Ovarian Toxicity Caused By Di-(2-Ethylhexyl)Phthalate And Its Molecular Mechanism

In the industrial production process of polyvinyl chloride(PVC) and other plastics, di(2-ethylhexyl)-phthalate(DEHP) is added to increase the flexibility and toughness of plastic. Currently DEHP has become a global environmental pollutant, many previous studies have shown that DEHP has reproductive toxicity, carcinogenicity, teratogenicity, mutagenicity, immunotoxicity, neurotoxicity, et al. The study intended to investigate the reproductive toxicity and the molecular mechanism from cell and animal levels. Firstly, CHO cells were exposed to different doses of DEHP for 24 h and 48 h, the cell viability were measured using CCK8 test, the apoptosis gene(Bcl-2, Caspase-8, Caspase-9) and oncogenes(c-fos, k-ras, p53) expression levels were detected by real-time PCR after 24 h exposure. Next, we carried out experiments on animals, a total of 40 healthy 5-week-old SPF female Sprague-Dawley rats were randomly divided into 4 groups with 10 rats in each group. The dose groups is control group(corn oil), low dose group(100 mg/kg/d DEHP), medium dose group(500 mg/kg/d DEHP) and high dose group(1500 mg/kg/d DEHP). Rats were exposed to DEHP via gavage administration for continuous six weeks, once a day and five times a week. After the exposure, rats were sacrificed for the further study. The weight of rats and the wet ovary were recorded, then the ovary coefficient were worked out. The pathological damage were observed by optical microscope after Hematoxylin-Eosin staining(HE staining), the ultrastructural changes in granulosa cells were observed by transmission electron microscopy(TEM). The apoptosis gene(Bcl-2,Caspase-3,Caspase-8,Caspase-9) and oncogenes(c-fos,k-ras,p53)expression levels were detected by real-time PCR. The protein expression levels of luteinizing hormone receptor(LHR) and gonadotropin releasing hormone receptor(Gn RHR) were detected by Western Blot. This study obtained the following main results:1. The IC50 to CHO cell treated with DEHP for 24 h was 612 μmol/L, while the IC50 to CHO cell treated with DEHP for 48 h was 336 μmol/L. The gene expression of apoptotic Bcl-2 were decreased significantly compared with control group at higher DEHP dose(150 μmol/L and 300 μmol/L)( p<0.05 or p<0.01), while the gene expression levels of Caspase-8 and Caspase-9 were significantly increased in 300μmol/L group compared with the control group(p<0.05 or p<0.01). The gene expression levels of p53 and k-ras were increased under 300 μmol/L DEHP exposure( p<0.05), there is no significant difference in the gene expression levels of c-fos in DEHP-treated groups compared with control group.2. The ovarian weight and ovarian coefficient in the DEHP-treated groups had no significant difference compaerd with control(p>0.05), pathological changes in the ovaries were observed by ordinary optical microscope, the oocytes in primary follicles disappeared in DEHP exposure group, follicular atresia were observed in DEHP middle-dose group, the granulosa cells surrounding oocytes showed a disordered arrangement and shedding. Ovarian granulosa cells in DEHP exposure group showed abnormal mitochondrial structure, nuclear chromatin condensation, formation of apoptotic bodies, et al. Ovarian granulosa cells in DEHP high dose group showed the pathological changes of degeneration and necrosis. 3. The gene expression level of Bcl-2 was significantly lower in the high-dose DEHP group, compared with the control group(p<0.01).The gene expression levels of Caspase-3, Caspase-8 in middle-dose and high-dose DEHP groups were increased significantly compared with the control group(p<0.05 or p<0.01).The gene expression levels of Caspase-9 were increased significantly in the high-dose group compared with the control group(p<0.05). The gene expression levels of c-fos in DEHP-treated groups has no significant difference compared with the control group(p > 0.05). The gene expression levels of k-ras and p53 were significantly higher than the control group in the medium and high DEHP dose groups(p<0.01). 4. The protein expression levels of LHR in the medium and high dose groups were decreased 25%~35%(p<0.05 or p<0.01, compared with the control group), while the protein expression levels of Gn RHR decreased 60%~80%(p <0.01,compared with the control group).This study obtained following conclusions through the cell experiment and the animal experiment:1.DEHP can induce decrease of the viability of CHO cell, it may cause the cell apoptosis through the intrinsic mitochondrial apoptosis pathway, while relatively high DEHP can induce elevated levels of oncogene expression, suggesting that it may have carcinogenic effect to some extent. 2.DEHP treatment caused structural damage in rat ovarian follicles, granulosa cell apoptosis, high doses of DEHP exposure induces cell necrosis. 3.At the animal level, DEHP also may induce granulosa cell apoptosis through intrinsic mitochondrial apoptosis pathway, it may play potential carcinogenic effects by inducing abnormal expression of oncogenes, on the other hand, DEHP can affect the expression levels of estrogen receptor, which promoted the ovarian toxicity.