Human AKR1A1 involves in metabolic activation of carcinogenic aristolochic acidⅠ  

人醛酮还原酶1A1参与致癌物马兜铃酸Ⅰ的代谢活化

作　　者：GAO Zhenna YOU Xinyue LIU Weiying WU Jiaying XI Jing CAO Yiyi ZHANG Xiaohong ZHANG Xinyu LUAN Yang 高振娜;尤馨悦;刘维映;吴佳颖;奚晶;曹易懿;张小红;张新宇;栾洋(上海交通大学医学院公共卫生学院,上海200025;沈阳药科大学无涯创新学院,辽宁沈阳110016)

机构地区：[1]School of Public Health,School of Medicine,Shanghai JiaoTong University,Shanghai 200025,China [2]Wuya College of Innovation,Shenyang Pharmaceutical University,Shenyang 110016,China

出　　处：《中国药理学与毒理学杂志》2024年第9期641-651,共11页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(81873081)。

摘　　要：OBJECTIVE To investigate whether aldo-keto reductases(AKRs)can act as a nitrore⁃ductase(NR)and bioactivate aristolochic acidⅠ(AA-Ⅰ)to produce AA-Ⅰ-DNA adducts.METHODS①Human-induced hepatocytes(hiHeps)and human bladder RT4 cells were used as tool cells and treated with AA-Ⅰ0,0.5,1.0 and 2μmol·L^(-1)for 24 h.Cell viability was detected using the CCK-8 method,and the half maximal inhibition concentration(IC_(50))was calculated using the CCK-8 method and the level of DNA adduct production was calculated.②hiHeps and RT4 cells were treated with AKR inhibitor luteotin(0,5,10 and 25μmol·L^(-1))+AA-Ⅰ0.2 and 1.0μmol·L^(-1)for 24 h,respectively,and the levels of DNA adducts were detected by a liquid chromatography-tandem mass spectrometer(LC-MS/MS).③hiHeps cells were incubated with 80 nmol·L^(-1)small interfering RNAs(si-AKRs)for 48 h and treated with AA-Ⅰ1.0μmol·L^(-1)for 24 h.Real-time qualitative PCR(RT-qPCR)method was used to detect the mRNA expression of AKRs gene and LC-MS/MS technology was used to investigate the effect of specific AKR gene knockdown on DNA adduct levels.④500 nmol·L^(-1)human AKR recombinant proteins AKR1A1 and AA-Ⅰwere incubated in vitro under anaerobic conditions and the formation of AA-Ⅰ-DNA adducts was detected.RESULTS①The IC_(50)of AA-Ⅰto hiHeps and RT4 cells was 1.9 and 0.42μmol·L^(-1),respec⁃tively.The level of DNA adduct production of the two cell lines was significantly different(P<0.01).②Luteolin≥5μmol·L^(-1)significantly inhibited the production of AA-Ⅰ-DNA adducts in both cells(P<0.05),and there was a concentration-dependent effect in hiHeps cells(P<0.01,R=0.84).③In the AKR family,the knockdown of AKR1A1 gene up to 80%inhibited the generation of AA-Ⅰ-DNA adducts by 30%-40%.④The AA-Ⅰ-DNA adducts were detected in the incubation of recombinant protein AKR1A1 and AA-Ⅰunder anaerobic conditions in vitro,approximately 1 adduct per 107 nucleotides.CONCLU⁃SION AKR1A1 is involved in AA-Ⅰbioactivation,providing a reference for elucid目的探究醛酮还原酶(AKR)对致癌物马兜铃酸Ⅰ(AA-Ⅰ)的硝基还原酶活性及代谢活化反应。方法①AA-Ⅰ0~5μmol·L^(-1)处理人诱导肝实质细胞(hiHeps)和RT4细胞24 h,CCK-8法检测细胞活力,计算半数抑制浓度(IC_(50)),测量DNA加合物生成水平。②AKR抑制剂木犀草素(0,5,10和25μmol·L^(-1))+AA-Ⅰ0.2和1.0μmol·L^(-1)分别处理hiHeps和RT4细胞24 h,液相色谱-串联质谱(LC-MS/MS)检测DNA加合物水平。③AKR的小干扰RNA(siAKR)80 nmol·L^(-1)孵育hiHeps细胞48 h后,加AA-Ⅰ1.0μmol·L^(-1)处理24 h,RT-qPCR检测AKR基因敲减效率,同时LC-MS/MS考察特定AKR基因敲低对DNA加合物水平的影响。④利用500 nmol·L^(-1)人源AKR重组蛋白AKR1A1和AA-Ⅰ在体外无氧条件下进行孵育,并检测AA-Ⅰ-DNA加合物生成。结果①AA-Ⅰ对hiHeps和RT4细胞的IC_(50)分别为1.90和0.42μmol·L^(-1)。2个细胞系生成DNA加合物水平存在明显差异(P<0.05)。②木犀草素≥5μmol·L^(-1)能明显抑制2种细胞中AA-Ⅰ-DNA加合物的生成(P<0.05),且在hiHeps细胞中存在一定浓度依赖性(P<0.01,R=0.84)。③基因AKR1A1被敲减达80%时,抑制30%~40%AA-Ⅰ-DNA加合物的生成。④重组蛋白AKR1A1和AA-Ⅰ体外无氧条件下孵育检测到AA-Ⅰ-DNA加合物的生成,约为每1×107个核苷酸中1个加合物。结论AKR1A1具有AA-Ⅰ硝基还原酶活性,可将AA-Ⅰ代谢活化并生成DNA加合物,为进一步阐明AA-Ⅰ的致癌机制提供参考。

关 键 词：metabolic activation nitro-reduction aldo-keto reductase superfamily aristolochic acidⅠ 

分 类 号：R96[医药卫生—药理学] R99[医药卫生—药学]