RADIOPROTECTION OF WISTAR RAT LYMPHOCYTES AGAINST MICROWAVE RADIATION MEDIATED BY BEE

Vera Garaj-Vrhovac and Goran Gajski Institute for Medical Research and Occupational Health, Zagreb, Croatia [email protected] , [email protected]

INTRODUCTION Microwave radiation is a type of non-ionising electromagnetic radiation present in the environment, and is a potential threat to human health. Today, non-ionising radiation has increasingly been used in industry, commerce, medicine, and for private purposes, especially in mobile telephone usage. Although average exposure levels are low compared to exposure limits, there is a growing public concern about the potential hazard of exposure to these frequencies for human health [1-3]. Cytogenetic studies of microwave radiation were conducted in vitro and in vivo, and have yielded contradictory and often intriguing experimental results. Some reports suggest that exposure of human cells to radiofrequency radiation does not result in increased cytogenetic damage. On the other hand, there is a range of studies showing that radiofrequency radiation can indeed induce genetic alteration after exposure to electric field. In addition to experiments with human cells, a number of studies have been conducted on animal models. These studies have also given contradictory results regarding exposure to microwave [4-6]. Bee venom is used in traditional medicine to treat variety of conditions, such as arthritis, rheumatism, back pain and skin disease. In recent years it has been reported that bee venom possesses antimutagenic, proinflammatory, anti-inflammatory, antinociceptive, and anticancer effects. In addition to the wide range of the bee venom’s activities, it also possesses a radioprotective capacity that was noted against X-ray and gamma radiation in various test systems [7,8]. Because of the lack of conclusive data on the genotoxic status induced by microwave radiation, the aim of the present study was assessment of the radioprotective effect of bee venom against 915 MHz microwave radiation- induced DNA damage in the Wistar rat’s lymphocytes in vitro. The possible genotoxic effect of bee venom alone was also assessed on non-irradiated

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lymphocytes. For that purpose the alkaline comet was used as a sensitive tool in detection of DNA damage. In addition to the standard protocol for the , the Fpg-modified comet assay was also used as a much more sensitive and specific technique in detection of DNA strand breaks and .

MATERIAL AND METHODS Adult male Wistar rats (11 weeks old, approximate body weight 350 g) were used in this study. The animals had passed through an accommodation period of 1 week. The animals were kept in steady-state microenvironment conditions, (22 ± 1) °C, and received standard laboratory food and water ad libitum, with alternating 12 h light and dark cycles. The whole blood samples were collected by cardiac puncture under sterile conditions in heparinized vacutainer tubes (Becton Dickinson, USA) containing lithium heparin as anticoagulant. After collection, blood was divided into 1 ml aliquots and placed into 24-well culture plates according to the exposure conditions. Lyophilized bee venom was dissolved in sterile redistilled water at 25 ºC and centrifuged at 12,000 rpm for 10 min to remove insoluble materials. Bee venom was added to lymphocyte cultures in final concentration of 1 µg/ml, 4 h prior to irradiation and immediately before irradiation. To test whether this concentration alone induces genotoxic effect lymphocytes were treated under the same conditions with bee venom in corresponding time periods. All experiments were conducted on whole blood samples at 37 ºC in an atmosphere of 5 % CO2 in air. An electromagnetic field was generated within the certified gigahertz transversal electromagnetic mode cell (GTEM-cell, USA). Whole blood was exposed to the carrier frequency of 915 MHz with GSM basic signal modulation for 30 minutes. Incident electromagnetic field strength of 30 V/m was uniform over the entire biological object throughout the exposure procedure. The power density of the field was 2.4 W/m2, corresponding to approximate whole body specific absorption rate (SAR) of 0.6 W/kg [9]. The comet assay was carried out under alkaline conditions, as described by Singh et al. [10] whereas the analysis of 8-oxo-7, 8-dihydro-2’- deoxyguanozine (8-oxodG) was performed using an Fpg FLARE™ assay kit (Trevigen Inc, USA). Statistical analysis was performed using Statistica 5.0 package (StaSoft, USA). The level of statistical significance was set at P < 0.05.

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RESULTS According to one-way ANOVA only parameter for tail length showed significant deviation from normal distribution in the alkaline comet assay. In the Fpg-modified comet assay all three parameters (tail length, tail intensity and tail moment) showed significant deviation from normal distribution for irradiated sample. When comparing mean values between different samples in both comet tests, difference was higher when the Fpg-modified comet assay was applied. All three parameters of the Fpg-modified comet assay showed statistically significant differences between irradiated group as compared to the control, to the BV treatment and to the MW/BV treatment groups (Figures 1 and 2).

20 Control BV (0.5h) 15 a,b,c BV (4.5h) MW (0.5h) 10 MW/BV (0.5h/0.5h) MW/BV (0.5h/4.5h) 5

0 Tail length Tail intensity Tail moment

Alkaline comet parameters Figure 1. Parameters of the alkaline comet assay (tail length, tail intensity and tail moment) measured in Wistar rat lymphocytes of controls, after exposure to microwave radiation (MW, 915 MHz), after treatment with bee venom (BV, 1 µg/ml) and after joint MW/BV treatment. a – statistically significant difference as compared to the control; b – statistically significant difference as compared to the BV treatment; c – statistically significant difference as compared to the MW/BV treatment; level of statistical significance was set at P < 0.05

When the standard comet assay was applied, the mean tail length for irradiated sample differed significantly from non-treated control samples, BV-treated samples, and from MW/BV-treated samples. For tail intensity and tail moment there was no significant difference found between the

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control sample, and samples treated only with BV or with MW/BV. In the Fpg-modified comet assay all three parameters differed significantly from non-treated control samples, BV-treated samples, and from MW/BV-treated samples. Generally, the means and medians of all three standard comet assay parameters were lower than of the Fpg-modified comet assay. These findings suggest that the Fpg-modified comet assay is more sensitive to the effects of microwave radiation than the standard comet assay.

20 a,b,c Control BV (0.5h) 15 BV (4.5h) MW (0.5h) 10 MW/BV (0.5h/0.5h) MW/BV (0.5h/4.5h) a,b,c 5

a,b,c 0 Tail length Tail intensity Tail moment Fpg-modified comet parameters Figure 2. Parameters of the Fpg-modified comet assay (tail length, tail intensity and tail moment) measured in Wistar rat lymphocytes of controls, after exposure to microwave radiation (MW, 915 MHz), after treatment with bee venom (BV, 1 µg/ml) and after joint microwave MW/BV treatment. a – statistically significant difference compared to the control; b – statistically significant difference compared to the BV treatment; c – statistically significant difference compared to the MW/BV treatment; The level of statistical significance was set at P < 0.05

CONCLUSION Results obtained by the Fpg-modified comet assay suggest that oxidative stress can be the cause for DNA damage in Wistar rat lymphocytes in addition to some basal DNA damage detected with the standard alkaline comet assay after exposure to 915 MHz microwave radiation in vitro. Bee venom in this study demonstrated radioprotective effects against low level of microwave radiation when administered prior to the radiation exposure and immediately before irradiation. This study also demonstrated that bee venom has radioprotective effect against basal and

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oxidative DNA damage. Furthermore, bee venom itself has no effect on induction of DNA breaks or on oxidative stress in the low concentrations used in this research. A combination of the standard and the Fpg-modified comet assay has proven usefulness in detecting of microwave radiation and radioprotection of bee venom.

Acknowledgment The study was supported by the Croatian Ministry of Science, Education and Sports (grant 022-0222148-2125). The authors thank Radiation Dosimetry and Radiobiology Unit, Institute for Medical Research and Occupational Health for the opportunity to use the GTEM-cell in this experiment.

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