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Research ArticleArticle

Involvement of Multiple UDP-glucuronosyltransferase 1A Isoforms in Glucuronidation of 5-(4′-hydroxyphenyl)-5-phenylhydantoin in Human Liver Microsomes

Miki Nakajima, Nozomi Sakata, Noriko Ohashi, Toshiyuki Kume and Tsuyoshi Yokoi
Drug Metabolism and Disposition November 2002, 30 (11) 1250-1256; DOI: https://doi.org/10.1124/dmd.30.11.1250

Abstract

In humans, orally administered phenytoin, 5,5-diphenylhydantoin, is mainly excreted as 5-(4′-hydroxyphenyl)-5-phenylhydantoin (4′-HPPH)O-glucuronide. Phenytoin is oxidized to 4′-HPPH by CYP2C9 and to a minor extent by CYP2C19, and then 4′-HPPH is metabolized to 4′-HPPH O-glucuronide by UDP-glucuronosyltransferase (UGT). In the present study, 4′-HPPHO-glucuronidation in human liver microsomes was investigated. The metabolite formed by incubation with human liver microsomes, 4′-HPPH, and UDP-glucuronic acid was identified as 4′-HPPHO-glucuronide by liquid chromatography-tandem mass spectrometry analysis. The 4′-HPPHO-glucuronosyltransferase activity in human liver microsomes was not saturated at concentrations up to 500 μM of 4′-HPPH. Any commercially available recombinant human UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells did not show detectable 4′-HPPHO-glucuronide. The 4′-HPPHO-glucuronidation in pooled human liver microsomes was inhibited by β-estradiol as a typical substrate for UGT1A1 (IC50 = 21.1 μM) and imipramine as a typical substrate for UGT1A4 (IC50 = 57.7 μM). The inhibitory effects of propofol as a specific substrate for UGT1A9 (IC50 = 167.1 μM) and emodin as a substrate for UGT1A8 and UGT1A10 (IC50 = 287.6 μM) were not prominent. The interindividual difference in the 4′-HPPHO-glucuronidation in 14 human liver microsomes was 28.5-fold (0.023–0.656 nmol/min/mg of protein). The 4′-HPPHO-glucuronosyltransferase activity in 11 human liver microsomes was significantly (r = 0.609,P < 0.05) correlated with the 4-nitrophenol glucuronosyltransferase activity, which is catalyzed by UGT1A6 and UGT1A9. These results suggest that multiple UGT1As such as UGT1A1, UGT1A4, UGT1A6, and UGT1A9 are involved in 4′-HPPHO-glucuronidation in human liver microsomes, although the percentage contribution of each UGT1A could not be estimated. Large interindividual differences in the glucuronidation of 4′-HPPH might be responsible for the nonlinearity of the phenytoin plasma concentration or adverse reactions in humans.

Phenytoin, 5,5-diphenylhydantoin, is widely used as an anticonvulsant drug. It has received much attention concerning its various toxicities, e.g., teratogenicity (Wells et al., 1989), carcinogenicity in animals (Diwan et al., 1993), hepatitis (Haruda, 1997), and autoantidody formation in humans (Leeder et al., 1992). Furthermore, there are many drug-drug interactions associated with phenytoin, including cases in which coadministered drugs modify the pharmacokinetics of phenytoin, and vice versa (Nation et al., 1990a,b). Since the therapeutic range is narrow and its blood concentration in humans has been shown to be nonlinear (Odani et al., 1997), therapeutic drug monitoring is usually recommended.

Phenytoin metabolism in humans has been extensively studied in our laboratory (Komatsu et al., 2000). Four oxidative metabolites of phenytoin, 5-(4′-hydroxyphenyl)-5-phenylhydantoin (4′-HPPH1), 5-(3′-hydroxyphenyl)-5-phenylhydantoin, 5-(3′,4′-dihydroxyphenyl)-5-phenylhydantoin, and 5-(3′,4′-dihydroxy-1′,5′-cyclohexadien-1-yl)-5-phenylhydantoin are reported in humans (Maguire, 1988; Szabo et al., 1990). The formation of 4′-HPPH is a major metabolic pathway. A number of studies suggest that both phenytoin and 4′-HPPH are bioactivated by peroxidase to free radical intermediates, which can oxidize lipids, proteins, and DNA (Winn and Wells, 1995; Parman et al., 1998). Phenytoin also produces hydroxyl radicals in vivo (Kim and Wells, 1996). It has also been reported that 5-(3′,4′-dihydroxyphenyl)-5-phenylhydantoin can be oxidized to semiquinone and quinone derivatives and lead to a mechanism-based inactivation of cytochrome P450s that may be involved in the initiation of drug hypersensitivity reactions (Munns et al., 1997). These reactive metabolic intermediates might be relevant not only to the teratogenicity of phenytoin (Winn and Wells, 1995) but also to the idiosyncratic drug reactions (fever, rash) and reversible lymphoma caused by phenytoin (Porter, 1989), although the underlying mechanisms have yet to be clarified.

The 4′-HPPH is presented in plasma almost entirely as glucuronic acid conjugate and is excreted in the urine (Maynert, 1960) (Fig.1). Some 75% of administered phenytoin appears in urine as the 4′-HPPH O-glucuronide (Maynert, 1960; Glazko et al., 1969). It has been reported that phenytoin could be metabolized to N-glucuronide with a minor extent (Smith et al., 1977). Up to 10% of oral phenytoin is excreted unchanged in the feces of humans and up to 5% is excreted unchanged in urine (Kutt and Louis, 1972). Thus, the majority of administered phenytoin appears in the urine mostly as the 4′-HPPH O-glucuronide. Glucuronidations of endobiotics and xenobiotics are catalyzed by UDP-glucuronosyltransferase (UGT) (Miners and Mackenzie, 1991). UGT-catalyzed glucuronidation and elimination may prevent the competing bioactivation of xenobiotics to toxic reactive intermediates. Therefore, the catalytic potency of the UGT enzymes in individuals may be an important determinant of the susceptibility to various phenytoin and 4′-HPPH toxicities.

Figure 1
Figure 1

Major metabolic pathways of phenytoin in humans.

It is well known that there are many isoforms of mammalian UGT enzymes (Tukey and Strassburg, 2000). To date, three UGT families have been identified in humans: UGT1, UGT2, and UGT8. Of these three families, UGT1 and UGT2 have been shown to catalyze the glucuronidation of xenobiotics in human livers. The UGT1 and UGT2genes appear to be structurally different in that the UGT1 proteins result from alternative splicing of different first exons with five shared exons encoded by the UGT1 gene complex, whereas UGT2 proteins appear to be encoded by unique genes. It has been reported that UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B11, and UGT2B15 are expressed in human livers (Tukey and Strassburg, 2000). However, it is unknown which UGT isoform(s) is responsible for 4′-HPPHO-glucuronidation in human livers. The concentration of 4′-HPPH O-glucuronide in human plasma or urine has been measured as aglycone by HPLC after hydrolysis with β-glucuronidase or acid treatment (Bochner et al., 1973; Vree, 1990). Furthermore, a large interindividual difference in the ratio of the concentrations of 4′-HPPH O-glucuronide and 4′-HPPH in 24 h accumulated urine samples of patients has been reported (1.83–10.75) (Vree, 1990). However, in vitro glucuronidation of 4′-HPPH in human liver microsomes has never studied until now. In the present study, the 4′-HPPHO-glucuronidation in human liver microsomes was thoroughly determined to characterize the kinetic properties and interindividual differences and to identify the UGT isoform(s).

Materials and Methods

Materials.

5-(4′-Hydroxyphenyl)-5-phenylhydantoin (4′-HPPH), UDP-glucuronic acid, alamethicin, β-estradiol, emodin, p-nitrophenyl β-glucuronide, and α-naphthyl β-glucuronide were purchased from Sigma-Aldrich (St. Louis, MO). 4-Nitrophenol and 1-naphthol were purchased from Wako Pure Chemical Industries (Osaka, Japan). Morphine hydrochloride was purchased from Takeda Chemical Industries (Osaka, Japan). Morphine-3-glucuronide was kindly provided by Dr. Kazuta Oguri of Kyushu University (Fukuoka, Japan). Pooled human liver microsomes (H161) and microsomes from 14 individual human livers (H003, H006, H023, H030, H042, H043, H056, H066, H070, H089, H093, H112, HK23, and HK34) were purchased from BD Gentest (Woburn, MA). Glucuronosyltransferase activities of β-estradiol, trifluoperazine, and propofol as typical substrates of UGT1A1, UGT1A4, and UGT1A9, respectively, in these human liver microsomes except for H006, H030, and H070 were provided by the manufacturer. Microsomes from baculovirus-infected insect cells expressing human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 (Supersomes) were from BD Gentest. All other chemicals and solvents were of the highest grade commercially available.

4′-HPPH O-Glucuronidation Assay.

A typical incubation mixture (250 μl of total volume) contained 100 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl2, 3 mM UDP-glucuronic acid, 50 μg/ml alamethicin, 1.0 mg/ml human liver microsomes (0.5 mg/ml for recombinant UGT), and 100 μM 4′-HPPH (25–500 μM for kinetic analysis). The 4′-HPPH was dissolved in CH3OH, and the final concentration of the organic solvent in the incubation mixture was <1%. The reactions were initiated by the addition of UDP-glucuronic acid and the reaction mixtures were incubated for 30 min. The reactions were then terminated by the addition of 250 μl of ice-cold acetonitrile. After removal of the protein by centrifugation at 10,000 rpm for 5 min, a 100-μl portion of the sample was subjected to the HPLC. Chromatography was performed using an LC-6A pump (Shimadzu, Kyoto, Japan), an SPD-6A UV detector (Shimadzu), an SIL-6B autosampler (Shimadzu), a C-R4A integrator (Shimadzu), and a CTO-6A column oven (Shimadzu) with a YMC-Pack ODS-A (6.0 × 300 mm; 5 μm) column (YMC Co., Ltd., Kyoto, Japan). The flow rate was 1.0 ml/min and the column temperature was 35°C. The eluent was monitored at 214 nm with a noise-base clean Uni-3 (Union, Gunma, Japan). The Uni-3 can reduce the noise by integrating the output, increase the signal 3-fold by differentiating the output, and 5-fold by further amplification with an internal amplifier, resulting in a maximum 15-fold amplification of the signal. The mobile phases were 20% CH3CN/0.1% HCOOH. Retention times of 4′-HPPH O-glucuronide and 4′-HPPH were 13 and 38 min, respectively. Assignment of the 4′-HPPHO-glucuronide peak was made by LC-MS/MS analysis described below. We present the activity for 4′-HPPH O-glucurnide formation on the basis of the chromatographic response using 4′-HPPH as a standard.

Identification of 4′-HPPH O-Glucuronide by LC-MS/MS Analysis.

LC-MS/MS analysis was performed using a LCQDeca (Thermoquest, San Jose, CA) under electrospray ionization (ESI) conditions. The operation conditions used were capillary temprature, 350°C; capillary volt, −6 V; tube lens volt, 25 V; ion spray volt, 4.5 V; sheath gas, N2; pressure, 80 psi; auxiliary gas, N2, 20 l/min; collision energy, 50%. Liquid chromatography was performed using an HP1100 (Agilent Technologies Inc., Palo Alto, CA) with a ODS-3 (2 × 150 mm; 3 μm) column (GL Science Inc., Tokyo, Japan). The flow rate was 0.2 ml/min, and the column temperature was 40°C. The mobile phase was 15% CH3CN/0.1% HCOOH. The retention times of 4′-HPPHO-glucuronide and 4′-HPPH were 23 and 27 min, respectively.

For the treatment with trimethylsilylimidazole, 4′-HPPHO-glucuronide was fractionated by HPLC and evaporated under N2 gas at room temperature. The samples were dissolved with 10 μl of CH3CN, and 5 μl of trimethylsilylimidazole was added. The mixtures were incubated for 30 min at 37°C. The resultant derivatives were measured by the method of flow injection and the speculated number of hydroxyl functional groups from the obtained mass spectra.

Other Glucuronidation Assays.

4-Nitrophenol glucuronosyltransferase activity in human liver microsomes was determined as described previously (Hanioka et al., 2001a). Briefly, a typical incubation mixture (200 μl of total volume) contained 50 mM potassium phosphate buffer (pH 7.4), 0.2% Triton N-101, 3 mM UDP-glucuronic acid, 1.0 mg/ml human liver microsomes, and 500 μM 4-nitrophenol. The reaction was initiated by the addition of UDP-glucuronic acid and was then incubated at 37°C for 5 min. The reaction was terminated by boiling at 100°C for 2 min and adding 2.8 ml of 0.2 M glycine buffer (pH 10.4). After removal of the protein by centrifugation at 2,000 rpm for 20 min, a 50-μl portion of the sample was subjected to HPLC. The HPLC instrument was the same as described above. Chromatographic separations were performed on a Mightysil RP-18 (4.6 × 150 mm; 5 μm) column (Kanto Chemical, Tokyo, Japan). The flow rate was 1.2 ml/min, and the column temperature was 35°C. The eluate was monitored at 302 nm by the UV detector. The mobile phase was 5% CH3OH/0.05M KH2PO4 (pH 6.5). The retention times of 4-nitrophenol glucuronide and 4-nitrophenol were 3.0 and 14.5 min, respectively. Formation of the metabolite was quantified by comparing the peak areas in the incubations to a standard curve containing known amounts of the metabolite.

The 1-naphthol glucuronosyltransferase activity in human liver microsomes was determined as described previously (Mackenzie and Hanninen, 1980). Briefly, a typical incubation mixture (200 μl of total volume) contained 50 mM potassium phosphate buffer (pH 7.4), 0.2% Triton N-101, 3 mM UDP-glucuronic acid, 0.5 mg/ml human liver microsomes, and 50 μM 1-naphthol. The reaction was initiated by the addition of UDP-glucuronic acid followed by incubation at 37°C for 2.5 min. The reaction was terminated by boiling for 2 min and adding 1.8 ml of phosphate-buffered saline. After removal of the protein by centrifugation at 2,000 rpm for 20 min, the fluorescence of the supernatant was measured at excitation 290 nm and emission 343 nm by an RF5000 fluorescence detector (Shimadzu). Formation of the metabolite was quantified by comparing the fluorescence in the incubations to a standard curve containing known amounts of the metabolite.

Morphine glucuronosyltransferase activity in human liver microsomes was determined as described previously (Milne et al., 1991). Briefly, a typical incubation mixture (250 μl of total volume) contained 50 mM potassium phosphate buffer (pH 7.4), 0.2% Triton N-101, 3 mM UDP-glucuronic acid, 1.0 mg/ml human liver microsomes, and 1 mM morphine. The reaction was initiated by the addition of UDP-glucuronic acid and was then incubated at 37°C for 30 min. The reaction was terminated by adding 250 μl of ice-cold acetonitrile. After removal of the protein by centrifugation at 2,000 rpm for 20 min, a 50-μl portion of the sample was subjected to HPLC. The HPLC instrument was the same as described above. Chromatographic separations were performed on a Mightysil RP-18 (4.6 × 150 mm; 5 μm) column (Kanto Chemical). The flow rate was 1.2 ml/min, and the column temperature was 35°C. The mobile phase was 20% CH3CN/0.8 mM sodium dodecyl sulfate/0.05 M KH2PO4 (pH 1.9). The eluate was monitored at excitation 210 nm and emission 350 nm by an 821-FPS fluorescence detector (Jasco, Tokyo, Japan). The retention times of morphine 3-glucuronide and morphine were 3.1 and 8.0 min, respectively. Formation of the metabolite was quantified by comparing the peak areas in the incubations to a standard curve containing known amounts of the metabolite.

Correlation Analyses.

Correlation analyses between 4′-HPPH O-glucuronidation and the other glucuronidation activities in microsomes from 11 human livers were determined by Spearman's rank method. A P value of less than 0.05 was considered statistically significant.

Results

4′-HPPH O-Glucuronide Formation in Human Liver Microsomes.

The formation of 4′-HPPH O-glucuronide increased in a microsomal protein concentration- and time-dependent manner. These formations were linear at least at 2.0 mg/ml microsomal protein and 60 min incubation. Unless specified, the standard incubation mixture contained 1.0 mg/ml microsomal protein and was incubated at 37°C for 30 min. None of these chromatograms showed any interfering peaks with 4′-HPPH O-glucuronide.

LC-MS/MS Analyses of 4′-HPPH O-Glucuronide.

The ESI mass spectrum of a peak typically formed by incubation of 4′-HPPH with human liver microsomes and UDP-glucuronic acid is shown in Fig. 2A. [M − H]− ion at m/z 443 corresponding to 4′-HPPH O-glucuronide was observed. The product ion spectrum of the peak showed [M − H]− ions at m/z 175 corresponding to glucuronic acid and [M − H]− ion at m/z 267 corresponding to 4′-HPPH (Fig. 2B). After the treatment with trimethylsilylimidazole, the ESI mass spectrum of the peak showed [M − H]− ion atm/z 485 corresponding to trimethylated 4′-HPPHO-glucuronide (Fig. 2C). From these observations, it was confirmed that the peak formed by the incubation of 4′-HPPH with human liver microsomes and UDP-glucuronic acid was 4′-HPPHO-glucuronide, not 4′-HPPH N-glucuronide.

Figure 2
Figure 2

ESI mass spectrum (A) and product ion spectrum (B) of the peak formed by the incubation of 4′-HPPH with human liver microsomes and UDP-glucuronic acid, and ESI mass spectrum (C) of the peak after the treatment with trimethylsilylimidazole.

A, the [M − H]− ion atm/z 443 corresponds to 4′-HPPHO-glucuronide. B, the fragment pattern of 4′-HPPHO-glucuronide is indicated. The product ion spectrum showed peaks at m/z 175 corresponding to glucuronic acid and at m/z 267 corresponding to 4′-HPPH. C, the [M − H]− ion atm/z 485 corresponds to trimethylated 4′-HPPH O-glucuronide.

4′-HPPH O-Glucuronidations in Recombinant Human UGT Isoforms.

Seven kinds of recombinant UGT isoforms expressed in baculovirus-infected insect cells that are commercially available were used to determine their 4′-HPPH O-glucuronosyltransferase activities. However, no recombinant UGT isoform exhibited detectable 4′-HPPH O-glucuronide formation. For recombinant UGT1A1 and UGT1A6, 4′-HPPH O-glucuronide was not detected, although the protein concentration was increased up to 3.0 mg/ml.

Kinetics of 4′-HPPH O-Glucuronidation in Human Liver Microsomes.

Kinetic analyses of 4′-HPPH O-glucuronidation in human liver microsomes were performed. As shown in Fig.3, the activity was not saturated at concentrations up to 500 μM of substrate, and the kinetics did not fit the Michaelis-Menten plot.

Figure 3
Figure 3

Kinetics of 4′-HPPH O-glucuronidation in human liver microsomes.

The 4′-HPPH O-glucuronosyltransferase activities in pooled human liver microsomes (H161) were determined with 4′-HPPH ranging from 25 to 500 μM. The 4′-HPPHO-glucuronosyltransferase activities were determined as described under Materials and Methods. Each data point represents the mean of duplicate determinations.

Inhibitory Effects of Typical Substrates for UGT Isoforms on 4′-HPPH O-Glucuronidations in Human Liver Microsomes.

The effects of β-estradiol (UGT1A1), imipramine (UGT1A4), propofol (UGT1A9), and emodin (UGT1A8 and UGT1A10) on 4′-HPPHO-glucuronidation in pooled human liver microsomes were determined (Fig. 4). The 4′-HPPHO-glucuronidation in the pooled human liver microsomes was inhibited by β-estradiol (IC50 = 21.1 μM) and imipramine (IC50 = 57.7 μM). The inhibitory effects of propofol (IC50 = 167.1 μM) and emodin (IC50 = 287.6 μM) were not prominent.

Figure 4
Figure 4

Inhibitory effects of typical substrates for UGT1A isoforms on 4′-HPPH O-glucuronosyltransferase activity in human liver microsomes.

The 4′-HPPH O-glucuronosyltransferase activity at 100 μM 4′-HPPH in pooled human liver microsomes (H161) was determined as described under Materials and Methods. β-Estradiol (UGT1A1), imipramine (UGT1A4), propofol (UGT1A9), and emodin (UGT1A8 and UGT1A10) were used as inhibitors. Each data point represents the mean of duplicate determinations. The 4′-HPPHO-glucuronosyltransferase activity in the pooled human liver microsomes in the absence of inhibitor was 0.327 nmol/min/mg of protein.

Interindividual Variability in 4′-HPPHO-Glucuronidation in Human Liver Microsomes and Correlation with Other Glucuronosyltransferase Activities.

The 4′-HPPH O-glucuronosyltransferase activities in microsomes from 14 human livers were determined (Fig.5). The interindividual difference in 4′-HPPH O-glucuronidation was 28.5-fold (0.023–0.656 nmol/min/mg of protein, 0.251 ± 0.201 nmol/min/mg of protein). The 4′-HPPH O-glucuronosyltransferase activities in the 11 human liver microsomes were significantly (r = 0.609,P < 0.05) correlated with the 4-nitrophenol glucuronosyltransferase activities (Fig.6). In contrast, no significant correlation was observed with the β-estradiol glucuronosyltransferase activities (r = 0.046), trifluoperazine glucuronosyltransferase activities (r = 0.155), propofol glucuronosyltransferase activities (r = 0.570), 1-naphthol glucuronosyltransferase activities (r = 0.450), and morphine glucuronosyltransferase activities (r = 0.178).

Figure 5
Figure 5

Interindividual variability in 4′-HPPH O-glucuronidation in human liver microsomes.

The 4′-HPPH O-glucuronosyltransferase activities in liver microsomes from 14 livers were determined. Human liver microsomes (1.0 mg/ml of microsomal protein) were incubated with 100 μM 4′-HPPH and 3 mM UDP-glucuronic acid at 37°C for 30 min. Each column represents the mean of duplicate determinations.

Figure 6
Figure 6

Correlation between 4′-HPPH O-glucuronosyltransferase activity and other glucuronosyltransferase activities in human liver microsomes.

β-Estradiol (UGT1A1), trifluoperazine (UGT1A4), and propofol (UGT1A9) glucuronosyltransferase activities in human liver microsomes except for H006, H030, and H070 were provided by the manufacturer. 4-Nitrophenol (UGT1A6 and UGT1A9), 1-naphthol (UGT1A1, UGT1A6, UGT1A8, and UGT1A9), and morphine (UGT2B7) glucuronosyltransferase activities were determined in the present study as described under Materials and Methods. The 4′-HPPH O-glucuronosyltransferase activity in microsomes from 11 human livers were significantly (r = 0.609, P < 0.05) correlated with the 4-nitrophenol glucuronosyltransferase activity.

Discussion

In humans, orally administered phenytoin is mainly excreted as 4′-HPPH glucuronide (Maynert, 1960; Glazko et al., 1969). This is the first study to investigate the glucuronidation of 4′-HPPH in human liver microsomes. We confirmed that the peak formed by the incubation of 4′-HPPH with human liver microsomes and UDP-glucuronic acid was 4′-HPPH glucuronide by the LC-MS/MS analyses. The 4′-HPPH glucuronide might be N-conjugate or O-conjugate. Trimethylsilylimidazole can methylate a hydroxy-group but not an aliphatic amine. If the formed 4′-HPPH glucuronide isO-glucuronide, three hydroxy-groups would be methylated. In contrast, if the formed 4′-HPPH glucuronide isN-glucuronide, four hydroxy-groups would be methylated. As the result of the treatment with trimethylsilylimidazole, the peak in the mass spectrometry spectrum shifted 42 mass, corresponding to three methylated 4′-HPPH O-glucuronide. Thus, it was confirmed that the formed metabolite is a 4′-HPPH O-glucuronide. The 4′-HPPH is a racemic mixture, and it is thought that 4′-HPPHO-glucuronide is also a racemic mixture (Vree, 1990).Hermansson et al. (1982) reported that the ratio of S-(−)- and R-(+)-4′-HPPH O-glucuronides in urine from phenytoin-treated patients is approximately 20:1. In our chromatographic condition, we could not separate the 4′-HPPHO-glucuronide diastereomers.

To identify the UGT isoform(s) involved in 4′-HPPHO-glucuronidation, the catalytic activity in human recombinant UGT was determined. However, no recombinant UGT isoform showed 4′-HPPH O-glucuronidation. We confirmed that these commercially available recombinant UGTs showed catalytic activities for other substrates such as imipramine (Nakajima et al., 2002), morphine (data not shown), and troglitazone (Watanabe et al., in press). However, the glucuronosyltransferase activities of imipramine and morphine in the recombinant UGTs were lower than those in human liver microsomes. Furthermore, we previously clarified that these recombinant UGTs could not also exhibit the detectable nicotine and cotinineN-glucuronidations (Nakajima et al., in press), although human liver microsomes did show these activities. Therefore, it was suspected that the recombinant UGTs have lower catalytic activities toward most substrates than human liver microsomes do. This fact might be partly due to the differences in the membrane circumstances in the expression system and in human liver microsomes, as it has been reported that the nature of the phopholipid environment influences the rate-limiting step of glucuronidation (Magdalou et al., 1982). Similarly, the absence of 4′-HPPH O-glucuronide formation in the recombinant UGTs might be due to the low capability of the expression systems.

The 4′-HPPH O-glucuronidation in human liver microsomes did not fit the Michaelis-Menten plot. The activity was not saturated at concentrations up to 500 μM 4′-HPPH (Fig. 3). Therefore, the apparent kinetic parameters could not be determined. The plasma concentration of 4′-HPPH in phenytoin-treated patients has been reported to be 5 to 20 μM (Bochner et al., 1973; Vree, 1990). We did not determine the activity at >500 μM 4′-HPPH, since the concentration of 4′-HPPH around enzymes could not reach so high in a clinical situation.

The 4′-HPPH O-glucuronosyltransferase activity in pooled human liver microsomes was strongly inhibited by β-estradiol, which is mainly glucuronidated by UGT1A1 (Senafi et al., 1994). Furthermore, the 4′-HPPH O-glucuronosyltransferase activity was weakly inhibited by imipramine, which is mainly glucuronidated by UGT1A4 (Nakajima et al., 2002). However, the inhibitory effects of propofol, which is mainly glucuronidated by UGT1A9 (Ebner and Burchell, 1993), and emodin, which is mainly glucuronidated by UGT1A8 and UGT1A10 (Cheng et al., 1999), were not prominent. These results suggest that UGT1A1 and UGT1A4 might be responsible for 4′-HPPHO-glucuronidation in humans. The 4′-HPPHO-glucuronosyltransferase activity in the 11 human liver microsomes was significantly correlated only with the 4-nitrophenol glucuronosyltransferase activity, which is catalyzed by UGT1A6 and UGT1A9 (Hanioka et al., 2001b). With four other relatively specific activities of β-estradiol glucuronosyltransferase activity (UGT1A1), trifluoperazine glucuronosyltransferase activity (UGT1A4) (Green and Tephly, 1996), propofol glucuronosyltransferase activity (UGT1A9) (Ebner and Burchell, 1993), morphine glucuronosyltransferase activity (UGT2B7) (Coffman et al., 1997) and nonspecific UGT1A activity of 1-naphthol glucuronosyltransferase activity (UGT1A1, UGT1A6, UGT1A8, and UGT1A9) (Hanioka et al., 2001b), the 4′-HPPHO-glucuronosyltransferase activity did not exhibit a significant correlation. Taking these results into consideration, 4′-HPPH O-glucuronidation in human liver microsomes would be catalyzed by multiple UGT1A isoforms. It has been reported that 4′-HPPHO-glucuronide formation is not detected in Gunn rat in which the UGT1 family is deficient (Kim et al., 1997). Therefore, the UGT isoform(s) responsible for 4′-HPPH O-glucuronidation would have a counterpart in rats and humans (i.e., UGT1A).

We first demonstrated that there is a large interindividual difference in 4′-HPPH glucuronidation in humans in vitro (28.5-fold). The results are consistent with a previous report of a large interindividual difference in the ratio of concentrations of 4′-HPPHO-glucuronide and 4′-HPPH in 24-h accumulated urine samples (Vree, 1990). It is known that there are genetic polymorphisms in UGT1A1 and UGT1A6 (Tukey and Strassburg, 2001). A mutation in UGT1A4 has also been found, although its clinical significance is unknown (Burchell et al., 1994). Therefore, the genetic polymorphisms in UGT1A isoforms might be a cause of the interindividual differences in 4′-HPPHO-glucuronidation in humans. UGT1A1 has been reported to be induced by phenobarbital, phenytoin, oltipraz, and 3-methylcholanthrene (Fisher et al., 2001). Furthermore, it has also been reported that the UGT1A6 and UGT1A9 are inducible by polycyclic aromatic hydrocarbons (Bock et al., 1999). Although limited information of smoking and medication history on the donors for the human liver microsomes are available, we could not find the relationship between the history and the large interindividual difference in 4′-HPPH glucuronidation.

It has been reported that 4′-HPPH is a mechanism-based inactivator of cytochrome P450 (Munns et al., 1997). The patients with low UGT activity would have higher plasma concentration of 4′-HPPH, resulting in an increased phenytoin plasma concentration. Alternatively, with regard to toxicity due to the formation of free radicals and reactive oxygen species, 4′-HPPH itself is as potent as phenytoin in causing macromolecular damage in both cell and embryo culture (Kim et al., 1997), in which case low UGT activity might enhance particularly the teratogenicity and idiosyncratic adverse reactions independent of any change in the concentration of phenytoin.

In conclusion, the 4′-HPPH O-glucuronidation in human liver microsomes appears to be catalyzed by multiple UGT1As such as UGT1A1, UGT1A4, UGT1A6, and UGT1A9. Unfortunately, the contributions of each UGT isoform to 4′-HPPH O-glucuronidation in human liver microsomes could not be directly estimated. The large interindividual variability of 4′-HPPH glucuronidation might be responsible for the nonlinearity of the phenytoin plasma concentrations or adverse reactions in humans.

Acknowledgments

The authors thank Dr. Kazuta Oguri of Kyushu University (Fukuoka, Japan) for providing morphine 3-glucuronide. We also acknowledge Brent Bell for reviewing the manuscript.

Footnotes

  • Abbreviations used are::
    4′-HPPH
    5-(4′-hydroxyphenyl)-5-phenylhydantoin
    UGT
    UDP-glucuronosyltransferase
    HPLC
    high performance liquid chromatography
    LC-MS/MS
    liquid chromatography-tandem mass spectrometry
    ESI
    electrospray ionization
    • Received June 13, 2002.
    • Accepted August 12, 2002.

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Involvement of Multiple UDP-glucuronosyltransferase 1A Isoforms in Glucuronidation of 5-(4′-hydroxyphenyl)-5-phenylhydantoin in Human Liver Microsomes

Miki Nakajima, Nozomi Sakata, Noriko Ohashi, Toshiyuki Kume and Tsuyoshi Yokoi
Drug Metabolism and Disposition November 1, 2002, 30 (11) 1250-1256; DOI: https://doi.org/10.1124/dmd.30.11.1250
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