Effects Of Radio Frequency Electromagnetic Radiation On Testosterone Secretion Of Male SD Rats And TM3 Cells

Background With the rapid development of communication, mobile telephone and other communication equipments are more and more popular. Nowadays, the exposed duration and intensity of human to mobile phones produced by radiofrequency electromagnetic fields(Radiofrequency Field, RF) are also increasing, therefore whether the harmful effects could induce by radiofrequency electromagnetic radiation on human health or not are concerned worldwide.Previous reports have shown that RF radiation may have adverse effects on the reproductive system, central nervous system and immune system. The reproductive system, which can produce reproductive cells, secret hormones, and maintain the important function such as secondary sex characteristic, is the important target organs of RF radiation. Therefore the relationship between RF radiation and the reproductive system became a hot issue of research in recent years. But as a result of these studies reported with different subjects and irradiation parameters, the experimental results could not be the same, and lacking of mechanism.Therefore, we choose the most commonly used radiation frequency of mobile phone such as 1840 MHZ and 1950 MHZ in our country at present, use SD rats and mice leydig cells(TM3) as the research object to analyze the effects of RF radiation on the reproductive system and make preliminary discussion on its possible mechanism from the morphology structure, hormone level, antioxidant system, energy metabolism enzyme and other aspects.Purpose To explore the effects of RF radiation on the male testosterone secretion, to further understand the biological effect of RF radiation produced by the mobile communication equipment produced on the reproductive system, to provide new theoretical basis and supported data for the protection of human health.Method Animal experiment: 24 adult male SD rats(6-week-old) were randomly divided into sham-exposure and exposure group according to the weight. SD rats were sham or exposed to the RF radiation with the frequency is 1840 MHz, the output power of 170 watts(W) and specific absorption rate(SAR) in testis of 3W/kg for continuous 5d, 30 min for every day. Rats were sacrificed to collect heart blood and testicular tissue specimens for the following indicators at immediately and 1week after radiation:1. Tissue morphology: Body and testicle weight of each rat was observed and testicle index was calculated. HE staining testicular tissue morphology was observed by measurement of cross seminiferous tubule diameter;2. Hormone secretion: Serum hormones such as Testosterone(T), gonadotrophin releasing hormone(Gn RH), Luteinizing hormone(LH) and follicle-stimulating hormone(FSH) were measured by radioimmunoassay;3. Antioxidant capacity: The activity of total antioxidant capacity(T-AOC), superoxide dismutase(SOD) and the contents of malondialdehyde(MDA) were determined by colorimetric method;4. Energy metabolic enzymes: The activity of lactic dehydrogenase(LDH), succinate dehydrogenase(SDH) and Ca-Mg-ATPase was determined by colorimetric method; Cell experiment:Mice Leydig cell(TM3) was used and irradiated by 1950 MHz RF radiation(s Xc irradiation system, GSM talk signal, the frequency 1950 MHz, SAR value: 3w/kg). Continuous irradiation for 24 h, at 0d, 1d, 2d, 3d, 4d and 5d after irradiation, cells and cell supernatant from the irradiated or sham-irradiated were collected for the following indicators:1. Cell proliferation: Cell proliferation was analysed by CCK-8 method and clony formation;2. Cell cycle and Cell apoptosis: Cell cycle and cell apoptosis were measured by flow cytometry;3. Testosterone: The testosterone level in TM3 cells was measured by ELISA; 4. ROS: The ROS level in TM3 cells was measured by flow cytometry and spectrometer;5. Antioxidant capacity: The activity of superoxide dismutase(SOD) and the contents of malondialdehyde(MDA) and glutathione(GSH) in TM3 cells were determined by ELISA;6. Testosterone secretion factor mRNA: Real-time polymerase chain reaction(PCR) was used to detect the steroidogenic acute regulatory protein(St AR), cholesterol-side-chain cleavage enzyme(P450scc) and Androgen Receptor(AR) m RNA expression level in TM3 cells.Result Animal experiment: 1. Effects of 1840 MHz RF radiation on the testicle of male SD rats: After 1840 MHz 3w/kg(30min/d) RF exposure for consecutive 5d, rat body weight, testicle weight, and testicle index in the irradiated group at 0d and 7d compared with the sham-irradiated group, the difference were not significant statistically. The length of seminiferous tubule diameter in the irradiated group was lower than the sham-irradiated group after radiation, but without any statistically significant.2. Effects of 1840 MHz RF radiation on the level of hormone in male SD rats: After 1840 MHz 3w/kg(30min/d) RF exposure for consecutive 5d, the level of serum testosterone(T) in the irradiated group was lower than the sham-irradiated group after radiation, and decrease significantly compare to the sham-irradiated group at 7d after exposure(p<0.05). There was no significant difference in the level of Gn RH, LH and FSH between the irradiated group and the sham-irradiated group at every time point after radiation.3. Effects of 1840 MHz RF radiation on the anti-oxidative capability of testicle in male SD rats: After 1840 MHz 3w/kg(30min/d) RF exposure for consecutive 5d, the activity of total antioxidant capacity(T-AOC) in the irradiated group increased significantly compare to the sham-irradiated group at 0d after exposure(p<0.05), and decreased slightly without any significant difference. The activities of SOD and the contents of MDA in the irradiation group did not change significantly compared with the sham-irradiated group after radiation.4. Effects of 1840 MHz RF radiation on the energy metabolic enzymes of testicle in male SD rats: After 1840 MHz 3w/kg(30min/d) RF exposure for consecutive 5d, there was no significant difference in the activity of lactic dehydrogenase(LDH) and Ca-Mg-ATPase between the irradiated group and the sham-irradiated group. But the activity of succinate dehydrogenase(SDH) in the irradiated group increased significantly compare to the sham-irradiated group at 7d after exposure(p<0.05).Cells experiment: 1. Testosterone secretion in mice testicular Leydig cells induced by 1950 MHz RF radiation. 1.1 TM3 cells were irradiated by 1950 MHz 3w/kg RF radiation(s Xc irradiation system, GSM talk signal, the frequency 1950 MHz, SAR value: 3w/kg). Compared to the sham-irradiated group, the cell proliferation in the irradiated group was inhibited from 2d to 9d after irradiation(p<0.05). Results from the clonogenic assay also proved that cells proliferation in the irradiation was slowlier than sham-irradiation cells, the difference was statistically significant(p<0.05).1.2 The cell cycle and cell apoptosis was measured by flow cytometry, the results indicated that cells in S phase in the irradiated group increased significantly compared with the sham-irradiated group at 1d, 2d, 3d, 4d and 5d after irradiation(p<0.05); percentage of cells in G1 phase in the irradiated group decreased significantly compare with the sham-irradiated group at 3d and 5d after irradiation(p<0.05). There was no significant difference in cell apoptosis between the irradiated group and the sham-irradiated group.1.3 The content of T in the cell supernatant in the irradiated group decreased compared to the sham-irradiated group at 1d, 2d, 3d, 4d and 5d after irradiation, and significantly lower than the sham-irradiated group at 1d, 2d and 4d after exposure(p<0.05). The content of T in cell lysates in the irradiated group decreased compared to the sham-irradiated group at 1d, 2d, 3d, 4d and 5d after irradiation, and significantly lower than the sham-irradiated group at 1d and 2d after exposure(p<0.05).2. Mechanisms of 1950 MHz RF radiation on Testosterone secretion in TM3 cells.2.1 There was no significant difference in the ROS level of TM3 cells between the irradiated group and the sham-irradiated group measured by flow cytometry. But the results from using spectrometer method, showed that the contents of ROS in the irradiated group were significantly lower than the sham-irradiated group at 2d, 3d, 4d and 5d after exposure(p<0.05).2.2 Compare with the sham-irradiated group, the activity of SOD in the irradiated group increased significantly at 1d after exposure(p<0.05), and there was no significant difference at 2d, 3d, 4d and 5d after exposure; The contents of GSH in the irradiation group did not change significantly at every time point after exposure; The contents of MDA in the irradiation group decreased after exposure, and decreased significantly at 4d and 5d after exposure(p<0.05).2.3 The content of mRNA expression level of St AR, P450 scc and AR were detected by Real time-PCR at every time point after continuous irradiation for 24 h. At 4d and 5d after irradiation, the m RNA expression level of AR in the irradiated group significantly decreased compared to the sham-irradiated group(p<0.05). But AR m RNA expression level didn’t change significantly in the 1-3d after irradiation. At 1d, 2d, 3d, 4d and 5d after irradiation, the St AR m RNA expression level in the irradiation group decreased compare to the sham-irradiated group. And at 5d after irradiation, the St AR m RNA expression level in the irradiation group decreased significantly in the sham-irradiated group(p<0.05). At 2d, 3d, 4d and 5d after irradiation, the P450 scc m RNA expression level in the irradiation group decreased significantly compare to the sham-irradiated group(p<0.05).Conclusion Results from present studies indicated that: 1.The level of testosterone(T) and some testicular energy metabolism enzyme changed in the irradiated SD male rats after 1840 MHz 3w/kg RF exposure 5 days. 2. TM3 cells were irradiated by 1950 MHz, 3w/kg RF 24 h. TM3 cells proliferation inhibited, capacity of testosterone synthesis and secretion reduced, and the gene related T synthesis such as P450 scc m RNA expression level changing induced by the present 1950 MHz RF exposure. The contents of free radical products MDA reduced and ROS level didn’t induced by the present exposure condition. Our results showed that oxidative stress couldn’t induced by present RF radiation in TM3 cells and the mechanisms remain to be explored further in future.