全氟辛酸通过芳香烃受体介导的氧化应激及细胞凋亡导致斑马鱼幼鱼心脏发育异常  

Perfluorooctanoic acid induces abnormal heart development via aryl hydrocarbon receptormediated oxidative stress and apoptosis in zebrafish larvae

作　　者：陈雪怡 马天驰 王康 陈涛[1] 姜岩 CHEN Xueyi;MA Tianchi;WANG Kang;CHEN Tao;JIANG Yan(School of Public Health,Soochow University,Suzhou,Jiangsu 215123,China;School of Basic Medical Science,Soochow University,Suzhou,Jiangsu 215123,China)

机构地区：[1]苏州大学公共卫生学院,江苏苏州215123 [2]苏州大学基础医学学院,江苏苏州215123

出　　处：《环境与职业医学》2024年第12期1354-1360,共7页Journal of Environmental and Occupational Medicine

基　　金：国家自然基金面上项目(82171689)。

摘　　要：[背景]近年来,越来越多的研究表明全氟辛酸(PFOA)暴露能影响心脏发育,但其具体机制尚不明确。芳香烃受体(AHR)是重要的环境感受器,可引起氧化应激及细胞凋亡。[目的]利用斑马鱼胚胎模型,探讨AHR在PFOA心脏发育毒性中的作用机制。[方法]将受精后2 h(2 hpf)的斑马鱼胚胎暴露于二甲亚砜(DMSO)对照以及1、10、100、1000μg·L^(-1)的PFOA中,并在高剂量组(1000μg·L^(-1))添加AHR抑制剂CH223191(CH)设置干预组。解剖镜下检测72 hpf斑马鱼幼鱼的存活率、死亡率、心率和心脏畸形率。以7-乙氧基试卤灵-O-脱乙基酶(EROD)染色方法检测AHR的活性。使用二氯二氢荧光素二乙酸酯和MitoSOX?Red检测斑马鱼幼鱼心脏部位的整体ROS及线粒体ROS水平。以吖啶橙(AO)染色及免疫荧光方法检测斑马鱼幼鱼心脏细胞凋亡。剖取各组胚胎心脏,提取总RNA,以荧光定量PCR方法检测氧化应激相关基因(sod2、cat)和凋亡相关基因(p53)mRNA表达水平。[结果]与DMSO对照组相比,低剂量(1μg·L^(-1))的PFOA即能引起72 hpf斑马鱼幼鱼心脏畸形率增加(P<0.05)和心率降低(P<0.01),并均有剂量-反应关系。添加AHR抑制剂CH后降低了高剂量(1000μg·L^(-1))PFOA引起的斑马鱼幼鱼心脏畸形率(P<0.001),且使心率恢复到对照组水平。EROD染色结果显示,高剂量PFOA(1000μg·L^(-1))能引起AHR活性升高(P<0.001)。进一步研究发现PFOA引起斑马鱼幼鱼心脏部位细胞内ROS升高(P<0.001)且存在剂量依赖性。高剂量PFOA(1000μg·L^(-1))会引起线粒体ROS增多(P<0.001)以及氧化应激相关基因sod2和cat的mRNA表达水平升高(P<0.05)。添加AHR抑制剂CH能有效拮抗高剂量PFOA(1000μg·L^(-1))引起的斑马鱼幼鱼心脏中的氧化应激(P<0.001)。PFOA暴露还会引起斑马鱼幼鱼心脏部位细胞凋亡小体剂量依赖性增多(P<0.05)。研究进一步确定高剂量PFOA(1000μg·L^(-1))引起cleaved-caspase 3免疫荧光染色增加(P<0.05)以及�[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 zebrafish 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 1000μg·L^(−1) of PFOA.The AHR inhibitor CH223191(CH)was added to the high-concentration group (1 000 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 using7-ethoxyresorufin-O-dealkylation (EROD) staining. The levels of intracellular and mitochondrial ROS in the heart of zebrafish larvae wereassessed 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 oxidativestress-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 relationship.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 1 000 μg·L^(−1) increased AHR activity (P < 0.001). Further studies revealed that PFOA caused a dosedependentincrease in intracellular ROS (P < 0.001) and an increase in mitochondrial ROS (P < 0.0

关 键 词：全氟辛酸 芳香烃受体 氧化应激 心脏发育 斑马鱼 

分 类 号：R11[医药卫生—公共卫生与预防医学]