Abstract
UDP-glucuronosyltransferase (UGT) 1A10 is an isoform of UGT1A, which is expressed in extrahepatic, biliary and aerodigestive/gastrointestinal tissues. We have previously reported two nonsynonymous single nucleotide polymorphisms in exon 1 of humanUGT1A10 gene; 177G>A and 605C>T resulting in amino acid alterations, M59I and T202I, respectively. In the present study, wild-type (WT) and these variant UGT1A10 cDNAs were transiently expressed in COS-1 cells for functional characterization. Glucuronidation activities in these COS-1 membrane fractions were assayed using 7-hydroxy-4-trifluoromethylcoumarin (HTFMC) and 17β-estradiol (E2) as substrates. WT and variant UGT1A10s catalyzed HTFMC glucuronidation with similar apparentKm values of approximately 5 μM, whereas the Vmax value of T202I normalized by the expressed UGT1A10 protein levels was nearly half of those of WT and M59I. High-performance liquid chromatography analysis of E2 glucuronide revealed that UGT1A10 catalyzed E2 3-O-glucuronidation but not 17-O-glucuronidation. Similarly, the three UGT1A10s catalyzed E2 3-O-glucuronidation with comparable apparent Km values (approximately 2 μM), whereas the normalized Vmax value of T202I was almost half that of WT and M59I. These results suggest that the lowered glucuronidation activity of T202I affects the gastrointestinal glucuronidation of orally administrated chemicals and the enterohepatic circulation of biliary excreted metabolites.
Footnotes
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This study was supported by the Program for Promotion of Fundamental Studies in Health Sciences (MPJ-1 and MPJ-6) of the Organization for Pharmaceutical Safety and Research of Japan.
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Hideto Jinno and Mayumi Saeki contributed equally to this article.
- Abbreviations used are::
- UGT
- UDP-glucuronosyltransferase
- HTFMC
- 7-hydroxy-4-trifluoromethylcoumarin
- E2
- 17β-estradiol
- PCR
- polymerase chain reaction
- MEM
- minimal essential medium
- UDPGA
- uridine diphosphoglucuronic acid
- HPLC
- high-performance liquid chromatography
- WT
- wild-type
- ANOVA
- analysis of variance
- Received November 12, 2002.
- Accepted January 21, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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