Effects of high intensity non-ionizing terahertz radiation on human skin fibroblasts

Data on the effects of high-intensity pulsed THz radiation (peak intensity ~30 GW/cm 2 , electric field strength ~3.5 MV/cm) on human skin fibroblasts have been obtained for the first time. A quantitative assessment of the number of histone H2AX phosphorylation foci in a cell as a function of irradiation time and THz pulse energy was obtained. It has been shown that the appearance of foci is not associated with either oxidative (cells retain their morphology, cytoskeleton structure, and the content of reactive oxygen species does not exceed the control values) or thermal stress. Long-term irradiation of cells did not reduce their proliferative index.


INTRODUCTION
Terahertz (THz) radiation is an electromagnetic radiation with a frequency of 0.1 to 10 THz (wavelength of 30 μm to 3 mm), which lies between microwave and infrared regions of spectrum. The energy of THz photon is not enough to cause ionization, i.e. THz is considered to be non-ionizing radiation (NIR). 1 Progress in developed of THz sources has extended the range of their applications, including medical, pharmacological, and security systems. 26 However, there is still a controversial issue on the safety of non-ionizing radiation. The developed safety standards for protection of human health from electromagnetic fields are varied greatly around the world and govern mainly tissue heating. 7 The action mechanism of NIR differs from that of ionizing radiation (IR) and is associated with oxidative stress (e.g. some effects of phone's range of radio frequencies cause the induction of reactive oxygen species), changing gene expression, as well as epigenetic and genetic processes leading to DNA damage. 7 Although, there are many studies related to the effect of THz radiation, the data is insufficient since opposite results are demonstrated for similar radiation parameters. For example, a qualitative analysis 8 showed the induction of histone γН2АХ when exposed to intense THz pulses for 10 min, while quantitative analysis did not reveal the same. [9][10][11] Some studies report no changes in cells, but others indicate alternations in gene expression, DNA damage, presence of micronuclei, and impaired membrane permeability [12][13][14][15][16] as well.
There is a detailed review 17 of possible mechanisms of the effect of THz radiation with biological objects. There is also evidence 18 that linear and nonlinear radiation can cause local breakdown of double-stranded DNA molecules, which indicates the genotoxicity of this radiation. One of the most sensitive markers of genotoxicity is the phosphorylation of histone H2AX. 19 The first report of the histone H2AX phosphorylation by serine 139 was discovered when cells were irradiated with IR. 20 The amount of histones depended on the dose of radiation and was associated with the presence of double-strand breaks. [21][22][23][24] In the case of NIR, an increase in γH2AX foci may be associated with a change in the chromatin structure due to the heating effect, and a number of endogenous processes in the cell 25 , including aging 26 , oxidative stress 27 , and processes in the cell cycle.
The aim of this work is to explain molecular and cellular response mechanisms of human skin fibroblasts to high-intensity pulsed THz radiation. For the first time, the dependence of the number of γH2AX foci on the duration of exposure and their post-radiation kinetics were obtained. We have analyzed a number of factors capable of initiating the formation of histone Н2АХ phosphorylation foci in human skin fibroblasts when exposed to high-intensity pulses of non-ionizing THz radiation.

THz exposure set-up
Optical rectification of femtosecond laser pulses is an efficient approach for generating THz radiation. A nonlinear OH1 [(2-(3-(4-hydroxystyryl)-5,5-dimethylcyclo-hex-2-enylidene) malononitrile] organic crystal (Rainbow Photonics, Switzerland) was used in the experimental setup 28 ( Figure 1A) to convert infrared laser pulses to THz ones. It was pumped by pulses from a Cr:forsterite laser system supplied with a multipass amplifier operating at 100 Hz (Avesta Project LLC) 29 and emitting 100 fs pulses at a wavelength of 1240 nm with the energy of 1.1 ± 0.05 mJ. The laser pump radiation was cut off by a LPF8.8-47 THz filter (Tydex LLC) with 70% transmission in the THz spectral range, placed behind the crystal. The energy of THz pulses after the filter, ETHz =18 ± 0.5 μJ was measured by a calibrated Golay cell (a GC-1D optoacoustic detector, Tydex LLC). To expand the THz beam, a telescope consisting of two off-axis parabolic mirrors was assembled after the OH1 crystal. The THz radiation was focused to a spot with d0.5 = 290 μm by an off-axis parabolic mirror with a reflected focal length of 50.8 mm. The pulse duration was measured using a well-known electro-optic sampling technique. A waveform and a spectrum (obtained by calculating the Fourier transform of the waveform) of a THz pulse are presented in Figure 1 B) and C). Duration of the Gaussian envelope approximation of the registered electric field waveform was fs as a full width at half maximum (FWHM). Thus, duration of a THz pulse by intensity was equal to fs. The cell exposure was performed by focusing the THz radiation through the bottom of a plastic dish (#80466, ibidi with a 180 μm thick polymer bottom) with a cell monolayer attached. A standard 35-mm Petri dish was placed in an incubating plate with a lid (heating system, Ibidi) for long-term cell irradiation mounted on a 3-dimentional motorized linear stage (8MT167-100 along X and Y axes, 8MT173-20 along Z axis, Standa). A video-channel consisting of a 20× microobjective with a numerical aperture NA=0.4 and a CCD-camera was assembled to control the position of the dish with respect to the focal plane of the focusing parabolic mirror. The Petri dish could be moved along X axis to either "THz" or "video" channels. To minimize the absorption of THz radiation by water vapour, the entire experimental setup was assembled in a sealed box purged with dry air. The relative humidity of the air in the box was locally reduced to 2-3%. Taking into account the transmission values of the air along the pathway, of the plastic dish and THz pulse fill-factor 30 , energy, peak intensity, and electric field strength of the THz pulses irradiating the cells were estimated to be = 15 μJ, = 32 GW/cm 2 , and = 3.5 MV/cm, correspondingly. 31 A distinguishing feature of the experimental setup is the combination of a high peak power of the THz source with a low average power. The former enables us to overcome the strong absorption of THz radiation by water, penetrate the culture medium (needed for the cell maintenance and viability) and reach the cells. The low average power enables us to minimize the thermal effects induced in cells.

Cell culture and exposure to THz radiation
This study was conducted in accordance with the Declaration of Helsinki and GCP guidelines. It was approved by the local Ethical Committee of the Federal Research and Clinical Center of Specialized Medical Care and Medical Technologies (protocol No. 4/5 from December 2, 2019). The patient signed an informed consent before enrolling in this study. The fibroblast primary cells culture was obtained by a biopsy of the behind-the-ear retroauricular skin regions as described previously. 3233 The biopsy was performed from a healthy volunteer donor who signed the informed consent.
THz radiation it was focused to a 480 μm-wide spot at the 1/e level to maximize intensity and electric field strength. The highest radiation intensity was reached the in the center of the beam and diminished along the radius to the level of 1/е from the maximum at the distance of 240 μm. Therefore, when evaluating the THz treatment effect, the analyzed field was limited by the 500 × 500 μm area. An area with a certain cell density was chosen. To simplify its identification, the corners of 500 × 500 μm square were marked using laser engraving. Tight focusing of THz radiation results in a small area of the irradiated surface and a low number of treated cells correspondingly. That's why flow cytometry, demanding high cell number (~10 6 ), could not be applied; the technique of immunocytochemical study was used instead.

Immunocytochemical assay
The cells were fixed 24 hours later or immediately after exposure in a Petri dish with 4% paraformaldehyde solution containing 0.1% saponin in PBS (pH 7.4) for 20 minutes at room temperature, followed by two washes in PBS and additional permeabilization of 0.5% Triton-X100 and 0.5% Tween 20 (in PBS, pH 7.4), supplemented with a 1% goat serum to block nonspecific antibody binding. Permeability-fixed cells were then incubated for 1 h at 37°C with primary rabbit anti-γH2AX polyclonal antibody (1 μg/ml, ab11174; abcam, USA) for double-strand break assay, with primary mouse anti-HSP70 monoclonal antibody (5 μg/ml, MS -482-B1, Thermo Fisher Scientific, USA) to determine the heat effect and primary rabbit anti-ki-67 polyclonal antibody (1 μg/ml, ab15580, Abcam, USA) to determine the proliferation index. After three washes with PBS, cells were incubated for 1 h with secondary goat antibodies against rabbit IgG (H + L) (conjugated to Alexa Fluor 488, 5 μg/ml; Invitrogen, USA) for H2AX and ki-67, goat secondary antibody Alexa Fluor 488 secondary goat anti-mouse IgG (H + L) antibodies and Hoechst 33342 for HSP70. Then the Petri dishes were washed three times with DPBS. Cell nuclei were labeled with Hoechst 33342 dye (Thermo Fisher Scientific, United States).
The proliferation index ki-67 was calculated as the ratio of the number of ki67-positive cells to the total number of cells stained with Hoechst 33342 (percentage).
An indicator based on H2DCFDA (C6827, Thermo Fisher Scientific, USA) was used to analyze reactive oxygen species (ROS) in cells exposed to THz radiation. A stock solution of CM-H2DCFDA (50 μg) was prepared in DMSO and diluted to a final concentration of 20 μM in 1 × PBS. Working solution of fluorochrome marker CM-H2DCFDA was added to serum-free culture medium incubated at 37°C in 5% CO2 for 1 hour. For a positive control, cells were incubated in serumfree medium supplemented with H2O2 for 30 min. The cells were then washed twice with DPBS and incubated at 37°C in 5% CO2 in serum-free medium containing 20 μM CM-H2DCFDA for 1 hour. After that, the cells of the experimental group and the cells of the positive control were fixed with 4% paraformaldehyde in DPBS for 20 min at room temperature. 34 Then the cells were washed twice in DPBS and permeabilized with 0.5% Triton-X100 and 0.5% Tween 20 (in PBS, pH 7.4) with the addition of phalloidin tetramethylrhodamine B isothiocyanate (P1951, Sigma Aldrich, USA) to visualize actin.
Immunofluorescence was analyzed using a Celena Digital imaging system (Logos biosystems, South Korea) and a Nikon A1 scanning laser confocal microscope (Nikon Co., Japan).

Statistical analysis
The experiment was repeated three times for each irradiation time to estimate the significance of the data. One-way variance Fisher's analysis (ANOVA) (p>0.05) was used for statistical analysis of the histone Н2АХ phosphorylation foci formation. ImageJ software was used to evaluate the fluorescence of stained cells. An image file was split into the color channels and the contour of each cell was delimited. The area, integrated density, and mean grey value were measured for each cell in the image and in the background as well. The corrected total cell fluorescence (CTCF) was calculated using the formula: CTCF = Integrated Density -(Area of the selected cell × Mean grey value of background readings).

Formation of foci of histone Н2АХ (γH2AX) in human skin fibroblast
In this study, the number of foci of phosphorylated histones H2AX in human skin fibroblasts was estimated after exposure to high-intensity pulses of THz radiation. Figure   . Immunofluorescence analysis of γH2AX foci in human skin fibroblasts exposed to THz radiation for 90 min and fixed on the next day after the irradiation. The area exposed to THz radiation is marked by the dotted circle. γH2AX were stained with goat-antirabbit secondary antibodies with Alexa Fluor 633 (red) and cell nuclei were stained with Hoechst (blue) in all panels. Scanning confocal microscopy. Bar size 100 μm (A) and 50 μm (B).

Graphs of THz radiation dependence on time
A series of experiments was carried out to determine the relation between the amount of histones and the irradiation time and the time after the radiation was turned off for energy of THz pulses = 15 mJ. The dynamics of the formation of histones γH2AX per cell when exposed to THz radiation at different times are shown in Figs. 3 A) and B). To study the dynamics of γH2AX foci formation, quantitative assessment was carried out immediately after cells exposure to THz radiation ( = 0 h, Fig. 3A) and 24 hours after switching off the THz source ( = 24 h, Fig. 3B).

Figure 3 A) demonstrates a correlation between
and exposure duration ; value in experimental group is much higher than that in control group for > 30 min. However, neither morphological changes nor foreign inclusions in cell were observed for any . Due to minor differences in observed for = 0 min and = 10 min this set of parameters was excluded from the subsequent experiments. Since the numbers of γH2AX foci in cells irradiated at = 30 and = 90 min do not statistically differ for both immediate fixation = 0 h (Fig.3A) and one day later = 24 h (Fig.3B), an additional study was carried out at = 180 min and = 24 h. This resulted in sixfold increase in γH2AX foci compared with the parallel control group and twofold growth compared with data for = 30 and = 90 min. No signs of apoptosis were however observed.

Determination of heat shock proteins in fibroblasts after irradiation
Thermal effect on the cell could be one of the possible reasons for formation of phosphorylated H2AX foci. 35 Since direct temperature measurement in biological experiments on cell exposure to THz radiation could sometimes be difficult, a theoretical model 36 based on solving the Kirchhoff equation for the heat capacity is widely applied. Theoretical estimations performed earlier 31,37 demonstrated that temperature increase in the center of the irradiated area does not exceed Δ = 0.7°C for a single THz pulse and Δ = 2.8°C for a series of pulses. The biological approach for the heat effect estimation is based on the expression of heat shock proteins. 38,39 To test the hypothesis of thermally induced increase in number of phosphorylated H2AX foci !/# , the expression of heat shock proteins (HSP70) was evaluated after fibroblasts exposure to THz pulses with energy $%& * = 15 μJ following with repetition rate of 100 Hz for ( = 30 min. The immunocytochemical analysis was performed for cells after the THz treatment (Fig. 4A) as well as for parallel (Fig.  4B) and positive control groups (not presented here). The corrected total cell fluorescence (CTCF) intensities for all the groups was calculated. There was no increase in the expression of heat shock proteins (HSP70) in experimental group compared to the group of parallel control, with the expression level much lower than the positive control group. The Mann-Whitney test demonstrated the statistical difference between the positive control group and the rest of the groups. No difference was observed between the experimental and parallel control groups (p<0.05). A detailed analysis of possible thermal effects of THz radiation on cells was performed earlier. 40 In this study, we confirm that histone foci are not associated with cells expressing HSP, that is, the origin of foci is not the thermal effect of THz radiation.

Assessment of the effect of oxidative stress caused by THz radiation on the human skin fibroblast proliferation index
An increase in the reactive oxygen species (ROS) level under the influence of exogenous factors (ionizing radiation, microwaves, heat exposure, chemicals, etc.) can lead to destabilization of the chromatin structure, appearance of double DNA-strain breaks (DNA DSB), and cell apoptosis. 41,42 This can result in formation of foci of γH2AX in the cell. The mechanisms of DNA damage by non-ionizing radiation are associated with the suppression of cellular repair mechanisms; this can lead to oxidative stress and the emergence of cancer due to damage to DNA and other cellular components 1. Disruption of the cell cycle also affects the formation of γH2AX foci. This can lead to a change in proliferative activity. 10,26 ROS levels was assessed after irradiation of fibroblasts for 30, 90, 180 min. Cells treated with hydrogen peroxide (H2O2) were used as a positive control. It was found that an increase in ROS levels and a change in F-actin filaments were found only in cells treated with H2O2. Moreover, no ROS was observed in all irradiated cells, and F-actin filaments retained their structure and maintained cell morphology. Moreover, for all irradiation times, the number of cells expressing ki67 (a marker of proliferation) was approximately the same in both control and experimental groups. Proliferation index of 50-65% suggests that THz radiation does not affect the proliferative activity of cells.

DISCUSSION
The formation of histone H2AX phosphorylation foci occurs spontaneously under normal conditions and an increase in their number occurs due to toxic substances, which can serve as one of the indicators of a genotoxic effect. Experimental studies with different types of cells, various irradiation parameters (frequency, average and peak intensities), and experimental conditions (exposure time, temperature) have been performed previously (see reviews 15,12,2,43 ).
An increase in γH2AX was detected 44 , with a qualitative analysis was performed. Unfortunately, no information on quantitative changes in the number of γH2AX foci was presented in both experimental and control samples of artificial skin. A quantitative analysis of γH2AX foci was reported. 10 However, it is difficult to calculate the exact number of foci per cell using the diagrams given. On average, the number varied from 0.3 to 1 and practically did not differ from the control group values. In this study, the effect of high-intensity THz radiation (the peak intensity of the source used was ~30 GW/cm 2 , which was much higher than typical values used in other studies) was estimated on the phosphorylation of histone H2AX foci in human skin fibroblasts. The cells were irradiated for 30, 90, and 180 min. The cells of the 3rd -5th passage from the one young and healthy donor were used in all experiments in order to exclude possible risks of an increase in γH2AX foci due to cell aging.
It has been demonstrated that THz radiation causes phosphorylation of histone Н2АХ in experimental group compared to the control one. The number of foci increased with the duration of THz irradiation. The foci formed after THz exposure for 30 and 90 min were observed right after the end of irradiation and persisted the next day. This is consistent with data 11,44 , which states that phosphorylation foci are formed immediately after exposure and reach their maximum in 30 min.
At the moment, there are studies reporting that γH2AX foci may indicate not only DNA double breaks 46 at relatively low levels of phosphorylation, but can be caused by either micro-heating or oxidative stress associated with THz exposure. In this regard, appropriate experiments were carried out.
It has been demonstrated earlier 37 that temperature in the center of THz beam (for an average power of 1.5 mW), does not exceed 40°C, which may cause microheating in cells. Semi-quantitative immunocytochemical analysis of heat shock proteins (HSP) showed that the level of HSP70 expression in cells in the focus of THz radiation did not differ from the level in control groups. Besides, the values were significantly lower than those in cells of the positive control, and foci of γH2AX were often found in cells not expressing HSP. However, due to semi-quantitative nature of performed analysis, it is impossible to say for sure about the absence of the effect of THz radiation on the genes responsible for the expression of chaperone proteins. There were also no signs of oxidative stress observed upon cell exposure to THz radiation. The cells retained their morphology and the structure of the cytoskeleton. The amount of ROS and the proliferation index (50-65%) did not differ from those in the control groups even for long-term irradiation (90 and 180 min).
The data obtained indicate a probable epigenetic mechanism of the effect of THz radiation on cells, which can lead to chromatin modification or the appearance of DNA DSB. Formation of DNA DSB upon exposure to THz radiation is still a controversial point, whereas changes in the expression of certain genes have been reported in many studies. 39,[47][48][49] A decrease in the expression of genes associated with the complex of epidermal differentiation in the region of 1q21 chromosome 4849 , dysregulation of 8 signaling pathways responsible for the development of many types of cancer in humans 47 and changes in the expression of genes regulating tumor growth have been observed. Aneugenic effects associated, according to authors, with the induction of aneuploidy have also been reported. 9,11,50,51 Understanding the mechanisms of interaction of non-ionizing THz radiation with biological objects would advance establishment of safety standards and further progress in applications of THz sources.
In the present study, it has been demonstrated that long-term exposure of cells to high-intensity THz radiation causes neither heating nor oxidative stress, as well as no changes in cell morphology and proliferative index. An increase in phosphorylated H2AX histones indicates a possible genotoxic effect, and long-term foci preservation testifies to an epigenetic nature of the effect, which may result in the cell sensibilization to other factors.
Further investigations aimed at studying the safety of THz radiation and the establishment of standards would also help in understanding the mechanisms of interaction of non-ionizing radiation with biological objects, as well as in advancement of potential use of THz radiation sources. In our study, it was shown that prolonged cell exposure to high-intensity THz radiation does not cause heating and oxidative stress and associated changes in the morphology and proliferative index of cells as well. An increase in number of phosphorylated histones H2AX indicates a possible genotoxic effect, and the longterm preservation of the foci indicates an epigenetic character, which could result in increasing the sensitivity of cells to other factors.

CONCLUSION
New data on the effect of high-intensity pulsed THz radiation (peak intensity of ~30 GW/cm 2 , electric field strength of ~3.5 МV/cm) on human skin fibroblasts have been obtained. It has been shown that the exposure of cells to high-intensity THz radiation does not affect their morphology or proliferative activity. However, such a treatment results in epigenetic changes of a cell due to histone Н2АХ phosphorylation. The formation of γН2АХ foci has a time-dependent effect, which persists for a long time in a cell.
The experiments were performed using the unique scientific facility "Terawatt Femtosecond Laser Complex" in the Center for Collective Usage "Femtosecond Laser Complex" of JIHT RAS. The reported study was funded by the Russian Fund for Basic Research (RFBR) according to the research project No. 19-02-00762.