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EMF research has suffered from the absence of a working experimental model (see Mechanistic models in EMF research) that defines relationships between all the physical exposure parameters required to produce a biological effect. For example, many investigators concern themselves only with average magnetic field intensity, while others attach importance to the direction of the time-varying field, its relationship to the geomagnetic (static) field, the waveform, the temperature of the biological test system, and other biological and physical parameters. As a result, many of these variables have not been controlled from one experiment to another, and this may be the reason why many experimental results have not yet been replicated.
Earlier studies, monitoring changes in calcium flux from brain tissue in vitro, demonstrated empirically that unusual but repeatable responses occur as functions of the intensity and frequency of the electric and magnetic fields, with the static (DC) magnetic field as a co-variable (Figures 1 and 2 ). These results were contrary to the commonly held notion that interactions between biological systems and electric or magnetic fields must be driven by energy deposited by the fields, with more energy creating more of an effect. Instead, researchers examining calcium efflux saw "windows" of effects, where increasing the magnitude of the applied magnetic field did not necessarily lead to increased effects. No theoretical model at the time could explain these results (see Models may support or limit research).
Lacking a theoretical model, researchers expected to see regular spacing of these windows of effects, citing harmonics of base frequencies as the explanation, or proportional spacing of windows as observed with some intensity effects. The IPR model suggests that while such expectations may be a good starting point for experimental efforts, there may be more complex, but predictable, roles for each controllable variable.
The IPR model is primarily derived from the earlier work of Podgoretskii and Khrustalev, originally extended to biological applications by Lednev (1991) who saw a possible link with Liboff's identification of ion resonance as a fundamental basis of biological interactions (1985). (Mathematically, there is an important distinction in the definition of resonance between the Liboff and IPR models: IPR resonance uses a relation that is inverse to Liboff's, so the only point of commonality is the n=1 case for the IPR model (exact resonance), with harmonics being defined differently for each model.) Both the IPR and Lednev models describe the detailed relationships between the intensity of the magnetic field (BAC), its frequency (fAC), and the static (BDC) magnetic field, which lead to predictions of transition probabilities of ions at resonance. The influence of the electric field, which is decoupled from the magnetic field at power frequencies, is not considered in these models.
Essentially the IPR model considers how an ion cofactor in a key molecular complex, for example an enzyme binding site, alters the conformation states of that molecular complex, resulting in an observable change in the biological system in which it is contained (Blanchard and Blackman, 1994). In an ion-enzyme complex, the result could be a change in reaction kinetics. The ionic influence is controlled by the restructuring of internal energy states resulting from the externally applied magnetic fields. The effectiveness of this restructuring in creating an observable biological change is in turn influenced by the resonance relationship between the frequency of the applied AC magnetic field, the flux density of the applied DC field, and the particular ion's charge-to-mass ratio, as well as by special features of candidate ion-molecular interactions that allow resonance response to occur for a sufficient time to affect a change.
While both the IPR and Lednev model require the AC and DC magnetic fields be applied in the same (parallel) direction, the IPR model differs from the Lednev model in the way it considers how the parallel AC and DC magnetic fields interact with each other to create changes within the biological system. This leads to the appearance of a factor of two in one of the fundamental equations of the IPR model, creating a substantially different predicted form of response from that predicted by the Lednev model. This difference is large enough that it can be easily explored experimentally, since it is ultimately experimental data that determines the strength of any theoretical interaction model. The results of such experimental tests are described in the following section.
The IPR model also considers possible effects when several ions are at or near resonance simultaneously, assuming an additive role for each ion's influence, unless there is evidence to the contrary. The Lednev model application is limited to calcium, and perhaps magnesium, ions. This distinction leads to minor changes in the predicted response forms, depending on the ions at resonance and the extent of their influence on the observed biological response.
The most extensive experimental test of the IPR model to date uses a cell differentiation response in vitro. PC-12 cells are a culture line derived from a rat adrenal medulla pheochromocytoma (Greene and Tischler, 1976). This cell system shows highly regular and dose-dependent responses to nerve growth factor (NGF) by producing neurites, or primitive neuronal-like projections. This system has been used throughout the world to study NGF-induced processes, and the optimum culture conditions and potential pitfalls of working with this cell line are relatively well understood. We used PC-12 cells that were first primed with NGF to evaluate possible effects of IPR magnetic field conditions in stimulating or inhibiting the process of differentiation.
PC-12 cells were primed, then washed and placed on dishes in medium containing fresh NGF at a concentration sufficient to stimulate the cells' production of neurites to approximately 75% of the maximum possible. One set of controls, however, was not given any more NGF after the original, priming dose was rinsed off. Under these conditions, PC-12 cells have repeatedly been shown to produce neurites at a rate dependent on the amount of nerve growth factor present.
We chose a length of time (23 hours) in which about 50% of the cells routinely produced neurites with the given a dose of NGF (5 ng/ml). In cell cultures without NGF, about 10% of the cells would display neurites. This assay, adapted from standard procedures used with these cells, was then used to compare the cell's response under magnetic field exposure conditions to their response when not exposed. Pseudo-randomly selected (nonoverlapping) areas of each dish, exposed or unexposed, were examined under a microscope. Cells or small clusters of cells in each microscopic area were scored positive or negative for neurite outgrowth by a counter who was unaware whether the dish had been exposed or not (i.e., scoring was done blind). If the cell/cluster showed a neurite outgrowth longer than a cell diameter, it was scored positive. Figure 3 shows photomicrographs of typical cells and clusters scored positive and negative: the distinction is even easier to make under phase contrast microscopy where the focus can be adjusted allow the observer to more easily follow the full length of a neuron. A minimum of 200 PC-12 cells or cell clusters were scored per dish. After all control and exposed dishes were examined, the codes indicating whether a dish was exposed or not were broken, and we calculated the percent of cells/clusters showing positive neurite outgrowth relative to the control values, and plotted it as a function of magnetic field exposure (Figure 4). Resonant conditions exhibited a reproducible pattern of neurite outgrowth inhibition, while nonresonant combinations of AC and DC magnetic fields had no effect.
Early work established that this neurite outgrowth process could be retarded directly by 50-Hz AC magnetic field intensity in an exposure-dependent manner, and showed that the inhibition was not directly related to the induced electric fields in the culture dish (Blackman et al., 1993). Subsequent experiments demonstrated that the retarded neurite-outgrowth responses as a function of magnetic field intensity also showed a dependence on the frequency of the magnetic fields between 15 and 70 Hz (Blackman et al., 1995b).
The predictions of the IPR model suggested three sets of experimental tests with the neurite outgrowth assay (Blackman et al., 1994) (see Nonlinear biological models):
Under standard toxicological models, one would expect to see a biological effect that, if anything, increased with BAC. Even the Lednev model, over the selected range, predicted a generally increasing effect if the magnesium ions were involved in neurite production. The IPR model, by contrast, predicted a "scalloped window" effect over the selected range, i.e., increased effect followed by decreasing effect with no effect (compared to controls) at the highest BAC in the selected range. The amount of PC-12 neurite outgrowth observed over this range repeatedly showed, in three separate tests, the distinct "scalloped window" effect predicted by the IPR model, indicating that higher intensity does not necessarily result in a bigger effect.
Traditional models might suggest a monotonically increasing exposure-response, but of smaller magnitude because of the reduction in range of AC intensities. The Lednev model and IPR model predictions would be the same, respectively, as in the first test. Over three different repetitions of this test, the distinctive PC-12 responses were exactly as predicted by the IPR model.
Models neglecting the influence of the DC magnetic field would predict the same results as in the first test. But both the Lednev and IPR models require ion resonance before changes in the AC field intensity can create an effect. Lacking resonance for any ion, the predicted response would not differ from controls over the entire range of AC magnetic field intensities. In fact, during each of three separate tests, the PC-12 cells demonstrated no change in neurite outgrowth from the control values, consistent with the predictions of both the Lednev and IPR resonance-based models.
Experimental tests of the IPR model have been performed which clearly distinguish it from the predictions of the Lednev model. The results of each of these tests have been reported in detail (Blackman et al., 1994). We subsequently extended the BAC range of these tests to check whether the nonlinear predictions of the IPR model would continue to match the data (Blackman et al., 1995a). Over this extended range, a distinct difference between on-resonance and off-resonance results was observed (Figure 4 ). The Lednev model's predictions, although nonlinear, did not predict the on-resonance pattern observed in the data very well (Figure 5). However, when we extended exactly the same fitting function derived from the initial set of tests (Blackman et al., 1994), we found it matched the data both in form and magnitude (Figure 6).
Subsequent tests by independent investigators in our laboratory confirmed a direct, hydrogen-ion-resonance involvement, suggested by the results of the original tests of the IPR model (Trillo et al., 1994). Later, the PC-12 cell response bandwidth at 45 Hz was determined by us, under a separate set of tests, to be ±10% (Blanchard et al., 1994).
The IPR model is the best heuristic model developed and tested to date to predict biological consequences from exposure to low-frequency AC and DC magnetic fields. It is highly predictive and can identify the involvement of various ions involved in the response. A more detailed understanding of the mechanistic basis for the underlying theory is currently in progress (See Applications of the IPR model).
Blackman CF, Benane SG, and House DE. "Evidence for direct effect of magnetic fields on neurite outgrowth." FASEB J., 7:801-806, 1993.
Blackman CF, Blanchard JP, Benane SG, House DE. "Empirical test of an ion parametric resonance model for magnetic field interactions with PC-12 cells." Bioelectromagnetics, 15:239-260, 1994.
Blackman CF, Blanchard JP, Benane SG, House DE. "The ion parametric resonance model predicts magnetic field parameters that affect nerve cells." FASEB J, in press, 1995a.
Blackman CF, Benane SG, and House DE. "Frequency-dependent interference by magnetic fields of nerve-growth-factor-induced neurite outgrowth in PC-12 cells." Accepted for publication in Bioelectromagnetics, 1995b.
Blanchard JP, Blackman CF. "Clarification and application of an ion parametric resonance model for magnetic field interactions with biological systems." Bioelectromagnetics, 15:217-238, 1994.
Blanchard JP, Blackman CF, Benane SG, House DE. "Resonance bandwidth under IPR model exposure conditions." Abstract, the 1994 Annual Review of Research on Biological Effects of Electric and Magnetic Fields from the Generation, Delivery and Use of Electricity, Albuquerque, NM, November 6-10, 1994.
Green LA, Tischler AS. "Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor." Proc. Natl. Acad. Sci. U S A, 73:2424-2428, 1976.
Lednev VV. "Possible mechanism for the influence of weak magnetic fields on biological systems." Bioelectromagnetics, 12:71-75, 1991.
Liboff AR. "Cyclotron resonance in membrane transport." In Chiabrera A, Nicoline C, Schwan HP (eds.), "Interactions between Electromagnetic Fields and Cells." NATO ASI Series A97, New York, Plenum Press, 1985: 281-296.
Trillo MA, Ubeda A, Blanchard JP, House DE, Blackman CF. "Magnetic fields at resonant conditions for the hydrogen ion affect neurite outgrowth in PC-12 cells: a test of the ion parametric resonance model." Abstract, Sixteenth Annual Meeting of the Bioelectromagnetics Society, June 12-17, 1994, Copenhagen, Denmark; Manuscript submitted to Bioelectromagnetics, 1994.
Podgoretskii MI, Khrustalev OA. "Interference effects in quantum transitions." Soviet Physics Uspeki 6: 682-700, 1964.