Study Of The Toxic Effects And Mechanism Targeted To Mitochondrial Damage In Leydig Cells Exposed To Lead

Objective: The toxic effects of lead acetate on Leydig cells were observed and the mechanismtargeted on mitochondria was studied.Method: Leydig cell lines R2C cells were exposed to100μM lead acetate in culture mediumsupplemented with25μM hydroxy cholesterol as progesterone synthesis substrate. Samples ofnormal cells and cells exposed to lead acetate were respectively collected at1,3,6,12and24h.The concentration of progesterone in the culture supernatant was detected by radiationimmunology technology. The expressions of progesterone synthesis enzymes (StAR, CYP11A1and3β-HSD) in gene and protein levels were measured by real time RT-PCR and Western blots.Mitochondrial membrane potential (ΔΨm) was detected by the technology of flow cytometryand intracellular levels of cAMP were measured by ELISA. The expressions of proteins and thephosphorylation level in cAMP-PKA/PKC-ERK1/2signaling were validated by Westernblots， and then the colocalization of MEK,ERK and mitochondria were validated byimmunofluorescence technology.Results: Compared with the normal control at the same time point, the content of progesteronesignificantly decreased in culture supernatant of R2C cell exposed to lead acetate in a timedependent manner (p<0.01at12h and p<0.001at24h). The gene expressions of StAR,CYP11A1and3β-HSD decreased at1h,6h and12h with exposure of lead acetate respectively.The protein expressions of StAR and CYP11A1decreased at1h, which were much earlier thanthe decreased expression of3β-HSD (decreased protein expression appeared at12h). The resultsof flow cytometry showed that mitochondrial membrane potential (ΔΨm) in cells exposed tolead acetate decreased about9.37%compared to the control at1h, and kept decreasing about10%at12h and24h. Transmissionelectronmicroscope reasults found lead acetate damaged themitochondria at1h, and it had a time effect on mitochondria from1h to24h. After exposure tolead acetate at different time points, intracellular levels of cAMP in R2C cells were also reducedsignificantly. Lead acetate downregulated the expressions of PKA at1h and PKC at3h, andinhibited the activity of MEK1/2and ERK1/2with the decreased phosphorylation level aftertreatment of1h. The reasults of immunofluorescence showed the colocalization of MEK,ERKand mitochondria decreased after the exposure of lead acetate.Conclusion: Lead acetate inhibits the progesterone secretion in Leydig cells withdownregulation of progesterone synthesis enzymes (StAR, CYP11A1and3β-HSD) in gene and protein levels. Lead acetate damaged mitochondria,and it significantly reduced mitochondrialmembrane potential (ΔΨm) and intracellular levels of cAMP in R2C cells. Lead acetatedownregulated the expressions of PKA at and PKC, and inhibited the activity of MEK1/2andERK1/2with the decreased phosphorylation level. All these results demonstrated that cAMP-PKA/PKC-ERK1/2signaling is related to the damage induced by lead acetate, andmitochondria is the target to effect. This mechanism likely contributed to a host ofpathophysiological events especially for Androgen Deficiency in Aging Male (PADAM) andMale hypogonadism.