Di(2-ethyl-hexyl) Phthalate Inhibits Proliferation Of Placental Trophoblast Cells By Arresting Cell Cycle At G2/M Phase

Objective: we investigated the mechanism of DEHP inhibits placental cell proliferation in terms of cell cycle arrest.Methods: Part I: Model of MEHP inhibits the proliferation of human placental trophoblast cells(HTR-8 cells): DEHP active metabolite MEHP(0,20,200,500 μM)was used to treat HTR-8 cells for 24 hours.Proliferation and cell cycle were detected by flow cytometry.DNA damage was detected by comet assay.Western Blot was used to detect the expression levels of DNA double-stranded marker protein(γ-H2AX),genotoxic stress-related proteins(ATM),DNA double-stranded repair protein(PARP-1,RAD51,DNA-PKcs)and cell cycle related proteins(CDK1,Cyclin B1).Part II: The effect of high level progesterone on HTR-8 cell cycle.HTR-8 cells were treated with MEHP(0,20,200,500 μM)for 24 hours.The contents of progesterone in the supernatant of HTR-8 cells were detected by ELISA.HTR-8 cells were treated with high level progesterone(0,50,250,500 n M)for 24 hours.Cell cycle was detected by flow cytometry.Western Blot was used to detect the expression levels of γ-H2 AX,Cyclin B1 and CDK1.Thymidine synchronizes HTR-8 cells in the S phase and releases them so that they can enter mitosis.The mitotic status of HTR-8 cells was determined by timing sampling,immunofluorescence and live cell observation.Part III: Verification of the genotoxicity of DEHP in vitro and in vivo.Adult ICR mice were randomly divided into four groups: control group(Tween 80+1% corn oil),DEHP group(5,50,500 mg/kg).Changes in body weight and blood glucose were measured before and after administration to evaluate the general condition of mice.The acute genotoxicity of DEHP was detected by bone marrow micronucleus assay.Pregnant mice were randomly divided into four groups: control group(Tween 80+1% corn oil)and(5,50,200 mg/kg)DEHP treatment group.The vaginal plug was found on the 0th day of pregnancy(GD0).From GD9 to GD15,DEHP were given by gavage and mice were sacrificed on GD16.The genotoxicity of DEHP was detected by micronucleus assay in bone marrow,fetal liver and placenta.HTR-8 cells were treated with MEHP(0,20,200,500 μM)for 24 hours.The genotoxicity of MEHP was verified in vitro by cytokinesis block micronucleus assay.The source of micronucleus was determined by immunofluorescence of ACA protein.HTR-8 cells were treated with high level progesterone(0,50,250,500 n M)for 24 hours.HTR-8 cells were treated with high level progesterone(0,50,250,500 n M)for 24 hours.Results: In vitro,we showed that MEHP,an active metabolite of DHEP,inhibited HTR-8 cell proliferation and resulted in G2/M cell cycle arrest,leading to multiple DNA damage(alkaline comet assay).DNA double strand breaks are the most serious DNA damage.Neutral comet assay and the expression level of DNA double strand break marker protein γ-H2 AX indicated that MEHP induced DNA double strand break damage.Parp-1,the promoter of DNA double-strand break damage repair,was slightly reduced,and Rad51 and DNA-PKCs,key proteins of homologous recombination repair and non-homologous end junction pathway,were decreased,suggesting that DNA double-strand break repair was inhibited.The expression of genetic toxic stress promoter protein ATM was decreased and the phosphorylation level was increased.The expression levels of CDK1 and Cyclin B1 in G2/M phase were decreased.Pregnancy DEHP exposure increased maternal serum progesterone level,however,MEHP treatment did not affect HTR-8 cell progesterone secretion,and the progesterone level was low.However,flow cytometry showed that high level of progesterone treatment could block HTR-8 cells in the G2/M phase.Then,the expression level of γ-H2 AX protein decreased,suggesting that progesterone did not cause DNA double strand break damage.Elevated expression of the G2/M phase associated protein Cyclin B1 suggests mitotic arrest.After synchronous cell treatment,it was found that the phosphorylation levels of Cyclin B1 and CDK1 were consistently higher in the progesterone-treated group than in the control group.Immunofluorescence counts showed an increase in the proportion of cells in metaphase and mitosis and an increase in delayed chromosomal abnormalities.Dynamic observation of living cells showed that the time of mitosis was significantly prolonged in the progesterone treatment group.Acute exposure to DEHP resulted in a dose-dependent increase in micronucleus rate in the bone marrow of mice in vivo.Exposure to DEHP during pregnancy resulted in increased micronucleus rates in maternal and placental sinuses and trophoblast cells,but did not affect the micronucleus in fetal rat liver.In vitro experiments showed that the micronucleus rate of HTR-8 cells increased after MEHP treatment,and the micronucleus containing ACA decreased,indicating that the micronucleus induced by MEHP mainly came from DNA double-strand breaks.Progesterone treatment also increased the micronucleus rate of HTR-8 cells.Conclusion: DEHP inhibits placental cell proliferation through two cell cycle arrest pathways.On the one hand,DEHP induces DNA double-strand break damage by inhibiting the classical DNA double-strand break repair pathway,activates genotoxic stress and leads to Cell cycle arrest in G2 phase.On the other hand,DEHP-induced high levels of progesterone affect the mitotic process,leading to the arrest of HTR-8 cells at metaphase of mitosis.DEHP induces placental cell cycle arrest in these two ways,and then inhibits placental cell proliferation.