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Fifty-hertz magnetic fields induce free radical formation in mouse bone marrow-derived promonocytes and macrophages
Biochimica et Biophysica Acta 1674 (2004) 231 – 238 http://www.elsevier.com/locate/bba
Fifty-hertz magnetic fields induce free radical formation in mouse bone marrow-derived promonocytes and macrophages Jana Rollwitz, Madeleine Lupke, Myrtill Simko´* Division of Environmental Physiology, Institute for Cell Biology and Biosystems Technology, University of Rostock, Albert-Einstein-Strasse 3, D-18057 Rostock, Germany Received 9 February 2004; received in revised form 9 June 2004; accepted 30 June 2004 Available online 28 July 2004
Abstract Our findings show a significant increase of free radical production after exposure to 50 Hz electromagnetic fields at a flux density of 1 mT to mouse bone marrow-derived (MBM) promonocytes and macrophages, indicating the cell-activating capacity of extremely low frequency magnetic fields (ELF-MF). We demonstrate that after exposure to ELF-MF mainly superoxide anion radicals were produced, both in MBM macrophages (33%) and also in their precursor cells (24%). To elucidate whether NADPH- or NADH-oxidase functions are target proteins for MF interaction, the flavoprotein inhibitor diphenyleneiodonium chloride (DPI) was used. MF-induced free radical production was not inhibited by DPI, whereas tetradecanoylphorbolacetate (TPA)-induced free radical production was diminished by about 70%. TPA is known to induce a direct activation of NADPH-oxidase through the PKC pathway. Since DPI lacks an inhibitory effect in MF-exposed MBM cells, we suggest that 50 Hz MF stimulates the NADH-oxidase pathway to produce superoxide anion radicals, but not the NADPH pathway. Furthermore, we showed an oscillation (1–10 days) in superoxide anion radical release in mouse macrophages, indicating a cyclic pattern of NADH-oxidase activity. D 2004 Elsevier B.V. All rights reserved. Keywords: Free radical; ELF-MF; ROS; NAD(P)H-oxidase; NADH-oxidase; Promonocyte macrophage
1. Introduction Epidemiological studies have indicated a correlation between extremely low frequency electromagnetic fields (ELF-EMF) and an increased incidence of some types of cancer [1–6]. Numerous publications have detected biological effects of ELF-EMF in vivo and vitro, although the basic interaction mechanisms between these fields and living systems are presently unclear. In our former studies [7], we reported an increase in the ability of phagocytosis in murine macrophages after exposure to 50 Hz magnetic fields (MF) at 1 mT. In addition, we also documented an increased formation of free radicals in macrophages after short-term (45 min) exposure * Corresponding author. Tel.: +49 381 498 6318; fax: +49 381 498 6302. E-mail address: [email protected] (M. Simko´). 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.06.024
to MF. Tao and Henderson [8] have shown that 60 Hz fields cause differentiation in haematopoietic progenitor cells, measured as phagocytosis, and Roy et al. [9] described an increased respiratory burst in neutrophils after exposure to 0.1 mT MF (60 Hz) and explained their data by the increased lifetime of free radicals. Macrophages play an essential role in the body’s defences and immune system. Activated macrophages release free radicals as reactive oxygen species (ROS), reactive nitrogen species (RNS), and also cytokines. ROS are unstable reactive molecules which are produced continuously in several cells. Free radicals including superoxide anion radicals, hydroxyl radicals, and hydrogen peroxides are formed as by-products in various metabolic processes. ROS are involved in intracellular signal transduction pathways and regulation of gene expression determining the anti-inflammatory response, cell growth, differentiation, proliferation and stress response [10].
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Enzymes such as NAD(P)H-oxidases, xanthine oxidases or arachidonic acid-metabolizing enzymes mediate the main production of ROS in macrophages. In phagocytic cells, the NADPH-oxidase is commonly associated with the brespiratory burstQ activity catalyzing the reduction of oxygen to superoxide anion radical. This high level of free radical formation is a primary host defence mechanism against any invading microorganism and is connected with cell activation. Isoforms of NADPH-oxidase are present in various non-phagocytic cells which were found to have similar characteristics. NADH-oxidase has been implicated in numerous cellular processes within signal transduction cascades and regulatory processes [11]. Alternatively, superoxide anion radical generation occur non-enzymatically by redoxreactive compounds such as the semi-ubiquinone compound of the mitochondrial electron transport chain [12]. In response to outside influence, superoxide anion radical is the primary one generated by phagocytes. They have a low bactericidal potency and are converted into other ROS that serve as mediators in many regulatory processes [13]. In cells, free radical concentration is determined by the balance between their rate of production and their rate of clearance, controlled by different enzymes and antioxidant compounds. These regulatory processes are important to reset the original state of redox homeostasis after temporary production of free radicals. High-level production of free radicals in the organism has shown an increased potential for cellular damage of substances such as DNA, proteins, and lipid-containing structures [14]. In contrast to molecules such as cytokines (large molecules signalling by docking with specific receptors and change molecular surfaces on the target cells) molecules such as ROS could react with diverse cell compounds in a non-specific mechanism. Therefore, free radicals play a decisive role in cytotoxicity and also as cellular messengers to control non-cytotoxic physiological responses. In this study, we investigated the effects of 50 Hz MF on free radical production, in mouse bone marrow-derived (MBM) macrophages and promonocytes. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and lipopolysaccharides (LPS) were used as positive controls, representing receptor-independent and receptor-dependent induced pathways, respectively. We assume that the activation of NADH-oxidase plays a major role also in phagocytic cells and is responsible for the MF-induced free radical production. To investigate this, we used the specific NADPHoxidase inhibitor diphenyleneiodonium chloride (DPI), which does not affect the NADH-oxidase activity, making it possible to distinguish between the two pathways. In addition, differentiated macrophages were investigated for extremely low frequency magnetic fields (ELF-MF) induced superoxide anion radical production over a period of 16 days to find out possible cell status-dependent sensitivity to field exposure.
2. Materials and methods 2.1. Cell culture Bone marrow cells were isolated from femur and tibia of Shoe-NMRI mice, resuspended and washed in phosphate buffer saline solution (PBS). Cells were used directly after preparation or seeded in tissue culture dishes (10020 mm, Biochrom-TPP, Berlin, Germany) in RPMI-1640 medium (PAA-Laboratories GmbH, Karlsruhe, Germany) supplemented with 30% conditioned medium from L929-cell line, 6% heat-inactivated fetal calf serum (FCS, Gibco BRL, Karlsruhe, Germany), 0.5% penicillin/streptomycin (10,000 U/10,000 Ag/ml), non-essential amino acids (Biochrom KG, Berlin, Germany) and 0.125% 2-mercaptoethanol. MBMderived macrophages were obtained by differentiation of promonocytes in vitro cultured for 2–3 days as a monolayer. Cells were incubated at 37 8C in a humidified atmosphere containing 5% CO2, medium was changed at regular intervals for 3 days. After differentiation to adherent and densely growing macrophages, cells were cooled to 20 8C in a freezer for 2–3 min until the cells rounded up. Macrophages were scraped off and re-plated into sterile culture dishes or prepared for experiments. 2.2. Electromagnetic field exposure For MF generation a Helmholtz coil system was used as described previously [7]. The Helmholtz coils were stationed in the middle of a 37 8C humidified incubator (Binder, Germany) containing 5% CO2. Non-exposed cell cultures were incubated in an identical incubator without a MF source. For all experiments, cells were exposed to horizontally polarized 50 Hz electromagnetic fields with a flux density of 1 mT. Cell culture plates were placed in the centre of the Helmholtz coils or in a control incubator for different time periods depending on experimental conditions. The magnetic flux density was measured with a precision Gauss/Tesla Meter (F.W. Bell Inc., USA, Model 6010) directly before cell exposure. 2.3. Production of ROS Directly after cell isolation promonocytes (15104 cells/ well/200 Al) were seeded in 96-well plates and cultured for 45 min in cell culture medium with or without 1 AM TPA (Sigma-Aldrich, Munich, Germany), 1 Ag/ml LPS (from E. coli, Serotype 026:B6 (Sigma-Aldrich) and/or 1 AM DPI (Sigma-Aldrich) in the presence or absence of MF. After exposure, cells were loaded with 1 AM dihydrorhodamine 123 (DHR, Molecular Probes, Leiden, Netherlands) in HBSbuffer (0.9% NaCl solution with 14 mM HEPES (Roth, Karlsruhe, Germany)) for 25 min at 37 8C in darkness. Fluorescence was subsequently measured using flow cytometer (Beckmann/Coulter Epics Altra, Germany) at 550 nm.
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2.4. Superoxide anion radical generation The nitro blue tetrazolium (NBT) test was used to measure the amount of superoxide anion radical in macrophages. From day 5 to day 16 of cell culture, cells were taken daily and seeded in 96-well flat-bottomed microtiter plates (Biochrom-TPP) at a concentration of 5104 cells per well and cultured overnight. Macrophages were incubated for 45 min in PBS supplemented with 1 mM NBT (Sigma-Aldrich), 1 AM TPA, 1 Ag/ml LPS and 1 AM DPI in the presence or absence of MF. In the same set of experiments, cells were additionally treated with DPI in parallel. After incubation for 45 min, supernatants were removed and the intracellular formazan crystals were solubilized in 100 Al dimethylsulfoxide (DMSO, SigmaAldrich). The absorbance was measured at 550 nm using a micro-ELISA reader (EL-808, BioTek Instruments, Inc., Vermont, USA). 2.5. Nitrogen generation Cells were cultured in 96-well plates (5104 cells/well) for at least 12 h prior to use. The production of NO2 during 45 min up to 24 h of MF and/or chemical exposure was determined in macrophages. For experiments, the following conditions were used: control cells were cultured with medium alone or medium containing 1 Ag/ml LPS; simultaneously, MF-exposed cells were cultured with medium alone or with the same additions as the controls. Nitrogen monoxide (NO) was measured as nitrite (NO2 ) using the colorimetric method based on the diazotization reaction of Griess reagent. Fifty-microliter aliquots of cell culture supernatants were mixed with an equal volume of 1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride (Griess reagent) and incubated for 10 min at room temperature (RT) in darkness. To abort the color reaction, 25 Al of 1 M H3PO4 was added and the absorbance
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at 550 nm was measured on an ELISA reader. Concentrations of NO2 were calculated using NaNO2 standard. 2.6. Flow cytometry For surface staining, freshly isolated bone marrow cells (15104 cells) were resuspended and incubated in 200 Al PBS supplemented with 2 mM EDTA, 0.1% BSA and 10 Al FITC-conjugated monoclonal rat anti-mouse CD11b-antibody or rat monoclonal immunoglobulin Isotype control (BD Bioscience, San Diego, USA) for 30 min at RT in darkness, followed by centrifugation (5 min, 200g). Cells were washed in 750 Al wash-buffer (PBS+0.1% NaN3, 1% FCS) before flow cytometric analysis (Beckmann/Coulter Epics Altra). 2.7. Statistical analysis Each independent experiment was performed 3–12 times. Data of exposed cells or chemically treated cells were analysed with reference to their respective controls. To compare the variability of each experiment, data were analysed as a ratio between experiment and respective control (E/C) and the 99% confidence interval level (CI) is given. For statistical analyses, Student’s t-test was used ( Pb0.01).
3. Results 3.1. Flow cytometric analysis in promonocytes The MBM-derived promonocytes used in these studies represent a homogeneous population of CD11b-positive cells (Fig 1A). The surface protein CD11b is considered to be a specific marker for murine promonocytes and macrophages. The population of CD11b+ cells were ascertained
Fig. 1. Flow cytometric results of normal MBM cells. The cells are stained with CD11b-FITC (A) and IgG2bn-FITC (B). The distribution of CD11b+ cells is shown in panel A. Fluorescence signal greater than of the isotypes were defined as positive cells. To compare isotypes and specific CD11b-binding, the fluorescence intensity of macrophages labelled with FITC-conjugated control antibody is presented in panel B.
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Control cells were not affected in ROS production after DPI-treatment. The production of free radicals in MF+DPIexposed cells is comparable to MF alone. Co-exposure of promonocytes with MF and TPA show no additional effects. Stimulation of mouse promonocytes with TPA yielded a 42fold increase whereas the presence of DPI resulted in a decrease to 14-fold of ROS release compared to controls. 3.2. Superoxide anion radical production in promonocytes
Fig. 2. Flow cytometric analysis of rhodamine fluorescence in CD11bpositive cells. The diagram shows the changes in rhodamine fluorescence in cells exposed to control conditions, MF or TPA for 45 min. Changes in rhodamine fluorescence intensity show the transformation of DHR leading to an increased free radical production after MF or TPA treatment of cells.
via antibody staining with FITC-conjugated rat anti-mouse CD11b or FITC-conjugated rat IgG2bn monoclonal isotype control. Bone marrow cells were investigated directly after isolation. Fig. 1A shows a typical distribution of MBM cells, whereby CD11b+ cells were gated. The specific and unspecific binding side of the antibody is shown in Fig. 1B. Production of ROS was analysed using the oxidation sensitive DHR assay. Directly after preparation promonocytes were incubated for 45 min in medium in the presence or absence of MF, TPA, LPS, DPI or in different combinations. After exposure, cells were loaded with DHR and changes in the fluorescence signal were measured using flow cytometry (Fig. 2). Promonocytes exposed to LPS or MF show a significantly increased production of free radicals (1.6-fold, 1.2-fold, respectively, Fig. 3). The NADPH-oxidase inhibitor DPI was used to determine whether the production of free radicals after MF exposure is connected to the NAD(P)H-enzyme activation process.
To identify the specific free radical species produced, we investigated the superoxide anion radical production after exposure to TPA, LPS and MF in promonocytes using the NBT-assay. Directly after preparation, cells were incubated for 45 min in NBT supplemented with TPA, LPS, DPI and in different combinations in the presence or absence of MF. Fig. 4 demonstrates a significant increase of superoxide anion radical release after exposure to TPA (3.5-fold), LPS (1.2-fold) and MF (1.2-fold) compared to control cells. After DPI addition to control cells, a reduction in superoxide anion radical production could be observed. Co-exposure to TPA and DPI results in a non-significant inhibition of reactive superoxide anion radical production when compared to TPA alone. A similar effect could be detected after co-exposure to DPI+MF, no inhibition of superoxide anion radical generation was observed. 3.3. Time course studies of superoxide anion radical production in MBM macrophages Promonocytes are able to differentiate into adherent macrophages within 2 days. Directly after preparation, cells were seeded in cell culture dishes and incubated at 37 8C. Differentiation of promonocytes results in morphological and functional changes. Promonocytes, monocytes and macrophages could be identified by the presence of extracellular membrane proteins such as CD11b. Differ-
Fig. 3. ROS production in promonocytes after exposure to 1 mT MF, 1 AM DPI, 1 AM TPA, 1 Ag/ml LPS and in different sets of co-exposures using DHR assay. Columns show mean data of experiments as a ratio between experiments and controls (E/C). Data represents the mean of triplicates of four independent experiments (n=12)Fconfidence interval (CI) and asterisks indicate statistically significant differences at the 99% level ( Pb0.01).
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Fig. 4. Superoxide anion radical generation in freshly isolated promonocytes after 45 min exposure in NBT supplemented with 1 AM TPA, 1 Ag/ml LPS, 1 AM DPI or TPA and DPI. Cells were incubated under control conditions or were exposed to 1 mT MF. Columns are means from triplicate measurements and six independent experiments (n=18)FCI, asterisks indicates statistically significant differences ( Pb0.01) to the respective control.
entiated cells represent a homogenous culture of CD11bpositive cells. Determinations of superoxide anion radical generation in MBM macrophages were performed in 24-h intervals starting at day 5 or 6 after cell adherence, and up to day 16 in culture using the NBT-assay. Macrophages were stimulated with TPA, LPS or MF for 45 min. Curves in Fig. 5 demonstrate the mean value of superoxide anion radical production of three different mice in three different experiments. The time series represents changes in the activation status of macrophages after chemical and MF treatment. In all cases, an oscillating ability to produce superoxide anion radical was found independently of culture condition. MF induced a significant superoxide anion radical release on days 7, 10, 12 and 14 in mouse 1 (Fig. 5A), and on days 7, 10, 14–16 in mouse 2 after preparation (Fig. 5B), whereas cells of mouse 3 showed a significant increased superoxide anion radical production on days 6 and 10–13 (Fig. 5C). This fact clearly shows that MF exposure enhances superoxide anion radical production at certain time points only, showing a correlation to LPS and TPA activation. However, this cyclic cell response changes in different animals. 3.4. Nitrogen oxide production in MBM macrophages We previously reported [7] that exposure to magnetic fields at 1 mT resulted in a significant increase of phagocytic uptake and ROS production in macrophages. To determine whether activation of macrophages is associated with the production of nitrogen oxide (NO), cells were exposed to 1 mT MF for 45 min up to 24 h. To activate the inducible nitric oxide synthase (iNOS) enzyme complex, we used LPS as a positive control. Fig. 6 shows the timedependent production of nitrite in macrophages after treatment with 1 Ag/ml LPS. In both LPS-treated and MF+LPS-
treated cells, a production of NO2 was seen after 8 h and longer times. MF-exposed cells show no detectable production of NO2 .
4. Discussion In this study, we demonstrate that 50 Hz magnetic fields (1 mT) affect cell function in murine macrophages and also in their precursor cells in vitro. Measurement of free radicals was used to indicate the cell-activating process in these immune relevant cells. The production of ROS has been recognized as a key chemical process that regulates signal transduction pathways leading to control of gene expression and posttranslational modification of proteins [14]. The measured ROS production using DHR includes the detection of hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and peroxy nitrite anions (ONOO ), whereas using NBT assay, superoxide anion radicals were detected directly. Our results show a significant increase in ROS production (20%) and in superoxide anion radical release (25%) in MF-exposed promonocytes. Furthermore, in differentiated macrophages, a significant increase of superoxide anion radical production up to 33% could be detected after MF exposure. Therefore, we suggest that MF induces cellular activation processes in murine promonocytes and macrophages. Identification of cellular sources of superoxide anion radical and ROS production is of major importance for investigating MF-mediated physiological processes in macrophages. We used DPI as a flavoprotein inhibitor to detect the potential source of free radical formation. DPI appears to inactivate flavoproteins at their flavin sites during electron-transfer reaction and is also known to inhibit K+ and Ca2+ channels [15]. In a preliminary series
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Fig. 5. Time course of superoxide anion radical generation in MBM macrophages in three independent examinations from three different animals (A, B, C). Curves represent the ability to produce superoxide anion radical after 45 min exposure to MF and chemical treatments such as 1 AM TPA and 1 Ag/ml LPS during a time course of 10–11 days. Determination of superoxide anion radical was performed in at least eight parallel probes at each time point using the NBTassay. n.s.: non-significant all other data are statistically significant to the control ( Pb0.01). Standard deviations (S.D.) are omitted for clarity.
of experiments, a concentration-dependent inhibition of TPA-stimulated NADPH-oxidase activity using DPI was studied and an optimal concentration of DPI was found at 1 AM (data not shown). Our results demonstrate that 1 AM DPI diminished ROS production by about 70% in TPA-treated promonocytes. On
the other hand, ELF-MF-induced formation of ROS and superoxide anion radicals was not inhibited by DPI in promonocytes. It is known that DPI inhibits flavoenzymes like NADPH oxidase, cytochrome P450 reductase and nitric oxide synthases leading to an inhibited free radical production [16–18]. The fact that DPI did not inhibit the
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Fig. 6. Production of nitrite oxide in MBM cells over 24 h, expressed in AM after exposure to 1 mT MF, 1 Ag/ml LPS or MF+LPS. Nitrogen levels were determined using Griess reagent assay as described in Materials and methods. Data are means from three separate experimentsFCI. Asterisk indicates Pb0.01.
MF-induced ROS production suggests that these flavoenzymes are not involved in the MF-mediated pathways. There are several cellular sources for free radical generation. One of the major sources of cellular superoxide anion radical production is the membrane-associated NAD(P)Hoxidase, a multi-component enzyme accepting electrons from the electron transport system. The free radical generation occurs at the flavin site of complex I, through reserved electron transfer [19,20] and is an unavoidable byproduct of cellular respiration. Another important source of superoxide anion radical formation is the NADH-oxidase. This enzyme was first found in membrane of nonphagocytic cells and plays a role in the regulation of intracellular signalling cascades in several cells [12,21]. This membrane-associated enzyme utilizes both NADH and NADPH as electron donor [10] and produces only onethird of the superoxide anion radicals compared to phagocytic NADPH-oxidase [11] responding to the respiratory burst. Our investigation demonstrates a low but significant induction of superoxide anion radical production (24% up to 33%) in promonocytes and macrophages after ELF-MF exposure. We assume therefore that activation of phagocytic NADPH-oxidase after exposure to MF should result in a higher production of superoxide anion radicals. A study from Morre´ [22] shows that inhibition of the plasma membrane NADH-oxidase activity by DPI was without any effect when the enzyme utilized NADH as electron donor in vascular cells. If NADPH was used as an electron donor, DPI inhibited the enzyme activity. Further, it is known that NADH-oxidase has a higher affinity to NADH than to NADPH [22]. It seems that MF induces the NADH- but not the NADPH-oxidase activity because it lacks the inhibitory effect of DPI in murine promonocytes and macrophages. This indicates the presence of two different pathways leading to free radical production in the same cell, namely through the activation of NADPHand/or NADH-oxidase. Interestingly, MF and TPA induced a superoxide anion radical release in an additive manner (360% TPA alone, 410% TPA+MF) in mouse promonocytes. Using DPI, the MF effect could be down-regulated to the TPA-induced
level, whereas the TPA effect was not affected. This fact indicates again the presence of the two independent acting pathways, the NADPH and the NADH in the same cell. Further, we investigated the influence of ELF-MF on differentiated macrophages and their nitrogen oxide production. Formation of NO could not be seen after exposure to ELF-MF for 24 h confirming the findings from Mnaimneh et al. [23] regarding to the production of NO in murine macrophages after exposure to MF. We found oscillations in the reactivity of differentiated macrophages regarding superoxide anion radical production. The ability to respond to external influences such as TPA, LPS or MF seems to be dependent on time after adherence in macrophages. Three independent examinations from three different mice show comparable alteration in the time course of superoxide anion radical generation (Fig. 5). Correlations between different exposure conditions give evidence for a fluctuating capacity of superoxide anion radical production. Morre´ et al. [24] investigated the biochemical basis for the biological clock involving the family of NAD(P)H-oxidase proteins. They demonstrated that this family of enzymes show a recurring pattern of oscillations with a period length of 24 min. Rosenspire et al. [25] found that the cytosolic concentration of NAD(P)H fluctuates in a periodic manner in neutrophils and in macrophages. We suggest that NADH-oxidase activity is responsible for the ELF-MF-induced superoxide anion radical production, which was found with maximal peaks between 7 and 12 days in a rhythmic pattern. However, the exact period length has to be investigated. An explanation of contradictory findings in the literature can thus be due to timedependent activating capacity of primary cells, where negative results of experiments are results of investigations performed at non-activating points of time. In summary, we reported in the present study about the cell-activating capacity of ELF-MF to induce superoxide anion radical and ROS production in murine MBM macrophages and their precursor cells. Furthermore, we detected the non-inhibitory capability of the flavoprotein inhibitor DPI in MF-exposed promonocytes to produce superoxide
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anion radicals, which indicates the activation of NADHoxidase. However, the induction of superoxide anion radicals using TPA was down-regulated by DPI, indicating inhibition of the NADPH-oxidase-mediated pathway. This suggests the independent action of two different pathways, namely the NADPH and the NADH pathways, leading to superoxide anion radical production in mouse macrophages and promonocytes and that MF stimulates the NADHoxidase only. Furthermore, we also showed the oscillating activation potential to produce superoxide anion radical in MBM cells, indicating the time-dependent capability of cell activity.
Acknowledgments The authors would like to thank Mats-Olof Mattsson, ¨ rebro, Sweden for helpful discussion. This University of O work is partly supported by the German Ministry of Education, Science and Technology (BMBF) and by the VERUM Foundation.
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Fifty-hertz magnetic fields induce free radical formation in mouse bone marrow-derived promonocytes and macrophages Jana Rollwitz, Madeleine Lupke, Myrtill Simko´* Division of Environmental Physiology, Institute for Cell Biology and Biosystems Technology, University of Rostock, Albert-Einstein-Strasse 3, D-18057 Rostock, Germany Received 9 February 2004; received in revised form 9 June 2004; accepted 30 June 2004 Available online 28 July 2004
Abstract Our findings show a significant increase of free radical production after exposure to 50 Hz electromagnetic fields at a flux density of 1 mT to mouse bone marrow-derived (MBM) promonocytes and macrophages, indicating the cell-activating capacity of extremely low frequency magnetic fields (ELF-MF). We demonstrate that after exposure to ELF-MF mainly superoxide anion radicals were produced, both in MBM macrophages (33%) and also in their precursor cells (24%). To elucidate whether NADPH- or NADH-oxidase functions are target proteins for MF interaction, the flavoprotein inhibitor diphenyleneiodonium chloride (DPI) was used. MF-induced free radical production was not inhibited by DPI, whereas tetradecanoylphorbolacetate (TPA)-induced free radical production was diminished by about 70%. TPA is known to induce a direct activation of NADPH-oxidase through the PKC pathway. Since DPI lacks an inhibitory effect in MF-exposed MBM cells, we suggest that 50 Hz MF stimulates the NADH-oxidase pathway to produce superoxide anion radicals, but not the NADPH pathway. Furthermore, we showed an oscillation (1–10 days) in superoxide anion radical release in mouse macrophages, indicating a cyclic pattern of NADH-oxidase activity. D 2004 Elsevier B.V. All rights reserved. Keywords: Free radical; ELF-MF; ROS; NAD(P)H-oxidase; NADH-oxidase; Promonocyte macrophage
1. Introduction Epidemiological studies have indicated a correlation between extremely low frequency electromagnetic fields (ELF-EMF) and an increased incidence of some types of cancer [1–6]. Numerous publications have detected biological effects of ELF-EMF in vivo and vitro, although the basic interaction mechanisms between these fields and living systems are presently unclear. In our former studies [7], we reported an increase in the ability of phagocytosis in murine macrophages after exposure to 50 Hz magnetic fields (MF) at 1 mT. In addition, we also documented an increased formation of free radicals in macrophages after short-term (45 min) exposure * Corresponding author. Tel.: +49 381 498 6318; fax: +49 381 498 6302. E-mail address: [email protected] (M. Simko´). 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.06.024
to MF. Tao and Henderson [8] have shown that 60 Hz fields cause differentiation in haematopoietic progenitor cells, measured as phagocytosis, and Roy et al. [9] described an increased respiratory burst in neutrophils after exposure to 0.1 mT MF (60 Hz) and explained their data by the increased lifetime of free radicals. Macrophages play an essential role in the body’s defences and immune system. Activated macrophages release free radicals as reactive oxygen species (ROS), reactive nitrogen species (RNS), and also cytokines. ROS are unstable reactive molecules which are produced continuously in several cells. Free radicals including superoxide anion radicals, hydroxyl radicals, and hydrogen peroxides are formed as by-products in various metabolic processes. ROS are involved in intracellular signal transduction pathways and regulation of gene expression determining the anti-inflammatory response, cell growth, differentiation, proliferation and stress response [10].
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Enzymes such as NAD(P)H-oxidases, xanthine oxidases or arachidonic acid-metabolizing enzymes mediate the main production of ROS in macrophages. In phagocytic cells, the NADPH-oxidase is commonly associated with the brespiratory burstQ activity catalyzing the reduction of oxygen to superoxide anion radical. This high level of free radical formation is a primary host defence mechanism against any invading microorganism and is connected with cell activation. Isoforms of NADPH-oxidase are present in various non-phagocytic cells which were found to have similar characteristics. NADH-oxidase has been implicated in numerous cellular processes within signal transduction cascades and regulatory processes [11]. Alternatively, superoxide anion radical generation occur non-enzymatically by redoxreactive compounds such as the semi-ubiquinone compound of the mitochondrial electron transport chain [12]. In response to outside influence, superoxide anion radical is the primary one generated by phagocytes. They have a low bactericidal potency and are converted into other ROS that serve as mediators in many regulatory processes [13]. In cells, free radical concentration is determined by the balance between their rate of production and their rate of clearance, controlled by different enzymes and antioxidant compounds. These regulatory processes are important to reset the original state of redox homeostasis after temporary production of free radicals. High-level production of free radicals in the organism has shown an increased potential for cellular damage of substances such as DNA, proteins, and lipid-containing structures [14]. In contrast to molecules such as cytokines (large molecules signalling by docking with specific receptors and change molecular surfaces on the target cells) molecules such as ROS could react with diverse cell compounds in a non-specific mechanism. Therefore, free radicals play a decisive role in cytotoxicity and also as cellular messengers to control non-cytotoxic physiological responses. In this study, we investigated the effects of 50 Hz MF on free radical production, in mouse bone marrow-derived (MBM) macrophages and promonocytes. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and lipopolysaccharides (LPS) were used as positive controls, representing receptor-independent and receptor-dependent induced pathways, respectively. We assume that the activation of NADH-oxidase plays a major role also in phagocytic cells and is responsible for the MF-induced free radical production. To investigate this, we used the specific NADPHoxidase inhibitor diphenyleneiodonium chloride (DPI), which does not affect the NADH-oxidase activity, making it possible to distinguish between the two pathways. In addition, differentiated macrophages were investigated for extremely low frequency magnetic fields (ELF-MF) induced superoxide anion radical production over a period of 16 days to find out possible cell status-dependent sensitivity to field exposure.
2. Materials and methods 2.1. Cell culture Bone marrow cells were isolated from femur and tibia of Shoe-NMRI mice, resuspended and washed in phosphate buffer saline solution (PBS). Cells were used directly after preparation or seeded in tissue culture dishes (10020 mm, Biochrom-TPP, Berlin, Germany) in RPMI-1640 medium (PAA-Laboratories GmbH, Karlsruhe, Germany) supplemented with 30% conditioned medium from L929-cell line, 6% heat-inactivated fetal calf serum (FCS, Gibco BRL, Karlsruhe, Germany), 0.5% penicillin/streptomycin (10,000 U/10,000 Ag/ml), non-essential amino acids (Biochrom KG, Berlin, Germany) and 0.125% 2-mercaptoethanol. MBMderived macrophages were obtained by differentiation of promonocytes in vitro cultured for 2–3 days as a monolayer. Cells were incubated at 37 8C in a humidified atmosphere containing 5% CO2, medium was changed at regular intervals for 3 days. After differentiation to adherent and densely growing macrophages, cells were cooled to 20 8C in a freezer for 2–3 min until the cells rounded up. Macrophages were scraped off and re-plated into sterile culture dishes or prepared for experiments. 2.2. Electromagnetic field exposure For MF generation a Helmholtz coil system was used as described previously [7]. The Helmholtz coils were stationed in the middle of a 37 8C humidified incubator (Binder, Germany) containing 5% CO2. Non-exposed cell cultures were incubated in an identical incubator without a MF source. For all experiments, cells were exposed to horizontally polarized 50 Hz electromagnetic fields with a flux density of 1 mT. Cell culture plates were placed in the centre of the Helmholtz coils or in a control incubator for different time periods depending on experimental conditions. The magnetic flux density was measured with a precision Gauss/Tesla Meter (F.W. Bell Inc., USA, Model 6010) directly before cell exposure. 2.3. Production of ROS Directly after cell isolation promonocytes (15104 cells/ well/200 Al) were seeded in 96-well plates and cultured for 45 min in cell culture medium with or without 1 AM TPA (Sigma-Aldrich, Munich, Germany), 1 Ag/ml LPS (from E. coli, Serotype 026:B6 (Sigma-Aldrich) and/or 1 AM DPI (Sigma-Aldrich) in the presence or absence of MF. After exposure, cells were loaded with 1 AM dihydrorhodamine 123 (DHR, Molecular Probes, Leiden, Netherlands) in HBSbuffer (0.9% NaCl solution with 14 mM HEPES (Roth, Karlsruhe, Germany)) for 25 min at 37 8C in darkness. Fluorescence was subsequently measured using flow cytometer (Beckmann/Coulter Epics Altra, Germany) at 550 nm.
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2.4. Superoxide anion radical generation The nitro blue tetrazolium (NBT) test was used to measure the amount of superoxide anion radical in macrophages. From day 5 to day 16 of cell culture, cells were taken daily and seeded in 96-well flat-bottomed microtiter plates (Biochrom-TPP) at a concentration of 5104 cells per well and cultured overnight. Macrophages were incubated for 45 min in PBS supplemented with 1 mM NBT (Sigma-Aldrich), 1 AM TPA, 1 Ag/ml LPS and 1 AM DPI in the presence or absence of MF. In the same set of experiments, cells were additionally treated with DPI in parallel. After incubation for 45 min, supernatants were removed and the intracellular formazan crystals were solubilized in 100 Al dimethylsulfoxide (DMSO, SigmaAldrich). The absorbance was measured at 550 nm using a micro-ELISA reader (EL-808, BioTek Instruments, Inc., Vermont, USA). 2.5. Nitrogen generation Cells were cultured in 96-well plates (5104 cells/well) for at least 12 h prior to use. The production of NO2 during 45 min up to 24 h of MF and/or chemical exposure was determined in macrophages. For experiments, the following conditions were used: control cells were cultured with medium alone or medium containing 1 Ag/ml LPS; simultaneously, MF-exposed cells were cultured with medium alone or with the same additions as the controls. Nitrogen monoxide (NO) was measured as nitrite (NO2 ) using the colorimetric method based on the diazotization reaction of Griess reagent. Fifty-microliter aliquots of cell culture supernatants were mixed with an equal volume of 1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride (Griess reagent) and incubated for 10 min at room temperature (RT) in darkness. To abort the color reaction, 25 Al of 1 M H3PO4 was added and the absorbance
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at 550 nm was measured on an ELISA reader. Concentrations of NO2 were calculated using NaNO2 standard. 2.6. Flow cytometry For surface staining, freshly isolated bone marrow cells (15104 cells) were resuspended and incubated in 200 Al PBS supplemented with 2 mM EDTA, 0.1% BSA and 10 Al FITC-conjugated monoclonal rat anti-mouse CD11b-antibody or rat monoclonal immunoglobulin Isotype control (BD Bioscience, San Diego, USA) for 30 min at RT in darkness, followed by centrifugation (5 min, 200g). Cells were washed in 750 Al wash-buffer (PBS+0.1% NaN3, 1% FCS) before flow cytometric analysis (Beckmann/Coulter Epics Altra). 2.7. Statistical analysis Each independent experiment was performed 3–12 times. Data of exposed cells or chemically treated cells were analysed with reference to their respective controls. To compare the variability of each experiment, data were analysed as a ratio between experiment and respective control (E/C) and the 99% confidence interval level (CI) is given. For statistical analyses, Student’s t-test was used ( Pb0.01).
3. Results 3.1. Flow cytometric analysis in promonocytes The MBM-derived promonocytes used in these studies represent a homogeneous population of CD11b-positive cells (Fig 1A). The surface protein CD11b is considered to be a specific marker for murine promonocytes and macrophages. The population of CD11b+ cells were ascertained
Fig. 1. Flow cytometric results of normal MBM cells. The cells are stained with CD11b-FITC (A) and IgG2bn-FITC (B). The distribution of CD11b+ cells is shown in panel A. Fluorescence signal greater than of the isotypes were defined as positive cells. To compare isotypes and specific CD11b-binding, the fluorescence intensity of macrophages labelled with FITC-conjugated control antibody is presented in panel B.
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Control cells were not affected in ROS production after DPI-treatment. The production of free radicals in MF+DPIexposed cells is comparable to MF alone. Co-exposure of promonocytes with MF and TPA show no additional effects. Stimulation of mouse promonocytes with TPA yielded a 42fold increase whereas the presence of DPI resulted in a decrease to 14-fold of ROS release compared to controls. 3.2. Superoxide anion radical production in promonocytes
Fig. 2. Flow cytometric analysis of rhodamine fluorescence in CD11bpositive cells. The diagram shows the changes in rhodamine fluorescence in cells exposed to control conditions, MF or TPA for 45 min. Changes in rhodamine fluorescence intensity show the transformation of DHR leading to an increased free radical production after MF or TPA treatment of cells.
via antibody staining with FITC-conjugated rat anti-mouse CD11b or FITC-conjugated rat IgG2bn monoclonal isotype control. Bone marrow cells were investigated directly after isolation. Fig. 1A shows a typical distribution of MBM cells, whereby CD11b+ cells were gated. The specific and unspecific binding side of the antibody is shown in Fig. 1B. Production of ROS was analysed using the oxidation sensitive DHR assay. Directly after preparation promonocytes were incubated for 45 min in medium in the presence or absence of MF, TPA, LPS, DPI or in different combinations. After exposure, cells were loaded with DHR and changes in the fluorescence signal were measured using flow cytometry (Fig. 2). Promonocytes exposed to LPS or MF show a significantly increased production of free radicals (1.6-fold, 1.2-fold, respectively, Fig. 3). The NADPH-oxidase inhibitor DPI was used to determine whether the production of free radicals after MF exposure is connected to the NAD(P)H-enzyme activation process.
To identify the specific free radical species produced, we investigated the superoxide anion radical production after exposure to TPA, LPS and MF in promonocytes using the NBT-assay. Directly after preparation, cells were incubated for 45 min in NBT supplemented with TPA, LPS, DPI and in different combinations in the presence or absence of MF. Fig. 4 demonstrates a significant increase of superoxide anion radical release after exposure to TPA (3.5-fold), LPS (1.2-fold) and MF (1.2-fold) compared to control cells. After DPI addition to control cells, a reduction in superoxide anion radical production could be observed. Co-exposure to TPA and DPI results in a non-significant inhibition of reactive superoxide anion radical production when compared to TPA alone. A similar effect could be detected after co-exposure to DPI+MF, no inhibition of superoxide anion radical generation was observed. 3.3. Time course studies of superoxide anion radical production in MBM macrophages Promonocytes are able to differentiate into adherent macrophages within 2 days. Directly after preparation, cells were seeded in cell culture dishes and incubated at 37 8C. Differentiation of promonocytes results in morphological and functional changes. Promonocytes, monocytes and macrophages could be identified by the presence of extracellular membrane proteins such as CD11b. Differ-
Fig. 3. ROS production in promonocytes after exposure to 1 mT MF, 1 AM DPI, 1 AM TPA, 1 Ag/ml LPS and in different sets of co-exposures using DHR assay. Columns show mean data of experiments as a ratio between experiments and controls (E/C). Data represents the mean of triplicates of four independent experiments (n=12)Fconfidence interval (CI) and asterisks indicate statistically significant differences at the 99% level ( Pb0.01).
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Fig. 4. Superoxide anion radical generation in freshly isolated promonocytes after 45 min exposure in NBT supplemented with 1 AM TPA, 1 Ag/ml LPS, 1 AM DPI or TPA and DPI. Cells were incubated under control conditions or were exposed to 1 mT MF. Columns are means from triplicate measurements and six independent experiments (n=18)FCI, asterisks indicates statistically significant differences ( Pb0.01) to the respective control.
entiated cells represent a homogenous culture of CD11bpositive cells. Determinations of superoxide anion radical generation in MBM macrophages were performed in 24-h intervals starting at day 5 or 6 after cell adherence, and up to day 16 in culture using the NBT-assay. Macrophages were stimulated with TPA, LPS or MF for 45 min. Curves in Fig. 5 demonstrate the mean value of superoxide anion radical production of three different mice in three different experiments. The time series represents changes in the activation status of macrophages after chemical and MF treatment. In all cases, an oscillating ability to produce superoxide anion radical was found independently of culture condition. MF induced a significant superoxide anion radical release on days 7, 10, 12 and 14 in mouse 1 (Fig. 5A), and on days 7, 10, 14–16 in mouse 2 after preparation (Fig. 5B), whereas cells of mouse 3 showed a significant increased superoxide anion radical production on days 6 and 10–13 (Fig. 5C). This fact clearly shows that MF exposure enhances superoxide anion radical production at certain time points only, showing a correlation to LPS and TPA activation. However, this cyclic cell response changes in different animals. 3.4. Nitrogen oxide production in MBM macrophages We previously reported [7] that exposure to magnetic fields at 1 mT resulted in a significant increase of phagocytic uptake and ROS production in macrophages. To determine whether activation of macrophages is associated with the production of nitrogen oxide (NO), cells were exposed to 1 mT MF for 45 min up to 24 h. To activate the inducible nitric oxide synthase (iNOS) enzyme complex, we used LPS as a positive control. Fig. 6 shows the timedependent production of nitrite in macrophages after treatment with 1 Ag/ml LPS. In both LPS-treated and MF+LPS-
treated cells, a production of NO2 was seen after 8 h and longer times. MF-exposed cells show no detectable production of NO2 .
4. Discussion In this study, we demonstrate that 50 Hz magnetic fields (1 mT) affect cell function in murine macrophages and also in their precursor cells in vitro. Measurement of free radicals was used to indicate the cell-activating process in these immune relevant cells. The production of ROS has been recognized as a key chemical process that regulates signal transduction pathways leading to control of gene expression and posttranslational modification of proteins [14]. The measured ROS production using DHR includes the detection of hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and peroxy nitrite anions (ONOO ), whereas using NBT assay, superoxide anion radicals were detected directly. Our results show a significant increase in ROS production (20%) and in superoxide anion radical release (25%) in MF-exposed promonocytes. Furthermore, in differentiated macrophages, a significant increase of superoxide anion radical production up to 33% could be detected after MF exposure. Therefore, we suggest that MF induces cellular activation processes in murine promonocytes and macrophages. Identification of cellular sources of superoxide anion radical and ROS production is of major importance for investigating MF-mediated physiological processes in macrophages. We used DPI as a flavoprotein inhibitor to detect the potential source of free radical formation. DPI appears to inactivate flavoproteins at their flavin sites during electron-transfer reaction and is also known to inhibit K+ and Ca2+ channels [15]. In a preliminary series
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Fig. 5. Time course of superoxide anion radical generation in MBM macrophages in three independent examinations from three different animals (A, B, C). Curves represent the ability to produce superoxide anion radical after 45 min exposure to MF and chemical treatments such as 1 AM TPA and 1 Ag/ml LPS during a time course of 10–11 days. Determination of superoxide anion radical was performed in at least eight parallel probes at each time point using the NBTassay. n.s.: non-significant all other data are statistically significant to the control ( Pb0.01). Standard deviations (S.D.) are omitted for clarity.
of experiments, a concentration-dependent inhibition of TPA-stimulated NADPH-oxidase activity using DPI was studied and an optimal concentration of DPI was found at 1 AM (data not shown). Our results demonstrate that 1 AM DPI diminished ROS production by about 70% in TPA-treated promonocytes. On
the other hand, ELF-MF-induced formation of ROS and superoxide anion radicals was not inhibited by DPI in promonocytes. It is known that DPI inhibits flavoenzymes like NADPH oxidase, cytochrome P450 reductase and nitric oxide synthases leading to an inhibited free radical production [16–18]. The fact that DPI did not inhibit the
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Fig. 6. Production of nitrite oxide in MBM cells over 24 h, expressed in AM after exposure to 1 mT MF, 1 Ag/ml LPS or MF+LPS. Nitrogen levels were determined using Griess reagent assay as described in Materials and methods. Data are means from three separate experimentsFCI. Asterisk indicates Pb0.01.
MF-induced ROS production suggests that these flavoenzymes are not involved in the MF-mediated pathways. There are several cellular sources for free radical generation. One of the major sources of cellular superoxide anion radical production is the membrane-associated NAD(P)Hoxidase, a multi-component enzyme accepting electrons from the electron transport system. The free radical generation occurs at the flavin site of complex I, through reserved electron transfer [19,20] and is an unavoidable byproduct of cellular respiration. Another important source of superoxide anion radical formation is the NADH-oxidase. This enzyme was first found in membrane of nonphagocytic cells and plays a role in the regulation of intracellular signalling cascades in several cells [12,21]. This membrane-associated enzyme utilizes both NADH and NADPH as electron donor [10] and produces only onethird of the superoxide anion radicals compared to phagocytic NADPH-oxidase [11] responding to the respiratory burst. Our investigation demonstrates a low but significant induction of superoxide anion radical production (24% up to 33%) in promonocytes and macrophages after ELF-MF exposure. We assume therefore that activation of phagocytic NADPH-oxidase after exposure to MF should result in a higher production of superoxide anion radicals. A study from Morre´ [22] shows that inhibition of the plasma membrane NADH-oxidase activity by DPI was without any effect when the enzyme utilized NADH as electron donor in vascular cells. If NADPH was used as an electron donor, DPI inhibited the enzyme activity. Further, it is known that NADH-oxidase has a higher affinity to NADH than to NADPH [22]. It seems that MF induces the NADH- but not the NADPH-oxidase activity because it lacks the inhibitory effect of DPI in murine promonocytes and macrophages. This indicates the presence of two different pathways leading to free radical production in the same cell, namely through the activation of NADPHand/or NADH-oxidase. Interestingly, MF and TPA induced a superoxide anion radical release in an additive manner (360% TPA alone, 410% TPA+MF) in mouse promonocytes. Using DPI, the MF effect could be down-regulated to the TPA-induced
level, whereas the TPA effect was not affected. This fact indicates again the presence of the two independent acting pathways, the NADPH and the NADH in the same cell. Further, we investigated the influence of ELF-MF on differentiated macrophages and their nitrogen oxide production. Formation of NO could not be seen after exposure to ELF-MF for 24 h confirming the findings from Mnaimneh et al. [23] regarding to the production of NO in murine macrophages after exposure to MF. We found oscillations in the reactivity of differentiated macrophages regarding superoxide anion radical production. The ability to respond to external influences such as TPA, LPS or MF seems to be dependent on time after adherence in macrophages. Three independent examinations from three different mice show comparable alteration in the time course of superoxide anion radical generation (Fig. 5). Correlations between different exposure conditions give evidence for a fluctuating capacity of superoxide anion radical production. Morre´ et al. [24] investigated the biochemical basis for the biological clock involving the family of NAD(P)H-oxidase proteins. They demonstrated that this family of enzymes show a recurring pattern of oscillations with a period length of 24 min. Rosenspire et al. [25] found that the cytosolic concentration of NAD(P)H fluctuates in a periodic manner in neutrophils and in macrophages. We suggest that NADH-oxidase activity is responsible for the ELF-MF-induced superoxide anion radical production, which was found with maximal peaks between 7 and 12 days in a rhythmic pattern. However, the exact period length has to be investigated. An explanation of contradictory findings in the literature can thus be due to timedependent activating capacity of primary cells, where negative results of experiments are results of investigations performed at non-activating points of time. In summary, we reported in the present study about the cell-activating capacity of ELF-MF to induce superoxide anion radical and ROS production in murine MBM macrophages and their precursor cells. Furthermore, we detected the non-inhibitory capability of the flavoprotein inhibitor DPI in MF-exposed promonocytes to produce superoxide
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anion radicals, which indicates the activation of NADHoxidase. However, the induction of superoxide anion radicals using TPA was down-regulated by DPI, indicating inhibition of the NADPH-oxidase-mediated pathway. This suggests the independent action of two different pathways, namely the NADPH and the NADH pathways, leading to superoxide anion radical production in mouse macrophages and promonocytes and that MF stimulates the NADHoxidase only. Furthermore, we also showed the oscillating activation potential to produce superoxide anion radical in MBM cells, indicating the time-dependent capability of cell activity.
Acknowledgments The authors would like to thank Mats-Olof Mattsson, ¨ rebro, Sweden for helpful discussion. This University of O work is partly supported by the German Ministry of Education, Science and Technology (BMBF) and by the VERUM Foundation.
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