B. Astroff

2,3,7,8-tetrachlorodibenzo-p-dioxin as an antiestrogen: Effect on rat uterine peroxidase activity

Treatment of 25-day-old female Sprague-Dawley rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly lowered constitutive uterine peroxidase activity and decreased uterine wet weights in a dose-response fashion. In cotreatment studies with 17 beta-estradiol, 2,3,7,8-TCDD antagonized the increase in uterine peroxidase activity and uterine wet weights, and these effects persisted for up to 156 hr. In the rat uterus, the antiestrogenic affects of two potent Ah receptor agonists, 2,3,7,8-TCDD and 2,3,4,7,8-pentachlorodibenzofuran, were comparable at a dose of 80 micrograms/kg, whereas the weaker Ah receptor agonist, 1,2,4,7,8-pentachlorodibenzo-p-dioxin, was relatively inactive at this dose. These results show that 2,3,7,8-TCDD antagonizes a well-characterized estrogen-induced response (uterine peroxidase activity), and the structure-activity data suggest that the Ah receptor is involved in mediating the antiestrogenic responses in target cells/organs.

2,3,7,8-tetrachlorodibenzo-p-dioxin inhibition of 17β-estradiol-induced increases in rat uterine epidermal growth factor receptor binding activity and gene expression

Abstract Treatment of immature female Sprague-Dawley rats with 17β-estradiol (5 μg/animal) resulted in an increase in uterine epidermal growth factor (EGF) receptor binding activity. Moreover, in a separate study it was also shown that 17β-estradiol increased steady-state levels of rat uterine EGF receptor mRNA as determined by Northern analysis. In contrast, 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) caused a dose-response decrease in constitutive rat uterine EGF receptor binding activity and this was paralleled by a decrease in steady-state levels of uterine EGF receptor mRNA. Cotreatment of the animals with both TCDD (16 nmol/kg) and 17β-estradiol (5 μg/rat) gave results which showed that TCDD significantly inhibited the estrogen-induced increases in rat uterine EGF receptor binding activity and EGF receptor mRNA levels. These results further extend the range of antiestrogenic properties of TCDD and suggest that the inhibition of growth factor expression may play a role in the growth-inhibiting properties of TCDD in estrogen-responsive tissues or cells.

Inhibition of the 17β-estradiol-induced and constitutive expression of the cellular protooncogene c-fos by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the female rat uterus

Acute administration of 17 beta-estradiol (5 micrograms/rat) to 25-day-old female Sprague-Dawley rats resulted in an increase of uterine mRNA for the cellular oncogene c-fos. The c-fos mRNA levels were significantly elevated 12 and 24 h after exposure to the hormone (232 and 164% of control values) and the elevation was not observed after 48 h. In contrast, treatment of the animals with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) resulted in a dose-dependent decrease in constitutive uterine c-fos mRNA levels. In rats co-treated with 17 beta-estradiol plus TCDD or MCDF, it was apparent from the results that the halogenated aromatic hydrocarbons significantly inhibited the estrogen-induced increases in uterine c-fos mRNA levels. These observations further extend the diverse spectrum of antiestrogenic effects caused by TCDD and related compounds and also show an interaction between TCDD and the constitutive expression of the c-fos proto-oncogene in the female rat uterus.

6-methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6 mice

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and TCDD plus MCDF were administered to C57BL/6 mice and their effects on several aryl hydrocarbon (Ah) receptor-mediated responses including hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction, immunotoxicity and teratogenicity were determined. MCDF did not induce hepatic microsomal AHH and EROD at doses up to 500 mumol/kg, however, co-administration of MCDF (50 mumol/kg) with a dose of TCDD which elicited a submaximal induction response (i.e. ED80-100, 15 nmol/kg) resulted in some small but significant inhibition of the induction of hepatic microsomal AHH and EROD (14 and 17%, respectively) compared to that observed with TCDD alone. Co-administration of TCDD and other doses of MCDF (10, 100, 200 or 500 mumol/kg) did not effect the induction response. These results were in contrast to the effectiveness of MCDF as an antagonist of the induction of AHH and EROD by TCDD in the rat (up to 50% inhibition of monooxygenase induction). Administration of MCDF (4, 20 and 40 mumol/kg) to C57BL/6 mice caused some inhibition of the splenic plaque-forming cell response to sheep erythrocytes only at the highest dose (26% decrease); the interaction of MCDF (4, 20 and 40 mumol/kg) and an immunotoxic dose of TCDD (3.7 nmol/kg) resulted in significant protection from the immunotoxic effects of TCDD at the 2 higher dose levels of MCDF. Similarly, MCDF (400 mumol/kg) did not cause cleft palate in mice but at this dose level MCDF afforded some protection from TCDD (20 micrograms/kg)-mediated cleft palate in mice. However, studies utilizing [3H]TCDD suggested that the protective effects may be due to modulation of TCDD reaching the palate in the co-treated animals (MCDF plus TCDD). Although both MCDF and Aroclor 1254 were both weak Ah receptor agonists in C57BL/6 mice, the former compound was much less effective as a TCDD antagonist. The observed species-specific effects for these 2 TCDD antagonists may be related species-dependent differences in receptor structure and receptor-ligand (i.e. agonist or antagonist) interactions.

6-substituted-1,3,8-trichlorodibenzofurans as 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonists in the rat: structure activity relationships

The activities of several 6-substituted-1,3,8-trichlorodibenzofurans (CDFs) as partial antagonists of the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities in the rat by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were structure-dependent. Treatment of the rats with TCDD (16 nmol/kg), the 6-substituted-1,3,8-triCDFs (50 mumol/kg) and TCDD plus the 6-substituted-1,3,8-triCDFs showed that most of the substituted congeners were either inactive (6-methyl, ethyl, propyl, i-propyl, t-butyl) or weak (6-cyclohexyl, nitro) inducers of AHH and EROD activities, whereas TCDD caused an 8.1- and 58-fold induction of these enzyme activities respectively. In the co-administration studies, the 6-methyl, propyl, ethyl, isopropyl and t-butyl analogs partially antagonized the induction of the monooxygenase enzyme activities by TCDD, whereas, the 6-cyclohexyl and 6-nitro-1,3,8-triCDFs exhibited minimal activity as TCDD antagonists. The Ah receptor binding affinities of the 6-substituted compounds were determined in a series of in vitro competitive binding studies using [3H]TCDD as the radioligand. Analysis of the data by Scatchard and Dixon plots showed that the avidities for the Ah receptor by the 6-substituted-1,3,8-triCDFs followed the order 6-methyl greater than 6-t-butyl greater than 6-i-propyl greater than 6-propyl approximately 6-ethyl greater than 6-cyclohexyl greater than 6-nitro-1,3,8-triCDF. In addition there was a good correlation between the in vitro binding avidities and Ki values for these compounds and their in vivo activity as partial antagonists of the induction of AHH and EROD activities by TCDD. The results suggested that the 6-substituted-1,3,8-triCDFs competitively displayed TCDD from the Ah receptor and this interaction may play a role in the mechanism of action of this class of TCDD antagonists.

Mechanism of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonists: Characterization of 6-[125I]methyl-8-iodo-1, 3-dichlorodibenzofuran-Ah receptor complexes☆

6-Methyl-8-iodo-1,3,-dichlorodibenzofuran (I-MCDF) and its radiolabeled analog [125I]MCDF have been synthesized and used to investigate the mechanism of action of 1,3,6,8-substituted dibenzofurans as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) antagonists. Like 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), I-MCDF partially antagonized the induction by TCDD of microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities in rat hepatoma H-4-II E cells and male Long-Evans rat liver. Incubation of rat liver cytosol with [125I]MCDF followed by velocity sedimentation analysis on sucrose gradients gave a specifically bound peak which sedimented at 9.6 S. This radioactive peak was displaced by coincubation with a 200-fold excess of unlabeled I-MCDF, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and benzo [a]pyrene. Based on the velocity sedimentation results and the elution profile from a Sephacryl S-300 gel permeation column, the Stokes radius and apparent molecular weights of the cytosolic [125I]MCDF-Ah receptor complex were 6.5 nm and 259,200, respectively. In addition, the nuclear [125I]MCDF-receptor complex eluted at a salt concentration of 0.29 M KCl from a DNA-Sepharose column. Velocity sediment analysis of the nuclear [125I]MCDF-Ah receptor complex from rat hepatoma H-4-II E cells gave a specifically bound peak at 5.6 +/- 0.8 S. All of these properties were similar to those observed using [3H]TCDD as the radioligand. In addition, there were several ligand-dependent differences observed in the properties of the I-MCDF and TCDD receptor complexes; for example, the [125I]MCDF rat cytosolic receptor complex was unstable in high salt buffer and was poorly transformed into a form with increased binding affinity on DNA-Sepharose columns; Scatchard plot analysis of the saturation binding of [3H]TCDD and [125I]MCDF with rat hepatic cytosol gave KD values of 1.07 and 0.13 nM and Bmax values of 137 and 2.05 fmol/mg protein, respectively. The nuclear extract from rat hepatoma H-4-II E cells treated with I-MCDF or TCDD interacted with a dioxin-responsive element in a gel retardation assay. These results suggest that the mechanism of antagonism may be associated with competition of the antagonist receptor complex for nuclear binding sites.

6-Alkyl-1,3,8-trichlorodibenzofurans as antiestrogens in female Sprague-Dawley rats

The comparative antiestrogenic effects of 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), 6-t-butyl-1,3,8-trichlorodibenzofuran (triCDF) and 6-cyclohexyl-1,3,8-triCDF were determined in immature female Sprague-Dawley rats. Treatment of the animals with 17 beta-estradiol (0.33 mumol/kg X 2) caused an increase in uterine cytosolic and nuclear estrogen and progesterone receptor levels, uterine peroxidase activity, uterine wet weights and uterine epidermal growth factor (EGF) receptor binding activity and steady state EGF receptor mRNA levels. MCDF and 6-t-butyl-1,3,8-triCDF, two compounds which exhibit moderate aryl hydrocarbon (Ah) receptor binding affinity were also administered (100 mumol/kg) to the female rats in the presence or absence of 17 beta-estradiol. The results of these studies show that both compounds decrease the constitutive and 17 beta-estradiol-induced responses noted above. In contrast, 6-cyclohexyl-1,3,8-triCDF, a congener which exhibits low Ah receptor binding, was inactive as an antiestrogen. These studies clearly demonstrate that selected 6-alkyl-1,3,8-triCDFs elicit a broad spectrum of antiestrogenic activity in immature female rats. Moreover, in contrast to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which also is a potent antiestrogen, the 6-alkyl-1,3,8-triCDFs are relatively non-toxic and can serve as prototypes for the future development of a new class of antiestrogens with potential for clinical applications.

Mechanism of action of 2,3,7,8-tcdd and 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) as antiestrogens in the female rat

Abstract Several studies have reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) acts as an antiestrogen in rodents and human breast cancer MCF-7 cells in culture. It has been proposed that these effects may be related to the induction of estradiol-2-hydroxylase activity which effects a decrease in cellular estradiol levels due to increased metabolism. This study reports the comparative antiestrogenic effects of TCDD and 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) on cytosolic and nuclear estrogen and progesterone receptor levels in both the rat uterus and liver. MCDF, like TCDD, acted as an antiestrogen in the female rat, causing a dose-response decrease in uterine and hepatic cytosolic and nuclear estrogen and progesterone receptor levels; both compounds also antagonized the estradiolinduced increase in uterine wet weights. TCDD was 300 to 700 times more potent than MCDF as an antiestrogen, however the TCDD/MCDF potency ratios as inducers of hepatic monooxygenases were > 150,000. Moreover, the antiestrogenic effects of MCDF were observed at doses which caused minimal induction of hepatic monooxygenases. These results suggest that the antiestrogenicity of TCDD and related compounds is not due to increased metabolism of estradiol.

6-methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-TCDD antagonist in C57BL/6 mice

MCDF exhibits moderate affinity for the Ah receptor in both mice and rats; however, it is only a weak Ah receptor agonist in both species. In contrast, to previous studies in rats, MCDF was a relatively poor antagonist of TCDD-induced monooxygenase activities in mice, and over a dose range of 10–500 μmol/kg, significant inhibition was observed only at a dose of 50 μol/kg. These dose-response interactive studies in C57BL/6 mice showed that MCDF significantly antagonized the TCDD-mediated induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH), 14% inhibition, and ethoxyresorufin-O-deethylase (EROD), 17% inhibition. MCDF was also a weak agonist for teratogenicity (cleft palate) and immunotoxicity (inhibition of the plaque-forming cell response to sheep erythrocytes) in C57BL/6 mice. Cotreatment of mice with an effective dose of 2,3,7,8-TCDD (ED70–100) and a subeffective dose of MCDF demonstrated that MCDF significantly antagonized 2,3,7,8-TCDD-induced teratogenicity and immunotoxicity in the mice.

Characterization of 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-TCDD antagonist in male rats: induction of monooxygenases

Abstract Treatment of male Long Evans rats with doses of MCDF up to 200 μmol/kg did not significantly induce hepatic microsomal aryl hydrocarbon hydroxylase (AHH) or ethoxyresorufin O-deethylase (EROD) activities, whereas administration of 2,3,7,8-TCDD (16 nmol/kg) caused up to a 10 to 40-fold induction of these enzymes. Cotreatment of the rats with 2,3,7,8-TCDD (16 nmol/kg) and MCDF (50 μmol/kg) resulted in the inhibition of the monooxygenase enzyme induction response over a 72 hour period. Treatment of the animals with 2,3,7,8-TCDD alone caused an initial decrease in the concentration of the cytosolic Ah receptor followed by an increase of these levels (∼2 times higher than in control rats) after 72 hours. In contrast, MCDF treatment did not alter hepatic cytosolic Ah receptor levels and in the cotreatment studies (MCDF + TCDD), the effects of 2,3,7,8-TCDD on receptor levels was inhibited. Using [ 3 H]-2,3,7,8-TCDD (+ MCDF), the time-course accumulation of nuclear [ 3 H]-2,3,7,8-TCDD-receptor complexes was also investigated. Surprisingly, MCDF did not decrease occupied nuclear 2,3,7,8-TCDD-Ah receptor levels.