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The ethoxyresorufin-O-deethylase (EROD) assay has been extensively used in whole animals and in cell culture as a biomarker of exposure to environmental contaminants such as dioxin-like compounds (DLCs). This paper addresses two controversial phenomena that arise when DLCs are examined by the EROD assay. Firstly, the maximum level of induced EROD activity varies with the identity of the inducing compound; secondly, the induced EROD activity reaches a concentration-dependent maximum level that is followed by an apparent reduction in activity when the concentration of inducer is further increased. These phenomena are completely explained by competitive inhibition of the EROD enzyme-substrate reaction by the dioxin-like compound. A kinetic model explains the biphasic appearance of EROD induction curves as a function of a compound's binding affinity with the Ah receptor (Kd) and its binding affinity to CYP 1A1 (Ki) which results in inhibition of the EROD enzyme-substrate reaction. These results limit the reliability of the information obtained from calibration curves of EROD activity versus concentration of a standard DLC such as 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Recent work on the metabolism of polychlorinated dibenzo-p-dioxins and related compounds such as polychlorinated dibenzofurans and coplanar polychlorinated biphenyls is reviewed. Most in vivo studies and epidemiological studies on humans have been concerned with rates of elimination of these compounds; in vitro work has shown that a wide variety of metabolites can be found, either in hydroxylated format (typically in feces) or as conjugates (in urine). Metabolism of this group of compounds seems always to represent a detoxification process rather than one of bioactivation.

We report a gel-filtration-based chromatographic method for separation of specific, nonspecific, and free radioligand in a protein receptor-ligand binding assay for the example of the estrogen receptor ERalpha. This assay affords relative binding affinities (RBAs) without the need for a separate determination of nonspecific binding. The probit method is recommended as the most satisfactory method of evaluating the data. The assay responds to both estrogen agonists and antagonists, mixtures respond additively, and the slopes of the probit plots indicate that all ligands bind to the same site on the estrogen receptor. RBAs obtained with rat and rainbow trout ERalpha were in good agreement, and also with those from other reported assays, consistent with the interspecies conservation of key regions of the ligand binding domain among estrogen receptors., (Copyright 1999 Academic Press.)

The in vitro metabolism of several chlorinated dibenzo-p-dioxin congeners (PCDDs) was studied using rat liver microsomes as a source of CYP 1 enzymes. The reactions were kinetically first order in both enzyme and substrate and showed a general trend toward decreasing reactivity with increasing chlorination. Michaelis-Menten kinetics were followed for 1-chlorodibenzo-p-dioxin (1-CDD); the reactivity of the enzyme preparation toward 1-CDD exactly paralleled its activity toward 7-ethoxyresorufin. The unreactive congeners 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) and 2,2'-dichlorobiphenyl (2,2'-DCB) acted as competitive inhibitors toward 1-CDD, with inhibition constants in the micromolar range, similar to the value of the Michaelis constant of 1-CDD. The inhibitory potency of furafylline, a mechanism-based inhibitor that is selective for CYP 1A2, declined in the order acetanilide (standard) > 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) > 1-CDD. We conclude that 1-CDD and 1,2,3,4-TCDD are oxidized almost exclusively by CYP 1A1, whereas 2,3,7,8-TCDD and 1,2,4,7,8-PeCDD are oxidized mainly by CYP 1A2. 1,2,3,7,8-PeCDD was oxidized too slowly for us to reach any conclusion about the P450 isozyme responsible.

The rate of thermal inactivation of the unliganded human Ah receptor, studied by sucrose density gradient centrifugation, with respect to loss of ligand binding ability, was found to be greater than those of most rodents at 20 degrees C, but the temperature coefficient of the rate constant was much smaller than for the rodent species. This implies that the unliganded human Ah receptor would be thermally more stable than the rodent analogs at physiological temperatures. The liganded form of the human Ah receptor was found to be less stable with respect to ligand release than the rodent receptors. These differences in behavior between human and rodent Ah receptors underline the difficulties in using rodent data in the development of receptor-based models of dioxin toxicity. Attempts to develop an alternative to sucrose density gradient centrifugation, comparable with the hydroxylapatite adsorption method used to assay rodent hepatic Ah receptor, were unsuccessful.

We describe a genetic analysis of the vitamin B12 receptor of Escherichia coli. Through the use of informational suppression, we have been able to generate a family of receptor variants, each identical save for a single, known substitution (Ser, Gln, Lys, Tyr, Leu, Cys, Phe) at a known site. We have studied 22 different mutants, 14 in detail, distributed throughout the length of the btuB gene. Most amino acid substitutions have a pleiotropic effect with respect to all ligands tested, the two colicins E1 and E3, the T5-like bacteriophage BF23, and vitamin B12. (The dramatic effect of a single amino acid substitution is also well exemplified by the G142A missense change which renders the receptor completely non-functional.) In some instances, however, we have been able to modify a subset of receptor functions (viz. Q62, Q150 and Q299 and the response to phage BF23). These data are summarized on a two-dimensional folding model for the BtuB protein in the outer membrane (devised using both amphipathic beta-strand analysis and sequence conservation amongst the TonB-dependent receptors). In addition, we report that the extreme C-terminus of BtuB is vital for receptor localization and provide evidence for it being a membrane-spanning beta-sheet with residue L588 situated on its hydrophobic surface. Two of the C-terminal btuB mutations are located within the region of overlap with the recently identified dga (murl) gene.

Fixed aliquots of both radiolabeled [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and hepatic Ah receptor from C57BL/6J mice were incubated competitively at 4, 23, and 30 degrees C with mixtures of 2,3,7,8-TCDD and several polychlorinated biphenyls (PCBs). The production of the radiolabeled receptor-ligand complex changed if the ligands were added sequentially, demonstrating that the competition between PCBs and TCDD for the Ah receptor in vitro is principally a kinetic rather than an equilibrium phenomenon and is irreversible on the time scale of our in vitro experiments. Examination of previous reports on the ability of TCDD, PCBs, and their mixtures to induce cleft palate in fetal mice suggests that the potency of receptor-ligand complexes is ligand-dependent. Receptor occupancy is not a sufficient condition for toxicity, and protection by one ligand against the toxic effect of a second, more potent one is only possible when a significant fraction of receptors is occupied.

Kinetic analysis of the time course of association of [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin with hepatic cytosol from five rodent species gave additional evidence for differences in the properties of the Ah receptor ligand binding subunit between species. A parallel study of the association of six tritiated polychlorinated dibenzo-p-dioxins and dibenzofurans with hepatic Ah receptor from Wistar rat and C57BL/6 mouse showed that their rank order for kinetic affinity did not correlate with the rank ordering of their toxic potency and may vary according to the source of the Ah receptor.

Immature male Sprague-Dawley rats were treated with phenobarbital (PB) at a dose of 50 mg/kg for 3 successive days and then sacrificed 1, 6, or 9 days after the last treatment. An increase in the concentration of hepatic cytosolic proteins able to bind specifically to [3h]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H])TCDD) was observed. The level of the induced protein fell in parallel with the elimination of PB in vivo. Readministration of PB 9 days after the last treatment, by which time only the constitutive receptor was present, induced additional protein at levels similar to those obtained upon the original administration of the drug. There was considerable variability within a given treatment group in both the ratio of constitutive to induced protein and the total amounts of these proteins. The kinetic properties of the induced protein were significantly different from those of the constitutive Ah receptor. In particular, [3H]TCDD was released faster from the liganded induced protein than from the constitutive Ah receptor. The protein induced by PB had properties very similar to those of the protein induced by a combination of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,2',4,4',5,5'-hexachlorobiphenyl.

The numerous toxic responses of dioxin-like compounds are mediated by the intracellular Ah (aryl hydrocarbon) receptor. It has been suggested that the regulation of dioxins and similar substances could be placed on a molecular foundation by considering the proportion of Ah-receptor sites occupied by toxicant molecules. The present work has shown that the following formation not yet available would be needed in order to develop this approach: correlation between dioxin exposure and human tissue levels; accurate determination of the association constants for human Ah-receptor with toxicant, and for human receptor-ligand complex with DNA; and knowledge of the intracellular concentrations of both receptor binding sites and DNA binding sites. Furthermore, since not all dioxin-like substances behave identically, this information would need to be gathered for a wide variety of substances.

Several tritiated chlorinated dibenzo-p-dioxins and dibenzofurans have been prepared with specific activities in the range of 30-50 Ci/mmol in order to investigate the effects of structure on the kinetics of their association with the rat cytosolic Ah receptor. The compounds were 2,3,7-trichlorodibenzo-p-dioxin (TrCDD), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentchlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,7,8-TCDF, and 1,2,3,7,8-pentachlorodibenzofuran (PeCDF). Although these congeners differed by up to 2 orders of magnitude in their biochemical and toxic potencies, their affinities for the Ah receptor as determined by conventional Scatchard analysis varied by less than 2-fold (range of KD values 5.0-9.3 nM). The rate of association of these ligands with the Ah receptor was studied at several temperatures, taking into account the competing thermal inactivation of the unbound receptor. The equilibrium constants (KD) were also obtained as the ratio of the rate constants for dissociation and formation, respectively, of the receptor-ligand complexes. The following conclusions were derived from the kinetic studies: (1) 2,3,7-TrCDD and 1,2,7,8-TCDF bound significantly more slowly to the Ah receptor than the other radioligands at all temperatures (13.5-37 degrees C), and this paralleled the lower biochemical potencies of the congeners; (2) the KD values obtained kinetically were in the subnanomolar range, with the smallest KD values observed for those ligands which bound most rapidly to the receptor; and (3) the temperature dependence of the KD values indicated that receptor-ligand association was favorable both enthalpically and entropically.

The individual pretreatment of Sprague-Dawley rats with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) has been previously shown to result in the "induction" of [3H]TCDD specific binding activity in hepatic tissue. In the present work, the coadministration of TCDD and HCB increased the concentration of hepatic proteins capable of binding [3H]TCDD specifically by at least 2-3-fold. This increase was shown not to be the result of activation, by HCB, of a form of the receptor having low affinity toward [3H]TCDD into a form with high affinity. Kinetic analysis of the time course of binding of [3H]TCDD to induced cytosol was consistent with the presence of an "inducible" binding protein in addition to the "constitutive" aryl hydrocarbon (Ah) receptor present in cytosol from untreated animals. The liganded ([3H]TCDD) form of the inducible binding component lost its ligand much faster than the liganded form of the constitutive Ah receptor at 37 degrees C; apparent first order rate constants for loss of [3H]TCDD were 0.55 min-1 and less than 0.0024 min-1, respectively. Conversely, the unliganded form of the induced binding component was slightly more stable (approximately 2-fold) toward thermal inactivation than the unbound constitutive Ah receptor. The [3H]TCDD-bound protein(s) in uninduced and induced cytosols behaved identically in a sucrose gradient; 8.7-8.9 S in the absence of salt, shifted to 5.5 S by 0.4 M KCl. They were also indistinguishable by gel permeation chromatography, and by photoaffinity labeling their TCDD-binding subunits, approximate molecular weights 105,000. These results show the hepatic TCDD-binding protein(s) induced upon pretreatment of Sprague-Dawley rats with TCDD/HCB to be kinetically distinct from the Ah receptor, but structurally very similar.

A series of exogenous chemicals was used as potential inducers for the hepatic Ah receptor in the Sprague-Dawley rat. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 2,2',4,4',5,5'-hexachlorobiphenyl and phenobarbital all induced an elevated level of 3H-2,3,7,8-tetrachlorodibenzo-p-dioxin specific binding, while 3,3',4,4'-tetrachloroazobenzene and trans-3,3',4,4'-tetrachlorostilbene caused a depression. Mixtures of these chemicals caused additive effects. Elevated levels of specific binding appeared to be heterologous, comprising a binding species having the normal high stability of the Ah receptor in its liganded form, and another less stable substance having a half-life of approximately 2 h at 37 degrees C.

Repeated freezing and thawing, the addition of salts, and elevated temperatures all promote the inactivation of the rat hepatic Ah receptor. The reduced availability of bulk water to solvate the protein is proposed to be the factor linking all these routes for inactivation. Prospective protocols for purification of unliganded Ah receptor should therefore minimize the number of freeze/thaw cycles; long-term freezing of cytosolic samples at -20 degrees C is inadequate to maintain long-term viability of the unliganded receptor. The stability of rat hepatic receptor is greatly increased upon binding the ligand, and the extent of ligand-induced stabilization is much greater than what is observed with steroid hormone receptors. Concentrations of NaCl and K2HPO4 up to 0.5 M inactivate the unbound Ah receptor irreversibly, with the loss of approximately 50% of the specific binding. At 2.0 M NaCl, a further reversible reduction in ligand binding activity is observed. The results at lower salt concentrations are interpreted in terms of the irreversible dissociation of a single binding unit from the trimeric cytosolic Ah receptor (which consists of two ligand-binding units and a 90-kDa heat shock protein), with the release of bound ligand from that subunit.

The thermal inactivation of the hepatic cytosolic Ah receptor was studied in vitro for several immature male rodents. The activation energies for receptor inactivation in C57BL/6 mice, Mongolian gerbils, golden Syrian hamsters, Hartley guinea-pigs, Sprague-Dawley rats and Wistar rats were 170, 142, 112, 131, 120 and 112 kJ/mol, respectively. The magnitude of the activation parameters pointed to a substantial change on inactivation, but not to complete unfolding of the protein. Statistical analysis indicated that attempts to interpret these results in terms of receptor heterology should be treated with caution. Among the species studied, the Ah receptor from the mouse offered the best possibility for in vitro studies at low temperature, free from the problem of thermal inactivation.

The constitution of chlorpromazine has been studied in the context of its phototoxicity. Electron transfer from the side chain to the aromatic nucleus of the drug contributes to its instability to light. Even without the side chain, however, chlorophenothiazines appear to be very photolabile, so that it is unlikely that nonphototoxic analogues of chlorpromazine can be prepared merely by altering the constitution of the side chain.

We have identified cDNA clones coding for the major sulphur-rich and sulphur-poor groups of barley storage proteins (the B- and C-hordeins, respectively). Hybridization studies have revealed unexpected homologies between B- and C-hordein mRNAs. Using a deletion mutant (Risø 56), we have mapped some C-hordein-related sequences within, or closely associated with, B-hordein genes at the Hor 2 locus. Nucleotide sequencing has shown that the primary structure of B-hordein polypeptides can be divided into at least two domains: domain 1 (repetitive, proline-rich, sulphur-poor), which is homologous to C-hordein sequences, and domain 2 (non-repetitive, proline-poor, sulphur rich), which makes up two-thirds of the polypeptide and is partially homologous to a 2S globulin storage protein found in dicotyledons. The coding sequences that are homologous in B- and C-hordein mRNAs have an asymmetric base composition (>80% C-A) and are largely composed of a degenerate tandem repeat based on a 24 nucleotide consensus that encodes Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln. We discuss the evolutionary implications of the domain structure of the B-hordeins and the unusual relationship between the two groups of barley storage proteins.

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.