Assessment of genetic effects on human fibroblasts induced by in vitro exposure to continuous and pulsed non-ionizing electromagnetic signals

Abstract

The human exposure to non-ionizing electromagnetic field in particular radio frequencies (RF) (3 kHz - 300 GHz), is growing over the last decades with the concomitant development of technological tools from both domestic and industrial uses, raising questions about their possible impact on human health. The most widely accepted RF mechanism of interaction with biological system is thermal increase caused by the excitation of electrons to a higher vibrational/rotatory energy state when passing through matter. Currently, safety standards for limiting human exposures to RF-EMF have been established on these effects. To date, based on the limited evidence of an association between mobile phone use and cancer in both human and animal investigations, the International Agency for Research on Cancer (IARC), classified RF exposure as a possible human carcinogen (class 2B). Several studies have been performed to investigate the biological and health non-thermal effects of RF-EMF, in particular many researchers evaluated the in vitro genotoxicity of these radiation, but the results are still controversial. Results reported in literature are very heterogeneous with some studies reporting direct DNA damage, indirect genotoxic effects and other showing no effect, giving no consistent or convincing evidence of a causal relation between exposure and any adverse health effect. An open question is related to possible different biological effects induced by signal modulation that occurs in a wide variety of RF applications (radar, wireless communications, broadcast communications and industrial processes). Few studies comparing the effects of continuous and modulated wave signals have been performed with no clear results, consequently modulation is a parameter poorly considered in most guidelines for limiting human exposure to RF-EMF. A new interesting field of research on biological response to RF radiation is related to the identification of sensitive genes which could modulate their expression profile after exposures. To date few studies have been performed on this topic with no clear results. Overall, the controversial outcomes from the studies reported in literature that often make difficult comparing the results obtained in the different laboratories, could mainly be related to heterogeneous exposure systems, different cellular models, frequencies, SAR, exposure times considered and insufficient number of experimental replicates. Moreover, many of the studies evaluating gene expression modulation suffers of methodological limitation related to the approach used (microarrays) and lack of validation through RT-PCR. In the light of these observations, the goal of this Ph.D. project was to perform a good quality in vitro investigation on the potential RF biological effects through a well designed experimental study and using a properly characterized exposure system. We chose as frequency of interest 2.45 GHz, because the widespread use of Wi-Fi technologies in everyday life is leading to concerns about their possible health consequences. The selected SAR value for these experiments was 2W/kg, which is the maximum value recommended by the guidelines for limiting human exposures to RF-EMF. Moreover, because of the growing interest on possible different biological effects related to signal modulation, the present study focused on the effects of 2.45 GHz with both continuous (CW) and pulsed (PW) signals. Since one of the most critical point in these studies is related to inaccurate dosimetry and uncharacterized exposure source and conditions, in this project particular time and emphasis was addressed to identify the appropriate exposure system. Comparing different design solutions, the system that better satisfied our research requirements was a WPC-based exposure system. During the set-up of the system, particular attention was paid to ensure that RF-energy was deposited in a homogeneous manner within the sample, avoiding the temperature rise in some part of the Petri dishes with the formation of “hot spots”. In order to evaluate the non-thermal effects of the studied frequency, before the in vitro experimental exposures a series of calibration measurements were performed to monitoring the temperature distribution inside the sample area using a fluoroptic thermometer, during the 2 hours of exposure. Since a temperature increase of 0.7°C was observed, an active cooling with forced air was used to reduce the temperature with a maximum thermal increase of 0.25°C. Moreover, to maintain cells under appropriate conditions (37°C, 5% CO2) the in vitro exposures were performed inside the incubator. We used as in vitro cellular model human dermal fibroblasts (HDF) and in order to obtain robust scientific results, several methodologies and at least three experimental replicates for end-points and type of exposures were performed. To evaluate chromosomal damage and to identify the clastogenic or aneugenic origin, CREST micronuclei test was carried out, while DNA double strand breaks (DSBs) were identified by γ-H2AX/53BP1 foci assay. Moreover the potential genotoxicity of the selected frequency was evaluated with cell cycle analysis, since cell cycle arrest is a consequence of DNA damage. As expected, the results from these end-points suggest that 2.45 GHz did not induce genotoxic effects, neither aneugenic nor clastogenic at the SAR exposure limits recommended by European guidelines for limiting the exposures to RF-EMF. Moreover, no significant differences between the two type of signal tested (CW and PW) were detected. This research is relevant especially with regards to the gene expression analysis, performed for the first time in human cells in vitro exposed to RF by the high-throughput RNA sequencing approach. The results of this analysis showed no evidence of altered gene expression profile in exposed fibroblasts, except one, with FDR-adjusted statistical analysis in CW exposed samples. However, using a less stringency statistical approach several genes with different expression profiles were detected. Among these genes no pathways seems to be particularly affected, but interestingly some cytoskeleton-related genes were identified. Nevertheless their biological response seems to be transitory, appearing differentially expressed only 2 hours after exposure, suggesting no effect on protein translation process. This consideration seems to be indirectly supported by the ultrastructural analysis, that showed no morphological changes in the polymerization of actin filaments in exposed cells, suggesting that proteins involved in the cytoskeletal structure may not be affected under the exposure conditions evaluated in this study. In conclusion the results of this Ph.D. project, based on well characterized exposure system and evaluating the potential biological effect using a multiparametric methodology approach, enhanced by the high-throughput NGS sequencing technology, aims at strengthening the scientific knowledge on the potential adverse effects of 2.45 GHz exposure. Moreover, it could be a procedure model for future researches on biological effects of non-ionizing electromagnetic radiation, which are essential for updated the guidelines for limiting RF exposures.