Effects of Simultaneous In-Vitro Exposure to 5G-Modulated 3.5 GHz and GSM-Modulated 1.8 GHz Radio-Frequency Electromagnetic Fields on Neuronal Network Electrical Activity and Cellular Stress in Skin Fibroblast Cells

Abstract

The widespread deployment of 5G wireless networks alongside existing GSM technologies has increased the need to assess potential biological effects of co-exposure to multiple radiofrequency electromagnetic fields (RF-EMF). This study evaluates the in-vitro impact of simultaneous exposure to 5G-modulated 3.5 GHz and GSM-modulated 1.8 GHz signals on neuronal electrical activity, mitochondrial reactive oxygen species (ROS) production, and cellular stress protein responses in neurons and skin fibroblasts. Primary cortical neurons and human immortalized skin fibroblasts were exposed to RF-EMF at specific absorption rates (SAR) of 1 or 4 W/kg for 15 min or 24 h, respectively. Neuronal activity was analyzed using multi-electrode arrays (MEAs), mitochondrial ROS production was measured using MitoSOX Red, and stress protein activity was assessed using bioluminescence resonance energy transfer (BRET) assays targeting RAS, PML, and HSF1 proteins. The results indicate no significant effects on the mean bursting rate (MBR) or mean firing rate (MFR) of cortical neurons, consistent with previous findings at similar SAR levels. Mitochondrial ROS production in fibroblasts also remained unaffected by RF-EMF co-exposure. BRET assays detected minor variations in the basal activity of RAS and PML and in the maximal efficacy of PMA and As₂O₃ to activate these pathways. However, these effects were small, near the detection threshold, and showed no consistent pattern across different tests or chemical treatments. No change was observed in HSF1 basal activity or responsiveness to MG132. These findings suggest that co-exposure to 5G- and GSM-modulated RF-EMF at SAR levels up to 4 W/kg does not produce conclusive evidence of marked biological effects under the tested conditions. Observed variations, when present, are of low amplitude and likely to fall within the range of experimental variability.