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Oxidative response of human monocytes and macrophages cultured under low oxygen culture conditions to ion parametric resonance magnetic fields.
BLACKMAN, C. F., G. N. Waite, H. O. Owegi, M. Bouwens, J. K. Page, AND S. J. Egot-Lemaire. Oxidative response of human monocytes and macrophages cultured under low oxygen culture conditions to ion parametric resonance magnetic fields. Presented at Bioelectromagnetics Society 33rd Annual Meeting, Halifax, NS, CANADA, June 12 - 17, 2011.
In the present study, we tested the IPR model with monocytes and macrophages. We used cells of the THP-l monocyte-derived macrophage cell line, monocytes isolated from human blood, and macrophages derived from human monocytes.
INTRODUCTION One proposed mechanism of action of electromagnetic fields (EMFs) on biological systems is the Ion Parametric Resonance (IPR) model, which has been experimentally validated in neuronal PC-12 cells [1, 2]. It proposes that when applied EMFs are tuned to resonate with the cyclotron motion of selected ions, there can be a measurable change in the biological response compared with the unexposed system. In the present study, we tested the IPR model with monocytes and macrophages. We used cells of the THP-l monocyte-derived macrophage cell line, monocytes isolated from human blood, and macrophages derived from human monocytes. All cultures were tested under two different culture conditions in that cells were either kept at regular 21% O2 or at 1% O2 concentration . The low 02 conditions simulate in vivo conditions, since cells typically experience a dramatic drop in O2 tension when leaving blood vessels and entering damaged or diseased tissue. The activity of monocytes/ macrophages was analyzed by hydrogenperoxide(H202)releaseasanindication of their oxidative burst activity. METHODS Cell Culture: Human monocytic leukemia THP-l cells were grown in suspension in a humidified normoxic atmosphere containing 5% C02 at 37°C. Culture medium was RPMI 1640, supplemented with 10% FBS, 10,000 units/ml penicillin, 10,000 ug/ml streptomycin and 25 mM glucose. Cells were differentiated to macrophages by treating them for 48 hours with 100 nM phorbol myristate acetate. Human blood monocytes were isolated from whole blood of voluntary donors (IRBnet 197455-3) and 50,000 cells per well were plated in 96 well plates. Cells were kept in culture for 24 hours or for 6 days before IPR EMF exposure. During the 6 days, cells were differentiated into macrophages by the addition of macrophage colony stimulating factor (20 ng/ml) and either polarized towards classically activated MI cells (by addition of 20 ng/ml IFN-y) or towards alternatively activated M2 cells (by addition of 20 ng/ml IL-4). Low Oxygen Exposure: During the 4-hour IPR exposure,cell cultures were either kept at normal 2I% O2 or they were exposed to steady-state 1% O2 generated by the enzymes glucose-l-oxidase and catalase . The O2 concentration in culture medium was modeled using Matlab and validated using an O2 micro electrode (Lazar Laboratories, DO-166 MT-I). Exposure to Magnetic Fields: The exposure system consisted of two Helmholtz coils. One coil with a vertical axis was used to produce the alternating magnetic field Bac. The other coil was used to cancel the horizontal component of the Earth static field(Bdc), so that Bdc was parallel to Bac in the center of the vertical coil. EMFs were measured with the FW Bell 5I80 meter and the MOS5I-3204 low-field probe. Cells were exposed to various Bdc and Bac conditions, which were optimized at the center of the vertical coil (Bdc = 42.2 ± 0.7 uT and Bac = 27.3 ± 0.0 uT RMS at 51.64 Hz) according to the IPR model predictions for H+, Mg2 + and Mnz2+. Cells kept at other places within the vertical coil served as controls. Additional controls were cells exposed to a frequency at which the model predicted no effect. Cells kept outside of the coil at a place with a similar Bdc and a background Bac < 0.4 uT served as sham exposure. Oxidative Burst: Cellular H2O2 release was quantified using a sensitive H2O2 assay . Briefly, cells were washed, stimulated with 1 ulM PMA and incubated at 37°C for 10 min. in the presence of 100 uM NaN3 and 50 uM luminol. Luminol is oxidized after injection of 10 uM sodium hypochlorite to emit a 2-second light flash that is amplified by H2O2. Chemiluminescence was quantified using a Lumistar Omega platereader (BMG Labtech Inc.) and calibrated with known amounts of H2O2. RESULTS THP-l macrophages showed up to 4-fold increased activity when exposed to optimized IPR conditions at the center of the vertical Helmholtz coil as compared to sham controls. The activity of control cultures that were exposed on top of the vertical coil at non-optimized IPR conditions was not significantly different from the activity of sham cells. Control cultures that were exposed at the vertical center of the coil, but close to the edge ofthe coil (Bj, and Bac not parallel) had an activity that was between the activities of sham exposed and IPR exposed cells. These effects were seen in cells exposed to 21% and 1% O2, with low oxygen cells being more sensitive to EMF exposure. Nine of 10 experiments showed a significant (p<0.05) effect, with an average increase of 60% of IPR exposed cells compared to sham cells. However, the magnitude of the effect varied between experiments. Ongoing experiments with human monocytes and macrophages indicate that untransformed cells can also respond to IPR magnetic fields. More details will be presented at the meeting. CONCLUSIONS The H202 release of a macrophage cell line is significantly increased when exposed to Ion Parametric Resonance magnetic field conditions, which are predicted to resonate with the cyclotron motion of H+, Mg2 + and Mn2 +. Experiments are underway to test this effect in untransformed monocytes and macrophages. ACKNOWLEDGMENTS The work was supported by Indiana University School of Medicine, the IRC Program at Rose-Hulman Institute of Technology, and CRADA #421-07 from the US Environmental Protection Agency. We thank Spencer Fox and his advisor Dr. Lee Waite for their contribution to this study.