Biological Effects of 25 to 150 GHz Radiation After In Vitro Exposure of Human Fibroblasts: a Comparison of Experimental Results
In this paper, we present a comprehensive discussion of the results obtained after in vitro exposure of human fetal fibroblasts and human adult fibroblasts to pulsed radiation in a wide band between 100 and 150 GHz and to continuous wave radiation at 25 GHz. In order to assess potential effects of exposure, the genome integrity, cell cycle, cytological ultrastructure, and proteins expression were evaluated.
KeywordsMillimeter waves Terahertz Biological effects Non-ionizing radiation In vitro exposure Human primary cells Fibroblasts
1 Introduction and Motivation of the Study
Millimeter waves and terahertz radiation are playing an increasing role in biophysics, biology, biochemistry, and biomedicine for their potential use in therapeutic and diagnostic applications . Through the years, several issues have been addressed, from the understanding of the interaction mechanisms with biological systems to the study of millimeter waves/THz-induced effects , which has been systematically conducted during the last decade [3, 4]. In spite of this effort, the variety of radiation sources employed by different research groups worldwide  have also implied quite different characteristics in terms of the radiation spectral content and its temporal structure, which may in turn affect the response of biological systems.
A feature common to all studies on the interaction of millimeter waves/THz radiation with biological systems and on any potential human exposure is the strong absorption of water and the small penetration depth in hydrated tissues, which has gradually steered the research interest toward the investigation of effects on skin cells and peripheral blood.
Pioneering studies, carried out in the frame of the European project THz-BRIDGE , indeed addressed the exposure of human lymphocytes, membrane model systems, and epithelial cell cultures. Biological end-points were the alteration of membrane permeability of liposomes , the induction of genotoxicity in lymphocytes [8, 9], and the changes in cell activity, differentiation, and barrier function in keratinocytes and neural cells . Few years later, a study addressed the exposure of primary human skin fibroblasts (HDF) and human keratinocyte cell lines (HaCaT) . Another series of experiments addressed the determination of death thresholds in skin cells, and the identification of gene expression signatures .
In the frame of a collaboration between ENEA, the Scientific Department of Army Medical Center - Rome, the Department of Science University of “Roma Tre,” and the Department of Clinical Sciences and Translational Medicine - University of Rome “Tor Vergata”, a project was carried out to evaluate potential genotoxic effects associated with the “in vitro” exposure of living cells to high-frequency electromagnetic radiation, such as microwave and THz radiation. In this paper, we present a summary and a comprehensive discussion of the results obtained in the frame of this project, after in vitro exposures of human fetal fibroblasts (human fetal foreskin fibroblasts cell line - HFFF2)  and human adult fibroblasts (human dermal fibroblasts cell line - HDF)  to pulsed radiation in a wide band between 100 and 150 GHz and to continuous wave (CW) radiation at 25 GHz . The observed differences between the effects of pulsed versus CW exposure are discussed in this paper.
2 Radiation Sources
Two radiation sources were employed in this study: an undulator-based free-electron laser (ENEA Compact-FEL) covering the spectral range from 100 to 150 GHz, and a continuous wave (CW) solid-state source tunable in the frequency range 18–40 GHz.
The CW source is an yttrium iron garnet (YIG) oscillator (Microlambda Wireless, Inc. - MLOS-1840), providing 20 mW output power in the spectral range between 18 and 40 GHz. In this study, the source was operated at a fixed frequency of 25 GHz.
3 Biological Targets
Due to the shallow penetration depth of millimeter waves/THz radiation in hydrated tissues, the absorption during any potential human exposure is located in the skin, which is the primary tissue target of non-ionizing electromagnetic radiation [2, 17]. More specifically, fibroblasts are particularly important in exposure studies since they are the most common cells of connective tissue in animals. They synthesize the extracellular matrix and collagen and play a critical role in wound healing. We have therefore chosen human fibroblasts as a cellular model and have investigated biological effects on HFFF2 and HDF cells in order to evaluate possible different responses according to the different developmental ages.
Both HFFF2 and HDF were cultured in DMEM medium (Euroclone) supplemented with 10% fetal bovine serum (Euroclone), 1% 2 mM l-glutamine and 1% penicillin/streptomycin (Gibco). For adult cell cultures, the medium was supplemented also with 1% non-essential amino acids (Euroclone). Cell cultures were grown incubated at 37 °C in humidified atmosphere, at 5% CO2. Twenty-four hours before millimeter wave/THz exposure, cells were seeded into 6-cm-diameter polystyrene Petri dishes (Corning 3295), in 5 ml of medium at the density of 2–2.5 × 105 cells. The thickness of the culture medium in the Petri dish was measured to be 0.21 cm. Cell cultures were exposed for 20 min using the exposure setups described below.
4 Exposure Setup
In vitro exposures of both HFFF2 and HDF were performed with radiation pulses in the band 100–150 GHz using the Compact-FEL described above. In addition to this, in order to investigate effects at lower frequencies, of potential interest in telecommunications, a CW exposure layout was also set up employing the YIG source described above. Based on currently allotted frequencies for telecommunications, the frequency of 25 GHz was chosen within the tuning range of the YIG source.
Material properties at 25 GHz for HFSS and CST-MS analyses: dielectric constant εr; conductivity σ (S/m); loss tangent tan δ, density ρ (kg/m3)
3.8 × 107
4.24 × 10−4
1.2 × 10−4
Distilled water @ 24 °C
Distilled water @ 37 °C
An average specific absorption ratio (SAR) of about 20 mW/g was calculated according to the simple model described in . The corresponding distribution of the SAR on the sample, computed with the HFSS code assuming an input power of 20 mW at 25 GHz, is shown in Fig. 7b.
In both setups, for each irradiation experiment, an unexposed sample, subjected to the same environmental conditions apart from the impinging radiation, was placed in the same working area and defined as “sham” sample.
Temperature measurements were performed by using a FLIR camera for the 100–150 GHz setup . In the case of the 25 GHz setup, they were performed by means of a miniaturized thermocouple temperature probe (Fluke type K) placed inside a Petri dish with the wire orthogonal to the electric field of the incident radiation. During the chosen 20-min irradiation time, both the “exposed” and “sham” samples asymptotically approached the room temperature within a temperature variation of approximately 0.3 °C for the 100–150 GHz exposure and 0.6 °C for the 25 GHz setup. In this latter case, the increment turned out to be due to the heat dissipated by the YIG DC electrical power in the YIG source itself, which is inherently located near the exposure setup.
5 End-points and Biological Assays
In order to assess potential effects of millimeter wave/THz exposure, we evaluated the genome integrity, cell cycle, cytological ultrastructure, and proteins expression in HFFF2 and HDF cells after radiation exposure. For each end-point, at least three experimental replicates were performed.
To evaluate the effects of radiation on cell cycle distribution of asynchronized log phase growing HFFF2 and HDF cells, fluorescence-activated cell sorting (FACS) analysis of DNA content was performed. Cells were incubated with propidium iodide that stains DNA and analyzed by flow cytometry using a FACSCalibur cytometer running CellQuest software. The fluorescence intensity of the stained nuclei correlates with the amount of DNA they contain and permits to examine distribution of cells in the different phases of the cycle.
The possible direct DNA damage was investigated using the comet assay and the H2AX phosphorylation foci analysis. Alkaline and neutral variants of the comet assay were performed to detect respectively single- and double-strand DNA breaks. The damage has been estimated as percentage of DNA contained in the tail of the comet-like cells after electrophoresis [19, 20]. Furthermore, we analyzed one of the earliest markers of DNA double-strand breaks, the phosphorylation of the histone variant H2AX, the so-called γ-H2AX. This phosphorylated form can be visualized as discrete foci using specific antibodies with fluorescent tags .
In order to evaluate possible chromosome alterations, we performed the micronuclei (MN)-CREST assay. Micronuclei are small nuclei in the cytoplasm formed by acentric fragments or whole chromosomes not included in the two daughter nuclei during mitosis . The CREST immunofluorescent technique, marking the centromere of all chromosomes, allows to identify the origin of the micronucleus. The presence (CREST-positive MN) or absence (CREST-negative MN) of the fluorescent signal inside the micronucleus indicates respectively an entire chromosome or a fragment. In the cytoplasm, the presence of a positive MN micronucleus corresponds to a chromosome that was not incorporated into one of the two daughter nuclei during mitosis (chromosome loss) due to a failure of mitotic spindle or to complex chromosomal configurations during anaphase.
We further assessed chromosome malsegregation induction in the exposed cells during mitosis analyzing the distribution between the two daughter nuclei of three homologous pairs (chromosomes 4, 10, and 17) by using centromeric probes labeled with three different fluorescent dyes. This phenomenon consists in an unequal division of the number of chromosomes in the two daughter cells during mitosis with one of the daughter nuclei lacking a chromosome copy and the other with an extra copy. Malsegregation events are due to the failure of homologous chromosomes on the metaphase plate during cell division. Chromosome loss and chromosome malsegregation are the main mechanisms involved in the induction of aneuploidy, which defines the alteration of cell normal chromosomes number.
Since the telomere is a functional structure involved in chromosomal stability and correct chromosome segregation , we analyzed the telomere modulation by Q-FISH to assess its possible role in the observed chromosomal instability.
The expression of heat shock and pro-survival signaling proteins has been evaluated by Western blot analysis. This technique allows to identify a target protein recognized by a specific antibody from a complex mixture of proteins. The target protein is first separated by gel electrophoresis according to its molecular weight and then transferred to a nitrocellulose membrane. The immune complexes are visualized by chemiluminescence. The intensity of the specific band correlates with its amount.
Ultrastructural analysis was performed to detect morphological cell changes after radiation exposure. This analysis consists in the observation of ultrathin section of cells by using an electron microscopy to examine cellular structures and organelles.
6 Comparison of Exposure Results
The results of the analyses performed on fetal and adult fibroblasts exposed to 100–150 GHz radiation suggest that this frequency does not induce direct DNA damage but indirect effects leading to aneuploidy.
The FACS analysis showed that the progression through the cell cycle was not affected by the 100–150 GHz irradiation both in adult and in fetal fibroblasts. Further, no modulation of the expression of heat shock and pro-survival signaling proteins, such as NF-kB, ERK1/2, and AKT, was observed after radiation exposure. In addition, markers of apoptosis activation, i.e., PARP-1 cleavage or differential expression of proapoptotic proteins, have not been modulated in irradiated compared to control cells. Similarly, no differences in the expression of cytoskeleton proteins such as actin and tubulin have been observed in irradiated as compared to control cells, both in adult and in fetal fibroblasts.
Similarly, the absence of direct DNA damage and the presence of aneuploidy effect were observed in both fetal and adult fibroblasts exposed to 25 GHz CW radiation. Indeed, comet assay, γ-H2AX foci, and CREST-negative MN frequency indicated no increase of DNA breakage after in vitro exposure.
Conversely, exposed cells showed a significant increase of CREST-positive MN frequency (Fig. 8c, d) and non-disjunction events indicating induction of aneuploidy associated to radiation exposure.
Telomere was not modulated after exposure and FACS analysis showed that the progression through the cell cycle was not affected by the 25 GHz radiation. We also found no modulation of the expression of heat shock and pro-survival signaling proteins after radiation exposure at this frequency.
7 Discussion and Perspectives
Biological assays performed on fibroblasts exposed to 100–150 GHz pulsed and 25 GHz CW radiation suggest that these frequencies can induce aneuploidy by chromosome loss and non-disjunction events. This effect was observed in both cell lines without great differences between fetal and adult fibroblasts. Since an altered polymerization of thin filaments in the cells was observed after irradiation at 100–150 GHz, the induction of aneuploidy could be due to a modified crosstalk between the spindle structure and the mitotic cortex or to the influence exerted on the mitotic apparatus by the relatively high electric field of the pulsed electromagnetic radiation. The normal polymerization of thin filaments in the cells after 25 GHz CW in vitro exposure suggests a different mechanism involved in the aneuploidy. In this case, the low electric field associated with CW exposure should not exert a significant influence on the spindle, while it is possible to hypothesize the alteration of one of the target of molecular events involved in the attachment of microtubules to chromosomal kinetochores.
Former studies carried out at ENEA-Frascati in the frame of the European project THz-BRIDGE indeed showed that short pulses of terahertz radiation generated by the Compact-FEL can yield a peak electric field greater than 2 kV/cm when the THz beam is focused to a spot size of about 0.5 cm2 at the sample. This relatively high value of the field amplitude is capable of inducing a voltage drop across a lipid bilayer, which cannot be considered completely negligible when compared to the natural membrane potential. The question then arises if rapidly oscillating short electromagnetic radiation pulses can be demodulated or “rectified” by biological systems. Interesting effects on the permeability of model membranes were observed on carbonic anhydrase (CA) loaded liposomes irradiated at 130 GHz for different values of the peak electric field and modulation conditions .
Other groups have shown that nanosecond pulsed electric fields greater than 20 kV/cm can penetrate in the interior of tumor cells and induce self-destruction . In a similar way, bursts of electromagnetic radiation could be used to reach specific cells or to trigger the release of pharmaceutical drugs at a desired site. Comparative studies of pulsed electric fields and pulsed electromagnetic radiation under similar amplitude and duration condition would help to clarify the issue of signal demodulation in biological systems and may provide a unifying view of these phenomena . A systematic investigation of the dependence of any effect on the above parameters and on the carrier frequency may prove to be crucial in the development of new diagnostic and therapeutic tools.
Recently, scientific attention has been focused on the identification of possible sensitive genes that change their expression profile after radiation exposure. The most used methodologies to study gene expression are microarray technology and the real-time PCR, techniques that imply the analysis of only preselected known genes. An innovative approach, based on the next-generation sequencing technology, permits to analyze the whole transcriptome, overcoming the limitation of previous techniques. This emerging and very promising methodology for the identification of new sensitive genes might contribute to clarify the biological pathways in response to non-ionizing radiation. We are currently investigating the biological response of cells exposed to 25 GHz radiation using the innovative NGS technology with the RNA-seq protocol and this will be object of a future work. To date, few data are available on the transcriptional response of cells exposed to MW radiation [25, 26, 27], although different frequencies, exposure duration, and cell lines have been used.
Finally, the variety of THz sources, detectors, components, and systems that are now available has also led to a high degree of interdisciplinary expertise in terahertz applications, which in turn can benefit from the cross-linking of the various fields. As an example, imaging devices originally developed for biological and environmental studies turned out to be an invaluable diagnostic tool in art conservation, which in turn provided new questions and new problems to be tackled in the understanding of biological degradation of work of arts. Similarly, safety issues of millimeter wave and terahertz radiation will be crucial in the implementation of 5G devices and in the development of active imaging systems for airport security.
We gratefully acknowledge the technical support of M. Aquilini, E. Campana, S. Di Giovenale, A. Fastelli, P. Petrolini, and B. Raspante in the design and realization of the exposure setup as well as their skillful assistance during the irradiation experiments.
This work was supported by the Italian Ministry of Defence, SEGREDIFESA/DNA – 5° Department of Technological Innovation (GREAM project).
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