Modifying effects of low-intensity extremely high-frequency electromagnetic radiation on content and composition of fatty acids in thymus of mice exposed to X-rays.

Just Accepted by International Journal of Radiation Biology

Modifying Effects of Low-Intensity Extremely High-Frequency Electromagnetic Radiation on Content and Composition of Fatty Acids in Thymus of Mice Exposed to X-Rays Andrew B. Gapeyev, Alexander V. Aripovsky, Tatiana P. Kulagina Doi: 10.3109/09553002.2014.980467

Int J Radiat Biol Downloaded from informahealthcare.com by Korea University on 12/27/14 For personal use only.

Abstract Purpose: To test the involvement of fatty acids (FA) in possible protective effects of extremely high-frequency electromagnetic radiation (EHF EMR) against ionizing radiation, the effects of EHF EMR on thymus weight and its FA content and FA composition in X-irradiated mice were studied. Materials and methods: Mice were exposed to low-intensity pulsemodulated EHF EMR (42.2 GHz, 0.1 mW/cm2, 20 min exposure, 1 Hz modulation) and/or X-rays at a dose of 4 Gy with different sequences of the treatments. In four-five hours, 10, 30, and 40 days after the last exposure, the thymuses were weighed; total FA content and FA composition of the thymuses were determined on days 1, 10, and 30 using a gas chromatography. Results: It was shown that after X-irradiation of mice the total FA content per mg of thymic tissue was significantly increased in 4-5 h and decreased in 10 and 30 days after the treatment. On days 30 and 40 after X-irradiation, the thymus weight remained significantly reduced. First and tenth days after X-rays injury independently of the presence and sequence of EHF EMR exposure were characterized by an increased content of polyunsaturated FA (PUFA) and a decreased content of monounsaturated FA (MUFA) with unchanged content of saturated FA (SFA). Exposure of mice to EHF EMR before or after X-irradiation prevented changes in the total FA content in thymic tissue, returned the summary content of PUFA and MUFA to the control level and decreased the summary content of SFA on the 30th day after the treatments, and promoted the restoration of the thymus weight of X-irradiated mice to 40th day of the observations. Conclusions: Changes in the content and composition of PUFA in the early period after treatments as well as at the restoration of the thymus weight under the combined action of EHF EMR and X-rays indicate to an active participation of FA in the acceleration of postradiation recovery of the thymus by EHF EMR exposure. © 2014 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.

Modifying Effects of Low-Intensity Extremely High-Frequency Electromagnetic Radiation on Content and Composition of Fatty Acids in Thymus of Mice Exposed to X-Rays

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Andrew B. Gapeyev1*, Alexander V. Aripovsky2, Tatiana P. Kulagina1 1

Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region,

Russia, 2State Scientific Center of Applied Microbiology and Biotechnology, Obolensk,

*Correspondence to: Prof. Andrew B. Gapeyev, Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia. Phone: +7 (965) 4405732; Fax: +7 (4967) 330509; E-mail: [email protected]

Short title: EHF EMR modifies the effects of X-rays on thymic FA

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Abstract

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Purpose: To test the involvement of fatty acids (FA) in possible protective effects of extremely high-frequency electromagnetic radiation (EHF EMR) against ionizing radiation, the effects of EHF EMR on thymus weight and its FA content and FA composition in X-irradiated mice were studied. Materials and methods: Mice were exposed to low-intensity pulsemodulated EHF EMR (42.2 GHz, 0.1 mW/cm2, 20 min exposure, 1 Hz modulation) and/or X-rays at a dose of 4 Gy with different sequences of the treatments. In four-five hours, 10, 30, and 40 days after the last exposure, the thymuses were weighed; total FA content and FA composition of the thymuses were determined on days 1, 10, and 30 using a gas chromatography. Results: It was shown that after X-irradiation of mice the total FA content per mg of thymic tissue was significantly increased in 4-5 h and decreased in 10 and 30 days after the treatment. On days 30 and 40 after X-irradiation, the thymus weight remained significantly reduced. First and tenth days after X-rays injury independently of the presence and sequence of EHF EMR exposure were characterized by an increased content of polyunsaturated FA (PUFA) and a decreased content of monounsaturated FA (MUFA) with unchanged content of saturated FA (SFA). Exposure of mice to EHF EMR before or after X-irradiation prevented changes in the total FA content in thymic tissue, returned the summary content of PUFA and MUFA to the control level and decreased the summary content of SFA on the 30th day after the treatments, and promoted the restoration of the thymus weight of X-irradiated mice to 40th day of the observations. Conclusions: Changes in the content and composition of PUFA in the early period after treatments as well as at the restoration of the thymus weight under the combined action of EHF EMR and X-rays indicate to an active

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Moscow Region, Russia

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participation of FA in the acceleration of post-radiation recovery of the thymus by EHF EMR exposure.

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Key words: extremely high-frequency electromagnetic radiation; X-rays; combined effects; fatty acids; thymus

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INTRODUCTION The relevance of search for the means and methods of protection of living organisms against the damaging effects of ionizing radiation is determined by a continuing risk of lesions due to potential accidents at the nuclear industry. Furthermore, there is a need of modification of clinical effects of ionizing radiation therapy by both chemical and physical means to achieve high therapeutic efficacy and to reduce toxic effects.

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It is known that the radioresistance of the organism may vary under the influence of non-radiation factors of different nature (Manti and D'Arco 2010, Vijayalaxmi et al. 2014). Mechanisms of stress, i.e. responses to extreme stimuli, were usually considered as the basis

the physical factors, which have pronounced effects on various biological processes

(Carpenter 2013, Pilla 2013). However, the mechanisms of the biological effects of low-

intensity non-ionizing EMF, in spite of their widespread application in the prevention and

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treatment of various diseases, remain in many respects unknown (Pakhomov and Murphy

2000, Lushnikov et al. 2002, Karu 2003, Vladimirov et al. 2004, Betskii and Lebedeva 2007, Gapeyev 2014), although the ability of magnetic fields (MF) to engender genomic instability has recently been demonstrated (Luukkonen et al. 2014). It was found that the non-ionizing

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EMF is capable of modifying the effects of ionizing radiation in vitro and in vivo (ArtachoCordón et al. 2013, Vijayalaxmi et al. 2014).

An increased frequency of binucleated cells with micronuclei was observed in rat tracheal cell lines exposed to 6 Gy of γ-rays and alternating MF (50 Hz, 0.1 mT) compared

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with γ-irradiation alone, when MF alone had no significant direct effect on micronucleus induction (Lagroye and Poncy 1997). It was shown that the apoptosis rate of BEL-7402 cells, a human hepatoma cell line, exposed to X-rays (2-8 Gy) could be significantly increased by alternating MF (100 Hz, 0.7 mT) (Jian et al. 2009). Pre-exposure of human peripheral blood lymphocytes to 1950-MHz UMTS (Universal Mobile Telecommunications

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of increase in non-specific resistance. Non-ionizing electromagnetic fields (EMF) are one of

System) radiofrequency (RF) field (for 20 h at 0.3 W/kg specific absorption rate (SAR)) significantly decreased the number of micronuclei after subsequent exposure of the cells to X-rays (1 and 1.5 Gy) as compared with those subjected to X-rays alone (Sannino et al. 2014). Using the comet assay technique there were no found any synergistic effects of 1.8-

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GHz RF EMF at 2 W/kg SAR for 24 h and X-rays at doses of 0.25, 0.5, 1.0, and 2.0 Gy on human leukocytes in vitro (Zhijian et al. 2009). Experiments carried out on hepatoma-implanted mice have shown that combined MF (100 Hz, 0.7 mT) and X-ray (4 Gy) irradiation could significantly increase the survival and reduce the tumor size compared to MF or X-ray treatment alone (Wen et al. 2011). A reduction of the growth rate of breast cancer xenografts, retarding tumor vascularization and

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metastasis in mice exposed to therapeutic EMF (15 mT amplitude, 120 pulses per second) and γ-radiation (total dose of 8 Gy) compared with γ-radiation therapy alone was found

(Cameron et al. 2005). Pre-exposure of mice to 900-MHz RF EMF (0.12 mW/cm2, average

produced by γ-rays as evidenced by a significant increase in survival time (after 8 Gy γ-

irradiation) and less severe pathological alterations in bone marrow and spleen (after 5 Gy γirradiation) (Cao et al. 2010, 2011). Pre-exposure of mice to 900-MHz RF EMF (0.12

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mW/cm2) for 4 h/day for 3, 5, 7, and 14 days significantly decreased DNA damage in

peripheral blood leukocytes (Jiang et al. 2012) and micronuclei formation in immature erythrocytes in peripheral blood and bone marrow (Jiang et al. 2013) induced by exposure to γ-rays at a dose of 3 Gy. Exposure of mice and rats to 900-MHz RF EMF (GSM (Global

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System for Mobile communications) signal from mobile phone simulator, intermittent exposure for 6 h/day for 4-5 days) at 2 W power followed by 8.8 Gy or 8 Gy γ-radiation significantly increased survival rate of animals compared with that exposed to γ-rays alone (Mortazaviet al. 2012). It should be noted that in spite of the different frequency bands of

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applied EMF from extremely low frequency MF to RF EMF, and apparently different mechanisms of their action, the revealed biological effects are very similar. All these findings may be regarded as an induction of the adaptive response by non-ionizing EMF. Among the possible mechanisms of the protective effects are considered an intensification of

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whole-body SAR of about 54.8 mW/kg) for 1 h/day for 14 days attenuated the damage

DNA repair, activation of specific gene expression and protein synthesis, activation of antioxidant systems, stimulation of proliferation of hematopoietic stem cells/hematopoietic progenitor cells and acceleration of repopulation of injured cells, stimulation of the immune system, lowering the level of radiotoxins and oxidative stress in tissues, etc.

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Reactive oxygen species (ROS) and lipoperoxides resulting from radiation injury can have toxic effects manifested as the damage to cellular membranes and associated suppression of protective immune functions. Fatty acids (FA) determine physical and chemical properties of cellular membranes, but also mediate intracellular signaling systems (Calder 2013), are involved in anti-tumor (Jing et al. 2013), pro- and anti-inflammatory responses (Volpe and Nogueira-Machado 2013). It is assumed that the specific content of

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certain FA and their composition can make a difference in radioresistance and

radiosensitivity of biological objects. It was shown that depending on a dose of ionizing

radiation, the content of both saturated and unsaturated FA may be increased (Ayari et al.

reaction of cells from lipid peroxidation. However, an increase in the degree of unsaturation of FA can be also considered as a protective mechanism against the damaging effects of

ROS, since unsaturated FA possess the ability to neutralize ROS (Imlay 2003). One of the

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most sensitive to the damaging effects of ionizing radiation is the immune system. Thymus and T-cells are the major targets for the action of ionizing radiation on the immune system (Kominami and Niwa 2006, Iarilin 1999). Biochemical changes including modification of the lipid composition in thymic cells are the basic morphological manifestations of radiation

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damage to the thymus (Kolomiytseva et al. 2002).

Recently, we have shown that the exposure of mice to low-intensity extremely highfrequency electromagnetic radiation (EHF EMR, 42.2 GHz, 0.1 mW/cm2, 20 min single exposure or quintuple exposure for 5 consecutive days) leads to the changes in FA

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composition of thymic cells and blood plasma (Gapeyev et al. 2011, 2013). The results obtained demonstrate the ability of EHF EMR to cause significant changes in the balance of monounsaturated and polyunsaturated FA (MUFA and PUFA) in the norm and at pathology (systemic inflammation and tumor growth). We hypothesized that changes in the FA composition of organs and tissues under the influence of EHF EMR may contribute to the

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2009). An increase in the content of saturated FA (SFA) can be regarded as a protective

mechanisms of nonspecific protection against the damaging effects of ionizing radiation. The purpose of the present work was to determine a correlation between changes in

thymus weight and its FA composition in mice irradiated with X-rays, as well as to study possible protective and therapeutic effects of EHF EMR under X-irradiation of mice. MATERIALS AND METHODS

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Animals and groups Adult male random-bred white Kv:SHK mice (2 months of age, 22–25 g in body weight) were purchased from the Animal Breeding Facility (Russian Academy of Medical Sciences, Kryukovo, Moscow Region, Russia) and used in all experiments. SHK mice originate from the parent strain of Swiss mice (Staats 1965). The mice were housed in a vivarium and fed a standard diet with free access to standard chow and tap water. All

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manipulations with the animals were conducted in accordance with experimental protocols approved by the Local Animal Care and Use Committee (Branch of Shemyakin-

Ovchinnikov Institute of Bioorganic Chemistry, Pushchino, Moscow Region, Russia).

we used six groups of animals with 25 mice in each group: 1) the control group of mice,

which were sham-exposed (sham-control); 2) a group of mice that were X-irradiated (X-

rays); 3) a group of mice exposed to EHF EMR (EHF EMR); 4) a group of mice that were

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pre-exposed to EHF EMR and then X-irradiated in 30 min (EHF EMR + X-rays); 5) a group of mice that were pre-exposed to X-rays and then exposed to EHF EMR in 30 min (X-rays + EHF EMR); and 6) a group of mice that were pre-exposed to EHF EMR, X-irradiated in 30 min and then again exposed to EHF EMR in 30 min (EHF EMR + X-rays + EHF EMR). The

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animals were randomly distributed across the groups. The experiments were conducted in November-December 2013.

Chemicals

Disodium ethylenediaminetetraacetate (EDTA), phosphate-buffered saline, 2,6-Di-

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tert-butyl-4-methylphenol, methanol, NaOH, BF3, n-heptane, margaric acid, and analytical standard of FA were purchased from Sigma (St. Louis, MO, USA). X-ray exposure

X-irradiation of mice was carried out on an RUT-15 therapeutic X-ray device

(Mosrentgen, Moscow, Russia) at a dose rate of 1 Gy/min (focal distance 37.5 cm, current

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To assess changes in thymus weight, FA content, and FA composition in thymus,

20 mA, voltage 200 kV). Mice were whole-body irradiated at room temperature at maximal non-lethal dose of 4 Gy (Gudkov et al. 2009). EHF EMR exposure High frequency generators (G4-141; Istok, Fryazino, Russia) were sources of EHF EMR. Because of a great number of mice and exposure procedures, we used three generators

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with the same physical parameters. The whole-body exposure of mice to EHF EMR was conducted in the far-field zone of a pyramidal horn antenna with an aperture of 32×32 mm2 at a distance of 300 mm from the radiating end of the antenna. The mice were exposed from the top in plastic containers (LaboratorSnab, Moscow, Russia) with a size of 100×100×130 mm3 where animals moved freely. The breadth of the directional diagram of the electric field vector for the pyramidal horn antenna was 2θ 0.1 ~ 25o. Accordingly, the major lobe width E

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(0.1 level) was about 130 mm at a distance of 300 mm from the antenna (Gapeyev and

Chemeris 2010). The bottom square of the animal container corresponded to the square of

the plane of an exposed object, an effective multilayer absorbent was placed between the

animal container and the floor; therefore, the conditions of exposure were close to free-field conditions. We used the exposure conditions and parameters of EHF EMR whose high

efficacy has been shown previously (Gapeyev et al. 2009; Gapeyev et al. 2014): carrier

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frequency of 42.2 GHz, incident power density (IPD) of 0.1 mW/cm2, pulse modulation by a meander signal, a positively defined rectangular wave signal with 50% duty factor, at fixed frequency of 1 Hz. The frequency of the output signal was controlled by a CH2-25

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wavemeter (Istok). The frequency stability of the generator in the continuous wave mode was ±15 MHz. The output power of the generator was controlled with a M5-49 thermistor head and a M3-22A wattmeter (Istok). The IPD distribution in the plane of the exposed object was approximately normal with a maximal value in the center of the exposure zone and a twofold smaller value near the walls of the animal container. The IPD in the plane of

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exposed objects and the surface SAR in the mouse skin were calculated according to previous dosimetric studies, which were carried out using microthermometric, infra-red thermographic, and calculation methods (Gapeyev et al. 2002). The surface SAR was about 1.5 W/kg at an IPD of 0.1 mW/cm2. Control animals were sham-exposed by placing the mice

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the exposed zone created by the major lobe of the antenna. To eliminate the interference in

into the exposure zone when the generator was turned on but the output power was maximally attenuated (to < 1 μW). In all these experiments, the duration of the exposure and

sham exposure was 20 min. Exposure procedures were carried out in the daytime between 10 and 12 h at room temperature (18–22 oC) under conditions of natural illumination. The background induction of the geomagnetic field was 45±3 μT.

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Collection and preparation of biological samples In four-five hours, 10, 30, and 40 days after the last exposure, six mice per group were randomly selected and decapitated. Thymuses were collected, weighed, and homogenized by a glass homogenizer in cold phosphate-buffered saline with 1 mM EDTA. Samples of thymus homogenates were stabilized immediately by mixing with 0.5% v/v of antioxidant solution (10% of 2,6-Di-tert-butyl-4-methylphenol in methanol) and stored in a

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refrigerator.

Lipid methylation method and gas chromatography

in volume) was placed into a screw top vial, 100 μl of internal standard solution was added (margaric acid, 320 μg/ml in methanol), and the mixture was dried exhaustively in a Savant SpeedVac vacuum concentrator (Savant Instruments, Farmingdale, NY, USA).

Saponification and methylation stages were performed in a Multiblock heater (Lab-Line,

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Melrose Park, IL, USA) following the usual procedures (Knapp 1979) that were previously described (Gapeyev et al. 2013). Briefly, a dry residue was treated first with 150 μl of 0.5 N methanolic NaOH for 2 min at 65°C. The saponified mixture was methylated with 1 ml of a 10% BF3 methanolic solution for 40 min at 75°C. Then methyl esters were extracted into the

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organic phase by vigorous shaking with 1 ml of water and 1 ml of n-heptane. Water and heptane layers were separated by centrifugation in a low-speed Savant SpeedVac centrifuge (Savant Instruments, Farmingdale, NY, USA). The upper organic phase was used for the chromatographic determination of FA methyl esters. Gas chromatographic analyses were

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performed using a GC 3900 analytical gas chromatograph (Varian, Walnut Creek, CA, USA) equipped with a fused silica SUPELCOWAX-10 polar capillary column (15 m × 0.25 mm × 0.3 μm; Supelco, Bellefonte, PA, USA) and a flame ionization detector at 260°C. The splitless mode of sample injection (2 μl) was used (helium purge delay time, 0.2–0.5 min,

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Sample derivatization was carried out in the following way. A liquid sample (100 μl

depending on the concentration of solution analyzed). The column temperature ranged from 90°C (held for 0.5 min) to 240°C (held for 5 min) by ramping it at a rate of 6°C/min. The data were collected and analyzed using Multichrom software version 1.5x (Ampersend, Moscow, Russia). Individual concentrations of FA in biological samples were determined by means of the internal standard method; the corresponding calibration coefficients were

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calculated from the chromatograms of a standard FA mixture with margaric acid. The FA results are given as a total FA content per 1 mg of thymus weight (in μg/mg) and as a percentage of the total 13 FA that were quantified. Statistical analysis All experiments were conducted utilizing the "blind" experimental protocol, when an investigator making the measurements did not know which treatments the animals

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received. The data were systematized according to the type of treatment. All data are given as the mean ± standard error of the mean (SEM). The normality of data was analyzed using the Kolmogorov-Smirmov test. All the data matched the normal distribution. Comparisons

followed by the Dunnett's multiple comparison test. The p-value was adjusted to the critical level of 0.001. RESULTS

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There was no mortality in all experimental groups for the observation period of 40 days.

In four-five hours after X-irradiation of mice, the significant increase in the total FA content per 1 mg of thymic tissue was observed accompanied by a trend toward

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reduction in thymus weight (Tables I and II). There were no significant changes compared to the control in the thymus weight and total FA content in the thymus in the groups of mice exposed to various combinations of X-rays and EHF EMR (Tables I and II). The FA composition of thymic tissue in normal (sham-exposed) mice is shown in Table III. On

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the first day of the experiment, changes in the ratio of individual FA were observed in Xirradiated mice and in all groups of combined exposure. An increase in the content of stearic acid while reducing the content of its precursor palmitic acid did not change the summary content of SFA (Figure 1). A decrease in the content of palmitoleic and oleic acids, and the summary content of MUFA was accompanied by an increase in the

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between different treatments were made using one-way analysis of variance (ANOVA)

summary content of PUFA. This increase is due to the elevation of both dihomo-γlinolenic and arachidonic n-6 acids, and docosapentaenoic and docosahexaenoic n-3 acids. It should be noted that the exposure of animals to EHF EMR did not change the thymus weight (Table I) and did not affect the FA content and composition in the thymus during all periods of the study (Table II, Figures 1-3).

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In 10 days after the irradiation, the significant decrease in the thymus weight as compared to the control was observed only in X-irradiated mice and in mice exposed to EHF EMR after X-irradiation (Table I). In the mice exposed to X-rays alone, in contrast to the first period of the observations, the significant reduction in the content of total FA per mg of thymic tissue was found. But in mice exposed to EHF EMR after X-irradiation, there were no significant changes in the content of total FA per mg of thymic tissue

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(Table II). Preliminary exposure of mice to EHF EMR before X-irradiation slowed the

reduction of the thymus weight and total FA content compared to animals exposed to Xrays alone (Tables I and II). During this period, there was an increase in the summary

docosahexaenoic, and docosapentaenoic FA (Figure 2) as well as it was at the first period of the study. The summary content of SFA did not differ from the control, and the

summary content of MUFA was significantly reduced in the groups of animals exposed to

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X-rays alone and to EHF EMR before X-rays (Figure 2).

On day 30 of the study, the significant decrease in the thymus weight was observed in all groups of animals after X-irradiation and combined exposure (Table I). But the total content of FA per mg of thymic tissue in mice exposed to EHF EMR before

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or after X-irradiation was maintained on the control level (Table II). The total content of FA per mg of thymic tissue in mice exposed to X-rays alone remained significantly reduced. In the group of mice exposed to EHF EMR before and after X-irradiation, the significant increase in the total content of FA per mg of thymic tissue and the decrease in

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the thymus weight were observed (Tables I and II). On day 30, the decreased content of MUFA at the expense of oleic acid, and the increased content of PUFA mainly due to an increase in the content of dihomo-γ-linolenic acid at unchanged content of SFA were found in the thymic tissue of mice exposed to X-rays alone (Figure 3). The decrease in the summary content of SFA was observed at all variants of combined exposure compared to

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content of PUFA caused by increased percentages of dihomo-γ-linolenic, arachidonic,

the control and to the X-irradiation alone; the summary MUFA content in these groups of mice was significantly higher compared to the case of X-irradiation (Figure 3). The exposure of mice to EHF EMR before and after X-irradiation caused the significant increase in the summary content of MUFA compared to the control, probably at the expense of reduction of the summary content of SFA (Figure 3).

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On day 40 after the irradiation, the thymus weight in mice exposed to EHF EMR before or after X-irradiation was not statistically different compared to the control (Table I). In the mice exposed to X-rays alone, the thymus weight was significantly decreased as compared to the control. A recovery of thymus weight was not also observed on day 40 of the study in mice twice exposed to EHF EMR before and after X-irradiation (Table I). DISCUSSION AND CONCLUSIONS

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Shrinkage of the thymus and features of internucleosomal chromatin fragmentation in thymocytes upon the whole-body exposure of animals to ionizing radiation are well-

known phenomena (Umansky et al. 1981, Ohyama et al. 1985). But lipid metabolism during

metabolism in the thymic cells under the action of ionizing radiation vary significantly depending on the dose, irradiation conditions, study periods, etc. (Miras et al. 1968, Kolomiytseva et al. 1987, Ahlers et al. 1992, Kulagina 1997, Park et al. 2006).

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In our experiments, a tendency of decreased thymus weight in mice in 4-5 h after Xirradiation may indicate the initial stage of massive cell death. This accompanied by a sharp increase in the total FA content per mg of thymic tissue. An increase in the content of nonesterified and total FA was first found in the blood plasma of animals exposed to γ-radiation

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at a lethal dose (Lomova 1963). According to the author, this increase is due to lipolytic processes in adipose tissue and is an adaptive response of the organism intended for the elimination of the energy deficit in tissues. Causes of the significant increase in the FA content in the thymic tissue are not clear; and the observed increase is an integral indicator.

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One of the reasons for increasing the FA content in thymocytes during the initial period of their massive death may be a need for constructing additional membranes during apoptosis, as apoptotic bodies formed are coated by bilayer membranes (Wyllie et al. 1980, Wyllie 1997). Additional lipids may be transported from both the adipose tissue and the liver. The own ability of thymic cells to synthesize lipids was found after γ-irradiation of rats at a dose

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the thymus atrophy has not been studied in detail. A few data on changes in the lipid

of 4 Gy (Kolomiytseva et al. 1987). On day 10 of the study, at decreased thymus weight in X-irradiated mice we

observed the significant decrease in the total FA content per mg of the thymus weight, which correlated well with data demonstrating a reduced number of thymocytes at this stage of post-radiation recovery (Takada et al. 1969, Iarilin et al. 1985). The decrease in the total FA

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content per mg of thymic tissue may be due to changes in the ratio of proteins and lipids in the thymic tissue, since the connective tissue of the thymus is composed mainly of collagen and elastin fibers and carbohydrates (Wyllie et al. 1980) that are more resistant to Xirradiation. Perhaps, components of connective tissue damaged by X-rays are more slowly eliminated from the thymus. The reduction of the thymus weight and the total FA content per mg of thymic tissue in 10 and 30 days after X-irradiation of mice is probably due to the lack

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of recovery of thymic cellularity. In contrast to the data demonstrating the recovery of

thymic cellularity on the 30th day after X- or γ-irradiation of mice (Takada et al. 1969, Iarilin et al. 1985), in our experiments the decreased thymus weight of X-irradiated mice even in

after irradiation. The recovery process of thymic cellularity apparently depends on the radiation source, dose rate, and a resistance of animal line to ionizing radiation.

The absence of significant changes in the thymus weight and in the content and

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composition of FA after a single exposure of mice to EHF EMR, in contrast to multiple exposure (Gapeyev et al. 2013), during all periods of the study indicates the absence of cell death under the influence of low-intensity pulse-modulated EHF EMR. The results

obtained in this study and in our previous experiments (Gapeyev et al. 2011) indicate the

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absence of significant changes in lipid metabolism of the thymus in early and in remote periods after the single exposure of mice to low-intensity EHF EMR. We have shown that the combined action of EHF EMR and X-rays regardless of the sequence of each of the treatments does not significantly change the total FA content per mg

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of thymic tissue. This is probably that the EHF EMR exposure, improving microcirculation, accelerates the elimination of damaged fragments of protein stroma, and the rates of thymocytes' death and thymic weight loss become approximately equal in contrast to the case of X-irradiation alone. This may provide the unchanged total FA content per mg of the thymus weight compared to the control. Pre-exposure of mice to EHF EMR before X-

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day 40 after the exposure shows the absence of recovery of thymic cellularity at this stage

irradiation in groups "EHF EMR + X-rays" and "EHF EMR + X-rays + EHF EMR" delays the reduction of the thymus weight, as it is not statistically different from the control on day 10 after exposure. Slower decline in the thymus weight in mice pre-exposed to EHF EMR before X-irradiation may be due to a later date of the thymocytes' death and/or inhibition of the phagocytic activity by the EHF EMR (Kolomytseva et al. 2002, Lushnikov et al. 2003).

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We suppose that the pre-exposure to low-intensity EHF EMR is capable of inducing an adaptive response that weakens the influence of X-rays. The detailed mechanisms of the adaptive response remain poorly understood and depend on EMF parameters (Manti and D'Arco 2010, Vijayalaxmi et al. 2014). The partial recovery of thymus weight in 40 days after the combined action of EHF EMR and X-rays in both groups of mice ("EHF EMR + Xrays" and "X-rays + EHF EMR") to a level that is not statistically different from the control

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indicates to the beneficial effect of EHF EMR on regenerative/reparative processes in the thymus.

The overall dynamics of the FA composition of the thymus in X-irradiated mice is

the content of MUFA is decreased at a constant content of SFA. Later on days 10 and 30 of the observations, this profile of FA is persisted in mice exposed to X-rays alone. All time

points (1, 10, and 30 days) are characterized by more than 1.5-fold increase in the levels of

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dihomo-γ-linolenic acid, probably due to its involvement in regulation of cell proliferation and apoptosis (Wang et al. 2012). Changes in the percentage content of both individual and summary SFA, MUFA, and PUFA under combined action of EHF EMR and X-rays on days 1 and 10 after the exposure are very close to the changes caused by X-rays alone.

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Apparently, at the combined action, the changes in the percentage FA composition at a constant total FA content per mg of thymic tissue are produced by X-rays. However, on the 30th day of the observations, MUFA and PUFA contents are returned to the control level, and the content of SFA is decreased in the groups of mice after the combined exposure to

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EHF EMR and X-rays. The recovery of the summary content of PUFA and MUFA in 30 days after the combined action of EHF EMR and X-rays indicates to the regeneration of the thymus. However, specific reasons for changes in the percentage composition of individual FA are still unknown.

In the group of mice exposed to EHF EMR before and after X-irradiation, the

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as follows. At the initial stage after radiation injury, the content of PUFA is increased and

decrease in the thymus weight with a significant increase in the total FA content per mg of the tissue on the 30th day after the exposure is very close to that observed in 4-5 h after Xirradiation alone. But in this group of mice there is a significant increase in the content of oleic and summary MUFA during this period, which may indicate to changes in a balance of anti-apoptotic/pro-apoptotic activity (Ahn et al. 2013). Reasons for the lack of recovery of

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the thymus weight even in 40 days after the combined irradiation in this group of mice are unclear and require further studies at a later date after irradiation. Thus, we have shown that X-irradiation of mice at a dose of 4 Gy causes a sharp increase in the total FA content per mg of thymic tissue in 4-5 h after the treatment. The exposure of mice to pulse-modulated EHF EMR (42.2 GHz, 0.1 mW/cm2, 20 min exposure, pulse modulated by a meander with a frequency of 1 Hz) before X-irradiation slows the

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reduction in the thymus weight compared to both the X-irradiation alone and exposure to EHF EMR after X-irradiation. The EHF EMR exposure facilitates the restoration of the

thymus weight of X-irradiated mice to day 40 of the observations, that is more expressed in

effects of EHF EMR may be associated with the induction of an adaptive response, positive effects on the regenerative/reparative processes in the thymus, as well as immunomodulatory effects. Changes in the content and composition of PUFA in the early period after the

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treatments as well as at the restoration of the thymus under the combined action of EHF EMR and X-rays indicate to an active participation of FA in the acceleration of postradiation recovery of the thymus by EHF EMR exposure. Declaration of interest

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The authors report no conflicts of interest.

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mice exposed to EHF EMR before X-irradiation. The mechanisms of the radiation protective

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Table I. Thymus weight (in mg) in normal mice (sham-control), in X-irradiated mice, in mice exposed to EHF EMR (42.2 GHz, 0.1 mW/cm2, 20 min, 1 Hz pulse-modulation), in mice exposed to EHF EMR and X-irradiated, in X-irradiated mice and exposed to EHF EMR, in mice exposed to EHF EMR and X-irradiated and exposed to EHF EMR at different

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time points after exposure.

Time points

Groups

10 days

30 days

40 days

47.0 ± 4.9

46.0 ± 4.2

45.0 ± 3.4

24.3 ± 3.9*

29.9 ± 3.8*

25.6 ± 3.6*

43.4 ± 4.7

36.5 ± 3.8

39.0 ± 4.6

35.4 ± 2.3

25.9 ± 4.2*

35.4 ± 5.1

44.3 ± 4.4

23.9 ± 3.3*

21.6 ± 2.5*

32.1 ± 4.6

50.5 ± 3.9

35.5 ± 3.8

24.8 ± 5.6*

26.2 ± 4.5*

48.8 ± 4.9

X-rays

33.7 ± 8.5

EHF EMR

49.5 ± 5.9

EHF EMR + X-rays

45.5 ± 4.7

X-rays + EHF EMR EHF EMR + X-rays +EHF EMR

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Sham-control

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Data are presented as mean ± SEM, n = 6 for each of the treatments.

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* p < 0.001 versus sham-control.

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4-5 h

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Table II. The total FA content of thymic tissue (in μg/mg) in normal mice (sham-control), in X-irradiated mice, in mice exposed to EHF EMR (42.2 GHz, 0.1 mW/cm2, 20 min, 1 Hz pulse-modulation), in mice exposed to EHF EMR and X-irradiated, in X-irradiated mice and exposed to EHF EMR, in mice exposed to EHF EMR and X-irradiated and exposed to EHF

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EMR at different time points after exposure.

Time points 10 days

30 days

Sham-control

14.6 ± 0.9

15.1 ± 1.0

14.2 ± 0.9

X-rays

22.7 ± 3.2*

10.6 ± 0.9*

9.3 ± 0.6*

EHF EMR

12.9 ± 0.9^

15.4 ± 0.3^

13.7 ± 1.2

EHF EMR + X-rays

15.0 ± 0.6^

13.3 ± 0.7

14.0 ± 0.9^

13.0 ± 0.6^

14.8 ± 1.5^

16.6 ± 0.8^

10.8 ± 0.2^

13.5 ± 1.3

22.1 ± 2.6*^

X-rays + EHF EMR

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4-5 h

EHF EMR + X-rays +EHF EMR

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Data are presented as mean ± SEM, n = 6 for each of the treatments.

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* p < 0.001 versus sham-control, ^ p < 0.001 versus X-irradiation.

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Groups

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Table III. Percentage content of FA in thymic tissue of normal mice (sham-control).

Fatty acids Pentadecanoic (C15:0)

0.099 ± 0.014 31.9 ± 0.7

Stearic (C18:0)

12.2 ± 0.7

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Palmitic (C16:0)

44.2 ± 0.8

Sum of SFA

6.0 ± 0.2

Oleic (C18:1; n-9)

22.8 ± 0.8 28.8 ± 0.9

Sum of MUFA

γ-Linolenic (C18:3; n-3) α-Linolenic (C18:3; n-3)

13.5 ± 0.6

0.049 ± 0.006

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Linoleic (C18:2; n-6)

0.28 ± 0.02

Dihomo-γ-linolenic (C20:3; n-6)

9.6 ± 0.6

Eicosapentaenoic (C20:5; n-3)

0.081 ± 0.016

Docosapentaenoic (C22:5; n-3)

0.53 ± 0.04

Docosahexaenoic (C22:6; n-3)

2.18 ± 0.23

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Arachidonic (C20:4; n-6)

0.75 ± 0.04

27.0 ± 0.6

Sum of PUFA

Data are presented as mean ± SEM, n = 6.

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Palmitoleic (C16:1, n-7)

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Figure 1. Changes in the FA content (in % of the control) in X-irradiated mice, in mice exposed to EHF EMR (42.2 GHz, 0.1 mW/cm2, 20 min, 1 Hz pulse-modulation), in mice exposed to EHF EMR and X-irradiated, in X-irradiated mice and exposed to EHF EMR, and in mice exposed to EHF EMR and X-irradiated and exposed to EHF EMR in 4-5 h after the exposure. Data are mean ± SEM, n = 6 for each of the

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treatments, * p < 0.001 versus sham-control.

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Figure 2. Changes in the FA content (in % of the control) in X-irradiated mice, in mice exposed to EHF EMR (42.2 GHz, 0.1 mW/cm2, 20 min, 1 Hz pulse-modulation), in mice exposed to EHF EMR and X-irradiated, in X-irradiated mice and exposed to EHF EMR, and in mice exposed to EHF EMR and X-irradiated and exposed to EHF EMR in 10 days after the exposure. Data are mean ± SEM, n = 6 for each of

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the treatments, * p < 0.001 versus sham-control.

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Figure 3. Changes in the FA content (in % of the control) in X-irradiated mice, in mice exposed to EHF EMR (42.2 GHz, 0.1 mW/cm2, 20 min, 1 Hz pulse-modulation), in mice exposed to EHF EMR and X-irradiated, in X-irradiated mice and exposed to EHF EMR, and in mice exposed to EHF EMR and X-irradiated and exposed to EHF EMR in 30 days after the exposure. Data are mean ± SEM, n = 6 for each of

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the treatments, * p < 0.001 versus sham-control, ^ p < 0.001 versus X-irradiation.

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