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Vol. 298, Issue 1, 49-56, July 2001
Institute of Experimental Physiology, School of Biochemical and Pharmaceutical Sciences, Rosario, Argentina (M.G.L., V.A.C., E.J.S.P., L.M.V., J.M.P., A.D.M.); Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky (T.H., M.V.); and Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo, Japan (S.I., Y.E., T.I.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The molecular basis of perinatal changes occurring in major
UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a
relevant isoform belonging to family 2, was analyzed in rat liver.
Nonpregnant, pregnant (19-20 days of pregnancy), and two groups of
postpartum animals corresponding to early and middle stages of
lactation (2-4 and 10-12 days after delivery, respectively) were
studied. UGT activity determined in
UDP-N-acetylglucosamine-activated microsomes revealed
that bilirubin, p-nitrophenol, and ethynylestradiol (17
-OH and 3-OH) but not androsterone and estrone glucuronidation rates, were decreased in pregnant rats. Decreased enzyme activities returned to control values after delivery.
p-Nitrophenol, androsterone, and estrone conjugation
rate increased in postpartum rats. Western blot analysis performed with
anti-peptide-specific (anti-1A1, 1A5, 1A6, and 2B1) antibodies revealed
decreased levels of all family 1 isoforms and UGT2B1 during pregnancy.
In postpartum animals, protein level recovered (1A5 and 2B1) or even
increased (1A1 and 1A6) with respect to control rats. Northern blot
analysis suggested that expression of UGT proteins is down-regulated at
a post-translational level during pregnancy and that increased levels
of 1A1 and 1A6 observed in postpartum rats were associated to increased
mRNA. To establish whether prolactin is involved in up-regulation of UGT1A1 and 1A6 postpartum, ovariectomized rats were treated with 300 µg of ovine prolactin per day for 7 days. The data indicated that
prolactin was able to increase expression of UGT1A6 (protein and mRNA)
but not 1A1. Thus, prolactin is the likely mediator of the increased
expression of UGT1A6 observed in maternal liver postpartum.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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UDP-glucuronosyltransferases
(UGTs) represent a superfamily of enzymes that catalyze the conjugation
of glucuronic acid to both endogenous compounds, including bilirubin,
bile acids, and steroid and thyroid hormones, and exogenous compounds,
including food additives, drugs, and environmental pollutants (for
review, see Tephly and Burchell, 1990
). Based on the nucleotide and
amino acid sequences, UGT isoforms in mammals are grouped into two
major families termed 1 and 2. Enzymes belonging to family 1, including a bilirubin cluster (UGT1A1 and UGT1A5) and a phenol cluster (UGT1A6 and UGT1A7), are formed by alternative splicing of an isoform-specific exon encoding a unique N-terminal region with common exons 2 to 5 encoding an identical C-terminal region. In contrast, UGT family 2 isoforms are each derived from an individual gene. The activity of UGT
is affected by many factors, including enzyme inducers, aging, diet,
diseases, and hormones (for review, see Tephly and Burchell, 1990;
Miners and Mackenzie, 1991; Burchell et al., 1994). Regulation may
occur either at the gene transcription level, resulting in changes in
mRNA and protein levels, or at the level of post-translational processing. Because of association of UGT with the lipid environment (Zakim and Dannenberg, 1992), restriction of the cosubstrate
UDP-glucuronic acid (UDPGA) to access the enzyme active site (Berg et
al., 1995; Bossuyt and Blanckaert, 1995) and protein-protein
interactions derived from oligomer formation (Peters et al., 1984;
Ikushiro et al., 1997; Meech and Mackenzie, 1997), enzyme activity
toward a specific substrate may also depend on the functional state of UGT.
Many studies have been conducted to determine UGT activity in
experimental animals during pregnancy. This subject is of particular interest because changes in metabolizing enzyme activities in maternal
liver may change the risk of exposure by the fetus. During pregnancy,
significant hormonal changes take place, mainly in sex steroids levels,
that may substantially affect disposition of potentially toxic
compounds. Most of the studies performed in rats reported a decrease in
liver UGT activity (expressed per milligram of microsomal protein)
compared with nonpregnant controls, affecting major family 1 substrates
such as bilirubin and phenol derivatives and some family 2 substrates
such as estradiol (Halac and Sicignano, 1969; Neale and Parke, 1973;
Vore and Soliven, 1979; Muraca et al., 1984; Borlakoglu et al., 1993).
It is reasonable to speculate that factors, such as those modulating
the functional state of UGT, may be affected in such a way that they
similarly decrease the activity of all the isoforms tested. We
previously reported that substantial changes in microsomal lipid
composition and membrane fluidity occur perinatally but, in contrast to
what is expected, they positively modulate UGT activity (Luquita et al., 1994). Consequently, a decrease in the expression of UGTs is more
likely involved in down-regulation of UGT activities during pregnancy.
At present, no studies have been conducted to clarify the molecular
basis of UGT regulation during pregnancy.
We reported that hepatic UGT activity toward planar phenols is
increased in female rats during lactation, particularly at the late
stage of lactation (19-21 days after delivery) (Luquita et al., 1994).
Using a polyclonal nonspecific antibody developed against a
phenol-conjugating isoform, we detected an increased content of UGT
protein in ovariectomized rats in response to ovine prolactin
treatment, suggesting that this hormone may be involved in the
regulation of UGT expression postpartum (Luquita et al., 1996). We
postulated that UGT induction, together with an increased bile
secretory function in lactating rats (Liu et al., 1992), may be an
adaptive response to increase the biliary excretion of toxic compounds,
thus preventing their secretion into breast milk. Taken together, the
evidence suggests a differential regulation of UGT in pregnancy and
postpartum. Immediately after delivery, levels of progesterone and
estrogens are dramatically decreased, and other hormones, i.e.,
prolactin, are greatly increased. In consequence, differences in UGT
activities in maternal liver between pregnant and lactating rats are
most reasonably associated with the various hormonal actions. The
activity and protein expression of the different UGT isoforms,
particularly those involving family 1 substrates, have not been
determined in postpartum rats or in response to prolactin administration.
In the present study we analyzed the molecular basis of changes occurring in UGT family 1 isoforms (1A1, 1A5, 1A6, and 1A7) and in UGT2B1, a relevant isoform belonging to family 2, in rat liver at late pregnancy and postpartum. For this purpose, enzyme activity toward the classical substrates of the different isoforms and protein and mRNA levels were systematically determined. The role of prolactin on activity and expression of UGTs from family 1 was also analyzed.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals.
[9,11-3H]Androsterone
(56.5 Ci/mmol), [2,4,6,7-3H]estrone (76.5 Ci/mmol), and 17
-[6,7-3H
(N)]ethynylestradiol (49.1 Ci/mmol) were from PerkinElmer Life Science
Products (Boston, MA). Bilirubin, UDPGA (ammonium salt), UDP-N-acetylglucosamine (UDP-N-AG),
p-nitrophenol, D-saccharic acid
1,4-lactone, and nonlabeled steroids (androsterone, estrone, and
ethynylestradiol) were purchased from Sigma Chemical Co. (St. Louis,
MO). Bromocriptine (2-Br--ergocriptine methane sulfate) was a gift
of Sandoz Research Institute (East Hanover, NJ). Ovine prolactin
(NIDDK-ovine prolactin-19; AFP-9221A) was kindly provided by National
Institute of Diabetes and Digestive and Kidney Diseases, National
Hormone and Pituitary Program, National Institute of Child Health and
Human Development, and U.S. Department of Agriculture. All other
reagents were of the highest grade commercially available.
Animals. Female Sprague-Dawley rats (Harlan Industries, Indianapolis, IN) were used throughout. All procedures involving animals were conducted in accordance with National Institutes of Health guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Kentucky. The pregnant and postpartum rats were timed according to the first day that sperm were detected (day 0). The rats had free access to Purina Rat Chow and water and were maintained on a 12-h automatically timed light and dark cycle. Nonpregnant rats (180-210 g) served as controls, and rats at 19 to 20 days of pregnancy (360-400 g) and at 2 to 4 days (240-270 g) and 10 to 12 days (250-280 g) postpartum [pp (2-4 days) and pp (10-12 days) groups] were used as late-pregnant and early- and mid-lactating rats, respectively. Litter size in postpartum animals ranged from 8 to 10 pups.
Two groups of ovariectomized rats weighing 190 to 230 g were implanted with osmotic minipumps (Alzet 2001; Alza, Palo Alto, CA) attached to an intravenous catheter as described by Liu et al. (1992).
The minipumps were filled with solvent (0.4 M
NaHCO3, 1.6% glycerol, 0.02% sodium azide)
(control group) or with solvent plus ovine prolactin to yield an
infusion rate of 300 µg of ovine prolactin per day for 7 days
(prolactin group). The minipumps were immersed in saline at 37°C to
ensure that flow had started. To suppress endogenous prolactin
secretion, all the rats were implanted with bromocriptine pellets (7.5 mg, 10 day-release) subcutaneously at the time of implantation of the minipumps.
Enzyme Assays.
All animals were killed by decapitation
between 9 and 11 AM, to avoid possible effects of diurnal variations.
Liver samples were collected (Luquita et al., 1994) and a portion snap
frozen in liquid nitrogen for RNA analysis. Liver homogenate (25% w/v) was prepared in 0.15 M Tris-HCl buffer, pH 7.4 and the corresponding microsomal fractions were obtained by ultracentrifugation at
105,000g for 1 h at 4°C as previously described
(Siekevitz, 1962). Protein concentration in microsomal preparations was
measured using the Lowry method (Lowry et al., 1951) with bovine serum
albumin as standard.
; Catania et al.,
1995). Glucuronidation rate of the neutral steroids androsterone and
estrone were determined according to Rao et al. (1977) and conjugating
activity of the synthetic derivative of estradiol ethynylestradiol
(3-OH and 17-OH conjugation) was determined under the same
incubation conditions (1.0 mM final concentration). In all cases, high
performance liquid chromatography was performed to separate the
corresponding steroid glucuronides. The mobile phase, consisting of
methanol/0.1 M KH2PO4, pH
4.5 (60:40, v/v) was the same as previously used for bile salts
separation (Tietz et al., 1984). Detection of radioactive unconjugated
and conjugated steroids was accomplished with an IN/US -RAM
flow-through detector (Pine Brook, NJ). The peaks corresponding to
glucuronides were confirmed by including a sample preincubated with
-glucuronidase. An ethynylestradiol 17-glucuronide standard
(generous gift of Dr. Brian Burchell, Department of Biochemical
Medicine, Ninewells Hospital, Dundee, UK) was used to discriminate
between 17- and 3-derivatives of ethynylestradiol.
D-Saccharic acid 1,4-lactone (2 mM) was
systematically included in all the incubation media to inhibit
enzymatic hydrolysis of glucuronides. Reactions were initiated with the
addition of UDPGA to the mixtures.
Western Blot Analysis.
Polyclonal anti-peptide antibodies
that specifically recognize the 1A1, 1A5, 1A6, and 1A7 isoforms
belonging to UGT family 1 as well as an antibody developed against a
peptide common to all isoforms of the same group (1A) (Ikushiro et al.,
1995) and a specific antibody against isoform 2B1 (UGT family 2)
(Ikushiro et al., 1997) were used in Western blot studies.
) and subjected to
electrophoresis. After electrotransfer onto nitrocellulose membranes
(Protran; Scheleicher & Schuell, Keene, NH), the blots were blocked at
least for 2 h at 4°C with Tris-buffered saline containing 0.1%
Tween 20 and 5% nonfat dry milk and then incubated overnight with the
primary antibodies 1A (1:4000), 1A1 (1:4000), 1A5 (1:1000), 1A6
(1:1000), or 1A7 (1:500). The immune complex was detected by incubation
with alkaline phosphatase-linked anti-rabbit secondary antibody
(1:2000; Sigma Chemical Co.) for 1 h. Immunoreactive bands were
detected by the alkaline phosphatase color reaction using
5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium and
quantified by densitometry (Shimadzu CS-9000; Shimadzu, Kyoto, Japan).
Northern Blot Analysis.
Probes consisting of 293-, 308-, and
317-base pair cDNA fragments were amplified from rat liver RNA by
reverse transcription-polymerase chain reaction (Access TR-PCR System;
Promega, Madison, WI) using the same primers and conditions reported in
Emi et al. (1995) to exons B1 (UGT1A1), B5 (UGT1A5), and A1 (UGT1A6),
respectively. Exon-specific oligonucleotide primers were synthesized by
Bio-Synthesis (Lewisville, TX). In preliminary experiments, the probes
thus obtained were able to detect specific induction of different UGT isoforms in response to treatment with clofibrate (UGT1A1 and 1A5) and
methylcholanthrene (UGT1A6), as was previously described (Emi et al.,
1995). The content of mRNA encoding the family 2 isoform UGT2B1 was
analyzed using a full-length rat probe (Mackenzie, 1986), which was
generously provided by Dr. Peter Mackenzie (Flinders University of
South Australia, Bedford Park, Australia). A single-stranded 26-mer
oligoprobe to 28S rRNA (Barbu and Dautry, 1989) was synthesized by
Integrated DNA Technologies (Coralville, IA).
).
Total RNA (15 µg) was denatured, electrophoresed through a 1.2%
agarose/formaldehyde gel, transferred to a nylon membrane (Hybond-N;
Amersham Pharmacia Biotech UK Limited, Little Chalfont,
Buckinghamshire, England) overnight by capillary blotting in 6× SCC
and baked at 80°C for 2 h. To analyze the content of mRNA
encoding UGTs, the membranes were prehybridized in 5× SSC, 0.02% SDS,
0.1% N-laurylsarcosine, and blocking reagent (catalog no.
1096176; Roche Molecular Biochemicals, Mannheim, Germany) at
68°C for at least 30 min. Hybridization was performed at 68°C for
16 h after addition of labeled cDNAs (UGT1A1, 1A5, 1A6, and 2B1).
The membranes were then washed in 2× SSC, 0.1% SDS twice at room
temperature for 5 min, followed by washing in 0.1× SSC, 0.1% SDS
twice at 68°C for 15 min under constant agitation. UGT probes were
labeled with digoxigenin-dUTP by a random-primed methodology and
detected with an alkaline phosphatase-conjugated antibody followed by
chemiluminescent reaction (kit 1585614; Roche Molecular Biochemicals).
Differences in RNA loading were corrected after reprobing the stripped
blots with 28S RNA. For this purpose, the membranes were prehybridized
in 5× SSC, 0.1% SDS, 0.5% dextran sulfate (mol. wt. = 500,000) and
blocking reagent (catalog no. RPN 5770; Amersham Pharmacia Biotech,
Piscataway, NJ) at 42°C for at least 30 min. Hybridization was
performed at 42°C for 2 h after addition of the labeled
oligoprobe. The membranes were then washed in 5× SSC, 0.1% SDS twice
at room temperature for 5 min, followed by washing in 1× SSC, 0.1%
SDS twice at 42°C for 15 min under constant agitation. The 28S
oligonucleotide was labeled with fluorescein-dUTP using a 3'-end
labeling kit (RPN5776) and detected with an alkaline
phosphatase-conjugated antibody followed by chemiluminescent reaction
(kit RPN3510; Amersham Pharmacia Biotech). In all cases the blots were
exposed to Bio-Max MR-2 film (Sigma Chemical Co.) for various time
periods until the autoradiographic signal was optimal. The
hybridization bands were then quantified by densitometry (Shimadzu
CS-9000; Shimadzu). The densitometry was done in the linear range of
the film.
Statistical Analysis. Data on enzyme activities and densitometric analysis of Western and Northern studies were presented as means ± standard deviation. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni test (pregnancy and postpartum studies) or Student's t test (prolactin studies). Values of p < 0.05 were considered to be statistically significant.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Expression of UGTs in Maternal Rat Liver
UGT Activities.
Enzyme activities toward the different
substrates were determined in the presence of UDP-N-AG, which is
considered a physiological activator of UGTs (Berg et al., 1995;
Bossuyt and Blanckaert, 1995). In addition, under this experimental
condition, no substantial disturbance of the membrane environment that
could affect catalytic activity of UGTs is expected. Figure
1 shows activity values for bilirubin,
p-nitrophenol, and ethynylestradiol conjugated in position 3-OH, substrates primarily associated with isoforms of the UGT1 family,
and for androsterone, estrone, and ethynylestradiol conjugated in
position 17-OH as substrates that react preferentially with isoforms
of the UGT2 family (Ebner et al., 1993; Mackenzie et al., 1997). All
substrates associated with UGT1 as well as ethynylestradiol in position
17-OH exhibited a decreased conjugating activity (55% in average)
in pregnant rats, whereas androsterone and estrone conjugation did not
change. The data clearly indicate that pregnancy differentially
affected conjugation of substrates associated with UGT family 2 isoforms.
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Immunoblotting Analysis.
Characterization of expression of the
different UGT isoforms by means of specific anti-peptide antibodies
showed a decrease in the intensity of immunoreactive bands in pregnant
rats for all the isoforms tested (Fig.
2). The data clearly indicate that the
decreased level of UGT protein in microsomes is the main cause of
decreased activities reported above. Within 2 to 4 days after delivery,
most of family 1 isoforms as well as UGT2B1 tended to increase their
levels, and within 10 to 12 days of delivery, they approached or
exceeded values in control rats. In fact, protein content corresponding
to 1A1 and 1A6 isoforms was significantly increased with respect to
controls in the pp (10-12 days) group. Under the current experimental
conditions, the 1A7 isoform was not detected in any of the groups
analyzed (data not shown).
|
-OH (Mackenzie et al., 1997). Assuming that these same
isoforms participate in the glucuronidation of ethynylestradiol at the
17-OH position, the decrease in content of the 2B1 isoform observed
during pregnancy (Fig. 2) may explain down-regulation of UGT activity
toward ethynylestradiol (Fig. 1). After delivery, both the expression
of UGT2B1 and glucuronidation of ethynylestradiol followed a parallel
recovery and gradually returned to control levels along the lactating
period. Glucuronidation of androsterone and estrone showed a different
pattern of variation along the different perinatal stages compared with
ethynylestradiol 17-OH conjugation (Fig. 1), indicating differential
regulatory features for the different family 2 isoforms in maternal
liver. Although pregnancy did not affect enzyme activity toward
androsterone and estrone, postpartum rats exhibited a significant
increase, particularly at mid-lactation. Additional studies are
necessary to confirm whether an increase in expression of the isoforms
involved in glucuronidation of these neutral steroids (e.g., UGT2B2) is responsible for the increased enzyme activity in microsomal membranes.
Analysis of mRNA Encoding UGTs.
Figure
3 shows liver content of mRNA encoding
the major UGT1 isoforms and the 2B1 isoform. Interestingly, no change
was observed in mRNAs from pregnant rats in contrast to what was
observed for protein levels. Within 10 to 12 days after delivery,
UGT1A1 and 1A6 mRNA content significantly increased with respect to
normal females (40 and 120%, respectively), agreeing well with
increased level of protein. Figure 3 also shows that the levels of mRNA encoding UGT1A5 and 2B1 did not change in maternal liver postpartum.
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Effect of Prolactin on Expression of UGTs
UGT Activities.
Because postpartum rats exhibited an increase
in the expression of the isoforms involved in conjugation of bilirubin
and p-nitrophenol, it was of interest to establish whether
the lactogenic hormone prolactin is involved. Prolactin was
administered at a dose that was shown to maximally increase the
expression of Na+-taurocholate cotransport
polypeptide (ntcp) (Liu et al., 1995) and p-nitrophenol UGT
(Luquita et al., 1996) in the rat liver and to increase the P1 subunit
of glutathione in the rat intestine (Luquita et al., 1999), thus
accounting for increases observed in postpartum rats. Figure
4 shows the activity of UGT toward bilirubin and p-nitrophenol in ovariectomized control and
ovine prolactin-treated rats. Only p-nitrophenol conjugation
was affected by hormone administration, exhibiting a 60% increase with
respect to controls.
|
Immunoblotting Analysis.
Figure
5 shows the effect of ovine prolactin on
the level of the isoforms of UGT involved in bilirubin (UGT1A1 and 1A5)
and p-nitrophenol (UGT1A6) conjugation. Only the content of
UGT1A6 was increased by the hormone (about 50% over controls), in
agreement with the increase in activity of p-nitrophenol
conjugation. Neither UGT1A1 nor UGT1A5 was affected by ovine prolactin.
As expected, the bands detected with anti-UGT1A, which mainly reflects
the level of the isoforms involved in bilirubin conjugation (Ikushiro et al., 1995), were not altered by the hormone. UGT1A7 was not detected
in any of the groups analyzed (data not shown).
|
Analysis of mRNA Encoding UGTs.
Figure
6 shows the level of mRNA encoding UGT1A1
and 1A6. Only UGT1A6 mRNA was increased in response to ovine prolactin
administration (about 80% over controls). The content of mRNA encoding
UGT1A5 was not affected by the hormone (data not shown).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Most studies of UGT activity toward several endogenous and
exogenous substrates report down-regulation of UGT-mediated reactions in liver in pregnancy (Halac and Sicignano, 1969; Neale and Parke, 1973; Vore and Soliven, 1979; Muraca et al., 1984; Borlakoglu et al.,
1993). Regulation of UGT activity in microsomes is multifactorial, and
depends not only on protein expression, but also on functional properties of the corresponding catalytic unit. Using polyclonal anti-peptide antibodies that specifically recognize the 1A1, 1A5, 1A6,
and 1A7 isoforms, we here demonstrate for the first time down-regulation of the expression of UGT family 1 isoforms as a main
cause of decreased UGT activity in maternal liver during pregnancy.
Expression of UGT2B1, an important member of UGT family 2, is also
decreased in pregnant rats, in agreement with impaired conjugation of
ethynylestradiol in position 17-OH.
Analysis of liver content of mRNA encoding UGT1 isoforms and UGT2B1 by
Northern blotting revealed that pregnancy did not substantially affect
their levels (Fig. 3). In consequence, an impairment in gene
transcription is unlikely the cause of protein down-regulation. Other
factors, such as impairment of protein synthesis, redistribution of
protein molecules into an additional membrane compartment (apart from
the endoplasmic reticulum), or an increase in protein degradation may
be involved. Additional studies are necessary to clarify our understanding of the mechanisms affecting UGTs regulation
post-translationally. It is interesting to note that expression of the
multidrug-resistant protein Mrp2, the main protein involved in
canalicular secretion of conjugated compounds (Keppler et al., 1997),
is also down-regulated in female rats during pregnancy (Cao et al.,
2001). In these rats, however, mRNA encoding Mrp2 is preserved. Phase
II enzymes acting coordinately with Mrp2-mediated secretion of
conjugated compounds across the apical domain of epithelial cells may
represent an important strategy to protect cells from chemical injury.
The molecular basis for the dissociation between protein and mRNA levels occurring during pregnancy for both UGT and Mrp2 is not known.
Treatment of male rats with 5 mg of ethynylestradiol per kilogram of
body weight for 5 days decreased Mrp2 protein level in liver plasma
membranes and was able to mimic dissociation between protein and mRNA
(Trauner et al., 1997). Treatment of female rats with twice this dose
of ethynylestradiol decreased UGT activity toward estradiol (3-OH and
17-OH groups) in a similar magnitude as was observed in pregnancy
(Connors and Vore, 1988). However, in contrast to what was reported for
pregnant rats, administration of estradiol or progesterone increased
bilirubin glucuronidation (Muraca et al., 1983). The same hormones
added to primary hepatocytes culture did not change the level of mRNA
encoding major isoforms of UGT belonging to family 1 and 2 (Li et al.,
1999). When administered simultaneously to ovariectomized rats,
estradiol and progesterone also did not decrease bilirubin UGT activity
(Muraca et al., 1984). Thus, hormonal treatments commonly used to mimic
hormonal changes occurring during pregnancy are not able to reproduce
alterations in UGT activity, indicating that complex and unknown
regulatory factors are involved.
The current data also indicate that during postpartum, UGT activities
recovered and even increased with respect to control females.
Particularly, p-nitrophenol-conjugating activity showed a
significant increase in the pp (10-12 days) group (Fig. 1). This is in
agreement with the previous finding in lactating rats at the late stage
of lactation (19-21 days postpartum) (Luquita et al., 1994). The
current study clearly demonstrates that increased expression of UGT1A6
is the likely explanation for the increase in planar phenol conjugation
observed in these rats. In fact, two major isoforms belonging to UGT
family 1, i.e., 1A6 and 1A7, are responsible for planar phenol
conjugation in the rat (Miners and Mackenzie, 1991; for review, see
Burchell et al., 1994). Using 1A6- and 1A7-specific antibodies, it was
previously demonstrated that 1A6, but not 1A7 isoform, is
constitutively expressed in rat liver (Ikushiro et al., 1995). In turn,
expression of UGT1A7 is more relevant in intestine, probably accounting
for conjugating activity toward planar phenols in this tissue. In
agreement with the previous observation, we did not find significant
expression of 1A7 isoform in liver from normal females and in addition,
neither pregnant nor postpartum animals exhibited any significant level of the 1A7 protein. The current study also demonstrates that an enhancement in the level of mRNA encoding UGT1A6 is involved in the
increased protein expression postpartum.
Using a polyclonal nonspecific antibody we previously demonstrated an
increased content of UGT protein in ovariectomized rats in response to
ovine prolactin treatment, suggesting that this hormone may be involved
in the regulation of UGT expression postpartum (Luquita et al., 1996).
In the second part of the current study, we demonstrated that prolactin
was able to specifically increase the expression of UGT1A6 isoform,
thus accounting for the increase in
p-nitrophenol-conjugating activity. The increase in the
level of UGT1A6 mRNA observed in these animals indicates increased gene transcription and/or stabilization of mRNA. Interestingly, ovine prolactin was not able to mimic the increase in expression of UGT1A1
observed in lactating rats. It is possible that other, unknown factors,
alone or in combination with prolactin, may be involved in
up-regulation of UGT1A1 postpartum. The mechanism of action of
prolactin on the liver tissue has been well characterized for
protein-mediated transport of taurocholate at the sinusoidal level. In
fact, it has been shown that prolactin increases the mRNA encoding the
ntcp in the rat liver (Liu et al., 1995) and that this occurs via
transcriptional regulation of the ntcp promoter by this
hormone (Ganguly et al., 1997). Thus, prolactin acts via the long form
of the hormone receptor in the hepatocyte to increase phosphorylation
and translocation of a signal transducer and activator of
transcription, Stat5, to the nucleus where it binds to
-interferon-activated sequence elements in the ntcp
promoter to increase gene transcription. Further studies are necessary
to determine whether a similar mechanism is involved in
prolactin-mediated increase in the expression of UGT1A6 in maternal rat
liver postpartum.
The analysis of regulation of bilirubin UGT in lactating rats revealed
a dissociation between protein and mRNA corresponding to UGT1A1, the
main isoform involved in pigment glucuronidation, and enzyme activity.
Particularly, it is noteworthy that even when UGT1A1 expression was
up-regulated (Figs. 2 and 3) in postpartum animals 10 to 12 days after
delivery, microsomal bilirubin glucuronidation was not affected (Fig.
1). This dissociation may be tentatively attributed to differences
between control and postpartum rats in either protein activity per
molecule or protein turnover. The former possibility would imply that
postpartum rats might express a number of immunologically reactive but
biologically inactive enzyme. Differences in catalytic activity of UGT
could result from complex interaction among different monomeric UGTs to
form active oligomers or from a particular influence of microsomal membrane environment in postpartum animals, since they critically affect the functional state of UGTs (Peters et al., 1984; Zakim and
Dannenberg, 1992; Ikushiro et al., 1997; Meech and Mackenzie, 1997).
In conclusion, we report a decrease in expression of UGT family 1 isoforms as a main cause of down-regulation of UGT-mediated reactions involving bilirubin and planar phenols during pregnancy. Down-regulation of UGT proteins likely occurs at the post-translational level, since mRNA levels were unchanged in control versus pregnant rats. After delivery, protein level from all isoforms gradually returned to control values or even increased (1A1 and 1A6) in association with increased mRNA levels. Prolactin administration was able to mimic the increase in UGT expression observed postpartum only for the 1A6 isoform.
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Acknowledgments |
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We thank Drs. Liyue Huang, Raquel Chan, Daniel Gonzalez, Silvina Fellitti, Lucrecia Alvarez, Cristian Magni, Silvina Pessino, and Javier Palatnik for technical assistance and valuable suggestions.
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Footnotes |
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Accepted for publication March 13, 2001.
Received for publication January 17, 2001.
This work was supported by Research Grants from Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario, and Subsecretaría de Investigación y Tecnología, Ministerio de Salud de la Nación, Argentina; and by U.S. Public Health Service Grants GM55343 and NS31220.
Address correspondence to: Aldo D Mottino, Ph.D., Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 570, 2000 Rosario, Argentina. E-mail: ifise1{at}citynet.net.ar
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Abbreviations |
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UGT, UDP-glucuronosyltransferase; UDPGA, UDP-glucuronic acid; UDP-N-AG, UDP-N-acetylglucosamine; pp, postpartum; SSC, standard saline citrate; ntcp, Na+-taurocholate cotransport polypeptide; Mrp, multidrug-resistant protein.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-estradiol in pregnancy and after treatment with phenobarbital or ethinyl-estradiol.
J Pharmacol Exp Ther
246:
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