Hanioka Nobumitsu, Obika Nobuhiko, Nishimura Masuhiro, Jinno Hideto, Tanaka-Kagawa Toshiko, Saito Keita, Kiryu Kimio, Naito Shinsaku, Narimatsu Shizuo
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan.
Food Chem Toxicol. 2006 Aug;44(8):1251-60. doi: 10.1016/j.fct.2006.01.019. Epub 2006 Mar 20.
UDP-glucuronosyltransferases (UGTs) are conjugation enzymes, which are regulated in a tissue-specific manner by endogenous and environmental factors. In this study, we focused on UGT1A isoforms (UGT1A1, UGT1A6 and UGT1A9), mainly expressed in the human liver, and examined the inducibility of UGT1As by beta-naphthoflavone (BNF) in human hepatoma HepG2 cells. The cells were pretreated for 72 h with BNF at concentrations of 25, 50 and 100 microM. 7-Ethyl-10-hydroxycamptothecin (SN-38) glucuronidation, used as a probe for UGT1A1, showed sigmoidal kinetics with a Hill coefficient (n) of 1.2-1.3 in control and BNF-pretreated HepG2 cells. The Vmax values were significantly increased 3.6- to 4.3-fold by BNF, whereas there was no significant change in the S50 values by BNF at any concentration examined. On the other hand, 4-methylumbelliferone (4-MU) glucuronidation as a probe for UGT1A6 and UGT1A9 in the control and BNF-pretreated HepG2 cells exhibited a biphasic kinetic pattern. Although Km1 values for the low-Km phase were similar between the control and BNF-pretreated HepG2 cells, Km2 values for the high-Km phase of BNF-pretreated HepG2 cells were reduced to 54-69% of control HepG2 cells. The values of Vmax1 and Vmax2 for the low- and high-Km phases, respectively, were significantly increased 1.9- to 2.6-fold by BNF at 25 and/or 50 microM but not 100 microM. With respect to Vmax (Vmax1 and Vmax2) and Vmax/Km (Vmax1/Km1 and Vmax2/Km2), the values were significantly increased 2.0- to 3.2-fold by BNF at all concentrations examined. Furthermore, real-time reverse transcription polymerase chain reaction using TaqMan probes demonstrated that BNF concentration-dependently induced mRNA levels of UGT1A1 but not UGT1A6 or UGT1A9 in HepG2 cells (1.3- to 6.0-fold). These results suggest that the inducibility of UGT1A isoforms in HepG2 cells by BNF is different from other aryl hydrocarbon receptor agonists previously reported, and should provide useful information for the prediction of drug-drug interactions and toxicological assessment of environmental chemicals.
UDP-葡萄糖醛酸基转移酶（UGTs）是结合酶，受内源性和环境因素以组织特异性方式调控。在本研究中，我们聚焦于主要在人肝脏中表达的UGT1A同工型（UGT1A1、UGT1A6和UGT1A9），并检测了β-萘黄酮（BNF）在人肝癌HepG2细胞中对UGT1A的诱导作用。细胞用浓度为25、50和100微摩尔的BNF预处理72小时。用作UGT1A1探针的7-乙基-10-羟基喜树碱（SN-38）葡萄糖醛酸化在对照和BNF预处理的HepG2细胞中呈现S形动力学，希尔系数（n）为1.2 - 1.3。Vmax值被BNF显著提高了3.6至4.3倍，而在所检测的任何浓度下，BNF对S50值均无显著影响。另一方面，在对照和BNF预处理的HepG2细胞中，用作UGT1A6和UGT1A9探针的4-甲基伞形酮（4-MU）葡萄糖醛酸化呈现双相动力学模式。尽管对照和BNF预处理的HepG2细胞中低Km相的Km1值相似，但BNF预处理的HepG2细胞高Km相的Km2值降至对照HepG2细胞的54 - 69%。低Km相和高Km相的Vmax1和Vmax2值分别在25和/或50微摩尔的BNF作用下显著提高了1.9至2.6倍，但在100微摩尔时未提高。就Vmax（Vmax1和Vmax2）和Vmax/Km（Vmax1/Km1和Vmax2/Km2）而言，在所检测的所有浓度下，BNF均使其值显著提高了2.0至3.2倍。此外，使用TaqMan探针的实时逆转录聚合酶链反应表明，BNF在HepG2细胞中浓度依赖性地诱导UGT1A1的mRNA水平，但不诱导UGT1A6或UGT1A9的mRNA水平（1.3至6.0倍）。这些结果表明，BNF对HepG2细胞中UGT1A同工型的诱导作用与先前报道的其他芳基烃受体激动剂不同，应为预测药物相互作用和环境化学物质的毒理学评估提供有用信息。
