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A B Okey

Publications and source records attributed to A B Okey.

At least 37 records · Page 2Linked to original sources
PubMed1993

Ah receptor binding properties of indole carbinols and induction of hepatic estradiol hydroxylation.

The effect of route of administration on the ability of indole-3-carbinol (13C), an anticarcinogen present in cruciferous vegetables, to induce estradiol 2-hydroxylase (EH) in female rat liver microsomes was investigated and compared to that of its main gastric conversion product, 3,3'-diindolylmethane (DIM). This dimer was more potent than 13C after either oral or intraperitoneal administration and was also a better in vitro inhibitor of EH in control and 13C-induced hepatic microsomes. The induction of both CYP1A1 and 1A2 in about equal amounts by 13C and DIM as well as of CYP2B1/2 was demonstrated using monoclonal antibodies. DIM, isosafrole, beta-naphthoflavone, 3-methylcholanthrene and naringenin added in vitro inhibited EH strongly in induced microsomes but gestodene was a better inhibitor of estrogen 2-hydroxylation in liver microsomes from untreated female rats. The binding affinities of 13C and DIM to the Ah receptor were compared to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by competition studies, and the IC50 values were shown to be 2.0 x 10(-9) M, 5.0 x 10(-5) M and 2.3 x 10(-3) M for TCDD, DIM and 13C, respectively. The ability of 13C or DIM to cause in vitro transformation of the Ah receptor to a form able to bind to the dioxin-responsive element-3 (DRE3) was compared to that of TCDD and shown to parallel their abilities to compete for binding of [3H]TCDD to the Ah receptor. These experiments confirm and extend the proposals that dietary indoles induce specific cytochrome P450s in rat liver by a mechanism possibly involving the Ah receptor. The induced monooxygenases, in turn, increase the synthesis of 2-hydroxylated estrogens in the competing pathways of 2- and 16 alpha-hydroxylation which decreases the levels of 16 alpha-hydroxyestrone able to form stable covalent adducts with proteins including the estrogen receptor. Such steroid-protein interaction has been correlated with mammary carcinogenesis.

Animals↗
PubMed1992

In vitro transformation of the human Ah receptor and its binding to a dioxin response element.

Many biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) are mediated by a soluble intracellular protein, the Ah receptor (AhR). After binding of TCDD to the cytoplasmic AhR there occurs a poorly understood "transformation" step, wherein the TCDD-AhR complex is converted to a form that can bind to DNA with high affinity. The binding of transformed AhR to a specific dioxin-responsive element (DRE) upstream of a given gene stimulates transcriptional activation of that gene. Using a gel retardation assay we examined the interaction of transformed human cytosolic TCDD-AhR complexes with a synthetic DNA oligonucleotide containing a single DRE site. Transformation and DNA binding of human AhR in vitro was ligand dependent and specific for DRE-containing DNA. Unlike rodent hepatic AhR, in vitro transformation of human AhR was completely temperature dependent. Although at 4 degrees AhR binds ligand, no transformation of human TCDD-AhR complex was observed at 4 degrees even after 24 h; however, rapid transformation as measured by DNA binding was detectable as early as 10 min after warming to 22 degrees, with maximal binding by about 60 min. Calf thymus DNA-Sepharose or DRE-Sepharose column chromatography showed that transformed human cytosolic AhR interacts with DNA as a single species. The absolute temperature dependency of human AhR transformation mimics that observed in vivo and provides a useful system to study the mechanism of AhR transformation in detail.

Animals↗
PubMed1991

The Ah receptor, cytochrome P450IA1 mRNA induction, and aryl hydrocarbon hydroxylase in a human lymphoblastoid cell line.

The immunosuppressive and carcinogenic effects of aryl hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (MC) on B lymphocytes of adult rodents and the induction of cytochrome P450IA1 and aryl hydrocarbon hydroxylase (AHH) in human mitogen-activated lymphocytes and B-lymphoblastoid cell lines are believed to be mediated by the Ah receptor. However, there has not been a direct demonstration or characterization of the Ah receptor in defined populations of any of these cells. We report here the detection and characterization of an abundant, high-affinity B lymphocyte Ah receptor in the AHH-inducible human B lymphoblastoid cell line BCR-5. Our results represent the first characterization of a human lymphocyte receptor in a well-defined lymphocyte population. Sucrose density gradient analysis of BCR-5 cytosols incubated with [3H]TCDD revealed a characteristic 9 S specific binding peak. The maximum concentration of Ah receptor was about 200 fmol/mg protein. Specific binding to the Ah receptor was also detected with [3H]MC and, to a lesser extent, with [3H]benzo[alpha]pyrene. The apparent binding affinity (Kd) for [3H]TCDD (determined by saturation analyses) was about 5 nM. A specific [3H]TCDD-Ah receptor complex which sedimented at 5 S was extracted from nuclei of BCR-5 cells incubated at 37 degrees with [3H]TCDD. The Ah receptor of BCR-5 cells is thus similar in characteristics to that identified in other cell lines. When BCR-5 cells were exposed in culture for 24 hr to increasing concentrations of benz[alpha]anthracene there was a concentration-dependent increase in induction and a good correlation (r = 0.98) between the level of induced AHH activity and the relative abundance of cytochrome P450IA1 mRNA. The human B lymphoblastoid cell line BCR-5, therefore, has a complete regulatory mechanism for Ah receptor-mediated induction of cytochrome P450IA1 that is essentially the same as that which has been well established in many rodent species. The accessibility of human blood lymphocytes and the ease of establishment of B lymphoblastoid cell lines from any donor provide a source of pure cultures of human B lymphocytes which can be grown continuously in vitro for the study of mechanisms related to Ah receptor-mediated cytochrome P450IA1 induction, immunosuppression and carcinogenesis.

Aryl Hydrocarbon Hydroxylases↗
PubMed1991

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.

Affinity Labels↗
PubMed1991

Detection and characterization of Ah receptor in tissue and cells from human tonsils.

Ah receptor was identified and characterized in cytosol and nuclear extracts from human tonsils obtained at surgery from children 2 to 6 years of age. Ah receptor was found in cytosol prepared from whole-tonsil homogenates as well as in cytosol and nuclear fractions prepared from tonsil lymphocytes or tonsil fibroblasts grown in primary culture. Cytosolic Ah receptor was detectable in tonsillar tissue with either halogenated (2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD)) or nonhalogenated (3-[3H]methylcholanthrene and [3H]benzo[a]pyrene) aromatic hydrocarbons and sedimented at approximately 9 S after velocity sedimentation on sucrose gradients. The apparent binding affinity (Kd) of [3H]TCDD for Ah receptor ranged from 3 to 12 nM in cytosols from seven different donors. The same analyses indicated a concentration of Ah receptor in human tonsils of approximately 100-300 fmol/mg cytosolic protein. Incubation of either tonsil lymphocytes or tonsil fibroblasts with [3H]TCDD resulted in transformation of cytosolic Ah receptor to a nuclear binding form which could be detected as a specifically labeled peak sedimenting at approximately 6 S on sucrose gradients. These data demonstrate the existence of Ah receptor in human tonsils and suggest that this immune organ may be an appropriate model for further studies on the mechanism and manifestation of aromatic hydrocarbon-induced immunotoxicity in man.

Cell Nucleus↗
PubMed1991

Downregulation of the Ah receptor in mouse hepatoma cells treated in culture with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The aromatic hydrocarbon (Ah) receptor behaves as a ligand-dependent transcription factor in the induction of cytochrome P450IA1. In cells exposed to the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the Ah receptor undergoes a transformation from a form with low affinity for nucleic acids (cytosolic receptor) into a form that preferentially associates with the cell nucleus (nuclear receptor). We followed the fate of the Ah receptor in mouse hepatoma cells during short-term exposure to [3H]TCDD by analyzing both cytosolic and nuclear fractions for specific binding. Nuclear Ah receptor levels increased over the first 2 h of treatment and then decreased to about 50% of maximal concentrations by 5 h after start of treatment. The decrease in nuclear receptor was not accompanied by a reappearance of detectable Ah receptor in the cytosolic fraction; further incubation with [3H]TCDD in cytosols from lysed cells did not label any additional receptor sites in cytosolic extract. By the 6th h of incubation, the total receptor population in the cell was only about 15-20% of that detected at the start of the incubation. The levels of specific binding detected were unaffected by up to 20 h of incubation with the vehicle DMSO, confirming that the presence of TCDD is required for the observed downregulation to occur. These results indicate that there is a substantial ligand-dependent loss in total Ah receptor during short-term exposure of cells to TCDD in culture.

Animals↗
PubMed1991

Ah receptor in mice genetically "nonresponsive" for cytochrome P4501A1 induction: cytosolic Ah receptor, transformation to the nuclear binding state, and induction of aryl hydrocarbon hydroxylase by halogenated and nonhalogenated aromatic hydrocarbons in embryonic tissues and cells.

The aromatic hydrocarbon (Ah) receptor mediates induction of cytochrome P4501A1 and associated aryl hydrocarbon hydroxylase (AHH) activity in tissues or cells exposed to polycyclic aromatic hydrocarbons. Strains of mice designated "nonresponsive" do not show increased hepatic AHH activity when exposed in vivo to nonhalogenated aromatic hydrocarbons such as 3-methylcholanthrene, benz[a]anthracene (BA), or benzo[a]pyrene and have reduced sensitivity to halogenated inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Recently, with a modified assay, we detected Ah receptor in hepatic cytosols from adult nonresponsive mice [Mol. Pharmacol. 35:823-830 (1989)]; the receptor was present in reduced amount, and the apparent affinity for TCDD was lower than in hepatic cytosol from responsive C57BL/6J mice. Using the same assay procedure, we now report detection of Ah receptor in cytosols prepared from embryonic tissue and from cultured embryo cells of both responsive (C57BL/6J) and nonresponsive mice (DBA/2J, AKR/J, and SWR/J). Cytosolic receptor in embryonic cells from nonresponsive as well as responsive strains was detectable both with [3H]TCDD and with [3H]3-methylcholanthrene. In addition, the receptor-ligand complex could be extracted from nuclei of embryo cells exposed to [3H]TCDD in culture. AHH activity was induced in embryo cell cultures incubated with either TCDD or BA. The EC50 values for AHH induction were virtually identical in cell cultures from nonresponsive (DBA/2J) and responsive (C57BL/6J) strains, using either TCDD or BA as the inducer. Moreover, the affinity with which [3H]TCDD bound to cytosolic Ah receptor was much more similar in cytosols from cell cultures from the two strains than in cytosols prepared from adult liver. Thus, embryonic cell cultures differ in at least three respects from the adult liver, as follows: (i) Ah receptor can be detected with [3H]3-methylcholanthrene in embryonic cell cytosols but not in cytosols from adult liver; (ii) the degree of difference between nonresponsive and responsive strains in the affinity with which [3H]TCDD binds to receptor is only about 2-fold in cytosol from embryonic cells, whereas it is almost 10-fold in adult liver; and (iii) induction of AHH activity (by either TCDD or by the nonhalogenated inducer BA) shows no significant difference between strains in embryonic cell culture, whereas there is at least a 15-fold difference in responsiveness between C57BL/6J and DBA/2J mice in adult liver in vivo. The mechanistic reason for the diminished degree of difference between responsive and nonresponsive mice during embryonic cell culture (compared with adult tissues) is not yet known.

Animals↗
PubMed1990

Equivalent molecular mass of cytosolic and nuclear forms of Ah receptor from Hepa-1 cells determined by photoaffinity labeling with 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin.

The structure of the Ah receptor previously has been extensively characterized by reversible binding of the high affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin. We report the use of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin as a photoaffinity ligand for Ah receptor from the mouse hepatoma cell line Hepa-1c1c9. Both cytosolic and nuclear forms of Ah receptor could be specifically photoaffinity-labeled, which allowed determination of molecular mass for the two forms under denaturing conditions. After analysis by fluorography of polyacrylamide gels run in the presence of sodium dodecyl sulfate, molecular mass for the cytosolic form of Ah receptor was estimated at 92,000 +/- 4,300 and that for the nuclear form was estimated at 93,500 +/- 3,400. Receptor in mixture of cytosol and nuclear extract (each labeled separately with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin) migrated as a single band. These results are consistent with the presence of a common ligand-binding subunit of identical molecular mass in both cytosolic and nuclear complexes.

Affinity Labels↗
PubMed1990

Competitive binding of 7-substituted-2,3-dichlorodibenzo-p-dioxins with human placental ah receptor--a QSAR analysis.

The competitive binding affinities of thirteen 7-substituted-2,3-dichlorodibenzo-p-dioxins to the human placental cytosolic aryl hydrocarbon (Ah) receptor were determined using [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin as the radioligand. Multiple parameter linear regression analysis of the competitive binding C50 values for these compounds gave the following equation: pEC50 (M) = 6.246 + 1.632 pi - 1.764 sigma 0m + 1.282 HB where pi, sigma m and HB are the physiochemical parameters for substituent lipophilicity, meta-directing electronegativity, and hydrogen bonding capacity respectively. The 7-t-butyl- and 7-phenyl-2,3-dichlorodibenzo-p-dioxins were treated as outliers for the derivation of this equation, and these results suggest that only substituents with van der Waals' volumes less than 40 cm3/mol were accommodated in the receptor binding site. The equations previously derived from the binding of the 7-substituted-2,3-dichlorodibenzo-p-dioxins to the rat, mouse, guinea pig, and hamster hepatic cytosolic receptor were different than the correlation obtained using human placental receptor and provide further evidence for the interspecies differences in the molecular and binding properties of the Ah receptor protein.

Binding, Competitive↗
PubMed1990

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.

Aryl Hydrocarbon Hydroxylases↗
PubMed1990

Nuclear Ah receptor from mouse hepatoma cells: effect of partial proteolysis on relative molecular mass and DNA-binding properties.

The nuclear Ah receptor from mouse hepatoma (Hepa-1c1c9) cells is a 176-kDa multimeric protein which is stable under conditions of up to 1 M KCl. Under denaturing conditions, the Hepa-1 nuclear receptor can be dissociated into a ligand-binding subunit of Mr approximately 91,000. The identity of subunits that compose the nuclear Ah receptor is currently unknown. We used partial proteolysis under nondenaturing conditions as an approach to study the domain organization of the nuclear form of Ah receptor from Hepa-1c1c9 cells treated with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in culture. Low concentrations of trypsin (0.5 microgram/mg nuclear protein) generated heterogeneous fragments with the main fragment having a Stokes radius (Rs) approximately 6 nm. More discrete ligand-binding fragments of Mr approximately 84,000 (Rs approximately 4 nm/approximately 5 S) and Mr approximately 16,000 (Rs approximately 2 nm/approximately 2 S) could be generated using higher concentrations of trypsin (5 micrograms/mg nuclear protein). The relative concentration of the 84 and 16-kDa fragment was dependent on duration of protease treatment; formation of the 16-kDa fragment was accompanied by some loss in [3H]TCDD binding. Treatment of nuclear Ah receptor with alpha-chymotrypsin (1 microgram/mg nuclear protein) generated a single, apparently homogeneous ligand-binding fragment of Mr approximately 101,000 (Rs approximately 5 nm/approximately 5 S). When analyzed by DNA-cellulose chromatography, the chymotryptic fragment eluted at a significantly higher KCl concentration (462 mM) compared to native untreated nuclear Ah receptor (385 mM). Despite this increased affinity for DNA-cellulose columns, the ligand-binding fragment generated by chymotrypsin treatment was unable to interact with a dioxin responsive element in a gel retardation assay. DNA-cellulose binding ability, therefore, does not appear to be a reliable indicator of specific DNA interactions for these protease-modified fragments.

Animals↗
PubMed1990

Detection and characterization of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin binding to Ah receptor in a rainbow trout hepatoma cell line.

Ah receptor was identified and characterized in cytosol and nuclear extracts from the rainbow trout hepatoma cell line RTH-149. The cytosolic receptor was detectable with both halogenated ([3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) and non-halogenated ([3H]3-methylcholanthrene and [3H]benzo[a]pyrene) aromatic hydrocarbons and sedimented at approximately 9 S after velocity sedimentation on sucrose gradients. The apparent binding affinity (kd) of cytosolic Ah receptor was always less than 1 nM as derived from Scatchard or Woolf plot analyses. The same analyses indicated a concentration of Ah receptor in the RTH-149 cells of approximately 20 fmol/mg cytosolic protein or approximately 4400 receptor sites per cell. Thus, this trout hepatoma cell line has a low concentration of high-affinity binding sites in comparison to Ah receptor concentrations in cytosol obtained from rodent tissues. Incubation of whole cells with the radioligand [3H]TCDD resulted in transformation of the cytosolic Ah receptor to a nuclear binding form which could be detected as a specifically labeled peak sedimenting at approximately 6 S on sucrose gradients. Aryl hydrocarbon hydroxylase was induced after exposure of RTH-149 cells to TCDD or benz[a]anthracene for 24 hr in culture. These data demonstrate the existence of the Ah receptor in a cell line derived from a nonmammalian species and provide an additional step toward understanding the mechanisms by which fish respond to specific aquatic contaminants.

Animals↗
PubMed1989

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 approximately 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 approximately 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 approximately 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.

Animals↗
PubMed1989

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.

Alkaline Phosphatase↗
PubMed1989

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.

Animals↗
PubMed1989

Detection and characterization of a low affinity form of cytosolic Ah receptor in livers of mice nonresponsive to induction of cytochrome P1-450 by 3-methylcholanthrene.

Ah "nonresponsive" mice (prototype, DBA/2) show no significant increase in hepatic P1-450 (P450IA1) when treated with 3-methylcholanthrene or other nonhalogenated polycyclic aromatic hydrocarbons. Potent halogenated aromatics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induce P1-450 in liver of nonresponsive mice, but the dose required is approximately 15-fold higher than in "responsive" mice (prototype, C57BL/6). It was postulated several years ago that the genetic basis of nonresponsiveness was a "defect" in the Ah receptor, which normally binds TCDD and other inducers and mediates the induction process. Cytosolic Ah receptor hitherto had not been detectable in hepatic cytosol from nonresponsive mice. Using a modified sucrose gradient assay that we developed in studies on human tissue [Cancer Res. 47:4861-4868 (1987)], we now have detected cytosolic Ah receptor in nonresponsive mice. By saturation analysis, the concentration of specific binding sites for [3H]TCDD in hepatic cytosol from DBA/2J mice was (mean +/- SE) 55 +/- 6.6 fmol/mg of cytosolic protein (n = 21) compared with 133 +/- 7.1 fmol/mg (n = 21) in responsive C57BL/6J mice. Ah receptor also was detected in significant concentrations in other nonresponsive strains; SWR/J, AKR/J, RF/J, and DBA/2N. The sedimentation coefficient on sucrose gradients was the same (approximately 9 S) in nonresponsive as in responsive strains. The major difference in nonresponsive mice is that hepatic cytosolic Ah receptor has an apparent affinity for [3H]TCDD that is about 10-fold lower than in responsive strains; Kd in DBA/2J mice = 16 +/- 2.5 nM (n = 21) and Kd in C57BL/6J mice = 1.8 +/- 0.2 nM (n = 21). Thus, nonresponsive mice do possess the cytosolic Ah receptor in liver. However, the receptor is present in reduced concentration and appears to be a low affinity form, possibly as the result of a mutation in the gene(s) coding for the receptor protein(s).

Animals↗
PubMed1988

Characterization of the Ah receptor and aryl hydrocarbon hydroxylase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin and benz(a)anthracene in the human A431 squamous cell carcinoma line.

Certain human cell lines previously have been shown to exhibit substantial induction of aryl hydrocarbon hydroxylase (AHH, cytochrome P450IA1) when treated in culture with aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz(a)anthracene. Yet the Ah receptor, which is known to mediate the AHH induction process in rodent cells and tissues, has not previously appeared to be present at a significant level in any human cell line. In the human A431 squamous cell carcinoma line we found that cytosolic Ah receptor was present in high concentration (approximately 200 fmol/mg cytosol protein at maximal saturation); this corresponds to approximately 10,000 Ah receptor sites per cell in the human A431 line compared with about 35,000 receptor sites per cell in the mouse Hepa-1 hepatoma cell line in which Ah receptor previously has been extensively characterized. Detection of Ah receptor in A431 cytosol required modification of assay techniques, especially reduction in the amount of charcoal used to adsorb nonspecifically bound radioligand. The specific binding peak from A431 cytosol sedimented approximately 9S on sucrose gradients, the same as the cytosolic receptor from the well-characterized mouse Hepa-1 hepatoma cell line. In addition to [3H]TCDD, specific binding to Ah receptor in A431 cytosol also was detected with [3H]3-methylcholanthrene and with [3H]benzo(a)pyrene as radioligands. A specific [3H]TCDD-Ah receptor complex was extracted from nuclei of A431 cells incubated in culture at 37 degrees C with [3H]TCDD. The nuclear form of Ah receptor sedimented approximately 5S, the same as the nuclear receptor from mouse Hepa-1 cells. AHH activity was induced in A431 cells treated in culture with TCDD or benz(a)anthracene. The maximum level of induced AHH activity that could be achieved in A431 cells was about 20% of the maximally induced level in the mouse Hepa-1 cell line. However, the dose-response curves for AHH induction by TCDD or benz(alpha)anthracene in A431 cells were shifted about one log unit to the right of the curves for Hepa-1 cells. The lower sensitivity of A431 cells to AHH inducers was in proportion to the lower affinity with which cytosolic Ah receptor in A431 cells bound [3H]TCDD. The saturation curve for binding of [3H]TCDD to cytosolic Ah receptor in A431 cells also was shifted about one log unit to the right of the curve for saturation of the cytosolic receptor from mouse Hepa-1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Aryl Hydrocarbon Hydroxylases↗