benzofurans and 6-methyl-1-3-8-trichlorodibenzofuran

Breast cancer is one of the most known cancer types caused to the women around the world. Dioxins on the other hand are a wide range of chemical compounds known to cause the effects on human health. Among them, 6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) is a relatively non toxic prototypical alkyl polychlorinated dibenzofuran known to act as a highly effective agent for inhibiting hormone-responsive breast cancer growth in animal models. In this study, we have developed a multi-level computational approach to identify possible new breast cancer targets for MCDF. We used PharmMapper Server to predict breast cancer target proteins for MCDF. Search results showed crystal Structure of the Antagonist Form of Glucocorticoid Receptor with highest fit score and AutoLigand analysis showed two potential binding sites, site-A and site-B for MCDF. A molecular docking was performed on these two sites and based on binding energy site-B was selected. MD simulation studies on Glucocorticoid receptor-MCDF complex revealed that MCDF conformation was stable at site-B and the intermolecular interactions were maintained during the course of simulation. In conclusion, our approach couples reverse pharmacophore analysis, molecular docking and molecular dynamics simulations to identify possible new breast cancer targets for MCDF.

Topics: Benzofurans; Binding Sites; Breast Neoplasms; Carcinogens; Cell Transformation, Neoplastic; Computer Simulation; Dioxins; Female; Humans; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Binding; Proteins

The aryl hydrocarbon receptor (AHR) was initially identified as a receptor that bound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related environmental toxicants; however, there is increasing evidence that the AHR is an important new drug target for treating multiple diseases including breast cancer. Treatment of estrogen receptor (ER)-negative MDA-MB-231 and BT474 breast cancer cells with TCDD or the selective AHR modulator 6-methyl-1,3,-trichlorodibenzofuran (MCDF) inhibited breast cancer cell invasion in a Boyden chamber assay. These results were similar to those previously reported for the antimetastic microRNA-335 (miR-335). Both TCDD and MCDF induced miR-335 in MDA-MB-231 and BT474 cells and this was accompanied by downregulation of SOX4, a miR-335-regulated (inhibited) gene. The effects of TCDD and MCDF on miR-335 and SOX4 expression and interactions of miR-335 with the 3'-UTR target sequence in the SOX4 gene were all inhibited in cells transfected with an oligonucleotide (iAHR) that knocks down the AHR, thus confirming AHR-miR-335 interactions. MCDF (40 mg/kg/d) also inhibited lung metastasis of MDA-MB-231 cells in a tail vein injection model, showing that the AHR is a potential new target for treating patients with ER-negative breast cancer, a disease where treatment options and their effectiveness are limited.

Topics: 3' Untranslated Regions; Animals; Benzofurans; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; MicroRNAs; Oligonucleotides; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Receptors, Estrogen; RNA Interference; RNA, Messenger; RNA, Small Interfering; SOXC Transcription Factors

The aryl hydrocarbon receptor (AhR) is a basic-helix-loop-helix transcription factor that binds halogenated aromatic hydrocarbons, polycyclic aromatic hydrocarbons, and endogenous compounds. We previously reported that AhR null (Ahr(-/-)) transgenic adenocarcinoma of the mouse prostate (TRAMP) mice on a C57BL/6J background develop prostate tumors with much greater frequency than AhR wild-type (Ahr(+/+)) TRAMP mice, suggesting that the AhR has tumor suppressor properties. Because AhR signaling pathway inactivation increased susceptibility to prostate tumorigenesis, we tested the hypothesis that a selective AhR modulator (SAhRM), 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), can protect against prostate tumorigenesis. TRAMP mice on the standard C57BL/6JxFVB genetic background were fed 0, 10, or 40mg 6-MCDF/kg diet beginning at 8 weeks of age. Tumor incidence, pelvic lymph node metastasis, and serum vascular endothelial growth factor (VEGF) concentrations were determined at 140 days of age. Prostate tumor incidence and size were not significantly reduced in mice fed 6-MCDF. However, the frequency of pelvic lymph node metastasis was reduced fivefold in mice fed the 40mg 6-MCDF/kg diet. Serum VEGF concentrations were also reduced by 6-MCDF treatment, particularly in mice without prostate tumors, and 6-MCDF was shown to act directly on cultured prostates to inhibit VEGF secretion. Together, these results suggest that 6-MCDF inhibits metastasis, in part, by inhibiting prostatic VEGF production prior to tumor formation. This is the first report that 6-MCDF can confer protection against prostate cancer in vivo.

Topics: Animals; Benzofurans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Organ Culture Techniques; Pelvic Neoplasms; Prostatic Neoplasms; Receptors, Aryl Hydrocarbon

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and the relatively non-toxic selective aryl hydrocarbon receptor (AhR) modulator 6-methyl-1,3,8-trichlorodibenzo-furan (MCDF) induced CYP1A1-dependent ethoxyresorufin O-deethylase activity and inhibited proliferation of seven estrogen receptor (ER) negative breast cancer cell lines. MCDF, TCDD and structurally related 2,3,7,8-tetrachlorodibenzofuran, 1,2,3,7,8-pentachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran, and 3,3',4,4',5-pentachlorobiphenyl induced CYP1A1 and inhibited proliferation of BT-474 and MDA-MB-468 cells. In BT474 and MDA-MB-468 cells transfected with a small inhibitory RNA for the AhR, the antiproliferative activity of the chlorinated aromatic compounds was reversed, whereas for MCDF, only partial reversal was observed, suggesting that this compound acts through both AhR-dependent and AhR-independent pathways in these two cell lines. MCDF also inhibited tumor growth in athymic nude mice in which MDA-MB-468 cells were injected directly into the mammary fat pad. These results suggest that the AhR is a potential drug target for treatment of ER-negative breast cancer.

Topics: Animals; Antineoplastic Agents; Benzofurans; Breast Neoplasms; Carrier Proteins; Cell Proliferation; Drug Delivery Systems; Female; Humans; Mice; Mice, Nude; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Receptors, Estrogen; RNA, Small Interfering; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The aryl hydrocarbon receptor (AhR) binds with high affinity to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatics, but also binds with lower affinity to structurally diverse exogenous and endogenous chemicals. One study reported that 3-methylcholanthrene (3MC) activated the estrogen receptor (ER) through the AhR, which acts as co-regulatory protein, whereas a recent report showed that 3MC directly bound and activated ERalpha. This study also shows that the AhR agonists benzo[a]pyrene, 3,3',4,4'-tetrachlorobiphenyl, chrysin, 6-methyl-1,3,8-trichlorodibenzofuran, and 3,3'-diindolylmethane also induce ERalpha-dependent transactivation. Moreover, in chromatin immunoprecipitation assays, these compounds induce binding of AhR and ERalpha to the CYP1A1 and pS2 gene promoters, which is consistent with their activities as both selective AhR modulators (SAhRMs) and selective ER modulators (SERMs).

Topics: Benzo(a)pyrene; Benzofurans; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cytochrome P-450 CYP1A1; Estrogen Receptor alpha; Flavonoids; Humans; Indoles; Ligands; Methylcholanthrene; Polychlorinated Biphenyls; Polychlorinated Dibenzodioxins; Presenilin-2; Promoter Regions, Genetic; Receptors, Aryl Hydrocarbon; Structure-Activity Relationship

LNCaP prostate cancer cells express the aryl hydrocarbon receptor (AhR), and treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and an Ah-responsive reporter gene. Similar results were obtained with the selective AhR modulator 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF); however, TCDD but not 6-MCDF induced degradation of the AhR protein. TCDD and 6-MCDF inhibited growth of LNCaP cells, and inhibitory AhR-androgen receptor (AR) crosstalk was investigated in cells transfected with constructs containing the androgen-responsive probasin promoter (-288 to +28) (pPB) or three copies of the -244 to -96 region of this promoter (pARR(3)). Ten nanomolar dihydrotestosterone (DHT) and 17 beta-estradiol (E2) induced transactivation in LNCaP cells transfected with pPB or pARR(3); however, inhibitory AhR-AR crosstalk was observed only with the latter construct. 6-MCDF and TCDD did not inhibit DHT- or E2-induced transactivation in ZR-75 human breast cancer cells, indicating that these interactions were promoter and cell context-dependent. Both E2 and DHT stabilized AR protein in LNCaP cells; however, cotreatment with TCDD or 6-MCDF decreased AR protein levels. These results indicate that inhibitory AhR-AR crosstalk in prostate cancer cells is complex and for some responses, AR protein stability may play a role.

Topics: Benzofurans; Breast Neoplasms; Cell Division; Cell Line, Tumor; Cytochrome P-450 CYP1A1; Dihydrotestosterone; Estradiol; Female; Genes, Reporter; Humans; Male; Polychlorinated Dibenzodioxins; Promoter Regions, Genetic; Prostatic Neoplasms; Receptors, Androgen; Receptors, Aryl Hydrocarbon; Time Factors; Transcriptional Activation

The polycyclic aromatic hydrocarbon 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) is related to the industrial byproduct dioxin and is a weak agonist and partial antagonist at the aryl hydrocarbon receptor (AhR). Tamoxifen is used for the treatment and prevention of breast cancer and interferes with the interaction of estrogen with estrogen receptor alpha (ER). The combination of MCDF and tamoxifen lowered the effective dose of both drugs required to inhibit 7,12-dimethylbenz(a)anthracene-induced mammary tumor growth in rats and protected against the estrogenic effects of tamoxifen on the uterus in rats (A. McDougal et al., Cancer Res 2001;61:3902-7), pointing to the potential use of MCDF in breast cancer treatment. Potential AhR-ER cross-talk is evidenced by the antiestrogenic activity of MCDF and the degradative effect of MCDF on ER protein levels. Our studies confirmed that MCDF degraded the ER. MCDF displayed antiestrogenic activity at higher concentrations in MCF-7 human breast cancer cells, but MCDF alone (10(-6) M) stimulated the growth of MCF-7 cells. MCDF also activated an estrogen response element (ERE)-luciferase reporter and increased mRNA levels of the estrogen-responsive gene transforming growth factor (TGF)-alpha. The estrogenic effects of MCDF are ER dependent because they were blocked by the pure antiestrogen ICI 182,780. MCDF induced ER-coactivator interaction in glutathione S-transferase pull-down assays and the formation of an ER.ERE complex in gel mobility shift assays, further indicating that the estrogenic actions of MCDF are mediated by the ER. In addition, knockdown of the AhR with small interfering RNA did not affect MCDF-induced ERE-luciferase activity. Overall, these data support the conclusion that MCDF is a partial agonist at the ER. This study provides the first evidence for the direct interaction of the ER with MCDF and challenges the view that MCDF is simply an AhR-specific ligand.

Topics: Basic Helix-Loop-Helix Transcription Factors; Benzofurans; Breast Neoplasms; Cell Division; Cell Line, Tumor; Estradiol; Estrogen Receptor alpha; Humans; Models, Molecular; Receptor Cross-Talk; Receptors, Aryl Hydrocarbon; Receptors, Estrogen; RNA, Messenger; RNA, Small Interfering; Transforming Growth Factor alpha

Regulation of gene expression by the aryl hydrocarbon (AHR) receptor is a much-studied pathway of molecular toxicology. Activation of AHR by the xenobiotic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is hypothesized as the mechanism by which TCDD exerts its toxic and carcinogenic effects. Paradoxically, some studies have shown that TCDD acts as an antiestrogen. This has led to the hypothesis that so-called selective aryl hydrocarbon receptor modulators (SAhRMs), AHR ligands that retain the antiestrogenic effects but lack the transcriptional effects of TCDD associated with toxicity, may be utilized as cancer chemotherapeutics in conjunction with other antiestrogenic compounds such as tamoxifen. The present study attempts to further define the molecular mechanism of action of the putative SAhRMs, 6-alkyl-1,3,8-trichlorodibenzofuran (6-MCDF), and diindolylmethane (DIM), focusing particularly on the former. We tested 6-MCDF and DIM for the recruitment of AHR and RNA polymerase II (pol II) to the regulatory region of the AHR responsive gene, cytochrome P4501A1 (CYP1A1), using the chromatin immunoprecipitation (ChIP) assay in the mouse hepatoma cell line Hepa-1c1c7 (Hepa-1). We also tested the level of CYP1A1 induction in Hepa-1 cells using quantitative real-time PCR. We show no difference in the recruitment of AHR or pol II to the regulatory region of CYP1A1 in response to TCDD, 6-MCDF, or co-treatment with both TCDD and 6-MCDF. Our results also show no antagonism of CYP1A1 induction with co-treatment of Hepa-1 cells with TCDD and 6-MCDF. These data suggest that 6-MCDF exhibits agonist activity with respect to induction of CYP1A1 in the Hepa-1 cell line.

Topics: Animals; Benzofurans; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytochrome P-450 CYP1A1; Liver Neoplasms; Mice; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; RNA, Messenger

Tamoxifen (TAM) is a highly effective selective estrogen receptor (ER) modulator used extensively for the treatment and prevention of breast cancer. However, prolonged treatment of women with TAM may be a risk factor for endometrial cancer, and research in our laboratory is focused on the development of selective aryl hydrocarbon receptor modulators that can be used in combination with TAM to improve its efficacy in the breast and inhibit TAM-induced endometrial effects. This study investigated the effects of the selective aryl hydrocarbon receptor modulators 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF) alone and in combination with TAM in the carcinogen-induced mammary tumor model and in the ovariectomized uterotropic assay using female Sprague Dawley rats. The lowest effective dose of 6-MCDF that inhibited tumor growth was 50 microg/kg/day, and TAM was antitumorigenic at a dose of 100 microg/kg/day. In animals cotreated with TAM + 6-MCDF at doses of 100, 50, or 25 microg/kg/day of each compound, complete inhibition of mammary tumor growth was observed at all doses, and the results are consistent with a more than additive antitumorigenic response for the low dose group (25 + 25 microg/kg) and additive interactions at the 50 and 100 microg/kg doses. In a separate experiment, 6-MCDF (800 microg/kg) inhibited TAM-induced peroxidase activity and progesterone receptor binding in the ovariectomized rat uterus but did not affect TAM-induced bone growth in ovariectomized rats. This study also investigated the effects of TAM and 6-MCDF alone and in combination on ERalpha protein levels in MCF-7 human breast cancer cells as a model for studying interactions between these compounds. The results show that 6-MCDF decreased TAM-induced ERalpha levels in the absence or presence of 17beta-estradiol through proteasome activation, and these interactions may contribute to the observed combined antitumorigenic effects of these compounds.

Topics: Animals; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Benzofurans; Bone Development; Cysteine Endopeptidases; Drug Synergism; Estrogen Receptor Modulators; Female; Mammary Neoplasms, Experimental; Multienzyme Complexes; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Receptors, Aryl Hydrocarbon; Tamoxifen; Uterus

Ishikawa endometrial cancer cells express the estrogen receptor (ER), and this study investigates aryl hydrocarbon receptor (AhR) expression and inhibitory AhR-ER crosstalk in this cell line. Treatment of Ishikawa cells with the AhR agonist [3H]2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) gave a radiolabeled nuclear complex that sedimented at 6.0 S in sucrose density gradients, and Western blot analysis confirmed that Ishikawa cells expressed human AhR and AhR nuclear translocator (Arnt) proteins. Treatment of Ishikawa cells with 10 nM TCDD induced a 9.7-fold increase in CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity and a 10.5-fold increase in chloramphenicol acetyltransferase (CAT) activity in cells transfected with pRNH11c containing an Ah-responsive human CYP1A1 gene promoter insert (-1142 to +2434). Inhibitory AhR-ER crosstalk was investigated in Ishikawa cells using E2-induced cell proliferation and transcriptional activation assays in cells transfected with E2-responsive constructs containing promoter inserts from the progesterone receptor and vitellogenin A2 genes. AhR agonists including TCDD, benzo[a]pyrene (BaP) and 6-methyl-1,3,8-trichlorodibenzofuran, inhibited 32-47% of the E2-induced responses. In contrast, neither estrogen nor progesterone inhibited EROD activity induced by TCDD in Ishikawa cells, whereas inhibitory ER-AhR crosstalk was observed in ECC-1 endometrial cells suggesting that these interactions were cell context-dependent.

Topics: Adenocarcinoma; Aryl Hydrocarbon Receptor Nuclear Translocator; Benzo(a)pyrene; Benzofurans; Chloramphenicol O-Acetyltransferase; Cytochrome P-450 CYP1A1; DNA-Binding Proteins; Endometrial Neoplasms; Estradiol; Female; Humans; Polychlorinated Dibenzodioxins; Promoter Regions, Genetic; Receptors, Aryl Hydrocarbon; Receptors, Estrogen; Receptors, Progesterone; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

The antitumorigenic activities of 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), 8-methyl-1,3,6-trichlorodibenzofuran (8-MCDF) and 6-cyclohexyl-1,3,8-trichlorodibenzofuran (6-CHDF) were investigated in the 7,12-dimethylbenz[a]anthracene (DMBA) rat mammary tumor model. At doses of 5, 10 or 25 mg/kg/week, both 6-MCDF and 8-MCDF significantly inhibited mammary tumor growth and at the 5 mg/kg/week dose >50% growth inhibition was observed. In contrast, 6-CHDF was inactive at the 5 mg/kg/week dose and the structure-antitumorigenicity relationships (6-MCDF/8-MCDF > 6-CHDF) correlated with structure-antiestrogenicity (rat uterus) studies and the relative binding affinities of these compounds for the aryl hydrocarbon receptor (AhR). The antitumorigenic activity of 6-MCDF or 8-MCDF in the mammary was not accompanied by any significant changes in liver/body weight ratios, liver morphology or induction of hepatic CYP1A1-dependent activity which is one of the most sensitive indicators of exposure to AhR agonists. RT-PCR and Western blot analysis of mammary tumor mRNA and protein extracts, respectively, confirmed the presence of AhR suggesting that AhR-mediated signaling pathways are functional in rat mammary tumors. These results define a relatively non-toxic group of AhR agonists which exhibit potent antitumorigenic activity in the DMBA-induced rat mammary tumor model (<1 mg/kg/day), and therefore represent a new class of indirect-acting antiestrogens which have potential for clinical treatment of mammary cancer.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antineoplastic Agents; Aryl Hydrocarbon Receptor Nuclear Translocator; Benzofurans; Cytochrome P-450 CYP1A1; DNA-Binding Proteins; Estrogen Antagonists; Female; Gene Expression Regulation, Neoplastic; Mammary Neoplasms, Experimental; Rats; Rats, Sprague-Dawley; Receptors, Aryl Hydrocarbon; Transcription Factors

Ligand-dependent differences in the molecular properties of the transformed cytosolic and nuclear aryl hydrocarbon receptor (AhR) were investigated using the proteolytic clipping band shift assay. AhR complexes were incubated with [32P]dioxin responsive element (DRE) (26-mer) or bromodeoxyuridine (BrdU)-DRE and the resulting protein-DNA or crosslinked protein-DNA complexes were treated with trypsin or V8 protease and analyzed by electrophoresis. The results showed that for several different AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran, 1,2,7,8-tetrachlorodibenzofuran and alpha-naphthoflavone, the pattern of degraded protein-DNA products were similar using transformed cytosolic or nuclear AhR complexes. In contrast, the proteolytic clipping band shift assay showed that there were significant differences in the pattern of degraded protein-DNA products using nuclear AhR complexes derived from mouse Hepa 1c1c7 cells treated with TCDD or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF). The differences detected in this in vitro assay parallel the in vivo and in vitro activities of these compounds in which TCDD is a potent AhR agonist whereas MCDF is a partial AhR agonist and antagonist.

Topics: Animals; Autoradiography; Base Sequence; Benzofurans; Cells, Cultured; Dioxins; Ligands; Liver Neoplasms, Experimental; Male; Mice; Molecular Sequence Data; Molecular Structure; Polychlorinated Dibenzodioxins; Protein Conformation; Proteins; Rats; Receptors, Aryl Hydrocarbon; Trypsin

Rat hepatoma H4IIE and mouse hepatoma Hepa 1c1c7 cells were transiently transfected with a plasmid construct that contained the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the mouse mammary tumor virus promoter and one copy of the dioxin responsive element. Treatment of transfected H4IIE and Hepa 1c1c7 cells with 10(-13) to 10(-6) M 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a concentration-dependent increase in transient CAT activity. Maximum CAT activity was induced in both cell lines by exposure to 10(-9) M TCDD. The induction of CAT activity correlated well with the TCDD-induced, P4501A1-dependent ethoxyresorufin O-deethylase activity. Cotreatment of transfected cells with 10(-9) M TCDD and 10(-8) to 10(-6) M alpha-naphthoflavone (alpha NF) or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) resulted in a concentration-dependent reduction of TCDD-induced CAT activity. Treatment of cells with 10(-6) M alpha NF or MCDF alone resulted in only minimal induction of CAT activity. Both antagonists inhibited the induction of genes under the control of the CYP1A1 and mouse mammary tumor virus promoters, which indicates that the alpha NF- and MCDF-mediated antagonism of TCDD-induced, aryl hydrocarbon receptor-dependent gene transcription does not depend on promoter context.

Topics: Animals; Benzoflavones; Benzofurans; Chloramphenicol O-Acetyltransferase; Cytochrome P-450 Enzyme System; Enzyme Induction; Gene Expression; Mice; Polychlorinated Dibenzodioxins; Rats; Receptors, Aryl Hydrocarbon; Tumor Cells, Cultured

alpha-Naphthoflavone (alpha NF) and 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 gene expression in MCF-7 human breast cancer cells and also decreased the accumulation of the nuclear [3H]TCDD-aryl hydrocarbon (Ah) receptor complex. Nuclear extracts from cells treated with 10(-6) M alpha NF and incubated with a dioxin responsive element (DRE, 26-mer) did not form a retarded band in a gel mobility shift assay. In contrast, incubation of nuclear extracts from cells treated with 10(-6) M MCDF and DRE gave a retarded band and this is consistent with the antiestrogenic and Ah receptor agonist activity of MCDF in human breast cancer cells. alpha NF was further investigated as an Ah receptor antagonist by determining the inhibition by alpha NF of TCDD-induced antiestrogenicity in MCF-7 cells. TCDD (10(-9) M) inhibited the 17 beta-estradiol-induced proliferation of MCF-7 cells and the secretion of the 52-kDa protein. In cotreatment studies, alpha NF (10(-8) to 10(-6) M) caused a concentration-dependent decrease in the antiestrogenic responses elicited by TCDD. In addition, alpha NF inhibited the TCDD-induced down-regulation of nuclear estrogen receptor levels in MCF-7 cells. alpha NF (10(-6) M) alone was inactive as an estrogen or antiestrogen and in cotreatment studies did not affect 17 beta-estradiol-induced responses in MCF-7 cells. Tamoxifen (10(-7) M), an antiestrogen which acts through the estrogen receptor, also inhibited 17 beta-estradiol-induced cell proliferation and alpha NF did not affect the tamoxifen-mediated antiproliferative response. Thus, alpha NF antagonized TCDD-induced CYP1A1 gene expression in MCF-7 cells and also acted as an anti-antiestrogen for TCDD-mediated antiestrogenicity in these cells. These results were consistent with the low levels of DRE binding observed with nuclear extracts from cells treated with 10(-9) M TCDD plus alpha NF (10(-8) to 10(-6) M) or 10(-6) M alpha NF alone. Thus, alpha NF appears to act as an Ah receptor antagonist in MCF-7 cells by decreasing the levels of transcriptionally active nuclear Ah receptor complexes.

Topics: Benzoflavones; Benzofurans; Breast Neoplasms; Cell Division; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Enzyme Induction; Gene Expression Regulation, Neoplastic; Humans; Oxidoreductases; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Receptors, Drug; Receptors, Estrogen; Tumor Cells, Cultured

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.

Topics: Animals; Benzofurans; Breast Neoplasms; Cell Division; Cell Line; Estradiol; Estrogen Antagonists; Female; Humans; Rats; Receptors, Aryl Hydrocarbon; Receptors, Drug; Receptors, Estrogen

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.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Base Sequence; Benzofurans; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Cytosol; DNA-Binding Proteins; Enzyme Induction; Kinetics; Microsomes, Liver; Molecular Sequence Data; Oligonucleotides; Oxidoreductases; Polychlorinated Dibenzodioxins; Protein Binding; Rats; Receptors, Aryl Hydrocarbon; Receptors, Drug; Regulatory Sequences, Nucleic Acid; Transcription Factors

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

Topics: Animals; Benzofurans; Dose-Response Relationship, Drug; Estradiol; Female; Gene Expression; Humans; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogenes; Rats; Rats, Inbred Strains; RNA, Messenger; Time Factors; Uterus

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

Topics: Animals; Benzofurans; Cell Nucleus; Cytosol; Estradiol; Estrogen Antagonists; Female; Rats; Rats, Inbred Strains; Receptors, Estrogen; Receptors, Progesterone

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.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Benzofurans; Dioxins; Enzyme Induction; Male; Polychlorinated Dibenzodioxins; Rats; Receptors, Aryl Hydrocarbon; Receptors, Drug

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (233 nmol/kg) causes a significant increase of hepatic uroporphyrin, heptacarboxyporphyrin, and total porphyrins in female C57BL/6 mice, ovariectomized C57BL/6 mice, male C57BL/10 mice, and male C57BL/6 mice 3 weeks after treatment. In contrast, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) was inactive at a dose of 750 mumol/kg. Cotreatment of the mice with TCDD (233 mol/kg) plus MCDF (750 mumol/kg) resulted in partial antagonism of TCDD-induced hepatic porphyrin accumulation only in the female mice. Parallel studies in female C57BL/6 mice showed that the TCDD-induced porphyria was accompanied by the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities and the depression of uroporphyrinogen decarboxylase (UROD). MCDF (750 mumol/kg) did not significantly affect these enzymes. In the cotreatment studies (MCDF plus TCDD), MCDF partially antagonized TCDD-induced hepatic porphyrin accumulation but did not affect the levels of hepatic AHH, EROD, or UROD. These results indicate that other factors, in addition to the induction of cytochrome P450-dependent monooxygenases and depressed UROD activity, are important in TCDD-induced porphyria in C57BL/6 female mice.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Benzofurans; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Dioxins; Drug Antagonism; Female; Liver Diseases; Male; Mice; Mice, Inbred C57BL; Ovariectomy; Oxidoreductases; Polychlorinated Dibenzodioxins; Porphyrias; Porphyrins; Uroporphyrinogen Decarboxylase

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.

Topics: Abnormalities, Drug-Induced; Animals; Antibody Formation; Aroclors; Aryl Hydrocarbon Hydroxylases; Benzofurans; Chlorodiphenyl (54% Chlorine); Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Dioxins; Enzyme Induction; Male; Mice; Mice, Inbred C57BL; Oxidoreductases; Polychlorinated Dibenzodioxins; Species Specificity

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)

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Benzofurans; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Enzyme Induction; Isoenzymes; Male; Oxidoreductases; Rats; Receptors, Aryl Hydrocarbon; Receptors, Drug; RNA, Messenger; Structure-Activity Relationship

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.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Benzofurans; Dioxins; Dose-Response Relationship, Drug; Enzyme Induction; Estradiol; Estrogen Antagonists; Female; Mesothelin; Microsomes, Liver; Polychlorinated Dibenzodioxins; Rats; Rats, Inbred Strains; Receptors, Aryl Hydrocarbon; Receptors, Drug; Receptors, Estrogen; Receptors, Progesterone