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A B Okey

Publications and source records attributed to A B Okey.

At least 55 records · Page 3Linked to original sources
PubMed1988

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)

Animals↗
PubMed1987

Ah receptor in human placenta: stabilization by molybdate and characterization of binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, and benzo(a)pyrene.

Aryl hydrocarbon hydroxylase (AHH, cytochrome P1-450) is highly inducible in several human cells and tissues exposed to specific halogenated and nonhalogenated aromatic chemicals of the "3-methylcholanthrene-type." In laboratory animals AHH induction is known to be regulated by binding of inducers to the Ah receptor, a soluble intracellular protein. However, the induction mechanism in the human species is incompletely understood largely because the Ah receptor, which seems to be essential to the induction process, has not previously been detectable in certain human cells and tissues (including placenta) that are highly responsive to AHH induction. We found that human placenta contains high concentrations of Ah receptor (comparable to the receptor concentrations in rat and mouse liver) but that special modifications were necessary in the assay techniques in order to detect and accurately quantitate receptor binding. Receptor was detected at concentrations approximately equal to 100 fmol/mg cytosol protein using [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as the radioligand. This high concentration of specific binding sites was present only if the placental tissue was initially homogenized in a buffer containing sodium molybdate (10 or 20 mM). Without molybdate in the homogenizing buffer, specific [3H]TCDD binding was only about 35 fmol/mg. Specific Ah receptor binding also was detectable with [3H]-3-methylcholanthrene and, to a lesser extent, with [3H]-benzo(alpha)pyrene. The receptor sedimented near 9S on sucrose gradients whether molybdate was present or not. About 80% of specific binding was lost if excessive charcoal was used to adsorb "nonspecifically bound" ligand from cytosol prior to gradient analyses. The apparent affinity with which [3H]TCDD bound to Ah receptor in human placental cytosol was relatively low (apparent Kd approximately equal to 5 to 8 nM) when compared with the affinity of [3H]TCDD binding in rat or mouse hepatic cytosols (Kd approximately equal to 1 to 3 nM). These data suggest that while molybdate has very little effect on the quantity or molecular size of the rodent Ah receptor assay, it is very important in stabilizing the human Ah receptor. Our experiments demonstrate that human placenta contains a high concentration of Ah receptor and suggest that AHH induction in placenta is mediated through a receptor mechanism analogous to that previously established in tissues and cells from laboratory animals.

Benzo(a)pyrene↗
PubMed1987

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.

Animals↗
PubMed1986

Hepatic Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Partial stabilization by molybdate.

In structure and general mode of action, the Ah receptor is very similar to the receptors for steroid hormones. Molybdate previously has been shown to be highly effective at preserving ligand-binding function in steroid receptors during their exposure to elevated temperature or high ionic strength and at stabilizing steroid receptors as high molecular weight oligomeric complexes. Since such stabilization by molybdate can be very useful during characterization and purification of receptors, we tested the effects of molybdate on the Ah receptor to determine if the Ah receptor, like the receptors for steroid hormones, might be stabilized. In hepatic cytosols from C57BL/6N mice and Sprague-Dawley rats, molybdate concentrations up to 30 mM in homogenizing and analysis buffers did not alter the concentration of specific Ah receptor sites detected by binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. However, inclusion of 20 mM molybdate in the homogenizing buffer did significantly protect unliganded Ah receptor from thermal inactivation at 20 degrees C and from KCl-induced loss of ligand-binding ability. In accord with previous reports, 20 mM molybdate in homogenizing and analysis buffers greatly increased the concentration of detectable glucocorticoid receptor in rat hepatic cytosol and estrogen receptor in rat uterine cytosol. Exposure to 0.4 M KC1 caused the glucocorticoid receptor from rat liver to shift sedimentation from approximately equal to 8 S to approximately equal to 4 S and caused a severe loss of specific glucocorticoid binding. Presence of 20 mM molybdate stabilized the glucocorticoid receptor as a single discrete peak sedimenting at approximately equal to 8 S. In contrast, the Ah receptor from rat liver exposed to 0.4 M KC1 in the presence of molybdate sedimented as biphasic peaks; one peak (approximately equal to 9.5 S) corresponded to the form of Ah receptor observed at low ionic strength, while the other peak (approximately equal to 5.5 S) corresponded to the form of Ah receptor seen in cytosol treated with 0.4 M KC1 in the absence of molybdate. Addition of heparin to hepatic cytosols from mice or rats shifted sedimentation of Ah receptor from approximately equal to 9.5 S to approximately equal to 5.5 S. Molybdate, again, provided stabilization in the approximately equal to 9.5 S form, but only for about one-half the total Ah receptor content in both rat and mouse hepatic cytosols. In sum, molybdate is far less effective at stabilizing rodent Ah receptors than it is at stabilizing steroid receptors in the same species.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Codistribution of unoccupied receptor with cytosolic marker enzymes during fractionation of mouse liver, rat liver and cultured Hepa-1c1 cells.

In early experiments Ah receptor appeared to be localized in cytosol when in its unoccupied state and it was thought that the receptor translocated into nuclei only when occupied by its ligands. However, a recent report [Whitlock and Galeazzi (1984) J. Biol. Chem. 259, 980-985] concluded that unoccupied Ah receptor in the intact cell was primarily located within the nucleus and that apparent 'cytosolic' Ah receptor was a redistribution artifact caused by fractionation of cells in large volumes of buffer. We examined the effect of buffer volume and ionic strength on apparent 'cytosolic' versus 'nuclear' distribution of unoccupied Ah receptor in liver from C57BL/6J mice and Sprague-Dawley rats as well as Hepa-1c1c9 cells in culture. In all three systems the Ah receptor appears to shift out of the nuclear fraction and into the cytosolic fraction as the volume of buffer is increased or when the ionic strength of the buffer is increased. In each system, however, the distribution of the Ah receptor was identical to the distribution of each of three standard cytosolic marker enzymes: aldolase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase. Co-distribution of unoccupied Ah receptor with these cytosolic marker enzymes during fractionation at varied buffer volumes and ionic strengths makes it seem unlikely that the unoccupied receptor is predominantly a 'nuclear' component in intact cells. Marker enzyme data favor an interpretation that unoccupied Ah receptor is primarily cytoplasmic or that this soluble protein is in equilibrium between cytoplasm and nucleus.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

Induction of drug-metabolizing enzymes: mechanisms and consequences.

The activity of many enzymes that carry out biotransformation of drugs and environmental chemicals can be substantially increased by prior exposure of humans or animals to a wide variety of foreign chemicals. Increased enzyme activity is due to true enzyme induction mediated by increased synthesis of mRNAs which code for specific drug-metabolizing enzymes. Several species of cytochrome P-450 are inducible as are certain conjugating enzymes such as glutathione S-transferases, glucuronosyl transferases, and epoxide hydrolases. Induction of drug-metabolizing enzymes has been shown in several instances to alter the efficacy of some therapeutic agents. Induction of various species of cytochrome P-450 also is known to increase the rate at which potentially toxic reactive metabolic intermediates are formed from drugs or environmental chemicals. Overall, however, induction of drug-metabolizing enzymes appears to be a beneficial adaptive response for organisms living in a "chemically-hostile" world.

Animals↗
PubMed1986

Ah receptor mediating induction of aryl hydrocarbon hydroxylase: detection in human lung by binding of 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin.

In laboratory animals and in mouse hepatoma cells in culture the Ah receptor previously has been shown to mediate induction of aryl hydrocarbon hydroxylase (cytochrome P1-450) by 3-methylcholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. We examined human lung cytosols to determine whether the Ah receptor was present in human tissues. Cytosol was prepared from grossly normal lung tissue obtained at pulmonary lobectomy for presumed lung cancer from 53 consecutive adult patients including 32 males (42-77 years old) and 21 females (18-81 years old). Ah receptor in the cytosols was identified and quantitated by specific binding of [3H]TCDD after separation by ultracentrifugation on sucrose gradients. Specific binding of [3H]TCDD to a component which met the criteria for Ah receptor was detected in 10 of the 53 specimens. As previously established in tissues from laboratory animals, the specific [3H]TCDD-binding component sedimented approximately 9S. Binding of [3H]TCDD to the 9S component was competitively inhibited by incubation in the presence of 2,3,7,8-tetrachlorodibenzofuran, dibenz(a,h)anthracene, and nonradioactive TCDD, all known to be potent agonists for Ah-receptor-mediated induction of aryl hydrocarbon hydroxylase. Specific Ah receptor also was detected in some specimens by direct binding of [3H]-3-methylcholanthrene. The human population studied exhibited striking heterogeneity in Ah receptor concentrations. Only 10 of the 53 individuals studied had detectable Ah receptor. In specimens with detectable specific binding, the mean concentration of binding sites was 6.9 +/- 1.2 (SE) fmol/mg cytosolic protein. These concentrations are approximately 10-30% of the concentrations of Ah receptor found in lung cytosols from laboratory animals. Our experiments indicate that the Ah receptor can be detected in lung cytosol from some humans and suggest that the regulatory mechanism mediating human cytochrome P1-450 induction may be similar to that in the murine model. Aryl hydrocarbon hydroxylase, the major enzyme induced under control of the Ah receptor, plays an important role in the metabolism of several carcinogens including polycyclic aromatic hydrocarbons such as benzo(a)pyrene. It is possible that differences in the Ah receptor content within the human population may be genetically based and that variation at the Ah receptor level may be an important determinant of individual susceptibility to certain chemically induced cancers.

Aryl Hydrocarbon Hydroxylases↗
PubMed1985

Effects of structure on binding to the 2,3,7,8-TCDD receptor protein and AHH induction--halogenated biphenyls.

The quantitative structure-activity relationships (QSARs) for polychlorinated biphenyl (PCB) congeners have been determined by comparing the EC50 values for three in vitro test systems, namely, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction in rat hepatoma H-4-II-E cells and competitive binding avidities to the rat cytosolic receptor protein (using 2,3,7,8-tetrachlorodibenzo-p-dioxin as a radioligand). For several PCB congeners that are in vivo inducers of rat hepatic microsomal AHH, there was a linear correlation between the -log EC50 values for receptor and the -log EC50 values for AHH (or EROD) induction; moreover, a comparable linear relationship was observed between the -log EC50 values for AHH and EROD induction. Previous in vivo studies have shown that the most active PCB congeners 3,3',4,4'-tetra-, 3,4,4',5-tetra-, 3,3',4,4',5-penta-, and 3,3',4,4',5,5'-hexachlorobiphenyl, cause many of the biologic and toxic effects reported for the highly toxic halogenated aryl hydrocarbon, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Moreover, the monoortho-substituted homologs of the four coplanar PCBs also elicit comparable in vivo biologic and toxic responses. It was evident from the QSARs for PCBs that there was an excellent correspondence between the in vivo and in vitro potencies of the individual PCB congeners. The effects of substituents on both receptor binding and AHH/EROD induction was determined for a series of 4'-substituted (X)-2,3,4,5-tetrachlorobiphenyls (where X = H, Cl, Br, I, OH, OCH3, NO2, COCH3, F, CF3, CH3, C2H5, i-C3H7, n-C4H9 and t-C4H9). Not unexpectedly, there was a linear relationship between the -log EC50 values for AHH and EROD induction, and these results confirm that both enzymatic oxidations are catalyzed by the same cytochrome P-450 isozyme(s). The effects of substituent structure on receptor binding for 12 substituents was subjected to multiple regression analysis which correlates the relative binding affinities of the compounds with the physical chemical characteristics of the substituents. The analysis gave the following equation: log (1/EC50) = 1.53 sigma + 1.47 pi + 1.09 HB + 4.08 for n = 12, s = 0.18, r = 0.978; where n is the number of substituents, s is the standard deviation, r is the correlation coefficient, and sigma = electronegativity, pi = hydrophobicity (log P) and HB = hydrogen bonding capacity for the substituent groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Elevated binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene to the Ah receptor in hepatic cytosols from phenobarbital-treated rats and mice.

Binding of 3-methylcholanthrene (MC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and other "MC-type" inducers to cytosolic Ah receptor sites is the first specific step in induction of aryl hydrocarbon hydroxylase (AHH; cytochrome P1-450) by these compounds. [3H]TCDD and [3H]MC were used as radioligands to quantitate and characterize Ah receptor in hepatic cytosols from genetically "responsive" C57BL/6J mice, genetically "nonresponsive" DBA/2J mice, and AHH-inducible Sprague-Dawley rats. Injection of 50-100 mg/kg of phenobarbital (PB) for 3 days more than doubled the concentration of Ah receptor in hepatic cytosol from Sprague-Dawley rats. In C57BL/6J mice, PB injection at 25 mg/kg X 3 days significantly increased (P less than 0.01) the Ah receptor concentration in hepatic cytosol. No cytosolic Ah receptor was detectable in hepatic cytosol from untreated DBA/2J mice, nor did any Ah receptor appear after PB treatment in this "nonresponsive" strain. Although PB significantly elevated Ah receptor in hepatic cytosols of responsive rodents, many previous studies have shown that the maximal level of AHH activity in animals given PB and an "MC-type" inducer simultaneously is additive rather than synergistic. Ah receptor concentrations can be doubled by PB treatment without doubling the subsequent AHH-induction response to "MC-type" compounds. Thus, the cytosolic Ah receptor concentration per se may not be the primary determinant of a given tissue's maximal capacity for AHH induction by "MC-type" compounds.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

Binding of benzo(a)pyrene and dibenz(a,h)anthracene to the Ah receptor in mouse and rat hepatic cytosols.

Binding of the polycyclic aromatic hydrocarbon (PAH) carcinogens [3H]benzo(a)pyrene (BP) and [3H]dibenz(a,h)anthracene [DB(a,h)A] to components in rodent hepatic cytosols was characterized by sucrose density gradient centrifugation and by gel permeation chromatography on Sephacryl S-300. In hepatic cytosols from Sprague-Dawley rats, [3H]BP and [3H]DB(a,h)A bound to a component which sedimented at 8 to 9S under low-ionic-strength conditions, and had a Stokes' radius of 4.7 nm. The 8 to 9S component also bound [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and [3H]-3-methylcholanthrene (MC), two compounds previously established as Ah receptor ligands. In hepatic cytosols from C57BL/6J mice [a strain genetically "responsive" for induction of aryl hydrocarbon hydroxylase (AHH, cytochrome P1-450) by PAHs], [3H]DB(a,h)A bound to a component sedimenting at 8 to 9S which had a Stokes' radius of approximately 6 nm. The 8 to 9S component also bound [3H]TCDD and [3H]MC, but no direct binding of [3H]BP could be detected at 8 to 9S in hepatic cytosol from C57BL/6J mice, nor could specific [3H]BP binding to the 6 nm component be detected in C57BL/6J cytosol by Sephacryl S-300 chromatography. The 8 to 9S component was not detectable in hepatic cytosol from DBA/2J mice (genetically "nonresponsive" to AHH induction by PAHs) with [3H]BP or [3H]DB(a,h)A, nor with the previously established Ah receptor ligands [3H]TCDD or [3H]MC. In addition to binding to the 8 to 9S-6 nm component in C57BL/6J cytosols, [3H]BP, [3H]DB(a,h)A, and [3H]MC also bound extensively to a cytosolic component sedimenting at 4 to 5S with a Stokes' radius of approximately 3 nm. The 4 to 5S-3 nm component was present in hepatic cytosols from genetically nonresponsive DBA/2J mice, as well as in genetically responsive C57BL/6J mice. Binding of [3H]BP, [3H]DB(a,h)A, and [3H]MC to the 8 to 9S component in rodent hepatic cytosols was eliminated by incubation in the presence of a 100-fold molar excess of nonradioactive TCDD, whereas TCDD had no effect on binding of [3H]BP, [3H]DB(a,h)A, or [3H]MC to the 4 to 5S-3 nm component. The 8 to 9S component segregates with the Ahb allele in genetic crosses with mice, but the 4 to 5S component is not associated with the Ahb allele which governs AHH responsiveness. Specificity of chemicals that bind to the 8 to 9S component and segregation of the 8 to 9S component with the Ahb allele identify it as the Ah receptor which functions to regulate AHH induction in animals treated with PAHs.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

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.

Animals↗
PubMed1982

Binding of polychlorinated biphenyls classified as either phenobarbitone-, 3-methylcholanthrene- or mixed-type inducers to cytosolic Ah receptor.

It has been postulated that reversible, high-affinity binding of 3-methylcholanthrene(MC)-type inducers to a receptor protein (the Ah receptor) in hepatic cytosol is essential for induction of aryl hydrocarbon hydroxylase (AHH) enzymic activity. To test this postulate, the binding affinities of 16 highly-purified, synthetic chlorinated biphenyl (PCB) congeners, which have been categorized either as phenobarbitone(PB)-, MC- or mixed (PB + MC)-type inducers of cytochrome P-450-dependent monooxygenase have been examined. The affinity of individual biphenyl congeners for the receptor was determined by their competition with 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin ([3H]TCDD) for specific cytosolic binding sites as measured by sucrose density gradient analysis following dextran-charcoal treatment. This assay demonstrates (a) that the receptor binds with highest affinity to 3,3', 4,4',5-pentachlorobiphenyl and 3,3',4,4'-tetrachlorobiphenyl ('pure MC-like' inducers); (b) mixed-type PCB inducers also bind to the receptor but with an affinity (average EC50-value of 8.6 microM) lower than that for 3,3',4,4',5-pentachlorobiphenyl; this corresponds with their relatively lower potencies as AHH inducers; (c) the receptor binds 2,2',4,4'-tetra-, 2,3,4,5-tetra-, 2,2',4,4',5,5'-hexa- and 2,3',4,4',5',6-hexachlorobiphenyl at high concentrations (0.1 mM), whereas PB fails to bind, even at a concentration of 10 mM. All PCBs tested competed with [3H]TCDD for Ah receptor, but there was a great variation in their relative binding affinities. The fact that two chlorinated biphenyls classed as 'PB-like' inducers and two chlorinated biphenyls which are neither 'PB- nor MC-type' inducers competed, coupled with the fact that PB did not bind to the receptor suggest that chemicals other than "pure MC-type' inducers can bind to the cytosolic receptor. Affinity of the binding dictates the relative potency of given PCB congeners as inducers of cytochrome P-448.

Animals↗