In Vivo Antagonism of AhR-Mediated Gene Induction by 3'-Methoxy-4'-nitroflavone in TCDD-Responsive lacZ Mice

Daniel A. Nazarenko, Stephen D. Dertinger,1 and Thomas A. Gasiewicz,2

Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York 14642

Received November 9, 2000; accepted February 7, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS:
 RESULTS
 DISCUSSION
 REFERENCES
 
The aryl-hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is a member of the bHLH-PAS family of proteins. The highest-affinity ligand of this receptor is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a potent immunological, reproductive, and developmental toxicant. The mechanism of TCDD-induced toxicity and the gene modulations that result in toxicity have not been fully defined. The majority of work to date exploring AhR function has focused on agonist-activated AhR signaling. However, it is expected that a better understanding of AhR antagonism will lead to an improved understanding of TCDD toxicity and other AhR-mediated events. This study contributes to such investigations by utilizing the AhR antagonist 3'-methoxy-4'-nitroflavone (3'M4'NF) and a dioxin-responsive lacZ transgenic mouse model to characterize antagonism of the receptor system in vivo. The dose-response and time course of TCDD-induced transgene activation were evaluated in transgenic mice to provide information necessary to design 3'M4'NF in vivo studies. TCDD induction of the transgene was noted as early as 8 h after exposure in the lung. 3-µg/kg body weight TCDD was the lowest dose found to induce the reporter transgene. Finally, experiments were performed to evaluate the in vivo efficacy of 3'M4'NF. We found that 3'M4'NF inhibits TCDD-mediated reporter gene activation and CYP1A1 induction in vivo. Based on these findings, it is clear that DRE-lacZ animals and the antagonist 3'M4'NF represent important tools which will help in the identification of tissues where AhR is active, and to further characterize AhR-mediated signaling.

Key Words: aryl hydrocarbon receptor (AhR); 3'methoxy-4'-nitroflavone (3'M4'NF); receptor antagonism; TCDD.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS:
 RESULTS
 DISCUSSION
 REFERENCES
 
The aryl hydrocarbon receptor (AhR) is a member of the bHLH-PAS family of proteins. This ligand-activated transcription factor has been shown to mediate most, if not all, of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), its most potent ligand. TCDD is an immunological, reproductive, and developmental toxicant. Binding of TCDD to the AhR represents the first step in a series of cellular and molecular changes that are believed to play a role in the toxicity observed. Unoccupied AhR exists in the cytoplasm complexed with two heat-shock protein 90 (hsp90) molecules and a 43-kDa protein (Chen and Perdew, 1994Go). Agonist binding to the AhR results in the dissociation of hsp90, localization of the AhR-ligand complex to the nucleus, and heterodimerization with aryl-hydrocarbon receptor nuclear translocator (ARNT) (Elferink et al., 1990Go; Gasiewicz et al., 1991Go; Henry and Gasiewicz, 1993Go; Pollenz et al., 1994Go; Reyes et al., 1992Go). This heterodimeric complex is then able to bind dioxin-responsive elements (DREs), which are cis-acting elements found in the 5' regulatory regions of dioxin-responsive genes. However, the mechanisms of TCDD-induced toxicity and the gene modulations that result in toxicity have not been conclusively identified. Exposure to TCDD has been shown to alter the differentiation and proliferation of a variety of cell types (Abbott et al., 1989Go; Blankenship et al., 1993Go). Furthermore, the phenotype of AhR -/- mice indicates that the AhR has a critical role in the development and maintenance of several tissues. In particular, these mice show liver abnormalities, vascular changes, premature aging, and reduced reproductive viability (Abbott et al., 1999Go; Fernandez-Salguero et al., 1997Go; Lahvis et al., 2000Go).

An endogenous ligand for the AhR has not been conclusively identified. Most efforts to identify such a ligand have focused on compounds that act as agonists of the AhR. However, it is possible that an endogenous ligand may function as an antagonist or partial agonist. Many exogenous AhR ligands express both agonist and antagonist activity (Biegel et al., 1989Go; Liu et al., 1993Go; Gasiewicz and Rucci, 1991Go; Gasiewicz et al., 1996Go; Harris et al., 1989Go; Kurl et al., 1993Go; Lu et al., 1995Go, 1996Go). Yet, the molecular factors involved in the determination of whether a compound will function as an AhR agonist or antagonist are not well defined. Receptor antagonists have been used to characterize the in vivo function of several signaling pathways including those of the estrogen and androgen receptors (Grese and Dodge, 1998Go; Kemppainen et al., 1999Go; Meyers et al., 1999Go; Pike et al., 1999Go; Sun et al., 1999Go). It is expected that studies with an antagonist of the AhR may also yield useful information regarding its in vivo function and may aid in the characterization of endogenous ligands.

Previous work in this laboratory to assess potential AhR antagonists has utilized Hepa1c1c7 cells stably transfected with a DRE-regulated luciferase reporter gene (Henry et al., 1999Go). This screening system avoids the complications introduced by the fact that several potential AhR antagonists inhibit CYP1A-associated enzyme activity, the induction of which is a common assay of AhR activation (Liu et al., 1993Go; Lu et al., 1996Go). Flavone compounds containing a 3'-methoxy substitution and a 4' substituent with one or more terminal atoms of high electron density have been found to have the highest affinity for the AhR and the greatest antagonist potential (Gasiewicz et al., 1996Go; Henry et al., 1999Go). Studies using cell-free systems and murine hepatoma cells, which are stably transfected with DRE-driven luciferase reporter genes, indicate that 3'-methoxy-4'-nitroflavone (3'M4'NF) possesses potent AhR antagonistic activity and very little or no agonist potential (Gasiewicz et al., 1996Go; Henry et al., 1999Go; Lu et al., 1995Go). Yet, the ability of 3'M4'NF to block TCDD-elicited DRE-binding and reporter gene activation under cell-free conditions in vitro or in isolated cells may not adequately represent the events that occur in intact animals. There is some evidence that 3'M4'NF is able to attenuate the metabolic activation and genotoxicity of the AhR ligand benzo[a]pyrene in mice (Dertinger et al., 2000Go), suggesting that this compound does have some activity in vivo. However, the importance of non-AhR-mediated events needs to be further explored before the significance of this protective effect can be completely explained. It is possible, for example, that 3'M4'NF may modulate benzo[a]pyrene toxicity by directly blocking CYP1A-mediated metabolic activation.

The focus of the current study was to specifically and directly evaluate 3'M4'NF for AhR antagonist activity in vivo. For these experiments, we used DRE-lacZ transgenic mice, which express the lacZ reporter gene under the control of two DREs and a minimal TATA box promoter region (Willey et al., 1998Go). This model was selected over other systems, such as CYP1A1 enzymatic activity, because of the potential for direct antagonist interference with such systems; the fact that many of the DRE-mediated genes also have non-DRE regulatory elements in their promoter regions (Vasiliou et al., 1995Go); and the lack of ubiquitous expression of these genes. Therefore these genes may not adequately represent AhR activity in all tissues. Initially, the dose responsiveness and time course of ß-galactosidase (ß-gal) induction were evaluated in tissues identified as sensitive to TCDD exposure in the adult mouse. The rate of metabolic clearance of the antagonist was also determined. The findings presented herein indicate that 3'M4'NF is an effective AhR antagonist in vivo, inhibiting TCDD-induced reporter expression, as well as CYP1A1.


    MATERIALS AND METHODS:
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS:
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
TCDD was purchased from Cambridge Isotopes (Cambridge, MA). The antagonist 3'M4'NF was synthesized and purified (>98%) by procedures previously described (Gasiewicz et al., 1996Go). Phenylmethylsulfonyl fluoride (PMSF), leupeptin, dithiothreitol (DTT), Triton X-100, and potassium phosphate were from Sigma (St. Louis, MO).

Animals.
Male C57Bl/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). DRE-lacZ animals were generated using the p2Dlac plasmid as previously described (Willey et al., 1998Go). This construct consists of 2 DRE-Ds, derived from the 5' flanking region of the murine CYP1A1 gene (Lusska et al., 1993Go), a TATA box region containing the chicken ovalbumin TATA box, the lacZ reporter, and the SV40 intron and polyadenylation signal. The mice were maintained as a heterozygous colony. This transgenic line was generated in C57Bl/6J x SJL F1 mice and has since been backcrossed to the C57Bl/6J background for more than 9 generations. A PCR-based assay was used to determine the transgene status of the animals. Purina Rodent Chow 5001 and water were available ad libitum.

Enzymatic assay for ß-galactosidase activity.
A biochemical assay based on the enzymatic cleavage of a ß-gal substrate to produce a luminescent product was used to measure transgene activation. Tissues were harvested from animals treated with TCDD, vehicle, or TCDD and 3'M4'NF, and were washed in PBS. Tissues were cleared of red blood cells by incubation on ice for five min in a solution of ammonium chloride, potassium bicarbonate, and EDTA. The tissue was then washed with PBS and resuspended in a cold lysis solution containing 100 mM potassium phosphate and 0.2% Triton-X 100, which was supplemented with 1 mM DTT, 0.2 mM PMSF, and 1 µg/ml leupeptin immediately before use. The tissue was allowed to incubate in this buffer for five min on ice and then homogenized between the ends of 2 frosted glass slides. The suspension was transferred to a microfuge tube and centrifuged for two min at 12,000 x g and 4°C. The supernatant was heated at 48°C for 60 min to inactivate mammalian (endogenous) ß-gal activity (Young et al., 1993Go). The lysate was stored at –20°C. The luminescence was assayed with the Galacto-Light PlusTM kit from Tropix (Bedford, MA), according to the manufacturer's instructions, and read on a Turner model 20e luminometer (Turner Designs, Sunnyvale, CA). Results were normalized to protein levels, which were determined using the NanoOrange fluoromentric protein assay (Molecular Probes, Eugene, OR).

TCDD dose response and time course evaluation in DRE-lacZ animals.
For the evaluation of dose response, young adult DRE-lacZ mice were treated with TCDD for 24 h with 0, 1.5, 3, 15, or 30 µg/kg delivered by intraperitoneal (ip) injection in equal volumes of olive oil. Four animals were included in each group. Liver and lung tissue was removed at 24 h and tissue lysates were generated for ß-gal analysis as described above.

To study the time course of transgene induction, young adult male DRE-lacZ animals were treated with 30 µg/kg TCDD for 8, 16, 20, or 24 h. Four animals were evaluated at each time point. Both liver and lung tissue was obtained and lysates generated for the determination of ß-gal reporter activity. The effect of treatment on enzymatic activity (RLU/mg protein) was evaluated by ANOVA and Fisher's PLSD test.

Plasma clearance of antagonist.
In order to devise an effective 3'M4'NF dosing schedule, it was necessary to determine the plasma half-life of the flavonoid. The bioassay which was used is based on the ability of 3'M4'NF in blood plasma from flavonoid-treated animals to inhibit TCDD-induced reporter activity in the Hepa2Dluc.3A4 (Hepa2Dluc) reporter cell line. Hepa2Dluc cells, which have been described previously (Henry et al., 1999Go; Willey et al., 1998Go), are stably transfected with a DRE-luciferase reporter that is responsive to AhR agonists such as TCDD. For this experiment, 12 male C57Bl/6J mice were treated with 20 mg 3'M4'NF/kg in 0.1ml olive oil by ip injection. At 30 min, 1, 2, and 4 h post-injection, 3 mice were warmed under a heat lamp and peripheral blood was collected from the tail artery and pooled from all 3 animals. Peripheral blood from 5 untreated male mice was also collected and pooled. Samples were centrifuged and plasma fractions stored at –20°C until analysis.

Two days prior to analysis, 1.5 x 106 Hepa2Dluc cells were combined with 60 mg Cytodex I microcarrier beads (Sigma) and were plated in minimal essential medium (MEM) (Gibco, Grand Island, NY) on non-tissue culture-treated, 100-mm dishes to promote attachment of the cells to the beads rather than the culture plates. The extremely high surface area provided by the beads increases the number of reporter cells per unit volume. The increased number of cells allows for the analysis of the AhR agonist and/or antagonist potential of compounds in a 96-well plate format. After 2 days of growth at 37°C and 5% CO2, the beads, now covered with near confluent cells, were transferred to microfuge tubes. The cell-covered beads were then allowed to settle and a 10% volume of MEM was removed without disturbing the beads. This volume was replaced by mouse plasma from the 0, 0.5, 1, 2, or 4-h antagonist treatment groups. Vehicle (DMSO, 0.1%) or a non-saturating concentration of TCDD (150 pM) was also added to the tubes. In this system, the presence of 3'M4'NF in blood plasma is detected by a reduction of TCDD-induced luciferase reporter activity. Luciferase reporter levels were compared to a standard curve generated with known concentrations of 3'M4'NF added to TCDD-treated beads/cells in the presence of 0-h mouse plasma. The beads/cells were mixed to achieve a homogeneous suspension, and 4 replicate 100-µl aliquots of each treatment were transferred to wells of a white, opaque 96-well plate (Packard, Meriden, CT). After 4 h at 37°C, 100 µl Steady-GloTM Reagent (Promega, Madison WI) was added to each well. Light emission was measured using a LumiCountTM microplate luminometer (Packard, Meridan, CT).

Antagonist evaluation in DRE-lacZ mice.
DRE-lacZ animals were divided into 4 groups for these experiments; vehicle (n = 8), TCDD (n = 12), antagonist (n = 6), and TCDD plus 3'M4'NF (n = 11). TCDD (15 µg/kg) or the corresponding vehicle (olive oil) was administered at time 0 only; vehicle or 2 mg/kg 3'M4'NF was administered to all groups at the –4-, 0-, and 4-h time points. All animals received the same total volume of vehicle and the same number of injections. At sixteen-h post-TCDD treatment, liver and lung tissue was removed, and lysates were generated for the evaluation of ß-gal reporter transgene induction using the Tropix kit. Results were normalized to total protein and expressed as fold-increase over vehicle alone. No outward signs of toxicity were observed in any of the treatment groups. The effect of treatment on enzymatic activity (RLU/mg protein) was evaluated by ANOVA and Fisher's PLSD test. A p value of < 0.05 was considered significant.

Western blot analysis for CYP1A1.
Lung tissue was obtained and approximately 100 mg (wet weight) of tissue was combined with 800 µl of lysing buffer (62.5 mM Tris (pH 6.8), 2% SDS, 10% glycerol, 5% ß-mercaptoethanol, and 0.005% bromophenol blue) and homogenized. The sample was centrifuged at 11,000 x g for 5 min and the supernatant transferred to a new tube. An aliquot of the supernatant was boiled for 5 min and assayed by SDS–PAGE (7.5% acrylamide gel) run at 15 mA for 14 h. The gel contents were then transferred to a PVDF membrane (Amersham, Piscataway, NJ), blocked, and probed overnight at room temperature with an anti-CYP1A1 antibody (Xenotech, Kansas City, KS), then with an HRP-conjugated secondary antibody for 2 h. The results were visualized using a chemiluminescent system (Kirkegaard and Perry, Gaithersburg, MD). Blots were stripped by washing in dH2O, 0.2 M NaOH, and again in dH2O, each for 5 min. The membrane was then blocked and reprobed with an anti-actin antibody (Sigma) for 2 h at room temperature to assure consistent loading of samples. The intensity of the bands was analyzed densitometrically, using Image Pro PlusTM software (Media Cybernetics, Silver Spring, MD).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS:
 RESULTS
 DISCUSSION
 REFERENCES
 
TCDD dose response in vivo.
The dose-responsiveness of the transgenic model was characterized by treatment of male DRE-lacZ mice with 0, 1.5, 3, 15, or 30 µg/kg TCDD. After 24 h of exposure, liver and lung tissue was removed and tissue lysates generated to assess ß-gal activity. The results of these studies were normalized to total protein (Fig. 1Go). In the lung, a 3.4-fold induction was observed at 15 µg/kg and a 13.3-fold change at 30 µg/kg, both of which were statistically significant. In the liver, a 2.7-fold induction was noted at 3 µg/kg TCDD, which was found to be significant. Additionally, a 4.4-fold induction was noted at 15 µg/kg in the liver and a 25-fold induction in the lung at 30 µg/kg, which were both significantly different from control.



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FIG. 1. TCDD dose responsiveness in DRE-lacZ transgenic mice. DRE-lacZ mice were treated with vehicle or 1.5, 3, 15, or 30 µg/kg TCDD (n = 4 per group), delivered in equal volumes of olive oil vehicle. ß-Gal reporter transgene induction was assessed in the lung and liver at 24 h after TCDD treatment. ß-Gal activity is presented as RLU/mg protein ± SE. The effect of treatment on enzymatic activity (RLU/mg protein) was evaluated by ANOVA and Fisher's PLSD test. #Statistically different from vehicle control (p < 0.05). *Statistically different from vehicle control (p < 0.01). **Statistically different from vehicle control (p < 0.001).

 
TCDD time course in vivo.
The time course of transgene induction was evaluated in DRE-lacZ animals treated with 30 µg/kg TCDD. At 0, 8, 16, 20, and 24 h post-TCDD treatment, lung and liver were obtained and lysates generated for ß-gal measurement using a chemiluminescence-based assay. Data were normalized to total protein. In the lung, significant induction was first observed at 8 h post-TCDD treatment. However, maximal induction was reached in both the liver and lung compartments by 16 to 20 h after treatment (Fig. 2Go). Similarly, CYP1A1 protein was found to increase in the lung by 8 h (Fig. 3Go). The time course data are important since the antagonist 3'M4'NF is cleared rapidly from the plasma (see below). Therefore, the best measurement of the flavone's AhR antagonist activity would be in the context of a sub-saturating TCDD challenge and an early tissue harvest, which would minimize the loss of antagonist activity due to metabolism. Sixteen hours was selected for subsequent studies.



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FIG. 2. Time course of transgene induction in DRE-lacZ mice. DRE-lacZ mice were treated with 30 µg/kg TCDD and at 0, 8, 16, 20, and 24-h post-TCDD treatment, lung and liver were obtained for ß-gal analysis. Four animals were utilized at each time point. Relative light units obtained were normalized to total protein. ß-Gal activity is presented as RLU/mg protein ± SE. The effect of treatment on enzymatic activity (RLU/mg protein) was evaluated by ANOVA and Fisher's PLSD test. *Statistically different from vehicle control (p < 0.01). **Statistically different from vehicle control (p < 0.05). ***Statistically different from vehicle control (p < 0.005).

 


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FIG. 3. Western blot analysis of CYP1A1 induction during TCDD treatment. Lung tissue from DRE-lacZ mice treated with TCDD for 8, 16, 20, or 24 h was processed for Western blot analysis and probed with an anti-CYP1A1 antibody. The blot was stripped and reprobed with an anti-actin antibody for normalization. Fold induction values are based on densitometric measurement of CYP1A1 normalized to actin, compared to zero h CYP1A1.

 
Blood plasma clearance of 3'M4'NF.
In order to adequately assess the in vivo efficacy of the antagonist 3'M4'NF, it was important to determine its relative half-life within the plasma compartment. This was evaluated by testing blood plasma from 3'M4'NF-treated mice for its ability to antagonize the activation of a TCDD-responsive reporter system (Hepa2Dluc) in the presence of TCDD. Flavone concentrations were calculated from a standard curve generated with known concentrations of 3'M4'NF added to plasma from untreated mice and assayed on the reporter cells in the presence of TCDD. Blood plasma levels of 3'M4'NF given as a single 20 mg/kg dose reached about 0.27 µM at 30 min and a maximum of 0.35 µM at one h. The level of antagonist dropped to approximately 0.15 µM by 4 h (Fig. 4Go). The data suggest that the half-life of 3'M4'NF in plasma is approximately 2 h.



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FIG. 4. Biossay of 3'M4'NF blood plasma levels. The stability of 3'M4'NF in plasma was estimated by evaluating the ability of plasma from antagonist-treated mice to antagonize the activation of a TCDD-responsive reporter system (Hepa2Dluc reporter cells). Plasma was collected from 3'M4'NF-treated animals at 30 min, and 1, 2, and 4 h after treatment and pooled for each treatment period. Flavone concentrations were calculated from a standard curve generated by the addition of known concentrations of 3'M4'NF added to plasma from untreated mice to the Hepa2Dluc reporter cells.

 
In vivo antagonism of TCDD-mediated reporter activity.
The plasma clearance data suggest that in order to characterize the ability of 3'M4'NF to antagonize AhR signaling in vivo, a multiple dosing schedule may be advantageous. DRE-lacZ mice were treated with either solvent, 15 µg/kg TCDD, 6 mg/kg 3'M4'NF, or TCDD and antagonist. Antagonist was administered as a split dose at –4, 0, and 4 h, relative to TCDD treatment (0 h). A dose of 15 µg/kg TCDD was chosen as it gave a sub-maximal induction in the lung and liver (Fig. 1BGo). Thus, saturation of receptor signaling, which could make modulations of the system difficult to detect, was avoided. Liver and lung were removed 16 h after TCDD treatment and tissue lysates were evaluated for ß-gal activity. Data were expressed as light units per milligram of total protein and represented as fold induction over vehicle control (Fig. 5Go). In the liver, TCDD significantly induced reporter activity above solvent control levels. Treatment with a split dose of antagonist and TCDD resulted in a level of transgene activity that was not significantly different from vehicle alone and significantly lower than observed in the TCDD-treated group (Fig. 5AGo). The lung showed a similar pattern of transgene induction (Fig. 5BGo). In this case the TCDD-treated group was again significantly different in comparison to the vehicle-only group. Although the flavone appeared to inhibit the transgene induction by TCDD, this decrease was not significantly different. However, the lung values for the TCDD + 3'M4'NF group were also not statistically different from vehicle control. No change was observed for 3'M4'NF alone. The protein levels of CYP1A1, a TCDD target gene, showed a slight increase with 3'M4'NF only (Fig. 6AGo) and a significant increase with TCDD treatment. The TCDD-induced level of CYP1A1 protein was moderately reduced in the co-treated animals (Fig. 6BGo).



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FIG. 5. In vivo antagonism of TCDD-mediated reporter activity. DRE-lacZ mice were treated with either vehicle (n = 8), 15 µg/kg TCDD (n = 12), 3 x 2 mg/kg 3'M4'NF (n = 6), or TCDD and 3'M4'NF. The antagonist, or corresponding vehicle was given as a split dose at –4, 0, and 4 h, relative to TCDD treatment. All animals received the same total volume of vehicle. At 16 h after TCDD treatment, lung and liver were obtained for ß-gal measurement. Results are presented as RLU/mg protein ± SE. The effect of treatment on enzymatic activity (RLU/mg protein) was evaluated by ANOVA and Fisher's PLSD test. *Statistically different from vehicle control (p < 0.05). **Statistically different from TCDD alone (p < 0.05).

 


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FIG. 6. (A) Western blot analysis for CYP1A1 in lung from DRE-lacZ mice treated with olive oil or 3 x 2mg/kg 3'M4'NF. (B) Western blot analysis for CYP1A1 induction after treatment with antagonist and TCDD. Lung tissue from DRE-lacZ mice treated with vehicle, TCDD (15 µg/kg), or TCDD and antagonist (6 mg/kg, given as 3 doses of 2 mg/kg at –4, 0, and 4 h relative to TCDD treatment) was prepared for Western blot analysis and probed with an anti-CYP1A1 antibody. Densitometric analysis found the CYP1A1 levels in the antagonist plus TCDD group to be reduced by about 65% in comparison to the TCDD only group. For both A and B, the blot was stripped and reprobed with an anti-actin antibody for normalization.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS:
 RESULTS
 DISCUSSION
 REFERENCES
 
The primary focus of this work was to characterize the in vivo antagonism of the AhR by 3M'4'NF. The study of receptor antagonism has been shown to be fruitful in many other receptor systems, including the androgen- and estrogen-receptor systems (Grese and Dodge, 1998Go), where, for example, inhibitors such as tamaoxifen have added to the study and treatment of breast cancer (Jordan et al., 1985Go; Kemppainen et al., 1999Go; Lerner and Jordan, 1990Go; Meyers et al., 1999Go; Pike et al., 1999Go; Sun et al., 1999Go). Antagonists have also been utilized in the study of the steroid receptor (Schapira et al., 2000Go). The flavonoid 3'M4'NF has been shown in vitro to have potent AhR antagonistic activity and minimal agonist activity. Using AhR contained in rat hepatic cytosol, a 1 µM concentration of 3'M4'NF shows only about 7% of the DRE binding activity of a 3 nM TCDD dose (Gasiewicz et al., 1996Go). Under these conditions, the IC50 for inhibition of TCDD-elicited DRE binding of AhR is 38 nM, which is lower than plasma levels obtained in the current studies (Gasiewicz et al., 1996Go; Henry et al., 1999Go). In murine hepatoma cells, a 50% reduction of luciferase reporter activity induced by TCDD is achieved at 6 nM 3'M4'NF. The antagonist alone has not been found to increase reporter activity in vivo in either adult or fetal mice.

The mechanism of AhR antagonism by 3'M4'NF has been studied in vitro, and is believed to involve competitive binding to the AhR and blocking of nuclear localization, likely by preventing the dissociation of hsp90 (Henry et al., 1999Go). In the absence of ligand, the AhR maintains its inactive state as a cytosolic complex with an immunophilin analogue protein (ARA9, AIP, Xap2) and 2 molecules of hsp90 (Carver and Bradfield, 1997Go; Chen and Perdew, 1994Go; Ma and Whitlock, Jr., 1997). Dissociation of at least one hsp90 is believed to allow import proteins, such as importin-{alpha}, access to the nuclear localization sequence of the AhR (Ikuta et al., 1998Go). An analysis of flavone derivatives with various substituent groups indicates that the presence of a 3' methoxy group and the 4' nitro group predict high receptor affinity and antagonist activity. This mechanism of antagonism is in contrast to that of some other AhR antagonists. For example, resveratrol has been shown to antagonize AhR-mediated gene activation. However, resveratrol appears to allow nuclear localization of the AhR and does not inhibit TCDD-mediated DNA binding (Casper et al., 1999Go). However, it does apparently inhibit CYP1A1 mRNA upregulation and TCDD-dependent reporter activation (Ciolino et al., 1998Go). Those data suggest that resveratrol binding alters an AhR conformation important in transactivation and that antagonism occurs at some point after DNA binding. On the other hand, with benzo[a]pyrene as the agonist and at higher concentrations of resveratrol, DNA binding is slightly reduced (Ciolino and Yeh, 1999Go). Resveratrol also seems to differentially antagonize AhR-dependent genes. Antagonism of cyp1a1 but not NAD(P)H quinone oxidoreductase induction has been shown (Casper et al., 1999Go). Another commonly utilized AhR antagonist is {alpha}-naphthoflavone ({alpha}-NF), which has been characterized as a mixed AhR agonist-antagonist (Blank et al., 1987Go; Gasiewicz and Rucci, 1991Go; Merchant et al., 1990Go, 1992Go). At a dose of 10–6 M, {alpha}-NF inhibited TCDD-mediated cyp1a1 gene expression and inhibited the formation of nuclear AhR complex (Merchant et al., 1993Go). In contrast, {alpha}-NF acts as an AhR agonist at higher concentrations (Santostefano et al., 1993Go).

In order to evaluate the in vivo efficacy of 3'M4'NF, we used a transgenic mouse model that allows for the quantitation of DRE-driven transcription on a tissue by tissue basis. This model incorporates a reporter construct consisting of the lacZ gene under the control of two DREs derived from the DRE-D of the cyp1a1 promoter (Lusska et al., 1991Go) and the chicken ovalbumin TATA box region (Willey et al., 1998Go). This model was selected for the in vivo analysis over other endpoints, such as the induction of CYP1A1 enzyme activity, because of the potential for direct antagonist interference with such systems. In addition, other endogenous AhR-responsive genes such as cyp1a1 are not ubiquitously expressed and may not adequately represent AhR transcriptional activity in all tissues. Finally, genes such as cyp1a1 and NAD(P)H quinone oxidoreductase have non-DRE regulatory elements in their promoter regions, which may secondarily influence the effect of TCDD on these genes (Vasiliou et al., 1995Go). The DRE-lacZ construct is therefore advantageous since the influence of these non-DRE regulators is minimized by the simplicity of the reporter construct. The DREs and TATA box elements were kept as small as possible to reduce the likelihood that other regulatory elements would influence reporter activation. A search of the construct for known recognition sequences found only the DRE and TATA binding regions (Willey et al., 1998Go). As a result of the simplicity of the construct, it is likely not as sensitive to induction as endogenous promoters, such as CYP1A1. This is also evidenced by the induction of CYP1A1 by doses of 3'M4'NF that had no effect on reporter activity. However, the model was designed to determine the temporal and spatial activation of the AhR and not to mimic the responsiveness of specific DRE-mediated genes.

Following a dosage of 20 mg/kg, blood plasma levels of 3'M4'NF were found to peak in the 350 nM range and to be at about 150 nM at 4 h. Assuming that the kinetics of 3'M4'NF are not dose-dependent at doses of 2 and 20 mg/kg, it is estimated that the lowest concentration in plasma achieved during the in vivo antagonist experiment was approximately 15 nM immediately before TCDD challenge and the second dose of 3'M4'NF. In comparison, 6 nM 3'M4'NF was found to be the IC50 for inhibition of 150 pM TCDD-induced luciferase activity in Hepa2Dluc cells. In this same cell system, 100 nM 3'M4'NF produced nearly complete inhibition of the TCDD-induced response (Henry et al., 1999Go). These findings support the hypothesis that a pharmacologically active concentration of 3'M4'NF, in terms of AhR antagonist activity, is likely present in the plasma. Under these conditions, we found that 3'M4'NF did antagonize TCDD-induced gene activity in vivo. No significant differences in reporter transgene activity were found after 16 h of treatment between the vehicle and TCDD plus antagonist groups in both the liver and lung indicating that antagonist had effectively blocked the significant induction by TCDD in both of these tissues. In the lung, CYP1A1 protein levels were observed to be partially antagonized in the presence of antagonist in comparison to TCDD alone. This incomplete antagonism is likely due to the partial agonist activity of 3'M4'NF, which is able to activate the more sensitive CYP1A1 promoter. Together, these studies are the first to show that 3'M4'NF is functioning as an AhR antagonist in vivo and that relatively rapid metabolism does not preclude the use of the compound for temporary inhibition of signal transduction.

The large degree of variability in the TCDD-treated group of the antagonist study is likely related to several factors. The use of a non-saturating dose of TCDD would make these data more sensitive to individual variation and slight differences in dose delivery. The use of heterozygous animals may also contribute to this variability. A gene dose effect (as much as 8- to 10-fold) has been found upon comparison of TCDD induction in homozygous and heterozygous animals (unpublished results). An additional factor that may contribute to this variability is the hemoglobin content of the tissues, the presence of which has been found to have a quenching effect on the luminescent ß-gal assay (Nazarenko et al., 2001Go). These factors also likely account for the reduced statistical significance between the TCDD and the TCDD plus antagonist groups in the lung.

Previous studies indicate that 3'M4'NF and other flavones are potential CYP1A1 inhibitors. Work in this laboratory has shown that the dose of 3'M4'NF used in the studies reported here is likely to have minimal or no direct effect on CYP1A activity (Dertinger et al., 2001Go). These studies utilized the zoxazolamine paralysis test to evaluate the ability of 3'M4'NF to inhibit CYP1A1/A2 activity in vivo. Zoxazolamine is a potent muscle relaxant which is metabolically inactivated by CYP1A1/A2 enzymatic activity and can be used to assess CYP1A1/A2 inhibition in vivo (Vancutsem and Babish, 1993Go; Atal et al., 1985Go). Doses of 3'M4'NF in the 0.2- to 2-mg/kg range, as used in the present studies, were found to have no measurable effect on zoxazolamine-induced paralysis time (Dertinger et al., 2001Go). This indicates that the antagonist alone did not inhibit CYP1A1 activity at these concentrations of 3'M4'NF.

The present studies are the first to identify 3'M4'NF as an AhR antagonist acting in vivo upon both a TCDD-mediated reporter transgene and also an endogenously regulated gene. Additionally, these studies show that the lacZ reporter transgene was capable of detecting the effects of this antagonist in vivo. Such findings suggest the possibility of future studies utilizing the DRE-lacZ animals in combination with 3'M4'NF to identify tissues where AhR is active with and without exogenous agonist exposure. This could be accomplished by the evaluation of changes in reporter activation with and without 3'M4'NF treatment in candidate tissues. Such studies may assist in the identification of areas where putative AhR endogenous ligands may be active and in the delineation of target tissues where such studies would be focused. However, to date, no effects of 3'M4'NF alone have been noted on reporter activity. This model will probably also contribute to the characterization of the mechanisms underlying TCDD toxicity. In particular, the use of antagonists may allow for the clarification of observations made in the AhR-null animals. For example, since AhR knockout animals express only 10% of basal level of CYP1A2 activity compared with wild-type controls (Lahvis and Bradfield, 1998Go), it is not clear whether the differential sensitivity of knockout animals to the genotoxic effects of AhR ligands such as benzo[a]pyrene is related to altered constitutive enzyme activity or to the lack of AhR-mediated gene regulation (Dertinger et al., 2001Go). The use of a pharmacological antagonist to block signaling may enable discrimination between these possibilities. Such studies would benefit from the design of a more persistent AhR antagonist that might eliminate the need for repeat dosing schedules. Efforts to develop such antagonists are underway.


    ACKNOWLEDGMENTS
 
The authors would like to thank the members of the Gasiewicz lab for their critical reviews of this manuscript. We also especially want to thank Andrew Kende for synthesizing 3'M4'NF and J. Jeff Willey for generating and initially characterizing the DRE-lacZ mice. We would also like to acknowledge Denise Hahn, Cheryl Hurley, and Jennifer LaRuffa for the expert animal care they provided. This work was supported by the National Institute of Environmental Health Sciences Grants ES09430 and ES09702, Center Grant ES01247, and Training Grant ES07026.


    NOTES
 
1 Present address: Litron Laboratories, Rochester, NY, 14620. Back

2 To whom correspondence should be addressed at the University of Rochester School of Medicine, Department of Environmental Medicine, 610 Elmwood Ave, Box EHSC, Rochester, NY 14642. Fax: (716) 256-2591. E-mail: Tom_Gasiewicz{at}urmc.rochester.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS:
 RESULTS
 DISCUSSION
 REFERENCES
 
Abbott, B. D., Diliberto, J. J., and Birnbaum, L. S. (1989). 2,3,7,8-Tetrachlorodibenzo-p-dioxin alters embryonic palatal medial epithelial cell differentiation in vitro. Toxicol. Appl. Pharmacol. 100, 119–131.[ISI][Medline]

Abbott, B. D., Schmid, J. E., Pitt, J. A., Buckalew, A. R., Wood, C. R., Held, G. A., and Diliberto, J. J. (1999). Adverse reproductive outcomes in the transgenic Ah receptor-deficient mouse. Toxicol. Appl. Pharmacol. 155, 62–70.[ISI][Medline]

Atal, C. K., Dubey, R. K., and Singh, J. (1985). Biochemical basis of enhanced drug bioavailability by piperine: Evidence that piperine is a potent inhibitor of drug metabolism. J. Pharmacol. Exp. Ther. 232, 258–262.[Abstract]

Biegel, L., Harris, M., Davis, D., Rosengren, R., Safe, L., and Safe, S. (1989). 2,2',4,4',5,5'-Hexachlorobiphenyl as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6J mice. Toxicol. Appl. Pharmacol. 97, 561–571.[ISI][Medline]

Blank, J. A., Tucker, A. N., Sweatlock, J., Gasiewicz, T. A., and Luster, M. I. (1987). {alpha}-Naphthoflavone antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced murine lymphocyte ethoxyresorufin-O-deethylase activity and immunosuppression. Mol. Pharmacol. 32, 169–172.[Abstract]

Blankenship, A. L., Suffia, M. C., Matsumura, F., Walsh, K. J., and Wiley, L. M. (1993). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) accelerates differentiation of murine preimplantation embryos in vitro. Reprod. Toxicol. 7, 255–261.[ISI][Medline]

Carver, L. A., and Bradfield, C. A. (1997). Ligand-dependent interaction of the aryl hydrocarbon receptor with a novel immunophilin homolog in vivo. J. Biol. Chem. 272, 11452–11456.[Abstract/Free Full Text]

Casper, R. F., Quesne, M., Rogers, I. M., Shirota, T., Jolivet, A., Milgrom, E., and Savouret, J. F. (1999). Resveratrol has antagonist activity on the aryl hydrocarbon receptor: Implications for prevention of dioxin toxicity. Mol. Pharmacol. 56, 784–790.[Abstract/Free Full Text]

Chen, H. S., and Perdew, G. H. (1994). Subunit composition of the heteromeric cytosolic aryl hydrocarbon receptor complex. J. Biol. Chem. 269, 27554–27558.[Abstract/Free Full Text]

Ciolino, H. P., Daschner, P. J., and Yeh, G. C. (1998). Resveratrol inhibits transcription of CYP1A1 in vitro by preventing activation of the aryl hydrocarbon receptor. Cancer Res. 58, 5707–5712.[Abstract]

Ciolino, H. P., and Yeh, G. C. (1999). Inhibition of aryl hydrocarbon-induced cytochrome P-450 1A1 enzyme activity and CYP1A1 expression by resveratrol. Mol. Pharmacol. 56, 760–767.[Abstract/Free Full Text]

Dertinger, S. D., Lantum, H., Silverstone, A. E., and Gasiewicz, T. A. (2000). Effect of 3'-methoxy-4'nitroflavone on benzo[a]pyrene toxicity. Aryl hydrocarbon receptor-dependent and -independent mechanisms. Biochem. Pharmacol. 60, 189–196.[ISI][Medline]

Dertinger, S. D., Nazarenko, D. A., Silverstone, A. E., and Gasiewicz, T. A. (2001). Aryl hydrocarbon receptor signaling plays a significant role in mediating benzo. Carcinogenesis 22, 171–.[Abstract/Free Full Text]

Elferink, C. J., Gasiewicz, T. A., and Whitlock, J. P., Jr. (1990). Protein-DNA interactions at a dioxin-responsive enhancer. Evidence that the transformed Ah receptor is heteromeric. J. Biol. Chem. 265, 20708–20712.[Abstract/Free Full Text]

Fernandez-Salguero, P. M., Ward, J. M., Sundberg, J. P., and Gonzalez, F. J. (1997). Lesions of aryl-hydrocarbon receptor-deficient mice. Vet. Pathol. 34, 605–614.[Abstract]

Gasiewicz, T. A., Elferink, C. J., and Henry, E. C. (1991). Characterization of multiple forms of the Ah receptor: Recognition of a dioxin-responsive enhancer involves heteromer formation. Biochemistry 30, 2909–2916.[ISI][Medline]

Gasiewicz, T. A., Kende, A. S., Rucci, G., Whitney, B., and Willey, J. J. (1996). Analysis of structural requirements for Ah receptor antagonist activity: Ellipticines, flavones, and related compounds. Biochem. Pharmacol. 52, 1787–1803.[ISI][Medline]

Gasiewicz, T. A., and Rucci, G. (1991). Alpha-naphthoflavone acts as an antagonist of 2,3,7, 8- tetrachlorodibenzo-p-dioxin by forming an inactive complex with the Ah receptor. Mol. Pharmacol. 40, 607–612.[Abstract]

Grese, T. A., and Dodge, J. A. (1998). Selective estrogen receptor modulators (SERMs). Curr. Pharm. Des. 4, 71–92.[ISI][Medline]

Harris, M., Zacharewski, T., Astroff, B., and Safe, S. (1989). Partial antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated induction of aryl hydrocarbon hydroxylase by 6-methyl-1,3,8-trichlorodibenzofuran: Mechanistic studies. Mol. Pharmacol. 35, 729–735.[Abstract]

Henry, E. C., and Gasiewicz, T. A. (1993). Transformation of the aryl hydrocarbon receptor to a DNA-binding form is accompanied by release of the 90 kDa heat-shock protein and increased affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochem. J. 294, 95–101.[ISI][Medline]

Henry, E. C., Kende, A. S., Rucci, G., Totleben, M. J., Willey, J. J., Dertinger, S. D., Pollenz, R. S., Jones, J. P., and Gasiewicz, T. A. (1999). Flavone antagonists bind competitively with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor but inhibit nuclear uptake and transformation. Mol. Pharmacol. 55, 716–725.[Abstract/Free Full Text]

Ikuta, T., Eguchi, H., Tachibana, T., Yoneda, Y., and Kawajiri, K. (1998). Nuclear localization and export signals of the human aryl hydrocarbon receptor. J. Biol. Chem. 273, 2895–2904.[Abstract/Free Full Text]

Jordan, V. C., Mittal, S., Gosden, B., Koch, R., and Lieberman, M. E. (1985). Structure-activity relationships of estrogens. Environ. Health Perspect. 61, 97–110.[ISI][Medline]

Kemppainen, J. A., Langley, E., Wong, C. I., Bobseine, K., Kelce, W. R., and Wilson, E. M. (1999). Distinguishing androgen receptor agonists and antagonists: Distinct mechanisms of activation by medroxyprogesterone acetate and dihydrotestosterone. Mol. Endocrinol. 13, 440–454.[Abstract/Free Full Text]

Kurl, R. N., DePetrillo, P. B., and Olnes, M. J. (1993). Inhibition of Ah (dioxin) receptor transformation by 9-hydroxy ellipticine. Involvement of protein kinase C? Biochem. Pharmacol. 46, 1425–1433.[ISI][Medline]

Lahvis, G. P., and Bradfield, C. A. (1998). Ahr null alleles: Distinctive or different? Biochem. Pharmacol. 56, 781–787.[ISI][Medline]

Lahvis, G. P., Lindell, S. L., Thomas, R. S., McCuskey, R. S., Murphy, C., Glover, E., Bentz, M., Southard, J., and Bradfield, C. A. (2000). Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 97, 10442–10447.[Abstract/Free Full Text]

Lerner, L. J., and Jordan, V. C. (1990). Development of antiestrogens and their use in breast cancer. Cancer Res. 50, 4177–4189.[Abstract]

Liu, H., Santostefano, M., Lu, Y., and Safe, S. (1993). 6-Substituted 3,4-benzocoumarins: A new structural class of inducers and inhibitors of CYP1A1-dependent activity. Arch. Biochem. Biophys. 306, 223–231.[ISI][Medline]

Lu, Y. F., Santostefano, M., Cunningham, B. D., Threadgill, M. D., and Safe, S. (1995). Identification of 3'-methoxy-4'-nitroflavone as a pure aryl hydrocarbon (Ah) receptor antagonist and evidence for more than one form of the nuclear Ah receptor in MCF-7 human breast cancer cells. Arch. Biochem. Biophys. 316, 470–477.[ISI][Medline]

Lu, Y. F., Santostefano, M., Cunningham, B. D., Threadgill, M. D., and Safe, S. (1996). Substituted flavones as aryl hydrocarbon (Ah) receptor agonists and antagonists. Biochem. Pharmacol. 51, 1077–1087.[ISI][Medline]

Lusska, A. E., Jones, K. W., Elferink, C. J., Wu, L., Shen, E. S., Wen, L. P., and Whitlock, J. P., Jr. (1991). 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces cytochrome P450IA1 enzyme activity by activating transcription of the corresponding gene. Adv. Enzyme Regul. 31, 307–317.[ISI][Medline]

Lusska, A., Shen, E., and Whitlock, J. P., Jr. (1993). Protein-DNA interactions at a dioxin-responsive enhancer. Analysis of six bona fide DNA-binding sites for the liganded Ah receptor. J. Biol. Chem. 268, 6575–6580.[Abstract/Free Full Text]

Ma, Q., and Whitlock, J. P., Jr. (1997). A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Biol. Chem. 272, 8878–8884.[Abstract/Free Full Text]

Merchant, M., Arellano, L., and Safe, S. (1990). The mechanism of action of alpha-naphthoflavone as an inhibitor of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 gene expression. Arch. Biochem. Biophys. 281, 84–89.[ISI][Medline]

Merchant, M., Krishnan, V., and Safe, S. (1993). Mechanism of action of alpha-naphthoflavone as an Ah receptor antagonist in MCF-7 human breast cancer cells. Toxicol. Appl. Pharmacol. 120, 179–185.[ISI][Medline]

Merchant, M., Morrison, V., Santostefano, M., and Safe, S. (1992). Mechanism of action of aryl hydrocarbon receptor antagonists: Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 gene expression. Arch. Biochem. Biophys. 298, 389–394.[ISI][Medline]

Meyers, M. J., Sun, J., Carlson, K. E., Katzenellenbogen, B. S., and Katzenellenbogen, J. A. (1999). Estrogen receptor subtype-selective ligands: Asymmetric synthesis and biological evaluation of cis- and trans-5,11-dialkyl-5,6,11,12-tetrahydrochrysenes. J. Med. Chem. 42, 2456–2468.[ISI][Medline]

Nazarenko, D. A., Dertinger, S. D., and Gasiewicz, T. A. (2001). Enhanced Detection of ß-galactosidase reporter activation by a luminescent assay in tissue lysates is achieved by a reduction of hemoglobin content. Biotechniques (in press).

Pike, A. C., Brzozowski, A. M., Hubbard, R. E., Bonn, T., Thorsell, A. G., Engstrom, O., Ljunggren, J., Gustafsson, J. A., and Carlquist, M. (1999). Structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist. EMBO J. 18, 4608–4618.[Abstract/Free Full Text]

Pollenz, R. S., Sattler, C. A., and Poland, A. (1994). The aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins show distinct subcellular localizations in Hepa 1c1c7 cells by immunofluorescence microscopy. Mol. Pharmacol 45, 428–438.[Abstract]

Reyes, H., Reisz-Porszasz, S., and Hankinson, O. (1992). Identification of the Ah receptor nuclear translocator protein (Arnt) as a component of the DNA binding form of the Ah receptor. Science 256, 1193–1195.[ISI][Medline]

Santostefano, M., Merchant, M., Arellano, L., Morrison, V., Denison, M. S., and Safe, S. (1993). {alpha}-Naphthoflavone-induced CYP1A1 gene expression and cytosolic aryl hydrocarbon receptor transformation. Mol. Pharmacol. 43, 200–206.[Abstract]

Schapira, M., Raaka, B. M., Samuels, H. H., and Abagyan, R. (2000). Rational discovery of novel nuclear hormone receptor antagonists. Proc. Nat. Acad. Sci. U.S.A. 97, 1008–1013.[Abstract/Free Full Text]

Sun, J., Meyers, M. J., Fink, B. E., Rajendran, R., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (1999). Novel ligands that function as selective estrogens or antiestrogens for estrogen receptor-alpha or estrogen receptor-beta. Endocrinology 140, 800–804.[Abstract/Free Full Text]

Vancutsem, P. M., and Babish, J. G. (1993). Effects of ciprofloxacin and enrofloxacin on zoxazolamine kinetics, plasma concentration, and sleeping times in mice. Toxicol. Lett. 69, 1–14.[ISI][Medline]

Vasiliou, V., Shertzer, H. G., Liu, R. M., Sainsbury, M., and Nebert, D. W. (1995). Response of [Ah] battery genes to compounds that protect against menadione toxicity. Biochem. Pharmacol. 50, 1885–1891.[ISI][Medline]

Willey, J. J., Stripp, B. R., Baggs, R. B., and Gasiewicz, T. A. (1998). Aryl hydrocarbon receptor activation in genital tubercle, palate, and other embryonic tissues in 2,3,7, 8-tetrachlorodibenzo-p-dioxin-responsive lacZ mice. Toxicol. Appl. Pharmacol. 151, 33–44.[ISI][Medline]

Young, D. C., Kingsley, S. D., Ryan, K. A., and Dutko, F. J. (1993). Selective inactivation of eukaryotic beta-galactosidase in assays for inhibitors of HIV-1 TAT using bacterial beta-galactosidase as a reporter enzyme. Anal. Biochem. 215, 24–30.[ISI][Medline]