Summary of Oral Evidence presented to IEGMP by Dr D de Pomerai, University of Nottingham, on Friday 21 January 2000

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Dr de Pomerai uses a system based on the transgenic strains of the nematode worm Ceanorhabiditis elegans to assess environmental pollutants such as metals and pesticides in samples of soil and water. He has tested the effects of radiofrequency fields (RF) in the same system. He believes that the system offers a number of advantages including ease of culture, short (three-day) life cycle, unrivalled genetics, and a transparent but fully differentiated animal. Dr de Pomerai uses two strains of transgenic C. elegans. In one, PC 72, the transgene consists of a lac Z reporter gene downstream of an hsp 16 promoter, whilst the other, PC 161, also carries a green fluorescent protein reporter.

Using in situ visualisation, Dr de Pomerai has found that an overnight exposure to 750 megahertz (MHz) radiation at 0.5 watts (W) increased expression of reporter products in 10–20% of the organisms, depending on the reporter system. The gut is a major organ in this organism and this explains the strong staining observed in gut nuclei (the reporter carries a nuclear localisation signal); strong staining is also observed in embryos within the adult organisms. Significant effects can be obtained following exposures as short as two hours although the effect is stronger following overnight exposures; Dr de Pomerai noted that it takes time for the organisms to produce the reporter products. He has tried using short RF exposures to induce the signal, but this has produced equivocal results. He feels that if changes are assayed at the transcriptional level, it may be possible to detect them after around 30 minutes.

Dr de Pomerai has examined the temperature dependence of the effect. In the absence of RF, the heat-shock response is induced at temperatures in excess of 27°C. Exposure to 0.5 W at 750 MHz effectively reduces the temperature required to elicit the response; the effect is observed at 25°C, but not at 24°C. There appears to be a linear temperature dependence that is parallel to the heat-shock response, but around 3°C lower. Dr de Pomerai has investigated the possibility that the effect could be due to heating by RF and now considers it unlikely, although it is difficult to guarantee no change in temperature. Using the measured electric field strength of 45 volts per metre (V/m) and a conductivity of 0.48 XX per metre (mho/m), determined from cavity perturbation at 615 MHz, he has estimated the specific energy absorption rate as 0.001 W/kg. Moreover, using a fibre optic temperature probe, he has been unable to detect any change in temperature during or after an overnight exposure. He was unable to measure the temperature inside the organisms, but he has not detected any change in the temperature of a concentrated suspension. The Group were concerned that the effect could be due to some form of selective heating effect. It was noted that as only 20% of the organisms responded it is possible that any heating is inhomogeneously distributed. Dr de Pomerai feels that the failure to observe an effect in all the organisms is unlikely to be due to selective heating of responders, and noted that similar or even lower response rates are observed following exposure to metals and pesticides. Although it is not possible to exclude localised heating within organisms, Dr de Pomerai noted that the tissues affected, gut and embryos, constituted over 50% of the body mass, and questioned whether it is possible to heat these organs without producing a detectable temperature change in the medium. It was suggested that one means of determining whether the effect was due to heating, would be to alter the exposure power; heating would be dependent on power, so if the effect is independent heating can be excluded. Dr de Pomerai explained that it is not possible to increase the power with his present system, although he can reduce it. The long exposure times used (18 hours) make it unlikely that substantial temperature differences could be maintained with heat diffusion.

The Group were keen to explore possible mechanisms that might be responsible for the effects observed and were generally impressed by Dr de Pomerai's work. Dr de Pomerai believes that the mechanism is unlikely to be thermal and suggested a number of possible alternatives. One possibility is that absorption of RF might produce limited protein denaturation by disrupting the weak interactions that are required to maintain complex three-dimensional polypeptide structures. Dr de Pomerai identified hydrophobic interactions as likely targets and noted that protein denaturation is well established as a trigger for the heat-shock response. Alternatively, RF exposure might enhance production of reactive oxygen species, which are also known to induce the heat-shock response. If this is the case the response should be inhibited by antioxidants such as ascorbic acid and glutathione. Dr de Pomerai also speculated that RF exposure might interfere with intracellular signalling, affecting calcium ions or kinase activation. Effects on post-translational modification of the heat-shock transcription factor appears less relevant as it is known that this does not affect its activity in Drosophila melanogaster. He believes that the effect on the heat shock response is linked to a stimulation of growth and indicated that he would like to explore this further. He noted that there is currently a programme to produce knockouts for every gene in C. elegans, and suggested that this would present an unrivalled opportunity to explore the mechanisms in detail. He believes that his system offers a unique experimental tool to investigate the mechanisms of non-thermal effects.

First issued 19 April 2000