Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York 14642
Received November 9, 2000; accepted February 7, 2001
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ABSTRACT |
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Key Words: aryl hydrocarbon receptor (AhR); 3'methoxy-4'-nitroflavone (3'M4'NF); receptor antagonism; TCDD.
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INTRODUCTION |
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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., 1989; Liu et al., 1993
; Gasiewicz and Rucci, 1991
; Gasiewicz et al., 1996
; Harris et al., 1989
; Kurl et al., 1993
; Lu et al., 1995
, 1996
). 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, 1998
; Kemppainen et al., 1999
; Meyers et al., 1999
; Pike et al., 1999
; Sun et al., 1999
). 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., 1999). 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., 1993
; Lu et al., 1996
). 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., 1996
; Henry et al., 1999
). 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., 1996
; Henry et al., 1999
; Lu et al., 1995
). 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., 2000
), 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., 1998). 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., 1995
); 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.
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MATERIALS AND METHODS: |
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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., 1998). This construct consists of 2 DRE-Ds, derived from the 5' flanking region of the murine CYP1A1 gene (Lusska et al., 1993
), 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., 1993). 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., 1999; Willey et al., 1998
), 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 SDSPAGE (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).
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RESULTS |
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DISCUSSION |
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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., 1999). 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, 1997
; Chen and Perdew, 1994
; Ma and Whitlock, Jr., 1997). Dissociation of at least one hsp90 is believed to allow import proteins, such as importin-
, access to the nuclear localization sequence of the AhR (Ikuta et al., 1998
). 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., 1999
). However, it does apparently inhibit CYP1A1 mRNA upregulation and TCDD-dependent reporter activation (Ciolino et al., 1998
). 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, 1999
). 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., 1999
). Another commonly utilized AhR antagonist is
-naphthoflavone (
-NF), which has been characterized as a mixed AhR agonist-antagonist (Blank et al., 1987
; Gasiewicz and Rucci, 1991
; Merchant et al., 1990
, 1992
). At a dose of 106 M,
-NF inhibited TCDD-mediated cyp1a1 gene expression and inhibited the formation of nuclear AhR complex (Merchant et al., 1993
). In contrast,
-NF acts as an AhR agonist at higher concentrations (Santostefano et al., 1993
).
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., 1991) and the chicken ovalbumin TATA box region (Willey et al., 1998
). 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., 1995
). 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., 1998
). 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., 1999). 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., 2001). 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., 2001). 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, 1993
; Atal et al., 1985
). 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., 2001
). 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, 1998), 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., 2001
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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.
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