Effects Of Nickel Sulfate On Steroidogenic Enzymes And Oxidative Stress Of Rat Leydig Cells In Vitro

Objective To explore the role of steroidogenic enzymes and oxidative stress in changes of testosterone production induced by nickel sulfate (NiSO4) in rat primary Leydig cells.Methods The rat Leydig cells were purified by techniques of Percoll density gradient centrifugation and differential adherent culture, and identified by3beta-hydroxysteroid dehydrogenase (3β-HSD) staining assay. The logarithmic phase of purified Leydig cells treated with0,250,500and1000μmol/L nickel sulfate and1IU/ml Human chorionic gonadotropin (hCG) for0,6,12or24h respectively in vitro. The concentration of testosterone (T) in culture supernatant was determined by enzyme-linked immunosorbant assay. The mRNA expression levels of steroidogenic acute regulatory protein (StAR), cytochrome P450cholesteral side-chain cleavage enzyme (P450scc),3β-hydroxysteroid dehydrogenase (3β-HSD), steroid17-alpha-Hydroxylase (P450c17) in Leydig cells were measured by real-time quantitative PCR technique. Reactive oxygen species (ROS) level of Leydig cells was analyzed by flow cytometry. The inhibiting capacity of hydroxyl free radical (Anti-OH) was detected by spectrophotometry assay. The content of8-hydroxyquinoline deoxyguanosine (8-OHdG) of Leydig cells was determined by enzyme-linked immunosorbant assay.Results (1) The cytoplasm of purified Leydig cells were stained the deep blue by using3β-HSD staining assay, and the purity of leydig cells was above90%.(2) After treatment Leydig cells for24h with hCG+NiSO4, compared with Oh group, the concentration of T significantly increased in hCG+0μmol/L NiSO4group (P<0.01), but the concentration of T significantly decreased in1000μmol/L NiSO4group (P<0.01). Compared with hCG+0μmol/L NiSO4group, the concentration of T significantly decreased in all hCG+NiSO4groups after treatment for24h (P<0.01).(3) After treatment Leydig cells with hCG+NiSO4for24h, compared with Oh group, the level of StAR mRNA significantly increased in0μmol/L NiSO4group (P<0.01), the mRNA expression levels of P450scc,3β-HSD and P450c17only decreased in250μmol/L NiSO4group (P <0.01), the mRNA expression levels of StAR, P450scc,3β-HSD and P450c17decreased significantly in500μmol/L and1000μmol/L NiSO4groups (P<0.01). After treatment Leydig cells with hCG+NiSO4for24h, compared with0μmol/L NiSO4group, the mRNA expression levels of StAR, P450scc,3β-HSD and P450c17decreased in all NiSO4groups (P<0.01).(4) Compared with Oh group, the content of ROS increased in12h group after treatment with250μmol/L NiSO4(P<0.05), the contents of ROS increased in6,12and24h groups after treatment with500μmol/L and1000μmol/L NiSO4tespectively (P<0.05). Compared with0μmol/L NiSO4group, the contents of ROS increased in500μmol/L and1000μmol/L NiSO4groups at6h, and500μmol/L and1000μmol/L NiSO4groups at12h (P<0.05), and all NiSO4groups at24h (P<0.01).(5) Compared with Oh group, the Anti-OH level decreased significantly in12and24h groups after treatment with250μmol/L NiSO4group and in6,12, and24h groups after treatment with500μmol/L and1000μmol/L NiSO4groups (P<0.05). Compared with0μmol/L NiSO4group, the Anti-’OH level decreased significantly in500μmol/L and1000μmol/L NiSO4groups at6h, and all NiSO4groups at12and24h (P<0.01).(6) Compared with Oh group, the contents of8-OHdG increased significantly in12h and24h groups after treatment with500μmol/L and1000μmol/L NiSO4groups (P<0.01). Compared with0μmol/L NiSO4group, the contents of8-OHdG increased significantly in500μmol/L and1000μmol/L NiSO4groups at12h and24h (P<0.05).Conclusion The results indicated that the nickel sulfate inhibited testosterone secretion of Leydig cells may be related with downregulation of steroid synthase genes and oxidative DNA damage in vitro.