Identification of human cytochrome P450s involved in the formation of all-trans-retinoic acid principal metabolites
Identification of human cytochrome P450s involved in the formation of all-trans-retinoic acid principal metabolites
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DOI：
10.1124/mol.58.6.1341
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发表时间：
2000-12-01
影响因子：
3.6
通讯作者：
Chabot, GG
中科院分区：
文献类型：
--
作者：
Marill, J;Cresteil, T;Chabot, GG
全反式维甲酸（atRA）的细胞色素P450（P450）依赖性代谢对其生物活性的表达具有重要意义。由于参与atRA主要代谢产物形成的人P450仅被部分鉴定，因此本研究的目的是鉴定参与atRA代谢的人P450。用16个人肝微粒体进行表型分析，发现2B 6、2C 8、3A 4/5和2A 6参与4-OH-RA和4-oxo-RA的形成，2B 6、2C 8和2A 6与18-OH-RA的形成有关;和2A 6、2B 6和3A 4/5活性与5，6-环氧-RA形成相关（30分钟温育，10 μ M atRA，HPLC分离，UV检测340 nm）。使用来自淋巴母细胞微粒体的15个cDNA表达的人P450，显示通过CYP 3A 7> CYP 3A 5> CYP 2C 18> CYP 2C 8> CYP 3A 4> CYP 2C 9形成4-OH-RA，而18-OH-RA形成涉及CYP 4A 11> 3A 7> 1A 1> 2C 9> 2C 8> 3A 5> 3A 4> 2C 18。动力学研究确定3A 7是形成三种代谢物中最活跃的P450：对于4-OH-视黄酸，3A 7显示127.7的V-max/K-m，随后是3A 5（V-max/ K-m = 25.6），2C8（V-max/K-m = 24.5），2C18（V-max/K-m = 15.8），3A4（V-max/K-m = 5.7），1A1（V-max/K-m = 5.0）和4A11（V-max/K-m = 1.9）;对于4-氧代-RA，3A 7显示13.4的V-max/K-m，随后对于2C 18和4A 11两者显示低10倍的活性（V-max/K-m = 1.2）;对于18-OH-RA，3A 7显示10.5的V-max/K-m，相比之下，4A 11的V-max/K-m为2.1，2C 8为2.0。5，6-Epoxy- RA仅在高底物浓度（>10 μ M）下检测到，P450 2C 8、2C 9和1A 1在其形成中最活跃.使用人P450 cDNA稳定转染的胚胎肾细胞（293）证实了P450 3A 7、1A 1和2C 8主要参与atRA的氧化，1A 2、2C 9和3A 4的参与程度较低。总之，已经鉴定了几种参与atRA代谢的人P450，其表达显示出指导atRA代谢朝向特定代谢物的形成。这些人类P450在atRA的生物学和抗癌作用中的作用仍有待阐明。
Cytochrome P450 (P450)-dependent metabolism of all-trans-retinoic acid (atRA) is important for the expression of its biological activity. Because the human P450s involved in the formation of the principal atRA metabolites have been only partially identified, the purpose of this study was to identify the human P450s involved in atRA metabolism. The use of phenotyped human liver microsomes (n = 16) allowed the identification of the following P450s: 2B6, 2C8, 3A4/5, and 2A6 were involved in the formation of 4-OH-RA and 4-oxo-RA; 2B6, 2C8, and 2A6 correlated with the formation of 18-OH-RA; and 2A6, 2B6, and 3A4/5 activities correlated with 5,6-epoxy-RA formation (30-min incubation, 10 muM atRA, HPLC separation, UV detection 340 nm). The use of 15 cDNA-expressed human P450s from lymphoblast microsomes, showed the formation of 4-OH-RA by CYP3A7> CYP3A5> CYP2C18> CYP2C8> CYP3A4> CYP2C9, whereas the 18-OH-RA formation involved CYPs 4A11> 3A7> 1A1> 2C9> 2C8> 3A5> 3A4>2C18. Kinetic studies identified 3A7 as the most active P450 in the formation of three of the metabolites: for 4-OH-retinoic acid, 3A7 showed a V-max/K-m of 127.7, followed by 3A5 (V-max/ K-m = 25.6), 2C8 (V-max/K-m = 24.5), 2C18 (V-max/K-m = 15.8), 3A4 (V-max/K-m = 5.7), 1A1 (V-max/K-m = 5.0), and 4A11 (V-max/K-m = 1.9); for 4-oxo-RA, 3A7 showed a V-max/K-m of 13.4, followed by a 10-fold lower activity for both 2C18 and 4A11 (V-max/K-m = 1.2); and for 18-OH-RA, 3A7 showed a V-max/K-m of 10.5 compared with a V-max/K-m of 2.1 for 4A11 and 2.0 for 2C8. 5,6-Epoxy- RA was only detected at high substrate concentrations in this system (>10 muM), and P450s 2C8, 2C9, and 1A1 were the most active in its formation. The use of embryonic kidney cells (293) stably transfected with human P450 cDNA confirmed the major involvement of P450s 3A7, 1A1, and 2C8 in the oxidation of atRA, and to a lesser extent, 1A2, 2C9, and 3A4. In conclusion, several human P450s involved in atRA metabolism have been identified, the expression of which was shown to direct atRA metabolism toward the formation of specific metabolites. The role of these human P450s in the biological and anticancer effects of atRA remains to be elucidated.