![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 30, Issue 2, 199-207, February 2002
Unité Mixte Recherche 7561 Centre National de la Recherche Scientifique-Université Henri Poincaré Nancy (L.A., J.-P.G., M.H.-P., M.O., S.F.-G., J.M.), Vandoeuvre-lès-Nancy, France; University of Helsinki, Department of Pharmacy (J.T.) and Finnish Institute of Occupational Health (E.E.), Helsinki, Finland; and Department of Molecular and Cellular Pathology (B.B., B.E.), Ninewells Hospital and Medical School, University of Dundee, Dundee, United Kingdom
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Catechols are a class of substances from natural or synthetic origin that contain a 1,2-dihydroxybenzene group. We have characterized the glucuronidation by rat liver microsomes and by the rat liver recombinant UDP-glucuronosyltransferase isoforms UGT1A6 and UGT2B1 of a series of 42 structurally diverse catechols, including neurotransmitters, polyphenols, drugs, and catechol estrogens. Small catechols (4-nitrocatechol, 2,3-dihydroxybenzaldehyde, 4-methylcatechol, and tetrachlorocatechol), tyrphostine A23, and octylgallate were glucuronidated at the highest rate by rat liver microsomes and the recombinant enzymes. By contrast, polyphenols from green tea (catechin and related compounds), 3,5-dinitrocatechol, the catechol-O-methyltransferase inhibitor drugs (entacapone, nitecapone, and tolcapone), the carboxyl catechols (gallic acid and dihydroxybenzoic acid derivatives), and the neurotransmitters and dopaminergic drugs, except dobutamine, were glucuronidated at low rate. Glucuronidation of most catechols was increased upon treatment of rats by 3-methylcholanthrene (3-MC) or Aroclor 1254. No induction was observed after administration of phenobarbital and clofibrate or treatment with catechols. Partial least-squares modeling was carried out to explain the variations of glucuronidation activity by liver microsomes of nontreated and 3-MC-treated rats. The model developed explained 82% and predicted 61% of the variations of glucuronidation activities. Among the 17 electronic and substructure parameters used that characterize the catechols, the hydrophobicity/molar volume ratio of catechols showed a strong positive correlation with the glucuronidation rate. The effect of the pKa of the catechol group was modeled to be nonlinear, the optimal pKa value for glucuronidation being between 8 and 9. Hydrogen bonding and steric effects also were important to account for to predict the glucuronidation rates.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Catechols
are a large group of compounds from natural or synthetic origin that
all contain the common 1,2-dihydroxybenzene (pyrocatechol) ring. Many
of the chemicals in the catechol class have antioxidant activity and
are able to prevent auto-oxidation via inhibition of radical formation
(Alanko et al., 1999
). Catechols exhibit a wide variety of
physiological and pharmacological properties. Several endogenous
catechols participate in normal functions of the cell, such as the
neurotransmitters, dopamine, and catecholamines. On the other
hand, although catechol estrogens have been reported to induce
carcinogenesis in rodents through the formation of DNA adducts (Wang
and Liehr, 1995), natural catechols extracted from plants (catechins
and other polyphenols from green tea) have been found to have
anticancer properties (Colic and Pavelic, 2000).
Upon administration, catechols undergo two main metabolic pathways, oxidation and conjugation. Oxidation of catechols generally leads to electrophilic quinone metabolites able to covalently bind proteins or DNA. In contrast, the conjugation reactions, sulfation, methylation, and glucuronidation, catalyzed by sulfotransferases, catechol-O-methyltransferases (COMTs2), and UGTs, respectively, are effective detoxication mechanisms and allow the excretion of the conjugates into bile and urine. Competition between conjugation reactions for the same catechol is dependent on the kinetic properties of the corresponding enzymes, their subcellular localization with regard to the lipophilicity of the substrate, and on several factors that affect the expression level of the enzymes, such as induction by food additives and environmental compounds or the physiopathological state. Therefore, it is important to determine the factors, especially the structural features, which guide the catechols toward different metabolic pathways and govern their biotransformation.
This work was focused on the glucuronidation pathway. UGTs are a
multigenic family of membrane-bound enzymes that catalyze the binding
of glucuronic acid from UDP-glucuronic acid on structurally unrelated
substances with an hydroxyl, carboxyl, amine, or thiol group
(Radominska-Pandya et al., 1999). At least 100 different cDNA encoding
distinct isoforms have been isolated in mammals, including humans
(Mackenzie et al., 1997). The expression of the cDNA coding for these
recombinant proteins in heterologous cells clearly indicated that
although each isoform exhibits a preference for a chemical class of
substrate, they all present an overlapping substrate specificity
(Burchell et al., 1995). We successfully stably expressed cDNA encoding
the UGT1A6 and UGT2B1 in V79 fibroblasts (Pritchard et al., 1994; Pless
et al., 1999). UGT1A6 exhibits strict substrate specificity toward
short and planar phenols, such as 1-naphthol or 4-methylumbelliferone.
UGT2B1 efficiently conjugates bulky phenols, alcohols, morphine, and
carboxylic acids, such as the nonsteroidal inflammatory drugs related
to 2-phenylpropionic acid (profens) and fatty acids.
The glucuronidation of catechols has not yet been well characterized with regard to their potential pharmacological and toxicological properties. The aim of this study is to investigate the structural features of catechols that govern their glucuronidation. For that purpose, we measured the glucuronidation rate of up to 42 catechols (small catechols, neurotransmitters, natural polyphenols, catechol estrogens, and catechol drugs) by rat liver microsomes. PLS modeling was undertaken to point out the electronic and structural features that are critical for glucuronidation. Moreover, the UGT isoforms involved in that process were investigated using the recombinant rat liver UGT isoforms UGT1A6 and UGT2B1 that have been stably expressed in V79 fibroblasts and with the aid of standard inducers, 3-MC, phenobarbital, or clofibrate known to stimulate the expression of distinct classes of UGTs.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Chemicals. Uridine 5'-diphosphate-glucuronic acid, sodium salt, and UDP-[U-14C]glucuronic acid (350 mCi/mmol) were purchased from Boehringer (Mannheim, Germany) and Isotopchim (Ganagobie-Peyruis, France), respectively. Adrenaline, apomorphine, 2,3-dihydroxybenzoic acid, 2-hydroxyestrone, 4-hydroxyestradiol, dobutamine, catechin, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, 4-methylcatechol, 4-tert-butyl-5-metoxycatechol, 4-nitrocatechol, 3,4-dihydroxymandelic acid, 5-hydroxydopamine, 6-hydroxydopa, carbidopa, 2,3-dihydroxybenzaldehyde, noradrenaline, and gallic acid were obtained from Sigma (Saint Quentin Fallavier, France). 4-tert-Butylcatechol, 3-fluorocatechol, tetrachlorocatechol, and methylgallate were purchased from Aldrich (Saint Quentin Fallavier, France).
Catechol, protocatechuic acid (3,4-dihydroxybenzoic acid), pyrogallol, L-dopa, dopamine, 6-hydroxydopamine, 3,4-dihydroxyphenyl acetic acid, and octylgallate were purchased from Fluka (Saint Quentin Fallavier, France). Tyrphostine A23, ethyl-3,4-dihydroxybenzylidenecyanoacetate, dihydrexidine [(±)-trans-10,11-dihydroxy-5,6,6
,7,8,12
-hexahydroxybenz[a]phenanthridine], 2-hydroxyestradiol, and
2-(1-thienyl)-ethyl-3,4-dihydroxybenzylidenecyanoacetate were obtained
from ICN (Orsay, France). The 3-nitrocatechol inhibitors of COMT,
entacapone, tolcapone, and nitecapone were kindly provided by Orion
Pharma (Espoo, Finland). 3,5-Dinitrocatechol was obtained from Tocris
Cookson (Bristol, UK). The chemical structure of the catechols is shown
in Table 1.
|
Enzyme Sources.
Male Wistar rats (Domaine des Oncins, St. Germain l'Arbresle, France)
were housed in an environmentally controlled room (a 12-h light cycle,
22-24°C) and fed rodent chow (UAR Alimentation, Villemoisson,
France). For induction studies, 3-MC, dissolved in corn oil, was
injected once intraperitoneally (100 mg/kg body weight; Sigma).
Phenobarbital, dissolved in 0.9% (w/v) NaCl (Fluka) was administered
once intraperitoneally (100 mg/kg body weight) and then was given with
drinking water (1 g/l) for 4 days. Animals were killed on the fifth
day. Clofibrate (0.3%, w/w; Sigma) was added to the rodent chow and
given to rats for 7 days. As previously reported by Haumont et al.
(1990), the doses of 3-MC, phenobarbital, and clofibrate were chosen
for selectively inducing the UGT isoforms that glucuronidate planar
phenols, bulky phenols, and bilirubin, respectively. In these
conditions, glucuronidation of 3'-azido-3'-deoxythymidine was
enhanced 4 times by phenobarbital, glucuronidation of 1-naphthol was
enhanced 6-fold by 3-MC, and glucuronidation of bilirubin was enhanced
2-fold by clofibrate. Aroclor 1254 (Foxboro, North Haven, CT),
2,3-dihydroxynaphthalene, tolcapone, and entacapone were dissolved in
olive oil and administered at the doses of 550, 300, 200, and 200 mg/kg
body weight, respectively. Aroclor 1254 was injected once
intraperitoneally. 4-Methylcatechol and 4-nitrocatechol were dissolved
in a sucrose solution and administered at two doses, 100 and 150 mg/kg
body weight and 100 and 200 mg/kg body weight, respectively. All
catechols were given for 3 days by oral gavage. Liver microsomes from
three individual rats treated by the inducers and from control rats
were prepared by differential centrifugations, according to Hogeboom
(1955), and stored at 
80°C in 5 mM Hepes buffer, pH 7.4, containing
0.25 M sucrose.
; Pritchard et al., 1994). The genetically engineered V79
cells expressing UGT2B1 and 1A6 were grown on Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) with 5% and 8% (v/v) Nu-serum (Tebu, Le Perray en Yvelines, France), respectively, and
containing 0.1 mg/ml streptomycin and 100 U/ml penicillin. One day
after replating, the cells were treated with 2 mM butyrate. Sodium
butyrate has been shown to increase the gene expression of recombinant
proteins in heterologous systems (Palermo et al., 1991). According to
our experiments, addition of sodium 2 mM butyrate in V79 cell cultures
enhanced the expression of recombinant UGTs 2.5 times, as previously
discussed (Fournel-Gigleux et al., 1991). Then, they were washed with
phosphate-buffered saline, scraped from the plates, and broken in 5 mM
Hepes buffer, pH 7.4, and 0.25 M sucrose by a 3 × 5-s sonication.
Microsomes were prepared by differential centrifugations. The
homogenate was centrifuged for 10 min at 10,000g. The
corresponding supernatant was centrifuged for 60 min at
100,000g on a RCM 120 GX micro-ultracentrifuge (Sorvall, Les
Ulis, France). The activity of the recombinant enzymes was tested with
marker substrates, 1-naphthol and 4-methylumbelliferone for UGT1A6
(Bock and White, 1974; Lilienblum et al., 1982) and morphine and
ketoprofen (Sigma) for UGT2B1 (Terrier et al., 1999), before their use
toward catechols. The protein concentration of liver microsomes and
cellular membranes was evaluated by the method of Bradford (1976), with
the Bio-Rad protein assay kit (Hercules, CA).
Screening of Glucuronidation Activity.
The thin-layer chromatography method of Bansal and Gessner (1980) was
used to measure the in vitro glucuronidation of catechols. The
catechols (0.5 mM) dissolved in dimethyl sulfoxide (2 µl) were added
to an incubation mixture containing 40 µg of microsomal proteins,
UDP-glucuronic acid (2.5 mM, 0.1 µCi), and 100 mM Tris-HCl buffer, pH
7.4, and 5 mM MgCl2 (final volume, 40 µl). A
control was simultaneously run without catechol. When rat liver
microsomes were used, they were previously treated by Triton X-100
(Sigma) at the final detergent/protein concentration of 0.4 (w/w) for 30 min on ice to obtain maximal enzyme activation.
Incubations were performed for 30 min at 37°C, and the reactions were
stopped by addition of 40 µl of ice-cold ethanol. The precipitated
proteins were discarded by centrifugation with a tabletop centrifuge
(5000g, 10 min), and the supernatant was placed onto silica
gel Si250 F-PA thin-layer chromatography plates (Whatman, Clifton, NJ). The products were separated with a mobile phase composed of
n-butanol/acetone/acetic acid/30% aqueous ammoniac/water
(70:50:18:1.5:60, v/v). After migration, the plates were dried and
exposed to X-Omat or BioMax films (Eastman Kodak, Rochester, NY; Sigma)
for 12 to 48 h. The films were developed, and the areas from the
spots corresponding to the radioactive glucuronides were scraped off
with the aid of a drop of water and put into 5 ml of Ultima Gold
scintillant cocktail (Packard Instrument Co., Meriden, CT). A similar
amount of silicagel from the control experiment was also scraped. The amount of radioactivity associated was measured on a LKB spectrometer (1214 Rack Beta; PerkinElmer Wallac, Turku, Finland). Activity values were expressed as nanomoles of glucuronides formed per minute
and milligram of protein after substraction of the value found in controls.
-glucuronidase (bovine liver; Sigma), as
previously reported (Pless et al., 1999). Data are the means ± S.D. (n = 3 rats; three determinations per rat and
catechol). Results were considered significant when p < 0.05 (Student's t test).
Kinetics of Rat Recombinant UGT.
Apparent kinetic constants Km and
Vmax toward catechols were determined
at the optimal pH, protein concentration, and reaction time yielding
linear product formation. Briefly, all assays mixtures (100 µl of
total volume) contained 100 mM Tris-maleate buffer, pH 7.4, 5 mM
MgCl2, 2 mM UDP-glucuronic acid with 0.1 µCi
radiolabeled UDP-glucuronic acid, 1 mM D-saccharic
acid 1,4-lactone, 100 µg of proteins, and increasing concentrations
of catechol (50 to 2500 µM) dissolved in a same volume (5 µl) of
dimethyl sulfoxide. After a 40-min incubation, the reaction was
terminated by the addition of 100 µl of prechilled methanol. The
precipitated proteins were removed by centrifugation at
5000g for 10 min. The resulting supernatant was directly
injected onto the high-performance liquid chromatography (HPLC) column
according to the method of Ethell et al. (1998). Briefly, the HPLC
system consisted of a binary acetonitrile gradient, 0 to 100% in 0.05 mM ammonium acetate, developed over 15 min on a 25-cm × 4.6-mm
i.d. Spherisorb 5ODS2 column (HPLC Technology, Macclesfield, UK) at a
flow rate of 1 ml/min. The radioactive UDP-glucuronic acid and
glucuronide were detected with a model 9701 radioactivity monitor
(Reeve Analytical, Glasgow, UK). Values were expressed as nanomoles of
glucuronide formed per minute and milligram of protein. The apparent
kinetic constants were calculated from nonlinear least-squares
regression analysis of the double-reciprocal plots of initial activity
versus substrate concentration.
PLS Modeling.
PLS analysis was made using the soft independent modeling by class
analogy Simca-P 8.1 program (Umetrics AB, Umeå, Sweden). Two
glucuronidation activities measured with liver microsomes of nontreated
and 3-MC-treated rats were used as y-variables after transformation to logarithmic scale. The detection limit 0.01 was used
instead of a zero activity value. Several physicochemical parameters
and substructure descriptors were used as x-variables. The
octanol-water partition coefficient, log P, was calculated using KOWWIN
v.1.62 program (Syracuse Research Institute, North Syracuse, NY). The
experimental log P value was used instead of the calculated one, when
reported by the program. The molar volume (Vm) and
pKa values for the most acidic
catecholic hydroxyl group were calculated using the ACD/log D 4.0 program (Advanced Chemistry Development, Inc., Richmond, Toronto,
Canada). For the COMT inhibitors (entacapone, tolcapone, and
nitecapone), experimental pKa values reported by Wikberg et al. (1993) were used.
(carboxylate group), HD (hydrogen
bond donor OH or NH other than N+). Depending on
the distance from the catechol ring, substituent atoms were classified
as proximal (p) or distal (d). In flexible side chains, the atoms one
or two bonds away from the catechol ring were considered proximal. In
addition, the substituent in the ortho position to one of the catechol
hydroxyls was specified in a separate class (o). In case of polycyclic
structures, all the atoms of the ring fused to the catechol ring were
considered proximal. Altogether the following 20 substructure
descriptors were considered: Spo, Upo, N+po,
Opo, HDpo, Sdo, Udo,
N+do, Odo, HDdo, Sp, Up,
N+p, Op, HDp, Sd, Ud,
N+d, Od, HDd. The
descriptor Opo showed nonzero value only for
one compound, and therefore it was combined with
Op. The descriptors N+po,
N+do, Odo and HDdo showed
zero value for all compounds and were therefore removed. Consequently,
14 substructure descriptors were used in the PLS analysis.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Glucuronidation of Catechols in Rat Liver Microsomes.
The glucuronidation activity measured upon incubation of rat liver
microsomes with the series of 42 catechols has been determined. In each
case, the catechol glucuronides formed were identified on the
thin-layer chromatography plate from 1) the associated radioactivity
when catechols were incubated with microsomes and UDP-[U-14C]glucuronic acid, 2) their absence in
control reactions in which UDP-glucuronic acid was omitted, and 3)
their susceptibility to hydrolysis by -glucuronidase. No attempt was
made to determine the position of the glucuronide on the
1,2-dihydroxybenzene ring.
|
Effect of Treatment with Inducers on the Glucuronidation of Catechols. Rats were administered with the inducing agents 3-MC, phenobarbital, or clofibrate, which are known to stimulate the expression of distinct classes of UGTs. The results reported in Figs. 1 and 2 and Table 2 clearly show that 3-MC only could markedly enhance the glucuronidation of catechols of different chemical structure (small catechols, catechins, cyanoacetate derivatives, and catechol estrogens). The increase in activity was particularly high (10×) for 3,5-dinitrocatechol, epicatechin, and epicatechin gallate. On the other hand, administration of the peroxisome proliferator clofibrate exhibited a moderate inducing effect (2×) on a limited number of catechols (octylgallate, catechins, and apomorphine). By contrast, phenobarbital failed to stimulate the glucuronidation of catechols, whatever the type of catechol (Fig. 1 and 2). Aroclor 1254 treatment caused an increase in the glucuronidation rate of catechols that was similar to that measured with 3-MC (results not shown). Moreover, no significant induction was observed when rats were treated by catechols, whatever the dose used (4-methylcatechol, 4-nitrocatechol, 2,3-dihydroxynaphthalene, entacapone, and tolcapone) (results not shown). This experiment indicates that the glucuronidation of catechols is catalyzed by UGT isoforms that are mainly sensitive to the inducing effect of polycyclic arylhydrocarbons (3-MC and Aroclor 1254). No autoinduction by catechols was observed.
|
|
Partial Least-Squares Model.
PLS analysis was carried out to model the variation of glucuronidation
activity based on the structural variation of the series of catechols
considered. Glucuronidation activities determined with liver microsomes
of noninduced and 3-MC-induced rats were used as y-variables
(Table 2). The x-variable block consisted of three
physicochemical descriptors (log P,
pKa,
Vm) (Table 2) and 14 substructure
descriptors. PLS modeling of the whole set of data from the 42 catechols resulted in a three-component cross-validated model
explaining 69% of the response variation. For two compounds, 6-hydroxydopa and 3,4-dihydroxyacetic acid, the activity predicted by
the PLS model deviated more than 1 log unit from the measured value.
After excluding these two compounds, a three-component model was
obtained explaining 82% (R2 = 0.82;
goodness of fit) and predicting 61%
(Q2 = 0.61; goodness of prediction) of
the response variation. The experimental versus the predicted
activities are shown in Fig. 3. Linear
relationships were obtained using the regression coefficients indicated
in Table 3. Individual
R2 and
Q2 values showed that the
glucuronidation activities with nontreated rats were modeled and
predicted better (R2 = 0.88;
Q2 = 0.70) than those obtained upon
3-MC induction (R2 = 0.75;
Q2 = 0.52; Fig. 3). The
glucuronidation activities in control and 3-MC-treated rats could be
calculated by inserting the coefficients of Table 2 in the following
regression equations:
|
(1) |
|
|
(2) |
|
p (indicator for the carboxyl group on the
catechol ring) and log P/Vm had a
strong influence (scattered at the greatest distance from the plot
origin) in all three dimensions of the model and were positively and
negatively correlated with the glucuronidation activities,
respectively. The pKa value of
catechol influenced mostly the second component of the model. The two
responses (log RLM, log 3-MC) were mainly separated in the third
dimension of the model, which was influenced by hydrogen bond donors in
ortho or distal positions (Fig. 4B).
|
|
|
Glucuronidation of Catechols by the Recombinant UGT1A6 and UGT2B1.
We previously reported that UGT1A6 exhibited a strict substrate
specificity toward short phenols (Harding et al., 1988), whereas UGT2B1
was very active in glucuronidating both phenol and carboxyl substances,
thus representing a vast panel of structures. These isoforms were
tested for their ability to glucuronidate catechols. Our results
indicated that only small catechols were glucuronidated by the two
isoforms to an appreciable extent (Table 2). The other catechols
tested, particularly the carboxyl catechols and the neurotransmitters,
were not substrates of these isoforms, except dobutamine for UGT2B1.
The apparent kinetic constants Km and
Vmax for the glucuronidation of small
catechols by the UGT1A6 and UGT2B1 were calculated to further predict
which isoform is likely to be involved in the glucuronidation in vivo
(Table 4). For the selected catechols,
the catalytic efficiency of UGT1A6 was higher than that of UGT2B1.
Catechol itself was glucuronidated by UGT1A6. Tetrachlorocatechol was a
good substrate for both isoforms. 4-Methylcatechol was preferentially
glucuronidated by UGT1A6, whereas octylgallate and dobutamine were
substrates of UGT2B1 only (Table 4). For dobutamine, two glucuronides
could be separated by the HPLC method. The apparent kinetic constants
show that one glucuronide was more efficiently formed than the other
(dobutamine 2; Table 4). Due to the limited number of catechols that
were substrates of the recombinant enzymes used in this study and the
low activity measured for some of them, no PLS model could be developed
on the series.
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The metabolism of catechols has not yet been well characterized, although they are widely represented in the living organisms and environment, and that some of them present potential valuable therapeutic properties. In this study, we investigated the potency of rat liver UGTs to glucuronidate a large variety of catechols from endogenous and exogenous origins. Liver microsomes that contain all expressed UGT isoforms were used as a first approach to evaluate their relative rates of conjugation in vitro. Furthermore, an attempt to determine the isoforms involved in that process was performed with the aid of recombinant UGT1A6 and 2B1 stably expressed into V79 cells and induction studies of the animals with standard inducers.
The glucuronidation rates of catechols by rat liver microsomes and recombinant UGT1A6 and 2B1 varied considerably according to the different structural classes and also within each class. To predict the glucuronidation activity of rat liver microsomes toward catechols, a PLS model was developed taking into account 17 different parameters that characterize the physicochemical properties of the molecules. The PLS modeling could not be attempted for activities measured with the recombinant UGT1A6 and 2B1; this was due to a strict substrate specificity of these isoforms toward small catechols and to low activities measured for other many catechols.
The modeling of the glucuronidation activity with rat liver microsomes
resulted in a three-component cross-validated model explaining 69% of
the response variation. When two compounds (6-hydroxydopa and
3,4-dihydroxyacetic acid) were excluded from the series, the model was
markedly improved, explaining 82% of the variations. Three
physicochemical parameters seemed to influence glucuronidation of
catechols. The data were best modeled using the ratio log
P/Vm for the effect of lipophilicity
and size and including the square term of
pKa for the effect of the ionization
of the catechol group. An appropriate series of selected substrate sets
would be required to confirm the nonlinearity of the effects. However,
both effects may be also related to the structure and function of UGTs.
Lipophilicity/size effect may reflect mainly the transport of
substrates to the active site, whereas the
pKa effect is more likely related to
the catalytic mechanism or electrostatic interactions close to the
reaction center. UGTs are integral endoplasmic reticulum glycoproteins with the substrate binding site on the luminal side of the membrane. Hydrophobicity is responsible for the accumulation of the catechols within the hydrophobic phospholipid bilayer of the endoplasmic reticulum. N-Terminal domains of UGTs that strongly interact with membrane phospholipids have been proposed to favor access of the hydrophobic substrates, such as tetrachlorocatechol, to the active site
(Ouzzine et al., 1999). By contrast, the hydrophilic carboxyl catechols
and some catechins or neurotransmitters cannot easily cross the
hydrophobic barrier to be glucuronidated.
The model also suggests a parabolic relationship between
pKa and glucuronidation activity, with
a maximum between 8 and 9. Partially ionized catechols were among the
substrates glucuronidated at the highest rates, whereas completely
ionized catechol (entacapone, tolcapone, nitecapone, and
3,5-dinitrocatechol) were glucuronidated to a lower extent. This result
can be related to the catalytic reaction mechanism accounting for the
glucuronidation of phenols. Recent results obtained in our laboratory
strongly suggest a general base (histidine or aspartic/glutamic acid
residues) for activation of the nucleophilic acceptor species by
deprotonation of the phenolic substrate according to a
SN2 mechanism (Ouzzine et al., 2000). This
process promotes its transfer to glucuronic acid, leading to the
release of UDP. A mechanistic explanation for the nonlinear pKa effect may be that an optimum of
the electron-withdrawing substituent effect may balance between the
stabilization of the transition-state charge and the decrease of the
nucleophilicity of the attacking oxygen.
On the catechol ring, the most acidic hydroxyl group is vicinal to the
nitro group (NO2 in ortho position). In this
condition, we have found that glucuronidation preferentially occurred
on the other less acidic hydroxyl group (Luukkanen et al., 1999). Another explanation may be that the nitro group or the ionized neighboring hydroxyl group hinders optimal binding. However, the effect
seems to be isoform-dependent. High glucuronidation activity was found
for 3,5-dinitrocatechol after 3-MC induction. This suggests that the
inducible UGT isoforms could accept both the nitro group and the
ionized catechol group. Human UGT1A9 has been reported to glucuronidate
entacapone at a high activity, and human UGT1A1 has been found to
glucuronidate the two hydroxyl groups of entacapone, although at a low
activity (Lautala et al., 2000).
Some of the catechols have other functional groups, such as alcohol, phenol, and carboxyl groups, that can be potentially acceptors for glucuronic acid instead of the catechol moiety. Most of these compounds, however, presented a very low glucuronidation rate. Therefore, this possible multisite glucuronidation does not interfere too much with the interpretation of the results. Two exceptions were observed for 2-hydroxyestradiol and dobutamine, which were glucuronidated at a high rate. However, glucuronidation activities with 2-hydroxyestradiol and 2-hydroxyestrone were similar, thus suggesting that glucuronidation takes place mainly on the catechol group. In the case of dobutamine, liquid chromatography/mass spectrometry analysis indicated that the catechol moiety of the molecule was more glucuronidated than the phenol end (H. Keski-Hynnilä, M. Kurkela, E. Elovaara, L. Antonio, J. Magdalou, L. Luukkanen, J. Taskinen, and R. Koistiainen, manuscript submitted).
Interestingly, from the standard inducers tested, only the polycyclic
aryl hydrocarbons 3-MC and Aroclor 1254 markedly enhanced the
glucuronidation rate of catechols. On the other hand, phenobarbital had
no significant effect. 3-MC and related polycyclic aryl hydrocarbons, such as dioxin, are known to specifically increase the expression of
UGT1A6 in rat (Bock et al., 1998). Such induction could account for the
stimulation effect of 3-MC found in rat liver microsomes. Indeed,
UGT1A6 was found to catalyze the glucuronidation of catechols, especially small catechols. This isoform presents strict substrate specificity toward planar and short phenols (Harding et al., 1988). Larger molecules, such as neurotransmitters, dopaminergic drugs, catechins, catechol estrogens, and COMT inhibitors, were, as expected, either not substrates or poor substrates of the enzyme. Concerning the
COMT inhibitors, Lautala et al. (1997, 2000) recently reported that
entacapone and tolcapone were not glucuronidated by the human orthologous UGT1A6. On the other hand, the phenobarbital-inducible UGT2B1 presents a broader substrate specificity when compared with
UGT1A6 since structurally unrelated alcohols, phenols, carboxylic acids
of the series of 2-phenylpropionic acid, and fatty acids were
glucuronidated by this isoform (Pritchard et al., 1994). Among the
series of catechols considered in this study, UGT2B1 competes with
UGT1A6 for the glucuronidation of small catechols. As for UGT1A6, the
carboxyl catechols, neurotransmitters, catechins, catechol estrogens,
or COMT inhibitors were not substrates of the UGT2B1 isoform. An
exception was found for octylgallate, dobutamine, and
ethyl-3,4-dihydroxybenzylidenecyanoacetate, which were specifically glucuronidated by UGT2B1. Phenobarbital was unable to stimulate the
glucuronidation of many catechols. This was the case of some catechols,
such as octylgallate and 4-tert-butyl-5-metoxycatechol, which are substrates of the phenobarbital-inducible UGT2B1. On the
other hand, glucuronidation of 4-tert-butylcatechol, a good substrate of UGT2B1, was indeed enhanced 2-fold upon phenobarbital treatment (Fig. 1; compound 8). It is likely that these compounds are
substrates of several other isoforms. Clofibrate stimulated the
glucuronidation of a restricted number of catechols, among those was
octylgallate. Clofibrate and other peroxisome proliferators are known
to enhance specifically the glucuronidation of bilirubin, supported by
the UGT1A1 isoform. Octylgallate has been reported to be a surrogate
substrate of UGT1A1 in humans (Senafi et al., 1994). This result would
explain why octylgallate glucuronidation was increased upon treatment
of rats with clofibrate. By contrast, the catechols themselves, and
particularly the COMT inhibitors, did not present any inducing effect
on UGT isoforms implicated in the glucuronidation of catechols,
indicating that no autoinduction occurred.
All catechols in which glucuronidation was strongly enhanced by 3-MC
were not effective substrates of UGT1A6. This was particularly observed
for bulky catechols, such as apomorphine, 4-hydroxyestradiol, octylgallate, or epicatechin gallate. The results suggest that other UGTs that are 3-MC-inducible could be involved in this process. A
potential candidate is UGT1A7, which has been recently characterized. The mRNA expression of this isoform has been shown to be increased by
3-MC (Emi et al., 1995). The enzyme catalyzes the glucuronidation of
bulky phenols chemically related to benzo [a]pyrene phenols (Grove et
al., 1997). In rabbit, UGT1A7 was reported to also glucuronidate some
catechols (octylgallate and catechol estrogens) (Bruck et al., 1997).
Our data showing that glucuronidation of octylgallate or
4-hydroxyestradiol was increased upon 3-MC treatment would support the
involvement of the rat UGT1A7. Work is in progress to investigate this point.
The PLS model was able to predict the glucuronidation values for many catechols in rats treated with 3-MC, although the model was better for activities from control animals. Glucuronidation of six compounds was increased more than 10-fold upon 3-MC treatment. This induction could be predicted for five of them. The PLS model implies that 3-MC may induce UGT isoforms that can tolerate larger structures and hydrogen bond donors, especially in the ortho and distal positions.
In conclusion, large differences in the glucuronidation rates were observed in the series of catechols considered in this study. Glucuronidation seems to be an efficient biotransformation pathway for selected catechols that are hydrophobic and noncharged molecules. Sulfotransferases and COMTs may relay the UGTs for the metabolism of other catechols.
| |
Footnotes |
|---|
Received June 21, 2001; accepted November 9, 2001.
1 Current address: Biologie Moléculaire Marine, LCA, Département Génie Biologique, Université de Toulon et du Var, France.
This work was supported by the Commission of the European Communities Grant BMH4-CT97-2621 and by the Région Lorraine.
Jacques Magdalou, UMR 7561 CNRS-Université Henri Poincaré Nancy, Faculté de Médecine, B.P. 184, 54505 Vandoeuvre-lès-Nancy cedex, France. E-mail: magdalou{at}facmed.u-nancy.fr
| |
Abbreviations |
|---|
Abbreviations used are: COMT, catechol-O-methyltransferase; UGT, UDP-glucuronosyltransferase; PLS, partial least squares; 3-MC, 3-methylcholanthrene; HPLC, high-performance liquid chromatography; p, proximal; d, distal; o, ortho; RLM, rat liver microsomes.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  H.-X. Liu, Y. Liu, J.-W. Zhang, W. Li, H.-T. Liu, and L. Yang UDP-Glucuronosyltransferase 1A6 Is the Major Isozyme Responsible for Protocatechuic Aldehyde Glucuronidation in Human Liver Microsomes Drug Metab. Dispos., August 1, 2008; 36(8): 1562 - 1569. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Elovaara, J. Mikkola, L. Luukkanen, L. Antonio, S. Fournel-Gigleux, B. Burchell, J. Magdalou, and J. Taskinen ASSESSMENT OF CATECHOL INDUCTION AND GLUCURONIDATION IN RAT LIVER MICROSOMES Drug Metab. Dispos., December 1, 2004; 32(12): 1426 - 1433. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Taskinen, B. T. Ethell, P. Pihlavisto, A. M. Hood, B. Burchell, and M. W. H. Coughtrie CONJUGATION OF CATECHOLS BY RECOMBINANT HUMAN SULFOTRANSFERASES, UDP-GLUCURONOSYLTRANSFERASES, AND SOLUBLE CATECHOL O-METHYLTRANSFERASE: STRUCTURE-CONJUGATION RELATIONSHIPS AND PREDICTIVE MODELS Drug Metab. Dispos., September 1, 2003; 31(9): 1187 - 1197. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Lu, X. Meng, C. Li, S. Sang, C. Patten, S. Sheng, J. Hong, N. Bai, B. Winnik, C.-T. Ho, et al. Glucuronides of Tea Catechins: Enzymology of Biosynthesis and Biological Activities Drug Metab. Dispos., April 1, 2003; 31(4): 452 - 461. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
