一种针对人细胞色素P450 2A6的抑制性单克隆抗体确定了其在人肝脏中对香豆素、7-乙氧基香豆素和4-硝基苯甲醚代谢中的作用。

An inhibitory monoclonal antibody to human cytochrome P450 2A6 defines its role in the metabolism of coumarin, 7-ethoxycoumarin and 4-nitroanisole in human liver.

作者信息

Sai Y, Yang T J, Krausz K W, Gonzalez F J, Gelboin H V

机构信息

Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institute of Health, Bethesda, Maryland 20892, USA.

出版信息

Pharmacogenetics. 1999 Apr;9(2):229-37.

PMID:10376770

Abstract

Cytochrome P450 (CYP) 2A6 is an important enzyme catalysing the metabolism of many drugs, procarcinogens and promutagens. Its role in human liver metabolism of coumarin, 4-nitroanisole, 4-nitrophenol and 7-ethoxycoumarin was analysed with an inhibitory monoclonal antibody (MAb) to CYP2A6. MAbs were derived from a panel of 16 hybridomas which yielded positive enzyme-linked immunosorbent assay (ELISA) results or immunoblots against CYP2A6. The hybridomas were selected from more than 500 clones generated by the fusion of myeloma cells with spleen cells of mice immunized with purified baculovirus-expressed human CYP2A6. The MAbs obtained from four of the 16 hybridomas exhibited strong inhibitory activity to CYP2A6-catalysed phenanthrene metabolism. MAb 151-45-4 was positive and highly specific to CYP2A6 as determined by ELISA and immunoblot, and showed no cross-reactivity with recombinant human CYP 1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5, as tested with ELISA and immunoblot analyses. MAb 151-45-4 specifically inhibited CYP2A6-catalysed metabolism of phenanthrene, 4-nitroanisole, 4-nitrophenol, coumarin and 7-ethoxycoumarin each by 94-99% and did not inhibit their metabolism catalysed by 10 other human CYPs. The potent inhibitory effect of MAb 151-45-4 was used to define the contribution of human CYP2A6 to the metabolism of coumarin, 4-nitroanisole and 7-ethoxycoumarin in seven human liver microsome samples. Coumarin metabolism in all of the seven samples was inhibited by greater than 94% by MAb 151-45-4 which indicates that essentially all microsome mediated coumarin metabolism in human liver is catalysed only by CYP2A6. Inhibition of 4-nitroanisole and 7-ethoxycoumarin metabolism by anti 2A6 MAb ranged from 22-65% and 8-24%, respectively. The degree of inhibition defines the contribution of CYP2A6 activity to the 4-nitroanisole and 7-ethoxycoumarin metabolism in human liver and the range reflects the variability among samples. The inhibitory antibody to CYP2E1 was used to determine its role in 4-nitroanisole and 7-ethoxycoumarin metabolism in seven human liver samples. The addition of both MAbs to CYP2A6 and 2E1 to the microsome samples defined combinatorially the relative role of CYP2A6 and 2E1 in the metabolism of 4-nitroanisole and 7-ethoxycoumarin.

摘要

细胞色素P450（CYP）2A6是一种重要的酶，可催化多种药物、前致癌物和前诱变剂的代谢。使用针对CYP2A6的抑制性单克隆抗体（MAb）分析了其在人肝脏中对香豆素、4-硝基苯甲醚、4-硝基苯酚和7-乙氧基香豆素的代谢作用。单克隆抗体源自一组16种杂交瘤，这些杂交瘤产生了阳性酶联免疫吸附测定（ELISA）结果或针对CYP2A6的免疫印迹。这些杂交瘤是从500多个克隆中筛选出来的，这些克隆是通过骨髓瘤细胞与用纯化的杆状病毒表达的人CYP2A6免疫的小鼠脾细胞融合产生的。从16种杂交瘤中的4种获得的单克隆抗体对CYP2A6催化的菲代谢表现出强烈的抑制活性。通过ELISA和免疫印迹测定，单克隆抗体151-45-4对CYP2A6呈阳性且高度特异性，并且如通过ELISA和免疫印迹分析测试所示，与重组人CYP 1A1、1A2、2B6、2C8、2C9、2C19、2D6、2E1、3A4和3A5无交叉反应。单克隆抗体151-45-4特异性抑制CYP2A6催化的菲、4-硝基苯甲醚、4-硝基苯酚、香豆素和7-乙氧基香豆素的代谢，每种代谢的抑制率均为94-99%，并且不抑制由其他10种人CYP催化的它们的代谢。单克隆抗体151-45-4的强效抑制作用用于确定人CYP2A6对7种人肝微粒体样品中香豆素、4-硝基苯甲醚和7-乙氧基香豆素代谢的贡献。单克隆抗体151-45-4对所有7个样品中的香豆素代谢的抑制率均大于94%，这表明人肝脏中基本上所有微粒体介导的香豆素代谢仅由CYP2A6催化。抗2A6单克隆抗体对4-硝基苯甲醚和7-乙氧基香豆素代谢的抑制率分别为22-65%和8-24%。抑制程度定义了CYP2A6活性对人肝脏中4-硝基苯甲醚和7-乙氧基香豆素代谢的贡献，而范围反映了样品之间的变异性。使用针对CYP2E1的抑制性抗体来确定其在7个人肝样品中对4-硝基苯甲醚和7-乙氧基香豆素代谢中的作用。将针对CYP2A6和2E1的两种单克隆抗体添加到微粒体样品中组合定义了CYP2A6和2E1在4-硝基苯甲醚和7-乙氧基香豆素代谢中的相对作用。