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Effects of Radio-Frequency Radiation on Mammalian Cells and Biomolecules In Vitro Stephen F. Cleary Physiology Department, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0551
In vitro biological systems, such as mammalian cells or biomole cules, provide the most direct means of assessing the biological ef fects of electromagneticfields.Such systems were exposed to radio-frequency electromagnetic radiation (RFER) under conditions that permitted differentiating indirect effects due to RFER-induced heat ingfromdirect exposure effects. This distinction is of importance be cause current RFER health protection guidelines were designed pri marily on the basis of limiting thermally induced physiological alterations. Studies reviewed here include RFER in vitro effects on cell proliferation, neoplastic transformation, cell membrane cation transport and binding, energy metabolism, and neuroelectrical ac tivity. Whereas the basic RFER biological interaction mechanisms remain elusive, in vitro data provide insight and direction for future studies. Such data also provide a perspective for emerging issues related to health effects of exposure to environmental electromag netic fields.
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N VITRO SYSTEMS afford a unique opportunity to investigate biological effects of radio-frequency electromagnetic radiation (RFER) under conditions of precise experimental control of variables including (1) induced electric (E) or magnetic (B)fieldstrength, (2) modulation, (3) dose rate or specific absorption 0065-2393/95/0250-0467$12.00/0 ©1995 American Chemical Society In Electromagnetic Fields; Blank, M.; Advances in Chemistry; American Chemical Society: Washington, DC, 1995.
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rate (SAR = 0.5σ£ , where σ is the effective conductivity), (4) temperature, and (5) composition of cell exposure medium. Temperature control is essential in studies designed to differentiate between direct effects of R F E R on biological systems as contrasted to indirect effects due to RFER-induced heating. Physical aspects of the interaction of R F E R with mammalian cells and biomolecules are also amenable to theoretical determination of the magnitude and spatial distribu tion of induced Ε and Β fields and hence cell or molecular level S A R distribu tions. Consequently, mammalian cells and biomolecules provide the most direct approach to determining basic interaction mechanisms of R F E R with biological systems. In vivo systems do not afford this opportunity because of inherent do simetric and densitometric complexities that introduce significant uncertainty regarding the accuracy of Ε field, Β field, or S A R determinations in tissue. The highly interactive nature of various organs and organ systems also impedes as sessments of cause-effect relationships in in vivo systems exposed to RFER. Although the relationship between S A R and tissue heating is complex, as a gen eral rule intensities somewhat greater than 1 W/kg are associated with some de gree of temperature elevation (1). In in vitro exposures active heat transfer sys tems may be used to control or eliminate field-induced temperature elevations over intensity ranges of 100 W/kg or greater (2). In vitro studies of the effects of R F E R on cell physiological end points include (1) cell proliferation and neoplastic transformation, (2) membrane cation transport and binding, (3) energy metabolism, (4) molecular-biochemical ef fects, and (5) effects on membrane ion channels. The results of these studies have been the subject of review articles (1-4). This chapter will review recent in vitro studies that provide additional insight regarding possible R F E R cellular interaction mechanisms and that present evidence of direct or athermal R F E R cellular effects. The potential relevance of such in vitro studies to human health effects, such as reported associations of R F E R exposure and cancer incidence, will be discussed. The purpose of this chapter is not to provide a comprehensive review of in vitro cellular effects of R F E R (for more comprehensive reviews, see references 1-4). The results of the studies reviewed here indicate that under certain expo sure conditions R F E R directly alters mammalian cell physiology in the absence of indirect thermal effects. Although basic interaction mechanisms are uncertain, the data suggest that the most likely interaction site for most of the reported cel lular effects of R F E R is the plasma membrane. Further insight regarding in vivo effects of RFER, such as cancer induction or promotion or effects on reproduc tion and development, may be provided by appropriately designed in vitro cell studies.
Cell Proliferation and Neoplastic Transformation A variety of in vitro functional and genomic cellular alterations have been at tributed to direct effects of R F E R exposure. Cleary et al. (5-7) reported altered proliferation of normal resting human peripheral lymphocytes and human or rat
In Electromagnetic Fields; Blank, M.; Advances in Chemistry; American Chemical Society: Washington, DC, 1995.
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glioma following a 2-h exposure to 27- or 2450-MHz continuous-wave (CW) or pulse-modulated (PM) R F E R at SARs in the range of 0.5 to 200 W/kg. Altered cell proliferation persisted for up to 5 days after R F E R exposure. The effect was biphasic; maximum increased proliferation occurred at 25 W/kg, whereas exposure at 50 W/kg or higher generally suppressed proliferation (7). Dose fractionation studies indicated exposure effects were persistent for at least 1 day, which suggested the possibility of cumulative or additive exposure effects on cell proliferation. Because cells were exposed under isothermal conditions (37 ± 0.2 °C), altered proliferation was attributed to a direct effect of RFER. A direct effect of pulsed R F E R on increased lymphoblastoid transformation was reported following a 5-day exposure to 2450-MHz R F E R at a maximum S A R of 12.3 W/kg (8). The relevance of RFER-induced alterations in cell proliferation to health effects is suggested by the following: (1) increased in vitro proliferation of cancer cells, such as glioma, is consistent with tumor promotion; (2) decreased in vitro proliferation of immune cells, such as lymphocytes, is consistent with immunosuppression; and (3) recent studies of cell-cycle control mechanisms indicate that alteration of the mammalian cell cycle per se may be associated with increased cancer incidence (9). Altered cell proliferation occurred under conditions that did not directly involve heating. The principle of dose reciprocity suggests the possibility that long-term low-intensity R F E R exposure could alter cell proliferation in vitro. Time-intensity thresholds for RFER-induced alterations in cell proliferation in vitro or in vivo have not been determined. Neoplastic cell transformation has been reported as a direct effect of lowintensity R F E R exposure. Mammalian embryonic fibroblasts were exposed for 24 h to 0.1-, 1-, or 4.4-W/kg, 2450-MHz R F E R pulse modulated at 120 H z (10). In the absence of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), R F E R did not affect cell survival or the rate of neoplastic transformation. Cells treated with T P A and R F E R experienced a statistically significant dosedependent increase in neoplastic transformation rate. Exposure to 4.4-W/kg R F E R in the presence of T P A had a neoplastic transformation effect equivalent to exposure to 1.5 Gy of X-radiation. It was determined that R F E R and X-rays acted independently in inducing neoplastic transformation (10). Evidence of direct genomic effects of R F E R on human somatic cells has also been reported (//, 12) and includes chromosomal aberrations (acentric fragments and dicentric chromosomes), micronuclei formation, and mutagenic characteristics typical of chemical mutagens. Studies of effects of R F E R on cell proliferation and the genome are summarized in Table I.
Membrane Cation Transport and Binding Studies of cell membrane cation permeability provided the first indication of direct nonthermal effects of RFER. Exposure of human, rabbit, and canine erythrocytes to 2450-, 3000-, and 3950-MHz C W microwave radiation at SARs of up to 200 W/kg resulted in intracellular K leakage and osmotic lysis. Temperature control studies conducted over the temperature range of 26 to 44 °C suggested +
In Electromagnetic Fields; Blank, M.; Advances in Chemistry; American Chemical Society: Washington, DC, 1995.
In Electromagnetic Fields; Blank, M.; Advances in Chemistry; American Chemical Society: Washington, DC, 1995. SAR
is maximum
Temperature-dependent increase in control and CW microwave-exposed samples; pulsed microwaves increased lym phoblastoid transformation without heating (ref. 8)
Higherfrequencyof chromosomal ab errations in all exposed samples; increasedfrequencyof micronuclei in exposed samples correlated with specific chromosomal aberrations (refs. 10 and 11)
Latent transformation revealed by TPA treatment of cells exposed to Xradiation and microwaves (ref. 9)
R F E R is radio-frequency electromagnetic radiation; C W is continuous wave; TPA is 12-
