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Vol. 290, Issue 1, 319-324, July 1999
Environmental Health and Occupational Medicine Center, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The ontogenic and hormonal regulation of a sulfotransferase,
SULT1B1, was examined. Hepatic RNA was isolated from rats of various
ages from 1 to 90 days. The mRNA for SULT1B1 is low for both sexes
until a dramatic increase (~6-fold) occurs between 15 and 30 days of
age in male rats. SULT1B1 expression then decreases to half of the
maximal level by 90 days of age. The increase in SULT1B1 mRNA in female
rats is less dramatic and occurs between 30 and 45 days of age. SULT1B1
mRNA expression plateaus from 45 to 90 days in female rats. Expression
of SULT1B1 mRNA is comparable in adult male and female rats. RNA was
isolated from hypophysectomized (HX) animals and HX animals treated
with growth hormone [by either male (injection) or female (infusion)
pattern], estradiol, progesterone, or testosterone. HX and HX plus
growth hormone, or HX plus steroid replacement, did not alter SULT1B1
mRNA expression. Pituitary-intact rats were treated with steroidal
compounds dexamethasone (DEX) and pregnenolone-16
-carbonitrile
(PCN). Both DEX and PCN increased expression of SULT1B1 mRNA in male
rats (4- and 3-fold, respectively). However, in female rats, only PCN
induced SULT1B1 mRNA (2-fold), whereas DEX did not induce SULT1B1 in
female rats. Analysis of SULT1B1 protein expression indicated that only
when SULT1B1 mRNA was markedly increased, that is in DEX-treated male
rats, was SULT1B1 protein increased. Thus, although adult male and
female rats have similar SULT1B1 mRNA expressions, the patterns develop ontogenically differently. SULT1B1 is not regulated by pituitary hormones and DEX induces SULT1B1 protein in male rats.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Sulfotransferases
(SULTs) are a family of phase II drug-metabolizing enzymes involved in
xenobiotic detoxication (e.g., acetaminophen), bioactivation of drugs
like minoxidil (Falany and Kerl, 1990
), and activation of certain
carcinogens such as N-hydroxy-2-acetylaminofluorene (DeBaun
et al., 1970), safrole (Borchert et al., 1973), and
hydroxymethylbenzanthracene (Surh et al., 1991). Additionally, SULTs
metabolize endogenous compounds, such as sex steroids and
glucocorticoid hormones, as well as some neurotransmitters (Brooks et
al., 1978; Penke et al., 1985; Rajkowski et al., 1997). SULTs use the
activated sulfate donor 3'- phosphoadenosine-5'-phosphosulfate to
catalyze the transfer of a sulfuryl functional group from the activated
sulfate donor to substrates (Klaassen and Boles, 1997), which leads to
an enhanced water solubility for these compounds to be excreted. In
contrast, the addition of a sulfuryl moiety can create highly reactive, electrophilic compounds that form covalent adducts with macromolecules such as nucleic acids (Okuda et al., 1989).
SULT nomenclature has previously been based on substrates. The
mammalian SULTs are divided into two major families: SULT1, "phenol" SULTs, and SULT2, "hydroxysteroid" SULTs (Fujita
et al., 1997). However, a new SULT nomenclature system modeled after
the cytochrome P-450 scheme is being reviewed. The new nomenclature will identify each SULT with respect to its unique cDNA sequence to remove the ambiguity that exists with substrate-based nomenclature.
A new member of the phenol SULT family of enzymes has recently been
identified. This isoform is designated as SULT1B1 and is extremely
similar to another enzyme, dopa/tyrosine SULT (Sakakibara et al., 1995;
Fujita et al., 1997). Only 1 amino acid difference results from 12 nucleotide differences between the two cDNAs, and the enzymes have very
similar substrate kinetics (Sakakibara et al., 1995; Fujita et al.,
1997). Thus, the physiological significance of the apparent sequence
differences between SULT1B1 and dopa/tyrosine SULT has not been
established. However, it is known that SULT1B1 is expressed at similar
levels in male and female rats and that this isoform is present in both
liver and kidney (Araki et al., 1997). The mRNA for SULT1B1 has also
been detected in the liver, intestine, and kidney of male and female
rats (Dunn and Klaassen, 1998).
The hormonal regulation of SULT1B1 expression is still unknown. Other
SULT mRNAs are responsive to growth hormone (GH) secretion patterns and
certain steroidal compounds, including dexamethasone (DEX) and
pregnenolone-16-carbonitrile (PCN) (Liu and Klaassen, 1996a,b,c) and
progesterone (PR) (Meyers et al., 1983). Certain SULT enzymes are also
responsive to xenobiotics, including 3-methylcholanthrene (Runge-Morris
and Wilusz, 1994).
Specific oligonucleotides that can distinguish between SULTs at the
level of their respective mRNAs are now available. Our previous work,
using specific oligonucleotides, has delineated hormonal responsiveness
of six major SULTs in male and female rats (Liu and Klaassen
1996a,b,c). In addition, oligonucleotides have been used as probes to
assess the tissue-specific distribution of SULT mRNAs in male and
female rats (Dunn and Klaassen, 1998). The purposes of the present
study were to analyze the ontogenic expression and to define the
hormonal responsiveness of SULT1B1 mRNA.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reagents and Buffers
All reagents were of molecular biology grade (Sigma Chemical
Co., St. Louis, MO) and were used as described previously (Liu and
Klaassen, 1996a,b,c). 3-(N-Morpholino)propanesulfonic acid buffer consisted of 0.2 M 3-(N-morpholino)propanesulfonic
acid, 0.05 M sodium acetate, and 0.01 M EDTA, pH 7.2, which was diluted 10-fold with diethylpyrocarbonate-treated ddH2O
before use. Prehybridization and hybridization solutions were obtained
from Sigma Chemical Co. Zetaprobe GT blotting membranes were from
Bio-Rad (Hercules, CA). Ultrapure agarose was purchased from GIBCO BRL
(Gaithersburg, MD). Rat GH (1.8 IU/mg AFP-87401) was a generous gift
from the National Institute of Diabetes and Digestive and Kidney
Disease (Bethesda, MD). Estradiol benzoate (EB) and PR were purchased from Sigma Chemical Co. Testosterone propionate (TP) was provided by
Dr. Donald Johnson (University of Kansas Medical Center). Alzet osmotic
minipumps (model 2001) were obtained from Alza Corp. (Palo Alto, CA).
Animals
Male and female Sprague-Dawley rats (200-250 g; 4 or
5/group/sex) (Harlan-Sprague-Dawley, Madison, WI) were used for these studies except where indicated. Rats were bred in an AAALAC-accredited facility for the ontogeny studies. For the pituitary studies, rats were
purchased at 22 days of age and surgically hypophysectomized (HX) at 25 days of age with IACUC-approved procedures. Glucose (5%) was provided
after surgery for 3 days. Incompletely HX rats, as determined by
monitoring body weight gain (>15 g/week), were not included in the
study. Age-matched rats with intact pituitaries were maintained under
identical conditions. At the conclusion of the study, rats were
anesthetized with CO2, and livers were removed
and flash frozen in liquid N2 and stored at
80°C until further use. Male New Zealand White rabbits were used
for the production of polyclonal antisera.
Treatments
To assess the role of certain physiological hormones in the regulation of SULT1B1 mRNA expression, HX rats were assigned to one of seven experimental groups 15 days after surgery. Treatments were for 5 days. 1) Rats received s.c. injections twice daily or were infused via osmotic minipumps with vehicle (0.15 M sodium chloride, 0.01 M sodium bicarbonate). 2) Rats received injections of GH twice daily (600 µg/kg s.c.). 3) Rats were implanted with a pump containing rat GH (5 µg/ml) and infused at a rate of 1 µl/h. 4) Rats received s.c. injections once a day with corn oil (2 mg/kg, vehicle for steroid hormones). 5) Rats received s.c. injections with EB (200 µg/kg). 6) Rats were administered PR (25 mg/kg s.c.) once per day. 7) Rats were given s.c. injections once daily with TP (10 mg/kg s.c.).
Pituitary-intact rats were administered single doses of the steroidal compounds DEX and PCN. Each compound was dissolved in corn oil (2 mg/kg, vehicle for steroids). Each rat (6/group) received an i.p. injection of DEX (50 mg/kg) or PCN (75 mg/kg). In this group, RNA was isolated from livers of rats 24 h after dosing.
Total RNA Isolation
Total RNA was isolated using RNAzol B reagent (Tel-Test Inc., Friendswood, TX) using instructions provided by the manufacturer. Isolation of total RNA has been previously described in detail (Dunn and Klaassen, 1998). RNA concentration and purity were assessed by UV absorbance at 260 nm and by A260/A280 ratio, respectively.
Oligonucleotide Probe
The SULT1B1 oligonucleotide probe was based on published
sequences and synthesized by the Biotechnology Support Facility at the
University of Kansas Medical Center. The oligonucleotide was assessed
for uniqueness by BLAST searches of the GenBank nucleotide sequence
databank. The oligonucleotide was designed to be complementary to a
certain divergent sequence of the respective cDNA. The SULT1B1-specific oligonucleotide was complementary to nucleotides 814-834 of the cDNA
sequence reported by Sakakibara et al. (1995) and complementary to
nucleotides 882-903 of the sequence reported by Fujita et al. (1997).
The SULT1B1 oligonucleotide detects both of the highly similar SULT
isoforms, SULT1B1 and dopa/tyrosine SULT.
The oligonucleotide was labeled with
[-32P]dATP (6000 Ci/mmol) (Amersham,
Arlington Heights, IL) by tailing with terminal deoxynucleotidyl transferase (Boehringer-Mannheim, Indianapolis, IN). Oligonucleotide labeling reaction was terminated by the addition of 5 µl (10% v/v)
of 0.5 M EDTA. Labeled oligonucleotide was chromatographically purified
using G-25 (fine) Sephadex (Pharmacia, Piscataway, NJ) spin columns
(Boehringer-Mannheim).
Northern Blot Analysis
RNA was transferred onto nylon membranes by capillary action in 10× standard sodium citrate (SSC) (1× SSC = 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0). Membranes were dried for 1 h at 70°C and then cross-linked under UV light, followed by prehybridization (4 h) and hybridization overnight (~18 h) with a [32P]-labeled oligonucleotide probe specific for SULT1B1 (5'-TGT TCC AGA CAA TTT CTT CTT-3'). Hybridization was performed at 46°C in 20% formamide. The membranes were washed twice in 2× SSC in 2% SDS for 20 min at 46°C and then washed once in 1× SSC in 2% SDS at 46°C, followed by a final wash in 1× SSC in 2% SDS at 50°C. Hybridization signals were detected and quantified after exposure to phosphor screens and analysis by phosphorautoradiography using Image Quant software (Molecular Dynamics, Sunnyvale, CA).
The value for the hybridization signal obtained for each SULT was divided by the value of the hybridization signal obtained for 28S rRNA. This calculation accounts for differences in RNA loading and efficiency of transfer of RNA from gels to nylon membranes. Data are reported as the ratio of SULT mRNA expression to 28S rRNA expression (×1000). Each animal's RNA was analyzed a minimum of four separate times in parallel Northern blots. Data are presented as the mean ± S.E.M. for the four or six rats.
Peptide-Specific Antibodies to SULT1B1
Selection of SULT1B1 Peptide.
To produce monospecific
antibodies directed against SULT1B1, the SULT1B1 amino acid sequence
was selected from the previously published sequence of the
"dopa/tyrosine" SULT (Sakakibara et al., 1995). A 12-amino-acid
peptide (GTAEDVFRKDLK) corresponding with the N-terminal
region of SULT1B1 (amino acids 2-13) was identified as a region of
SULT1B1 nonhomologous to other SULT family members by BLASTp analysis.
This peptide appeared sufficient for use in generating polyclonal
antibodies by computational Kyte-Doolittle analysis (0.75 mean
hydrophobicity score) (Kyte and Doolittle, 1982). The peptide was
synthesized and HPLC-purified by the University of Kansas Medical
Center Biotechnology Support Facility.
Generation of SULT1B1 Peptide-Carrier Protein Conjugates.
The SULT1B1 peptide contains an artificial N-terminal
cysteine residue that was added (CGTAEDVFRKDLK; MW = 1427 g/mol;
71% recovery) to facilitate cross-linkage of the peptide to the
carrier protein. To generate carrier protein-peptide hapten conjugates, the N-C-SULT1B1 peptide (1.2 mg; 0.6 µmol) was
dissolved in 100 µl of PBS, pH 7.4, mixed with maleimide-activated
keyhole limpet hemocyanin (2 mg; 0.6 µmol of maleimide; Imject
Activated SuperCarrier System; Pierce, Rockford, IL) and incubated for
2 h at room temperature (Sharp et al., 1995). After the
incubation, the sample was filtered through a Centricon concentrator
(30,000-Da cutoff; Amicon, Inc., Beverly, MA), the filtrate was
retained for analysis of residual peptide-derived cysteine content, and
the carrier protein-hapten conjugate was washed (three times) with PBS.
Washed and concentrated samples (100 µl/1 mg conjugate) were diluted
in PBS (0.4 ml). Cysteine content in the initial filtrate was assayed
using Ellman's reagent (Dimonte et al., 1984), and the total
incorporation of SULT1B1 peptide into KLH was determined by back
calculating from residual cysteine in the filtrate. At least 90% of
SULT1B1 peptide was incorporated into the carrier protein, yielding the
SULT1B1-KLH conjugate. This conjugate was used as the immunogen for
antibody production in rabbits.
Immunization of Rabbits and Generation of Antisera.
For
production of anti-SULT1B1 sera, carrier-protein-SULT1B1 conjugates
(1.0 mg/0.5 ml PBS) were suspended in Freund's complete adjuvant (0.5 ml) for the priming injection and Freund's incomplete adjuvant for
subsequent booster injections. To initiate an immune response, New
Zealand White rabbits (two rabbits per antigen) were given primary
injections of the antigen in eight sites (s.c.; 100 µg
antigen/site) as described by Harlow and Lane (1988). After the initial
priming injection, animals were given two booster injections at 28-day
intervals and test bled 10 to 14 days after each booster injection.
Blood samples (5-10 ml) were obtained from the rabbit's ear vein. All
animals were given two booster injections, and after assessment of
SULT1B1 peptide hapten-specific antibody titers by enzyme-linked
immunosorbent assay (ELISA), the animals were exsanguinated. Whole
blood was immediately refrigerated and allowed to coagulate, followed
by serum separation via centrifugation and storage at 80°C.
Verification of Antibody Titers to SULT Peptides with Antibody-Capture ELISAs. To validate the generation of antibodies to the various SULT peptides, the SULT1B1 peptide was used in ELISA and in initial Western analyses. Because these peptides do not bind to microtiter plates efficiently or separate electrophoretically on standard polyacrylamide gel systems, each peptide was cross-linked to maleimide-activated BSA (mBSA; Pierce). Briefly, mBSA (2.0 mg; 0.51 nmol maleimide; 17 mol maleimide/mol BSA mg/ml) was incubated with the SULT1B1 peptide (0.7 mg; 0.51 nmol peptide) at 4°C overnight. Excess reactants were removed by filtration as above. These peptide-BSA conjugates were used in ELISA analyses (1 µg SULT1B1-BSA conjugate/well).
The SULT1B1-BSA conjugates were applied to Immunolon 4 96-well Microtiter Immunoassay Plates (Dynatech Laboratories, Inc., Chantilly, VA) at a concentration of 0.5 µg/50 µl PBS/well, and the peptide-BSA conjugate was allowed to bind to the well overnight at 4°C. Subsequently, the conjugate solution was removed, wells were rinsed three times with PBS containing 0.05% Tween-20 (PBS-T), and protein-binding sites in each microtiter well were blocked with a solution of 1% BSA (w/v) in PBS-T (300 µl/well) overnight at 4°C. Blocking solution was removed, the wells were rinsed (three times) with PBS-T, and serial dilutions of each antibody (1:25 to 1:25,000 in PBS-T) were aliquoted into appropriate wells (50 µl/well) and allowed to incubate for 2 h at room temperature. Plates were rinsed (three times) with PBS-T. Secondary goat anti-rabbit IgG conjugated to horseradish peroxidase were added to the wells and incubated for 30 min (1:5000 in PBS-BSA). Plates were rinsed (three times) with PBS-T, and horseradish peroxidase ABTS (2,2'-azino-di[3-ethoxybenzyl thiazoline sulfonate])/McIlwain's substrate solution was added to each well. ABTS/McIlwain's solution consists of 1.0 ml of 10 mg/ml ABTS in water, 8.95 ml of McIlwain's solution (13.3 ml of 0.1 M citric acid and 11.7 ml of 0.2 M sodium phosphate, pH 4.6), and 50 µl of 1% hydrogen peroxide. The substrate solution was added to each well (50 µl/well). The reaction was allowed to develop for 15 min at 37°C. Antibody-dependent absorbance was measured spectrophotometrically at 405 nm with a 96-well microplate reader (Molecular Devices Corp.), and the data were analyzed using Softmax Version 2.32 software (Molecular Devices).Western Blot Analysis
Samples of liver tissue from the DEX- and PCN-treated rats were
homogenized in buffer (5× volume of 20 mM Tris, 6 mM
2-mercaptoethanol, pH 7.5) using a Teflon pestle and a 15-ml glass
homogenizing vessel (Wheaton, Millville, NJ). Each homogenate was
centrifuged for 1 h at 100,000g to obtain the cytosolic
fraction. After centifugation, the protein concentration of each
cytosol was assessed by the bicinchoninic acid procedure (Smith et al.,
1985) using a kit supplied by Pierce. Cytosolic proteins (50 µg/lane)
and molecular weight markers (Bio-Rad) were separated by
SDS-polyacrylamide gel electrophoresis (running buffer: 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.4) using 15% polyacrylamide gels (1 mm × 8.8 cm × 8.1 cm) (Sigma Chemical). After separation,
protein was transferred onto nitrocellulose membranes for 1 h at
25 V (transfer buffer: 12 mM Tris base, 96 mM glycine, 20% methanol).
Each membrane was blocked for 1 h at room temperature in 1% BSA
in PBS-T. After blocking, the membranes were incubated in the presence
of SULT1B1 antiserum or preimmune serum (1:100 in PBS-T) for 1 h
with gentle rocking. Each blot was then washed in PBS-T (three washes,
5 min/wash). The blots were incubated in the presence of secondary
antibody (1:30,000) (goat anti-rat: GIBCO BRL, Grand Island, NY) for 30 min with gentle rocking. The blots were washed in PBS-T (15 min) and
then in PBS (three washes of 15 min each). Detection of antibody interaction was through enhanced chemiluminescence detection (ECL; Amersham, Arlington Heights, Il). Blots were exposed to X-ray film
(X-OMAT AR; Kodak) for 30 s. The film was developed and examined by densitometric analysis using a personal densitometer (Molecular Dynamics) and quantified with Image Quant software.
Statistical Analysis
Data (four rats per group for the ontogeny studies, six rats per group for the DEX and PCN studies, and four rats per group for the studies with HX rats) were analyzed by one-way ANOVA followed by Duncan's post hoc test. The accepted level of statistical significance was set at P < .05.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The developmental expression of SULT1B1 mRNA was assessed in male
and female rats (Fig. 1). Expression of
SULT1B1 mRNA is low in male rats until 15 days of age. However, between
15 and 30 days of age, there is a dramatic increase in SULT1B1
expression (~6 fold), which is maximal by 30 to 45 days in male rats.
Expression of SULT1B1 mRNA gradually decreased in male rats until 90 days of age.
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The developmental expression of SULT1B1 was low in females less than 30 days of age but increased about 2-fold from 30 to 45 days of age. Expression of SULT1B1 mRNA in female rats was steady from 45 to 90 days of age.
The influence of pituitary hormones on SULT1B1 mRNA expression was
examined (Fig. 2). HX resulted in a
slight decrease in SULT1B1 mRNA expression in both sexes, but the
decrease was not statistically significant. Replacement of GH by either
male or female pattern did not affect SULT1B1 expression from that
observed in HX rats.
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The influence of sex steroid hormones on SULT1B1 expression in HX rats
was assessed. Replacement of EB, PR, or TP did not result in
significant changes in expression from control or HX male and female
rats (Fig. 3).
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The influence of the steroidal compounds DEX and PCN was examined in
pituitary-intact male and female rats (Fig.
4). Treatment of male rats with a single
dose of 50 mg/kg DEX resulted in an approximate 4-fold increase in
SULT1B1 mRNA expression. Similarly, PCN (75 mg/kg) induced SULT1B1
expression in male rats about 3-fold over control. The steroidal
compounds were also administered to female rats. DEX was ineffective at
inducing SULT1B1 mRNA in female rats. PCN treatment, however, did
result in a 2-fold enhancement of SULT1B1 mRNA expression over control
rats.
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Peptide-specific antibodies were developed to enable detection
of SULT1B1 protein. SULT1B1 immunoreactive protein was
detected in both male and female rats (Fig.
5). Comparison of the expression of
SULT1B1 protein between control and DEX- and PCN-treated rats revealed that DEX induced SULT1B1 protein in male rats but not female rats. PCN, however, did not induce of SULT1B1 protein in either
male or female rats.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we assessed the ontogenic expression and hormonal
regulation of SULT1B1 mRNA and protein expression. Studies of SULT gene
regulation have revealed that the expression of these enzymes is sex
dependent in rats, with the phenol SULTs being predominant in male rats
and hydroxysteroid SULTs being predominant in female rats. In addition,
it has also been demonstrated that pituitary hormones (e.g., GH) exert
control over SULT gene expression and that sex-specific secretion of GH
(pulsatile in males and continuous in females) is important for the
expression of most SULT isoforms (Liu and Klaassen, 1996a,b).
The ontogenic expression of SULT1B1 is unique among SULT isoforms. In
contrast to other SULT isoforms, SULT1B1 is expressed at similar levels
in adult male and female rats, despite differences in the developmental
expression pattern. Other SULT1 family members (SULT1A1, SULT1C1, and
SULT1E2) are expressed predominantly in adult male rats with virtually
no expression of SULT1C1 or SULT1E2 in adult female rats (Liu and
Klaassen, 1996a,b).
Although the expression of SULT1B1 is similar in adult male and female rats, the developmental expression profile of SULT1B1 mRNA is different between male and female rats; male rats exhibit higher levels of expression between 30 and 60 days of age than female rats but return to female levels at 60 days of age.
The lack of a sex difference in SULT1B1 expression suggests that this
isoform has a parallel role in both sexes of rats. SULT1B1 has
enzymatic activity toward thyroid hormone substrates
T3 and T4 (Sakakibara et
al., 1995; Fujita et al., 1997). Another SULT1 family isoform, SULT1C1,
also has activity toward thyroid hormones. The findings reported here
and by Fujita et al. (1997) imply that SULT1B1 may be more important
than SULT1C1 for thyroid hormone homeostasis because SULT1C1 is almost
exclusively expressed in male rats and because thyroid hormone does not
exhibit sex-specific regulation.
The hormonal regulation of SULT1B1 mRNA expression is also distinct
from other SULT isoforms. Phenol SULT and hydroxysteroid SULT gene
expression are under the control of numerous hormones, including GH,
certain sex steroids, and thyroid hormones. Runge-Morris and Wilusz
(1984) demonstrated that hydroxysteroid SULT40/41 is negatively
regulated by 3-methylcholanthrene. Thus, regulation of SULT expression
is complex. SULT1B1 gene regulation is distinct from the other rat
SULTs in that it is not dramatically altered by HX. Replacement of GH,
which restores sex-specific SULT expression of certain isoforms (Liu
and Klaassen, 1996a,b) does not affect SULT1B1 mRNA expression in HX
rats. In addition, sex steroids do not appear to be major regulatory
hormones of SULT expression and do not alter SULT1B1 gene expression.
SULT gene expression can be altered by pharmacologic chemicals. The
synthetic glucocorticoid DEX has been shown to alter SULT mRNA
expression and enzyme activity (Liu and Klaassen, 1996c). In addition,
the antiglucocorticoid compound PCN induces SULT mRNA expression and
enzyme activity (Liu and Klaassen, 1996c). Both DEX and PCN are known
to induce expression of the cytochrome P-450 3A subfamily (Elshourbagy
et al., 1981; Heuman et al., 1982; Scheutz et al., 1984). Dexamethasone
induced SULT1A1 mRNA in both male and female rats and had
isoform-specific effects on expression of hydroxysteroid SULTs in male
and female rats. SULT1B1 mRNA, like SULT1A1, was markedly inducible
(5-fold) by DEX in male rats, and the marked induction of SULT1B1 mRNA
was reflected by a subsequent significant induction of SULT1B1 protein.
However, in contrast to the other phenol SULTs, SULT1B1 mRNA was also
inducible by PCN in male rats. SULT1B1 mRNA was not inducible by DEX in
female rats, but PCN did elicit a small (2-fold) but significant
increase in SULT1B1 mRNA. The smaller increase in SULT1B1 mRNA caused
by PCN was not mimicked at the protein level.
In conclusion, SULT1B1 is unique among the phenol SULT family of enzymes (SULT1 family). SULT1B1 is present at similar levels in both adult male and female rats and does not exhibit sex-specific expression like other SULT isoforms. SULT1B1 is not regulated by hormones that are controlled at the level of the pituitary. Despite the differences in physiological hormone control of SULT1B1, this isoform is inducible by the synthetic glucocorticoid DEX (male rats). SULT inducibility by synthetic glucocorticoids (e.g., DEX) implies that endogenous glucocorticoids may play a role in the control of SULT1B1 expression, and this possibility is currently under investigation.
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Acknowledgment |
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We thank the Center for Environmental and Occupational Health at the University of Kansas Medical Center for the use of their instruments and equipment.
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Footnotes |
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Accepted for publication March 1, 1999.
Received for publication July 24, 1998.
1 This work was supported by National Institutes of Health Grant ES03192 and, in part by National Institutes of Health Training Grant ES07079 (R.T.D. and D.P.H.).
Send reprint requests to: Curtis D. Klaassen, Ph.D., Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7417. E-mail: cklaasse{at}kumc.edu
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Abbreviations |
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SULT, sulfotransferase;
SSC, standard sodium
citrate;
GH, growth hormone;
HX, hypophysectomy;
ELISA, enzyme-linked
immunosorbent assay;
PBS-T, PBS containing 0.05% Tween-20;
EB, estradiol benzoate;
TP, testosterone propionate;
PR, progesterone;
DEX, dexamethasone;
PCN, pregnenolone-16-carbonitrile;
ABTS, 2,2'-azino-di[3-ethoxybenzyl thiozoline sulfonate].
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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gene expression by 3-methylcholanthrene.
Toxicol Appl Pharmacol
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 J. Orans, D. G. Teotico, and M. R. Redinbo The Nuclear Xenobiotic Receptor Pregnane X Receptor: Recent Insights and New Challenges Mol. Endocrinol., December 1, 2005; 19(12): 2891 - 2900. [Abstract] [Full Text] [PDF]
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 M. H. A. Kester, E. Kaptein, T. J. Roest, C. H. van Dijk, D. Tibboel, W. Meinl, H. Glatt, M. W. H. Coughtrie, and T. J. Visser Characterization of rat iodothyronine sulfotransferases Am J Physiol Endocrinol Metab, September 1, 2003; 285(3): E592 - E598. [Abstract] [Full Text] [PDF]
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 W. Teubner, W. Meinl, and H. Glatt Stable expression of rat sulfotransferase 1B1 in V79 cells: activation of benzylic alcohols to mutagens Carcinogenesis, November 1, 2002; 23(11): 1877 - 1884. [Abstract] [Full Text] [PDF]
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 S. A. Kliewer, B. Goodwin, and T. M. Willson The Nuclear Pregnane X Receptor: A Key Regulator of Xenobiotic Metabolism Endocr. Rev., October 1, 2002; 23(5): 687 - 702. [Abstract] [Full Text] [PDF]
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 J. M. Maglich, C. M. Stoltz, B. Goodwin, D. Hawkins-Brown, J. T. Moore, and S. A. Kliewer Nuclear Pregnane X Receptor and Constitutive Androstane Receptor Regulate Overlapping but Distinct Sets of Genes Involved in Xenobiotic Detoxification Mol. Pharmacol., September 1, 2002; 62(3): 638 - 646. [Abstract] [Full Text] [PDF]
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 S. A. Kliewer and T. M. Willson Regulation of xenobiotic and bile acid metabolism by the nuclear pregnane X receptor J. Lipid Res., March 1, 2002; 43(3): 359 - 364. [Abstract] [Full Text] [PDF]
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N. R. Vansell and C. D. Klaassen Effect of Microsomal Enzyme Inducers on the Biliary Excretion of Triiodothyronine (T3) and Its Metabolites Toxicol. Sci., February 1, 2002; 65(2): 184 - 191. [Abstract] [Full Text] [PDF]
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.05).