Study On The Mechanisms Of Steroidogenesis Induced By Mono-butyl Phthalate In Mouse Leydig Tumor Cells

Phthalate acid esters (PAEs), a large group of industrial andpharmaceutical chemicals distributed diffusely in the environment, caninterfere with reproduction and development in an endocrine-mediatedprocess. Dibutyl phthalate (DBP), one of the most dominant PAEs, isused mainly as a plasticizer in PVC products as well as in cosmetics andother personal care products. Examination of urinary PAEs metabolitesrevealed that the general people appeared to be exposed todisproportionately higher amounts of DBP than other PAEs. DBP and itsactive metabolite, mono-butyl phthalate (MBP), display no bindingaffinity for the androgen receptor, yet exert anti-androgenic effects byaltering steroid biosynthesis. However, the mechanisms underlying thisobserved effect are not known. The purpose of this study was todetermine the site of MBP action on steroidogenesis in vitro in mouseLeydig tumor cells (MLTC-1). Partâ… Effect of MBP on Steroidogenesis in MLTC-1 CellsObjective: To examine the response of MLTC-1 cells to somestimulators and the effects of MBP on steroidogenesis in intro. Methods:MLTC-1 cells were employed as a cellular model. Cell viability wasevaluated by measuring the level of ³H-thymidine incorporation(³H-TDR) and MTT assay. To assess the response of MLTC-1 cell tosome stimulators, the cells were stimulated by human chorionicgonadotrophin (hCG), cholera toxin (CT) and forsklin for 4h afterpreincubation for 24h. Then the medium was collected for progesteronemeasurement. In another experiment, various concentrations of MBPdetermined by cell viability test and the solvent DMSO were added tothe medium for 24, 48 and 72h followed by 4h-stimulation by hCG toexplore the time-and dose-effect of MBP on stroidogenesis. Results:Data showed that progesterone synthesis stimulated by hCG, CT andforskolin was in a dose-response manner. Furthermore, the MBPdosages below 10^-3 mol/L did not have any effects on cell viability. Theprogesterone levels induced by MBP exhibited biphasic model. Therewere rapid increases in progesterone production under MBP treatment at10^-7 to 10^-6 mol/L. Conclusion: This cell line displayed an excellentresponse to hCG, CT and forskolin. There was a stimulatory effect ofMBP on steroid hormone synthesis at relative low concentratin. MBPdosages used in this part, i.e. 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4 mol/L and 50,100, 200, 400 and 800μmol/L, could be used in the followingexperiments. Objective: To investigate the role of hCG-cAMP-PKA pathway andtransport of cholesterol in steroidogenesis induced by MBP. Methods:Various concentrations of MBP determined by partâ… were added to themedium for 24h followed by stimulation of some compounds such ashCG, CT, cAMP analog 8-Br-cAMP, 22R-HC and pregnenolone.Progesterone levels in the medium and amounts of intracellular cAMPwere measured by RIA. Expression of steroidogenic acute regulatoryprotein (StAR) was monitored by real-time PCR and Western blot.Results: The increases in progesterone production in the presence ofhCG, CT and 8-Br-cAMP were augmented by MBP at 10^-7 and 10^-6mol/L while suppressed at 200μmol/L and higher. In contrast, the levelsof intracellular cAMP exhibited no statistical significance under MBPtreatment. Moreover, 22R-HC and pregnenolone resulted in no alterationin progesterone production, making clear that MBP did not influence theP450scc and 3β-HSD activity but on the rate-limiting step insteroidogenesis pathway, cholesterol transportation into mitochondria. Infact, these results were confirmed by StAR expression in MBP-treatedcells as StAR is a key protein in the process of cholesterol transportation.Conclusion: Effect of this chemical on steroidogenesis shows aninvert-U dose-response curve. Furthermore, MBP promotes steroidhormone production by facilitating StAR expression while MBP inhibitssteroid hormone production by down-regulating StAR expression inMLTC-1 cells. Partâ…¢Role of Protein kinase C (PKC) and Ca²⁺ signalin Steroidogenesis Induced by MBPObjective: To explore the role of PKC and Ca²⁺ in steroidogenesisinduced by MBP. Methods: Various concentrations of MBP determinedby partâ… were added to the medium for 24h followed by co-treatmentwith hCG and PMA, PKI, AA, EGTA, VER, A23187 and TGrespectively. Progesterone levels were measured by RIA andintracellular Ca²⁺ concentration ([Ca²⁺]i) was monitored with fluorescentprobe for Ca²⁺, fluo3/AM. Results: Progesterone production in thepresence of hCG was seriously inhibited in the presence of PMA andPKI. Moreover, effect of MBP at the doses lower than 10^-5 mol/L on theprogesterone synthesis stimulated by hCG disappeared when the cellswere co-treated with PMA and hCG. Similar results could be seen withPKI in the medium at the doses of 10^-6 mol/L MBP and below. AAcould offset MBP effect at 10^-4 mol/L and lower. On the other hand,progesterone synthesis was thoroughly counteracted in the Ca²⁺-fleemedium or in the medium supplemented with Ca²⁺ chelator EGTA.Opposite result was obtained in the presence of A23187 which allowsextracellular Ca²⁺ entry. VER, a Ca²⁺ channel blocker, dramaticallydiminished MBP-induced progesterone biosynthesis in the presence ofhCG, but these two trends were similar. On the contrary, TG, aCa²⁺-ATPase inhibitor which increases [Ca²⁺]i, prevented the MBPeffects at doses of 200μmol/L and lower. Results from [Ca²⁺]imeasurement showed that 10^-6 mol/L MBP could markedly augmented[Ca²⁺]i under hCG treatment in Ca²⁺-free buffers. But, this dose didn't increase [Ca²⁺]i in Ca²⁺-supplemented buffers. In addition, [Ca²⁺]i wassuppressed by 10^-3 mol/L MBP in both Ca²⁺-free and Ca²⁺-supplemented buffers. Conclusion: Data from this part support thatPKC, AA and Ca²⁺ signal was involved in the changes of steroidhormone production affected by low concentrations of MBP in MLTC-1cells. Ca²⁺ signal is a key factor implicated in adjusting steroidogenesisaffected by MBP at relative high doses.