Luca Massaro ^1,2* Stefania De Sanctis ² Valeria Franchini ² Elisa Regalbuto ² Gaetano Alfano ² Chiara Focaccetti ¹ Monica Benvenuto ¹ Loredana Cifaldi ¹ Antonella Sgura ³ Francesco Berardinelli ³ Jessica Marinaccio ³ Federica Barbato ³ Erica Rossi ³ Daniela Nardozi ⁴ Laura Masuelli ⁴ Roberto Bei ¹ Florigio Lista ²

• ¹ Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Roma, Italy
• ² Defence Institute for Biomedical Sciences, Rome, Sicily, Italy
• ³ Department of Science, Roma Tre University, Rome, Lazio, Italy
• ⁴ Department of Experimental Medicine, Sapienza University of Rome, Rome, Lazio, Italy

The widespread use of radiofrequency (RF) sources, ranging from household appliances to telecommunications devices and military equipment, raises concerns among people and regulatory agencies about the potential health risks of RF exposure. Consequently, several in vitro and in vivo studies have been done to investigate the biological effects, in particular non-thermal, of this non-ionizing radiation. To date, this issue is still being debated due to the controversial results that have been reported. Furthermore, the impact of different RF signal modulations on biological systems remains poorly investigated. The present in vitro study aims to evaluate the cytotoxicity and genotoxicity of continuous or pulsed 1.6 GHz RF in human dermal fibroblasts (HDF).Methods: HDF cultures were exposed to continuous and pulsed 1.6 GHz RF, for 2 hours, with Specific Absorption Rate (SAR) of 0.4 W/kg. The potential biological effects of 1.6 GHz RF on HDF were assessed with a multi-methodological approach, analyzing the effects on cell cycle, ultrastructure, protein expression, mitotic spindle, CREST stained micronuclei, chromosome segregation and γ-H2AX/53BP1 foci. Results: 1.6 GHz RF exposure modified proteins expression and morphology of HDF. Specifically, the expression of different heat-shock proteins (HSP) (i.e. HSP-90, HSP-60 and HSP-25) and phospho-AKT were affected. In addition, both continuous and pulsed RF modified the cytoskeletal organization in HDF and increased the number of lysosomes, while the formation of autophagosomes was observed only after pulsed RF exposure. Mitotic spindle anomalies were also found after exposure. However, no significant effect was observed on cell cycle, chromosome segregation, CREST-stained micronuclei and γ-H2AX/53BP1 foci.The results of the present study show the absence of genotoxic damage in 1.6 GHz RF exposed HDF and, although mitotic spindle alterations were observed, they did not have an aneugenic effect. On the other hand, changes in some proteins expression and cell ultrastructure in exposed HDF suggest that RF can potentially induce cell alterations at the morphological and molecular levels.