EFFECTS OF HIGH ELF MAGNETIC FIELDS ON ENZYME-CATALYZED DNA AND RNA SYNTHESIS IN VITRO AND ON A CELL-FREE DNA MISMATCH REPAIR.

Harada, S.; Yamada, S.; Kuramata, O.; Gunji, Y.; Kawasaki, M.; Miyakawa, T.; Yonekura, H.; Sakurai, S.; Bessho, K.; Hosono, R.; Yamamoto, H.

Lab. of Magnetic Field Control and Applications, Faculty of Engineering, Kanazawa, Univ., 2-40-20 Kodatsuno, Kanazawa 920-8667, Japan, e-mail: yamada@magstar.ec.t.kanazawa-u.ac.jp (RR/S.Y., O.K., Y.G., M.K., T.M., K.B.); Center for Biomedical Res. and Education, Sch. of Medicine, Kanazawa, Univ., 2-40-20 Kodatsuno, Kanazawa 920-8667, Japan (S.H., S.S.); Dept. of Biochemistry, Sch. of Medicine, Kanazawa, Univ., 2-40-20 Kodatsuno, Kanazawa 920-8667, Japan (H.Yo., H.Ya.); Dept. of Physical Information, Faculty of Medicine, Kanazawa, Univ., 2-40-20 Kodatsuno, Kanazawa 920-8667, Japan (R.H.)

The authors investigated the effects of a strong extremely low-frequency (ELF) magnetic field on enzyme-catalyzed DNA and RNA synthesis in cell-free bacterial and HeLa cell extracts. The effects on a cell-free DNA mismatch repair process with defined sequences were also examined. Sixty-Hz magnetic fields with flux densities of 1.0 mT (10 G) were produced using a custom-built exposure system. The system consists of two iron cores of the same size, each shaped like the letter E. The two outer yokes of the cores are in contact with each other, while the central yokes of the two cores face each other, separated by a 13-mm gap. The cores are encircled by current-carrying coils and sinusoidal magnetic fields are generated in the central air gap space by an eddy current mechanism, as described by Bessho et al. (Experientia 51:284-288, 1995; BENER Abstract No. 11791). The system can produce fields with a peak flux density as high as 1.2 T when a 60-Hz, 190-A rms current is applied to the coils. Temperatures in the exposure volume were maintained within +/- 0.5 C of the target (physiological) temperature by circulating water at a flow rate of about 11 l/min. In a preliminary experiment in which a 150-A rms 60-Hz AC was used to generate a 1.0-T magnetic field in the exposure volume, the field in the exposure volume was found to be homogeneous to better than 2%, based on measurements made with a Bell Industries model 615 gaussmeter. In experiments examining the effects of magnetic field exposure on enzyme-catalyzed DNA synthesis, reaction mixtures containing 67-mM potassium phosphate buffer, tritium-labeled deoxyribonucleoside thymidine-5'-triphosphate (3H-dTTP) at a specific activity of 0.5 Curies (Ci)/mmol, 2 mM each of unlabeled deoxyribonucleosides dATP, dCTP, and dGTP, 100-pM of poly(dA) template and oligo(dT) primer, and 0.15 units of Klenow enzyme or its des-3'-exonuclease equivalent in a volume of 50 ul were placed in the magnetic field generator for 1 to 15 min at a temperature of 37 C. Control reactions were run in the generator without applying any magnetic fields. After stopping the reaction with 10% ice cold trichloroacetic acid (TCA), the resultant TCA-insoluble material was washed with ice cold 5% TCA, followed by ethanol, and 3H activity in the material was measured by liquid scintillation counting. To obtain an estimate of the fidelity of DNA synthesis, experiments were run with 1,250 Ci/mmol of sulfur-35 (35S) labeled dCTP in the reaction mixture. Incorporation of 35S activity into the TCA insoluble material was determined by liquid scintillation counting. In experiments examining the effects of magnetic field exposure on enzyme-catalyzed RNA synthesis, reaction mixtures contained 40-mM Tris-hydrochloride, 150-mM potassium chloride, 10-mM magnesium chloride, 0.1-mM EDTA, 0.1-mM dithiothreitol, 0.5-mg/ml bovine serum albumin, 400-uM each of ATP, CTP, GTP, and UTP, which included 1.0-Ci/mmol 3H-UTP, 40-ug/ml poly(dA)-poly(dT), and 60-units/ml Escherichia coli (E-coli) RNA polymerase in a final volume of 50 ul. To estimate errors in the transcription reaction, 20 uCi of 35S-CTP was also included in the reaction mixtures. The mixtures were exposed or sham exposed to the magnetic fields for 1 to 30 min at 37 C then the reactions were stopped with 10% ice cold TCA and processed as above. The effects of magnetic field exposure on DNA mismatch repair were evaluated by measuring the effects on heteroduplex repair processes in extracts of human HeLa cells using the assay described by Thomas et al. (J Biol Chem 266:3744-3751, 1996) in which 5-ng of heteroduplex DNA were incubated with or without 50-ug of nuclear extracts from repair-proficient HeLa cells while being exposed or sham exposed to a 1-T magnetic field for 15 min at a temperature of 37 C. After the reaction was stopped with proteinase-K and sodium dodecyl sulfate, the DNA was phenol extracted, precipitated with ethanol, and used to transfect NR9162 cells, a mutant E. coli strain, by electroporation. The transfected cells were then incubated with IPTG, X-gal, and a log phase culture of XL1-blue cells, an alpha-complementation E. coli strain, and then incubated at 37 C to develop plaques. Three types of plaques typically formed: blue, colorless, or mixed, and the efficiency of mismatch repair was determined from the proportion of mixed plaques in cells transfected with magnetic field-exposed vs. unexposed extracts. All experimental data were tested statistically using Student's t-test. Results were presented in the form of plots of incorporation as a function of incubation time and as histograms of blue, colorless, and mixed plaques for the mismatch repair assays. Exposure to 60-Hz, 0.25- or 0.5-T magnetic fields did not significantly affect enzyme-catalyzed DNA or RNA synthesis. Exposure to a 1-T 60-Hz field did not significantly affect the efficiency of mismatch repair in the DNA mismatch repair assay. The authors concluded that these results suggest that core processes related to the transmission of genetic information are not affected by strong ELF magnetic fields. Since the reactions studied in these experiments were taken out of cellular systems and utilized synthetic homopolymeric templates and pure prokaryotic enzymes, they have not dealt with possible "hot spot" areas for mutations in specific genes that could respond to magnetic fields, nor have they addressed events that occur in the genome of exposed cells. These are issues that require further investigation. (15 Refs). [Copyright 2001, Information Ventures, Inc.]

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