Binding characteristics of Ah receptors from rats and mice before and after separation from hepatic cytosols

Abstract

The Ah receptor, a soluble protein implicated in the mechanism of action of the toxic halogenated aryl hydrocarbons has been examined in rodent livers. Due to the difficulty of making reliable quantitative determinations on binding parameters for hydrophobic compounds in cytosols that contain several components, Ah receptors from livers of Sprague-Dawley rats and C57BL/6 mice have been separated, in a preparative manner, using sucrose density gradient centrifugation in a vertical rotor. The rationale of this approach is supported by the results obtained and the major conclusions are as follows.1. The intrinsic binding characteristics of Ah receptors were dependent on the presence or absence of other cytosolic binding components (light-density component and 4-S carcinogen-binding protein).2. In contrast with many previous unsuccessful attempts, the separation of the C57BL/6 Ah receptor allowed the unambiguous detection of a 9-S binding peak with [3H]benzo[a]pyene as a radioligand.3. The intrinsic binding characteristics of the separated Ah receptors of Sprague-Dawley rats and C57BL/6 mice were similar if not identical.4. A good correlation exists between the competitive potency (IC,o) of chemicals and their ability to induce aryl hydrocarbon hydroxylase activity, except for 7-hydroxyellipticine which binds to the Ah receptors of rat and mouse liver (IC50 z 5 -10 pM) without inducing aryl hydrocarbon hydroxylase.5. When coadministered with various inducers, 7-hydroxyellipticine antagonizes (from about 20% to 65%) the inducing ability of chemicals displaying similar (ellipticines) or weaker (chlorpromazine, phenothiazine) binding affinities for the Ah receptor.The cytosol from liver or other organs of rodents contains several components that bind halogenated aromatic compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [l -31 and polycyclic aromatic hydrocarbons such as 3-methylcholanthrene and benzo[a]pyrene [4 -61. For some ten years many laboratories have focused attention on the Ah receptor (aromatic hydrocarbon), one function of which is the regulation of induction of aryl hydrocarbon hydroxylase and other drug-metabolizing enzymes associated with the Ah gene locus [7,8]. Most investigations concern the physico-chemical properties of this protein [2, 9 -111, its mechanism of action [7] (which is probably similar to those of receptors for steroid hormones [12]), the possible existence of an endogenous ligand and the role of the receptor in the toxicity of dioxin, dibenzofuran and biphenyl derivatives [13,14]. More recently, several laboratories have investigated the properties and function(s) of another soluble protein, the 4-S carcinogen-binding

The Anticancer Drug Ellipticine Is a Potent Inducer of Rat Cytochromes P450 1A1 and 1A2, Thereby Modulating Its Own Metabolism

“…Dioxins and related compounds induce CYP1A by binding to the ligand-activated transcription factor, aryl hydrocarbon receptor (AhR). Binding of ellipticine to this transcription factor has already been described previously (Fernandez et al, 1988), suggesting that this drug may induce CYP1A expression (Cresteil et al, 1982;Gasiewicz et al, 1996). Indeed, the previous studies of Cresteil et al (1982) have shown that ellipticine is a CYP1A inducer, although these authors did not determine which members of this subfamily were affected.…”

“…This induction might be a consequence of the ellipticine-binding to AhR described by several authors (Fernandez et al, 1988;Gasiewicz et al, 1996). Ellipticine binding allows the cytosolic AhR to translocate into the nucleus and to dimerize with AhR nuclear translocator (ARNT).…”

Polycyclic Aromatic Hydrocarbons: Metabolism, Activation and Tumour Initiation

“…NEBERT and GELBOIN (1969) found that certain strains of mice could be classified as being "responsive" to induction of so-called aryl hydrocarbon hydroxylase (ARR) activity in liver by PAR, whereas others were non-responsive. This variation in induction has been extensively studied in the mouse and has been ascribed to the expression, or otherwise, of a cytosolic protein, molecular weight 95K, which is capable of binding PAR within the cell (POLAND and GLOVER 1988;FERNANDEZ et al 1988), an event postulated to result in expression of a number of genes encoding cytochrome P-450 and other enzyme activities (NEBERT and JENSEN 1979;NEBERT and GONZALEZ 1987). Thus it could be predicted that responsive strains would demonstrate increased susceptibility to PAR-induced tumorigenesis compared with equivalent non-responsive animals.…”

Microspectrofluorimetric study of the kinetics of cellular uptake and metabolization of benzo(a)pyrene in human T 47 D mammary tumor cells: evidence for cytochrome P1450 induction

“…Thus the extracellular release of the primary metabolite after conjugation becomes the rate limiting step of ihe detoxification process. Interpretation of this sharp feature must be related to previous biochemical work in which authors have provided: i) evidence for AHH and Cyt-Pt450 mRNA induction after binding of B(a)P to a soluble cytoplasmic protein (namely the Ah receptor) in hepatic cytosols of various mammalian species (Denison and Wilkinson 1985;Okey et al 1984;Fernandez et al 1988). ii) evidence for the presence of the Ah receptor in human T 47D mammary tumor cells (Pasanen et al 1988;Harris et al 1989).…”