Cell phone & the cell

On recent studies in Finland, the land of Nokia.

THE widespread use of mobile, or cell, phones has been accompanied by controversy over the adverse health effects of the electromagnetic field (EMF) radiation emitted by them. The controversy remains despite years of research. Most of the claimed effects, and the consequent research that these have triggered, have to do with genotoxicity, mutations, cancer and male sterility. But there are also reports about mobile phone radiation, whose frequencies (9001,800 megahertz, or MHz) are in the microwave region of the EM spectrum (see diagram), causing such non-life-threatening ailments as sleep disorders, headaches and allergic symptoms.

A series of studies by a research team led by Dariusz Leszczynski, at STUK, the Finnish Radiation and Nuclear Safety Authority, Helsinki, has now demonstrated that the radiation emitted by ordinary mobile phones which is essentially a low-energy radio-frequency-modulated EMF (RF-EMF) can cause subtle biological effects in tissues, such as alteration in the protein expression of certain genes active in the exposed tissues and increased phosphorylation (which is the addition of phosphate groups to proteins and is a measure of their biochemical activity). Following earlier in vitro experiments with human cell lines, Leszczynskis team has now demonstrated for the first time that such molecular level changes occur in human tissues in vivo too.

In this experiment, a small area of skin on the forearm of 10 female volunteers was exposed for one hour to a 900 MHz GSM (Global System for Mobile communication) signal of strength similar to a normal mobile phones signal. The response of the skin tissue to this RF-EMF was studied by carrying out biopsies for all extractable proteins on exposed and non-exposed areas of skin. This was done with a technique known as two-dimensional electrophoresis (2-DE). The technique came up with 579 proteins, an analysis of which identified eight proteins, two of which were common to all the volunteers, that were statistically significantly altered. The results of the experiment were published on February 11 in BioMed Centrals open access online journal BMC Genomics. According to the authors, the results were similar to those seen in their earlier in vitro experiments. Since findings in vitro do not necessarily imply that similar responses would be seen in living tissues as well, this in vivo study strengthens their earlier findings.

It is generally believed that radiation from mobile phones is unlikely to cause any biological effects. A major reason for this belief is that the energy deposited in a tissue by a mobile phone is far lower than the energy needed to break a chemical bond, which is about one electronvolt (eV). The energy required to knock off electrons from a molecule is an order of magnitude higher, about 10 eV. Electromagnetic energy is carried by photons. The higher the frequency, the higher the energy in each photon (see diagram). The frequency at which the radiation becomes ionising is around the upper end of the ultraviolet (UV) range, a little below the frequency where X-rays begin.

Thus, the energy carried by a 900 MHz GSM mobile phone (4 10-6 eV) or by a 1,800 MHz GSM mobile phone (7 10-6 eV) is about a million times less than that required to cause chemical reactions. Nevertheless, at high intensities, all non-ionising radiation for instance, extreme low frequency (ELF) fields such as those produced by high voltage electrical wires, RF, microwaves, infrared, visible light and low-frequency UV can cause heating of the material on which the energy is deposited, including tissues. For this reason, safety regulations for RF and microwave devices fix their exposure levels on the basis of their well-understood thermal effects when sufficient energy is deposited to cause a measurable increase in the temperature of the sample. Therefore, many question whether this low energy would be able to induce biological effects at all, and since the physical mechanism remains unknown, the biological effects reported are often dismissed as artefacts of the experiment, Leszczynski points out in a paper.

But there are many animal and in vitro studies on mobile phone exposure that suggest the possibility of non-thermal biological effects such as levels of expression and activity of certain proteins, which get activated by external stress even when it is too small to cause a rise in temperature. The well-known British report (2000) by the Independent Expert Group on Mobile Phones of the erstwhile National Radiological Protection Board, headed by Sir William Stewart, and the report of the expert panel of the Royal Society of Canada (1999) took cognisance of such findings and advocated a precautionary approach to the use of wireless devices pending definitive proof of their potential health risks. The Finnish study should, therefore, be viewed as one that has gone a step further through its in vivo findings in humans.

In the first of the earlier studies done in 2002, and reported in the journal Differentiation, Leszczynskis team exposed a human endothelial cell line (a single layer of smooth, flattened cells characteristic of the linings of the heart, blood vessels and lymph vessels) called EA.hy926 to a 900 MHz GSM mobile phone for one hour to see whether the signal activated a stress response much like the higher energy EMFs. The unit that is commonly used to measure the energy deposited in biological systems is the specific absorption rate (SAR), which is defined in watts per kilogram (W/kg) and is the rate of absorption of EM energy in a unit mass of tissue. In the experiment, the GSM-mobile-phone-simulating signal was maintained at a SAR of 2.4 W/kg, which is slightly above the European safety limit of 2.0 W/kg.

One of the proteins affected in the study was identified as heat-shock protein-27 (Hsp27). It was found that mobile phone exposure caused a transient increase (two- to sevenfold) in the phosphorylation of Hsp27, which was prevented by a specific inhibitor called SB203580 derived from a protein kinase designated as p38 mitogen-activated protein kinase (p38 MAPK). (Protein kinases are enzymes that activate phosphorylation and MAPKs are protein kinases that respond to external stimuli and regulate cellular processes.) It was also found that the exposure caused transient changes in the protein expression levels of Hsp27 (twofold) and p38 MAPK.

The observed increase in the expression and phosphorylation of Hsp27 in cells led the researchers to suggest that the cells in EA.hy926 had recognised RF-EMF as an external stress factor and this is what elicited an Hsp27-dependent defence. All these effects were non-thermal because, as was determined using temperature probes, irradiation intensity was maintained at levels so as to not alter the temperature of the cell culture, which remained throughout the irradiation period at 37 0.3 C. On the basis of this finding, the researchers suggested that mobile phone radiation activated a variety of cellular signal transduction pathways, the stress response pathway involving Hsp27 and p38 MAPK being one of them.

Heat-shock proteins are a class of proteins that get expressed as a result of cell response to external stress factors, such as heat and radiation, including intense sunlight. The response is designed to be the cells defence mechanism against the external stress. Changes in protein expression and phosphorylation levels can be detected within seconds of exposure, according to Leszczynski. The difference between these detectable cellular responses and RF-EMF-induced stress is that, as noted earlier, the energy deposited by heat (infrared), sunlight (visible light) or UV radiation is far greater than that of microwave or radio frequency radiation.

In a paper on Activation of cellular stress response by RF-EMF and its possible impact on cell physiology, Leszczynski noted: Comparison of the extent of RF-EMF-induced stress response (weak stimulus) with the extent of stress response induced by heat (strong stimulus) for the purpose of claims that the RF-EMF-induced stress is negligible is incorrect; it would be like comparing response to tickling with feeder with response to hitting with hammer. In an e-mail communication, Leszczynski pointed out that such a quantitative comparison was difficult because classical heating and heating with microwaves had different kinetics. The new finding of Leszczynskis group is that even the weak stimulus of RF-EMF-induced stress is sufficient to elicit a cellular response in Hsp27 and associated biochemical pathways.

Investigating further whether these changes had any impact on cellular physiology, the scientists examined the status of stress fibres in the exposed cells because phosphorylation of Hsp27 was known to regulate the stability of certain (F-actin) stress fibres.

It was found that the stability of the stress fibres increased after the period of irradiation and did not decline during the subsequent one-hour incubation period. This induction of increased stability of stress fibres, which could be prevented by the inhibitor SB203580, was found to cause cells to visibly shrink. On the basis of this finding, the researchers hypothesised that increased stabilisation of stress fibres and the cell shrinking caused by it, if they occurred in the endothelial lining of brains blood vessels, might have an effect on the functioning of the blood-brain barrier. This they observed could explain the increased permeability of the blood-brain barrier in certain animal experiments under RF-EMF exposure.

They further noted that Hsp27s overexpression and increased phosphorylation also resulted in inhibition of programmed cell death, or apoptosis, owing to the formation of a new chemical complex with the protein complex apoptosome. This, they hypothesised, could support the survival of cells that transformed spontaneously or were damaged by external factors. The study also found changes in certain proteins of the cytoskeleton (the scaffolding or the skeleton of the cytoplasm in the cells).

The cell line showed up as many as 1,300 altered proteins by the 2-DE technique, of which 49 were found to be statistically significantly altered. Among these 49 were certain cytoskeletal proteins known as vimentins. Thus, the upshot of the experiment is that there is a possibility that RF-EMF-induced molecular events could lead to alteration in cell physiology. Whether any impact on organs or whole body will be exerted by this change remains to be determined by in vivo studies, the researchers noted. This set the stage for the human volunteer project, and as a first step, human skin cells were exposed to low-energy RF-EMF from mobile phones.

To an e-mail query whether exposure to higher energy EM radiation, such as heat or sunlight, would result in similar blood-brain-barrier-related and apoptotic effects, Leszczynski said: Obviously, sunlight has no effect on the blood-brain barrier, at least directly. He further emphasised that the effects on stress proteins and stress fibres induced by mobile phone radiation were transient and cells returned to their normal state after a few hours. Whether repeated exposures could lead to a permanent up-regulation of the stress status of cells, we simply do not know, he added.

Since we know that continuous exposure to higher energy sunlight and heat (particularly in tropical and desert climates) causes no other long-term remarkable physiological changes in general populations (except in the cases of heat strokes, which can be fatal and in cases where UV exposure of non-melanin-rich populations can cause skin cancer in the long term), one could argue a priori that mobile phones are unlikely to cause any long-term health impact. No, Leszczynski said in his e-mail response. It is not correct because mobile phone radiation might induce non-thermal effects. Furthermore, body physiology has mechanisms to adjust to increased temperature caused by slow heating (sunshine). Heating with microwaves is fast and the body has no time to adjust physiology so fast. Therefore, the responses in respect to health might be different. However, this is only a hypothesis, and we do not know whether it is correct. We simply do not have studies that would examine such a possibility.

In another related study, Leszczynski and colleagues carried out the same experiment on two closely related variants of the human endothelial cell line: EA.hy296 and EA.hy296v1. While they found that gene and protein expression were altered in both the cell lines in response to a one-hour radiation exposure at an average SAR of 2.8 W/kg, it was found that the same genes and proteins were affected by the exposure differently. Therefore, the researchers observed an important facet of the biological effects seen, it is likely that different types of cells and from different species might have different sensitivity to this weak stimulus. Our findings might also explain, at least in part, the origin of discrepancies in replication studies between different laboratories.

In all these studies, Leszczynski and associates made a paradigm shift in the technique used for such analyses. Conventional methods are not suited for detecting the small changes in protein expression or phosphorylation that could occur because of weak stimuli, such as RF-EMF-induced stress, and which might be insufficient to cause any physiological changes or potential health effects, as against effects due to strong stimuli. The researchers employed the new approach of high throughput screening techniques (HTST) that have come into use with the increasing automation in biological laboratory methods. This allows a researcher to carry out thousands of biochemical, pharmacological or genetic tests rapidly on samples at hand. Using this technique, one can quickly identify active compounds, antibodies or genes that are involved in a particular biomolecular pathway. This technique has come particularly into vogue in the fields of proteomics (the study of all protein expressions in a given population of cells) and transcriptomics (the study of the expression of all the genes at a time), which have emerged as the natural next steps to derive maximum knowledge out of genomics (the deciphering of complete genomes of organisms).

In an editorial in Proteomics in 2006, before the latest in vivo experiment, Leszczynski pointed out that finding and validating any potential health hazard using the normal epidemiological approach might not be possible because of the low sensitivity of the methodology, which would be insufficient to reliably detect the weak biological effects of low-energy EMF. Further, he pointed out that most epidemiological studies are focussed on the induction of cancer. Therefore, independent of their outcome, these studies will provide information only about cancer, he wrote.

The research executed so far, he said in the editorial, has not produced convincing or robust evidence about the possibility of induction of biological effects by mobile phone radiation.The use of HTSTmight be a useful approach to determine possible molecular targets of EMF on the sub-cellular level. The HTST approach seems to be particularly well suited for studying biological effects of EMF because it might reveal effects that are not possible to predict based on the presently available knowledge. [I]t is necessary to remember that HTST can pick up small changes in protein or gene expression which might be of insufficient magnitude to alter cell physiology. [T]he use of the HTST approach will likely lead to the identification of cellular signalling pathways that respond to EMF exposure. This will allow the formulation of much better, knowledge-based hypotheses aimed at determining whether there might be any health hazard associated with the EMF exposures. Thus, the method of using genome-wide and proteome-wide screening techniques was aimed at quickly identifying all the genes and proteins that respond to low-energy RF-EMF rather than detecting any possible health effects. The human volunteer project has demonstrated that living human skin does respond to mobile phone radiation.

The human volunteer studies conducted so far, the authors point out in the paper, have focussed on cognitive responses to RF-EMF and there is no information available about the proteome, as well as the transcriptome, response to mobile phone radiation in humans. Their findings imply that the response of human skin to RF-EMF results in the alteration of protein expression. However, as they emphasise, this does not necessarily mean that these observed biochemical changes would have any effect on health or cell physiology. This, they point out, requires a further, larger study. Accordingly, a more extensive study with 50-100 volunteers is now planned at STUK. Depending upon the availability of funding, they plan to launch the study in 2009. The present study was funded by Tekes (the Finnish Funding Agency for Technology and Innovation) and STUK, and it was a part of the Finnish research programme on Health Risk Assessment of Mobile Communications (HERMO).