Flavone antagonists bind competitively with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor but inhibit nuclear uptake and transformation.

Previous analyses suggested that potent aryl hydrocarbon receptor (AhR) antagonists were planar, with a lateral electron-rich center. To further define structural requirements and mechanism for antagonism, ten additional flavone derivatives were synthesized. Based on their ability to 1) compete with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) for binding to the AhR; 2) inhibit TCDD-elicited binding of AhR to dioxin-responsive elements (DRE) in vitro; and 3) inhibit TCDD-induced transcription of DRE-dependent luciferase in stably transfected hepatoma cells, the most potent flavones contained a 3'-methoxy group and a 4'-substituent having one or more terminal atoms of high electron density (-N3, -NO2, or -NCS). Furthermore, these had low agonist activity as assessed by their inability to elicit AhR. DRE binding or to induce luciferase. Compounds containing bulkier 3' or 4'-substituents, or a 3'-OH group were less potent antagonists, and some were partial agonists. In rat liver cytosol, 3'-methoxy-4'-azido- and 3'-methoxy-4'-nitroflavones bound competitively (with TCDD) to the AhR, indicating that they bind to the TCDD-binding site. When hepatoma cells were exposed to these flavones, AhR complexes were primarily immunoprecipitable from the cytosol and contained 90 kDa heat shock protein. In contrast, AhR in TCDD-treated cells was primarily immunoprecipitated from nuclear extracts and was associated with Arnt but not 90 kDa heat shock protein. Immunocytofluorescence analysis in intact cells further indicated that the potent antagonist inhibited nuclear uptake of AhR and blocked TCDD-dependent down-regulation of AhR. Together, these data indicate that the most potent antagonists bind the AhR with high affinity but cannot initiate receptor transformation and nuclear localization.

Analysis of structural requirements for Ah receptor antagonist activity: ellipticines, flavones, and related compounds.

A number of studies have examined the structure-activity relationships for the agonist activity of Ah receptor (AhR) ligands. Fewer studies have considered the structural basis for potential antagonist properties. Certain ellipticine derivatives have been reported to bind to the AhR and inhibit the ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to transform the AhR to a form that recognizes a dioxin-responsive enhancer element (DRE) upstream of the cytochrome P4501A1 gene. In the present study, over 30 ellipticine derivatives and structurally related compounds were examined for their ability to bind to the AhR, activate it to a DRE-binding form, induce the luciferase gene under control of a DRE-containing enhancer, and block activation of the AhR by TCDD. The ability of several ellipticine derivatives to inhibit TCDD-elicited DRE binding and TCDD-induced luciferase activity was inversely related to their ability to alone stimulate these responses. The most potent antagonist activity was related to good AhR binding characteristics in terms of conforming to previously predicted 14 x 12 x 5 A van der Waals dimensions and the presence of an electron-rich ring nitrogen at or near a relatively unsubstituted X-axis terminal position. Based on these data, a number of flavone derivatives were synthesized and tested for their relative agonist/antagonist activity. These additional data were consistent with the hypothesis that an electron-rich center near or along a lateral position of the van der Waals binding cavity is a characteristic that enhances AhR antagonist activity.

Weanling female Sprague-Dawley rats are not sensitive to the antiestrogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Investigators have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can inhibit certain estrogenic events in vivo and in vitro. To further investigate this phenomenon, the effects of estradiol (E2) alone or TCDD plus estradiol on several estrogen-dependent parameters were evaluated in weanling female Sprague-Dawley rats. E2 (10 micrograms/kg/day, Postnatal Days (PND) 21 and 22) caused significant increases in relative uterine weight and keratinization of the vaginal epithelium (PND 23). E2 significantly reduced uterine estrogen receptor (ER) protein levels and serum FSH levels, with a trend toward reduction of ER mRNA levels. None of these parameters were affected by pretreatment with 20, 40, or 80 micrograms/kg TCDD (PND 19). Uterine progesterone receptor levels were not affected by E2 or TCDD in the present study. In contrast, TCDD significantly decreased body weight (40 or 80 micrograms/kg) by PND 21, significantly decreased relative thymic weights, and significantly increased relative hepatic weights (20, 40, and 80 micrograms/kg, by PND 23). In addition, TCDD dramatically induced CYPIA1 hepatic mRNA levels, indicating that TCDD was properly delivered and could mediate other well-documented Ah receptor-dependent events. Thus, weanling female Sprague-Dawley rats are not sensitive to the antiestrogenic effects of TCDD at doses which cause overt toxicity. The results provide evidence that the previously reported antiestrogenic effects of TCDD are probably species, strain, and age dependent.

Purification to homogeneity of the heteromeric DNA-binding form of the aryl hydrocarbon receptor from rat liver.

The aryl hydrocarbon receptor (AhR) is a transcriptional enhancer that is activated by the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related toxic xenobiotics, as well as some naturally occurring compounds. Ligand binding initiates 1) dissociation of the ligand-bound monomeric AhR from the ligand-unoccupied multimeric complex and 2) biochemical and/or conformational changes that enable association of the ligand-bound monomer with other proteins. This heteromeric complex has high affinity for specific elements [dioxin-responsive elements (DREs)] in the regulatory regions of a number of structural genes, the induction and/or repression of which may be a mechanism of toxicity of TCDD. We have developed a relatively simple and rapid procedure that enables purification to homogeneity of a TCDD-bound receptor complex. The final step of purification is based on binding to an oligonucleotide containing the specific DRE sequence that is found in the upstream region of the CYP1A1 structural gene. The purified complex retains in vitro DRE-binding function. Silver staining and Western blot analyses demonstrate that the complex consists of the AhR ligand-binding monomer of approximately 104 kDa, plus two proteins (94 and 96 kDa) that are recognized by antibodies prepared against the AhR nuclear translocator protein. Previous attempts to purify a DRE-binding form of the AhR were unsuccessful because of dissociation of the complex during chromatography; this is the first report of an isolated functional complex. The purified preparation will be valuable in further studies of receptor regulation and function.

Alpha-naphthoflavone acts as an antagonist of 2,3,7, 8-tetrachlorodibenzo-p-dioxin by forming an inactive complex with the Ah receptor.

alpha-Naphthoflavone (ANF) has previously been shown to compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for binding to the Ah receptor under conditions in vitro. However, ANF also prevents TCDD-elicited cytochrome P450lA1 induction, immunosuppression, and down-regulation of the estrogen receptor in vivo and within intact isolated cells. These data suggest that ANF is a TCDD antagonist. This study investigated the ability of ANF to transform the Ah receptor contained in rat hepatic cytosol or mouse hepatoma cells to a form that recognizes the dioxin-responsive enhancer element (DRE) upstream of the cytochrome P450lA1 gene. Gel retardation analysis indicated that TCDD- or beta-naphthoflavone (BNF)-bound receptor was able to bind to the DRE, whereas essentially no receptor-DRE complexes were observed using cytosol incubated with ANF concentrations as high as 1000 nM. Furthermore, an excess of ANF, when added to cytosol just before TCDD, blocked, in a concentration-dependent manner, the ability of TCDD to transform the receptor to a form that bound to the DRE. These studies indicated that ANF binds to the receptor and confers on it a conformation that cannot recognize the DNA recognition sequence contained in the DRE. Although an excess of the agonist 2,3,7,8-tetrachlorodibenzofuran (TCDF) readily reversed the inhibitory actions of ANF, ANF was unable to reverse the effects of TCDD, TCDF, or BNF on the receptor. These studies suggested that TCDD binding, unlike that of ANF, results in a receptor conformation that has higher affinity for the ligand. Treatment of mouse hepatoma Hepa 1c1c7 cells with TCDD or BNF resulted in receptor contained in nuclear extracts that bound to the DRE. Only a very minor ligand-dependent protein-DNA complex was detected when cells were treated with ANF. These data indicated that ANF acts as an antagonist of TCDD by directly binding to the Ah receptor and eliciting a protein conformation that has very low affinity for DNA.

Characterization of multiple forms of the Ah receptor: comparison of species and tissues.

Biochemical and toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) appear to be mediated via the Ah receptor, a gene-regulatory protein that, like steroid hormone receptors, undergoes a ligand-dependent acquisition of affinity for nuclei and DNA. Since responses to TCDD are highly species- and tissue-specific, we compared DNA-binding properties of Ah receptor from several tissues of rat, C57BL/6 mouse, hamster, and guinea pig, using DNA-Sepharose chromatography. Hepatic cytosol from all species contained TCDD.receptor complexes that eluted at approximately 0.15 (peak 1) and approximately 0.33 M NaCl (peak 2). The relative proportions of these forms as well as of TCDD-receptor that did not bind to DNA (i.e., was present in flowthrough fractions) varied among species. In each case, the yield of the higher affinity form (peak 2) increased with time or temperature of incubation. Cytosol from lung, thymus, kidney, and testis contained the same two forms; peak 2 was the major DNA-binding form only in thymus. In KCl extracts of hepatic nuclei from animals treated with [3H]TCDD, only the higher affinity form (peak 2) was found. Peak 1 isolated from cytosol by DNA-Sepharose and incubated with hepatic cytosol from D2 mouse (which contains no detectable receptor) transformed into peak 2, suggesting that these two forms are different conformations of the same protein. Sucrose density gradient and gel filtration analyses of peaks 1 and 2 isolated from DNA-Sepharose indicated that (i) the untransformed form (peak 1) was smaller than the unoccupied and the transformed forms, (ii) 0.4 M KCl in the density gradients had little effect on these isolated forms, and (iii) nuclear receptor sedimented like peak 2. On the basis of these results, we hypothesize that the Ah receptor exists in several forms: When occupied, it has no affinity for DNA. Ligand binding initially yields a smaller form with low DNA affinity (i.e., peak 1), as well as, in some cases, a form with no DNA affinity (flowthrough fractions); further incubation in the presence of cytosolic factor(s) induces a change conferring higher DNA affinity and faster sedimentation (i.e., peak 2). The latter form is likely the transcriptionally active form in vivo. Species and tissue differences in this scheme are quantitative rather than qualitative.

In vivo kinetics and DNA-binding properties of the Ah receptor in the golden Syrian hamster.

The in vivo long-term cytosolic-nuclear kinetics and DNA-binding properties of the Ah receptor were examined in liver from the golden Syrian hamster. For the kinetic studies, a dose of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) that has been previously shown to produce maximal and sustained hepatic enzyme induction without substantial toxicity was used. Following an intraperitoneal dose of 10 micrograms/kg of [3H]TCDD, occupied cytosolic receptor levels reached a peak within 8 h and then decreased rapidly to a level that was approximately 2% of the total receptor. Throughout the 35-day period, unoccupied cytosolic receptor represented from 65 to 80% of the total receptor content. At 8 h following dosing, less than 30% of the total amount of receptor was associated with the nuclear fraction; this percentage declined slowly to less than 5% of the total at Day 35. The half-life for the decline in detectable nuclear receptor levels was 13 days and was similar to the half-life for the decline in [3H]TCDD content of the whole liver, cytosol, and nuclear extract. The Ah receptor contained in hamster hepatic cytosol underwent a ligand-dependent transformation in vitro to two forms having affinity for DNA-Sepharose, one of which was isolated from nuclei of animals treated with [3H]TCDD in vivo. A comparison of the specific binding recovered following various analytical procedures revealed that the binding of [3H]TCDD to the form not found in nuclear extracts was more labile under certain experimental conditions. These studies indicate the heterogeneity of the Ah receptor in hamster hepatic cytosol and suggest that DNA binding in vitro and nuclear uptake in vivo occur through a ligand-dependent transformation process. The maintenance of maximal hepatic enzyme induction is, in part, a consequence of the sustained presence in the nucleus of only a small percentage of the total receptor content. The whole-tissue kinetics of TCDD appears to be a major factor regulating the long-term retention of the TCDD-receptor complex in the nucleus.

Changes in hamster hepatic cytochrome P-450, ethoxycoumarin O-deethylase, and reduced NAD(P): menadione oxidoreductase following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Partial dissociation of temporal and dose-response relationships from elicited toxicity.

The temporal and dose-related characteristics of hepatic enzymes induced in the hamster by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were examined. Male Syrian golden hamsters received a single intraperitoneal injection of TCDD at a dose of 0-500 micrograms/kg. At various times up to 35 days, a number of variables were determined and compared: whole body, liver, and thymus weights; hepatic concentrations of cytochrome P-450 (P-450); and activities of 7-ethoxycoumarin O-deethylase (ECOD) and reduced NAD(P): menadione oxidoreductase (NMOR). Increased liver weights and decreased thymus weights were observed to be dose related. At day 7 following treatment, the approximate ED50 values for these responses were 15 and 100 micrograms/kg respectively. The ED50 values for the increase in hepatic P-450 concentrations and activities of ECOD and NMOR ranged from 0.5 to 2.0 micrograms/kg. At 10 and 500 micrograms/kg, NMOR activity remained maximally induced for up to 35 days. This was also the case for P-450 and ECOD activity at a dose of 10 micrograms/kg. At 500 micrograms/kg, both P-450 and ECOD demonstrated an induction up to day 4 followed by a decrease to near control levels by day 14. This decrease appeared to correlate with changes in hepatic morphology. These results demonstrate a dissociation of the induction of these hepatic enzymes from TCDD-induced lethality, in this species.

Cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Evidence for a homologous nature among various mammalian species.

The presence and properties of the Ah receptor were examined in the guinea pig, rat, hamster, monkey, and three different strains of mice. These species and strains have demonstrated differences in sensitivity and variability of response to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. All species examined, with the exception of DBA/2J mice, possess similar amounts of binding protein with high affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin in hepatic tissue. Numerous dibenzo-p-dioxin congeners and polycyclic aromatic hydrocarbons demonstrated a similar rank order ability to bind to receptor molecules from these species. When analyzed by gel-exclusion high-performance liquid chromatography, hepatic cytosolic receptors from all species eluted at volumes corresponding to a similar molecular weight range. Association of the hepatic Ah receptor with the nuclear fraction was observed in all cases following the i.p. treatment of guinea pig, rat, C57BL/6J mouse, or hamster with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. In all species and tissues examined, with the exception of hamster duodenum and thymus, the highest concentrations of receptor were localized in the liver, lung, thymus, intestine, and kidney. Exceptionally high concentrations of receptors were also observed in guinea pig testes. These findings indicate that, despite species and tissue specific differences in the biochemical and toxicological responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds, a number of different mammalian species possess Ah receptors with similar properties. Thus, the correlative differences between certain strains of mice in terms of altered specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and sensitivity to this compound may be unique and not necessarily applicable to other species. Although all data indicate that the receptor mediates these responses, it appears that species- and tissue-specific differences may be determined by a number of additional factors. These results also suggest the conservation of some, as yet unknown, functional role of the receptor molecule.

Examination and rapid analysis of hepatic cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using gel-permeation high performance liquid chromatography.

Gel-permeation high performance liquid chromatography was used to examine the molecular characteristics of the cytosolic and nuclear receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin. The physical properties for this receptor previously established by sucrose-density gradient analysis have been maintained by the technique. The high performance liquid chromatography method offers the advantages of rapid separation and reproducibility thus minimizing possible effects of proteolytic enzymes or dissociation of the ligand-receptor complex. The specific binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin as measured by this system also corresponds quantitatively to that determined by the hydroxyapatite method. These methods will facilitate a further understanding of the biochemical actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds, as well as an understanding of the physiological function of this receptor molecule.

Ontogeny of the cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver, lung, and thymus.

The ontogeny of the cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin was studied in Sprague-Dawley rats by quantitation of the receptor in hepatic, lung, and thymic cytosol. Concentrations of hepatic and lung cytosolic receptors increased rapidly after birth and remained at the highest levels from days 2 to 21. After this time, receptor levels in these tissues slowly declined with age. In the thymus, cytosolic receptor concentrations remained high from days 2 to 42 following birth. In these tissues and at all times examined, the receptors demonstrated very high affinities for [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. From days 15 to 42 following birth, no consistent sex related differences in receptor content or affinity were observed in any of these tissues.

Distribution, excretion, and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin in C57BL/6J, DBA/2J, and B6D2F1/J mice.

The strains of mice, C57BL/6J, DBA/2J, and B6D2F1/J, have been used as models to study the mechanism of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The distribution, excretion, and metabolism of this compound was studied in male C57BL/6J, DBA/2J, and B6D2F1/J mice following the intraperitoneal administration of radiolabeled TCDD at a dose of 10 micrograms/kg. In all strains, the liver and adipose tissue were the major sites for the accumulation of 3H-TCDD, with more 3H-TCDD being distributed to the livers of the C57BL/6J and B6D2F1/J strains as compared to the DBA/2J strain. While in all strains the feces were the major route of elimination, the total amount of 3H-TCDD-derived radioactivity excreted in the feces amounted to approximately 72% of the original dose in the C57BL/6J and B6D2F1/J strains whereas this was only 54% in the DBA/2J strain. The half-lives for the cumulative excretion of radioactivity in the feces were similar in all strains. The half-life for the excretion of radioactivity in the urine was considerably greater in the DBA/2J strain as compared to the C57BL/6J and B6D2F1/J strains. The estimated half-lives for the total cumulative excretion of 3H-TCDD-derived radioactivity by all routes was 11.0, 24.4, and 12.6 days for the C57BL/6J, DBA/2J, and B6D2F1/J strains, respectively. Greater than 85% of the total radioactivity excreted in urine, bile, and feces from all three mouse strains was present as metabolites of TCDD.