WITHDRAWN: Toxicity and SOS response to ELF magnetic field and nalidixic acid in E. coli cells

Mutation Research 722 (2011) 84–88

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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

Toxicity and SOS response to ELF magnetic field and nalidixic acid in E. coli cells Igor Belyaev a,b,∗ a b

Department of Genetics, Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden Laboratory of Molecular Genetics, Cancer Research Institute, Bratislava, Slovak Republic

a r t i c l e

i n f o

Article history: Received 9 August 2010 Accepted 21 March 2011 Available online 29 March 2011 Keywords: ELF magnetic fields Genotoxicity RecA-lacZ fusion SOS response

a b s t r a c t Extremely low frequency (ELF) magnetic fields have previously been shown to affect conformation of chromatin and cell proliferation. Possible genotoxic and carcinogenic effects of ELF have also been discussed and tested. In this study, we analyzed the effect of ELF on chromatin conformation in E. coli GE499 cells by the anomalous viscosity time dependence (AVTD) technique. Possible genotoxic ELF effects at the specific combination of static and ELF magnetic fields, that has been proven to have effects on chromatin conformation, were investigated by clonogenic assay, cell growth kinetics, and analysis of SOS-response using inducible recA-lacZ fusion and the ␤-galactosidase assay. Genotoxic agent nalidixic acid (NAL) was used as positive control and in combination with ELF. Nalidixic acid at 3–30 ␮g/ml decreased the AVTD peaks and induced cytotoxic effect. In contrast to NAL, ELF increased AVTD, stimulated cell growth, and increased cloning efficiency. These effects depended on frequency within the frequency range of 7–11 Hz. While NAL induced SOS response, ELF exposure did not induce the recA-lacZ fusion. Exposure to ELF did not modify the genotoxic effects of NAL either. All together, the data show that ELF, under specific conditions of exposure, acted as nontoxic but cell growth stimulating agent. © 2011 Elsevier B.V. All rights reserved.

1. Introduction There is growing evidence for biological effects of weak magnetic fields of extremely low frequency (ELF, 1–300 Hz) in cellular systems [1]. Biochemical studies associated with ELF include effects on gene expression [2], calcium signaling [3,4], enzymatic activity [5,6], growth factor receptors [7,8], melatonin synthesis [9] and events involving generation of free radicals [10]. ELF effects on cell proliferation have been described and both inhibition and stimulation have been observed, depending on cell type and exposure conditions [11–13]. Exposure to ELF at low intensities has consistently been reported to be associated with increased childhood leukemia [14]. On the other hand, no association of ELF exposure with leukemia was found in adults. While this discrepancy has only partially been clarified at the mechanistic basis [15], ELF was classified as a possible carcinogen based on these studies [16]. Previous results have shown that ELF can both modify [17–19] and induce genotoxic

Abbreviations: AC, Alternating current; AVTD, anomalous viscosity time dependence; CFU, colony forming units; DC, direct current; DSB, DNA double strand breaks; EMF, electromagnetic field; ELF, extremely low frequency; NAL, nalidixic acid. ∗ Correspondence address: Department of Genetics, Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden. Tel.: +46 8 16 41 08; fax: +46 8 6129552. E-mail address: [email protected] 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.03.011

effects [20–25]. However, these observations were not supported by other investigations [26–28]. Experimental data have indicated that the ELF effects occur depending on several physical parameters including frequency and magnetic flux density [9,29]. Difference in these physical parameters and biological variables including genetic background, cell type and physiological state may explain various outcomes of studies with ELF effects [10,30,31]. In particular, the role of static magnetic (direct current, DC) fields during exposure to ELF alternating magnetic (alternating current, AC) field was shown in several studies [32–36]. Therefore, it seems plausible to take into account the values of DC geomagnetic field in investigations of potential genotoxic ELF effects [37]. The changes in chromatin conformation were found using the method of anomalous viscosity time dependence (AVTD) after exposure of E. coli cells to ELF at specific frequencies and combinations of AC/DC fields [38,39]. These specific frequencies varied between different E. coli strains and ELF effects depended on several physiological parameters as phase of cell growth and presence of radicals and ion scavengers [30,31,40]. In this study, we examined the E. coli GE499 cells for induction of recA-lacZ fusion, cell survival and cell growth under conditions of ELF exposure, which induced changes in chromatin conformation. We used recA-lacZ protein fusion to estimate possible induction of the DNA damage inducible recA gene by ELF. The recA-lacZ protein fusion is under transcriptional and translational control of recA gene, which is involved in a postreplication DNA repair system that allows DNA replication to bypass lesions or errors in the DNA (SOS

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response) [41]. The recA-lacZ protein fusion is very sensitive to DNA damage induced by various genotoxic agents such as nalidixic acid, ionizing radiation, ultraviolet, and chemicals [42–44]. The combined effect of nalidixic acid and ELF was also analyzed. 2. Materials and methods 2.1. Chemicals Reagent grade chemicals were from Sigma–Aldrich (St. Louis, MO, USA), Merck (Darmstadt, Germany) and Boehringer Mannheim (Scandinavia AB, Bromma, Sweden). 2.2. Cells The recA-lacZ fusion strain of E. coli GE499 provided by Dr. M. Näslund (Stockholm University, Stockholm, Sweden) was described previously [30]. The cells were grown for 20 h in 5 ml of Luria broth (“Sigma”, USA; tryptone 10 g/l, yeast extract 5 g/l, NaCl 0.5 g/l) as described previously [38]. Under these conditions, the cells reached an optical density of 0.83–0.89 at 550 nm, which corresponded to early stationary phase of growth. The cells were harvested by centrifuging and diluted at the concentration of 4 × 107 cells/ml in the M9 buffer (3 g/l KH2 PO4 , 0.2 g/l NH4 Cl, 6 g/l Na2 HPO4 , 1 g/l MgSO4 , 0.5 g/l NaCl, pH 7.0). 2.3. Exposure setup, ELF exposure and co-exposure In each independent experiment, GE499 cells in 5 ml aliquots were exposed to ELF at several different frequencies within 7–11 Hz for 15 min and then incubated for 75 min. Under these conditions of exposure, ELF resulted in changes of chromatin conformation in the GE499 cells as previously described [30]. A vertical ELF magnetic field was applied using a pair of 1200 turns, 17.6 cm diameter Helmholtz coils, mounted horizontally. Each coil had a resistance of 384.5  and an inductance of 251 mH. An AC generator supplied an alternating sine current signal. The intensities of static and alternating magnetic fields were controlled by means of a SAM 3 magnetometer (Dowty Electronics Ltd., UK) and a one-dimensional G-79 microteslometer (NPO Mikroprovod, Russia). Before, during and after exposure the horizontal and vertical components of the static magnetic field were 19 ± 1 ␮T and 43 ± 1 ␮T, respectively. The ambient ELF magnetic field was measured by means of a Combinova MFM10 three-dimensional microteslometer (Field dosimeter 3, Combinova, Sweden). This field was less then 0.05 ␮T during cell exposure and post-exposure incubation. During exposure, the temperature was measured with an accuracy of 0.1 ◦ C. No measurable heating was induced by exposure of cells in the Helmholtz coils. The control cells were concurrently subjected to the same manipulations as exposed cells including sham-exposure inside the Helmholtz coils while AC generator was disconnected. 2.4. Survival For estimation of cloning efficiency and survival, 100 cells were seeded on 90mm plastic plates with Luria agar in triplicate. The colonies were grown overnight. The number of colony forming units (CFU) was used to quantify the results. 2.5. ˇ-Galactosidase assay The assay for ␤-galactosidase activity was performed essentially as described previously by using the chloroform–sodium dodecyl sulfate lysis procedure [42]. 2.6. AVTD measurements The changes in chromatin conformation were measured in lysates using the method of anomalous viscosity time dependencies (AVTD) as described previously [30]. The cell suspension was distributed to the polyallomer test tubes, 1 ml in each. Solutions of lysozyme (“Sigma”, 1.5 mg/ml) – 0.3 ml, sarcosyl (“Serva”, 2%) – 1 ml and papain–glycerol (“Merck”, 3 mg/ml – 10%) – 0.7 ml were added to each test-tube. All solutions were prepared in a lysing buffer (0.25 M Na2 EDTA, 0.01 M Tris–base, pH 7.1). The lysates were kept in the dark for 40–45 h, at 33 ◦ C and the AVTD was measured using an AVTD-analyzer (Archer-Aquarius, Ltd., Russia) at a shear rate of 5.8 s−1 and a shear stress of 0.0065 N/m2 . Maximum relative viscosity was used to quantify the ELF effect on chromatin conformation. 2.7. Statistical analysis Statistica 8.0 (StatSoft Inc, Tulsa, OK) and SPSS Statistics 17.0 (SPSS Inc., Chicago, IL) software were used for statistical analysis. Some data did not fulfill the Poisson distribution as analyzed using the Kolmogorov–Smirnov test. Therefore, we analyzed data arrays using both non-parametric (the Kruskal–Wallis ANOVA by ranks, the Wilcoxon matched pairs test) and parametric statistics (ANOVA). Bonferroni adjustment was used in multiple comparisons. In general, both parametric and non-parametric statistics provided similar results and conclusions. Results were considered as significantly different at p < 0.05.

Fig. 1. Effects of ELF (A) and NAL (B) on chromatin conformation in E. coli GE499 cells. Cells were exposed to ELF (21 ␮T r.m.s., 15 min) or treated with NAL at various concentrations and lysed 75 min after exposure. Mean of four independent experiments and SD are shown in each data point.

3. Results In accordance to previously published data [30], exposure to ELF at specific frequencies caused increased maximal relative viscosity (Fig. 1A). Statistically significant differences from control were observed at 8.5 Hz, 8.7 Hz, 9 Hz and 9.5 Hz (ANOVA and Kruskal–Wallis ANOVA by ranks, p < 0.05). Thus, ELF effect depended on frequency. Contrary to ELF, NAL significantly decreased the AVTD peaks (Fig. 1B). This decrease is in line with previously published data showing that DNA breakage induced by ionizing radiation in E. coli cells resulted in decreased maximal relative viscosity [45,46]. In parallel with AVTD measurements, we analyzed survival by cloning efficiency and induction of recA-lacZ fusion by ␤-galactosidase assay. For this analysis, we have chosen five frequencies, from which two, 8.5 and 9 Hz, were effective and

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Table 1 ELF effects on cell growth and RecA-lacZ fusion in E. coli GE499 cells. Comparison

ELF and sham NAL + ELF and NAL (3 ␮g/ml) NAL + ELF and NAL (30 ␮g/ml)

Cell growth

RecA-lacZ fusion

Relative effect (% ± SD)

Wilcoxon test (p)

Relative effect (% ± SD)

Wilcoxon test (p)

107.2 ± 2.4 102.6 ± 4.1 101.3 ± 1.9

0.01 NS NS

97.7 ± 4.1 103.4 ± 6.6 102.1 ± 3.9

NS NS NS

three, 7, 8, and 10 Hz, were ineffective according to the AVTD data. ELF exposure was not toxic (Fig. 2A). On the contrary, statistically significant increase, about 12%, was observed in cloning efficiency at the frequencies of 8.5 Hz and 9 Hz (ANOVA and Kruskal–Wallis ANOVA by ranks p < 0.01). The level of ␤-galactosidase was very low both in control and ELF-exposed cells (around 300 units) and no statistically significant ELF effects on recA-lacZ induction were observed (Fig. 2B). To increase sensitivity of ␤-galactosidase detection, fresh medium was added to the cell suspension (1:9) 75 min after exposure and cells were further incubated at 37 ◦ C. The effective frequencies of 8.5 Hz or 9 Hz were used in these experiments. Each 30 min, the aliquots were taken from control and exposed suspensions for measurements of cell growth and ␤galactosidase. As expected, NAL inhibited cell growth (Fig. 3A) and induced SOS-response (Fig. 3B) in dependence on dose. On the other hand, ELF exposure neither induced ␤-galactosidase nor modified SOS-response induced by NAL. The data of four independent experiments with combined treatment of cells to ELF and NAL are given in Table 1. Interestingly, the cells grew slightly faster after exposure to ELF in comparison to unexposed cells (Fig. 3A, Table 1). This effect was relatively low, around 7%, but statistically significant (p < 0.01, Wilcoxon matched pairs test). From the obtained data, we conclude that ELF weakly stimulated cell division but neither induced SOSresponse alone nor modified toxic effects of NAL under combined treatment. 4. Discussion In this paper, we studied cell survival, cell growth and induction of DNA damage inducible recA-lacZ fusion in E. coli cells under conditions of ELF exposure, which resulted in changes of chromatin conformation as measured by the AVTD technique. The AVTD technique is sensitive to various types of genotoxic stress including ionizing and no-ionizing radiation, chemicals and heat shock [30,47–51]. The AVTD changes can be caused by DNA breaks as well as by other changes in chromatin conformation such as based on redistribution of proteins which are bound to DNA and condensation or decondensation of DNA domains of supercoiling (DNA-loops) [52,53]. Of note, toxic agents always resulted in decreasing the maximum relative viscosity in the E. coli cell lysates that is caused by transition of circular chromosomal DNA to linear form following by fragmentation and condensation of chromosomal DNA [48]. Similar to other genotoxic agents, nalidixic acid resulted in dose-dependent reduction of the maximum relative viscosity in the E. coli cell lysates (Fig. 1B). Such dose-dependent reduction in maximum relative viscosity was observed previously after exposure of E. coli cells to ionizing radiation at high doses [45,46]. Nalidixic acid is known to affect DNA by means of blocking a DNA-gyrase [54]. This decrease in gyrase activity was shown to result in accumulation of physiological DNA breaks [54]. Due to this DNA breakage, significant induction of SOS-response was observed (Fig. 3) concordantly with decrease in maximal relative viscosity (Fig. 1B). Contrary to genotoxic effects of NAL and ionizing radiation, ELF increased maximal relative viscosity in GE499 cells (Fig. 1A). The ELF-induced changes in chromatin conformation measured by the AVTD method as increase in maximal relative viscosity (Fig. 1A) may be connected to changes in rigidity, hydro-

Fig. 2. Effect of ELF on cloning efficiency (A) and induction of the recA-lacZ fusion on frequency of ELF exposure. E. coli GE499 cells were exposed to ELF (21 ␮T r.m.s., 15 min) at various frequencies and plated in Petri dishes 75 min after exposure. Mean of four independent experiments and SD are shown at each data point.

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References

Fig. 3. Effect of ELF and NAL on growth of GE499 cells (A) and induction of the recAlacZ fusion (B). The cells were treated with NAL (3 or 30 ␮g/ml) and/or exposed to ELF (9 Hz, 21 ␮T r.m.s., 15 min), incubated in M9 media for 75 min at room temperature, and then incubated in M9-LB media (9:1) at 37 ◦ C. NAL was added 45 min before ELF exposure. The data from one typical experiment out of four are shown.

dynamic radius, and compactness of nucleoids. Such changes can be caused by redistribution of proteins bound to DNA [52,53]. The correlation between ELF-induced AVTD changes with changes in the spectrum of DNA-bound proteins in E. coli cells has previously been described [53]. In contrast to NAL, ELF did not induce toxicity (Fig. 2A) or SOSfunction in cells (Fig. 3, Table 1). Therefore, ELF, under those specific conditions of exposure that induce changes in genome conformational state as measured with AVTD, neither damaged DNA alone nor modified the NAL-induced toxic effects. Contrary to nalidixic acid, ELF stimulated cell growth. ELFinduced stimulation was observed by both CFU assay (Fig. 2) and optical measurements (Table 1). These data support observations about ELF-induced cell growth stimulation reported previously for E. coli [38,39,55,56] and other cell types [20,57–60]. Interestingly, ELF under conditions of exposure that induce cell division have been used for medical treatments [60,61]. Of note, ELF inhibited cell proliferation and development in some other studies [62,63]. Dependence of ELF effects on physical and biological variables may account for these discrepancies between studies [30,31]. Therefore, our data cannot exclude possibility of ELF genotoxic effects under other conditions of exposure than was used in present study. In conclusion, the data suggest that ELF exposure is not toxic and do not affect DNA-damage inducible SOS-functions under specific conditions of exposure. To the contrary, we observed ELF stimulating effects measured by survival, cell growth kinetics and analyzing chromatin conformation.

Conflict of interest None.

Acknowledgements The author is thankful to Dr. M. Näslund (Stockholm University, Sweden) for kind donation of the recA-lacZ fusion strain and valuable technical advises. The Swedish Radiation Safety Authority and the National Scholarship Program of the Slovak Republic supported.

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