Independent And Combined Steroid Hormone Effects Of P,p’-DDE And PCB-153on The Rat Pituitary Cells In Vitro Via ERK1/2Singal Pathway

As two kinds of persistent organic pollutants (POPs) and environmental endo-crine disrupting chemicals (EEDs)， Organochlorine pesticides DDT and PCBs arewidely existed in the kinds of environmental media and organisms. PCB-153is one ofthe homologous series of PCBs, while p, p’-DDE is the main metabolite of DDT, theendocrine ndisrupting effects of them attract an extensive attention.Therefore, from the point of view of the ERK signaling pathway, the research ofthat the molecular mechanisms of PCB-153and p, p’-DDE and their combined effectson the pituitary cells is important，and can provide some new academic ideas abouthow to prevent thyroid hormone disorders from EECs.Part I Culture and identification of rat pituitary cells in vitro andthe independent and combined effects of p, p’-DDE,PCB-153on ratPituitary cellsObjective：①To find out the best method for culture of rat pituitary cells in vitro．②To explore the effects ofp, p’-DDE, PCB-153and their combinationonpituitarycells.Methods：①Pituitary of adult SD female rats were isolated for cell primary culture,immunocytochemistry method was used to identify expression of luteinizing hor-mone(LH)．②The cells were treated withp, p’-DDE, PCB-153and their combinationfor48h, then MTT method was used to test their absorbencies under490nmwave-length.Results:①identify expression of rat pituitary cells: Positive product was brown incytoplasm. And LH positive cells were big, less and scattered growing.②Effects ofPCB-153, p, p’-DDE and their combination in rat pituitarycells after 48h, the difference of Absorbance between DMSO control group and the negativecontrol group was not statistically significant (P>0.05),and with the increasing of theconcentrationofp, p’-DDE, PCB-153and their combination, the absorbance decreasedgradually.Conclusion:①Pituitary cells in a single cell can be cultured with application of thiscell culture method.②p, p’-DDE, PCB-153and their combination could do harm topituitary cells when the concentrationofp, p’-DDE, PCB-153and their combinationwere above70μmol/L and125mol/L, and the combination had greater impact, indicating that the combined exposure of two kinds of poison have a certain synergy.Part II p, p’-DDE and PCB-153, and their combined effects on theERK1/2signaling pathway in the pituitary cellsObjective: To explore the effects of PCB-153, p, p’-DDE and their combination onthe ERK1/2signaling pathway in the rat pituitary cellsMethod: The real-time fluorescence quantitative PCR method was used to test thek-ras、raf-1、MEK1、ERK1、ERK2、c-fos、c-myc and ELK-1mRNA and Westernblottng method was used to test the ERK1/2and p-ERK1/2protein expressions inthe rat pituitary cells in vitro. The immunohistochemical method was used todetermin the localization and express of the ERK1/2protein in the cellsResults:①The pituitary cells were treated with0、10、30、50μmol/Lp, p’-DDE,0、1、5、25μmol/LPCB-153and their combination for48h. Compared with thecontrol group, k-ras、MEK1、ERK1、ERK2、c-fos、c-myc mRNA expressions wassignificantly increased；And ELK-1mRNA expression also was significantlyincreased.②ERK1/2total protein expression was signifycantly increased.And thep-ERK1/2protein expression was significantly decreased. Compared with the sameconcentration of separated group,the two poisons present combined effects. As well asthe results show that the two poisons had synergies effects in combination groups.③ERK1/2protein was located in the cytoplasm, and the cell morphology of the exposed groups was affected.Conclusion: p, p’-DDE and (or) PCB-153exposure can activate ERK1/2signalingpathway through ras-MAPK,then increase the expression of nuclear genes and tra n-scription factors, and may eventually lead to changes in cell viability and morpholo-gy.However the sustained activation of ERK1/2may decrease raf-1expression by negative feedback regulation,which may lead to reduce the p-ERK1/2protein ex-pression,which indicated that cell proliferation was obstructed. And the results alsoshow that the two poisons had synergies effects in combination groups.Part III p, p’-DDE and PCB-153and combined effects on hormonereceptors in pituitary cellsObjective: To investigate hormone receptor gene and protein expression levels afterPCB-153and p, p’-DDE exposure, and its combined exposure to pituitary cells,.Method:The real-time fluorescence quantitative PCR method was used to test theTRα1,TRβ1,TRβ2mRNA and Western blottng method was used to test the TRβ1protein expression in the rat pituitary cells in vitro.Results：Compared with the control group, TRa1mRNA expressions was signify-cantly decreased; and TRβ1and TRβ2mRNAexpression was significantlyincreased. as well as TRβ1proteinexpressionwas significantly increased. the resultsalso show that the two poisons had synergies effects in combination groups.Conclusion: p, p’-DDE, PCB-153can produce the combined effects on thyroidhormone receptor, thereby interfere with the hypothalamic-pituitary-thyroidaxis(HPT axis).