Radiofrequency electromagnetic fields (RF-EMF) are now widespread due to the rapid growth of wireless technologies, leading to a steady rise in environmental exposure from both new and existing sources. While epidemiological studies have suggested a potential link between RF-EMF exposure and glioma risk, leading IARC to classify RF-EMFs as “possibly carcinogenic to humans” (Group 2B), mechanistic evidence supporting this association remains limited. To address this knowledge gap, the EU-funded NextGEM project (Grant Agreement Number 101016567) investigates the impact of 5G-NR modulated RF-EMF on a range of biological endpoints in vitro, including genotoxicity, oxidative stress, and gene expression alterations.

HaCaT human keratinocyte cells were selected due to the relevance of the skin as a target tissue for RF-EMF in the 5G frequency range. Genotoxicity and transcriptomics analyses were performed in both 3.5 GHz (5G-FR1) and 26.5 GHz (5G-FR2) exposure conditions. The effects on oxidative stress were evaluated only under 3.5 GHz exposure conditions. Cells were exposed to 5G-modulated 3.5 GHz signals using the validated sXc3500 exposure system (IT’IS Foundation) and a validated reverberation chamber-based system for 26.5 GHz, with specific absorption rates (SAR) ranging from 0.4 to 1 W/kg. Each experiment included sham-exposed, temperature, and incubator controls, as well as positive controls (methyl methanesulfonate (MMS) for micronucleus; ethyl methanesulfonate (EMS) for comet; menadione and H₂O₂ for oxidative stress).

To explore the impact on oxidative stress, intracellular reactive oxygen species (ROS) levels were quantified post-exposure using a DCFDA fluorescence assay. In parallel, transcriptomic profiling was conducted via TempO-Seq®, a high-throughput targeted RNA sequencing platform, to identify potential gene expression changes associated with stress response pathways, DNA repair, and inflammation. Genotoxicity was assessed using both the in vitro cytokinesis-block micronucleus (CBMN) assay and the in vitro alkaline comet assay. For all assays, quality assurance measures were taken, including blinding, inclusion of replicates and proper controls, and – in the case of the genotoxicity assays — automated scoring (Metafer 4 system).

Validated protocols for the micronucleus, comet, oxidative stress, and TempO-Seq assays were developed and applied under controlled exposure conditions. Our data suggest no significant cytotoxicity or increase in DNA damage under the tested conditions. Preliminary data for oxidative stress also show no significant difference between sham and exposed conditions. Preliminary TempO-Seq results suggest only modest changes in genes across exposed and non-exposed conditions, with no conserved genes between different conditions.