ISSN 0006-3509, Biophysics, 2017, Vol. 62, No. 3, pp. 444–449. © Pleiades Publishing, Inc., 2017. Original Russian Text © A.B. Gapeyev, D.A. Yurshenas, A.A. Manokhin, R.N. Khramov, 2017, published in Biofizika, 2017, Vol. 62, No. 3, pp. 552–558.

CELL BIOPHYSICS

The Protection of DNA in Blood Leukocytes from Damaging Action of Ultraviolet Radiation Using the “Useful Sun” Strategy A. B. Gapeyeva, *, D. A. Yurshenasb, A. A. Manokhina, and R. N. Khramovb, c a Institute

of Cell Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, Pushchino, Moscow oblast, 142290 Russia b Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, Pushchino, Moscow oblast, 142290 Russia c ZAO Polisvetan, ul. Donskaya 4, Moscow, 119049 Russia *e-mail: [email protected] Received June 7, 2016

Abstract—The damaging effects of light that was emitted by a DRSh250-3 mercury lamp on the DNA of mouse blood leukocytes was studied in vitro. It was shown that the main DNA damage is due to the action of UVB radiation (280–320 nm). Under the combined effects of the UV radiation and the orange–red fluorescent component it was found that the additional fluorescent light with the spectral maximum at 625 nm from nanoluminophore materials (quantum dots that are based on CdSe/ZnS, CdSe/CdS/ZnS) protected the cellular DNA from the damaging effect of UV radiation. Using nanomolar concentrations of hydrogen peroxide, the hypothesis of the role of reactive oxygen species in the protective effects of the red–orange light was tested in vitro. It was shown for the first time that the mechanisms of the protective effects are associated with the induction of an adaptive response by nanomolar concentrations of hydrogen peroxide that are induced by the orange–red light. Keywords: ultraviolet radiation, orange–red light, mouse blood leukocytes, DNA damage, comet assay, hydrogen peroxide, adaptive response, "Useful Sun" strategy DOI: 10.1134/S0006350917030058

The negative effects of sunlight on biological systems are mainly caused by ultraviolet (UV) radiation. Due to the absorption of UVC (<280 nm) radiation and the most part of the UVB (280–320 nm) radiation by the stratospheric ozone layer, the main damaging action is due to the UVA (320–400 nm) (95%) and UVB (5%) radiation. It was demonstrated that solar radiation can damage cellular DNA by several mechanisms [1]. The direct excitation of a DNA molecule leads to generation of cyclobutane pyrimidine dimers, 6-4 photoproducts of pyrimidine, and thymine dimers, which are of importance for cytotoxic, mutagenic, and cancerogenic effects of UVC and UVB radiation. Within the UVA and the visible region of the spectrum the DNA molecule absorbs very weakly [2] and the mechanisms of the DNA damage and genotoxic effects are caused by the action of reactive oxygen species, which occur due to the light–oxygen effect [3]. The use of the comet assay revealed that DNA damage that is induced by sunlight in monocytes and lymphocytes of human blood is higher during the summer period compared to the winter period [4, 5]. These results indicate that sunlight penetrates sufficiently deeply and reaches the microcirculatory bed of Abbreviations: UV, ultraviolet.

the skin and can have both damaging and photostimulating effects on blood cells. The “Useful Sun” strategy of photobiomodulation was formulated for the first time for living systems [6] and based on the use of light-converting photoluminophore-containing materials that absorb short-wave solar or artificial radiation and transform it into biostimulating orange–red and/or infrared radiation. The prerequisite for the development of the Useful Sun strategy was studies where artificial irradiation (laser, LED, lamp) of red and near infrared light was found to have therapeutic and prophylactic actions; a decrease in the intensity of inflammatory responses; and an increase of tissue regeneration, local resistance, and anti-infective defense; this leads to a number of other positive changes in living systems [7, 8]. Previously, it was demonstrated in animals and humans for the first time that the Useful Sun strategy (transformation of UV radiation) in contrast to the Safe Sun strategy (absorption or screening of UV radiation without conversion) has the following positive effects: (a) it reduces the healing time of trophic ulcers, chronic nonhealing wounds, and burn wounds of human skin to 30% [9]; (b) it increases the physical performance of animals that are adapted to stress with a cumulative

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Energy irradiance, W/(m2 nm)

30 25 20 15 10 5 0 250

300

350

400 450 500 Wavelength, nm

550

600

650

Fig. 1. The energy spectrum of a DRSh250-3 mercury lamp.

effect by a factor greater than 1.5 with improvement of morphological characteristics of the myocardium [10]; (c) it increases the number of mouse embryos that develop in the culture in vitro by a factor of 2.8 [11]; (d) it reduces chemically induced DNA damage in white blood cells of rats [12, 13]; and (e) it improves the functional state of cells in vitro and in vivo [14]. These data demonstrate the versatility of the Useful Sun strategy and the biostimulatory effect of the conversed orange–red light for living systems. The purpose of this study was to assess the damaging effects of UVA and UVB radiation on DNA in the white blood cells of mice in vitro and the possibility of protection of the cellular DNA from damage under the effects of orange–red light and to reveal the possible mechanisms of the protection. MATERIALS AND METHODS Obtaining biological samples. Adult male Kv:SHK mice (2-month-old, 20–23 g in weight) were used in all experiments. The mice were grown and kept under controlled conditions at a 12 : 12 h light–dark cycle; the animals had a standard laboratory diet and water ad libitum. The peripheral blood of the mice was sampled from the tail vein into tubes that contained phosphate buffer with 1 mM EDTA as an anticoagulant. Whole blood was used to make the preparations, which was diluted by a factor of 10 in order for the final leukocyte concentration in the composition of agarose slides to be approximately 0.5 million/mL. Exposure to ultraviolet and optical radiation. A DRSh250-3 lamp was used for irradiation, with the radiation spectrum being given in Fig. 1. The DRSh250-3 lamp was chosen as a model light source, since it has a line spectrum in the UV region and estimation of the damaging action of the UV components BIOPHYSICS

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of light was one of the problems under study. To select individual regions of the spectrum UVC5 (280– 400 nm), UVC6 (300–395 nm), combinations of UVC5 and ZhS20 (260–340 nm) filters, and a polycarbonate filter that is not transparent to UV radiation were used. To obtain an additional orange–red component in the light flux, a luminescent screen with a QD625 nanoluminophore, which is based on CdSe/ZnS and CdSe/CdS/ZnS (active screen) and has an excitation spectrum in the UV, violet, and blue regions, was used [14]. The wavelength of the maximum of the additional luminescent flow corresponded to 625 nm. The reference control in these experiments was the same light-converting screen, but with the inactivated luminophore; the structure of its quantum dots was impaired due to treatment with 3% hydrogen peroxide (passive screen). The thickness of the passive screen was chosen so that the transmittance spectra of the both light-converting screens coincide in the UV region. The filters and light-converting screens were placed between the light source (lamp) and object (agarose slides with immobilized cells). The slides were put in glass Petri dishes 60 mm in diameter that were filled with 15 mL phosphate buffer to compensate for possible heating during irradiation. The spectral characteristics of the light filters and light fluxes that are obtained upon their use were determined by an automated spectrometric complex on the basis of an MDR-41 monochromator (ZAO OKB SPEKTR, Russia) within the range from 200 to 1000 nm, and the energy characteristics were determined by an CMP-3 pyranometer (Kipp & Zonen, Neitherlands) with the permanent spectral sensitivity within the range from 310 to 2800 nm. The energy irradiance of the total light flux from the DRSh250-3 lamp (without filters) was 100 mW/cm2 at a distance of 45 cm from the lamp. The durations of exposure of white blood cells were chosen as 10, 20, 40, and 60 s, which corresponded to

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the radiant exposures of 1, 2, 4, and 6 J/cm2. The energy irradiance of the light flux in the visible region of the spectrum (>400 nm) was approximately 70 mW/cm2 at a distance of 45 cm from the lamp. To achieve the corresponding radiant exposures (1, 2, 4, 6 J/cm2), the time of irradiation was 15, 30, 60, and 90 s. According to these estimates, the fraction of the UV component in the general energy irradiance was approximately 30 mW/cm2 at a distance of 45 cm from the lamp. The actual fraction of UV in the total radiant exposure upon irradiation for 10, 20, 40, and 60 s was 0.3, 0.6, 1.2, and 1.8 J/cm2, respectively. The energy irradiances of the light flux in the spectrum regions of 280–320 and 320–400 nm were determined similarly and constituted approximately 4 and 26 mW/cm2 at a distance of 45 cm from the lamp. Upon irradiation for 10 s, the radiant exposures were 0.04 and 0.26 J/cm2, respectively. When different filters were used, their transmittance characteristics were taken into account and the duration of the exposure was chosen to provide the correspondence of the radiant exposures. Treatment with hydrogen peroxide. Cell treatment with H2O2 at a concentration of 0.1–30 µM was performed in the composition of the microscopic slides, which were incubated in the presence of corresponding H2O2 concentrations for 10 min at 37°C. For the combined effects of H2O2 and UV radiation, the cells were exposed to the UV radiation immediately after treatment with H2O2 at concentrations of 0.1–2 µM for 10 min at 37°C. Analysis of DNA damage in the cells was carried out using an alkaline version of the comet assay with some modifications [15]. The method is based on the analysis of the electrophoresis pattern of individual cells, whose DNA is stained with a fluorescent dye [16]. The name of the method is due to the visual similarity of electrophoregrams that are obtained with comets, i.e., a comet head and tail that brightly fluoresce are observed, with the tail being formed due to the migration of damaged or unwound DNA regions after agarose electrophoresis. The microscopic slides were prepared from three layers of 0.5% low-melting-point agarose (Serva, Germany) with the cells being immobilized in the middle layer. After different exposures the slides were subjected to the comet assay: cell lysis in a lysing solution (1% sodium lauroyl sarcosinate, 2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris-HCl, pH 10.0, 1% Triton X-100) for 25 min at 37°C; alkaline denaturation of DNA in an alkaline solution (0.3 M NaOH, 0.001 M EDTA, pH > 13) for 20 min at 4°C; electrophoresis in a fresh portion of the alkaline solution for 20 min at 4°C in an SE-1/S-1N electrophoresis chamber (OOO Helikon, Moscow) at an electric field strength of 2 V/cm and a current strength of 300 mA; alkaline neutralization by distilled water; and DNA staining in a phosphate buffer with 1 μg/mL of ethidium bromide for 1 h. Prior to the analysis, every slide was washed in distilled water for 5 min and cov-

ered with a cover slip. All the procedures were carried out under artificial illumination using incandescent lamps in order to avoid additional DNA damage in the cells. The preparations were analyzed by a Comet Expert hardware and software system (OOO Gen Expert, Pushchino). The percentage content of DNA in the comet tail was used as an indicator of DNA damage [17]. Each blood sample was used to prepare the necessary number of slides according to the number of exposures. From 30 to 50 comet images were registered in each slide, which were used to calculate the average percentage content of DNA in the comet tail. The mean values and standard errors of the mean for each variant of the exposure were calculated according to the results of independent experiments (n ≥ 9). Statistical analysis. All the experiments were blind, i.e., the experimenter that made measurements was not aware of which exposure was used. All the data are presented as the mean value ± standard error. Since all the data were normally distributed (by the Kolmogorov–Smirnov test), the statistical analysis was carried out using ANOVA and Dunnett’s test for multiple comparisons (p < 0.01); the Student’s t-test was used for paired comparisons of the groups of data (p < 0.05). RESULTS AND DISCUSSION The exposure of the white blood cells of mice to the total irradiation of the DRSh250-3 lamp without filters was found to result in the damage of the cellular DNA, which enhanced at an increase in the radiant exposure within the range from 1 to 6 J/cm2 (Fig. 2). To construct light-converting screens and to choose a specific luminophor it was of importance to find out, which spectrum regions had the most damaging effect on the leukocyte DNA. By using the polycarbonate filter, we selected the spectrum region above 400 nm. Exposure to the radiation with these characteristics did not induce DNA damage in the cells (Fig. 2). It should be noted that the light flux within the range of 400–650 nm did not damage DNA even at an increase in the exposure to 20 min (data are not given), i.e., the DNA damage under the effect of the total irradiation of the DRSh250-3 lamp was likely to be induced by the light flux within 280–400 nm. In a separate series of experiments with application of the UVC5 filter, which selected the UV region of the spectrum, this assumption was tested and proved; the results of exposure to the total irradiation and those of exposure to the UV radiation actually coincide within the error of measurements for the corresponding radiant exposures (Fig. 2). To study the effect of different components of the UV radiation the UVA region was selected by the UVC6 filter and the UVB region was selected by the combination of the UVC5 and ZhS20 filters. When the cells were exposed to the UVA region (320– BIOPHYSICS

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*

20

* DNA content in the comet tail, %

20

15 * 10

5

*

0 0

1 2 3 4 5 (0.3) (0.6) (1.2) Radiant exposure, J/cm2

400 nm) weak DNA damage was observed. When the cells were exposed to the UVB region (280–320 nm), the level of DNA damage was high and enhanced with an increase in the radiant exposure. Thus, it was found that the main damage via the radiation of the DRSh250-3 lamp in white blood cells of mice was induced by the radiation of the UVB region (Fig. 3). Light-converting screens that are based on nanoluminophores have been effectively used recent years for protection from UV radiation. The Useful Sun strategy is widely applied in biotechnologies for stimulation of the development of plants [18] and stimulation of the dynamics of populations of microorganisms in contaminated soils [19]; it is promising in biomedicine and sports for enhancement of physical performance and treatment of skin wounds and retinal burns [9]. To estimate the protective effect of the orange–red light on the DNA damage of cells by UV radiation we used luminescent screens (with active and inactivated luminophores). The energy spectra of light fluxes that passed through these screens were made identical in the UV regions by selecting the thickness of the passive screen and differed in the visible region of the spectrum including by the presence of a small addition in the 625 ± 20 nm band that did not exceed 1%. The energy irradiance of the light flux in the UV region (280–400 nm) for both screens was approximately 8 mW/cm2. The comparative analysis of the DNA damage of the cells under irradiation through these Vol. 62

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*

10

5 * 0

6 (1.8)

Fig. 2. DNA damage in white blood cells of mice under the irradiation of a DRSh250-3 mercury lamp without filters (circles) using a polycarbonate filter (triangles) and UVC5 (squares). The polycarbonate filter is transparent to light in the spectral region above 400 nm, and UVC5 is transparent in the region of 280–400 nm. The radiant exposures for the UV region are given in brackets along the X-axes. * p < 0.001 according to the Dunnett’s test with respect to the control and damage under the effect of the light of the visible region.

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0

1.0 0.2 0.4 0.6 0.8 (0.03) (0.06) (0.12) (0.15) Radiant exposure, J/cm2

Fig. 3. The DNA damage in white blood cells of mice under the irradiation of a DRSh250-3 mercury lamp using a UVC6 filter (circles) and UVC5 + ZhS20 filters (triangles). The radiant exposures for the UVB radiation are given in brackets along the X-axes. * p < 0.001 according to the Dunnett’s test with respect to the control and damage under the effect of the UVA radiation.

screens revealed the protective effect for the active luminophore: the DNA damage was reduced, on average, by 30%, when compared to the inactivated luminophore (Fig. 4). We proposed that the protective effect of the orange–red light can be due to the effect of reactive oxygen species at small regulatory concentrations. It 35 DNA content in comet tail, %

DNA content in comet tail, %

447

30

Inactivated luminophore Active luminophore

25 * 20 15

*

10 5

*

0 1.0 1.5 3.0 Radiant exposure, J/cm2 Fig. 4. The DNA damage in white blood cells of mice under the irradiation of a DRSh250-3 mercury lamp using screens with active and inactivated nanoluminophores; the radiant exposures for the UV region of light (280–400 nm) are given. * p < 0.03 the differences are significant according to the Student’s t-test.

GAPEYEV et al.

was found previously that orange–red light in aqueous solutions produces hydrogen peroxide and hydroxyl radicals at nanomolar concentrations, which might mediate the protective effect [13]. To directly test this assumption, the effect of hydrogen peroxide at concentrations from 100 nm to 30 μm on the DNA of white blood cells was tested in mice. It was found that H2O2 at concentrations less than 1 μM did not result in DNA damage to blood leukocytes in mice and at concentrations greater than 1 μM DNA damage in cells was enhanced with an increase in the concentration (Table 1). Taking hydrogen peroxide production at nanomolar concentrations under the effect of the orange–red light and the presence of the protective effect of the orange–red light into account, we studied the combined effects of the low concentrations of hydrogen peroxide (100–2000 nM) and the irradiation of the UVB region at a damaging dose of 0.1 J/cm2, which did not induce a very high level of DNA damage. The preliminary incubation of the cells in the presence of exogenous H2O2 at concentrations of 300–500 nm for 10 min at 37°C led to a decrease in the DNA damage that was induced by the UVB radiation, on average, by 25–30% (Fig. 5). The adaptive response is one of the mechanism of the protective effect and universal response of cells to low-dose exposures, which is manifested by an increase in the resistance to the damaging action at high doses [20, 21]. Preconditioning in biomedicine can induce the adaptive response, which underlies the protective effects of different agents with mutagenic and genotoxic properties [22, 23]. Many researchers believe that the mechanisms of the adaptive response are due to the functioning of repair systems of the cell [24, 25]. The adaptive response can be triggered and switched by different stress inducers including an increase in the temperature and other physical effects, biologically active substances, and reactive oxygen species. Unfortunately, the detailed mechanisms of the induction of the adaptive response has not been revealed as yet. It has been found that micromolar concentrations of hydrogen peroxide are able to induce de novo synthesis of a great number of proteins that are involved in energy metabolism, signaling, translation, transcription, reparation of DNA, regulation of the redox potential, stress responses, apoptosis, protein folding, etc. [23, 26]. It has been demonstrated that micromolar concentrations of hydrogen peroxide are able to activate systems of the antioxidant defense, including enhancement of the activities of such enzymes as superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase [27]. Hydrogen peroxide at low concentrations is capable of stimulating the expression of the antiapoptotic protein Bcl-2 [23], the increase of which in the cell can suppress the cleavage of poly(ADP–ribose) polymerase 1, which is activated upon the occurrence of single-

Table 1. The dependence of DNA damage in white blood cells of mice on the H2O2 concentration H2O2 concentration, μM

DNA content in the comet tail, %

Control 0.1 0.3 1.0 2.0 5.0 10.0 20.0 30.0

0.12 ± 0.06 0.26 ± 0.08 0.30 ± 0.08 0.37 ± 0.08* 0.42 ± 0.05* 0.76 ± 0.18* 1.54 ± 0.33* 5.60 ± 0.34* 6.84 ± 0.57*

Cells were incubated in the presence of hydrogen peroxide at different concentrations for 10 min at 37°C. * p < 0.05, the differences are significant when compared to the control, according to the Student’s t-test.

strand DNA breaks and is involved in DNA repair [28]. Thus, an increase in the level of Bcl-2 and suppression of the cleavage of poly(ADP–ribose) polymerase 1 can protect cells from apoptosis and DNA damage that is induced by more potent genotoxicants. It has been found that hydrogen peroxide at nanomolar concentrations can have a radioprotective effect during DNA damage of blood leukocytes of mice under X-ray radiation [29]. These and other experimental data indicate that the adaptive response is a complex reaction that involves a wide range of cellular functions. 10 DNA content in the comet tail, %

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9 8 7 *

6 * 5 0

0 500 1000 1500 2000 The priming concentration of H2O2, nM Fig. 5. The DNA damage in white blood cells of mice under the combined effects of exogenous hydrogen peroxide at concentrations of 100–2000 nm and UVB irradiation of a DRSh250-3 lamp at a dose of 0.1 J/cm2. * p < 0.05 the differences are significant compared to the control (0 nM), according to the Student’s t-test. BIOPHYSICS

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The results we obtained and the literature data make it possible to suggest that reactive oxygen and nitrogen species that are formed under the effect of the orange–red light may have a regulatory effect. The mechanisms of the protective effect can be connected with the additional expression and enhancement of the activity of enzymes of DNA repair. The effects we found may be important from the point of view of novel possibilities and methods for protection of cellular DNA from the damaging effects of genotoxic factors. ACKNOWLEGMENTS The study was supported by the Russian Foundation for Basic Research and Government of the Moscow oblast (project no. 14-44-03672-r_center_a). REFERENCES 1. J. Cadet, E. Sage, and T. Douki, Mutat. Res. 571, 3 (2005). 2. C. A. Jones, E. Huberman, M. L. Cunningham, and M. J. Peak, Radiat. Res. 110, 244 (1987). 3. S. V. Gudkov, O. E. Karp, S. A. Garmash, et al., Biophysics (Moscow) 57 (1), 1 (2012). 4. P. Moller, H. Wallin, E. Holst, and L. Knudsen, FASEB J. 16, 45 (2002). 5. S. I. Tsilimigaki, N. Messini-Nikolaki, M. Kanariou, and S. M. Piperakis, Mutagenesis 18, 139 (2003). 6. R. N. Khramov, L. R. Bratkova, A. B. Gapeyev, et al., in Biological Effects of Light – 1995, Ed. by M. F. Holick and E. G. Jung (Walter de Gruyter, Berlin, 1996), pp. 192–194. 7. Low-Intensity Laser Therapy, Ed. by S. V. Moskvin and V. A. Builin (TOO Tekhnika, Moscow, 2000) [in Russian]. 8. N. A. Zhevago and K. A. Samoilova, Photomed. Laser Surg. 24, 129 (2006). 9. A. V. Vorob’ev, R. N. Shchelokov, R. N. Khramov, et al., RF Patent No. RU97103974 (April 20, 1999). 10. R. N. Khramov, I. M. Santalova, L. I. Fakhranurova, et al., Biophysics (Moscow) 55 (3), 447 (2010).

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Translated by E. Berezhnaya