Exposure To Benzo(a)pyrene Suppresses Ovarian Mitophagy Via ANT1-PARKIN During Early Pregnancy

Objective:Benzo（a）pyrene is a widespread environmental chemical carcinogen.Studies have reported that BaP exposure impairs embryo implantation.This process is associated with the estrogen and progesterone synthesised in ovary.Autophagy is involved in regulating the generation of steroid hormones and maintaining homeostasis.Mitophagy,as a special type of autophagy,plays important role in clearing damaged mitochondria and maintaining mitochondrial function.Mitochondria are the source of steroid hormone synthesis,and dysfunction of mitochondria will disrupt normal ovarian function.Therefore,the purpose of this study was to investigate the effects and the potential molecular mechanisms of BaP on ovarian mitophagy during early pregnancy.Methods:（1）Animals and treatment:The pregnant mice were randomly divided into the control group and BaP-treated group,and then received corn oil and 0.2 mg/kg/d BaP daily by oral gavage at 0.1 ml/10 g of body weight respectively from pregnant day 1（D1）to D7.The pregnant mice were sacrificed by cervical dislocation on D4 and D7,and the ovaries were collected immediately.（2）Cell culture and treatment:The cultured human ovarian granule cell（KGN）line were divided into 3 groups and treated with vehicle alone（control group,0.1%DMSO）,1.0 IU/ml hCG（hCG group）and 1.0 IU/ml hCG plus 0.5μmol/L BPDE（hCG+BPDE group）simultaneously for 24h at 37℃with 5%CO₂.（3）The effects of BaP and BPDE on autophagy:The autophagosomes was observed by TEM in vivo and in vitro.Western blot,q RT-PCR and IF were used to test the expression of autophagy markers ULK1、BECLIN1、ATG5、ATG7、LC3 and P62.（4）The effects of BaP and BPDE on mitophagy:Western blot and IF were used to detect the expression of mitophagy markers PINK1、PARKIN、TOM20 and LC3.（5）The role of ANT1 on BPDE-inhibited mitophagy:The expression of ANT1 was detected by Western blot and q RT-PCR both in vivo and in vitro.Overexpression of ANT1 was used to investigate whether ANT1 is involved in the regulation of the BPDE-inhibited mitophagy in luteinic KGN cells.Results:（1）BaP exposure inhibited ovarian autophagy during early pregnancy.（2）BPDE exposure inhibited autophagy in luteinic KGN cells.（3）Exposure to BaP and BPDE suppressed PINK1/PARKIN-mediated mitophagy in vivo and in vitro respectively.（4）Exposure to BaP and BPDE inhibited the expression of ANT1 in vivo and in vitro respectively and affected mitochondrial function.（5）Overexpression of ANT1 induced mitophagy,which was suppressed by BPDE exposure,improved mitochondrial function and increased the mRNA levels of rate-limiting enzymes of steroid hormone.Conclusion:This study indicates that BaP and its metabolites BPDE can inhibit mitophagy by suppressing ANT1-PINK1-PARKIN signaling,affect mitochondrial function,and decrease the levels of estrogen and progesterone in vivo and in vitro respectively.While overexpression of ANT1 can restore mitophagy,improve mitochondrial function,and increase the mRNA levels of rate-limiting enzymes of steroid hormone.