Cytotoxic Mechanism Related To Dihydrolipoamide Dehydrogenase In Leydig Cells Exposed To Heavy Metals

Objective: To study the cytotoxic mechanism related to dihydrolipoamide dehydrogenase(DLD), a mitochondrial protein, in Leydig cells exposed to heavy metals. Method: In vivo,the rats were divided into different groups: Pb treatment rats were treated with Pb by gavage in doses of 10 mg/kg and 25 mg/kg body weight for 10 w; Cd treatment rats were treated with Cd by gavage in doses of 5 mg/kg and 25 mg/kg body weight for 7 w; Mn treatment rats were injected with Mn intraperitoneally in doses of 5 mg/kg and 10 mg/kg body weight for 7 w. In vitro, R2 C cells were exposed to Pb(100 and 500 m M), Cd(10 and 20 m M) and Mn(500 and 1000 m M) in culture medium supplemented with 25 m M hydroxy cholesterol for 24 h. The concentration of steroid hormones was detected by radiation immunology technology. The expressions of steroid synthesis enzymes(St AR, CYP11A1 and 3β-HSD) in protein levels were measured by Western blottings. Mitochondrial membrane potential(ΔΨm) was detected by the technology of flow cytometry. Expressions of proteins and the phosphorylation level related to c AMP-PKA/PKC-ERK1/2 signaling pathway were validated by Western blottings. Our previous study reported that dihydrolipoamide dehydrogenase(DLD) located on the mitochondria was significantly decreased in Leydig cells exposed to cadmium, which suggested that DLD might be involved into the cytotoxic effects. Therefore, the altered expression of DLD was validated in rats and R2 C cells exposed to cadmium, manganese and lead. DLD was knockdown by si RNA to study the role of DLD in the steroid synthesis pathway c AMP-PKA-ERK1/2. Mitochondrial membrane potential(ΔΨm) was also detected by the technology of flow cytometry. Results: In vivo and vitro, steroid hormones were significantly decreased in Leydig cells after exposure to heavy metals. The results of flow cytometry showed that mitochondrial membrane potential(ΔΨm) in cells exposed to heavy metals was decreased notably compared to that in control cells. The protein expressions of St AR, CYP11A1 and 3β-HSD were decreased too. Heavy metals downregulated the expressions of PKA, PKC, and inhibited the activity of ERK1/2 with a decreased phosphorylation level.After knockdown of DLD, progesterone synthesis was reduced in cells compared to the normal synthesis level; ΔΨm had no alteration; the protein expression of steroidogenic enzyme St AR and 3β-HSD was dramatically reduced; and the concentration of c AMP, protein expression of PKA, and the phosphorylation of ERK1/2 were all decreased. Conclusion: In conclusion, heavy metals exposure dramatically reduced the expression of DLD in Leydig cells by inhibiting the activation of c AMP/PKA pathway and the steroidogenic enzymes St AR, CYP11A1 and 3β-HSD. DLD is related to steroid biosynthesis in the cytotoxic effects of heavy metals exposure in Leyig cells. DLD also regulates the expression of St AR and 3b-HSD enzymes. c AMP is involved in the process as downstream effector molecules of DLD, which activate phosphorylation of ERK1/2 to phosphorylate St AR and initiate the steroid biosynthesis.