Effects Of Trichlorfon On Steroidogenesis

Since 1990s, environmental endocrine disruptors (EEDs) orendocrine-disrupting chemicals (EDCs), which do harm to human healthand environment, have been drawing attention to academic and public ofmany countries. Pesticide is one of the big categories among EEDs.Trichlorfon, one of the most popular organophosphorus pesticides, hasalso been listed as a potential endocrine disrupter by the German FederalEnvironment Agency. With the increase in the application of trichlorfonin recent years, many reports have shown that trichlorfon has adverseeffects on reproductive system of human and animals. Therefore, it isnecessary to explore the adverse effects of trichlorfon from thereproductive and endocrine point of view. The present study wasperformed to determine the effects of trichlorfon on steroidogenesis in ovary and testis using cultured cells in vitro. Another purpose was toelucidate the possible roles of cAMP-dependent cascade involved in theeffects.PartⅠEffects of Trichlorfon on Steroidogenesis in OvaryObjectiveTo investigate the effect of trichlorfon on steridogenesis in ovary.Methods1. The primary culture of human granulosa-lutein cells (hGLCs) was usedas the experimental model.2. The cell viability of hGLCs affected by trichlorfon was analyzed usingthe method of MTT.3. hGLCs were treated with different concentrations of trichlorfon for24h. In some conditions, 200ng/ml FSH, 1mmol/L 8-bromo-cAMP or25μmol/L 22R-HC were added into the medium. The level ofprogesterone in the medium was measured by RIA.4. cAMP was extracted with absolute alcohol, and determined by RIA.5. The level of StAR mRNA expression in hGLCs was determined byreal-time RT-PCR.Results 1. The doses of trichlorfon were selected in virtue of the MTT result, thatwas, there was nontoxic effect of trichlorfon on hGLCs viability. Theywere control (0.1% solvent), 1, 5, 25 and 125μmol/L.2. In basal condition, trichlorfon inhibited progesterone production in thedose-dependent and time-dependent manners.3. The inhibitory trend of trichlorfon on progesterone production in200ng/ml FSH condition was similar as in the basal condition. At25μmol/L and 125μmol/L, progesterone yield was significantly declinedby 44.4% and 51.3%, respectively. However, the 8-bromo-cAMPtreatment did not abolish the suppression of trichlorfon on progesteroneproduction.4. There were no significant changes in cAMP levels induced bytrichlorfon with or without FSH-stimulation.5. The alteration of progesterone level induced by trichlorfon disappearedwhen 22R-HC was added into the medium.6. Either in basal or FSH-stimulated condition, trichlorfon induced StARmRNA abundance reduced in a dose-dependent manner. In bothconditions, at 125μmol/L, trichlorfon could cause about 85% of StARgene expression decreased.ConclusionTrichlorfon significantly inhibited steroidogenesis in hGLCs, at least in part, by obstructing the delivery of cholesterol over mitochondrialmembranes, and the cAMP-dependent cascade may not be involved.PartⅡEffects of Trichlorfon on Steroidogenesis in TestisObjectiveTo investigate the effect of trichlorfon on steridogenesis in testis.Methods1. The MTT method was employed to assess the viability of mouseLeydig tumor cells (MLTC-1) and ensure the appropriate dose oftrichlorfon.2. MLTC-1 cells were treated with different concentrations of trichlorfonfor 24h. In some conditions, 0.1U/L hCG, 30ng/ml CT, 10μmol/Lforskolin, 25μmol/L 22R-HC or 10μmol/L pregnenolone were added intothe medium for additional 4h. The level of progesterone and cAMP wasmeasured by RIA.3. mRNA expression of StAR and P450scc in MLTC-1 cells wasdetermined by real-time RT-PCR. 4. Protein expression of StAR and P450scc in MLTC-1 cells wasassessed by western blot.Results1. The doses of trichlorfon were selected in virtue of the MTT result, thatwas, there was nontoxic effect of trichlorfon on MLTC-1 cell viability.They were control (0.1% solvent), 0.04, 0.2, 1, 5 and 25μmol/L.2. Whether in hCG, CT or forskolin stimulated condition, theprogesterone production gradually decreased in dose-dependent manneras the concentrations of trichlorfon in medium increased. At 25μmol/L,progesterone yield declined by about 85%.3. There were no significant changes in cAMP levels induced bytrichlorfon with any of the activators (hCG, CT or forskolin).4. The expressions of StAR mRNA and protein gradually decreased inMLTC-1 cells as the concentrations of trichlorfon increased, and theinhibition to StAR protein was even more significant than StAR mRNA.5. Trichlorfon significantly suppressed 22R-HC-driven progesteroneproduction, and the inhibitory effect of trichlorfon on progesteroneformation disappeared in the presence of pregnenolone.6. Treated with trichlofon for 24h, P450scc mRNA and proteinexpressions were significantly decreased in dose-dependent manners. ConclusionTrichlorfon significantly inhibited steroidogenesis in MLTC-1 cells,and the inhibition of StAR expression and P450scc activity may be themechanisms of this response. However, cAMP-dependent cascade maynot be involved.