Kinetic models for association of 2,3,7,8-tetrachlorodibenzo-p-dioxin with the Ah receptor

Saturation binding studies of the interaction between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the Ah receptor obtained from the hepatic cytosol of Wistar rats have been carried out. The conventional Scatchard analysis for determination of the equilibrium constant for ligand-receptor binding has been shown to be inappropriate due to thermal inactivation of the unoccupied receptor. Simulation models of the receptor-ligand binding kinetics which take into account receptor degradation have been developed and the results are consistent with two alternative kinetic models. In Model 1, reversible 2,3,7,8-TCDD-receptor binding occurs in parallel with inactivation of the unbound receptor; analysis of the observed data using this model suggests that the previously determined equilibrium constants (Kass) for association of the ligand with the receptor are orders of magnitude too low and the total initial receptor concentrations are somewhat underestimated. In Model 2, the unbound receptor is converted unimolecularly to an activated state which then undergoes competitive degradation or entrapment by ligand. Experiments have been carried out over the temperature range 4-37 degrees C, enabling activation parameters to be obtained. According to Scheme 1, the activation enthalpies for association of receptor with ligand and for thermal inactivation of the unoccupied receptor are high, and numerically almost identical (delta H++ ca 125 kJ mol-1). These reactions are strongly entropically driven and this is consistent with association being accompanied by a conformational change in the receptor protein, and the previously postulated binding of the ligand to a hydrophobic pocket. According to Scheme 2, there is only one enthalpy of activation because both inactivation and entrapment by 2,3,7,8-TCDD are fast processes which follow the same slow activation step. On the basis of this latter model, a 10(-9) M concentration of 2,3,7,8-TCDD is sufficient to trap roughly two-thirds of the activated receptors.     

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.     

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

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

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)     

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

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

Ah receptor 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.     

Effects of thyroidectomy on the Ah receptor and enzyme inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat liver

Thyroidectomy of rats confers some protection, by an unknown mechanism, from the weight loss, immunotoxicity, and mortality induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Since at least some of the many effects of TCDD appear to be mediated by the Ah receptor, perhaps the thyroid plays a role in regulation of this receptor, thereby modifying the toxicity of TCDD. We tested this hypothesis by comparing TCDD-binding characteristics of the receptor and hepatic enzyme inducibility by TCDD (a receptor-mediated response) in thyroidectomized (ThX) and euthyroid rats. There were no significant differences in levels of TCDD binding in vitro in hepatic cytosol, in receptor affinity, nor in the molecular size of the TCDD-bound receptor in untreated ThX rats compared to controls fed ad libitum or pair-fed. Total hepatic cytochrome P-450 (P-450) levels and NADPH-menadione oxidoreductase (NMOR) activity were unaffected by thyroid status, whereas 7-ethoxycoumarin O-deethylase (ECOD) activity was approx. 50% lower in ThX animals than in ad libitum or pair-fed controls. At 3 and 10 days after TCDD administration (10 micrograms/kg, i.p.), P-450 concentrations and NMOR and ECOD activities were induced by approximately the same proportions in ThX and pair-fed intact rats; however, the absolute levels of the induced activities were lower in ThX than in pair-fed controls. It was concluded that hypothyroidism does not regulate Ah receptor concentration or function in the liver. Therefore, the modulation of TCDD toxicity by hypothyroidism appears not to involve changes in the hepatic Ah receptor.     

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.     

Ah receptor involvement in mediation of pyruvate carboxylase levels and activity in mice given 2,3,7,8-tetrachlorodibenzo-p-dioxin

The arylhydrocarbon receptor (AhR) plays a central role in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity in animals. The investigations described here provide evidence that support a role for the AhR in TCDD-mediated pyruvate carboxylase (PC) level/activity reductions in mice. Pyruvate carboxylase plays a pivotal role in gluconeogenesis and in supplying carbon units for the citric acid cycle. Delivered ip in a corn oil carrier, TCDD suppresses PC activity/amount at doses as low as 1 μg/kg in responsive C57BL/6J(Ah^b/b) mice. Corn oil alone injected ip into mice at 4 mL/kg appears to be an inducer that increases the amount and activity of PC. However, TCDD suppresses this induction. In the Ah^b/b mouse, PC levels and activity are reduced to 10% of control values at a dose of 75 μg/kg. A time-course experiment shows that the PC reductions are apparent within 16 hours post-TCDD exposure. Here we report investigations on the PC/TCDD response using a congenic C57BL/6J(Ah^d/d) mouse strain having an AhR with a low affinity for TCDD. If the PC/TCDD response is AhR mediated, the congenic mouse strain (Ah^d/d) would require much higher doses of TCDD to suppress PC. In the Ah^d/d mice, we observe that an approximately 60-fold increase in TCDD dose is necessary to produce a PC/TCDD effect. We also find that in Ah^d/d mice, corn oil does not induce an increase in PC activity/amounts, as reported for Ah^b/b mice.     

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.     

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.     

Interaction of hexachlorobenzene with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro and in vivo. Evidence that hexachlorobenzene is a weak Ah receptor agonist

Hexachlorobenzene (HCB) produces hepatic porphyria and induces the hepatic cytochrome P450 isozymes P450c (P450IA1) and P450d (P450IA2) in rodents. These and other effects of HCB resemble those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which acts via its binding to the aromatic hydrocarbon (Ah) receptor. We therefore examined the ability of HCB to interact with this receptor in vitro and in vivo. HCB, at concentrations of 1 microM or higher, inhibited the specific binding of [3H]TCDD (0.3 nM) to the Ah receptor in vitro, whereas the solubility of [3H]TCDD was affected only at 100 microM HCB. The inhibition was competitive, with a KI of approximately 2.1 microM. In rats fed a diet containing 3000 ppm HCB for varying times (4 h to 7 days), the specific binding of [3H]TCDD in hepatic cytosol was reduced by up to 40%, as observed previously for known Ah receptor agonists. The decrease in [3H]TCDD specific binding in cytosol of HCB-treated rats was due principally to a decrease in the number of binding sites for [3H]TCDD rather than competition from residual HCB. As shown by immunoblotting and radioimmunoassay, HCB induced the cytochrome P450 isozymes P450c and P450d, which are regulated by the Ah receptor, as well as the phenobarbital-inducible isozymes P450b and P450e. Together these results indicate that HCB is a weak agonist for the Ah receptor, and suggest that some of its effects may be mediated by its interaction with this gene-regulatory protein.     

Influence of proteinase inhibitors and substrates on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-binding capacity of the rat hepatic Ah receptor

These studies investigated the effects of various serine proteinase inhibitors and substrates on the TCDD-binding capacity of the rat hepatic Ah receptor. TCDD binding to the Ah receptor was inhibited by serine proteinase inhibitors phenylmethylsulfonyl fluoride (PMSF), tosyl-lysine chloromethyl ketone (TosLysCH2Cl), tosylamide-phenylethyl chloromethyl ketone (TosPheCH2Cl) and substrates tosyl-L-arginine methyl ester (TosArgOMe) and D-tryptophan methyl ester (TrpOMe). The order of potency was TosPheCH2Cl greater than TosLysCH2Cl much greater than PMSF approximately equal to TosArgOMe approximately equal to TrpOMe. Reactivity of the chloromethyl ketones with sulfhydryl groups was suggested by their steep inhibition curves above the concentration of nonprotein sulfhydryl groups, and the partial mitigation of inhibition by 1 mM dithiothreitol. Inhibition by these reagents was irreversible, while that by TosArgOMe and TrpOMe was completely reversible by gel filtration. The mechanism of inhibition by TosArgOMe and TrpOMe was formally competitive, with inhibition constants similar to those reported in steroid hormone receptor systems. Neither inhibitors nor substrates displaced previously bound TCDD.     

Response of murine epidermis to 2,3,7,8-tetrachlorodibenzo-p-dioxin: Interaction of the Ah and hr loci

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons produce epidermal hyperplasia, hyperkeratosis and sebaceous gland metaplasia in the skin of mice bearing the recessive mutation (hr/hr) hairless. This response is mediated through the cytosol receptor protein: the structure-activity relationship for receptor binding corresponds to that for production of the skin lesion, and these histopathological changes segregate with the genetic polymorphism at the Ah locus, the locus determining the cytosol receptor. In HRS/J mice, an inbred strain segregating for the hr locus, both hairless (hr/hr) and haired (hr/ +) mice possess the high-affinity cytosol receptor and respond to TCDD with the induction of epidermal aryl hydrocarbon hydroxylase activity, a receptor-mediated biochemical response; however, only hr/hr mice develop the proliferative/metaplastic skin response. We propose a genetic model for the interaction of the Ah and hr loci, to account for the differential response to TCDD observed in the skin of HRS/J hr/hr and hr/ + mice.     

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 (K_d 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.     

Polyanionic-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. A comparison with the glucocorticoid receptor

The interaction of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) with immobilized heparin (heparin-Sepharose) or DNA (DNA-cellulose) has been compared to the polyanionic-binding properties of the rat hepatic glucocorticoid receptor. Both the nonoccupied and in vitro occupied forms of the receptors interacted with heparin-Sepharose but with varying strength, as determined by ligand binding assays or an enzyme-linked immunosorbent assay based on a monoclonal antibody against the steroid- and DNA-binding Mr approximately 94,000 glucocorticoid receptor protein. In the absence of ligand, both the dioxin and glucocorticoid receptors eluted from heparin-Sepharose at 0.1-0.2 M KCl, in contrast to the in vitro occupied receptor forms which eluted at 0.3-0.4 M KCl. Following elution of the in vitro occupied dioxin receptor from heparin-Sepharose, it was efficiently retained on DNA-cellulose and eluted at an ionic strength of approximately 0.2 M KCl. In the presence of 20 mM sodium molybdate which is known to inhibit the activation of steroid hormone receptors to a DNA-binding form, both the dioxin and glucocorticoid receptors eluted at 0.1-0.2 M KCl from heparin-Sepharose. In analogy to what has previously been shown for the glucocorticoid receptor, sodium molybdate stabilized a large dioxin-receptor complex with a sedimentation coefficient, S20,w, of 9-10 S, a Stokes radius of approximately 7.5 nm, and a calculated Mr of 290,000-310,000. Limited proteolysis of both the dioxin and glucocorticoid receptors with trypsin which is known to eliminate the DNA-binding property of both receptor forms also resulted in a decreased strength in the interaction of both in vitro occupied receptors with heparin-Sepharose (elution at 0.1-0.2 M KCl). In line with these data, calf thymus DNA in solution competed for receptor binding to heparin-Sepharose. In conclusion, the chromatographic properties of the dioxin receptor on heparin-Sepharose are indistinguishable from those of the glucocorticoid receptor, and both receptors appear to be structurally and functionally closely related proteins.     

Compounds in urban air compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin for binding to the receptor protein

Acetone extracts of filter-collected urban airborne particulate matter contain compounds which can competitively inhibit 2,3,7,8-[1,6-3H]tetrachlorodibenzo-p-dioxin (TCDD) binding to the rat liver TCDD-receptor protein. The concentration of conventional polycyclic aromatic hydrocarbons (PAHs) or chlorinated dioxins and dibenzofurans cannot account for more than 1-30% of the observed competition for [3H]TCDD binding to the receptor protein. The difference in potency between samples collected in urban areas during different periods of the year and a background sample is 25-400-fold. Collecting samples in the presence of increased concentrations of nitrogen dioxide, nitrous acid, nitric acid or ozone did not increase the amount of compounds with receptor affinity. However, with nitrogen dioxide and especially with nitric acid, a substantial increase of the mutagenic effects in the Ames Salmonella assay in the absence of mammalian activation as well as a degradation of several PAHs were noted. Affinity for the TCDD-receptor protein, mutagenicity in the absence of mammalian metabolic activation in the Ames Salmonella assay and PAH-content are characteristics of urban particulate matter showing the presence of compounds, that represent potential health risks. The compounds with affinity for the receptor may constitute a group of substances different from both conventional PAHs and direct-acting mutagens.     

Actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human epidermal keratinocytes in culture

Summary In humans, the skin is a particularly sensitive target for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and certain halogenated analogs. Reported lesions include a thickening of the epidermis (acanthosis), hyperkeratosis, and squamous metaplasia of the epithelial lining of the sebaceous glands. In this report we describe ongoing studies on the actions of TCDD on cultured human epidermal cells. This system has been established as an in vitro model for interfollicular epidermal hyperkeratinization. Treatment of newly confluent cultures with TCDD results in enhanced differentiation as judged by histologic examination of the cultures, a decrease in the number of basal proliferating cells, and an increase in the number of envelope competent (differentiating) cells and terminally differentiated cells with highly cross-linked cornified envelopes. Changes in the differentiation program are preceded by a decrease in epidermal growth factor (EGF) binding. The concentration dependence and stereospecificity for these responses suggest the involvement of theAh receptor. We propose that TCDD modulates normal patterns of epidermal differentiation through direct actions on proliferating basal cells, modulating the responsiveness of these cells to growth factors such as EGF. This paper was presented at the Session-In-Depth on In Vitro Applications in Toxicology at the 34th Annual Meeting of the Tissue Culture Association, Orlando, FL, June 12–16, 1983. Rosemarie Osborne was a Chemical Industry Institute of Toxicology Postdoctoral Fellow.     

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.