2,4,4′三氯联苯和2,2′,5,5′-四氯联苯诱发重组中国地鼠V79-hCYP2E1-hSULT1A1细胞系Hprt基因的分子突变谱研究  

Spectrum of mutation in the Hprt gene of V79-hCYP2E1-hSULT1A1 induced by 2,4,4’-Trichlorobiphenyl and 2,2’,5,5’-Tetrachlorobiphenyl

作　　者：金桂芳 胡克岐 潘德顺 杨智承 刘云岗[2] JIN Gui-fang;HU Ke-qi;PAN De-shun;YANG Zhi-cheng;LIU Yun-gang(School of Pharmacy,Guangdong Pharmaceutical University,Guangzhou,Guangdong 510006,China;不详)

机构地区：[1]广东药科大学药学院,广东广州510006 [2]南方医科大学公共卫生学院

出　　处：《现代预防医学》2021年第23期4349-4355,共7页Modern Preventive Medicine

基　　金：广东省医学科研基金(A2016138);国家自然科学基金(21577054)。

摘　　要：目的了解多氯联苯化合物诱发哺乳动物细胞次黄嘌呤鸟嘌呤磷酸核糖转移酶(Hprt)基因的分子突变谱。方法采用自行构建的重组中国仓鼠肺细胞株(V79-hCYP2E1-hSULT1A1),用空2,4,4′-三氯联苯(PCB 28)(10、20、40μmol/L)、2,2′,5,5′-四氯联苯(PCB 52)(80μmol/L)和阳性对照N-二乙基亚硝胺(NDEA,200μmol/L)染毒,实验结束后收集突变克隆进行细胞扩增,提取细胞总RNA,逆转录得cDNA,采用PCR扩增Hprt基因片段,并进行基因测序,统计分析各化学物诱发突变发生率及分子突变谱。结果对照组细胞Hprt基因的自发突变中单个碱基置换占88.2%,PCB 28诱发的突变除单碱基置换外,呈现多类型突变特征,包括两个以上碱基置换、外显子缺失、移码突变及小缺失;随着PCB 28浓度增加,单碱基置换所占比例呈下降趋势,即由68.8%下降到33.3%;并且高浓度时以小缺失为主要突变类型(占66.7%)。PCB 52和NDEA诱发突变类型则类似自发突变组。自发突变的单碱基置换主要为A:T→G:C改变,而PCB 28(20μmol/L)诱发的碱基置换主要为G:C→A:T。PCB 28组(20μmol/L)突变克隆细胞平均碱基置换次数高于自发突变组。结论 PCB 28诱发突变呈现多类型突变特征,与自发突变的分子突变谱有差异,可能与该化合物特异的致突变机理有关。Objective To investigate the molecular mutation profile of Hprt gene induced by polychlorinated biphenyls(PCBS) in mammalian cells. Methods Using recombinant Chinese hamster lung cell line(V79-HCYP2e1-HSULT1A1) constructed by ourselves, 2,4,4’-trichlorobiphenyl(PCB 28)(10, 20, 40 μmol/L), 2,2’,5,5 ’-tetrachlorobiphenyl(PCB 52)(80 μmol/L) and positive control n-diethyl nitrosamine(NDEA, 200 μmol/L) were used. At the end of the experiment, mutant clones were collected for cell amplification, total RNA was extracted and cDNA was obtained by reverse transcription. PCR was used to amplify Hprt gene fragments, and gene sequencing was conducted to statistically analyze the incidence of chemical induced mutations and molecular mutation spectrum. Results Analysis of spontaneous mutations in the Hprt gene treated with DMSO(control) revealed that the proportion of base substitutions was 88.2%. Sequence changes caused by PCB 28 indicated multiple mutation types, including tandem mutation, exon deletion, frameshifts and small deletion, besides of base substitution. A decreasing trend was observed for the proportion of the base substitutions, from 68.8% to 33.3%, with the increasing concentration of PCB 28. Furthermore, small deletion dominated at the highest concentration, accounting for 66.7%. Mutants in the PCB 52 and NDEA groups demonstrated a spectrum of mutations similar to that of spontaneous mutations. The groups of DMSO and low concentration of PCB 28(10 μmol/L) displayed dominance for A:T→G:C transitions, while in the intermediate PCB 28 concentration(20 μmol/L) the mutation type of G:C→A:T dominated. The number of base substitutions per mutant clone in the group pf PCB 28 at 20 μmol/L was more than that in the vehicle group. Conclusion The mutation profiles of human CYP2 E1-activated, PCB 28-induced Hprt mutations in V79-derived cells exhibit a wide spectrum, significantly different from spontaneous mutations, which may suggest specific mechanism of PCB 28 mutagenicity.

关 键 词：CYP2E1 PCB 28 PCB 52 HPRT 基因测序 

分 类 号：R114[医药卫生—卫生毒理学]