The Machanism Of MBP And MEHP Co-infected In Leydig Cells Mitochondrial Damage And Its Effect For Testosterone Synthesis

Objective: To study the mitochondrial injure mechanism of phthalic acid monobutyl ester(monobutyl phthalate,MBP)and phthalic acid(2-ethylhexyl)ester(mono(2 ethylhexyl)phthalate,MEHP)joint effect to testosterone production level in vitro cultured mouse leydig cells(TM-3).Methods: 1.Mouse testicular stromal cells(TM-3)were cultured in complete medium containing 10% fetal bovine serum and DMEM high glucose.2.The cells were treated with different concentrations of 0(control),1,10,100,500,1000 μmol/L MBP and MEHP,which was applied to mouse testicular stromal cells for 24 hours.The cell viability or inhibition rate was detected by MTT method(3,4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide.Detect the effects of MBP and MEHP toxicity on cell structure change.The modified K?ber method was used to get the median lethal doses of both MBP and MEHP,and the dose of each groups in subsequent experiments were determined by which.3.Transmission electron microscopy was used to observe the change of mitochondrial ultrastructure.4.Mitochondrial membrane potential detection kit was used to detect mitochondrial membrane potential changes.5.Western blot was used to detect the expression level of dihydrolipoamide dehydrogenase(DLD)and steroidogenic acute regulatory protein(StAR)in cell lysis buffer.6.The method of enzyme-linked immune insorbent assay(ELISA)detects cAMP levels.7.The method of enzyme-linked immune insorbent assay(ELISA)detects testosterone levels.Results: 1.MBP,MEHP and MBP+MEHP combination could all inhibit the proliferation of TM-3 cells,damaged the cell structure and decreased the cell survival rate.The difference was statistically significant(P<0.05).2.Compared with the control group,mitochondrial structural damage can be caused by MBP,MEHP and MBP+MEHP.The mitochondrial edema,swelling and rounding,mitochondrial lysis,disappearance,and even mitochondrial vacuoles disappear.3.MBP,MEHP and MBP+MEHP combination can cause mitochondrial membrane potential decrease,compared with the control group,the difference was statistically analyze by 2×2 Factorial design ANOVA,The interaction effect of MBP+MEHP combined exposure on the mitochondrial membrane potential of TM-3 cells was statistically significant(P<0.05)and showed an antagonistic effect.4.Testosterone levels decreased in the group of MBP,MEHP and MBP+MEHP,compared with the control group,the difference was statistically significant(P<0.05).After the analysis of variance of 2×2 factorial designs,the interaction effect of MBP+MEHP combined on testosterone levels was statistically significant(P<0.05),and showed an antagonistic effect.5.MBP,MEHP and MBP+MEHP combination could reduce the expression of DLD,StAR and cAMP in testicular stromal cells,compared with the control group,the difference was statistically significant(P<0.05).By 2×2 factorial design analyses of variance,the interaction of MBP+MEHP on the expression of DLD,StAR,and cAMP were statistically significant(P<0.05),showing a synergistic effect,and the difference was statistically significant(P<0.05).Conclusions: 1.MBP,MEHP and MBP+MEHP combination can inhibit the proliferation of Leydig cells(TM-3).2.MBP,MEHP and MBP+MEHP combination had mitochondrial damage effect on mouse testicular stromal cells.3.MBP,MEHP and MBP+MEHP combination can inhibit the production of DLD,StAR and cAMP.MBP+MEHP combination has a combined effect on the production of DLD,StAR and cAMP,and it was showed synergistic effect.4.MBP,MEHP and MBP+MEHP can lead to the decrease of mitochondrial membrane potential and the decrease of testosterone level,and MBP+MEHP combination on mitochondrial membrane potential and testosterone has the interact effect,and it was antagonistic.5.MBP,MEHP and MBP+MEHP combination may damage the mitochondria of mouse testicular stromal cells,inhibit the upstream indicators of the testosterone synthesis pathway in mitochondria,and influence the synthesis of testosterone.