Radiofrequency fields at 2.45 GHz reprogram mitochondria–lysosome crosstalk and modulate the survival/death of macrophages exposed to LPS and/or the SARS-CoV-2 Spike Protein.

Macrophages are a key component of the innate immune response and are sensitive to oxidative stress. However, the ability of non-ionizing radiofrequency electromagnetic fields (RF-EMFs) to alter the immune response to bacterial or viral stimuli remains largely unexplored. Bacterial lipopolysaccharide (LPS) activates the production of nitric oxide (NO) via the TLR4 receptor, which regulates mitochondria, lysosomes, and cell survival. The SARS-CoV-2 spike protein (CSP) can amplify these same signaling pathways. Since communication between mitochondria and lysosomes involves ROS and RNS signaling pathways, as well as degradation processes, these organelles are considered sensitive receptors of combined radiofrequency radiation and inflammatory stimuli in macrophages. This study examines the impact of exposure to 2.45 GHz RF radiation and LPS/CSP proteins on RAW 264.7 macrophages.

The RAW 264.7 murine macrophage cell line was exposed to unmodulated 2.45 GHz radiation in a GTEM chamber for 6, 24, or 48 hours. The temperature was maintained at 37 ± 0.2°C to prevent thermal artifacts. The mean specific absorption rate (SAR) was 0.174 W/kg, corresponding to a field strength of 102.8 V/m and a power density of 28 W/m². During this time, the cells were treated with LPS, CSP, or both (hereafter referred to as "treated cells," as opposed to "exposed cells;" editor’s note). The following parameters were analyzed: cell viability (using trypan blue and propidium iodide), lysosomal and mitochondrial activity (using fluorometry), nitric oxide (NO) production (using the Griess reaction), and the levels of apoptotic and necrotic cells. Statistical analysis was performed using two-factor ANOVA with Bonferroni or Holm–Sidak post hoc test.

After 6 hours of exposure, all three viability assays demonstrated a consistent reduction in viability in the untreated controls (including one trypan blue assay and two propidium iodide assays. The propidium iodide assay was performed in parallel with the lysosomal and mitochondrial activity assays, editor’s note.) After 24 hours, a treatment-dependent effect was observed in the trypan blue assay. The combination of CSP and LPS reduced viability compared to the control group (CSP+LPS sham-exposed). However, after 48 hours, this effect reversed under identical treatment conditions. The viability of the exposed CSP+LPS cells was significantly higher than that of the sham-exposed CSP+LPS cells, whose viability plummeted. Initially, exposure had a damaging effect, but then it had a protective effect. Nitric oxide production relative to cell count increased significantly after 24 hours in the LPS and CSP+LPS groups compared to the untreated control group. A field-dependent effect was observed only in the LPS group. The exposed group produced significantly more nitric oxide than the non-exposed group. This pattern changed after 48 hours. In all treated and exposed groups, nitric oxide production decreased significantly. Lysosomal activity increased sharply and significantly in all groups after 6 hours of exposure, regardless of treatment. The lysosomal inhibitor blocked exposed and sham-exposed cells equally, confirming that the effect of exposure is specific. Mitochondrial activity decreased significantly only in the LPS group after 6 hours. After 24 hours, mitochondrial activity decreased across all groups, regardless of exposure. After 24 hours, exposure provided statistically significant protection against apoptosis in all three treatment groups. (As shown by the bar charts, the number of apoptotic cells decreased by at least half, indicating an exceptional level of protection; editor’s note). A similar trend was observed for necrosis after 24 hours. However, RF radiation demonstrated a statistically significant protective effect only in the LPS group. After 48 hours, the effect of the field was inconsistent. In some cases, the number of necrotic cells increased to over 60%, occurring independently of treatment and unrelated to RF exposure.

The authors concluded that exposure to 2.45 GHz does not act as a non-specific toxin but rather reprograms the interaction between mitochondria, lysosomes, nitric oxide (NO), and cell death (apoptosis and necrosis) in a time- and stimulus-dependent manner. They also found that a drop in NO levels after 48 hours indicates an overwhelmed antioxidant defense system due to the interaction between electromagnetic fields (EMFs) and inflammatory stimuli. The shift in thresholds at mitochondrial and lysosomal checkpoints, which initially favors apoptosis and later necrosis, demonstrates that RF-EMFs influence the life-or-death decision of the immune cell lines rather than directly damaging the cells.

Editor’s note:

This study stands out due to its carefully controlled exposure system, defined dosimetry in the GTEM chamber, and active thermoregulation. These features eliminate any room for objections regarding thermal effects. Furthermore, the use of two orthogonal viability assays and several functional markers, measured at three time points in some cases, is noteworthy. The authors' key finding – that exposure modulates the cells' response in conjunction with the inflammatory stimulus rather than non-specifically killing cells – is supported by the increase in lysosomal activity after 6 hours and the reversal of the vitality trend during CSP+LPS co-treatment (damage after 24 hours and protection after 48 hours). The authors describe this phenomenon as "time- and stimulus-dependent," yet they do not propose a mechanism of action. The nitric oxide analysis replicated the scientists' 2020 results [1], showing maximum NO production after 24 hours. These results were reported in ElektrosmogReport 01/2021. The results suggest that RF radiation can affect nitric oxide signaling pathways, as other scientists have previously postulated [2]. However, the reference to mitochondrial activity is overstated because only one significant field effect is documented. Another interpretive error concerns the early vitality data. The selective loss of vitality described is invalidated by the fact that all untreated controls (not exposed to bacterial or viral stressors) are also affected. The most relevant finding for the debate on non-thermal interactions is that exposure to 2.45 GHz EMFs can interfere with the cellular survival response. (RH)

1.        Sueiro-Benavides RA, Leiro-Vidal JM, Salas-Sánchez AÁ, Rodríguez-Gonzalez JA, Ares-Pena FJ, Martín MEL (2021). Radiofrequency at 2.45GHz increases toxicity, pro-inflammatory and pre-apoptotic activity caused by black carbon in the RAW 264.7 macrophage cell line. Science of the Total Environment, 765, 142681. https://doi.org/10.1016/j.scitotenv.2020.142681

2.        Pall ML (2018). Wi-Fi is an important threat to human health. Environmental Research, 164, 405–16. https://doi.org/10.1016/j.envres.2018.01.035