Disposition of flavonoids via enteric recycling: Determination of the UDP-Glucuronosyltransferase Isoforms responsible for the metabolism of flavonoids in intact caco-2 TC7 cells using siRNA
Disposition of flavonoids via enteric recycling: Determination of the UDP-Glucuronosyltransferase Isoforms responsible for the metabolism of flavonoids in intact caco-2 TC7 cells using siRNA
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DOI：
10.1021/mp0601190
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发表时间：
2007-11-01
影响因子：
4.9
通讯作者：
Hu, Ming
中科院分区：
文献类型：
--
作者：
Liu, Xing;Tam, Vincent H.;Hu, Ming
我们最近的研究表明，微粒体葡萄糖醛酸化率不能预测细胞葡萄糖醛酸排泄率，需要整个细胞系统来准确测定代谢率。本研究旨在利用siRNA确定完整Caco-2细胞和细胞裂解物中负责类黄酮代谢的UGT异构体的贡献。结果表明，经过siRNA处理，UGT1A6的活性（通过对硝基苯酚葡萄糖醛酸化测定）和表达通常降低了60-80%。通过siRNA介导的沉默，我们还发现，在完整细胞中，siRNA处理在低浓度和高浓度下显著降低了芹菜素葡糖苷的排泄率（bb0 35%, p < 0.05），尽管在高浓度下仅适度降低了染料木素葡糖苷的排泄率（29%）。结果还表明，在Caco-2细胞中表达良好的UGT亚型UGT1A1、UGT1A3、UGT1A6和UGT2B7能够比染料木素更快地代谢芹菜素，而UGT1A6的沉默并没有显著提高染料木素代谢UGT亚型的表达水平。我们还测定了UGT1A6对芹菜素和染料木素代谢的贡献与浓度的关系，结果表明，芹菜素和染料木素的代谢是饱和的，siRNA处理大大降低了芹菜素的代谢速率，但没有降低染料木素的代谢速率。总之，我们首次证明siRNA可以有效地用于确定完整细胞中哪种UGT异构体有助于其底物的代谢。结果还表明，在完整的Caco-2细胞和细胞裂解物中，UGT1A6是芹菜素葡萄糖醛酸化的主要贡献者，而不是染料木素。
Our recent study indicates that microsomal glucuronidation rates are not predictive of the cellular glucuronide excretion rates and whole cell systems are needed to accurately determine the metabolic rates. This study aims to determine the contribution of UGT isoforms responsible for the metabolism of flavonoids in intact Caco-2 cells and cell lysates using siRNA. The results showed that UGT1A6 activities (as measured by p-nitrophenol glucuronidation) and expression were typically decreased 60-80% by siRNA treatment. Using siRNA-mediated silencing, we also showed that in intact cells, siRNA treatment substantially decreased the rate of excretion of apigenin glucuronide at low and high concentrations (>35%, p < 0.05), although it only moderately decreased the rate of excretion of genistein glucuronide at a high concentration (29%). The results also indicated that well-expressed UGT isoforms in the Caco-2 cells, UGT1A1, UGT1A3, UGT1A6, and UGT2B7, were capable of metabolizing apigenin faster than genistein and that UGT1A6 silencing did not substantially increase the level of expression of genistein-metabolizing UGT isoforms. We also determined the contribution of UGT1A6 to the apigenin and genistein metabolisms as a function of concentration, and the results indicated that metabolism of apigenin and genistein was saturable and siRNA treatment greatly reduced the rate of metabolism of apigenin but not that of genistein. In conclusion, we show for the first time that siRNA can be used effectively to determine which UGT isoform contributes to the metabolism of its substrate in intact cells. The results also indicate that UGT1A6 is a major contributor to glucuronidation of apigenin but not genistein in intact Caco-2 cells and in cell lysates.