Research Of Joint Effects On Nerve Cells Of Different Types Of Endocrine Disrupting Chemicals And The Mechanism

Objective:1.Study of Dose-effect Relationship of diethylstilbestrol(DES),dibutyl phthalate(DBP),propyl thiouracil(PTU)on the proliferation of GT1-7 cells.2.The joint toxicity of DES,DBP and PTU to GT1-7 cells which secrete Gn RH was investigated.3.The possible mechanism of secreting Gn RH of GT1-7 cells that was exposed to DES,DBP and PTU jointly.Methods:1.The immortalized hypothalamic GT1-7 neuron model was applied to carry out the experiment after the three kinds of subjects were exposed for 24 hours.The concentration of the subjects: The dose of DES was 0.1、1、10、100、1000 mmol/L,the dose of DBP was 0.01、1、10、100、1000 mmol/L,the dose of PTU was 0.0001、0.01、1、100、1000 mmol/L.DES(0.1-1000mmol/L),DBP(0.01mmol/L),PTU(0.0001mmol/L)were used for joint toxicity.2.The cell counting Kit-8 was used for detecting the cell viability of DES,DBP and PTU while they were exposed to GT1-7cells individually or jointly.3.Gn RH RIA Kit was used for evaluating the secretion of Gn RH in alone or combined cell culture medium4.Western Blot was used for evaluating the protein expression of PLC,GPR54,PKC,and Gn RH in combined exposure group.5.RT-PCR was used for evaluating the Gene expression of PLC,GPR54,PKC,and Gn RH in combined exposure group.Results:1.When it was exposed to DES alone,the cell viability of GT1-7 cells in each group was reduced from 110% to about 4%.The cell viability of each group was reduced from130% to about 60% when exposed to DBP.It showed that the cell viability increased first and then decreased with the concentration increasing.Compared with the control group,the cell viability of GT1-7 cells exposed to PTU was increased and maintained at a certain level of about 120%-135%.When the concentration of DES,DBP,PTU was below 10 mmol/L、100 mmol/L、1000 mmol/L respectively,the cell viability of GT1-7cells was higher than 90%.The secretion of Gn RH from GT1-7 cells was increased first and then decreased with the increase of the concentration of DES.The control group was 2.36 μg/ml,and the exposure group decreased from 6.26 μg/ml to 2.19μg/ml.With the increase of DBP concentration,the secretion showed a downward trend,the control group decreased to 2.60 μg/ml while the exposure group was down to 1.66 μg/ml,1.95 μg/ml.With the increase of PTU concentration,it was first increased and then decreased and the control group was 2.3 μg/ml while the exposure group decreased from 3.01 μg/ml to 1.46 μg/ml2.When GT1-7 cells were exposed to DES and PTU,DBP and PTU,the cell viabilities were increased first and then decreased with the increase of the concentration.Compared with exposed to DBP individually,the cell viability was increased about126%-160% when 0.01 m mol/L DBP and 0.0001 m mol/L PTU was added to0-10μmol/L DES.Added 0.01 mmol/L DBP and 0.0001 mmol/L PTU into DES,Gn RH secretion decreased with the increase of DES concentration,control group was 4.23μg/ml,exposure group was 0.75 μg/ml and 1.50 μg/ml.3.Compared with control group,the expression of PKC protein in the Kisspeptins/GPR54 signaling pathway was 1.8 and 1.3 respectively,the expression of PLC protein in each concentration group was 1.11 and 1.14,and protein expression level of GPR54 was 1.1 with 0.1μmol/L DES.It showed the expression of these proteins was increased compared with the control group.The expression of Gn RH protein in each concentration group was 0.9 and 0.8,GPR54 with 1 μmol/L DES protein expression level was 0.7.It meant the expression of the protein was decreased compared with the control group.The expression of PKC gene was 3.9 and 2.8,the expression of GPR54 gene was 2.2 and 1.0,and the expression of PLC gene was 2.1and 1.7.The expression of these genes was increased compared with the control group while the expression of Gn RH gene was 0.8 and 0.6.The PLC,PKC and GPR54 Gene expression showed an increase trend while Gn RH Gene expression was decrease.In addition,the expression of Gn RH protein and its gene expression in cells were consistent with the changes of Gn RH secretion in the culture medium,which was contrary to the changes in the expression of several major proteins in the signaling pathway.Conclusion:1.Exposed to DES,DBP,and PTU jointly in a certain concentration,it had a joint effect on cell proliferation.According to the concentration of DES,the synergistic and antagonistic effects were observed.It was synergistic effects when exposed to 0.01μmol/L DBP,0.0001 μmol/L PTU and 0-10 μmol/L DES while it was antagonistic effects when exposed to 0.01 μmol/L DBP,0.0001 μmol/L PTU and 100 μmol/L DES.2.When a certain concentration of DES,DBP and PTU were exposed to GT1-7 cells,it showed a synergistic effect on the secretion of Gn RH.3.The combined exposure of DES,DBP and PTU may promote the protein and gene expression of GPR54,PKC,PLC in Kisspeptins/GPR54 signaling pathway while inhibit the protein and gene expression of Gn RH.The results suggest that although Kisspeptins/GPR54 signaling pathway is involved in the regulation of Gn RH secretion,there may be other significant signaling pathways involved.The specific regulatory mechanisms and other unknown mechanisms need to be further studied.