Human Fibroblasts In Vitro Exposed to 2.45 GHz Continuous and Pulsed Wave Signals: Evaluation of Biological Effects with a Multimethodological Approach.

The increasing exposure to radiofrequency electromagnetic fields (RF-EMF), especially from wireless communication devices, raises questions about their possible adverse health effects. So far, several in vitro studies evaluating RF-EMF genotoxic and cytotoxic non-thermal effects have reported contradictory results that could be mainly due to inadequate experimental design and lack of well-characterized exposure systems and conditions. Moreover, a topic poorly investigated is related to signal modulation induced by electromagnetic fields. The aim of this study was to perform an analysis of the potential non-thermal biological effects induced by 2.45 GHz exposures through a characterized exposure system and a multimethodological approach. Human fibroblasts were exposed to continuous (CW) and pulsed (PW) signals for 2 h in a wire patch cell-based exposure system at the specific absorption rate (SAR) of 0.7 W/kg. The evaluation of the potential biological effects was carried out through a multimethodological approach, including classical biological markers (genotoxic, cell cycle, and ultrastructural) and the evaluation of gene expression profile through the powerful high-throughput next generation sequencing (NGS) RNA sequencing (RNA-seq) approach. Our results suggest that 2.45 GHz radiofrequency fields did not induce significant biological effects at a cellular or molecular level for the evaluated exposure parameters and conditions.

Study of genotoxic and cytotoxic effects induced in human fibroblasts by exposure to pulsed and continuous 1.6 GHz radiofrequency.

Background: The widespread use of radiofrequency (RF) sources, ranging from household appliances to telecommunications devices and military equipment, raises concerns among people and regulatory agencies about the potential health risks of RF exposure. Consequently, several in vitro and in vivo studies have been done to investigate the biological effects, in particular non-thermal, of this non-ionizing radiation. To date, this issue is still being debated due to the controversial results that have been reported. Furthermore, the impact of different RF signal modulations on biological systems remains poorly investigated. The present in vitro study aims to evaluate the cytotoxicity and genotoxicity of continuous or pulsed 1.6 GHz RF in human dermal fibroblasts (HDF). Methods: HDF cultures were exposed to continuous and pulsed 1.6 GHz RF, for 2 h, with Specific Absorption Rate (SAR) of 0.4 W/kg. The potential biological effects of 1.6 GHz RF on HDF were assessed with a multi-methodological approach, analyzing the effects on cell cycle, ultrastructure, protein expression, mitotic spindle, CREST stained micronuclei, chromosome segregation and γ-H2AX/53BP1 foci. Results: 1.6 GHz RF exposure modified proteins expression and morphology of HDF. Specifically, the expression of different heat-shock proteins (HSP) (i.e., HSP-90, HSP-60, and HSP-25) and phospho-AKT were affected. In addition, both continuous and pulsed RF modified the cytoskeletal organization in HDF and increased the number of lysosomes, while the formation of autophagosomes was observed only after pulsed RF exposure. Mitotic spindle anomalies were also found after exposure. However, no significant effect was observed on cell cycle, chromosome segregation, CREST-stained micronuclei and γ-H2AX/53BP1 foci. Conclusion: The results of the present study show the absence of genotoxic damage in 1.6 GHz RF exposed HDF and, although mitotic spindle alterations were observed, they did not have an aneugenic effect. On the other hand, changes in some proteins expression and cell ultrastructure in exposed HDF suggest that RF can potentially induce cell alterations at the morphological and molecular levels.

Characterization of Primary Human Dermal Fibroblasts to Ensure for Instance EMF Exposure Experiments under Comparable Cell Culture Condition.

HDFa (human dermal fibroblasts) are used as cellular models for EMF exposure. To ensure reproducible in vitro experiments, comparable proliferation and differentiation cell conditions must exist, and different donors, passage numbers, culture time, and growth media must be considered. In this study, the authors cultured fibroblasts in DMEM or 106 medium. Growth curves, vitality, morphology, and gene expression of genes coding for proliferation (PCNA, CDKN2A, CDKN1A, SFN), differentiation (PDGFRA, TGM2, ACTA2, PDPN, NTN1, MGP, PPP1R14), and SFN target genes (TP63, MMP1, MMP3) were examined in both media and passage numbers 3-4, 5-6 and >6. At passages 3-4, proliferating cells can be observed in both media. While cells cultured in DMEM proliferate over the passages, from passage 5, cells in 106 medium persisted around the seeded number. TGM2 down-regulation over all passages in both media and cells morphology suggest papillary-type fibroblasts. Downregulation of SFN (negative regulator of mitotic translation and cell differentiation) coincided with proliferating fibroblasts over all examined conditions. Downstream SFN target genes in proliferating cells appeared upregulated (TP63) and downregulated (MMP1/MMP3), suggestive for a status characterized by increased stemnesses (upregulated TP63) and wound healing capacity (downregulated MMP1, MMP3). Resting cells (SFN control values) were associated with control values of TP63 and MMP1/MMP3 expression, suggesting a reduced stemness and wound healing capacity. In conclusion, a set of markers related to proliferation (SFN), differentiation (TGM2), stemnesses (TP63), and wound healing (MMP1/MMP3) allow a culture characterization so that cells under two different conditions can be exposed, thus enabling reproducible EMF experiments or experiments with other exposures.

Bringing Radiation Exposures and Associated Health Risks into Perspective-Development of an App.

The NATO HFM 291 research task group (RTG) on "Ionizing Radiation Bioeffects and Countermeasures" represents a group of scientists from military and civilian academic and scientific institutions primarily working in the field of radiobiology. Among other tasks, the RTG intends to extend their work on risk estimation and communication to bridge the gap in appropriate judgment of health risks given a certain radiation exposure. The group has no explicit psychological background but an expertise in radiobiology and risk assessment. The group believes that, as one of the essential first steps in risk communication, it is required to put radiation risk into perspective. Radiation risk requires a weight in comparison to already-known risks. What we envision is to Compare Radiation exposure-associated health Risks (CRRis App) with daily life health risks caused by other common exposures such as cigarette smoking, driving a car, etc. Within this paper, we provide (1) an overview of health risks after radiation exposure, (2) an explanation of the task and concept of an envisioned CRRis App, (3) an overview of existing software tools related to this issue, (4) a summary of inputs and discussions with experts in the field of radiation protection and risk communication during the ConRad conference, and finally, (5) identification of the next steps in the development of the App.

Study of the effects of 0.15 terahertz radiation on genome integrity of adult fibroblasts.

The applications of Terahertz (THz) technologies have significantly developed in recent years, and the complete understanding of the biological effects of exposure to THz radiation is becoming increasingly important. In a previous study, we found that THz radiation induced genomic damage in fetal fibroblasts. Although these cells demonstrated to be a useful model, exposure of human foetuses to THz radiation is highly improbable. Conversely, THz irradiation of adult dermal tissues is cause of possible concern for some professional and nonprofessional categories. Therefore, we extended our study to the investigation of the effects of THz radiation on adult fibroblasts (HDF). In this work, the effects of THz exposure on HDF cells genome integrity, cell cycle, cytological ultrastructure and proteins expression were assessed. Results of centromere-negative micronuclei frequencies, phosphorylation of H2AX histone, and telomere length modulation indicated no induction of DNA damage. Concordantly, no changes in the expression of proteins associated with DNA damage sensing and repair were detected. Conversely, our results showed an increase of centromere-positive micronuclei frequencies and chromosomal nondisjunction events, indicating induction of aneuploidy. Therefore, our results indicate that THz radiation exposure may affect genome integrity through aneugenic effects, and not by DNA breakage. Our findings are compared to published studies, and possible biophysical mechanisms are discussed. Environ. Mol. Mutagen. 59:476-487, 2018. © 2018 Wiley Periodicals, Inc.

Gene Expression Analysis in Human Peripheral Blood Cells after 900 MHz RF-EMF Short-Term Exposure.

Radiofrequency electromagnetic fields (RF-EMF) are a basic requirement of modern wireless communication technology. Statutory thresholds of RF-EMF are established to limit relevant additional heat supply in human tissue. Nevertheless, to date, questions concerning nonthermal biological effects have yet to be fully addressed. New versions of microarrays (8 × 60K v2) provide a higher resolution of whole genome gene expression to display adaptive processes in cells after irradiation. In this ex vivo/ in vitro study, we irradiated peripheral blood cells from five donors with a continuous wave of 900 MHz RF-EMF for 0, 30, 60 and 90 min. Gene expression changes ( P ≤ 0.05 and ≥twofold differences above or below the room temperature control exposed samples) were evaluated with microarray analysis. The results were compared with data from room temperature + 2°C samples. Verification of microarray results was performed using bioinformatic analyses and qRT-PCR. We registered a lack of an EMF-specific gene expression response after applying the false discovery rate adjustment (FDR), using a high-stringency approach. Low-stringency analysis revealed 483 statistically significant deregulated transcripts in all RF-EMF groups relative to the room temperature exposed samples without an association with their corresponding room temperature + 2°C controls. Nevertheless, these transcripts must be regarded as statistical artefacts due to the absence of a targeted biological response, including enrichment and network analyses administered to microarray expressed gene subset profiles. Correspondingly, 14 most promising candidate transcripts examined by qRT-PCR displayed an absence of correlation with respect to the microarray results. In conclusion, these findings indicate that 900 MHz EMF exposure establishing an average specific absorption rate of 9.3 W/kg to whole blood cells is insufficient to induce nonthermal effects in gene expression during short-time exposure up to 90 min.

Genotoxic Effects in Human Fibroblasts Exposed to Microwave Radiation.

In the last decades, technological development has led to an increasing use of devices and systems based on microwave radiation. The increased employment of these devices has elicited questions about the potential long-term health consequences associated with microwave radiation exposure. From this perspective, biological effects of microwave radiation have been the focus of many studies, but the reported scientific data are unclear and contradictory. The aim of this study is to evaluate the potential genotoxic and cellular effects associated with in vitro exposure of human fetal and adult fibroblasts to microwave radiation at the frequency of 25 GHz. For this purpose, several genetic and biological end points were evaluated. Results obtained from comet assay, phosphorylation of H2AX histone, and antikinetochore antibody (CREST)-negative micronuclei frequency excluded direct DNA damage to human fetal and adult fibroblasts exposed to microwaves. No induction of apoptosis or changes in prosurvival signalling proteins were detected. Moreover, CREST analysis showed for both the cell lines an increase in the total number of micronuclei and centromere positive micronuclei in exposed samples, indicating aneuploidy induction due to chromosome loss.

Biological effects of in vitro THz radiation exposure in human foetal fibroblasts.

In recent years, terahertz (THz) radiation has been widely used in a variety of applications: medical, security, telecommunications and military areas. However, few data are available on the biological effects of this type of electromagnetic radiation and the reported results, using different genetic or cellular assays, are quite discordant. This multidisciplinary study focuses on potential genotoxic and cytotoxic effects, evaluated by several end-points, associated with THz radiation. For this purpose, in vitro exposure of human foetal fibroblasts to low frequency THz radiation (0.1-0.15THz) was performed using a Compact Free Electron Laser. We did not observe an induction of DNA damage evaluated by Comet assay, phosphorylation of H2AX histone or telomere length modulation. In addiction, no induction of apoptosis or changes in pro-survival signalling proteins were detected. Moreover, our results indicated an increase in the total number of micronuclei and centromere positive micronuclei induction evaluated by CREST analysis, indicating that THz radiation could induce aneugenic rather than clastogenic effects, probably leading to chromosome loss. Furthermore, an increase of actin polymerization observed by ultrastructural analysis after THz irradiation, supports the hypothesis that an abnormal assembly of spindle proteins could lead to the observed chromosomal malsegregation.

Dose estimation using dicentric chromosome assay and cytokinesis block micronucleus assay: comparison between manual and automated scoring in triage mode.

In cases of an accidental overexposure to ionizing radiation, it is essential to estimate the individual absorbed dose of a potentially radiation-exposed person. For this purpose, biological dosimetry can be performed to confirm, complement or even replace physical dosimetry when this proves to be unavailable. The most validated biodosimetry techniques for dose estimation are the dicentric chromosome assay, the "gold standard" for individual dose assessment, and cytokinesis-block micronucleus assay. However, both assays are time consuming and require skilled scorers. In case of large-scale accidents, different strategies have been developed to increase the throughput of cytogenetic service laboratories. These are the decrease of cell numbers to be scored for triage dosimetry; the automation of procedures including the scoring of, for example, aberrant chromosomes and micronuclei; and the establishment of laboratory networks in order to enable mutual assistance if necessary. In this study, the authors compared the accuracy of triage mode biodosimetry by dicentric chromosome analysis and the cytokinesis block micronucleus assay performing both the manual and the automated scoring mode. For dose estimation using dicentric chromosome assay of 10 blind samples irradiated up to 6.4 Gy of x-rays, a number of metaphase spreads were analyzed ranging from 20 up to 50 cells for the manual and from 20 up to 500 cells for the automatic scoring mode. For dose estimation based on the cytokinesis block micronucleus assay, the micronucleus frequency in both 100 and 200 binucleated cells was determined by manual and automatic scoring. The results of both assays and scoring modes were compared and analyzed considering the sensitivity, specificity, and accuracy of dose estimation with regard to the discrimination power of clinically relevant binary categories of exposure doses.