AHR/oxidative Stress-mediated Cardiac Developmental Toxicity Of Trichloroethylene And The Protective Effect Of Resveratrol In Zebrafish Embryos

Objective:Trichloroethylene(TCE)is a widely used industrial organic chemical and frequently detected in the environment.Studies show that TCE has cardiac developmental toxicity,but the underlying molecular mechanisms remain to be elucidated.Based on the importance of aryl hydrocarbon receptor(AHR)and oxidative stress in cardiac development,we hypothesised that AHR and oxidative stress mediated the developmental toxicity of TCE,and resveratrol(RSV)as an AHR antagonist or a ROS scavenger could exert protective effect.Methods:Zebrafish embryos were exposed to TCE and its oxidative metabolism intermediate trichloroacetic acid(TCA)at different concentritions within three hours of fertilization.AHR inhibitors CH223191(CH)(0.125μM)and StemRegenin 1(SRI,0.25μM),reactive oxygen species(ROS)scavenger N-acetyl-L-cysteine(NAC,0.25 μM)and resveratrol(1μg/ml)were supplemented according to the experimental design.The development of zebrafish embryos were observed under a stereo microscope,and heart malformation rates and survival rates were recorded.CYP1A1 activity was detected by in vivo EROD method.ROS level was measured by in vivo ROS staining assay using 2,7-Dichlorodi-hydrofluorescein diacetat(DCFH-DA)as a probe.The distribution of lipid droplet was detected by in vivo ORO staining assay.Apoptosis was measured by acridine orange(AO)staining.Embryonic heart was isolated,and the mRNA expression of genes involved in AHR pathway,Nrf2 pathway,oxidative stress and cardiac development were detected by qPCR.Protein expression of AHR and 8-OHdG in zebrafish embryonic heart were detected by immunofluorescence.Results:(1)TCE at 1 ppb had little effect on zebrafish heart development,but TCE at 10 and 100 ppb significantly increased the rates of cardiac malformation.TCA at 10 ppb also increased heart defects,but the cardiac malformation rate was significantly lower than TCE at the same concentration.AHR inhibitors CH and SR1 significantly reduced the heart defects caused by TCE at 10 ppb,and the rate of cardiac malformation was even decreased to control level in TCE plus CH group.CH also antagonized the TCE-induced overexpression of AHR target gene cyplbl.TCE signiifcantly increased ROS and 8-OHdG levels in the heart of zebrafish embryos,which were counteracted by both CH and NAC.We used morpholine antisense oligonucleotide to knock down AHR and found that the TCE-induced ROS and 8-OHdG elevation were significantly inhibited.TCE had no significant effect on lipid consumption and apoptosis in the heart of zebrafish embryos.TCE significently up-regulated the expression levels of oxidative stress related genes sod2,ho1,nmRNA qo1 and inhibited the mRNA expression level of nrf2b,which were attenuated by CH supplemention.In addition,TCE increased the activities of Total-SOD,Cu.Zn-SOD(SOD 1)and Mn-SOD(SOD2)superoxide dismutase,while the addition of CH has an inhibitory effect.For genes essential in cardiac development,TCE decreased the mRNA expression levels of gata4 and hand2,and increased the mRNA expression levels of c-fos and sox9b,and CH supplemention antagonized the gene expression changes caused by TCE.(2)RSV significantly alleviated the cardiac defects caused by TCE and abrogated the TCE-induced increase of sox9b and decrease of gata4.Furthermore,RSV antagonized the TCE-induced overexpression of AHR target gene cyp1b1 and the upregulation of ROS and 8-OHdG in the heart of zebrafish embryos.For oxidative stress related genes,RSV antagonized the TCE-induced mRNA expression changes of nrf2b,ho1,nqol and sod2.RSV also inhibits the hyperactivities of Total-SOD,SOD1 and SOD2 superoxide dismutase caused by TCE.Conclusion:TCE induced dose-dependent heart defects in zebrafish embryos.AHR mediated the oxidative stress induced by TCE,and inhibition of either AHR or ROS reduced the cardiac defects caused by TCE.RSV can act as both an AHR antagonist and an antioxidant to counteract the cardiac developmental toxicity of TCE in zebrafish embryos.Our results contribute to the understanding of the molecular mechanism of the cardiac developmental toxicity of TCE,and provide scientific basis for the prevention and treatment of TCE-induced cardic developmental diseases.