苯并[k]荧蒽(BkF)致雄性生殖细胞损害的初步研究  

Benzo[k] fluoranthene induces male germ cell damage

作　　者：王丹丹 王芙蓉 杨晓燕[3] 周妮娅 杨惠芳 曹佳[2] WANG Dandan;WANG Furong;YANG Xiaoyan;ZHOU Niya;YANG Huifang;CAO Jia(Department of Toxicology,School of Public Health and Management,Ningxia Medical University,Yinchuan,Ningxia Hui Autonomous Region,750000;Institute of Toxicology,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038;Chongqing Health Center for Women and Children,Chongqing,400021,China)

机构地区：[1]宁夏医科大学公共卫生与管理学院卫生毒理学教研室,银川750000 [2]陆军军医大学(第三军医大学)军事预防医学系毒理学研究所,重庆400038 [3]重庆市妇幼保健院,重庆400021

出　　处：《陆军军医大学学报》2022年第8期740-748,共9页Journal of Army Medical University

基　　金：国家自然科学基金重点项目(81630087);重庆市科卫联合医学科研项目(2021MSXM123)。

摘　　要：目的 建立高环多环芳烃的代表性化合物苯并[k]荧蒽(BkF)致小鼠精母细胞(GC-2spd)损伤模型,初步探讨BkF引起GC-2细胞的损伤效应及机制。方法 通过比较毒理基因组学数据库(Comparative Toxicogenomics Database, CTD)筛选与BkF相关的男性生殖系统疾病的基因,对其进行功能和通路富集,探索可能的通路机制。以终浓度为0、10、20、40、80μmol/L BkF处理GC-2细胞,检测细胞周期、氧化应激水平及DNA损伤效应,采用Western blot检测关键基因及相关通路核心分子的蛋白表达水平。结果 CTD数据库中筛选出与BkF相关生殖系统疾病的关键基因为CYP1A1和CYP17A1。GO富集分析发现其在男性生殖系统、细胞周期阻滞、氧化应激等生物过程中明显富集,KEGG富集分析发现其在细胞色素P450通路中富集明显(P<0.01)。BkF染毒GC-2细胞72 h后,细胞增殖活力较对照组明显下降,且存在剂量-效应关系。染毒剂量高于40μmol/L后细胞周期阻滞明显(P<0.05),且染毒组细胞内ROS水平(P<0.05)、DNA损伤标志物γ-H2AX蛋白表达量(P<0.01)呈剂量依赖性增加。与对照组相比,染毒组细胞CYP1A1和CYP17A1的蛋白表达显著增加(P<0.05)。加入AhR抑制剂CH223191后,染毒组CYP1A1和CYP17A1的表达明显下调(P<0.05)。结论 BkF通过诱导AhR受体激活,引起下游基因CYP1A1和CYP17A1的表达升高、细胞内ROS增加,从而导致GC-2细胞氧化应激,造成遗传损伤。Objective To establish a damage model of mouse spermatocyte GC-2 spd cells induced by benzo[k] fluoranthene(BkF), a representative compound of high-molecular weight polycyclic aromatic hydrocarbons, so as to investigate the effect and mechanism of BkF on GC-2 cells. Methods Related genes of male reproductive diseases associated with BkF were screened in Comparative Toxicogenomics Database(CTD), and functional and pathway enrichment analysis is conducted subsequently to explore the possible pathway mechanism. Moreover, GC-2 cells were treated with BkF at a final concentration of 0, 10, 20, 40 or 80 μmol/L for 72 h, and then the cell cycle, oxidative stress and DNA damage effect were detected. Western blotting was used to determine the protein levels of the key genes as well as the core molecules of related pathways. Results The key genes CYP1 A1 and CYP17 A1 associated with BkF-related reproductive system diseases were screened out from the CTD database. Gene Ontology(GO) enrichment analysis found that they were involved in male reproductive system and cell cycle arrest, obviously enriched in biological processes such as oxidative stress. Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis showed that these genes were significantly enriched in cytochrome P450 pathway(P<0.01). After GC-2 cells were exposed to BkF for 72 h, the proliferation was significantly decreased in a dose-dependent manner, as compared with the control group. When the exposure dose was higher than 40 μmol/L, the cell cycle arrest was obvious(P<0.05);Both the intracellular reactive oxygen species(ROS) level(P<0.05) and the protein level of DNA damage marker γ-H2 AX(P<0.01) were remarkably increased in a dose-dependent manner. In addition, the protein levels of CYP1 A1 and CYP17 A1 were greatly higher in the exposure group than the control group(P<0.05), while treatment of AhR inhibitor CH223191 resulted in notably down-regulated the expression of the 2 proteins(P<0.05). Conclusion BkF induces the activation of AhR receptors, then

关 键 词：苯并[k]荧蒽 GC-2spd细胞 氧化损伤 AhR-CYP1A1信号通路 生信分析 

分 类 号：R-33[医药卫生] R321.1