Study On Effects Of Fenvalerate On Steroidogenesis And The Ralated Signal Pathways

In recent years,the endocrine disruption of environmental endocrine disruptors(EEDs)or endocrine-disrupting chemicals(EDCs)on human beings has caused comprehensive attention of many governments and researchers.Among the EDCs announced by USA Enviromental Protection Agency(USAEPA),two-thirds are pesticides.China,as a big agriculture country,the output and consumption of pesticides increase year after year.Among the pesticides,fenvalerate which belongs to the family of synthetic pyrethroid is widely used for its limited soil persistence.However,Many studied have showed that fenvalerate may cause reproductive dysfunction.In the present study,mouse Leydig tumor cells(MLTC-1)were used to determine the effects of fenvalerate on progesterone production and study the possible roles of cAMP-dependent cascade and IGF signal pathway involved in the effects.PartⅠEffects of fenvalerate on SteroidogenesisObjectiveTo investigate the effect of fenvalerate on steridogenesis.Methods1.The mouse Leydig tumor cells(MLTC-1)were used as the experimental model.2.The cell viability of MLTC-1 cells affected by fenvalerate was analyzed using the method of MTT.3.MLTC-1 cells were treated with different concentrations of fenvalerate for 24h.Then hCG(0.1U/L)was added into the medium.The level of progesterone in the medium was measured by RIA.4.MLTC-1 cells were treated with different concentrations of fenvalerate for 24h.Medium was then replaced by fenvalerate-free media for 24h following the stimulation with hCG.Results1.The doses of fenvalerate were selected in virtue of the MTT result. They were control(0),1,5,25 and 125μmol/L.2.Fenvalerate inhibited progesterone production in dose-dependent manner with the stimulation of hCG.3.After fenvalerate was removed,the progesterone production was similar in the cells previously treated by various concentrations of fenvalerate.ConclusionFenvalerate significantly inhibited steroidogenesis in MLTC-1 cells, and the cells could recover most of their steroidogenic activity after the removal of fenvalerate.PartⅡRole of cAMP-dependent cascade in the inhibited progesterone production of MLTC-1 cells by fenvalerateObjectiveTo investigate the role of cAMP-dependent cascade in the inhibited progesterone production of MLTC-1 cells by fenvalerate.Methods 1.MLTC-1 cells were treated with different concentrations of fenvalerate for 24h.Then 0.1U/L hCG,30ng/ml CT or 10μmol/L forskolin were added into the medium for additional 4h.The level of progesterone and cAMP was measured by RIA.2.MLTC-1 cells were treated with different concentrations of fenvalerate for 24h.Then 500μmol/L 8-Br-cAMP,25μmol/L 22R-HC or 10μmol/L pregnenolone were added into the medium for additional 4h.The level of progesterone was measured by RIA.3.mRNA expression of StAR and P450scc in MLTC-1 cells was determined by real-time RT-PCR.4.Protein expression of StAR and P450scc in MLTC-1 cells was assessed by western blot.Results1.The progesterone production declined in dose-dependent manner as the concentrations of fenvalerate increased when stimulated by CT or forskolin.2.Dose-dependent attenuation of cAMP formation was observed after 24h exposure to fenvalerate when MLTC-1 cells were stimulated with hCG.However,in the presence of CT or forskolin,fenvalerate treatment resulted in almost equivalent amounts of cAMP production at different doses. 3.When stimulated with 8-Br-cAMP,the progesterone production was significantly inhibited by fenvalerate.4.Fenvalerate significantly suppressed 22R-HC-driven progesterone production,and the inhibitory effect of fenvalerate on progesterone formation disappeared in the presence of pregnenolone.5.The expressions of StAR mRNA and protein decreased in MLTC-1 cells as the doses of fenvalerate increased.6.Fenvalerate treatment inhibited P450scc mRNA and protein levels in a dose-dependent manner.ConclusionFenvalerate inhibited progesterone production in MLTC-1 cells,and this action involved the impairment of the cAMP-dependent cascade by attenuating cAMP formation.Also,fenvalerate suppressed StAR and P450scc expression,protein levels and/or its activity which might partly be explained by the involvement of the cAMP-dependent cascade.PartⅢRole of IGF-I signal pathway in the inhibited progesterone production of MLTC-1 cells by fenvalerate ObjectiveTo investigate the role of IGF-I signal pathway in the inhibited progesterone production of MLTC-1 cells by fenvalerate.Methods1.MLTC-1 cells were treated with different concentrations of fenvalerate for 24h.Then hCG was added into the medium for 4h.IGF-I mRNA expression was determined by RT-PCR.3.MLTC-1 cells were treated with different concentrations of fenvalerate for 24h.Then hCG,hCG plus LY294002 or hCG plus U0126 were added into the medium for additional 4h.4.MLTC-1 cells were treated with different concentrations of fenvalerate for 24h.Then hCG was added into the medium.The expression of ERK1/2 phosphorylation was assessed by western blot.Results1.The expressions of IGF-I mRNA decreased in MLTC-1 cells as the doses of fenvalerate increased.2.The progesterone production gradually declined stimulated by hCG or hCG plus LY294002.And the two trends were similar;When U0126 was added to the medium,a more significant declining trend was observed than without U0126.3.Fenvalerate treatment inhibited ERK1/2 phosphorylation.ConclusionFenvalerate inhibited progesterone production in MLTC-1 cells,and this action might involved the impairment of the IGF signal pathway by attenuating the expression of IGF-I mRNA and ERK1/2 phosphorylation.