The Cardiac Developmental Toxicity Of PM2.5 And The Protective Machanisms Of Folic Acid In Zebrafish Embryos

Objective: Epidemiological studies indicated that atmospheric fine particulate matter(PM2.5)is associated with heart developmental diseases,and animal experiments showed that PM2.5 could induce heart defects,but the underlying molecular mechanisms remain to be elucidated.Using zebrafish embryo as a research model,we studied the cardiac developmental toxicity of extractable organic matter(EOM)from PM2.5 and the protective effects of folic acid(FA).To explore the molecular mechanisms,we focus on the interactions between Ahr and wnt/?-catenin signaling pathways,and the involvement of DNA methylation.Methods:(1)PM2.5 collection: PM2.5 samples were collected from Suzhou using a PM2.5 sampler,and organic matters were extracted by a Soxhlet extractor apparatus.(2)Whole embryonic experiment: Embryos were exposed to EOM at different concentrations in the absence or presence of CH(0.5 mM)or CHIR(1 mM)in glass petri dishes,and were observed under a stereomicroscope to calculate embryonic hatching rate,heart rate and heart malformation.mRNA expression levels of Ahr and wnt/?-catenin pathway related genes were examined by qPCR;protein expression level of ?-catenin was measured by Western Blot;Cyp1a1 activity was measured by a modified in vivo EROD assay.(3)Isolated hearts experiment: The zebrafish embryos were exposed to EOM at a concentration of 5 mg/L with or without folic acid(0.05 ?M),and were observed under a stereomicroscope to calculate embryonic hatching rate and heart malformation.Hearts isolated from embryos were confirmed under a florescent microscope;mRNA expression levels of genes related to Ahr,wnt/?-catenin signal pathways and DNA methylation modifications were examined by q PCR;protein expression levels of Ahr and ?-catenin were measured by Western Blot.The DNA methylation level of c-fos promoter region was detected by COBRA.Cyp1a1 activity was measured by a modified in vivo EROD assay.Results:(1)Extractable organic matter(EOM)form PM2.5 increased cardiac malformations and decreased heart rate in zebrafish embryos.Ahr antagonist CH and ?-catenin agonist CHIR rescued the EOM-induced heart defects in zebrafish embryos.EOM upregulated mRNA expression of Ahr targeted genes(Cyp1a1,Cyp1b1 and Ahrra)while downregulated mRNA levels of ?-catenin targeted genes(Axin2,Nkx2.5 and Sox9b)and a ?-catenin upstream gene(Rspo2).Supplementation with CH or CHIR attenuated most of the EOM-induced gene expression changes.However,the mRNA expression level of Ahr inhibitor Ahrrb,which did not change by EOM treatment alone,was increased in embryos co-exposed to EOM with CH or CHIR.(2)Supplementation with FA rescued the EOM-induced heart malformation in zebrafish embryos,and both Ahr antagonist CH and ?-catenin agonist CHIR could enhance the protective efficiency of FA.For genes related to Ahr and Wnt/?-catenin signal pathways,EOM induced similar mRNA expression changes in isolated heart as in whole embryos.In addition,DNA methylation status and mRNA expression levels of genes involved in DNA methylation were also modulated by EOM,which can be alleviated by FA.In particularly,EOM induced DNA hypermethylation in the promoter region of c-fos correlating with m RNA downregulation,which were significantly attenuated by supplementation with folic acid.EROD experiments further confirmed that the EOM-activated Cyp1a1 was suppressed by FA.Furthermore,EOM-induced changes in protein expression of ?-catenin and Ahr were also attenuated by FA.Conclusion: Our data indicated that EOM activated Ahr and inhibited Wnt/?-catenin signal pathway in zebrafish embryos,leading to heart defects.Folic acid might attenuate EOM-induced changes in Ahr and Wnt/?-catenin pathways through stabilization of DNA methylation.Our study suggested that folic acid can be used for preventing PM2.5-induced congenital heart diseases.