TOXICOLOGICAL SCIENCES 78, 41– 49 (2004)
DOI: 10.1093/toxsci/kfh045
Advance Access publication December 22, 2003
Modulation of AhR-Mediated CYP1A1 mRNA and EROD Activities by
17␤-Estradiol and Dexamethasone in TCDD-Induced H411E Cells
K. P. Lai, M. H. Wong, and Chris K. C. Wong 1
Institute for Natural Resources and Environmental Management, and Department of Biology, Hong Kong Baptist University, Hong Kong, PR China
Received August 13, 2003; accepted November 17, 2003
human beings. One well-recognized toxic potency for TCDD is
mediated via its interaction with cytosolic aryl hydrocarbon
receptor (AhR), followed by heterodimerization with an Ah
receptor nuclear translocator, and finally binding on the cisacting dioxin responsive element (DRE) (Matsushita et al.,
1993; Reyes et al., 1992; Safe, 1995; Schmidt and Bradfield,
1996). In addition, interventions on the hypoxia or PKC signaling pathways were reported (Matsumura et al., 1997; Nie et
al., 2001). On a molecular level, the activation of DRE located
in the 5⬘-flanking region of the CYP1A1 gene can stimulate the
expression of CYP1A1 mRNA and/or its associated phase I
enzyme, 7-ethoxyresorufin-O-deethylase (EROD) activity
(Denison et al., 1989; Fujisawa-Sehara et al., 1988; Jones et
al., 1986a,b; Neuhold et al., 1986). On a pathological level,
TCDD-activated AhR pleiotropic responses could lead to a
variety of species- and organ-specific toxic consequences, including a wasting syndrome, hepatotoxicity, thymic atrophy,
immune suppression, perturbations of the endocrine system,
reproductive alternation, and malignant cell transformation
(Kociba et al., 1978; Murray et al., 1979; Poland and Knutson,
1982; Umbreit et al., 1988), in which de novo hormonal
modulation of TCDD-mediated pathways were suggested to be
essential (MacKenzie et al., 1992; Peterson et al., 1993; Umbreit and Gallo, 1988; Umbreit et al., 1989). The hormonal
modulation of TCDD action has been demonstrated in many
cell-line models. Direct or indirect interventions on TCDDelicited signal pathways were reported (Celander et al., 1997;
Wiebel and Cikryt, 1990; Wolfle et al., 1993a). To elucidate
the interactive regulation, numerous studies have reported a
variety of natural compounds or ligands that produced agonistic and/or antagonistic effects on TCDD-elicited pathways,
including tryptophan and indole-containing compounds (Chen
et al., 1995; Miller, 1997; Wei et al., 1998, 2000), bilirubin
(Sinal and Bend, 1997), lipoxin A4 (Schaldach et al., 1999),
flavones (Reiners, et al., 1999), glucocorticoids, ␤-estradiol,
tamoxifen (Celander et al., 1997; Silverstone et al., 1994;
Umbreit et al., 1988, 1989; Wiebel and Cikryt, 1990; Wormke
et al., 2000), indirubin (Adachi et al., 2001) and 7-ketocholesterol (Savouret et al., 2001). Collectively, TCDD-stimulated
AhR pleiotropic responses seemed to be modulated by many
different factors. The elucidation of the underlying modulating
TCDD elicits a variety of species- and organ-specific pathological consequences. The differential toxicities are thought to relate
to the de novo modulation of TCDD action by endogenous hormones. Previous studies from this laboratory demonstrated a doseand time-dependent induction of CYP1A1 expression and
7-ethoxyresorufin-O-deethylase (EROD) activities in H4IIE cells
by picomolar levels of TCDD treatment. In this study, we examined the hormonal modulation of TCDD-elicited AhR-mediated
biochemical responses. Lipid-soluble hormones, 17␤-estradiol
(E 2), diethylstilbestrol (DES), testosterone (T), 5␣-dihydrotestosterone (DHT), dexamethasone (DEX), and T 3, were studied for
their possible interactions with the TCDD-mediated effects. Our
results showed that CYP1A1 expression and EROD activities
induced by TCDD were potentiated or suppressed, respectively, by
DEX or E 2/DES treatment. Other tested hormones, however, had
no significant effect. Using a receptor antagonist (RU486), DEXmediated potentiation of TCDD-elicited EROD activity was completely abolished. E 2-mediated suppression, however, was not affected by cotreatment with the estrogen receptor antagonists,
4-hydroxytamoxifen or ICI 182780. Taking a step further to dissect the possible mechanisms involved, with the aid of cycloheximide (CHX), DEX-mediated potentiation was found to depend on
the posttranscriptional process. The DEX pretreatment study indicated that the potentiation was a time-dependent process. In
contrast, E 2-mediated suppression did not rely on the synthesis of
protein factors. Presumably it might hinder the formation of the
activated TCDD/AhR complex and so the subsequent binding on
DRE.
Key Words: CYP1A1 mRNA; EROD; 17␤-estradiol; dexamethasone; H411E cells.
An anthropogenic compound, 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), is one of the most toxic human-made chemicals. Like most of persistent organic pollutants, the fat-seeking
and ubiquitous features of TCDD can lead to its bioaccumulation and biomagnification along food chains, finally reaching
1
To whom correspondence should be addressed at Department of Biology,
Hong Kong Baptist University, Kowloon Tong, Hong Kong. Fax:(852)-34115995. E-mail: [email protected].
Toxicological Sciences vol. 78 no. 1 © Society of Toxicology 2004; all rights
reserved.
41
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LAI ET AL.
mechanism can provide a better understanding of AhR-mediated pathways as well as TCDD-mediated toxicity.
The major physiological function of AhR is not well defined;
however its physicochemical properties have been characterized, and it is suggested to be a member of the ligand transcriptional factor superfamily that includes retinoic acid, steroids, and thyroid hormone receptor proteins (Cuthill et al.,
1987; Denis et al., 1988; Evans, 1988; Gustafsson et al., 1987;
Henry et al., 1989). The resemblance in receptor properties
shed light on the possible interventions between TCDD and
steroid/lipid-soluble hormones inside the cells. Based on this,
the present study addressed the possible interactions between
TCDD and lipid-soluble hormones, including 17␤-estradiol
(E 2), diethylstilbestrol (DES), testosterone (T), 5␣-dihydroxytestosterone (DHT), dexamethasone (DEX), and triiodothyronine (T 3), on CYP1A1 expression and EROD activity in
rat hepatoma H4IIE cell. The outcome of this study would be
useful for our better understanding of the biochemical interactions of TCDD with natural hormones, shedding light on the
issue of de novo modulation of TCDD-elicited responses.
MATERIALS AND METHODS
Effects of natural hormones on EROD activities in H4IIE cells. Rat
hepatoma cells, H4IIE, grown in Dulbecco’s minimum essential medium and
supplemented with 10% fetal calf serum, 50U/ml penicillin and 50 ␮g/ml
streptomycin (GIBCO/BRL, Carlsbas, CA), were subjected to one of the
following treatments for 24 h: (a) 0.001– 8 pg/ml 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) (Cambridge Isotope Laboratories, Inc.), (b) 0.1 nM to 100
␮M 17␤-estradiol (E 2), diethylstilbestrol (DES) (Sigma), or 4-hydroxytamoxifen (Calbiochem), (c) 0.01 nM to 100 ␮M testosterone (T) or 5␣-dihydroxytestosterone (DHT) (Sigma), (d) 10 fM to 1 nM dexamethasone (DEX)
(Calbiochem), (e) 0.01 nM to 1 ␮M triiodothyronine (T 3) (Calbiochem), or (f)
dimethyl sulphoxide (DMSO, solvent control) (Sigma). For CYP1A1 mRNA
quantification, the cells were dissolved in TRIZOL reagent (GIBCOL/BRL)
for total RNA isolation and subjected to real-time PCR quantification. For
EROD assay, the medium was removed, and 100 ␮l of fresh medium containing 8 ␮M 7-ethoxyresorufin (Sigma) and 10 ␮M dicumarol (Sigma) was added
for additional 60 min of incubation at 37°C. Afterwards, the medium was
transferred to a new 96-well plate and mixed with 130 ␮l of absolute ethanol.
Resorufin-associated fluorescence was measured in the solution on a multiwell
fluorescence reader, with excitation/emission wavelengths of 530/590 nm
(FluroStar) (Lai et al., 2004). Protein content was measured by Bio-Rad
protein assay kit.
Effects of the hormones on TCDD-mediated EROD and CYP1A1 levels.
For TCDD interactions with lipid-soluble hormones, cells were exposed for
24 h to one of the following treatments: (a) 1.6 pg/ml TCDD, (b) 1.6 pg/ml
TCDD ⫹ 0.1 nM to 100 ␮M E 2, (c) 1.6 pg/ml TCDD ⫹ 0.2– 40 ␮M
4-hydroxytamoxifen, (d) 1.6 pg/ml TCDD ⫹ 0.2– 40 ␮M ICI 182780, (e) 1.6
pg/ml TCDD ⫹ 10 –50 ␮M E 2 ⫹ 0.2– 40 ␮M 4-hydroxytamoxifen or ICI
182780, (f) 1.6 pg/ml TCDD ⫹ 0.01 nM to 100 ␮M T or DHT, (g) 1.6 pg/ml
TCDD ⫹ 0.01 nM to 1 ␮M T 3, (h) 1.6 pg/ml TCDD ⫹ 0.1 pM to 0.1 ␮M
DEX, (i) 1.6 pg/ml TCDD ⫹ 0.01–1 ␮M RU486, or (j) 1.6 pg/ml TCDD ⫹
0.01 ␮M DEX ⫹ 0.01–1 ␮M RU486. Total RNA was isolated for CYP1A1
mRNA quantification. EROD assay was conducted as described above.
Preparation of CYP1A1 and actin standards for real-time PCR.
CYP1A1 and GAPDH PCR products were generated by PCR of total RNA
derived from H4IIE cells. The primers were designed on the basis of the
published sequence of CYP1A1: CCTCTTTGGAGCTGGGTTTG-forward
and 5⬘-TGCTGTGGGGGATGGTGAAG-reverse, GAPDH: ATGGTGAAGGTCGTGTGAAC-forward and TCCACCACCCTGTTGCTGTA-reverse. The
PCR fragments for CYP1A1 (230 bp) and GAPDH (200 bp) were purified,
subcloned into pCR威II-TOPO威 (Invitrogen), and subjected to dideoxy sequencing for verification. The purified plasmids were quantified, and the
respective copy numbers were calculated.
Real-time PCR. The treated cells were dissolved in TRIZOL Reagent
(GIBCO/BRL) and total RNA was extracted according to the manufacturer’s
instructions. Purified RNA with a ratio of 1.6 –1.8 at A 260/A 280 ratio was used
in this study. Real-time PCR was conducted for mRNA quantification. Briefly,
5 ␮g of total cellular RNA was mixed with 0.5 ␮g of pd(T) 12–18 to a final
volume of 28 ␮l, incubated at 70°C for 10 min, and finally added into 22 ␮l
reverse transcription buffer containing 200 U of MMLV (Invitrogen, Netherlands). Quantitated standards (10 4–10 8) and sample cDNA were analyzed by
iCycler iQ real-time PCR detection system using iQ™ SYBR威 Green Supermix (Bio-Rad). The copy number for each sample was calculated, and the data
were normalized using the expression level of GAPDH mRNA. The PCR
conditions were 95°C for 3 min and 40 cycles of 95°C for 30 s, 56°C for 30 s,
and 72°C for 1 min. Fluorescent signals were captured at 82°C; the occurrence
of primer– dimers and secondary products was detected using melting curve
analysis. Control amplifications were done either without RT or without RNA.
Following PCR amplification, the reaction products were run at 100 V on a 1%
agarose gel with 0.5 ␮g/ml ethidium bromide to determine products specificity.
All glass- and plastic-ware was treated with diethyl pyrocarbonate and autoclaved.
Western blot analysis. The treated cells were washed with two or three
changes of cold PBS. Adherent cells were scraped from the plastic surface and
transferred to a microcentrifuge tube. The cells were pelleted and resuspended
in 30 –50 ␮l of cold lysis buffer containing 250 mM Tris/HCl, pH 8.0, 1%
NP-40 and 150 mM NaCl. After 10 min incubation on ice, the lysed cells were
pelleted, and the supernatant was assayed for protein concentration (DC
Protein Assay Kit II; Bio-Rad Pacific Ltd) and finally mixed with LDS sample
buffer, which was then subjected to electrophoresis in NuPage 4 –12% Bis-Tris
gradient gel (Invitrogen). The gel was blotted onto a PVDF membrane.
Western blot was conducted using a WesternBreeze TM Chemiluminescent kit
(Invitrogen). Briefly, the membrane was incubated with a blocking solution
containing rabbit anti-CYP1A1 (Chemicon Int.), followed by alkaline phosphatase-conjugated goat anti-rabbit antibody. The membrane was developed
with chemiluminescence reagent.
Effects of cycloheximide on DEX or 17␤-estradiol modulated TCDD
activated CYP1A1 mRNA and EROD activities in H4IIE cells. The cells
were incubated at 37°C in a humidified 5% CO 2 incubator. For the EROD
assay, cells were seeded in a density of 2 ⫻ 10 4/well in a 96-well plate. The
cells were exposed for 5 and 24 h to one of the following treatments: (a) 1.6
pg/ml TCDD, (b) 1–10 ␮g/ml cycloheximide (CHX), (c) 1.6 pg/ml TCDD ⫹
2 ␮g/ml CHX, (d) 1.6 pg/ml TCDD ⫹ 50 –100 ␮M E 2, (e) 1.6 pg/ml TCDD ⫹
50 –100 ␮M E 2 ⫹ 2 ␮g/ml CHX, (f) 1.6 pg/ml TCDD ⫹ 0.01– 0.1 ␮M DEX,
or (g) 1.6 pg/ml TCDD ⫹ 0.01– 0.1 ␮M DEX ⫹ 2 ␮g/ml CHX. No treatment
and DMSO-treated cells were used as the controls.
In the DEX pretreatment study, the cells were preincubated with 0.01 mM
DEX for 0, 4, or 8 h before the addition of 1.6 pg/ml TCDD. The cells were
then incubated for another 24 h and were assayed for EROD activities.
Statistical analysis. Drug treatments were performed in triplicate in the
same experiments, and individual experiments were repeated at least three
times. All data are represented as mean ⫾ SE. Statistical significance is tested
by Student’s t-test or one-way analysis of variance (ANOVA) followed by
Duncan’s Multiple Range Test. Groups were considered significantly different
if p ⬍ 0.05.
RESULTS
CYP1A1 mRNA and EROD induction profiles produced by
TCDD treatments are shown in Figure 1a. Dose-dependent
MODULATION OF AHR-MEDIATED CYP1A1 mRNA AND EROD ACTIVITIES
43
FIG. 1. The potency of EROD induction. The cells were subjected to one
of the following treatments for 24 h: (a)
TCDD, (b) dexamethasone and 17␤-estradiol, diethylstilbestrol, 4-hydroxytamoxifen, testosterone, 5␣-dihydrotestosterone, and T 3. Dose-dependent inductions of EROD and CYP1A1 mRNA
were found in TCDD-treated H4IIE
cells.
inductions of CYP1A1 mRNA and EROD activities were
observed in cells exposed to 0.2– 4 pg/ml and 0.001– 8 pg/ml
TCDD, respectively. For cells exposed to different concentrations of the lipid-soluble hormones, a mild induction of EROD
activities was found in cells exposed to the higher end of the
concentration range for DEX (10 –100 nM) (Fig. 1b). The
agonistic effect was about 10% of maximum TCDD-induced
EROD activity. Other hormones such as E 2, DES, 4-hydroxytamoxifen, T, DHT, and T 3 had no effect on the basal EROD
activities.
To study the effects of the hormones on TCDD-mediated
EROD induction, TCDD-treated (1.6 pg/ml) H4IIE cells were
exposed to different concentrations of the hormones (Fig. 2).
The cotreatment studies indicated that TCDD-induced EROD
activities were suppressed by E 2 and DES in a dose-dependent
manner at the concentration range of 1–100 ␮M; no effect was
found at the submicromolar range. DEX exerted a suppressive
effect at concentrations of 0.1 pM to 1 nM. However, a strong
potentiated effect on TCDD-induced EROD activity at concen-
trations of 0.01– 0.1 ␮M DEX was found. T, DHT, and T 3 had
no effect on TCDD-induced EROD activities.
To eliminate the possibility that the EROD-modulating effect was due to the direct antagonistic or agonistic action of E 2
and DEX on the CYP1A1 enzyme system, the effects of the
hormones on CYP1A1 expression level of TCDD-induced
cells were examined using real-time PCR and western blot
analysis. In agreement with the EROD results, CYP1A1
mRNA and protein were significantly reduced or induced by E 2
or DEX treatment, respectively (Fig. 3).
To test whether the modulation of TCDD-mediated EROD
activities by E 2 and DEX were mediated by their innate receptors, the effect of receptor antagonists including 4-hydroxytamoxifen, ICI 182780 (for estrogen receptor), and RU486 (for
glucocorticoid receptor, GR) were examined. The treatments of
the cells with the receptor antagonist alone had no significant
effect on the basal EROD activity of the untreated control cells.
In the cotreatment studies, ICI 182780 had no effect on E 2mediated suppression of TCDD-stimulated EROD activity (re-
44
LAI ET AL.
transcript in TCDD/CHX/DEX treatment at 24 h was due to the
potentiation effect of DEX or the action of CHX. To determine
if the DEX-mediated potentiation required the preceding synthesis of protein factors, a DEX pretreatment study was conducted. The results indicated 8-h DEX pretreatment produced
cells with a significantly higher EROD activity than the cells
with no pretreatment (Fig. 7).
DISCUSSION
FIG. 2. The modulation of TCDD-induced EROD activities. TCDD
treated cells (1.6 pg/ml) were exposed for 24 h to different concentrations of
the following treatments: (a) 17␤-estradiol and (b) dexamethasone. Results
shown were from more than three independent experiments.
sults not shown). The observation suggested that the suppressive effect of E 2 might not be mediated by its receptor.
Moreover, in the cotreatment study of the cells with TCDD, E 2,
and 4-hydroxytamoxifen, the suppressive effect on TCDDinduced EROD activity was further enhanced (Fig. 4a). The
observation was not completely unanticipated as it was observed that 4-hydroxytamoxifen alone could reduce TCDD
mediated EROD induction. In the cotreatment study of TCDD,
DEX and RU486, the DEX-potentiated TCDD induced EROD
activity was completely abolished (Fig. 4b).
In order to investigate whether the DEX- and E 2-mediated
modulating effects on the steady-state mRNA levels were
acting at the posttranscriptional level, the effect of the protein
synthesis inhibitor, CHX, on TCDD-mediated expression of
CYP1A1 mRNA accumulation and EROD activities was examined. Different doses (1–10 ␮g/ml) of CHX were tested in
preliminary studies, and a dose of 2 ␮g/ml CHX was selected
in which very low cell toxicity was detected. The cotreatment
of CHX with TCDD reduced the EROD activities to the
steady-state level, as measured in the untreated and control
cells (Fig. 5). The accumulated CYP1A1 mRNA level, however, was significantly increased as compared with the TCDDtreated cells at 5 and 24 h posttreatment (Fig. 6). CHX had no
effect on E 2-mediated suppression of the transcription while
modulated the DEX potentiation effect in TCDD-treated cells.
In the first 5 h of treatment, CHX-elicited “superinduction”
was not found in TCDD/CHX/DEX cotreated cells. In the 24-h
treatment, TCDD/CHX/DEX cotreatment produced cells with
significantly higher levels of CYP1A1 mRNA than the TCDD
or TCDD/DEX treatments, although considerably lower than
that of the TCDD/CHX “superinduced” cells. On this basis, it
is not possible to identify if the “increase” in the CYP1A1
Previous studies from this laboratory demonstrated a doseand time-dependent induction of CYP1A1 gene expression and
EROD activities by TCDD treatment. In agreement with the
preceding study (Lai et al., 2004), CYP1A1 expression and
EROD activity were highly induced by treatment with TCDD.
The present study demonstrated the interactions of steroid and
thyroid hormones with TCDD/AhR-mediated responses. Because the major activated biochemical activity involved was
the increase in CYP1A1 expression and EROD activities, we
evaluated the effect by measuring the level/activity of the
corresponding mRNA and protein. In the first part of the study,
FIG. 3. Messenger RNA (a) and protein (b) levels of CYP1A1 in H4IIE
cells. Cells were incubated for 24 h in 10% FBS/DMEM containing TCDD,
TCDD ⫹ E 2, or TCDD ⫹ DEX. Total RNA of each sample was reversetranscribed and analyzed by iCycler iQ real-time PCR detection system using
iQ™ SYBR威 Green Supermix. For western blot, dose-dependent suppression
and induction of CYP1A1 protein by E 2 and DEX, respectively, were noted.
Bars with the same letter are not significantly different according to the results
of one-way ANOVA followed by Duncan’s multiple range test (p ⬍ 0.05).
Results shown were from more than three independent experiments.
MODULATION OF AHR-MEDIATED CYP1A1 mRNA AND EROD ACTIVITIES
45
its suppressive effect. To our knowledge, this is the first report
to demonstrate the dual effects of DEX on modulating TCDDinduced EROD activity. The possibility for ligands to function
as both AhR agonists and antagonists has been well documented (Biegel et al., 1989; Gasiewicz et al., 1996; Harris et
al., 1989; Kurl et al., 1993; Liu et al., 1993; Lu et al., 1996).
However, the molecular factors for the determination of either
activity are still not known. Nevertheless, DEX-mediated potentiation of TCDD-induced responses can also be detected in
the levels of CYP1A1 mRNA and protein expression. To
elucidate some of the underlying mechanisms, a glucocorticoid
receptor (GR) antagonist (RU486) was used to investigate in
more detail. We found that the effects of DEX on TCDDmediated EROD induction was concentration dependent and
was reversible by RU486, which strongly indicated GR involvement.
Exposure of the cells to T, DHT, and T 3 did not show any
modulation of TCDD induction of EROD activity. However,
down-modulation of the TCDD-induced CYP1A1 expression
and EROD activity was observed in E 2 or DES treatments. In
this study, E 2-mediated suppressive effect cannot be blocked
by using two different estrogen receptor antagonists, 4-hydroxytamoxifen and ICI 182780. The observation was explicable because the effective doses of E 2 were in the micromolar
range, which were considerably above the physiological level.
The nonresponsiveness of the TCDD-mediated EROD activity
to physiological doses of E 2 treatment revealed that the receptor-mediated interactive effect reported in other studies was
specific to estrogen-regulated cells, depending on various transcriptional factors and coactivators (Glass et al., 1997; Ricci
FIG. 4. Effects of (a) 4-hydroxytamoxifen (Tam) on 17␤-estradiol (E 2)mediated suppressive effect and (b) RU486 on DEX-mediated potentiation of
TCDD-induced EROD activities in H411E cells. TCDD treated cells (1.6
pg/ml) were exposed for 24 h to different concentrations of the following
treatments (a) E 2, Tam, or E 2 ⫹ Tam, (b) DEX, RU486, DEX ⫹ RU486.
Results shown were from more than three independent experiments.
the effect of E 2, DES, T, DHT, DEX, or T 3 treatment on the
basal EROD activity was examined. At the tested doses, DEX
showed a weak agonist effect at the higher end of the concentration range. In the cotreatment studies, DEX potentiated
TCDD-induced EROD activity at nanomolar concentration;
however, a suppressive effect was detected at subnanomolar
concentration. Similar biphasic effects of DEX on AhR-mediated glutathione S-transferase A2 (GSTA2) gene expression
were reported (Falkner et al., 2001; Prough et al., 1996). The
study indicated that low concentrations of DEX (⬍10 ␮M)
suppressed GSTA2 gene expression via a classical GR-dependent pathway, whereas high concentrations of DEX (⬎10 ␮M)
induced expression via a pregnane X receptor (PXR)-dependent mechanism. Whether the PXR pathway was involved in
mediating DEX biphasic effects in this study, however, has not
been addressed. Nevertheless, the DEX-mediated potentiation
was considered to be much more significant as compared with
FIG. 5. Effects of CHX on TCDD-stimulated EROD activity in H4IIE
cells. TCDD-treated cells (1.6 pg/ml) were exposed for 24 h to 1.6 pg/ml
TCDD, 2 ␮g/ml CHX or TCDD ⫹ CHX. A significant reduction of EROD
activity in TCDD ⫹ CHX treatment was noted. Results shown were from more
than three independent experiments.
46
LAI ET AL.
FIG. 6. Effect of CHX on CYP1A1 mRNA levels of TCDD, TCDD ⫹ E 2,
and TCDD ⫹ DEX treated cells. The cells were exposed for (a) 5 or (b) 24 h
to different concentrations of the following treatments: TCDD, TCDD ⫹ E 2,
TCDD ⫹ DEX, TCDD ⫹ CHX, TCDD ⫹ CHX ⫹ E 2, or TCDD ⫹ CHX ⫹
DEX. Total RNA of each sample was reverse-transcribed and analyzed by
iCycler iQ real-time PCR detection system using iQ™ SYBR威 Green Supermix. Results shown were from more than three independent experiments.
et al., 1999; Wormke et al., 2000). Taken together, the E 2mediated suppression was possibly not mediated by its innate
receptor; however it might interact with AhR and cause an
interference on TCDD-activated pathways (Gallo et al., 1986).
It was interesting to note that in the cotreatment study of
TCDD and 4-hydroxytamoxifen, a suppressive effect on
TCDD-induced EROD activity was observed. In addition,
4-hydroxytamoxifen exerted an additive effect on E 2-mediated
suppression. The added suppression by 4-hydroxytamoxifen
was demonstrated to be dose dependent. In most species,
tamoxifen can act as a partial agonist-antagonist of estrogen in
reproductive tissue; however it acts mostly as a full agonist in
the liver (Furr and Jordan, 1984). Therefore, the observation
suggested that in this study 4-hydroxytamoxifen, a nonsteroidal selective estrogen receptor modulator (SERM), might
mimic the nonspecific action of E 2 in the cell, hindering the
formation of the activated TCDD/AhR complex. In addition to
its role as a SERM, 4-hydroxytamoxifen has been identified to
be a new member of protein kinase C (PKC) inhibitors
(O’Brian et al., 1986). While a considerable number of studies
indicated that the activation of PKC activities was required for
AhR-mediated signal pathway (DePetrillo and Kurl, 1993;
Kramer et al., 1987; Kurl et al., 1993; Long et al., 1998;
Stephen et al., 1997; Weber et al., 1996; Wolfle et al., 1993b),
the possible involvement of PKC inhibition in 4-hydroxytamoxifen-mediated down-modulation of EROD activities cannot be excluded.
The DEX-mediated potentiation, as well as the E 2-mediated
suppression of TCDD-induced responses can be detected in the
CYP1A1 mRNA, protein, and EROD levels. The DEX potentiation was a GR-dependent process, while E 2 suppressive
effect was not mediated by its innate receptor. To ascertain the
mechanism of the action, we investigated more specifically to
determine whether the modulating effects on the steady-state
CYP1A1 mRNA levels were acting at the posttranscriptional
level. The effect of the protein synthesis inhibitor CHX on
TCDD-mediated expression of CYP1A1 mRNA accumulation
and EROD activities was examined. The results demonstrated
that CHX blocked the newly synthesized proteins, which were
required for the degradation of TCDD-activated AhR complex,
and so led to the superinduction of CYP1A1 mRNA in the
cells. The observation was in agreement with the studies of Ma
et al. (Ma and Baldwin, 2000; Ma et al., 2000). In this study,
CHX had no significant effect on E 2-mediated suppression. In
addition, CHX-mediated “superinduction” was abolished in the
TCDD/E 2-cotreated cells even if the AhR degradation process
in the cells was already attenuated by CHX treatment. The
results supported our earlier assumption that E 2 might hinder
the formation of activated TCDD/AhR complex. Hence, the
FIG. 7. The effect of DEX pretreatment on TCDD induced EROD activity. The TCDD (1.6 pg/ml) stimulated cells were pretreated with DEX for 0,
4, and 8 h. A significant increase in EROD activity was noted in 8 h
pretreatment as compared with the unpretreated cells.
MODULATION OF AHR-MEDIATED CYP1A1 mRNA AND EROD ACTIVITIES
E 2-mediated suppressive effect was independent of the posttranscriptional process.
In the first 5 h of TCDD/CHX/DEX cotreatment, the
CYP1A1 transcript level of the cell was similar to that of the
TCDD/DEX or TCDD treatments. No DEX potentiation effect
was observed at all. The results suggested that the potentiation
was time dependent. Comparable results were observed in the
DEX pretreatment study, in which 8-h pretreatment produced
cells with significantly higher EROD activities. Similar observations have been demonstrated by other studies using
H4IIEC3/T (Wiebel and Cikryt, 1990), porcine, and human
aorta endothelial cells (Celander et al., 1997). In 24 h of
treatment, DEX induced potentiation in TCDD and “possibly”
on TCDD/CHX-treated cells. It is interesting to note that the
transcript level in TCDD/CHX/DEX cells was significantly
lower than that of the TCDD/CHX-superinduced cells and
higher than that of the DEX-potentiated cells, indicating that
the CHX “superinductive” effect was attenuated or the DEXmediated potentiation was enhanced. On this basis, we cannot
draw a conclusive statement, because it is not possible to
identify if the “increase” in the CYP1A1 transcript in TCDD/
CHX/DEX treatment resulted from the action of DEX, CHX,
or both. Considerable number of studies indicated DEX had
stimulatory effect on ubiquitin expression (Chrysis et al., 2002;
Du et al., 2000; Hong and Forsberg, 1995; Marinovic et al.,
2002; Mitch et al., 1999; Price et al., 1994; Thompson et al.,
1999; Vugmeyster et al., 2002; Wang et al., 1998). The DEXstimulated ubiquitin expression might compensate for the
CHX inhibitory effect on the activities of ubiquitin proteosome
(Ma and Baldwin, 2000, 2002; Ma et al., 2000). It is particularly true in this study because the dose of 2 ␮g/ml CHX used
is considerably lower than that in Ma et al. (2000) study
(10 ␮g/ml).
The present study provided a comprehensive screening of
the modulating effect of different lipid-soluble hormones on
TCDD induction of CYP1A1 expression. We are the first to
demonstrate the biphasic effects of DEX on TCDD-induced
EROD activities, providing an insight on the complexity of
GR-modulated AhR-mediated pathways. In addition, two distinctive regulatory mechanisms that were mediated by DEX
and E 2 were identified and characterized. DEX-mediated upmodulation was a GR- and time-dependent process. The induction process was sensitive to protein synthesis inhibitors.
Furthermore, this study provided evidence to support an E 2elicited non-receptor-mediated pathway to reduce TCDD-stimulated CYP1A1 expression. We hypothesized that the regulation involved the hindrance of TCDD/AhR complex formation.
The hypothesis was different from the proposed estrogen receptor mediated mechanism reported by others (Ricci et al.,
1999), but they are not contradictory with each other. This
indicated that the two E 2-mediated pathways are possibly involved in AhR-mediated gene regulation. However the regulation seems to be cell-type specific and E 2 concentration
dependent. Other hormones, such as T, DHT, and T 3 had no
47
effect on TCDD-induced CYP1A1 expression. The results of
this study are beneficial for our better understanding on the
biological interaction of TCDD with other ligands at the cellular level, shedding light on the similar modulating effect on
TCDD-mediated toxicity occurring in in vivo systems.
ACKNOWLEDGMENTS
This work was supported by Group Research–Central Allocation of the
Research Grants Council, University Grants Committee of Hong Kong.
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