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Oncology and Molecular Endocrinology, Research Center, 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 or Dr. Alain Bélanger, Oncology and Molecular Endocrinology Research Center, CHUL Research Center, 2705 Laurier boulevard, Québec, Canada G1V 4G2. E-mail: dean.hum@crchul.ulaval.ca and alain.belanger{at}crchul.ulaval.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-androstane-3,17ß-diol,
androsterone, and 4-hydroxyestradiol was measured using tissue
homogenates and radiolabeled [14C]UDP-glucuronic acid.
All tissues contained conjugation activity on these substrates, but
glucuronidation rates were significantly lower in steroid target
tissues than in liver, kidney, or gut. However, the ratio of steroid
glucuronidation vs. eugenol glucuronidation was higher
in steroid target tissues, suggesting a differential expression of
steroid-conjugating enzymes in these tissues. Taken together, these
results clearly demonstrate the presence of steroid glucuronidation
enzymes in extrahepatic steroid target tissues and support the
hypothesis that steroid glucuronidation is an important intracrine
pathway involved in termination of steroid signaling. |
Introduction |
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Steroid hormones are specific ligands of steroid nuclear receptors that regulate the expression of many genes to control physiological processes. The enzymes and mechanisms regulating the synthesis of steroid hormones in humans have been studied extensively; however, the pathways involved in steroid catabolism (including glucuronidation), which has an equal potential to regulate steroid levels, has not been well characterized.
To date, more than 60 different UGT enzymes have been isolated in several mammalian species and, on the basis of sequence similarities, have been divided into two families, UGT1 and UGT2 (4). In humans, the UGT1 gene family is located on chromosome 2q37 where the gene locus contains 12 different exons 1 (including three pseudogenes) and 4 common exons, exons 2–5 (5, 6). UGT2 proteins are further categorized into 3 subfamilies, UGT2A, UGT2B, and UGT2C (4). The UGT2B subfamily is composed of separate genes that share the same organization of 6 exons and 5 introns, and several human UGT2B genes have been located on chromosome 4q13 (7, 8). Six human UGT2B complementary DNA (cDNA) clones that encode steroid-conjugating enzymes have been characterized. It is apparent that the UGT2B enzymes can glucuronidate xenobiotics such as eugenol; however, their conjugation of steroid substrates is relatively more specific. On the other hand, previous studies of the UGT1A enzymes demonstrated their activities on xenobiotics and some endogenous substrates, such as bilirubin. However, several recent reports demonstrate that UGT1A enzymes can also glucuronidate steroids (9, 10, 11, 12, 13, 14).
In humans, the plasma contains significant levels of steroid
metabolites in the form of glucuronidated conjugates. Moreover, a
recent study suggested that the level of circulating androgen
glucuronides [androsterone-glucuronide (ADT-G) and
5-androstane-3,17ß-diol-glucuronide (3-Diol-G)] is
correlated with the total androgen pool in men, more so than the level
of unconjugated C19 steroids (15). In addition,
the plasma level of steroid glucuronides is increased in some
hyperandrogenic pathologies, such as acne or hirsutism, which are
related to the increased production of 5-reduced
C19 steroids (16).
It is widely accepted that the liver is a major site of
glucuronidation; however, it is now clear that extrahepatic tissues are
also involved in the conjugation of compounds to which these tissues
are exposed. Glucuronidation activity has been demonstrated in the
human liver, kidney, gut, skin, and prostate (17, 18, 19, 20). In addition,
high levels of 5-reduced C19 steroid
glucuronides were found in the human prostate, breast cyst fluid, and
ovarian follicular fluid (21, 22, 23). These results are consistent with
the expression of UGT1A and UGT2B transcripts in steroid target tissues
and indicate that UGT enzymes can contribute to terminate the steroid
response and modulate steroid levels in extrahepatic steroid target
tissues (9, 24).
Comparison of the circulating levels of 5-reduced
C19 steroid glucuronides among mammalian species
showed that the human and monkey are unique in having high levels of
circulating ADT-G and 3-Diol-G (25). The similarities of steroid
glucuronidation between humans and monkey are further supported by the
high sequence homology and similar biochemical characteristics between
the human enzymes and the 6 UGT1A and 5 UGT2B simian enzymes cloned to
date (9, 26, 27, 28, 29, 30) (Albert, C., et al., unpublished data). To
further understand the role of steroid glucuronidation in extrahepatic
tissues, the present study examined the expression of UGT1A and UGT2B
transcripts and proteins in 24 monkey tissues. To ascertain the
capacities of the tissues to conjugate steroids, the activities of
tissue homogenates were assessed on androgens (ADT, 3-Diol) an
estrogen [4-hydroxyestradiol (4OHE2)], and a
nonsteroidal substrate, eugenol.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monkey tissues
The monkeys were maintained for research study according to the
Guidelines for Care and Use of Experimental Animals. Tissues from adult
male and female cynomolgus monkey (Macaca fascicularis) were
collected and freed from fat and connective tissue immediately after
death. A part of each tissue was immediately homogenized (see
glucuronidation assays), and then each homogenate was quickly frozen in
liquid nitrogen and kept at -80 C for subsequent RNA and microsome
isolation.
RNA isolation
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., Cincinnati, OH).
Quantification was made by OD at 260 nm.
RT-PCR analysis
The tissue distributions of monkey UGT1A and UGT2B were
determined using RT-PCR analysis. Ten micrograms of total RNA from
monkey tissues were predigested by ribonuclease-free deoxyribonuclease
(Roche Molecular Biochemicals, Indianapolis, IN) for 60
min at 37 C according to the manufacturer’s instructions. Reverse
transcriptase reactions were achieved using 2 µg
oligo(deoxythymidine) primer in the presence of the Moloney murine
leukemia virus reverse transcriptase in a final volume of 40 µl
according to the manufacturer’s instructions (Roche Molecular Biochemicals). 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 deoxy-NTP, and 2.5 U AmpliTaq DNA polymerase
(Perkin-Elmer Corp./Cetus) in a total volume of 100 µl.
The reaction was carried out using 100 pmol of the UGT1A-specific sense
primer (5'-AAGCTATGGCAATTGCTGATGC-3') and antisense primer
(5'-TCTCAATGGGTCTTGGATTTGTGGGC-3'), leading to the amplification of a
653-bp specific monkey UGT1A. For UGT2B, the reaction was carried out
using 100 pmol of the monkey UGT2B-specific sense primer
(5'-GGAGTTGTGGAAAGGTGCTGGTGT-3') and antisense primer
(5'-CAATCCAGAAGACTGCTCGATCCAGG-3'), leading to the amplification of a
1350-bp specific monkey UGT2B cDNA. Both PCR reactions were performed
for 35 cycles [1 min at 94 C, 1 min at 62 C (UGT1A) or 60 C (UGT2B), 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 agarose gel, and the DNA fragments obtained were
visualized under UV light. The specificity of primer pairs was verified
by amplification using monkey UGT1A or UGT2B cDNA (10 ng) as template
in the same conditions. All RT reactions were controlled by using
specific oligonucleotides for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). The identity of all PCR products for UGT1A was verified by
direct sequencing PCR products (31).
Preparation of microsomal proteins
Microsomes were prepared by differential centrifugation. Monkey
tissues were homogenized in 0.1 M
K2HPO4-0.1 M
KH2PO4 (pH 7.4),
20%glycerol, 1 mM EDTA, 1 mM dithiothreitol,
2.5 µg/ml pepstatin, and 0.5 µg/ml leupeptin using a
Potter-Glas-col (Terre Haute, IN) homogenizer with a Teflon pestle at 4
C. The homogenates were centrifuged at 12,000 x g for
20 min at 4 C to remove nuclei, unbroken cells, and mitochondria. The
pellet was discarded, and the supernatant was centrifuged at
105,000 x g for 60 min at 4 C to obtain the microsomal
pellet, which was resuspended in homogenization buffer at about 10 mg
protein/ml and stored at -80 C. The protein content of microsomal
fraction was measured by the method of Bradford (Bio-Rad Laboratories, Inc., Richmond, CA).
Western blot analysis
To ascertain the expression of UGT1A and UGT2B enzymes in monkey
tissues, 20 µg microsomal protein from liver, jejunum, colon, kidney,
uterus, mammary gland, prostate, testis, HK293 cells, and from HK293
cells stably expressing monUGT1A09 or UGT2B19 were separated by 10%
SDS-PAGE. The gel was transferred onto a nitrocellulose membrane and
probed with the antihuman UGT1A common carboxyl-terminus region
antiserum RC71 (1:2000 dilution) as previously reported (9). The same
blot was subsequently probed with the EL93 anti-UGT2B antiserum (1:3000
dilution) as previously reported (27). A goat
antirabbit IgG antibody conjugated with horseradish peroxidase
(Amersham Pharmacia Biotech, Oakville, Canada) was used as
the second antibody, and the resulting immunocomplexes were visualized
using an enhanced chemiluminescence kit (Renaissance, Québec,
Canada) following the manufacturer’s instructions and exposed on
Hyperfilm (Eastman Kodak Co., Rochester, NY).
Glucuronidation assay using tissue homogenate preparations
Fresh tissues were homogenized with a Polytron (Brinkmann Instruments, Inc., Westbury, NY) in phosphate buffer (20
mM KH2PO4, 0.25
M sucrose, and 1 mM EDTA, pH 7.5) containing protease
inhibitors (1 mM dithiothreitol, 2.5 µg/ml pepstatin, and
0.5 µg/ml leupeptin) and centrifuged at 1,000 x g
for 15 min at 4 C to remove cell debris. Then tissue homogenates were
stored at -80 C at about 10 mg protein/ml. The protein content of
tissue homogenates was measured by the method of Bradford. 3-Diol,
ADT, and 4OHE2 were solubilized in ethanol, and
eugenol was solubilized in chloroform. Glucuronidation activity was
determined with 24 tissues in duplicate with the 4 substrates in the
presence of 15 µM
[14C]UDPGA, 500 µM
unlabeled UDPGA, 200 µM aglycone, 50
mM Tris-HCl (pH 7.5), 10 mM
MgCl2, and 100 µg/ml phosphatidylcholine in a
final volume of 100 µl. All tissues were from male monkeys, except
for female liver, ovary, mammary gland, uterus, and vagina. Incubation
times at 30 C were 15 min for liver, gall bladder, kidney, stomach,
duodenum, jejunum, ileum, and cecum and 2 h for ovary and adipose
tissues. For all other tissues the reaction time was 1 h.
Concentrations of homogenate proteins during the assays were 10 µg
for the liver; 50 µg for gall bladder, kidney, stomach, duodenum,
jejunum, ileum, cecum, and adipose tissue; and 150 µg for all other
tissues. Some preliminary experiments were performed to assess the time
of the reaction and the protein quantity conducting to linear
conjugation activity with these conditions. Assays were terminated by
adding 100 µl 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 to TLC plates (0.25-mm-thick silica gel;
Whatman, Maidstone, UK) and chromatographed in a solvent
composed of toluene-methanol-acetic acid (7:3:1). The TLC plates were
exposed for 4 days, and the extent of glucuronidation was assessed by
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale,
CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Also, the oligonucleotides used for
amplification of UGT2B mRNAs were designed to be specific for all of
the monkey UGT2B transcripts identified to date (Fig. 1C). By this very
sensitive method, the expression of UGT1A and UGT2B transcripts was
detected in all of the tissues analyzed, including steroid target
tissues such as prostate, ovary, mammary gland, skin, and testis (Fig. 1A). Oligonucleotides specific for the GAPDH transcript were used as a
positive control for the RT-PCR reactions and yielded a product of
appropriate length with each RNA sample (Fig. 1).
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). The polyclonal antibody that was
raised against the common carboxyl region of the human UGT1A proteins
demonstrated UGT1A expression in the microsome preparations of all
tissues examined, except testis, HK293 cells, and HK293 cells
expressing UGT2B19. As expected, the antiserum recognized proteins of
55 kDa, which is the appropriate size for UGT1A enzymes (9).
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Glucuronidation activity in monkey tissue homogenates
To determine the capacities of different monkey tissues to express
glucuronidation activity, enzyme assays were performed using tissue
homogenates and the four substrates: ADT and 3-Diol (androgen
metabolites), 4OHE2 (estrogen metabolite), and
eugenol, which is an exogenous compound conjugated by all of the
mammalian UGT enzymes characterized to date. In this study ADT and
3-Diol were chosen as substrates because they are the predominant
glucuronidated androgen metabolites found in human plasma; and
4OHE2 was used because it is apparent that all
mammalian UGT enzymes characterized to date that can conjugate
4OHE2 can also conjugate other
C18 steroids (estrogens). All of the tissues
examined demonstrated detectable glucuronidation activity with the four
substrates (Table 1 and Fig. 3). The liver contains the highest
enzymatic activity for the four substrates, followed by the
gastrointestinal tract and kidney, which have activities at least
5-fold lower than the liver. The other tissues express low
glucuronidation activities, which were less than 2% of the levels
found in liver.
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). To
further ascertain the capacity of the tissues to glucuronidate the
steroid substrates, the ratio of steroid-glucuronide formation
vs. eugenol-glucuronide (eugenol-G) formation was assessed
(Fig. 4 and Table 1). In general,
nonsteroidal tissues such as liver, kidney, and gastrointestinal tract,
have low ratios due to the low level of steroid glucuronidation
relative to the higher eugenol conjugation, whereas the steroid target
tissues have higher ratios. The ratio of 3-Diol-G/eugenol-G is
elevated in homogenates of pancreas, heart, scrotum, testis, prostate,
seminal vesicle, ovary, mammary gland, and uterus compared with that in
liver and gut. A very similar pattern was obtained for the ratio of
ADT-G/eugenol-G, with the exception of mammary gland, vagina, and
uterus, which had lower ratios than female liver. The ratio of
4OHE2-glucuronide
(4OHE2-G)/eugenol-G shows significant differences
compared with the two androgen metabolites because many more tissues
had a higher ratio than the liver, including steroid target tissues
(skin, uterus, vagina, mammary gland, ovary, seminal vesicle, prostate,
and testis) and other tissues (kidney, cecum, colon, pancreas, spleen,
lung, heart, and brain). Interestingly, the ratio of ADT-G/eugenol-G
was higher than the ratio of 4OHE2-G/eugenol-G in
homogenates of scrotum, testis, prostate, and ovary.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reduced
C19 steroid glucuronides among nine mammalian
species, it was found that humans and simians are unique in having
significant levels of ADT-G and 3-Diol-G (25). Both species were
demonstrated to express UGT1A and UGT2B transcripts, which encode
steroid-conjugating enzymes, in extrahepatic tissues (9, 29). The
cloning and characterization of human and monkey UGT1A and UGT2B cDNAs
demonstrated that the encoded proteins have conserved primary
structures, which range from 74–95% identity. Orthologous UGT1A and
UGT2B proteins have been identified from the two species, which have
similar substrate specificities, and biochemical properties (9, 27, 28, 29, 30). In addition to the glucuronidation proteins, the cloning and
characterization of other steroidogenic enzymes, such as 5-reductase
or 3ß-hydroxysteroid dehydrogenase, from the rhesus monkey
demonstrated high homology in primary structure and very similar
biochemical properties compared with the human proteins (32, 33).
Similarly to humans, monkeys express the same enzymes capable of
converting dehydroepiandrosterone sulfate to the active sex steroids
dihydrotestosterone and estradiol in steroid target tissues (34, 35).
Thus, taken together these data indicate that the monkey is an
appropriate animal model in which to study steroidogenesis and the role
of steroid glucuronidation in extrahepatic steroid target tissues. In this study the expressions of UGT1A and UGT2B transcripts were assessed by RT-PCR using two pairs of oligonucleotides designed to amplify all simian UGT mRNAs known to date, and both families of UGT transcripts were detected in the 24 different monkey tissues examined. It is interesting that previous tissue distribution experiments also performed by RT-PCR using oligonucleotides specific for each of the known UGT2B transcripts did not detect any expression in lung and spleen (26, 27, 28, 29, 30). Therefore, these results suggest that there exists novel uncharacterized simian UGT2B transcripts expressed in these 2 tissues.
As previously demonstrated in humans (17, 18, 36), the liver, digestive tract, and kidney are major glucuronidation tissues that express high levels of UGT proteins. These results are consistent with the important roles of these organs in the elimination of endogenous and exogenous compounds in detoxification processes. Interestingly, the jejunum and colon express readily detectable levels of UGT1A protein, but significantly lower levels of UGT2B protein in Western blot analysis. This is in contrast to tissues such as liver, kidney, uterus, breast, and prostate, which express detectable levels of protein from both families. Therefore, it is apparent that the majority of the glucuronidation activities seen in the jejunum and colon involve UGT1A enzymes. However, it is noteworthy that the expression of UGT2B proteins is demonstrated in specific cell types of the simian jejunum and colon by immunohistochemistry analysis (Barbier, O., et al., unpublished data).
Although the liver, digestive tract, and kidney are major sites of
glucuronidation, it has become evident that steroid target tissues such
as ovary and prostate express steroid-specific UGT enzymes and excrete
glucuronidated steroids (17, 22, 24). The tissue distribution of human
(UGT2B7, UGT2B15, and UGT2B17) and simian (UGT2B9, UGT2B18, UGT2B19,
UGT2B20, and UGT2B23) UGT2B transcripts demonstrated the expression of
specific mRNAs in various extrahepatic steroid target tissues (24, 26).
The encoded proteins of the various UGT2B transcripts in these tissues
have overlapping, but distinct, patterns of specificity for steroid
glucuronidation. As found in humans, there exist to date more monkey
UGT2B than UGT1A enzymes that are active on the glucuronidation of
C19 steroids, whereas there are more UGT1A than
UGT2B proteins active on C18 steroids. For
example, all five of the monkey UGT2B enzymes characterized to date
catalyze the glucuronidation of 3-Diol, and three of them (UGT2B9,
UGT2B18, and UGT2B23) are highly active on ADT. However, UGT2B9 and
UGT2B19 can conjugate 4OHE2, but with only
moderate activity (26, 27, 28, 29). In contrast, human UGT1A3, UGT1A8, and
UGT1A9 can glucuronidate 4OHE2 with high
activity; only UGT1A4 is moderately active on ADT or 3-Diol, whereas
UGT1A3 and UGT1A9 have low activity on these C19
steroids. Despite having different specificities for steroid
substrates, all of the human and monkey UGT enzymes that have been
characterized to date are active on eugenol.
It is apparent that extrahepatic steroid target tissues have less glucuronidation capacity than the liver, and although all of the tissues we examined can conjugate eugenol, it is striking that some of these tissues, such as heart, scrotum, prostate, seminal vesicle, and ovary, can more readily conjugate steroid metabolites than eugenol, in contrast to liver, kidney, and gastric tract. To compare the preponderance of a given tissue to specifically conjugate steroid substrates relative to the overall glucuronidation capacity of the tissue, the ratio of steroid-G/eugenol-G formation in tissue homogenates was obtained. This ratio is higher in steroid target tissues such as seminal vesicle, prostate, and ovary than in liver and gut. One possible explanation for these results is a higher relative expression of steroid-conjugating UGT enzymes in steroid target tissues, whereas the other tissues express UGT enzymes less involved in steroid metabolism. However, it is also possible that steroid target tissues express specific modulators or contain an environment to posttranslationally modify and increase or induce the steroid-conjugating capacity of UGT enzymes. The formation of dimers between two UGT polypeptides has been reported, and it has been suggested that these interactions may alter UGT enzyme substrate specificity and/or kinetic properties (37, 38, 39).
The steroid-G/eugenol-G ratio is highly elevated, particularly in the
heart, seminal vesicles, and ovary, where steroids have several
physiological roles. Androgens synthesized in thecal cells of the
ovaries are trophic factors for granulosa cell growth, and they also
serve as precursors for estrogen synthesis via P450 aromatase. In
addition, it has been shown that high androgen levels in the ovary are
associated with pathologies such as polycystic ovary syndrome. The
finding of significant glucuronidation activity in ovary homogenate is
consistent with previous results showing high levels of ADT-G and
3-Diol-G in human follicular fluid (22). In heart and seminal
vesicles, the ratio of 4OHE2-G/eugenol-G is
higher than the ratios with 3-Diol-G and ADT-G. Although the role of
estrogens in these two tissues is not well understood, they both have
been found to express estrogen receptors (ER and ERß). In the rat,
antiestrogen treatment decreased the size of the seminal vesicles
(40, 41, 42), whereas treatment of guinea pig cardiomyocytes with estradiol
led to a significant decrease in Ca2+ current
across the cell membrane (43, 44). In all three of these tissues, the
elimination of estrogens and androgens by glucuronidation is a
potentially important process required to maintain normal
homeostasis.
A comparison between the ratios of 4OHE2-G/eugenol-G and C19 steroid-G/eugenol-G, demonstrates that 18 of the 24 tissues examined have a greater capacity to glucuronidate 4OHE2 (C18 steroid, and catechol estrogen) than the androgen metabolites. This was observed in tissues of the gastrointestinal tract and may reflect the higher expression of UGT1A proteins relative to UGT2B in these tissues, and the fact that more of the UGT1A enzymes conjugate C18 rather than C19 steroids. However, the greater capacity to glucuronidate 4OHE2 was also observed in estrogen-responsive tissues such as the mammary gland, uterus, and vagina, where a potential physiological role of estrogen glucuronidation in steroid target tissues is to terminate the estrogen response and eliminate C18 steroids from the tissue. However, another potential role of catechol estrogen glucuronidation is to inactivate and eliminate these potentially genotoxic steroid metabolites from a given tissue and prevent cell damage (45, 46, 47). Catechol estrogens can undergo metabolic redox cycling catalyzed by P450 enzymes, where the hydroperoxide-dependent oxidation of catechol estrogens to quinones and the NADPH-dependent reduction of the quinones back to hydroquinones yield semiquinone-free radical intermediates and superoxide radicals (48). The continuous generation of free radicals by the redox cycle has 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 (45, 48, 49). This potential problem of catechol estrogens is particularly relevant in estrogen-sensitive tissues such as breast, ovary, and uterus, which express steroidogenic enzymes, including aromatase required for estrogen synthesis, and enzymes such as cytochrome P4501B1, which yield catechol estrogens (50, 51, 52, 53).
When comparing the ratios obtained with ADT and 3-Diol, the results
show that ADT-G/eugenol-G is higher in 13 tissues,
3-Diol-G/eugenol-G is higher in 8 tissues, and the 2 androgen
metabolites have similar ratios in 4 tissues. In tissues from the
gastrointestinal tract, the ratio of 3-Diol-G is higher or at least
equal to that of ADT-G. The same was observed with estrogen target
tissues such as the mammary gland, uterus, and vagina. It is tempting
to speculate that the higher capacity to conjugate 3-Diol than ADT
is due to the relatively greater expression of UGT1A than UGT2B
proteins in these tissues, and the fact that a protein such as UGT1A4
has a 4-fold higher activity on 3-Diol than ADT (13). However, it
cannot be excluded that this can also be due to the expression of UGT2B
enzymes such as UGT2B19 and UGT2B20, which conjugate 3-Diol and not
ADT (27, 30).
The ratio of ADT-G/eugenol-G is higher than that of
3-Diol-G/eugenol-G in gallbladder, pancreas, scrotum, testis, and
prostate. There are several human (UGT2B17) and monkey (UGT2B9,
UGT2B18, and UGT2B23) enzymes that conjugate both ADT and 3-Diol and
are expressed in these tissues, and it is interesting that they all
have a higher activity on ADT than 3-Diol. The prostate demonstrates
a greater capacity to conjugate ADT (a C19
steroid) than 4OHE2 and 3-Diol, which is in
agreement with previous results demonstrating that the prostate
produces more ADT-G than 3-Diol-G (21). Androgens have multiple
physiological roles in the human prostate, which include regulation of
cell proliferation and expression of prostate-specific antigen.
Therefore, a potential role for androgen glucuronidation in this tissue
would be to regulate the androgen response and promote androgen
elimination into the circulation. The role of UGT enzymes remains to be
determined in pathologies such as androgen-responsive prostate cancer,
where androgens have a deleterious effect and abnormally increase cell
proliferation.
Using the monkey as an animal model, this study demonstrates the expression of UGT1A and UGT2B transcripts, proteins, and enzyme activities in the liver and many extrahepatic tissues. The expression of these proteins in steroid target tissues such as ovary, mammary gland, skin, and prostate is consistent with the hypothesis that steroid conjugation by UGT enzymes plays an important physiological role to terminate the steroid response, increase steroid removal from the tissue, and ultimately increase conjugated steroid elimination from the circulation via the hepatic and urinary systems. By comparing the capacities of different tissues to conjugate steroids vs. their capacities to conjugate eugenol, it is apparent that steroid target tissues have a greater preponderance to conjugate steroids than a nonsteroid target tissue such as liver or kidney. In agreement with this concept, UGT2A enzymes that are more specific for conjugating odorant compounds are expressed predominantly in the olfactory epithelium. However, due to the expression of multiple UGT enzymes in a given tissue, it will be important to determine the contribution of a given enzyme to the overall glucuronidation capacity and specificity of a tissue. There is strong evidence of cell type-specific expression (Barbier, O., et al. unpublished results) and the differential regulation of expression of different UGT enzymes with overlapping substrate specificities (8, 54) in a single tissue. In addition to the expression of specific UGT enzymes in a tissue, it is possible that the glucuronidation capacity and specificity of a tissue may be dependent on heterologous dimer formation and posttranslational modifications such as N-glycosylation.
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Acknowledgments |
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Footnotes |
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2 Recipient of a scholarship from the Medical Research Council of
Canada.
Received December 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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