The Effects And Mechanism Of PCB118 And 4-OH-CB107 On GnRH Secretion In GT1-7 Cells

PCB118 and its hydroxyl metabolite 4-OH-CB107 are both typical endocrine disrupting chemicals which have been found to have endocrine disrupting effects on animals and humans.In the past,Researches on endocrine disruption of PCBs and OH-PCBs pay much more attention to the disruption of thyroxine and estrogen.Studies on neuroendocrine system,however,is relatively few.As a consequence,this study was conducted to investigate the effects of PCB118 and 4-OH-CB107 on GnRH secretion and the underlying mechanism in GT1-7 cells,a model of mouse immortalized hypothalamus GnRH neuron in vitro.GT1-7 cells were exposed to different concentrations of PCB118(0~50000 nM)and 4-OH-CB107(0~500 nM)at different time points.Cell viability,LDH activity and cell apoptosis were respectively performed by CCK-8 assay,LDH release assay and Annexin V-FITC/PI double staining methods.Then,GT1-7 cells were treated with PCB118 in different concentrations of 0,0.05,5,500 and 50000 nM,while 4-OH-CB107 in different concentrations of 0,0.05,5 and 500 nM.After 6 h and 24 h in culture,the concentration of GnRH in cell culture media was determined by mouse GnRH enzyme-linked immunosorbent assay(ELISA)kit.The expression of associated proteins of GnRH,GPR54,PLC and PKC in Kisspeptin/GPR54 signal pathway was detected by Western blot and further detected the related gene expression of GnRH,GPR54,PLC and PKC by Real-time quantitative PCR assay.The results showed that PCB118(0.05~50000 nM)significantly reduced cell viability of GT1-7 cells in dose-and time-dependent manners after exposure to PCB118 for 6 h,12 h,24 h and 48 h.Interestingly,increased release of lactate dehydrogenase(LDH)and the increase of early apoptotic cells was only observed under the treatment of 50000 nM PCB118.Exposure to 5,500 and 50000 nM PCB118 for 6 h significantly decreased the concentration of GnRH in the culture media.The expressions of GnRH,GPR54,PLC and PKC proteins were significantly inhibited.And the expressions of PLC and PKC genes were down-regulated while the expressions of GnRH and GPR54 genes were not changed significantly;Similarly,GnRH expression levels were decreased significantly after exposure to 500 or 50000 nM PCB118 for 24 h,the expressions of GnRH,GPR54,PLC and PKC proteins and genes were also significantly decreased.4-OH-CB107(0.05~500 nM)significantly reduced cell viability of GT1-7 cells after exposure for 48 h.0.05 nM 4-OH-CB107 could stimulate GnRH release after 6 h treatment with the increase of protein and gene expressions of GnRH,GPR54,PLC and PKC;Only 500 nM 4-OH-CB107 could stimulate GnRH release after 24 h treatment.And it could also increase the expressions of these proteins and genes.Although there is no significant difference on the secretion of GnRH after exposure to 5 nM 4-OH-CB107,the protein and gene expressions of GnRH,GPR54,PLC and PKC were increased.Above all,PCB118(0.05~50000 nM)and 4-OH-CB107(0.05~500 nM)both have the cytotoxic effect of inhibiting cell proliferation in dose-and time-dependent manners.50000 nM PCB118,which has significant cytotoxicity,could increase the release of LDH and promote early apoptosis of GT1-7 cells;PCB118 could inhibit the synthesis and secretion of GnRH through down-regulating the expressions of GPR54,PLC and PKC proteins and genes in Kisspeptin/GPR54 signal pathway at the gene and protein level.And its main metabolite 4-OH-CB107 could stimulate the secretion of GnRH via promoting the expressions of GPR54,PLC and PKC proteins and genes.Kisspeptin/GPR54 signal pathway is partly involved in effects of PCB118 and 4-OH-CB107 on GnRH synthesis and secretion of GT1-7 cells.