Zhang Yan, Gaikwad Nilesh W, Olson Kevin, Zahid Muhammad, Cavalieri Ercole L, Rogan Eleanor G
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA.
Metabolism. 2007 Jul;56(7):887-94. doi: 10.1016/j.metabol.2007.03.001.
Accumulating evidence suggests that specific metabolites of estrogens, namely, catechol estrogen quinones, react with DNA to form adducts and generate apurinic sites, which can lead to the mutations that induce breast cancer. Oxidation of estradiol (E(2)) produces 2 catechol estrogens, 4-hydroxyestradiol (4-OHE(2)) and 2-OHE(2) among the major metabolites. These, in turn, are oxidized to the quinones, E(2)-3,4-quinone (E(2)-3,4-Q) and E(2)-2,3-Q, which can react with DNA. Oxidation of E(2) to 2-OHE(2) is mainly catalyzed by cytochrome P450 (CYP) 1A1, and CYP3A4, whereas oxidation of E(2) to 4-OHE(2) in extrahepatic tissues is mainly catalyzed by CYP1B1 as well as some CYP3As. The potential involvement of CYP isoforms in the further oxidation of catechols to semiquinones and quinones has, however, not been investigated in detail. In this project, to identify the potential function of various CYPs in oxidizing catechol estrogens to quinones, we used different recombinant human CYP isoforms, namely, CYP1A1, CYP1B1, and CYP3A4, with the scope of oxidizing the catechol estrogens 2-OHE(2) and 4-OHE(2) to their respective estrogen quinones, which then reacted with DNA. The depurinating adducts 2-OHE(2)-6-N3Ade, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua were observed in the respective reaction systems by ultraperformance liquid chromatography/tandem mass spectrometry. Furthermore, more than 100-fold higher levels of estrogen-glutathione (GSH) conjugates were detected in the reactions. Glutathione conjugates were observed, in much smaller amounts, when control microsomes were used. Depurinating adducts, as well as GSH conjugates, were obtained when E(2)-3,4-Q was incubated with CYP1B1 or control microsomes in a 30-minute reaction, further demonstrating that GSH is present in these recombinant enzyme preparations. These experiments demonstrated that CYP1A1, CYP1B1, and CYP3A4 are able to oxidize catechol estrogens to their respective quinones, which can further react with GSH, protein, and DNA, the last resulting in depurinating adducts that can lead to mutagenesis.
越来越多的证据表明，雌激素的特定代谢产物，即儿茶酚雌激素醌，会与DNA反应形成加合物并产生脱嘌呤位点，这可能导致诱发乳腺癌的突变。雌二醇（E(2)）氧化产生2种主要代谢产物，即4-羟基雌二醇（4-OHE(2)）和2-羟基雌二醇（2-OHE(2)）。这些代谢产物进而被氧化为醌，即E(2)-3,4-醌（E(2)-3,4-Q）和E(2)-2,3-醌，它们可与DNA发生反应。E(2)氧化为2-OHE(2)主要由细胞色素P450（CYP）1A1和CYP3A4催化，而在肝外组织中E(2)氧化为4-OHE(2)主要由CYP1B1以及一些CYP3A催化。然而，CYP同工型在儿茶酚进一步氧化为半醌和醌过程中的潜在作用尚未得到详细研究。在本项目中，为了确定各种CYP在将儿茶酚雌激素氧化为醌中的潜在功能，我们使用了不同的重组人CYP同工型，即CYP1A1、CYP1B1和CYP3A4，目的是将儿茶酚雌激素2-OHE(2)和4-OHE(2)氧化为各自的雌激素醌，然后它们与DNA发生反应。通过超高效液相色谱/串联质谱在各自反应体系中观察到了脱嘌呤加合物2-OHE(2)-6-N3Ade、4-OHE(2)-1-N3Ade和4-OHE(2)-1-N7Gua。此外，在反应中检测到雌激素-谷胱甘肽（GSH）结合物水平比对照高100多倍。当使用对照微粒体时，观察到的GSH结合物量要少得多。当E(2)-3,4-Q与CYP1B1或对照微粒体在30分钟反应中孵育时，获得了脱嘌呤加合物以及GSH结合物，这进一步证明这些重组酶制剂中存在GSH。这些实验表明，CYP1A1、CYP1B1和CYP3A4能够将儿茶酚雌激素氧化为各自的醌，这些醌可进一步与GSH、蛋白质和DNA反应，最后导致产生可导致诱变的脱嘌呤加合物。
