Mechanism Of Human Trophoblast Swan 71 Cells Mitochondrial Damage Induced By Benzo(a)Pyrene-7,8-9,10 Diol Epoxide

Objective Benzo(a)pyrene[B(a)P] has been identified as a human teratogen,mutagen,carcinogen and endocrine disruptor.Numerous studies have shown that B(a)P causes trophoblast-related diseases,such as preeclampsia,growth restriction or miscarriages.However,the underlying mechanism of mitochondrial-related BPDE-induced trophoblast dysfucntion is unclear.In this study,we investigated the functions of trophblast cell line Swan 71 treated with Benzo(a)pyrene-7,8-diol-9,10-epoxide(BPDE)which is the ultimate mutagen of B(a)P in mammal.We studied the changes in cell and mitochondrial functions,futher explored their corresponding molecular mechanism.Studies on the effects of mitochondrial damage in the trophoblast cell dysfunction provide further understanding for effective and specific biological treatment in trophoblastic disease.Methods:1.MTT assay was adopted to select BPDE concentrations for cell treatment.Cells invasion ability was determined using Transwell method,HCG secretion was test by ELISA and Western blot methods and cell apoptosis was test by flow cytometry method.Then,ROS,MDA and SOD levels in the cells were determined using fluorescence or ultraviolet photometer.Futhermore,the protein expression levels of pro-inflammatory(IL-6 and TNF-а)in different groups were determined by RT-PCR method.Mitochondrial membrane potential and cell constructure were detected by fluorescence microscope and transmission electron microscope.Mitochondrial DNA copy number was detected by Real time quantitative PCR.2.First: The protein and m RNA levels of mitochondrial fusion and fission genes(Mfn1,Mfn2,OPA1,Drp1 and Fis1)were determined by quantitative Real-time PCR analysis and Western Blot analysis.Second: The apoptosis-related proteins,Cyt c in mitochondrial or cytoplasm,Pro-Caspase 3 and Actived-Caspase 3 were determined by Western Blot analysis.Results 1.Effect of BPDE on Swan 71 cell function.The invasion ability of Swan 71 cells exposed to 0.1 μmol/L,0.2 μmol/L,0.4μmol/L or 0.8 μmol/L BPDE was attenuated compared with control group,the differences were statistically significant(P < 0.001);HCG in cellular supernatant and protein expression of choriogonadotropin β were gradually decreased with increasing BPDE concentrations in 0.5,1 and 2 μmol/L BPDE(P < 0.05).Cell apoptosis was increased significantly after exposure to 0.5,1 and 2 μmol/L BPDE(P < 0.001).2.Effect of BPDE on Swan 71 cell oxidative stress.The ROS and MDA levels in Swan 71 cells were significantly increased,while SOD activities were significantly decreased compared with the control group after exposure to 0.25μmol/L,0.5μmol/L,1μmol/L and 2μmol/L BPDE(P < 0.05).IL-6 and TNF-α m RNA expression levels were significantly increased after treatment with 1μmol/L and 2 μmol/L BPDE(P < 0.001).3.Effect of BPDE on Swan 71 cell mitochondrial constructure and functionThe mitochondrial membrane potential was reduced with increasing BPDE concentration.In 0.5μmol/L group,most of mitochondria were swelling,the mitochondrial cristae were arranged in disorder,the mitochondrial membrane was fragmentation and the endoplasmic reticulum was swelling,determiend by transmission electron microscopy.In 1μmol/L group,mitochondria showed serious injuries,orderly mitochondrial breach,cristae dissolved and endoplasmic reticulum presented vacuole-shape.The mtDNA copy number was significatly increased compared with control group after exposure to 0.25μmol/L,0.5μmol/L and 1μmol/L BPDE(P < 0.05),then it was decreased at 2μmol/L BPDE(P > 0.05).4.Effect of BPDE on apoptosis-related proteinsWith increasing BPDE concentration,protein levels of P53,Bax and Bak1 were gradually increased but those of Bcl-2 were gradually decreased.All groups were statistically significant compared to control group except P53 in 0.25 μmol/L group(P < 0.05).5.Effect of BPDE on Swan 71 cell mitochondrial fusion and fission genes.The mRNA level of fusion-related OPA1,Mfn1 and Mfn2 were significantly decreased while fission-related Drp1 were significantly increased relative to the control group after exposure to 0.5 μmol/L,1 μmol/L and 2 μmol/L BPDE(P < 0.05).The m RNA level of fission-related Fis1 were significantly increased relative to control after exposure to 1 μmol/L and 2 μmol/L BPDE(P < 0.05).The protein level of OPA1 were significantly increased relative to control at 0.5 μmol/L,1 μmol/L and2 μmol/L BPDE(P < 0.05).The protein level of Mfn1 and Mfn2 were significantly decreased(P < 0.05)while Drp1 and Fis1 were significantly increased(P < 0.001)relative to control at 0.25 μmol/L,0.5 μmol/L,1 μmol/L and 2 μmol/L BPDE.6.Effect of BPDE on Swan 71 cell Cyt c release and Caspase 3The amount of Cyt c in mitochondrial was decreased but that in cytoplasm was increased with increasing BPDE concentration.Pro-Caspase 3 was decreased but the the Activated-Casepase-3 was increased in BPDE-treated Swan 71 cells.Conclusion: This study reveal that BPDE exposure leads to increase human trophoblast Swan 71 cells oxidative damage due to increase mitochondrial fission and decrease fusion,leading to mitochondrial membrane fragmentation.Subsequently,Cyt c is released from mitochondrial to cytoplasm and lead to a Mitochondrial pathway apoptosis.Finally,the apoptosis of Swan 71 cells reduces the abilities of migration,invasion and HCG secretion.Providing experimental evidence for trophoblast related diseases resulting from BPDE.