| Budget Amount *help |
¥3,950,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2007: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2006: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
Since glucuronidation is an important metabolic reaction for xenobiotic elimination, information on the induction or suppression of UDP-glucuronosyltransferase (UGT) enzymes at the isoform level is beneficial in drug therapy and the toxicological assessment of environmental chemicals. 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 β-naphthoflavone (BNF) in human hepatoma HepG2 cells. The cells were pretreated for 72 h with BNF at concentrations of 25, 50 and 100 μM. 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 V_<max> values were significantly increased 3.6- to 4.3-fold by BNF, whereas there was no significant change in the S_<50> values by BNF at any concentration examined. On the other hand, 4-methylumbelliferone (4-MU) glucuronida
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tion as a probe for UGT1A6 and UGT1A9 in the control and BNF-pretreated HepG2 cells exhibited a biphasic kinetic pattern Although K_<ml> values for the low-K_m phase were similar between the control and BNF-pretreated HepG2 cells, K_<m2> values for the high-K_m phase of BNF-pretreated liepG2 cells were reduced to 54-69% of control HepG2 cells. The values of V_<max> and V_<max2> for the low- and high-K_m phases, respectively, were significantly increased 1.9- to 2.6-fold by BNF at 25 and/or 50μM but not 100 μM With respect to V_<max> (V_<max1> and V_<max2>) and V_<max>/K_m (V_<max1>/K_<m1> and V_<max2>/K_<m2>), 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 (RT-PCR) using TaqMan probes demonstrated that BNF concentration-dependently induced mRNA levels of UGTIA1 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 (AhR) agonists previously reported, and should provide useful information for the prediction of drug-drug interactions and toxicological assessment of environmental chemicals. Less
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