HBP介导的O-GlcNAc修饰在PFOS暴露致斑马鱼胚胎发育异常中的作用  

Role of hexosamine biosynthesis pathway-mediated O-GlcNAcylation in abnormal zebrafish embryonic development induced by perfluorooctane sulfonate exposure

作　　者：袁琴 范赟 钱鸿 韩舒雯 汤静雅 徐俏俏 吴炜[1,2] 姜子燕 秦玉峰[1,2] 陆春城 YUAN Qin;FAN Yun;QIAN Hong;HAN Shu-wen;TANG Jing-ya;XU Qiao-qiao;WU Wei;JIANG Zi-yan;QIN Yu-feng;LU Chun-cheng(School of Public Health,Nanjing Medical University,Nanjing 211166,China;Key Laboratory of Modern Toxicology of Ministry of Education,School of Public Health,Nanjing Medical University;State Key Laboratory of Reproductive Medicine,Nanjing Medical University;The First Affiliated Hospital with Nanjing Medical University/Jiangsu Province Hospital)

机构地区：[1]南京医科大学公共卫生学院,南京211166 [2]南京医科大学公共卫生学院现代毒理学教育部重点实验室 [3]南京医科大学生殖医学国家重点实验室 [4]南京医科大学第一附属医院江苏省人民医院

出　　处：《环境卫生学杂志》2023年第11期809-817,829,共10页JOURNAL OF ENVIRONMENTAL HYGIENE

基　　金：国家自然科学基金(82271691)。

摘　　要：目的通过构建全氟辛烷磺酸(perfluorooctane sulfonate,PFOS)斑马鱼染毒模型,探讨PFOS暴露对胚胎发育的影响及可能机制。方法基于人群内暴露浓度设置PFOS暴露浓度梯度(0、0.02、0.2、2和20μmol/L),对斑马鱼胚胎染毒至120 hpf受精后小时(hourspost-fertilization,hpf),检测斑马鱼胚胎的孵化率、畸形率、死亡率、心率及运动分析等基础指标;采用非靶向代谢组学技术分析PFOS暴露后斑马鱼胚胎整体代谢水平的变化,并结合实时荧光定量(RT-qPCR)和蛋白质免疫印迹(Western blot)观察差异代谢酶及蛋白表达水平。结果暴露于20μmol/L的PFOS导致斑马鱼胚胎畸形率升高(P<0.05)、心率升高(P<0.05)、行为分析异常(P<0.05);代谢组学结果发现20μmol/L PFOS暴露组斑马鱼胚胎中葡萄糖-6-磷酸(Glu-6-P)、乙酰氨基葡萄糖(GlcNAc)和N-乙酰氨基葡萄糖-6-磷酸(GlcNAc-6-P)水平显著升高(P<0.05),提示己糖胺生物合成途径(hexosamine-biosynthesis pathway,HBP)被过度激活;RT-qPCR结果显示20μmol/L PFOS组HBP关键代谢酶slc2a1b、hk1、gfat1基因表达水平显著上调(P<0.05);Western blot结果显示斑马鱼胚胎整体O-连接-N-乙酰葡萄糖胺糖基化(O-GlcNAc)水平在20μM PFOS暴露组与对照组之间存在差异。结论PFOS暴露可能通过干扰HBP途径,介导O-GlcNAc修饰水平改变导致斑马鱼胚胎发育畸形率升高及运动行为学异常,提示PFOS具有胚胎发育毒性。Objective To investigate the effects of perfluorooctane sulfonate(PFOS)exposure on embryonic development and its possible mechanism by constructing a model of PFOS zebrafish infection.Methods The PFOS concentration gradients(0,0.02,0.2,2,and 20μmol/L)were established based on human internal exposure levels,and exposed to zebrafish embryos for 120 hours post fertilization,then the basic indicators of zebrasish(hatching rate,malformation rate,mortality rate,heart rate,and motor ability)were measured.The global metabolic changes of zebrafish embryos exposed to PFOS were measured through non-targeted metabolomics technology.The expression of differential metabolic enzymes and proteins was determined by real-time quantitative PCR(RT-qPCR)and western blot.Results Exposure to 20μmol/L PFOS significantly increased the malformation rate(P<0.05),heart rate(P<0.05),and motor abnormalities(P<0.05)of zebrafish embryos.The metabolomics analysis showed that in the 20μmol/L PFOS exposure group,the levels of glucose-6-phosphate(Glu-6-P),N-acetylglucosamine(GlcNAc),and N-acetylglucosamine-6-phosphate(GlcNAc-6-P)were significantly increased,indicating the hyperactivation of hexosamine biosynthesis pathway(HBP).RT-qPCR demonstrated that the gene expression levels of HBP-related key metabolic enzymes(slc2a1b,hk1,and gfat1)were significantly upregulated in 20μmol/L PFOS group.Western blot showed that there was a significant difference in the global O-linked N-acetylglu-cosaminylation(O-GlcNAc)level between the 20μmol/L PFOS group and the control group.Conclusion PFOS exposure may interfere with HBP to mediate change of O-GlcNAcylation level,resulting in increased malformation and motor abnormalities in zebrafish embryos,suggesting that PFOS is toxic to embryonic development.

关 键 词：全氟辛烷磺酸(PFOS) 斑马鱼胚胎发育 代谢组学 O-连接-N-乙酰葡萄糖胺糖基化(O-GlcNAc)修饰 

分 类 号：R114[医药卫生—卫生毒理学]