Study On The Effects Of Combined Effect Of P, P'-DDE, β-BHC On JNK MAPK Signal Transduction Pathway Of Rat Sertoli Cells

Large numbers of studies show that lots of persistent environment contaminants had biological effects of environmental hormone, which mimick or disrupt normal endocrine function of animals, especially disrupt the process of developing and reproduction of the reptiles and wild mammals, birds and fish. These natural or synthetic chemical substances are called endocrine disrupting chemicals (EDCs). The pesticides such as DDT and BHC that have the characteristics of environmental persistence, wide spreading, bioaccumulation and high toxicity were called Persistent organochlorine pollutants (POPs). p,p'-DDE andβ-BHC are the main metabolic products of DDT and BHC that have been always detected in adipose tissue and blood samples of human.Recently, studies on the effects of organochlorine pesticides on signal transduction pathway became hotspot. Some research reported that Mitogen-activated protein kinase (MAPK)play a major role in the maintenance and control of spermatogenesis, which can regulate cell proliferation, differentiation and apoptosis in testicle tissues of mammal animals. The previous studies of our laboratory have demonstrated that p,p'-DDE could result in the phosphorylation of ERK and induce the activation of the downstream pathway in order to regulate rat Sertoli cells. But the combined effect of p,p'-DDE,β-BHC on JNK MAPK signal transduction pathway of rat Sertoli cells remains unclear. The aim of this research is to investigate the possible mechanism of combined effects of p,p'-DDE andβ-BHC on rat testicle Sertoli cells. Part I The toxicity of Combined effect of p,p'-DDE andβ-BHC on rat Sertoli cellsSertoli cells were separated from testicular tissue of SD rats of 18-20 days age, then they were cultured in DMEM media with 5% fetal bovine serum at 35℃in a humidified incubator of 5% CO2. After that Sertoli cells were treated with 10μmol/L, 30μmol/L, 50μmol/L, 70μmol/L p,p'-DDE,β-BHC for 24h, then we used MTT method to determine their absorbencies under the 490nm wave-length. In the same way Sertoli cells were treated with 10μmol/L, 30μmol/L, 50μmol/L of combined effects of p,p'-DDE andβ-BHC for 24h, we also used MTT method to determine their absorbencies under the 490nm wave-length. The results show that with the increase of concentration of p,p'-DDE,β-BHC and their combination, the absorbency all decreased. at the concentration of 50μmol/L p,p'-DDE,β-BHC and their combined effect all have a significant differences compared with DMSO(P<0.05)group. The results also show that among p,p'-DDE,β-BHC and their combination group, there have a significant diference at the concentration of 50μmol/L(P<0.05). In a word, the results indicated that p,p'-DDE,β-BHC and their combination could do harm to Sertoli cell when the concentration of p,p'-DDE,β-BHC and thire combinated effect were above 50μmol/L , It also indicated that the adverse effects of p,p'-DDE is more obvious thanβ-BHC, and the coexistence of p,p'-DDE andβ-BHC has a synergistic effect.Part II Study on the mechanisms of signal transduction pathway of Combined effect of p,p'-DDE,β-BHC on the express of JNK mRNA of rat Sertoli cellsSertoli cells were cultured 2 days after separating from testicular tissue of rats, and then were divided into four groups respectively incubated with DMSO (used as control group), p,p'-DDE,β-BHC and their combination under the concentration of 10, 30, 50μmol/1 for 24h, After that we used two-step RT-PCR method to study the combined effect of p,p'-DDE,β-BHC on the expression of jnk mRNA of rat Sertoli cells. The results show that p,p'-DDE,β-BHC and their combination can all enhance the expression of jnk mRNA and the effects of p,p'-DDE group is more obvious thanβ-BHC group, otherwise the combined effects is more obvious than separated effects, the coexistence of p,p'-DDE andβ-BHC has a synergistic effect.PartⅢStudy on the mechanisms of signal transduction pathway of Combined effect of p,p'-DDE,β-BHC on the express of p-JNK1/2, JNK1/2 of rat Sertoli cellsSertoli cells were cultured 2 days after separating from testicular tissue of rats, and then were divided into four groups respectively incubated with DMSO (used as control group), p,p'-DDE,β-BHC and their combination under the concentration of 10, 30, 50μmol/1 for 24h, then we used western blotting method to study the combined effects of p,p'-DDE,β-BHC on expression of p-JNK1/2, JNK1/2 proteins in different concentrations firstly. Otherwise Sertoli cells were incubated at the high concentration of 50μmol/1, we used western blotting method to study the combined effects of p,p'-DDE,β-BHC on expression of p-JNK1/2, JNK1/2 proteins in different time. The researches show that in different concentrations of p,p'-DDE,β-BHC and their combination, p-JNK1/2 protein expression all increased while JNK1 and JNK2 had no differences and the effects of p,p'-DDE group is more obvious thanβ-BHC group, the combined effect is more obvious than separated effect, the coexistence of p,p'-DDE andβ-BHC has a synergistic effect. Sertoli cells were cultured in different time with the same high concentration, the expressions of p-JNK1/2 protein were actived temporally in both p,p'-DDE and their combination group , then descended, after that there was a persistent increasing. In theβ-BHC group, the expressions of p-JNK1/2 protein were inhibited temporally, after that there was a persistent increasing. The results indicated that p,p'-DDE,β-BHC and their combination can result in the phosphorylation of JNK protein and induce the activation of JNK MAPK pathway ,The activation of JNK MAPK pathway could be a kind of adverse effect on male reproductive function.PartⅣStudy on the expression of c-jun mRNA of rat Sertoli cells treated with p,p'-DDE,β-BHC and their combined effectsSertoli cells were cultured 2 days after separating from testicular tissue of rats, and then were divided into four groups respectively incubated with DMSO (used as control group), p,p'-DDE,β-BHC and their combination under the concentration of 10, 30, 50μmol/1 for 24h, After that we used two-step RT-PCR method to study the combined effect of p,p'-DDE,β-BHC on the express of c-jun mRNA of rat Sertoli cells. The results show that p,p'-DDE,β-BHC and their combination can all enhance the expression of c-jun mRNA (the downstream gene of jnk gene)and the effects of p,p'-DDE group is more obvious thanβ-BHC group, otherwise the combined effects is more obvious than separated effect, the coexistence of p,p'-DDE andβ-BHC has a synergistic effect.