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.
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.
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发表时间：
1999-04
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通讯作者：
Yang Sai;T. Yang;K. Krausz;F. J. Gonzalez;H. Gelboin
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作者：
Yang Sai;T. Yang;K. Krausz;F. J. Gonzalez;H. Gelboin
细胞色素P450（CYP）2A 6是一种重要的酶，催化许多药物、前致癌物和前诱变剂的代谢。用CYP 2A 6抑制性单克隆抗体（MAb）分析了它对香豆素、4-硝基苯甲醚、4-硝基苯酚和7-乙氧基香豆素在人肝代谢中的作用。单克隆抗体来源于一组16个杂交瘤，这些杂交瘤对CYP 2A 6产生阳性酶联免疫吸附试验（ELISA）结果或免疫印迹。从骨髓瘤细胞与纯化的杆状病毒表达的人CYP 2A 6免疫小鼠的脾细胞融合产生的500多个克隆中选择杂交瘤。从16个杂交瘤中的4个杂交瘤中获得的单克隆抗体对CYP 2A 6催化的菲代谢表现出较强的抑制活性。经ELISA和免疫印迹分析确定，MAb 151-45-4对CYP 2A 6呈阳性且高度特异性，经ELISA和免疫印迹分析检测，MAb 151-45-4与重组人CYP 1A 1、1A 2、2B 6、2C 8、2C 9、2C 19、2D 6、2 E1、3A 4和3A 5无交叉反应性。MAb 151-45-4特异性抑制CYP 2A 6催化的菲、4-硝基苯甲醚、4-硝基苯酚、香豆素和7-乙氧基香豆素代谢，抑制率分别为94-99%，且不抑制其他10种人CYP催化的代谢。在7份人肝微粒体样品中，使用MAb 151-45-4的强效抑制作用来确定人CYP 2A 6对香豆素、4-硝基苯甲醚和7-乙氧基香豆素代谢的贡献。MAb 151-45-4对所有7份样品中香豆素代谢的抑制率均超过94%，这表明人肝脏中基本上所有微粒体介导的香豆素代谢均仅由CYP 2A 6催化。抗2A 6单抗对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CYP 2A 6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，范围反映了样品间的变异性。CYP 2 E1的抑制性抗体用于确定其在7个人肝脏样品中4-硝基苯甲醚和7-乙氧基香豆素代谢中的作用。向微粒体样品中加入CYP 2A 6和2 E1的两种MAb，组合定义了CYP 2A 6和2 E1在4-硝基苯甲醚和7-乙氧基香豆素代谢中的相对作用。
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.