The rapid evolution of electromagnetic field-based telecommunications applications introduced various global challenges, including environmental, health, technical, and regulatory concerns. Concerning the potential health implications due to cumulative radiofrequency (RF) exposure, the existence and significance of possible health effects remain uncertain, as scientific studies have produced conflicting results, making it challenging to establish a clear consensus within the research community1. Moreover, the 5G New Radio (5G-NR) rollout further exacerbated the debate, and very few studies are available in the literature dealing with the effects of 5G modulated signals in the different frequency ranges of FR1 (sub-7 GHz) and FR2 (above 24 GHz). These concerns are particularly pronounced among certain activist groups, which view fifth-generation (5G) networking as potentially posing a greater public health risk than previous wireless technologies1. Still, even among experts, there is an opinion that the body of evidence, especially in FR2, is lacking2.
; Currently, scientific evidence demonstrating adverse health effects from RF-EMF exposure is limited. In 2011, the International Agency for Research on Cancer (IARC) classified RF-EMF as “possibly carcinogenic to humans” (Group 2B)3. This classification was based on epidemiological studies suggesting a possible link between wireless phone use and an elevated risk of glioma, a type of brain cancer. Beyond carcinogenicity4, RF-EMF exposure has been associated with possible neurological5,6, reproductive7–10, and physiological effects11 and oxidative stress12. Moreover, the potential underlying mechanisms behind these alleged health effects remain unclear and are still under investigation, primarily due to common methodological problems in experimental design within the literature13. New approaches for elucidating the mechanism behind these effects could provide new insights.
; A whole transcriptome gene expression study could significantly enhance our understanding of the biological effects of RF-EMF exposure by identifying molecular changes in cells and tissues following exposure based on the cell’s transcriptomic response. This approach allows researchers to examine how RF-EMF influences gene expression across the entire genome, providing insights into its potential impact on pathways involved in cellular stress responses, inflammation, DNA damage, or other biological processes that could be linked to health effects. Such a study will help us provide mechanistic insights into if and how RF-EMF interacts with biological systems by identifying differentially expressed genes and revealing affected pathways, including but not limited to the processes described above. This could help assess dose-response relationships, bridge contradictory findings between studies, and refocus future research. Finally, it could identify biomarkers of exposure with possible
; applications in monitoring long-term health effects in populations with high RF-EMF exposure.
; Here, we present the results of an in vitro study investigating the effects of 5G modulated signal in the FR1 and FR2 frequency bands in the framework of the EU-funded NextGEM project. In particular, the frequencies of 3.5 GHz and 26.5 GHz were used at Sciensano and CNR-IREA, respectively, to evaluate the effects on the transcriptomics profile of a human keratinocyte cell model (HaCaT). These cells were chosen since the skin is a primary target organ for RF-EMF absorption in the 5G-NR FR2 band—due to the reduced penetration depth of these frequencies.
; In summary, this study aims to examine the impact of 5G-NR modulated RF-EMF at two different frequencies on gene expression in an in vitro cell system, paying particular attention to high-quality experimental design.
; METHODS
; Cell culture
; HaCaT cells were cultured at 95% humidity, 5% CO2, and 37°C in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine. Cells were maintained in culture until passage 16 (P16), after which they were discarded.
; Exposure
; Exposure was performed at both 3.5 GHz and 26.5 GHz with a 5G-NR modulated signal. For the 3.5 GHz exposure, a sXc3500 5G-NR exposure system (IT’IS foundation) was used, allowing for test blinding in a temperature-controlled environment. The system consists of two chambers: one in which cells are exposed to radiation and one for sham exposure. The 26.5 GHz exposure was performed using a reverberation chamber-based system designed, manufactured, and characterized by the University of Cassino and Southern Lazio, Italy. Both exposure systems consist of two chambers hosted in standard cell culture incubators: RF exposure and sham exposure. Both RF devices assured well-controlled RF exposure regarding field distribution, SAR levels, and temperature inside the exposed samples.
; Several quality control mechanisms were included in this study. For example, all exposures were performed in a blinded fashion in a validated exposure system at known dosimetric parameters. For both frequencies, cell cultures were exposed to SAR values of 0.4 W/Kg and 1 W/Kg for 1 hour, 3 hours, and 24 hours.
; Sample preparation
; After exposure, cell lysates were collected using the 2x enhanced lysis buffer (Biospyder) and PBS (Thermofisher). Samples were frozen at -80°C until shipped to a company specializing in TempO-Seq.
; Templated Oligo Sequencing (TempO-Seq)
; To ensure high-quality data generation and reproducibility, the TempO-Seq assay and sequencing were outsourced to BioClavis Ltd. (Glasgow, UK), a specialized contract research organization experienced in TempO-Seq applications. The assay used a Whole Transcriptome Panel, allowing for comprehensive transcriptomic profiling14.
; Data analysis
; TempO-Seq sequencing data were processed using the BioClavis analytical pipeline. Gene expression counts were normalized, and differential expression analysis was performed using DESeq2 (Bioconductor, R). Pathway enrichment analysis was conducted to identify key biological pathways affected by RF-EMF exposure.
; RESULTS AND DISCUSSION
; Data from both Sciensano and CNR-IREA laboratories are currently available, but further work is needed in the upcoming months to perform the analysis and evaluate whether these can be confirmed in qPCR experiments. Initial results are expected to be available by the time of the conference and will allow a direct comparison between the effects of a 5G modulated signal in two different frequency bands on the transcriptomics of HaCaT cells. The results will be discussed based on current knowledge.
; NEXT STEPS
; Based on the differential gene expression analysis results, genes of interest will be selected. These selected genes will be validated using RT-qPCR as a more targeted approach that can also be used for a more targeted dose-response analysis, which can be used for more advanced risk assessment tools (such as the Benchmark Dose approach). After RF-EMF exposure, the cells designated for RT-qPCR will be lysed, followed by RNA extraction and purification. The concentration and quality of the extracted RNA will be assessed using a Nanodrop 2000C spectrophotometer (Thermo Scientific). Only RNA samples with A260/280 absorbance ratios of ≥ 2.0 will be used for further analysis. For each sample, the collected RNA will be used for complementary DNA (cDNA) synthesis, which will be performed using the iScript cDNA Synthesis Kit (BioRad) according to the manufacturer’s protocol. The qPCR assay, including the appropriate controls, will be conducted in a 96-well plate format. On the qPCR plate, purified cDNA (GenElute™ PCR Clean-Up Kit, Sigma) will be used in the reaction mix, prepared with TaqMan® Gene Expression Master Mix (Applied Biosystems™). For the qPCR experiments, mRNA expression levels will be normalized using the geometric mean of five housekeeping genes to calculate ΔΔCq values15. The log₂ fold changes for each exposure condition relative to the control will be determined using each sample’s 2^-ΔΔC(T) method.
; CONCLUSIONS
; A whole transcriptome gene expression study has the potential to significantly advance the field of RF-EMF health research by providing molecular-level insights into the biological responses triggered by exposure by including a high-quality experimental design. By identifying differentially expressed genes and affected cellular pathways, this approach can help clarify potential mechanisms of action, resolve inconsistencies between previous studies, and contribute to a more comprehensive risk assessment. Furthermore, it may aid in identifying biomarkers for exposure and susceptibility, ultimately guiding public health policies and regulatory frameworks to ensure the safe integration of emerging wireless technologies.