Dioxin Response Element Research Articles

Identification of a Critical Amino Acid in the Aryl Hydrocarbon Receptor

Two aryl hydrocarbon receptors (rtAHR2alpha and rtAHR2beta) have been identified in the rainbow trout (Oncorhynchus mykiss). These receptors share 98% amino acid identity, yet their functional properties differ. Both rtAHR2alpha and rtAHR2beta bind 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dimerize with rainbow trout ARNTb (rtARNTb), and recognize dioxin response elements in vitro. However, in a transient transfection assay the two proteins show differential ability to recognize enhancers, produce transactivation, and respond to TCDD. To identify the sequence differences that confer the functional differences between rtAHR2alpha and rtAHR2beta, we constructed chimeric rtAHRs, in which segments of one receptor form was replaced with the corresponding part from the other isoform. This approach progressively narrowed the region being examined to a single residue, corresponding to position 111 in rtAHR2beta. Altering this residue in rtAHR2beta from the lysine to glutamate found in rtAHR2alpha produced an rtAHR2beta with the properties of rtAHR2alpha. All other known AHRs resemble rtAHR2alpha and carry glutamate at this position, located at the N terminus of the PAS-A domain. We tested the effect of altering this glutamate in the human and zebrafish AHRs to lysine. This lysine substitution produced AHRs with transactivation properties that were similar to rtAHR2beta. These results identify a critical residue in AHR proteins that has an important impact on transactivation, enhancer site recognition, and regulation by ligand.

Unique property of some synthetic retinoids: activation of the aryl hydrocarbon receptor pathway.

Potential pharmacological applications in the areas of oncology, dermatology, diabetes, and atherosclerosis of synthetic analogs of retinoic acid that target a specific nuclear receptor and/or biological response have generated great interest in the development of new retinoid and rexinoid drugs. The pan-retinoic acid receptor antagonist AGN 193109 has been previously reported to elevate CYP1A1 levels, implicating the aryl hydrocarbon receptor (AhR) as an additional target for this retinoid. AhR is a cytosolic ligand-dependent transcription factor that, in conjunction with the AhR nuclear translocator (Arnt), binds to dioxin response elements (DREs) located in the promoter region of target genes, such as CYP1A1, and induces their transcription. The purpose of these studies was to determine whether additional synthetic retinoids were capable of elevating CYP1A1 levels and to examine the mechanism of this increase in CYP1A. Two additional retinoids, AGN 190730 and AGN 192837, were found to be potent inducers of DRE-driven transcriptional activity; AGN 190730 was the most potent. Moreover, electrophoretic mobility-shift assays demonstrate that AGN 190730 can transform AhR into its active DNA recognition form. In addition, trypsin digestion of AGN 190730-treated AhR reveals a conformational change in the protein similar to the conformational change of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-bound AhR. Finally, competitive binding studies demonstrate that AGN 190730 can inhibit the binding of TCDD to AhR. The sum of the data demonstrates that some synthetic retinoids in addition to activating the retinoic acid receptor/retinoid X receptor pathway are capable of binding to AhR and activating the AhR/Arnt pathway.

The Q-rich subdomain of the human Ah receptor transactivation domain is required for dioxin-mediated transcriptional activity.

The aryl hydrocarbon receptor (AhR), a basic helix-loop-helix/Per-Arnt-Sim transcription factor, mediates many of the toxic and biological effects of the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin, which include the transcriptional activation of dioxin-responsive genes such as CYP1A1. Many aspects of this process are known; however, the mechanism of transcriptional activation and the proteins that are key to this process remain to be determined. The hAhR has a complex transactivation domain, composed of three potentially distinct subdomains. Deletional analysis of the hAhR transactivation domain indicates that removal of the P/S/T-rich subdomain enhances transcriptional activity, whereas the Q-rich subdomain is critical for hAhR transactivation potential, and the acidic subdomain by itself fails to activate a dioxin response element-driven reporter gene. Deletional analysis of the Q-rich subdomain identified a critical stretch of 23 amino acids between residues 666 and 688 of the hAhR, which are required for transactivation potential. Alanine scanning mutagenesis of this region identified a leucine residue (Leu-678), which is required for hAhR activity. Functional analysis of this point mutant revealed that it is capable of binding ligand, heterodimerization, and subsequent binding to dioxin response elements. Further, when hAhR/L678A and hAhR containing only the acidic subdomain were overexpressed they acted as dominant negative receptors and repressed wild-type hAhR activity. In addition, the hAhR/L678A failed to activate CYP1A1 gene transcription in transfected BP-8 cells and exhibited reduced binding to RIP140 in vitro. Thus, Leu-678 appears to be critical for efficient transactivation activity of the hAhR and appears to disrupt recruitment of co-regulators.

Transient Expression of CYP1A1 in Rat Epithelial Cells Cultured in Suspension

Suspension of human epidermal cells in methylcellulose-containing medium induces CYP1A1 by a mechanism requiring functional Ah receptor (AhR). In present work CYP1A1 mRNA was induced in a variety of cultured rat epithelial cells by suspension, but the induction was transient, with CYP1A1 mRNA reaching maximal levels by 5 h and disappearing by 12 h. Though the methylcellulose itself contained no detectable ligand, (a) suspension activated the AhR, as judged by mobility shift assays, (b) the AhR competitive inhibitor α-naphthoflavone inhibited suspension-mediated induction, and (c) induction was dependent upon dioxin responsive transcriptional elements in the CYP1A1 promoter. The rapid disappearance of CYP1A1 mRNA after 5 h of suspension was unaffected by the addition of TCDD but was prevented by the inclusion of the protein synthesis inhibitor cycloheximide. Thus the downregulation appears to be mediated by a novel short-lived protein induced or activated by suspension.

Interactions between aryl hydrocarbon receptor (AhR) and hypoxia signaling pathways

Most if not all of the toxic responses of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) are mediated through the AhR, which requires ARNT to regulate gene expression. ARNT is also required by HIF-1α to enhance the expression of various genes in response to hypoxia. Since both the AhR and hypoxia transcriptional pathways require ARNT, some of the effects of TCDD and similar types of ligands could be explained by interaction between the AhR and hypoxia pathways involving ARNT. The studies on which we report here were conducted to test the hypothesis that there is cross talk between AhR- and HIF-1-mediated transcription pathways. TCDD significantly reduced the hypoxia-mediated reporter gene activity in B-1 cells. Reciprocally, the hypoxia response inducers desferrioxamine or CoCl 2 inhibited AhR-mediated CYP1A1 enzyme activity in B-1 and Hepa 1 cells, and the AhR-mediated luciferase reporter gene activity in H1L1.1c2 cells. The inhibition of AhR-mediated transcription by hypoxia inducers, however, was not observed in H4IIE-luc cells. The interaction between the AhR- and HIF-1-mediated transcription can be attributed to changes in DNA binding activities. TCDD-induced protein binding to dioxin responsive element (DRE) was diminished by desferrioxamine, and TCDD reduced the binding activity to HIF-1 binding site in desferrioxamine-treated Hepa 1 cells. This mutual repression may provide an underlying mechanism for many TCDD-induced toxic responses. The results reported here indicate that there is cross talk between ARNT-requiring pathways. Since ARNT is possibly required by a number of pathways, this type of interaction may explain some of the pleiotropic effects caused by TCDD.

Physiological modeling of a proposed mechanism of enzyme induction by TCDD

A physiological model was previously constructed to facilitate extrapolation of surrogates for the effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in rat liver to doses comparable to human environmental exposures. The model included induction of P450 isozymes and suggested the presence of multiple binding sites with different affinities for the TCDD-liganded Ah receptor at CYP1A1 dioxin responsive elements. The model also indicated that protein synthesis on the mRNA template exhibited saturation kinetics with respect to message levels. In the present work the earlier model was revised to include the increased proteolysis of the Ah receptor on binding TCDD, more realistic representations of gene transcription and mRNA translation, and different stability for each mRNA. The revised model includes multiple TCDD-liganded Ah receptor binding sites for CYP1A1 and CYP1B1 genes, a lag of 0.2 day for production of mRNA and induced proteins, and stabilization of mRNA by a poly(A) tail. The model reproduced the transient depletion of the Ah receptor subsequent to binding ligand and the dose–response of the receptor in rats treated with biweekly oral doses of TCDD in corn oil. The model reproduced tissue TCDD concentrations observed for several dosing scenarios. Such robustness indicates the utility of the model in estimating internal dose. The model also reproduced the observed dose–response patterns for mRNA and protein for CYP1A1, CYP1A2, and CYP1B1 after repeated dosing. Neither of the two dissociation constants for the Ah receptor bound to the CYP1B1 gene is negligible, supporting the assumption of multiple response elements for this gene. The poorer induction of CYP1B1 was predicted to be due to lower affinity of the dioxin responsive elements for binding the liganded Ah receptor, suggesting the involvement of other regulatory factors, and a shorter poly(A) tail on CYP1B1 mRNA, leading to a shorter lifetime. Saturation in the kinetics of protein synthesis was linked to the limited number of ribosomes that could bind to each message molecule, resulting in fewer ribosomes bound per message at higher doses. Predicted induction at low doses was found to vary widely with the assumptions used in the construction of a model. More detailed descriptions of biological processes might provide more reliable predictions of enzyme induction.

Suppression of CYP1A1 expression by 4-nonylphenol in murine Hepa-1c1c7 cells

This study investigated the effects that 4-nonylphenol (NP) has on CYP1A1 expression in Hepa-1c1c7 cell cultures. NP alone did not affect CYP1A1-specific 7-ethoxyresorufin- O-deethylase (EROD) activity. In contrast, the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-inducible EROD activities were markedly reduced upon concomitant treatment with TCDD and NP in a dose-dependent manner. Treatment with tamoxifen, an anti-estrogen that acts through the estrogen receptor, did not affect the suppressive effects that NP has on TCDD-inducible EROD activity. The TCDD-inducible CYP1A1 mRNA levels were markedly suppressed upon concomitant treatment with TCDD and NP that is consistent with their effects on EROD activity. A transient transfection assay using dioxin-response element (DRE)-linked luciferase and an electrophoretic mobility shift assay revealed that NP reduced the transformation of the aryl hydrocarbon (Ah) receptor to a form capable of binding specifically to the DRE sequence of the CYP1A1 gene promoter. These results suggest that the down-regulation of CYP1A1 gene expression by NP in Hepa-1c1c7 cells might be an antagonism of the DRE-binding potential of the nuclear Ah receptor, but is not mediated through the estradiol receptor.

Molecular biology of the Ah receptor and its role in carcinogenesis

The aryl hydrocarbon receptor (AhR) is a ligand-activated nuclear transcription factor that mediates responses to toxic halogenated aromatic toxins such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), polynuclear aromatic hydrocarbons, combustion products, and numerous phytochemicals such as flavonoids and indole-3-carbinol (I3C). The nuclear AhR complex is a heterodimer containing the AhR and AhR nuclear translocator (Arnt) proteins, and the molecular mechanism of AhR action is associated with binding of the heterodimer to dioxin responsive elements (DREs) in regulatory regions of Ah-responsive genes. TCDD, a ‘xenodioxin’, is a multi-site carcinogen in several species and possibly in humans, whereas natural AhR ligands including I3C and flavonoids tend to protect against cancer. Both TCDD and phytochemicals inhibit estrogen-induced breast and endometrial cancer, and the molecular mechanisms of this common response will be described.

Transcriptional activation of heat shock protein 27 gene expression by 17beta-estradiol and modulation by antiestrogens and aryl hydrocarbon receptor agonists.

Heat shock protein 27 (Hsp 27) is expressed in mammary tumors and may play a role in tumor growth and response to anti-neoplastic drug therapy. 17beta-Estradiol (E2) induces Hsp 27 mRNA levels in MCF-7 human breast cancer cells, and we have investigated the comparative inhibitory mechanisms using the aryl hydrocarbon receptor (AhR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the direct-acting antiestrogen ICI 164,384. TCDD inhibited E2-induced Hsp 27 gene expression and analysis of the Hsp 27 gene promoter showed that the inhibitory response was associated with AhR interactions with a pentanucleotide motif at -3 to +2 in the promoter that corresponded to the core sequence of a dioxin responsive element. In contrast, ICI 164,384 induced Hsp 27 gene expression and reporter gene activity in MCF-7 cells and this represents one of the few examples of the estrogen receptor-alpha (ERalpha) agonist activity of the 'pure' antiestrogen ICI 164,384.

Transcriptional activation of cathepsin D gene expression by 17β-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition

17β-estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast cancer cells and this response is inhibited by aryl hydrocarbon receptor (AhR) agonists, such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Analysis of the cathepsin D gene promoter initially identified a pentanucleotide GCGTG core dioxin responsive element (DRE) that blocked E2 action by inhibiting formation of a transcriptionally active estrogen receptor (ER)-Sp1 complex. A second functional downstream inhibitory DRE (iDRE2) (−130 to −126) has now been identified in the cathepsin D gene promoter and inhibition of E2-induced transactivation involves inhibitory AhR crosstalk with the E2-responsive adenovirus major late promoter element (MLPE) at −124 to −104 in the cathepsin D gene promoter. The MLPE site primarily binds USF1/USF2 and ERα, and gel mobility shift and DNA footprinting assays show that the AhR complex decreases binding of these transcription factors to the MLPE.

The murine IL-2 promoter contains distal regulatory elements responsive to the Ah receptor, a member of the evolutionarily conserved bHLH-PAS transcription factor family.

Signaling through the TCR and costimulatory signals primarily control transcription of the IL-2 gene in naive T cells. The minimal promoter necessary for this expression lies proximal, between -300 and the transcription start site. We had previously shown that activation of the arylhydrocarbon receptor (AHR), a member of the bHLH-PAS family of transcription factors, leads to increased mRNA expression of IL-2 in murine fetal thymocytes. The AHR is abundant in the thymus and may play a role for the development of the immune system. Moreover, its overactivation by chemicals such as dioxins leads to immunosuppression and thymic involution. Binding motifs for the liganded AHR can be identified in the distal region -1300 to -800 of the mouse IL-2 promoter. We show here that these DNA motifs, the so-called dioxin response elements, after binding to the liganded AHR are sufficient to transactivate luciferase expression in a reporter gene system. The IL-2 gene can be induced by the AHR also in thymocytes in vivo after injection of 2,3,7, 8-tetrachlorodibenzo-p-dioxin, a potent ligand of the AHR. The AHR mediates the IL-2 induction as shown with AHR-deficient mice. However, in spleen cells in vitro costimulation via the TCR is necessary for optimal IL-2 gene induction. Thus, the IL-2 promoter region contains novel distal regulatory elements that can be addressed by the AHR to induce IL-2 and can cooperate with the proximal promoter in this.

Down-Regulation of Murine Cyp1a-1 in Mouse Hepatoma Hepa-1c1c7 Cells by Bisphenol A

Cultured mouse hepatoma Hepa-1c1c7 cells were treated with either bisphenol A or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or in combination to assess the role of bisphenol A in the process of Cyp1a-1 induction. Treatment of Hepa-1c1c7 cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced Cyp1a-1, as determined by analysis of 7-ethoxyresorufin O-deethylase (EROD) activities. Bisphenol A alone did not affect the activity of Cyp1a-1-specific EROD; in contrast, TCDD-induced EROD activities were markedly reduced in the concomitant treatment of TCDD and bisphenol A in a dose-dependent manner. Treatment with tamoxifen, an antiestrogen that acts through the estrogen receptor, did not affect the suppressive effects of bisphenol A on TCDD-induced EROD activity. TCDD-induced Cyp1a-1 mRNA levels were markedly suppressed in the concomitant treatment of TCDD and bisphenol A consistent with their effects on EROD activity. Transient transfection assay using dioxin-response element (DRE)-linked luciferase revealed that bisphenol A reduced transformation of the aryl hydrocarbons (Ah) receptor to a form capable of specifically binding to the DRE sequence in the promoter of the Cyp1a-1 gene. These results suggest the down-regulation of the Cyp1a-1 gene expression by bisphenol A in Hepa-1c1c7 cells might be antagonism of the DRE binding potential of nuclear Ah receptor but not mediated through estradiol receptor.

Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions

In order to further establish zebrafish as a vertebrate model for studying the mechanism of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) toxicity it is necessary to characterize the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AhR/ARNT) signaling pathways in this species. In this study, three zfARNT2 cDNAs were isolated, expressed, and characterized and named zfARNT2b, zfARNT2c, and zfARNT2a. zfARNT2b, zfARNT2c, and zfARNT2a encode proteins with theoretical molecular weights of 81, 79, and 45 kDa, respectively. zfARNT2b and zfARNT2a proteins are identical over the first 403 amino acids but differ in their C-terminal domains as a result of alternative mRNA splicing. zfARNT2c is nearly identical to zfARNT2b, with the exception of an in frame 15 amino acid deletion adjacent to the basic region of zfARNT2c. Using quantitative RT-PCR methods the tissue distribution of each zfARNT2 isoform was determined. In COS-7 cells expressing zfARNT2b and zfAhR2, 10 nM TCDD causes a nine-fold induction of a dioxin responsive reporter gene. In COS-7 cells expressing zfARNT2a or zfARNT2c, TCDD does not induce reporter gene expression. In contrast, all three zfARNT2 proteins induce reporter gene activity under control of hypoxia responsive elements when cotransfected with the zebrafish endothelial specific PAS protein 1. DNA gel shift analysis suggests that the decreased function of zfARNT2a is due to inefficient binding of zfARNT2a/zfAhR2 complexes to dioxin responsive elements. These results also indicate that alternative mRNA splicing results in formation of ARNT proteins with distinct functional properties.

The application of reporter gene assays for the determination of the toxic potency of diffuse air pollution

Diffuse air pollution consists of a mixture of numerous compounds. It is emitted by many distributed sources and is omnipresent due to atmospheric transport. Risk assessment of the complex mixture of air pollutants on the basis of the toxicity of the individual compounds is not yet possible because the chemical identity and/or toxicity of the constituencies of a substantial fraction is unknown. In addition, no adequate procedures are available to integrate toxicity data of such complex mixtures, so that an individual risk assessment of the constituents of air pollution disregards possible combination effects. In the present study, an approach has been developed to assess the toxic potency by using in vitro bio-assay techniques. Genotoxicity was assessed in the umu-assay, a reporter gene assay using a strain of Salmonella typhimurium stably transfected with a plasmid (pSK1002) carrying the SOS-gene umuC fused to the reporter gene lacZ. Arylhydrocarbon-receptor activation was assessed in the DR-CALUX-assay, using a stably transfected H4IIE hepatoma cell line containing a plasmid for the luciferase gene under transcriptional control of dioxin-responsive elements. Samples of airborne particulate matter (APM) were collected with a high volume sampler next to a highway and in a natural conservation area. Both assays proved to be applicable to quantify genotoxicity and the presence of polycyclic aromatic hydrocarbons (PAHs) in small extracts from air-filter samples. Results indicate that PAHs from traffic exhausts seem to be largely responsible for an increased genotoxic activity of APM collected down-wind from the highway (western wind). APM collected at eastern wind directions seems to have a different composition of compounds, with a higher genotoxic activity that is less related to highway-emitted PAH-like compounds. At northern wind directions, APM is relatively less genotoxic and contains less PAHs than at other wind directions. Dioxin-like compounds contribute negligibly to the Ah-receptor agonistic potency of APM. Airborne pollutants with genotoxic and/or PAH-like characteristics form an undesired mutagenic risk, which will be evaluated in further in vivo studies.

Regulation of DNA Binding Activity of the Ligand-Activated Aryl Hydrocarbon Receptor by Tyrosine Phosphorylation

Aryl hydrocarbon receptor (AhR), a member of the bHLH–PAS family, is a ligand-activated transcription factor which plays an important role in normal liver development and in mediating the toxicity of polycyclic and halogenated aromatic hydrocarbon pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Phosphorylation is known to regulate the transformation process of unliganded AhR into functionally active AhR/ARNT heterodimer that has high affinity for dioxin-responsive elements (DRE) and transactivation activity. Here, we report that DRE binding activity of the AhR is regulated by phosphorylation on the AhR/ARNT complex itself. Studies with specific protein phosphatases indicated that tyrosine phosphorylation is involved in this modulation. In addition, the AhR is phosphorylated at tyrosine residue(s) as determined by anti-phosphotyrosine immunoblot analysis. These results suggest that tyrosine phosphorylation on the AhR is required for its DNA binding activity and may provide mammalian cells with another layer of control mechanism that allows cell type specific and developmental stage specific induction of the AhR target genes.

Molecular characterization and developmental expression of the aryl hydrocarbon receptor from the chick embryo

The aryl hydrocarbon receptor (AhR) was cloned from the chick embryo and its function and developmental expression characterized. Chicken AhR cDNA coded for 858 amino acid protein and 396 bp of 3' UTR. The basic helix loop helix domain exhibited 87-100% amino acid identity to avian, mammalian, and amphibian AhR, and 69-74% to piscine AhR. The PAS (Per-ARNT-Sim) region was slightly less well conserved with (a) 97% identity to other avian sequences, (b) 81-86% to amphibian and mammalian AhR, and (c) 64-69% with piscine AhR. The carboxy terminus diverged the most among species with less than 53% amino acid identity between chicken and any available mammalian and piscine AhR sequences. The chicken AhR RNA and protein were 6.1 kb and 103 kDa, respectively. Chicken AhR dimerized with human AhR nuclear translocator and bound the mammalian dioxin-response element in a ligand-dependent manner. AhR protein was detected in neural ganglia; smooth, cardiac, and skeletal muscle; and epithelium involved in epithelial-to-mesenchymal transformations, such as pituitary, gastrointestinal tract, limb apical-ectodermal ridge, and kidney collecting ducts. AhR mRNA was detected in all tissues expressing protein, except myocardium. Cytochrome P4501A4 mRNA was highly induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a subset of tissues expressing AhR, including small intestine, liver, kidney, blood vessels, and outflow tract myocardium. In conclusion, the AhR sequence and function is highly conserved between birds and mammals, and although many tissues express AhR during chick embryo development, only a subset are responsive to TCDD induction of CYP1A4.

Demonstration of 2,3,7,8-tetrachlorodibenzo- p-dioxin attenuation of P450 steroidogenic enzyme mRNAs in rat granulosa cell in vitro by competitive reverse transcriptase-polymerase chain reaction assay

We investigated the effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), in prepubertal (PP) and adult (A) rat granulosa cells (GC) in vitro by examining the changes in estrogen secretion, aromatase enzyme activity and mRNAs for steroidogenic enzymes P450scc, 3β-HSDI, P450arom; and for components of the AHR signaling pathway-CYP1A1, aromatic hydrocarbon receptor (AHR), and the AHR nuclear translocator protein (ARNT). In PP and A rat GC, TCDD (3.1 nM) reduced estrogen secretion at 48 h without altering aromatase enzyme activity. Addition of FSH (50 ng/ml) increased aromatase activity in GC with or without TCDD. FSH-induced aromatase activity was significantly reduced by TCDD (3.1 nM) at 48 h. Semi-quantitative RT-PCR showed a significant increase in CYP1A1 mRNA both at 24 and 48 h with TCAP, while a significant reduction in P450scc and P450arom mRNA was observed with competitive RT-PCR. All steroidogenic enzyme mRNAs were significantly lower in adults than in PP GC. We conclude that in rat GC, TCDD modulates the level of cytochrome P450 enzymes involved in the steroid biosynthetic cascade. This effect may be attributable to AHR interaction with dioxin-responsive elements present in the genes encoding these enzymes.

Transcriptional Regulation of the Human CYP1B1 Gene: EVIDENCE FOR INVOLVEMENT OF AN ARYL HYDROCARBON RECEPTOR RESPONSE ELEMENT IN CONSTITUTIVE EXPRESSION

The cytochrome P450 1B1 gene (CYP1B1) is expressed constitutively and is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the human breast adenocarcinoma cell line MCF-7 but not in the human hepatoma cell line HepG2. Genomic DNA isolated from both cell lines was digested with the methylation-sensitive restriction enzyme isoschizomers MspI and HpaII, and subjected to Southern analysis with a probe for the CYP1B1 promoter/enhancer region. Although differences were observed in methylation patterns for the CYP1B1 gene from MCF-7 and HepG2 cells, treatment with the demethylating agent 5-azacytidine (10 microM for 6 days) did not activate CYP1B1 mRNA expression in HepG2 cells. Furthermore, treatment with the histone deacetylase inhibitor trichostatin A (100 nM for 24 h) did not activate CYP1B1 mRNA expression in HepG2 cells. Comparative analysis of the constitutive expression of luciferase/1B1 reporter constructs containing a series of deletions in the 5' enhancer region indicated that in MCF-7 cells the region from -987 to -732 (relative to the transcription start site) was necessary for maximal levels of activity. Mutation of the aryl hydrocarbon receptor response elements (dioxin response elements) in this region showed that the dioxin response elements located at -833 is essential for constitutive gene expression in MCF-7 cells. In HepG2 cells, reporter gene activity was at least equal or greater than the activity observed in MCF-7 cells, which is in marked contrast to the expression of the native CYP1B1 gene. Taken together these findings indicate that the observed cell-specific differences in CYP1B1 constitutive expression are not mediated by DNA promoter/enhancer methylation, but are likely due to either 1) inaccessibility of the 5'-enhancer region in HepG2 cells to transcriptional activators due to a higher order chromatin structure that does not involve histone acetylation, or 2) the action of a repressor protein at cis-elements located outside of the -2296 to +25 region examined with the CYP1B1 reporter constructs. Furthermore, at least one of the dioxin response elements in the enhancer region is required for constitutive expression of CYP1B1.

Mechanisms of inhibitory aryl hydrocarbon receptor-estrogen receptor crosstalk in human breast cancer cells.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that forms a functional heterodimeric complex with the AhR nuclear translocator (Arnt) protein. The environmental toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a high affinity ligand for the AhR and has been extensively used to investigate AhR-mediated biochemical and toxic responses. TCDD modulates several endocrine pathways including inhibition of 17beta-estradiol-induced responses in the immature and ovariectomized rodent uterus and mammary gland and in human breast cancer cell lines. TCDD inhibits formation and growth of mammary tumors in carcinogen-induced rodent models and relatively nontoxic selective AhR modulators (SAhRMs) are being developed for treatment of breast cancer. The mechanisms of inhibitory AhR-estrogen receptor (ER) crosstalk have been investigated in MCF-7 breast cancer cells by analysis of promoter regions of genes induced by E2 and inhibited by TCDD. AhR-mediated inhibition of E2-induced cathepsin D, pS2, c-fos, and heat shock protein 27 gene expression involves direct interaction of the AhR complex with inhibitory pentanucleotide (GCGTG) dioxin responsive elements (iDREs) resulting in disruption of interactions between proteins binding DNA elements required for ER action and the basal transcription machinery. Mechanisms of inhibitory AhR-ER crosstalk indicate that functional iDREs are required for inhibition of some genes; however, results indicate that other interaction pathways are important including AhR-mediated proteasome-dependent degradation of the ER.

Suppressive effects of flavonoids on dioxin toxicity.

Dioxin type chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause a variety of toxicity. Most of the toxicity of TCDD has been attributed to a mechanism by which TCDD is bound to aryl hydrocarbon receptor (AhR) and transforms the receptor. Thus, suppression of the AhR transformation by food factors can suppress the dioxin toxicity. In this study, flavonoids at various concentrations were treated to a rat cytosolic fraction containing AhR before adding 1 nM TCDD. The transformed AhR was detected by an electrophoretic mobility shift assay with a DNA oligonucleotide consensus to dioxin response element. As the results, flavones and flavonols at dietary levels act as the antagonists for AhR and suppress the transformation. The antagonistic IC50 values were in a range between 0.14 and 10 microM, which are close to the physiological levels in human. These results suggest that a plant-based diet can prevent the dioxin toxicity.