Mutagenicity and co-mutagenicity of static magnetic fields detected by bacterial mutation assay

Mutation Research 427 Ž1999. 147–156 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres

Mutagenicity and co-mutagenicity of static magnetic fields detected by bacterial mutation assay Masateru Ikehata a

a,)

, Takao Koana a , Yuji Suzuki b, Hidesuke Shimizu b, Masayoshi Nakagawa c,1

EnÕironmental Biotechnology Laboratory, Railway Technical Research Institute, 2-8-38, Hikari, Kokubunji City, Tokyo 185-8540, Japan b Department of Public Health and EnÕironmental Medicine, Jikei UniÕersity School of Medicine, 3-25-8, Nishishinbashi, Minato-ku, Tokyo 105-8461, Japan c Fundamental Research DiÕision, Railway Technical Research Institute, 2-8-38, Hikari, Kokubunji city, Tokyo 185-8540, Japan Received 6 October 1998; received in revised form 31 March 1999; accepted 7 April 1999

Abstract Possible mutagenic and co-mutagenic effects of strong static magnetic fields were estimated using bacterial mutagenicity test. Mutagenic potential of static magnetic fields up to 5T ŽT:1Ts 10,000 G. was not detected by the bacterial mutagenicity test using four strains of Salmonella typhimurium ŽTA98, TA100, TA1535 and TA1537. and Escherichia coli WP2 uÕrA either in the pre-incubation method or in the plate incorporation method. In the co-mutagenicity test, E. coli WP2 uÕrA cells were treated with various chemical mutagens and were simultaneously exposed to a 2T or a 5T static magnetic field. Mutation rate in the exposed group was significantly higher than that in the non-exposed group when cells were treated with N-ethyl-N X-nitro-N-nitrosoguanidine ŽENNG., N-methyl-N X-nitro-N-nitrosoguanidine ŽMNNG., ethylmethanesulfonate ŽEMS., 4-nitroquinoline-N-oxide Ž4-NQO., 2-amino-3-methyl-3H-imidazow4,5-fxquinoline ŽIQ. or 2-Ž2-furyl.-3-Ž5-nitro-2furyl. acrylamide ŽAF-2.. The mutagenicity of 2-aminoanthracene Ž2-AA., 9-aminoacridine Ž9-AA., N 4-aminocytidine and 2-acetoamidofluorene Ž2-AAF. was not affected by the magnetic field exposure. Possible mechanisms of the co-mutagenicity of magnetic fields are discussed. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Magnetic fields, static; Bacterial mutation assay; Mutant frequency

1. Introduction In developed countries, exposure to various types of EMFs are commonly encountered; extremely low frequency ŽELF. EMF from power lines, high fre) Corresponding author. Tel.: q81-42-573-7316; Fax: q81-42573-7349; E-mail: [email protected] 1 Present address: Otsuki Public Health Center, 1608-3, Hanasaki, Otsuki, Otsuki city, Yamanashi 401-0015, Japan.

quency EMF from cellular phones and computers and static field from magnetic resonance imaging ŽMRI. are the most familiar sources of exposure. Current research on the biological effects of EMFs has been largely focused on weak power frequency Ž50r60 Hz. fields. The results suggest that these have some biological effects, and NIEHS working group report w1x of EMF Research and Public Information Dissemination ŽEMFRAPID, United States. stated that ELF EMFs are possibly carcinogenic to

0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 9 . 0 0 0 8 7 - 1

148

M. Ikehata et al.r Mutation Research 427 (1999) 147–156

humans ŽGroup 2B.. Recently, the focus has shifted to radiofrequency fields as the use of cellular phones exploded globally. World Health Organization conducted an international EMF project to evaluate the health effects of EMFs ranging from static Ž0 Hz. to radiofrequency Ž300 GHz. fields. Repacholi w2x emphasized the necessity of research on biological effects of exposure to radiofrequency fields. Although high density Ž) 2T. static magnetic fields are believed to have potential to affect some biological systems, the health effects of strong static magnetic fields have not been evaluated sufficiently. With the development of superconducting technology, application of a 4T or a 7T magnetic field is being considered to obtain higher resolutions for MRI. Therefore, biological studies on the effects of strong static magnetic fields are necessary to evaluate the safety of exposure to static magnetic fields. There are a large number of studies on the biological effect of static magnetic fields, however the results often contradict each other. Some of the effects that include an increase or decrease in the rate of cell division at different physiological conditions in Escherichia coli w3x and an alteration of human tumor cell adhesion w4x were reported in in vitro studies. However, other studies have not detected any effects w5,6x. In a study on the genotoxicity of magnetic fields exposure, Kiranmai w7x reported that 200 mT static magnetic field exposure caused mutations in dry seeds of Helianthus annus. Chronic exposure to 0.7 mT static magnetic field resulted in an increase in wing length and in sex-linked recessive lethals in Drosophila melanogaster w8x. In contrast, no increase in mutation frequency was observed among the progeny of D. melanogaster males exposed to 1.3–3.7T homogeneous static magnetic fields w9x. A 3.0T homogeneous static magnetic fields did not increase DNA damage in several E. coli mutant strains w10x. As a variety of test procedures and exposure systems have been used in studying the effect of magnetic fields on biological systems, it is difficult to compare or confirm results. To be able to compare experimental data from many laboratories, experiments should conform to standard methods. For this reason, we performed bacterial mutation assay w11x which is a standard test commonly used to screen chemicals and environmental contaminants for muta-

genic activity. Previous reports on bacterial mutation assay for evaluation of safety of EMFs are mainly in time-varying ELF fields. A 100Hz 0.2 mT EMF and a 0.3Hz triangular wave EMFs Ž0.08 mT. did not affect the mutagenic frequency of histidine auxotroph Salmonella typhimurium TA100 w12,13x, or a 0.3 mT of 60, 600, 6000 Hz EMFs did not induce genotoxicity in S. typhimurium TA97a, TA98, TA100 and TA102 w14x. However, EMFs used in the above experiments were below 1 mT and thus, these results do not prove the absence of genotoxic effects of strong static magnetic fields. The present study was performed in higher density fields Žup to 5T. to evaluate the mutagenicity and the co-mutagenicity of static magnetic fields.

2. Materials and methods 2.1. Exposure system A superconducting magnet ŽSCM, Toshiba JS500. with a horizontal bore of 20 cm diameter that generates a homogeneous static magnetic field up to 5T was used. Eighteen bacterial plates Ž90 mm diameter. could be exposed simultaneously to a homogeneous magnetic field in this superconducting magnet. To avoid temperature fluctuation, the SCM was located in a constant temperature room ŽMCU-3000, Sanyo, Japan. and the bacterial plates were maintained at 37 " 0.58C. For shaking cultures, we used reciprocal shaker incubators to maintain the bacterial suspension tubes in the magnetic field and in sham space simultaneously at 37 " 0.28C using circulating hot water. 2.2. Chemicals N-ethyl-N X-nitro-N-nitorosoguanidine ŽENNG, CAS No. 4245-77-6., 4-nitroquinoline-N-oxide Ž4NQO, 56-57-5. and 2-acetoamidofluorene Ž2-AAF, 53-96-3. were purchased from Sigma ŽSt. Louis, MO, USA.. N-methyl-N X-nitro-N-nitrosoguanidine ŽMNNG, 70-25-7. and 9-aminoacridine Ž9-AA, 90-45-9. were purchased from Aldrich Chemical ŽMilwaukee, WI, USA.. 2-Ž2-furyl.-3-Ž5-nitro-2furyl.acrylamide ŽAF-2, 3688-53-7. and 2-aminoanthracene Ž2-AA, 613-13-8. were purchased from

M. Ikehata et al.r Mutation Research 427 (1999) 147–156

Wako Pure Chemical Industries ŽOsaka, Japan.. N 4aminocytidine and 2-amino-3-methyl-3H-imidazow4, 5-fxquinoline ŽIQ, 76180-96-6. were purchased from Funakoshi ŽTokyo, Japan.. Ethyl methanesulfonate ŽEMS, 62-50-0. was purchased from TCI ŽTokyo, Japan.. Nutrient broth ŽOxoid, Nutrient broth No. 2. was purchased from Unipath ŽHampshire, UK.. Bactoagar was purchased Difco Laboratories ŽDetroit, MI, USA.. The S9 mix, prepared from liver of phenobarbital and 5,6-benzoflavone pretreated male Sprague–Dawley rat, was purchased from Kikkoman ŽChiba, Japan.. Other reagents were laboratory-grade materials purchased from Wako. 2.3. Bacterial strains Four routinely used Ames tester strains of S. typhimurium ŽTA98, TA100, TA1535 and TA1537. were used to test the mutagenicity of static magnetic fields. S. typhimurium strains were originally provided by Dr. B.N. Ames of the University of California, Berkeley and E. coli WP2 uÕrA was provided by Dr. T. Matsushima of the University of Tokyo, Tokyo ŽTable 1.. 2.4. Determination of cytotoxicity of a static magnetic field Two L-tubes Ž20 mm diameter. containing 12 ml nutrient broth were inoculated with 10 ml inoculum from 8.75% dimethylsulfoxide ŽDMSO. stock stored

149

at y808C. One of the tube was placed in a polypropylene tube stand on one side of the reciprocal shaker incubator Ž378C, 50 rpm. while the other was placed in the stand on the opposite side of the shaker incubator. One tube stand was placed in a 5T magnetic field and the other in a sham space. Viable cell concentration was determined as colony forming units ŽCFUs.. From each culture, a 100-ml sample was taken at each incubation time. The aliquot was diluted appropriately to obtain a concentration of 10 2 –10 4 cellsrml with 0.1 M phosphate buffer, and 100 ml of the diluted cell suspension was mixed with 2 ml of 0.6% molten agar and poured on to nutrient agar plate. Viable cell number was counted after 24 h incubation at 378C and viable cell concentration in each culture at each time point was calculated. E. coli WP2 uÕrA was used in this experiment and three independent tests were performed. 2.5. Mutagenicity assay of magnetic field Experiments were carried out with plate incorporation method and also with preincubation method w11,15x. Four strains of S. typhimurium TA98, TA100, TA1535 and TA1537 and E. coli WP2 uÕrA were used in these experiments. In the preincubation method, cell suspension Ž0.1 ml of preculture, 1–3 = 10 9 cellsrml. with phosphate buffer Ž0.5 ml. were preincubated in a magnetic field or in a sham space for 20 min using reciprocal shaker incubator Ž378C, 50 rpm. in test tubes. Contents were mixed with top agar Ž2 ml., plated onto minimal glucose agar plates and incubated at 378C in a conventional incubator for 48 h. Revertant colonies were counted with

Table 1 Genotypes of bacterial 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 TA100 TA1535 TA1537

his D 3052 his G 46 his G 46 his C 3076

Frameshift Base pair substitution Base pair substitution Frameshift

GC GC GC GC

uÕrB uÕrB uÕrB uÕrB

rfa rfa rfa rfa

pKM101 pKM101 – –

E. coli WP2 uÕrA

trp E 56

Base pair substitution

AT

uÕrA

q

–

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M. Ikehata et al.r Mutation Research 427 (1999) 147–156

Colony Analyzer CA-90S ŽToyo Sokki, Kanagawa, Japan. after 48 h incubation at 378C. In the plate incorporation method, cell suspension Ž0.1 ml of preculture, 1–3 = 10 9 cellsrml., phosphate buffer Ž0.5 ml., top agar Ž2 ml. were mixed and plated onto minimal glucose agar plates. The plates were incubated in a 2 or a 5T magnetic field, or in a conventional incubator as control for 48 h, then revertant colonies were scored. Each experiment was run in triplicate and conducted at least three times. Three extra plates were treated with chemical mutagens as positive control in each experiment. AF-2 was used for S. typhimurium TA98, TA100 and E. coli WP2 uÕrA. Sodium azide was used for S. typhimurium TA1535. 9-Aminoacridine was used for S. typhimurium TA1537.

ENNG Ž1 mg dissolved in DMSO., cell suspensions Ž0.1 ml of preculture, 1–3 = 10 9 cellsrml., phosphate buffer Ž0.5 ml. and top agar Ž2 ml. were made and randomly divided into seven groups. Control plates were incubated for 48 h in a conventional incubator, and experimental plates were incubated in a 5T static magnetic field for different time periods Ž1.5, 3, 6, 15, 24 or 48 h., transferred to a conventional incubator and then incubated for a total of 48 h. The revertant colonies were counted by colony analyzer. Three independent tests were done in this time course study.

3. Results and discussion 3.1. Mutagenicity of static magnetic fields

2.6. Co-mutagenicity assays of magnetic field and chemical mutagens Experiments were conducted in the plate incorporation method of Ames et al. w11x, Maron and Ames w16x and McMahon et al. w17x with a slight modification. Mutagens were dissolved in DMSO ŽENNG, MNNG, 4-NQO, AF-2, 2-AA, 2-AAF, IQ and EMS. or in distilled water Ž9-AA and N 4-aminocytidine.. Bacterial culture was rinsed twice with 0.1 M phosphate buffer to remove all traces of the growth medium. Cell suspensions Ž0.1 ml of preculture, 1–3 = 10 9 cellsrml., mutagen solution or solvent Ž0.1 ml. and phosphate buffer or S9 mixture Ž0.5 ml., were diluted with top agar Ž2 ml., and plated on to minimal glucose agar plates Ž90 mm diameter.. Six plates were made at each point and randomly divided into two groups; three plates were placed at the center of the magnet bore while the other three were placed in a conventional incubator. Revertant colonies were scored after 48 h of incubation. Each experiment was repeated at least three times. Student’s t-test was used for the statistical analysis of the result between the number of revertant colonies in the exposed plates to that of control. 2.7. Time course study of co-mutagenicity Time course study was performed with E. coli WP2 uÕrA in a 5T magnetic field in the presence of ENNG. Twenty-one plates Ž90 mm diameter. with

Mutagenicity of 2 and 5T static magnetic fields were tested using five different tester strains; E. coli WP2 uÕrA, S. typhimurium TA98, TA100, TA1535 and TA1537. Either in plate method ŽTable 2. or in preincubation method ŽTable 3., there was no significant difference in the frequency of revertant colonies between exposed Ž2T. and unexposed control groups in all tester strains. A 5T static magnetic field was

Table 2 Mutagenicity of a 5T static magnetic field by plate incorporation methoda Strain

Revertant coloniesrplate Control

E. coli WP2 uÕrA

18"5 b

S. typhimurium TA98 40"6 TA100 138"7 TA1535 13"2 TA1537 6"2 a

Exposure

Positive control c

21"4

173"15

39"5 143"13 13"2 6"2

619"16 358"12 355"23 285"54

This assay was performed in the absence of rat liver S9 mix. Data are given as mean"S.D. from the four independent tests of triplicate plates at each point. c Chemical mutagen was used as positive control. For E. coli WP2 uÕrA and S. typhimurium TA100, 0.01 mgrplate of AF-2 was used while 0.1 mgrplate was used for S. typhimurium TA98. For S. typhimurium TA1535, 0.5 mgrplate of sodium azide was used. For S. typhimurium TA1537, 80 mgrplate of 9-aminoacridine was used. b

M. Ikehata et al.r Mutation Research 427 (1999) 147–156

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Table 3 Mutagenicity of a 5T static magnetic field by pre-incubation methoda

Table 5 Mutagenicity of a 2T static magnetic field by pre-incubation methoda

Strain

Strain

Revertant coloniesrplate Control

E. coli WP2 uÕrA

20"4b

S. typhimurium TA98 28"3 TA100 107"10 TA1535 11"1 TA1537 7"2

Exposure

Positive control

c

Mutant coloniesrplate Control

Exposure

Positive control c

19"5

219"18

E. coli WP2 uÕrA

27"4b

29"4

175"13

32"3 104"12 11"2 9"4

321"25 449"39 289"22 358"27

S. typhimurium TA98 30"6 TA100 120"10 TA1535 10"1 TA1537 9"1

28"7 122"8 11"1 9"1

401"20 469"23 574"24 423"20

a

a

This assay was performed in the absence of rat liver S9 mix. Data are given as mean"S.D. from the four independent tests of triplicate plates at each point. c Chemical mutagen was used as positive control. For E. coli WP2 uÕrA and S. typhimurium TA100, 0.01 mgrplate of AF-2 was used, while 0.1 mgrplate was used for S. typhimurium TA98. For S. typhimurium TA1535, 0.5 mgrplate of sodium azide was used. For S. typhimurium TA1537, 80 mgrplate of 9-aminoacridine was used.

This assay was performed in the absence of rat liver S9 mix. Data are given as mean"S.D. from the four independent tests of triplicate plates at each point. c Chemical mutagen used as positive control. For E. coli WP2 uÕrA and S. typhimurium TA100, 0.01 mgrplate of AF-2 was used while 0.1 mgrplate was used for S. typhimurium TA98. For S. typhimurium TA1535, 0.5 mgrplate of sodium azide was used. For S. typhimurium TA1537, 80 mgrplate of 9-aminoacridine was used.

also revealed to have no mutagenic potential in both the plate method and preincubation method in all tester strains ŽTables 4 and 5.. To test the cytotoxicity of a 5T static magnetic field, cell growth in liquid culture was monitored by colony forming unit

till late log phase. No significant difference between exposed and control group was observed. This result indicated that growth rate was not affected by exposure to a 5T magnetic field ŽFig. 1.. The thermodynamic energy of the interaction between an electron spin and a 5T static magnetic

b

b

Table 4 Mutagenicity of a 2T static magnetic field by plate incorporation methoda Strain

Revertant coloniesrplate Control

E. coli WP2 uÕrA

21"2 b

S. typhimurium TA98 29"2 TA100 107"8 TA1535 14"1 TA1537 5"2

Exposure

Positive control c

22"2

132"7

26"4 126"12 12"1 5"1

780"27 336"23 426"10 251"7

a

This assay was performed in the absence of rat liver S9 mix. Data are given as mean"S.D. from the four independent tests of triplicate plates at each point. c Chemical mutagen was used as positive control. For E. coli WP2 uÕrA and S. typhimurium TA100, 0.01 mgrplate of AF-2 was used while 0.1 mgrplate was used for S. typhimurium TA98. For S. typhimurium TA1535, 0.5 mgrplate of sodium azide was used. For S. typhimurium TA1537, 80 mgrplate of 9-aminoacridine was used. b

Fig. 1. Growth of E. coli WP2 uÕrA in a 5T static magnetic field. Growth was determined as colony forming unit ŽCFU. on nutrient agar plate. Data are given as mean from three independent tests and standard deviation are within symbols.

152

M. Ikehata et al.r Mutation Research 427 (1999) 147–156

field is only 6.7 = 10y3 kcalrmol which is much lower than the energy of X-rays, UV light and even of thermal noise. The bonding energy of weak chemical bonds in organisms such as van der Waals bonding Žapproximately 1 kcalrmol., hydrogen bonding Žapproximately 3–7 kcalrmol. and ion bonding Žapproximately 5 kcalrmol. are much higher. Therefore, it is expected that chemical bonds will not be altered by exposure to a 2T or a 5T magnetic field. Moreover, the absence of any effect on growth rate of E. coli WP2 uÕrA by exposure to magnetic field suggests that static magnetic field Žup to 5T. has no acute toxicity for bacteria. These results suggest that a static magnetic field of up to 5T has very weak or no mutagenic effect on the five tester strains used in this study. 3.2. Co-mutagenicity of strong magnetic fields Mutagenicity tests of 10 chemical mutagens at three different concentration with or without exposure to static magnetic fields were carried out. When E. coli WP2 uÕrA cells were incubated for 48 h in a 2T magnetic field with ENNG Ž0.5, 1 or 2 mgrplate., the rate of mutation increased approximately two-fold compared with that of control Žchemical treatment without magnetic field exposure. experiment. Mutagenesis by MNNG, EMS, AF-2, 4-NQO and procarcinogen IQ was also affected significantly by exposure to a magnetic field. On the other hand, mutagenicity of 9-AA, N 4-aminocytidine, 2-AA and 2AAF was not affected by exposure to a 2T static magnetic field. Table 6 summarizes the result of the co-mutagenicity tests of a 2T static magnetic field exposure with each chemical. Some chemicals were also tested in a 5T magnetic field to confirm the effect of magnetic field exposure. The results were similar to that of the 2T experiment. Mutagenesis by AF-2, 4-NQO and ENNG were significantly enhanced by exposure to a 5T magnetic field for 48 h and that of N 4-aminocytidine remained unchanged. However, as the extent of enhancement was similar to that of a 2T field, dose–response relationship was unclear ŽTables 7–9.. In Tables 6 and 7, the ratio of the average number of colonies between exposed and control plates is shown. The degree of enhancement of the mutation frequency did not depend on the concentration of

Table 6 Co-mutagenic activity of a 2T static magnetic field with chemical mutagen in the absence of rat liver S9 mix Mutagen dose Žmgrplate.

Revertant coloniesrplate Control Exposure

Ratio ŽExrCon.

None ŽDMSO.

18"3

18"3

1.0

9-AA 20 40 80

19"3 22"4 21"5

19"3 19"4 25"3

1.0 0.9 1.2

N 4 -Aminocytidine 0.025 206"20 0.05 316"28 0.1 522"15

197"5 325"24 509"23

1.0 1.0 1.0

4-NQO 0.25 0.5 1

66"7 338"18 540"30

98"14 500"80 773"42

1.5U 1.5U 1.4UU

AF-2 0.005 0.01 0.02

56"5 135"12 331"24

62"2 164"15 411"47

1.1 1.2UU 1.2U

ENNG 0.5 1 2

50"1 106"1 203"10

101"17 203"25 417"20

2.0U 1.9U 2.1UU

MNNG 6.25 12.5 25

152"16 295"14 505"42

189"2 379"11 668"50

1.2 1.3UU 1.3U

EMS 250 500 1000 2000

61"0 116"4 213"19 407"40

77"1 154"5 281"10 511"50

1.3UU 1.3UU 1.3U 1.3UU

Each value is a mean of three independent experiments with triplicate plates. U P - 0.05, UU P - 0.01 compared to the revertant colonies observed in control ŽStudent’s t-test..

chemicals. It is possible that the magnetic field has a synergetic rather than an additive effect. Background bacterial lawn on plates suggested that cytotoxicity of chemicals was not grossly altered by the exposure to magnetic fields. We also observed that there was no difference in growth or size of colonies between the exposed and control assay plates in each experi-

M. Ikehata et al.r Mutation Research 427 (1999) 147–156 Table 7 Co-mutagenic activity of a 2T static magnetic field with chemical mutagen in the presence of rat liver S9 mix Mutagen dose Žmgrplate. None ŽDMSO.

Revertant coloniesrplate Control Exposure

Ratio ŽExrCon.

23"5

22"3

1.0

210"17 567"32 975"46

220"16 573"38 952"68

1.0 1.0 1.0

2-AAF 2.5 5 10

39"3 60"5 92"6

36"2 69"7 94"8

0.9 1.2 1.0

IQ 1.25 2.5 5

73"11 176"3 336"13

95"14 251"17 445"11

2-AA 2.5 5 10

1.3U 1.4U 1.3UU

Each value is a mean of three independent experiments with triplicate plates. U P - 0.05, UU P - 0.01 compared to the revertant colonies observed in control ŽStudent’s t-test..

153

forces Žcalled Lorentz Forces., on moving electrolytes giving rise to induced electric fields and currents. This means that electric fields and currents can be induced in bodies moving through a static magnetic field. However, in our study, cells were exposed to a magnetic field in fixed condition on the agar plate and there was, practically, no electrolyte flow. Therefore, the above mechanism does not seem to relate to our observation. Žii. Magneto-mechanical effects lead to orientation of paramagnetic and ferromagnetic materials along the magnetic field gradients. Anisotropic diamagnetic materials can also be oriented in strong magnetic fields. Magnetotactic bacterium use magnetite to orient themselves within a geomagnetic field w19x. Meanwhile, almost all biomolecules have diamagnetic susceptibility. Orientation of erythrocyte and polymerizing fibrin in strong magnetic fields were reported and this phenomenon was explained as the anisotropic diamagnetic susceptibility w20,21x. It is possible that double strand DNA also has

ment Ždata not shown.. These results suggest that the strength of the magnetic field or concentration of chemical mutagens used in our experiments was manifestly non-toxic. In the time course study of the exposure period, it was noted that the enhancement of the mutagenic activity of ENNG depended on the period of exposure to the magnetic field ŽFig. 2.. Increase in the mutation frequency became saturated after 24-h exposure. It is inferred that all the supplied tryptophan was used up in 24 h of incubation, and thereafter, the cells could not divide and therefore the mutations could not be fixed.

Table 8 Co-mutagenic activity of a 5T static magnetic field with chemical mutagen in the absence of rat liver S9 mix

3.3. Possible mechanism of the co-mutagenicity of magnetic fields Static magnetic fields can alter the spin orientation of electron and proton and hence their energy levels. Static magnetic fields also exert forces on biological molecules depending on its anisotropic magnetic susceptibility. The biological effect of static magnetic fields has been explained by the following mechanisms w18x. Ži. Static magnetic fields exert

Mutagen dose Žmgrplate. None ŽDMSO.

Revertant coloniesrplate Control Exposure 21"5 18"4

Ratio ŽExrCon. 0.9

4

N -Aminocytidine 0.01 100"10 0.05 281"33 0.1 503"38

92"17 284"25 508"32

0.9 1.0 1.0

4-NQO 0.25 0.5 1

72"2 242"4 618"42

90"9 310"51 759"90

1.3U 1.3U 1.2U

AF-2 0.02

381"38

449"43

1.2UU

ENNG 0.5 1 2

75"5 167"22 341"25

149"20 304"44 626"35

2.0U 1.8U 1.8UU

Each value is a mean of at least three independent experiments with triplicate plates. U P - 0.05, UU P - 0.01 compared to the revertant colonies observed in control ŽStudent’s t-test..

M. Ikehata et al.r Mutation Research 427 (1999) 147–156

154

Table 9 Co-mutagenic activity of a 5T static magnetic field with chemical mutagen in the presence of rat liver S9 mix Mutagen dose Žmgrplate. None ŽDMSO.

Revertant coloniesrplate Control Exposure 22"3 22"5

Ratio ŽExrCon.

2-AA 10

953"7

1030"46

1.1

2-AAF 2.5 5 10

32"3 62"3 91"3

33"1 62"4 90"4

1.0 1.0 1.0

IQ 1.25 2.5 5

89"14 196"19 379"34

105"8 243"18 455"24

1.0

1.2U 1.2UU 1.2UU

Each value is a mean of three independent experiments with triplicate plates. U P - 0.05, UU P - 0.01 compared to the revertant colonies observed in control ŽStudent’s t-test..

anisotropic susceptibility, however, as genomic DNA is condensed into a nucleoid in the cytosol. It is expected that strong static magnetic fields will not affect its structure. Consequently, magneto-mechanical effects are not responsible for the observations in our study. Žiii. Magnetic fields can effect chemical reactions by influencing the electronic spin states of reaction intermediates. This electronic interactions has been observed in photochemical reactions. Studies on the decay rate of benzophonone ketyl radical and on the hydrogen abstraction reaction of 2-naphthylphenylcarbene have shown that some radical recombinations in a micelle were affected by a static magnetic field w22,23x. These studies indicated that magnetic field effect on intersystem crossing ŽISC. of the radical pair might be observed at several 10 mT and dose–response relationship between flux density and the effect of ISC is non-linear with a saturation plateau around 1T. Meanwhile, Nagata et al. w24x reported that MNNG, 4-NQO and various other chemicals could form radical intermediate during metabolization in cells and this process is related to chemical mutagenesis or carcinogenesis. These results suggest that electronic interactions with static magnetic fields could affect mutagenesis of such

chemicals. We have previously reported the recombinogenic effect of a 5T static magnetic field in the wing spot test of D. melanogaster w25x. This study suggested that the life time of spontaneously produced radical pairs in living cells was affected by static magnetic field because its effect was suppressed by antioxidant vitamin E. These studies suggest that reactions of DNA with chemical mutagens can be affected by exposure to strong static magnetic fields. Several studies also suggest that strong static magnetic fields cause some kind of oxidative stress for living organism. Chignell and Sik w26x reported that an application of a 335 mT static magnetic field during UV-irradiation reduced the time for 50% photohemolysis of human erythrocytes by the phototoxic drug ketoprofen. This study suggests the reaction of triplet state ketoprofen which induced by UV-irradiation with erythrocyte componentŽs. was affected by exposure to static magnetic fields. Besides reacting with DNA, mutagens also react with lipid, protein, peptides, etc., in bacterial cell and some products of these reactions have mutagenic potential for bacteria. For example, malondialdehyde, a product of lipid peroxidation is mutagenic in S. typhimurium w27x. Watanabe et al. w28x reported enhancement of lipid peroxidation in the liver of mice exposed to 4.7T static magnetic field. It is

Fig. 2. Time course study of the effect of 5T static magnetic field on ENNG-induced mutation frequency in E. coli WP2 uÕrA. Symbol shows the ratio of mutation frequency of each exposure period group as colony number per plate to control. Data are given as mean"S.D. from three independent tests with triplicate plates.

M. Ikehata et al.r Mutation Research 427 (1999) 147–156

suggested that strong static magnetic fields could affect metabolic pathways of chemical mutagens and this can cause an increase in the number of revertant colonies. These studies suggest that the increase in mutagenicity of some chemical mutagens observed on exposure to strong static magnetic fields in our study might be related to the effect of the magnetic field on electronic interactions. Besides the possible mechanisms listed above, it is possible that magnetic fields can affect the mutation pathway of chemical mutagens. Alterations of membrane permeability by exposure to static magnetic fields have been reported in liposome vesicles w29x. Although the effect was observed at a temperature near the phase transition point of the lipid bilayer, it is possible, if not probable, that exposure to magnetic fields causes similar changes in membrane permeability at physiological temperature and that mutagen uptake is increased. It is also possible that alkylation process in the bacterial cell is affected by magnetic field exposure, since we found that all three alkylating agents used in this study were affected by exposure to a static magnetic field. Alkylating agents are strong electrophilic agent and react on carbon or oxygen of DNA by electrophilic substitution and modify DNA to alkyl-DNA. It is possible that magnetic field affects this reaction. It is also possible that additional radical reactions, besides electrophilic substitution, may be enhanced by exposure to static magnetic fields since alkylating agent may produce free radicals w24x. However, all of the radicals do not always show mutagenicity on the strains used in this study. In vitro reaction study and other experiments should be made to confirm the relation of radicals and effect of the magnetic field more directly. As another possibility, effects of magnetic fields on DNA repair process should be considered. Grissom and Harkins w30x showed that ethanolamine ammonia lyase was affected by exposure to static magnetic fields. Nossol et al. w31x reported the effect of weak static and 50-Hz magnetic fields on the redox activity of cytochrome-C oxidase. It is possible that repair enzymes might be affected by exposure to static magnetic fields. In future studies, experiments involving dosage of the magnetic field density, in vitro reaction of DNA and chemicals, mutant strains which are defective in

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