From the Department of Environmental Health Sciences and Center for Bioenvironmental Research, Tulane University School of Public Health and Tropical Medicine, New Orleans, Louisiana 70112-2699
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ABSTRACT |
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2,3,7,8-Tetrachlorodibenzo-p-dioxin
(TCDD) exerts its toxic action via the aryl hydrocarbon (Ah) receptor,
which induces a battery of xenobiotic-metabolizing enzymes, including
the cytochrome P450 isozyme, CYP1A1. TCDD-induced
7-ethoxycoumarin-O-deethylase activity was reduced 75% in
cultured human endometrial ECC-1 cells exposed to various
concentrations of 17 TCDD,1 also known as
dioxin, is the archetype of a family of related polychlorinated
compounds found ubiquitously in the environment. Exposure of animals to
TCDD results in many toxic actions (see Ref. 1 for a review). Dramatic
differences in dioxin toxicity have been observed between the sexes of
some animal species, suggesting hormonal modulation of dioxin
action (2).
Dioxins are lipophilic compounds that readily pass through membranes
and bind to an intracellular receptor with no known enzymatic function,
called the Ah receptor (for a review, see Ref. 3). The Ah receptor is a
ligand-activated transcription factor that stimulates gene expression
when coupled with another structurally related protein, Ah receptor
nuclear translocator (4). The heterodimer binds specific DNA sequences,
AhREs, and initiates transcription of various genes, including those
for xenobiotic metabolizing enzymes (5).
Estrogen regulates the proliferative cycle of the endometrium and
mammary glands by binding to estrogen receptor and stimulating transcription, in part, by inducing growth factors and growth factor
receptors (for a review, see Ref. 6). Like the Ah receptor, estrogen
receptor is a transactivating enhancer protein. Upon ligand binding,
estrogen receptor forms a homodimer that recognizes specific DNA
sequences, estrogen response elements, located in target genes.
Estrogen-responsive tissues are particularly sensitive to dioxin
actions, some of which are not toxic. For example, TCDD exerts
protective effects against the appearance of benign mammary and uterine
tumors in Sprague-Dawley rats (7). An epidemiology study showed that
breast and endometrial cancers were slightly reduced in women exposed
to dioxin as a result of an industrial accident that occurred in
Seveso, Italy (8). In contrast, rhesus monkeys exposed to TCDD showed a
concentration-dependent increase in the incidence and
severity of endometriosis (9), a painful condition correlated with
infertility (10). The association of endometriosis with TCDD exposure
has resulted in several medical hypotheses linking its incidence in
humans with environmental exposure to dioxin (11, 12).
A mechanism explaining the interaction between estrogen and dioxin
signaling has yet to be defined. Some investigators have argued that
TCDD is anti-estrogenic (13) because it antagonizes many estrogen
actions, including estrogen-stimulated proliferation of cultured
mammary cells and estrogen-stimulated increase in uterine weights (for
a review, see Ref. 14). TCDD is not a typical estrogen antagonist,
however, because it does not compete with estrogen binding to its
receptor (15). Instead, TCDD induces three known cytochrome P450
isozymes that hydroxylate 17 Conversely, estrogen appears to mitigate Ah receptor-mediated
cytochrome P450 monooxygenase activity in many different systems. This
was first observed by Nebert et al. (20) almost 30 years ago, when reduced cytochrome P450-catalyzed aryl hydrocarbon
hydroxylase activity in both animals and cultured cells was observed
after exposure to 17 In this study, we used an estrogen-responsive clonal endometrial
epithelial cell line, ECC-1, to examine the mechanism by which estrogen
receptor modulates dioxin-responsive genes. We chose ECC-1 cells as our
model system because they contain functional estrogen and Ah receptors
(26, 27). We report that TCDD-induced CYP1A1 was diminished at the
transcriptional level when ECC-1 cultures were also exposed to
17 Materials--
TCDD was obtained from Cambridge Isotopes
Laboratory (Andover, MA). Dr. A. Wakeling (Zeneca Pharmaceuticals)
kindly provided ICI 182,780. The restriction enzymes RsaI,
HindIII, PvuII, SacI, PstI,
PvuII, SacI XhoI, BspEI,
NheI, and Klenow fragment (3' Cells and Culture Conditions--
ECC-1 cells were generously
provided by Dr. P. G. Satyaswaroop (Milton S. Hershey Medical
School, Pennsylvania State University, Hershey, PA), who derived the
cell line from a human adenocarcinoma of endometrial epithelium. HuE, a
primary keratinocyte cell line, was derived from human neonatal
foreskin in this laboratory (28). Hep-3B (ATCC HB-8064) human liver
cells were from Dr. Ali Scandurro, Tulane Medical School. Dr. Louise
Nutter (University of Minnesota, Minneapolis, MN) provided MCF-7 breast
cells. For culturing ECC-1 cells, an in vitro method
developed for dermal epithelium (28) was adapted to model the
morphology of the endometrial epithelium. ECC-1 cells were grown on a
layer of lethally irradiated murine 3T3 fibroblasts, which mimic
endometrial stroma and enhance attachment. ECC-1 cells grown in this
manner typically reached confluence after 8 days. ECC-1, MCF-7, and HuE
cells were all cultivated in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.), containing 5% iron-supplemented bovine calf serum
(Hyclone, Salt Lake City, UT); ECC-1 cell medium contained 1 nM insulin (Sigma). Cells were grown in an atmosphere of
5% CO2/95% air under saturating humidity at 37 °C.
Prior to chemical exposure, cells were grown in 5% charcoal-stripped
calf serum in phenol red-free Dulbecco's modified Eagle's medium
containing 1 nM insulin for a minimum of 5 days. This
procedure was necessary to eliminate steroids normally found in serum
(29). When cultures reached confluence, they were rinsed with
Dulbecco's modified Eagle's medium and exposed to either TCDD,
17 Protein Assay--
Protein was estimated using the procedure of
Lowry as modified by Peterson (30). Bovine serum albumin was the
standard. The minimum detectable protein level was 1 ng.
ECOD Assay--
The method used to measure cytochrome P450
activity is a modification of published procedures from this laboratory
(31). Briefly, cells were rinsed with phosphate-buffered saline and removed from 100-mm culture dishes by scraping with a rubber policeman. Cells were harvested by centrifugation at 500 × g for
3 min, suspended in 10 mM Tris-HCl (pH 7.5), and broken
using a Dounce homogenizer. The broken cell suspension was collected by
centrifugation (150 × g at 4° C for 10 min). The
supernatant containing the microsomal fraction (100 µl) was added to
a reaction mixture containing 32.5 µmol of potassium phosphate, pH
7.2, 250 nmol each of NADPH and NADH, 2.4 µmol of MgCl2,
and 250 nmol of 7-ethoxycoumarin (Aldrich) in a total volume of 1 ml.
The reaction mixture was incubated for 45 min with shaking at 37° C.
The reaction was terminated by the addition of 125 µl of 15% (w/v)
trichloroacteic acid. 7-Hydroxycoumarin formed by the catalytic
dealkylation reaction was extracted into chloroform (2 ml) by vigorous
shaking followed by centrifugation to break emulsions (1000 × g for 5 min). 7-Hydroxycoumarin from the organic phase was
extracted using 2 ml of alkaline salt solution (1 N NaCl,
0.01 N NaOH). 7-Hydroxycoumarin in the aqueous phase was
measured fluorometrically by a Shimadzu spectrofluorometer RF-5301PC
(Tokyo, Japan) ( Radiolabeling of Antisense RNA Probes for Northern Blot
Analysis--
A plasmid containing the human CYP1A1 gene
(pBS1A1) was the generous gift of Dr. Robert Tukey (Cancer Genetics
Program, University of California, San Diego, CA) (32, 33). A
1.6-kilobase CYP1A1 DNA fragment containing the T7 RNA
polymerase promoter was removed from pBS1A1 by digesting with
RsaI restriction enzyme. This fragment produced a 586-base
pair antisense ribonucleotide probe (riboprobe) when placed in an
in vitro transcription reaction with T7 RNA polymerase. A
plasmid containing the 36B4 cDNA (p36B4) was the generous gift of
Dr. Gary Fisher (University of Michigan, Ann Arbor, MI). The 36B4
cDNA codes for human acidic ribosomal phosphoprotein PO and was
used as a loading control because it is not regulated by 17- Radiolabeling of cDNA for Northern Blot Analysis--
The
pBS1A1 plasmid was digested with HindIII to obtain a
1.5-kilobase fragment of human CYP1A1 cDNA. A plasmid containing the human CYP1B1 gene (phCYP1B1-clone 1) was the generous
gift of Dr. Thomas Sutter (The Johns Hopkins University) (18). A 1.3-kilobase CYP1B1 cDNA fragment was removed from phCYP1B1 by digestion with PvuII and SacI. The p36B4 plasmid
was digested with PstI to remove a 760-base pair fragment of
human ribosomal phosphoprotein PO cDNA. The fragments were labeled
using a published procedure for random oligonucleotide primed DNA
synthesis (35). The DNA fragments were denatured in the presence of
random DNA hexamers (2 µg) by boiling for 3 min and immediately
submerging into ice. A mixture containing (final concentration) 0.05 mM dATP/dGTP/dTTP, 50 µCi [ Northern Blot Analysis--
Total RNA was isolated using Tri
Reagent® RNA isolation reagent (Molecular Research Center, Inc.,
Cincinnati, OH). Aliquots of total RNA were separated
electrophoretically on denaturing 1% agarose/6.4 M
formamide gels and stained with ethidium bromide to examine the quality
and quantity of RNA. Gels were equilibrated in 20× SSPE (3 M NaCl, 200 mM sodium phosphate, 20 mM EDTA, pH 7.4) and RNA was transferred onto Zeta Probe
membranes (Bio-Rad) by capillary action. RNA was cross-linked to
membranes with UV radiation using a GS-Genelinker (Bio-Rad) and
prehybridized for 1 h in a solution containing 50% formamide, 5×
SSPE; 1% SDS; sonicated, boiled herring sperm (10 µg/ml); and 5×
Denhardt's solution (0.1% each of bovine serum albumin,
polyvinylpyrrolidone, and Ficoll (36)). Membranes were hybridized for
16 h with 106 cpm/ml of the riboprobes at 60° C or
at 43° C with the DNA probes in prehybridization solution. Membranes
were washed using standard procedures (37) and exposed to Kodak
BiomaxTM MS double emulsion film at Plasmid Construction and Transient Transfections--
A plasmid
containing 7600 base pairs of the 5'-untranslated region of the human
CYP1A1 cDNA (the generous gift of Dr. Y. Fujii-Kuriyama) was
digested with PvuII to obtain a 1904 base pair fragment
(
One day prior to transfection, ECC-1 cells were plated on 6-well dishes
at a density of 5 × 106/well. Cells were transfected
using a cationic lipid-mediated system with 1 µg of
pGL3-5í1A1 and 6 µl of LipofectAMINE (Life Technologies,
Inc.) overnight. Transfections were performed according to
manufacturer's protocol. In experiments using NF-1, cells were transfected with either 1 µg of pCMV-NF1 or an equimolar amount of
the empty expression vector, pCMV, plus pBluescriptSK(+) to equalize
DNA concentration. After transfection, cells were exposed to the
chemical(s) indicated for 48 h. Cells were rinsed twice in
phosphate-buffered saline and lysed on the plate by addition of 150 µl of lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM DTT, 2 mM EDTA, 10% glycerol, 1% Triton
X-100). Cell lysates were collected by scraping into microcentrifuge
tubes and centrifuged for 20 s at 12,000 rpm. One hundred
microliters of luciferin-containing buffer (0.3 mM beetle
luciferin, 0.5 mM acetyl-CoA, 2 mM ATP in 30 mM glycylglycine, pH 7.8, containing 15 mM
MgCl2, 0.5 mM DTT) were added to 10 µl of
supernatant. Luciferase activity was measured in a luminometer
(Monolight 2000, Analytical Luminescence Laboratory, Ann Arbor, MI).
Luciferase assays were performed in triplicate, and relative light
units were normalized to protein content.
Nuclear Run-off Transcription Assay--
Nuclei were collected
by sucrose gradient centrifugation, and the run-off assay was performed
essentially as described by Marzluff and Huang (38) with some
modifications. Five dishes (100 mm) of confluent cells were exposed to
the chemical(s) for the time indicated in the legends to Figs. 5, 6,
and 10, washed twice with phosphate-buffered saline, collected into a
swelling buffer (200 µl of 25 mM KCl, 10 mM
HEPES, pH 7.6, 2 mM magnesium acetate, 1 mM
EDTA, 3 mM CaCl2, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 µM
leupeptin) using a rubber policeman, and placed directly into a glass
homogenizer. An equal amount of a sucrose-containing buffer was added
(0.32 M sucrose, 3 mM CaCl2, 2 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris, pH 8.0, 1 mM DTT, 1 mM
phenylmethylsulfonyl fluoride, and 10 µM leupeptin), and
cells were allowed to swell for 5 min. Cells were broken in a Dounce
homogenizer, and the cell homogenate was layered onto 20 ml of sucrose
cushion buffer (0.88 M sucrose, 2 mM magnesium
acetate, 0.1 mM EDTA, 10 mM Tris, 3 mM CaCl2). The sucrose layers were centrifuged
at 1500 × g for 15 min. The resulting pellet contained
nuclei, which were suspended in an equal volume of a glycerol buffer
(40% glycerol, 20 mM Tris, pH 8.0, 10 mM
MgCl2 and 1 mM DTT). More than 1 × 107 nuclei were used in each run-off reaction. The reaction
mixture consisted of 10 mM Tris, pH 7.5, 0.1 mM
EDTA, 80 mM KCl, 5 mM MgCl2, 1 mM DTT, 100 units/ml creatine kinase, 5 mM each
of ATP, CTP, and GTP, and 100 µCi of [ Ah Receptor Gel Shift Assay--
Dioxin-induced binding of Ah
receptor to DNA was performed using a published method (40). The DNA
probe was made by annealing two oligonucleotides,
5'-GATCCGGCTCTTCTCACGCAACTCCGAGCTCA-3' and 5'-GATCTGAGCTCGGAGT-TGCGT-GAGAAGAGCCA-3', previously shown to bind AhR, and end-labeled with [ Statistical Analysis--
Significant differences between
treatment groups were determined by analysis of variance using the
ANOVA1 macro sheet from Microsoft Excel® software package (Microsoft
Corp.).
Ligand-bound Ah receptor mediates induction of cytochrome P450
monooxygenase(s) by increasing the rate of transcription of these genes
(44). Cytochrome P450-catalyzed O-dealkylation of 7-ethoxycoumarin was used to assess induction of enzymatic activity by
TCDD (45). ECOD activity is specific for CYP1A1, one of three known
cytochrome P450 isozymes induced by TCDD (46). ECC-1 endometrial cells
contain functional Ah receptors and respond to TCDD, which induces
CYP1A1 activity in both a time- and concentration-dependent manner (27).
Estrogen Reduced CYP1A1 Activity--
We examined whether
17 Estrogen Action Was Specific--
Exposing ECC-1 cultures to other
steroid hormones did not mediate a decrease in TCDD-induced ECOD
activity (Fig. 2). Cultures exposed to
TCDD plus 4-androstene-3,17-dione, an estrogen precursor, had no effect
on the level of induced CYP1A1. Cultures exposed to TCDD plus
dexamethasone, a glucocorticoid receptor ligand, and cultures exposed
to TCDD plus progesterone, a progesterone receptor ligand, showed a
20% increase in induced ECOD activity. Both the glucocorticoid
receptor and the progesterone receptor are present in ECC-1 cells (27).
Glucocorticoids tend to enhance Ah receptor induction of CYP1A1,
presumably by glucocorticoid receptor binding at
glucocorticoid-responsive elements located within the CYP1A1
gene (47). Progesterone and glucocorticoid receptors bind to the same
DNA sequence; therefore, it is possible that the progesterone receptor
interacts with the same response elements located within the CYP1A1
gene (48). These data suggest that the steroid hormone-mediated
decrease in Ah receptor action was estrogen-specific.
Reversibility of 17 Cellular Specificity of 17 17 Estrogen Inhibited Dioxin-mediated Transcription of CYP1A1--
To
investigate whether estrogen exerts control over CYP1A1 mRNA levels
by affecting its transcription, we performed transient transfections of
ECC-1 with a reporter plasmid containing a portion of the 5' regulatory
sequence for the human CYP1A1 gene (
To establish whether the decrease in CYP1A1 mRNA occurred at the
transcriptional level and whether transcription varied over time, we
performed nuclear run-off experiments using nuclei collected from ECC-1
cultures exposed to TCDD alone, or TCDD plus 17 Gel Mobility Shift Assay--
We performed a gel mobility shift
assay to determine whether the observed alteration in CYP1A1
transcription resulted from reduced binding of Ah receptor to AhREs
using nuclear extracts from cultures exposed to TCDD alone or to TCDD
and 17 17 Recovery of TCDD-mediated Transcription--
The promoter
sequences of CYP1A1 and CYP1B1 contain several
AhRE sites and binding sites for the SP-1 general transcription factor
(56-58). Transcription of CYP1A1 is regulated, in part, by
the NF-1 transcription factor, but CYP1B1 is not (56). In addition, NF-1 functions synergistically with estrogen receptor to
activate transcription driven by that receptor (59). Because the
estrogen receptor and NF-1 interact, we hypothesized that activated
estrogen receptor may sequester NF-1 thereby reducing ability of the Ah
receptor complex to induce CYP1A1. To test this hypothesis,
we examined whether overexpression of NF-1 in ECC-1 cells would
overcome the inhibitory effect of 17 Induction of CYP1A1 monooxygenase activity is a hallmark of dioxin
alteration of gene expression. In this study, we demonstrated that
cultured human endometrial cells exposed to 17 That dioxin decreases many actions mediated by the estrogen receptor is
well established (60-62). Reciprocal interaction between estrogen
receptor and Ah receptor was reported in MCF-7 human cells and Hepa
1c1c7 murine liver cells (25). The authors suggested that each receptor
acted to reduce the ability of the other to bind their respective
response elements. A subsequent report, however, disputed whether
estrogen affected dioxin action and showed that estrogen does not
affect Ah receptor function in the same cell systems (63). Our findings
support the observation that estrogen receptor disrupts dioxin-induced
CYP1A1, not by altering Ah receptor binding to DNA but by a
mechanism involving the general transcription factor NF-1. Using gel
mobility shift analysis, we showed that 17 Two regions of the CYP1A1 5'-untranslated region control its
transcription. The AhRE-containing dioxin-responsive enhancer begins
several hundred base pairs upstream of the transcriptional start site,
and the promoter region, containing binding sites for general
transcription factors (SP-1 and NF-1), is located immediately upstream
of the transcriptional start site (Ref. 64 and references therein).
Like CYP1A1, CYP1B1 contains multiple Ah receptor
binding sites and binding sites for SP-1 (57). One apparent difference
between these two genes is that the promoter for CYP1A1
contains two NF-1 binding sites, of the sequence CCAAT, but
CYP1B1 does not (58). In fact, deletion of the NF-1 site proximal to the transcriptional start site of CYP1A1 reduces
transcriptional activation by the Ah receptor 80% (56). We deduced
that estrogen inhibition of CYP1A1 activity could be mediated by loss
of NF-1 function. When NF-1 was overexpressed in ECC-1 cells, we
observed reversal of estrogen action on TCDD-activated CYP1A1 (Fig.
11). We also examined whether overexpression of SP-1 or the
co-activating protein p300, both of which are involved in Ah receptor
and estrogen receptor mediated transcription (61, 65, 66), could
reverse estrogen action, but we did not observe an
effect.2
NF-1 is associated with both Ah receptor-mediated and estrogen
receptor-mediated transcription. NF-1 synergizes with the estrogen receptor to mediate transcription (59, 67, 68). Our data suggest that
when 17 The observation that CYP1A1 induction was affected by exposing ECC-1
cultures to 17 If dietary ligands are important directors of Ah receptor action, then
it is possible that estrogen metabolism is governed, in part, by CYP1A1
induced by these ligands. For example, in human subjects exposed to
indole-3-carbinol, estradiol 2-hydroxylation was increased 1.5-fold
(72). The 2-C derivative of 17 The data presented here show that estrogen exerted significant,
immediate, and reversible action on reducing TCDD-induced CYP1A1 transcription and subsequent activity in human
endometrial cells. Our data suggest that the ligand-activated estrogen
receptor can down-regulate a pathway of estrogen metabolism, and this
effect has significant implications for understanding both estrogen
receptor-associated disease and Ah receptor-mediated toxicity.
-estradiol for up to 72 h, with a
half-maximal effective concentration (EC50) of 0.9 nM. Reduced enzyme activity was correlated with decreased
CYP1A1 mRNA levels, and transcription. Exposure to TCDD plus
17
-estradiol also reduced CYP1A1 activity in MCF-7 breast cancer
cells but not in Hep-3B human liver cells or HuE primary human
keratinocytes, suggesting that the effect was specific to
estrogen-regulated cells. Estrogen receptor antagonists
4-hydroxytamoxifen and
7
-[9-(4,4,5,5,5-pentafluoro-pentylsulfinyl)nonyl]estra-1,3,5(10)-triene3, 17
-diol
restored TCDD-induced CYP1A1 transcription, steady-state mRNA levels, and enzymatic activity in ECC-1 cells. Gel mobility shift assay showed that 17
-estradiol had little effect on Ah receptor binding to its DNA-responsive element. 17
-Estradiol did not
alter the induction of another Ah receptor-regulated gene, CYP1B1, suggesting that altered Ah receptor binding to DNA
does not mediate reduced CYP1A1 transcription. Transfecting
ECC-1 cells with a general transcription factor involved in CYP1A1
induction, nuclear factor-1, reversed 17
-estradiol antagonism of
dioxin induced-CYP1A1. The data suggest that 17
-estradiol reduced
CYP1A1 expression at the transcriptional level by squelching available nuclear factor-1, a transcription factor that interacts with both Ah and estrogen receptors.
INTRODUCTION
Top
Abstract
Introduction
References
-estradiol, the most biologically potent
estrogen, to various catechols (16). These isozymes, CYP1A1, CYP1A2,
and CYP1B1, are under direct transcriptional regulation by Ah receptor
interacting at AhREs (17-19). These cytochromes play important roles
in xenobiotic metabolism but also appear to mediate 17
-estradiol
hydroxylation in some cells.
-estradiol. This observation has appeared
periodically in the literature, but no mechanism has unequivocally
defined the effect (21-25).
-estradiol. We present data showing restoration of dioxin-induced
CYP1A1 message and activity in cultures exposed to two estrogen
receptor antagonists. We also show that reduction of CYP1A1 activity
after exposure to 17
-estradiol was specific to estrogen-regulated
cells. We present gel mobility shift data showing no reduction of Ah
receptor binding to AhRE and demonstrate reversal of 17
-estradiol
mediated reduction of TCDD-induced transcription by transient
transfection of NF-1, a general transcription factor involved in
CYP1A1 induction.
EXPERIMENTAL PROCEDURES
5' exo
) and
T4 DNA ligase were purchased from New England Biolabs, Beverly, MA.
Restriction enzymes BglII and BamHI and T7 RNA
polymerase, DNA polymerase I large Klenow fragment, and pGL3-Basic
vector were from Promega (Madison, WI). [
-32P]ATP
(SA = 4000 Ci/mmol), [
-32P]UTP (SA = 3000 or
600 Ci/mmol), and [
-32P]dCTP (SA = 3000 Ci/mmol)
were purchased from ICN (Costa Mesa, CA). Unless specified otherwise,
all other reagents were purchased from commercial sources and used
without further purification.
-estradiol, or their analogs, dissolved in Me2SO
unless otherwise stated. Me2SO never exceeded 0.1% in the culture medium.
ex = 368 nm;
em = 456 nm), and the concentration was estimated using a standard curve
generated from known quantities of 7-hydroxycoumarin. Specific activity
was expressed as pmol of 7-hydroxycoumarin formed/mg of protein/min.
Assays were carried out under conditions where 7-hydroxycoumarin
formation was linear with respect to protein and incubation time.
estradiol (34). A 2.3-kilobase 36B4 DNA fragment containing the T7 RNA
polymerase promoter was removed from p36B4 by digesting with
RsaI. This fragment produced a 250-base pair riboprobe when transcribed with T7 RNA polymerase. Riboprobes were synthesized by
incubating 1 µg of linearized DNA template at 37° C for 1 h with 4 µl of 5× transcription buffer (40 mM Tris-HCl, pH
7.5; 6 mM MgCl2; 2 mM spermidine;
10 mM NaCl); 2 µl 100 mM DTT; 20 units of
ribonuclease inhibitor (RNAsin, Promega); ATP, GTP, and CTP (2.5 mM each); 100 µM UTP; 50 µCi
[
-32P]UTP (specific activity of 600 Ci/mmol); 20 units
of T7 RNA polymerase; and nuclease free water to a final volume of 20 µl. The labeled RNA fragments were purified from unincorporated
[
-32P]UTP by mini-column chromatography using Bio-Gel
P60 (Bio-Rad) and used in hybridization procedures.
-32P]dCTP
(SA = 3000 Ci/mmol), 20 units of Klenow fragment (3'
5' exo
), 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and 7.5 mM DTT was added to the DNA, and the fill-in reaction was carried out for 2 h at 37° C. The labeled DNA fragments were purified from unincorporated [
-32P]dCTP by mini-column chromatography using Bio-Gel
P60 (Bio-Rad).
70° C for 1-2
days. Autoradiographs were scanned using a 670 imaging densitometer
(Bio-Rad), and densitometric volumes of the mRNA bands were
calculated using Molecular AnalystTM software (Bio-Rad).
1612/+292) and ligated into the SmaI site of the
BluescriptSK(+) vector (Strategene, La Jolla, CA) using T4 DNA ligase.
This portion of the 5'-untranslated region contains the promoter, exon
1, and at least three functional AhREs (32, 39). The AhR-responsive
fragment was removed by digestion with SacI and
XhoI and inserted into the pGL3-Basic vector upstream of the
cDNA encoding firefly luciferase. This plasmid, pGL3-5'1A1, was
used to examine Ah receptor function by transiently transfecting it
into ECC-1 cells. In experiments examining the action of NF-1 on CYP1A1
transcription, cells were transfected with an expression plasmid
containing the cDNA for NF-1 under transcriptional control of the
cytomegalovirus promoter (pCMV-NF1). Dr. Gordon Hager (National
Insititutes of Health, Bethesda, MD) generously supplied a plasmid
containing NF-1 (pEGFP-NF1). pEGFP-NF1 was digested with
BspEI and NheI to remove the cDNA encoding
green fluorescent protein from pEGFP-C1 (CLONTECH,
Palo Alto, CA). The ends were filled using DNA polymerase I large
Klenow fragment and ligated. The plasmid was designated pCMV-NF1. The cDNA encoding NF-1 was removed from pCMV-NF1 by digestion with BglII and BamHI, ligated, and designated pCMV.
-32P]UTP
(SA = 3000 Ci/mmol) and was incubated for 30 min at 30° C.
Total RNA was isolated using Tri Reagent® RNA isolation reagent (Molecular Research Center, Inc.). RNA was hybridized to 5 µg of a
linearized plasmid containing CYP1A1 cDNA, human
-actin, or the plasmid vector alone, that was immobilized on Zeta
Probe® GT nylon membrane (Bio-Rad), according to manufacturer's
instructions, using a slot blot apparatus (Schleicher & Schuell).
Hybridization, washing, and autoradiography was performed as described
for Northern blot analysis, except that hybridization temperature was
42° C and was continued for 72 h. Films were exposed for up to
3 weeks.
-32P]ATP and T4
polynucleotide kinase (41, 42). Cells were exposed to TCDD (10 nM) in the presence or absence of 17
-estradiol (10 nM) in 0.1% Me2SO for 1 h. Nuclear
extracts (60 µg) were incubated with 100,000 cpm of
32P-labeled AhRE oligonucleotide (43) for 15 min in the
presence of 6 µg of herring sperm DNA. Samples were analyzed using
nondenaturing gel electrophoresis (40). Phosphorimaging was performed
using a Fuji FLA-200 phosphorimager for quantitative analysis of
shifted bands, and gels were also exposed to Kodak BiomaxTM
MS double emulsion film for 2 days in order to obtain a permanent copy.
RESULTS
-estradiol altered TCDD-mediated induction of CYP1A1 activity.
ECOD activity was measured in cell extracts from ECC-1 cultures exposed
to saturating levels of TCDD (10 nM) and various
concentrations of 17
-estradiol. Estrogen reduced CYP1A1 activity in
a concentration-dependent manner (Fig.
1A). The EC50 for
17
-estradiol modulation of ECOD activity was 0.9 nM,
which correlates well with the apparent KD for
17
-estradiol in ECC-1 cells (0.7 nM) (27). CYP1A1
activity in the presence of 17
-estradiol was reduced by 75%
compared with that in cells exposed to TCDD alone. The time dependence
of 17
-estradiol action was also examined. Exposing ECC-1 cultures to
17
-estradiol resulted in a decrease in ECOD activity from the
earliest indication of activity (2 h) that lasted until the experiment
was terminated at 65 h (Fig. 1B). Maximal reduction of
CYP1A1 activity after exposing ECC-1 cultures to 17
-estradiol in the
presence of TCDD occurred after 8 h of exposure and was maintained
throughout the time course analysis.
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Fig. 1.
17 -Estradiol
decreased TCDD-induced cytochrome P450 activity in a
concentration-dependent manner. A,
confluent cultures of ECC-1 cells were exposed for 48 h to either
10 nM TCDD alone (CYP1A1 specific activity = 109 ± 11.7 pmol/mg of protein/min) or 10 nM TCDD plus
17
-estradiol (
) at the concentrations indicated. Cells were
harvested and homogenized, and a crude preparation containing the
microsomal fraction was assayed for CYP1A1 activity as described under
"Experimental Procedures." Each data point represents
the mean from three separate cultures performed in triplicate.
B, confluent cultures of ECC-1 cells were exposed to either
10 nM TCDD alone (
) or TCDD plus 17
-estradiol (
)
(10 nM) for the times indicated. Data are presented as a
percentage of the maximum value of CYP1A1 induction (65 h exposure = 73.5 ± 7.5 pmol of 7-hydroxycoumarin formed/mg of
protein/min).
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Fig. 2.
Other steroid hormones and steroid receptor
agonists do not mediate a decrease in TCDD-induced cytochrome P450
activity. Confluent cultures were exposed for 48 h to 10 nM TCDD plus 10 nM of the chemical indicated
(A, 4-androstene-3,17-dione; D, dexamethasone;
E, 17 -estradiol; P, progesterone). Enzyme
activity was assayed as described in Fig. 1. Each column
represents the mean from three separate cultures performed in
triplicate ± S.E.
-Estradiol Action--
To further establish
involvement of the estrogen receptor, we examined whether estrogen
receptor antagonists could reverse 17
-estradiol reduction of CYP1A1
induction. Two structurally different "anti-estrogens,"
4-hydroxytamoxifen and ICI 182,780, alter 17
-estradiol binding to
estrogen receptor and were used in these experiments (49, 50). ECC-1
cultures were exposed to combinations of TCDD, 17
-estradiol, and
either 4-hydroxytamoxifen or ICI 182,780, and ECOD activity was
measured after 48 h of exposure. Anti-estrogens reversed estrogen
attenuation of TCDD-induced ECOD activity in a
concentration-dependent manner (Fig.
3). The EC50 values for
4-hydroxytamoxifen and ICI 182,780 were 750 and 100 nM,
respectively. Reversal of estradiol action by estrogen receptor antagonists indicated a role for estrogen receptor in reduction of TCDD
induction of CYP1A1 by 17
-estradiol.
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Fig. 3.
Estrogen receptor antagonists reversed
17 -estradiol action and reinstated TCDD
induction of cytochrome P450 activity. Confluent cultures were
exposed to a combination of TCDD (10 nM) plus
17
-estradiol (10 nM) in the presence of various
concentrations of 4-hydroxytamoxifen (
) or ICI 182,780 (
) for
48 h. Results are expressed as percentage of activity induced by
TCDD (10 nM) plus the antagonist (1000 nM).
Enzyme activity in cultures exposed to TCDD plus tamoxifen was
49.78 ± 2.95 pmol/mg of protein/min, and TCDD plus ICI 182,780 was 74.9 ± 1.36 pmol/mg of protein/min. CYP1A1 activity was
measured as described in Fig. 2. Each data point represents
the mean from three separate cultures performed in triplicate.
-Estradiol Action--
To determine
whether the observed reduction in CYP1A1 activity was specific to ECC-1
endometrial cells, we examined the effect of 17
-estradiol on several
human cell lines. We selected MCF-7 mammary carcinoma cells because
they, like ECC-1 cells, are derived from estrogen-sensitive tissue and
are regulated by 17
-estradiol (51-53). As a comparison, we selected
Hep-3B human liver cells, and HuE, a primary human keratinocyte cell,
neither of which is derived from estrogen-regulated tissues. Similar to
ECC-1 cells, 17
-estradiol reduced TCDD-induced CYP1A1 activity in
MCF-7 cells, but induction of CYP1A1 in Hep-3B and HuE cells by TCDD
was unaffected by the presence of 17
-estradiol (Fig.
4). To confirm that estrogen receptor
level could be a factor in determining the mitigation response by
17
-estradiol, we examined each cell line for estrogen receptor
content by radioreceptor assay (27), followed by Scatchard analysis
(54). We were unable to detect estrogen receptors in either HuE or
Hep-3B cells by this method, but both ECC-1 and MCF-7 cells contained
similar quantities of estrogen receptor with comparable apparent
Kd values (MCF-7: Kd = 0.74 ± 0.08 nM, Bmax = 418 ± 54 fmol/mg; ECC-1: Kd = 0.74 ± 0.07 nM, Bmax = 282 ± 38.5 fmol/mg).2
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Fig. 4.
17 -Estradiol reduced
CYP1A1 activity only in estrogen-regulated cells. Confluent
cultures of ECC-1 human endometrial cells, MCF-7 mammary carcinoma
cells, Hep-3B human liver cells, and HuE primary human keratinocyte
cells were exposed for 48 h to 10 nM TCDD, with or
without 10 nM 17
-estradiol. The solvent control was
0.1% Me2SO (DMSO). CYP1A1 activity was assayed
as described in the legend to Fig. 1. The data shown are the mean from
three separate cultures performed in triplicate ± S.E.
-Estradiol Treatment Decreased CYP1A1 mRNA Levels--
To
determine whether reduced monooxygenase activity reflected altered
CYP1A1 mRNA expression, we examined steady-state levels of CYP1A1
mRNA over time in ECC-1 cells exposed to TCDD and 17
-estradiol by Northern blot analysis. Total RNA collected from cells exposed to
saturating concentrations of TCDD and 17
-estradiol showed a
reduction of TCDD-induced CYP1A1 mRNA when compared with cells exposed to TCDD alone (Fig. 5). Cells
that were not exposed to TCDD did not display detectable CYP1A1
mRNA. Densitometric analysis of the autoradiograph showed that
estrogen reduced CYP1A1 mRNA to 35% of TCDD-induced levels after
24 h, in close agreement with the observed decrease in enzymatic
activity (Fig. 1B). To determine whether this effect could
be reversed, we exposed cultures of ECC-1 cells to combinations of
TCDD, 17
-estradiol, and the anti-estrogens used earlier. The
estrogen receptor antagonists ICI 182,780 and 4-hydroxytamoxifen each
reversed estrogen action at the level of CYP1A1 mRNA and did not
affect CYP1A1 induction by TCDD (Fig. 6).
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Fig. 5.
17 -Estradiol
decreased TCDD-induced CYP1A1 mRNA. Cells were exposed to TCDD
(10 nM) in the presence or absence of 17
-estradiol (10 nM) for the times indicated. Total RNA (5 µg) was
separated by electrophoresis on a denaturing agarose gel. The RNA was
transferred to a charged nylon membrane and hybridized to an antisense
RNA probe for CYP1A1 mRNA. The quantity of RNA loaded was assessed
using a probe for the ribosomal protein, 36B4. The time course of
mRNA accumulation is in good agreement with induction of enzymatic
activity. A, autoradiograph of the Northern blot incubated
with RNA probes for both CYP1A1 and 36B4 simultaneously. Film was
developed after 48 h exposure to blot. B, densitometric
analysis of Northern blot autoradiograph shown, where the ratio of the
quantity of CYP1A1 mRNA (in arbitrary units) induced by TCDD (
)
or TCDD plus 17
-estradiol (
), normalized to the quantity of 36B4
mRNA for the sample loaded, is plotted against time of exposure.
Ratios for the solvent control (0.1% Me2SO) and
17
-estradiol (10 nM) were 0.018 and 0.012, respectively.
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Fig. 6.
Estrogen receptor antagonists reversed
17 -estradiol reduction of TCDD-induced CYP1A1
mRNA. Confluent cultures of ECC-1 cells were exposed for
16 h to the chemicals indicated. Cells were exposed to the
following concentrations: 10 nM TCDD, 10 nM
17
-estradiol, 1000 nM 4-OH-tamoxifen, 1000 nM ICI 182,780. Cells were harvested and Northern analysis
was performed as described under "Experimental Procedures."
1612/+292). This
segment contains the promoter and at least three functional AhREs (32,
39, 55). When activated by the Ah receptor complex, the promoter drives
transcription of the cDNA sequence encoding firefly luciferase.
Exposing transiently transfected cells to TCDD increased luciferase
activity 24-fold above that measured in the Me2SO control
(2.1 × 103 versus 49.6 × 103 relative light units/mg of protein). Exposing
transfected cells to both TCDD and 17
-estradiol resulted in a 74%
decrease in TCDD-induced luciferase activity (Fig.
7). Addition of estrogen receptor
antagonists reversed the estrogen-mediated decrease of TCDD-induced
transcriptional activation of the reporter plasmid.
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Fig. 7.
Transient transfection of ECC-1 cells showed
17 -estradiol-mediated reduction of Ah receptor
transcription is reversible by estrogen receptor antagonists.
ECC-1 cells were transfected with a plasmid containing a portion of the
5'-regulatory sequence of the CYP1A1 gene (
1612/+292) that
drives transcription of firefly luciferase when activated by TCDD.
Cells were transfected with 1 µg of reporter plasmid overnight and
exposed to the chemical(s) indicated in 0.1% Me2SO
(DMSO) (10 nM TCDD, 10 nM
17
-estradiol (E2), 1 µM ICI 182,780, 1 µM 4-OH-tamoxifen (TAM)) for 48 h. Cells
were lysed, and relative luciferase activity was measured and
normalized to protein content. Each column represents the
mean from three separate cultures performed in triplicate ± S.E.
Statistical analysis comparing TCDD alone to TCDD plus the chemicals
indicated was performed by ANOVA. Significant difference (*) was
observed only between TCDD plus 17
-estradiol and TCDD alone
(p < 0.004).
-estradiol, after
1.5, 4, and 12 h. 17
-Estradiol reduced CYP1A1 transcription at
each time point examined (Fig.
8A). Densitometric analysis of
autoradiographs from three separate experiments showed that TCDD
induced CYP1A1 mRNA maximally by 1.5 h of exposure, which was
maintained for at least 12 h (Fig. 8B). Exposure of
ECC-1 cultures to 17
-estradiol reduced transcription by 60%. The
data suggest a linear relationship exists between CYP1A1
transcription, steady-state message levels, and expression of
functional enzyme that was uniformly affected by exposure of ECC-1
cultures to 17
-estradiol.
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Fig. 8.
Estrogen decreased transcription rate of
TCDD-induced CYP1A1. Confluent cultures were exposed to either
TCDD (10 nM) or TCDD plus 17 -estradiol (10 nM) for the times indicated. Nuclei were isolated, and
in vitro transcription of nascent RNA proceeded in the
presence of [
-32P]UTP. Newly transcribed mRNA was
isolated and hybridized to CYP1A1 cDNA and human
-actin
cDNA. A, autoradiograph of membranes after 3 weeks of
exposure. B, quantitation of densitometric analysis from
three separate experiments was carried out as described under
"Experimental Procedures." Relative units refer to normalized
densitometric volumes (CYP1A1 mRNA/
-actin mRNA) expressed as
fold induction above background for TCDD (
) and TCDD plus
17
-estradiol (- -
- -).
-estradiol (Fig. 9). The data
indicate a minimal decrease in AhRE binding by nuclear proteins from
ECC-1 cultures exposed to TCDD and 17
-estradiol compared with
cultures exposed to TCDD alone. Statistical analysis of areas of
shifted bands from densitometry indicated no significant difference in
DNA binding from nuclear extracts of ECC-1 cells exposed either to TCDD
alone or to TCDD plus 17
-estradiol. This observation suggests that
the estrogen receptor does not interfere with the ability of the Ah
receptor to interact with AhRE.
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Fig. 9.
17 -Estradiol reduced
Ah receptor complex binding to AhRE. ECC-1 cells were exposed to
TCDD (T) (10 nM) and 17
-estradiol
(E2) (10 nM) alone or in combination, or with
solvent alone (0.1% Me2SO (DMSO)), for 1 h, after which nuclear protein extracts were prepared. Sixty
micrograms of nuclear extract were incubated with
32P-labeled AhRE oligonucleotide probe and subjected to
electrophoresis on polyacrylamide gels under nondenaturing conditions.
The arrow indicates bound AhRE. Statistical analysis of
areas obtained from densitometric scans by one-way ANOVA showed no
significant differences between the groups exposed to TCDD alone or in
the presence of TCDD and 17
-estradiol.
-Estradiol Does Not Affect CYP1B1 mRNA Levels--
To
examine whether the observed decrease in CYP1A1 activity, message, and
transcription rate was specific to CYP1A1, or a generalized
action, we examined whether 17
-estradiol affected the mRNA level
of another TCDD-regulated gene, CYP1B1. We used Northern
blot analysis to assess steady-state levels of CYP1B1 message in RNA
extracted from ECC-1 cultures exposed to TCDD alone, or TCDD plus
various concentrations of 17
-estradiol for 24 h (Fig.
10). The level of CYP1B1 mRNA was
not altered by exposure of the cells to 17
-estradiol, but the CYP1A1
message levels decreased as a function of 17
-estradiol
concentration, indicating the effect was specific to the
CYP1A1 gene. This experiment was performed twice with
similar results.
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Fig. 10.
17 -Estradiol does
not reduce TCDD-induced CYP1B1 mRNA. Confluent cultures of
ECC-1 cells were exposed to 10 nM TCDD plus the indicated
concentration of 17
-estradiol for 24 h. Cells were harvested
and Northern analysis was performed as described under "Experimental
Procedures," except DNA probes rather than riboprobes were used. The
blot shown was first incubated with probes for CYP1B1 plus 36B4, placed
on film for autoradiography. Later, the blot was stripped and incubated
with a CYP1A1 probe and placed on film for autoradiography.
-estradiol. ECC-1 cells were
co-transfected with the luciferase reporter plasmid containing the 5'
regulatory region of human CYP1A1, described above, and a
plasmid containing the cDNA for NF-1 under transcriptional control
of the cytomegalovirus promoter (pCMV-NF1). Overexpression of NF-1
reversed 17
-estradiol mediated reduction of TCDD-induced transcription (Fig. 11).
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Fig. 11.
Transfection of ECC-1 cells with NF-1
cDNA reverses estrogen receptor-mediated reduction of CYP1A1
transcription. ECC-1 cells were transfected with 1 µg of NF-1 in
a pCMV vector or an equivalent quantity of empty vector as described
under "Experimental Procedures." Transfecting medium was replaced
with medium containing 17 -estradiol (10 nM); TCDD (10 nM); TCDD (10 nM) plus 17
-estradiol (10 nM); or 0.1% Me2SO (DMSO) as a
vehicle control. Cultures were incubated for 48 h, and luciferase
activity was assayed as described under "Experimental Procedures."
The data shown represent the mean of three separate experiments assayed
in triplicate ± S.E. Differences between groups were significant
as analyzed by one-way ANOVA (p < 0.025).
DISCUSSION
-estradiol reduced
dioxin-induced CYP1A1 transcription, mRNA steady-state levels, and enzymatic activity compared with cultures exposed to TCDD
alone. The reduction by estrogen was observed in conjunction with the
first appearance of CYP1A1 and was persistent. Estrogen action was
concentration-dependent and was reversible by estrogen receptor antagonists, which strongly indicates estrogen receptor involvement. Exposure of ECC-1 cultures to other steroid hormones did
not show an inhibitory action on TCDD induction of CYP1A1 activity.
Estrogen down-modulation of CYP1A1 activity was also cell
type-specific. MCF-7 breast cells and ECC-1 endometrial cells, derived
from estrogen-sensitive tissues and containing comparable levels of
estrogen receptor, both showed decreased CYP1A1 activity in the
presence of 17
-estradiol. 17
-Estradiol did not, however, affect
CYP1A1 activity in either Hep-3B human liver cells or HuE human
keratinocytes, cells that are not normally regulated by estrogen and
that had undetectable levels of estrogen receptor.
-estradiol did not
significantly alter TCDD-activated Ah receptor binding to its
DNA-responsive element. To corroborate the functional significance of
this observation, we examined whether 17
-estradiol altered
expression of another dioxin-regulated gene, CYP1B1.
Estrogen modulation of dioxin was specific to CYP1A1 but did
not affect CYP1B1 mRNA levels, which suggests that Ah receptor
binds to AhREs unhampered by 17
-estradiol.
-estradiol activates the estrogen receptor, recruitment of
NF-1 by Ah receptor to CYP1A1 is altered, and the factor is
directed to 17
-estradiol-responsive genes, resulting in reduction of
CYP1A1 induction. Similar to the action of NF-1 with estrogen and other
steroid receptors, Ah receptor activates CYP1A1
transcription by modification of chromatin structure in the promoter
region, allowing access of NF-1 and other factors to bind and initiate
transcription (44, 56, 64, 69). Our observation raises the question of
how estrogen receptor appears to sequester available NF-1. It is
possible that the nuclear location of ligand-free estrogen receptor
versus cytosolic location of ligand-free Ah receptor
provides access for the estrogen receptor to direct NF-1 away from
dioxin-sensitive CYP1A1 before the Ah receptor complex
enters the nucleus or is able to initiate transcription. The relative
abundance of estrogen receptor (280 fmol/mg of cytosolic protein)
compared with Ah receptor (2 fmol/mg of cytosolic protein) (27) in
ECC-1 cells may also contribute to this response.
-estradiol but CYP1B1 was not may be important to the
physiology of endometrial cell function. Down-modulation of CYP1A1 by
17
-estradiol could account for the observation that the major
endometrial metabolite of 17
-estradiol is the 3,4-catechol derivative, a product of CYP1B1 monooxygenase activity (70, 71). It is
possible that Ah receptor is present in the endometrium to act as a
mediator of estrogen metabolism. If so, our observation that estrogen
can reduce Ah receptor-mediated transcription of CYP1A1
suggests communication exists between the estrogen receptor and the Ah
receptor to maintain estrogen homeostasis in estrogen-sensitive tissue.
We used TCDD in our study because enzymes induced by the Ah receptor do
not metabolize it. Therefore, its effects are persistent and pronounced
compared with those induced by other metabolizable ligands for the Ah
receptor, which include compounds of dietary origin, such as
substituted carbazoles and indole carbinols found in various
vegetables, as well as polycyclic aromatic compounds (72-74). The
discovery of nontoxic Ah receptor agonists has added to growing
evidence that Ah receptor may have other important physiological
functions, in addition to mediating xenobiotic metabolism (see Ref. 19
and references therein).
-estradiol hydroxylation is the major
estrogen metabolite of CYP1A1 activity (75, 76). This derivative is a
less potent estrogen receptor ligand than 17
-estradiol and is a
precursor to the major urinary estrogen metabolite, 2-methoxyestrone
(16). Therefore, dietary ligands may exert normal homeostatic control
over estrogen function by inducing enzymes that regulate estrogen
levels. When a toxic, nonmetabolizable ligand, such as TCDD, binds Ah
receptor, overproduction of CYP1A1 could occur, leading to observed
decreases in estrogen action (77-79). Presence of high levels of
estrogen receptor in cells from estrogen-sensitive tissue could
mitigate this Ah receptor-induced response, thereby maintaining levels
of 17
-estradiol and preserving estrogen homeostasis.
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ACKNOWLEDGEMENTS |
---|
We thank Amy Lavin for expert advice in carrying out gel mobility shift assays. We thank Wayne Backes and John McLachlan for generous use of their fluorometers. We thank Karl Kelsey for his expert assistance in carrying out the NF-1 recovery experiment. We also thank Charles Miller and John McLachlan for critical comments regarding this work.
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FOOTNOTES |
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* This work was supported in part by the Cancer Association of Greater New Orleans and by funds from the Department of Defense.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work id dedicated to Dr. Richard A. Hartline
Recipient of a predoctoral fellowship from the Center for
Bioenvironmental Research Fund.
§ To whom correspondence should be addressed: Dept. of Environmental Health Sciences, Tulane School of Public Health & Tropical Medicine SL-29, 1430 Tulane Ave., New Orleans, LA 70112-2699. Tel.: 504-582-7904; Fax: 504-585-6939; E-mail: wtoscan{at}tmc.tulane.edu.
The abbreviations used are:
TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; Ah, aryl hydrocarbon; AhRE, Aryl hydrocarbon response element; CYP1A1, cytochrome P450 1A1; CYP1B1, cytochrome P450 IB1; ECOD, 7-ethoxy-coumarin-O-deethylase; ICI 182, 780,
7-[9-(4,4,5,5,5-pentafluoro-pentylsulfinyl)nonyl]estra-1,3,5(10)-triene-3,17
-diol; NF-1, nuclear factor 1; SA, specific activity; DTT, dithiothreitol; ANOVA, analysis of variance.
2 M. S. Ricci and W. A. Toscano, unpublished observations.
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REFERENCES |
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