Study On The Female Genital Toxicities And Its Related Mechanism By HF-EMF And The Protection Effect Of Isoflavone

Radiofrequency radiation (RF) is a kind of non-ionising radializations, which contains microwave (300MHz–300GHz) and high-frequency electromagnetic field (HF-EMF, 100kHz-300MHz). HF-EMF are used in many situations, including radio frequency gluing in shoes factory, plastic heated combine in plastic factory, quenching metal smelting in machinery factory and oscillight manufactural procedure in electronic factory. Epidemiological survey and animal experiment show that genital system is the organ most frequently damaged by electromagnetic radiation, which had arisen scientific and public concern. HF-EMF is one of electromagnetic radiation. However, the conclusions of the investigations related to genital functions varied, since the difference of frequency and intensity. Further investigation is in need to illustrated relation between adverse genital effects and exposure to high-frequency electromagnetic radiation.The purpose of this study was to assess the female genital toxicities of HF-EMF, the molecular mechanism of apoptotic on the ovaries and the protective effect of isoflavone against the female genital toxicities of HF-EMF by labour hygiene on the scene, epidemiologic survey and animal experiment. The theoretical basis will be able to provide to the precaution and control of healthy harm of HF-EMF. PartⅠStudy on the relationship between toxicities of female gonads and occupational exposures to HF-EMFObjectives: The purpose of this study was to assess the relationship between adverse reproductive effects of female workers and occupational exposures to high-frequency electromagnetic radiation in the shoes factories, and the relationship between toxicities of female rats gonads and short period or inferior chronicity exposures to HF-EMF. The destination was to demonstrate the toxicities of female gonads.Methods: 1. 118 busywork spot's exposure levels of the female workers exposed to high-frequency electromagnetic radiation were detected by field intensity indicador in the shoes factories in Fuzhou urban field, China. 180 female workers exposure to high-frequency electromagnetic radiation in the shoes factories were selected as exposure group, and 349 female workers exposure to non- radiation in the marketplace were selected as control group. Then, evaluate their menstrual status and pregnancy outcomes with cross-sectional study. Meanwhile, the serum levels of reproductive hormones were detected by the radioimmunoassay. 2. 60 Wistar female adult rats were randomly divided into five groups based on body weight and radiated with different levels of 30MHz electromagnetic field (0; 25; 100; 400; 1600V/m) for eight hours daily exposure on anestrus stage. After lasting three days distinct follicle on ovaries were observed with the HE staining and the ultramicrostructure were observed with the transmission electron microscope. Meanwhile, the serum levels of sexual hormones were detected with the radioimmunoassay. 3. 60 Wistar female adult rats were randomly divided into five groups based on body weight and radiated with different levels of 30MHz electromagnetic field (0; 25; 100; 400; 1600V/m) for eight hours daily. Weekly the rats were continuous exposed five days. After lasting forty-eight days the four stage of estrus cycle were observed with smear method of the vaginal cell. After lasting fifty-six days ovarial humid weight and organ coefficientthe were observed on estrus stage. The constituent ratio of the distinct follicle number on ovaries was observed with the HE staining and the ultramicrostructure were observed with the transmission electron microscope. Meanwhile, the serum levels of sexual hormones were detected with the radioimmunoassay.Results: 1.Study the effect of HF-EMF on reproductive and endocrine functions of female workers.①97.46% exposure levels of high-frequency electric field intensity went far higher than those permitted under the human exposure guidelines.②the incidence of menstrual disorder was increase significantly in the both exposure group (p<0.01). However, the incidence of menorrhagia was increased just in the high exposure group (p<0.05), not in the low exposure group. There was no significant difference on menstrual cycle and menstruation and menalgia and menostasis between the exposure groups and the control group.③With the increase of exposure levels of electric field intensity, the incidence of menstrual disorder had a significantly increase among three groups (p<0.05). The incidence of menorrhagia had a significantly increase between the 500~ exposure group and the >1000 exposure group compare to the control group (p<0.05).④In comparison with the control group, serum progesterone (P4) was decrease significantly in the exposure group (p<0.01). 2. Study the effects of short period exposure to HF-EMF on toxicities of female rats gonads.①The constituent ratio of the corpus luteum/albicans number in the 100V/m group were increase significantly (P<0.05), however which were decrease significantly in the 400V/m and 1600V/m group (P<0.01). The constituent ratio of the pri/sec follicle and the atresic follicle were increase significantly, and the primordial follicle were decrease significantly in the 1600V/m group (P<0.01).②Serum estradiol (E2) were increase significantly in the 1600V/m group and progesterone (P4) were increase significantly in the every exposure group (P<0.05). Serum luteinizing hormone (LH) in the 25V/m and 100V/m group was increase significantly (P<0.05), however which were decrease significantly in the 400V/m and 1600V/m group (P<0.01).③There was no significant difference on ultramicrostructure of cell granule between the exposure and the control groups. 3. Study the effects of inferior chronicity exposure to HF-EMF on toxicities of female rats gonads.①There was no significant difference on ovarial humid weight and organ coefficientthe between the exposure and the control groups.②A long with the increase of radiation dose, the time of the preoestrus were decurtated, and the time of anestrum was extended obviously. The time of the preoestrus in the 100V/m group, the 400V/m group and the 1600V/m group were decrease significantly (P<0.01), and the time of the anestrum in the 400V/m group and the 1600V/m group were increase significantly (P<0.01).③The constituent ratio of the corpus luteum/albicans number in the 400V/m group and the 400V/m group was increase significantly (P<0.05), and which of the atresic follicle number were increase significantly in the 100V/m, the 400V/m and the 1600V/m group (P<0.01). The constituent ratio of the mature follicle and the pri/sec follicle were decrease significantly in the 100V/m, the 400V/m and the 1600V/m group (P<0.01).④Serum LH in the 400V/m and 1600V/m group were increase significantly (P<0.01), and serum E2 were decrease significantly (P<0.01).⑤Along with the increase of radiation dose, the ultramicrostructure of cell on the ovaries appear more abnormal cells.PartⅡStudy on the mechanism of HF-EMF-induced apoptotic signal transductionObjective: The destination was to demonstrate the relationship between cell apoptotic on the ovaries and short period (or inferior chronicity) exposure to HF-EMF, and observe the mechanism mediated the apoptotic signal transduction on the ovaries.Methods: 120 Wistar female adult rats were randomly divided into ten groups based on body weigh. Five groups rats were radiated with a different levels of HF-EMF (0; 25; 100; 400; 1600V/m) for three days, and other five groups rats were radiated for fifty-six days. The apoptotic rate of the granulosa cell on the ovaries was detected with the DeadEndTM Fluorometric TUNEL System, respectively. Moreover, the mRNA expression of Caspase-3, Caspase-8, Caspase- 9 were detected by RT-PCR, and the protein expression of Fas, Bcl-2, bax were detected by western-blot on the ovaries.Results:①There was no significant difference on the apoptotic rate of the granulosa cell on the between the exposure and the control groups.②Along with the increase of radiation dose, the apoptotic rate of the granulosa cell on the corpus luteum/albicans and which of cellula intersitialis on the ovaries were increase significantly (P<0.01).③The mRNA expression of Caspase-3 on the ovaries were appeared in the every groups. Compared with control group, the mRNA expression of Caspase-3 on the ovaries was increase significantly in the exposure groups (P<0.01).④The mRNA expression of Caspase-8 on the ovaries were appeared in the every groups. But compared with control group, the mRNA expression of Caspase-8 on the ovaries did not change obviously.⑤The expression of Fas protein on the ovaries were appeared in the every groups. But there was no significant difference on the expression of Fas protein between the control and the exposure groups.⑥The mRNA expression of Caspase-9 on the ovaries were appeared in the every groups. There was significantly difference the mRNA expression of Caspase-9 in the 100V/m, the 400V/m and the 1600V/m exposure groups (P<0.01).⑦The expression of Bcl-2 protein on the ovaries were appeared in the every groups. Along with the increase of radiation dose, the expression of Bcl-2 protein on the ovaries was reduced. There was significantly difference between the control and the exposure groups.⑧The expression of Bax protein on the ovaries were appeared in the every groups. But compared with control group, the expression of Bax protein on the ovaries was increase significantly in the 400V/m and the 1600V/m exposure groups (P<0.01).PartⅢStudy on the protection effect and its related mechanism of SIFObjective: The purpose of this study was to assess the protection effect of SIF and its related mechnism. Methods: 60 Wistar female adult rats were randomly divided into five groups based on body weight. Pre-irradiation the each rat were intragastric administration with SIF (150mg/Kg.d dose) and radiated with a different levels of 30MHz electromagnetic field (0; 25; 100; 400; 1600V/m) for eight hours daily. Weekly the rats were continuous exposed five days. After lasting forty-eight days the four stage of estrus cycle were observed with smear method of the vaginal cell. After lasting fifty-six days ovarial humid weight and organ coefficientthe were observed on estrus stage. The constituent ratio of the distinct follicle number on ovaries was observed with the HE staining and the ultramicrostructure were observed with the transmission electron microscope. Meanwhile, the serum levels of sexual hormones were detected with the radioimmunoassay and the apoptotic rate on the ovaries were detected with the DeadEndTM Fluorometric TUNEL System. The mRNA expression of Caspase-3, Caspase-8, Caspase-9 were detected by RT-PCR. The protein expression of Fas, Bcl-2, bax was detected by western-blot.Results: 1. There was no significant difference on ovarial humid weight and organ coefficientthe between the exposure and the control groups. Along with the increase of radiation dose, the time of the anestrum were extended obviously. Compared with control group, the time of the anestrum was increase significantly in the 1600V/m exposure groups (P<0.01). The constituent ratio of the corpus luteum/albicans numbers in the 1600V/m group were increase significantly (P<0.01), and which of the atresic follicle numbers were increase significantly in the 400V/m and the 1600V/m group (P<0.01). The constituent ratio of the mature follicle and the pri/sec follicle were decrease significantly in the 1600V/m group (P<0.01). Along with the increase of radiation dose, the level of serum LH, E2 and P4 were stepped up obviously. Compared with control group, the level of serum LH in the 1600V/m group was increase significantly (P<0.01). Along with the increase of radiation dose, the ultramicrostructure of cell on the ovaries appear more abnormal cells. But compared with exposure group (no SIF), the ultramicrostructure of cell on the ovaries appear improve. 2. Compared with control group, the apoptotic rates of the granulosa cell on the corpus luteum/albicans in the 400V/m and the 1600V/m exposure group were increase significantly (P<0.01), the apoptotic rate of the cellula intersitialis in the exposure groups were increase significantly (P<0.01). The expression of Caspase-3, -8, -9 mRNA and Fas, Bax, Bcl-2 protein on the ovaries was appeared in the every group. Compared with control group, the mRNA expression of Caspase-3 on the ovaries was increase significantly in the 100V/m, the 400V/m and the 1600V/m exposure groups (P<0.01). The mRNA expression of Caspase-8 on the ovaries was increase significantly in the 1600V/m exposure groups (P<0.01). The expression of Fas protein on the ovaries was reduced significantly in the 400V/m and the 1600V/m exposure groups (P<0.01).⑤T he mRNA expression of Caspase-9 on the ovaries was appeared in the every group. Along with the increase of radiation dose, the mRNA expression of Caspase-9 on the ovaries was enhanced. Compared with control group, there was significantly difference the mRNA expression of Caspase-9 in the 1600V/m exposure group (P<0.01). The expression in the 25V/m, the 100V/m and the 400V/m exposure group did not change obviously.⑥The expression of Bcl-2 protein on the ovaries was appeared in the every group. Along with the increase of radiation dose, the expression of Bcl-2 protein on the ovaries was reduced. Compared with control group, the expression of Bcl-2 protein in the 400V/m and the 1600V/m exposure group were reduced significantly (P<0.01). There was no significantly difference in the 25V/m and the 100V/m exposure groups.⑦The expression of Bax protein on the ovaries was appeared in the every group. Along with the increase of radiation dose, the expression of Bax protein on the ovaries was enhanced. But compared with control group, the expression of Bax protein on the ovaries was increase significantly in the every exposure group (P<0.01).Conclusions: 1. The toxicities of female gonads were closely associated with exposures to HF-EMF under our experimental. 2. The nonage damage of female gonadal toxicities might emerge on the ovaries. 3. There was the association between cell apoptotic on the ovaries and short period exposure to HF-EMF. Howerve inferior chronicity exposures to HF-EMF may induce cell apoptotic on the ovaries. The Fas-mediated cell apoptotic signal transduction was existed on the ovaries. The cytochondriome pathway of Bcl-2 family mediated may be one of important mechanism on the cell apoptosis of HF-EMF-induced. 4. There was the relationship between protection effect of SIF and low dose (25-400V/m) exposure to HF-EMF, but no association between protection effect of SIF and high dose (1600V/m) exposure to HF-EMF. 5. SIF might inhibit apoptosis through Bcl-2 family-mediated apoptotic signal transduction.