Effects Of Plasticizer DEHP On Human Thyroid Function And The Mediating Role Of MiR-454-3p/ERα/Notch Pathway

Di(2-ethylhexyl)phthalate(DEHP)is widely used as a plasticizer and can enter the human body through various pathways,rapidly metabolized into mono-(2ethylhexyl)phthalate(MEHP),which has toxic effects on the body,and DEHP can affect the fetus through the placental barrier.DEHP and MEHP have estrogenic effects and can bind to estrogen receptors(ERs),affecting thyroid function,The Notch signaling pathway plays an important role in the progression of thyroid diseases,ERs transcription factors can bind to the Notch promoter,so ERs may play a role in DEHP induced thyroid dysfunction by regulating the Notch signaling pathway.DEHP can affect microRNAs(miRNAs)to regulate the ERs.However,the role of miRNAs regulating the ERs/Notch pathway in effect of DEHP on human thyroid function is still unclear.Human are widely exposed to DEHP,and DEHP metabolites have been detected in the urine of newborns,adolescents,and adults.Our research group has already completed the detection and analysis of DEHP levels in adolescent urine in the early stage,so this study focused on the population of puerpera-newborns and adults,DEHP level in puerpera’s urine was used to reflect intrauterine DEHP exposure for newborns,clarified the association between intrauterine DEHP exposure in newborns and adult DEHP exposure and thyroid hormones(THs).It was found that miR-454-3p may play a potential role in the association between DEHP exposure and THs in the population through the ERs/Notch pathway.Besides,human normal thyroid cells(Nthyori3-1)were cultivated in vitro and exposed to MEHP,ERa silence,Notch pathway inhibition and miR-454-3p overexpression in cell models were used to elucidate the role of miR454-3p regulating the ERs/Notch pathway in effect of DEHP on human thyroid function.This study can provide scientific basis for reducing exposure to DEHP and preventing and treating thyroid related diseases caused by DEHP.Part I Effects of DEHP exposure on thyroid function of populationObjective:Clarify the exposure levels and influencing factors of intrauterine DEHP exposure of newborns and DEHP exousure to adults.Assess the risk of DEHP exposure in adults.Explore the association between intrauterine DEHP exposure and THs of newborns,as well as between adult DEHP exposure and self THs.Methods:Convenient sampling was used to select puerpera-newborns(N=300)who gave birth from 2020-2021 and adults(N=312)who underwent health examinations in 2022 as the research subjects in a hospital in Jilin Province.General demographic,behavioral,dietary habits,and living environment data of the research subjects,as well as biological samples such as blood and urine were collected.HPLC-MS/MS was used to detect DEHP metabolites(MEHP,MEOHP,MEHHP,MCMHP,and MECPP)in the urine of study subjects.The estimated daily intake(EDI)and hazard quotient(HQ)of adult calculated by DEHP.ELISA was used to detect serum levels of FT3,FT4,TT3,TT4,TSH,and TRH.Multiple linear regression was used to analyze the association between DEHP metabolites in urine and THs in the study population.Results:1.DEHP metabolites have been detected in the urine of puerpera and adults.The detection rate of DEHP metabolites in urine of puerpera was 100.0%;The detection rate of DEHP metabolites in urine of adult was 97.1%to 100.0%.2.Before creatinine adjustment,the quartile levels of ΣDEHP in urine of puerpera and adults were 29.56(17.20,55.11)and 45.52(26.16,69.58)μg/L,respectively.After creatinine adjustment,the quartile levels of ΣDEHP in urine of puerpera and adults were 51.06(35.35,84.56)and 37.00(23.57,58.16)μg/g creatinine,respectively.3.In the volume-based calculation model,the median level of adult EDI was 3.61μg/kg·bw/day.The proportion of HQRfDHQRfD AA,and HQTDI greater than 1 was 7.05%,5.13%and 1.92%,respectively.In the creatinine-based calculation model,the median level of adult EDI was 3.44 μg/kg·bw/day.Adults with HQRfD,HQRfD AA,and HQTDI greater than 1 accounted for 7.37%,5.77%,and 2.56%,respectively.4.There was a significant positive correlation between the use of plastic water bottles,high-frequency of color cosmetics,high-frequency of aromatic cosmetics,use of fragrance beads,presence of industrial chimneys within 500 meters of residence or workplace,and exposure to wooden flooring and the levels of DEHP metabolites in urine of puerpera(P<0.05).There was a significant positive correlation between highfrequency use of beauty cosmetics and insecticides and the levels of DEHP metabolites in the urine of the adults(P<0.05).5.There was a significant negative association between maternal urine MEHHP and FT3 level cord blood,a significant positive association with TRH(P<0.05).There was a significant negative association between MECPP in urine and FT3 in peripheral blood,MEHHP and TT4,MECPP and FT3/TT3 in adults(P<0.05),and a significant negative association between MEHP in urine and FT4,TT3,TT4,and TRH levels in peripheral blood(P<0.05).Conclusions:1.Factors such as behavioral patterns,dietary habits,and living environment may affect the intrauterine DEHP exposure of newborns and adult DEHP2.Newborns were generally exposed to intrauterine DEHP,which may affect the levels of FT3 and TRH in newborns.3.Adults were generally exposed to DEHP,exposure to DEHP in adults may affect their own levels of FT3,FT4,TT3,TT4,and TRH.DEHP exposure poses potential health risks to the population.Part Ⅱ Roles and mechanisms of the ERs/Notch pathway in the association between urinary DEHP and THsObjective:Clarify the association between intrauterine DEHP exposure or adult DEHP exposure and THs-related genes in newborns and adults.Explore the mediating role of the ERs/Notch pathway in the association between urinary DEHP metabolites and human THs.Methods:The research population was the same as the first part.Real-Time PCR was used to detect THs-related genes TBG,TTR,TPO,TG,TSHR,NIS,TTF1,PAX8,DIO1,DIO2,DI03 and ER/Notch pathway genes ERα,ERβ,GPR30,Notch1,Notch4 and Dll4 mRNA expression levels in the blood of the study subjects.Multiple linear regression method was used to analyze the association between DEHP metabolites in urine and THs-related genes in the population.Parallel mediation and chain-mediation models to analyze the mediating role of the ERs/Notch pathway in effect of DEHP on human THs.Results:1.The level of MEHHP in maternal urine is negatively correlated with the expression level of TG mRNA in neonatal umbilical cord blood,and significantly positively correlated with the expression levels of TTR and DIO3 mRNA(P<0.05).There was a significant negative association between urine ΣDEHP and TTF-1 mRNA levels in cord blood of newborns(P<0.05).MCMHP in urine were significantly positively associated with the expression level of DIO3 mRNA(P<0.05).2.There was a significant negative association between urinary MEHP and TG,TSHR and NIS mRNA expression levels in peripheral blood of adults(P<0.05).MECPP in urine was significantly negatively associated with TG,TTF-1,and PAX8 mRNA expression levels in peripheral blood(P<0.05).MEOHP in urine was significantly negatively associated with DIO1 and DIO2 mRNA expression levels peripheral blood(P<0.05).3.Maternal urine MECPP levels and ERα and ERβ mRNA in cord blood of newborns was significantly positively associated(P<0.05).Urinary ΣDEHP level was significantly negatively associated with ERβ and GPR30(P<0.05).Urinary MEOHP levels were significantly positively associated with Notch1(P<0.05).4.MEOHP levels in urine was significantly positively associated with ERα mRNA expression level in peripheral blood of adults(P<0.05).MEHP levels in urine were significantly positively associated with Notch1(P<0.05).MECPP levels in urine were significantly positively associated with Dll4(P<0.05).5.The results of the parallel mediation model showed that ERα played a mediating role between MEHHP in urine and FT4 in peripheral blood of adults,with a mediating effect ratio of 25.22%.6.There were multiple binding sites between transcription factors ERα and the promoter regions of Notch1,Notch4,and Dll4 genes.7.The results of the chain mediation model showed that:ERα/Notch4 played a chain mediated role between MEOHP,MEHHP ΣDEHP in urine and FT4 in peripheral blood of adults.ERα/Dll4 played a chain mediated role between MEHP,MEOHP,MEHHP,MCMHP,ΣDEHP in urine and FT4 in peripheral blood of adults.Conclusions:1.Intrauterine DEHP exposure may affect the mRNA expression of TG,TTF-1,TTR and DIO3 in newborns.DEHP exposure to adults may affect the mRNA expression of TG,TBG,TSHR,TTF-1,PAX8,NIS DIO1 and DIO2.2.Intrauterine DEHP exposure to newborns may affect the mRNA expression of ERα,ERβ,GPR30 and Notch1.DEHP exposure to adults may affect the mRNA of ERα,Notch1 and Dll4.3.Transcription factors ERα may regulate the expression of Notch1,Notch4,and Dll4 genes,activating the Notch signaling pathway.The ERα/Notch pathway exhibited a chain mediated effect in the association between DEHP exposure and serum FT4 in adults,DEHP exposure to adults may interfere with the ERs/Notch pathway and affect serum FT4 levels.Part III MEHP affects human thyroid function in Nthyori3-l cells through miR-454-3p regulating ERα/Notch pathwayObjective:Clarify the association between population DEHP exposure and miRNAs expression levels.Clarify the effects of MEHP exposure on ERα/Notch pathway and miRNAs in Nthyori3-1 cells.Elucidate the effect on human thyroid function through miR-454-3p regulating ERα/Notch pathway after MEHP exposure.Methods:45 individuals were randomly selected from two populations as the research subjects,puerpera-newborns and adults.Potential miRNAs regulating ERα regulation were searched through online databases,such as miRTarBase,miRDB,mirDIP,and mirwalk.Real-Time PCR was used to detect the expression levels of miR-19b-3p,miR-22-3p,and miR-454-3p in the blood of the study subjects,and to analyze the relationship between three types of miRNAs and urinary DEHP metabolites and ERαexpression levels.Nthyori3-l cells were cultured in vitro and treated with MEHP for 24 hours.ERα was silenced by siRNA,groups were divided into blank control group(Con),negative control group(NC),inhibition group(siERα),250 μM MEHP group(MEHP),inhibition of ERα+250 μM MEHP group(siERα+MEHP).Notch signaling pathway was inhibited byγ-secretion enzyme inhibitor DAPT.The groups were blank control group(Con),solvent control group(VCon),inhibition group(DAPT),and 250 μM MEHP group(MEHP)and DAPT+250 μM MEHP group(DAPT+MEHP).miR-454-3p was overexpressed using mimics,groups were divided into blank control group(Con),negative control group(NC),mimics group(mimics),and 250 μM MEHP group(MEHP),mimics+250 μM MEHP group(mimics+MEHP).Annexin V-FITC/PI staining was used to detect the level of cell apoptosis.ELISA was used to detect the levels of FT3 and FT4 in the cell supernatant.Real-Time PCR and Western Blot were used to detect the expression levels of THs related genesResults:1.Maternal urine MEOHP,MEHPP,and ΣDEHP levels were significantly negatively correlated with miR-22-3p expression in cord blood(P<0.05),MEHP levels were significantly negatively correlated with miR-454-3p expression(P<0.05),and miR-19b-3p,miR-454-3p were significantly negatively correlated with ERα in cord blood(P<0.05).2.MEHP,MEOHP,MCMHP,MECPP,and ΣDEHP in urine was significantly negatively correlated with the expression of miR-454-3p in peripheral blood of adults(P<0.05).The expression level of miR-454-3p was significantly negative correlated with ERα in peripheral blood(P<0.05).3.In Nthyori3-1 cells,MEHP group showed a marked rise in apoptosis rate compared to Con and VCon groups(P<0.05).FT3 and FT4 in 50 μM and 250 μM MEHP groups showed decline than VCon and 10 μM MEHP group(P<0.05).TG mRNA and protein in 250 μM MEHP group showed decrease in comparision to Con group(P<0.05).The TTF-1 mRNA in 50 μM and 250 μM MEHP were lower than else group(P<0.05).4.The ERα,Notchl,Notch4 and D114 in 250 μM MEHP group showed the significant increase compared to Con and VCon group(P<0.05).There was a lower expression of miR-454-3p in 50 μM and 250 μM MEHP group compared to Con and VCon groups(P<0.05).5.In the cell model of ERα silence,the cell apoptosis rate in the siERα+MEHP group showed a decrease compared to MEHP group,increase compare to Con,NC and siERα group(P<0.05).In comparision to siERα+MEHP group,the Notch1 and Notch4 mRNA in MEHP group was higher(P<0.05).The levels of FT3 and FT4 in siERα+MEHP group were significantly higher than those in MEHP group(P<0.05).6.In the cell model of Notch pathway inhibition,the DAPT+MEHP group exhibited a higher level of FT3 and FT4 secreation than MEHP group(P<0.05).Addationally,the cell apoptosis in DAPT+MEHP group was lower compared to MEHP group(P<0.05).The protein of TG and TTF-1 in DAPT+MEHP showed a marked increase compared to MEHP group(P<0.05).7.In the cell model of miR-454-3p overexpression,FT3 and FT4 secreted by cells in mimics+MEHP group were higher than MEHP group(P<0.05).the mimics+MEHP group exhibited a lower cell apoptosis rate than MEHP group(P<0.05).The ERαprotein in the mimics+MEHP group displayed a marked increase compared to mimics group,a decrease compared to else group(P<0.05).Conclusions:1.DEHP exposure to human may downregulate the expression level of miR-454-3p,while MEHP exposure could reduce the expression level of miR-454-3p in Nthyori3-l cells.2.MEHP exposure could promote apoptosis of Nthyori3-l cells,reduce the secretion levels of FT3 and FT4,and decreased the expression levels of TG and TTF-1 genes in cells.3.MEHP may activated the ERα/Notch pathway in Nthyori3-1 cells,and ERα/Notch pathway played a role in MEHP affecting cell apoptosis and THs secretion.4.ERα may be the potential binding target for miR-454-3p,and miR-454-3p regulating ERα may play a role in DEHP affecting human thyroid function.