AHR-Mediated Mitochondrial Damage Caused Cardiac Developmental Toxicity Of Benzo(a)pyrene And The Protective Mechanism Of Resveratrol

Objective:Benzo(a)pyrene(BaP),a widespread polycyclic aromatic hydrocarbon in the environment,can activate aromatic hydrocarbon receptor(AHR),cause oxidative stress,and has cardiac developmental toxicity,but its molecular mechanism remain unclear.Resveratrol(RSV)is a natural polyphenol compound,which is an antioxidant and antagonist of AHR.We hypothesized that AHR activated by Benzo(a)pyrene leads to oxidative stress,which interferes with cardiac development by causing mitochondrial damage,while resveratrol plays a protective role by inhibiting AHR activity and ROS production.Methods:Zebrafish embryos were infected with Benzo(a)pyrene,and separately added with AHR inhibitor CH223191(CH),reactive oxygen species(ROS)antagonist N-acetylL-cysteine(NAC)or resveratrol.The embryo survival rate of zebrafish in each group at 24 hours post-fertilization(24hpf)and the malformation rate at 72 hours postfertilization(72hpf)were calculated.7-ethoxy-3-isophenoxazolone-deethylase(EROD),dichlorodihydrofluorescein diacetate(DCFH-DA)and acridine orange(AO)staining were used to detect AHR activity,ROS production and apoptosis.Hearts of zebrafish embryos in each group were isolated and stained with MitoSOX red and Mitotracker to determine the levels of mitochondrial reactive oxygen species and mitochondria membrane potentials,qPCR was used to detect the expression of mRNA,and immunofluorescence was used to detect cleaved-caspase 3/9.Results:(1)Benzo(a)pyrene at dose levels of 0.1μM,0.2μM and 0.4μM caused concentrationdependent heart malformations in zebrafish embryos,while decreased survival was only found in embryos exposed to the highest dose level(0.4μM).We further found that Benzo(a)pyrene increased AHR activity and the expression of its target genes,and significantly increased ROS level.Both CH,an inhibitor of AHR,and NAC,a ROS scavenger,could antagonize Benzo(a)pyrene-induced ROS overproduction,but NAC has no effect on Benzo(a)pyrene-induced AHR activation.Benzo(a)pyrene also decreased the expression of genes key to cardiac development,increased mitochondrial ROS,decreased mitochondrial membrane potential,and caused endogenous apoptosis.All these effects can be counteracted by either CH or NAC.(2)Resveratrol(1 Omg/L)can significantly reduce the heart malformations caused by Benzo(a)pyrene.The addition of resveratrol also attenuates Benzo(a)pyrene-induced ROS overproduction,mitochondrial damage and endogenous apoptosis.However,resveratrol had no significant effect on the Benzo(a)pyrene-induced AHR activition and the overexpression of AHR target genes.Conclusion:Activation of AHR by Benzo(a)pyrene increases ROS production,which causes mitochondrial damage and promotes endogenous apoptosis,resulting in zebrafish heart defects.Resveratrol has a protective effect on Benzo(a)pyrene-induced aberrant heart development in zebrafish embryos mainly by inhibiting oxidative stress rather than antagonizing AHR activity.