ID Number		1258
Study Type		In Vitro
Model		900 MHz (GSM) exposure to cells and analysis of mitosis and endocytosis of fluorescent dyes
Details		Mouse melanoma (B16-F1), human carcinoma (A253), and Chinese hamster fibroblast (DC-3F) cells in 35 mm Petri dishes were exposed to 900 MHz (GSM or CW) for 10 to 60 minutes at average SARs ranging from 0.08 to 4.55 W/kg (peak from 0.6 to 36.4 W/kg) in an in vitro exposure system employing a signal generator (RF S 900-60) and a wire patch antenna (described in Laval et al, Bioelectromagnetics (2000) 21:255-263). Exposures lasting between 5 to 90 minutes were also performed using a low-frequency (50 to 400 Hz) EMF at field strengths of 1.2 to 8 V/cm and pulse durations between 75 and 580 microseconds. During exposure, cells were assessed for endocytotic activity by examining uptake of fluorescent dyes (Lucifer Yellow and fluorescent FITC-Dextran). Temperature in the samples exposed to higher field strengths did increase 2-3 degrees C. Increased endocytosis was observed with GSM exposure at SARs of 2.6 - 4.5 W/kg but not below 1.3 W/kg, suggesting a threshold between 1.3 to 2.6 W/kg, depending upon the cell line. The effect did not appear to be due to antioxidant production, as the presence of an antioxidant mixture did not affect the result. The authors also suggested the effects were not due to electrophoresis of charged particles, acute resonance interactions such as calcium oscillations, or conformational modifications of enzymes or proteins. Effects were also observed using the low frequency pulsed exposure, with a threshold for effect between 1.3 and 2.3 V/cm depending upon the cell line. In contrast, no effect was reported with CW exposure at similar dose. There was no effect of any exposure on cell viability, and the effects on endocytotic activity were reversible. They conclude by stating that the observed effect thresholds appear to be lower than average peak SAR associated with the use of mobile phones (< 2 W/kg over 10g). In a subsequent study, the authors constructed a novel TEM exposure system attached to an inverted microscope and housed within a cell culture incubator allowing them to observe cells in real time during growth. Cells were exposed to 900 MHz (GSM)for 1 h at between 2.2 - 4.7 W/kg). Temperature was held at a constant 30 (+/- 1) degrees C. The inverted mocroscope allowed the authors to observe the cells during exposure in real time. The authors again confirmed that the rate of endocytosis was increased with RF exposure (at 4.2 and 4.7 W/kg), although there was no change in mitosis (at 2.2 W/kg).In a subsequent study, mouse melanoma (B16-F10) cells were again exposed in 35 mm Petri plates to 900 MHz (GSM) for 20 minutes at an SAR of 3.2 +/- 0.8 W/kg using the wire patch cell exposure system placed inside of a cell culture incubator. The authors again report an increased endocytotic activity with GSM exposure, and further report that when cells were pre-incubated with either Chlorpromazine (a cationic amphiphilic drug that disrupts the assemblydisassembly of clathrin) or ethanol (also known to block clatherin mediated endocytosis), the uptake of fluorescent dyes was blocked. In contrast, incubation with the drug Filipin III (which binds to cholesterol in the caveolae membrane micro-domains and disrupts the smaller caveolar membrane invaginations on the cell surface that are also involved in membrane trafficking, sorting, transport and signal transduction) did not reverse the effects of GSM exposure. There was no effect of GSM signal exposure on fluorescent dye release from the cell endosomes. The authors conclude their results & suggest that GSM modulated EMF could affect the uptake of molecules not only by fluid phase endocytosis but also by receptor-mediated endocytosis, as clathrin-coated vesicles are instrumental in this uptake.
Findings		Effects
Status		Completed With Publication
Principal Investigator		Institute Gustave-Roussy, France
Funding Agency		Private/Instit.
Country		FRANCE
References		Moisescu, MG et al. Bioelectromagnetics, (2008) 30:222-232 Moisescu, MG et al. Bioelectrochemistry, (2008) 74:9-15 Mahrour , N et al. Biochimica et Biophysica Acta, (2005) 1668:126-137
Comments		The authors do acknowledge that the endocytotic process can be significantly influenced by temperature, and that small temperature changes (up to 1oC) did occur within the culture during exposure, but they conclude that the observed effects were non-thermal in nature. Temperature during the exposure was monitored at a single point within the culture media during exposure using a Luxtron optical probe as well as immediately before and after using a fast digital thermometer P600 with a thin thermocouple probe. Since the uptake of LY dye was described as having a significant temperature dependence at temperatures above 32oC, but remains relatively constant between 27-31oC, the cells were maintained at 29 oC during dye loading and RF exposure periods. However for experiments with the endocytosis inhibitors, cells were pre-incubated for 30 min at 37 oC prior to dye loading and RF exposure, and equilibrated to & about 29 +/- 0.5 oC as follows: & inside the incubator the air temperature was set at 30 oC and the humidity was provided by an open large container filled with demineralized water with a stabilized temperature of 29.529.8 oC; (b) the [wire patch cell] with its protective walls was put inside the incubator for at least 12 h before the experiments took place; (c) the lower sample holders with 8 ml of water were placed on wire patch plate 2 hours before the experiment; (d) the direct holders of the samples with 5ml of water were heated in a block heater for 20 min before use. The authors report a culture temperature before exposure of 29 +/- 0.5 oC and after exposure between 28.5 and 29.5 oC.