ID Number		872
Study Type		In Vitro
Model		1.9, 10 GHz (CW, AM-50Hz) exposure to cells and isolated enzymes and analysis of gene and protein expression
Details		In initial studies, in vitro solutions of beta-galactosidase enzyme from Bacillus acidocaldarius (thermophillic bacteria) were exposed to 10.4 GHz microwaves for 15, 30, 45, or 60 minutes at SARs of 1.1 and 1.7 W/g in a waveguide system. MW exposure resulted in a dose dependent decrease in enzyme activity (20% of control values after 60 minutes using 1.7 W/kg) although non-RF heating (using a water bath) to 70 degrees did not affect bGal activity. The MW inhibition was irreversible, although it was only observed with enzyme concentrations between 10 and 50 ug/ml, and not when bGal concentrations were greater than 50 ug/ml. In an earlier study, isolated solutions of S-Adenosylhomocysteine hydrolase (AdoHcy) and 5'-methylthioadenosine phosphorylase (MTA) from another thermophilic bacteria, Sulfolobus solfataricus, were exposed as above for up to 90 minutes at SARs of 1.5-3.1 W/kg (sufficient to bring the solution temperature to 70-90 degrees). Again, MW exposure resulted in an irreversible and time-dependent inhibition of both enzymes. Both enzymes retained full enzyme activity after 90 minutes of non-MW heating at 70 C or 30 minutes at 90 C in a water bath. Exposure to solutions of acid phosphatase with 9 GHz (CW and PW) MW for 10 minutes at 160 up to 480 W/kg also resulted in decreased enzyme activity. The authors did not provide a mechanism to explain the observed effects, but they did suggest that MW exposure may result in an irreversible and non-thermal effect on protein structure. In a related study, myoglobin protein (isolated from bluefin Tuna heart ventricles) was exposed to 1.95 GHz RF for 3 hours at 51 mW/kg (calculated using absorbed power and mass of the sample) in an acidic pH 3.0 solution (to induce fluctuation between folded and partially folded states). Refolding was induced during exposure with 100 mM sodium phosphate to adjust the pH to 7.0. A water jacket surrounded the waveguide exposure system to control temperature, although temperature was reported to increase from 25 to 30 degrees C after 30 minutes and then stabilize for the remainder of the experiment. The authors report that RF exposure slowed the speed of refolding (by spectrophotometric analysis) and suggest "the collapsing process of reconstruction of the heme pocket and the binding of heme" as a possible mechanism. In a recent publication (2005), human mouth skin cancer (KB) cells were exposed to 1.95 GHz (CW) at 3.6 +/- 0.2 W/kg for 1-3 hours and analyzed for protein expression (via western blot), DNA fragmentation, surface antigens (via flow cytometry), and fluorescence microscopy to evaluate effects on the ras-to-Erk (MAPK)cell signaling pathway, including hsp proteins. Exposure for 3 hours increased hsp70 and hsp27 levels, and decreased ras and Raf-1 expression / activity in a time-dependent manner, and decreased activity of anti-apoptosis enzymes Erk-1 and Erk-2. Exposure also increased apoptosis and proteasome-dependent degradation. Exposure did not, however, affect Erk-1 or Erk-2 protein levels themselves. The authors speculate that RF may inhibit the ras-to-Erk-mediated gene cascade by decreasing hsp90 expression and interrupting proper folding of proteins. Whether RF effects normal cells in this way, however, is not known.
Findings		Effects
Status		Completed With Publication
Principal Investigator		ICEmB, CNR-IREA, Univ Naples, Italy
Funding Agency		Private/Instit.
Country		ITALY
References		Bismuto, E et al. Eur Biophys J., (2003) 32:628-634 Caraglia, M et al. Am J Physiol Cell Physiol , (2005) 204:539-548 Mancinelli , F et al. J Cell Biochem, (2004) 93:188-196 La Cara, F et al. Bioelectromagnetics, (1999) 20:172-176 Porcelli, M et al. FEBS Lett., (1997) 402:102-106
Comments		The effects of local temperature on the enzyme preparations were not well characterized, and local thermal effects could have been a source of error. In the J Cellular Biochem (2004 93(1):188-96 paper, no modeling or experimental data to determine how uniform the exposure was (SAR was simply calculated using absorbed power and mass of the sample), and no actual temperature measurements to determine peak hot spots. In the 2005 study, no detailed information on the cell culture exposure system or exposure assessment was provided. Not clear whether unexposed were sham-exposed or simply held nearby and not handled - authors seem to indicate a single exposure chamber, so experiments may not have been conducted in a blinded manner. Exposure time listed as eithr 1, 2, or 3 hours in some parts and 48 hours in others.