Liu Hong, Qin Zhihui, Thatcher Gregory R J, Bolton Judy L
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, M/C 781, Chicago, Illinois 60612-7231, USA.
Chem Res Toxicol. 2007 Nov;20(11):1676-84. doi: 10.1021/tx7001367. Epub 2007 Jul 13.
Long-term usage of the selective estrogen receptor modulator (SERM) tamoxifen has been associated with an increased risk of endometrial cancer. One potential mechanism of tamoxifen-induced carcinogenesis involves metabolism to reactive intermediates, such as an o-quinone, quinone methide, and carbocations. We have previously shown that the benzothiophene SERMs, raloxifene and desmethylated arzoxifene (DMA), can also be bioactivated to electrophilic quinoids by rat/human liver microsomes and rat hepatocytes [(2006) Chem. Res. Toxicol. 19, 1125-1137]. Because the uterus is a major target tissue of estrogens and antiestrogens, it was of interest to determine if quinoids could be formed from SERMs in uterine tissue potentially producing cytotoxic effects. Incubations with rat uterine microsomes showed that both raloxifene and DMA could be oxidized to electrophilic diquinone methides that were trapped as the corresponding GSH conjugates. A new raloxifene GSH-dependent conjugate was identified as raloxifene Cys-Gly that was formed from the hydrolysis of 7-glutathinyl raloxifene by gamma-glutamyl transpeptidase. Interestingly, the metabolism of raloxifene and DMA in rat uterine microsomes was not NADPH-dependent and could be inhibited by cyanide and NADPH or enhanced by H2O2. In addition, coincubations with the peroxidase substrates guaiacol or o-phenlyenediamine inhibited diquinone methide GSH conjugate formation from both SERMs. Incubations of raloxifene and DMA with horseradish peroxidase (HRP) were studied as models of the interaction between benzothiophene SERMs and peroxidase. The results showed that HRP could directly oxidize raloxifene and DMA to the corresponding dimers via the formation of phenoxyl radicals in the absence of exogenous hydrogen peroxide. In addition, GSH appears to be involved in multiple peroxidase-catalyzed oxidative metabolic pathways of benzothiophene SERMs. Finally, COATag (covert oxidatively activated tag) methodology, which involves the utilization of biotin-conjugated raloxifene and DMA, was used to identify target proteins by affinity chromatography. Incubations of raloxifene and DMA COATags with rat uterine microsomes showed several modified proteins by Western blot analysis. The protein modification could be enhanced by the addition of H2O2 and decreased by the addition of NADPH, suggesting that unlike liver metabolism the formation of quinoids in the uterus could be mediated by uterine peroxidases.
选择性雌激素受体调节剂（SERM）他莫昔芬的长期使用与子宫内膜癌风险增加有关。他莫昔芬诱导致癌的一种潜在机制涉及代谢为反应性中间体，如邻醌、醌甲基化物和碳正离子。我们之前已经表明，苯并噻吩类SERM雷洛昔芬和去甲基阿佐昔芬（DMA）也可被大鼠/人肝微粒体和大鼠肝细胞生物活化成亲电醌类物质[（2006年）《化学研究毒理学》19卷，1125 - 1137页]。由于子宫是雌激素和抗雌激素的主要靶组织，因此确定SERM在子宫组织中是否能形成可能产生细胞毒性作用的醌类物质很有意义。用大鼠子宫微粒体进行孵育表明，雷洛昔芬和DMA均可被氧化为亲电二醌甲基化物，并被捕获为相应的谷胱甘肽（GSH）共轭物。一种新的雷洛昔芬GSH依赖性共轭物被鉴定为雷洛昔芬半胱氨酰甘氨酸，它是由γ-谷氨酰转肽酶水解7-谷胱甘肽基雷洛昔芬形成的。有趣的是，雷洛昔芬和DMA在大鼠子宫微粒体中的代谢不依赖于NADPH，可被氰化物和NADPH抑制，或被H2O2增强。此外，与过氧化物酶底物愈创木酚或邻苯二胺共同孵育可抑制两种SERM形成二醌甲基化物GSH共轭物。研究了雷洛昔芬和DMA与辣根过氧化物酶（HRP）的孵育，作为苯并噻吩类SERM与过氧化物酶相互作用的模型。结果表明，在没有外源过氧化氢的情况下，HRP可通过形成苯氧自由基将雷洛昔芬和DMA直接氧化为相应的二聚体。此外，GSH似乎参与了苯并噻吩类SERM的多种过氧化物酶催化的氧化代谢途径。最后，利用涉及生物素共轭雷洛昔芬和DMA的COATag（隐蔽氧化活化标签）方法，通过亲和色谱法鉴定靶蛋白。用雷洛昔芬和DMA COATag与大鼠子宫微粒体孵育，通过蛋白质印迹分析显示有几种蛋白质被修饰。添加H2O2可增强蛋白质修饰，添加NADPH则降低蛋白质修饰，这表明与肝脏代谢不同，子宫中醌类物质的形成可能由子宫过氧化物酶介导。
