Effects of magnetic fields on the accumulation of thiobarbituric acid reactive substances induced by iron salt and H(2)O(2) in mouse brain homogenates or phosphotidylcholine.

In this study, we examined the effects of magnetic fields (MFs) on the generation of thiobarbituric acid reactive substances (TBARS) in the mouse brain homogenates or phosphotidylcholine (PC) solution, incubated with FeCl(3) and/or H(2)O(2). Active oxygen species were generated and lipid peroxidation was induced in mouse brain homogenates by incubation with iron ions, resulting in the accumulation of TBARS. Lipid peroxidation was induced in PC by incubation with iron ions and H(2)O(2). Exposure to sinusoidal MFs (60 Hz, 0.2-1.2 mT), symmetric sawtooth-waveform MFs (50 Hz, 25-600 mT/s), rectangular MFs (1/0.4-1/16 Hz, 3.3 mT) and static MFs (1, 5-300 mT) had no effect on the accumulation of TBARS in brain homogenates induced by FeCl(3). In contrast, when the homogenates were incubated with FeCl(3) in static MFs (2-4 mT), the accumulation of TBARS was decreased. However, this inhibitory effect disappeared when EDTA was added to the homogenate and incubated with H(2)O(2). The accumulation of TBARS in PC solution incubated with FeCl(3) and H(2)O(2) was also inhibited by the static MF. These results indicate that only static MFs had an inhibitory effect on iron-induced lipid peroxidation and the effectiveness of this magnetic field on iron ion-induced active oxygen species generation is restricted to a so called 'window' of field intensity of 2-4 mT.

Environmental magnetic fields inhibit the antiproliferative action of tamoxifen and melatonin in a human breast cancer cell line.

We have previously reported that environmental-level magnetic fields (1.2 microT [12 milligauss], 60 Hz) block the growth inhibition of the hormone melatonin (10(-9) M) on MCF-7 human breast cancer cells in vitro. We now report that the same 1.2 microT, 60 Hz magnetic fields significantly block the growth inhibitory action of pharmacological levels of tamoxifen (10(-7) M). In biophysical studies we have taken advantage of Faraday's Law of Current Induction and tested whether the 1.2 microT magnetic field or the associated induced electric field is responsible for this field effect on melatonin and tamoxifen. We observe that the magnetic field component is associated with the field blocking effect on melatonin and tamoxifen function. To our knowledge the tamoxifen studies represent the first experimental evidence for an environmental-level magnetic field modification of drug interaction with human breast cancer cells. Together, these findings provide support to the theory that environmental-level magnetic fields can act to modify the action of a drug or hormone on regulation of cell proliferation. Melatonin and tamoxifen may act through different biological pathways to down-regulate cell growth, and further studies are required to identify a specific biological site of interaction for the 1.2 microT magnetic field.

Experimental evidence for 60 Hz magnetic fields operating through the signal transduction cascade. Effects on calcium influx and c-MYC mRNA induction.

We tested the hypothesis that early alterations in calcium influx induced by an imposed 60 Hz magnetic field are propagated down the signal transduction (ST) cascade to alter c-MYC mRNa induction. To test this we measured both ST parameters in the same cells following 60 Hz magnetic field exposures in a specialized annular ring device (220 G (22 mT), 1.7 mV/cm maximal E(induced), 37 degrees C, 60 min). Ca2+ influx is a very early ST marker that precedes the specific induction of mRNA transcripts for the proto-oncogene c-MYC, an immediate early response gene. In three experiments influx of 45Ca2+ in the absence of mitogen was similar to that in cells treated with suboptimal levels of Con-A (1 micrograms/ml). However, calcium influx was elevated 1.5-fold when lymphocytes were exposed to Con-A plus magnetic fields; this co-stimulatory effect is consistent with previous reports from our laboratory [FEBS Lett. 301 (1992) 53-59; FEBS Lett. 271 (1990) 157-160; Ann. N.Y. Acad. Sci. 649 (1992) 74-95]. The level of c-MYC mRNA transcript copies in non-activated cells and in suboptimally-activated cells was also similar, which is consistent with the above calcium influx findings. Significantly, lymphocytes exposed to the combination of magnetic fields plus suboptimal Con-A responded with an approximate 3.0-fold increase in band intensity of c-MYC mRNA transcripts. Importantly, transcripts for the housekeeping gene GAPDH were not influenced by mitogen or magnetic fields. We also observed that lymphocytes that failed to exhibit increased calcium influx in response to magnetic fields plus Con-A, also failed to exhibit an increase in total copies of c-MYC mRNA. Thus, calcium influx and c-MYC mRNA expression, which are sequentially linked via the signal transduction cascade in contrast to GAPDH, were both increased by magnetic fields. These findings support the above ST hypothesis and provide experimental evidence for a general biological framework for understanding magnetic field interactions with the cell through signal transduction. In addition, these findings indicate that magnetic fields can act as a co-stimulus at suboptimal levels of mitogen; pronounced physiological changes in lymphocytes such as calcium influx and c-MYC mRNA induction were not triggered by a weak mitogenic signal unless accompanied by a magnetic field. Magnetic fields, thus, have the ability to potentiate or amplify cell signaling.

ELF magnetic fields, breast cancer, and melatonin: 60 Hz fields block melatonin's oncostatic action on ER+ breast cancer cell proliferation.

In this study we investigated whether a 60 Hz magnetic field can act at the cellular level to influence the growth of human estrogen-dependent breast cancer cells. Our experimental design assessed cell proliferation of a human breast cancer cell line, MCF-7, in the absence or the presence of melatonin which inhibits growth at a physiological concentration of 10(-9) M. In three experiments, continuous exposure to average sinusoidal 60 Hz magnetic fields of 1.90 +/- 0.01, 2.40 +/- 0.70, and 2.53 +/- 0.50 mG, or simultaneous exposure in matched incubators to average 60 Hz magnetic fields of 10.4 +/- 2.12, 11.95 +/- 2.73, and 11.95 +/- 3.28 mG, respectively, had no effect on cell proliferation in the absence of melatonin. When MCF-7 cells were cultured in the presence of 10(-9) M melatonin, an 18% inhibition of growth was observed for cells in a 2.40 +/- 0.70 mG field. This effect was blocked by a 60 Hz magnetic field of 11.95 +/- 2.75 mG. In a second experiment, a 27% inhibition of MCF-7 cell growth was observed for cells in a 2.53 +/- 0.50 mG magnetic field, and this was blocked by a 60 Hz magnetic field of 11.95 +/- 3.28 mG. These results provide the first evidence that ELF frequency magnetic fields can act at the cellular levels to enhance breast cancer cell proliferation by blocking melatonin's natural oncostatic action. In addition, there appears to be a dose threshold between 2 and 12 mG. The mechanism(s) of action is unknown and may involve modulation of signal transduction events associated with melatonin's regulation of cell growth.

Calcium signaling in lymphocytes and ELF fields. Evidence for an electric field metric and a site of interaction involving the calcium ion channel.

Calcium influx increased during mitogen-activated signal transduction in thymic lymphocytes exposed to a 22 mT, 60 Hz magnetic field (E induced = 1.7 mV/cm, 37 degrees C, 60 min). To distinguish between an electric or a magnetic field dependence a special multi-ring annular cell culture plate based on Faraday's Law of Induction was employed. Studies show a dependence on the strength of the induced electric field at constant magnetic flux density. Moreover, exposure to a pure 60 Hz electric field or to a magnetically-induced electric field of identical strength resulted in similar changes in calcium transport. The first real-time monitoring of [Ca2+]i during application of a 60 Hz electric field revealed an increase in [Ca2+]i observed 100 s after mitogen stimulation; this suggests that the plateau phase rather than the early phase of calcium signaling was influenced. The hypothesis was tested by separating, in time, the early release of calcium from intracellular stores from the influx of extracellular calcium. In calcium-free buffer, 60 Hz field exerted little influence on the early release of calcium from intracellular stores. In contrast, addition of extracellular calcium during exposure enhanced calcium influx through the plasma membrane. Alteration of the plateau phase of calcium signaling implicates the calcium channel as a site of field interaction. In addition, an electric field exposure metric is mechanistically consistent with a cell-surface interaction site.

Time-varying and static magnetic fields act in combination to alter calcium signal transduction in the lymphocyte.

We have tested the hypothesis that extremely low frequency (ELF) time-varying magnetic fields act in combination with static magnetic fields to alter calcium signalling in the lymphocyte. Results indicate that a 60-min exposure of thymic lymphocytes at 37 +/- 0.05 degrees C to a 16 Hz, 421 mG (42.1 microT) magnetic field simultaneously with a colinear static magnetic field of 234 mG (23.4 microT) (a.c./d.c. field intensity ratio = 1.8) inhibits calcium influx triggered by the mitogen Concanavalin A. Significantly, resting lymphocytes do not respond to the fields, thus, only mitogen-activated cells undergoing calcium signalling exhibit a field response. These results indicate that signal transduction involving calcium is an important biological constraint which operates to mediate this field interaction. Additional split field exposures show that the presence of the a.c. field or the d.c. field alone does not produce an effect. This is consistent with a proposed parametric resonance theory of interaction of low intensity magnetic fields with biological systems (L.L. Lednev (1991) Bioelectromagnetics 12, 71-75), which predicts the occurrence of biological effects at specific values for the frequency and field intensity of the ELF and static magnetic fields.

Nonthermal 60 Hz sinusoidal magnetic-field exposure enhances 45Ca2+ uptake in rat thymocytes: dependence on mitogen activation.

The effect of a 60 Hz sinusoidal magnetic field of nonthermal intensity on Ca2+ metabolism in rat thymic lymphocytes (thymocytes) was assessed in resting cells and in cells activated with the mitogen Concanavalin A (Con A). A 60 min exposure at 37 degrees C to an induced electric field of 1.0 mV/cm produced an average 2.7-fold increase in Con A-dependent 45Ca2(+)-uptake compared to non-exposed, isothermal control cells. In contrast, 45Ca2+ uptake remained unaltered during exposure of resting thymocytes. It was also found that thymocytes with a diminished ability to mobilize Ca2+ in response to Con A were most sensitive to the 60 Hz magnetic field. Although the precise mechanism of field interaction is at present unknown, modulation of Ca2+ metabolism during cell activation may represent a common pathway for field coupling to cellular systems.

Quantitative HPLC analysis of human plaque proteins in coronary and thoracic aorta arteries.

Quantitative HPLC analysis of saline-soluble proteins obtained from human coronary and thoracic aorta plaque and from whole internal mammary artery were performed. Protein extracts were characterized by anion exchange and reverse-phase HPLC and the integrated chromatographs revealed significant differences in both peak retention times and areas for protein species from coronary artery compared to thoracic aorta artery plaque. Coronary artery plaque proteins possessed a high degree of cationic charge and polarity compared to those present in thoracic aorta plaque and normal mammary artery. This suggests that specific protein markers may be expressed in plaque of different anatomical origin, and that the processing of protein may be distinct to plaque sites. In contrast, characterization of molecular weight by gel electrophoresis resolved no major differences between plaque types. These findings indicate that proteins in human plaque lesions of different anatomical origin can be resolved by HPLC methodology and that they exhibit different charge and polarity. Such an HPLC approach may prove useful in the quantitative identification and ultimate isolation of specific protein markers present in plaque during atherogenesis, and in the study of mechanisms of protein involvement in plaque formation.

Microwaves and the cell membrane. IV. Protein shedding in the human erythrocyte: quantitative analysis by high-performance liquid chromatography.

The erythrocyte responds to microwave fields by shedding at least 11 low-molecular-weight proteins of less than or equal to 31,000 Da, with components of 28,000-31,000 Da released during the destabilization of divalent calcium-protein bridges [R.P. Liburdy and P.F. Vanek, Radiat. Res. 109, 382-395 (1987)]. Significantly, protein shedding was shown to be restricted to exposure temperatures coinciding with the cell membrane phase/structural transition temperature, Tc, of 17-25 degrees C. We report here a further characterization of protein shedding at Tc using high-performance liquid chromatography and membrane-associated blood group antigen testing. Proteins shed from human erythrocytes in microwave fields (2450 MHz, CW) compared to sham-heating displayed a twofold increase in total protein mass released concomitant with the appearance of unique protein species during reverse-phase, hydrophobic interaction, and anion-exchange HPLC. These HPLC analyses indicate that microwaves result in the shedding of proteins which are relatively nonpolar and hydrophobic and which carry a net positive electrostatic charge compared to those released during sham-heat treatment. Assessment of 23 blood group antigens that represent integral protein markers on the erythrocyte cell surface indicates that microwave fields do not result in the exhaustive loss of these proteins. The class of proteins that is shed in response to microwave fields most likely is the loosely bound "peripheral" or extrinsic proteins associated with the exterior of the cell surface. Such proteins play a major role in the transduction of signals to integral membrane proteins which span the bilayer. That this class of proteins is susceptible to release by microwave fields is discussed in relation to microwave absorption at the cell surface by membrane-associated bound water, field interaction with dipolar side groups, and the disruption of divalent cation bridges known to stabilize peripheral membrane proteins.

Microwaves and the cell membrane. III. Protein shedding is oxygen and temperature dependent: evidence for cation bridge involvement.

Microwaves (2450 MHz, 60 mW/g) are shown to result in the release or shedding of at least 11 low-molecular-weight proteins (less than or equal to 31,000 Da) from rabbit erythrocytes maintained in physiological buffer. Protein release was detected by gel electrophoresis of cell-free supernatants using sensitive silver staining. This release is oxygen dependent and occurs in 30 min for exposures conducted within the special temperature region of 17-21 degrees C, which is linked to a structural or conformational transition in the cell membrane. Shedding of 26,000 and 24,000 Da proteins is unique to microwave treatment, with enhanced release of 28,000 and less than or equal to 15,000 Da species during microwave compared to sham exposures. Two-dimensional isoelectric focusing further reveals that proteins of less than or equal to 14,000 Da shed during microwave treatment exhibit a pI of 6.8-7.3 not seen in sham-treated cells. Treatment of erythrocytes with a serine-directed protease inhibitor does not prevent release of proteins. However, when erythrocytes are maintained at 17-21 degrees C by conventional heating in the absence of divalent cations, release of 28,000-31,000 and less than or equal to 14,000 Da components is detected. This indicates that cation-bridge stability may be important for release of these proteins. The above results provide evidence that microwaves alter erythrocyte protein composition at temperatures linked to a transition in the cell membrane and that destabilization of salt bridges may play a role in an interaction mechanism for protein release.

Magnetic field-induced drug permeability in liposome vesicles.

Liposome vesicles maintained in a uniform static magnetic field release a chemotherapeutic drug (ARA-C, MW = 243) at temperatures approaching the phase-transition region where these liposomes are not normally leaky. Drug release is rapid, and a maximum difference between treated and unexposed liposomes of 30% of the total maximal release of ARA-C was observed within 1 min in a magnetic field. Dose-effect studies conducted between 0.01 and 7.5 T (1 T = 10(4) G) reveal that this permeability effect has a sigmoidal dependence on magnetic flux density. The ED50 is 15 mT, with a 95% confidence interval of 6.50-34.9 mT. Magnetic field exposures were conducted using a superconducting magnet with the liposomes maintained at +/- 0.08 degrees C. For comparison, samarium-cobalt permanent magnets induced a comparable drug release at 0.4 T. These results indicate that a static magnetic field of 10 mT or greater can increase passive transport in phospholipid membrane bilayers maintained at or near their membrane phase-transition temperature. Lipid clustering which occurs at prephase-transition temperatures may predispose phospholipid domains to diamagnetic orientation in a magnetic field and thereby facilitate drug release.

Microwave-stimulated drug release from liposomes.

Microwaves (2450 MHz) are shown to stimulate the release of an aqueous chemotherapeutic drug from phospholipid vesicles. This effect occurs at temperatures below the membrane phase transition temperature of 41 degrees C where these liposomes are normally not leaky. In buffered saline, microwave exposure (60 mW/g) triggers the onset of drug release at 33 degrees C, whereas in plasma a near maximal release is observed as low as 27 degrees C. Significantly, this drug release is enhanced by oxygen and is attenuated by antioxidants. These results demonstrate that phospholipids in artificial membranes devoid of protein are influenced by nonionizing electromagnetic radiation, and that this interaction can be modulated by two physiologically important factors, plasma and oxygen. Such a permeability effect may provide a means for investigating microwave interactions with ordered membrane bilayers.

Microwaves and the cell membrane. II. Temperature, plasma, and oxygen mediate microwave-induced membrane permeability in the erythrocyte.

Microwaves (2450 MHz) are shown to increase 22Na permeability of rabbit erythrocytes for exposures only within the narrow temperature range of 17.7 to 19.5 degrees C (Tc) which coincides with a nonlinearity in the Arrhenius plot reflecting an apparent membrane phase transition. Significantly, this response is not observed for cholesterol-loaded erythrocyte membranes which exhibit a linear Arrhenius plot and no apparent phase transition at Tc. The permeability increase at Tc is a nonlinear function of absorbed power but is a linear function of the internal electric field strength of the sample and saturates at approximately 400 mW/g and 600 V/m, respectively. The permeability increase was found to be reversible and transient in that immediately following termination of exposure sodium influx is significantly reduced but returns to normal within 60 min. Extracellular factors exert a significant influence on the microwave effect. The presence of plasma markedly potentiates the increase in 22Na permeability at Tc. Oxygen also modulates the microwave effect with relative hypoxia (5 mm Hg) and hyperoxia (760 mm Hg) enhancing the permeability increase. In contrast, the presence of two antioxidants, ascorbic acid or mercaptoethanol, inhibits the effect. These findings raise important questions about the physical and chemical nature of microwave interactions with cell membranes and also shed light on earlier studies reporting either positive or negative effects on membrane permeability.

Radiofrequency radiation and the immune system. Part 3. In vitro effects on human immunoglobin and on murine T- and B-lymphocytes.

Radiofrequency radiation (RFR) altered the physical separation of immunoglobulin (Ig) and of T- and B-lymphocytes during liquid gel chromatography. Exposure of human serum to a 10 MHz electric field (8500 V/m, less than or equal to 0.134 W kg-1) during chromatography resulted in accelerated elution of the IgM, IgA and IgG fractions. This effect is consistent with an increase in steric resistance of Ig molecules to the gel pores resulting in rapid elution. The low level of absorbed power employed did not lead to measurable heating of the gel media (25.00 +/- 0.05 degrees C). Effects on lymphocyte separation were investigated by performing immunoaffinity cell chromatography during exposure to 2500 MHz RFR (194 V/m, less than or equal to 0.117 W kg-1). Murine spleen lymphocytes were fractionated at 4.0 degrees C over Ig-derivatized agarose beads into Ig- and Ig+ lymphocyte subpopulations. RFR exposures resulted in premature elution of 19 per cent of the Ig+ (B-cell) population indicating an alteration of Ig binding. Temperature excursions in excess of +/- 0.05 degree C were not observed during exposures. These in vitro results demonstrate that Ig, whether freely diffusing in solution or bound to the lymphocyte cell surface, is influenced by RFR at absorbed power levels below the current recommended safety limit of 0.4 W kg-1 (U.S.A.). A possible mode of interaction for these effects as well as the relevance of these findings to in vivo biological processes are discussed.

Microwave bioeffects in the erythrocyte are temperature and pO2 dependent: cation permeability and protein shedding occur at the membrane phase transition.

Microwave exposure (2450 MHz, 60 mW/g, CW) of rabbit erythrocytes increases Na passive transport only at membrane phase transition temperatures (Tc) of 17-19 degrees C. This permeability effect is enhanced for relative hypoxia which is characteristic of intracellular oxygen tension (pO2 less than or equal to 5 mm Hg). Neither the permeability nor the pO2 effects are observed in temperature-matched (+/- 0.05 degrees C), sham-exposed controls. In addition, at Tc, microwave exposure is observed to induce the shedding or release of two erythrocyte proteins not seen in sham-exposed controls. Moreover, the enhanced shedding of at least seven other proteins all of molecular weight less than or equal to 28,000 D was detected in the microwave-treated samples. Using sensitive silver staining we estimate that approximately 450 fg of protein were shed per erythrocyte. These results demonstrate that temperature and pO2 are important influences on both functional and structural responses of cell membranes to microwave radiation.