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Vol. 30, Issue 11, 1250-1256, November 2002
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan (M.N., N.S., T.Y.); and Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., Saitama, Japan (N.O., T.K.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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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'-HPPH
O-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'-HPPH
O-glucuronide by liquid chromatography-tandem mass
spectrometry analysis. The 4'-HPPH
O-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'-HPPH
O-glucuronide. The 4'-HPPH
O-glucuronidation in pooled human liver microsomes was
inhibited by 
-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'-HPPH
O-glucuronidation in 14 human liver microsomes was
28.5-fold (0.023-0.656 nmol/min/mg of protein). The 4'-HPPH O-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'-HPPH
O-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.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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.
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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 UGT2 genes 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'-HPPH
O-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 -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'-HPPH
O-glucuronidation in human liver microsomes was thoroughly
determined to characterize the kinetic properties and interindividual
differences and to identify the UGT isoform(s).
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Materials.
5-(4'-Hydroxyphenyl)-5-phenylhydantoin (4'-HPPH), UDP-glucuronic acid,
alamethicin, -estradiol, emodin, p-nitrophenyl
-glucuronide, and 
-naphthyl -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 -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'-HPPH O-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'-HPPH
O-glucuronide and 4'-HPPH were 23 and 27 min, respectively.
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.
). 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.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 at
m/z 485 corresponding to trimethylated 4'-HPPH
O-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'-HPPH
O-glucuronide, not 4'-HPPH N-glucuronide.
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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.
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Inhibitory Effects of Typical Substrates for UGT Isoforms on
4'-HPPH O-Glucuronidations in Human Liver Microsomes.
The effects of -estradiol (UGT1A1), imipramine (UGT1A4), propofol
(UGT1A9), and emodin (UGT1A8 and UGT1A10) on 4'-HPPH
O-glucuronidation in pooled human liver microsomes were
determined (Fig. 4). The 4'-HPPH
O-glucuronidation in the pooled human liver microsomes was
inhibited by -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.
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Interindividual Variability in 4'-HPPH
O-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 -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).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 is
O-glucuronide, three hydroxy-groups would be methylated. In
contrast, if the formed 4'-HPPH glucuronide is
N-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'-HPPH
O-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'-HPPH O-glucuronide diastereomers.
To identify the UGT isoform(s) involved in 4'-HPPH
O-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 cotinine
N-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 -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'-HPPH
O-glucuronidation in humans. The 4'-HPPH
O-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 -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'-HPPH O-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'-HPPH O-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'-HPPH
O-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'-HPPH O-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.
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Acknowledgments |
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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.
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Footnotes |
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Received June 13, 2002; accepted August 12, 2002.
Address correspondence to: Tsuyoshi Yokoi, Ph.D., Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi 13-1, Kanazawa 920-0934, Japan. E-mail: tyokoi{at}kenroku.kanazawa-u.ac.jp
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Abbreviations |
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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.
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-phenyl-N-t-butylnitrone: implications for bioactivation by prostaglandin synthetase.
Toxicol Appl Pharmacol
97:
192-202[CrossRef][Medline].This article has been cited by other articles:
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Y. Watanabe, M. Nakajima, N. Ohashi, T. Kume, and T. Yokoi Glucuronidation of Etoposide in Human Liver Microsomes Is Specifically Catalyzed by UDP-Glucuronosyltransferase 1A1 Drug Metab. Dispos., May 1, 2003; 31(5): 589 - 595. [Abstract] [Full Text] [PDF]
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