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Laboratory of Molecular Endocrinology (C.A., M.V., G.B., A.B., D.W.H.), Medical Research Council Group in Molecular Endocrinology (A.B.), CHUL Research Center, Laval University, Québec, Canada, G1V 4G2
Address all correspondence and requests for reprints to: Dr. Dean W. Hum, Molecular Endocrinology Laboratory, CHUL Research Center, 2705 Laurier Boulevard, Québec, G1V 4G2, Canada. E-mail: Dean.Hum{at}crchul.ulaval.ca
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Introduction |
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UGT enzymes catalyze the transfer of the sugar group, from UDP-glucuronic acid to small hydrophobic molecules (aglycones), which include the steroid hormones, having functional groups of oxygen, nitrogen, sulfur or carbon (13, 14). UGTs are important phase II detoxifying enzymes that conjugate xenobiotics, as well as endogenous compounds such as bilirubin, bile acids, thyroxin, biogenic amines, fat-soluble vitamins, and steroids (13, 14, 15). Glucuronidated compounds are more polar, less toxic, and are generally eliminated from the body through the bile or urine.
On the basis of sequence similarities, UGT enzymes are divided into two major families, UGT1 and UGT2; and UGT2 proteins are further categorized into two subfamilies, UGT2A and UGT2B (13, 16). In humans, the UGT1 gene family is located on chromosome 2q37, where the gene locus contains the necessary exons for 12 UGT1 transcripts, which include three pseudogenes (17, 18). Each UGT1 protein is encoded by a gene composed of a unique first exon, and four common exons 2 to 5, which are shared among all the UGT1 proteins characterized to date. Thus, it is apparent that the region encoded by the first exon, which entails half of the protein, confers the aglycone substrate specificity.
The physiological importance of UGT1 enzymes is demonstrated by genetic mutations, which cause several diseases with varying degrees of hyperbilirubinemia (Crigler-Najjar Type-I and II, and Gilbert’s disease) (17, 19). These mutant genes demonstrate that decreased or absent levels of some UGT activities can lead to toxic levels of substrate accumulation in the body. Initial studies on UGT1A enzymes demonstrated their activity on bilirubin and xenobiotics; however, several recent reports demonstrate that UGT1 can also glucuronidate steroids (20, 21, 22, 23, 24, 25, 26).
Although it is widely accepted that the liver is a major site of glucuronidation, it is now clear that extrahepatic tissues are also involved in the conjugation of compounds to which these tissues are exposed (13, 14, 27). Glucuronidation has been demonstrated in the kidney, gut, lung, skin, brain, adipose, thymus, prostate, and breast (14, 27, 28, 29), and the expression of UGT1 transcripts have also been demonstrated in many of these tissues.
To obtain an appropriate animal model to study the role of steroid
glucuronidation in humans, a comparison of the circulating levels of
5
-reduced C19 steroid glucuronides among mammalian species, showed
that human and monkey are unique in having high levels of circulating
ADT-G and 3-Diol-G (30). In separate studies, humans and simians
were found to be unique in having adrenals that secrete large amounts
of the precursor steroids dehydroepiandrosterone (DHEA)
and DHEA-sulfate (DHEA-S), which are
converted into potent androgens and/or estrogens in peripheral tissues
(31). As found in humans, extrahepatic steroid target tissues, which
include the prostate, testis, skin, and breast, of the monkey also
express steroid conjugating UGT transcripts (32). All these data
indicate that the monkey is the most appropriate animal model for
studying the role of steroid glucuronidation in extrahepatic
tissues.
The similarities of steroid glucuronidation between humans and monkey are further supported by the high sequence homology, and similar enzyme characteristics of UGT1A9 described in this study. MonUGT1A09 (GenBank Accession Number AF104336) is the first simian protein of the UGT1A family to be cloned and characterized. The enzyme from both species have a high activity for estrogens and catechol estrogens, especially 4-hydroxyestradiol and 4-hydroxyestrone. As well, UGT1A9 transcript is expressed in several extrahepatic steroid target tissues, including the estrogen-responsive tissues such as the prostate, breast, and ovary.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA isolation
Total human RNA was isolated from human adipose tissue, skin,
placenta, and ovary, according to the Tri reagent acid phenol protocol
as specified by the supplier (Molecular Research Center, Inc., Cincinnati, OH). Total monkey RNA was isolated from
various tissues according to the Tri reagent acid phenol protocol as
specified by the supplier (Molecular Research Center, Inc.). Quantification was made by optical density at 260 nm.
Isolation of a novel UGT1 complementary DNA (cDNA) from monkey
liver
To isolate monkey UGT1 cDNA clones for further characterization,
we synthesized a cynomolgus monkey liver cDNA library with ZAP Express
cDNA synthesis kit as specified by the supplier
(Stratagene) using affinity purified monkey liver
messenger RNAs. We screened this amplified monkey liver cDNA library
with a human probe consisting of the human carboxyl terminus common
region of the UGT1 family of enzymes. This probe, consisting of a part
of the exon 2, the exon 3 and 4 and a part of the exon 5, was obtained
by RT-PCR amplification of human liver RNA using Pfu polymerase
(Stratagene) and specific common carboxyl terminus region
sense primer 5'-AAGCTATGGCAATTGCTGATGC-3' and antisense primer
5'-CCCACTTCTCAATGGGTCTTGG-3'. The DNA fragment obtained (657 bp) was
purified and radiolabeled by the random primer technique in the
presence of [-32P]dCTP. The filters were prehybridized
in 40% formamide, 5 x Denhardt’s solution, 5 x SSPE,
0.1% SDS, and 100 µg/ml salmon sperm DNA for 4 h at 42 C. The
hybridization was performed in the same solution for 16 h at 42 C
with 2.0 x 106 cpm/ml of probe. The filters were
washed twice in 2 x SSC, 0.1% SDS at 42 C for 15 min, and
exposed for 16 h at -80 C on XAR5 film with an intensifying
screen (Eastman Kodak Co., Rochester, NY). After screening
approximately 1 x 106 recombinants, 5000 positive
clones were obtained. One hundred positive clones were randomly chosen
in all positive clones obtained. To specifically isolate a clone
homologous to human UGT1A9, we screened these selected clones with a
human probe consisting of a part of the first exon of UGT1A9
[nucleotides (nt) 68 to 749]. This probe was obtained by RT-PCR
amplification of human liver RNA using Pfu polymerase
(Stratagene) and specific human UGT1A9 sense primer
5'-CCGAGGCAGGGAAGCTAG-3' and antisense primer
5'-ATTGATGTGTGGCTGTAGAGATCATACT-3'. Sequence analysis revealed that two
clones were identical and code for the novel UGT1 cDNAs monUGT1A09.
MonUGT1A09 cDNA clone was sequenced in both directions using specific
UGT1 oligonucleotides.
Isolation of human UGT1A9 by RT-PCR
Human UGT1A9 cDNA was isolated from human liver total RNA by
RT-PCR using M-MLV reverses transcriptase (Boehringer Mannheim) and Pfu DNA polymerase
(Stratagene). The RT reaction was carried out using ten
µg total kidney RNA and 2 µg of oligo-deoxythymidine primer
in the presence of the M-MLV reverse transcriptase according to the
manufacturer’s instructions (Boehringer Mannheim). One
microliter of the RT product was used as a template in a PCR, the PCR
was performed according to the manufacturer’s instructions with 5%
DMSO and 1% glycerol. The reaction was carried out using 100 pmol of
the hUGT1A9 specific sense primer 5'-TCCGGATCCCAGTTCCCCAACTCACCTCTGG-3'
containing a BamHI restriction site and specific UGT1 common
region antisense primer 5'-AGAGCTCGAGCCCACTTCTCAATGGGTCTTGG-3'
containing XhoI restriction site, leading to the
amplification of about 1.6 kb specific hUGT1A9 DNA. The PCR was
performed for 35 cycles (1 min at 94 C, 4 min at 62 C, 1 min at 72 C),
the protocol was preceded by an incubation of 5 min at 94 C and
followed by an extended elongation time of 10 min at 72 C. The PCR
product was then purified and digested with BamHI and
XhoI restriction enzymes and inserted in the
BamHI-XhoI site of the pBK-CMV vector downstream
the CMV promoter. UGT1A9 cDNA clone was sequenced in both directions
using specific oligonucleotides. The sequence corresponds to hUGT1A9
(GenBank Accession Number AF056188).
Stable expression of the monkey and human UGT1A9 protein
HK293 cells were grown in DMEM containing 4.5 g/l glucose, 10
mM HEPES, 110 µg/ml sodium pyruvate, 100 IU of
penicillin/ml, 100 µg/ml of streptomycin and 10% FBS in a humidified
incubator, with an atmosphere of 5% CO2, at 37 C. Five
micrograms of pBK-CMV-monUGT1A09 and pBK-CMV-hUGT1A9 were used to
transfect HK293 cells using Lipofectin according to the manufacturer’s
instructions (Gibco BRL). Forty-eight hours post
transfection, stable transfectants were selected in media containing 1
mg/ml G418. After colony selection, ten monoclonal cell lines stably
expressing human or monkey UGT1A9 were isolated. The clone
demonstrating the highest activity for each enzyme was used for
glucuronidation assay and kinetic analysis.
Production and purification of a UGT1 common carboxyl terminus
region protein
Expression of a 24-kDa protein composed of amino acids 312 to
531 of human UGT1 common carboxyl terminus region was achieved by
RT-PCR amplification of this region from human liver RNA using Pfu
polymerase (Stratagene), specific UGT1 common carboxyl
terminus region sense primer 5'-AAGCTATGGCAATTGCTGATGC-3', and
antisense primer 5'-CCCACTTCTCAATGGGTCTTGG-3'. The DNA fragment
obtained (658 bp) was subcloned into the SmaI digest PQE 31
prokaryotic expression vector (Qiagen) which produced a
24-kDa protein, 6 x His-tagged at the amino terminus. The
sequence of the UGT1 common carboxyl terminus region obtained was
similar to the sequence expected. Escherichia coli ME15
cells harboring the recombinant vector were grown at 25 C in LB culture
medium supplemented with ampicillin (100 µg/ml), kanamycin (25
µg/ml), and 2% glucose. When the absorbency of the cells reached an
OD600 of 0.5–0.6, production of the protein was induced
with 1 mM isopropyl ß-D-thiogalactopyranoside
for 6 h at 25 C. The cells were harvested by centrifugation at
5,000 x g for 10 min at 4 C. The bacterial cell pellet
was resuspended in lysis buffer containing 6M guanidium-HCl and
sonicated to homogeneity. The recombinant protein was purified in
denaturing conditions using Ni-NTA gel according to the manufacturer’s
instructions (Qiagen).
Immunization procedure
Rabbits from Charles River Laboratories, Inc.,
(Québec, Canada) were injected sc at multiple sites with 500 µl
of a total of 100 µg of purified protein in PBS, 0.1% SDS, in the
presence of 500 µl of complete Freund’s adjuvant. Two booster
injections were administered at 4-week intervals with the same quantity
of protein in the presence of incomplete Freund’s adjuvant. The
production of antibodies in the rabbit’s serum was checked 12 days
after the second injection, and the rabbits are killed 10 days after
the third injection.
Preparation of the microsomal fraction
Microsomes were prepared by differential centrifugation. HK293
cells expressing human or monkey UGT1A9 were homogenized in
K2HPO4 0.1 M,
KH2PO4 0.1 M, pH 7.4, glycerol
20%, EDTA 1 mM, dithiothreitol 1 mM, 2.5
µg/ml pepstatin, 0.5 µg/ml leupeptin using a Potter-Glas-col (Terre
Haute, IN) type homogenizer with a Teflon pestle. The homogenate was
centrifuged at 12,000 x g for 20 min to remove nuclei,
unbroken cells, and mitochondria. The pellet was discarded and the
supernatant was centrifuge at 105,000 x g for 60 min
to obtain the microsomal pellet, which were resuspended in
homogenization buffer at 10 mg protein per ml and stored at -80 C.
Western blot analysis
To ascertain the expression of the human and monkey UGT1A9
protein, 20 µg of microsomal protein from HK293 cells stably
expressing human UGT1A9 or monkey UGT1A09, HK293 cells, and human and
monkey liver homogenates were separated by 10% SDS-PAGE. The gel was
transferred onto a nitrocellulose membrane and probed with the
antihuman UGT1 common carboxyl terminus region antiserum RC71 (1:2000
dilution). The same blot was subsequently probed with an
anticalnexin CT antibody (StressGen Biotechnologies Corp.,
Victoria, Canada, 1:1000 dilution) as control. A donkey antirabbit IgG
antibody conjugated with the horseradish peroxidase (Amersham Corp., Oakville, Canada) was used as the second antibody, and
the resulting immunocomplexes were visualized using enhanced
chemiluminescence kit (Renaissance, Québec, Canada) following the
manufacturer’s instructions and exposed on hyperfilm for 2 min
(Eastman Kodak Co.) and quantified by Phosphoimager using
Imagequant software (Molecular Dynamics, Inc.).
Glucuronidation assay using microsome preparations
To screen for substrates that react with UGT1A9, assays were
performed using 15 µM [14C]UDP-glucuronic
acid (UDPGA), 100 µM unlabeled UDPGA, 200
µM aglycone, and 25 µg of proteins from microsome
preparations in 50 mM Tris-HCl (pH 7.5), 10 mM
MgCl2, 100 µg/ml phosphatidylcholine and 8.5
mM saccharolactone in a final volume of 100 µl. Assays
were performed for 16 h at 30 C, and were terminated by adding 100
µl of methanol. Samples were centrifuged at 14,000 rpm for 2 min in
an Eppendorf microcentrifuge to remove the precipitated
proteins. One hundred microliters of the aqueous phase were applied
onto TLC plates (0.25 mm-thick silica gel, Whatman,
Maidstone, UK) and chromatographed in a solvent of
toluene:methanol:acetic acid (7:3:1). The TLC plates were exposed for
24 h and the extent of glucuronidation was assessed by
Phosphorimager (Molecular Dynamics, Inc.).
Compounds that demonstrated reactivity with UGT1A9 in the screening assay were subsequently reassayed to assess activity in the same buffer for 60 min at 30 C, containing 15 µM [14C]UDP-glucuronic acid, 500 µM unlabelled UDPGA and 200 µM aglycone. Under these saturating conditions, both enzyme reactions are linear for 90 min, and the Km of UDPGA conjugation is 190 µM and 113 µM for hUGT1A9 and monUGT1A09 respectively.
Kinetic analysis
When determining the apparent Km using
microsomes from HK293 cells stably expressing human or monkey UGT1A9,
the reactions were performed by incubation with aglycones at 0.1
µM to 100 µM at 30 C for 1 h.
Vmax and Km were then calculated using
Lineweaver-Burk plot.
RT-PCR
Total RNA was digested with RNase free DNase (Boehringer Mannheim) for 60 min at 37 C according to the manufacturer’s
instructions. The tissue distribution of human and monkey UGT1A9 were
achieved using a RT-PCR technique. Ten micrograms of total RNA from
human and monkey tissues were predigested by RNase free DNase were
used. Reverse transcriptase reactions were achieved using 2 µg of
oligo-deoxythymidine primer in the presence of the M-MLV
reverse transcriptase in a final volume of 40 µl according to the
manufacturer’s instructions (Boehringer Mannheim). One
microliter of the RT product was used as a template in a PCR containing
10 mM Tris-HCl pH 8.3, 50 mM KCl, 2
mM MgCl2, 0.2 mM dNTP and 2.5 U of
Amplitaq DNA polymerase (Perkin-Elmer Cetus Corp.) in a
total volume of 100 µl. The reaction was carried out using 100
pmol of the human UGT1A9 specific sense primer
5'-GATATATTCTCTATTAATGGGTTCATACA-ATGAC-3' and antisense primer
5'-ATTGATGTGTGGCTGAGAGATCATACT-3', leading to the amplification of a
438-bp specific human UGT1A9. The reaction was carried out using 100
pmol of the monkey UGT1A9 specific sense primer
5'-TCAAATTGCAGGAGTTTGTTAAGGAC-3' and antisense primer
5'-CCAAATTGATATTTGTCTGTGAAGATCATAT-3' leading to the amplification of a
390-bp specific UGT1A09 DNA. Both PCR reaction were performed for 40
cycles (1 min at 94 C, 1 min at 63 C (human) or at 60 C (monkey), 1 min
at 72 C); the protocol was preceded by an incubation of 5 min at 94 C
and followed by an extended elongation time of 10 min at 72 C. One
fifth of the PCR product was electrophoresed on an ethidium bromide
stained 2% agarose gel and the DNA fragments obtained were visualized
under UV light. All RT reactions were controlled by using specific
oligonucleotides for glyceraldehyde phosphate dehydrogenase (GAPDH).
The identity of all PCR products was verified by direct sequencing PCR
product (33).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The same blot was subsequently
hybridized with a cDNA probe for GAPDH, to assess the RNA level in each
lane (Fig. 1B).
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The longer of the two cDNA clones isolated is comprised of 2396
bp, and contains an open reading frame of 1590 nt flanked by a
5'-untranslated region of 59 bp, and a 3'-untranslated region of 747
bp. A putative polyadenylation signal with the consensus sequence
AATAAA is located 20 nt upstream of the poly(A+) tail,
which starts at position 2379. The open reading frame of 1590 nt
starting at the first AUG (initiator) codon encodes a
polypeptide of 530 amino acids. The encoded protein contains a
characteristic hydrophobic putative signal peptide at the amino
terminal end, proposed for targeting UGT1A proteins into the
endoplasmic reticulum (35). Also as characteristic of other UGT
proteins, a hydrophobic putative transmembrane region is found between
amino acids 488 to 504, which anchors the protein to the ER. Similar to
most UGT enzymes, the novel monkey protein contains three potential
asparagine-linked glycosylation sites (NXS/T) present at residues 71,
292, and 344. (Fig. 2).
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Primary structure comparisons with human UGT1A enzymes reveal that the
variable region of the monkey protein is most homologous to the region
encoded by exon one of hUGT1A9 and hUGT1A8, demonstrating 90% and 84%
identity respectively (Table 1). This
monkey protein is most probably the ortholog of hUGT1A9, and is thus
named monUGT1A09. Comparison of the conserved carboxyl region of the
monkey and human proteins demonstrate 97% identity, where only 8 out
of the 245 residues are different (Fig. 2
). The high homology of this
region is accentuated by an identical stretch of 115 amino acids
between residues 380 to 495, which is interrupted by only one
difference at amino acid 441.
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), which is absent in untransfected
HK293 cells. As well, microsomal proteins from liver tissue yielded a
broad band, corresponding to the presence of hepatic UGT1 proteins in
both species. The same blot was subsequently probed with an
anticalnexin antibody to ensure that equal amounts of protein from the
control (untransfected) and stably transfected cells were loaded in
each lane (Fig. 3B). Levels of the 88-kDa calnexin protein are
comparable in the microsome preparations. The absence of calnexin in
the human and monkey liver extracts is most likely due to the lower
relative abundance in total cell extracts. The relative levels of
hUGT1A9 and monUGT1A09 expressed in the cells were the same when
normalized to the level of calnexin (Fig. 3C).
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, Tables 2 and 3).
Of the 34 exogenous compounds tested, 26 were conjugated by monUGT1A09,
and 24 were conjugated by hUGT1A9 (Table 2). Glucuronidation of these
compounds was absent in control HK293 cell microsomes, not containing
the 55 kDa UGT1A9 proteins. MonUGT1A09 conjugates a variety of
exogenous compounds including planar and bulky phenols, nonsteroidal
antiinflammatory drugs, anthraquinone, benzodiazepines, coumarin
derivatives, and flavonoids. The hUGT1A9 enzyme is active on many of
the same compounds, but conjugation of benzodiazepines was not
detected. Despite the comparable expression level of the hUGT1A9 and
monUGT1A09 proteins in the stable cell lines, the human enzyme is more
active on the flavonoide compounds than is the monkey protein.
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and Figs. 4 and 5). Similarly,
the hUGT1A9 enzyme is active on the C18 steroids, but it also had low
activity for C19 steroids. However, the enzyme from both species did
not conjugate C21 steroids, bile acids, and the neurotransmitters
tested. As expected, the control HK293 cell microsomes did not
glucuronidate any of the aglycones tested. Despite the comparable
expression level of the hUGT1A9 and monUGT1A09 proteins in the cell
lines, the monkey enzyme is more active on the 4-hydroxy and 2-hydroxy
estrogens than is the human protein.
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and Table 4). As well, the
apparent Km values are remarkably similar for both
proteins. However, for each of the four substrates, the
Vmax of the monkey enzyme is consistently and significantly
higher than the human protein, despite a comparable amount of the UGT
proteins expressed. Although both proteins conjugate the C18 steroids
with similar apparent Km values, they have the highest
activity for 4-hydroxyestrogens, followed by 2-hydroxyestradiol and
then estradiol.
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).
In the cynomolgus monkey, monUGT1A09 transcript is expressed in the
same tissues; however, expression was also found in other tissues
including the seminal vesicle, epididymis, vagina, foreskin, adrenal,
heart, colon, and gall bladder. In both species, UGT1A9 transcript was
not detected in the uterus, lung, spleen, and brain. Specificity of the
oligonucleotides used was confirmed by direct sequencing of the PCR
product, and demonstrated amplification of UGT1A9 transcript. RT-PCR
amplification of the GAPDH transcript, which yielded a product of
appropriate length in each RNA sample, was used as a positive control
for the reactions (Fig. 6).
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-OH position, whereas both enzymes can glucuronidate steroids
such as testosterone and DHT at the 17ß-OH position. It has also been
shown that, although both UGT2B15 and UGT2B17 are found in the same
tissues, their expressions are differentially regulated where the level
of transcript for UGT2B17 but not UGT2B15 is down-regulated by
physiological effectors such as EGF, IL-1a, and FGF (43, 44). As well,
the UGT2B17 protein is significantly more labile than UGT2B15, which
suggests that inhibition of UGT2B17 gene expression would also lead to
a rapid decrease of protein and enzyme activity (43). While the characterization of UGT2B proteins have demonstrated the ability of several of these enzymes to conjugate C19 steroids in particular, recent studies on UGT1A proteins have also demonstrated the specificity of these enzymes for steroid molecules. Human UGT1A4 conjugates progestins, androgens, and estrogens (25), whereas initial studies on the UGT1A1 (24), UGT1A3 (23), UGT1A7 (22), UGT1A8 (26), UGT1A9 (20), and UGT1A10 (22) have demonstrated their ability to glucuronidate estrogens.
It is well established that UGT enzymes can conjugate steroids, that these proteins are expressed in extrahepatic tissues, and that these tissues secrete glucuronidated steroids. Monkeys have been used as an animal model to study the physiological role of phase I P450 enzymes (45, 46, 47, 48), and some studies suggest that simians may be the most relevant model in which to study steroid conjugation by UGT enzymes (30, 49). Previous studies have demonstrated the high homology between several UGT2B enzymes, which express overlapping substrate specificities (39, 50). Some differences in specificities and enzyme kinetics were also observed between the human and simian UGT2B proteins; however, it is unclear if these variations are due to different properties of orthologous or separate proteins (32, 51). From the analysis in this study, it is apparent that the novel UGT1A protein characterized is the simian ortholog of hUGT1A9. The primary structure of monkey UGT1A09 is most homologous to human UGT1A9 (93% identical), and both proteins conjugate the same exogenous (bulky and planar phenols, coumarins, and flavonoids) and endogenous (estrogens and catecholestrogens) substrates. Some substrates such as C19 steroids and benzodiazepines were apparently conjugated by only either one of the proteins; however, in each case the activity was very low and may reflect experimental variations close to the limit of detection. It is interesting that the mouse UGT1A9 isoform is somewhat less homologous (76% identity) with the human protein, and the rat UGT1A9P is a pseudogene, which further supports the monkey as the most relevant model to study steroid glucuronidation.
The activity of the human and monkey UGT1A9 proteins on estrogens, and their expression in extrahepatic estrogen-sensitive tissues such as the breast, ovary, and prostate, is consistent with this enzyme playing a role in steroid metabolism. Conjugation of estrogens by UGT1A9 in these tissues may be required to promote steroid elimination to maintain homeostasis, or where increased elimination may be required following increased estrogen synthesis in response to physiological conditions. In addition, it has been proposed that glucuronidated steroids cannot interact with their receptors, thus suggesting that glucuronidation may play a role in regulating the steroid response (39, 40, 42).
The high activity of UGT1A9 on 2-hydroxyestradiol, 4-hydroxyestradiol, and 4-hydroxyestrone, and the conjugation of these substrates with apparent Km values in the micromolar range, suggest a physiological role of the enzyme on catecholestrogens. The glucuronidation of catecholestrogens such as 4-hydroxyestrone, is potentially an important catabolic pathway required to eliminate these genotoxic steroid metabolites from a given tissue, and prevent cell damage (5, 11, 12). Catecholestrogens can undergo metabolic redox cycling catalyzed by P450 enzymes. The hydroperoxide-dependent oxidation of catecholestrogens to quinones, and the NADPH-dependent reduction of the quinones back to hydroquinones, yield semiquinone free radical intermediates and superoxide radicals (8). The continuous generation of the free radicals by the redox cycle have been postulated to mediate DNA damage such as single strand breaks, 8-hydroxylation of guanine bases, and depurination of adenine-guanine adducts, leading to tumor development (5, 7, 8). This potential problem of catecholestrogens is particularly relevant in estrogen-sensitive tissues such as the breast, ovary, and uterus, which express steroidogenic enzymes including aromatase required for estrogen synthesis, and the enzymes such as cytochrome P4501B1, which yield catecholestrogens (52, 53, 54, 55). It is interesting that several steroid-specific UGT enzymes including UGT1A9 are also expressed in these tissues; thus, it will be important to determine the role these proteins may have in regulating the level of estrogens and subsequently influencing their effects.
The monkey has been used extensively as an animal model for numerous studies on steroid metabolism and related pathologies (30, 49, 56, 57, 58). The finding that the simian UGT1A09 enzyme is structurally and functionally similar to the human protein further establishes the relevance of using the monkey as a model to ascertain the physiological role of steroid glucuronidation in extrahepatic steroid target tissues. It is apparent that UGT1A9 is active on several xenobiotic compounds, which correlates with its expression in the liver and kidney, which are the major detoxification tissues. Demonstration that the predominant endogenous substrates for UGT1A9 are estrogens and catecholestrogens correlates with its expression in several estrogen-responsive tissues. In vivo studies will be required to ascertain the function of UGT1A9 in influencing the steroid levels or the steroid response in these tissues under normal and pathological states.
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Acknowledgments |
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Footnotes |
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2 Holder of a scholarship from the MRC of Canada.
Received November 9, 1998.
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References |
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-reduced C19 steroid glucuronides in
primates, rodents, and domestic animals. Am J Physiol
271:E348–E353
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 R. Obligacion, M. Murray, and I. Ramzan Drug-Metabolizing Enzymes and Transporters: Expression in the Human Prostate and Roles in Prostate Drug Disposition J Androl, March 1, 2006; 27(2): 138 - 150. [Full Text] [PDF]
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 S. Chouinard, M. Tessier, G. Vernouillet, S. Gauthier, F. Labrie, O. Barbier, and A. Belanger Inactivation of the Pure Antiestrogen Fulvestrant and Other Synthetic Estrogen Molecules by UDP-Glucuronosyltransferase 1A Enzymes Expressed in Breast Tissue Mol. Pharmacol., March 1, 2006; 69(3): 908 - 920. [Abstract] [Full Text] [PDF]
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 F. Labrie, A. Belanger, V. Luu-The, C. Labrie, J. Simard, L. Cusan, J. Gomez, and B. Candas Gonadotropin-Releasing Hormone Agonists in the Treatment of Prostate Cancer Endocr. Rev., May 1, 2005; 26(3): 361 - 379. [Abstract] [Full Text] [PDF]
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 J. Hukkanen, P. Jacob III, and N. L. Benowitz Metabolism and Disposition Kinetics of Nicotine Pharmacol. Rev., March 1, 2005; 57(1): 79 - 115. [Abstract] [Full Text] [PDF]
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N. Picard, D. Ratanasavanh, A. Premaud, Y. Le Meur, and P. Marquet IDENTIFICATION OF THE UDP-GLUCURONOSYLTRANSFERASE ISOFORMS INVOLVED IN MYCOPHENOLIC ACID PHASE II METABOLISM Drug Metab. Dispos., January 1, 2005; 33(1): 139 - 146. [Abstract] [Full Text] [PDF]
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O. Barbier, H. Girard, Y. Inoue, H. Duez, L. Villeneuve, A. Kamiya, J.-C. Fruchart, C. Guillemette, F. J. Gonzalez, and B. Staels Hepatic Expression of the UGT1A9 Gene Is Governed by Hepatocyte Nuclear Factor 4{alpha} Mol. Pharmacol., January 1, 2005; 67(1): 241 - 249. [Abstract] [Full Text] [PDF]
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 J. Lepine, O. Bernard, M. Plante, B. Tetu, G. Pelletier, F. Labrie, A. Belanger, and C. Guillemette Specificity and Regioselectivity of the Conjugation of Estradiol, Estrone, and Their Catecholestrogen and Methoxyestrogen Metabolites by Human Uridine Diphospho-glucuronosyltransferases Expressed in Endometrium J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5222 - 5232. [Abstract] [Full Text] [PDF]
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 Y. Duguay, M. McGrath, J. Lepine, J.-F. Gagne, S. E. Hankinson, G. A. Colditz, D. J. Hunter, M. Plante, B. Tetu, A. Belanger, et al. The Functional UGT1A1 Promoter Polymorphism Decreases Endometrial Cancer Risk Cancer Res., February 1, 2004; 64(3): 1202 - 1207. [Abstract] [Full Text] [PDF]
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 L. Villeneuve, H. Girard, L.-C. Fortier, J.-F. Gagne, and C. Guillemette Novel Functional Polymorphisms in the UGT1A7 and UGT1A9 Glucuronidating Enzymes in Caucasian and African-American Subjects and Their Impact on the Metabolism of 7-Ethyl-10-hydroxycamptothecin and Flavopiridol Anticancer Drugs J. Pharmacol. Exp. Ther., October 1, 2003; 307(1): 117 - 128. [Abstract] [Full Text] [PDF]
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O. Barbier, D. Duran-Sandoval, I. Pineda-Torra, V. Kosykh, J.-C. Fruchart, and B. Staels Peroxisome Proliferator-activated Receptor {alpha} Induces Hepatic Expression of the Human Bile Acid Glucuronidating UDP-glucuronosyltransferase 2B4 Enzyme J. Biol. Chem., August 29, 2003; 278(35): 32852 - 32860. [Abstract] [Full Text] [PDF]
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 D. Turgeon, S. Chouinard, P. Belanger, S. Picard, J.-F. Labbe, P. Borgeat, and A. Belanger Glucuronidation of arachidonic and linoleic acid metabolites by human UDP-glucuronosyltransferases J. Lipid Res., June 1, 2003; 44(6): 1182 - 1191. [Abstract] [Full Text] [PDF]
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H. Jinno, M. Saeki, T. Tanaka-Kagawa, N. Hanioka, Y. Saito, S. Ozawa, M. Ando, K. Shirao, H. Minami, A. Ohtsu, et al. Functional Characterization of Wild-type and Variant (T202I and M59I) Human UDP-glucuronosyltransferase 1A10 Drug Metab. Dispos., May 1, 2003; 31(5): 528 - 532. [Abstract] [Full Text] [PDF]
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D. Turgeon, J.-S. Carrier, S. Chouinard, and A. Belanger Glucuronidation Activity of the UGT2B17 Enzyme toward Xenobiotics Drug Metab. Dispos., May 1, 2003; 31(5): 670 - 676. [Abstract] [Full Text] [PDF]
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O. Barbier, L. Villeneuve, V. Bocher, C. Fontaine, I. P. Torra, C. Duhem, V. Kosykh, J.-C. Fruchart, C. Guillemette, and B. Staels The UDP-glucuronosyltransferase 1A9 Enzyme Is a Peroxisome Proliferator-activated Receptor alpha and gamma Target Gene J. Biol. Chem., April 11, 2003; 278(16): 13975 - 13983. [Abstract] [Full Text] [PDF]
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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]
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K. F. Ilett, B. T. Ethell, J. L. Maggs, T. M. E. Davis, K. T. Batty, B. Burchell, T. Q. Binh, L. T. A. Thu, N. C. Hung, M. Pirmohamed, et al. Glucuronidation of Dihydroartemisinin in Vivo and by Human Liver Microsomes and Expressed UDP-Glucuronosyltransferases Drug Metab. Dispos., September 1, 2002; 30(9): 1005 - 1012. [Abstract] [Full Text]
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J.-F. Gagne, V. Montminy, P. Belanger, K. Journault, G. Gaucher, and C. Guillemette Common Human UGT1A Polymorphisms and the Altered Metabolism of Irinotecan Active Metabolite 7-Ethyl-10-hydroxycamptothecin (SN-38) Mol. Pharmacol., September 1, 2002; 62(3): 608 - 617. [Abstract] [Full Text] [PDF]
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 C. Guillemette, I. De Vivo, S. E. Hankinson, C. A. Haiman, D. Spiegelman, D. E. Housman, and D. J. Hunter Association of Genetic Polymorphisms in UGT1A1 with Breast Cancer and Plasma Hormone Levels Cancer Epidemiol. Biomarkers Prev., June 1, 2001; 10(6): 711 - 714. [Abstract] [Full Text] [PDF]
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 O. Barbier, C. Girard, L. Berger, M. El Alfy, A. Belanger, and D. W. Hum The Androgen-Conjugating Uridine Diphosphoglucuronosyltransferase-2B Enzymes Are Differentially Expressed Temporally and Spatially in the Monkey Follicle throughout the Menstrual Cycle Endocrinology, June 1, 2001; 142(6): 2499 - 2507. [Abstract] [Full Text] [PDF]
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O. Barbier, C. Albert, I. Martineau, M. Vallée, K. High, F. Labrie, D. W. Hum, C. Labrie, and A. Bélanger Glucuronidation of the Nonsteroidal Antiestrogen EM-652 (SCH 57068), by Human and Monkey Steroid Conjugating UDP-Glucuronosyltransferase Enzymes Mol. Pharmacol., March 1, 2001; 59(3): 636 - 645. [Abstract] [Full Text]
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Q. Ren, S. E. Murphy, Z. Zheng, and P. Lazarus O-Glucuronidation of the Lung Carcinogen 4-(methylnitrosamino)-1- (3-Pyridyl)-1-Butanol (nnal) by Human Udp-Glucuronosyltransferases 2b7 and 1a9 Drug Metab. Dispos., November 1, 2000; 28(11): 1352 - 1360. [Abstract] [Full Text]
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C. Albert, O. Barbier, M. Vallee, G. Beaudry, A. Belanger, and D. W. Hum Distribution of Uridine Diphosphate-Glucuronosyltransferase (UGT) Expression and Activity in Cynomolgus Monkey Tissues: Evidence for Differential Expression of Steroid-Conjugating UGT Enzymes in Steroid Target Tissues Endocrinology, July 1, 2000; 141(7): 2472 - 2480. [Abstract] [Full Text] [PDF]
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 R. Raftogianis, C. Creveling, R. Weinshilboum, and J. Weisz Chapter 6: Estrogen Metabolism by Conjugation J Natl Cancer Inst Monographs, July 1, 2000; 2000(27): 113 - 124. [Abstract] [Full Text] [PDF]
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O. Barbier, D. Turgeon, C. Girard, M. D. Green, T. R. Tephly, D. W. Hum, and A. Bélanger 3'-azido-3'-deoxythimidine (AZT) is glucuronidated by human UDP-glucuronosyltransferase 2B7 (UGT2B7) Drug Metab. Dispos., May 1, 2000; 28(5): 497 - 502. [Abstract] [Full Text]
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