Perfluorooctanoic acid induces abnormal heart development via aryl hydrocarbon receptor-mediated oxidative stress and apoptosis in zebrafish larvae
[Background]In recent years,a growing number of studies have indicated that perfluorooctanoic acid(PFOA)exposure can impact heart development,though the specific mechanisms remain elusive.The aryl hydrocarbon receptor(AHR)is a critical environmental sensor capable of inducing oxidative stress and cell apoptosis.[Objective]To explore the role of AHR in the cardiac developmental toxicity of PFOA by using ze-brafish embryo as an in vivo model.[Methods]Zebrafish embryos at 2 h post-fertilization(2 hpf)were exposed to dimethyl sulfoxide(DMSO)control,1,10,100,and 1000 μg·L-1 of PFOA.The AHR inhibitor CH223191(CH)was added to the high-concentration group(1000 μg·L-1)to form an intervention group.Under a dissecting microscope,the survival rate,mortality rate,heart rate,and heart malformation rate of 72 hpf zebrafish larvae were assessed.The activity of AHR was measured using 7-ethoxyresorufin-O-dealkylation(EROD)staining.The levels of intracellular and mitochondrial ROS in the heart of zebrafish larvae were assessed using dichloro-dihydro-fluorescein diacetate and MitoSOX™ Red,respectively.Apoptosis was examined using acridine orange(AO)staining and Immunofluorescence method.Total RNA was extracted from dissected hearts,and mRNA expression levels of oxidative stress-related genes(sod2,cat)and apoptosis-related gene(p53)were analyzed using quantitative PCR.[Results]Compared to the DMSO control group,PFOA at even the lowest concentration(1 μg·L-1)increased heart malformation rate(P<0.05)and reduced heart rate(P<0.01)in the zebrafish larvae at 72 hpf,and the results showed a clear concentration-response rela-tionship.Adding the AHR inhibitor CH significantly decreased heart malformation rate and restored heart rate to the control group level.The EROD results showed that PFOA at 1000 μg·L-1 increased AHR activity(P<0.001).Further studies revealed that PFOA caused a dose-dependent increase in intracellular ROS(P<0.001)and an increase in mitochondrial ROS(P<0.001)in the heart region of zebrafish lar-vae.A high dose of PFOA(1000 μg·L-1)also induced elevated mRNA expression levels of oxidative stress-related genes sod2 and cat(P<0.05).Addition of the AHR inhibitor CH effectively antagonized PFOA-induced(1000 μg·L-1)oxidative stress in the heart of zebrafish larvae(P<0.001).In addition,PFOA exposure induced a concentration-dependent increase in apoptotic bodies in the heart of zebrafish larvae(P<0.05).Moreover,PFOA at 1000 μg·L-1 caused an increase in the cleaved-caspase 3 immunofluorescence signal(P<0.05)as well as overexpression of the apoptosis-associated gene p53(P<0.001).which were attenuated by the CH supplementation.[Conclusion]PFOA triggers oxidative stress and apoptosis via AHR activation in the heart of zebrafish larvae,resulting in cardiac defects.
国家科技期刊平台
NSTL
万方数据
