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T Zacharewski

Publications and source records attributed to T Zacharewski.

At least 37 records · Page 2Linked to original sources
PubMed1992

Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently.

We have purified and cloned a HeLa cell nuclear protein that strongly stimulates binding of retinoic acid and thyroid hormone receptors (RARs and TRs) to response elements. The purified protein is a human retinoid X receptor beta (hRXR beta). Three murine members of the RXR family (mRXR alpha, beta, and gamma) have also been cloned, and their interactions with RARs and TRs have been investigated. Under conditions where RAR, RXR, and TR bound poorly as homodimers to various response elements, strongly cooperative RAR-RXR and TR-RXR binding was observed. The binding efficiency was dependent on the sequence, relative orientation, and spacing of the repeated motifs of response elements. We show also that unstable RAR-RXR heterodimers were formed in solution, and that C-terminal sequences and the DNA-binding domains of both receptors were required for efficient formation of stable heterodimers on response elements. These findings suggest a convergence of the signaling pathways of some members of the nuclear receptor superfamily.

Amino Acid Sequence↗
PubMed1992

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) as an antiestrogen in human and rodent cancer cell lines: evidence for the role of the Ah receptor.

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) is a relatively nontoxic analog of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Treatment of aryl hydrocarbon (Ah)-responsive MCF-7 human breast cancer cells with 100 nM MCDF resulted in the inhibition of 17 beta-estradiol-induced proliferation and the secretion of the 34-, 52-, and 160-kDa proteins. After treatment of the cells with 17 beta-[3H]estradiol, 100 nM of MCDF caused a decrease in the accumulation of the radiolabeled nuclear estrogen receptor (ER) complex in these cells. In parallel experiments, the antiestrogenic effects of MCDF were also determined in Ah-responsive wild-type Hepa 1c1c7 cells and Ah-nonresponsive class 1 and class 2 mutant cells. Treatment of the wild-type cells with 17 beta-[3H]estradiol and 100 nM MCDF caused a decrease in the accumulation of radiolabeled nuclear ER complex in these cells whereas no significant effects were observed in the mutant cells as determined by velocity sedimentation analysis. Comparable results were obtained using ER antibodies to measure the decrease in immunoreactive nuclear ER. In addition, both actinomycin D and cycloheximide inhibited the MCDF-mediated decrease of nuclear ER levels in the Hepa 1c1c7 wild-type cells. Although 100 nM MCDF did not induce cytochrome P-450-dependent monooxygenases in the MCF-7 or Hepa 1c1c7 cell lines, incubation of nuclear extracts from the MCF-7 cells treated with 100 nM MCDF with a synthetic consensus dioxin responsive element (an oligonucleotide duplex of 26 bases) gave a retarded band in a gel-retardation assay. The data suggest that the antiestrogenic effects of MCDF does not require the induction of the CYP1A1 gene expression but may involve the induction of other genes.

Animals↗
PubMed1991

DNA binding properties of the Ah receptor in wild-type and variant mouse hepatoma cells.

Mouse hepatoma Hepa 1c1c7 cells and nonresponsive mutants have been extensively used as models for investigating the molecular mechanism of induction of CYP1A1 gene transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Incubation of cytosolic [3H]TCDD-aryl hydrocarbon (Ah) receptor from wild-type Hepa 1c1c7 cells for 16 h at 4 degrees C in 0.4 M KCl resulted in the formation of transformed liganded receptor which exhibited increased binding affinity on DNA-Sepharose columns. The elution properties of the peak with the highest DNA binding affinity were similar to the elution profiles of the nuclear receptor complex isolated from wild-type cells. TAOc1BPrcl (class I) nonresponsive mutant cells were characterized by relatively low levels of the cytosolic and nuclear Ah receptor complex. The BPrcl (class II) variant cell line contained levels of cytosolic receptor which were comparable to those observed in the wild-type cells; however, significantly reduced levels of nuclear receptor complex were observed in the class II variant cell line. Incubation of the nuclear or transformed liganded cytosolic Ah receptor from wild-type cells with a consensus 32P-labeled dioxin responsive element (DRE) in a gel shift assay gave a retarded band associated with the receptor-DRE complex. Incubation of the cytosolic receptor complex from the class I and II mutant cells for 16 h at 4 degrees C in 0.4 M KCl or for 2 h at 20 degrees C did not yield complexes with increasing binding affinities on DNA-Sepharose columns. Moreover, incubation of these complexes with 32P-labeled DRE did not give a retarded band in a gel shift assay. However, coincubation of the liganded class II mutant cytosol with cytosol from class I cells resulted in transformation of the liganded receptor and this was confirmed in both the DNA-Sepharose and gel retardation assays. These results suggest that the failure of class II mutant cells to respond to TCDD is due to a defect in the factors responsible for transformation of the cytosolic receptor complex.

Animals↗
PubMed1991

Evidence for the mechanism of action of the 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated decrease of nuclear estrogen receptor levels in wild-type and mutant mouse Hepa 1c1c7 cells.

Treatment of wild-type Hepa 1c1c7 cells with 1 nM [3H]-17 beta-estradiol resulted in the rapid accumulation of the nuclear estrogen receptor complex whose levels were maximized within 1 hr. Cotreatment of the cells with 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and [3H]-17 beta-estradiol did not affect the nuclear estrogen receptor levels 1 hr after addition of the radioligand; however, pretreatment of the cells for 1, 6, 24 or 42 hr with 10 nM TCDD prior to the addition of the radiolabeled hormone caused a greater than 50% decrease in nuclear estrogen receptor levels (determined by velocity sedimentation analysis) 1 hr after the addition of [3H]-17 beta-estradiol. In parallel experiments in which 10 nM TCDD was added 6 hr prior to the radiolabeled hormone, TCDD caused a 63 and 74% decrease in immunodetectable cytosolic and nuclear estrogen receptor protein levels, respectively, in the wild-type Hepa 1c1c7 cells. The nuclear estrogen receptor was also detected in two Hepa 1c1c7 mutant (class 1 and class 2) cell lines which have been characterized previously as TCDD non-responsive due to either decreased aryl hydrocarbon (Ah) receptor levels or a defect in the accumulation of transcriptionally active nuclear Ah receptor complexes, respectively. Treatment of these mutant cell lines with TCDD and [3H]-17 beta-estradiol (as described above) caused only a minimum (class 1) or non-detectable (class 2) decrease in nuclear estrogen receptor binding activity or immunodetectable protein levels. These results, coupled with the structure-dependent differences in the activities of TCDD (a strong Ah receptor agonist) and 2,8-dichlordibenzo-p-dioxin (a weak Ah receptor agonist) in this assay system, support a role for the Ah receptor in the TCDD-mediated decrease of the nuclear estrogen receptor in mouse Hepa 1c1c7 cells. In addition, actinomycin D and cycloheximide both inhibited the TCDD-mediated decrease of nuclear estrogen receptor levels in the Hepa 1c1c7 wild-type cells, and these results suggest that TCDD may induce specific gene products which are involved in this process.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds as antioestrogens: characterization and mechanism of action.

In the female Sprague-Dawley rat uterus 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds exhibited a broad spectrum of antioestrogenic responses. For example 2,3,7,8-TCDD inhibited the 17 beta-oestradiol-induced uterine wet weight increase, peroxidase activity, oestrogen and progesterone receptor levels, epidermal growth factor (EGF) receptor binding, and EGF receptor and c-fos protooncogene mRNA levels. The aryl hydrocarbon (Ah) receptor was identified in the rat uterus and the antioestrogenic activities of TCDD and related compounds were structure-dependent. In parallel studies, the effects of TCDD as an antioestrogen in MCF-7 human breast cancer cells was also investigated. TCDD inhibited the 17 beta-oestradiol-induced proliferation of these cells and the secretion of the 34-, 52- and 160-kDa proteins. Treatment of MCF-7 cells with 1 nM [3H]-17 beta-oestradiol resulted in a rapid accumulation of nuclear oestrogen receptor (ER) complexes. Pretreatment of the cells with TCDD caused a rapid decrease in nuclear ER binding activity and immunoreactive protein; moreover, the structure-dependent potencies of TCDD and related compounds as antioestrogens were similar to their Ah receptor binding affinities. TCDD also caused a decrease in nuclear ER levels in wild-type Ah-responsive Hepa 1c1c7 cells but was inactive in Ah non-responsive mutant Hepa 1c1c7 cells. Moreover, in the wild-type cells, both actinomycin D and cycloheximide blocked the effects of TCDD. 6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) has previously been characterized as a TCDD antagonist in rodents and in transformed rodent cell lines. However, like TCDD, MCDF also exhibited a broad spectrum of antioestrogenic activities in both the female Sprague-Dawley rat uterus and MCF-7 cells. MCDF is relatively non-toxic compared to TCDD and is being investigated as a compound which may be clinically useful for the treatment of mammary cancer.

Animals↗
PubMed1990

Structure-dependent induction of aryl hydrocarbon hydroxylase activity in C57BL/6 mice by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related congeners: mechanistic studies.

The time- and dose-dependent induction of murine hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities by five polychlorinated dibenzo-p-dioxin and dibenzofuran congeners showed that the order of induction potency was 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) greater than 2,3,7,8-tetrachlorodibenzofuran (TCDF) greater than 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PCDD) greater than 1,2,3,7,8-pentachlorodibenzofuran (PCDF) greater than 2,3,7-trichlorodibenzo-p-dioxin (TrCDD). These structure-induction relationships were comparable to the structure-toxicity and competitive structure-receptor binding relationships previously reported for these compounds. However, using the corresponding radiolabeled congeners, the direct binding Kd values for dissociation of the cytosolic receptor-ligand complexes were 9.52, 7.96, 1.27, 3.10, and 8.31 nM for the 2,3,7,8-TCDD, 2,3,7,8-TCDF, 2,3,7-TrCDD, 1,2,3,7,8-PCDD, and 1,2,3,7,8-PCDF congeners and these data were clearly not structure dependent (i.e., similar to the structure-activity relationships). Some of the molecular properties for several radioligand-receptor complexes were similar; for example, the sedimentation coefficients for the cytosolic and nuclear receptor complexes varied from 8.8-10.4 S and 5.98-7.0 S, respectively, and the nuclear receptor complexes for all the radioligands eluted from a DNA-Sepharose column at salt concentrations of 0.27-0.29 M. Treatment of the mice with a maximum inducing dose of 2,3,7,8-[3H]TCDD resulted in a time-dependent formation of the nuclear receptor complex which was maximized between 16-24 hr and subsequently decreased up to 72 hr after initial exposure. In parallel studies, the nuclear receptor complex levels were determined 16 hr after treatment of the mice with different doses (2.25, 4.5, and 45 micrograms/kg) of all five radioligands. The results showed that at submaximal induction of the monooxygenase enzyme activities there was a linear correlation between the induced AHH or EROD activities (after 32 hr) and the corresponding nuclear receptor complex levels. It was also apparent from the data that the relative levels of nuclear receptor complex were structure dependent and this suggests that the transformation or activation of cytosolic receptor complexes may be a ligand structure-dependent process which correlates with the observed structure-activity relationships for 2,3,7,8-TCDD and related compounds.

Animals↗
PubMed1990

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds on the occupied nuclear estrogen receptor in MCF-7 human breast cancer cells.

Treatment of MCF-7 human breast cancer cells with 17 beta-[3H]estradiol resulted in a rapid accumulation of occupied nuclear estrogen receptor complex in which levels were maximized within 1 h and decreased after 3 h. Pretreatment of the cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 6 or 12 h prior to the addition of the radiolabeled hormone resulted in a 38 and 63% reduction in the levels of the occupied nuclear estrogen receptor, respectively, whereas addition of TCDD 1 h prior to the radioligand did not cause any significant change in the levels of the occupied nuclear receptor using velocity sedimentation analysis. Moreover, it was also shown with estrogen receptor antibodies that the TCDD-mediated dose-dependent decrease in occupied nuclear receptor levels was paralleled by a comparable decrease in immunoreactive protein at concentrations of 10(-8) to 10(-11) M TCDD. The reduction in levels of the occupied nuclear estrogen receptor was not due to increased estradiol metabolism since a significant reduction was observed at TCDD concentrations (10(-11) to 10(-13) M) which do not induce cytochrome P-450-dependent monooxygenase enzyme activities in MCF-7 cells. Treatment of the MCF-7 cells with actinomycin D or cycloheximide resulted in a greater than 2-fold increase in levels of the occupied nuclear estrogen receptor, and cotreatment of the cells with both TCDD and these inhibitors significantly decreased levels of the nuclear estrogen receptor complex. The structure-activity relationships for TCDD and several congeners were similar for both the reduction of occupied nuclear estrogen receptor levels and several aryl hydrocarbon (Ah) receptor agonist activities, and these results support a role of the Ah receptor in these processes.

Breast Neoplasms↗
PubMed1990

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on I-compounds in hepatic DNA of Sprague-Dawley rats: sex-specific effects and structure-activity relationships.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds on the specific patterns of age-dependent I-compound DNA adducts in the liver of male and female Sprague-Dawley rats were determined by the 32P-postlabeling assay. In female rats, TCDD causes a dose-dependent decrease of several individual and total hepatic I-compound levels after administration of 1 and 5 micrograms/kg per week for 4 weeks. In contrast, no such effects were observed in male Sprague-Dawley rats treated with the 5 micrograms/kg dose level of TCDD. The relative effects of TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PCDD) and 1,2,4,7,8-PCDD on hepatic I-compound levels in the susceptible female Sprague-Dawley rats were determined using a dose of 5 micrograms/kg per week for 4 weeks. The two compounds which are substituted in all four lateral positions, namely TCDD and 1,2,3,7,8-PCDD, caused a significant decrease in hepatic I-compound levels, whereas 1,2,4,7,8-PCDD which is substituted in only three lateral positions was inactive. The structure-activity relationships observed for the effects of these compounds on hepatic I-compounds correlated with their corresponding structure-Ah receptor binding and structure-toxicity relationships. The results are therefore consistent with a role for the Ah receptor in the TCDD-mediated reduction in hepatic I-compound levels in female Sprague-Dawley rats. These results and data from previous studies demonstrate a correlation between the susceptibility of an organ/species to the carcinogenic effects of TCDD and the reduction of I-compound levels. The significance of this correlation in the development of TCDD-induced carcinogenesis has not been delineated.

Age Factors↗
PubMed1989

Photoaffinity labeling of the nuclear Ah receptor from mouse Hepa 1c1c7 cells using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin.

The photoinduced formation of the covalently labeled cytosolic and nuclear aryl hydrocarbon (Ah) receptors was studied using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the photoaffinity label. Irradiation of TCDD alone at wavelengths of greater than 300 nm resulted in rapid degradation of this compound (t 1/2 = 8 min). In a separate experiment, the unliganded cytosolic Ah receptor was only slowly inactivated (t 1/2 = 48 min) using the greater than 300 nm light source. Preliminary experiments with rat hepatic cytosol did not result in significant formation of specifically bound [3H]TCDD-protein covalent adducts which could be visualized by autoradiography. Irradiation of [3H]TCDD-nuclear Ah receptor complexes isolated from mouse Hepa 1c1c7 cells for 15 min gave approximately a 40% overall yield of the radiolabeled Ah receptor protein adduct. Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]TCDD-nuclear Ah receptor photoadduct gave a single major radiolabeled protein with an apparent molecular size of 91 kDa. The chromatographic properties of the control (dark) and photolabeled nuclear Ah receptor complexes were comparable using Sephacryl S-300 and DNA-Sepharose columns. Velocity sedimentation of both the control (dark) and irradiated nuclear Ah receptor complexes gave specifically bound peaks which sedimented at 6.5 S. However, the trichloroacetic acid-precipitable (buffer-reconstituted) [3H]TCDD-nuclear Ah receptor photo-covalent adduct was eluted from the Sephacryl S-300 column in the void volume and did not exhibit a specifically bound peak after velocity sedimentation analysis due to protein aggregate formation. In contrast, the elution profile of the aggregate on a DNA-Sepharose column was similar to that observed for the control (dark) and photolabeled complexes, which were eluted from the column with salt concentrations between 0.24 and 0.28 M. These photolabeling studies show that [3H] TCDD can act as a photoaffinity label for the Ah receptor and can be utilized as photoligand to probe further the structure and function of this protein.

Affinity Labels↗
PubMed1989

Structure-dependent induction of aryl hydrocarbon hydroxylase in human breast cancer cell lines and characterization of the Ah receptor.

The structure-dependent induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and 1,2,4,7,8-pentachlorodibenzo-p-dioxin was determined in the MCF-7, T47-D, and MDA-MB-231 human breast cancer cell lines. Both the MCF-7 and T47-D cells were responsive to the induction effects of the halogenated aryl hydrocarbons and the structure-induction relationships were comparable to the reported structure-activity (induction, receptor binding, and toxicity) relationships observed in rodents and rodent cells in culture. The induction of ethoxyresorufin O-deethylase in the T47-D cells was the most sensitive aryl hydrocarbon (Ah) receptor-mediated response in both cell lines and this enzyme activity was more inducible than aryl hydrocarbon hydroxylase. In contrast, the three congeners were inactive as monooxygenase enzyme inducers in the MDA-MB-231 cells. Despite the differential Ah responsiveness of the cell lines, incubation of the cells with [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin followed by extraction of the nuclei with high salt and velocity sedimentation analysis of the extracts showed that specifically bound nuclear Ah receptor complexes were present in the three cell lines. The sedimentation coefficients (and levels) for the nuclear receptors were 6.6 S (32.1 fmol/mg protein/mg DNA), 6.9 S (61.6 fmol/mg protein/mg DNA), and 7.4 S (38.2 fmol/mg protein/mg DNA) in the T47-D, MCF-7, and MDA-MB-231 cell lines, respectively. Cytosolic receptor was also detected in the MCF-7 and MDA-MB-231 cells. Thus, despite the differences in Ah responsiveness of the T47-D and MDA-MB-231 cells, comparable levels of nuclear receptor were detected in both cell lines. Furthermore, the elution profiles of the nuclear receptors from DNA-Sepharose columns by using a salt gradient were similar and this suggested that defects in the DNA-binding activity of MDA-MB-231 nuclear receptor complexes were not major factors associated with their failure to respond to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds.

Aryl Hydrocarbon Hydroxylases↗
PubMed1989

Induction of cytochrome P450-dependent monooxygenase activities in rat hepatoma H-4-IIE cells in culture by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds: mechanistic studies using radiolabeled congeners.

Treatment of rat hepatoma H-4-IIE cells in culture with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF), 1,2,7,8-TCDF, and 2,3,7-trichlorodibenzo-p-dioxin (TrCDD) resulted in the structure-dependent induction of aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase activities. The induction potencies followed the order 2,3,7,8-TCDD greater than 2,3,7,8-TCDF greater than 1,2,3,7,8-PeCDD approximately 1,2,3,7,8-PeCDF greater than 1,2,7,8-TCDF greater than 2,3,7-TrCDD and were comparable to structure-toxicity relationships which have previously been reported. In contrast, many of the properties of these compounds were structure-independent. For example, using tritiated congeners of high specific activity (greater than 30 Ci/mmol) the sedimentation coefficients (S) for the nuclear and cytosolic aryl hydrocarbon (Ah) receptor complexes were 5-6 and 9-10 S, respectively, for all the radioligands. Moreover, examination of the processing of nuclear Ah receptor complexes for the radiolabeled congeners showed that after 6 h, the rates of nuclear processing were very low and varied between 0.006 and 0.0385 fmol degraded/mg protein/mg total DNA. These results were consistent with the reported stability and persistence of the nuclear Ah receptor complexes and in addition, there were no apparent structure-dependent differences in the processing rates. Inspection of the nuclear receptor levels and the corresponding induced enzyme activities for the congeners showed that there was a linear correlation between average nuclear receptor complex levels (18-42 h) and induced enzyme activities (32-42 h) for all six radioligands; these data indicated that the rates of cytochrome P450-dependent gene expression correlated with the levels of nuclear Ah receptor complex. In contrast, the accumulation of occupied nuclear receptor complexes in rat hepatoma H-4-IIE cells was structure-dependent and appeared to be one of the factors which governed the observed structure-induction and the previously reported structure-toxicity relationships for 2,3,7,8-TCDD and related halogenated aryl hydrocarbons.

Animals↗
PubMed1989

Development and validation of in vitro induction assays for toxic halogenated aromatic mixtures: a review.

Halogenated aromatic industrial compounds, typified by the polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) have been identified as residues in almost every component of the global ecosystem. Risk assessment of the complex mixtures of halogenated aromatics found in environmental samples is complicated by analytical problems and the lack of toxicological information on individual compounds and mixtures. Research in our laboratory has focused on the development and vadidation of the in vitro aryl hydrocarbon hydroxylase (AHH) induction assay in rat hepatoma H-4-II E cells in culture for quantitating individual toxic halogenated aryl hydrocarbons and their mixtures. For several PCB, PCDD, PCDF congeners, their mixed bromo/chloro analogs and reconstituted mixtures there was an excellent linear correlation between their -log ED50 values for AHH induction in rat hepatoma cells and their -log ED50 values for in vivo hepatic microsomal AHH induction, inhibition of body weight gain and thymic atrophy in the rat. It has also been shown for selected compounds that there was a good correlation between their in vitro AHH induction potencies and their effects in guinea pigs (AHH induction, inhibition of body weight gain) and mice (immunotoxicity). This assay system has been utilized to quantitative the "2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents" present in extracts from diverse sources including fly ash from a municipal incinerator and pyrolyzed brominated flame retardants which contain a complex mixture of halogenated dibenzo-p-dioxins and dibenzofurans.

Animals↗
PubMed1989

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.

Animals↗
PubMed1988

Applications of the in vitro aryl hydrocarbon hydroxylase induction assay for determining "2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents": pyrolyzed brominated flame retardants.

The pyrolysis of brominated flame retardants FR 300 BA (decabromobiphenyl) ether, FireMaster BP-6 (polybrominated biphenyls), Bromkal 70-5-DE (primarily pentabromodiphenylether), Bromkal 70-DE (primarily penta and tetrabromodiphenylether) and Bromkal G1 (pentabromodiphenylether) resulted in the formation of relatively high levels of polybrominated dibenzofurans (PBDFs) and dibenzo-p-dioxins (PBDDs as determined by gas chromatography-mass spectrometric analysis. The dose response EC50 values for the induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) by the flame retardant pyrolysates was determined in rat hepatoma H-4-II E cells and compared to the relative induction activities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the concentrations of "2,3,7,8-TCDD equivalents" were calculated. The range of "2,3,7,8-TCDD equivalents" levels (micrograms/g or ppm) derived from values obtained from the AHH and EROD bioassays for each of the pyrolyzed flame retardant samples was: 174-194, 480-1400, 2140-4680, 6740-8780 and 3920-5260 ppm for FR 300 BA, FireMaster BP-6, Bromkal 70 DE, Bromkal 70-5 DE and Bromkal G1, respectively. The in vivo dose-response effects of 2 pyrolyzed flame retardants were determined in immature male Wistar rats and compared to the dose-response activities of 2,3,7,8-TCDD. The in vivo responses which were measured included hepatic microsomal AHH and EROD induction, body weight loss and thymic atrophy. For the pyrolyzed FireMaster BP-6 and Bromkal 70-5 DE samples, the range of calculated in vivo "2,3,7,8-TCDD equivalents" (ppm in sample) for the 4 in vivo bioassays was 520-1780 ppm and 3860-8960 ppm, respectively. The excellent overlap between the in vivo and in vitro 2,3,7,8-TCDD equivalents for the 2 flame retardant pyrolysate extracts supports the utility of the in vitro induction bioassay for quantitatively determining "2,3,7,8-TCDD equivalents" for mixtures containing toxic halogenated aryl hydrocarbons.

Animals↗
PubMed1988

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)

Animals↗
PubMed1987

Aroclor 1254 as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: effects on enzyme induction and immunotoxicity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor 1254 induced the cytochrome P-450 dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells and C57BL/6J mice. It has been proposed that both Aroclor 1254 and 2,3,7,8-TCDD induce these enzymes via a common mechanism which features initial binding to the aryl hydrocarbon (Ah) cytosolic receptor protein. The major difference between these compounds was the relative potency (i.e. 2,3,7,8-TCDD much greater than Aroclor 1254). Cotreatment of rat hepatoma H-4-II E cells or C57BL/6J mice with a dose of 2,3,7,8-TCDD which submaximally induces AHH and EROD and a dose of Aroclor 1254 which exhibited little or no induction activity resulted in significant antagonism of the induction effects of 2,3,7,8-TCDD. For example, cotreatment of C57BL/6J mice with 2,3,7,8-TCDD (15 nmol/kg) and Aroclor 1254 (25, 75 and 150 mumol/kg) resulted in up to 23% antagonism of AHH induction by 2,3,7,8-TCDD. Moreover, cotreatment with a higher dose of the 2,3,7,8-TCDD agonist (30 or 50 nmol/kg) partially reversed some of the antagonism by Aroclor 1254. In vivo antagonism was observed only at Aroclor 1254/2,3,7,8-TCDD molar ratios of 1667:1, 5000:1 and 10,000:1. Administration of 2,3,7,8-TCDD (3.72 nmol/kg) to C57BL/6J mice resulted in a 76% decrease in the splenic plaque forming cell response to sheep red blood cells. This T-cell mediated immunotoxic effect of 2,3,7,8-TCDD segregates with the Ah locus. In contrast, administration of 5, 15, 75 and 150 mumol/kg of Aroclor 1254 resulted in impairment of the immune response only at the highest dose level. However, cotreatment of mice with 2,3,7,8-TCDD (3.72 nmol/kg) and Aroclor 1254 (5, 15 or 75 mumol/kg) resulted in no significant decrease in the plaque forming cell response and complete protection from the immunotoxicity of 2,3,7,8-TCDD. Cotreatment of the mice with Aroclor 1254 (75 mumol/kg) and a higher dose of the 2,3,7,8-TCDD agonist resulted in partial reversal of the protective effects of Aroclor 1254. The in vitro and in vivo data suggest that within specific antagonist/agonist dose ratios, Aroclor 1254 can antagonize at least 2 Ah receptor-mediated effects of 2,3,7,8-TCDD, namely AHH induction and immunotoxicity.

Animals↗