Effects Of P,p'-DDE And β-BHC On The Apoptosis Of Sertoli Cells In Vitro Via Caspase Pathway

More and more evidences proved that lots of environmental contaminants could disturb endocrine, especially influence the male reproduction, attracting highly attention in the public. DDT and BHC, the representative organochlorine pesticides in the few decades, are persistent organic pollutants (POPs) with the features of widely polluting, huge harm, and longtime persistence in environment, and entering the living system via biomagnification of food chain, may persist mainly by the forms of metabolite p,p'-DDE andβ-BHC in the blood fat and adipose tissue for several decades. Respecting their long-term great effect, environmental antiandrogen and studies of correlated environmental and reproductive problems become hot spots of researches. Relevant reports about endocrine disturbance of p,p'-DDE andβ-BHC are increasing gradually. However, seldom studies about the respective and combined effect of p,p'-DDE andβ-BHC on reproductive system are reported, especially reports of p,p'-DDE andβ-BHC effecting on Sertoli cells. And the main purpose of this study is to explore the possible mechanism of p,p'-DDE andβ-BHC on cell apotosis in target cells, Sertoli cells of rat testis in vitro. So it is the innovative point of this expriment.PartⅠRat Sertoli cell culture in vitro and effects on viability of Sertoli cellSertoli cells were treated with 10μmol/L, 30μmol/L, 50μmol/L, 70μmol/L p,p'- DDE,β-BHC and their mixture which were cultured in vitro and determined the absorbency on 490 nm by MTT method after 24 h treatment. The results were that the absorbency has significant difference at the concentration of 50μmol/L and 70μmol/L of p,p'-DDE andβ-BHC, and 50μmol/L of the mixture of p,p'-DDE andβ-BHC compared with DMSO(P <0.05). It also indicated that p,p'-DDE andβ-BHC would do harm to Sertoli cell when concentration was above 50μmol/L.PartⅡEffects on Sertoli cell apoptosisWith a series of concentration p,p'-DDE (10, 30 and 50μmol/L),β-BHC(10, 30 and 50μmol/L) and p,p'-DDE +β-BHC(10, 30 and 50μmol/L) co-incubating the Sertoli cells in vitro,and the group with 100μmol/L Caspase-3 inhibitor Ac-DEVD-CHO treated Sertoli cells for 2 h before 50μmol/L p,p'-DDE+50μmol/Lβ-BHC 24 h-treatment, there was apoptosis in Sertoli cells treated with p,p'-DDE,β-BHC and their mixture compared by acridine orange/ethidium bromide (AO/EB ) double staining method by fluorescence microscopy. Compared with the Caspase-3 inhibitor Ac-DEVD-CHO group, apoptosis rate of Sertoli cells significantly increased with increasing concentration of p,p'-DDE,β-BHC and their mixture (P <0.05).It demonstrated that p,p'-DDE,β-BHC and their mixture could cause apoptosis of Sertoli cells in vitro, and apoptosis occurred via Caspase-3 pahtway. PartⅢEffects on Caspase-3,Caspase-8 and Caspase-9 of p,p'-DDE andβ-BHC We determined the mRNA expression of Caspase-3, Caspase-8 and Caspase-9 with different concentration of p,p'-DDE,β-BHC and their mixture, and different time treated at high dose and to explore the effects on functional protein of p,p'-DDE,β-BHC and their mixture by RT-PCR on the level of mRNA. The results were that mRNA expressions of Caspase-3, Caspase-8 and Caspase-9 in Sertoli cells in vitro increased with the increasing concentration of p,p'-DDE,β-BHC and their mixture by means of dose-dependent relationship.PartⅣInfluence to the express of protein Caspase-3, Caspase-8 and Caspase-9 treated with p,p'-DDE,β-BHC and their mixtureWe cultulred Sertoli cells separated from testicular tissue of rats and examined the quantity of Caspase-3, Caspase-8 and Caspase-9 by Western Blotting method. On one hand, we poisoned Sertoli cells with 10μmol/L, 30μmol/L and 50μmol/L p,p'-DDE,β-BHC and their mixture for 24 h before detecting the Caspase-3, Caspase-8 and Caspase-9. On the other hand, we treated cells with 50μmol/L p,p'-DDE,β-BHC and their mixture at different time(1.5 h, 3 h, 6 h, 12 h and 24 h) and determined the protein expression of Caspase-3, Caspase-8 and Caspase-9. We can see that with the concentration and treated time increasing proenzyme expression of Caspase-3, Caspase-8 and Caspase-9 decreased, and effects on Caspase-3, Caspase-8 and Caspase-9 of combined dose are more obvious than of p,p'-DDE or ofβ-BHC, suggesting that p,p'-DDE andβ-BHC might activate the apoptosis via Caspase pathway, and their combined effect may be synergistic effect.We can see from the above experiments: 1. p,p'-DDE,β-BHC and their mixture induced apoptosis of Sertoli cell. Sertoli cells play an important role in spermatogenesis since they foster the development of germ cells and maintain viability of those cells by secreting hormonal and nutritive factor into a specialized compartment. p,p'-DDE is an environmental antiandrogen andβ-BHC is an environmental pseudo-estrogen. They both affected apoptosis resulting in the changes of function of germ cells and spermatogenesis disorder. 2. p,p'-DDE,β-BHC and their mixture caused the mRNA expressions of Caspase-3,Caspase-8 and Caspase-9 significantly increased. 3. p,p'-DDE,β-BHC and their mixture reduced proenzyme expressions of Caspase-3, Caspase-8 and Caspase-9. Also, 50μmol/L p,p'-DDE,β-BHC and their mixture decreased proenzyme expressions of Caspase-3, Caspase-8 and Caspase-9 by time-dependent manner.From the above three points that except for the mechanisms of p,p'-DDE andβ-BHC competing hormone receptors with gonadal hormone there was other pathways which did not rely on receptor. The structure changes of apoptotic cells are mainly induced by Caspases. Once these intracellular enzymes are activated, cells enter apoptotic process. Activated Caspase effects on related substrate and apoptosis is irreversible. In the Caspase cascade, Caspase-3 ,which is an important effective factor downstream, plays a key part in the final stage of apoptosis process induced by various kinds of agents. Cell apoptotic rate increased with the concentrations of p,p'-DDE,β-BHC and their mixture treated on Sertoli cells increasing, and the apoptosis could be inhibited by Caspase-3 inhibitor Ac-DEVD-CHO, suggesting that p,p'-DDE,β-BHC and their mixture could activate Caspase-3 pathway, resulting in Sertoli apoptosis. The mRNA expressions of Caspase-3, Caspase-8 and Caspase-9 were increased with the concentrations of p,p'-DDE,β-BHC and their mixture increasing, and proenzyme expressions of Caspase-3, Caspase-8 and Caspase-9 were in dose- and time-dependent manner, indicating that apoptosis was carried out by activating Caspase-8 and Caspase-9, and subsequently Caspase-3 downstream. This study provided basis for the concrete mechanism of it.