Abstract
To clarify the UDP-glucuronosyltransferase (UGT) isoform(s) responsible for the glucuronidation of the thyroid hormone thyroxine (T4) in the human liver, the T4 glucuronidation activities of recombinant human UGT isoforms and microsomes from seven individual human livers were comparatively examined. Among the 12 recombinant human UGT1A and UGT2B subfamily enzymes examined, UGT1A1, UGT1A3, UGT1A9, and UGT1A10 showed definite activities for T4 glucuronidation. These UGT1A enzymes, with the exception of UGT1A10, were detected in all of the human liver microsomes examined. Interindividual differences in T4 glucuronidation activity were observed among the microsomes from the seven individual human livers, and the T4 glucuronidation activity was closely correlated with β-estradiol 3-glucuronidation activity. Furthermore, Spearman correlation analysis for a relationship between the T4 glucuronidation activity and the level of UGT1A1, UGT1A3, and UGT1A9 in the microsomes revealed that levels of UGT1A1 and UGT1A3, but not that of UGT1A9, were closely correlated with T4 glucuronidation activity. T4 glucuronidation activity in human liver microsomes was strongly inhibited by 26,26,26,27,27,27-hexafluoro-1α,23(S),25-trihydroxyvitamin D3 (an inhibitor of UGT1A3), moderately inhibited by either bilirubin (an inhibitor of UGT1A1) or β-estradiol (an inhibitor of UGT1A1 and UGT1A9), but not inhibited by propofol (an inhibitor of UGT1A9). These findings indicated strongly that glucuronidation of T4 in the human liver was mediated by UGT1A subfamily enzymes, especially UGT1Al and UGT1A3, and further suggested that the interindividual differences would come from differences in the expression levels of UGT1A1 and UGT1A3 in individual human livers.
Thyroid hormone, a thyroxine (T4), is metabolized via deiodination, O-glucuronidation, O-sulfation, ether bond cleavage, and/or oxidative deamination (Visser, 1996; Wu et al., 2005). Among these metabolisms, O-glucuronidation is important, because it is responsible for the metabolism of many endogenous and exogenous chemicals (Radominska-Pandya et al., 1999; Iyanagi, 2007). Visser (1996) had first reported that T4 glucuronidation was mediated by UDP-glucuronosyltransferase (UGT) 1A subfamily enzymes, in particular UGT1A1 and UGT1A6, in the rat liver. On the other hand, it had been reported that T4 glucuronidation in the human liver was mediated mainly by UGT1A1 and UGT1A9 (Visser et al., 1993; Findlay et al., 2000). Quite recently, Yamanaka et al. (2007) reported that the T4 glucuronidation activity in human liver is catalyzed mainly by UGT1A1.
However, these previous studies on the contribution of the UGT subfamily enzymes responsible for T4 glucuronidation in rats and humans were performed using only limited samples and/or techniques. The UGT isoform(s) for the T4 metabolism has not been clearly determined. In addition, the UGT genes are divided into two families, UGT1 and UGT2, based on a homology of the amino acid sequence (Mackenzie et al., 2005).
In the present study, to further clarify the human UGT isoform(s) responsible for T4 glucuronidation, we examined comparatively the activities of the 12 recombinant human UGTs, including UGT1 and UGT2 family enzymes, and further determined a relationship between T4 glucuronidation activity and levels of UGT subfamily enzymes in microsomes from seven individual human livers.
Materials and Methods
Materials. UDP-glucuronic acid, alamethicin, and propofol were purchased from Sigma-Aldrich (St. Louis, MO). 26,26,26,27,27,27-Hexafluoro-1α,23(S), 25-trihydroxyvitamin D3 (ST232), a probe substrate for UGT1A3 (Kasai et al., 2005), was kindly donated by Sumitomo Pharmaceuticals (Osaka, Japan) and used as a selective inhibitor of UGT1A3. Its chemical structure is shown in Fig. 1. [125I]T4 (116 Ci/mmol), radiolabeled with 125I at the 5′ position of the outer ring, was obtained from PerkinElmer Life and Analytical Sciences (Boston, MA).
Human liver microsomes from seven individual human livers (HH2, HH13, HH47, HG3, HG32, HG64, and HG74), including data on UGT activities toward β-estradiol, trifluoperazine, and propofol, were purchased from BD Gentest (Woburn, MA). The recombinant human UGT isoforms, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17, which are expressed in insect cells (Supersomes) infected with the corresponding UGT gene-inserted baculovirus, were purchased from BD Gentest. Likewise, the microsomes from the insect cells infected with wild-type baculovirus (without a UGT gene) were purchased and used as a control (without UGTs).
Preparation of Anti-Human UGT Antibody. The peptide KWLPQNDLLGHPKA deduced from a UGT2B15 cDNA sequence (356–369) (Green et al., 1994) was used as a UGT antigen and immunized to rabbits as described previously (Ikushiro et al., 1995). The established antibody, which is cross-reactive with all of the UGT1A and UGT2B subfamily isoforms examined, was designated as an anti-human UGT.
Immunoblot Analysis. The amount of each UGT isoform expressed in insect cells was determined by Western blotting with an anti-human UGT. To obtain a clear band on the immunoblot, a portion (20 μl) of the microsomal fraction containing human UGT(s) was treated with endoglycosidase H (200 units) for 60 min at 37°C, and the reaction was terminated by heating at 98°C for 5 min. The endoglycosidase H-treated microsomal fraction was subjected to SDS-polyacrylamide gel electrophoresis (PAGE). SDS-PAGE was performed using a 4% stacking and a 10% separating gel. After SDS-PAGE, the separated proteins on a gel were transferred to a nitrocellulose sheet by a semidry blotting method. The proteins bound to an anti-human UGT on a sheet were detected using chemical luminescence (ECL detection kit; GE Healthcare, Piscataway, NJ). Amounts of UGT1A and UGT2B proteins in the microsomes were estimated using maltose binding protein (MBP)-UGT1AC and MBP-UGT2B7C fusion proteins (New England Biolabs, Ipswich, MA), respectively, as standards. In addition, amounts of the fusion proteins were determined by a bicinchoninic acid protein assay.
Levels of UGT isoforms in human liver microsomes were measured by Western blotting with either anti-h1AC (anti-human UGT1A antibody) or isoform-specific antibodies (anti-h1A1, anti-h1A3, anti-h1A9, and anti-2B7), as described previously (Ikushiro et al., 2006). In addition, a portion (20 or 40 μg of protein/lane) of the microsomal preparation was treated with endoglycosidase H and then used for the Western blot analysis. Levels of UGT isoforms in individual human microsomes were shown as a ratio to that of the respective isoforms expressed in a human sample HG3.
Glucuronidation Assay. Microsomal T4 glucuronidation activity was determined according to the method of Barter and Klaassen (1992). Briefly, the microsomal preparation (2.0 mg of protein) with each human recombinant UGT or human liver microsomal preparation (2.0 mg of protein) was added to the reaction medium (final volume of 1 ml) containing 0.1 mg of alamethicin, 13.2 mM MgCl2, 66 mM Tris-HCl (pH 7.4), 1.26 mM saccharic acid-1,4-lactone, and 4 mM UDP-glucuronic acid. The reaction was initiated by the addition of 90 μMT4 solution containing [125I]T4 (40–65 μCi/μmol), performed at 37°C for 4 h, and terminated by the addition of ice-cold ethanol (500 μl). After centrifugation of the reaction mixture, the resultant supernatant was used for the assay. An aliquot (50 μl) of a supernatant fraction was applied to an LK6DF silica gel-coated thin layer chromatography plate (Whatman, Clifton, NJ) and then developed with the solution containing ethyl acetate, methyl ethyl ketone, formic acid, and water (50:30:10:10). After separation by thin layer chromatography, the entire plate was scraped in 5-mm fractions, and radioactivity on each fraction was measured by gamma scintillation spectrometry (PerkinElmer Life and Analytical Sciences).
Effects of Several UGT Inhibitors on Microsomal T4 Glucuronidation. Bilirubin, ST232, and propofol were used as typical inhibitors of UGT1A1 (Senafi et al., 1994; King et al., 1996), UGT1A3 (Kasai et al., 2005), and UGT1A1/UGT1A9 (Burchell et al., 1995), respectively. A human liver microsomal preparation (HH13), which contained a lot of UGT1A1, UGT1A3, and UGT1A9, was selected as an enzyme source, and inhibitory effects of bilirubin, ST232, and propofol on the microsomal activity for T4 glucuronidation were examined by the method described under Glucuronidation Assay.
Correlation Analysis. Correlation analyses between the glucuronidation activity toward T4 or other substrates and the level of UGT1A isoforms in microsomes from seven individual human livers were performed by linear regression.
Results
Expression Levels of the UGT1A and UGT2B Isoforms in the Insect Cell-Transfected Human UGT Genes. The quantitative immunoblot analyses with the anti-UGT cross-reactive with microsomal UGT1A and UGT2B isoforms were performed using the corresponding MBP-UGT fusion proteins as standards. As judged by Western blot analysis, amounts of microsomal UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 in the insect cells expressing the corresponding human recombinant UGT isoforms were 1.6, 0.9, 1.2, 1.4, 1.3, 1.1, 1.1, 1.2, 0.8, 0.6, 0.6, and 0.8 nmol/mg of protein, respectively.
T4 Glucuronidation by Recombinant Human UGT Isoforms. T4 glucuronidation activities of 12 human UGTs, including 8 UGT1A subfamily and 4 UGT2B subfamily isoforms, were examined. All of the UGT isoforms examined showed definite activities for T4 glucuronidation (Fig. 2). As judged by glucuronidation activity [v/E0 (per minute)], UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT1A10, and UGT2B7 showed higher capacities than the other UGT isoforms examined. In addition, UGT1A8, an extrahepatic enzyme (Cheng et al., 1998; Gregory et al., 2004), showed the highest activity.
Interindividual Variation of T4 Glucuronidation Activity in Human Livers. In general, microsomal glucuronidation activity is increased by perturbation of microsomal membranes (Guéraud and Paris, 1998). Therefore, the effect of alamethicin, a pore-forming oligopeptide, on the T4 glucuronidation activity of human liver microsomes (HH13) was first examined. As shown in Table 1, the addition of alamethicin (0.04–0.20 mg/ml reaction mixture) resulted in an increase in the glucuronidation activity of the human microsomes. Therefore, in the present experiment, the T4 glucuronidation activity was measured in the assay system with alamethicin (0.1 mg/ml reaction mixture).
Microsomal fractions were independently prepared from seven individual human livers, HH2, HH13, HH47, HG3, HG32, HG64, and HG74, and their T4 glucuronidation activities were comparatively examined. Although all of the human liver microsomes examined showed definite activities for T4 glucuronidation, there are interindividual differences (Fig. 3). The microsomes from HH13, HH64, and HH74 had the highest activities among the samples examined.
UGT Activities of Human Liver Microsomes Toward T4, β-Es-tradiol, Trifluoperazine, and Propofol. A correlation between T4-UGT activity and the UGT activities of human liver microsomes toward other typical UGT substrates, such as β-estradiol, trifluoperazine, and propofol was examined. For this purpose, the data on the human UGT activities toward β-estradiol, trifluoperazine, and propofol provided by BD Gentest were used. As shown in Fig. 4, T4-UGT activity was closely correlated with the activity of β-estradiol 3-glucuronidation (r = 0.729, P < 0.05), whereas it showed no correlation with the UGT activities for trifluoperazine and propofol.
Western Blot Analysis of UGT1A Subfamily Enzymes in Human Liver Microsomes. Western blot analyses with anti-h1AC and several anti-UGT1A isoform-specific antibodies were performed to determine the UGT isoforms expressed in human liver microsomes. The anti-h1AC could cross-react with the human UGT1A isoforms, whereas anti-h1A1, anti-h1A3, anti-h1A9, and anti-2B7 antibodies are specifically reactive with UGT1A1, UGT1A3, UGT1A9, and UGT2B7, respectively (Ikushiro et al., 2006).
The amount of total UGT1A isoforms (UGT1As) detected with an anti-h1AC was greatest in the human liver sample HH47 among the seven individual human samples (Fig. 5). On the other hand, the amounts of UGT1A1 determined with anti-UGT1A1 in the samples were in the following order: HH13 > (HG74, HH2, HH47, HG64) > HH32, HG3. For UGT1A3, the order was HG64 > (HG74 > HH13, HG32, HH47) > HH2, HG3. In addition, amounts of UGT1A9 and UGT2B7 were not significantly different among the human samples examined.
UGT Isoforms Responsible for T4 Glucuronidation in Human Liver Microsomes. The relative expression levels of UGT1A, UGT1A1, UGT1A3, and UGT1A9 in individual human liver microsomes were determined, and a relationship between the level of each UGT isoform and T4-UGT activity was examined. T4-UGT activity was closely correlated with the level of either UGT1A1 (r = 0.738, P < 0.05) or UGT1A3 (r = 0.804, P < 0.01) (Fig. 6). On the other hand, no correlation between the T4-UGT activity and the level of UGT1A9 or total UGT1As was observed.
Inhibition of T4-UGT Activity by UGT Inhibitors in Human Liver Microsomes. Effects on microsomal T4-UGT activity of the UGT inhibitors with different specificities were examined using human liver sample HH13, which had definite levels of UGT1A1, UGT1A3, and UGT1A9. The inhibitors, with the exception of propofol, significantly decreased T4-UGT activity (Fig. 7). In particular, addition of ST232 (0.4 mM) to the reaction mixture resulted in a 75% decrease in T4-UGT activity. In contrast, propofol (0.4 mM) showed little inhibitory effect on the microsomal T4-UGT activity.
Discussion
In the present experiments, T4-UGT activities of human UGT isoforms, such as UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 were comparatively examined. The results revealed that all of the UGTs examined showed definite T4 glucuronidation activities. Among the UGTs examined, UGT1A8 showed the strongest activity. However, UGT1A8 was not detected in human liver microsomes, as reported previously (Cheng et al., 1998; Gregory et al., 2004). Among the UGT isoforms detected in the human liver microsomes, recombinant UGT1A1, UGT1A3, and UGT1A9 enzymes had considerable T4-UGT activities. Furthermore, microsomal T4 glucuronidation activity in the human liver was closely correlated with the activity of β-estradiol 3-glucuronidation activity, which is mediated by either UGT1A1 or UGT1A9 (Senafi et al., 1994), whereas it showed no correlation with the glucuronidation activities toward trifluoperazine, a typical substrate of UGT1A4 (Dehal et al., 2001; Court, 2005), and propofol, a typical substrate of UGT1A9 (Burchell et al., 1995). In addition to these results, T4 glucuronidation activity in human liver microsomes was significantly inhibited by either bilirubin, an inhibitor of UGT1A1 (Senafi et al., 1994; King et al., 1996), or ST232, an inhibitor of UGT1A3 (Kasai et al., 2005), but not by propofol, an inhibitor of UGT1A9 (Burchell et al., 1995), indicating strongly that UGT1A1 and UGT1A3 enzymes, but not UGT1A9, mainly mediated the T4 glucuronidation in human microsomes. All of these findings indicate that T4-UGT activity in the human liver is dependent mainly on the levels of UGT1A1 and UGT1A3 and further suggest that the interindividual differences among humans in T4 glucuronidation activity would come from differences in the levels of the UGT isoforms.
Although UGT1A1 had been reported to be an important enzyme for hepatic T4 glucuronidation in humans (Visser et al., 1993; Findlay et al., 2000; Yamanaka et al., 2007), the present findings further confirm this report. On the other hand, the importance of UGT1A3 for hepatic T4 glucuronidation in humans was first demonstrated in the present experiments, whereas Yamanaka et al. (2007) had reported that UGT1A3 would contribute little to T4 glucuronidation. Thus, differences concerning the significance of UGT1A3 for T4 glucuronidation would come from differences in the experimental methods rather than from those in human samples. Yamanaka et al. (2007) came to their conclusion on the basis of their results showing that microsomal activity for chenodeoxycholic acid 24-O-glucuronidation, which is thought to be catalyzed by UGT1A3 (Trottier et al., 2006), was not correlated with the T4 glucuronidation activity and was not inhibited by imipramine, an inhibitor of UGT1A3 and UGT1A4 (Nakajima et al., 2002). However, the results obtained only by enzyme assays would not be enough to establish the conclusion that UGT1A3 would not contribute to T4 glucuronidation. In the present experiments, the importance of UGT1A3 for T4 glucuronidation was more directly demonstrated not only by the inhibition assays with a selective inhibitor (ST232) of UGT1A3 but also by the correlation analysis between the microsomal T4 glucuronidation activity and level of UGT1A3 in microsomes. In addition, the band detected by Western blotting with anti-h1A3 antibody, which might show a cross-reactivity with UGT1A5, was judged to be a UGT1A3 protein, because UGT1A3 mRNA, but not UGT1A5 mRNA, is detected in human liver (Tukey and Strassburg, 2000).
UGT1A8 and UGT1A10 are also reported to be T4 glucuronidation enzymes (Visser et al., 1993; Findlay et al., 2000). However, these UGT isoforms would hardly contribute to hepatic T4 glucuronidation, because they are expressed in the intestine but little in the liver (Cheng et al., 1998; Gregory et al., 2004). In addition, UGT1A8 and UGT1A10 are considered to contribute to intestinal T4 glucuronidation (Yamanaka et al. (2007).
In conclusion, we demonstrate herein that UGT1Al and UGT1A3 are important enzymes for hepatic T4 glucuronidation in humans and further suggest that interindividual differences in hepatic T4 glucuronidation activity would come from differences in the levels of UGT1A1 and UGT1A3.
Footnotes
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This work was supported in part by Grant-in-Aid for Scientific Research (C) 18510061 (Y.K.) and Grant-in-Aid for Scientific Research (B) 19310042 (Y.K.) from the Japan Society for the Promotion of Science and by Health and Labour Sciences Research Grants for Research on Risk of Chemical Substances from the Ministry of Health, Labour and Welfare of Japan.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.018184.
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ABBREVIATIONS: T4, thyroxine; UGT, UDP-glucuronosyltransferase; ST232, 26,26,26,27,27,27-hexafluoro-1α,23(S),25-trihydroxyvitamin D3; PAGE, polyacrylamide gel electrophoresis; MBP, maltose binding protein.
- Received August 9, 2007.
- Accepted September 27, 2007.
- The American Society for Pharmacology and Experimental Therapeutics