Ah Receptor Complex Research Articles

Human ovarian cancer cells: interaction with Tetrachlorodibenzo- p-dioxin (TCDD)

Langdon and coworkers (1990) have characterized a series of estrogen receptor (ER)-positive and ER-negative human ovarian carcinoma cell lines. Previous studies have demonstrated that TCDD exhibits a broad spectrum of antiestrogenic activities in ER-positive human breast cancer cell lines. Studies in this laboratory have shown that after treatment with 10 nM [ 3H]TCDD, specifically-bound aryl hydrocarbon (Ah) receptor complexes were identified in the cytosolic and nuclear fractions of the ER-positive PE01, PE04 and PE06 human ovarian carcinoma cell lines. Preliminary studies indicate that the molecular properties of the nuclear and cytosolic Ah receptor complexes in the ovarian carcinoma cells were similar to those reported for the Ah receptor from other human tissues and cells. The effects of TCDD on cytochrome P-4501A1 and the growth of these cell lines will be discussed.

Characterization of the human Ah receptor

Using [ 3 H]2,3,7,8- tetrachlorodibenzo-p- dioxin ([ 3H]TCDD) or 7-[ 125 I]- iodo-2,3- dibromodibenzo-p- dioxin ([ 125I]DBDD) as high affinity photoaffinity ligands, the photoaffinity-labeled nuclear Ah receptor complexes from human cell lines have been analyzed by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results show that the apparent molecular mass for the Ah receptor from several of these cell lines was 110-kDa and no significant variations were observed. However, there were significant differences in the apparent molecular weights of the nuclear Ah receptor complexes. For example, in the estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) human breast cancer cell lines, the apparent molecular weights of the nuclear receptor complexes were 171,000 and 192,000, respectively. These results indicate that there are differences in the molecular weights of the human nuclear Ah receptor complexes and the intraspecies heterogeneity may be associated with the non-ligand binding protein and not the Ah receptor.

Effects of ligand structure on the in vitro transformation of the rat cytosolic aryl hydrocarbon receptor

Incubation of radiolabeled 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF),1,2,3,7,8-pentachlorodibenzo- p-dioxin(PeCDD), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF), 1,2,7,8-TCDF, and 2,3,7-trichlorodibenzo- p-dioxin (TrCDD) with rat hepatic cytosol for 2 h at 0 °C gave liganded aryl hydrocarbon (Ah) receptor complexes which were indistinguishable as determined by velocity sedimentation analysis and DNA-Sepharose column chromatography. Incubation of the cytosol plus the different radioligands for 2 h at 20 °C resulted in the formation of Ah receptor complexes which exhibited increased retention times on DNA-Sepharose columns. It was apparent that the amount of specifically bound Ah receptor complex or the levels of the transformed Ah receptor complex which eluted from the column with 0.2–0.3 m salt were dependent on the structure of the radioligand. For example, after incubation for 2 h at 20 °C the overall yields of the specifically bound transformed Ah receptor complex were 3.4, 2.0, 1.2, 1.9, 0.3, and 0.1%, respectively, using 2,3,7,8-TCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDD, 1,2,3,7,8-PeCDF, 1,2,7,8-TCDF, and 2,3,7-TrCDD as radioligands. A more quantitative measure of the structure-dependent transformation of the liganded cytosolic Ah receptor complex was determined using a gel retardation assay with a consensus synthetic dioxin-responsive element (DRE) (26-mer, duplex). The EC 50 values obtained for the concentration-dependent formation of the retarded DRE-Ah receptor complex using 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 2,3,7-TrCDD, and 1,2,7,8-TCDF as ligands were 0.26, 0.35, 0.78, 1.75, 27.0, and 220 n m, respectively. The structure-dependent differences in these values were similar to their different potencies

Characterization of the aryl hydrocarbon receptor in the human C-4II cervical squamous carcinoma cell line

Treatment of C-4II human cervical squamous carcinoma cells with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) gave a concentration-dependent increase in ethoxyresorufin O-deethylase (EROD) activity. The EC 50 for this response was approximately 1 nM and the maximum induced activity was 27 pmol/min/mg protein. The molecular properties of the cytosolic and nuclear aryl hydrocarbon (Ah) receptor complex were determined by velocity sedimentation analysis, photoaffinity labeling, gel retardation using a consensus dioxin responsive element (DRE), and DNA-Sepharose, DRE-Sepharose and Sephaeryl S-300 gel permeation column chromatography. The apparent molecular masses of the cytosolic and nuclear photoaffinity-labeled Ah receptor complexes were 110kDa and differed from the corresponding values obtained for the Ah receptor from other animal species. In contrast, most of the other molecular properties of the Ah receptor were not significantly different from those previously reported for other species. The relative Ah-responsiveness of the C-4II cells was assessed by determining the ratio of the induced EROD activity/nuclear Ah receptor levels for a submaximal inducing dose of [ 3H]TCDD. The induced activity/binding ratio for the human C-4II cells was 0.77 and was at least one order of magnitude lower than the corresponding value for the Ah-responsive rat hepatoma H-4-II E cells.

Heterogeneity of rat hepatic Ah receptor: identification of two receptor forms which differ in their biochemical properties.

In cytosol, the rat hepatic Ah receptor (AhR) appears to exist in two distinct forms (AhR alpha, AhR beta) in similar concentration. The binding of ligand (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) to AhR alpha requires the receptor be in its oligomeric 8-10 to S conformation (bound to other protein subunits), while ligand binding to AhR beta can occur with the dissociated 5-6 S form. Occupancy of AhR alpha by ligand (TCDD) protects it from salt-dependent inactivation; AhR beta is not inactivated by high salt conditions. The addition of molybdate to cytosol during tissue homogenization stabilized AhR alpha against salt-dependent inactivation and subunit dissociation but did not prevent dissociation of AhR beta by high salt. Although the presence of molybdate appears to stabilize AhR alpha in its oligomeric 8-10 S, it had no significant effect on the overall amount of TCDD:AhR complex which bound to its specific DNA recognition site, the dioxin responsive element (DRE). These results suggest that AhR alpha, unlike AhR beta, is either unable to transform or bind to the DRE with high affinity.

Mechanism of benzo[ a]pyrene-induced Cyp1a-1 gene expression in mouse Hepa 1c1c7 cells: Role of the nuclear 6 s and 4 s proteins

Treatment of wild-type (wt) aryl hydrocarbon (Ah)-responsive mouse Hepa 1c1c7 cells with benzo[ a]pyrene (B[ a]P) caused a concentration-dependent induction of ethoxyresorufin O-deethylase (EROD) activity. In contrast, B[ a]P was inactive as an inducer in Ah nonresponsive class 1 and class 2 mutant cell lines. In parallel experiments, the nuclear fractions from wt cells treated with 10 −7 m [ 3H]B[ a]P contained both the 4 s carcinogen binding protein and the 6 s (Ah receptor) complexes, whereas only the 4 s complex was present in the nuclear fraction of the class 2 mutant cells. The results obtained from cotreatment of wt Hepa 1c1c7 cells with 10 −6 or 10 −7 m B[ a]P and 5 × 10 −7 or 10 −7 m 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) showed that MCDF inhibited the induction of EROD activity and Cyp1a-1 mRNA levels by B[ a]P. Moreover, using 10 −7 m [ 3H]B[ a]P and unlabeled MCDF, it was shown that MCDF not only inhibited the induction response but also caused a concentration-dependent decrease in levels of the nuclear 6 s complex but not the 4 s complex. In contrast, in situ competition studies with unlabeled 10 −6 m] benzo[ ghi]-perylene (B [ ghi]P) resulted in the elimination of the nuclear [ 3H]B[ a]P 4 s complex (but not the 6 s complex); however, the EROD activity and Cyp1a-1 mRNA levels in cells treated with 10 −7 m B[ a]P in the presence or absence of 10 −6 m B[ ghi]P were not significantly different. These results indicate that the 4 s binding protein is not required for the induction of Cyp1a-1 gene expression in Hepa 1c1c7 cells and suggest that B[ a]P and 2,3,7,8-tetrachlorodibenzo- p-dioxin induce Cyp1a-1 gene expression via a common mechanism which involves binding to the Ah receptor.

DNA binding properties of the Ah receptor in wild-type and variant mouse hepatoma cells

Mouse hepatoma Hepa 1c1c7 cells and nonresponsive mutants have been extensively used as models for investigating the molecular mechanism of induction of CYP1A1 gene transcription by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and related compounds. Incubation of cytosolic [ 3H]TCDD-aryl hydrocarbon (Ah) receptor from wild-type Hepa 1c1c7 cells for 16 h at 4 °C in 0.4 m KCl resulted in the formation of transformed liganded receptor which exhibited increased binding affinity on DNA-Sepharose columns. The elution properties of the peak with the highest DNA binding affinity were similar to the elution profiles of the nuclear receptor complex isolated from wild-type cells. TAOc1BP rcl (class I) nonresponsive mutant cells were characterized by relatively low levels of the cytosolic and nuclear Ah receptor complex. The BP rcl (class II) variant cell line contained levels of cytosolic receptor which were comparable to those observed in the wild-type cells; however, significantly reduced levels of nuclear receptor complex were observed in the class II variant cell line. Incubation of the nuclear or transformed liganded cytosolic Ah receptor from wild-type cells with a consensus 32P-labeled dioxin responsive element (DRE) in a gel shift assay gave a retarded band associated with the receptor-DRE complex. Incubation of the cytosolic receptor complex from the class I and II mutant cells for 16 h at 4 °C in 0.4 m KCl or for 2 h at 20 °C did not yield complexes with increasing binding affinities on DNA-Sepharose columns. Moreover, incubation of these complexes with 32P-labeled DRE did not give a retarded band in a gel shift assay. However, coincubation of the liganded class II mutant cytosol with cytosol from class I cells resulted in transformation of the liganded receptor and this was confirmed in both the DNA-Sepharose and gel retardation assays. These results suggest that the failure of class II mutant cells to respond to TCDD is due to a defect in the factors responsible for transformation of the cytosolic receptor complex.

Evidence for the mechanism of action of the 2,3,7,8-tetrachlorodibenzo- p-dioxin-mediated decrease of nuclear estrogen receptor levels in wild-type and mutant mouse hepa 1c1c7 cells

Treatment of wild-type Hepa 1c1c7 cells with 1 nM [ 3H]-17β-estradiol resulted in the rapid accumulation of the nuclear estrogen receptor complex whose levels were maximized within 1 hr. Cotreatment of the cells with 10 nM 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and [ 3H]-17β-estradiol did not affect the nuclear estrogen receptor levels 1 hr after addition of the radioligand; however, pretreatment of the cells for 1, 6, 24 or 42 hr with 10 nM TCDD prior to the addition of the radiolabeled hormone caused a greater than 50% decrease in nuclear estrogen receptor levels (determined by velocity sedimentation analysis) 1 hr after the addition of [ 3H]-17β-estradiol. In parallel experiments in which 10 nM TCDD was added 6 hr prior to the radiolabeled hormone, TCDD caused a 63 and 74% decrease in immunodetectable cytosolic and nuclear estrogen receptor protein levels, respectively, in the wildtype Hepa 1c1c7 cells. The nuclear estrogen receptor was also detected in two Hepa 1c1c7 mutant (class 1 and class 2) cell lines which have been characterized previously as TCDD non-responsive due to either decreased aryl hydrocarbon ( Ah) receptor levels or a defect in the accumulation of transcriptionally active nuclear Ah receptor complexes, respectively. Treatment of these mutant cell lines with TCDD and [ 3H]-17β-estradiol (as described above) caused only a minimum (class 1) or non-detectable (class 2) decrease in nuclear estrogen receptor binding activity or immunodetectable protein levels. These results, coupled with the structure-dependent differences in the activities of TCDD (a strong Ah receptor agonist) and 2,8-dichlorodibenzo- p-dioxin (a weak Ah receptor agonist) in this assay system, support a role for the Ah receptor in the TCDD-mediated decrease of the nuclear estrogen receptor in mouse Hepa 1c1c7 cells. In addition, actinomycin D and cycloheximide both inhibited the TCDD-mediated decrease of nuclear estrogen receptor levels in the Hepa 1c1c7 wild-type cells, and these results suggest that TCDD may induce specific gene products which are involved in this process.

In situ and in vitro photoaffinity labeling of the nuclear aryl hydrocarbon receptor from transformed rodent and human cell lines

The photoaffinity labeling of the nuclear aryl hydrocarbon (Ah) receptor from mouse Hepa 1c1c7, rat hepatoma H-4-II E, and human liver Hep G2 cells was investigated using two high affinity ligands, namely 2,3,7,8-[ 3H]tetrachlorodibenzo- p-dioxin (TCDD) and 7-[ 125I] iodo-2,3-dibenzo- p-dioxin ([ 125I]DBDD). Irradiation of nuclear [ 3H]TCDD-Ah receptor complexes from the three cell lines for 5 min gave 47, 38, and 62% yields of trichloroacetic acid-precipitable photoadducts from the Hepa 1c1c7, H-4-II E, and Hep G2 cell lines, respectively; denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation followed by autoradiography gave one major Ah receptor photoadduct for each cell line with apparent molecular masses at 97, 100, and 110 kDa, respectively. [ 125I]DBDD could also be used as a photoaffinity label for the nuclear Ah receptor from the three cell lines; although the maximum net yield of photoaffinity labeled nuclear Ah receptor from the rodent nuclear Ah receptor preparations was relatively low (0.5–2.5%), a >15% yield of photoadduct was obtained from the human Hep G2 cells. Both [ 3H]TCDD and [ 125I]DBDD were utilized to photoaffinity label the nuclear Ah receptor in Hepa 1c1c7 cells in suspension and the net yield of photoadducts with these ligands was 94.6 and 3.0%, respectively. The cytosolic Ah receptor from the three cell lines was photolabeled with [ 125I]DBDD and the net yield of photoadducts varied from 3.3 to 14.7%. The functional activity of the photoaffinity-labeled nuclear TCDD-Ah receptor complexes from the cell lines was also determined by comparing relative binding affinities of the photolyzed and unphotolyzed complexes with a synthetic dioxin-responsive element (DRE) using a gel retardation assay. The photolyzed and unphotolyzed complexes from the three cell lines all bound with the DRE in the gel shift assay; however, the gel mobilities of the rodent and human nuclear receptor-DRE complexes were different. Quantitative analysis of the DRE binding showed that there were no significant differences between the photolyzed and unphotolyzed nuclear receptor complexes from the rodent cells, whereas there was a significant 27% decrease in the DRE binding of the photolyzed versus the unphotolyzed nuclear receptor complex from the human Hep G2 cells. These studies demonstrate the utility of [ 3H]TCDD and [ 125I]DBDD as photoaffinity labels for the Ah receptor and illustrate the structural and photochemical differences between the rodent and the human nuclear Ah receptor complexes.

Characterization of the interaction of transformed rat hepatic cytosolic Ah receptor with a dioxin responsive transcriptional enhancer

Many of the biological and toxic effects of 2,3,7,8-tet-rachlorodibenzo- p-dioxin (TCDD, dioxin), a highly toxic environmental contaminant, are mediated by a soluble intracellular protein (the Ah receptor (AhR)). Following a poorly defined process of transformation, during which the TCDD:AhR complex acquires the ability to bind to DNA with high affinity, TCDD:AhR complexes activate gene transcription by binding to dioxin responsive enhancers (DREs) adjacent to the responsive gene. Here we have utilized gel retardation analysis to study the interaction of rat hepatic cytosolic TCDD:AhR complexes, transformed in vitro, with dioxin responsive enhancer DNA. Cytosol contains a protein(s) that binds to the DRE in a TCDD-inducible, sequence-specific, time- and temperature-dependent manner and exhibits AhR ligand binding specificity. These results imply that this inducible protein—DNA complex represents the binding of liganded: AhR complex to the DRE. The TCDD:AhR complex bound to the DRE with an equilibrium dissociation constant of 1.2 ± 0.1 n m, an affinity at least 3800-fold stronger than that for binding to nonspecific DNA. Assuming one DNA binding site per AhR molecule, the total concentration of transformed AhR in these studies was approximately 56.1 ± 6.6 fmol/mg protein (representing transformation of 45% of the total amount of AhR present in the same cytosolic preparations). Inhibition of AhR transformation, but not ligand or DNA binding, by EDTA and EGTA suggests that a chelatable divalent cation(s) may play a critical role in the transformation process.

Lightly chlorinated ortho-substituted PCB congeners decrease dopamine in nonhuman primate brain and in tissue culture

Exposure of the nonhuman primate, Macaca nemestrina, to Aroclor 1016, a commercial mixture of 26 lightly chlorinated PCB congeners, decreased dopamine concentrations in the caudate, putamen, substantia nigra, and hypothalamus. Only three ortho-substituted nonplanar PCB congeners (2,4,4′, 2,4,2′,4′, and 2,5,2′,5′) were detected in these brain regions, suggesting that these congeners may be responsible for the observed decreases in dopamine. The ability of these and other PCB congeners to alter dopamine function was tested directly by applying them to dopamine-synthesizing cells in culture, PC-12 pheochromocytoma cells. In vitro testing demonstrated that these three congeners reduced cellular dopamine concentrations while planar, dioxin-like congeners, e.g., 3,4,3′,4′ and 3,4,5,3′,4′, did not. Thus, these ortho-substituted nonplanar congeners may be directly responsible for the observed changes in in vivo neurochemistry. Furthermore, these results suggest that the observed decreases in both in vivo and in vitro dopamine concentrations may occur through a novel mechanism and not through the Ah-receptor complex thought to mediate immunotoxic and hepatotoxic changes following exposure to dioxin and dioxin-like PCBs.

The hepatic Ah receptor for 2,3,7,8-Tetrachlorodibenzo- p-dioxin: Species differences in subunit dissociation

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD, dioxin) produces many of its biological effects by binding to a soluble, intracellar protein (the Ah receptor (AhR)). The hepatic AhR, from a variety of species, is present in low salt cytosol as a form which sediments at 8–10 S. High salt (0.4 m KCL) dissociates the rat, guinea pig, and rabbit cytosolic TCDD:AhR complex to a form which sediments at 5–6 S. In contrast, high salt conditions failed to dissociate the 8–10 S TCDD:AhR complex present in any of the mouse strains studied. Incubation of cytosol with heparin resulted in a shift of the [ 3H]TCDD:AhR complex to a smaller sedimenting form in all species. Mouse TCDD:AhR complex sedimented at 8–10 S when cytosol was simultaneously incubated with high salt and heparin, indicating that the interaction of heparin with the AhR was electrostatic in nature. Incubation of heparin-dissociated mouse TCDD: AhR complex (5–6 S) with high salt resulted in reassociation of AhR to a form which sediments at 8–10 S. Our data suggests that the resistance of mouse AhR to salt-mediated dissociation may be due to a property of the receptor protein itself and also indicates that mouse hepatic cytosolic AhR is distinctly different from that present in all other species examined to date.

Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2

The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 ± 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [ 3H]TCDD and 3-[ 3H]methylcholanthrene. 3-[ 3H]Methylcholanthrene did not bind to any component besides that at ∼9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [ 3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [ 3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant ( K d ) for binding of [ 3H]TCDD to Hep G2 Ah receptor was 9 n m by Woolf plot analysis, about an order of magnitude weaker than the affinity of [ 3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [ 3H]TCDD·Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [ 3H]TCDD at 37 °C in culture, sedimented at ∼6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC 50 for AHH induction was 5.3 /gm m for benz( a)anthracene and 1.3 μ m for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 m m sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drugmetabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.

The binding of transformed aromatic hydrocarbon (Ah) receptor to its DNA recognition site is not affected by metal depletion

The biological effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD, dioxin), a potent environmental contaminant, are mediated by a soluble intracellular protein, the aromatic hydrocarbon (Ah) receptor (AhR). TCDD:AhR complexes activate gene transcription by binding to specific DNA sequences termed dioxin-responsive elements adjacent to TCDD-responsive genes. Analogies between the AhR and receptors for steroid hormones imply similarities in their mechanism of action. The presence of chelatable, protein-bound metal(s), presumably zinc, is required for DNA binding of several proteins, including steroid hormone receptors and the transcription factor SP1. Utilizing gel retardation and DNA-cellulose binding assays we have investigated the importance of metal in DNA binding of transformed TCDD:AhR complexes. Here, we report that although 1,10-phenanthroline, a metal ion chelating agent, inhibited the DNA binding of SP1 and transformed glucocorticoid receptor, no inhibition of transformed AhR was observed. EDTA was similarly ineffective in inhibiting DNA binding of transformed AhR. Our findings suggest that the AhR, although similar to steroid receptors, appears not to require metals for binding to its specific DNA recognition sequence.

Oxidative stress induced by TCDD is mediated by the Ah-receptor complex

We describe a family of seven boys affected by Lesch–Nyhan disease with various phenotypes. Further investigations revealed a mutation c.203T > C in the gene encoding HGprt of all members, with substitution of leucine to proline at residue 68 (p.Leu68Pro). Thus patients from this family display a wide variety of symptoms although sharing the same mutation. Mutant HGprt enzyme was prepared by site-directed mutagenesis and the kinetics of the enzyme revealed that the catalytic activity of the mutant was reduced, in association with marked reductions in the affinity towards phosphoribosylpyrophosphate (PRPP). Its Km for PRPP was increased 215-fold with hypoxanthine as substrate and 40-fold with guanine as substrate with associated reduced catalytic potential. Molecular modeling confirmed that the most prominent defect was the dramatically reduced affinity towards PRPP. Our studies suggest that the p.Leu68Pro mutation has a strong impact on PRPP binding and on stability of the active conformation. This suggests that factors other than HGprt activity per se may influence the phenotype of Lesch–Nyhan patients.

Molecular properties of cytosolic and nuclear TCDD receptor complexes from mouse hepatoma cell lines

Wild-type mouse Hepa 1c1c7 cells are responsive to the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. This cell line and nonresponsive mutants (TAOc1BP rc1 and BP rc1) have been used to study properties of cytosolic (unactivated and activated) and nuclear Ah receptor-[ 3H]TCDD complexes as a model for investigating the molecular mechanism of action of halogenated aromatic hydrocarbons. Incubation of cytosolic [ 3H]TCDD-aryl hydrocarbon (Ah) complexes from wild-type Hepa 1c1c7 cell for 16h at 4° C in 0.4 M KCl resulted in the formation of transformed specifically-bound receptor complexes which exhibited increased binding affinity on DNA-Sepharose columns. The elution properties of the peak with the highest DNA binding affinity were similar to the elution profiles of the nuclear receptor complex isolated from wild-type cells. Comparable in vitro transformation and DNA binding properties were also observed using hepatic cytosol from C57BL/6J mice. TAOc1BP rc1 (class I) nonresponsive mutant cells were characterized by relatively low levels of the cytosolic and nuclear Ah receptor complexes. The cytosolic TCDD-Ah receptor complex did not undergo in vitro transformation. The BP rc1 (class II) variant cell lines contained levels of cytosolic receptor which were comparable to those observed in the wild type cells, but there were significantly reduced levels of nuclear receptor complex in the class II variant cell line. Incubation of the cytosolic receptor complex from these mutant cells at 4° C for 16 h or 20° C for 2 h did not result in transformation of the cytosolic receptor complex into forms which exhibit increased retention on DNA-Sepharose columns.

Characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo- p-dioxin: Use of chemical crosslinking and a monoclonal antibody directed against a 59-kDa protein associated with steroid receptors

The Ah receptor regulates induction of cytochrome P450IA1 (aryl hydrocarbon hydroxylase) by “3-methylcholanthrene-type” compounds and mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin and related halogenated aromatic hydrocarbons. Hepatic Ah receptor from untreated rodents is localized in the cytosol and has an apparent molecular mass of 250 to 300 kDa. This large form can be dissociated into a smaller ligand-binding subunit upon exposure to high ionic strength. The Ah receptor displays many structural similarities to the receptors for steroid hormones. Two non-ligand-binding proteins have been identified to be associated with the cytosolic forms of the steroid hormone receptors. The first is a 90-kDa heat shock protein (hsp 90); the second is a 59-kDa protein (p59) of unknown function. The cytosolic Ah receptor ligand-binding subunit previously has been shown to be associated with hsp 90. In the present study, we used a monoclonal antibody, KN 382/EC1, generated against the 59-kDa protein which is associated with rabbit steroid receptors to determine if p59 also is a component of the large cytosolic Ah receptor complex. Cytosolic forms of rabbit progesterone receptor, glucocorticoid receptor, and Ah receptor were analyzed by velocity sedimentation on sucrose gradients under low-ionic-strength conditions and in the presence of molybdate. Progesterone receptor from rabbit uterine cytosol and glucocorticoid receptor from rabbit liver each had a sedimentation coefficient of ≈9 S. In the presence of KN 382/EC1 antibody the progesterone receptor and the glucocorticoid receptor both underwent a shift in sedimentation to a value of ≈11 S. The increase in sedimentation velocity is an indication that the receptor-protein complexes are interacting with the antibody. Under low-ionic-strength conditions the Ah receptors from rabbit uterine cytosol and liver cytosol had a sedimentation coefficient of ≈9 S. However, in contrast to the steroid receptors, the Ah receptor showed no change in its sedimentation properties in either tissue in the presence of KN 382/EC1, indicating that the antibody is not interacting with the Ah receptor. Multimeric Ah receptor complexes that were chemically crosslinked still did not show any interaction with KN 382/EC1. These data indicate that the 59-kDa protein either is not associated with the Ah receptor or is present in an altered form which the antibody cannot recognize.

Photoaffinity labeling of the nuclear Ah receptor from mouse Hepa 1c1c7 cells using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin

The photoinduced formation of the covalently labeled cytosolic and nuclear aryl hydrocarbon (Ah) receptors was studied using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the photoaffinity label. Irradiation of TCDD alone at wavelengths of greater than 300 nm resulted in rapid degradation of this compound (t 1/2 = 8 min). In a separate experiment, the unliganded cytosolic Ah receptor was only slowly inactivated (t 1/2 = 48 min) using the greater than 300 nm light source. Preliminary experiments with rat hepatic cytosol did not result in significant formation of specifically bound [3H]TCDD-protein covalent adducts which could be visualized by autoradiography. Irradiation of [3H]TCDD-nuclear Ah receptor complexes isolated from mouse Hepa 1c1c7 cells for 15 min gave approximately a 40% overall yield of the radiolabeled Ah receptor protein adduct. Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]TCDD-nuclear Ah receptor photoadduct gave a single major radiolabeled protein with an apparent molecular size of 91 kDa. The chromatographic properties of the control (dark) and photolabeled nuclear Ah receptor complexes were comparable using Sephacryl S-300 and DNA-Sepharose columns. Velocity sedimentation of both the control (dark) and irradiated nuclear Ah receptor complexes gave specifically bound peaks which sedimented at 6.5 S. However, the trichloroacetic acid-precipitable (buffer-reconstituted) [3H]TCDD-nuclear Ah receptor photo-covalent adduct was eluted from the Sephacryl S-300 column in the void volume and did not exhibit a specifically bound peak after velocity sedimentation analysis due to protein aggregate formation. In contrast, the elution profile of the aggregate on a DNA-Sepharose column was similar to that observed for the control (dark) and photolabeled complexes, which were eluted from the column with salt concentrations between 0.24 and 0.28 M. These photolabeling studies show that [3H] TCDD can act as a photoaffinity label for the Ah receptor and can be utilized as photoligand to probe further the structure and function of this protein.

Ah receptor for 2,3,7,8-tetrachlorodibenzo- p-dioxin in rat liver: Lack of sensitivity to alkaline phosphatase when compared with that of glucocorticoid receptor

Rat hepatic cytosol was treated with alkaline phosphatase in order to determine if dephosphorylation altered the ability of Ah receptor to bind 2,3,7,8-[ 3H]tetrachlorodibenzo- p-dioxin (TCDD). Glucocorticoid receptor was studied for comparison. As previously had been shown in other laboratories, treatment of cytosol with purified alkaline phosphatase dramatically reduced the subsequent ability of glucocorticoid receptor to bind hormone. However, alkaline phosphatase had no effect on the ability of Ah receptor to bind [ 3H]TCDD. If either glucocorticoid receptor or Ah receptor was occupied by its ligand prior to exposure to alkaline phosphatase there was no loss in ligand binding capacity. Crude alkaline phosphatase (containing some protease activity) substantially reduced the ability of glucocorticoid receptor to bind hormone and shifted the sedimentation position of the glucocorticoid receptor from ≈8 S to ≈2 S. Crude alkaline phosphatase did not reduce the ability of Ah receptor to bind [ 3H]TCDD and did not alter sedimentation of the 9 S [ 3H]TCDD. Ah receptor complex. Although the Ah receptor appears to be a member of the steroid receptor superfamily, the lack of effect of alkaline phosphatase on Ah receptor (compared to the sensitivity of glucocorticoid receptor) highlights another significant difference in molecular characteristics between the Ah receptor and the receptors for steroid hormones.

Induction of cytochrome P450-dependent monooxygenase activities in rat hepatoma H-4-IIE cells in culture by 2,3,7,8-tetrachlorodibenzo- p-dioxin and related compounds: Mechanistic studies using radiolabeled congeners

Treatment of rat hepatoma H-4-IIE cells in culture with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzo- p-dioxin (PeCDD), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF), 1,2,7,8-TCDF, and 2,3,7-trichlorodibenzo- p-dioxin (TrCDD) resulted in the structure-dependent induction of aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase activities. The induction potencies followed the order 2,3,7,8-TCDD > 2,3,7,8-TCDF > 1,2,3,7,8-PeCDD ~ 1,2,3,7,8-PeCDF > 1,2,7,8-TCDF > 2,3,7,-TrCDD and were comparable to structure-toxicity relationships which have previously been reported. In contrast, many of the properties of these compounds were structure-independent. For example, using tritiated congeners of high specific activity (>30 Ci/mmol) the sedimentation coefficients (S) for the nuclear and cytosolic aryl hydrocarbon (Ah) receptor complexes were 5–6 and 9–10 S, respectively, for all the radioligands. Moreover, examination of the processing of nuclear Ah receptor complexes for the radiolabeled congeners showed that after 6 h, the rates of nuclear processing were very low and varied between 0.006 and 0.0385 fmol degraded/mg protein/mg total DNA. These results were consistent with the reported stability and persistence of the nuclear Ah receptor complexes and in addition, there were no apparent structure-dependent differences in the processing rates. Inspection of the nuclear receptor levels and the corresponding induced enzyme activities for the congeners showed that there was a linear correlation between average nuclear receptor complex levels (18–42 h) and induced enzyme activities (32–42 h) for all six radioligands; these data indicated that the rates of cytochrome P450-dependent gene expression correlated with the levels of nuclear Ah receptor complex. In contrast, the accumulation of occupied nuclear receptor complexes in rat hepatoma H-4-IIE cells was structure-dependent and appeared to be one of the factors which governed the observed structure-induction and the previously reported structure-toxicity relationships for 2,3,7,8-TCDD and related halogenated aryl hydrocarbons.