George Rucci

Analysis of Structural Requirements for Ah Receptor Antagonist Activity: Ellipticines, Flavones, and Related Compounds

A number of studies have examined the structure-activity relationships for the agonist activity of Ah receptor (AhR) ligands. Fewer studies have considered the structural basis for potential antagonist properties. Certain ellipticine derivatives have been reported to bind to the AhR and inhibit the ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to transform the AhR to a form that recognizes a dioxin-responsive enhancer element (DRE) upstream of the cytochrome P4501A1 gene. In the present study, over 30 ellipticine derivatives and structurally related compounds were examined for their ability to bind to the AhR, activate it to a DRE-binding form, induce the luciferase gene under control of a DRE-containing enhancer, and block activation of the AhR by TCDD. The ability of several ellipticine derivatives to inhibit TCDD-elicited DRE binding and TCDD-induced luciferase activity was inversely related to their ability to alone stimulate these responses. The most potent antagonist activity was related to good AhR binding characteristics in terms of conforming to previously predicted 14 x 12 x 5 A van der Waals dimensions and the presence of an electron-rich ring nitrogen at or near a relatively unsubstituted X-axis terminal position. Based on these data, a number of flavone derivatives were synthesized and tested for their relative agonist/antagonist activity. These additional data were consistent with the hypothesis that an electron-rich center near or along a lateral position of the van der Waals binding cavity is a characteristic that enhances AhR antagonist activity.

Ontogeny of the cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver, lung, and thymus

The ontogeny of the cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin was studied in Sprague-Dawley rats by quantitation of the receptor in hepatic, lung, and thymic cytosol. Concentrations of hepatic and lung cytosolic receptors increased rapidly after birth and remained at the highest levels from days 2 to 21. After this time, receptor levels in these tissues slowly declined with age. In the thymus, cytosolic receptor concentrations remained high from days 2 to 42 following birth. In these tissues and at all times examined, the receptors demonstrated very high affinities for [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. From days 15 to 42 following birth, no consistent sex related differences in receptor content or affinity were observed in any of these tissues.

Changes in hamster hepatic cytochrome p-450, ethoxycoumarin o-deethylase, and reduced nad(p):menadione oxidoreductase following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin: Partial dissociation of temporal and dose-response relationships from elicited toxicity

The temporal and dose-related characteristics of hepatic enzymes induced in the hamster by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were examined. Male Syrian golden hamsters received a single intraperitoneal injection of TCDD at a dose of 0-500 micrograms/kg. At various times up to 35 days, a number of variables were determined and compared: whole body, liver, and thymus weights; hepatic concentrations of cytochrome P-450 (P-450); and activities of 7-ethoxycoumarin O-deethylase (ECOD) and reduced NAD(P): menadione oxidoreductase (NMOR). Increased liver weights and decreased thymus weights were observed to be dose related. At day 7 following treatment, the approximate ED50 values for these responses were 15 and 100 micrograms/kg respectively. The ED50 values for the increase in hepatic P-450 concentrations and activities of ECOD and NMOR ranged from 0.5 to 2.0 micrograms/kg. At 10 and 500 micrograms/kg, NMOR activity remained maximally induced for up to 35 days. This was also the case for P-450 and ECOD activity at a dose of 10 micrograms/kg. At 500 micrograms/kg, both P-450 and ECOD demonstrated an induction up to day 4 followed by a decrease to near control levels by day 14. This decrease appeared to correlate with changes in hepatic morphology. These results demonstrate a dissociation of the induction of these hepatic enzymes from TCDD-induced lethality, in this species.

Examination and rapid analysis of hepatic cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using gel-permeation high performance liquid chromatography

Gel-permeation high performance liquid chromatography was used to examine the molecular characteristics of the cytosolic and nuclear receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin. The physical properties for this receptor previously established by sucrose-density gradient analysis have been maintained by the technique. The high performance liquid chromatography method offers the advantages of rapid separation and reproducibility thus minimizing possible effects of proteolytic enzymes or dissociation of the ligand-receptor complex. The specific binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin as measured by this system also corresponds quantitatively to that determined by the hydroxyapatite method. These methods will facilitate a further understanding of the biochemical actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds, as well as an understanding of the physiological function of this receptor molecule.

In vivo kinetics and DNA-binding properties of the Ah receptor in the golden Syrian hamster.

The in vivo long-term cytosolic-nuclear kinetics and DNA-binding properties of the Ah receptor were examined in liver from the golden Syrian hamster. For the kinetic studies, a dose of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) that has been previously shown to produce maximal and sustained hepatic enzyme induction without substantial toxicity was used. Following an intraperitoneal dose of 10 micrograms/kg of [3H]TCDD, occupied cytosolic receptor levels reached a peak within 8 h and then decreased rapidly to a level that was approximately 2% of the total receptor. Throughout the 35-day period, unoccupied cytosolic receptor represented from 65 to 80% of the total receptor content. At 8 h following dosing, less than 30% of the total amount of receptor was associated with the nuclear fraction; this percentage declined slowly to less than 5% of the total at Day 35. The half-life for the decline in detectable nuclear receptor levels was 13 days and was similar to the half-life for the decline in [3H]TCDD content of the whole liver, cytosol, and nuclear extract. The Ah receptor contained in hamster hepatic cytosol underwent a ligand-dependent transformation in vitro to two forms having affinity for DNA-Sepharose, one of which was isolated from nuclei of animals treated with [3H]TCDD in vivo. A comparison of the specific binding recovered following various analytical procedures revealed that the binding of [3H]TCDD to the form not found in nuclear extracts was more labile under certain experimental conditions. These studies indicate the heterogeneity of the Ah receptor in hamster hepatic cytosol and suggest that DNA binding in vitro and nuclear uptake in vivo occur through a ligand-dependent transformation process. The maintenance of maximal hepatic enzyme induction is, in part, a consequence of the sustained presence in the nucleus of only a small percentage of the total receptor content. The whole-tissue kinetics of TCDD appears to be a major factor regulating the long-term retention of the TCDD-receptor complex in the nucleus.

Temporal - and dose-related characteristics of biochemical and morphological alterations in the hamster induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin

The temporal - and dose-related characteristics of hepatic enzymes induced and toxic responses elicited in the hamster by 2,3,7,8-tetrachlorodibenzo-p-dioxin were examined. The results demonstrate a dissociation of the induction of these hepatic enzymes from TCDD-elicited lethality, but not necessarily hepatic damage, in this species. Furthermore, the hyperthyroidism observed is suggestive of a role of these hormones in the relatively insensitive nature of this species to TCDD treatment.