Intestinal microbiota via NLRP3 inflammasome dependent neuronal pyroptosis mediates anxiety-like behaviour in mice exposed to 3.5 GHz radiofrequency radiation.

The rapid development of 5G communication technology has increased public concern about the potential adverse effects on human health. Till now, the impacts of radiofrequency radiation (RFR) from 5G communication on the central nervous system and gut-brain axis are still unclear. Therefore, we investigated the effects of 3.5 GHz (a frequency commonly used in 5G communication) RFR on neurobehavior, gut microbiota, and gut-brain axis metabolites in mice. The results showed that exposure to 3.5 GHz RFR at 50 W/m2 for 1 h over 35 d induced anxiety-like behaviour in mice, accompanied by NLRP3-dependent neuronal pyroptosis in CA3 region of the dorsal hippocampus. In addition, the microbial composition was widely divergent between the sham and RFR groups. 3.5 GHz RFR also caused changes in metabolites of feces, serum, and brain. The differential metabolites were mainly enriched in glycerophospholipid metabolism, tryptophan metabolism, and arginine biosynthesis. Further correlation analysis showed that gut microbiota dysbiosis was associated with differential metabolites. Based on the above results, we speculate that dysfunctional intestinal flora and metabolites may be involved in RFR-induced anxiety-like behaviour in mice through neuronal pyroptosis in the brain. The findings provide novel insights into the mechanism of 5G RFR-induced neurotoxicity.

Effects of radiofrequency field from 5G communication on fecal microbiome and metabolome profiles in mice.

With the rapid development of 5G networks, the influence of the radiofrequency field (RF) generated from 5G communication equipment on human health is drawing increasing attention in public. The study aimed at assessing the effects of long-term exposure to 4.9 GHz (one of the working frequencies of 5G communication) RF field on fecal microbiome and metabolome profiles in adult male C57BL/6 mice. The animals were divided into Sham group and radiofrequency group (RF group). For RF group, the mice were whole body exposed to 4.9 GHz RF field for three weeks, 1 h/d, at average power density (PD) of 50 W/m2. After RF exposure, the mice fecal samples were collected to detect gut microorganisms and metabolites by 16S rRNA gene sequencing and LC-MS method, respectively. The results showed that intestinal microbial compositions were altered in RF group, as evidenced by reduced microbial diversity and changed microbial community distribution. Metabolomics profiling identified 258 significantly differentially abundant metabolites in RF group, 57 of which can be classified to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Besides, functional correlation analysis showed that changes in gut microbiota genera were significantly correlated with changes in fecal metabolites. In summary, the results suggested that altered gut microbiota and metabolic profile are associated with 4.9 GHz radiofrequency exposure.

Effects of X-ray cranial irradiation on metabolomics and intestinal flora in mice.

Cranial radiotherapy is an important treatment for intracranial and head and neck tumors. To investigate the effects of cranial irradiation (C-irradiation) on gut microbiota and metabolomic profile, the feces, plasma and cerebral cortex were isolated after exposing mice to cranial X-ray irradiation at a dose rate of 2.33 Gy/min (5 Gy/d for 4 d consecutively). The gut microorganisms and metabolites were detected by 16 S rRNA gene sequencing method and LC-MS method, respectively. We found that compared with sham group, the gut microbiota composition changed at 2 W and 4 W after C-irradiation at the genus level. The fecal metabolomics showed that compared with Sham group, 44 and 66 differential metabolites were found to be annotated into metabolism pathways at 2 W and 4 W after C-irradiation, which were significantly enriched in the arginine and proline metabolism. Metabolome analysis of serum and cerebral cortex showed that, at 4 W after C-irradiation, the expression pattern of metabolites in serum samples of mice was similar to that of sham group, and the cerebral cortex metabolites of the two groups were completely separated. KEGG functional analysis showed that serum and brain tissue differential metabolites were respectively enriched in tryptophan metabolism, and arginine proline metabolism. The correlation analysis showed that the changes of gut microbiota genera were significantly correlated with the changes of metabolism, especially Helicobacter, which was significantly correlated with many different metabolites at 4 W after C-irradiation. These data suggested that C-irradiation could affect the gut microbiota and metabolism profile, even at relatively long times after C-irradiation.

Quantitative proteomics reveals effects of environmental radiofrequency electromagnetic fields on embryonic neural stem cells.

The effects of environmental radiofrequency electromagnetic fields (RF-EMF) on embryonic neural stem cells have not been determined, particularly at the proteomic level. This study aims to elucidate the effects of environmental levels of RF-EMF radiation on embryonic neural stem cells. Neuroectodermal stem cells (NE-4C cells) were randomly divided into a sham group and an RF group, which were sham-exposed and continuously exposed to a 1950 MHz RF-EMF at 2 W/kg for 48 h. After exposure, cell proliferation was determined by a Cell Counting Kit-8 (CCK8) assay, the cell cycle distribution and apoptosis were measured by flow cytometry, protein abundance was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and mRNA expression was evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). We did not detect differences in cell proliferation, cell cycle distribution, and apoptosis between the two groups. However, we detected differences in the abundance of 23 proteins between the two groups, and some of these differences were consistent with alterations in transcript levels determined by qRT-PCR (P < 0.05). A bioinformatics analysis indicated that the differentially regulated proteins were mainly enriched in 'localization' in the cellular process category; however, no significant pathway alterations in NE-4C cells were detected. We conclude that under the experimental conditions, low-level RF-EMF exposure was not neurotoxic but could induce minor changes in the abundance of some proteins involved in neurodevelopment or brain function.

Combined effects of EMP and RF field on emotional behavior in mice.

Background: Recently, concerns about the combined effects of electromagnetic field (EMF) in daily living and occupational environment are rapidly growing. Methods: In this study, we investigated the combined effects of 1-week exposure to electromagnetic pulse (EMP) at 650 kV/m for 1,000 pulses and 4.9 GHz radiofrequency (RF) at 50 W/m2 for 1 h/d in male mice. Open field test, tail suspension test and Y-maze were applied to evaluate anxiety, depression-like behaviors and spatial memory ability, respectively. Results: It was found that compared with Sham group, combined exposure to EMP and RF induced anxiety-like behavior, increased the level of serum S100B and decreased the level of serum 5-HT. The results of quantitative proteomic and KEGG analysis showed that the differentially expressed proteins in hippocampus were enriched in Glutamatergic and GABAergic synapse after combined exposure group, which were verified by western blot. In addition, an obvious histological alteration and autophagy-associated cell death were observed in amygdala instead of hippocampus after combined exposure to EMP and 4.9 GHz RF. Conclusion: Combined exposure to EMP and 4.9 GHz RF could induce emotional behavior alteration, which might be associated with Glutamatergic and GABAergic synapse system of hippocampus and autophagy in amygdala.

Effects of radiofrequency field from 5G communications on the spatial memory and emotionality in mice.

The rapid development of 5G network technology has gained much popularity as well as concerns about its adverse effects. In this study, we investigated the effects of 4.9 GHz (one of working frequencies of 5G communication) radiofrequency (RF) field on emotional behaviours and spatial memory in adult male mice. Open field test (OFT), tail suspension test (TST) and Y maze were used to evaluate anxiety, depression-like behaviour and spatial memory ability, respectively. It was found that the anxiety-like behaviour and spatial memory ability of mice did not change, but the depression-like behaviour was induced in mice after 4.9 GHz RF exposure. In addition, the number of neurons significantly reduced and the level of pyroptosis obviously increased in amygdala rather than hippocampus. These results suggested that 4.9 GHz RF exposure could induce depression-like behaviour, which might be associated with the neuronal pyroptosis in amygdala.

Abscopal effects of thoracic X-ray radiation on spermatogenesis in mice.

The study aimed to elucidate abscopal effects of thoracic X-ray irradiation on spermatogenesis in mice. Male C57BL/6 mice were randomly divided into sham group and radiation group, and subjected to thorax fractionated X-ray irradiation or sham irradiation with the total dose of 5 Gy/day for each animal for four consecutive days. After irradiation, sperm morphology was observed, and sperm number was counted under microscope, and sperm apoptosis was detected by flow cytometry. Meanwhile, testis index was calculated, testicular morphology was observed using haematoxylin-eosin (HE) staining, and testicular ultrastructure was observed under transmission electron microscopy. The permeability of blood-testis barrier (BTB) was detected by Evans Blue fluorescence colorimetry. The protein levels of Bcl-2 associated X protein (Bax), B-cell leukemia-lymphoma-2 (Bcl-2) and Cleaved caspase 3, promyelocytic leukaemia zinc finger (PLZF) and c-kit proto-oncogene (c-kit) in testes were determined by western blotting (WB). The location of apoptotic cells was confirmed by terminal deoxynucleotidyl transferase (TdT) enzymaticated dUTP nick end labelling (TUNEL) assay. The levels of tumor necrosis factor alpha (TNF-α), transforming growth factor-ß1 (TGF-ß1), interleukin 10 (IL-10) were measured by enzyme-linked immunosorbent assay (ELISA). The levels of Total superoxide dismutase (T-SOD) and malondialdehyde (MDA) were measured by the biochemical assay kit. Compared with sham group, the sperm quality of mice in radiation group showed decreased number and survival rate, along with increased abnormality and total apoptosis rate. The testis index of irradiated mice was lower, the testicular apoptosis was increased, and their testicular histology and ultrastructure was severely damaged. The permeability of BTB was increased, the level of PLZF in testis was decreased, and the level of c-kit was increased by irradiation. After irradiation, the levels of TNF-α, TGF-ß1, IL-10, T-SOD and MDA in testes were significantly changed. Taken together, abscopal effects of thoracic X-ray irradiation on spermatogenesis were obvious, which could decrease sperm quality and damage testicular morphology and increase the permeability of BTB, and a series of inflammation and oxidative stress factors were involved in the process. These findings provide novel insights into prevention and treatment for male reproductive damage induced by clinical thoracic irradiation.

Effects of 5.8 GHz Microwaves on Testicular Structure and Function in Rats.

Objective: To investigate the effects of exposure to 5.8 GHz microwaves on testicular structure and function of male adult rats. Methods: After 30 days of exposure, we evaluated sperm quality by determining sperm concentration and quantifying the number of abnormal sperm. Testicular morphology was investigated by hematoxylin-eosin (HE) staining. The levels of testosterone (T), follicle-stimulating hormone (FSH), luteinizing hormone (LH), glial cell line-derived neurotrophic factor (GDNF), stem cell factor (SCF), and transferrin (TRF) were determined by enzyme-linked immunosorbent assays (ELISAs). We also used western blotting to determine the levels of GDNF and SCF and apoptosis-related protein (caspase-3) in the testis. Results: Compared with the sham group, there were no significant differences in terms of sperm count, sperm abnormality, and the levels of T, FSH, LH, GDNF, SCF, and caspase-3 in the microwave group. Conclusion: Under the experimental conditions, 5.8 GHz microwave exposure has no obvious effect on testicular structure and function of rats.

The preventive and therapeutic effect of repetitive transcranial magnetic stimulation on radiation-induced brain injury in mice.

PURPOSE: To clarify the preventive and therapeutic effects of repetitive transcranial magnetic stimulation (rTMS) on brain injury induced by X-ray cranial irradiation, preliminarily identify the mechanism and provide a novel clinical approach for the prevention and treatment of radiation-induced brain injury (RBI). MATERIALS AND METHODS: Male C57BL/6 mice were randomly divided into the sham group, large fractionated dose (5 Gy × 4 d) group, large fractionated dose + rTMS (5 Gy × 4 d + rTMS) group, conventional fractionated dose (2 Gy × 10 d) group and conventional fractionated dose + rTMS (2 Gy × 10 d + rTMS) group. After cranial irradiation and rTMS, behavioral experiments, morphological staining and molecular biology experiments were performed. We further determined the mechanism of rTMS on the prevention and treatment of RBI, including changes in hippocampal neuronal apoptosis, neural stem cell (NSC) proliferation and differentiation, and neuronal synaptic plasticity. RESULTS: rTMS alleviated the negative effects of cranial radiation on the general health of mice and promoted their recovery. rTMS ameliorated the impairment of spatial learning and memory induced by cranial radiation, and this beneficial effect was more robust in the conventional fractionated dose group than the large fractionated dose group. Moreover, rTMS alleviated the alterations in hippocampal structure and neuronal death and had preventive and therapeutic effects against RBI. In addition, rTMS reduced hippocampal cell apoptosis, promoted NSC proliferation and differentiation in the hippocampus after cranial irradiation, and enhanced neuronal synaptic plasticity in the hippocampus. Subsequent studies showed that rTMS upregulated the expression of learning- and memory-related proteins. CONCLUSION: rTMS could alleviate learning and memory impairment caused by RBI, and the preventive and therapeutic effects of rTMS were better for the conventional fraction radiation paradigms.

The Abscopal Effects of Cranial Irradiation Induce Testicular Damage in Mice.

To investigate whether the abscopal effects of cranial irradiation (C-irradiation) cause testicular damage in mice, male C57BL/6 mice (9weeks of age) were randomly divided into a sham irradiation group, a shielded group and a C-irradiation group and administered sham/shielded irradiation or C-irradiation at a dose rate of 2.33Gy/min (5Gy/d for 4 d consecutively). All mice were sacrificed at 4weeks after C-irradiation. We calculated the testis index, observed testicular histology by haematoxylin-eosin (HE) staining and observed testicular ultrastructure by transmission electron microscopy. Western blotting was used to determine the protein levels of Bax, Bcl-2, Cleaved caspase 3, glial cell line-derived neurotrophic factor (GDNF) and stem cell factor (SCF) in the testes of mice. Immunofluorescence staining was performed to detect the expression of Cleaved caspase 3 and 3ß hydroxysteroid dehydrogenase (3ßHSD), and a TUNEL assay was used to confirm the location of apoptotic cells. The levels of testosterone (T), GDNF and SCF were measured by ELISA. We also evaluated the sperm quality in the cauda epididymides by measuring the sperm count, abnormality, survival rate and apoptosis rate. The results showed that there was no significant difference in testicular histology, ultrastructure or sperm quality between the shielded group and sham group. Compared with the sham/shielded group, the C-irradiation group exhibited a lower testis index and severely damaged testicular histology and ultrastructure at 4weeks after C-irradiation. The levels of apoptosis in the testes increased markedly in the C-irradiation group, especially in spermatogonial stem cells. The levels of serum T and testicular 3ßHSD did not obviously differ between the sham group and the C-irradiation group, but the levels of GDNF and SCF in the testes increased in the C-irradiation group, compared with the sham group. In addition, the sperm count and survival rate decreased in the C-irradiation group, while the abnormality and apoptosis rate increased. Under these experimental conditions, the abscopal effects of C-irradiation induced testicular damage with regard to both structure and function and ultimately decreased sperm quality in mice. These findings provide novel insights into prevention and treatment targets for male reproductive damage induced by C-irradiation.

Effects of pulsed electromagnetic fields on tumor cell viability: a meta-analysis of in vitro randomized controlled experiments.

Malignant tumor treatment remains a big challenge till now, and expanding literature indicated that pulsed electromagnetic fields (PEMF) is promising in tumor treatment with the advantage of safety and being economical, but it is still controversial on whether PEMF could affect the tumor cell viability. Therefore, we conducted the meta-analysis to evaluate effects of PEMF on tumor cell viability. The PubMed, EMBASE, Web of Science, and Cochrane Library databases were searched for studies published up to February 2021. Studies on the direct effects of PEMF on tumor cell viability, determined using colorimetric analysis, were included. Two authors extracted the data and completed the quality assessment. A meta-analysis was performed to calculate the absorbance values and 95% confidence intervals (CIs) using random-effects models. Seven studies, including 32 randomized controlled experiments, were analyzed. Compared with the control group, tumor cell viability in the PEMF exposure group was obviously lower (SMD, -0.67; 95% CI: -1.12 to -0.22). The subgroup meta-analysis results showed that PEMF significantly reduced epithelial cancer cell viability (SMD, -0.58; 95% CI: -0.92 to -0.23) but had no influence on stromal tumor cell viability (SMD, -0.93; 95% CI: -0.21 to 0.15). Our study demonstrated that PEMF could inhibit tumor cell proliferation to some extent, but the risk of bias and high heterogeneity (I2 > 75%) weakened the strength of the conclusions drawn from the analysis.