Effect Of Dibutyl Phthalate On Growth And Secretory Function Of Rat Ovarian Granular Cells

PURPOSE: Environmental pollution is one of the negative effects of the rapid development of industry,especially chemical industry in recent decades.Among these pollutions,some has estrogen-like effects,so they are named Environmental Estrogenic hormones(EEs).The main sources of EEs are artificial pesticides,fertilizers,growth regents,food additives,etc.Dibutyl phthalate(DBP)is a major chemical matrial as plasticizers and is also widely used in cosmetics,automotive glass and paint industries.The annual consumption of dibutyl phthalate of the world is between 3 and 4 million tons.In Europ and USA,DBP was listed in suspected teratogens and is forbidden in cosmetics,toys for children and child care articles,etc.DBP has estrogen-like effects and anti-androgen effects.Like other EEs,the activity of DBP is very low compared with endogenous estrogen.But DBP exists widely in environment,so chance of people's contact with DBP is much more than other EEs,so DBP is widely enriched in organism,especially for those people working in DBP-related industries whose bodies are checked and found being carrying much higher volume of DBP than E2.Previous studies in animals revealed that DBP could cause endocrine-disorder and damage in reproductive system,and also could induce tumors to hormone-dependent organs.Those researches mainly focused on male reproductive system damage cause by DBP,including spermatogenesis failure,seminiferous tubules dysgenesis and interstitial cell hyperplasia,etc.However,there are few studies of DBP toxicity to the female reproductive system.On the basis of rat ovarian Granular Cells isolation and culture,this research established a model of dibutyl phthalate exposured Granular Cells,focused on the female reproductive system damage caused by DBP,to study the effects of DBP on ovarian Granular Cells' growth and secretory functions,and further analysed of its impact on the maturation of oocytes and the mechanism that DBP may cause reproductive toxicity for females.METHOD: Firstly,granular cells(GCs)were isolated and cultured from ovaries of female rat which were stimulated by pregnant mare serum(PMS).The activity of GC was verified by Trypan blue dyeing,AKP dyeing and c-kit dyeing.GCs were treated by DBP with different concentration which simulated the concentration of DBP in organisms,to establish a model of DBP exposured Granular Cells.Cell proliferation and toxicity were measured by method of CCK-8 and clone formation experiment.Then the effect of different concentrations of DBP on ovarian granular cells in terms of secretion for estradiol E2 and progesterone P were checked.P450 aromatase and P450 cholesterol side-chain cleavage enzyme,which are important for generating of E2 and P,were checked in terms of m RNA level and protein level by RT-q PCR and Western Blot method.At last,the expression levels of two cytokines,Insulin-like growth factor-1(IGF-1)and tumor necrosis factor alpha(TNF?),were also measured by RT-q PCR and Western Blot method,in order to study the effects of different DBP concentrations to the expression levels of cytokines in ovarian granular cells.RESULT: We found that cell proliferation was elevated when long-time treated GCs with low-concentration DBP,while decreased when high-concentration DBP was used for treatment.Secretion of Estradiol E2 and progesterone increased when GCs were long-time treated with DBP,meantime P450 aromatase and P450 cholesterol side-chain cleavage enzyme were both increased in m RNA and protein levels.In terms of the effects to the expression level of cell factors,we also found the expression of IGF-1 increased when GCs were treated by DBP,while the expression of TNF? did not change remarkably.CONCLUSION: Our study revealed cell proliferation mode of ovary granular cells were affected by long-time DBP treatment.Meanwhile,DBP impact on the secretion of estradiol E2,progesterone,and also expression level of P450 aromatase and P450 cholesterol side-chain cleavage enzyme.The mechanism may be: DBP induced increasing of IGF-1 expression level,and then promoted activity of the P450 aromatase and P450 cholesterol side-chain cleavage enzyme,thus contributed to the secretion of estradiol E2 and progesterone P.