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Reinhold J Hutz - One of the best experts on this subject based on the ideXlab platform.

  • Familiar and novel reproductive endocrine disruptors: xenoestrogens, dioxins and nanoparticles.
    Current trends in endocrinology, 2020
    Co-Authors: Reinhold J Hutz, Monika G Baldridge, Carvan Mj, Jeremy K. Larson, Liu Q, Stelzer Rv, King-heiden Tc, Shahnoor N, Julien K
    Abstract:

    Environmental contaminants are known to exert endocrine-disrupting effects on the reproductive axis of animals. Many of these molecules can affect steroid biosynthesis or estrogen-Receptor signaling by behaving as estrogen-like molecules (“xenoestrogens”), or by exerting estrogenmodulatory effects. Exposure to some compounds has been correlated with the skewing of sex ratios in aquatic species, feminization and demasculinization of male animals, declines in human sperm counts, and overall diminution in fertility of birds, fish, and mammals. We herein devote space to several classes of endocrine-disrupting compounds (EDCs), including estrogenic substances such as bisphenol A (BPA), molecules that can behave at times anti-estrogenically while activating the Aromatic Hydrocarbon Receptor (AHR), such as dioxins (a known human carcinogen), and novel, ubiquitous molecules such as nanoparticles, particularly gold nanoparticles (GNPs), that appear to alter the sexsteroid biosynthetic pathway.

  • autoradiographic localization of Aromatic Hydrocarbon Receptor ahr in rhesus monkey ovary
    American Journal of Primatology, 2007
    Co-Authors: Monika G Baldridge, Reinhold J Hutz
    Abstract:

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener of a large class of manmade pollutants that persist in the environment. TCDD exerts its toxic effects, in part, by binding to its Receptor known as the Aromatic Hydrocarbon Receptor (AHR). TCDD is estrogen modulatory and in some systems its Receptor associates directly with estrogen Receptors via co-activator molecules. TCDD inhibits steroid synthesis in human ovarian granulosa cells and AHR is found in these cells. We have previously shown that AHR is found in whole rhesus monkey ovary, but have yet to establish its location. In the present study, we set out to show that radiolabeled TCDD binds to monkey ovarian follicles and that this binding is Receptor mediated. Ovaries from Macaca mulatta were sectioned on a cryostat at 10 µ m; and sections were incubated with either control vehicle, 3H-TCDD, or 3H-TCDD plus alpha-naphthoflavone (ANF), a known Receptor-blocking agent. Here, we show for the first time specific binding of TCDD to the granulosa cells of antral follicles and other regions of the rhesus monkey ovary. Our data indicate a 60-fold increase in binding with 3H-TCDD over that of control, and that this binding is reduced to the levels seen in controls with the addition of the competitive antagonist ANF. These findings support the hypothesis that TCDD directly affects primate ovarian function via the AHR. Am. J. Primatol. 69:681–691, 2007. © 2006 Wiley-Liss, Inc.

  • Estrous cycle-regulated expression of CYP1B1 mRNA in the rat ovary.
    Comparative Biochemistry and Physiology B, 2002
    Co-Authors: A K Dasmahapatra, Amanda L Trewin, Reinhold J Hutz
    Abstract:

    CYP1B1, a member of the cytochrome P450 superfamily, is expressed constitutively in the steroidogenic tissues of mammals and is inducible by peptide hormones, cAMP and Aromatic Hydrocarbon Receptor (AHR) ligands. The mechanism of induction of this cytochrome P450 is similar to that for CYP1A1, i.e. through the Aromatic Hydrocarbon Receptor (AHR) signaling pathway. We have recently reported that CYP1B1, but not CYP1A1, is expressed in rat granulosa cells (GC) in the absence of any external stimulus. The induction of CYP1B1 mRNA in rat GC by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vitro was followed by an increase in AHR and estrogen Receptor (ER-β) RNA levels. Estrous cycle-dependent expression of AHR, AHR-nuclear translocator (ARNT) and ER-mRNAs in the rat ovary was reported. We suggest that CYP1B1 may play a major role in the regulation of rat ovarian function/cycle but until now this has been unexplored experimentally. The present study was therefore aimed at examining the expression of CYP1A1, CYP1B1 and ER-mRNA in rat ovarian tissues throughout the estrous cycle to establish any correlation in the expressions of these mRNAs in rat ovary. Total RNA was extracted from the ovary and liver of cycling adult rats and the mRNAs were analyzed using relative RT-PCR with gene-specific primers for the target mRNA and for RPL 19 or S16 primers as an internal control. The results indicated that in the ovary, CYP1B1 mRNA increased significantly on the evening of proestrus and dramatically decreased on the morning of estrus, while ER-mRNA remained unaltered throughout the estrous cycle. CYP1A1 mRNA in the ovary and both CYP1A1 and CYP1B1 mRNAs in the liver were undetectable. That the sudden decrease of ovarian CYP1B1 mRNA on the morning of estrus was not an effect of the LH surge was verified in vitro using our short-term GC culture model. GC prepared from rats super-stimulated with equine chorionic gonadotropin (eCG) were cultured for 6 h with or without LH and TCDD. It was observed that both CYP1A1 and CYP1B1 mRNAs were induced by TCDD with no apparent effect of LH. It is suggested that the high level of CYP1B1 mRNA expression on the evening of proestrus in rat ovary might be involved in metabolism of estrogens to catecholestrogen (a known effect of CYP1B1), and that expression is unaffected in GC by LH.

  • Effects of dioxin, an environmental pollutant, on mouse blastocyst development and apoptosis
    Fertility and Sterility, 2001
    Co-Authors: Michelle M Matthews, Reinhold J Hutz, Amanda L Trewin, Ira Heimler, Mary M. Fahy, Ewa Radwanska, Richard G Rawlins
    Abstract:

    Abstract Objective: To evaluate the effects of 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD; dioxin) on mouse embryo development and apoptosis. Design: Controlled animal study. Setting: Academic research environment. Animal(s): Female mice (CB6F1) at 3 to 6 weeks of age and proven breeders (C578B46). Intervention(s): Mouse embryos were obtained at the morula stage and cultured to the blastocyst stage in a pharmacologic dose of TCDD (3.1 μM) or a control medium. The morphology was assessed, and staining for apoptosis was performed. Immunohistochemistry for the presence of Aromatic Hydrocarbon Receptor (AhR) was performed in another set of morula-stage embryos. Main Outcome Measure(s): The number of embryos developing from the morula to the blastocyst stage and number of apoptotic blastomeres in control vs. TCDD culture conditions. Result(s): No statistically significant differences were observed in the percentage of embryos reaching the blastocyst stage: 80.9% (115 of 142) in the TCDD-treated group, vs. 82.9% (121 of 146) in the control group. There was also no difference in the degree of apoptosis: 22.6 ± 7.3% apoptotic cells (TCDD) vs. 25.3 ± 9.7% (controls). Staining indicated the slight presence of Aromatic Hydrocarbon Receptor in the morula-stage mouse embryos. Conclusion(s): TCDD at 3.1 μM did not alter the development of early mouse morula to blastocysts and did not significantly induce apoptosis in vitro.

  • Lack of effect of beta-naphthoflavone on induction of Nramp genes in adult rainbow trout Oncorhynchus mykiss.
    Marine Environmental Research, 2000
    Co-Authors: A K Dasmahapatra, Barbara A B Wimpee, K J Budsberg, M O Dorschner, R B Phillips, Reinhold J Hutz
    Abstract:

    Abstract Natural resistance-associated macrophage protein (Nramp) genes in rainbow trout, Oncorhynchus mykiss, were identified and characterized. The greatest mRNA level encoding these genes was in the developing ovary of rainbow trout. We evaluated the response of these genes to a certain Aromatic Hydrocarbon Receptor (AHR) agonist. Adult rainbow trout were treated with β-naphthoflavone (BNF) (50 and 100 mg/kg) for 48 h. Using reverse-transcriptase polymerase chain reaction with ovary and head kidney RNA and specific α and β Nramp primers, a 400 bp Nramp-α- and a 400 bp Nramp-β-specific cDNA were obtained. There were no changes in the α and β Nramp mRNA levels in the ovary following BNF administration. CYP1A1 mRNA was increased in the ovary and kidney, suggesting the presence of AHR in rainbow trout ovary, while the AHR agonist produced no effect on Nramp mRNAs.

James P Whitlock - One of the best experts on this subject based on the ideXlab platform.

  • Myb-binding Protein 1a Augments AhR-dependent Gene Expression
    Journal of Biological Chemistry, 2002
    Co-Authors: Letetia C. Jones, Steven T Okino, Thomas J. Gonda, James P Whitlock
    Abstract:

    We have studied the mechanism by which an acidic domain (amino acids 515-583) of the Aromatic Hydrocarbon Receptor (AhR) transactivates a target gene. Studies with glutathione S-transferase fusion proteins demonstrate that the wild-type acidic domain associates in vitro with Myb-binding protein la, whereas a mutant domain (F542A, 1569A) does not. AhR-defective cells reconstituted with an AhR containing the wild-type acidic domain exhibit normal AhR function; however, cells reconstituted with an AhR containing the mutant acidic domain do not function normally. Transient transfection of Myb-binding protein la into mouse hepatoma cells is associated with augmentation of AhR-dependent gene expression. Such augmentation does not occur when Myb-binding protein la is transfected into AhR-defective cells that have been reconstituted with an AhR that lacks the acidic domain. We infer that 1) Myb-binding protein la associates with AhR, thereby enhancing transactivation, and 2) the presence of AhR's acidic domain is both necessary and sufficient for Myb-binding protein la to increase AhR-dependent gene expression.

  • Accessibility and Activity of the Promoter for a Dioxin-Inducible Ecto-ATPase Gene
    Archives of Biochemistry and Biophysics, 2001
    Co-Authors: James P Whitlock
    Abstract:

    We have analyzed the core promoter for a dioxin-inducible ecto-ATPase gene in mouse hepatoma cells. The transcriptional initiation site maps to a region that contains neither a TATA sequence nor a consensus initiator sequence nor a downstream promoter element. The core promoter has constitutive activity that does not require either the Aromatic Hydrocarbon Receptor or its heterodimerization partner Arnt. Two GC-rich regions contribute approximately equally to the constitutive activity. Proteins constitutively occupy the GC-rich regions in chromatin. The promoter assumes a non-nucleosomal configuration in its native chromosomal setting in both uninduced and dioxin-induced cells. Our findings imply that the GC-rich regions together with their cognate binding proteins carry out core promoter functions for the ecto-ATPase gene. The promoter is constitutively accessible in situ, and chromatin structure is not a limiting factor for dioxin-inducible ecto-ATPase transcription in intact cells.

  • the Aromatic Hydrocarbon Receptor transcription and endocrine aspects of dioxin action
    Vitamins and Hormones Series, 2000
    Co-Authors: Steven T Okino, James P Whitlock
    Abstract:

    Abstract The widespread and persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo- p -dioxin elicits adaptive and adverse biological responses by inducing changes in gene transcription. Some of dioxin's effect reflect disruption of endocrine homeostatis. The Aromatic Hydrocarbon Receptor protein, together with its heterodimerization partner, the Aromatic Hydrocarbon Receptor nuclear translocator protein, mediates dioxin action. There are notable similarities between the mechanism of dioxin action and the mechanisms of steroid/retinoid/thyroid hormone action. Studies of dioxin action may provide insights into the regulation of hormone-responsive genes and endocrine physiology.

  • A Novel Response to Dioxin INDUCTION OF ECTO-ATPase GENE EXPRESSION
    Journal of Biological Chemistry, 1998
    Co-Authors: Liqun Dong, James P Whitlock
    Abstract:

    Abstract We used differential display to discover a new gene that the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) regulates in mouse hepatoma cells. Its predicted amino acid sequence suggests that the gene encodes an ecto-ATPase that contains multiple glycosylation sites, conserved cysteine residues, and apyrase conserved regions. cDNA expression experiments in mouse hepatoma cells confirm that the new gene encodes an ecto-ATPase. Wild-type mouse hepatoma cells contain both constitutive and TCDD-inducible ecto-ATPase activity. Induction of ecto-ATPase gene expression by TCDD is direct and occurs at the transcriptional level. Studies in mutant hepatoma cells indicate that induction requires both the Aromatic Hydrocarbon Receptor (AhR) and the AhR nuclear translocator (Arnt). Furthermore, induction requires AhR’s transactivation domain, but not that of Arnt. Our findings reveal new aspects of dioxin’s biological effects and TCDD-dependent gene regulation.

  • Down-regulation of Major Histocompatibility ComplexQ1bGene Expression by 2,3,7,8-Tetrachlorodibenzo-p-dioxin
    Journal of Biological Chemistry, 1997
    Co-Authors: Liqun Dong, Qiang Ma, James P Whitlock
    Abstract:

    : We analyzed mouse hepatoma cells using differential display to discover new genes that respond to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We identified a class I major histocompatibility complex (MHC) gene, which we designated as MHC Q1b, whose expression decreases in the presence of TCDD. TCDD-induced down-regulation of MHC Q1b requires both the Aromatic Hydrocarbon Receptor and the Aromatic Hydrocarbon Receptor nuclear translocator, transcription factors that up-regulate other genes in response to TCDD. Down-regulation of MHC Q1b by TCDD appears to involve both transcriptional and post-transcriptional regulatory events; the post-transcriptional destabilization of MHC Q1b mRNA is probably a secondary response to TCDD. Our findings reveal new mechanistic aspects of gene regulation by TCDD. In addition, our observations suggest a mechanism that might account for some of TCDD's immunotoxic effects.

David H Sherr - One of the best experts on this subject based on the ideXlab platform.

  • Aryl Hydrocarbon Receptor (AhR) agonists suppress interleukin-6 expression by bone marrow stromal cells: an immunotoxicology study
    Environmental Health, 2003
    Co-Authors: Brenda A Jensen, Jennifer J Schlezinger, Rebecca J Leeman, David H Sherr
    Abstract:

    Background Bone marrow stromal cells produce cytokines required for the normal growth and development of all eight hematopoietic cell lineages. Aberrant cytokine production by stromal cells contributes to blood cell dyscrasias. Consequently, factors that alter stromal cell cytokine production may significantly compromise the development of normal blood cells. We have shown that environmental chemicals, such as Aromatic Hydrocarbon Receptor (AhR) agonists, suppress B lymphopoiesis by modulating bone marrow stromal cell function. Here, we extend these studies to evaluate the potential for two prototypic AhR agonists, 7,12-dimethylbenz [a]anthracene (DMBA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to alter stromal cell cytokine responses.

  • bax caspase 2 and caspase 3 are required for ovarian follicle loss caused by 4 vinylcyclohexene diepoxide exposure of female mice in vivo
    Endocrinology, 2003
    Co-Authors: Yasushi Takai, Gloria I Perez, Jennifer J Schlezinger, Stanley J Korsmeyer, David H Sherr, Jacqueline Canning, Richard Kolesnick, Junying Yuan, Richard A Flavell, Jonathan L Tilly
    Abstract:

    The industrial chemical, 4-vinylcyclohexene diepoxide (VCD), kills oocytes within immature follicles in the ovaries of mice and rats and is considered a potential occupational health hazard. It has been reported that VCD-induced follicle loss occurs via a cell death process involving elevated expression of Bax, a proapoptotic Bcl-2 family member, and increased caspase-3-like activity. We have previously shown that oocytes lacking acid sphingomyelinase (ASMase; an enzyme that generates the proapoptotic stress sensor ceramide), the Aromatic Hydrocarbon Receptor (Ahr), Bax, or caspase-2 are resistant to apoptosis induced by other chemical toxicants. Therefore, this study was designed to investigate the functional importance of ASMase, Ahr, Bax, and caspase-2 as well as the related executioner enzyme caspase-3 to VCD-induced ovotoxicity in mice using gene knockout technology. For each gene mutant mouse line, wild-type and homozygous-null female siblings derived from heterozygous matings were given once-daily ...

  • ligand activation of the Aromatic Hydrocarbon Receptor transcription factor drives bax dependent apoptosis in developing fetal ovarian germ cells
    Endocrinology, 2002
    Co-Authors: Tiina Matikainen, Gloria I Perez, Stanley J Korsmeyer, David H Sherr, Toshitake Moriyama, Yutaka Morita, Jonathan L Tilly
    Abstract:

    We recently reported that a targeted disruption of the gene encoding the Aromatic Hydrocarbon Receptor (AHR) in mice reduces fetal oocyte apoptosis, leading to a 2-fold increase in the number of primordial follicles endowed at birth. Although the identity of the natural ligand(s) for the AHR remains to be unequivocally established, these findings indicate that the level of AHR function is an important physiological determinant of how many oocytes will succumb to apoptosis during development of the fetal ovaries. Furthermore, the AHR is a well established Receptor for polycyclic Aromatic Hydrocarbons (PAHs), a class of ubiquitous environmental chemicals known to cause the death of female germ cells in fetal life. Given the possibility that the AHR serves as a key mediator of fetal oocyte death under both physiological and pathological situations, this study was conducted to more fully examine the impact of PAH-AHR interaction on fetal ovarian germ cells. In addition, experiments were designed to begin identification of the mechanism(s) by which ligand activation of the AHR induces prenatal oocyte depletion after transplacental exposure of fetuses to PAHs in vivo. Embryonic d 13.5 murine fetal ovaries cultured in the presence of PAHs exhibited a high level of germ cell loss via apoptosis that was prevented by the selective AHR antagonist, -napthoflavone (ANF). Immunohistochemical analysis revealed an accumulation of Bax protein in germ cells of fetal ovaries exposed to PAHs before the onset of apoptosis, whereas cotreatment with ANF inhibited the induction of Bax expression. The functional importance of increased Bax expression to the cytotoxic response was confirmed by findings that fetal ovarian germ cell loss caused by in utero exposure of wild-type female fetuses to PAHs was not observed in Bax-deficient female fetuses exposed in parallel. We conclude that a central role exists for the AHR in transducing the actions of PAHs in fetal ovarian germ cells, and that the proapoptotic Bcl-2 family member, Bax, is a required mediator of PAH-induced oocyte loss in female fetuses exposed to PAHs in utero. (Endocrinology 143: 615– 620, 2002)

  • Aromatic Hydrocarbon Receptor driven bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals
    Nature Genetics, 2001
    Co-Authors: Tiina Matikainen, Gloria I Perez, Andrea Jurisicova, Jennifer J Schlezinger, Jarmo Laine, Toshiyuki Sakai, Stanley J Korsmeyer, Robert F Casper, David H Sherr
    Abstract:

    Polycyclic Aromatic Hydrocarbons (PAHs) are toxic chemicals released into the environment by fossil fuel combustion. Moreover, a primary route of human exposure to PAHs is tobacco smoke1,2. Oocyte destruction and ovarian failure occur in PAH-treated mice1,2, and cigarette smoking causes early menopause in women1,3. In many cells, PAHs activate the Aromatic Hydrocarbon Receptor (Ahr), a member of the Per-Arnt-Sim family of transcription factors4,5. The Ahr is also activated by dioxin, one of the most intensively studied environmental contaminants. Here we show that an exposure of mice to PAHs induces the expression of Bax in oocytes6, followed by apoptosis. Ovarian damage caused by PAHs is prevented by Ahr or Bax inactivation. Oocytes microinjected with a Bax promoter–reporter construct show Ahr-dependent transcriptional activation after PAH, but not dioxin, treatment, consistent with findings that dioxin is not cytotoxic to oocytes. This difference in the action of PAHs versus dioxin is conveyed by a single base pair flanking each Ahr response element in the Bax promoter. Oocytes in human ovarian biopsies grafted into immunodeficient mice also accumulate Bax and undergo apoptosis after PAH exposure in vivo. Thus, Ahr-driven Bax transcription is a novel and evolutionarily conserved cell-death signaling pathway responsible for environmental toxicant-induced ovarian failure.

  • fluoranthene induced apoptosis in murine t cell hybridomas is independent of the Aromatic Hydrocarbon Receptor
    Toxicology and Applied Pharmacology, 1996
    Co-Authors: Koichi Yamaguchi, Richard I Near, Alexander M Shneider, Shyrte Ju, David H Sherr
    Abstract:

    Abstract Recent studies suggest that environmental chemicals such as polycyclic Aromatic Hydrocarbons (PAH) compromise the immune system in part through the induction of programmed cell death (apoptosis). Nevertheless, mechanisms through which PAH induce apoptosis remain elusive. In particular, the role of the 8S AhR remains controversial and the nature of intracellular signal transduction in PAH-induced apoptosis remains largely undefined. To extend previous studies to the T cell compartment and to develop a clonal system in which intracellular signals leading to PAH-induced apoptosis can be dissected, the ability of fluoranthene, a ubiquitous, but less well-studied PAH, to induce apoptosis in murine T cell hybridomas was evaluated. Particular emphasis was placed on the role of the 8S AhR. The data indicate that (1) three of four hybridomas studied undergo apoptosis within 8 hr of fluoranthene exposure; (2) fluoranthene induces growth arrest concurrent with apoptosis; (3) at doses sufficient to induce lymphocyte apoptosis, fluoranthene does not induce AhR nuclear translocation in cells expressing high AhR levels; (4) fluoranthene-responsive hybridomas do not express AhR mRNA or protein; (5) the Ca 2+ chelating agent EGTA partially inhibits fluoranthene-induced apoptosis. These results (1) indicate the immunosuppressive potential of fluoranthene; (2) support a role for apoptosis in PAH immunotoxicity; (3) demonstrate that fluoranthene-mediated T cell death and growth arrest are AhR independent; and (4) illustrate similarities between PAH- and antigen-specific Receptor-mediated apoptosis. These findings encourage consideration of AhR-independent events in PAH risk assessment.

Thomas A Gasiewicz - One of the best experts on this subject based on the ideXlab platform.

  • influence of Aromatic Hydrocarbon Receptor mediated events on the genotoxicity of cigarette smoke condensate
    Carcinogenesis, 1998
    Co-Authors: Stephen D Dertinger, Allen E Silverstone, Thomas A Gasiewicz
    Abstract:

    : The role of Aromatic Hydrocarbon Receptor (AhR)-mediated events on the genotoxicity of mainstream cigarette smoke condensate was investigated. In vitro studies with mouse hepatoma cells stably transfected with a DRE-dependent luciferase reporter indicate that cigarette smoke condensate is able to transform AhR to an active form which is capable of initiating gene transcription. Micronucleus formation in two hepatoma cell lines was used as an index of genotoxicity. Cigarette smoke condensate was observed to induce a higher frequency of micronuclei in Hepa1c1c7 cells relative to TAOc1BP(r)c1 cells, which express approximately 10-fold less AhR. Furthermore, the frequency of micronuclei was potentiated when Hepa1c1c7 cells were pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin, a high affinity ligand of AhR. These in vitro studies were followed by an in vivo experiment with Ahr+/+ and Ahr-/- mice. Animals were dosed for three consecutive days with cigarette smoke condensate (0.5-10 microg/kg/day, i.p. injection). The frequency of micronuclei in reticulocytes and total erythrocytes was determined in peripheral blood samples collected 24 h after the last administration. While condensate was found to increase the incidence of micronucleated reticulocytes in Ahr+/+ mice, no increase was observed in the null allele animals. Furthermore, the frequency of micronucleated erythrocytes, a measure of basal chromosome-damaging activity, was slightly but significantly higher in Ahr+/+ relative to Ahr-/- mice. Together, these data suggest that cigarette smoke contains chemicals which transform the AhR to an active transcription factor and AhR-regulated enzyme induction plays an important role in mediating the genotoxicity of this complex environmental pollutant.

  • expression of functional Aromatic Hydrocarbon Receptor and Aromatic Hydrocarbon nuclear translocator proteins in murine bone marrow stromal cells
    Archives of Biochemistry and Biophysics, 1998
    Co-Authors: Amy L Lavin, Denise J Hahn, Thomas A Gasiewicz
    Abstract:

    Abstract 2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) acting through the Aromatic Hydrocarbon Receptor (AhR) and its dimerization partner, the AhR nuclear translocator protein (arnt), elicits numerous toxicological effects including immunosuppression and thymic atrophy. Previous work has shown that TCDD alters bone marrow prothymocyte populations. These effects could be mediated at the lymphocyte level directly and/or through effects on bone marrow stromal cells, a population important in the support of lymphopoiesis. The purpose of this study was to characterize AhR and arnt expression in three murine bone marrow stromal cell lines (S17, M2-10B4, and BMS2) and in primary stromal cell cultures. Immunoblot analysis detected AhR protein in M2-10B4 and BMS2 cells. AhR protein was also detected in the primary cultures. Arnt protein could be detected in all cell cultures. Electrophoretic mobility shift assays detected TCDD-dependent dioxin-responsive element (DRE) binding in all three cell lines. DNA binding was sequence-specific and dependent on AhR, as demonstrated by the addition of unlabeled DRE DNA or of anti-AhR antibody. Results obtained with the primary cultures paralleled those seen with the stromal cell lines. The ED 50 for induction of TCDD-dependent DRE binding in M2-10B4 cells was 0.21 nM. TCDD treatment did not induce stromal P4501A1 mRNA expression but did increase P4501B1 mRNA levels in all three cell lines and in the primary cultures. These results indicate that murine bone marrow stromal cells express AhR and arnt proteins. Furthermore, these proteins are functional in terms of their DRE-binding ability and potential to regulate mRNA levels in a gene-specific fashion.

Donato F Romagnolo - One of the best experts on this subject based on the ideXlab platform.

  • genistein prevents brca1 cpg methylation and proliferation in human breast cancer cells with activated Aromatic Hydrocarbon Receptor
    Current developments in nutrition, 2017
    Co-Authors: Donato F Romagnolo, Andreas J Papoutsis, Micah G Donovan, Thomas Doetschman, Ornella I Selmin
    Abstract:

    Background: Previous studies have suggested a causative role for agonists of the Aromatic Hydrocarbon Receptor (AhR) in the etiology of breast cancer 1, early-onset (BRCA-1)–silenced breast tumors, for which prospects for treatment remain poor. Objectives: We investigated the regulation of BRCA1 by the soy isoflavone genistein (GEN) in human estrogen Receptor α (ERα)–positive Michigan Cancer Foundation-7 (MCF-7) and ERα-negative sporadic University of Arizona Cell Culture-3199 (UACC-3199) breast cancer cells, respectively, with inducible and constitutively active AhR. Methods: In MCF-7 cells, we analyzed the dose- and time-dependent effects of GEN and (–)-epigallocatechin-3-gallate (EGCG) control, selected as prototype dietary DNA methyltransferase (DNMT) inhibitors, on BRCA-1 expression after AhR activation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and in TCDD-washout experiments. We compared the effects of GEN and EGCG on BRCA1 cytosine-phosphate-guanine (CpG) methylation and cell proliferation. Controls for DNA methylation and proliferation were changes in expression of DNMT-1, cyclin D1, and p53, respectively. In UACC-3199 cells, we compared the effects of GEN and α-naphthoflavone (αNF; 7,8-benzoflavone), a synthetic flavone and AhR antagonist, on BRCA1 expression and CpG methylation, cyclin D1, and cell growth. Finally, we examined the effects of GEN and αNF on BRCA1 , AhR-inducible cytochrome P450 ( CYP )-1A1 ( CYP1A1 ) and CYP1B1 , and AhR mRNA expression. Results: In MCF-7 cells, GEN exerted dose- and time-dependent preventative effects against TCDD-dependent downregulation of BRCA-1. After TCDD washout, GEN rescued BRCA-1 protein expression while reducing DNMT-1 and cyclin D1. GEN and EGCG reduced BRCA1 CpG methylation and cell proliferation associated with increased p53. In UACC-3199 cells, GEN reduced BRCA1 and estrogen Receptor-1 ( ESR1 ) CpG methylation, cyclin D1, and cell growth while inducing BRCA-1 and CYP1A1 . Conclusions: Results suggest preventative effects for GEN and EGCG against BRCA1 CpG methylation and downregulation in ERα-positive breast cancer cells with activated AhR. GEN and flavone antagonists of AhR may be useful for reactivation of BRCA1 and ERα via CpG demethylation in ERα-negative breast cancer cells harboring constitutively active AhR.

  • brca 1 promoter hypermethylation and silencing induced by the Aromatic Hydrocarbon Receptor ligand tcdd are prevented by resveratrol in mcf 7 cells
    Journal of Nutritional Biochemistry, 2012
    Co-Authors: Andreas J Papoutsis, Ornella I Selmin, Jamie Borg, Donato F Romagnolo
    Abstract:

    Abstract Epigenetic mechanisms may contribute to reduced expression of the tumor suppressor gene BRCA-1 in sporadic breast cancers. Through environmental exposure and diet, humans are exposed to xenobiotics and food compounds that bind the Aromatic Hydrocarbon Receptor (AhR). AhR-ligands include the dioxin-like and tumor promoter 2,3,7,8 tetrachlorodibenzo- p -dioxin (TCDD). The activated AhR regulates transcription through binding to xenobiotic response elements (XREs=GCGTG) and interactions with transcription cofactors. Previously, we reported on the presence of several XREs in the proximal BRCA-1 promoter and that the expression of endogenous AhR was required for silencing of BRCA-1 expression by TCDD. Here, we document that in estrogen Receptor-α-positive and BRCA-1 wild-type MCF-7 breast cancer cells, the treatment with TCDD attenuated 17β-estradiol-dependent stimulation of BRCA-1 protein and induced hypermethylation of a CpG island spanning the BRCA-1 transcriptional start site of exon-1a. Additionally, we found that TCDD enhanced the association of the AhR; DNA methyl transferase (DNMT)1, DNMT3a and DNMT3b; methyl binding protein (MBD)2; and trimethylated H3K9 (H3K9me3) with the BRCA-1 promoter. Conversely, the phytoalexin resveratrol, selected as a prototype dietary AhR antagonist, antagonized at physiologically relevant doses (1 μmol/L) the TCDD-induced repression of BRCA-1 protein, BRCA-1 promoter methylation and the recruitment of the AhR, MBD2, H3K9me3 and DNMTs (1, 3a and 3b). Taken together, these observations provide mechanistic evidence for AhR agonists in the establishment of BRCA-1 promoter hypermethylation and the basis for the development of prevention strategies based on AhR antagonists.

  • Epigenetic Programming of Breast Cancer and Nutrition Prevention
    2012
    Co-Authors: Donato F Romagnolo, Ornella Selmin
    Abstract:

    Abstract : The purpose of this project is to investigate whether epigenetic mechanisms may contribute to reduced expression of the tumor suppressor gene BRCA-1 in sporadic breast cancers. The scope is to test the role of xenobiotics and food compounds that bind the Aromatic Hydrocarbon Receptor (AhR). AhR-ligands include the dioxin-like and tumor promoter 2,3,7,8 tetrachlorobenzo-p-dioxin (TCDD). The activated AhR regulates transcription through binding to xenobiotic response elements (XRE=GCGTG) and interactions with transcription cofactors. Major findings: Gestational exposure to TCDD increased levels of methylation of a CpG region comprising an XRE harbored in the BRCA-1 promoter. This enrichment was paralleled by increased recruitment of DNMT1, decreased BRCA-1 mRNA and protein expression, reduced mammary gland structures associated with differentiation, and mRNA increases of cell cycle regulatory protein cyclin D1 and cdk4. Conversely, cotreatment with the dietary compound resveratrol (Res) had protective effects on TCDD-induced BRCA-1 methylation, BRCA- 1 expression, and changes in mammary gland development. Significance. These data provide new insight into the impact of gestational exposure on epigenetic events induced by the AhR on the BRCA-1 gene.

  • resveratrol prevents epigenetic silencing of brca 1 by the Aromatic Hydrocarbon Receptor in human breast cancer cells
    Journal of Nutrition, 2010
    Co-Authors: Andreas J Papoutsis, Sarah D Lamore, Georg T Wondrak, Ornella I Selmin, Donato F Romagnolo
    Abstract:

    The BRCA-1 protein is a tumor suppressor involved in repair of DNA damage. Epigenetic mechanisms contribute to its reduced expression in sporadic breast tumors. Through diet, humans are exposed to a complex mixture of xenobiotics and natural ligands of the Aromatic Hydrocarbon Receptor (AhR), which contributes to the etiology of various types of cancers. The AhR binds xenobiotics, endogenous ligands, and many natural dietary bioactive compounds, including the phytoalexin resveratrol (Res). In estrogen Receptor- alpha (ER alpha )-positive and BRCA-1 wild-type MCF-7 breast cancer cells, we investigated the influence of AhR activation with the agonist 2,3,7,8 tetrachlorobenzo(p)dioxin (TCDD) on epigenetic regulation of the BRCA-1 gene and the preventative effects of Res. We report that activation and recruitment of the AhR to the BRCA-1 promoter hampers 17 beta -estradiol (E2)–dependent stimulation of BRCA-1 transcription and protein levels. These inhibitory effects are paralleled by reduced occupancy of ER alpha , acetylated histone (AcH)-4, and AcH3K9. Conversely, the treatment with TCDD increases the association of mono-methylated-H3K9, DNA-methyltransferase-1 (DNMT1), and methyl-binding domain protein-2 with the BRCA-1 promoter and stimulates the accumulation of DNA strand breaks. The AhR-dependent repression of BRCA-1 expression is reversed by small interference for the AhR and DNMT1 or pretreatment with Res, which reduces TCDD-induced DNA strand breaks. These results support the hypothesis that epigenetic silencing of the BRCA-1 gene by the AhR is preventable with Res and provide the molecular basis for the development of dietary strategies based on natural AhR antagonists.

  • cyclooxygenase 2 promoter activation by the Aromatic Hydrocarbon Receptor in breast cancer mcf 7 cells repressive effects of conjugated linoleic acid
    Nutrition and Cancer, 2007
    Co-Authors: Stephanie C Degner, Michael Q Kemp, Jennifer K Hockings, Donato F Romagnolo
    Abstract:

    Abstract The role of the Aromatic Hydrocarbon Receptor (AhR) in transcriptional regulation of the human cyclooxygenase-2 (COX-2) gene remains elusive. We report that the AhR-ligands benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced transcription activity of COX-2 in breast cancer MCF-7 cells. The TCDD-dependent activation of the COX-2 promoter was abrogated by mutation of 2 xenobiotic response elements (XREs) = CGTG). We found that TCDD stimulated the binding of the AhR to COX-2 and cytochrome P4501A1 (CYP1A1) oligonucleotides containing consensus XREs. Conversely, the cotreatment with TCDD plus a mixture of conjugated linoleic acid (CLA) or selected CLA isomers prevented (CLAmix = t10,c12-CLA > c9,t11-CLA) the induction of transcription from the COX-2 promoter. The TCDD-induced binding of the AhR to COX-2 and CYP1A1 oligonucleotides was repressed by cotreatment with CLA (t10,c12-CLA > c9,t11-CLA), and the AhR antagonists, 3-methoxy-4-naphthoflavone, and resveratrol. We conclude that t...