Introduction

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UDP-glucuronosyltrasferases (UGTs¹) play important roles in the metabolism of xenobiotics and endogenous compounds. UGTs have been classified into two families, UGT1 and UGT2, the latter being subdivided into the UGT2A and UGT2B subfamilies (Mackenzie et al., 1997). So far, 17 human functional UGT genes including seven UGT2B genes have been identified.

Remarkable interindividual differences in UGT activities have been reported (Burchell et al., 2000; Mackenzie et al., 2000). Most interindividual variations in the activities of drug-metabolizing enzymes such as cytochrome P450 and N-acetyltransferase have been explained by genetic polymorphisms of the genes (Weber, 1997). Genetic polymorphisms of UGT have also been reported to account for interindividual differences of the activity of this enzyme. In particular, defects in the UGT1A1 gene have been reported to be responsible for the absent or impaired glucuronidation of bilirubin in the Crigler-Najjar and Gilbert syndromes (Mackenzie et al., 1997).

UGT2B7 is one of the most important UGT2B isozymes expressed in the human liver (Tukey and Strassburg, 2000) and is known to catalyze the glucuronidation of S-oxazepam (Patel et al., 1995) and 3'-azido-3'-deoxythymidine (Barbier et al., 2000). The genetic polymorphisms of the UGT2B7 gene in a coding region arise from the substitution of tyrosine for histidine at residue 268 (Jin et al., 1993), whereas both UGT2B7(Y268) and UGT2B7(H268) have not accounted for the interindividual differences because of the lack of differences in the activities of the mutants (Coffman et al., 1998). Although remarkable interindividual differences in the glucuronidation activities of S-oxazepam and 3'-azido-3'-deoxythymidine have been presented (Pacifici et al., 1996; Furlan et al., 1999), the molecular mechanism(s) involved in the interindividual differences has not been fully understood.

The expression level of mRNA is altered by the genetic polymorphisms of the 5'-flanking region and the amounts of transcription factors that cause interindividual differences. In fact, we recently reported that the amounts of HNFs presented in human livers determined the expression level of a dihydrodiol dehydrogenase mRNA (T. Ozeki, Y. Takahashi, K. Nakayama, M. Funayama, K. Nagashima, T. Kodama, and T. Kamataki, submitted for publication).

In this paper, the expression levels of UGT2B7 mRNA in 12 human livers were quantified. We found that the expression level of HNF-1, which regulates the UGT2B7 gene, rather than a mutation(s) of genes of the UGT2B7 gene, was a causal factor determining the interindividual variation of UGT2B7.

	Materials and Methods

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Human Liver Samples. Liver samples used in this study were described previously (Pearce et al., 1996). This study was approved by the ethics committee of Hokkaido University.

Preparation of Total RNA and Genomic DNA. Total RNA and genomic DNA were prepared from 12 human livers using Trizol (Invitrogen, Carlsbad, CA) or DNA Extraction kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions.

Reverse Transcriptase Reaction. The RT reaction was performed according to the method of Carninci et al. (2000) with minor modifications. Briefly, 50 µl of a reaction mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 0.7 M trehalose, 0.4 mM each dNTP, 10 µg of total RNA, 1 µg of oligo(dT)₁₅ primer (Promega, Madison, WI), and 400 units of Maloney murine leukemia virus reverse transcriptase (Promega) was used. The RNA and the primer mixture were initially heated at 70°C for 1 min before addition of the other reagents. The temperature-cycle reactions were performed in an MJ Research (Waltham, MA) thermal cycler as follows: after annealing of the sample at 35°C for 2 min and extension at 45°C for 5 min, the samples were incubated at 60°C for 2 min followed by 55°C for 2 min, for 10 cycles.

Quantitative PCR. The quantitative PCR was performed in an ABI-Prism 7700 (Applied Biosystems, Foster City, CA) thermal cycler using a SYBR Green PCR core reagent kit (Applied Biosystems) according to the manufacturer's instructions. Primers for the amplification of the HNF-1 were HNF1RT-Fw, 5'-TACACCTGGTACGTCCGCAA-3' and HNF1RT-Rv, 5'-CACTTGAAACGGTTCCTCCG-3'. Primers used to amplify UGT2B7 or -actin were 2B7RT-Fw, 5'-AGACTTGCTGAATGCATTGAAGAG-3'; 2B7RT-Rv, 5'-GGCTTCACTGGTTGATCATGTT-3'; actinRT-Fw, 5'-ATTGCCGACAGGATGCAGA-3' and actinRT-Rv, 5'-GCTCAGGAGGAGCAATGATCTT-3'. PCR amplifications were performed 40 cycles with melting for 15 s at 95°C, annealing and extension for 1 min at 58, 68, and 65°C for -actin, HNF-1 and UGT2B7, respectively, after preheating for 10 min at 95°C.

	Results and Discussion

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To quantify the expression levels of UGT2B7 mRNA in human livers, a quantitative RT-PCR was performed. Sense and antisense primers were designed in exon 5 and in exon 6, respectively. As shown in Fig. 1A, the amounts of UGT2B7 mRNA in total RNA prepared from human livers ranged from 0.22 to 2.63 copies/10³ copies of -actin, indicating that there was a remarkable interindividual difference in the expression levels of UGT2B7 mRNA. These results suggest that the interindividual variation in the activities of UGT2B7 occurs at a transcription level.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   A, the expression levels of UGT2B7 mRNA; B, relationships between UGT2B7 genotype and expression level of UGT2B7 mRNA. These data represent relative to the amount of -actin mRNA. For each subject, two sets of quantitative RT-PCR reaction were performed. The bars (in B) show the positions of the means for each genotype.

The proximal promoter of the UGT2B7 gene locates up to 275 base pairs (Ishii et al., 2000). To examine the possibility of whether the interindividual variation of the expression of the UGT2B7 mRNA was caused by a mutation(s) in a 5'-upstream region of the gene, the 5'-franking sequences of the UGT2B7 gene from 12 human livers were analyzed. One base substitution (253G to A) was found in seven subjects (three hetero- and four homozygotes) as a new mutation. The relationship between the UGT2B7 gene genotype and the expression level of UGT2B7 mRNA is shown in Fig. 1B. The average amounts of the mRNA in subjects with the G/G (wild type), G/A (heterozygote), and A/A (mutant type) genotypes were 1.27 ± 0.97, 0.90 ± 0.43, 1.41 ± 1.09 copies/10³ copies of -actin, respectively, suggesting no apparent relationship between the genotypes and the expression level of UGT2B7 mRNA.

It is postulated that the expression level of UGT2B7 mRNA might be affected by the amounts of a transcription factor(s). Thus, the binding of a regulatory factor(s) to a promoter region of the UGT2B7 gene was then investigated. The UGT2B7 gene has a promoter region containing a consensus sequence of HNF-1 adjacent to the transcription start site (Radominska-Pandya et al., 2001). HNF-1 has been shown to bind to the UGT2B7 gene promoter to activate (Ishii et al., 2000). In this study, HNF-1 mRNA was expressed at levels ranging from 2.99 to 24.76 copies/10⁶ copies of -actin in these subjects. To show a relationship between the amounts of HNF-1 and the expression levels of UGT2B7, linear regression analysis was performed by the method of least-squares. Figure 2 shows the correlation of the amounts of HNF-1 mRNA with UGT2B7 mRNA. The amounts of HNF-1 mRNA clearly correlated with the expression levels of UGT2B7 mRNA (r = 0.786, p = 0.002). In contrast, the amounts of HNF-1 mRNA did not correlate with the expression levels of UGT2B15 mRNA (data not shown). These data indicate that UGT2B7 mRNA is expressed depending on the amount of HNF-1 mRNA. Consequently, it is strongly suggested that the interindividual variation in the amount of HNF-1 mRNA would be a factor causing interindividual differences in the activity of UGT2B7 enzyme in human livers.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Relationship between the level of HNF-1 mRNA and UGT2B7 mRNA; r = 0.786, p = 0.002.  These data represent relative to the amount of -actin mRNA. For each subject, two sets of quantitative RT-PCR reaction were performed. Significant correlation was observed between the level of HNF-1 and UGT2B7 mRNA (p = 0.002).

HNF-1 has been reported to be a member of the nuclear receptor superfamily that regulates the mouse and human UGT1A1 gene (Bernard et al., 1999), the mouse UGT1A6 gene (Vasiliou et al., 1997), the rat UGT1A7 gene (Metz et al., 2000), the rat UGT2B1 gene (Hansen et al., 1997), and the human UGT2B17 gene (Gregory et al., 2000). Since the reported 5'-flanking sequence of the UGT2B15 gene does not contain a putative HNF-1 binding site (Turgeon et al., 2000), it is reasonable that the amount of HNF-1 mRNA does not affect the expression levels of UGT2B15 mRNA. In fact, no relationship was observed between the amounts of HNF-1 mRNA and the expression levels of UGT2B15 mRNA in our study (data not shown).

The UGT1A1 isoform is likely to be the major one involved in bilirubin glucuronidation in humans (Bosma et al., 1994). The inactivation of the HNF-1 gene has been reported to cause a moderate hyperbilirubinemia, via an altered UGT1A1 expression (Pontoglio et al., 1996). Taken together with our data, it is possible that the subjects expressing a low amount of HNF-1 mRNA in the liver show higher plasma concentration of bilirubin and lower urinary elimination of bilirubin glucuronide.

In conclusion, in this paper we demonstrated that the interindividual variation in the UGT2B7 enzyme activity occurred by the variation in the amounts of HNF-1 mRNA.