Binding characteristics of Ah receptors from rats and mice before and after separation from hepatic cytosols. 7-Hydroxyellipticine as a competitive antagonist of cytochrome P-450 induction

The Ah receptor, a soluble protein implicated in the mechanism of action of the toxic halogenated aryl hydrocarbons has been examined in rodent livers. Due to the difficulty of making reliable quantitative determinations on binding parameters for hydrophobic compounds in cytosols that contain several components, Ah receptors from livers of Sprague-Dawley rats and C57BL/6 mice have been separated, in a preparative manner, using sucrose density gradient centrifugation in a vertical rotor. The binding characteristics of Ah receptors, before and after separation, were assessed by competition of various chemicals as 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, 3-methylcholanthrene, benzo[a]pyrene, beta-naphthoflavone and ellipticines with [3H]3-methylcholanthrene and 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin as radioligands. The rationale of this approach is supported by the results obtained and the major conclusions are as follows. 1. The intrinsic binding characteristics of Ah receptors were dependent on the presence or absence of other cytosolic binding components (light-density component and 4-S carcinogen-binding protein). 2. In contrast with many previous unsuccessful attempts, the separation of the C57BL/6 Ah receptor allowed the unambiguous detection of a 9-S binding peak with [3H]benzo[a]pyrene as a radioligand. 3. The intrinsic binding characteristics of the separated Ah receptors of Sprague-Dawley rats and C57BL/6 mice were similar if not identical. 4. A good correlation exists between the competitive potency (IC50) of chemicals and their ability to induce aryl hydrocarbon hydroxylase activity, except for 7-hydroxyellipticine which binds to the Ah receptors of rat and mouse liver (IC50 approximately 5-10 microM) without inducing aryl hydrocarbon hydroxylase. 5. When coadministered with various inducers, 7-hydroxyellipticine antagonizes (from about 20% to 65%) the inducing ability of chemicals displaying similar (ellipticines) or weaker (chlorpromazine, phenothiazine) binding affinities for the Ah receptor.     

The induction of cytochrome P-450 by isosafrole and related methylenedioxyphenyl compounds

Using sucrose gradients, the Ah receptor and a 3-4S binding peak were measured in hepatic cytosol from Dub: ICR, C57BL/6, and DBA/2 male mice. Isosafrole, piperonyl butoxide, and 5-t-butyl-1,3-benzodioxole were unable to displace 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3-methylcholanthrene from either the Ah receptor or the 3-4S binding peak, in vitro. In in vivo experiments, treatment of C57BL/6 mice with 3-methylcholanthrene caused a 4-fold reduction in Ah receptor binding 2 h after i.p. injection; whereas, isosafrole caused a 2-fold enhancement of the Ah receptor after 24 h. This increase in the Ah receptor binding following isosafrole treatment may be due to induction. 3-Methylcholanthrene treatment of C57BL/6 mice also caused a 3-fold reduction in the 3-4S binding peak 2 h after i.p. injection; isosafrole treatment had little or no effect on the 3-4S peak in C57BL/6 or DBA/2 mice. Both in vivo and in vitro data appear to demonstrate that there is no direct role for the Ah receptor or the 3-4S protein in the regulation of cytochrome P-450 by methylenedioxyphenyl compounds. Using Sephadex G-100 chromatography, a cytosolic protein fraction was obtained from C57BL/6 and Dub:ICR mice which was previously implicated by others as a carrier in the metabolism of benzo[a]pyrene (B[a]P). This fraction was applied to sucrose gradients and sedimented in the 3-4S region. Hence it appears that the 3-4S binding peak may be the carrier described by these workers.     

Radioligand-dependent differences in the molecular properties of the mouse and rat hepatic aryl hydrocarbon receptor complexes

A comparison of the molecular properties of the male Long-Evans rat and male C57BL/6 mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor complex was determined using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-[3H]tetrachlorodibenzofuran (TCDF) as radioligands. In low salt buffer, the sedimentation coefficients, Stokes radii, relative molecular masses, frictional ratios, axial ratios and gel permeation chromatographic properties of the rat receptor complexes were ligand independent. In contrast, there were several ligand-dependent differences in the mouse Ah receptor complexes formed after incubation in low salt buffer and these include: sucrose density gradient analysis of the 2,3,7,8-[3H]TCDF receptor complex gave a 9.5 S specifically bound peak and a 2.6 S nonspecifically bound peak whereas the corresponding 2,3,7,8-[3H]TCDD receptor complex gave a single 9.6 S specifically bound peak; sucrose density gradient analysis of the two major peaks eluted from a Sephacryl S-300 column chromatographic separation of the 2,3,7,8-[3H]TCDF receptor complex gave two specifically bound peaks at 9.2 and 5.1 S. The molecular properties of the rat hepatic cytosolic receptor complexes incubated in high salt (0.4 M KCl) buffer were ligand independent with one exception, namely the significant difference in the sedimentation coefficient of the specifically bound disaggregated 2,3,7,8-[3H]TCDD receptor complex (6.8 S) and the corresponding 2,3,7,8-[3H]TCDF receptor complex (5.0 S). The major ligand-dependent differences in the mouse receptor complexes incubated in high salt (0.4 M KCl) were associated with the sedimentation coefficients of the complexes derived after direct incubation and after gel permeation chromatography. For example, both ligands gave two specifically bound complexes after chromatography on Sephacryl S-300 column and centrifugation of these fractions gave both the approximately 9 and approximately 5 S peaks; this suggested that there was some equilibration between the aggregated and disaggregated receptor complexes. The behavior of the 2,3,7,8-[3H]TCDF mouse receptor complex was similar after incubation in low or high salt buffer except that sucrose density gradient analysis of the gel permeation chromatographic fractions gave an additional specifically bound peak which sedimented at 7.2 S. These studies demonstrate that the molecular properties of the Ah receptor were dependent on the source of the cytosolic receptor preparation, the ionic strength of the incubation media, and the structure of the radioligand.     

Induction of cytochrome P-450 in congenic C57BL/6J mice by isosafrole: lack of correlation with the Ah locus

Isosafrole induction of cytochrome P-450 was compared in congenic strains of C57BL/6J mice, one of which expresses normal levels of the Ah receptor [B6(Ahb)], and another that does not contain a measurable receptor concentration [B6(Ahd)]. Using sucrose gradient analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding, an Ah receptor concentration of 69.1 +/- 3.8 fmol/mg protein was measured in the hepatic cytosol from B6(Ahb) mice, while no receptor could be detected in the cytosol from B6(Ahd) mice. Isosafrole treatment (75 mg/kg X 3 days) increased the total hepatic microsomal cytochrome P-450 content to the same extent in the two congenic strains. The level of microsomal monooxygenase induction in the isosafrole-treated B6(Ahd) mice was greater than that of B6(Ahb) mice for ethylmorphine N-demethylase and isosafrole metabolite-complex formation, the latter a measure of cytochrome P2-450. In the case of 7-ethoxycoumarin O-deethylase only the isosafrole-treated B6(Ahd) mice had elevated microsomal activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) also revealed a similar induction pattern for the two congenic strains, following isosafrole treatment. Thus, the isosafrole treated B6(Ahd) mice produced an equivalent or slightly larger induction of cytochrome P-450 than the B6(Ahb) mice, suggesting that there is no direct role for the Ah receptor in the regulation of these cytochrome P-450 monooxygenase activities by isosafrole.     

High-affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin in cell nuclei from rat liver

The intranuclear binding of radioactive 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat liver has been studied both in vivo and in vitro. Following the intravenous administration of [1,6-3H]TCDD, a maximum uptake by cell nuclei could be observed at 2 h after injection with a concurrent decrease in the cytosolic uptake. Using linear sucrose density gradient centrifugation, dextran-coated charcoal adsorption assay, DEAE-Sepharose ion-exchange chromatography, competition, enzymatic and saturation studies, a high-affinity binding protein for TCDD in liver cell nuclei could be demonstrated both in vivo and after an exchange in vitro of intravenously administered unlabelled 2,3,7,8- tetrachlorodibenzofuran (TCDBF) for [3H]TCDD. Sucrose density gradient analysis showed a size of 4-5 S for both the cytosolic and nuclear TCDD binding entity. The specific binding of [3H]TCDD to nuclear components was heat labile and saturable and had an equilibrium dissociation constant of 1.05 nM. Based on a differential susceptibility to specific hydrolases, i.e. DNAase, RNAase, trypsin and pronase, the binding entity appears to be a 4-5 S salt-extractable protein.     

Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: ontogeny in chick embryo liver

Aryl hydrocarbon hydroxylase (AHH, cytochrome P1-450) is induced in chick liver very early during embryonic development if embryos are treated with 3-methylcholanthrene-type compounds such as 3,4,3'4'-tetrachlorobiphenyl. In mammals, AHH induction is known to be mediated by the Ah receptor. Liver from embryonic and newly hatched chicks was found to contain a cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which has properties that are very similar to properties of the Ah receptor previously characterized in mammalian tissues. In chick embryo liver, cytosolic binding sites for TCDD were of high affinity (Kd for [3-H]-TCDD = 0.2 nM) and were specific for 3-methylcholanthrene-type inducers. The specific binding component sedimented at about 9S on sucrose density gradients prepared at low ionic strength. A high level of Ah receptor was detected in chick embryo liver by the fifth day of incubation (5 DI); this is at least 24 hours prior to the onset of AHH inducibility. The Ah receptor concentration increased from 5 DI to 8 DI, the period when chick liver is undergoing early morphological differentiation. After 8 DI, Ah receptor levels dropped substantially and remained low into the posthatching period. In contrast, AHH inducibility was high by 7 DI and remained high throughout embryonic development and into the posthatching period. The discrepancy between Ah receptor levels and the degree of AHH inducibility suggests that only a small fraction of the Ah receptor population is required for maximal AHH induction.     

Ah receptor in spleen of rodent and primate species: detection by binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin

In many species systemic toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is manifested by a generalized wasting syndrome accompanied by a variety of specific organ changes including atrophy of the thymus and spleen. TCDD toxicity in most tissues is thought to be mediated by the Ah receptor. Although the spleen is a prime target for TCDD toxicity, the possible presence of Ah receptor in the spleen has not previously been investigated. Specific binding of [3H]TCDD to Ah receptor in spleen cytosols was assessed by velocity sedimentation on sucrose gradients. Ah receptor was detected in spleen cytosols from adult Rhesus monkeys (mean +/- SEM, 36 +/- 8 fmol/mg cytosol protein), fetal Rhesus monkeys (9 +/- 6), Sprague-Dawley rats (20 +/- 5), C57BL/6J mice (18 +/- 2), New Zealand white rabbits (19 +/- 2), and Hartley guinea pigs (15 +/- 2). Ah receptor was not detectable in spleen cytosol from genetically "nonresponsive" DBA/2J mice or from Golden Syrian hamsters, a species resistant to toxicity of TCDD. Molecular properties of Ah receptor from spleen were similar to those of the receptor from liver of the same species. The high Ah receptor content in spleen cytosols from those species that are most susceptible to TCDD toxicity is consistent with the view that the Ah receptor mediates TCDD toxicity in spleen as well as in other tissues.     

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.     

Omeprazole, an inducer of human CYP1A1 and 1A2, is not a ligand for the Ah receptor.

Omeprazole is a benzimidazole derivative which induces both P450 1A1 and 1A2 in human liver in vitro and in vivo. Northern blot analysis of polyA RNA prepared from primary cultures of human hepatocytes indicates that both 1A1 and 1A2 messages are induced by beta-naphthoflavone and omeprazole. Co-treatment of cells with these inducers and with actinomycin D or cycloheximide results in no accumulation of both mRNA or superinduction of 1A1 mRNA, respectively. 9S enriched fraction of cytosol was prepared either from human hepatocytes in culture or from human liver tissue and analyzed by sucrose density gradient sedimentation for its capacity to bind 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), omeprazole or omeprazole sulfone (a metabolite of omeprazole in man). Whereas 2 microM TCDD displaced almost totally [3H]TCDD from the Ah receptor, both omeprazole and omeprazole sulfone did not, even at 5000-fold molar excess. In addition, when [14C] omeprazole was incubated with 9S enriched fraction of human liver or hepatocyte cytosol, no interaction could be detected in sucrose density gradient. These experiments suggest that omeprazole is not a ligand for the human liver Ah receptor.     

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.     

Use of [125I]4'-iodoflavone as a tool to characterize ligand-dependent differences in Ah receptor behavior

We have synthesized [(125)I]4'-iodoflavone to study Ah receptor (AhR)-ligand interactions by a class of AhR ligands distinct from the prototypic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This radioligand allows the comparison of AhR-ligand interactions using a ligand that differs in AhR affinity, and yet has the same radiospecific activity as [(125)I]2-iodo-7,8-dibromodibenzo-p-dioxin. Specific binding of [(125)I]4'-iodoflavone with the AhR was detected as a single radioactive peak ( approximately 9.7 S) following density sucrose gradient analysis. Cytosolic extracts from both Hepa 1 and HeLa cells were used as the source of mouse and human AhR, respectively. A approximately 6.7 S form of radioligand-bound Ah receptor was detected in the high salt nuclear extracts of both cell lines. In HeLa cells approximately twofold more [(125)I]4'-iodoflavone-AhR 6 S complex, compared with [(125)I]2-iodo-7,8-dibromodibenzo-p-dioxin, was recovered in nuclear extracts. A comparison of the ability of 4'-iodoflavone and TCDD to cause time-dependent translocation of AhR-yellow fluorescent protein revealed that 4'-iodoflavone was more efficient at enhancing nuclear accumulation of the receptor. These results suggest that [(125)I]4'-iodoflavone is a particularly useful and easily synthesized ligand for studying the AhR.     

Dioxin alters the human low-density and very low-density lipoprotein structure with evidence for specific quenching of Trp-48 in apolipoprotein C-II

The intercellular transport of cholesterol and triglycerides via lipoproteins interacting with their receptors is a critical component in human lipid metabolism. The delivery of cholesterol to cells is accomplished primarily through low-density lipoproteins (LDLs), while the transport of fatty acids to adipose and muscle tissue is accomplished primarily through the actions of very low-density lipoproteins (VLDLs). Disruption of lipoprotein structure leading to impaired binding between these lipoproteins and their obligate receptors is a known risk factor for cardiovascular disease. Because of recent investigations linking 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in humans with coronary artery disease, investigations have been carried out by fluorescence and circular dichroism to evaluate conformational changes in LDL and VLDL structure upon binding of TCDD. These studies demonstrate that, at a molar ratio of three TCDD molecules to one lipoprotein molecule, TCDD binds and disrupts the secondary and tertiary lipoprotein structure. Circular dichroism studies show that residues within the inner core of apoC-II, which compose a four-alpha-helix bundle when this apolipoprotein is associated with VLDL, are directly affected upon binding TCDD. Fluorescence also indicates the specific interaction of Trp-48 within apoC-II upon TCDD binding. We found that the TCDD/apoC-II complex suffers a 5-fold reduction in its ability to bind lipoprotein lipase compared to untreated apoC-II. The interaction of TCDD with LDL markedly altered the secondary structure of apoB reducing its alpha-helical content. These cumulative responses in lipoprotein structure may impair the LDL and VLDL cellular uptake leading to a buildup of serum lipoproteins and fats thus hastening the development of coronary artery disease.     

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.     

Isosafrole-induced cytochrome P2-450 in DBA/2N mouse liver. Characterization and genetic control of induction

Mouse "cytochrome P2-450" is defined as that form of isosafrole-induced P-450 in DBA/2N liver most specifically correlated with isosafrole metabolism. Isosafrole pretreatment does not induce aryl hydrocarbon hydroxylase activity ("cytochrome P1-450") in C57BL/6N or DBA/2N mice, induces acetanilide 4-hydroxylase activity ("cytochrome P3-450") more than 3-fold in C57BL/6N but not in DBA/2N mice, and induces isosafrole metabolite formation more than 3-fold in both C57BL/6N and DBA/2N mice. P2-450 was, therefore, purified from isosafrole-treated DBA/2N liver microsomes having negligible amounts of contaminating P1-450 and P3-450. The apparent molecular weight of P2-450 is 55,000, and the protein appears homogeneous on sodium dodecyl sulfate-polyacrylamide gels. The Soret peak of the reduced purified cytochrome X CO complex is 448 nm. Purified P2-450, reconstituted in vitro, metabolizes acetanilide poorly and benzo[a]pyrene hardly at all. Anti-(P2-450) inhibits (90 to 100%) liver microsomal isosafrole metabolite formation, yet has no effect on aryl hydrocarbon hydroxylase, acetanilide 4-hydroxylase, biphenyl 2- or 4-hydroxylase, or 7-ethoxycoumarin O-de-ethylase activities. 3-Methylcholanthrene induces anti-(P2-450)-precipitable protein about 12-fold in C57BL/6N and 2-fold in DBA/2N liver; 2,3,7,8-tetrachlorodibenzo-p-dioxin (10 micrograms/kg), about 12-fold in both C57BL/6N and DBA/2N liver; isosafrole, more than 3-fold in both C57BL/6N and DBA/2N. Benzo[a]anthracene at maximal doses induces anti-(P2-450)-precipitable protein in C57BL/6N liver no more than 2-fold, yet is known to be a highly potent inducer of P1-450 mRNA in C57BL/6N liver. The sensitivity of the P2-450 induction process to isosafrole is inherited as an autosomal additive trait; studies of offspring from the C57BL/6N(DBA/N)F1 X DBA/2N backcross confirm involvement of the Ah locus or s closely segregating gene. In contrast, among crosses between C57BL/6N and DBA/2N, sensitivity of the P1-450 and P3-450 induction process to 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin is inherited as an autosomal dominant trait. These data suggest that, although P1-450, P2-450, and P3-450 proteins are controlled by the Ah locus, either a P-450 protein polymorphism exists between C57BL/6N and DBA/2N mice or subtle differences may exist in the interaction of various inducers with Ah receptor.     

Heterogeneity of the rat hepatic Ah receptor and evidence for transformation in vitro and in vivo

The characteristics of the Ah receptor from rat liver were investigated following the incubation of cytosol with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) under various conditions, and using DEAE- and DNA-Sepharose chromatography and sucrose density gradient centrifugation. These studies indicated that the Ah receptor can exist in three distinct forms in vitro that are dependent on the presence or absence of TCDD and the duration and temperature of incubation. The unoccupied receptor was distinguished by its elution from DEAE-Sepharose columns at 0.20-0.23 M NaCl and lack of affinity for DNA-Sepharose. Following the incubation of the unoccupied receptor with [3H]TCDD, two occupied forms were distinguished based on their overall surface charges and affinities for DNA. One of these forms was predominant following short incubations (2 h) with [3H]TCDD at a low temperature (0 degree C) and was characterized by having the same elution profile on DEAE-Sepharose as the unoccupied form, but demonstrated some affinity for DNA. Another occupied form was predominant following an incubation for a longer time (20 h, 0 degree C) or at an elevated temperature (2 h, 20 degrees C). This form had an overall surface charge that was less negative and a greater affinity for DNA. These changes in receptor characteristics were dependent on the presence of TCDD and were not accompanied by apparent changes in the sedimentation coefficients of the two occupied forms. Anion exchange chromatography of the [3H]TCDD-receptor complex extracted from hepatic nuclei of [3H]TCDD-treated rats indicated that the ligand-induced change of the unoccupied receptor to a less negatively charged form had occurred in vivo. These results indicated a biochemical heterogeneity of the Ah receptor and suggested the occurrence of a ligand- and temperature-dependent transformation process in vivo and in vitro.     

Evidence that 2,3,7,8-tetrachlorodibenzo-p-dioxin induces NADPH cytochrome c (P-450) reductase in rat hepatoma cells in culture

The lack of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) (EC 1.14.14.1) induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a clone of rat hepatoma (HTC cl-1) cells is not caused by the lack of nuclear Ah receptor or by a deficiency in the activity of NADPH-cytochrome c (P-450) reductase. Treatment of HTC cl-1 cell line with TCDD for 18 h in culture resulted in a reproducible 500-600% increase in reductase activity without concomitant expression in AHH activity. These data suggests that TCDD induces cytochrome c reductase activity and that the lack of inducible AHH activity in rat hepatoma cells could reflect a defect in the structural gene (s) encoding for cytochrome P1-450, or an Ah receptor with a faulty DNA binding domain.     

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.