ID Number		230
Study Type		In Vivo
Model		750, 1800 MHz (CW, GSM) exposure to nematodes (C.elegans) and analysis of hsp and other gene expression and ±-synuclein (marker for Parkinson's disease).
Details		C. elegans strain PC72 carrying a stress inducible hsp16-beta galactosidase reporter gene were exposed in 24 well plates to 750 MHz (CW) or 1 GHz RF at 500 mWatts (SAR ~0.015-0.020 W/kg) in a TEM cell held at a constant temperature of 25 C. An initial study by Power et al (J Bioscience (1998) 23:513-519) reported that after 2 or 26 hours of RF exposure, hsp-driven beta galactosidase expression increased ~20% above controls (as determined by homogenization of the worms and performing a MUG fluorescence assay). After 4 and 8 hours, however, no increase in beta-gal reporter was observed using the same assay. A highly significant clustering of beta-gal reporter activity was observed in nematodes cultured in the wells closest to the microwave source, while the wells furthest away from the source did not show significant beta-gal elevation. In addition, Power et al reported an increase in proliferation in the nematode cells that accelerated development. In a subsequent study (de Pomerai et al, Nature 2000, 405:417-418), findings of a non-thermal increase in hsp gene expression were repeated. A subsequent report (FEBS Lett, 2003, 543:93-97) exposed an aqueous solution of BSA in 24-well plates using 1.0 GHz at 500 milliwatts (SAR ~0.015-0.020 W/kg) in the same TEM cell for 3 to 48 hours, with resulting temperatures ranging from 25 to 45°C. The authors report that exposure enhanced aggregation of BSA in a time- and temperature-dependent manner. Exposure also promoted amyloid fibril formation by bovine insulin at 60°C. The alterations in protein conformation were not accompanied by measurable temperature changes, and were suppressed by the addition of anti-hsp RNA. The authors speculated that the denaturation of cellular proteins might explain the modest heat-shock responses observed in previous studies using Caenorhabditis elegans. However, subsequent reports (FGF / COST 281 -sponsored heat shock meeting in Helsinki Finland, 4/29/04, Bioelectromagnetics 2006, 2008, 2009) involving multiple replication attempts showed that the results could not be replicated after improvements in the exposure system and culture handling were implemented. Specifically, the authors report SARs were actually 5x higher in the initial studies at the edges of the incubator wells as compared to the center (i.e., SAR ~0.075  0.100 W/kg). Further, an 8.5% power loss in the system was measured resulting in a heating effect of 0.3 degrees C per 1.0 W. The group showed that slight heating (from 26.0 to 26.2 degrees C) was able to induce the hsp reporter ~ 20%, the same amount as had been observed in earlier experiments. The original handling conditions were also suspected of possibly shocking the worms. The reports further suggest that small temperature rises during the above exposure conditions might have contributed to initial observations of hsp gene induction. Further studies using Affymetrix chips to evaluate all 1900 genes in nematodes with both RF as well as mild heating reported no consistent effects of non-thermal RF exposures. In recent studies performed in collaboration with Dobson of Keele University, the authors report biogenic magnetite in the nematode C elegans (citation listing #2466), and suggest that the presence of a ferrimagnetic iron oxide represents a possible confounder in EMF experiments, providing a physically plausible explanation for heat shock protein (HSP) responses with RF. Reports from the group also demonstrate that with more control of exposure to eliminate heating effects (1.8 GHz at 1.8 W/kg for 2.5 hours at 25 degrees C), neither pulsed GSM with DTx modulations or CW RF had any effect on increased hsp16 promoter activity in transgenic nematodes (although RF exposure did result in a ~15% decrease in hsp16 promoter activity with both CW and GSM +/- DTx, which the authors could not explain). AUTHORS' ABSTRACT: de Pomerai et al. 2016 (IEEE #6295): Potential health effects of radiofrequency (RF) radiation from mobile phones arouse widespread public concern. RF fields from handheld devices near the brain might trigger or aggravate brain tumors or neurodegenerative diseases such as Parkinson's disease (PD). Aggregation of neural ±-synuclein (S) is central to PD pathophysiology, and invertebrate models expressing human S have helped elucidate factors affecting the aggregation process. We have recently developed a transgenic strain of Caenorhabditis elegans carrying two S constructs: SC tagged with cyan (C) blue fluorescent protein (CFP), and SV with the Venus (V) variant of yellow fluorescent protein (YFP). During S aggregation in these SC+SV worms, CFP, and YFP tags are brought close enough to allow Foerster Resonance Energy Transfer (FRET). As a positive control, S aggregation was promoted at low Hg(2+) concentrations, whereas higher concentrations activated stress-response genes. Using two different exposure systems described previously, we tested whether RF fields (1.0 GHz CW, 0.002-0.02 W kg(-1) ; 1.8 GHz CW or GSM, 1.8 W kg(-1) ) could influence S aggregation in SC+SV worms. YFP fluorescence in similar SV-only worms provided internal controls, which should show opposite changes due to FRET quenching during S aggregation. No statistically significant changes were observed over several independent runs at 2.5, 24, or 96 h. Although our worm model is sensitive to chemical promoters of aggregation, no similar effects were attributable to RF exposures.
Findings		Effects (only at thermal levels)
Status		Completed With Publication
Principal Investigator		Medical Countermeasures Dept, CBD, UK
Funding Agency		MMF, MTHR (NRPB), UK
Country		UNITED KINGDOM
References		Dawe, AS et al. Bioelectromagnetics., (2009) 30:602-612 Dawe, AS et al. Bioelectromagnetics, (2008) 29:92-99 Dawe, AS et al. Bioelectromagnetics, (2006) 27:88-97 de Pomerai, D et al. FEBS Letters, (2003) 543:93-97 de Pomerai, D et al. Nature, (2000) 405:417-418 de Pomerai, D et al. IEEE Trans. Microwave Theory Techniques, (2000) 48:2076-2081 de Pomerai, DI et al. Bioelectromagnetics., (2016) 37:116-129
Comments		In the initial paper by Power et al, the clustering of beta-gal reporter activity closest to the microwave source did not correspond to the RF exposure gradient following a detailed characterization of the exposure system, suggesting no obvious dose-response effect. Significant variability was also observed in the controls from different experiments. A non-significant elevation at the lowest power level used (p=0.06) was taken as evidence that an effect did occur, and that this effect was not due to generalized heating. The nematode heat shock promoter activation temperature is 28 degrees (only 3 degrees above the ambient temperature of 25 degrees during exposure and assay). It is impossible from the description given in the paper to determine the actual RF dose level that any given nematode received, although it is approximated between 0 and 5 W/kg, with the authors subsequently reporting an exposure of 0.015 - 0.02 W/kg. The authors do not supply the temperature inside the TEM cell during exposures (they simply report the final temperature of the large 25 degree incubator, inside which the TEM cell exposure system was placed). Sham exposed worms were not held in the TEM cell, but were wrapped in foil and placed along side the TEM cell within the incubator during exposures, and significant differences between the exposed and sham atmosphere and temperature conditions may have existed. Although the study reported increased B-Gal activity with RF exposure (~ 4-5 fold), the statistical analysis of these findings was barely significant (p = 0.05). Figure 3 in the 1998 Mutation Research paper, the variability is large, and in Figure 3 of the IEEE Trans 2000 paper, the variability between worms cannot be readily determined as the values are given as SEM and not SD. In the Mutation Research study two actual experiments were performed (although the paper refers to numerous replicate enzyme assays for each of the experiments). At the highest power level, statistically significant increases in B-Gal with exposure were observed in one experiment, but were not observed in a second. Further, the difference between the groups exposed at higher SAR between the two experiments was itself statistically significant, demonstrating the high level of variability in the data. At the middle exposure level, statistically significant variability between the two experiments was also observed, and this variability was greater than between exposed and control groups at any other level. The B-Gal increases were observed only in exposed groups after 2 or 16 hours of exposure, and not after 4 or 8 hours of exposure, and this is more easily explained by variability within the data set than by a complex non-thermal RF effect. Finally, the non-uniform induction of B-gal enzyme activity on different sides of the nematode petri dish cannot be explained by variations within the RF field. This study is NOT supported by Leszczynski (#762) because the present study reported induction of hsp at very low SAR (on the order of uW/kg), while the SAR in the Leszczynski study showed a threshold for hsp phosphorylation of ~2W/kg. A new study funded by the MTHR / UK program will expand the current findings and determine which of the nematode's 19000 genes are switched on or off via microarray assay. It will also compare the effects of mild heating with that of RF exposure on gene expression. The ongoing study will further confirm the original findings and analyze possible mechanisms of stress response gene activation.