Evaluation of mitochondrial stress following ultraviolet radiation and 5G radiofrequency field exposure in human skin cells.

Whether human cells are impacted by environmental electromagnetic fields (EMF) is still a matter of debate. With the deployment of the fifth generation (5G) of mobile communication technologies, the carrier frequency is increasing and the human skin becomes the main biological target. Here, we evaluated the impact of 5G-modulated 3.5 GHz radiofrequency (RF) EMF on mitochondrial stress in human fibroblasts and keratinocytes that were exposed for 24 h at specific absorption rate of 0.25, 1, and 4 W/kg. We assessed cell viability, mitochondrial reactive oxygen species (ROS) production, and membrane polarization. Knowing that human skin is the main target of environmental ultraviolet (UV), using the same read-out, we investigated whether subsequent exposure to 5G signal could alter the capacity of UV-B to damage skin cells. We found a statistically significant reduction in mitochondrial ROS concentration in fibroblasts exposed to 5G signal at 1 W/kg. On the contrary, the RF exposure slightly but statistically significantly enhanced the effects of UV-B radiation specifically in keratinocytes at 0.25 and 1 W/kg. No effect was found on mitochondrial membrane potential or apoptosis in any cell types or exposure conditions suggesting that the type and amplitude of the observed effects are very punctual.

Effects of Nanosecond Pulsed Electric Field (nsPEF) on a Multicellular Spheroid Tumor Model: Influence of Pulse Duration, Pulse Repetition Rate, Absorbed Energy, and Temperature.

Cellular response upon nsPEF exposure depends on different parameters, such as pulse number and duration, the intensity of the electric field, pulse repetition rate (PRR), pulsing buffer composition, absorbed energy, and local temperature increase. Therefore, a deep insight into the impact of such parameters on cellular response is paramount to adaptively optimize nsPEF treatment. Herein, we examined the effects of nsPEF ≤ 10 ns on long-term cellular viability and growth as a function of pulse duration (2-10 ns), PRR (20 and 200 Hz), cumulative time duration (1-5 µs), and absorbed electrical energy density (up to 81 mJ/mm3 in sucrose-containing low-conductivity buffer and up to 700 mJ/mm3 in high-conductivity HBSS buffer). Our results show that the effectiveness of nsPEFs in ablating 3D-grown cancer cells depends on the medium to which the cells are exposed and the PRR. When a medium with low-conductivity is used, the pulses do not result in cell ablation. Conversely, when the same pulse parameters are applied in a high-conductivity HBSS buffer and high PRRs are applied, the local temperature rises and yields either cell sensitization to nsPEFs or thermal damage.

In vitro exposure of neuronal networks to the 5G-3.5 GHz signal.

Introduction: The current deployment of the fifth generation (5G) of wireless communications raises new questions about the potential health effects of exposure to radiofrequency (RF) fields. So far, most of the established biological effects of RF have been known to be caused by heating. We previously reported inhibition of the spontaneous electrical activity of neuronal networks in vitro when exposed to 1.8 GHz signals at specific absorption rates (SAR) well above the guidelines. The present study aimed to assess the effects of RF fields at 3.5 GHz, one of the frequencies related to 5G, on neuronal activity in-vitro. Potential differences in the effects elicited by continuous-wave (CW) and 5G-modulated signals were also investigated. Methods: Spontaneous activity of neuronal cultures from embryonic cortices was recorded using 60-electrode multi-electrode arrays (MEAs) between 17 and 27 days in vitro. The neuronal cultures were subjected to 15 min RF exposures at SAR of 1, 3, and 28 W/kg. Results: At SAR close to the guidelines (1 and 3 W/kg), we found no conclusive evidence that 3.5 GHz RF exposure impacts the activity of neurons in vitro. On the contrary, CW and 5G-modulated signals elicited a clear decrease in bursting and total firing rates during RF exposure at high SAR levels (28 W/kg). Our experimental findings extend our previous results, showing that RF, at 1.8 to 3.5 GHz, inhibits the electrical activity of neurons in vitro at levels above environmental standards.

Effects of 5G-modulated 3.5 GHz radiofrequency field exposures on HSF1, RAS, ERK, and PML activation in live fibroblasts and keratinocytes cells.

The potential health risks of exposure to radiofrequency electromagnetic fields from mobile communications technologies have raised societal concerns. Guidelines have been set to protect the population (e.g. non-specific heating above 1 °C under exposure to radiofrequency fields), but questions remain regarding the potential biological effects of non-thermal exposures. With the advent of the fifth generation (5G) of mobile communication, assessing whether exposure to this new signal induces a cellular stress response is one of the mandatory steps on the roadmap for a safe deployment and health risk evaluation. Using the BRET (Bioluminescence Resonance Energy-Transfer) technique, we assessed whether continuous or intermittent (5 min ON/ 10 min OFF) exposure of live human keratinocytes and fibroblasts cells to 5G 3.5 GHz signals at specific absorption rate (SAR) up to 4 W/kg for 24 h impact basal or chemically-induced activity of Heat Shock Factor (HSF), RAt Sarcoma virus (RAS) and Extracellular signal-Regulated Kinases (ERK) kinases, and Promyelocytic Leukemia Protein (PML), that are all molecular pathways involved in environmental cell-stress responses. The main results are (i), a decrease of the HSF1 basal BRET signal when fibroblasts cells were exposed at the lower SARs tested (0.25 and 1 W/kg), but not at the highest one (4 W/kg), and (ii) a slight decrease of As2O3 maximal efficacy to trigger PML SUMOylation when fibroblasts cells, but not keratinocytes, were continuously exposed to the 5G RF-EMF signal. Nevertheless, given the inconsistency of these effects in terms of impacted cell type, effective SAR, exposure mode, and molecular cell stress response, we concluded that our study show no conclusive evidence that molecular effects can arise when skin cells are exposed to the 5G RF-EMF alone or with a chemical stressor.

In Vivo Functional Ultrasound (fUS) Real-Time Imaging and Dosimetry of Mice Brain Under Radiofrequency Exposure.

This study aims to analyze in real-time the potential modifications induced by low-level continuous-wave and Global System for Mobile Communications radiofrequency (RF) exposure at 1.8 GHz on brain activation in anesthetized mice. A specific in vivo experimental setup consisting of a dipole antenna for the local exposure of the brain was fully characterized. A unique neuroimaging technique based on a functional ultrasound (fUS) probe was used to observe the areas of mice brain activation simultaneously to the RF exposure with unprecedented spatial and temporal resolution (~100 µm, 1 ms) following manual whisker stimulation using a brush. Numerical and experimental dosimetry was carried out to characterize the exposure and to guarantee the validity of the biological results. Our results show that the fUS probe can be efficiently used during in vivo exposure without interference with the dipole. In addition, we conclude that exposure to brain-averaged specific absorption rate levels of 2 and 6 W/kg does not introduce significant changes in the time course of the evoked fUS response in the left barrel field cortex. The proposed technique represents a valuable instrument for providing new insights into the possible effects induced on brain activation under RF exposure. For the first time, brain activity under mobile phone exposure was evaluated in vivo with fUS imaging, paving the way for more realistic exposure configurations, i.e. awake mice and new signals such as the 5 G networks. © 2022 Bioelectromagnetics Society.

Millimeter-Wave Heating In Vitro: Local Microscale Temperature Measurements Correlated to Heat Shock Cellular Response.

OBJECTIVE: Cellular sensitivity to heat is highly variable depending on the cell line. The aim of this paper is to assess the cellular sensitivity of the A375 melanoma cell line to continuous (CW) millimeter-waves (MMW) induced heating at 58.4 GHz, between 37 °C and 47 °C to get a deeper insight into optimization of thermal treatment of superficial skin cancer. METHODS: Phosphorylation of heat shock protein 27 (HSP27) was mapped within an area of about 30 mm 2 to visualize the variation of heat-induced cellular stress as a function of the distance from the waveguide aperture (MMW radiation source). A multiphysics computational approach was then adopted to yield both electromagnetic and thermal field distributions as well as corresponding specific absorption rate (SAR) and temperature elevation. Induced temperature rise was experimentally measured using a micro-thermocouple ( µTC). RESULTS: Coupling of the incident electromagnetic (EM) field with µTC leads was first characterized, and optimal µTC placing was identified. HSP27 phosphorylation was induced at temperatures ≥ 41 °C, and its level increases as a function of the thermal dose delivered, remaining mostly focused within 3 mm 2. CONCLUSION: Phosphorylation of HSP27 represents a valuable marker of cellular stress of A375 melanoma cells under MMW exposure, providing both quantitative and spatial information about the distribution of the thermal stress. SIGNIFICANCE: These results may contribute to the design of thermal treatments of superficial melanoma through MMW-induced heating in the hyperthermic temperature range.

A nanosecond pulsed electric field (nsPEF) can affect membrane permeabilization and cellular viability in a 3D spheroids tumor model.

Three-dimensional (3D) cellular models represent more realistically the complexity of in vivo tumors compared to 2D cultures. While 3D models were largely used in classical electroporation, the effects of nanosecond pulsed electric field (nsPEF) have been poorly investigated. In this study, we evaluated the biological effects induced by nsPEF on spheroid tumor model derived from the HCT-116 human colorectal carcinoma cell line. By varying the number of pulses (from 1 to 500) and the polarity (unipolar and bipolar), the response of nsPEF exposure (10 ns duration, 50 kV/cm) was assessed either immediately after the application of the pulses or over a period lasting up to 6 days. Membrane permeabilization and cellular death occurred following the application of at least 100 pulses. The extent of the response increased with the number of pulses, with a significant decrease of viability, 24 h post-exposure, when 250 and 500 pulses were applied. The effects were highly reduced when an equivalent number of bipolar pulses were delivered. This reduction was eliminated when a 100 ns interphase interval was introduced into the bipolar pulses. Altogether, our results show that nsPEF effects, previously observed at the single cell level, also occur in more realistic 3D tumor spheroids models.

High-voltage 10 ns delayed paired or bipolar pulses for in vitro bioelectric experiments.

Recent studies proved that classical bio-effects induced by nanosecond pulsed electric field (nsPEF) can be reduced by the delivery of a negative polarity pulse generated immediately after a positive polarity pulse. This phenomenon is known as "bipolar cancellation" and it was reported for a wide range of bipolar pulses with pulse duration from 2 ns to 900 ns. On the contrary, paired pulses, i.e., two identical pulses with the same polarity, increased traditional nsPEF outcomes. Herein, we propose a novel robust and flexible generator, based on the frozen-wave concept, able to produce a broad range of pulses with the duration of 10 ns and delay between 17 and 360 ns. Numerical simulations and experimental measurements were performed to fully characterize the proposed generator. YO-PROTM-1 uptake was investigated in the U87-MG human glioblastoma cell line as a marker of membrane permeabilization in response to 10 ns, 11.5MV/m nsPEF. Our results showed that bipolar cancellation occurred for delays of 0-30 ns and decreased as a function of the interphase interval. In addition, we observed that cellular response following the application of paired nsPEF was more than two-fold compared to the unipolar pulse response and was independent from the interphase interval.

Millimeter-wave pulsed heating in vitro: cell mortality and heat shock response.

Millimeter wave (MMW)-induced heating represents a promising alternative for non-invasive hyperthermia of superficial skin cancer, such as melanoma. Pulsed MMW-induced heating of tumors allows for reaching high peak temperatures without overheating surrounding tissues. Herein, for the first time, we evaluate apoptotic and heat shock responses of melanoma cells exposed in vitro to continuous (CW) or pulsed-wave (PW) amplitude-modulated MMW at 58.4 GHz with the same average temperature rise. Using an ad hoc exposure system, we generated 90 min pulse train with 1.5 s pulse duration, period of 20 s, amplitude of 10 °C, and steady-state temperature at the level of cells of 49.2 °C. The activation of Caspase-3 and phosphorylation of HSP27 were investigated using fluorescence microscopy to monitor the spatial variation of cellular response. Our results demonstrate that, under the considered exposure conditions, Caspase-3 activation was almost 5 times greater following PW exposure compared to CW. The relationship between the PW-induced cellular response and SAR-dependent temperature rise was non-linear. Phosphorylation of HSP27 was 58% stronger for PW compared to CW. It exhibits a plateau for the peak temperature ranging from 47.7 to 49.2 °C. Our results provide an insight into understanding of the cellular response to MMW-induced pulsed heating.

Millimeter-Wave Heating in In Vitro Studies: Effect of Convection in Continuous and Pulse-Modulated Regimes.

Shallow penetration of millimeter waves (MMW) and non-uniform illumination in in vitro experiments result in a non-uniform distribution of the specific absorption rate (SAR). These SAR gradients trigger convective currents in liquids affecting transient and steady-state temperature distributions. We analyzed the effect of convection on temperature dynamics during MMW exposure in continuous-wave (CW) and pulsed-wave (PW) amplitude-modulated regimes using micro-thermocouples. Temperature rise kinetics are characterized by the occurrence of a temperature peak that shifts to shorter times as the SAR of the MMW exposure increases and precedes initiation of convection in bulk. Furthermore, we demonstrate that the liquid volume impacts convection. Increasing the volume results in earlier triggering of convection and in a greater cooling rate after the end of the exposure. In PW regimes, convection strongly depends on the pulse duration that affects the heat pulse amplitude and cooling rate. The latter results in a change of the average temperature in PW regime. Bioelectromagnetics. 2019;40:553-568. © 2019 Bioelectromagnetics Society.

Decreased spontaneous electrical activity in neuronal networks exposed to radiofrequency 1,800 MHz signals.

The rapid development of wireless communications has raised questions about their potential health risks. So far, the only identified biological effects of radiofrequency fields (RF) are known to be caused by heating, but the issue of potential nonthermal biological effects, especially on the central nervous system (CNS), remains open. We previously reported a decrease in the firing and bursting rates of neuronal cultures exposed to a Global System for Mobile (GSM) RF field at 1,800 MHz for 3 min (Moretti D, Garenne A, Haro E, Poulleier de Gannes F, Lagroye I, Lévêque P, Veyret B, Lewis N. Bioelectromagnetics 34: 571-578, 2013). The aim of the present work was to assess the dose-response relationship for this effect and also to identify a potential differential response elicited by pulse-modulated GSM and continuous-wave (CW) RF fields. Spontaneous bursting activity of neuronal cultures from rat embryonic cortices was recorded using 60-electrode multielectrode arrays (MEAs). At 17-28 days in vitro, the neuronal cultures were subjected to 15-min RF exposures, at specific absorption rates (SAR) ranging from 0.01 to 9.2 W/kg. Both GSM and CW signals elicited a clear decrease in bursting rate during the RF exposure phase. This effect became more marked with increasing SAR and lasted even beyond the end of exposure for the highest SAR levels. Moreover, the amplitude of the effect was greater with the GSM signal. Altogether, our experimental findings provide evidence for dose-dependent effects of RF signals on the bursting rate of neuronal cultures and suggest that part of the mechanism is nonthermal. NEW & NOTEWORTHY In this study, we investigated the effects of some radiofrequency (RF) exposure parameters on the electrical activity of neuronal cultures. We detected a clear decrease in bursting activity, dependent on exposure duration. The amplitude of this effect increased with the specific absorption rate (SAR) level and was greater with Global System for Mobile signal than with continuous-wave signal, at the same average SAR. Our experiment provides unique evidence of a decrease in electrical activity of cortical neuronal cultures during RF exposure.