Toxicology and Applied Pharmacology 174, 153–159 (2001) doi:10.1006/taap.2001.9209, available online at http://www.idealibrary.com on The Synthetic Retinoid AGN 193109 but Not Retinoic Acid Elevates CYP1A1 Levels in Mouse Embryos and Hepa-1c1c7 Cells Dianne Robert Soprano,* ,† Carlo J. Gambone,* Sabina N. Sheikh,‡ Jerome L. Gabriel,* Roshantha A. S. Chandraratna,§ Kenneth J. Soprano,† ,‡ and Devendra M. Kochhari *Department of Biochemistry, †Fels Institute for Cancer Research and Molecular Biology, and ‡Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140; §Retinoid Research, Department of Chemistry and Department of Biology, Allergan, Inc., Irvine, California 92623; and iDepartment of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Received February 5, 2001; accepted April 20, 2001 The Synthetic Retinoid AGN 193109 but Not Retinoic Acid Elevates CYP1A1 Levels in Mouse Embryos and Hepa-1c1c7 Cells. Soprano, D. R., Gambone, C. J., Sheikh, S. N., Gabriel, J. L., Chandraratna, R. A. S., Soprano, K. J., and Kochhar, D. M. (2001). Toxicol. Appl. Pharmacol. 174, 153–159. The synthetic retinoid AGN 193109 is a potent pan retinoic acid receptor (RAR) antagonist. Treatment of pregnant mice with a single oral 1 mg/kg dose of this antagonist on day 8 postcoitum results in severe craniofacial (median cleft face or frontonasal deficiency) and eye malformations in virtually all exposed fetuses. Using differential display analysis, we have determined that CYP1A1 mRNA levels are elevated in mouse embryos 6 h following treatment with AGN 193109. Similarly, an elevation in CYP1A1 mRNA levels, protein levels, and aryl hydrocarbon hydoxylase activity occurs in Hepa-1c1c7 cells, with the maximal elevation observed when the cells were treated with 10 2 5 M AGN 193109 for 4 to 8 h. Elevation in CYP1A1 mRNA levels in mouse embryos and Hepa-1c1c7 cells does not occur upon treatment with the natural retinoid, all-trans-retinoic acid. Finally, elevation in CYP1A1 mRNA levels was not observed when mutant Hepa1c1c7 cells, which are defective in either the aryl hydrocarbon receptor (AhR) or aryl hydrocarbon receptor nuclear translocator (ARNT), were treated with AGN 193109. This suggests that the AhR/ARNT pathway and not the RAR/RXR pathway is mediating the elevation of CYP1A1 mRNA levels by AGN 193109, at least in the Hepa-1c1c7 cells. This is the first example of a retinoid that displays the abililty to regulate both the RAR/RXR and AhR/ARNT transcriptional regulatory pathways. © 2001 Academic view, see Chambon, 1996), we have developed an animal model for mechanistic studies through intervention in receptor function with the help of receptor antagonists. The synthetic retinoid AGN 193109 is a potent pan RAR antagonist (Johnson et al., 1995; Agarwal et al., 1996). Prior studies have demonstrated that the treatment of pregnant mice with a single oral 1 mg/kg dose of this antagonist on 8 days postcoitum (dpc) results in severe craniofacial (median cleft face or frontonasal deficiency) and eye malformations in virtually all exposed fetuses (Kochhar et al., 1998). These anomalies induced by AGN 193109 are strikingly similar to the craniofacial defects that occurred in double knockout mutants lacking both RARa and RARg (Lohnes et al., 1994; Mendelsohn et al., 1994). Here we report our attempts to monitor early molecular events associated with the exposure of the embryo to this antagonist. Using mRNA differential display (Liang and Pardee, 1992), we have identified an mRNA, CYP1A1, whose expression is elevated by AGN 193109 exposure in both mouse embryos and Hepa-1c1c7 hepatoma cells. Surprisingly, this elevation in CYP1A1 mRNA levels in Hepa-1c1c7 cells appears to be mediated by the aryl hydrocarbon receptor (AhR)/ aryl hydrocarbon receptor nuclear translocator (ARNT) pathway rather than the RAR/RXR signaling pathway. This leads to the possibility that AGN 193109 is capable of modulating simultaneously two distinct signaling pathways, RAR/RXR and AhR/ARNT. MATERIALS AND METHODS Press Vitamin A deficiency during pregnancy can cause a number of congenital anomalies in the fetus (Wilson and Warkany, 1950; Wilson et al., 1953; Underwood, 1984; Wolf, 1996). Since major vitamin A signals are transduced by retinoic acid receptors (RARs) 1 and retinoid X receptors (RXRs) (for re- Animals. Mature male and virgin female CD-1 mice were housed in environmentally controlled rooms and acclimatized to a 12-h light/dark cycle. All animals were maintained on Purina Lab Chow and tap water ad libitum. A group of three or four females was caged with a single male for 4 h. Presence of a vaginal plug immediately afterward was regarded as evidence of successful mating and this day was designated as 0 dpc. On 8 dpc dams were orally intubated with either 1 mg/kg AGN 193109 or 100 mg/kg all-trans-RA. AGN 193109 was dissolved in DMSO:soybean oil 1 Abbreviations used: AHH, arylhydrocarbon hydroxylase; AhR, arylhydrocarbon receptor; ARNT, arylhydrocarbon receptor nuclear translocator; CYP1A1, cytochrome P1– 450; dpc, days postcoitum; RA, retinoic acid; RAR, 153 retinoic acid receptor; RARE, retinoic acid response element; RXR, retinoid X receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. 0041-008X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. 154 SOPRANO ET AL. vehicle (1:19, v/v) and all-trans-RA was dissolved in ethanol (10 mg/ml) from which fresh dilutions were made in soybean oil. Control animals were given an appropriate volume of either DMSO/soybean oil or ethanol/soybean oil carrier alone. Dams were euthanzied at the indicated times following treatment (0, 3, 6, 12, or 24 h) and the embryos without extraembryonic membranes were removed in cold phosphate-buffered saline. All embryos from one litter were combined as a single sample, rapidly frozen in liquid nitrogen, and stored at 270 o C until the time of RNA isolation. Cell culture. Hepa-1c1c7 (CRL-2026), taoBpRc1 (CRL-2218), and BpRc1 (CRL-2217) (termed BP rc1 in Miller et al., 1983) cell lines were obtained from the American Type Culture Collection (Rockville, MD). Stock cells were maintained in a-DMEM (Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 2 mM glutamine (Gibco BRL), 100 units/ml penicillin (Cellgro), and 100 mg/ml streptomycin (Cellgro) at 37°C in a 98% humidified, 5% CO 2 atmosphere. AGN 193109 was prepared as a 10 2 3 M stock solution in DMSO and all-trans-RA was prepared as a 10 2 3 M stock solution in ethanol. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was obtained as a 50 mg/ml stock solution in nonane from ChemSyn Laboratories (Lenexa, KS) and was diluted in DMSO. Control cells were treated with an equal volume of either DMSO or ethanol carrier. RNA isolation and differential display. Total cellular RNA was isolated by the RNAzol method (Rappolee et al., 1989) (Cinna/Biotecx Laboratories). RNA was quantitated by absorbance at 260 nm, and purity was assessed by absorbance at 280 nm. Differential display analysis of RNA was performed using the RNAimage mRNA differential display system (GenHunter Corporation) essentially as described by Liang and Pardee (1992) using two total RNA samples prepared from a pooled sample of 8-dpc embryos obtained from four dams each, one treated with 1 mg/kg AGN 193109 and the other with vehicle only for 6 h. DNA from the differentially displayed band was isolated, amplified by PCR using the primers H-T 11C and H-AP21 (GenHunter Corporation), and cloned into the pCR-TRAP cloning vector using the pCR-TRAP cloning system (GenHunter Corporation). DNA sequence was determined by the Sanger Dideoxynucleotide method (Sanger et al., 1977). Northern blot. The steady state level of CYP1A1 mRNA was determined by Northern blot analysis (Thomas, 1980). Briefly, 25 mg of total RNA for each sample was electrophoresed on a 1% agarose–formaldehyde gel and then transferred to nitrocellulose. Prehybridization, hybridization, and washing conditions were performed as previously described (Soprano et al., 1986; Harnish et al., 1990). The 394-bp 3C21 DNA fragment, which contains only sequences within the 39 untranslated region of the mouse CYP1A1 mRNA from base 2226 to the poly(A) tail, was used as a probe for CYP1A1 mRNA levels. BLAST analysis demonstrated that the 3C21 DNA sequence has no significant homology with any cytochrome P450 mRNA transcript or gene except CYP1A1. A normalizing probe, 28S rRNA cDNA, was used to reprobe the blots to confirm that equal amounts of RNA from each sample were loaded. All cDNA fragments were labeled with a[ 32P]dCTP (3000 Ci/mmol, New England Nuclear) by random priming using the Prime-a-Gene Labeling System (Promega Corp.). For the studies involving the Hepa-1c1c7 cells, the Northern blots were performed in duplicate using independently isolated RNA samples. Western blot. Total cellular protein extracts were prepared from cells by homogenization in 1 volume/packed cell volume of resuspension buffer (50 mM NaPO 4, pH 7.4; 0.1 mM EDTA; 10% glycerol), followed by centrifugation in a microfuge for 15 min at 4 oC. The supernatant was removed and the protein concentration was determined using the Bio-Rad Protein Assay reagent (Bio-Rad). Samples were stored at 270 oC. The Western blot was performed in duplicate using independently isolated protein samples. CYP1A1 protein levels were measured by Western blot analysis essentially as described previously (Tairis et al., 1994). Typically, 50 mg of total cellular protein was fractionated by discontinuous sodium dodecyl sulfate–polyacrylamide gel electrophoresis using a 5% polyacrylamide stacking gel and a 9% polyacrylamide separating gel. Proteins were electroblotted to polyvinylidene difluoride membranes (Millipore Immobilon-P) according to the method of Burnette (1981). The membranes were blocked for at least 1 h at room temperature in 5% (w/v) nonfat dry milk in TBST (20 mM Tris–HCl, pH 7.4; 150 mM NaCl; 0.1% (v/v) Tween-20). After blocking, the membrane was incubated with goat anti-CYP1A1 polyclonal primary antibody (DAIICHI), which was diluted 1:500 in 5% (w/v) nonfat dry milk in TBST for 30 min at room temperature. Following removal of the primary antibody, the membrane was washed three times with TBST and then was incubated for 30 min with rabbit anti-goat IgG conjugated horseradish peroxidase secondary antibody (Santa Cruz Biotechnology), which was diluted 1:5000 in 5% (w/v) nonfat dry milk in TBST. After incubation with the secondary antibody, the membrane was again washed three times with TBST. The protein was visualized using Enhanced Chemiluminescence (Amersham). Arylhydrocarbon hydroxylase activity assay. Total cellular extracts were prepared by homogenization of the cells in harvesting buffer (0.25 M sucrose; 50 mM Tris–HCl, pH 7.5). Cellular debris was discarded following centrifugation in a microfuge for 15 min at 4 oC. The protein concentration of the supernatant was determined using the Bio-Rad Protein Assay reagent and the samples were stored at 270 oC. Arylhydrocarbon hydroxylase activity was quantitated using the radioactive method as described by Van Cantfort et al. (1977) and based on the spectrophotofluorometric method described by Nebert and Gelboin (1968). Briefly, the total reaction volume of 0.5 ml contained 50 mM Tris–HCl, pH 7.5; 0.36 mM NADPH; 5 mM MgCl 2; 800 mg of protein extract; and 0.6 mM (0.02 mCi) [ 3H]benzo[a]pyrene. The stock [ 3H]benzo[a]pyrene(54 Ci/mmol, Amersham) was diluted to a final specific activity of 30 mCi/mmol with unlabeled benzo[a]pyrene in hexane, purified by extraction six times with 1 M KOH/DMSO (65/85; v/v), followed by evaporation under a stream of nitrogen and stored dry at 220 oC. The reaction mixture was incubated with shaking at 37 oC for 30 min in air and was stopped by the addition of 1 ml of 0.15 M KOH in 85% DMSO. The unmetabolized substrate was extracted twice into 5 ml hexane (mixed for 5 min by revolving agitation) and the phases were separated by centrifugation at 3000 rpm in an Eppendorf Model 5810R centrifuge. Each time the upper phases and the interphases were eliminated and the aqueous layer was retained. After the second extraction, 0.5 ml of the aqueous layer was placed in a liquid scintillation vial, acidified by the addition of 50 ml of 1 M HCl, and 5 ml of Scintisafe liquid scintillation cocktail (Fisher) was added. The radioactivity in the samples was quantitated by liquid scintillation counting using a Beckman LS6500 liquid scintillation counter. Enzymatic activity was calculated as nmol [ 3H]3-hydroxybenzo[a]pyrene formed/min/mg protein. Blank values were obtained by incubating harvesting buffer alone in place of the protein extract. RESULTS CYP1A1 mRNA level is elevated by AGN 193109 in mouse embryos. Differential display, utilizing RNA isolated from 8-dpc mouse embryos treated with either 1 mg/kg AGN 193109 or carrier for 6 h, resulted in the identification of a band, designated 3C21, which was very intense in the AGN 193109-treated sample while it was barely detectable in the DMSO control sample. Northern blot analysis using 3C21 cDNA as a probe demonstrated an intense band, which migrated between the 28S and 18S marker, in the AGN 193109treated RNA sample while it was slightly detectable in the control RNA sample (Fig. 1). Analysis of the level of 3C21 mRNA in 8-dpc mouse embryos treated for various periods of time with 1 mg/kg AGN 193109 demonstrated that 3C21 mRNA levels rose rapidly following treatment for 6 h, remained slightly elevated after 12 h treatment, and returned to basal level by 24 h (Fig. 2). On the other hand, treatment of 8-dpc mouse embryos with a dose of 100 mg/kg all-trans-RA (a dose that causes fetal malformations in essentially all ex- AGN 193109 AND CYP1A1 EXPRESSION 155 FIG. 3. Time-course effect of AGN 193109 on CYP1A1 mRNA levels in Hepa-1c1c7 cells. Hepa-1c1c7 cells were treated with 10 2 5 M AGN 193109 (AGN193109) or 10 2 9 M TCDD (TCDD) for the indicated periods of time (h). Total RNA was isolated and the level of CYP1A1 mRNA level was determined by Northern blot analysis using 3C21 cDNA as a probe. The blot was reprobed with a 28S ribosomal RNA cDNA. FIG. 1. 3C21 (CYP1A1) mRNA levels in AGN 193109-exposed mouse embryos. Pregnant mice were treated on 8 dpc with a single oral dose of 1 mg/kg AGN 193109 (AGN193109) or DMSO carrier (DMSO) for 6 h, the embryos were collected, and total RNA was isolated. (A) The level of 3C21 mRNA was determined by Northern blot analysis using cDNA obtained by PCR amplification of the DNA in the 3C21 band following elution from the differential display gel. (B) The blot was reprobed with a 28S ribosomal RNA cDNA. posed embryos (Kochhar et al., 1984; Kochhar, 1967)) resulted in no induction in CYP1A1 mRNA levels in mouse embryos treated for 0 to 24 h (see Fig. 2 for 6-h time point and data not shown). DNA sequence analysis of 3C21 DNA demonstrated that it was 394 bp long and that is was 100% homologous to the 39 untranslated region of mouse CYP1A1 (cytochrome P1– 450) (accession no. K02588) mRNA extending from base 2226 to the poly(A) tail (Kimura et al., 1984). Since all-trans-RA treatment did not regulate CYP1A1 mRNA levels in mouse FIG. 2. Time-course effect of AGN 193109 and retinoic acid on 3C21 (CYP1A1) mRNA levels in mouse embryos. Pregnant mice were treated on 8 dpc with a single oral dose of 1 mg/kg AGN 193109 (AGN193109) or 100 mg/kg all-trans-retinoic acid (RA) for the indicated periods of time (h), the embryos were collected, and total RNA was isolated. The level of CYP1A1 mRNA was determined by Northern blot analysis using 3C21 cDNA as a probe. The blot was reprobed with a 28S ribosomal RNA cDNA embryos and analysis of the DNA sequence of the mouse CYP1A1 promoter (Jones et al., 1985) indicated that there were no DNA sequences resembling a retinoic acid response element (RARE), we hypothesized that the elevation in CYP1A1 mRNA levels observed with AGN 193109 treatment was not directly mediated by RARs. CYP1A1 mRNA level, protein level, and enzymatic activity are elevated by AGN 193109 in Hepa-1c1c7 cells. Since CYP1A1 mRNA levels have been demonstrated to be regulated by various xenobiotic agents, including TCDD, in Hepa- 1c1c7 cells and because Hepa-1c1c7 mutant cell lines are available (Miller et al., 1983), we examined the effect of AGN 193109 on CYP1A1 mRNA levels in these cells. Figure 3 shows CYP1A1 mRNA levels in Hepa-1c1c7 cells treated with 10 2 5 M AGN 193109 for various periods of time from 0 to 72 h. The level of CYP1A1 mRNA rose rapidly following treatment of the cells, with AGN 193109 peaking at 4 h and returning to near basal level by 16 h. Similarly, the level of CYP1A1 protein and its enzymatic activity, arylhydrocarbon hydroxylase, showed a similar pattern of elevation in Hepa-1c1c7 cells following treatment with 10 2 5 M AGN 193109 (Figs. 4 and 5, respectively). Finally, Fig. 6 shows a comparison of the levels of CYP1A1 mRNA following treatment of Hepa-1c1c7 cells with 10 2 7 to 10 2 5 M AGN 193109 and 10 2 9 M TCDD. A small elevation in CYP1A1 mRNA levels was seen when the cells were treated with 10 2 6 M AGN 193109, with the maximum elevation, similar to that observed when cells were treated with 10 2 9 M TCDD, was obtained when cells were treated with 10 2 5 M AGN 193019. Comparison of the fold elevation in CYP1A1 mRNA levels between TCDD-treated cells and AGN193109-treated cells suggests that AGN 193109 is approximately 10,000-fold less potent than TCDD. However, as was observed in mouse embryos, no elevation in CYP1A1 mRNA levels was observed in the Hepa-1c1c7 cells treated with all-trans-RA up to a concentration of 10 2 5 M (Fig. 6). 156 SOPRANO ET AL. FIG. 6. Dose response of AGN 193109 and all-trans-retinoic acid on CYP1A1 mRNA levels in Hepa-1c1c7 cells. Hepa-1c1c7 cells were treated with the indicated concentrations of AGN 193109 (AGN193109) for 4 h, all-trans-RA for 4 h, or TCDD for 24 h. Total RNA was isolated and the level of CYP1A1 mRNA was determined by Northern blot analysis using 3C21 cDNA as a probe. The blot was reprobed with 28S ribosomsal RNA cDNA. FIG. 4. Time-course effect of AGN 193109 on CYP1A1 levels in Hepa1c1c7 cells. Hepa-1c1c7 cells were treated with 10 2 5 M AGN 193109 for the indicated times and total cellular protein was isolated. Fifty-microgram samples of total cellular protein were separated on a discontinuous SDS–9% polyacrylamide gel and the level of CYP1A1 protein was determined by Western blot analysis using goat anti-CYP1A1 polyclonal antibody followed by rabbit anti-goat IgG-horseradish peroxidase-conjugated secondary antibody. Proteins were visualized using enhanced chemiluminescence. CYP1A1 mRNA level is elevated by AGN 193109 only in AhR- and ARNT- positive cells. To begin our investigation of the mechanism responsible for the elevation in CYP1A1 mRNA levels by AGN 193109, we utilized two mutant cell lines, taoBpRc1 and BpRc1, that are derived from the parental Hepa-1c1c7 cells (Miller et al., 1983). TaoBpRc1 cells have a defect in AhR rendering them AhR negative and BpRc1 cells have a defect in ARNT rendering them ARNT negative. Both functional AhR and ARNT are required for the induction of CYP1A1 mRNA levels by xenobiotic agents via the AhR/ ARNT pathway; therefore CYP1A1 mRNA is not induced in FIG. 5. Time-course effect of AGN 193109 on arylhydrocarbon hydroxylase (AHH) activity (CYP1A1) in Hepa-1c1c7 cells. Hepa-1c1c7 cells were treated with10 2 5 M AGN 193109 for the indicated times. Total cellular protein extracts were prepared and AHH activity was assayed by measuring the conversion of [ 3H]benzo[a]pyrene substrate to [ 3H]3-hydroxybenzo[a]pyrene product. Data points are means 6 SD. these two mutant cells lines upon treatment with TCDD and other xenobiotic agents (Israel and Whitlock, 1983). As shown in Fig. 7, both TCDD and AGN 193109 elevated CYP1A1 mRNA levels only in the parental Hepa-1c1c7 cells and not in the two mutant cell lines. This provides evidence that the AhR/ARNT pathway and not the RAR/RXR pathway is mediating the elevation in CYP1A1 mRNA levels by AGN 193109 in Hepa-1c1c7 cells. DISCUSSION These data demonstrate that the conformationally restricted nonisoprenoid retinoid AGN 193109 but not the natural retinoid all-trans-RA can elevate the level of CYP1A1 mRNA protein and enzymatic activity in a time- and concentrationdependent manner in Hepa-1c1c7 cells and can elevate CYP1A1 mRNA levels in mouse embryos. Studies utilizing mutant cells suggest that this elevation in CYP1A1 mRNA levels in Hepa-1c1c7 cells is mediated by an AhR/ARNTdependent pathway and not by a RAR/RXR-dependent pathway. This is the first example of a retinoid that displays the FIG. 7. The effect of AGN 193109 on CYP1A1 mRNA levels in wildtype Hepa-1c1c7 cells, AhR-negative taoBpRc1 cells, and ARNT-negative BpRc1 cells. Hepa-1c1c7 cells, taoBpRc1 cells, and BpRc1 cells were treated with DMSO carrier (DMSO), 10 2 9 M TCDD (TCDD) for 24 h or 10 2 5 M AGN 193109 (AGN193109) for 4 h. Total RNA was isolated and the level of CYP1A1 mRNA was determined by Northern blot analysis using 3C21 cDNA as a probe. The blot was reprobed with 28S ribosomal RNA cDNA. AGN 193109 AND CYP1A1 EXPRESSION ability to regulate both the RAR/RXR and AhR/ARNT transcriptional regulatory pathways. Although AGN 193109 appears to be approximately 10,000fold less potent than TCDD in the elevation of CYP1A1 mRNA levels, its potency is similar to that of other reported inducers of CYP1A1 mRNA levels, including a-napthoflavone, YH439, 3-methylcholanthrene, benz[a]anthracene, and omeprazole (Poland and Glover, 1974; Quattrochi and Tukey, 1993; Postlind et al., 1993; Garrison et al., 1996; Lee et al., 1996). Some of the difference in potency between TCDD and AGN 193109 may lie in the metabolism of the two compounds within cells; TCDD is poorly metabolized (Whitlock et al., 1997), while AGN 193109 is rapidly metabolized (Kochhar et al., 1998). The transient increase in CYP1A1 mRNA levels in both the Hepa-1c1c7 cells and the mouse embryos is consistent with the rapid pharmacokinetics of AGN 193109 observed in maternal plasma and whole embryos (Kochhar et al., 1998). A peak in AGN 193109 levels in maternal plasma and whole embryos was observed 4 h after a single oral dose and was followed by a decline over the next 20 h to a level 100-fold less than the peak value. Finally, an increase in CYP1A1 mRNA levels was observed in embryos of dams exposed to a single oral dose of 1 mg/kg AGN 193109. Hence, it is possible that CYP1A1 mRNA levels could also be elevated in humans treated with a pharmacological dose of AGN 193109. Many xenobiotic agents have been demonstrated to induce the expression of one or more of the cytochrome P450 genes resulting in their own metabolism (Savas et al., 1999; Waxman et al., 1999). The “orphan” nuclear receptors (those for which the natural ligand have not yet been demonstrated), peroxisome proliferator-activated receptor-a, constitutively active receptor, and pregnane X receptor/steroid and xenobiotic receptor, have been demonstrated to bind xenobiotics and activate the expression of cytochrome P450 genes including CYP2B, CYP3A, and CYP4A (Waxman, 1999; Wei et al., 2000; Xie et al., 2000; Lee et al., 1995). In each case the induction of cytochrome P450 gene expression has been demonstrated to be mediated by one of these “orphan” nuclear receptors as a heterodimeric partner with RXR (Waxman, 1999). In contrast, the AGN 193109-induced increase in CYP1A1 expression reported here appears to be mediated by the AhR/ARNT receptor pathway and is a distinct mechanism from the regulation by xenobiotics of other cytochrome P450 genes by “orphan” nuclear receptors and RXR. CYP1A1 mRNA levels are generally very low or undetectable in cells. However, a variety of xenobiotic agents transcriptionally elevate CYP1A1 mRNA levels by liganding AhR and activating the AhR/ARNT transcriptional regulatory pathway. The best characterized inducers of CYP1A1 mRNA expression and high affinity AhR ligands include a variety of synthetic toxic chemicals, such as halogenated aromatic hydrocarbons (e.g., TCDD) and polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene and 3-methylcholanthrene) (Safe, 1990). In addition, several naturally occurring chemicals have been dem- 157 FIG. 8. Overlay of benzo[a]pyrene, indole derivative compound IV, AGN 193109, and all-trans-RA with TCDD. Three-dimensional space filling molecular models of benzo[a]pyrene, indole derivative compound IV (Gillner et al., 1985), AGN 193109, all-trans-RA, and TCDD were created using a Silicon Graphics Personal Iris Workstation and the Organic Builder Module within the Biograf Molecular Modeling Software. The structures with the lowest energy conformation for benzo[a]pyrene (A), indole derivative compound IV (B), AGN 193109 (C), and all-trans-RA (D) were superimposed on that of TCDD (black in each panel). The arrow in C indicates the position of the benzoate group of AGN 193109 while the arrows in A and B indicate a similar position in benzo[a]pyrene and indole derivative compound IV, respectively. onstrated to elevate CYP1A1 mRNA levels at relatively high concentrations and to be low affinity AhR ligands (Chen et al., 1995; Ciolino et al., 1998, 1999; Heath-Pagliuso et al., 1998; Washburn et al., 1997; Sinal and Bend, 1997; Phelan et al., 1998; Denison et al., 1998). However, to date, a high affinity natural ligand for AhR has not been described. The structure of the majority of these AhR ligands is polycyclic, aromatic, planar, and hydrophobic. In addition, on the basis of computer modeling of AhR agonists, it has been suggested that compounds that interact tightly with AhR fit into a hypothetical planar rectangle with the dimensions of 6.8 Å 3 13.7 Å (Gillner et al., 1985). However, more recently, several AhR ligands have been described that do not fit these specifications (Lee et al., 1996; Schaldach et al., 1999; Heath-Pagliuso et al., 1998; Washburn et al., 1997; Denison et al., 1998). AGN 193109, like the prototypical AhR ligand, is polycyclic, aromatic, relatively planar, and hydrophobic. Molecular modeling of AGN 193109 demonstrates that it too can fit into the hypothetical rectangle lengthwise but is slightly wider than the 6.8 Å width. However, superimposition of AGN 193109 with TCDD shows that the fused ring system of the backbone of AGN 193109 is directly superimposable onto the fused ring structure of TCDD (Fig. 8C). In addition, the benzoate group of AGN 193109, which extends slightly out of the 6.8 Å limit on the width of the rectangular box, occupies a similar position as in benzo[a]pyrene and indole derivative compound IV (Gillner et al., 1985) (Figs. 8A–C). Competition binding assays with [ 3H]TCDD and rat liver cytosol have demonstrated that benzo[a]pyrene has an IC50 value of 42 nM and indole derivative compound IV has an IC50 value between 150 and 1500 nM (Gillner et al., 1985). Finally, note that, as shown in Fig. 8D, all-trans-RA is nonpolycyclic, is not coplanar, and it does not fill the same space as TCDD, benzo[a]pyrene, and indole 158 SOPRANO ET AL. derivative compound IV. This is consistent with the possibility that AGN 193109 but not all-trans-RA is an AhR ligand. Future studies are required to directly determine if AGN 193109 is indeed an AhR ligand. Exposure of mouse embryos to TCDD has been demonstrated to result in fetal malformations including cleft palate and hydronephrosis (Courtney and Moore, 1971). Recent studies utilizing AhR-null mice clearly demonstrate that AhR is required to mediate TCDD-induced developmental defects (Peters et al., 1999; Mimura et al., 1997), however, no specific role has been demonstrated for the elevation in CYP1A1 in mediating these developmental defects. The question arises as to what role if any the potential activation of AhR by AGN 193109 and the concomitant elevation in CYP1A1 levels might be playing in the ultimate phenotype observed in AGN 193109-exposed mouse embryos (Kochhar et al., 1998). Since synergism between TCDD and all-trans-RA has been previously described (Birnbaum et al., 1989; Weston et al., 1995), it is possible that both the activation of the AhR/ARNT pathway and the antagonism of RAR/RXR pathway by AGN 193109 maybe contributing to the complex phenotype observed in these embryos. Comparison of the fetal defects observed in wild-type and AhR-null mice exposed to AGN 193109 may help to determine what role AhR may be playing in mediating the developmental defects caused by AGN 193109 treatment. ACKNOWLEDGMENT This work was supported by National Institutes of Health Grants DE11954 (to D.M.K. and D.R.S.) and CA82770 (to D.R.S.). REFERENCES Agarwal, C., Chandraratna, R. A. S., Johnson, A. T., Rorke, E. A., and Eckert, R. L. (1996). AGN193109 is a highly effective agonist of retinoid action in human ectocervical epithelial cells. J. Biol. Chem. 271, 12209 –12212. Birnbaum, L. S., Harris, M. W., Stocking, L. M., Clark, A. M., and Morrissey, R. E. (1989). Retinoic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin selectively enhances teratogenesis in C57 BL/6 mice. Toxicol. Appl. Pharmacol. 98, 487–500. Burnette, W. N. (1981). “Western blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195–203. Chambon, P. (1996). A decade of molecular biology of retinoic acid receptors. FASEB J. 10, 940 –954. Chen, Y-H., Riby, J., Srivastava, P., Bartholomew, J., Denison, M., and Bjeldanes, L. (1995). Regulation of CYP1A1 by indolo[3,2-b]carbazole in murine hepatoma cells. J. Biol. Chem. 270, 22548 –22555. Ciolino, H., Daschner, P., Wang, T., and Yeh, G. (1998). Effect of curcumin on the aryl hydrocarbon receptor and cytochrome P450 1A1 in MCF-7 human breast carcinoma cells. Biochem. Pharmacol. 56, 197–206. Ciolino, H., Daschenr, P. J., and Yeh, G. C. (1999). Dietary flavonols quercetin and kaempferol are ligands of the aryl hydrocarbon receptor that affect CYP1A1 transcription differentially. Biochem. J. 340, 715–722. Courtney, K. D., and Moore, J. A. (1971). Teratology studies with 2,4,5- trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 20, 396 – 403. Denison, M. S., Phelan, D., Winter, G. M., and Ziccardi, M. H. (1998). Carbaryl, a carbamate insecticide, is a ligand for the hepatic Ah (Dioxin) receptor. Toxicol. Appl. Pharmacol. 152, 406 – 414. Garrison, P. M., Tullis, K., Aarts, J. M., Brouwer, A., Giesy, J. P., and Denison, M. S. (1996). Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Fundam. Appl. Toxicol. 30, 194 –203. Gillner, M., Bergman, J., Cambillau, C., Fernstrom, B., and Gustaffson, J. A. (1985). Interactions of indoles with specific binding sites for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver. Mol. Pharmacol. 28, 357–363. Harnish, D. C., Barua, A. B., Soprano, K. J., and Soprano, D. R. (1990). Induction of b-retinoic acid receptor mRNA by teratogenic doses of retinoids in murine fetuses. Differentiation 45, 103–108. Heath-Pagliuso, S., Rogers, W. J., Tullis, K., Seidel, S. D., Cenijn, P. H., Brouwer, A., and Denison, M. S. (1998). Activation of the Ah receptor by tryptophan and tryptophan metabolites. Biochemistry 37, 11508 –11515. Israel, D. I., and Whitlock, J. P. (1983). Induction of mRNA specific for cytochrome P 1-450 in wild type and variant mouse hepatoma cells. J. Biol. Chem. 258, 10390 –10394. Johnson, A. T., Klein, E. S., Gillett, S. J., Wang, L. M., Song, T. K., Pino, M. E., and Chandraratna, R. A. S. (1995). Synthesis and characterization of a highly potent and effective antagonist of retinoic acid receptors. J. Med. Chem. 38, 4764 – 4767. Jones, P. B., Galeazzi, D. R., Fisher, J. M., and Whitlock, J. P. (1985). Control of cytochrome P1– 450 gene expression by dioxin. Science 227, 1499 –1502. Kimura, S., Gonzalez, F. J., and Nebert, D. W. (1984). The murine Ah locus. J. Biol. Chem. 259, 10705–10713. Kochhar, D. M. (1967). Teratogenic activity of retinoic acid. Acta Pathol. Microbiol. 70, 289 –298. Kochhar, D. M., Penner, J. D., and Tellone, C. I. (1984). Comparative teratogenic activities of two retinoids: Effect on palate and limb development. Teratogen. Carcinog. Mutagen. 4, 377–387. Kochhar, D. M., Jiang, H., Penner J. D., Johnson, A. T., and Chandraratna, R. A. S. (1998). The use of retinoid receptor antagonist in a new model to study vitamin A-dependent developmental events. Int. J. Dev. Biol. 42, 601– 608. Lee, I. J., Jeong, K. S., Roberts, B. J., Kallarakal, A. T., Fernandez-Salguero, P., Gonzalez, F. J., and Song, B. J. (1996). Transcriptional induction of the cytochrome P4501A1 gene by a thiazolium compound, YH439. Mol. Pharmacol. 49, 980 –988. Lee, S. S., Pineau, T., Drago, J., Lee, E. J., Owens, J. W., Kroetz, D. L., Fernandez-Salguero, P. M., Westphal, H., and Gonzalez, F. J. (1995). Targeted disruption of the alpha isoform of the peroxisome proliferatoractivated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol. Cell. Biol. 15, 3012–3022. Liang, P., and Pardee A. B. (1992). Differential display of eukaryotic mRNA by means of the polymerase chain reaction. Science 257, 967–971. Lohnes, D., Mark, M., Mendelsohn, C., Dolle, P., Dierich, A., Gorry, P., Gansmuller, A., and Chambon, P. (1994). Function of the retinoic acid receptors (RARs) during development. 1. Craniofacial and skeletal abnormalites in RAR double mutants. Development 120, 2723–2748. Mendelsohn, C., Lohnes, D., Decimo, D., Lufkin, T., Lemeur, M., Chambon, P., and Mark, M. (1994). Function of the retinoic acid receptors (RARs) during development. 2. Multiple abnormalites at various stages of organogenesis in RAR double mutants. Development 120, 2749 –2771. Miller, A. G., Israel, D. I. and Whitlock, J. P. (1983). Biochemical and genetic analysis of variant mouse hepatoma cells defective in the induction of benzo[a]pyrene-metabolizing enzyme activity. J. Biol. Chem. 258, 3523– 3527. AGN 193109 AND CYP1A1 EXPRESSION Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T. N., Nakao, K., Sogawa, E. M., Sogawa, K., Yasuda, M., Katsuki, M., and FujiiKuriyama, Y. (1997). Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes Cells 2, 645– 654. Nebert, D. W., and Gelboin, H. V. (1968). Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. II. Cellular responses during enzyme induction. J. Biol. Chem. 243, 6250 – 6261. Peters, J. M., Narotsky, M. G., Elizondo, G., Fernandez-Salguero, P. M., Gonzalez, F. J., and Abbott, B. D. (1999). Amelioration of TCDD-induced teratogenesis in aryl hydrocarbon receptor (AhR)-null mice. Toxicol. Sci. 47, 86 –92. Phelan, D., Winter, G. M., Rogers, J., Lam, J. C., and Denison, M. S. (1998). Activation of the Ah receptor signal transduction pathway by bilirubin and biliverdin. Arch. Biochem. Biophys. 357, 155–163. Poland, R. S., and Glover, E. (1974). Comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcholanthrene. Mol. Pharmacol. 10, 349 –359. Postlind, H., Vu, T. P., Tukey, R. H., and Quattrochi, L. C. (1993). Response of human CYP-1 luciferase plasmids to 2,3,7,8-tetrachlorodibenzo-p-dioxin and polycyclic aromatic hydrocarbons. Toxicol. Appl. Pharmacol. 118, 255–262. Quattrochi, L. C., and Tukey, R. H. (1993). Nuclear uptake of the Ah (dioxin) receptor to omeprazole: Transcriptional activation of the human CYP1A1 gene. Toxicol. Appl. Pharmacol. 43, 504 –508. Rappolee, D. A., Wang, A., Mark, D., and Werb, Z. (1989). Novel method for studying mRNA phenotypes in single or small numbers of cells. J. Cell. Biochem. 39, 1–11. Safe, S. (1990). Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and related compounds: Environmental and mechanistic considerations which support the development of toxic equivalency factors (TEFs). Crit. Rev. Toxicol. 21, 51– 88. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. Savas, U., Griffin, K. J., and Johnson, E. F. (1999). Molecular mechanisms of cytochrome P-450 induction by xenobiotics: An expanded role for nuclear hormone receptors. Mol. Pharmacol. 56, 851– 857. Schaldach, C. M., Riby, J., and Bjeldanes, L. F. (1999). Lipoxin A 4: A new class of ligand for the Ah receptor. Biochemistry 38, 7594 –7600. Sinal, C. J., and Bend, J. R. (1997). Aryl hydrocarbon receptor-dependent induction of Cyp1a1 by bilirubin in mouse hepatoma 1c1c7 cells. Mol. Pharmacol. 52, 590 –599. Soprano, D. R, Soprano, K. J., and Goodman, D. S. (1986). Retinol-binding 159 protein messenger RNA levels in the liver and in extrahepatic tissues of the rat. J. Lipid Res. 27, 166 –171. Tairis, N., Gabriel, J. L., Gyda, M., Soprano, K. J., and Soprano, D. R. (1994). Arg 269 and Lys 220 of retinoic acid receptor-b are important for the binding of retinoic acid. J. Biol. Chem. 269, 19516 –19522. Thomas, P. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77, 5201– 5205. Underwood, B. A. (1984). Vitamin A in animal and human nutrition. In The Retinoids (M. B. Sporn, A. B. Roberts, and D. S. Goodman, Eds.), Vol. 1, pp. 281–392. Academic Press, New York. Van Cantfort, J., De Graeve, J., and Gielen, J. E. (1977). Radioactive assay for aryl hdrocarbon hydroxylase. Improved method and biological importance. Biochem. Biophys. Res. Commun. 79, 505–512. Washburn, B. S., Rein, K. S., Baden, D. G., Walsh, P. J., Hinton, D. E., Tullis, K., and Denison, M. S. (1997). Brevetoxin-6 (PbTx-6), a nonaromatic marine neurotoxin, is a ligand of the aryl hydrocarbon receptor. Arch. Biochem. Biophys. 343, 149 –156. Waxman, D. J. (1999). P450 gene induction by structurally diverse xenochemicals: Central role of nuclear receptors CAR, PXR and PPAR. Arch. Biochem. Biophys. 369, 11–23. Wei, P., Zhang, J., Egan-Hafley, M., Liang, S., and Moore, D. D. (2000). The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature 407, 920 –923. Weston, W. M., Nugent, P., and Greene, R. M. (1995). Inhibition of retinoicacid-induced gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochem. Biophys. Res. Commun. 207, 690 – 694. Whitlock, J. P., Chichester, C. H., Bedgood, R. M., Okino, S. T., Ko, H. P., Ma, Q., Dong, L., Li, H., and Clarke-Katzenberg, R. (1997). Induction of drug-metabolizing enzymes by dioxin. Drug Metab. Rev. 29, 1107–1127. Wilson, J. G., and Warkany, J. (1950). Cardiac and aorta anomalies in offspring of vitamin A deficient rats correlated with similar human anomalies. Pediatrics 5, 708 –725. Wilson, J. G., Roth, C. B., and Warkany, J. (1953). An analysis of the syndrome of malformations induced by maternal vitamin A deficiency. Effects of restoration of vitamin A at various times during gestation. Am. J. Anat. 92, 189 –217. Wolf, G. (1996). A history of vitamin A and retinoids. FASEB J. 10, 1102– 1107. Xie, W., Barwick, J. L., Downs, M., Blumberg, B., Simon, C. M., Nelson, M. C., Neuschwander-Tetri, B. A., Grunt, E. M., Guzelian, P. S., and Evans, R. M. (2000). Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature 406, 435– 439.