Synergistic interactions of 2,3,7,8-TCDD and 2,2',4,4',5,5'-hexachlorobiphenyl in C57BL/6J and DBA/2J mice: role of the Ah receptor

Treatment of C57BL/6J mice with 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP, 500 mumol/kg) elevated hepatic cytosolic Ah receptor levels 82-107% for up to 14 days. Scatchard analysis of the [3H]2,3,7,8-TCDD (TCDD)-Ah receptor saturation binding curves from corn oil and HCBP treated rats gave KD values of 0.80 and 0.90 nM, respectively and confirmed that treatment with HCBP did not significantly alter receptor-radioligand affinities. Administration of HCBP to DBA/2J mice did not result in detectable hepatic cytosolic Ah receptor levels. Cotreatment of C57BL/6J mice with HCBP (500 mumol/kg) at a dose level of TCDD (1 nmol/kg) which elicited less than 10% of the maximum induction response resulted in significant synergistic induction of hepatic EROD and AHH [compared to animals treated only with TCDD (1 nmol/kg)]. In contrast, cotreatment of C57BL/6J mice with HCBP (500 mumol/kg) and maximally inducing dose levels of TCDD (100 or 500 nmol/kg) resulted in either a slight or no difference in the induction of AHH or EROD compared to the induction responses observed in mice treated only with TCDD. In contrast, cotreatment of DBA/2J mice with TCDD and HCBP (500 mumol/kg) resulted in significant synergistic induction of AHH and EROD at both submaximal (10-500 nmol/kg) and maximal (5000 nmol/kg) induction levels of TCDD. The only significant interactive effect of HCBP (500 mumol/kg) on the toxicity of TCDD in C57BL/6J and DBA/2J was protection from body weight loss observed after cotreatment of HCBP and TCDD in DBA/2J mice.     

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the hepatic estrogen and glucocorticoid receptors in congenic strains of Ah responsive and Ah nonresponsive C57BL/6J mice.

The present study examines the role of the Ah receptor in the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the binding capacity of the hepatic glucocorticoid (GC) and estrogen (E) receptors in female congenic C57BL/6J mice differing only at the Ah (aromatic hydrocarbon responsiveness) locus. The Ah locus is thought to encode the Ah receptor, which regulates the effects of TCDD and related compounds on cytochrome P450IA1 and appears to mediate most of the toxic effects of TCDD. The differences between Ah responsive (Ahb/b) and nonresponsive (Ahd/d) mice appear to reflect differences in the affinity of the Ah receptor in the two strains for ligands such as TCDD. Administration of a single oral dose of TCDD (30 micrograms/kg) to Ahb/b mice produced approximately a 30% decrease in the maximum binding capacities of both the hepatic GC and E receptors, as well as 50-fold induction of a P450IA1-mediated enzymatic activity, ethoxyresorufin-O-deethylase (EROD). Tyrosine aminotransferase (TAT) activity, which is mediated by the GC receptor, was also decreased approximately 30% by TCDD. Dose-response curves indicated that Ah responsive mice are 10-fold more sensitive to induction of EROD than Ah nonresponsive mice (ED50 1.6 vs 15 micrograms/kg), as would be expected for an effect mediated by the Ah receptor. Dose-response curves also indicated that there was a statistical difference in the responsiveness of the hepatic E receptor to TCDD in the two congenic strains of mice (p less than 0.01). Surprisingly, no significant differences in the dose-response curves for the effect of TCDD on hepatic GC receptor binding or TAT activity were observed in the two strains of mice in two separate experiments. These results indicate that the Ah receptor regulates the effects of TCDD on the binding of estrogen to the hepatic estrogen receptor, but suggest that the decrease in the binding capacity of the hepatic GC receptor does not appear to be mediated directly by the Ah locus.     

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.     

Influence of the Ah locus on the humoral immunotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: evidence for Ah-receptor-dependent and Ah-receptor-independent mechanisms of immunosuppression

There are conflicting reports in the literature regarding the role of the Ah locus in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) immunotoxicity. The present studies have utilized two congenic strains of C57Bl/6 mice that differ only at this locus to assess its influence on TCDD-induced suppression of antibody responses. Mice were given a single oral dose of TCDD 2 days prior to challenge with sheep red blood cells (SRBC) or trinitrophenyl-lipopolysaccharide (TNP-LPS). The subsequent dose-dependent effects of TCDD on the amount of antibody produced by splenic plasma cells were measured using the hemolytic antibody isotope release assay. In addition, the relative importance of the Ah genotype of lymphoid versus nonlymphoid tissue was examined in adoptive transfer experiments. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced in Ahbb mice by a dose of 0.5 micrograms/kg TCDD and maximally induced by a dose of 2 micrograms/kg. Ahdd mice required 10-fold higher doses of TCDD to induce comparable levels of AHH. The degree of thymic involution and liver hypertrophy induced by TCDD was also influenced by the Ah genotype of the animals. Both Ahbb and Ahdd mice exhibited dose-dependent suppression of the anti-TNP response following TCDD exposure. The ID50 was 7.0 micrograms/kg in Ahbb mice and 30.8 micrograms/kg in Ahdd mice. Suppression of the antibody response to SRBC was also dependent on the Ah locus. The ID50 in Ahbb mice was 0.6 micrograms/kg TCDD. However, an apparent biphasic dose response for suppression of the anti-SRBC response in Ahdd mice suggested the involvement of an Ah-independent component of suppression as well. In adoptive transfer studies, lymphocytes were identified as an Ah-dependent component of the response. The Ah-independent component of the response was not identified, and could be either lymphoid or nonlymphoid in nature. The possibility that T helper cells represent the Ah-independent component is discussed.     

6-Methyl-1,3,8-trichlorodibenzofuran as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: inhibition of the induction of rat cytochrome P-450 isozymes and related monooxygenase activities

In addition to being one of the most toxic chemicals known, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent inducer of rat liver microsomal cytochrome P-4501A1 (P-450c). Previous studies have demonstrated that a high affinity, low capacity cytosolic receptor (the Ah receptor) mediates the activity of TCDD to induce cytochrome P-4501A1, which catalyzes benzo[a]pyrene hydroxylation [aryl hydrocarbon hydroxylase (AHH]) and 7-ethoxyresorufin O-dealkylation (EROD). The results of the present study indicate that 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) effectively competes with [3H]TCDD for binding to the Ah receptor in rat liver cytosol. The concentration of MCDF effecting 50% displacement of [3H]TCDD was 4.9 X 10(-8) M, which is approximately 50 times greater than the EC50 for unlabeled TCDD (approximately 1 X 10(-9) M). However, in contrast to TCDD, MCDF was only a weak inducer of AHH and EROD activity in rat hepatoma H-4-II cells in culture. When co-incubated, MCDF diminished in a concentration-dependent manner the ability of TCDD to induce AHH and EROD activity in vitro. Treatment of rats with 20-200 mumol/kg MCDF in vivo had little or no effect on liver microsomal AHH and EROD activity, whereas treatment of rats with 16 nmol/kg TCDD caused a 6- and a 70-fold induction of AHH and EROD activity, respectively. When co-administered, MCDF diminished by approximately 50% the ability of TCDD to induce AHH and EROD activity in vivo. The partial antagonism produced by 50 mumol/kg MCDF could be partially overcome by doubling the dosage of TCDD from 16 to 32 nmol/kg. Immunochemical analysis of rat liver microsomes revealed that treatment of rats with 20-200 mumol/kg MCDF caused little or no induction of cytochromes P-4501A1 and P-4501A2 (P-450d), whereas these isozymes were induced 33- and 5-fold, respectively, in rats treated with 16 nmol/kg TCDD. When co-administered, MCDF diminished by approximately 50% the ability of TCDD to induce cytochrome P-4501A1 in vivo, which established that MCDF was not simply acting as an inhibitor of AHH and EROD activity. MCDF also antagonized the ability of TCDD to induce cytochrome P-4501A2, which suggests that the induction of both cytochromes P-4501A1 and P-4501A2 is regulated by the Ah receptor. These results indicate that MCDF binds with high affinity to the Ah receptor in rat liver cytosol and competitively blocks the binding of TCDD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative antiestrogenic activities of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 6-methyl-1,3,8-trichlorodibenzofuran in the female rat

The ED50s for the dose-response induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the female Sprague-Dawley rat were 3.3 and 2.7 nmol/kg, respectively. In contrast, the corresponding ED50 values for induction by the nontoxic 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) were 524 and 578 mumol/kg for AHH and EROD, respectively, and TCDD was greater than 1.5 X 10(5) more potent than MCDF as an agonist for this response. Cotreatment of the female rats with MCDF (20, 50, or 100 mumol/kg) and TCDD (6.4 nmol/kg) showed that MCDF partially antagonized the induction of AHH and EROD by TCDD and this corresponded with results previously reported in the male rat. Like TCDD, MCDF also caused a dose-response decrease in uterine and hepatic cytosolic and nuclear estrogen (ERc and ERn) and progesterone (PRc and PRn) receptor levels. The relative TCDD/MCDF potencies for the reduction of uterine ERc, ERn, PRc, and PRn levels were 293, 569, 560, and 459, respectively, and comparable potency ratios (693, 409, 405, and 424, respectively) were observed in the liver. Since MCDF was active as an antiestrogen at dose levels which caused only minimal induction of hepatic monooxygenases, it is unlikely that induction of these enzymes and the subsequent increased metabolism of estradiol play a role in the antiestrogenic effects of MCDF (or TCDD). The reasons for the differences in the relative potency of MCDF for the "traditional" Ah receptor-mediated response (i.e., AHH induction) and the modulation of steroid hormone receptor binding levels are unknown. MCDF, a compound which exhibits relatively high TCDD receptor binding activity and low toxicity, represents a new class of nontoxic halogenated aromatic antiestrogens that can be utilized to further probe the mechanism of this response in model systems.     

Biphasic response for hepatic microsomal enzyme induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin in C57BL/6J and DBA/2J mice

The induction of the murine hepatic microsomal cytochrome P-450 monooxygenase system by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied over a wide range of doses, including those associated with acute toxicity. Studies were conducted in two inbred strains of mice which vary at the Ah receptor and at a number of other genetic loci. C57BL/6J mice possess a high-affinity Ah receptor and are responsive to enzyme inductive effects of TCDD, whereas DBA/2J mice do not possess a high-affinity receptor and are less responsive to TCDD. In a dose-response study, 7-ethoxyresorufin O-deethylase (EROD) activity appeared to be maximally induced in C57BL/6J and DBA/2J mice at 7 days following exposure to 3 and 30 micrograms of TCDD/kg respectively. Very similar results were reported previously for the induction of aryl hydrocarbon hydroxylase activity in these strains of mice. However, at higher doses of TCDD (at least 45 micrograms/kg for C57BL/6J and 300 micrograms/kg for DBA/2J), EROD activity was further increased (2-fold) from the apparent maximal (plateau) level, resulting in an unusual biphasic log dose-response relationship. EROD activity remained at these elevated rates in both strains for doses approaching and exceeding the respective LD50 values for each strain. To further characterize this biphasic induction phenomenon, cytochrome P-450 content, benzo[a]pyrene metabolism, and EROD and NADPH-cytochrome P-450 reductase activities were measured 1, 3 and 7 days after TCDD administration to C57BL/6J (3 and 150 micrograms/kg) and DBA/2J (30 and 600 micrograms/kg) mice. Maximal responses occurred in both strains at 3 days for all doses. In both strains, TCDD produced a dose-dependent increase in cytochrome P-450 content, EROD, and benzo[a]pyrene metabolism. Furthermore, a 2-fold induction of reductase activity was observed in each strain following exposure to the respective high doses. Induction of cytochrome P1-450 and P3-450 was also measured by Western immunoblot, using antisera raised against the homologous rat isozymes. In both strains, TCDD produced a dose-related increase in two protein-staining bands recognized by anti-P-450BNF-B (P1-450) and anti-P-450BNF/ISF-G (P3-450) respectively. The extended induction of hepatic microsomal monooxygenase activities at the respective high doses of TCDD appears to be due, in part, to increases in NADPH-cytochrome P-450 reductase activity and cytochromes P1-450 and P3-450 content. Significant alterations in the expression of the cytochrome P-450 monooxygenase system following exposure to high doses of TCDD may be associated, in part, with the delayed acute toxicity reported at this level of exposure.     

Comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated hepatotoxicity in C57BL/6J and DBA/2J mice

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined by clinical chemistry and liver histopathology in Ah-responsive C57BL/6J (C57) and Ah-nonresponsive DBA/2J (DBA) mice. Hepatotoxicity was assessed at 1, 3, and 7 d following a single ip injection of TCDD at doses that maximally induce hepatic aryl hydrocarbon hydroxylase (AHH) activity (3 micrograms/kg for C57 and 30 micrograms/kg for DBA mice) and at doses approaching the LD50 (150 micrograms/kg for C57 and 600 micrograms/kg for DBA mice). Histological examination of liver sections was found to be a more sensitive detection method for TCDD-induced hepatic changes than clinical chemistry analyses. Dramatic differences in the development and type of liver injury were observed between TCDD-treated C57 and DBA mice. C57 mice given 3 micrograms TCDD/kg developed mild to moderate hepatic lipid accumulation in the absence of both inflammation and necrosis. Severe fatty change and mild inflammation and necrosis occurred in C57 mice that received 150 micrograms TCDD/kg. In contrast, DBA mice exposed to 30 micrograms TCDD/kg developed hepatocellular necrosis and inflammation without any fatty change. Only slight hepatic lipid accumulation occurred with some necrosis and inflammation in DBA mice given 600 micrograms TCDD/kg. The Ah locus may play a role in determining the sensitivity of C57 mice to the steatotic effects of TCDD.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on serum concentrations and the uterotrophic action of exogenous estrone in rats

Experiments were conducted to determine whether the short-term administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in low doses alters the disposition and action of exogenous estrogen. Ovariectomized rats were pretreated with TCDD, 1 μg/kg body wt/day for 5 days, or with vehicle. Following the administration of estrone, 10 μg/100 g body wt/day for 4 days, serum estrone concentrations were 57% higher in TCDD-pretreated animals. Aryl hydrocarbon hydroxylase (AHH) activity in liver microsomes was increased 20-fold 5 days following the last dose of TCDD, data which confirm that monooxygenase activity was altered during the course of estrone administration. Since estrone disposition and/or metabolism appears to be decreased by TCDD exposure, experiments were performed to determine if TCDD altered the uterotrophic response to estrone. When various doses of estrone were administered to control animals, serum estrone values increased with the administered dose without any apparent conversion to 17β-estradiol. Estrone administered in doses of 0.4, 2.0, and 10.0 μg/100 g for 4 days elicited a dose-dependent increase in uterine wet weight. Control and TCDD-pretreated animals showed no differences in the uterine weight increase observed at each dose of estrone. Thus, the uterine growth response to exogenous estrone was not altered by any change in estrogen disposition elicited by short-term administration of TCDD.     

Aryl hydrocarbon hydroxylase activity levels in the hepatic microsomal fraction from MC- or TCDD-treated mice: a comparison between aromatic hydrocarbon responsive and non-responsive strains

The effects of in vivo administration of polycyclic aromatic hydrocarbons on the levels of aryl hydrocarbon hydroxylase (AHH) activity in aromatic hydrocarbon (Ah) responsive and non-responsive strains of mice were studied using the hepatic microsomal fraction. Injection of 3-methylcholanthrene (MC; 42 mg/kg body wt.) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 120 micrograms/kg body wt.) into both strains produced marked enhancement of AHH activity except for MC treatment of Ah non-responsive strains. Addition of 7,8-benzoflavone (BNF) to the microsomal AHH assay mixture prepared from mice previously injected with vehicle (olive oil) alone caused an increase in activity when the mice were responsive, while BNF lowered the activity in non-responsive strains. With regard to MC-injected mice, BNF and 3-methyl-sulphonyl-4,5,3',4'-tetrachlorobiphenyl (3-MSF-TCB) decreased microsomal AHH activity in Ah-responsive mice, whereas these drugs enhanced the activity in Ah-non-responsive strains. 3-MSF-TCB also had inhibitory potency on AHH activity, but the mechanism of inhibition seems to be somewhat different from that of BNF. It may also suggest that cytochrome P-450 isozymes inhibited by BNF are different from those inhibited by 3-MSF-TCB.     

Dose-dependent elevation of Ah receptor binding by TCDD in rat liver

Changes in [3H]TCDD binding to unoccupied liver Ah receptor were examined following chronic or acute administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to adult female Sprague-Dawley rats. Chronic biweekly administration of TCDD equivalent to 0, 10, 30, or 100 ng/kg/day TCDD for 22 weeks caused dose-dependent increases in liver TCDD concentration, aryl hydrocarbon hydroxylase (AHH) induction, and [3H]TCDD binding to unoccupied cytosolic Ah receptor sites. Maximal increases (twofold) of cytosolic receptor binding occurred at an estimated dose of slightly greater than 30 ng/kg/day. The increase in [3H]TCDD binding was half-maximal at an estimated dose of approximately 17 ng/kg/day which produced a liver concentration of 1.5 ppb TCDD. Cytosolic [3H]TCDD binding in control and treated animals sedimented mainly in the 8-9 S region of sucrose density gradients with a minor peak sedimenting in the 4-5 S region. Binding was markedly elevated in the 8-9 S region of cytosols from the TCDD-induced rats; however, TCDD treatment had no affect on [3H]TCDD binding in the 4-5 S region. Saturation and Scatchard analyses of Ah receptor binding showed no apparent changes in Kd following chronic TCDD treatment; however, a twofold increase in the number of unoccupied Ah receptor binding sites was observed. Neither aging nor ovariectomy significantly changed measurable cytosolic Ah receptor binding in control animals. When adult female rats were administered a single dose of TCDD (6 micrograms/kg) an initial drop (approximately 40%) in cytosolic receptor binding was observed at 30 and 60 min, followed by a steady increase in binding up to 250% of controls 9 days after TCDD treatment.     

Hepatic Aryl Hydrocarbon Hydroxylase and Cytochrome P450 Induction following the Transpulmonary Absorption of TCDD from Intratracheally Instilled Particles

Hepatic Aryl Hydrocarbon Hydroxylase and Cytochrome P450 Inductionfollowing the Transpulmonary Absorption of TCDD from IntratracheallyInstilled Particles. NESSEL, C. S., AMORUSO, M. A., UMBREIT,T. H., AND GALLO, M. A. (1990). Fundam. Appl. Toxicol. 15, 500-509.Inhalation of particles contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) will be an increasingly important route of human exposurein light of the increased utilization of municipal waste incinerationand the resultant emission of contaminated materials into theenvironment. The potential for pulmonary absorption of the compoundfrom respirable particles was assessed in the present studyfollowing the intratracheal instillation of TCDD (1) as a contaminantof gallium oxide particles and (2) in a corn oil vehicle. Groupsof five female Sprague-Dawley rats received 0, 0.005, 0.055,0.55, or 5.5 µg/kg TCDD in a single instillation and wereeuthanized 4 days later. Absorption was characterized by enzymeinduction [aryl hydrocarbon hydroxylase (AHH) activity and totalcytochrome P450] and histopathological examination of the liver.Induction of hepatic enzymes was dose-dependent with both treatmentregimes. Up to an 18-fold increase in AHH and an 80% increasein cytochrome P450 were observed in treated animals. Inductionwas slightly higher when animals received TCDD in com oil thanwhen animals received TCDD-contaminated particles and was relativelycomparable to induction following oral exposure. Similar resultswere obtained when animals were treated with particles contaminatedup to 4 weeks prior to instillation. Characteristics of TCDD-inducedhepatotoxicity, including enlarged hepatocytes and fatty infiltration,were apparent in treated rats, but were not present in vehicle-instilledanimals. These results indicate that systemic effects occurfollowing pulmonary exposure to TCDD and that inhalation maybe an important route Of exposure for TCDD.     

Alteration of the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by estradiol and tamoxifen

We have hypothesized that part of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is mediated by interaction with the estrogen receptor complex. The experiments reported here investigate the interactions of TCDD with agonists and antagonists of the estrogen receptor. CD-1 female mice were observed for 2 months after treatment with various combinations of corn oil, estradiol, or tamoxifen, and/or TCDD in corn oil on 3 consecutive days. Estradiol had little effect on acute TCDD lethality but increased severity of TCDD-induced ascites and antagonized TCDD-induced uterine suppression. Severe liver damage did occur in TCDD and estradiol:TCDD treatment groups. Tamoxifen, a competitive inhibitor and a mixed agonist of the mouse estrogen receptor, antagonized the estrogenic effects of estradiol and estradiol:TCDD. Tamoxifen or tamoxifen:TCDD treatment greatly slowed body weight gain in comparison to controls and estrogen-treated animals. While the dose of tamoxifen used was otherwise non-toxic, tamoxifen greatly increased toxicity of TCDD as measured by time to death and percent lethality while having no effect on relative liver weight or relative uterine weight changes induced by TCDD. These findings are consistent with the hypothesis that a portion of the toxicity of TCDD is manifest through activity of the estrogen receptor complex.     

Assessment of polystyrene extract for estrogenic activity in the rat uterotrophic model and an in vitro recombinant receptor reporter gene assay

The purpose of the study was to determine whether polystyrene used in food-contact applications would elicit an estrogenic response when extracts simulating exaggerated conditions of use were subjected to in vivo and in vitro tests. A sample of polystyrene was subjected to extraction conditions that simulate, or exaggerate, the actual food-contact uses of polystyrene to maximize the amount of low molecular weight polystyrene extractables. The food-simulating solvent and the time and temperature conditions recommended by the Food and Drug Administration (FDA) were selected to maximize the level of extractable components from polystyrene. The extract was examined for its estrogenic response in vivo using the immature rat uterotrophic assay and in vitro using an estrogen receptor (ER)-mediated recombinant receptor reporter gene assay. In vivo, the uterine weights of juvenile female Sprague Dawley rats (10 rats/group) were determined after oral gavage exposure to the extract (two dosage levels: one represents the maximum potential daily human exposure to polystyrene extractables and the other represents one-tenth of the maximum exposure level), vehicle control (sesame oil), or positive control [diethylstilbestrol (DES), at 200 micrograms/kg body weight]. In addition, five treatment groups were dosed by subcutaneous injection of either estradiol (1, 50, and 500 micrograms/kg body weight) or DES (2 and 200 micrograms/kg body weight). Dosing began on postnatal day (pnd) 21 and continued daily through pnd 23. Body weights were collected at study initiation (pnd 21) and at necropsy (pnd 24). Body weights were not different statistically between treatment groups at study initiation or at necropsy. Uterine wet weights and uterine weights relative to body weights were significantly increased (p < 0.05) for estradiol at 50 and 500 micrograms/kg, DES at 2 and 200 micrograms/kg, and DES at 200 micrograms/kg (oral) over vehicle control. The polystyrene extract had no effect on uterine wet weight or uterine weights relative to body weights at either level tested. An in vitro recombinant estrogen receptor/reporter gene assay that involved transiently transfecting MCF-7 human breast cancer cells with the chimeric human ER, Ga14-HEGO, consisting of the yeast Ga14 DNA binding domain linked to the ligand binding domain of the human ER and a Ga14 response element (17mer)-regulated reporter gene (17m5-G-Luc) was employed. Dose-dependent induction of the reporter gene, 17m5-G-Luc, was observed with the positive control, 17 beta-estradiol (E2). Induction of greater than 100-fold was obtained following incubation of transfected MCF-7 cells with 10 nM E2 for 24 hours. No induction of reporter gene activity was observed with the polystyrene extracts dissolved in dimethylsulfoxide (0.01, 0.1 or 0.01 mg/ml) using the same assay conditions. These results indicate that polystyrene extract does not elicit ER-mediated activity using the Ga14-HEGO/17m5-G-Luc recombinant receptor/reporter gene assay. In conclusion, extracts from polystyrene produced no estrogenic response in either the rat uterotrophic assay or the MCF-7 cell assay for estrogen receptor-mediated activity.     

Elevated binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene to the Ah receptor in hepatic cytosols from phenobarbital-treated rats and mice

Binding of 3-methylcholanthrene (MC),2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and other “MC-type” inducers to cytosolic Ah receptor sites is the first specific step in induction of aryl hydrocarbon hydroxylase (AHH; cytochrome P₁?450) by these compounds. [₃H]TCDD and [₃H]MC were used as radioligands to quantitate and characterize Ah receptor in hepatic cytosols from genetically “responsive” C57BL/6J mice, genetically “nonresponsive” DBA/2J mice, and AHH-inducible Sprague-Dawley rats. Injection of 50–100 mg/kg of phenobarbital (PB) for 3 days more than doubled the concentration of Ah receptor in hepatic cytosol from Sprague-Dawley rats. In C57BL/6J mice, PB injection at 25 mg/kg × 3 days significantly increased (P < 0.01) the Ah receptor concentration in hepatic cytosol. No cytosolic Ah receptor was detectable in hepatic cytosol from untreated DBA/2J mice, nor did any Ah receptor appear after PB treatment in this “nonresponsive” strain. Although PB significantly elevated Ah receptor in hepatic cytosols of responsive rodents, many previous studies have shown that the maximal level of AHH activity in animals given PB and an “MC-type” inducer simultaneously is additive rather than synergistic. Ah receptor concentrations can be doubled by PB treatment without doubling the subsequent AHH-induction response to “MC-type” compounds. Thus, the cytosolic Ah receptor concentration per se may not be the primary determinant of a given tissue's maximal capacity for AHH induction by “MC-type” compounds.     

Relationship between the murine Ah phenotype and the hepatic uptake and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin

The influence of the Ah locus on the hepatic uptake and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied using isolated hepatocytes from Ah responsive C57BL/6J (C57) and nonresponsive DBA/2J (DBA) mice. Hepatocytes from control and TCDD-pretreated C57 and DBA mice were incubated with purified [14C] TCDD (2.2 microM) for 8 hr in the metabolism studies or 2 hr in the uptake studies. Mice were pretreated 7 days prior to hepatocyte isolation with TCDD at doses that maximally induce aryl hydrocarbon hydroxylase activity (C57: 3 micrograms/kg, ip; DBA: 30 micrograms/kg, ip) or at doses approaching the LD50 value (C57: 150 micrograms/kg, ip; DBA: 600 micrograms/kg, ip). Hepatocytes isolated from untreated C57 and DBA mice had similar uptake of [14C]TCDD, and, at all doses, TCDD pretreatment increased [14C]TCDD uptake. The rates of hepatic TCDD metabolism over the first 2 hr of incubation were similar for control C57 and DBA mice, although some qualitative differences in metabolites were detected by HPLC. TCDD pretreatment at doses of 3 and 30 micrograms/kg for C57 and DBA mice, respectively, produced no detectable quantitative or qualitative changes in TCDD metabolism, despite increases in cytochrome P-450 content, 7-ethoxyresorufin O-deethylase (EROD) activity, and benzo[a]pyrene (BaP) metabolism. Pretreatment of C57 and DBA mice with the respective LD50 doses of TCDD decreased the rate of TCDD metabolism by hepatocytes, although cytochrome P-450 content, EROD activity, and BaP metabolism were increased. These results suggest that the uptake and the rate of hepatic metabolism of TCDD do not correlate with genetic differences at the murine Ah locus.     

Dioxin inhibition of estrogen-induced mouse uterine epithelial mitogenesis involves changes in cyclin and transforming growth factor-beta expression.

A single dose of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD; 5 microg/kg, ip) inhibits 17beta-estradiol (E2)-induced uterine epithelial mitogenesis, apparently through disruption of stromal-epithelial interactions. To understand if TCDD alters early uterine (Ut) responses to E2, young adult C57BL/6J mice were ovariectomized and given (i.p.) either oil or 5 microg/kg TCDD. After 24 h, TCDD-treated mice received E2, and oil-treated mice were given E2 or oil. Body and Ut weights were collected 6 and 18 h later. Ut were flash-frozen at 6 h. E2 increased Ut weight (p < 0.0001) and Ut/body weight ratio (p < 0.0001), compared to mice given oil alone. Ut cyclin expression was assessed by an RNase protection assay. E2 increased mRNA expression for cyclin A2 and B1 (p < 0.05), in addition to D1, D2, and D3 (p < 0.001), while cyclin C was unchanged from oil controls and cyclins A1 and B2 were undetectable. In contrast, TCDD completely abolished E2-induced cyclin A2, which has been associated with S phase initiation, and reduced B1 and D2 (p < 0.05). Interestingly, TCDD did not alter E2-induced Ut weight increases at 6 h, but inhibited E2-induced Ut weight gain at 18 h. A 10-microg/kg TCDD dose was necessary for attenuation of the early E2-induced Ut weight increases (p < 0.01). Since TGF-beta regulates cyclins, Ut TGF-beta was also assessed in TCDD + E2-treated and control mice. TGF-beta mRNA levels were increased after TCDD compared to E2 alone (p < 0.01), suggesting a possible mechanism for TCDD inhibition of Ut cyclin A2. Thus, TCDD alters specific E2-regulated Ut G(1) phase activities and may inhibit E2-induced Ut epithelial mitogenesis by disrupting specific cell signaling mechanisms necessary for S phase initiation in vivo.     

Role of the aryl hydrocarbon receptor and Cyp1b1 in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin

The role of aryl hydrocarbon receptor (AhR) and cytochrome P450 (Cyp) 1 family in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in vivo. Immature (21 days old) AhR, Cyp1a2, or Cyp1b1 knockout (–/–) mice were treated intraperitoneally with estradiol (E2, 20 ng/mouse per day, for 14 consecutive days) and/or TCDD (200 ng/mouse per day, on days 7, 9, 11, and 13). Uterine wet weight and uterine peroxidase activity (UPA) were measured as markers of estrogen responsiveness. UPA was a better marker of estrogen responsiveness than the uterine wet weight. In AhR wild-type (+/+) mice, UPA (208.1±81.6 units/g tissue) was increased by the administration of E2 (to 297.2±178.7 units/g). The administration of TCDD significantly (p<0.01) decreased the UPA (10.5±3.4 units/g) compared with that in the control mice. Co-administration of TCDD with E2 also significantly (p<0.05) decreased the UPA (18.8±19.9 units/g) compared with that in E2-treated mice. In AhR(–/–) mice, UPA (162.9±146.7 units/g) was significantly (p<0.01) increased by the administration of E2 (486.8±108.2 units/g). In contrast to the results in AhR(+/+) mice, UPA was not affected by the administration of TCDD (51.8±70.6 units/g) compared with control, and co-administration of TCDD with E2 (545.8±189.4 units/g) compared with that in E2-treated mice. In Cyp1a2/1b1(+/+) mice, UPA was significantly (p<0.05) increased by the administration of E2 (70.0±36.4 units/g). Co-administration of TCDD with E2 significantly (p<0.05) decreased the UPA (29.6±22.2 units/g) compared with that in E2-treated mice. In Cyp1a2(–/–) mice, co-administration of TCDD with E2 significantly (p<0.01) decreased the UPA (6.8±5.1 units/g) compared with that in E2-treated mice. In Cyp1b1(–/–) mice, UPA (5.5±8.1 units/g) was significantly (p<0.05) increased by the administration of E2 (56.6±34.1 units/g). In contrast to the results in Cyp1a2/1b1(+/+) mice or Cyp1a2(–/–) mice, UPA was not affected by the co-administration of TCDD and E2 (52.6±30.1 units/g) compared with that in E2-treated mice. This is the first demonstration that Cyp1b1 as well as AhR is involved in the antiestrogenic effects of TCDD.     

Induction of cytochrome P450IA1 in mouse hepatoma cells by several chemicals. Phenobarbital and TCDD induce the same form of cytochrome P450

The mouse hepatoma cell line Hepa-1 was studied for aryl hydrocarbon hydroxylase (AHH) inducibility by sixteen compounds known to be inducers of cytochrome P450 of different "classes". Both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and sodium phenobarbital induced AHH activity. A cytochrome P450IA1-specific (P1-450) mouse cDNA probe was used to quantitate mRNA induction. There was a good correlation between the amount of cytochrome P450IA1 mRNA induced and AHH activity. Immunoblots with monoclonal antibody 1-7-1, which recognizes rat liver P450IA1 and P450IA2 (P450c and P450d, respectively), showed that both phenobarbital and TCDD increase the amount of a P450 isozyme immunorelated to P450IA1 in this cell line. Hepa-1 mutants with no AHH inducibility (no functional P450IA1 structural gene; no Ah receptor; no nuclear translocation of the inducer-receptor complex; and presence of dominant repressor) did not respond to phenobarbital. The cytosolic receptor for TCDD (Ah receptor) was characterized to see if phenobarbital induced cytochrome P450IA1 mRNA and the hydroxylase enzyme through the same mechanism as TCDD. 20 mM Phenobarbital almost completely abolished the binding of 3H-TCDD to the cytosolic receptor. These data indicate that phenobarbital can be a weak ligand for the Ah receptor and thus induce cytochrome P450IA1 and AHH activity. The observation increases the list of different P450 forms inducible by phenobarbital.