Mechanism of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonists: characterization of 6-[125I]methyl-8-iodo-1,3-dichlorodibenzofuran-Ah receptor complexes.

6-Methyl-8-iodo-1,3,-dichlorodibenzofuran (I-MCDF) and its radiolabeled analog [125I]MCDF have been synthesized and used to investigate the mechanism of action of 1,3,6,8-substituted dibenzofurans as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) antagonists. Like 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), I-MCDF partially antagonized the induction by TCDD of microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities in rat hepatoma H-4-II E cells and male Long-Evans rat liver. Incubation of rat liver cytosol with [125I]MCDF followed by velocity sedimentation analysis on sucrose gradients gave a specifically bound peak which sedimented at 9.6 S. This radioactive peak was displaced by coincubation with a 200-fold excess of unlabeled I-MCDF, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and benzo [a]pyrene. Based on the velocity sedimentation results and the elution profile from a Sephacryl S-300 gel permeation column, the Stokes radius and apparent molecular weights of the cytosolic [125I]MCDF-Ah receptor complex were 6.5 nm and 259,200, respectively. In addition, the nuclear [125I]MCDF-receptor complex eluted at a salt concentration of 0.29 M KCl from a DNA-Sepharose column. Velocity sediment analysis of the nuclear [125I]MCDF-Ah receptor complex from rat hepatoma H-4-II E cells gave a specifically bound peak at 5.6 +/- 0.8 S. All of these properties were similar to those observed using [3H]TCDD as the radioligand. In addition, there were several ligand-dependent differences observed in the properties of the I-MCDF and TCDD receptor complexes; for example, the [125I]MCDF rat cytosolic receptor complex was unstable in high salt buffer and was poorly transformed into a form with increased binding affinity on DNA-Sepharose columns; Scatchard plot analysis of the saturation binding of [3H]TCDD and [125I]MCDF with rat hepatic cytosol gave KD values of 1.07 and 0.13 nM and Bmax values of 137 and 2.05 fmol/mg protein, respectively. The nuclear extract from rat hepatoma H-4-II E cells treated with I-MCDF or TCDD interacted with a dioxin-responsive element in a gel retardation assay. These results suggest that the mechanism of antagonism may be associated with competition of the antagonist receptor complex for nuclear binding sites.     

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.     

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 degrees 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 degrees 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 degrees 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 2,3,7,8-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 EC50 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 nM, respectively. The structure-dependent differences in these values were similar to their different potencies as Ah receptor agonists and these data suggest that the structure-dependent transformation of the liganded cytosolic Ah receptor may significantly contribute to the structure-activity relationships observed for 2,3,7,8-TCDD and related compounds.     

Synthesis and aryl hydrocarbon receptor binding properties of radiolabeled polychlorinated dibenzofuran congeners

Microchlorination of 1,4,9[3H]dibenzofuran gave several polychlorinated dibenzofuran (PCDF) products and 2,3,7,8-[3H]tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-[3H]pentachlorodibenzofuran (PeCDF), and 1,2,3,6,7,8-/1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) of high specific activity (57, 34, and 32.5 Ci/mmol, respectively) were purified by preparative high-pressure liquid chromatography. These compounds were investigated as radioligands for the rat liver cytosolic aryl hydrocarbon (Ah) receptor protein. Like 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD), the radiolabeled PCDF congeners exhibited saturable binding with the receptor protein and sucrose density gradient analysis of the radiolabeled ligand-receptor complexes gave specific binding peaks with comparable sedimentation profiles. The rank order of radioligand binding affinities (Kd values) was 2,3,7,8-TCDD greater than 2,3,7,8-TCDF greater than 1,2,3,6,7,8-HCDF greater than 1,2,3,7,8-PeCDF and the maximum difference in Kd values for the four radioligands was less than 13-fold (0.44-5.9 nM). The interactions of the PCDF radioligands with the cytosolic receptor all exhibited saturable binding curves and linear Scatchard plots and the slopes of their Hill plots were in the range 1.0-1.1, thus indicating that cooperativity was not a factor in these binding interactions. The relative stabilities and dissociation kinetics of the radioligand-receptor complexes were highly dependent on the structure of the radioligand. The dissociation curves of the 2,3,7,8-[3H]TCDD and PCDF receptor complexes were biphasic and this suggests that there may be a temporal shift in ligand binding affinities. However, the rates of dissociation did not correlate with the rank order of ligand binding affinities. The stabilities of the radioligand-receptor complexes were also dependent on the structures of the radioligands; for example, the 2,3,7,8-[3H]TCDD-receptor complex degraded more rapidly than the PCDF-receptor complex and these relative stabilities were clearly not related to the Kd values or the relative in vivo or in vitro biologic potencies of these halogenated aryl hydrocarbons.     

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 approximately 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 (Kd) for binding of [3H]TCDD to Hep G2 Ah receptor was 9 nM 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 degrees C in culture, sedimented at approximately 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 EC50 for AHH induction was 5.3 microM for benz(a)anthracene and 1.3 microM for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 mM sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drug-metabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.     

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, intracellular 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.     

Structure and function of the Ah receptor: sulfhydryl groups required for binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to cytosolic receptor from rodent livers

Cytosol from rodent liver was exposed to a variety of sulfhydryl-modifying reagents to determine if the cytosolic Ah receptor contained reactive sulfhydryl groups that were essential for preservation of the receptor's ligand binding function. At a 2 mM concentration in rat liver cytosol, all sulfhydryl-modifying reagents tested (except iodoacetamide) both blocked binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to unoccupied receptor and caused release of [3H]TCDD from receptor sites that had been labeled with [3H]TCDD before exposure to the sulfhydryl-modifying reagent. Exposure of cytosol to iodoacetamide before labeling with [3H]TCDD prevented subsequent specific binding of [3H]TCDD, but iodoacetamide was not effective at displacing previously bound [3H]TCDD from the Ah receptor. The mercurial reagents, mersalyl, mercuric chloride, and p-hydroxymercuribenzoate, were more effective at releasing bound [3H]TCDD from previously labeled sites than were alkylating agents (iodoacetamide, N-ethylmaleimide) or the disulfide compound 5,5'-dithiobis(2-nitrobenzoate). Presence of bound [3H]TCDD substantially protected the Ah receptor against loss of ligand binding function when the cytosol was exposed to sulfhydryl-modifying reagents. This may indicate that the critical sulfhydryl groups lie in or near the ligand binding site on the receptor. Subtle differences exist between the Ah receptor and the receptors for steroid hormones in response to a spectrum of sulfhydryl-modifying reagents, but the Ah receptor clearly contains a sulfhydryl group (or groups) essential for maintaining the receptor in a state in which it can bind ligands specifically and with high affinity.     

Defective binding of 3-methylcholanthrene to the Ah receptor within C3H/1OT1/2 clone 8 mouse fibroblasts in culture

C3H/1OT1/2 clone 8 mouse fibroblasts (C3H/1OT1/2 cells) exhibit induction of aryl hydrocarbon hydroxylase (cytochrome P1-450) when exposed in culture to benzo(a)pyrene, benz(a)anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but do not display the induction response when treated with 3-methylcholanthrene (MCA), the classical inducer of cytochrome P1-450. Induction of cytochrome P1-450 is regulated by the Ah receptor which initially binds inducing chemicals in the cytoplasm, after which the inducer x receptor complex translocates into the nucleus. Cytosolic and nuclear forms of the Ah receptor can be detected in C3H/1OT1/2 cells using [3H]TCDD as the radioligand in culture, but specific Ah receptor binding is not detectable within C3H/1OT1/2 cells incubated with [3H]MCA. In contrast, in Hepa-1c1 cells, which exhibit cytochrome P1-450 induction when treated with MCA, cytosolic and nuclear Ah receptor can be detected by incubation of the cells either with [3H]MCA or with [3H]TCDD. Nonradioactive MCA is able to compete with [3H]TCDD for Ah receptor sites in C3H/1OT1/2 cells, but the relative potency of MCA as a competitor is lower within C3H/1OT1/2 cells than in C3H/1OT1/2 cytosol during extracellular incubation. Specific binding of [3H]MCA to Ah receptor can be detected by incubation of [3H]MCA with C3H/1OT1/2 cytosol outside the cell. The selective loss of response to MCA as a cytochrome P1-450 inducer (while retaining response to other inducers) appears to be due to defective interaction of MCA with the Ah receptor within the intracellular environment. The specific molecular alteration which makes the MCA x receptor complex ineffective within C3H/1OT1/2 cells is unknown. Some fibroblast lines other than C3H/1OT1/2 also selectively fail to respond to MCA; thus, this variation in Ah receptor function may not be due to a mutational change in the Ah regulatory gene which codes for the Ah receptor.     

Structure and function of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Species difference in molecular properties of the receptors from mouse and rat hepatic cytosols

Molecular properties of cytosolic Ah receptors from livers of Sprague-Dawley rats and C57BL/6N mice were assessed by velocity sedimentation on sucrose gradients and by gel permeation chromatography on Sephacryl S-300. Analyses were done under conditions of both moderate ionic strength (presence of 0.1 M KCl) and high ionic strength (0.4 M KCl). [3H] 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was used as the radioligand. In conditions of moderate ionic strength the receptor from Sprague-Dawley rat liver sedimented at 8.8 +/- 0.05 S, had a Stokes radius of 7.0 +/- 0.21 nm, and an apparent relative molecular mass (Mr) of 257,000 +/- 7,700. In conditions of high ionic strength the Ah receptor from rat hepatic cytosol dissociated to a [3H]TCDD-binding subunit which sedimented at 5.6 +/- 0.58 S, had a Stokes radius of 5.2 +/- 0.24 nm, and an apparent Mr of 121,000 +/- 5,600. The Ah receptor from liver of C57BL/6N mice, in moderate ionic strength conditions, sedimented at 9.4 +/- 0.54 S, had a Stokes radius of 7.1 +/- 0.12 nm, and an apparent Mr of 277,000 +/- 4,800. Whereas the Ah receptor from rat liver readily dissociated into a [3H]TCDD-binding subunit during brief exposure to 0.4 M KCl, the mouse Ah receptor resisted dissociation. When exposed to 0.4 M KCl for 2 h, the mouse Ah receptor remained at the same molecular size that it had exhibited in moderate ionic strength conditions. Prolonged exposure (16 h) to 0.4 M KCl prior to analysis partially converted the mouse Ah receptor into a smaller [3H]TCDD-binding subunit which sedimented at 4.9 +/- 0.07 S, had a Stokes radius of 5.2 +/- 0.19 nm, and an apparent Mr of 105,000 +/- 3,800. The potency of seven different Ah receptor agonists in competing with [3H]TCDD for specific receptor sites was slightly different in mouse cytosol than in rat cytosol. By criteria of size, response to high ionic strength environments, and ligand binding preferences the mouse and rat Ah receptors appear to be similar but not identical molecular species.     

Identification of the Ah receptor in selected mammalian species and induction of aryl hydrocarbon hydroxylase

The Ah receptor protein, important in the mechanism of induction of aryl hydrocarbon hydroxylase activity, has been identified and partially characterized in hepatic cytosolic preparations from rat, BALB/c mouse, gerbil, hamster, rabbit, ferret and guinea-pig by means of sucrose density centrifugation analysis and hydroxyapatite binding assays. Using 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin (TCDD) as the ligand, total specific binding capacities ranged over 74-691 fmol [3H]TCDD/mg cytosolic protein and apparent dissociation constants ranged over 0.30-7.8 nM. There was no quantitative correlation between the concentration of cytosolic Ah receptors and the 3-methylcholanthrene-mediated induction of aryl hydrocarbon hydroxylase activity in the species studied. Competitive binding studies with a series of monohydroxylated benzo[a]pyrene derivatives suggested the importance of electronic character in their ability to bind to the Ah receptor and to compete with TCDD for specific binding sites on the receptor.     

Characterization of an inducible aryl hydrocarbon receptor-like protein in rat liver.

The individual pretreatment of Sprague-Dawley rats with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) has been previously shown to result in the "induction" of [3H]TCDD specific binding activity in hepatic tissue. In the present work, the coadministration of TCDD and HCB increased the concentration of hepatic proteins capable of binding [3H]TCDD specifically by at least 2-3-fold. This increase was shown not to be the result of activation, by HCB, of a form of the receptor having low affinity toward [3H]TCDD into a form with high affinity. Kinetic analysis of the time course of binding of [3H]TCDD to induced cytosol was consistent with the presence of an "inducible" binding protein in addition to the "constitutive" aryl hydrocarbon (Ah) receptor present in cytosol from untreated animals. The liganded ([3H]TCDD) form of the inducible binding component lost its ligand much faster than the liganded form of the constitutive Ah receptor at 37 degrees C; apparent first order rate constants for loss of [3H]TCDD were 0.55 min-1 and less than 0.0024 min-1, respectively. Conversely, the unliganded form of the induced binding component was slightly more stable (approximately 2-fold) toward thermal inactivation than the unbound constitutive Ah receptor. The [3H]TCDD-bound protein(s) in uninduced and induced cytosols behaved identically in a sucrose gradient; 8.7-8.9 S in the absence of salt, shifted to 5.5 S by 0.4 M KCl. They were also indistinguishable by gel permeation chromatography, and by photoaffinity labeling their TCDD-binding subunits, approximate molecular weights 105,000. These results show the hepatic TCDD-binding protein(s) induced upon pretreatment of Sprague-Dawley rats with TCDD/HCB to be kinetically distinct from the Ah receptor, but structurally very similar.     

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 greater than 2,3,7,8-TCDF greater than 1,2,3,7,8-PeCDD approximately 1,2,3,7,8-PeCDF greater than 1,2,7,8-TCDF greater than 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 (greater than 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.     

Detection and characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the human colon adenocarcinoma cell line LS180.

The Ah (aromatic hydrocarbon) receptor mediates induction of aryl hydrocarbon hydroxylase (AHH; an enzyme activity associated with cytochrome P450IA1) by polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene (MC) and benzo[a]pyrene (BP) and the halogenated toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Until recently the AhR seemed to be present only at very low levels in human cells and tissue. With a modified assay (the presence of sodium molybdate and a reduction in the amount of charcoal used to adsorb "excess" ligand) we found that cytosol from LS180 cells contains a high concentration of AhR (400-500 fmol/mg cytosolic protein) when detected by [3H]TCDD or [3H]MC. Cytosolic receptor also was detected with [3H]BP but at a level that was 35% of that detected with [3H]TCDD or [3H]MC. These levels are similar to those found in mouse Hepa-1 hepatoma cells in which AhR has been extensively characterized. The apparent binding affinity (Kd) of the cytosolic receptor for [3H]TCDD and for [3H]MC was about 5 nM. As with Hepa-1, the human LS180 cytosolic AhR sedimented at about 9 S on sucrose gradients when detected with [3H]TCDD, [3H]BP or [3H]MC. The nuclear-associated ligand.receptor complex recovered from cells incubated in culture with [3H]TCDD sedimented at about 6.2 S. The 9.8 S cytosolic form corresponds to a multimeric protein of a relative molecular mass (Mr) of about 285,000 whereas the 6.2 S nuclear receptor corresponds to a multimeric protein of Mr 175,000. The smallest specific ligand-binding subunit (detected by sodium dodecyl sulfate-polyacrylamide electrophoresis under denaturing conditions of receptor photoaffinity labeled with [3H]TCDD) was about Mr 110,000. AHH activity was induced in cells exposed in culture to TCDD or benz[a]anthracene (BA). The EC50 was 4 x 10(-10) M for TCDD and 1.5 x 10(-5) M for BA. For both inducers the EC50 in LS180 cells was shifted about one log unit to the right as compared to the EC50 for AHH induction in mouse Hepa-1 cells. The lower sensitivity of the LS180 cells to induction of AHH activity by TCDD or BA is consistent with the lower affinity of TCDD and MC for binding to human AhR. The ligand-binding properties, physicochemical properties, and mode of action of the AhR in this human cell line are therefore very similar to those of the extensively characterized AhR in rodent cells and tissues.     

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 approximately 8 S to approximately 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.     

Physicochemical characterization of the nuclear form of Ah receptor from mouse hepatoma cells exposed in culture to 2,3,7,8-tetrachlorodibenzo-p-dioxin

Molecular properties of nuclear aromatic hydrocarbon (Ah) receptor from Hepa-1c1c9 (Hepa-1) cells were assessed by velocity sedimentation on sucrose gradients and by gel permeation chromatography on Sephacryl S-300. Nuclear Ah receptor was obtained by exposing intact cells to [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 1 h at 37 degrees C in culture followed by extraction of receptor from nuclei with buffers containing 0.5 M KCl. The nuclear Ah receptor was compared to the cytosolic Ah receptor from the same cells. Under conditions of low ionic strength, the Ah receptor from Hepa-1 cytosol sedimented as a single 9.4 +/- 0.63 S binding peak that had a Stokes radius of 7.1 +/- 0.12 nm and an apparent relative molecular mass of 271,000 +/- 16,000. After prolonged (24 h) exposure to high ionic strength (0.5 M KCl), cytosol labeled with [3H]TCDD exhibited two specific binding peaks. The large form of cytosolic Ah receptor seen under high ionic strength conditions sedimented at 9.4 +/- 0.46 S, had a Stokes radius of 6.9 +/- 0.19 nm, and an apparent Mr 267,000 +/- 15,000. The smaller ligand-binding subunit generated by exposing cytosol to 0.5 M KCl sedimented at 4.9 +/- 0.62 S, had a Stokes radius of 5.0 +/- 0.14 nm, and an apparent Mr 104,000 +/- 12,000. Nuclear Ah receptor, analyzed under high ionic strength conditions, sedimented at 6.2 +/- 0.20 S, had a Stokes radius of 6.8 +/- 0.19 nm, and an apparent Mr 176,000 +/- 7000. Nuclear Ah receptor from rat H4IIE hepatoma cells was analyzed and found to have physicochemical characteristics identical to those of nuclear Ah receptor from the mouse Hepa-1 cells. The molecular mass of Hepa-1 nuclear Ah receptor was found to be statistically different from both the Mr approximately 267,000 cytosolic Ah receptor and the Mr approximately 104,000 subunit which were present in cytosol under high ionic strength conditions. Hepa-1 nuclear Ah receptor could not be converted to a smaller ligand-binding subunit by treatment with alkaline phosphatase, ribonuclease, or sulfhydryl-modifying reagents or prolonged exposure to 1.0 M KCl. Cytosolic Ah receptor from Hepa-1 cells was "transformed" by heating at 25 degrees C in vitro into a form with high affinity for DNA-cellulose. The transformed cytosolic Ah receptor, when analyzed under conditions of high ionic strength, sedimented at approximately 6 S, had a Stokes radius of approximately 6.7 nm, and an apparent Mr approximately 167,000.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protamine sulfate precipitation: a new assay for the Ah receptor

The ability of protamine sulfate to effect the quantitative precipitation of 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD):Ah receptor complexes from rat liver cytosol has been developed into a new assay for the identification, quantitation, and characterization of the Ah receptor. The method is reliable, uncomplicated, and rapid, and can be applied to large numbers of samples. The major advantage of the assay is that protamine sulfate appears to selectively precipitate the Ah receptor protein and does not precipitate a number of other proteins that bind [3H]TCDD nonspecifically.     

Ah receptor in primate liver: binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and carcinogenic aromatic hydrocarbons

Ah receptor in hepatic cytosols from adult cynomolgus monkeys (Macaca fasicularis) was identified and quantitated by its binding of the highly toxic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the carcinogens 3-methylcholanthrene, benzo[a]pyrene, and dibenz[a,h]anthracene. The concentration of Ah receptor in cynomolgus hepatic cytosols (approximately 10 fmol/mg cytosol protein) was about one-quarter of that typically detected in rodent hepatic cytosols. Receptor concentrations were equal in male and female cynomolgus. [3H]TCDD bound to cytosolic receptor with high affinity (Kd approximately 3 nM). In rodents, Ah receptor is known to play a central role in toxicity caused by halogenated aromatic compounds and in carcinogenesis caused by polycyclic aromatic hydrocarbons. Existence of Ah receptor in monkeys indicates that the receptor also may mediate such responses in primates.