Effects of substituents on the cytosolic receptor-binding avidities and aryl hydrocarbon hydroxylase induction potencies of 7-substituted 2,3-dichlorodibenzo-p-dioxins. A quantitative structure-activity relationship analysis

The binding affinities of 16 7-substituted 2,3-dichlorodibenzo-p-dioxins for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cytosolic receptor protein from male Wistar rats have been determined. The EC50 value for each compound was estimated by competitive displacement of [3H]TCDD and the data illustrated that the differences between competitive ligands were dependent on the substituent (X) group. The EC50 value for 7-trifluoromethyl-2,3-dichlorodibenzo-p-dioxin was 1.95 X 10(-8) M and was greater than 1000-fold more active than 7-amino-2,3-dichlorodibenzo-p-dioxin (EC50 = 2.88 X 10(-5) M). Multiple parameter linear regression analysis of the data for 14 different compounds gave the following equation: log (1/EC50) = 1.24 pi + 6.11. This demonstrated that the binding affinity was linearly dependent on the lipophilicity (pi) of the 7-X-group. This contrasted with a comparable analysis of the substituent effects on the binding of 13 4'-substituted 2,3,4,5-tetrachlorobiphenyls to the cytosolic receptor which showed that the lipophilicity, electronegativity, and hydrogen-bonding capacity were important physicochemical determinants which facilitated binding to the receptor protein. These data suggest that the halogenated dibenzo-p-dioxins and biphenyls may interact with different binding sites on the receptor or they may bind to the same site but exert different conformational effects on the receptor protein. For the 7-X-2,3,-dichlorodibenzo-p-dioxins, there was not a rank order correlation between receptor-binding EC50 values and the induction of aryl hydrocarbon hydroxylase (AHH) or ethoxyresorufin O-deethylase in rat hepatoma H-4-II E cells in culture. However, the data could be correlated with an estimate of substituent width, the STERIMOL factor (B5), i.e., log (AHH) = 1.29 log (binding) + 2.19 delta B5 - 1.31 (delta B5)2 - 1.48. The importance of a steric factor in the correlation between receptor binding and AHH induction for substituted dibenzo-p-dioxins and halogenated biphenyls is consistent with a structure-dependent conformational change(s) in the receptor protein:ligand complex after the initial binding event. Presumably, this latter process is associated with the steps involving interactions between the ligand:receptor complex and nuclear binding sites.     

Competitive binding to the cytosolic 2,3,7,8-tetrachlorodibenzo-p-dioxin receptor. Effects of structure on the affinities of substituted halogenated biphenyls--a QSAR analysis

The proposed mechanism of action of the toxic halogenated aromatics, typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), involves the initial binding to a high-affinity, low-capacity, cytosolic receptor protein. Previous studies have shown that several 4'-halo-2,3,4,5-tetrachlorobiphenyls bind to the TCDD receptor and that a lateral substituent on both phenyl rings is required for activity. Using an extended series of eighteen 4'-substituted-2,3,4,5-tetrachlorobiphenyls as probes, the effects of a variable lateral substituent on receptor binding affinity and the induction of aryl hydrocarbon hydroxylase (AHH) in vivo and in rat hepatoma H-4-II E cells have been determined. For most substituents, there was an excellent correlation between the rank-order potency for receptor binding and the rank-order potency for AHH induction. Based on in vitro binding affinities (EC50 values) of the 4'-substituted tetrachlorobiphenyls, a multiparameter regression equation was formulated correlating the binding constants to physicochemical substituent parameters. For thirteen compounds out of the present series, multiple regression analysis of the binding data led to the following equation: log(1/EC50) = 1.53 sigma + 1.47 pi + 1.09HB + 4.08, r = 0.978. The results suggest that halogen substitution on both phenyl rings is not a requirement for binding and that hydrophobic (pi) and electronic (sigma) substituent constants and a variable for hydrogen bond (HB) formation are significant parameters describing relative binding avidities of this series of substituted biphenyls for the TCDD receptor.     

Partial antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated induction of aryl hydrocarbon hydroxylase by 6-methyl-1,3,8-trichlorodibenzofuran: mechanistic studies

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) binds with moderate affinity to the aryl hydrocarbon (Ah) receptor protein (4.9 x 10(-8) M) but is a weak Ah receptor agonist. Cotreatment of male Long Evans rats with MCDF (50 mumol/kg) and a dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that causes a near-maximal induction of hepatic microsomal aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase activities resulted in a significant inhibition of these activities for up to 96 hr. Comparable results were obtained with MCDF (10(-7) M) and TCDD (10(-8) M) in rat hepatoma H-4-II E cells in culture over 36 hr. TCDD treatment of rats resulted in an initial decrease of hepatic cytosolic Ah receptor within 6 hr and this was followed by a subsequent 138% increase in cytosolic receptor levels 72 hr after treatment. Although MCDF (50 mumol/kg) did not significantly alter rat hepatic cytosolic Ah receptor levels in animals cotreated with TCDD plus MCDF, the latter compound significantly inhibited TCDD-mediated replenishment of the cytosolic Ah receptor. In contrast, treatment of rat hepatoma H-4-II E cells with TCDD (10(-8) M) resulted in the rapid (within 1 hr) depletion of cytosolic Ah receptor, which remained undetectable for up to 36 hr; cotreatment of the cells with MCDF (10(-7) M) and TCDD (10(-8) M) resulted in cytosolic Ah receptor levels that were similar to those observed after treatment with TCDD alone. The effects of MCDF on the uptake and persistence of nuclear [3H]TCDD-Ah receptor complex levels were also determined in rat liver and rat hepatoma H-4-II E cells in culture. MCDF did not significantly decrease levels of occupied nuclear Ah receptor complexes in the rat or rat hepatoma cells. Moreover, using the sucrose density gradient assay procedure, the sedimentation coefficients of the cytosolic and nuclear TCDD-Ah receptor complexes in the presence or absence of MCDF were comparable. The results of these and other related studies with 6-substituted-1,3,8-trichlorodibenzofurans suggest that MCDF may act as a partial TCDD antagonist by competing with TCDD for nuclear binding sites.     

Nondetectable concentrations of human placental Ah receptors are associated with potent induction of microsomal benzo[a]pyrene hydroxylase in individuals exposed to polychlorinated biphenyls, quaterphenyls, and dibenzofurans

Human exposure to polychlorinated biphenyls, quaterphenyls, and dibenzofurans occurred in an accidental food poisoning episode in Taiwan in 1979. Our objective was to evaluate the role of Ah receptors in the production of biochemical effects detected in placentas from exposed subjects. Placentas were obtained from pregnant women who were identified from a registry of exposed individuals. Benzo[a]pyrene (BaP) hydroxylase was quantified in placental microsomes and Ah receptor was investigated in placental cytosolic and nuclear fractions by a variety of receptor-binding techniques using 2,3,7,8-[1,6-3H]tetrachlorodibenzo-p-dioxin ([3H]TCDD) as the ligand. Microsomal BaP hydroxylase was dramatically elevated in placentas of exposed subjects compared to those of nonexposed, nonsmoking control subjects. However, concentrations of displaceable [3H]TCDD binding in placental preparations from exposed or control subjects were uniformally low; approximately 1.0 fmol/mg cytosolic protein. Further evaluation of displaceable TCDD binding by sucrose-gradient sedimentation and hydroxylapatite column chromatography revealed that binding properties were different than those for the Ah receptor. These data suggest that extremely low concentrations of Ah receptors in human placenta may be sufficient to markedly elevate microsomal BaP hydroxylation. Alternatively, induction in human placenta may be mediated by a mechanism not requiring Ah receptor.     

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.     

Characterization of the aryl hydrocarbon receptor in the human C-4II cervical squamous carcinoma cell line.

Treatment of C-4II human cervical squamous carcinoma cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) gave a concentration-dependent increase in ethoxyresorufin O-deethylase (EROD) activity. The EC50 for this response was approximately 1 nM and the maximum induced activity was 27 pmol/min/mg protein. The molecular properties of the cytosolic and nuclear aryl hydrocarbon (Ah) receptor complex were determined by velocity sedimentation analysis, photoaffinity labeling, gel retardation using a consensus dioxin responsive element (DRE), and DNA-Sepharose, DRE-Sepharose and Sephacryl S-300 gel permeation column chromatography. The apparent molecular masses of the cytosolic and nuclear photoaffinity-labeled Ah receptor complexes were 110 kDa and differed from the corresponding values obtained for the Ah receptor from other animal species. In contrast, most of the other molecular properties of the Ah receptor were not significantly different from those previously reported for other species. The relative Ah-responsiveness of the C-4II cells was assessed by determining the ratio of the induced EROD activity/nuclear Ah receptor levels for a submaximal inducing dose of [3H]TCDD. The induced activity/binding ratio for the human C-4II cells was 0.77 and was at least one order of magnitude lower than the corresponding value for the Ah-responsive rat hepatoma H-4-II E cells.     

Muscarinic receptor binding profile of para-substituted caramiphen analogues

Para-substituted analogues of the antimuscarinic agent caramiphen were synthesized and evaluated for their ability to bind to the M1 and M2 subtypes of the muscarinic receptor. The purpose of the set was to look for a possible relationship in binding affinity or receptor subtype selectivity with aromatic substituent parameters such as Hammett's sigma or Hansch's pi values. It is felt this could be determined initially with only four properly chosen substituents. In this approach, substituents were chosen which have an extreme value for sigma and for pi, in a positive and negative direction, in all combinations. The substituents chosen for examination were amino (-sigma, -pi); 1-pyrrolidinyl (-sigma, +pi); 1-tetrazolyl (+sigma, -pi), and iodo (+sigma, +pi). It was determined in this research that caramiphen binds with high affinity (Ki = 1.2 nM) and is selective for the M1 over M2 muscarinic receptor subtype (26-fold). An examination of para-substitution reveals that compounds with electron-withdrawing (+sigma) substituents showed M1 selectivity, while the derivatives with electron-donating groups (-sigma) were nonselective in the binding assays. On the basis of this finding, the nitro and cyano derivatives were prepared and found to be M1 selective. The + sigma derivatives showed a decrease in M2 affinity while the p-nitro and p-iodo derivatives retained approximately equal affinity as caramiphen for the M1 site. The nitro- and iodocaramiphen derivatives were as potent (M1, Ki = 5.52 and 2.11 nM, respectively) and showed a greater selectivity of M1 over M2 binding than the M1 prototypical agent pirenzepine (M1, Ki = 5.21 nM).     

Detection of the Ah receptor in rainbow trout: use of 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin in cell culture.

The Ah receptor was detected in RTG-2 cells (rainbow trout embryonic gonad cells) following the addition of the photoaffinity ligand, [125I]2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, to cells in culture. Cytosolic and nuclear extracts were prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed one radiolabeled band. Very little non-specific binding was observed under the conditions employed when compared to photoaffinity labeling RTG-2 cytosolic extracts in vitro. The photoaffinity-labeled Ah receptor in RTG-2 cytosol was analyzed by sucrose density centrifugation. The cytosolic form was observed to sediment at approximately 9.8S and the high salt nuclear extract form at approximately 7.5S. The relative molecular weight of the Ah receptor was determined to be 145 kDa under denaturing conditions and is considerably larger than the Ah receptor from mammalian sources. Inhibition of photoaffinity ligand binding to the RTG-2 cytosolic Ah receptor by competing ligands revealed the same rank order of ligand affinity as that previously demonstrated for the mouse Ah receptor.     

Comparison of aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase induction by polycyclic aromatic compounds in human and mouse cell lines

The human MCF-7 and the mouse Hepa-1 cell culture lines were compared for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]anthracene (BA) and TCDD- and BA-specific binding in the cytosol and nucleus. The effective concentration of BA in the growth medium required to induce either enzyme to 50% of its maximally inducible activity (EC50) was the same (5-11 microM) in both MCF-7 and Hepa-1 cells. On the other hand, the EC50 for TCDD in MCF-7 cells (5-25 nM) was more than 40-fold greater than that in Hepa-1 cells (0.4 to 0.6 nM). P1-450- and P3-450-specific mouse cDNA probes were used to quantitate mRNA induction in the Hepa-1 cell line. P1-450 mRNA was induced markedly by TCDD and benzo[a] anthracene, whereas P3-450 mRNA was induced negligibly. A P1-450-specific human cDNA probe was used to quantitate P1-450 mRNA induction in the MCF-7 cell line. Aryl hydrocarbon hydroxylase inducibility by TCDD or BA always paralleled P1-450 mRNA inducibility in either the mouse or human line. Although the cytosolic Ah receptor in Hepa-1 cells was easily detected by sucrose density gradient centrifugation, gel permeation chromatography, and anion-exchange high-performance liquid chromatography, the cytosolic receptor cannot be detected in MCF-7 cells. Following in vivo exposure of cultures to radiolabeled TCDD, the intranuclear concentration of inducer-receptor complex was at least fifty times greater in Hepa-1 than MCF-7 cultures. The complete lack of measurable cytosolic receptor and almost totally absent inducer-receptor complex in the nucleus of MCF-7 cells was, therefore, out of proportion to its capacity for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility. This MCF-7 line should provide an interesting model for a better understanding of the mechanisms of drug-metabolizing enzyme induction by polycyclic aromatic compounds, including the Ah receptor-mediated mechanism.     

Ah receptor mediating induction of cytochrome P450IA1 in a novel continuous human liver cell line (Mz-Hep-1). Detection by binding with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin and relationship to the activity of aryl hydrocarbon hydroxylase.

The Ah receptor regulates induction of cytochrome P450IA1 and mediates certain toxicities of polyhalogenated aromatics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It has been characterized previously in continuous cell lines, notably the mouse hepatoma line Hepa 1, the human squamous cell carcinoma line A431, and the human liver cell line Hep G2. The present work extends our knowledge of the Ah receptor in continuous human liver cell lines. Ah receptor can be detected in Mz-Hep-1, a hepatitis B virus-negative cell line derived from a Thorotrast-induced hepatocellular carcinoma. The mean concentration of Ah receptor in Mz-Hep-1 cells was 341 +/- 22 fmol/mg cytosol protein (mean +/- SEM, nine separate determinations). This is equivalent to approximately 30,000 sites per cell. The concentration of Ah receptor in Mz-Hep-1 cells is similar to that in Hepa 1 cells and approximately three times higher than that in Hep G2 cells. The Mz-Hep-1 Ah receptor sedimented in continuous sucrose gradients at approximately 9 S. Specificity of binding by [3H]TCDD was demonstrated by competitive binding of non-radiolabeled 2,3,7,8-tetrachlorodibenzofuran, 3-methylcholanthrene (MC), and dibenz[a,h]anthracene in 50-fold molar excess. Phenobarbital, which is not a substrate for P450IA1, did not compete with [3H]TCDD for binding to Mz-Hep-1 Ah receptor. Dexamethasone and estradiol also did not compete with [3H]TCDD for binding, suggesting non-identity of Ah receptor with glucocorticoid or estrogen receptor. In separate experiments, glucocorticoid receptor was identified in Mz-Hep-1 cells. By Scatchard plot analysis, the apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Mz-Hep-1 Ah receptor was estimated to be 4.4 nM, compared to 0.8 nM in Hepa 1 cells. By Woolf plot analysis the Kd was 5.4 nM, compared to 1.2 nM in Hepa 1 cells. The [3H]TCDD.Ah receptor complex extracted from nuclei of Mz-Hep-1 cells incubated with [3H]TCDD in culture at 37 degrees sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was detectable in Mz-Hep-1 cells after pretreatment with inducing chemicals. Mz-Hep-1 cells have the highest concentrations of Ah receptor in any continuous human liver cell line thus far investigated. The Mz-Hep-1 Ah receptor is similar physicochemically to that described in murine systems. AHH activity is inducible in Mz-Hep-1 cells.     

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.     

Detection and characterization of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin binding to Ah receptor in a rainbow trout hepatoma cell line

Ah receptor was identified and characterized in cytosol and nuclear extracts from the rainbow trout hepatoma cell line RTH-149. The cytosolic receptor was detectable with both halogenated ([3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) and non-halogenated ([3H]3-methylcholanthrene and [3H]benzo[a]pyrene) aromatic hydrocarbons and sedimented at approximately 9 S after velocity sedimentation on sucrose gradients. The apparent binding affinity (kd) of cytosolic Ah receptor was always less than 1 nM as derived from Scatchard or Woolf plot analyses. The same analyses indicated a concentration of Ah receptor in the RTH-149 cells of approximately 20 fmol/mg cytosolic protein or approximately 4400 receptor sites per cell. Thus, this trout hepatoma cell line has a low concentration of high-affinity binding sites in comparison to Ah receptor concentrations in cytosol obtained from rodent tissues. Incubation of whole cells with the radioligand [3H]TCDD resulted in transformation of the cytosolic Ah receptor to a nuclear binding form which could be detected as a specifically labeled peak sedimenting at approximately 6 S on sucrose gradients. Aryl hydrocarbon hydroxylase was induced after exposure of RTH-149 cells to TCDD or benz[a]anthracene for 24 hr in culture. These data demonstrate the existence of the Ah receptor in a cell line derived from a nonmammalian species and provide an additional step toward understanding the mechanisms by which fish respond to specific aquatic contaminants.     

A Human Aryl Hydrocarbon Receptor Signaling Pathway Constructed in Yeast Displays Additive Responses to Ligand Mixtures

An optimized signal transduction pathway that reproduces the response of human aryl hydrocarbon (Ah) receptor to ligands has been established in Saccharomyces cerevisiae. Ligand treatment induced a 50-fold increase in beta-galactosidase activity from a reporter plasmid in yeast engineered to express human Ah receptor and Ah nuclear translocator (Arnt) proteins. The archetypal Ah receptor ligand, 2,3,7,8-tetrachlorodibenzo(p)dioxin, activated Ah receptor and induced lacZ reporter activity at concentrations of >/=0.3 nM. Mixtures of halogenated and nonhalogenated Ah receptor ligands produced additive signaling responses in this yeast bioassay. These results were consistent with the existence of a common binding site and mechanism of ligand-mediated Ah receptor activation. Although yeast have no natural counterpart to the Ah receptor pathway, expression of human Ah receptor and Arnt under the appropriate conditions provides a functional model system for studying Ah receptor activation and signal transduction.     

2,2',4,4',5,5'-hexachlorobiphenyl as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6J mice

At doses as high as 750 to 1000 mumol/kg, 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP) did not cause fetal cleft palate, suppress the splenic plaque-forming cell response to sheep red blood cells, or induce hepatic microsomal ethoxyresorufin O-deethylase (EROD) in C57BL/6J mice. Despite the lack of activity of HCBP in eliciting any of these aryl hydrocarbon (Ah) receptor-mediated responses, competitive binding studies indicated that HCBP competitively displaced 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) from the murine hepatic cytosolic receptor. Cotreatment of C57BL/6J mice with TCDD (3.7 nmol/kg) and HCBP or 4,4'-diiodo-2,2',5,5'-tetrachlorobiphenyl (I2-TCBP) (400 or 1000 mumol/kg) showed that both compounds partially antagonized TCDD-mediated cleft palate and immunotoxicity (i.e., suppression of the splenic plaque-forming cell response to sheep red blood cells), and HCBP antagonized TCDD-mediated hepatic microsomal EROD induction. Thus, HCBP and I2-TCBP, like the commercial polychlorinated biphenyl mixture Aroclor 1254, were partial antagonists of TCDD action in C57BL/6J mice; however, it was also apparent from the results that Aroclor 1254 was the more effective antagonist at lower doses. Using [3H]TCDD, it was also shown that some of the effects of HCBP on TCDD-mediated cleft palate may be due to the decreased levels of TCDD found in the fetal palates after cotreatment with TCDD and HCBP. 4,4'-[125I2]diiodo-2,2',5,5'-tetrachlorobiphenyl ([125I2]TCBP) of high specific activity (3350 Ci/mmol) was synthesized and used to investigate the direct binding of this compound to the murine hepatic Ah receptor or other cytosolic proteins. No direct specific binding was observed between 125I2-TCBP and any cytosolic proteins using a sucrose density gradient assay procedure. These results contrasted with previous studies with Aroclor 1254 that suggested that this mixture acted as a competitive Ah receptor antagonist.     

An in vitro model for studying the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to human thymus

A coculture system of human thymic epithelial (HuTE) cells and thymocytes (T lymphocyte precursors) has been established and characterized as an in vitro model for assessing the potential toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to human thymus. HuTE cells in culture were responsive to TCDD as judged by induction of the cytochrome P1-450 monooxygenase activities, 7-ethoxycoumarin O-deethylase (ECOD) and 7-ethoxyresorufin O-deethylase (EROD). Measurement of the responsiveness of thymocytes cocultured on TCDD-pretreated HuTE monolayers to the mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) indicated that TCDD can act directly on HuTE cells to suppress thymocyte maturation (at a concentration of 10 nM, TCDD produced a 25 to 50% inhibition of thymocyte responsiveness to Con A and PHA). Both the induction of cytochrome P1-450 monooxygenase activity (EC50 values approximately 1 nM) and immunosuppressive responses elicited by TCDD in HuTE cells were concentration-dependent and stereospecific (as judged by the relative activities of chlorinated dibenzo-p-dioxin and dibenzofuran isomers), indicating involvement of the Ah receptor which was detected in all HuTE strains examined. Initial characterization of these Ah receptor-mediated responses in several strains of HuTE cells indicated marked interstrain differences in maximally inducible ECOD and EROD activities which did not appear to directly correlate with measured concentrations of the cytosolic Ah receptor, and in certain strains examined, differences in sensitivity and magnitude were observed for TCDD-evoked immunotoxic responses but not always for the induction response. These data on the actions of TCDD on cultured HuTE cells suggest that human thymus is a target for TCDD and related halogenated aromatic compounds. In HuTE cells, measurement of either the Ah receptor concentration or of marker responses such as the induction of cytochrome P1-450 alone cannot provide an accurate quantitative assessment of susceptibility to TCDD-induced thymus toxicity.     

Cytostatic and antiestrogenic effects of 2-(indol-3-ylmethyl)-3,3'-diindolylmethane, a major in vivo product of dietary indole-3-carbinol.

Under acidic conditions, indole-3-carbinol (13C) is converted to a series of oligomeric products thought to be responsible for the biological effects of dietary 13C. Chromatographic separation of the crude acid mixture of 13C, guided by cell proliferation assay in human MCF-7 cells, resulted in the isolation of 2-(indol-3-ylmethyl)-3,3'-diindolylmethane (LTr-1) as a major antiproliferative component. LTr-1 inhibited the growth of both estrogen-dependent (MCF-7) and -independent (MDA-MB-231) breast cancer cells by approximately 60% at a non-lethal concentration of 25 microM. LTr-1 had no apparent effect on the proliferation of MCF-7 cells in the absence of estrogen. LTr-1 was a weak ligand for the estrogen receptor (ER) (IC50 70 microM) and efficiently inhibited the estradiol (E2)-induced binding of the ER to its cognate DNA responsive element. The antagonist effects of LTr-1 also were exhibited in assays of endogenous pS2 gene expression and in cells transiently transfected with an estrogen-responsive reporter construct (pERE-vit-CAT). LTr-1 activated both binding of the aryl hydrocarbon (Ah) receptor to its cognate DNA responsive element and expression of the Ah receptor-responsive gene CYP1A1. LTr-1 was a competitive inhibitor of CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity. In summary, these results demonstrated that LTr-1, a major in vivo product of I3C, could inhibit the proliferation of both estrogen-dependent and -independent breast tumor cells and that LTr-1 is an antagonist of estrogen receptor function and a weak agonist of Ah receptor function.     

Detection and characterization of Ah receptor in tissue and cells from human tonsils.

Ah receptor was identified and characterized in cytosol and nuclear extracts from human tonsils obtained at surgery from children 2 to 6 years of age. Ah receptor was found in cytosol prepared from whole-tonsil homogenates as well as in cytosol and nuclear fractions prepared from tonsil lymphocytes or tonsil fibroblasts grown in primary culture. Cytosolic Ah receptor was detectable in tonsillar tissue with either halogenated (2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD)) or nonhalogenated (3-[3H]methylcholanthrene and [3H]benzo[a]pyrene) aromatic hydrocarbons and sedimented at approximately 9 S after velocity sedimentation on sucrose gradients. The apparent binding affinity (Kd) of [3H]TCDD for Ah receptor ranged from 3 to 12 nM in cytosols from seven different donors. The same analyses indicated a concentration of Ah receptor in human tonsils of approximately 100-300 fmol/mg cytosolic protein. Incubation of either tonsil lymphocytes or tonsil fibroblasts with [3H]TCDD resulted in transformation of cytosolic Ah receptor to a nuclear binding form which could be detected as a specifically labeled peak sedimenting at approximately 6 S on sucrose gradients. These data demonstrate the existence of Ah receptor in human tonsils and suggest that this immune organ may be an appropriate model for further studies on the mechanism and manifestation of aromatic hydrocarbon-induced immunotoxicity in man.     

Measurements of the cytosolic Ah receptor among four strains of Drosophila melanogaster

Four strains of Drosophila melanogaster exhibit differences in aryl hydrocarbon hydroxylase (AHH) inducibility by phenobarbital or Aroclor 1254, yet do not show the typical AHH induction response when exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[a]anthracene. Adult flies were nevertheless examined for the presence of cytosolic TCDD-specific binding (Ah receptor). Berlin-K and Haag 79 exhibit AHH induction by Aroclor 1254 and possess detectable amounts of Ah receptor. Hikone-R has negligible AHH inducibility by Aroclor 1254, yet possesses measurable amounts of the receptor. Oregon-K displays AHH induction by Aroclor 1254 but has no detectable levels of the cytosolic receptor. Specific (high-affinity, low-capacity and saturable) binding of [³H-1,6]TCDD to the Ah receptor in D. melanogaster was shown to be similar to that observed in C57BL/6 mouse liver. Similar specific binding of generally labeled [³H]benzo[a]anthracene in D. melanogaster cytosol was not found. These data suggest that the presence of the Ah receptor per se, or quantity of receptor, does not guarantee AHH inducibility by TCDD or benzo[a]anthracene in adults of these four fruit fly strains.     

1,4-Dihydropyridine antagonist activities at the calcium channel: a quantitative structure-activity relationship approach

The effect of 46 1,4-dihydropyridine-type calcium channel antagonists on the tonic contractile response of longitudinal muscle strips of guinea pig ileum was determined. 2,6-Dimethyl-3,5-dicarbomethoxy-4-phenyl-1,4-dihydropyridine (13) and 13 ortho-, 15 meta-, and seven para-monosubstituted and 10 polysubstituted aromatic derivatives of 13 were studied. The pharmacological activities of the monosubstituted derivatives were best correlated by eq 10, log 1/C = 0.68 pi + 2.50 sigma m -0.47Lmeta -3.40B1para + 11.31, which had a correlation coefficient of 0.89. The full data set was best correlated by eq 11, log 1/C = 0.62 pi + 1.96 sigma m -0.44Lmeta -3.26B1para -1.51Lmeta' + 14.23, which had a correlation coefficient of 0.90. Equations of similar form but involving an ortho steric term were found to correlate the radioligand binding data for this class of compounds.     

Induction of aryl hydrocarbon hydroxylase and demonstration of a specific nuclear receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in two human hepatoma cell lines

Two established human hepatoma cell lines, Hep3B and HepG2, were examined for aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) induction and for the presence of the murine-equivalent aromatic hydrocarbon (Ah) receptor. Both cell lines demonstrated polycyclic aromatic hydrocarbon (PAH)-induced AHH activity; however, assay conditions for induction were different than those established for the control mouse hepatoma cell line, Hepa c1-9. When cytosols from either cell line were exposed to tritiated 2,3,7,8-tetrachlorodibenzo-p-dioxin [( 3H]TCDD) and analyzed on sucrose gradients with or without prior charcoal treatment, two peaks were observed at positions corresponding to 4-5 S and 8-9 S. The 8-9 S peak was identified as the probable human Ah receptor equivalent since, like the mouse Ah receptor, this peak: (a) was eliminated only by cytochrome P1-450 inducers; (b) was sensitive to protease digestion; and (c) was thermolabile. Levels of TCDD specifically bound in the 8-9 S peak for HepG2 and Hep3B were 27 and 34 fmol/mg cytosolic protein respectively. The level of TCDD specifically bound was not affected by charcoal treatment or by the addition of sodium molybdate, which is known to stabilize ligand binding to steroid receptors. Incubation of Hep3B or HepG2 cells with [H]TCDD at 37 degrees for 1 hr effected a redistribution of binding from the cytosol 8-9 S peak to a nuclear 6 S peak. The nuclear peaks from both human cell lines demonstrated similar sedimentation properties, temperature-dependence and inducer-specificity, as for the mouse nuclear Ah receptor. Appearance of nuclear 6 S binding is consistent with a temperature-dependent translocation process, supporting the observation that these human hepatoma cell lines contain a binding component which is similar to the mouse Ah receptor in structure and function during AHH induction.