Neuroblastoma

ABSTRACT Despite growing concern about electromagnetic radiation, the interaction between 50- to 60-Hz fields and biological structures remains obscure. Epidemiological studies have failed to prove a significantly correlation between exposure to radiation fields and particular pathologies. We demonstrate that a 50- to 60-Hz magnetic field interacts with cell differentiation through two opposing mechanisms: it antagonizes the shift in cell membrane surface charges that occur during the early phases of differentiation and it modulates hyperpolarizing K channels by increasing intracellular Ca. The simultaneous onset of both mechanisms prevents alterations in cell differentiation. We propose that cells are normally protected against electromagnetic insult. Pathologies may arise, however, if intracellular Ca regulation or K channel activation malfunctions.

INTRODUCTION

There is growing concern about the increase in environmental pollution due to the emission of electromagnetic waves. The mechanism of interaction between extremely low frequency electromagnetic fields (ELF-EMF) and biological structures, if it exists, is still obscure. Epidemiological studies have failed to find a correlation in live subjects between the continuous presence of ELF-EMFs at different intensities and the appearance of any particular pathology (Reipert et al., 1997; Lacy-Hulbert et al., 1998; Hatch et al., 1998; Day, 1999). However, several epidemiological studies have demonstrated increases in childhood leukemia and other related diseases in children from populations exposed to extremely low (50-60 Hz) frequency electromagnetic fields (Thomson et al., 1988) such as those produced by major power lines in the proximity of residential areas. However, none of these studies has found a significant correlation between the presence of ELF-EMFs and increases in pathological conditions. Likewise, not only do macroscopic analyses of cell survival using homogeneous primary cultures obtained from humans and animals show conflicting results, but investigations at subcellular level have also failed to explain the sporadic alterations observed after treatment with an ELF-EMF source (Reipert et al., 1997; Feychting et al., 1998). In general, if a detectable modification does exist, it is not constant and always occurs after activation of a complex cell mechanism (Hojevik et al., 1995; Grynkiewicz et al., 1985; Eichwald and Walleczek, 1996). The most evident effects induced by magnetic waves are the mobilization of intracellular Ca and occasionally morphological changes, although cell signals when present are extremely variable (Liburdy et al., 1993; Karabakhtsian et al., 1994; Goodman et al., 1995; Barbier et al., 1996; Loscher and Liburdy, 1998).

Our study is based on the possibility that ELF-EMFs could interfere with dynamic cell conditions such as division, differentiation, and membrane voltage fluctuation as well as changes in intracellular Ca concentrations. During our experiments we used field intensities of the same order of magnitude as those measured in the proximity of household appliances or electric power lines (source http://www. hsph.harvard.edu/organizations/canprevent/emf.html).

All of our experiments, from cell culture to patch-clamp, were performed in the constant presence of a magnetic field. We were not expecting to find that exposure to magnetic fields produces drastic effects on cell physiology. Our aim was to reveal sudden alterations that, in most cases, could well be buffered by alternative cytosolic pathways without triggering degenerative damage to cells.

In the present study we show that ELF-EMFs interfere with the differentiation of NG108-15 neuroblastomaX glioma cells in vitro. The differentiating agent, by acting on the surface charges, modulates the normal "depolarization-- repolarization" response to growth factors. The resulting change in cell membrane potential represents the cell's commitment to differentiation (Arcangeli et al., 1987; Olivotto et al., 1996). The mechanism of interaction that we propose exists between electromagnetic fields and chemically induced differentiation is based on two antagonistic cellular events. An ELF-EMF is able to prevent the shift in the surface charge potential and, thereby, hyperpolarization. However, it simultaneously stimulates an increase in intracellular Ca in a dose-dependent manner. By opening Cadependent potassium channels, the increase in cytoplasmic divalent ions acts as a rescue agent in cell membrane hyperpolarization, reestablishing the cell's commitment to differentiation. In NG108-15 cells exposed to low intensity ELF-EMFs the two mechanisms appear separate where the magnetic field continues to counteract hyperpolarization but intracellular Ca is not sufficiently raised. The simultaneous onset of the two mechanisms probably prevents major damage during cell differentiation. Problems with exposure to ELF-EMFs may be linked with pathological situations in the event of a malfunctioning of intracellular Ca control mechanisms.

METHODS

Cell cultures and FACS analysis

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R. Tonini,* M. D. Baroni,* E. Masala,* M. Micheletti,^ A. Ferroni,* and M. Mazzanti^^

*Dipartimento di Fisiologia e Biochimica Generali, l^sup a^ Universita di Milano, 1-20133 Milano; ^DIBIT, HSR, 1-20100 Milano; and ^^Dipartimento di Biologia Cellulare e dello Sviluppo, University "La Sapi^enza," 1-00185 Roma, Italy

Received for publication 28 November 2000 and in final form 25 July 2001.

Address reprint requests to Prof. Michele Mazzanti, Dipartimento di Biologic Cellulare e dello Sviluppo University "La Sapienza," P.le Aldo Moro 5,I-00185, Roma, Italy. Tel.: 39-6-49912683; Fax: 39-2-70263884 / 39-- 6-49912351; E-mail: michele.mazzanti@uniromal.it.

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