GENOTOXICITY EVALUATION OF RADIOFREQUENCY EXPOSURE AT 1800 MHz GSM IN OVARIAN TISSUES OF ADULT FEMALE RATS Dr. ALI SAEED AL-CHALABI Dr. Erkihun Aklilu Dr. Abdul Rahman Bin Aziz Dr. Mohd Azam Khan B. Goriman Khan Faculty of Veterinary Medicine, Universiti Malaysia Kelantan (UMK)

Introduction Exposure to electromagnetic fields from mobile communications has been increasing during the past 10 years. A lot of concerns have been raised due to the introduction of the new technologies without the provisions of adequate public information about the potential health consequences. Damages to the reproductive organs can cause subfertility and abnormality in embryonic development. , Several non-infective factors, including exposures to non-ionizing EMF, especially RF fields used in mobile communications have been proposed as possible risk factors contributing to infertility (Poulletier de Gannes et al., 2013). Human infertility and its correlation to ovarian function and cytological changes linked to ever-increasing and diversity of mobile phone technologies, and all kinds of wireless communications become a critical subject due to increasing number of studies in this field with controversial outcomes. This is perhaps due to variations in the experimental design in terms of a source of EMWs, frequency, exposure duration and the specific absorption rate (SAR) used. .

The aim of the study To evaluate the genotoxic effect of electromagnetic field of GSM frequency on ovarian tissue through assessing oxidative stress status and DNA damage biomarkers (8-OHdG enzyme and micronuclei formation) in ovarian tissues.

Methodology Forty female Sprague-Dawley rats were distributed over the four groups (control and three exposure groups(, whole-body exposure for 2h/day, seven days / week for 15, 30 and 60 continuous days were applied during the experiment at 1800 MHz GSM-like frequency of mobile phone using PSG vector signal generator at calculated SAR level 0.974 W/kg. Figure 1. It shows the EMF exposure setup using GSM like radiofrequency generator system.

Histopathological changes Experimental design RF - EMW Control group Exposed groups 15-Days exposure 30-Days exposure 60-Days exposure Whole body irradiation, 2h/day at SAR value 0.974 W/Kg B.W Oxidative stress MDA GSH-Px Mt Genotoxicity 8-OHdG Microneuclie formation Histopathological changes Statistical Analysis ANOVA One-way, Post Hoc LSD at P<0.05

Results 1. Effect of RF-EMW on oxidative stress biomarkers * * * * Figure 1: effect of RF-EMF at 1800 MHz frequency on ovarian MDA level Figure 2: Effect of RF=EMF at 1800MHz frequency on ovarian GSH-Px level * * Figure 3: Effect of RF=EMF at 1800 MHz serum Melatonin level

2. Effect of RF-EMW on DNA damage biomarkers Figure 4: Effect of GSM-like signal at 1800 MHz frequency on a serum 8-OHdG enzyme level Table 1: Effect of RF-EMW on micronuclei formation in ovarian tissue sections Period 15 days of exposure 30 days of exposure 60 days of exposure Groups Positive rate (%) Chi-square value P value control 14 1.261 0.532 12 5.577 0.062 11.331 0.003 2h/day exposure 19 25 33 *

3. Effect of RF-EMW on histopathological changes in ovarian sections Figure 5: Show pre-ovulatory belongs to ovarian section of the 30-days exposure group with follicle micronucleus formation in oocyte and autophagy granulosa cells strongly contact with the oocyte which characterized by a thin zona pellucida. Vacuolation of ooplasm and granulosa cell's layer with separation of granulosa cells was indicated by arrows. H&E stain 100X Figure 6: 60-day’s exposure group show mature ovarian follicle with vacuolation in theca cell and granulosa cell layers in addition to separation of granulosa cell with micronucleus formation was indicated by arrows. H&E stain 100X

Figure 7: 60-day’s exposure group show mature ovarian follicle with numerous granulosa cells separated from granulosa cell layer had micronuclei formation and indicated by arrows. Vacuolation in granulosa cell layer and theca cell layer was clear. H&E stain 100X Figure 8: ovarian section of the ovary belongs to the 60-days exposure group shows enlarged luteal cells with micronuclei formation indicated by arrows. Numerous vacuolated luteal cells present in this section. H&E stain 100X

DISCUSSION & CONCLUSION Imbalance in the oxidant and antioxidant system leads to enhanced production of free radicals who have the ability to attack large molecules such as proteins and lipids and nucleic acids (DNA), micronuclei appearance is one the DNA damages shapes, leading to occur apoptosis, which results in a defect in the function of cells. Theca interstitial cells are sensitive to free radicals' level and reduction in antioxidant triggers apoptosis and antioxidants with precisely different mechanisms of action stimulate a course of actions consistent with the apoptosis mechanism in an ovarian mesenchyme. Our data indicate that chronic RF-EMW exposure lead to the genotoxic effect in ovarian tissues via oxidative stress mechanism which is related to the pathogenesis and progression of ovarian disorders leading to infertility.

References Avci, Bahattin, Ayşegül Akar, Birşen Bilgici, and Özgür Korhan Tunçel. 2012. “Oxidative Stress Induced by 1.8 GHz Radio Frequency Electromagnetic Radiation and Effects of Garlic Extract in Rats.” International journal of radiation biology 88(11): 799–805. Dasdag, Suleyman et al. 2012. “Effect of 900 MHz Radio Frequency Radiation on Beta Amyloid Protein, Protein Carbonyl, and Malondialdehyde in the Brain.” Electromagnetic Biology and Medicine 31(1): 67–74. Gharib, Ola Ali, Usama Abdelnaby Abd Ellatif, Nadia Mohammad Abdellah, and Marwa Abdelhameed Mohammad. 2012. “Radio-Protective Response on the Environmental Pollutant Induced Oxidative Stress.” Advances in Bioscience and Biotechnology 03(07): 989–96. Halliwell1, B. 2007. “Biochemistry of Oxidative Stress.” inflammation 35(5): 1147–50. Kerman, Memduh, and Nilgun Senol. 2012. “Oxidative Stress in Hippocampus Induced by 900 MHz Electromagnetic Field Emitting Mobile Phone : Protection by Melatonin.” Biomedical Research 23(1): 147–51. Kismali, Gorkem et al. 2012. “The Influence of 1800 MHz GSM-like Signals on Blood Chemistry and Oxidative Stress in Non-Pregnant and Pregnant Rabbits.” International journal of radiation biology 88(5): 414–19. Mattsson, Mats-Olof, and Myrtill SimkÃ3. 2014. “Grouping of Experimental Conditions as an Approach to Evaluate Effects of Extremely Low-Frequency Magnetic Fields on Oxidative Response in in Vitro Studies.” Frontiers in Public Health 2(September): 1–11. Ozgur, Elcin et al. 2013. “Effects of Prenatal and Postnatal Exposure to GSM-like Radiofrequency on Blood Chemistry and Oxidative Stress in Infant Rabbits, an Experimental Study.” Cell biochemistry and biophysics 67(2): 743–51. Poulletier de Gannes, Florence et al. 2013. “Rat Fertility and Embryo Fetal Development: Influence of Exposure to the Wi-Fi Signal.” Reproductive toxicology 36: 1–5. Tök, Levent et al. 2014. “Effects of Melatonin on Wi-Fi-Induced Oxidative Stress in Lens of Rats.” Indian journal of ophthalmology 62(1): 12–15. Yakymenko, Igor, Evgeniy Sidorik, Diane Henshel, and Sergiy Kyrylenko. 2014. “Low Intensity Radiofrequency Radiation: A New Oxidant for Living Cells.” Oxid Antioxid Med Sci 3(1): 1–3.