Muta,!ion Research, 282 (1992) 25-29 © 1902 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
25
MU'fLET 0651
Electric a n d / o r m a g n e t i c field effects on D N A structure and function in cultured h u m a n cells M. Fiorani a, O. Cantoni b p. Sestili b R. Conti c, p. Nicolini c, F. Vetrano a and M. Dachh a Centro di Metodologie Biochimiche Applicate and b Istituto di Farmacologia e Farmacognosia, Unit~ersith degli Studi di Urbino, Urbino, and ~ ENEL, Centro di Ricerca Elettrica, Cologno Monzese, Milan (Italy) (Received 2 December 1991) (Accepted 23 December 1991)
Keywords: ELF; Electric and magnetic fields; DNA damage; Cytotoxicity; Cultured mammalian cells
Summary Exposure of cultured K562 cells to 50 Hz electric (0.2-20 k V / m ) , magnetic (0.002-2 G), or combined electric and magnetic fields for up to 24 h did not result in the production of detectable D N A lesions, as assayed by the filter elution technique. The rate of cell growth was also unaffected as well as the intracellular A T P and N A D + levels. These results indicate that, under the experimental conditions utilized in this study, 50 Hz electric, magnetic and electromagnetic fields are not geno- and cyto-toxic in cultured mammalian cells.
Exposure to electromagnetic fields has been reported to increase the risk of certain types of cancer, such as leukemia, cancer of the central nervous system and lymphoma (Wertheimer and Leel~er, 1979; Savitz et al., 1988). The results from these studies, however, were not confirmed by other investigators (Fulton et al., 1980), and thus it is generally believed that more epidemiological research is needed in order to either prove or confute the association between exposure to the fields and the risk of cancer. Since most carcinogenic agents display genotoxic effects, it would be extremely important to
Correspondence: Dr. Orazio Cantoni, Istituto di Farmacologia e Farmacognosia, Universith degli Studi di Urbino, Via S. Chiara 27. 1-61029 Urbino (Italy).
gather information on whether or not field exposure induces genetic alterations. The results from these studies would certainly provide relevant information for either questioning or affirming the carcinogenicity of the fields. There are some preliminary reports in the literature (Miller et al., 1976; Cohen et al. 1986a, b; Benz and Carsten, 1986) indicating that, with few exceptions (Eberle and May, 1982), field exposure does not induce genetic alterations. In particular, in one study, the alkaline elution assay, a very sensitive technique for measuring D N A single- and doublestrand breakage and D N A - D N A crosslinks as well as DNA-protein crosslinks, has been used to test the effect of 60 Hz magnetic, electric and superimposed electric and magnetic fields (Reese et al., 1988) on the D N A of Chinese hamster ovary cells. Results from this study have indicated
26
that field exposure does not produce damage at the DNA level. It should be noted, however, that these experiments covered a very limited range of intensities and, importantly, cell exposure was only for 1 h (Reese et al., 1988). This experimental detail may have important implications since some effects of the fields in cellular systems have been observed only under specific a m p l i t u d e / frequency criteria (window effects) (Parkinson and Hanks, 1989). We now report the results of a study that was performed to investigate the effect of power frequency electric a n d / o r magnetic on the DNA of a human tumor cell line (K562 cells). In this study, the experiments involved field exposure for various lengths of time and have covered a wide range of intensities. In addition, other biochemical parameters such as NAD + and ATP levels as well as the rate of cell replication were evaluated. Materials and methods Field exposure system
The field exposure apparatus was developed in our laboratories and consisted of four units allowing uniform exposure to magnetic B-field (unit 1), electric E-field (unit 2), mutually orthogonal Efield and B-field (unit 3) and no field (unit 4). Cells were suspended in complete growth medium and exposed to 50 Hz electric (0.2-20 k V / m ) , magnetic (0.002-2 G), or combined electric and magnetic fields (see Table 1) for 1, 4, 6, 12 or 24 h at 37°C. Incubations were performed under conditions of continuous shaking. In some experiments, cells were treated with increasing concentrations of methyl methanesulfonate (MMS) and then assayed as described below. Materials
Radiolabeled compounds were from New England Nuclear Corp. (Boston, MA, U.S.A.). Tetraethylammonium hydroxide was from MerckSchuchardt (Munich, Germany). Free acid EDTA, disodium EDTA, tetrasodium EDTA, sodium dodecyl sulfate and most reagent-grade biochemicals were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Polycarbonate filters were from Nuclepore (Pleasanton, CA, U.S.A.). RPMI 1640
medium, fetal bovine serum and trypsin were from Gibco (Grand Island, NY, U.S.A.). Cells"
K562 cells (a human tumor cell line) were routinely grown in our laboratory in RPMI 1640 medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum at 37°C in an atmosphere of 5% CO 2 and 95% air. Cells were subcultured (2.5 × 105 cells/ml) and exposed for various time intervals to the fields (or for 30 min to MMS). Following exposure cells were analyzed for cell number, NAD + and ATP intracellular contents and DNA damage. Alkaline elution assay
Cells were labeled for 24 h with 14C-thymidine (0.05 p~Ci/ml) and then, following a 6-h incubation in a label-free medium, were exposed to the fields. At specific time intervals, samples were taken and analyzed by the filter elution assay. This procedure was carried out as described by Kohn et al. (1981) with minor modifications (Cantoni et al., 1986). Briefly, 5 x 105 cells were gently loaded onto 25-mm, 2 /zm pore polycarbonate filters and then rinsed twice with 10 ml of ice-cold saline A containing 5 mM E D T A (disodium salt). Cells were then lysed with 5 ml of 2% sodium dodecyl sulfate, 0.025 M E D T A (tetrasodium salt), pH 10.1. Lysates were rinsed with 7 ml of 0.02 M E D T A (tetrasodium salt) and the DNA was eluted in the dark with 1.5% tetraethyl ammonium hydroxide/0.02 M E D T A (free acid)/0.1% sodium dodecyl sulfate (pH 12.1), at a flow rate of about 0.04 ml/min. Fractions of approximately 3.5 ml were collected and counted in 7 ml of Lumagel containing 0.7% glacial acetic acid. DNA remaining on the filters was recovered by heating for 1 h at 60°C in 0.4 ml of 1 N HC1 followed by the addition of 0.4 N N a O H (2.5 ml) and was again determined by scintillation counting. D N A was also recovered from the interior of the membrane holders after vigorous flushing with 3 ml of 0.4 N NaOH. This solution was processed for scintillation counting as described above. A TP and N A D + assay
Cells were rinsed with saline A and extracted with 2.5% perchloric acid. Samples were then
27 n e u t r a l i z e d with K 2 C O 3 and, following centrifugation, a q u e o u s solutions o f t h e n u c l e o t i d e s w e r e f i l t e r e d t h r o u g h 0.22 p~m p o r e size microfilters a n d a n a l y z e d for A T P a n d N A D + c o n t e n t s by r e v e r s e d - p h a s e h i g h - p e r f o r m a n c e liquid chromatography. Chromatographic apparatus and c o n d i t i o n s have b e e n d e s c r i b e d (Stocchi et al., 1985).
r e s u l t e d in a progressive i n c r e a s e in the D N A elution rate, i n d i c a t i n g a d o s e - d e p e n d e n t g e n e r a tion of D N A s t r a n d breaks. S t r a n d scission factors w e r e c a l c u l a t e d f r o m alkaline elution profiles such as t h o s e in Fig. 1A, a n d the resulting d o s e r e s p o n s e curve relating the d e g r e e of s t r a n d b r e a k a g e to the initial c o n c e n t r a t i o n of the alkylating a g e n t in the e x t r a c e l l u l a r m e d i u m (shown in Fig. 1B) was l i n e a r over the r a n g e o f M M S conc e n t r a t i o n s studied. W h e n cells w e r e e x p o s e d to the fields ( u n d e r all the e x p e r i m e n t a l c o n d i t i o n s s u m m a r i z e d in T a b l e 1), the D N A e l u t i o n p r o files were not significantly d i f f e r e n t from those o f the s h a m - e x p o s e d cells, suggesting that field exp o s u r e d o e s not i n d u c e d a m a g e at the D N A level. S o m e s a m p l e s ( e x p o s e d to 50 H z electric, 20 k V / m , m a g n e t i c , 2 G, or c o m b i n e d electric and m a g n e t i c fields, 20 k V / m - 2 G, for 24 h at 37°C) were assayed by a m o d i f i c a t i o n o f the filter e l u t i o n t e c h n i q u e in o r d e r to m a k e sure that no D N A - p r o t e i n crosslinks were g e n e r a t e d by the
Results and discussion
12,NA s i n g l e - s t r a n d b r e a k s w e r e r e a d i l y det e c t c d by m o n i t o r i n g the r a t e of D N A elution w h e n c r u d e cellular lysates w e r e e l u t e d with an alkaline solution t h r o u g h m e m b r a n e s with 2-p~m pores. Fig. 1A shows that t r e a t m e n t of K562 cells for 30 min with v a r i o u s c o n c e n t r a t i o n s (0.25-1 m M ) of M M S , a known D N A - d a m a g i n g agent, significantly i n c r e a s e d the elutability of the D N A , thus suggesting that s i n g l e - s t r a n d b r e a k s w e r e p r o d u c e d . I n c r e a s i n g the c o n c e n t r a t i o n o f M M S
100 - ~
12
,,.,.,,0-..~ O~r.....0...._~0."
J t-
121 u.i
o
z
25
MU'fLET 0651
Electric a n d / o r m a g n e t i c field effects on D N A structure and function in cultured h u m a n cells M. Fiorani a, O. Cantoni b p. Sestili b R. Conti c, p. Nicolini c, F. Vetrano a and M. Dachh a Centro di Metodologie Biochimiche Applicate and b Istituto di Farmacologia e Farmacognosia, Unit~ersith degli Studi di Urbino, Urbino, and ~ ENEL, Centro di Ricerca Elettrica, Cologno Monzese, Milan (Italy) (Received 2 December 1991) (Accepted 23 December 1991)
Keywords: ELF; Electric and magnetic fields; DNA damage; Cytotoxicity; Cultured mammalian cells
Summary Exposure of cultured K562 cells to 50 Hz electric (0.2-20 k V / m ) , magnetic (0.002-2 G), or combined electric and magnetic fields for up to 24 h did not result in the production of detectable D N A lesions, as assayed by the filter elution technique. The rate of cell growth was also unaffected as well as the intracellular A T P and N A D + levels. These results indicate that, under the experimental conditions utilized in this study, 50 Hz electric, magnetic and electromagnetic fields are not geno- and cyto-toxic in cultured mammalian cells.
Exposure to electromagnetic fields has been reported to increase the risk of certain types of cancer, such as leukemia, cancer of the central nervous system and lymphoma (Wertheimer and Leel~er, 1979; Savitz et al., 1988). The results from these studies, however, were not confirmed by other investigators (Fulton et al., 1980), and thus it is generally believed that more epidemiological research is needed in order to either prove or confute the association between exposure to the fields and the risk of cancer. Since most carcinogenic agents display genotoxic effects, it would be extremely important to
Correspondence: Dr. Orazio Cantoni, Istituto di Farmacologia e Farmacognosia, Universith degli Studi di Urbino, Via S. Chiara 27. 1-61029 Urbino (Italy).
gather information on whether or not field exposure induces genetic alterations. The results from these studies would certainly provide relevant information for either questioning or affirming the carcinogenicity of the fields. There are some preliminary reports in the literature (Miller et al., 1976; Cohen et al. 1986a, b; Benz and Carsten, 1986) indicating that, with few exceptions (Eberle and May, 1982), field exposure does not induce genetic alterations. In particular, in one study, the alkaline elution assay, a very sensitive technique for measuring D N A single- and doublestrand breakage and D N A - D N A crosslinks as well as DNA-protein crosslinks, has been used to test the effect of 60 Hz magnetic, electric and superimposed electric and magnetic fields (Reese et al., 1988) on the D N A of Chinese hamster ovary cells. Results from this study have indicated
26
that field exposure does not produce damage at the DNA level. It should be noted, however, that these experiments covered a very limited range of intensities and, importantly, cell exposure was only for 1 h (Reese et al., 1988). This experimental detail may have important implications since some effects of the fields in cellular systems have been observed only under specific a m p l i t u d e / frequency criteria (window effects) (Parkinson and Hanks, 1989). We now report the results of a study that was performed to investigate the effect of power frequency electric a n d / o r magnetic on the DNA of a human tumor cell line (K562 cells). In this study, the experiments involved field exposure for various lengths of time and have covered a wide range of intensities. In addition, other biochemical parameters such as NAD + and ATP levels as well as the rate of cell replication were evaluated. Materials and methods Field exposure system
The field exposure apparatus was developed in our laboratories and consisted of four units allowing uniform exposure to magnetic B-field (unit 1), electric E-field (unit 2), mutually orthogonal Efield and B-field (unit 3) and no field (unit 4). Cells were suspended in complete growth medium and exposed to 50 Hz electric (0.2-20 k V / m ) , magnetic (0.002-2 G), or combined electric and magnetic fields (see Table 1) for 1, 4, 6, 12 or 24 h at 37°C. Incubations were performed under conditions of continuous shaking. In some experiments, cells were treated with increasing concentrations of methyl methanesulfonate (MMS) and then assayed as described below. Materials
Radiolabeled compounds were from New England Nuclear Corp. (Boston, MA, U.S.A.). Tetraethylammonium hydroxide was from MerckSchuchardt (Munich, Germany). Free acid EDTA, disodium EDTA, tetrasodium EDTA, sodium dodecyl sulfate and most reagent-grade biochemicals were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Polycarbonate filters were from Nuclepore (Pleasanton, CA, U.S.A.). RPMI 1640
medium, fetal bovine serum and trypsin were from Gibco (Grand Island, NY, U.S.A.). Cells"
K562 cells (a human tumor cell line) were routinely grown in our laboratory in RPMI 1640 medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum at 37°C in an atmosphere of 5% CO 2 and 95% air. Cells were subcultured (2.5 × 105 cells/ml) and exposed for various time intervals to the fields (or for 30 min to MMS). Following exposure cells were analyzed for cell number, NAD + and ATP intracellular contents and DNA damage. Alkaline elution assay
Cells were labeled for 24 h with 14C-thymidine (0.05 p~Ci/ml) and then, following a 6-h incubation in a label-free medium, were exposed to the fields. At specific time intervals, samples were taken and analyzed by the filter elution assay. This procedure was carried out as described by Kohn et al. (1981) with minor modifications (Cantoni et al., 1986). Briefly, 5 x 105 cells were gently loaded onto 25-mm, 2 /zm pore polycarbonate filters and then rinsed twice with 10 ml of ice-cold saline A containing 5 mM E D T A (disodium salt). Cells were then lysed with 5 ml of 2% sodium dodecyl sulfate, 0.025 M E D T A (tetrasodium salt), pH 10.1. Lysates were rinsed with 7 ml of 0.02 M E D T A (tetrasodium salt) and the DNA was eluted in the dark with 1.5% tetraethyl ammonium hydroxide/0.02 M E D T A (free acid)/0.1% sodium dodecyl sulfate (pH 12.1), at a flow rate of about 0.04 ml/min. Fractions of approximately 3.5 ml were collected and counted in 7 ml of Lumagel containing 0.7% glacial acetic acid. DNA remaining on the filters was recovered by heating for 1 h at 60°C in 0.4 ml of 1 N HC1 followed by the addition of 0.4 N N a O H (2.5 ml) and was again determined by scintillation counting. D N A was also recovered from the interior of the membrane holders after vigorous flushing with 3 ml of 0.4 N NaOH. This solution was processed for scintillation counting as described above. A TP and N A D + assay
Cells were rinsed with saline A and extracted with 2.5% perchloric acid. Samples were then
27 n e u t r a l i z e d with K 2 C O 3 and, following centrifugation, a q u e o u s solutions o f t h e n u c l e o t i d e s w e r e f i l t e r e d t h r o u g h 0.22 p~m p o r e size microfilters a n d a n a l y z e d for A T P a n d N A D + c o n t e n t s by r e v e r s e d - p h a s e h i g h - p e r f o r m a n c e liquid chromatography. Chromatographic apparatus and c o n d i t i o n s have b e e n d e s c r i b e d (Stocchi et al., 1985).
r e s u l t e d in a progressive i n c r e a s e in the D N A elution rate, i n d i c a t i n g a d o s e - d e p e n d e n t g e n e r a tion of D N A s t r a n d breaks. S t r a n d scission factors w e r e c a l c u l a t e d f r o m alkaline elution profiles such as t h o s e in Fig. 1A, a n d the resulting d o s e r e s p o n s e curve relating the d e g r e e of s t r a n d b r e a k a g e to the initial c o n c e n t r a t i o n of the alkylating a g e n t in the e x t r a c e l l u l a r m e d i u m (shown in Fig. 1B) was l i n e a r over the r a n g e o f M M S conc e n t r a t i o n s studied. W h e n cells w e r e e x p o s e d to the fields ( u n d e r all the e x p e r i m e n t a l c o n d i t i o n s s u m m a r i z e d in T a b l e 1), the D N A e l u t i o n p r o files were not significantly d i f f e r e n t from those o f the s h a m - e x p o s e d cells, suggesting that field exp o s u r e d o e s not i n d u c e d a m a g e at the D N A level. S o m e s a m p l e s ( e x p o s e d to 50 H z electric, 20 k V / m , m a g n e t i c , 2 G, or c o m b i n e d electric and m a g n e t i c fields, 20 k V / m - 2 G, for 24 h at 37°C) were assayed by a m o d i f i c a t i o n o f the filter e l u t i o n t e c h n i q u e in o r d e r to m a k e sure that no D N A - p r o t e i n crosslinks were g e n e r a t e d by the
Results and discussion
12,NA s i n g l e - s t r a n d b r e a k s w e r e r e a d i l y det e c t c d by m o n i t o r i n g the r a t e of D N A elution w h e n c r u d e cellular lysates w e r e e l u t e d with an alkaline solution t h r o u g h m e m b r a n e s with 2-p~m pores. Fig. 1A shows that t r e a t m e n t of K562 cells for 30 min with v a r i o u s c o n c e n t r a t i o n s (0.25-1 m M ) of M M S , a known D N A - d a m a g i n g agent, significantly i n c r e a s e d the elutability of the D N A , thus suggesting that s i n g l e - s t r a n d b r e a k s w e r e p r o d u c e d . I n c r e a s i n g the c o n c e n t r a t i o n o f M M S
100 - ~
12
,,.,.,,0-..~ O~r.....0...._~0."
J t-
121 u.i
o
z
