Extractable Organic Matters Of PM_2.5 Induces Cardiac Malformations Via AHR-mediated Endoplasmic Reticulum Stress In Zebrafish Larvae

Objective:Recent studies have shown that maternal exposure to ambient fine particulate matter(PM2.5)is associated with congenital heart disease in the offspring,but the underlying molecular mechanisms remain to be elucidated.We previously reported that extractable organic matters(EOM)from PM2.5 induces excess reactive oxygen species(ROS)by activating aryl hydrocarbon receptors(AHR),leading to apoptosis and inhibition of the Wnt signaling,resulting in heart development in zebrafish embryos.Given that endoplasmic reticulum(ER)stress can cause apoptosis and abnormal Wnt signaling inhibition,we hypothesized that AHR/ROS might mediate PM2.5-elicited cardiac developmental toxicity through ER stress.In this study,we examined the effects of EOM on ER stress,apoptosis and Wnt signaling in zebrafish embryo,and explored their roles in EOM-induced heart defects.Methods:PM2.5 was collected in Suzhou urban area,and EOM was extracted by a Soxhlet extraction method.Zebrafish embryos were exposed to EOM,in the presence or absence of 4-phenylbutyric acid(4-PBA),a pharmaceutical inhibitor of ER stress,AHR inhibitor CH223191(CH),and reactive oxygen species(ROS)scavenger N-acetyl-L-cysteine(NAC).Knockdown of AHR or CHOP was achieved by microinjecting morpholine oligonucleotide(MO)or antisense 2’-O-methyloligonucleotide(antago),respectively.Survival rate and cardiac malformation rate of embryos at 72 hours post fertilization(hpf)were counted under a dissecting microscope.Dichlorodihydrofluorescein diacetate(DCFH-DA)and acridine orange(AO)staining were used to detected ROS and apoptosis levels in the heart area.Hearts were dissected from zebrafish embryos at 72 hpf,and MitoSOXMT was used to measured mitochondrial ROS.qPCR was used to evaluate mRNA expression levels.Immunofluorescence staining was used to measured levels of ER stress,apoptosis and βcatenin phosphorylation.Results:Our results showed that 4-PBA at a dose of 5 nM,significantly inhibited the EOMelevated heart malformation rates.In addition,EOM upregulated mRNA and protein expression levels of ER stress marker genes,including C/EBP Homologous Protein(CHOP)and PDI,and interfered with the mRNA expression levels of other ER stress genes(hspa5,atf6,ern1,elfak3,and atf4b1).Inhibition of AHR activity by CH and AHR genetic knockdown abolished EOM-induced ER stress.ROS scavenger NAC also inhibited EOMinduced ER stress.We further demonstrated that 4-PBA rescued PM2.5-induced intracellular ROS and mitochondrial ROS overproduction.AO staining and Cleaved caspase-3 immunofluorescence results indicated that 4-PBA rescued PM2.5-induced apoptosis.4-PBA also rescued PM2.5-induced Wnt signaling inhibition(manifested as upregulation of phosphorylated β-catenin,downregulation of non-phosphorylated β-catenin,and decreased expression of Wnt signaling downstream genes nkx2.5 and sox9b).We also found that genetic knockdown of CHOP significantly reduced the EOM-induced heart malformation,while dramatically abolishing apoptosis and Wnt signaling inhibition.Conclusion:Our results indicate that PM2.5 induces AHR/ROS-mediated ER stress,which leads to apoptosis and Wnt signaling inhibition,ultimately resulting in heart defects.Our results provide promising molecular targets for the prevention and treatment of cardiac developmental toxicity caused by PM2.5.