Effects Of Pentachlorophenol On Early Spontaneous Abortion And Its Mechanism

Spontaneous abortion is the spontaneous termination of pregnancy before 20weeks of gestation,and is a common complication of pregnancy outcome.Environmental endocrine disruptors have been reported to cause reproductive development.Pentachlorophenol?PCP?,an environmental endocrine compounds,was widely used as herbicides,insecticides and preservatives,which has carcinogenic,teratogenic,reproductive toxicity.The aim of this study was to evaluate the association between PCP and spontaneous abortion,the developmental toxicity of PCP and its potential mechanism.Based on a case-control study conducted in Nanjing Medical University Affiliated Hospital,we explored the association between levels of PCP exposure in maternal urinary levels and spontaneous abortion in a non-occupational exposure population including 70 cases with medically unexplained spontaneous abortion and 171 controls.The concentration of PCP was detected by Ultra Performance Liquid Chromatography electrospray tandem Masspectrometry?UPLC-MS/MS?.The results showed that high PCP exposure group is related to the risk of spontaneous abortion?P<0.05?.To gain insight into the PCP molecular mechanism,56 pregnancy ICR mice were randomly assigned to 0,0.02,0.2 and 2 mg/kg PCP.No significance was observed in the number of total fetus per liter and the weight of placenta.The fetal loss rate,the number of resorbed fetus and abnormal fetus were higher in 0.2 and 2 mg/kg PCP exposure groups than that in control.The structure of placental chorionic villi of existing fetus was disorder in hematoxylin-eosin staining?HE?on gestation day?GD?16.5.Autophagosomes in chorionic villi were observed through Transmission Electron Microscope?TEM?.Moreover,mRNA and/or protein expression of P62,LC3-??/LC3-?and Beclin1 were increased in placenta,indicating the occurrence of autophagy.However,mRNA and protein expression of Bcl 2 were not significantly different.Then,to further explore the autophagy mechanism,microRNA?miR?-30a-5p,an expression inhibitor of Beclin 1,was predicted through bioinformatics predictions and RT-PCR,and it was reduced in PCP-treated mice.Transfection and luciferase reporter gene test were used to verify the interaction between Beclin1 and miR-30a-5p.These results firstly indicate that,PCP exposure can downregulate the expression of miR-30a-5p,and then induce autophagy through upregulation of Beclin1 to result in fetal loss.Our study laid a foundation for understanding the PCP developmental toxicity through autophagy.Part ?:The association between non-occupational exposure to PCP of pregnant women and spontaneous abortionObjectiveTo study the association between PCP non-occupational exposures in pregnancy and spontaneous abortion based on the case-control study.Methods1.UPLC-MS/MS was used to detect concentration of urinary PCP in pregnancy;2.According to the level of pentachlorophenol exposure,the population were divided into three groups;3.Binary logistic regression analysis was used to discuss the correlation between PCP exposure and spontaneous abortion.Results1.The pentachlorophenol detection rates of the control and case were 16.96%and32.86%,respectively.The females in case were significantly higher than that in control in the level of pentachlorophenol exposure?P=0.007?;2.Compared to the lowest exposure,the risk of spontaneous abortion in high pentachlorophenol exposure significantly increased with the crude OR?95%CI?5.71?2.38,13.66?(P _{for trends}<0.001)and adjusted OR?95%CI?3.79?1.17,12.31?(P _{for trends}=0.046).ConclusionRelatively high PCP exposure in early pregnancy is related to increase the risk of spontaneous abortion.Our study provides an epidemiological evidence of reproductive toxicity of pentachlorophenol.Part ?: Maternal pentachlorophenol exposure induces developmental toxicity mediated by autophagy on pregnancy miceObjective Based on pregnancy mice model,to explore the potential mechanism of PCP to induce fetus loss.Methods 1.To investigate the effect of PCP on mice developmental toxicity,pregnancy mice were exposure on different doses of PCP?0,0.02,0.2,2 mg / kg?by gavage from GD 0.5 to GD 8.5;2.Placental structure was observed by HE stained and TEM on GD 16.5;3.The level of autophagy was detected by RT-PCR and Western Blot;4.According to Target Scan,mi Randa and RNA22 websites,we screened the mi RNA of the target genes.Cell transfection experiments and dual luciferase reporter assay were used to observe the binding of mi RNAs to target genes.Results 1.We found that the rates of fetus loss,the numbers of abnormal and resorption fetus per litter in uterus were higher in middle and high PCP exposure group compared with the control group;2.The pathological change of placenta on GD 16.5 was observed by HE staining in PCP exposure groups.TEM results showed that the percentage of autophagosome raised in the PCP middle and high exposure mice placenta;3.RT-PCR and Western Blot results showed that the level of autophagy was increased in the PCP middle and high exposure mice placenta.Moreover,Beclin1 played a vital role in autophagy signaling pathway.4.Through bioinformatics,mi R-30 family was predicted to be the target mi-RNA of Beclin1.5.RT-PCR results showed that the relative expression of mi R-30a-5p was significantly lower in middle and high PCP-exposure group.6.Cell transfection experiments and dual luciferase reporter assay were used to testify the binding of mi R-30 a to Beclin1.Conclusion 1.PCP exposure increased fetus loss in ICR pregnant mice.2.PCP caused spontaneous abortion through the downregulation of mi R-30-5p to increase the Beclin1 expression leading to the high level of autophagy in placenta.