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.     

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.     

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.     

The aromatic hydrocarbon receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin and variable levels of induced aryl hydrocarbon hydroxylase activity in clones of mouse hepatoma cells

Induction of aryl hydrocarbon hydroxylase (AHH) activity was studied in clones and subclones of mouse hepatoma (Hepa-lcl) cells. When maximally induced, one clone had significantly lower (p less than 0.005), two had approximately the same, and two had significantly higher (p less than 0.005) levels of AHH activity compared with Hepa-lcl. The maximal level of induced activity, relative to the parent population, in two clones chosen for further analysis was 0.14 +/- 0.09 for clone 1 (Hs-1) and 0.94 +/- 0.28 for clone 9 (Hs-9). These relative levels were stable over a period of 10 months and were similar when activity was induced either with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz[a]anthracene. Subclones of Hepa-lcl cells, derived from the Hs-9 clone, also demonstrated variation in induced AHH activity. When maximally induced with TCDD, six subclones had significantly lower AHH activity (p less than 0.005), two had approximately the same, and one had significantly higher levels (p less than 0.005) compared with the progenitor Hs-9 population. Comparative analysis of Ah receptor characteristics in two unselected clones of Hepa-lcl with significantly different levels of AHH activity demonstrated that there was no apparent correlation between relative level of induced AHH activity and (i) total quantity of Ah receptor (cytosol and nuclear), (ii) receptor affinity for TCDD and number of receptor sites in each cell, (iii) subcellular distribution of [3H]TCDD, or (iv) specificity and saturable nature of binding. Coordinate measurement of the concentration of nuclear receptor and absolute induced AHH activity in Hepa-lcl and its clones had a positive correlation (r = 0.79).     

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.     

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.     

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.     

Photoaffinity labeling of the nuclear Ah receptor from mouse Hepa 1c1c7 cells using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin

The photoinduced formation of the covalently labeled cytosolic and nuclear aryl hydrocarbon (Ah) receptors was studied using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the photoaffinity label. Irradiation of TCDD alone at wavelengths of greater than 300 nm resulted in rapid degradation of this compound (t 1/2 = 8 min). In a separate experiment, the unliganded cytosolic Ah receptor was only slowly inactivated (t 1/2 = 48 min) using the greater than 300 nm light source. Preliminary experiments with rat hepatic cytosol did not result in significant formation of specifically bound [3H]TCDD-protein covalent adducts which could be visualized by autoradiography. Irradiation of [3H]TCDD-nuclear Ah receptor complexes isolated from mouse Hepa 1c1c7 cells for 15 min gave approximately a 40% overall yield of the radiolabeled Ah receptor protein adduct. Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]TCDD-nuclear Ah receptor photoadduct gave a single major radiolabeled protein with an apparent molecular size of 91 kDa. The chromatographic properties of the control (dark) and photolabeled nuclear Ah receptor complexes were comparable using Sephacryl S-300 and DNA-Sepharose columns. Velocity sedimentation of both the control (dark) and irradiated nuclear Ah receptor complexes gave specifically bound peaks which sedimented at 6.5 S. However, the trichloroacetic acid-precipitable (buffer-reconstituted) [3H]TCDD-nuclear Ah receptor photo-covalent adduct was eluted from the Sephacryl S-300 column in the void volume and did not exhibit a specifically bound peak after velocity sedimentation analysis due to protein aggregate formation. In contrast, the elution profile of the aggregate on a DNA-Sepharose column was similar to that observed for the control (dark) and photolabeled complexes, which were eluted from the column with salt concentrations between 0.24 and 0.28 M. These photolabeling studies show that [3H] TCDD can act as a photoaffinity label for the Ah receptor and can be utilized as photoligand to probe further the structure and function of this protein.     

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.     

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

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

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.     

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.