Modulation of AhR-Mediated CYP1A1 mRNA and EROD Activities by 17ß-Estradiol and Dexamethasone in TCDD-Induced H411E Cells

K. P. Lai, M. H. Wong and Chris K. C. Wong1

Institute for Natural Resources and Environmental Management, and Department of Biology, Hong Kong Baptist University, Hong Kong, PR China

1 To whom correspondence should be addressed at Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong. Fax: (852)-3411-5995. E-mail: ckcwong{at}hkbu.edu.hk.

Received August 13, 2003; accepted November 17, 2003

ABSTRACT

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 dose- and 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, 17ß-estradiol (E2), diethylstilbestrol (DES), testosterone (T), 5{alpha}-dihydrotestosterone (DHT), dexamethasone (DEX), and T3, 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 E2/DES treatment. Other tested hormones, however, had no significant effect. Using a receptor antagonist (RU486), DEX-mediated potentiation of TCDD-elicited EROD activity was completely abolished. E2-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, E2-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; 17ß-estradiol; dexamethasone; H411E cells.

An anthropogenic compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin (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 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 cis-acting dioxin responsive element (DRE) (Matsushita et al., 1993Go; Reyes et al., 1992Go; Safe, 1995Go; Schmidt and Bradfield, 1996Go). In addition, interventions on the hypoxia or PKC signaling pathways were reported (Matsumura et al., 1997Go; Nie et al., 2001Go). 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., 1989Go; Fujisawa-Sehara et al., 1988Go; Jones et al., 1986aGo,bGo; Neuhold et al., 1986Go). 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., 1978Go; Murray et al., 1979Go; Poland and Knutson, 1982Go; Umbreit et al., 1988Go), in which de novo hormonal modulation of TCDD-mediated pathways were suggested to be essential (MacKenzie et al., 1992Go; Peterson et al., 1993Go; Umbreit and Gallo, 1988Go; Umbreit et al., 1989Go). The hormonal modulation of TCDD action has been demonstrated in many cell-line models. Direct or indirect interventions on TCDD-elicited signal pathways were reported (Celander et al., 1997Go; Wiebel and Cikryt, 1990Go; Wolfle et al., 1993aGo). 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., 1995Go; Miller, 1997Go; Wei et al., 1998Go, 2000Go), bilirubin (Sinal and Bend, 1997Go), lipoxin A4 (Schaldach et al., 1999Go), flavones (Reiners, et al., 1999Go), glucocorticoids, ß-estradiol, tamoxifen (Celander et al., 1997Go; Silverstone et al., 1994Go; Umbreit et al., 1988Go, 1989Go; Wiebel and Cikryt, 1990Go; Wormke et al., 2000Go), indirubin (Adachi et al., 2001Go) and 7-ketocholesterol (Savouret et al., 2001Go). Collectively, TCDD-stimulated AhR pleiotropic responses seemed to be modulated by many different factors. The elucidation of the underlying modulating 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., 1987Go; Denis et al., 1988Go; Evans, 1988Go; Gustafsson et al., 1987Go; Henry et al., 1989Go). 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 17ß-estradiol (E2), diethylstilbestrol (DES), testosterone (T), 5{alpha}-dihydroxytestosterone (DHT), dexamethasone (DEX), and triiodothyronine (T3), 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, 50 U/ml penicillin and 50 µg/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-tetrachlorodibenzo-p-dioxin (TCDD) (Cambridge Isotope Laboratories, Inc.), (b) 0.1 nM to 100 µM 17ß-estradiol (E2), diethylstilbestrol (DES) (Sigma), or 4-hydroxytamoxifen (Calbiochem), (c) 0.01 nM to 100 µM testosterone (T) or 5{alpha}-dihydroxytestosterone (DHT) (Sigma), (d) 10 fM to 1 nM dexamethasone (DEX) (Calbiochem), (e) 0.01 nM to 1 µM triiodothyronine (T3) (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 µl of fresh medium containing 8 µM 7-ethoxyresorufin (Sigma) and 10 µM 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 µl 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., 2004Go). 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 µM E2, (c) 1.6 pg/ml TCDD + 0.2-40 µM 4-hydroxytamoxifen, (d) 1.6 pg/ml TCDD + 0.2-40 µM ICI 182780, (e) 1.6 pg/ml TCDD + 10-50 µM E2 + 0.2-40 µM 4-hydroxytamoxifen or ICI 182780, (f) 1.6 pg/ml TCDD + 0.01 nM to 100 µM T or DHT, (g) 1.6 pg/ml TCDD + 0.01 nM to 1 µM T3, (h) 1.6 pg/ml TCDD + 0.1 pM to 0.1 µM DEX, (i) 1.6 pg/ml TCDD + 0.01-1 µM RU486, or (j) 1.6 pg/ml TCDD + 0.01 µM DEX + 0.01-1 µM 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®II-TOPO® (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 A260/A280 ratio was used in this study. Real-time PCR was conducted for mRNA quantification. Briefly, 5 µg of total cellular RNA was mixed with 0.5 µg of pd(T)12-18 to a final volume of 28 µl, incubated at 70°C for 10 min, and finally added into 22 µl reverse transcription buffer containing 200 U of MMLV (Invitrogen, Netherlands). Quantitated standards (104-108) and sample cDNA were analyzed by iCycler iQ real-time PCR detection system using iQTM SYBR® 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 µg/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 µl 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 WesternBreezeTM 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 17ß-estradiol modulated TCDD activated CYP1A1 mRNA and EROD activities in H4IIE cells.
The cells were incubated at 37°C in a humidified 5% CO2 incubator. For the EROD assay, cells were seeded in a density of 2 x 104/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 µg/ml cycloheximide (CHX), (c) 1.6 pg/ml TCDD + 2 µg/ml CHX, (d) 1.6 pg/ml TCDD + 50-100 µM E2, (e) 1.6 pg/ml TCDD + 50-100 µM E2 + 2 µg/ml CHX, (f) 1.6 pg/ml TCDD + 0.01-0.1 µM DEX, or (g) 1.6 pg/ml TCDD + 0.01-0.1 µM DEX + 2 µg/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 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 E2, DES, 4-hydroxytamoxifen, T, DHT, and T3 had no effect on the basal EROD activities.



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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 17ß-estradiol, diethylstilbestrol, 4-hydroxytamoxifen, testosterone, 5{alpha}-dihydrotestosterone, and T3. Dose-dependent inductions of EROD and CYP1A1 mRNA were found in TCDD-treated H4IIE cells.

 
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 E2 and DES in a dose-dependent manner at the concentration range of 1-100 µM; 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 concentrations of 0.01-0.1 µM DEX was found. T, DHT, and T3 had no effect on TCDD-induced EROD activities.



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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) 17ß-estradiol and (b) dexamethasone. Results shown were from more than three independent experiments.

 
To eliminate the possibility that the EROD-modulating effect was due to the direct antagonistic or agonistic action of E2 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 E2 or DEX treatment, respectively (Fig. 3).



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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 + E2, or TCDD + DEX. Total RNA of each sample was reverse-transcribed and analyzed by iCycler iQ real-time PCR detection system using iQTM SYBR® Green Supermix. For western blot, dose-dependent suppression and induction of CYP1A1 protein by E2 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.

 
To test whether the modulation of TCDD-mediated EROD activities by E2 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 E2-mediated suppression of TCDD-stimulated EROD activity (results not shown). The observation suggested that the suppressive effect of E2 might not be mediated by its receptor. Moreover, in the cotreatment study of the cells with TCDD, E2, and 4-hydroxytamoxifen, the suppressive effect on TCDD-induced 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).



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FIG. 4. Effects of (a) 4-hydroxytamoxifen (Tam) on 17ß-estradiol (E2)-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) E2, Tam, or E2 + Tam, (b) DEX, RU486, DEX + RU486. Results shown were from more than three independent experiments.

 
In order to investigate whether the DEX- and E2-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 µg/ml) of CHX were tested in preliminary studies, and a dose of 2 µg/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 TCDD-treated cells at 5 and 24 h posttreatment (Fig. 6). CHX had no effect on E2-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 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).



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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 µg/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.

 


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FIG. 6. Effect of CHX on CYP1A1 mRNA levels of TCDD, TCDD + E2, 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 + E2, TCDD + DEX, TCDD + CHX, TCDD + CHX + E2, or TCDD + CHX + DEX. Total RNA of each sample was reverse-transcribed and analyzed by iCycler iQ real-time PCR detection system using iQTM SYBR® Green Supermix. Results shown were from more than three independent experiments.

 


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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.

 
DISCUSSION

Previous studies from this laboratory demonstrated a dose- and time-dependent induction of CYP1A1 gene expression and EROD activities by TCDD treatment. In agreement with the preceding study (Lai et al., 2004Go), 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, the effect of E2, DES, T, DHT, DEX, or T3 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., 2001Go; Prough et al., 1996Go). The study indicated that low concentrations of DEX (<10 µM) suppressed GSTA2 gene expression via a classical GR-dependent pathway, whereas high concentrations of DEX (<10 µM) 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 its suppressive effect. To our knowledge, this is the first report to demonstrate the dual effects of DEX on modulating TCDD-induced EROD activity. The possibility for ligands to function as both AhR agonists and antagonists has been well documented (Biegel et al., 1989Go; Gasiewicz et al., 1996Go; Harris et al., 1989Go; Kurl et al., 1993Go; Liu et al., 1993Go; Lu et al., 1996Go). 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 TCDD-mediated EROD induction was concentration dependent and was reversible by RU486, which strongly indicated GR involvement.

Exposure of the cells to T, DHT, and T3 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 E2 or DES treatments. In this study, E2-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 E2 were in the micromolar range, which were considerably above the physiological level. The nonresponsiveness of the TCDD-mediated EROD activity to physiological doses of E2 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., 1997Go; Ricci et al., 1999Go; Wormke et al., 2000Go). Taken together, the E2-mediated 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., 1986Go). 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 E2-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, 1984Go). Therefore, the observation suggested that in this study 4-hydroxytamoxifen, a nonsteroidal selective estrogen receptor modulator (SERM), might mimic the nonspecific action of E2 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., 1986Go). While a considerable number of studies indicated that the activation of PKC activities was required for AhR-mediated signal pathway (DePetrillo and Kurl, 1993Go; Kramer et al., 1987Go; Kurl et al., 1993Go; Long et al., 1998Go; Stephen et al., 1997Go; Weber et al., 1996Go; Wolfle et al., 1993bGo), 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 E2-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 E2 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, 2000Go; Ma et al., 2000Go). In this study, CHX had no significant effect on E2-mediated suppression. In addition, CHX-mediated "superinduction" was abolished in the TCDD/E2-cotreated cells even if the AhR degradation process in the cells was already attenuated by CHX treatment. The results supported our earlier assumption that E2 might hinder the formation of activated TCDD/AhR complex. Hence, the E2-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, 1990Go), porcine, and human aorta endothelial cells (Celander et al., 1997Go). 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 DEX-mediated 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., 2002Go; Du et al., 2000Go; Hong and Forsberg, 1995Go; Marinovic et al., 2002Go; Mitch et al., 1999Go; Price et al., 1994Go; Thompson et al., 1999Go; Vugmeyster et al., 2002Go; Wang et al., 1998Go). The DEX-stimulated ubiquitin expression might compensate for the CHX inhibitory effect on the activities of ubiquitin proteosome (Ma and Baldwin, 2000Go, 2002Go; Ma et al., 2000Go). It is particularly true in this study because the dose of 2 µg/ml CHX used is considerably lower than that in Ma et al. (2000) study (10 µg/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 E2 were identified and characterized. DEX-mediated up-modulation was a GR- and time-dependent process. The induction process was sensitive to protein synthesis inhibitors. Furthermore, this study provided evidence to support an E2-elicited 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., 1999Go), but they are not contradictory with each other. This indicated that the two E2-mediated pathways are possibly involved in AhR-mediated gene regulation. However the regulation seems to be cell-type specific and E2 concentration dependent. Other hormones, such as T, DHT, and T3 had no 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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