Life Sciences

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Effects of extremely low frequency electromagnetic ﬁ eld (ELF-EMF) on catalase, cytochrome P450 and nitric oxide synthase in

erythro-leukemic cells☆

Antonia Patruno

^a

, Shams Tabrez

^b

, Mirko Pesce

^a

, Shazi Shakil

^c

, Mohammad A. Kamal

^b

, Marcella Reale

^d,

⁎

aDepartment of Medicine and Aging Science, University‘G. d'Annunzio’of Chieti-Pescara, Via dei Vestini, Chieti, Italy

bKing Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia

cDepartment of Bioengineering, Integral University, Kursi Road, Lucknow, UP, India

dDepartment of Experimental and Clinical Sciences, University "G.d'Annunzio" Chieti-Pescara, Chieti, Italy

a b s t r a c t a r t i c l e i n f o

Article history:

Received 14 September 2014 Accepted 1 December 2014 Available online 11 December 2014

Keywords:

K562 Catalase Cytochrome P450 INOS

Aims:Extremely low frequency electromagneticﬁelds (ELF-EMFs) are widely employed in electrical appliances and different equipment such as television sets, mobile phones, computers and microwaves. The molecular mechanism through which ELF-EMFs can inﬂuence cellular behavior is still unclear. A hypothesis is that ELF- EMFs could interfere with chemical reactions involving free radical production. Under physiologic conditions, cells maintain redox balance through production of ROS/RNS and antioxidant molecules. The altered balance between ROS generation and elimination plays a critical role in a variety of pathologic conditions including neurodegenerative diseases, aging and cancer. Actually, there is a disagreement as to whether there is a causal or coincidental relationship between ELF-EMF exposure and leukemia development. Increased ROS levels have been observed in several hematopoietic malignancies including acute and chronic myeloid leukemias.

Main methods:In our study, the effect of ELF-EMF exposure on catalase, cytochrome P450 and inducible nitric oxide synthase activity and their expression by Western blot analysis in myelogenous leukemia cell line K562 was evaluated.

Keyﬁndings:A signiﬁcant modulation of iNOS, CAT and Cyt P450 protein expression was recorded as a result of ELF-EMF exposure in both phorbol 12-myristate 13-acetate (PMA)-stimulated and non-stimulated cell lines.

Modulation in kinetic parameters of CAT, CYP-450 and iNOS enzymes in response to ELF-EMF indicates an interaction between the ELF-EMF and the enzymological system.

Signiﬁcance:These new insights might be important in establishing a mechanistic framework at the molecular level within which the possible effects of ELF-EMF on health can be understood.

Introduction

There has been considerable concern and controversy about the effects related with extremely low-frequency electromagneticﬁelds (ELF-EMFs) on the health of human populations[36]. Power lines and almost all kinds of household electrical appliances such as television sets, computers, hair dryers, mobile phones and many more emit ELF- EMFs. In view of its large application in everyday life, great attention is focused on the effects of the ELF-EMFs. The biological effects of ELF- EMFs have been the subject of more extensive studies since they can penetrate deeper into tissues[6,13,14,55]. Several epidemiological

studies linked ELF-EMFs with an increased risk of cancer, for instance childhood leukemia, brain cancer, breast cancer, kidney cancer, cancer of the nervous system, lymphoma as well as cardiovascular diseases [7,32,45,48].

Despite the large number of studies performed, a causal relationship and biological mechanisms for potential effects of ELF-EMFs on carcinogenesis have not been clearly identiﬁed as yet. The main cause of skepticism is the ability of low amount of energy transfer by theseﬁelds to DNA[28]. Moreover, epidemiological associations observed between ELF-EMFs and cancer are believed to be mainly due to promoter, co- promoter or progressor effects rather than initiator[31].

On the other hand, appropriately controlled application of ELF-EMFs have therapeutic applications as well. Low frequency and low intensity ﬁelds have been used extensively for the treatment of non-union fractures and can accelerate wound healing[20,50,51]. Pain and spastic- ity reduction is another area in which pulsed electromagnetic therapy has been reported to be very effective[8,16].

☆ This research was partially supported by the grants from the Italian MIUR (60% in 2009).

⁎ Corresponding author at: Dept. of Experimental and Clinical Sciences, University“G d'Annunzio”Chieti-Pescara, Via Dei Vestini, 31 66100 Chieti, Italy.

E-mail address:[email protected](M. Reale).

Contents lists available atScienceDirect

Life Sciences

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Currently, very little is known about how ELF-EMF modiﬁes the biological systems. It can initiate a number of biochemical and physiological alterations in biological systems of different species[18,21,40].

The biological effects in cell lines exposed to ELF-EMFs have been frequently noted[19,24,35,33,42]. However, despite the large number of studies, an understanding is still lacking[12,25,46]. The time span of application is an important factor which governs the physiological response of cells towards ELF-EMF exposure. In our earlier study, we have reported that ELF-EMFs applied at different time lengths modulate chemokine production and keratinocyte growth via inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway and might inhibit inﬂammatory processes[54].

Another study reported that a single exposure to ELF-EMF results in a decrease in K562 differentiation, while continous ELF-EMF exposure caused an increase in differentiation[2].

In this study, we used the K562 cell line, that are considered to be a reliable in vitro model of the hematopoietic system and of oxidative stress to assess the biological effects of ELF-EMF exposure. The purpose of this study was to evaluate activity and expression of catalase, cytochrome P450 and inducible nitric oxide synthase and their kinetic parameters in K562 cell line exposed to a well-deﬁned and controlled ELF-EMF to add new knowledge about the mechanisms responsible for the biological effects of ELF-EMFs.

Material and methods

Magneticﬁeld exposure system and exposure conditions of cell cultures

The experimental setup and ELF-EMF exposure system have been previously described by[54]. Briefly, the oscillating magnetic field (AC MF) consisted of: 1) a generator of sinusoidal signal at 50 Hz (Agilent mod. 33220A, Loveland, USA); 2) a power amplifier (216;

NAD Electronics, London, UK); 3) an oscilloscope (ISR658; ISO-TECH,

Vicenza, Italy) dedicated to monitoring output signals from the gaussmeter (MG-3D, Walker Scientific, Worcester, MA, USA); 4) a 160 turn solenoid (22 cm in length, 6 cm in radius, copper wire diameter of 1.25 × 10⁻⁵cm) generating a horizontal magneticfield. The solenoid was then placed inside the incubator. The geomagnetic field and magneticfield generated by solenoid have the same orientation. The achieved MF intensity (1 mT (rms)) was measured continuously using the hall-effect probe, situated adjacent to the specimen located in the central part of the solenoid, and connected to the gaussmeter.

Exposure condition of cell cultures

Cell culture was located in the central part of the solenoid, which was characterized by the greatestﬁeld homogeneity (98%). This setting was placed inside the incubator with a 5% CO2atmosphere. The incubator built-in digital thermometer monitored the internal temperature, which was set constant at 37 ± 0.3 °C. In addition, another digital thermometer (HD 2107.2, Delta OHM, Padova, Italy) was placed inside the solenoid and near the cell culture to record local temperature varia- tions, while the temperature of the cell medium was measured using a specially designed thermoresistor (HD 9216; Delta OHM, Padova, Italy).

No signiﬁcant temperature changes related to ap; °C). However, no thermal effects on cells can be hypothesized for temperatures around 37 °C, since ELF-EMF interactions with biological molecules are known to be non-thermal in nature[53]. The low-level Joule heating was efﬁ- ciently dissipated by the fan system inside the incubator and was b0.1308 °C in the medium of exposed cells.

K562 cell culture

Human erythro-leukemic cell line K562 was grown in RPMI-1640 containing 10% heat inactivated fetal calf serum (FCS, Sigma, Milano, Italy), 5 mM of (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 1.5 mg/ml of gentamicin, 200μg/ml of penicillin and streptomycin and maintained at 37 °C in an atmosphere of 5% CO2in air. Before each experiment, cell viability was assessed by Trypan blue exclusion method. K562 cells were incubated with or without PMA (100 nM) at 37 °C, 5% CO2for 1, 3, 6, 9, 12, 18 and 24 h and exposed or not to ELF-EMF. Control non-exposed cells were placed in a different incubator, located in the same room. At the end of the incubation time, the cells were harvested and viability was evaluated by Trypan blue dye exclusion and counted in a Burker chamber. For each experiment the cultures were carefully matched in terms of all conditions including cell density, passage number and batch of medium. Each measurement was performed blind.

Measurement of catalase (CAT) activity

Catalase activity was measured spectrophotometrically as followed by[52]at different time points (1–3–6–9–12–18–24 h). The decompo- sition of H2O2was monitored continuously at 240 nm for 3 min. The assay mixture in aﬁnal volume of 3 ml contained 10 mM of potassium phosphate buffer (pH 7.4), 10 mM of H2O2 and 10μg of protein of enzymatic extract. CAT units were deﬁned as 1 μmol H2O2

decomposed/min at 25 °C.

Determination of O2−

Production of O2−was determined spectrophotometrically (Hewlett Packard 8452 A, Palo Alto, CA, USA) by monitoring the reduction of ferricytochrome c (Type VI, Sigma, Milano, Italy) at 550 nm, as described by [39] at different time points (1–3–6–9–12–18–24 h). Brieﬂy, ferricytochrome c (50μmol/l) was added to the cuvette containing the cells and PBS (ﬁnal volume 1 ml), either in the presence or absence of superoxide dismutase (SOD, 350 U/ml), subsequent changes in absor- bance were followed for 10 min. Rates of O2−production were calculated Fig. 1.CAT activity (υ) versus exposure time for control (C), PMA, ELF-EMF and PMA +

ELF-EMF. A spline curve was created with 28 points calculated with the x-values ranging from 1.0 to 24.0.

Table 1

Kinetic characterization of CAT activity in K562 cell line.

Condition Totalυ ↑® ↓® рТ(h) Рυ

Control 122 ± 2 6.9 0.74 ± 0.3 3.5 24.2

PMA 279 ± 13 0.83 0.18 ± 0.04 11.4 9.4

ELF-EMF 148 ± 2 2.5 0.92 ± 0.3 3.5 8.9

PMA + ELF-EMF 291 ± 10 1.5 0.49 ± 0.1 7.8 11.4

υ, activity;↑®, Upwardυrate;↓®, downwardυrate;рТ, peak time;рυ, peak activity.

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on the basis of the molar extinction coefﬁcient of reduced ferricytochrome c (ε= 21,000 cm⁻¹(mol/l)⁻¹). Cell counts were used to calculate results as nmol O₂⁻/10⁶cells/min.

Analyses of iNOS activity

iNOS activity was assayed by measuring the conversion ofL-(2,3-3H) arginine toL-(2,3-3H) citrulline in cell homogenates[34]at different time points (1–3–6–9–12–18-24 h). Brieﬂy, 1μl of radioactive arginine,

L-(2,3,4,5)-(³H) arginine monohydrochloride 64 Ci/mM, 1 μCi/μl (Amersham, Arlington Heights, IL, USA), and 5μl of nicotinamide adenine dinucleotide phosphate [NADPH] (10 mM), 5μl CaCl2(6 mM) (Calbiochem, CA, USA) were added to each sample and incubated for 30 min at room temperature. The reaction was stopped by the addition of 400μl of stop-buffer (50 mM HEPES, pH 5.5 and 5 mM EDTA).

Unreacted arginine was removed by the addition of equilibrated cation- ic exchange resin (Dowex AG50WX-8, Sigma-Aldrich, St. Louis, MO, USA). After centrifugation, the radioactivity, corresponding toL-(³H)- citrulline in the eluate, was measured with liquid scintillation spectrom- etry and expressed as pmol³H/min/mg protein.

Western blot analysis

Total protein extracts were prepared by treating cells with lysis buffer (RIPA). Proteins were quantiﬁed using the Bradford method.

Western blot analysis was performed as described previously by[34]

using the following primary antibodies: anti-iNOS (NOS2 (N-20), sc- 651), anti-Catalase [(F17), sc-34285] and anti-cytochrome P450 (ab19140). A rabbit anti-human polyclonal antibody recognizing the human GAPDH (G9545; Sigma-Aldrich) was used as control. The nitrocellulose membrane was then washed in TBS and incubated with sec- ondary antibody HRP-conjugated (dilution 1:10,000 Pierce) for 1 h, washed again, and developed. The nitrocellulose was scanned using a

computerized densitometric system (Bio-Rad Gel Doc 1000, Milan, Italy).

Estimation of new kinetic constants

Various kinetic constants such as total velocity (Totalυ), rate of increase (↑®), rate of decrease (↓®), pT (hr) and peak activity (рυ) of speciﬁc enzymes were estimated in K562 cell line exposed to ELF- EMFs in the presence or absence of PMA stimulation and in control using linear and non-linear regression analyses.

Graphics

Graphs were plotted using prism software (version 4, GraphPad, San Diego, CA) as described in[22,23]and speciﬁc kinetic constants were obtained by the regression analysis (linear and several different non- linear forms) computed by this packages.

Results

Kinetically characterization of CAT activity in K562 cell line

The characterization of the kinetic constant of CAT activity in K562 cell line has been described inTable 1. In the presence of PMA and PMA + ELF-EMF, a significant rise of 128% and 138% was recorded in totalυrespectively. Moreover, ELF-EMF exposure alone resulted in a significant increase of 21% in total velocity of CAT enzyme. Phorbol- 12-myristate-13-acetate is known as a direct activator of protein kinase C (PKC). The activation of PKC leads to the phosphorylation of the cytosolic components of the NADPH oxidase and thus to the release of ROS. Therefore, we used PMA as a positive control to show the activating capacity of the used cells. Moreover, the combined treatment group of ELF-EMF and PMA resulted in an increase of 9.3 fold in total υcompared with the control. A steep decline in the rate of increase (↑®) of enzymatic reaction was recorded in the presence of its stimulator i.e. PMA (6.9 vs 0.83). ELF-EMF exposure exhibited a comparatively lower decline in CAT enzymatic reaction (6.9 vs 2.5). However, combined ELF-EMF and PMA exposure leads to a reduction from 6.9 to 1.5 in↑® of enzymatic reaction. Rate of decrease (↓®) in CAT reaction was found to be reduced by 76% as a result of PMA exposure to this cell line. However, it was found to be elevated by 24% in the presence of ELF-EMF. Moreover, combined exposure of ELF-EMF and PMA resulted a reduction in↓® of CAT by approx 34%. Time at which maximum activity was recorded (pT) in response to PMA, ELF-EMF and PMA plus ELF-EMF exposure was recorded to be 11.4, 3.5 and 7.8 h respectively compared to control (3.5) (Fig. 1). This result highlighted the significant change in peak time in response of PMA and PMA plus ELF-EMF exposure. A major reduction in the peak velocity was recorded from 24.2 to 9.4, 8.9 and 11.4 as a result of exposure to stimulator ELF-EMF and PMA plus ELF-EMF respectively (Fig. 1).

Kinetically characterization of CYP-450 activity in K562 cell line

The kinetic constants of O2−production by CYP-450 in K562 cell line has been described inTable 2. A significant rise of approx 23% and 52% in total velocity of CYP-450 was recorded in response to independent exposure of PMA and ELF-EMF respectively. Moreover, the combined exposure group of PMA and ELF-EMF resulted in a 70% increase in CYP-450 total velocity. A significant rise in rate of increase (↑®) of enzymatic reaction by 11%, 65% and 104% was recorded in response to PMA, ELF-EMF and PMA plus ELF-EMF exposure respectively. Rate of decrease (↓®) in CYP-450 enzymatic activity did not show any significant change in response to stimulator. However, it was found to be elevated by 109%

and 195% in response to ELF-EMF and PMA + ELF-EMF exposure respectively. Time at which CYP-450 activity attended peak was found to be decreased from 15.9 to 15.6 in response to PMA plus ELF-EMF Fig. 2.CYP-450 activity (υ) versus exposure time for control (C), PMA, ELF-EMF and

PMA + \ELF-EMF. A spline curve was created with 28 points calculated with the x-values ranging from 1.0 to 24.0.

Table 2

Kinetic characterization of CYP450 activity in K562 cell line.

Control 386 ± 9 1.06 0.61 15.9 16.9

PMA 474 ± 10 1.18 0.64 16.03 18.9

ELF-EMF 588 ± 8 1.75 1.28 16.04 28.1

PMA + ELF-EMF 655 ± 8 2.17 1.80 15.6 33.9

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exposure (Fig. 2). However, it was found to be similarly elevated from 15.9 to 16.04 as a result of independent exposure of PMA or ELF-EMF.

Peak activity was also found to be increased from 16.9 to 18.9, 28.1 and 33.9 in response to PMA, ELF-EMF and PMA plus ELF-EMF exposure respectively (Fig. 2).

Kinetic characterization of iNOS activity in K562 cell line

The kinetic parameters related with iNOS activity have been described inTable 3. Total velocity was found to have signiﬁcantly declined by 58%, 73% and 75% in response to PMA, ELF-EMF and PMA + ELF-EMF exposures respectively. Moreover, the rate of increase of iNOS reaction was found to be increased by more than 8 and 4 fold as a result of PMA and PMA + ELF-EMF exposure respectively. However, rate of increase in enzymatic reaction was found to be reduced by 68%

in response to ELF-EMF exposure only. A steep decline in the rate of decrease (↓®) by 89%, 82% and 95% in iNOS reaction was recorded in response to PMA, ELF-EMF and PMA plus ELF-EMF exposures respectively.

The change in peak time (pT) was recorded from 14.6 h to 13.9 h in response to ELF-EMF (Fig. 3). However, an equally steep fall in pT value was recorded in response to PMA and PMA plus ELF-EMF compared with control (14.6 vs 1.9). A more than 5 and 3 fold increase in peak activity (Рυ) was recorded in response to PMA and PMA + ELF- EMF exposure respectively. However, ELF-EMF exposure alone resulted in a 77% decline in peak activity (Fig. 3).

Effect of ELF-EMF on iNOS, catalase and cytochrome P450 protein expression in K562 cells

To conﬁrm the effect of ELF-EMF on the expression of iNOS, catalase and cytochrome P450, Western blot analysis was performed. The time- course of iNOS, catalase and cytochrome P450 expression in unexposed or ELF-EMF-exposed K562 cells treated or untreated with PMA is reported inFig. (4).Fig. 4A shows that iNOS protein is up-regulated in both exposed and non-exposed cells from 1 h and reached a peak level at 18 h with higher reduction in ELF-EMF-exposed cells. At 24 h, iNOS protein levels were found to be declined in both exposed and non-exposed K562 cells. In PMA-stimulated cells, a signiﬁcantly decreased iNOS protein expression was recorded in ELF-EMF-exposed cells with respect to non exposed cells, at all time points studied.

Because ROS are naturally produced as a consequence of aerobic metabolism, cells have developed a sophisticated antioxidant strategy to prevent the toxic accumulation of these species. In our study, densitometric analysis of catalase protein showed a peak of expression after 3 h of incubation, which overlap with the peak of activity. This is the ﬁrst study that evaluated CAT activity after short term (1 to 3 h).

Previous studies have evaluated CAT activity at 24–48 h in leukemia cells and both over-expression and suppression of catalase have been observed[4].

We showed that in K562 cells exposed to ELF-EMF, levels of catalase were reduced with respect to non-exposed cells, at all studied time points. Conversely, in ELF-EMF exposed and PMA-treated K562 cells, the expression of catalase was found to be significantly higher compared with nonexposed cells (Fig. 4B). Moreoever,Fig. (4C) shows that ELF-EMF exposure significantly increase cytochrome P450 protein levels in non-stimulated cells, at all time points studied. While in PMA-treated cells, only after 18 h of exposure to ELF-EMF, we observed a significant increase in cytochrome P450 expression. We have also added a representativefigure displaying the effect of ELF-EMF on the total activities of CAT, CYP450 and iNOS enzymes aimed at a quick understanding of this article for readers from all thefields (Fig. 5).

Discussion

Oxidative stress is a condition arising from an increased production of reactive oxygen species (ROS) associated with a decreased antioxidant capability of the cell. ROS are constantly generated in aerobic cells by the incomplete reduction of molecular O2 to H2O during mitochondrial oxidative phosphorylation, as well as during a number of processes such as inﬂammation, infections, mechanical and chemical stresses, exposure to UV and to ionizing irradiation[29]. Evidence for chronic oxidative stress has been found in several hematopoietic malignancies such as myeloid leukemia. Moreover, ROS play an important role in the regulation of signal transduction causing erythroid, neuronal, or monocytic differentiation as well[47].

Nitric oxide (NO) is a highly reactive free radical that acts as inter/

intracellular mediator in physiological and pathological processes.

Nitric oxide is produced by a family of NO synthases (NOSs) which catalyze the conversion of the amino acidL-arginine toL-citrulline in a NADPH and O2-dependent process. Several differential cellular responses towards NO depend on the concentration and duration of exposure. Nitric oxide is part of anti-oxidative defenses by its diffusion-controlled reaction with O2−[1]. This prevents the reductive chemistry of O2−and inhibits H2O2formation. Lower levels of NO have been demonstrated to exert a protective function in leukemic and melanoma cells and to inhibit effector caspases by S-nitrosylation. On the other hand, in the excess of the superoxide radical, peroxynitrite are generated in the interaction with NO. The resulting nitrosative stress causes the inactivation of critical cellular enzymes by the nitrosylation of thiol groups and iron-sulfur clusters, which directly affects redox-sensitive transcription factors implied in carcinogenesis or modulates accessibility of promoters via increased DNA methylation or histone deacetylation[26]. These demonstrate that ROS generation and NO signaling mutually regulate each other's signaling mechanism.

Yoshikawa et al.[57]reported an increased production of NO under certain circumstances in response to ELF-EMF exposure. A possible mechanism proposed for the effect of ELF-EMF on biological systems has been its effect on the cellular oxidative machinery that leads to the production of ROS, and a earlier study also showed that electromagnetic radiation is capable of producing alterations in the antioxidant defenses of the cell[9,10,49]. Increased oxidative stress is also well documented in transformed cells[27]and ROS regulation is crucial for transformed cells that counteract their accumulation by up regulating antioxidant systems[5].

In another study, Farina[11]reported the generation of ROS and mitochondrial membrane potential on PC12, glioblastoma GL15 and Fig. 3.iNOS activity (υ) versus exposure time for control (C), PMA, ELF-EMF and PMA +

ELF-EMF. A spline curve was created with 28 points calculated with the x-values ranging from 1.0 to 24.0.

Table 3

Kinetic characterization of iNOS activity in K562 cell line.

Control (C) 402 ± 20.4 1.8 ± 0.45 3.61 14.6 31

PMA 169 15.28 0.42 ± 0.23 1.95 160

ELF-EMF 110 ± 6 0.58 ± 0.07 0.66 13.9 7.1

PMA + ELF-EMF 100 8.54 0.20 ± 0.13 1.90 102

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C2C12 myocyte cellular models, in response to ELF-EMF exposure. For these cell lines, they concluded that the production of ROS strictly depends on cell model rather than on the utilized ELF-EMF intensity or time of exposure.

In one study, Garip-İnhan et al.[15]reported the inﬂuence of ELF- EMF exposure on different cellular parameters such as proliferation, cell cycle distribution, superoxide radical anion and HSP70 protein levels in the human leukemia cell line K562. In another study, Ayşe et al.[2]reported increased ROS levels and differentiation in K562 cells in response to ELF-EMF exposure.

The enzyme activated by ELF-EMF carries on a feedback control of the signal process that modulates the calciumflux and also influenced other calcium-dependent cellular processes. Walleczek and Budinger [56]demonstrated that ELF-EMF exposure resulted a significant increase in serum alanine aminotransferase and aspartate aminotransferas activities compared with the control.

A search of the literatures revealed only three related articles by Harkins and Grissom[17]and Prashanth et al.[37,38]on the effect of ELF-EMF in which enzyme kinetics had been partially studied.

Prashanth et al.[37,38]reported an increase inα-amylase activity and velocity of reaction in response to 50 Hz ELF-EMF exposure and in another study they performed a kinetic analysis on the effect of ELF- EMF on acid phosphatase. Harkins and Grissom[17]demonstrated the effects of magneticﬁelds greater than 50 mT on the enzyme B12 ethanolamine ammonia lyase and observed a decrease in the ratio Vmax/Kmof the said enzyme.

In this work, we evaluated the cellular redox state by measuring the level of O2−production by cytocrome c reduction assay and CAT activity that is the main enzyme involved in redox homeostasis. In order to determine whether the modulation of iNOS, CAT and Cyt P450 activity is also related to their expression, the effects of ELF-EMF exposure on protein levels were also examined by Western blot assay. The data presented here show that exposure to ELF-EMFs modulates iNOS, CAT

and CYP-450 expression and enzyme activity, associated with a recov- ery of the antioxidant activity shown by an increase in total velocity of CAT and CYP 450. However, iNOS showed a significant decline in total velocity. For thefirst time, we have also proposed a computational imaginary image of stress caused by ELF-EMF on the catalase, cytochrome P450 and inducible nitric oxide synthase activity, aimed at an easy understanding of this manuscript for the readers from all thefields (Fig. 5). Although, several papers demonstrated that ELF-EMFs could act on membrane bound enzymes[30,41,43,44], it is possible to presume that the variation in enzymatic activities observed in our studies in response to ELF-EMF exposure, could depend upon the ability of ELF- EMF to stimulate transcription by interacting with DNA, as suggested by Blank and Goodman[3]or by activation of signaling pathways.

Our data suggest a possible action of ELF-EMFs on the redox state of cells and thus establish a plausible link between ELF-EMF exposure, the expression and activity of speciﬁc enzymes and their kinetic activities.

The analysis of enzyme kinetics could help in understanding cellular metabolism, and the alteration of rate constants could interfere with biological functions.

Conclusion

We hypothesize that ELF-EMF could trigger protein activation mediated by ligands, such as Ca²⁺, activating NADH oxidase that alter ROS-regulated pathways, particularly pro-proliferative and/or survival pathways. In addition, modulation of kinetic parameters of CAT, CYP- 450 and iNOS enzymes in response to ELF-EMF indicate an interaction between ELF-EMF and enzymological system. Our results confirm that the ELF-EMF affects not only the ROS product but also the enzymatic activity. Taken together, these results demonstrate the interplay between ROS and antioxidant molecules influenced by ELF-magnetic fields and underscore the subtle effects of low-frequency magnetic Fig. 4.Relative expression of iNOS (A) Catalase (B) and Cytochrome P450 (C) protein expression in ELF-EMF-exposed or not and PMA-treated or not, K562 cells. Each immune-reactive band was analyzed by densitometry and normalized to GAPDH levels. Each value represents the mean±SD of 4 different experiments. Each experiment was performed in triplicate (*P \b0.05).

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ﬁelds on oxidative metabolism and ROS signaling, that may be particularly important in hematologic malignancies.

Conﬂict of interest None to declare.

Acknowledgement

The authors gratefully acknowledge King Fahd Medical Research Center, King Abdulaziz University, Kingdom of Saudi Arabia and Integral University, Lucknow, India for providing the IT facility and research service opportunities for the completion of partial writing and data analysis tasks of this study.

References

[1] T. Andoh, P.B. Chock, C.C. Chiueh, Preconditioning-mediated neuroprotection: role of nitric oxide, cGMP, and new protein expression, Ann. N. Y. Acad. Sci. 962 (2002) 1–7.

[2] I.-G. Ayşe, A. Zafer, O. Sule, I.-T. Işil, T. Kalkan, Differentiation of K562 cells under ELF- EMF applied at different time courses, Electromagn. Biol. Med. 29 (2010) 122–130.

[3] M. Blank, R. Goodman, Initial interactions in electromagneticﬁeld-induced biosyn- thesis, J. Cell. Physiol. 199 (2004) 359–363.

[4] E. Coe, A.D. Schimmer, Catalase activity and arsenic sensitivity in acute leukemia, Leuk. Lymphoma 49 (10) (2008) 1976–1981.

[5] R.A. Cairns, I.S. Harris, T.W. Mak, Regulation of cancer cell metabolism, Nat. Rev.

Cancer 11 (2011) 85–95.

[6] M. Cifra, J.Z. Fields, A. Farhadi, Electromagnetic cellular interactions, Prog. Biophys.

Mol. Biol. 105 (2011) 223–246.

[7] C. D'Angelo, E. Costantini, M.A. Kamal, M. Reale, Experimental model for ELF-EMF exposure: concern for human health, Saudi J. Biol. Sci. (2014),http://dx.doi.org/

10.1016/j.sjbs.2014.07.006.

[8] S. Das, S. Kumar, S. Jain, V.D. Avelev, R. Mathur, Exposure to ELF- magneticﬁeld promotes restoration of sensori-motor functions in adult rats with hemisection of thoracic spinal cord, Electromagn. Biol. Med. 31 (3) (2012) 180–194.

[9] S. Di Loreto, S. Falone, V. Caracciolo, P. Sebastiani, A. D'Alessandro, A. Mirabilio, V.

Zimmitti, F. Amicarelli, Fifty hertz extremely low-frequency magneticﬁeld exposure elicits redox and trophic response in rat-cortical neurons, J. Cell. Physiol. 219 (2009) 334–343.

[10]S. Falone, M.R. Grossi, B. Cinque, B. D'Angelo, E. Tettamanti, A. Cimini, C. Di Ilio, F.

Amicarelli, Fifty hertz extremely low-frequency electromagneticﬁeld causes changes in redox and differentiative status in neuroblastoma cells, Int. J. Biochem. Cell Biol. 39 (2007) 2093–2106.

[11] M. Farina, ELF-EMFS induced effects on cell lines: Controlling elf generation in laboratory, Prog. Electromagn. Res. B 24 (2010) 131–153.

[12] K.R. Foster, Mechanisms of interaction of extremely low frequency electricﬁelds and biological systems, Radiat. Prot. Dosim. 106 (2003) 301–310.

[13] R. Funk, T. Monsees, N. Özkucur, Electromagnetic effects—from cell biology to medicine, Prog. Histochem. Cytochem. 43 (2009) 177–264.

[14]R.H.W. Funk, T.K. Monsees, Effects of electromagneticﬁelds on cells: physiological and therapeutical approaches and molecular mechanisms of interaction. A review, Cells Tissues Organs 182 (2006) 59–78.

[15] A. Garip-İnhan, I.İşal-Tugut, M.T. Kalkan, Effect of ELF-EMF on K562 cell differentiation in the presence or absence of quercetin and heat- shock, Biotechnol. Biotechnol.

Equip. 21 (2007) 182–185.

[16] A. Guseo, Pulsing electro-magneticﬁeld therapy of multiple sclerosis by the gyuling- bordas device: double blind, cross over and open studies, J. Bioelectricity 6 (1987) 23–35.

[17]T.T. Harkins, C.B. Grissom, Magneticﬁeld effects on B12 ethanolamine ammonia lyase: evidence for a radical mechanism, Science 263 (1994) 958–960.

[18] A.H. Hashish, M.A. El-Missiry, H.I. Abdelkader, R.H. Abou-Saleh, Assessment of biological changes of continuous whole body exposure to static magneticﬁeld and extremely low frequency electromagneticﬁelds in mice, Ecotoxicol. Environ.

Saf. 71 (2008) 895–902.

[19]C.Y. Huang, C.W. Chang, C.R. Chen, C.Y. Chuang, C.S. Chiang, W.Y. Shu, T.C. Fan, I.C.

Hsu, Extremely low-frequency electromagneticﬁelds cause G1 phase arrest through the activation of the ATM-Chk2-p21 pathway, PLoS One 9 (8) (2014) e104732 (11).

[20]R. Huo, Q. Ma, J.J. Wu, K. Chin-Nuke, Y. Jing, J. Chen, M.E. Miyar, S.C. Davis, J. Li, Noninvasive electromagneticﬁelds on keratinocyte growth and migration, J. Surg.

Res. 162 (2010) 299–307.

[21] A. Jelenković, B. Janać, V. Pesić, D.M. Jovanović, I. Vasiljević, Z. Prolić, Effects of extremely low-frequency magneticﬁeld in the brain of rats, Brain Res. Bull. 68 (2006) 355–360.

[22] M.A. Kamal, M. Reale, A.A. Al-Jafari, Multiple approaches to analyse the data for rat brain acetylcholinesterase inhibition by cyclophosphamide, Neurochem. Res. 35 (2010) 1501–1509.

[23]M.A. Kamal, Y. Tan, J.P. Seale, X. Qu, Targeting BuChE-inﬂammatory pathway by SK0506 to manage type 2 diabetes and Alzheimer disease, Neurochem. Res. 34 (2009) 2163–2169.

[24] J. Kaszuba-Zwoińska, I. Ciećko-Michalska, D. Madroszkiewicz, T. Mach, Z.

Słodowska-Hajduk, E. Rokita, W. Zaraska, P. Thor, Magnetic field anti- inflammatory effects in Crohn's disease depends upon viability and cytokine profile of the immune competent cells, J. Physiol. Pharmacol. 59 (2008) 177–187.

[25]R. Kavet, M.A. Stuchly, W.H. Bailey, T.D. Bracken, Evaluation of biological effects, dosimetric models, and exposure assessment related to ELF electric- and magnetic-ﬁeld guidelines, Appl. Occup. Environ. Hyg. 16 (2001) 1118–1138.

[26] K.-D. Kröncke, Nitrosative stress and transcription, Biol. Chem. 384 (2003) 1365–1377.

[27]T.B. Kryston, A.B. Georgiev, P. Pissis, A.G. Georgakilas, Role of oxidative stress and DNA damage in human carcinogenesis, Mutat. Res. 711 (2011) 193–201.

[28] Q. Ma, P. Deng, G. Zhu, C. Liu, L. Zhang, Z. Zhou, X. Luo, M. Li, M. Zhong, Z. Yu, C. Chen, Y. Zhang, Extremely low-frequency electromagneticﬁelds affect transcript levels of neuronal differentiation-related genes in embryonic neural stem cells, PLoS One 9 (3) (2014) e90041,http://dx.doi.org/10.1371/journal.pone.0090041.

[29] G. Manda, M.T. Nechifor, T.-M. Neagu, Reactive oxygen species, cancer and anti-cancer therapies, Curr. Chem. Biol. 3 (2009) 22–46.

[30] A. Morelli, S. Ravera, I. Panfoli, I.M. Pepe, Effects of extremely low frequency electro- magneticﬁelds on membrane-associated enzymes, Arch. Biochem. Biophys. 441 (2005) 191–198.

[31]M. Moretti, M. Villarini, S. Simonucci, C. Fatigoni, G. Scassellati-Sforzolini, S.

Monarca, R. Pasquini, M. Angelucci, M. Strappini, Effects of co-exposure to extremely low frequency (ELF) magneticﬁelds and benzene or benzene metabo- lites determined in vitro by the alkaline comet assay, Toxicol. Lett. 157 (2005) 119–128.

[32] National Radiological Protection Board, Electromagneticﬁelds and the risk of cancer: Supplementary report by the advisory group on non-ionising radiation, DOC. NRPB, 1994, pp. 77–81.

[33] J. Park, Y. Seo, H. Yoon, C. Kim, J. Park, S. Jeon, Electromagneticﬁelds induce neural differentiation of human bone marrow derived mesenchymal stem cells via ROS mediated EGFR activation, Neurochem. Int. 62 (2013) 418–424.

[34]A. Patruno, P. Amerio, M. Pesce, G. Vianale, S. Di Luzio, A. Tulli, S. Franceschelli, A.

Grilli, R. Muraro, M. Reale, Extremely low frequency electromagneticﬁelds modulate expression of inducible nitric oxide synthase, endothelial nitric oxide synthase and cyclooxygenase-2 in the human keratinocyte cell line HaCat: potential therapeutic effects in wound healing, Br. J. Dermatol. 162 (2010) 258–266.

[35] A. Patruno, S. Tabrez, P. Amerio, M. Pesce, G. Vianale, S. Franceschelli, A. Grilli, M.A.

Kamal, M. Reale, Kinetic study on the effects of extremely low frequency electromagneticﬁeld on catalase, cytochrome P450 and inducible nitric oxide synthase in human HaCaT and THP-1 cell lines, CNS Neurol. Disord. Drug Targets 10 (2011) 936–944.

[36]A. Pilla, Nonthermal electromagneticﬁelds: Fromﬁrst messenger to therapeutic applications, Electromagn. Biol. Med. 32 (2) (2013) 123–136.

Fig. 5.A representativeﬁgure displaying the effects of extremely low frequency electromagneticﬁeld (ELF-EMF) on the total activities of catalase (CAT), cytochrome P450 (CYP450) and nitric oxide synthase (iNOS) enzymes. Here,‘υ’stands for‘enzyme activity’. A symbolic‘electromagnet’shown at the left hand side represents the‘ELF- EMF’while the right hand side is representative of the‘Control’conditions (i.e. the enzyme activities when no ELF-EMF is applied). The ribbon structures of the enzymes were Constructed from the crystal structures available with the‘Protein Data Bank’(PDB).

PDB IDs for catalase, cytochrome P450 and nitric oxide synthase enzymes were 1F4J, 1W0E and 4NOS, respectively.

(7)

[37] K.S. Prashanth, T.R.S. Chouhan, S. Nadiger, Effect of 50 Hz electromagneticﬁelds on alpha amylase activity, Rom. J. Biophys. 18 (2008) 255–263.

[38] K.S. Prashanth, T.R.S. Chouhan, S. Nadiger, Effect of 50 Hz electromagneticﬁelds on acid phosphatase activity, Afr. J. Biochem. Res. 3 (2009) 060–065.

[39] K.A. Pritchard, L. Groszek, D.M. Smalley, W.C. Sessa, M. Wu, P. Villalon, M.S. Wolin, M.B. Stemerman, Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion, Circ. Res. 77 (1995) 510–518.

[40] F. Raggi, G. Vallesi, S. Ruﬁni, S. Gizzi, E. Ercolani, R. Rossi, ELF magnetic therapy and oxidative balance, Electromagn. Biol. Med. 27 (2008) 325–339.

[41] S. Ravera, B. Bianco, C. Cugnoli, I. Panfoli, D. Calzia, A. Morelli, I.M. Pepe, Sinusoidal ELF magneticﬁelds affect acetylcholinesterase activity in cerebellum synaptosomal membranes, Bioelectromagnetics 31 (2010) 270–276.

[42]S. Ravera, C. Falugi, D. Calzia, I.M. Pepe, I. Panfoli, A. Morelli, First cell cycles of sea urchin paracentrotus lividus are dramatically impaired by exposure to extremely low-frewquency electromagneticﬁeld, Biol. Reprod. 75 (2006) 948–953.

[43]S. Ravera, E. Repaci, A. Morelli, I.M. Pepe, R. Botter, D. Beruto, Effects of extremely low frequency electromagneticﬁelds on the adenylate kinase activity of rod outer segment of bovine retina, Bioelectromagnetics 25 (2004) 545–551.

[44] S. Ravera, E. Repaci, A. Morelli, I.M. Pepe, R. Botter, D. Beruto, Electromagneticﬁeld of extremely low frequency decreased adenylate kinase activity in retinal rod outer segment membranes, Bioelectrochemistry 63 (2004) 317–320.

[45] M. Reale, M.A. Kamal, A. Patruno, E. Costantini, C. D'Angelo, M. Pesce, N. Greig, Neuronal cellular responses to extremely low frequency electromagneticﬁeld exposure: implications regarding oxidative stress and neurodegeneration, PLoS One 9 (8) (2014) e104973.

[46]M.T. Santini, G. Rainaldi, P.L. Indovina, Cellular effects of extremely low frequency (ELF) electromagneticﬁelds, Int. J. Radiat. Biol. 85 (2009) 294–313.

[47] H. Sauer, M. Wartenberg, J. Hescheler, Reactive oxygen species as intracellular messengers during cell growth and differentiation, Cell. Physiol. Biochem. 11 (2001) 173–186.

[48]D. Stratton, S. Sigrun Lange, J.M. Inal, Pulsed extremely low-frequency magnetic ﬁelds stimulate microvesicle release from human monocytic leukaemia cells, Biochem. Biophys. Res. Commun. 430 (2013) 470–475.

[49] M. Simkó, M.-O. Mattsson, Extremely low frequency electromagneticﬁelds as effectors of cellular responses in vitro: possible immune cell activation, J. Cell.

Biochem. 93 (2004) 83–92.

[50] B. Strauch, M.K. Patel, J.A. Navarro, M. Berdichevsky, H.-L. Yu, A.A. Pilla, Pulsed magneticﬁelds accelerate cutaneous wound healing in rats, Plast. Reconstr. Surg.

120 (2007) 425–430.

[51]V.G. Sunkari, B. Aranovitch, N. Portwood, A. Nikoshkov, Effects of a low-intensity electromagneticﬁeld onﬁbroblast migration and proliferation, Electromagn. Biol.

Med. 30 (2011) 80–85.

[52] S. Tabrez, M. Ahmad, Some enzymatic/nonenzymatic antioxidants as potential stress biomarkers of trichloroethylene, heavy metal mixture, and ethyl alcohol in rat tissues, Environ. Toxicol. 26 (2011) 207–216.

[53]T.S. Tenforde, Biological interactions and potential health effects of extremely-low frequency magneticﬁelds from power lines and other common sources, Annu.

Rev. Public Health 13 (1992) 179–196.

[54] G. Vianale, M. Reale, P. Amerio, M. Stefanachi, S. Di Luzio, R. Muraro, Extremely low frequency electromagneticﬁeld enhances human keratinocyte cell growth and decreases proinﬂammatory chemokine production, Br. J. Dermatol. 158 (2008) 1189–1196.

[55] P. Volpe, Interactions of zero-frequency and oscillating magneticﬁelds with biostructures and biosystems, Photochem. Photobiol. Sci. 2 (2003) 637–648.

[56] J. Walleczek, T.F. Budinger, Pulsed magneticﬁeld effects on calcium signaling in lymphocytes: dependence on cell status andﬁeld intensity, FEBS Lett. 314 (1992) 351–355.

[57] Yoshikawa, Tanigawa, Imai, Hongo, Kondo, Enhancement of nitric oxide generation by low frequency electromagneticﬁeld, Pathophysiology 7 (2000) 131–135.