1-nitropyrene induces ovarian granulosa cell dysfunction through mitochondrial damage

To investigate the effect of 1-nitropyrene（1-NP）on the proliferation, cycle progression and sex hormone synthesis of human ovarian granulosa cell line KGN via inducing mitochondrial damage.

After KGN cells were treated with 1-NP at different concentrations（0.625, 1.25, 2.5, 5 and 10 μmol/L）for 48 h, cell proliferation, cell cycle, and contents of estradiol and pregnenolone in the supernatant were detected by EdU assay, flow cytometry and ELISA, respectively. And the levels of reactive oxygen species（ROS）, mitochondrial superoxide（MitoSOX）and mitochondrial membrane potential were examined with specific fluorescent probes including DCFH-DA probe, MitoSOX probe and JC-1 probe, respectively. In addition, ATP kit was adopted to measure the cellular ATP level, and Western blotting was also conducted to determine the expression of proteins related with the cell proliferation, cycle progression, DNA damage response and synthesis of sex hormones. Finally, the mitochondrial antioxidant idebenone（IDE, 1 μmol/L）and 1-NP（10 μmol/L）were combined to treat the cells for 48 h, and then the above indicators were determined correspondingly again.

As compared with the control cells, 1-NP（0.625~10 μmol/L）treatment significantly inhibited cell proliferation（P<0.05）. The expression of PCNA, a marker of cell proliferation, was reduced after treatment with 1-NP（2.5, 5 or 10 μmol/L）, and the percentage of cells at G₂/M phase was elevated at 5 and 10 μmol/L of 1-NP（P<0.05）. Western blotting showed that the expression of γ-H2AX began to rise after 1-NP treatment（10 μmol/L）for 3 h（P<0.01）, and the levels of DNA damage pathway proteins, p-ATM, p53 and p21^cip1 were also obviously increased（P<0.05）, while those of G₂/M phase block-related proteins, CDK-1 and CyclinB1, were notably decreased at different doses of 1-NP（P<0.05）. In addition, the contents of estradiol and pregnenolone in the supernatant of KGN cells were remarkably diminished after 1-NP treatment（P<0.05）, and the expression levels of sex hormone synthesis-related proteins CYP19 and CYP11A1 were inhibited as well（P<0.05）. The detection of mitochondrial and oxidative stress-related indicators indicated that the levels of ROS and MitoSOX in KGN cells were greatly up-regulated due to 1-NP treatment, while the mitochondrial membrane potential and ATP level were decreased in a dose-dependent manner. However, the combination of 1-NP and IDE significantly declined the percentage of G₂/M-phase cells, elevated the contents of estradiol and pregnenolone, and restored the expression levels of proteins related to DNA damage, cell cycle as well as sex hormone synthesis when compared with the cells treated with 10 μmol/L 1-NP. Moreover, the combination treatment also ameliorated the mitochondrial damage induced by 1-NP, including lowering ROS and MitoSOX levels and improving the mitochondrial membrane potential and ATP level.

1-NP can cause mitochondrial damage, which leads to proliferation inhibition, cycle arrest and disorder of sex hormone synthesis, improve mitochondrial function, and thus partially alleviate 1-NP-induced dysfunction in KGN cells.