Epigenetic Mechanisms Of Hepatic Metabolic Disorder And Accelerated Preadipocyte Differentiation Resulting From EDCs Exposure

Bisphenol A(BPA),as a typical environmental endocrine disrupting chemical,has been worldwide used in both industrial manufacture and daily life supplies.It can be detected in multiple environmental mediums and food packages to which most people are exposed.Both epidemiology and animal studies have given of the association between BPA exposure and diabetes,abnormal liver-enzyme and cardiovascular diseases.Furthermore,BPA was found to be able to pass through the placental barrier and affect the development of the fetus via maternal exposure.The"Developmental origins of health and disease" theory has suggested that,exposure of environmental pollutants during early growth might program long time effects on state of health and the risk for diseases in adult life.Recently,increasing evidence has been accumulated indicating the important role of epigenetic modifications in the developmental origins of adult diseases,which is due to the epigenetic modifications on the regulation of multiple gene expression.For example,DNA methylation is the most common epigenetic modification which has been widely studied.Hepatic glucokinase(GCK)is a key enzyme in the progress of glucose utilization.The downregulation of its activity and mRNA expression is associated with metabolic disorders,insulin resistance and type 2 diabetes.This study had investigated the developmental origins of metabolic disorders in adult rats induced by perinatal BPA exposure.The levels of global DNA methylation in liver tissue and the degree of DNA methylation in hepatic Gck promoter were determined to explore the epigenetic mechanism of metabolic disorders during adulthood induced by perinatal BPA exposure.Perfluorooctanoic acid(PFOA)is an environmental endocrine disrupting chemicals and persistent organic pollutant.Due to its stability,surface activity,repellent properties and lipotropy,it has been used extensively in various commercial applications including industrial manufacture and personal care products.For more than half a century,the widely use and production of PFOA has resulted in environmental presence and detectable in wild animals and human beings,which are highly persistent in long-term.Furthermore,PFOA was found to be able to penetrate the placental barrier.As a result,increasing attentions have been driven to its developmental toxicity.Multiple scientific researches have proven that PFOA exposure during early development could trigger toxic effects on liver,cardiovascular system and metabolic system.From a recent birth cohort study,prenatal PFOA exposure has been founded to be associated with obesity and abnormal lipid metabolism in adulthood.A growing body of evidence has demonstrated that environmental pollutants exposure early in development may program risks for obesity,diabetes and other metabolic diseases in adult life.Environmental endocrine disrupting chemicals have been founded to affect the differentiation of preadipocyte resulting in obesity in adulthood.Peroxisome proliferator-activated receptor γ(PPARγ)and CCAAT/enhancer binding protein(C/EBPα),the two families of transcription factor play an important role in adipogenesis.In this study,3T3-L1 preadipocyte were treated with different doses of PFOA to investigate whether PFOA affects the differentiation and proliferation of 3T3-L1 preadipocyte.The morphological changes and the expression levels of PPARγ and C/EBPα were determined.The effects of PFOA combined with GW9662,a chemical inhibitor of PPARy,on adipogenesis were tested to investigate whether PFOA could act through the PPARy.And we also examined the role of epigenetic mechanism in contributing to increased differentiation and obesity by PFOA exposure.Part I:Hepatic DNA methylation modifications in early development of rats resulting from perinatal BPA exposure contribute to metabolic disorder in adult offsprings.Objectives:To investigate the association between the metabolic disorder in adult rat offsprings inducing by perinatal BPA exposure and the hepatic global DNA methylation and the degree of GCK promoter methylation in weaned and adult rats.Methods:The day when sperm-positive smears appeared was considered to be gestation day 0(GD0).Pregnant Wistar rats were administered to BPA(50 μg/kg/day)or corn oil by oral gavage throughout gestation and lactation.Variables associated with insulin resistance and hepatic DNA methylation were examined at postnatal 3 weeks(week 3)and week 21 in male offspring.Results:In BPA-treated offspring,serum insulin and HOMA-insulin resistance were increased(P<0.01,P<0.05),and the insulin sensitivity index and hepatic glycogen storage were decreased compared with controls at week 21(P<0.05,P<0.01).At week 3,none of these variables were significantly changed.However,hepatic global DNA methylation was decreased(P<0.05,P<0.01),accompanied by overexpression of DNA methyltransferase 3B mRNA at week 3(P<0.05,P<0.01).Meanwhile,perinatal exposure to BPA induced promoter hypermethylation and a reduction in gene expression of hepatic glucokinase(P<0.05.P<0.01).Moreover,increased promoter hypermethylation of Gck became more pronounced in BPA-treated offspring at week 21.Conclusion:Perinatal BPA exposure induced hepatic global DNA hypomethylation and GCK promoter hypermethylation resulting decreased expression of its mRNA.Abnormal DNA methylation in hepatic tissue precedes development of insulin resistance induced by perinatal BPA exposure.These findings support the potential role of epigenetics in fetal reprogramming by BPA-induced metabolic disorders.Part II:Study of the effects of PFOA exposure on 3T3-L1 preadipocyte differentiation and associated epigenetic mechanism.Objectives:3T3-L1 preadipocyte was treated with different doses of PFOA to observe the effects of PFOA exposure on adipocyte proliferation and differentiation and associated mechanism.Methods:3T3-L1 preadipocyte was subcultured and passaged from frozen storage.Confluent cells were cultured in the absence or presence of different doses of PFOA(0.1%DMSO,0.1,1.0,10.0,100.0 μg/mL)for 8 days.GW9662(20 μM)as an inhibitor of PPARy combined with PFOA(100 μg/mL).were also applied.MTT reduction assay was used to reflect the proliferation of the cells.Flow cytometry was used to determine the effects of PFOA exposure on cell cycle.3T3-L1 preadipocyte was treated with different doses of PFOA for 8 days.The adipocytes were stained with oil red and observed by the electron microscope to identify whether the cells were differentiated.The accumulation of triglyceride in cells was measured and DNA,mRNA and protein was isolated for further determinations.The degree of methylation of PPARy promoter was tested and the levels of expression of PPARγ and C/EBPa were measured.Western blot was used to determine the expression levels of protein.Results:PFOA(at doses of 0.1,1.0,10.0,100.0 μg/mL)promoted the proliferation and differentiation of 3T3-L1 preadipocyte.The accumulation of lipid and triglyceride was increased in treated cells(P<0.05,P<0.01).The expression levels of PPARγ,C/EBPa and Ap2,LPL target genes of PPARy were all increased after exposed to PFOA.Furthermore,GW9662 blocked the inducing effects of PFOA on adipogenesis.In addition,the CpG sites of PPARy promoter were hypomethylated consisting with its mRNA and protein expression in PFOA treated cells(P<0.01).Conclusion:PFOA promoted the proliferation and differentiation of 3T3-L1 preadipocyte through PPARy.And the increased expression of PPARy was associated with its hypomethylation of promoter.