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J Biol Chem, Vol. 275, Issue 10, 6908-6914, March 10, 2000
From the It is suggested that formation of more polar
metabolites of all-trans-retinoic acid (atRA) via oxidative
pathways limits its biological activity. In this report, we
investigated the biotransformation of oxidized products of atRA via
glucuronidation. For this purpose, we synthesized 4-hydroxy-RA
(4-OH-RA) in radioactive and nonradioactive form, 4-hydroxy-retinyl
acetate (4-OH-RAc), and 5,6-epoxy-RA, all of which are major products
of atRA oxidation. Glucuronidation of these retinoids by human liver
microsomes and human recombinant UDP-glucuronosyltransferases (UGTs)
was characterized and compared with the glucuronidation of atRA. The
human liver microsomes glucuronidated 4-OH-RA and 4-OH-RAc with 6- and
3-fold higher activity than atRA, respectively. Analysis of the
glucuronidation products showed that the hydroxyl-linked glucuronides
of 4-OH-RA and 4-OH-RAc were the major products, as opposed to the
formation of the carboxyl-linked glucuronide with atRA, 4-oxo-RA, and
5,6-epoxy-RA. We have also determined that human recombinant UGT2B7 can
glucuronidate atRA, 4-OH-RA, and 4-OH-RAc with activities similar to
those found in human liver microsomes. We therefore postulate that this
human isoenzyme, which is expressed in human liver, kidney, and
intestine, plays a key role in the biological fate of atRA. We also
propose that atRA induces its own oxidative metabolism via a cytochrome P450 (CYP26) and is further biotransformed into glucuronides via UGT-mediated pathways.
atRA1 is a major
metabolite of vitamin A (all-trans-retinol) that undergoes
isomerization and metabolism in vivo, yielding 13-cis-retinoic acid (13-cis-RA),
9-cis-retinoic acid (9-cis-RA) (1-3),
5,6-epoxy-RA, 4-oxo-RA (4), 3,4-didehydro-RA, 4- and 18-hydroxy-RA
(4-OH-, 18-OH-RA) (5-9), and
all-trans-retinoyl- Recently, a novel atRA-inducible cytochrome P450 (CYP26) that
specifically metabolizes atRA to 18-OH-RA and 4-OH-RA has been identified (5-9). The 4-OH-RA is subsequently oxidized to 4-oxo-RA by
an alcohol dehydrogenase(s) (20). Such polar metabolites of atRA are
thought to limit its biological activity (5-9). Characterization of
the enzymatic metabolism of atRA will provide significant insight into
the regulation of retinoid homeostasis.
The purpose of the present work was to examine phase II metabolism of
atRA and identify the human UGTs (EC 2.4.1.17) involved in the
glucuronidation of atRA, 4-OH-RA, 4-oxo-RA, 5,6-epoxy-RA, and 4-OH-RAc
(structures shown in Fig. 1). UGTs catalyze the conjugation of
endogenous and xenobiotic compounds to D-glucuronic acid
derived from UDP-glucuronic acid (UDP-GlcUA). This converts the
metabolites into a more polar form, promoting their excretion in urine
and/or bile. However, several examples of bioactivation of parent
compounds via glucuronidation have been reported (21-25). UGTs can be
divided into two families, UGT1A and UGT2B, based on their sequences. In this work, human liver microsomal UGTs and recombinant isoforms from
both families were evaluated for their ability to catalyze glucuronidation of retinoids in various stages of oxidation.
To obtain the appropriate substrates for these studies, we synthesized
4-OH-RAc, 5,6-epoxy-RA, and both radioactive and nonradioactive forms
of 4-OH-RA. The studies in this report center on examining the
glucuronidation of atRA, 4-OH-RA, 4-oxo-RA, 5,6-epoxy-RA, and 4-OH-RAc
by human liver microsomes and the human recombinant UGTs, 1A3 and 2B7.
Human liver UGT2B7 is involved in the glucuronidation of a large
variety of endogenous and exogenous substrates including bile acids,
steroids, and a variety of xenobiotics (26-30). This report identifies
UGT2B7 as the major isoform catalyzing the formation of carboxyl- and
hydroxyl-linked glucuronides of retinoids at high levels of activity.
Materials
[14C]UDP-GlcUA was purchased from American
Radiolabeled Chemicals (St. Louis, MO). atRA; 9-cis-RA,
13-cis-RA, retinol, 13-cis-retinol, Brij 58, UDP-GlcUA, and saccharolactone were purchased from Sigma. (E)-Retinoic acid (atRA) and RAc were purchased from ACROS
Organics (Pittsburgh, PA), and
(E)-[11,12-3H]RA, ([3H]RA,
specific activity 35.8 Ci/mmol), and sodium borotritiide (NaB3H4, 204 mCi/mmol), were purchased from NEN
Life Science Products. All retinoids were stored at Synthesis of Radioactive and Nonradioactive Retinoid
Substrates
Synthesis of (±)-5,6-Epoxy-(E)-retinoic Acid (Scheme
1)--
Racemic 5,6-epoxy-RA
(2) was synthesized by epoxidation of atRA (31) with
monoperoxyphthalic acid, which was synthesized by a reaction of
phthalic anhydride with alkaline H2O2 as
described elsewhere (32) and quantitated iodometrically. 1 (1.0 g, 3.3 mmol) was reacted with a 15-fold molar excess of
monoperoxyphthalic acid in diethyl ether (50 ml) for 1 h at room
temperature. The formation of the oxirane was quantitative as evidenced
by TLC analysis using ethyl acetate-hexane (40:60 or 50:50, v/v) as the mobile phase. The reaction mixture was washed with saturated sodium bicarbonate, water, and saturated NaCl. The ethereal layer was dried
over anhydrous MgSO4, and solvent was removed in
vacuo. The oxirane was recrystallized from diethyl ether/hexane
(84% yield). Mass and 1H NMR spectra were obtained for the
corresponding methyl ester, which was synthesized by reaction of the
free acid with ethereal diazomethane prepared as described below: EI-MS
m/z 330 (M+), 315 (M Synthesis of 4-Oxo-(E)-retinyl Acetate (4) (Scheme
2)--
RAc (1.13 g, 3.44 mmol)
(3) was stirred with 30 g of MnO2 in 120 ml
of CH2Cl2 for 90 h at room temperature.
The MnO2 was removed by filtration, and the solvent was
removed in vacuo. The product was purified by silica gel
chromatography using ethyl acetate-hexane (10:90, v/v) as the mobile
phase, which afforded a yield of 78%: EI-MS m/z 342 (M+), 327 (M Synthesis of (±)-4-Hydroxy-(E)-retinyl Acetate (5)
(Scheme 2)--
4 (0.5 g, 1.5 mmol) was dissolved in 50 ml
of methanol, an equal volume of water was added, and the mixture was
stirred at 4 °C. An excess of NaBH4 was added, and the
reaction was allowed to proceed at 4 °C for 15 min and then for
1 h at room temperature. The reaction mixture was neutralized with
phosphate buffer and extracted with ethyl acetate. The pooled ethyl
acetate extracts were washed with water and subsequently dried over
MgSO4. 5 was subsequently purified by silica gel
chromatography using ethyl acetate as the mobile phase, which afforded
a 75% yield (EI-MS m/z 344 (M+), 326 (M Synthesis of (E)-Methylretinoate (6) (Scheme
3)--
atRA (1.0 g, 3.3 mmol) was
dissolved in 25 ml of anhydrous diethyl ether (note: not all of the
atRA may dissolve) to which distilled ethereal diazomethane (~50-100
mmol) was added, and the reaction was allowed to sit for 1 h at
room temperature, after which the ether phase was removed under a
stream of argon. The ethereal diazomethane was prepared by reaction of
Diazald® with NaOH using a Diazald® kit
(Aldrich). The reaction was shown to be quantitative as evidenced by
TLC analysis using ethyl acetate/hexane or benzene (20:80 v/v) as
mobile phases. EI-MS of 6 demonstrated a molecular ion of
m/z 314 (M+) (base peak), as well as
fragmentation ions of 299 (M Synthesis of 4-Oxo-(E)-methylretinoate (7) and
4-Oxo-(E)-retinoic Acid (10) (Scheme 3)--
6
(1.04 g, 3.3 mmol) was reacted with MnO2 (30 g) by stirring
at room temperature in CH2Cl2 (200 ml) for
72 h. The MnO2 was removed by filtration, the solvent
was removed in vacuo, and the product was purified by silica
gel chromatography using ethyl acetate/hexane (10:90 v/v) as the mobile
phase. The yield was 68%. EI-MS m/z 328 (M+) (base peak);
1H NMR (300 MHz, CDCl3) d (ppm) 1.21 (s, 6H), 1.84 (s,
3H), 2.04 (s, 3H), 2.37 (s, 3H),
2.52 (t, 2H), 3.47 (dd, 2H), 5.84 (s, 1H), 6.27 (d, 1H), 6.33 (s,
3H), 6.38 (d, 1H), 6.98 (dd, 1H).
The corresponding free acid (10) was prepared by hydrolysis
of 4-oxo-methylretinoate (7). A solution of 7 (0.5 g, 1.5 mmol) in methanol (40 ml) was reacted with aqueous NaOH (6 N, 40 ml) for 4 h at room temperature and then added
to aqueous H2SO4 (500 ml, 1 N). The
mixture was extracted with ethyl acetate, and the pooled ethyl acetate
extracts were washed with water and saturated NaCl, dried over
MgSO4, and solvent was removed in vacuo.
10 was subsequently purified by recrystallization from
diethyl ether/hexane (86% yield), and its purity was verified by
analytical reverse phase HPLC analysis and UV spectroscopy as described
previously (31).
Synthesis of (±)-4-Hydroxymethylretinoate (8) and
(±)-4-Hydroxyretinoic Acid (9) (Scheme
3)--
7 (0.6 g, 1.8 mmol) was dissolved in 50 ml of
methanol, an equal volume of water was added, and the mixture was
stirred at 4 °C. An excess of NaBH4 was added, and the
reaction was allowed to proceed at 4 °C for 15 min and then for
1 h at room temperature. The reaction mixture was neutralized with
phosphate buffer and extracted with ethyl acetate. The pooled ethyl
acetate extracts were washed with water and subsequently dried over
MgSO4. 8 was subsequently purified by silica gel
chromatography using 20% ethyl acetate/hexane as the mobile phase,
which afforded a 61% yield. EI-MS m/z 330 (M+),
312 (M
The corresponding free acid (9) was obtained by hydrolysis
of 8. A solution of 8 (0.35 g, 1.1 mmol) in methanol (40 ml) was reacted with aqueous NaOH (6 N, 40 ml)
for 4 h at room temperature and then acidified to pH 5.0 with
citrate buffer (3 M). This buffer was used for
acidification because we have shown that higher proton concentrations
result in significant acid-catalyzed dehydration of the alcohol (data
not shown). The mixture was extracted with ethyl acetate, the pooled
ethyl acetate extracts were washed with water and dried over
MgSO4, and solvent was removed in vacuo.
9 was subsequently purified by recrystallization from
diethyl ether/hexane (81% yield). The purity of the latter was
verified by analytical reverse phase HPLC analysis and UV spectroscopy
as described previously (33).
Synthesis of Racemic 4-Hydroxy-4-[3H]retinoic Acid
(11) (Scheme 3)--
50 mg of 10 (0.159 mmol)
was dissolved in 1.5 ml of methanol and 0.5 ml of 2-propanol, followed
by the addition of 0.2 ml of 1 M NaOH (0.2 mmol). To this
mixture, 20 µl of 0.1 M NaBH4 (2 µmol, in
0.01 N NaOH) was added. After 2 h, the mixture was
transferred to a vial containing 5 mCi of
NaB3H4 (204 mCi/mmol, 24.5 µmol). The
reaction was allowed to proceed overnight, and the course of the
reaction was followed by TLC using ethyl acetate-hexane (40:60, v/v) as
the mobile phase. Subsequently, NaBH4 (30 mg) was added in
order to assure reduction of any unreacted ketone. After 2 h, the
mixture was acidified to pH 6 using 1 M acetic
acid and extracted with ethyl acetate. The organic phase was washed
with water and dried over Na2SO4, and the
solvent was removed in vacuo. The yield was 64% (36 mg)
with a specific activity of 32 mCi/mmol. The product was shown to be
pure by analytical reverse phase HPLC, as described in the previous section.
Human Liver Microsomes--
The human liver microsomes used in
the experiments were from a 56-year-old man who had died of cerebral
bleeding (HLM15) and from a 13-year-old girl who died from brain damage
(HLM18). These samples were obtained from the University of Groningen,
Groningen, The Netherlands. The HLM served as a control for the
glucuronidation assays by providing a basis of comparison for
recombinant UGTs.
Human Recombinant UGTs--
Human recombinant UGT1A3 was
expressed in a mammalian expression system as described previously
(34). UGT2B7 was expressed in human embryonic kidney (HK293) cells as
reported previously (28, 29). Enriched endoplasmic reticulum membrane
fractions were prepared as described previously (35). The membrane
fractions were stored at Enzyme Assays--
UGT activity was measured with both
radioactive and unlabeled forms of atRA and 4-OH-RA as the aglycons
with UDP-GlcUA serving as the sugar donor (retinoid structures shown in
Fig. 1). All retinoid substrates were prepared in the form of mixed
micelles with Brij 58 (0.12%). The Brij 58 micelles both activated the enzyme and solubilized the retinoids. Human liver microsomes and recombinant UGTs (50 µg of protein) were used in the assays. All enzymatic assays were performed under yellow light. The amount of
product formed was less than 10% of total substrate added and was
linearly proportional to the amount of microsomal protein added.
The retinoid derivatives (0.10 mM final
concentration) were incubated in 100 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, 5 mM saccharolactone,
and 0.05% Brij 58 in a final volume of 60 µl. The reaction mixture
was preincubated with the proteins at room temperature for 10 min
before starting the reaction with the addition of either 50 mM UDP-GlcUA for radioactive retinoids (4.17 mM
final concentration) or 20 mM [14C]-UDP-GlcUA
(3.33 mM final concentration) for unlabeled retinoids. The
reactions were incubated for 30 min at 37 °C. Reactions were stopped
with 20 µl of ethanol, vortexed and placed on ice. For TLC, 60 µl
of the reaction mix was applied to the preadsorbent layer of a
19-channeled silica gel TLC plate (Baker Si250-PA (19C); VWR
Scientific) after which the plates were dried and developed twice in
chloroform-methanol-glacial acetic acid-water (65:25:2:4, v/v). These
TLC conditions allowed for separation of carboxyl- and hydroxyl-linked
glucuronides. After development, the plates were dried and subjected to
autoradiography for 3-7 days at -80 °C.
TLC Identification of the Type of Glucuronide
Biosynthesized--
For the identification of the type of glucuronide
biosynthesized (structures shown in Fig. 2), two methods were used.
First, in addition to TLC in the acidic system described above, samples were chromatographed in an alkaline solvent system (ethanol-ethyl acetate-30% NH4OH, 45:45:10, v/v) to hydrolyze carboxyl
glucuronides in the course of TLC. Alternatively, after completion of
the incubations, 8 N NaOH was added to the incubation
mixtures and the samples were incubated for an additional 30 min at
room temperature to hydrolyze the carboxyl glucuronide. Controls
without 8 N NaOH were run concurrently. Hydroxyl-linked
glucuronides are stable under these experimental conditions. This
procedure allowed clear determination of the formation of hydroxyl-
and/or carboxyl-linked glucuronides (structures shown in Fig. 2). After
development in chloroform-methanol-glacial acetic acid-water 65:25:2:4
(v/v), plates were dried and subjected to autoradiography for 3-7 days at Determination of Kinetic Constants--
To determine
Km and Vmax for HLM15 and
UGT2B7 with atRA and 4-OH-RA as substrates, the concentrations of atRA
and 4-OH-RA were varied (1-200 µM [3H]RA
or unlabeled 4-OH-RA), while the concentration of UDP-GlcUA was held
constant (4 mM). Data were analyzed and Michaelis-Menten kinetic parameters were determined using EnzymeKinetics software (Trinity Software, Campton, NH).
Synthesis of Retinoid Substrates--
Many of the retinoids used
in the current study were synthesized because they are not commercially
available. These include compounds 2-7 and
9-11 (Schemes 1-3). Racemic 5,6-epoxyretinoic
acid (2) was synthesized by reacting atRA (1)
with monoperoxyphthalic acid (Scheme 1). (±)-4-Hydroxyretinyl acetate
(5) was synthesized by first oxidizing retinyl acetate
(3) with manganese dioxide to afford 4-oxoretinyl acetate
(4) and reduction of the latter with sodium borohydride to
yield (5) (Scheme 2). atRA (1) was esterified to
the corresponding methyl ester (6) using ethereal
diazomethane which was subsequently oxidized with manganese dioxide to
produce 4-oxomethylretinoate (7) (Scheme 3). The free acid
(10) was prepared by saponification of the ester. Racemic
4-hydroxymethylretinoate (8) was prepared by reduction of
(7) with sodium borohydride and (±)-4-hydroxyretinoic acid
(9) was prepared by hydrolysis of (8). Tritiated
(±)-4-hydroxyretinoic acid (11) was synthesized by
reduction of (10) with NaB[3H]4.
Enzymatic Glucuronidation--
Human liver microsomes and human
recombinant UGTs were assayed for glucuronidation activity toward atRA,
9-cis-RA, 13-cis-RA, 4-OH-RA, 4-OH-RAc, 5,6-epoxy-RA, and 4-oxo-RA
(structures shown in Fig. 1). Two human
recombinant UGTs were available; UGT1A3 and UGT2B7. The enzymatic
activities of HLM15 and the recombinant proteins toward all retinoid
substrates used are summarized in Table
I. UGT2B7 had high activity toward the
retinoid substrates (with the exception of 4-oxo-RA), with 4-OH-RA and
4-OH-RAc being the optimal substrates for glucuronidation by UGT2B7.
UGT2B7 glucuronidated the retinoid substrates on the same level of
activity as HLM15. atRA was glucuronidated by HLM15 and UGT2B7 at rates
of 227 and 275 pmol/mg × min, respectively, and 4-OH-RA was
glucuronidated at 4-6-fold higher rates by both HLM15 and UGT2B7.
UGT1A3 also showed activity toward retinoid substrates.
With human liver microsomes, 5,6-epoxy-RA was the best substrate for
generating carboxyl-linked glucuronides. Comparison of the rates of
glucuronidation of 5,6-epoxy-RA by microsomes and recombinant UGT2B7,
however, indicates that other UGT isoforms might be involved in the
glucuronidation of this retinoid.
In separate studies designed to compare the rates of glucuronidation
for isomeric forms of atRA, the substrates atRA, 9-cis-RA, and
13-cis-RA were incubated with HLM18. Although, this microsomal preparation had much lower activity toward isomeric forms of atRA than
did HLM15, the limited amount of the latter preparation necessitated its use. The specific activities indicated that 9-cis-RA was the best
substrate for human liver microsomes, followed by atRA and 13-cis RA.
The activities were 590 ± 102, 110 ± 9, and 88 ± 31 pmol/mg protein × min, respectively. All three substrates were glucuronidated at their carboxyl function.
Kinetic Analysis--
Kinetic parameters were determined for the
glucuronidation of the carboxyl-function of atRA and the
hydroxyl-function of 4-OH-RA using HLM15 and recombinant UGT2B7 (Table
II). The Km for atRA
was 182-fold lower than for 4-OH-atRA with both microsomes and
recombinant UGT2B7. This indicated that both microsomal and recombinant
UGTs have a much higher affinity for free atRA than for the
4-hydroxylated derivative.
Product Identification--
Fig. 3 is an autoradiogram of a
TLC plate from a representative assay of glucuronidation of
[3H]atRA, and unlabeled 4-OH-RA and 4-OH-RAc by
HLM15 and UGT2B7. The metabolites formed by glucuronidation of
retinoids containing carboxyl and/or hydroxyl functional groups were
either carboxyl- or hydroxyl-linked glucuronides (Fig.
2). Biosynthesis of double glucuronides
was not observed. Double development of the TLC plate with
chloroform-methanol-glacial acetic acid-water 65:25:2:4 (v/v) solvent
effectively separated the mixture of the two glucuronides (Fig.
3). An unidentified
14C-labeled endogenous product was detected (Fig. 3,
lanes 1 and 4) when radiolabeled
UDP-GlcUA was used.
To identify which glucuronides were produced with a given retinoid
substrate, an alkaline solvent system was used to develop the TLC
plate. Under these conditions, carboxyl-linked glucuronides were
hydrolyzed as has been described previously (36), whereas the 4-OH-RA
glucuronide was resistant to alkaline hydrolysis (data not shown).
Additional proof of the identity of the glucuronide biosynthesized was
achieved by alkaline hydrolysis (8 N NaOH, 30 min room
temperature) performed prior to TLC separation.
The TLC analysis demonstrated that free atRA and the
cis-isomers, as expected, exclusively formed carboxyl-linked
glucuronide. 4-OH-RA or 4-OH-RAc were glucuronidated exclusively at the
hydroxyl function, producing alkaline-resistant ether glucuronides.
This indicated that the presence of the hydroxyl function on the
The important roles that atRA and RAG play in cellular
differentiation, proliferation, apoptosis, and other cellular events necessitate studies directed at understanding the metabolism of atRA.
Additionally, atRA is an important chemotherapeutic agent for skin,
breast, endometrial, and hematopoietic cancers. Therefore, knowledge of
atRA metabolism is essential to understand its limitations as an
anti-cancer compound.
Isomers of atRA, such as 9-cis-RA and 13-cis-RA,
are considered to be as biologically active as atRA (37). Experimental results from in vitro and in vivo systems
indicate that atRA oxidative metabolism involves both cytochrome P450
and prostaglandin H synthase-mediated processes. We have previously
demonstrated that atRA and 13-cis-RA are hydroxylated at the
C-4 position by prostaglandin H synthase (33, 38, 39). Recent reports
suggest that the generation of 4-OH- and 18-OH-RA relies on a
cytochrome P450-dependent process. Members of a new
cytochrome P450 family, CYP26, can be induced by atRA and can also
inactivate biologically active atRA (5-9).
A major unanswered question is whether oxidized retinoic acid
metabolites are only intermediates in the catabolic degradation of
atRA, or if these oxidized metabolites possess biological functions similar to those of atRA. If the formation of more polar metabolites of
atRA via hydroxylation is indeed deleterious to its biological activity, then the role of atRA and RAG will not be completely understood without identifying the sequence of events in the metabolism of atRA. To gain insight into these questions, two major issues must be
addressed: 1) the physiological mechanisms that promote atRA-induced RA
hydroxylation by CYP26, and 2) the metabolic fate of these
oxidized-derivatives. In this report, we focused our investigation on
the glucuronidation by UGTs of oxidized retinoids in comparison to
atRA.
Several commercially unavailable compounds needed for these studies,
such as 4-OH-RA, 4-OH-RAc, and 5,6-epoxy-RA, were synthesized for use
as substrates for human UGTs. Metabolites such as these are involved in
mediating atRA function. For example, 4-oxo-RA binds to retinoid
receptors with an affinity comparable to that of atRA and can regulate
the expression of genes (4). 4-oxo-RA can also bind to cellular atRA
binding proteins (40). All of this suggests that this derivative
possesses biological activity similar to that of atRA.
Since limited information exists on the biosynthesis and
biotransformation of 5,6-epoxy-RA, we were particularly interested in
characterizing the metabolism of this important compound. 5,6-Epoxy-RA has been identified as a major in vivo metabolite of atRA
(as well as retinol and RAc) in rodents (12, 41-43). We have recently identified 5,6-epoxy-RA as a major in vivo metabolite of
13-cis RA in skin,2 and we
have detected the glucuronide of 5,6-epoxy-RA in human bile.2 5,6-Epoxy-RA also exhibits potent activity in
various biological assays. For example, 5,6-epoxy-RA was more effective
than RAc at producing potent growth effects in vitamin A-deficient rats (44). In addition, the oxirane exhibited inhibitory effects similar to
those of atRA on
12-O-tetradecanoylphorbol-13-acetate-dependent tumor promotion in the two-stage (initiation-promotion) mouse skin
carcinogenesis assay (45). Additionally, 5,6-epoxy-RA is more potent
than atRA in opposing the effects of
12-O-tetradecanoylphorbol-13-acetate on the induction of
tumor promotional markers in bovine lymphocytes (31) and
polymorphonuclear leukocytes (46).
These previous findings imply that the retinoid response may involve
metabolic activation of atRA to the 5,6-epoxide with the oxirane
representing the pharmacologically active agent. Epoxidation of these
retinoids does not appear to occur through a cytochrome P450-catalyzed
mechanism (47). We have unequivocally demonstrated that the
5,6-epoxides of atRA and 13-cis-RA are generated via peroxyl
radical-dependent mechanisms (33, 38, 39, 48-50). In
addition, we have recently demonstrated that 5,6-epoxy-RA is generated
during prostaglandin H synthase-catalyzed oxidation of atRA
involving a direct oxene transfer mechanism analogous to cytochrome
P450.2
Table I shows the rates of glucuronidation and identification of the
type of glucuronide formed for various retinoids. These data
demonstrate that glucuronidation of atRA, 4-oxo-RA, and 5,6-epoxy RA is
directed toward the carboxyl function of these substrates, whereas the
4-hydroxylated derivatives are glucuronidated almost exclusively at the
4-OH position. This gives rise to three observations. 1)
4-OH-derivatives of atRA and RAc are glucuronidated at their hydroxyl
function by human liver microsomes and UGT2B7 with significantly higher
activities than those observed for the formation of carboxyl-linked glucuronides of atRA and the 4-oxo- and 5,6-epoxy derivatives of atRA.
A comparison of hydroxylated versus non-hydroxylated compounds suggests that the 4-hydroxy group directs glucuronidation to
the hydroxyl function and that the carboxyl group is not required for
glucuronidation to occur at the 4-OH position. However, the significantly lower enzymatic activity toward 4-OH-RAc indicates that a
free carboxyl function is required for optimal glucuronidation. 2)
atRA, 5,6-epoxy RA, and 4-oxo-RA are glucuronidated exclusively at the
carboxyl function. 3) Although the glucuronidation of 4-OH substrates
by UGT2B7 is similar to that found in microsomes, the significantly lower activity of the recombinant protein toward 4-oxo-RA
and 5,6-epoxy-RA might suggest the involvement of other UGT isoforms in
the glucuronidation of these retinoids. Other isomeric forms of atRA,
such as 9-cis- and 13-cis-RA, were also actively glucuronidated by
human liver microsomes. Of the three compounds, atRA, 9-cis-RA, and
13-cis-RA, the 9-cis derivative is the best substrate for microsomal
glucuronidation at the carboxyl position and, in comparison to the
other retinoids studied, is second only to 5,6-epoxy-RA.
The Km for the formation of the carboxyl-linked
glucuronide of atRA is in the low micromolar range (1.3-1.5
µM) both for HLM15 and recombinant UGT2B7. The maximal
catalytic rates (Vmax) for atRA, as determined
with HLM15 and recombinant UGT2B7, are 764 and 523 pmol
glucuronidated × min Of the UGT isoforms investigated to date, human recombinant UGT2B7 has
the highest capacity to glucuronidate atRA and 4-OH-RA. UGT2B7
activities toward atRA and 4-OH-RA are similar to the reported activities in human liver microsomes, suggesting that UGT2B7 plays a
key role in metabolizing atRA and 4-OH-RA to the carboxyl-linked RAG
and the hydroxyl-linked 4-OH-RAG. UGT2B7 is capable of catalyzing the
biosynthesis of the hydroxyl-linked glucuronide when 4-OH-RA is the
substrate or the carboxyl-linked glucuronide when atRA is the
substrate. Recent studies on glucuronidation of steroid hormones and
fatty acids by UGT2B7 have shown that this isoform is actively involved
in the formation of both hydroxyl- and carboxyl-linked glucuronides of
those lipophilic substrates (30). Taken collectively, retinoids,
steroid hormones, and fatty acids are important ligands involved in
initiating cellular signaling events. We postulate that UGT2B7 may be
involved in controlling intracellular levels of ligands, such as
steroids and atRA. If this is the case, it may also be involved in a
feedback loop that controls the amounts of ligands available for
steroid and retinoid receptors.
In summary, we speculate that atRA induces its own oxidative metabolism
via a cytochrome P450, CYP26, mechanism followed by a
UGT-dependent mechanism. The hydroxyl-linked glucuronide of 4-OH-RA is the directed product of atRA metabolism by CYP26. Thus, 4-OH
glucuronidation of 4-OH-RA terminates the biological activity of atRA,
while the carboxyl-linked glucuronide of atRA might be a biologically
active compound involved in cellular processes. Thus, CYP26 and UGT2B7
may together play a crucial role in the metabolism and biological fate
of atRA.
We thank Joanna Little and Pat Rabjohn for
their thorough editing of this paper.
*
This work was supported in part by National Institutes of
Health Grants DK51971, DK45123, and DK49715 (to A. R.-P.) and
R29ES06765 (to V. M. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, University of Arkansas for Medical Sciences,
4301 W. Markham, Slot 516, Little Rock, AR 72205. Tel.: 501-686-5414; Fax: 501-603-1146; E-mail:
radominskaanna@exchange.uams.edu.
2
V. M. Samokyszyn, unpublished data.
The abbreviations used are:
atRA, all-trans-retinoic acid;
13-cis-RA, 13-cis-retinoic acid;
9-cis-RA, 9-cis-retinoic acid;
4-OH-RA, 4-hydroxy-all-trans-retinoic acid;
4-OH-RAc, 4-hydroxy-all-trans-retinyl acetate;
5, 6-epoxy-RA,
5,6-epoxy-all-trans-RA;
UGT, UDP-glucuronosyltransferase;
UDP-GlcUA, UDP-glucuronic acid;
RAG, all-trans-retinoyl-
4-Hydroxyretinoic Acid, a Novel Substrate for Human Liver
Microsomal UDP-glucuronosyltransferase(s) and Recombinant UGT2B7*
,,
, and Departments of Toxicology and Pharmacology
and the § Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences, Little Rock, Arkansas
72205, the Department of Pharmacology, University of Iowa, Iowa
City, Iowa 52242, and the ¶ Department of Clinical Pharmacology,
Flinders University School of Medicine, Bedford Park,
South Australia 5046, Australia
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucuronide (RAG) (10-12). These
metabolites are thought to be involved in mediating atRA function. It
has been well established that atRA and some of its metabolites
modulate expression of a set of genes involved in apoptosis, cellular
growth and differentiation, and embryonic development (13, 14).
Retinoids are also effective in preventing carcinogenesis and can
inhibit proliferation of a large variety of normal and neoplastic
cells. In addition, atRA is currently used as a chemotherapeutic agent
against promyelocytic leukemia (15), various endothelial cancers (16),
breast cancer (17), and endometrial cancer (18). Retinoid metabolism
and atRA-induced growth inhibition in head and neck squamous cell carcinoma cell lines has also been documented (16). Furthermore, the
glucuronidated atRA derivative, RAG, has been shown to be less
cytotoxic than the parent atRA, while retaining its potency for driving
cell growth, differentiation, and proliferation (12, 19). This
highlights the importance of elucidating the physiological role of
glucuronide conjugates of atRA and oxidized derivatives of atRA.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C under
argon, and all synthetic reactions were also carried out under argon.
Stock solutions of the retinoids were prepared fresh in methanol
(Fisher Scientific, Pittsburgh, PA), and all procedures involving these
compounds were performed in the dark or under yellow light.
Diazald®
(N-methyl-N-nitroso-p-toluenesulfonamide),
sodium borohydride, bis(trimethylsilyl)trifluoroacetamide, all
deuterated NMR solvents, and hydrogen peroxide (~30%) were purchased
from Aldrich, and the H2O2 solution was
quantified by iodometric titration. Phthalic anhydride was purchased
from Eastman Kodak Co., and manganese dioxide (MnO2) was
purchased from Fluka (Milwaukee, WI). All other reagents were of the
highest commercially available quality.
CH3)+, 299 (M OCH3)+, 271 (M CO2CH3)+; 1H NMR (500 MHz, Me2SO-d6) d (ppm) 6.97 (dd,
J = 15.0, 15.3 Hz, 1H), 6.42 (d, J = 15.1 Hz, 1H), 6.25 (d,
J = 6.5, 11.1 Hz, 2H), 6.04 (d, J = 15.7 Hz, 1H), 5.78 (s,
1H), 2.26 (s, 3H), 1.95 (s, 3H), 1.73 (brs,
2H), 1.37 (brs., 2H), 1.09 (s, 3H), 1.08 (s,
3H), 0.85 (s, 3H).
View larger version (14K):
[in a new window]
Scheme 1.
Synthesis of racemic
5,6-epoxy-(E)-retinoic acid. 1,
(E)-retinoic acid; 2,
(±)-5,6-epoxy-(E)-retinoic acid.
CH3)+, 298 (M CO2)+, 282, 267, 249, 145 (base
peak); IR (cm
1) 1685 (
,-unsaturated carbonyl),
1242, 1735 (ester), 1380-1365 (CH3 [acetate]); 1H NMR
(300 MHz, CDCl3) d (ppm) 1.19 (s, 6H), 1.86 (s,
3H), 1.84 (m, 2H), 1.90 (s, 3H), 1.99 (s,
3H), 2.07 (s, 3H), 2.51 (t, 2H), 4.74 (d, 2H),
5.66 (t, 1H), 6.22 (d, 1H), 6.27 (d, 1H), 6.32 (d, 1H), 6.37 (d,
1H), 6.64 (dd, 1H).
View larger version (14K):
[in a new window]
Scheme 2.
Synthesis of
4-oxo-(E)-retinyl acetate and racemic
4-hydroxy-(E)-retinyl acetate. 3,
(E)-retinyl acetate; 4,
4-oxo-(E)-retinyl acetate; 5,
(±)-4-hydroxy-(E)-retinyl acetate.
H2O)+). In order to verify the presence of a
hydroxyl group using mass spectrometry, the trimethylsilyl derivative
was prepared by reaction of the acetate with
bis(trimethylsilyltrifluoroacetamide): EI-MS m/z 416 (M+).
CH3)+,
282, 267, 159, 119, and 105.
View larger version (18K):
[in a new window]
Scheme 3.
Synthesis of
4-oxo-(E)-retinoic acid,
(±)-4-hydroxy-(E)-retinoic acid, and
(±)-4-hydroxy-4-[3H]-(E)-retinoic acid.
1, (E)-retinoic acid; 6,
methyl-(E)-retinoate; 7,
4-oxo-methyl-(E)-retinoate; 8,
(±)-4-hydroxymethyl-(E)-retinoate; 9,
(±)-4-hydroxy-(E)-retinoic acid; 10,
4-oxo-(E)-retinoic acid; 11,
(±)-4-[3H]4-hydroxy-(E)-retinoic acid.
H2O)+; IR (cm1)
3500 (O-H stretch), 1092 (C-O stretch); 1H NMR (300 MHz,
methanol-d4) d (ppm) 1.01 (s, 3H), 1.05 (s,
3H), 1.41 (m, 2H), 1.69 (m, 2H), 1.80 (s, 3H),
2.02 (s, 3H), 2.34 (s, 3H), 3.68 (s,
3H), 3.95 (t, 1H), 5.82 (s, 1H), 6.16 (d, 1H), 6.23 (d,
1H), 6.29 (d, 1H), 6.38 (d, 1H), 7.10 (dd, 1H).
80 °C in 5 mM HEPES, 0.25 M sucrose, 20 mM MgCl2 (pH 7.4).
The enzymatic activity of the recombinant UGT proteins was sustained
for up to 6 months under these conditions.
80 °C. When tritium-labeled substrates were used, plates were sprayed with En3Hance (NEN Life Science Products) before
autoradiography. Glucuronide bands were localized using the
autoradiographs, and silica gel containing labeled metabolites (or from
corresponding areas in control lanes) was scraped into vials and
radioactivity was determined by scintillation counting (LKB RackBeta
1214; Wallac Inc., Gaithersburg, MD). Specific activities of enzymes
are expressed as picomoles/min × mg of protein; where applicable,
means ± S.E. are reported.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
Structures of the retinoid substrates used in
these studies.
Glucuronidation of atRA, 4-OH-RA, 4-OH-RAc, 5,6-epoxy-RA, and
4-oxo-RA by human liver microsomes and recombinant UGT1A3 and
UGT2B7
Kinetics of glucuronidation of atRA and 4-OH-RA using human liver
microsomes (HLM15) and recombinant UGT2B7
View larger version (16K):
[in a new window]
Fig. 2.
Structures of carboxyl and hydroxyl-linked
glucuronides of retinoids.
View larger version (22K):
[in a new window]
Fig. 3.
TLC separation of radioactive glucuronides of
atRA, 4-OH-RA, and 4-OH-RAc. The leftmost two
panels (lanes 1-6) show the
glucuronidation of [3H]RA with unlabeled UDP-GlcUA by
human liver microsomes (HLM15) and UGT2B7 membranes. Lanes
1 and 4 represent incubations where UDP-GlcUA was
omitted. Lanes 2 and 3 and
lanes 5 and 6 represent duplicate
determinations of atRA glucuronidation as described under
"Experimental Procedures." With human liver microsomes, two
products were obtained: upper band-carboxyl-linked glucuronide of atRA;
lower band-unidentified glucuronidated product of atRA
biotransformation. The third through sixth
panels (lanes 7-18) represent
glucuronidation of 4-OH-RA and 4-OH-RAc with
[14C]UDP-GlcUA. Lanes 7 and
13 show an unidentified glucuronide of an endogenous
substrate biosynthesized under these experimental conditions. Double
development of the TLC plate is necessary to achieve separation of all
biosynthesized retinoid glucuronides.
-ionine ring redirects the glucuronidation from the carboxyl to the
hydroxyl function.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 × mg protein1,
respectively. These correspond to catalytic efficiencies
(Vmax/Km) of 509 µl x
min1 × mg -1 for HLM15 and 402 µl × min1 × mg-1 for recombinant UGT2B7,
revealing significant efficiency of formation of atRA carboxyl-linked
glucuronide. The Km values for 4-OH-directed
glucuronidation are 273 and 221 µM for HLM15 and recombinant UGT2B7, respectively. Typical Vmax
values were determined to be in the low nanomolar range (2176 and 1709 pmol × mg1 × min1 for HLM15 and
recombinant 2B7, respectively), leading to a much lower efficiency of
formation for the hydroxyl-linked glucuronides, as shown by a
Vmax/Km of 8 µl x
min1 × mg-1 for both HLM15 and recombinant
UGT2B7. In general, the presence of the hydroxyl group in the retinoid
moiety switches the site of glucuronidation from carboxyl to hydroxyl
and reverses the affinity of the UGT(s) involved. The corresponding
catalytic efficiencies (Vmax/Km) were several
hundredfold higher for glucuronidation of the carboxyl function of atRA
than for glucuronidation of the 4-OH moiety of the hydroxylated retinoid.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
-glucuronide;
4-OH-RAG, all-trans-retinoic acid-4-O--glucuronide;
HLM, human liver microsome;
HPLC, high performance liquid chromatography;
EI-MS, electron impact mass spectrometry.
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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J. V. Chithalen, L. Luu, M. Petkovich, and G. Jones HPLC-MS/MS analysis of the products generated from all-trans-retinoic acid using recombinant human CYP26A J. Lipid Res., July 1, 2002; 43(7): 1133 - 1142. [Abstract] [Full Text] [PDF]
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 S. A. Gestl, M. D. Green, D. A. Shearer, E. Frauenhoffer, T. R. Tephly, and J. Weisz Expression of UGT2B7, a UDP-Glucuronosyltransferase Implicated in the Metabolism of 4-Hydroxyestrone and All-Trans Retinoic Acid, in Normal Human Breast Parenchyma and in Invasive and in Situ Breast Cancers Am. J. Pathol., April 1, 2002; 160(4): 1467 - 1479. [Abstract] [Full Text] [PDF]
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B. L. Coffman, W. R. Kearney, M. D. Green, R. G. Lowery, and T. R. Tephly Analysis of Opioid Binding to UDP-Glucuronosyltransferase 2B7 Fusion Proteins Using Nuclear Magnetic Resonance Spectroscopy Mol. Pharmacol., June 1, 2001; 59(6): 1464 - 1469. [Abstract] [Full Text]
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A. R. Jude, J. M. Little, A. W. Bull, I. Podgorski, and A. Radominska-Pandya 13-Hydroxy- and 13-Oxooctadecadienoic acids: Novel Substrates for Human UDP-Glucuronosyltransferases Drug Metab. Dispos., April 13, 2001; 29(5): 652 - 655. [Abstract] [Full Text]
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J. Marill, T. Cresteil, M. Lanotte, and G. G. Chabot Identification of Human Cytochrome P450s Involved in the Formation of All-trans-Retinoic Acid Principal Metabolites Mol. Pharmacol., April 13, 2001; 58(6): 1341 - 1348. [Abstract] [Full Text]
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P. J. Czernik, J. M. Little, G. W. Barone, J.-P. Raufman, and A. Radominska-Pandya Glucuronidation of Estrogens and Retinoic Acid and Expression of UDP-Glucuronosyltransferase 2B7 in Human Intestinal Mucosa Drug Metab. Dispos., October 1, 2000; 28(10): 1210 - 1216. [Abstract] [Full Text] [PDF]
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