.

Table 2 Effect of magnetic field on mutagenicity in S. typhimurium (TA1535, TA1537, TA98, and TA100), and E. coli (WP2 uvrA and WP2 uvrA/pKM101)a

Strain Experiment Revertant colonies per plate Ratio (exposure/control)

Strain Experiment Revertant colonies per plate Ratio (exposure/control)

Control Exposure Control Exposure

a Values represent mean S.D. No statistical difference was found in all experiments. b Each test used at least nine plates. c Each test used at least three plates.

2.7. Statistical analysis

We used the Student t-test to determine statistical significance.

3. Results 3.1. Mutagenicity of power frequency MFs

Table 2 shows the results for the MF-exposed groups and the unexposed control groups. The number of revertant colonies was almost the same in the replicate tests for each strain. The ratio of the number of revertant colonies in the exposed group to the number in the control group approximated unity in all strains, and statistical analysis indicated no significant differences between the groups (Table 2). The revertant colonies in the exposed and unexposed groups were similar in shape and size (data not shown).

3.2. Co-mutagenicity of power frequency MFs

Table 3 shows the ratios of the number of revertants in the exposed groups to the number in the control groups. Except for AC, which was not mutagenic for TA 98, all of the chemicals were mutagenic to the four strains tested. E. coli WP2 uvrA/pKM101 was the most sensitive strain for all the chemicals (data not shown). The ratios of the revertant number in the MF-exposed group to the revertant number in the unexposed group ranged from 0.6 to 1.4. Some significant differences were found between the MF-exposed and MF-unexposed groups, but they were not reproducible in replicate experiments. The largest apparent effect was observed in TA98 with AC, and that was not statistically significant. Positive results were observed in TA100 with BH and in WP2 uvrA/pKM101 with AC, but neither of those results was reproducible. Moreover, tests using other strains with the same chemicals were negative. The results suggest that the two positive tests were false, and that the MF was not co-mutagenic.

In the case of AO, some statistically significant results were observed for each strain, with the largest effect seen in WP2 uvrA (Table 3). The effect was an inhibitory one. Reproducibility of the results was carried out in the next well-controlled experiment (shown in the next section).

3.3. Effect of power frequency MF on mutagenicity of light irradiation with and without AO

We used the most mutagen-sensitive strain, WP2 uvrA/pKM101, to test the effect of MF exposure on

Table 3 Effect of magnetic field on co-mutagenicity in S. typhimurium (TA98 and TA100), and E. coli (WP2 uvrA and WP2 uvrA/pKM101)

Strain Mutagen Dose (mg per plate) Experimenta 12 34

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