Hu Ming, Krausz Kristopher, Chen Jun, Ge Xia, Li Jianqi, Gelboin Harry L, Gonzalez Frank J
LAboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Healh, Bethesda, Maryland, USA.
Drug Metab Dispos. 2003 Jul;31(7):924-31. doi: 10.1124/dmd.31.7.924.
This study determined the cytochrome P450 (P450) isoforms responsible for metabolism of isoflavones using human liver microsomes (HLM) and expressed P450s. The primary metabolite of genistein is 3'-OH-genistein, as identified with an authentic chemically synthesized standard. CYP1A2 was predominantly responsible for 3'-OH-genistein formation since its formation was inhibited (>50%, p < 0.05) by a monoclonal antibody specific for CYP1A2, was correlated with CYP1A2 activities of HLM, and was catalyzed by expressed CYP1A2. In addition to CYP1A2, CYP2E1 also catalyzed, although to a lesser extent, its formation. The contribution of these P450s to the formation of 3'-OH-genistein was also confirmed with a panel of expressed enzymes. Methylated isoflavones biochanin A, prunetin, and formononetin (10-100 microM) were rapidly converted by HLM and expressed CYP1A2 to more active genistein and daidzein. The conversion of biochanin A to genistein appears to be mainly mediated by CYP1A2 because of the strong correlation between the conversion rates and CYP1A2 activities in HLM. Thus, CYP1A2 is an effective prodrug-converting enzyme for less active methylated isoflavones. CYP1A2-catalyzed conversion of biochanin A to genistein (Km, 7.80 microM; Vmax, 903 pmol/min/mg of protein; Vmax/Km, 116 microl/min/mg of protein) was much faster than 3'-hydroxylation of genistein (Km, 12.7 microM and Vmax, 109 pmol/min/mg of protein; Vmax/Km, 8.6 microl/min/mg of protein). The interaction studies showed that genistein inhibited formation of acetaminophen from phenacetin with an IC50 value of 16 microM. Additional studies showed that phenacetin and genistein were mutually inhibitory. In conclusion, CYP1A2 and CYP2E1 metabolized genistein and CYP1A2 acted as prodrug-converting enzymes for other less active methylated isoflavones.
本研究利用人肝微粒体（HLM）和表达的细胞色素P450（P450）同工酶，确定了负责异黄酮代谢的P450同工酶。用化学合成的标准品鉴定出，染料木黄酮的主要代谢产物是3'-羟基染料木黄酮。CYP1A2主要负责3'-羟基染料木黄酮的形成，因为其形成被CYP1A2特异性单克隆抗体抑制（>50%，p<0.05），与HLM的CYP1A2活性相关，且由表达的CYP1A2催化。除CYP1A2外，CYP2E1也催化其形成，不过程度较小。一组表达酶也证实了这些P450对3'-羟基染料木黄酮形成的贡献。甲基化异黄酮鹰嘴豆芽素A、樱黄素和芒柄花黄素（10 - 100微摩尔）被HLM和表达的CYP1A2迅速转化为活性更高的染料木黄酮和大豆苷元。鹰嘴豆芽素A向染料木黄酮的转化似乎主要由CYP1A2介导，因为转化率与HLM中CYP1A2活性之间存在很强的相关性。因此，CYP1A2是一种将活性较低的甲基化异黄酮转化为前药的有效酶。CYP1A2催化鹰嘴豆芽素A向染料木黄酮的转化（米氏常数，7.80微摩尔；最大反应速度，903皮摩尔/分钟/毫克蛋白质；最大反应速度/米氏常数，116微升/分钟/毫克蛋白质）比染料木黄酮的3'-羟基化反应（米氏常数，12.7微摩尔，最大反应速度，109皮摩尔/分钟/毫克蛋白质；最大反应速度/米氏常数，8.6微升/分钟/毫克蛋白质）快得多。相互作用研究表明，染料木黄酮抑制非那西丁生成对乙酰氨基酚，半数抑制浓度值为16微摩尔。进一步研究表明，非那西丁和染料木黄酮相互抑制。总之，CYP1A2和CYP2E1代谢染料木黄酮，且CYP1A2作为其他活性较低的甲基化异黄酮的前药转化酶。
