Reproductive Toxicity Of DEHP And Its Metabolites MEHP In Female Rats And Related Mechanism

Environmental endocrine disruptor pollution and its impact on the humanreproductive system had become the attention focus of environmental hygieology,also related to the sustainable development of human and nature. DEHP asenvironmental endocrine disruptor was widely present in the environment, though itwas included in the monitoring of priority environmental pollutants in China, butliquor plasticizer incident, PVC plastic wrap and DEHP excess in export toys showedthat plasticizer contamination is a serious problem in recent years, which shouldattract wide attention from scholars.Objective:This study intends to systematically investigate mechanism of DEHP and MEHPon sex hormones level and granulosa cells apoptosis from the angle of the tissue,cellular and molecular levels, clarify the effects on the female reproductive organ andreproductive function, explore the disrupting role of DEHP on the reproductiveendocrine, provided the scientific basis for the comprehensive evaluation of thetoxicology of DEHP and its effect on potential hazards of human reproduction system.Methods:PartⅠ Female reproductive toxicity of DEHP and MEHP1.48Wistar female rats were randomly divided into4groups （0,300,1000,3000mg/kg/d） by gavage for4weeks,6days/week. Observe the general growthconditions and record water, diet and weight change during the period of theexperimental study. Observe the estrous cycle in2weeks before death at6:00am and6:00pm by vaginal smears, studying the effect of DEHP on estrous cycle. Execute themice during anoestrum after the weighing. Sample Blood in mice eyes, take the uterusand ovaries, calculate organ coefficient of the uterus and ovaries, and detect hormonelevels of FSH, LH, P4, E2and T in serum by radioimmunoassay. Observe pathologicalchanges by HE staining of uterus and ovaries, detect expression of FSHR and LHR byimmunohistochemistry. 2. Immature granulosa cells in Wistar rats were cultured, and HE staining andimmunocytochemistry of the cells obtained were identified. Observe cell generalgrowth status and sex hormone secretion levels, measure growth curve by MTT assayand medium P4and E2hormone levels by radioimmunoassay. Select DEHPmetabolites MEHP was expose to the GC test, dosed with0（control group）,25,50,100μmol/L. After exposure cell relative activity was assayed by MTT, and medium P4and E2hormone levels were measured by radioimmunoassay, FSH receptorexpression of granulosa cells was measured by immunocytochemistry staining.Part Ⅱ Effects of DEHP and MEHP on critical gene and related receptorexpression in sex hormone synthesis pathway and its mechanism1. Animal grouping and exposure were the same as the first part. Extractingovarian tissue RNA, the STAR, P₄₅₀scc,3β-HSD,17β-HSD Ι,17β-HSD II and P₄₅₀arom gene expression differences in rat ovarian sex hormone synthesis pathway weremeasured by RT-PCR assay. Receptor PR and ER expression related with sexhormone synthesis in ovarian and uterine tissue were observed byimmunohistochemical staining.2. Ovarian granulosa cells culture, identification methods and exposure were thesame as the first part. Separating ovarian granulosa cells, the STAR, P₄₅₀scc,3β-HSD,17β-HSD Ι,17β-HSD II and P₄₅₀arom gene expression differences in rat ovarian sexhormone synthesis pathway were measured by RT-PCR assay. Receptor PR and ERexpression related with sex hormone synthesis were observed byimmunohistochemical staining.Part Ⅲ Effects of DEHP and MEHP on ovarian granulosacell apoptosis and its mechanism1. Animal grouping and exposure were the same as the first part. Observe ratovarian granulosa cell apoptosis by Annexin V-FITC/PI double staining method.Caspase-3activity was assayed by spectrophotometer. Apoptosis-related proteins Bax,Bcl-2expression levels were measured by Western Blot.2. Ovarian granulosa cells culture, identification methods and exposure were thesame as the first part. Observe rat ovarian granulosa cell apoptosis by AnnexinV-FITC/PI double staining method. Caspase-3activity was assayed by spectrophotometer. Apoptosis-related proteins Bax, Bcl-2expression levels weremeasured by Western Blot.Result:PartⅠ Female reproductive toxicity of DEHP and MEHP1．The metaestrus, anestrus, and estrous cycle duration were significantly longerthan that of control group（P＜0.05）; the proestrus and estrus in each group had nosignificant difference（P＞0.05）.2．The water intake in each group was not statistically significant （P＞0.05）;DEHP exposure increased the food intake, the difference was significant （P＜0.05）;DEHP exposure significantly decreased body weight in female rats, and the differencewas significant （P＜0.05）.3. The rat ovarian organ coefficient significantly reduced, the difference wassignificant （P＜0.05）; the uterine organ coefficient in each group was no significantdifference （P＞0.05）. The ovarian tissue of rats in the control group was normal, thegranule cells of ovarian follicles were acutely damaged, and the structure of granulecells appeared loose, degeneration, loss, the gap between granule cells and membranecells widened, atretic follicles increased significantly in experimental groups. Theuterine tissue of rats in the control group was normal, the nuclei of endometriumappeared pseudostratified phenomenon, the number of glands in the lamina propriareduced, the epithelial hyperplasia and endothelial fibrosis was observed in theendometrium, the number of glands in the lamina propria reduced, the part of theglands were depauperate.4. Serum FSH, LH, P4, T and E2hormone secretion levels decreased significantly（P＜0.05）.5. The MOD of endometrial LHR in the experimental group was significantlylower that control the difference was significant （P＜0.05）; Other receptor expressionlevels had a downward trend, but the differences were not significant （P＞0.05）.6. MEHP decreased the relative activity of the granulosa cells, and the differencewas significant （P＜0.05）.7. MEHP stimulated P4and E2hormone secretion in cell culture medium, and thedifference was significant （P＜0.05）. 8. Semi-quantitative analysis showed that FSHR expression levels of ovariangranulosa cells were significantly increased compared with the control group, and thedifference was significant （P＜0.05）.Part Ⅱ Effects of DEHP and MEHP on critical gene and related receptorexpression in sex hormone synthesis pathway and its mechanism1. STAR, P₄₅₀scc,3β-HSD,17β-HSD Ι,17β-HSD II and P₄₅₀arom gene relativeexpression levels decreased with the increase of DEHP doses in the rat ovarian sexhormone synthesis pathway, and the difference was significant （P＜0.05）.2. Semi-quantitative analysis showed that PR, ER expression levels in ovariantissue decreased with the increase of DEHP doses, and the difference was significant（P＜0.05）. PR, ER expression levels of the uterine tissue in each group had nosignificant difference （P＞0.05）.3. STAR, P₄₅₀scc,17β-HSD Ι and17β-HSD II gene relative expression levels ofovarian granulosa cells increased with the increase of MEHP doses in vitro, and thedifference was significant （P＜0.05）.3β-HSD, P₄₅₀arom relative gene expressionlevels had no significant difference （P＞0.05）.4. Semi-quantitative analysis showed that PR, ER expression levels in ovariangranulosa cells increased with the increase of DEHP doses, and the difference wassignificant （P＜0.05）.Part Ⅲ Effects of DEHP and MEHP on ovarian granulosa cell apoptosisand its mechanism1. DEHP could increase early, late and total apoptosis rate of rat ovariangranulosa cells significantly（P＜0.05） Caspase-3activity of ovarian granulosa cells ineach group was not significant（P＞0.05）. The ratio of Bax/Bcl-2was graduallyincreased with the increase of DEHP doses, and the difference was significant （P＜0.05）.2. MEHP could increase early, late and total apoptosis rate of rat ovariangranulosa cells significantly in vitro（P＜0.05）, the ratio of Bax/Bcl-2was graduallyincreased with the increase of MEHP doses（P＜0.05）, Caspase-3activity of ratovarian granulosa cells in exposure groups significantly increased compared with thecontrol（P＜0.05）. Conclusion:1. DEHP could prolong metaestrus, anestrus and estrous cycle duration of thefemale rats, and the duration of estrous cycle was mainly due to that metaestrus andanestrus were significantly prolonged; DEHP can decrease ovarian organ coefficient,and the granule cells of ovarian follicles were acutely damaged, suggesting that ovarymay be the main target organ of DEHP.2. Successfully establish culture system of rat ovarian granulosa cells in vitro,MEHP could decrease the relative activity of the granulosa cells, and inhibit cellsproliferation.3. DEHP could reduce STAR, P₄₅₀scc,3β-HSD,17β-HSD Ι,17β-HSD II and P₄₅₀arom gene relative expression levels in the rat ovarian sex hormone synthesis pathway,decrease sex hormone-related receptor expression levels, inhibit FSH, LH, P4, T andE2hormone secretion, interfering with normal ovarian reproductive endocrinefunction.4. MEHP could promote STAR, P₄₅₀scc,17β-HSD Ι and17β-HSD II gene relativeexpression levels of ovarian granulosa cells, increase sex hormone-related receptorexpression levels, stimulated P₄and E₂hormone secretion in cell culture medium,interfering with normal reproductive endocrine function of granulosa cells in vitro.5. DEHP could increase the ratio of Bax/Bcl-2, and induce apoptosis of ovariangranulosa cells; MEHP could activate the Caspase-3of granulosa cells, increase theratio of Bax/Bcl-2, and induce apoptosis of ovarian granulosa cells in vitro inmitochondrial pathway.In vitro and in vivo results show that DEHP and its metabolites MEHP have animpact on female reproductive organs and reproductive endocrine function, induceapoptosis of ovarian granulosa cells, may have female reproductive toxicity on femalerats, and have disrupting role on the reproductive endocrine.