AHR-mediated Oxidative Stress Contributes To Cardiac Devdopmental Toxicity Of PM2.5 And The Protective Effect Of Resveratrol

Objective:Epidemiological studies indicate a correlation between maternal exposure to atmospheric fine particulate matter(PM2.5)and congenital heart diseases,but the underlying molecular mechanism is unclear.It has been well documented that PM2.5 induces oxidative stress,and we previously found that extractable organic matter(EOM)from PM2.5 causes heart defects in zebrafish embryos by activating aryl hydrocarbon receptor(AHR).Resveratrol(RSV),which is mainly derived from grapes,Polygonum cuspidatum and other plants,is a natural polyphenolic compound with antioxidant and AHR antagonistic effects.Therefore,we hypothesized that AHR mediates the PM2.5 induced overproduction of reactive oxygen species(ROS),leading to heart defects,and RSV would have protective effect by suppressing ROS production and AHR activity.Methods:Zebrafish embryos were treated with EOM from PM2.5 in the presence or absence of AHR inhibitor CH223191(CH),ROS scavenger N-acetyl-L-hemi Cystine(NAC)or RSV.AHR knockdown was achieved by using Morpholino(MO)antisense oligonucleotides.Zebrafish embryos at 72 hours post fertilization(hpf)were observed under microscope,and cardiac malformation rate and heart rate were counted.AHR activity,ROS,and apoptosis levels in the heart region of zebrafish embryos were detected by EROD,DCFH-DA and AO staining.Hearts were dissected from embryos of each group.mRNA expression level was examined by using qPCR,and DNA damage and apoptosis by immunofluorescence.Results:(1)AHR inhibitor CH(0.5 μM)and ROS scavenger NAC(0.25 μM)significantly attenuated EOM(5 mg/L)-induced cardiac malformations in zebrafish embryos.At the same time,both CH and NAC can reduce EOM-induced increase of ROS,DNA damage and apoptosis in zebrafish embryonic heart region.In addition,both CH and NAC attenuated EOM-induced expression changes of genes involved in cardiac development(nkx2.5,sox9b),oxidative stress(ho-1,sod2,gstp1,gstp2,cat,nrf2a,nrf2b)and apoptosis(p53,bax).We further validated that AHR is required for EOM-induced ROS generation,DNA damage and apoptosis by AHR knockdown.NAC had no detectable effect on EOM-induced AHR activity.(2)RSV at 0.1,1 and 10 mg/L has little effect on heart development in zebrafish embryos,but RSV at 100 mg/L significantly elevated heart malformation rate.Therefore,10 mg/L RSV was selected in subsequent experiments.We found that RSV significantly attenuated EOM-induced cardiac malformations in zebrafish embryos.At the same time,supplementation with RSV also suppressed EOM-induced ROS production,DNA damage and apoptosis in zebrafish embryo hearts.In addition,RSV attenuated EOM-induced expression changes of genes involved in oxidative stress(sod1,sod2,cat,nrf2a,nrf2b,gstp1,gstp2),apoptosis(p53,bax)and cardiac development(nkx2.5,sox9b,axin2).We further found that EOM increased the activities of total Sod(Total-Sod),Sodl and Sod2 in zebrafish embryos,and these effects were counteracted by RSV supplementation.However,RSV did not inhibit EOM-induced AHR activity and the elevated expression of AHR target genes(cyp1a1 and cyp1b1).Conclusion:Our study demonstrated that 1)AHR meidates PM2.5-induced ROS production,which induces DNA damage and apoptosis,leading to heart malformation in zebrafish embryos;2)AHR antagonist CH,antioxidant NAC and RSV all significantly attenuated PM2.5-induced DNA damage and apoptosis;3)Inhibition of AHR reduced PM2.5-induced oxidative stress,but inhibition of ROS had no effect on PM2.5-induced AHR activity;4)Resveratrol protects against PM2.5-induced heart defects by suppressing oxidative stress rather than through AHR antagonism.