Study of high- and low-current-configuration homes from the 1988 Denver Childhood Cancer Study

An epidemiological study conducted by Savitz et al. reported that residential wire codes were more strongly associated with childhood cancer than were measured magnetic fields, a peculiar result because wire codes were originally developed to be a surrogate for residential magnetic fields. The primary purpose of the study reported here, known as the Back to Denver (BTD) study, was to obtain data to help in the interpretation of the original results of Savitz et al. The BTD study included 81 homes that had been occupied by case and control subjects of Savitz et al., stratified by wire code as follows: 18 high current configuration (HCC) case homes; 20 HCC control homes; 20 low current configuration (LCC) case homes; and 23 LCC control homes. Analysis of new data acquired in these homes led to the following previously unpublished conclusions. The home-averaged (i.e., mean of fields measured in subjects' bedrooms, family/living rooms, and rooms where meals normally eaten) spot 60 Hz, 180 Hz, and harmonic (i.e., 60-420 Hz) magnetic fields were associated with wire codes. The 180 Hz and harmonic components, but not the 60 Hz component, were associated with case/control status. Measured static magnetic fields were only weakly correlated (rapproximately 0.2) between rooms in homes. The BTD data provide little support for, but are too sparse to definitively test, the 1995 resonance hypothesis proposed by Bowman et al. Case and control homes had similar concentrations of copper in their tap water. Copper concentration was not associated with wire codes nor with the level of electric current carried by a home's water pipe. These results of the BTD study suggest that future case/control studies investigating power frequency magnetic fields might wish to include measurements of 180 Hz or harmonic magnetic fields in order to examine their associations (if any) with disease status.     

Effect of magnetic field exposure on calcium channel currents using patch clamp technique

Calcium influxes through the membrane of PC-12D cells were measured under exposure to DC biased AC magnetic fields in resonant conditions of the ion cyclotron and the ion parametric resonance hypotheses and compared with influxes in cells without exposure to the magnetic field. After cancellation of the geomagnetic field, the cells were exposed to the horizontal fields generated by a current sheet, a planar sheet of conductor which generated a satisfactorily homogeneous horizontal magnetic field on the stage of a microscope without hindering treatment of a cell under observation. At or near any resonant conditions, no change in calcium influx could be detected under standard patch clamp conditions.     

Exposure to AC and DC magnetic fields induces changes in 5-HT1B receptor binding parameters in rat brain membranes

The binding properties of the G-protein coupled receptor (GPCR) serotonin 5-HT1B receptor were studied under exposure to AC (50 and 400 Hz) and DC magnetic fields (MF) in rat brain membranes. This was an attempt at replicating the positive findings of Massot et al. In saturation experiments using [3H]5-HT, 1-h exposures at 1.1 mT(rms) 50 Hz caused statistically significant increases in both the K(D) and B(max) binding parameters, from 1.74 +/- 0.3 to 4.51 +/- 0.86 nM and from 1428 +/- 205 to 2137 +/- 399 CPM, respectively, in good agreement with previous results. Exposure of the membranes at 400 Hz 0.675 mT(rms) did not elicit a larger increase in K(D) in spite of a much larger induced current density. DC fields (1.1 and 11 mT) had a lesser effect compared to AC fields at low values of K(Dsham), but decreased the affinity at higher values of K(Dsham). Modeling of the receptor-ligand-G protein interactions using the extended ternary complex model yielded good fits for all our data and that of Massot et al., showing that the AC field may act by decreasing the ability of the G-protein to alter the ligand-receptor affinity. The hypothesis is that the bipolar nature of the AC field explains the different nature of the effects observed with AC and DC exposures. These findings constitute one of the few documented pieces of evidence for cell-free effects of DC and extremely low frequency (ELF) AC MFs in the mT range.     

Interaction of gentamicin and spermine with bilayer membranes containing negatively charged phospholipids

We measured the electrophoretic mobility of multilamellar phospholipid vesicles, the 31P NMR spectra of both sonicated and multilamellar vesicles, and the conductance of planar bilayer membranes to study the binding of spermine and gentamicin to membranes. Spermine and gentamicin do not bind significantly to the zwitterionic lipid phosphatidylcholine. We measured the concentrations of gentamicin and spermine that reverse the charge on vesicles formed from a mixture of phosphatidylcholine and either phosphatidylserine or phosphatidylinositol. From these measurements, we determined that the intrinsic association constants of the cations with these negative lipids are all about 10 M-1. This value is orders of magnitude lower than the apparent binding constants reported in the literature by other groups because the negative electrostatic surface potential of the membranes and the resultant accumulation of these cations in the aqueous diffuse double layer adjacent to the membranes have not been explicitly considered in previous studies. Our main conclusion is that the Gouy-Chapman-Stern theory of the aqueous diffuse double layer can describe surprisingly well the interaction of gentamicin and spermine with bilayer membranes formed in a 0.1 M NaCl solution if the negative phospholipids constitute less than 50% of the membrane. Thus, the theory should be useful for describing the interactions of these cations with the bilayer component of biological membranes, which typically contain less than 50% negative lipids. For example, our results support the suggestion of Sastrasinh et al. [Sastrasinh, M., Krauss, T. C., Weinberg, J. M., & Humes, H. D. (1982) J. Pharmacol. Exp. Ther. 222, 350-358] that phosphatidylinositol is the major binding site for gentamicin in renal brush border membranes.     

Power line frequency electromagnetic fields do not increase the rate of protein synthesis in human skin fibroblasts as previously reported

Rodemann et al. [Rodemann et al. (1987): Biochem Biophys Res Commun 145:1-9] reported that human skin fibroblasts increase their rate of protein synthesis by as much as over ninefold in response to long term exposure to 20 Hz, 8.4 mT (84 G) magnetic fields. Here we report studies of protein synthesis using an identical cell type, exposure conditions, and the same means of measuring protein synthetic rates. Our initial goal was to determine if the earlier results could be replicated, but we found an inconsistency in the earlier protocol. It exposed cells to [(3)H]leucine for 6 h prior to measuring incorporation into protein. We found, however, that 24 h is required for [(3)H]leucine to reach a steady state distribution across the cells' plasma membranes. In addition, we typically measured 100-200 cpm/thousand cells. This is four- to eightfold higher than the 19-28 cpm/1000 cells previously reported. Using these conditions, we could find no significant difference in protein synthesis rates between control cells and cells exposed for up to three weeks in an identical electromagnetic field. In addition, we investigated the effects of a 60 Hz field since that is the frequency used for electric power distribution in the United States. Again, we could find no significant effect of this field on rates of protein synthesis, even after 21 days of exposure.     

Calcium binding to metallochromic dyes and calmodulin in the presence of combined, AC-DC magnetic fields.

The possibility that weak, ac and dc magnetic fields in combination may affect binding equilibria of calcium-ions (Ca2+) was investigated with two metallochromic dyes as calcium-binding molecules: murexide and arsenazo III. Calcium-dye equilibria were followed by measuring solution absorbances with a fiber-optic spectrophotometer. A Ca(2+)-arsenazo solution was also used indirectly to monitor the binding of Ca2+ to calmodulin. Parallel, ac and dc magnetic fields were applied to each preparation. The ac magnetic field was held constant during each of a series of experiments at a frequency in the range between 50 and 120 Hz (sine wave) or at 50 pps (square wave) and at an rms flux density in the range between 65 and 156 microT. The dc magnetic field was then varied from 0 to 299 microT at 1.3 microT increments. The magnetic fields did not measurably affect equilibria in the binding of metallochromic dyes or calmodulin to Ca2+.     

Some problems in modern bioelectromagnetics

One of the main problems of bioelectromagnetics - the unbelievable narrow resonance peaks at the cyclotron frequency of the alternating magnetic field - was considered. Modern electrodynamics of condensed matter clearly brings out that the reason of this phenomenon is extremely low viscosity within coherence domains of aqueous electrolytic solutions. The electrochemical model of action of combined static and alternating magnetic fields on aqueous solutions of amino acids is proposed. The possibility of arising a succession of changes in ionic forms in these processes was revealed. The dipole ions (zwitterions) together with water molecules electrostatically forming joint groups in the solution, create favorable conditions for arising mixed coherence domains there. Simultaneously with evolution of the coherent processes in these domains, the amino acid zwitterions are transforming into the usual ionic form, fit for cyclotron resonance. The development of cyclotron resonance under action of combined magnetic fields increases the ion kinetic energy, and the ions leave the domains for the incoherent component of the solution according to Del Giudice pattern (Comisso et al., 2006; Del Giudice et al., 2002), creating the peak current through the solution. Then the ions are transforming little by little into zwitterionic form again; after that, the solution becomes ready to react on exposure of magnetic fields again. The possibilities for formation of coherence domains composed of water molecules together with peptide molecules or protein ones are discussed.     

Studies on the anorectic effect of N-acylphosphatidylethanolamine and phosphatidylethanolamine in mice

N-acyl-phosphatidylethanolamine is a precursor phospholipid for anandamide, oleoylethanolamide, and other N-acylethanolamines, and it may in itself have biological functions in cell membranes. Recently, N-palmitoyl-phosphatidylethanolamine (NAPE) has been reported to function as an anorectic hormone secreted from the gut and acting on the brain (Gillum et al., [5]). In the current study, two of our laboratories independently investigated whether NAPE metabolites may be involved in mediating the anorectic action of NAPE i.p. injected in mice. Thus, the anorectic activity of a non-hydrolysable NAPE analogue, having ether bonds instead of ester bonds at sn1 and sn2 was compared with that of NAPE in molar equivalent doses. Furthermore, the anorectic effect of NAPE in NAPE-hydrolysing phospholipase D knockout animals was investigated. As negative controls, the NAPE precursor phosphatidylethanolamine and the related phospholipids phosphatidylcholine and phosphatidic acid were also tested. All compounds except one were found to inhibit food intake, raising the possibility that the effect of NAPE is non-specific.     

Role of afferent input in muscle atrophy.

It is well known that soleus muscle of rat atrophies following spaceflight or hindlimb suspension (Ohira et al., 1992). It is, further, reported that the electromyogram (EMG) of soleus muscle disappears immediately in response to unloading by exposure to actual micro-g environment (Kawano et al., 2002; Leterme and Falempin, 1998) and by hindlimb suspension of rats (Alford et al., 1987; Ohira et al., 2000). However, the EMG level is increased gradually to the control level following 7-10 days of continuous hindlimb suspension (Alford et al., 1987; Ohira, 2000), while muscle atrophy is progressing (Winiarski et al., 1987). We previously reported that reduction of the EMG level of rat soleus in response to actual micro-g environment, created by a parabolic flight of a jet airplane, was closely associated with a decrease of the afferent input recorded at the L5 segmental level of spinal cord (Kawano et al., 2002). However, it is still unclear how the EMG level of soleus muscle adapts to unloading condition. The current study was performed to investigate the responses of soleus EMG and both afferent and efferent neurogram at the L5 segmental level of spinal cord to acute (20 seconds) and chronic (14 days) unloading.     

Organotin compounds promote the formation of non-lamellar phases in phosphatidylethanolamine membranes.

Organotin compounds are important contaminants in the environment. They are membrane active molecules with broad biological toxicity. We have studied the interaction of tri-n-butyltin chloride and tri-n-phenyltin chloride with model membranes composed of different phosphatidylethanolamines using differential scanning calorimetry, X-ray diffraction, 31P-nuclear magnetic resonance and infrared spectroscopy. Organotin compounds laterally segregate in phosphatidylethanolamine membranes without affecting the shape and position of the lamellar gel to lamellar liquid-crystalline phase transition thermogram of the phospholipid. This is in contrast with their reported effect on phosphatidylcholine membranes [Chicano et al. (2001) Biochim. Biophys. Acta 1510, 330-341] and emphasises the importance of the nature of the lipid headgroup in determining how the behaviour of lipid molecules is affected by these toxicants. Interestingly, we have found that organotin compounds disrupt the pattern of hydrogen-bonding in the interfacial region of dielaidoylphosphatidylethanolamine membranes and have the ability to promote the formation of hexagonal H(II) structures in this system. These results open the possibility that some of the specific toxic effects of organotin compounds might be exerted through the alteration of membrane function produced by their interaction with the lipidic component of the membrane.     

Ca2+ ion transport through patch-clamped cells exposed to magnetic fields

The total current of Ca²⁺ ions through patch-clamped cell membranes was measured while exposing clonal insulin-producing β-cells (RINm5F) to a combination of DC and AC magnetic fields at so-called cyclotron resonance conditions. Previous experimental evidence supports the theory that a resonant interaction between magnetic fields and organisms can exist. This experiment was designed to test one possible site of interaction: channels in the cell membrane. The transport of Ca²⁺ ions through the protein channels of the plasma membrane did not show any resonant behavior in the frequency range studied. © 1995 Wiley-Liss, Inc.     

Diatom response to extremely low-frequency magnetic fields.

Reports that extremely low-frequency magnetic fields can interfere with normal biological cell function continue to stimulate experimental activity as well as investigations into the possible mechanism of the interaction. The "cyclotron resonance" model of Liboff has been tested by Smith et al. (Bioelectromagnetics 8, 215-227, 1987) using as the biological test system the diatom Amphora coffeiformis. They report enhanced motility of the diatom in response to a low-frequency electromagnetic field tuned to the cyclotron resonance condition for calcium ions. We report here an attempt to reproduce their results. Following their protocol diatoms were seeded onto agar plates containing varying amounts of calcium and exposed to colinear DC and AC magnetic fields tuned to the cyclotron resonant condition for frequencies of 16, 30, and 60 Hz. The fractional motility was compared with that of control plates seeded at the same time from the same culture. We find no evidence of a cyclotron resonance effect.     

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.     

Reconstitution of the proton translocating ATPase from bovine heart mitochondria into planar phospholipid bilayer membranes

Proton translocating ATPase (F0F1) from bovine heart mitochondria was reconstituted into planar phospholipid bilayers, and its electrogenicity was directly demonstrated. The F0F1 ATPase was solubilized using 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS) as a detergent followed by sucrose density gradient centrifugation according to the method originally described by McEnery et al. for rat liver mitochondria (McEnery et al. (1986) J. Biol. Chem. 259, 4642-4651), with minor modifications. The purified ATPase was reconstituted into proteoliposomes and then reconstituted into planar phospholipid bilayers by the modified fusion method (Hirata et al. (1986) J. Biol. Chem. 261, 9839-9843). A short-circuit current of up to 0.4 pA was induced by adding ATP, and this current was suppressed by the F1 ATPase inhibitor NaN3 or by a specific mitochondrial F0 inhibitor, oligomycin. The direction of the current corresponded to the flow of positive charges from the F1 side to the F0 side. All these facts clearly demonstrate that the mitochondrial F0F1 ATPase was successfully reconstituted into planar phospholipid bilayers, and the current was generated by the ATPase.     

The energy level scheme for the ferryl heme in compound II of the peroxidase-catalase family as determined from analysis of low-temperature magnetic circular dichroism

The expressions for temperature-dependent magnetic circular dichroism (MCD) of the ferryl heme (Fe(4+)Por, S=1), which is a model of an intermediate product of the catalytic cycle of heme enzymes (compound II), have been derived in the framework of a two-term model. Theoretical predictions for the temperature and magnetic field dependence of MCD intensity of the ferryl heme are compared with those of the high-spin and low-spin ferric heme. Analysis of reported MCD spectra of myoglobin peroxide [Foot et al., Biochem. J. 2651 (1989) 515-522] and compound II of horseradish peroxidase [Browett et al., J. Am. Chem. Soc. 110 (1987) 3633-3640] has shown the presence in the samples of approximately 1% of a low-spin ferric component, which, however, should be taken into account in simulating observed temperature dependences of MCD intensity. The values of two adjustable parameters are estimated from the fit of the observed and simulated plots of MCD intensity against the reciprocal of the absolute temperature. One of them, the energy gap between the ground and excited terms, predetermines the axial zero-field splitting. The other parameter is correlated with the energy of splitting of excited quartets arising from either the porphyrin pi-->pi* transition or the spin-allowed charge-transfer transition.     

A simple model for calculating residential 60-Hz magnetic fields

A model is presented that permits the calculation of densities of 60-Hz magnetic fields throughout a residence from only a few measurements. We assume that residential magnetic fields are produced by sources external to the house and by the residential grounding circuit. The field from external sources is measured with a single probe. The field produced by the grounding circuit is calculated from the current flowing in the circuit and its geometry. The two fields are combined to give a prediction of the total field at any point in the house. A data-acquisition system was built to record the magnitude and phase of the grounding current and the field from external sources. The model's predictions were compared with measurements of the total magnetic field at a single location in 23 houses; a correlation coefficient of .87 was obtained, indicating that the model has good predictive capability. A more detailed study that was carried out in one house permitted comparisons of measurements with the model's predictions at locations throughout the house. Again, quite reasonable agreement was found. We also investigated the temporal variability of field readings in this house. Daily magnetic field averages were found to be considerably more stable than hourly averages. Finally, we demonstrate the use of the model in creating a profile of the magnetic fields in a home.     

A note on an interaction between Rhinopithecus bieti and a buzzard at Baima Snow Moutain

Predation avoidance relies primarily on behavioural mechanisms [van Schaik and van Hooff, 1983]. Primates alarm call at predators, including most birds and mammals [Cheney and Wrangham, 1987]. Alarm calls could be used to signal to the predator that it has been spotted [Zuberbühler et al., 1999], thereby probably decreasing the likelihood of an attack [Schultz, 2001], and they also inform prey of the presence of the predator, thereby increasing overall attention levels [Schülke, 2001]. Although eagles are reported to be one of the predators of Rhinopithecus bieti [Bai et al., 1987], few interactions between these monkeys and raptors have been documented to date. Here I document an interaction witnessed between R. bieti and a buzzard [Buteo sp., Yang X-J, pers. comm.].     

Organotin compounds promote the formation of non-lamellar phases in phosphatidylethanolamine membranes

Organotin compounds are important contaminants in the environment. They are membrane active molecules with broad biological toxicity. We have studied the interaction of tri-n-butyltin chloride and tri-n-phenyltin chloride with model membranes composed of different phosphatidylethanolamines using differential scanning calorimetry, X-ray diffraction, ³¹P-nuclear magnetic resonance and infrared spectroscopy. Organotin compounds laterally segregate in phosphatidylethanolamine membranes without affecting the shape and position of the lamellar gel to lamellar liquid-crystalline phase transition thermogram of the phospholipid. This is in contrast with their reported effect on phosphatidylcholine membranes [Chicano et al. (2001) Biochim. Biophys. Acta 1510, 330-341] and emphasises the importance of the nature of the lipid headgroup in determining how the behaviour of lipid molecules is affected by these toxicants. Interestingly, we have found that organotin compounds disrupt the pattern of hydrogen-bonding in the interfacial region of dielaidoylphosphatidylethanolamine membranes and have the ability to promote the formation of hexagonal H_II structures in this system. These results open the possibility that some of the specific toxic effects of organotin compounds might be exerted through the alteration of membrane function produced by their interaction with the lipidic component of the membrane.     

Induction of conductance heterogeneity in gramicidin channels

In previous work from our laboratory, 5-10% of the channels formed by [Val1]gramicidin A have conductances that fall outside the narrow range that conventionally has defined the standard gramicidin channel [e.g., see Russell et al. (1986) Biophys. J. 49, 673]. Reports from other laboratories, however, show that up to 50% of [Val1]gramicidin channels have conductances that fall outside the range for standard channels [e.g., see Prasad et al. (1986) Biochemistry 25, 456]. This laboratory-to-laboratory variation in the distribution of gramicidin single-channel conductances suggests that the conductance variants are induced by some environmental factor(s) [Busath et al. (1987) Biophys. J. 51, 79]. In order to test whether extrinsic agents can induce such conductance heterogeneity, we examined the effects of nonionic or zwitterionic detergents upon gramicidin channel behavior. In phospholipid bilayers, detergent addition induces many changes in gramicidin channel behavior: all detergents tested increase the channel appearance rate and average duration; most detergents decrease the conductance of the standard channel; and all but one of the detergents increase the conductance heterogeneity. These results show that the conductance heterogeneity can result from environmental perturbations, thus providing a possible explanation for the laboratory-to-laboratory variation in the heterogeneity of gramicidin channels. In addition, the differential detergent effects suggest possible mechanisms by which detergents can induce the conformational perturbations that result in gramicidin single-channel conductance variations.