4.0 THE CELL MEMBRANE, ION EXCHANGE AND CELLULAR EFFECTS OF EMR

SUMMARY

The cell membrane is considered as the primary site for EMR interaction with cellular systems. Interference with membrane-mediated signal detection, transduction, or amplification processes may underlie many of the biological non-thermal effects reported in the literature. The mobilization of cellular calcium ion (Ca2+ ) by electromagnetic radiation, or other stimuli, is an important biological response in the regulation of cellular activities.

A common finding is that calcium ion concentrations and Ca-dependent cellular processes are affected by EM fields. While the data on calcium efflux is equivocal, it cannot be ignored that independent research groups have reported an increase in 45Ca2+ efflux from brain tissue exposed to low levels of microwave or RF radiation, usually modulated around 16 Hz. The positive effects were supported by a further report of altered calcium ion efflux in human neuroblastoma cells exposed to 915 MHz modulated at 16 Hz. As the effective SAR in all of these studies was below 0.05 W/kg this suggests that a non-thermal mechanism of interaction exists that depends on the extremely low frequency modulation component. The efflux assay system requires further research which may yield useful information in determining the means by which EMR exposure conditions can sensitise and affect cell membrane responses. The significance for human health of such transient ionic changes is uncertain.

Apart from gross effects on metabolism and membrane structure that may result from substantial bulk heating, there is good supporting evidence of discrete changes in cell membrane permeability where heating does not occur. There is gathering evidence of non-thermal effects of EMR. Molecular lipid composition of bilaminar membranes is altered at specific structural phase-transitional temperatures. Evidence is given of enhanced permeability of lymphocytes to sodium at a specific temperature rather than due to a temperature increase (Liburdy 1992). Membrane stability is reduced by a downward shift in the lipid phase-transition temperature. Microwave radiation (unmodulated) has been shown to reduce the phase-transition temperature. Calcium ions are implicated in providing structural integrity by cationic bridges in the cell membrane.

It is unlikely that any single interactive mechanism is responsible for the range of effects observed at the cellular level. Research is needed at the level of ionic channels to demonstrate the mechanism of ion transport into and out of cells during exposure to EMR.

4.1 Introduction

RF radiation at frequencies up to several hundred GHz are known to be nonionizing forms of energy, because their quantum energy is too low to cause physicochemical or biological effects by ionization of molecules. Ionizing radiation readily produces chromosomal damage in cells. This is the major reason why biologists and physicists have long held the view that non-ionizing electromagnetic energy can produce detectable effects only via mechanisms involving significant heating in cells, such as the non-excitable cells of the immune system. During the past 10 years, however, bioelectromagnetics research has shown that nonionizing electromagnetic energy can induce a variety of biological effects not only by thermal interactions but also through interaction mechanisms that do not involve any macroscopic heating. Low-intensity field effects, which apparently are not induced by thermal interactions, are referred to in the literature as athermal, or alternatively, non-thermal field effects.

4.1.1 Cell Membrane

Cell membranes have a high content of fat molecules (phospholipids) that partition each cell from its neighbour. This plasma membrane is comprised of a double layer of phospholipid molecules, approximately 40 Å thick. A steady state potential of approximately 0.1V (equivalent to an electrical gradient 100kV/cm) exists across the membrane of most cells. Glycoprotein molecules protrude through the cell membrane and form a strongly negatively charged glycocalyx on its outer surface which provides a receptor site for hormones, antibodies, neurotransmitter molecules and cancer promoters. Tissues comprise of aggregate of cells separated by narrow fluid channels, approximately 150 Å wide, through which these substances travel to reach binding sites on cell membrane receptors (Adey 1992). This intracellular space provides preferred strongly conducting pathways for electromagnetic fields, having considerably lower electrical impedance than cell membranes. Cell to cell communication occurs via this route.

Within cells, molecular systems mediate essential processes of metabolism, reproduction and responses to environmental stimuli. Cells interact primarily with their physical and chemical environment and communicate through the cell membrane. The enveloping cell membrane acts as both sensor and effector. As a sensor, it detects altered chemistry in the surrounding fluid. It offers a path for inward signals generated on its surface by a range of stimulating ions and molecules, including hormones, antibodies and neurotransmitters. As effectors, cell membranes may secrete substances synthesized internally, including hormones, antibodies and structural proteins such as collagen. Both the sensor and effector functions are susceptible to manipulation by natural or imposed electromagnetic fields. The interaction triggers a cascade of events at the biomolecular level that may profoundly alter cell growth activity and proliferation. It is suggested that receptor-mediated influx of calcium in epidermal cells or mast cells is due to molecular vibration at the receptor sites and is not voltage-activated. Such an influx is observed, for example, following binding of the epidermal growth factor (EGF) to its specific receptor resulting in a 4-fold increase in intracellular calcium while the membrane potential was unchanged (Moolenaar et al 1986).

4.1.2 Ion Channels

Present in the cell membrane are specialised channels for ion transport that regulate ion fluxes required to regulate proper cell function. Important examples of such signal transduction events at the cell membrane are the binding of extra-cellular ligands (e.g. hormones, proteins) to cell surface receptor sites, and ion-channel transport across the lipid bilayer. Both events result in structural changes in the bilayer organisation that initiate the activation of diverse biochemical pathways transducing signals to internal cellular sites (Gardner 1989). Calcium plays a key role in this signalling process.

Such an effect is among the earliest detectable events triggered by binding of a ligand (e.g. antigen, receptor antibody or mitogenic lectin) to an appropriate receptor on the outer cell surface. The subsequent cascade of cellular reactions in lymphoid cells is best understood for T- cells (Weiss & Imboden 1987). Ligand induced Ca2+ mobilization is reflected by an initial rise in the cell's internal concentration of calcium ions {Ca2+}i which is caused by inositol 1,4,5-triphosphate-induced release of Ca2+ from intracellular stores and followed by a sustained receptor-mediated Ca2+ influx from the extracellular medium. Perturbation of these events with chemical agents (such as Ca2+ channel blockers, Ca2+ - specific ionophores) or lowering the extracellular Ca2+ concentration by using chelators can alter Ca2+ membrane fluxes and subsequently modify cellular activity. Effects produced include altered cell proliferation, secretion, motility, or cytotoxicity (Harris et al 1988; Lichtman et al 1983). Ca2+ regulation in lymphoid cells of the immune system could be similarly affected by appropriate EMR interaction.

The interaction of EMR fields depends on the efficiency of energy transfer to components in the cell membranes. Dipolar components, such as polar amino-acid side chains and cell surface bound water molecules will undergo rotational field orientation at microwave frequencies. At sufficiently high levels of specific absorption rates (SAR > 1-10 mW/gm) this motion will result in heating (ANSI 1982). Localised heating at the cell membrane can alter the phase of phospholipid molecules. The phase transition temperature may be altered under such conditions.

4.2 Experimental Evidence

According to Cleary (1990) there is definite evidence, from in vitro studies, of direct, frequency-dependent and field-strength dependent alterations of various types of mammalian cells by RF radiation. The variety of effects suggests multiple macromolecular mechanisms. There is some evidence to suggest that EMF-altered Ca2+ regulation is an early trigger of field effects in cells of the immune system. Given the established role of Ca2+ in the regulation of lymphocyte proliferation it has been proposed that EMR-altered Ca2+ regulation can modify lymphocyte DNA synthesis (Walleczek 1992). Most of this research has been carried out at ELF where, for mitogen-activated cells, EMF signals were effective modulators of both Ca2+ uptake and DNA synthesis. These results suggest that activation of transmembrane Ca2+ signaling is required in order to obtain field effects on both Ca2+ uptake and DNA synthesis. Similarly, increase in calcium uptake and DNA synthesis has been reported following single exposures at microwave frequencies (Cleary 1990). The use of an agonist co-promoter further reduced the exposure threshold level.

Evidence is given of enhanced permeability of lymphocytes to sodium at a specific temperature rather than due to a temperature increase (Liburdy 1992). Exposure for 90 min to 2450 MHz at 6 mW/gm power density produced no effect at 40°C, but resulted in a two-fold increase in accumulation of 22Na+ in rat lymphocytes at 37°C. This cell type is critical to the immune system and is reported to exhibit a loss of cell surface proteins and alteration of membrane permeability when exposed to microwaves at normal body temperature. Inhibition of the intracellular Na+/K+ pump (and consequent accumulation of Na+ ions) has also been reported in human erythrocytes exposed to 2450 MHz frequency at 37°C (Allis & Sinha-Robinson 1987).

The findings that microwave fields influence both passive and active sodium transport in eukaryotic lymphocytes and erythrocytes at 37°C may have important implications for the immune system. Na+/K+ transport is critically involved in intracellular enzyme function, and regulation of cellular growth and functions. The transport of sodium, potassium and calcium is vital in lymphocyte proliferation and maturation, and antibody production. It is possible that microwave-induced alterations of cation transport can perturb nuclear processes such as DNA synthesis. It is noteworthy that increased DNA synthesis has been reported in human lymphocytes following a single exposure for 2 h to 2450 MHz, at SAR of 5 to 50 W/kg at 37°C (Cleary et al 1990 a). This laboratory has also reported increased proliferation and transcription of glioma (human brain tumour) cells using similar exposure conditions (Cleary et al 1990 b). The altered rate of growth was maintained for up to five days after the irradiation. <

4.2.1 Ion Fluxes

In many types of cells (neuronal, cardiac, secretory) fluctuations occur in membrane potential and are accompanied by oscillations in the concentration of intercellular ions. The most commonly studied effect is that of altered concentration of intracellular free ionized calcium [Ca2+]i resulting from Ca2+ influx via voltage-sensitive calcium channels in the cell membrane. So called oscillatory Ca2+ responses also occur in non-excitable cells (Fewtrell 1993) in response to a stimulus at the cell membrane receptors. Early experiments reported alteration of Ca2+ efflux from avian brain tissue irradiated with RF modulated at 16 Hz (Bawin et al 1975; Adey 1981; Blackman et al 1982).

The use of sensitive endpoints for bioeffects research seems to be inevitably accompanied by publication of contradictory findings. The very nature of highly sensitive systems involving fluxes in ionic composition suggests that a response may be elicited by some aspect of experimental conditions that might differ between separate laboratories testing the same biological endpoint. The issue of calcium efflux response to RF fields is no exception. Early studies exposed isolated chick brain tissue to power densities 10-20 W/m2 at 147 MHz and reported statistically significant increases in labelled calcium ion (45Ca2+ ) efflux when the field was modulated at frequencies from 6-20 Hz. The SAR was estimated as 0.002 W/kg and the effect considered to be non-thermal with the maximum effect at 16 Hz modulation. No effect was observed from unmodulated fields (Bawin et al, 1975). This work was replicated in other laboratories at 450 MHz carrier wave frequencies where the effect was observed at specific modulation frequencies and at specific power density windows (Sheppard et al 1979). Enhanced efflux of calcium ion from chick brain tissue was also reported for power density windows at a frequency of 147 MHz modulated at 16 Hz, but was subsequently found to be related to the temperature of the preparation (WHO 1993; Blackman et al 1991).

Calcium ion efflux was increased in rat synaptosomal preparation by exposure to 450 MHz, amplitude modulated at 16 Hz with a power density of 10 W/m2 (Lin-Liu & Adey 1982). Meanwhile, a number of negative results were reported in rat brain tissue preparations exposed in vitro to 1-2 GHz, pulse modulated at 16 Hz and other ELF frequencies with power densities of 10 to 150 W/m2 (Shelton & Merritt 1981; Merritt et al 1982). However, the negative studies were not exact replications of the exposure conditions and experimental protocol used by Bawin or Blackman.

Positive effects have been reported at carrier frequency relevant to cellular telephones when human neuroblastoma cells in culture were exposed to 915 MHz at SAR of 0.005 and 0.05 W/kg modulated around 16 and 60 Hz (Dutta et al 1984, 1989).

Detection of changes in calcium concentration can be confounded by complicated dynamics of calcium cytoplasmic concentration and intracellular stores of free Ca2+ . The location of these stores varies with different types of cells. Muscle cells are known to store calcium in a specialised organelle, the sarcoplasmic reticulum, however, it is not understood how Ca2+ ions are transported across its membrane into the cytoplasm. The structure and intracellular location of Ca2+ stores in other cells is still a subject of debate (Rossier et al 1991; Krause 1991). It is known that intracellular Ca2+ concentration changes as calcium is released from stores or as Ca2+ traverse the cell plasma membrane in response to stimuli. Excitable cells are thought to respond when depolarisation of the cell membrane potential activates voltage-sensitive Ca2+ channels and allows influx of Ca2+. Non-excitable cells are thought to lack voltage-sensitive Ca2+ channels, nevertheless variations in extra cellular Ca2+ concentration modulates the frequency Ca2+ oscillations in many cells (Kawanishi et al 1989).

Despite the difficulties in interpretation of reported results, the fact exists that the literature contains a number of reports of imbalance of ionic concentration resulting from exposure to EMR. A recent attempt to verify a report of enhanced calcium efflux (Schwartz et al 1990) in amphibian cardiac muscle was unsuccessful (Wood et al 1993) and demonstrated the wide variability in intracellular calcium levels. The excised hearts were irradiated with RF 240 MHz amplitude modulated by 16 Hz at SARs up to 0.36 W/kg. The studies are important as calcium plays a critical role in the physiology and contractile operation of cardiac muscle. It is important that these studies are repeated with more sensitive ionic detection procedures. A study is currently being developed to use computer acquired data from the confocal microscope and fluorescent molecules to obtain information in real time on Ca2+ movements.

4.3 Possible Mechanism

It is thought that Ca ions form cationic bridges with protein/phospholipid moieties on the cell surface. When the Ca ions bind to the polar, anionic head groups of phospholipids in the membrane they provide stability to the membrane by raising its structural phase transition temperature (to 40°C). Exposure to microwave fields reduces the phase transition temperature to around 37°C resulting in destabilisation of the bridges and release of protein from the cell surface (Liburdy 1992). At the same time membrane permeability is altered. This effect of protein shedding in lymphocytes and erythrocytes has been recently reported after brief exposure of _ 30 min at 2450 MHz (cw) and 60 mW/kg (Liburdy 1992, 1994).

It is thought that loosely bound proteins play an important role in the transduction of signals to integral proteins that span the bilipid membrane. Exposure of liposomes to 2450 MHz at only 0.6 mW/kg for 5 min resulted in a reduction in the main structural phase transition temperature from 39.5 to 38°C (Liburdy 1994). Radiofrequency radiation fields have also caused the release of immuno-globulin (Ig) from antibody receptor sites on the surface of B-lymphocytes (Liburdy & Wyant 1984) at non-thermal exposure conditions (2450 MHz, SAR = 0.117 mW/gm).

Free Radicals

Due to its extreme reactivity towards macromolecules, hydroxl radical (OH*) is a highly toxic moiety that is probably implicated in many molecular biological effects. It is known to cause strand breakage and base modification in DNA, crosslinking of nucleic acids and proteins, enzyme inactivation and lipid peroxidation. Thus, it has the capability to significantly intefere with and alter the growth and development processes of cells. Exposure to many chamical compounds and non-ionizing radiation are capable of inducing free radical production at the cellular level.

There is increasing support for the theory that free radicals play an important role in discrete, important sub-cellular events during exposure to microwaves. The field of magneto-chemistry is beginning to have an impact on the understanding of subtle effects in molecular biology of cell systems. Chemical bonds consist of paired electrons with opposite spins. Free radicals are highly charged and can only form bonds between radicals of opposite spins. Electron spins may be altered by EM fields and radicals prevented from uniting. Recent information on the small unstable molecule, nitric oxide (NO), as a physiological mediator has shown the importance of oxygen free radicals in biological systems. NO is understood to modulate neurotransmission and regulate cerebral arterial blood flow and has been implicated in the pathogenesic of Alzheimer’s disease.

The microwave-induced lowering of phase transition temperature and increasing membrane permeability is inhibited by the presence of antioxidants, thereby implicating free radical involvement (Liburdy 1993). A number of laboratories have reported enhanced permeability to sodium cation in erythrocytes during exposure to microwave fields (Liburdy & Penn 1984; Clearly et al 1982; Allis & Sinha-Robinson 1987; Lotz & Saxton 1989; Liburdy 1992).

4.4 Implications

Studies on alterations in calcium flow generally measure the end result of an interaction, and mostly speculate on the actual mechanism of flow and its initiation process. Studies with RF and microwave exposures tend to present phenomenological data. The process of trying to correlate between studies that often use different exposure protocols, different cell types and subsequent variation in dosimetry is complicated further by the absence of an accepted mechanism by which the effect occurs.

Much of the literature on cellular effects of EMR lacks a dose-response and reported effects are considered to be due to some non-thermal mechanism. Biochemical pathways in signal transduction are becoming better understood, but much of these so-called non-thermal mechanisms are speculative. There are serious gaps in the knowledge which need to be studied rather than dismissed as fanciful ideas. Studies that are directed towards understanding the underlying principles in cellular non-thermal reactions need urgent support, particularly where there are genuine health implications, such as in the control and development of neural proteins, or alteration in the cell cycle kinetics in brain glioma cells. Some interesting data has been published on the effects of microwave radiation (27 and 2450 MHz, single exposure for 2 h), on glioma cell proliferation, assayed by 3H-thymidine incorporation in DNA (Cleary 1990). Similar accelerated growth effects were demonstrated for human lymphocytes exposed to SAR of 5 W/kg. The altered growth rates lasted for up to 5 days after the irradiation.

There is a flaw in the notion that applies “safety” standards that only refer to measurable gross physical quantities. When considering biological consequences it is essential to understand that the extreme lethal levels can be less harmful to the organism/individual than a seemingly negligible change that modifies the behaviour and development of cells. For example, it is now well understood that during early pregnancy, at the time of closure of the mammalian neural tube, hyperthermia can have devastating effects for the developing embryo and fetus. Large temperature increase results in embryonic death and abortion. However, modest temperature increase can interfere with the critical developmental processes and result in a range of severe abnormalities of the central nervous system including exencephaly, micrencephaly, microphthalmia (Edwards 1993).

While some opinions might consider that apparently esoteric studies on a specific cell reaction in a petri dish has little relevance to the "real world" of human health hazards from irradiation with EMR, the advantages of these studies cannot be overlooked. Analysis of molecular and ionic behaviour in EM fields is fundamental to understanding whether EMR can perturb enzymatic and biochemical control pathways and interfere with cell growth and development. Of course, reactions in a simplified controlled in vitro environment cannot be directly extrapolated to the in vivo situation where homeostatic humoral and thermal feedback control systems dominate. In vitro studies are far more sensitive than animal studies and allow more precise quantification of dosage and control of environmental variables. Any safety recommendation or guideline needs to take account of the possibility of risk from all mechanisms. The current ANSI standard apparently only considers data based on animal behavioural changes resulting from a gross thermal effect that elevates the whole body temperature by at least 1°C. Meanwhile, there are many reports of biological effects that cannot be attributed to heating and are, consequently, ignored by the Safety Standard. The reported biological effects are clearly responses in some, as yet, undefined way to the EM field.

At the other end of the spectrum human epidemiology studies are extremely crude, by comparison. There are enormous difficulties in gaining high statistical power due to the wide range of environmental factors and complicated behavioural patterns of mobile populations. It would need to be an extremely robust effect that could be detected above the background “noise” level. Many environmental factors, chemical, UV, power lines, stress, airborne pollutants, are said to be associated with human cancers and will mask the effects of another potential agent such as EMR. Without knowledge gained from laboratory research such surveys would not have a realistic hypothesis to test and, therefore, would have little chance of uncovering useful information. It is rather optimistic to propose that a population survey will provide scientifically acceptable information when no known mechanism exists and a probable outcome cannot be predicted.