Effects Of P, P'-DDE And β-BHC On The Expression Of FasL And Activation Of NF-κB In Rat Sertoli Cells

Under normal circumstances, human and other organisms could synthesize endocrine hormones according to their own needs of growth and development. But recently, scholars found some substances that could mimic natural hormones, inhibit the action of hormones, and/or alter the normal regulation function of the endocrine system, they were called environmental estrogens or endocrine disrupting chemicals (EDCs). It was reported that there is reproductive and developmental abnormalities in human and wildlife. The scholars believed that environmental estrogen and other EDCs caused the reproductive abnormalities. DDT (2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane) and BHC (benzene-hexachloride) were persistent organic pollutants(POPs), these organochlorine pesticides were left over in the adipose tissue and blood lipids through biomagnifications of the food chain for decades. p,p'-DDE andβ-BHC are the major metabolites of DDT and BHC respectively, they have a high level in human and other animals. The researches indicate that these organochlorine pesticides and their metabolites have the interference of endocrine, especially to male reproductive function.Fas and FasL can induce cell apoptosis. When the amounts of apoptotic spermatogenic cell increases, the expression of Fas and FasL increase too. NF-kappaB transcription factors have been shown to have a role in regulating the apoptotic program, either as essential for the induction of apoptosis, or perhaps more commonly, as blockers of apoptosis. The aim of research is to primarily explore the possible mechanisms whose target cell is Sertoli cell (the only body cell in testicular seminiferous tubule) and treated with p,p'-DDE,β-BHC and their combination to male reproductive system. Especially there is rare reports about the effects of p,p'-DDE,β-BHC and their combination on Sertoli cell Fas/FasL system and nuclear factor-κB, so it is the innovative point of the experiments.PartⅠRat Sertoli cell culture in vitro and effects of p,p'-DDE,β-BHC and their combination on viability of rat Sertoli cellsCultured Sertoli cells for 2 days after separating from testicular tissue of SD rats, then incubated with DMSO as a control group, with a series of concentrations p,p'-DDE,β-BHC and their combination (10, 30, 50, 70μmol/L) co-incubating the Sertoli cells respectively in vitro and determined the absorbency on 490nm by MTT method after 24h treatment. The results were that the absorbency had significent difference only at the concentration of 70μmol/L compared with DMSO (P<0.05). It was also indicated that p,p'-DDE,β-BHC and their combination would do harm to Sertoli cells when the concentration of p,p'-DDE,β-BHC and their combination was above 50μmol/L.PartⅡEffects of p,p'-DDE,β-BHC and their combination on the expression of FasL in rat Sertoli cellsTo study the effects of p,p'-DDE,β-BHC and their combination on the expression of FasL in rat Sertoli cells. The expression of FasLmRNA was detected by two-step RT-PCR, the FasL protein was detected by Western Blot. The expression of FasLmRNA was increased with the increasing of toxicant concentrations in cell culture, there was statistical significance between the concentration of 50μmol/L and control group separately (P<0.05) , there was statistical significance between the concentration of 50μmol/L and 10, 30μmol/L separately (P<0.05). The expression of FasL protein increased with the increasing of toxicant concentrations in cell culture, there was statistical significance between the concentration of 50μmol/L and control group separately (P<0.05) , there was consistent with FasLmRNA and protein on the concentration of 50μmol/L. on the time point, the expression of FasL protein presented the trend of firstly increased then decreased in 24h.PartⅢEffects of p,p'-DDE,β-BHC and their combination on the expression of NF-κB in rat Sertoli cellsTo study the effects of p,p'-DDE,β-BHC and their combination on the expression of NF-κB in rat Sertoli cells. NF-κB activation state was examined with laser confocus microscope. The transposition of NF-κB was obviously increased, the activation of NF-κB enhanced with the increase of concentration of p,p'-DDE,β-BHC and their combination, the combined effects of p,p'-DDE andβ-BHC on Sertoli cells were more obvious. In conclusion, the results suggested that: 1. the primary cultured Sertoli cells in vitro model could be used to explore the disrupting mechanism of EDCs in vitro; 2. the expression of FasLmRNA was increased with the increasing of toxicant concentrations in cell culture; 3. the expression of FasL protein was increased with the increasing of toxicant concentrations in cell culture, the expression of FasL protein presented the trend of firstly increased then decreased in 24h; 4. the transposition of NF-κB was obviously increased. the activation of NF-κB enhanced with the increase of concentration of p,p'-DDE,β-BHC and their combination, the combined effects of p,p'-DDE andβ-BHC on Sertoli cells were more obvious. These studies indicated that p,p'-DDE,β-BHC and their combination could increase the expression of FasLmRNA, and then induced the protein increasing, thus activating Fas/FasL system. NF-κB might regulate the expression of FasL, then regulate the apoptosis of germ cells, eventually affect the normal spermatogenesis. We also found that combined effects of p,p'-DDE,β-BHC was more obvious, the combination effects have interactions on rat Sertoli cells, Meanwhile there were still questions remained to be elucidated such as how they affect the caspase and the apoptosis of Sertoli cells.