Role of mitochondrial damage in BPDE-induced inhibition of testosterone production in mouse Leydig cells

Abstract

Objective To observe the effect of benzo[a]pyrene-r-7, t-8-dihydrodiol-t-9, 10-epoxide(±), (anti) (BPDE) on testosterone synthesis from mouse Leydig cells, and investigate the potential mechanism involved mitochondrial damage. Methods TM3 mouse Leydig cells were treated with various doses of BPDE for 48 h. Cell viability was assessed by trypan blue assay. Then the doses under the lethal dose were selected according to results of cell viability. After TM3 cells were treated with 0, 25, 50, or 100 nmol/L BPDE for 48 h, testosterone concentration in the medium, mitochondrial ultrastructure of TM3 cells, mitochondrial membrane potential, ATP level, mitochondrial DNA copy number, and expression levels of PGC-1α, COX Ⅳ, StAR and TSPO were measured through ELISA kit for testosterone, transmission electron microscopy, JC-1 probe, ATP detection kit, quantitative PCR and Western blotting, respectively. Furthermore, TM3 cells were pre-treated with 5 μmol/L ZLN005, a regulator to protect mitochondrial function and stimulate mitochondrial biogenesis. Then, after 24 h, these pre-treated cells were treated with 100 nmol/L BPDE for 48 h, the indicators above were determined in the same ways. Results BPDE at the doses of 25, 50, 100 nmol/L had no significantly effect on cell viability of TM3 cells. Compared to control cells, 50 and 100 nmol/L BPDE reduced testosterone synthesis by 21.15% and 34.96%, respectively (P < 0.01), and also induced mitochondrial structure and function, including alterations of mitochondrial ultrastructure, and appreciable decreases in mitochondrial membrane potential, cellular ATP production and mitochondrial DNA copy number (P < 0.05). What's more, the levels of proteins related to mitochondrial function, COX Ⅳ and to mitochondrial biogenesis, PGC-1α, and the key proteins for testosterone synthesis, StAR and TSPO, were all reduced in a dose-dependent manner. Compared to TM3 cells treated with 100 nmol/L BPDE alone, pre-treatment with 5 μmol/L ZLN005 for 24 h significantly elevated testosterone level in the medium (P < 0.05). Notable restores of the indicators above were observed in the TM3 cells pre-treated with ZLN005, including mitochondrial membrane potential, cellular ATP production, mitochondrial DNA copy number, and protein levels of COX Ⅳ, PGC-1α, StAR and TSPO (P < 0.05). Conclusion BPDE induces reduction of testosterone synthesis via mitochondrial compromise. Protecting mitochondria could alleviate BPDE-induced mitochondrial damage and reduction of testosterone synthesis.

Keywords

• benzo[a]pyrene-r-7 t-8-dihydrodiol-t-9 10-epoxide (±) (anti)
• leydig cells
• testosterone
• mitochondria