Abstract Cytochrome P450 (CYP) enzymes are the major metabolizers of xenobiotics, e.g. drugs, and environmental toxins. Thus, changes in CYP expression have an important impact on drug metabolism and susceptibility to chemical toxicity. In the present study, the transcriptional mechanisms of both constitutive and inducible regulation of the Cyp2a5 gene in mouse liver were investigated. Mouse primary hepatocyte cultures were used as the main model system together with cell and molecular biology methods. The key activation regions of the Cyp2a5 5' promoter were determined using reporter gene assays. Two major transcription activation sites of the Cyp2a5 5' promoter, called the proximal and the distal, were found. Transcription factors hepatocyte nuclear factor-4 (HNF-4) and nuclear factor I were shown to bind to the proximal promoter. Aryl hydrocarbon receptor nuclear translocator (ARNT) and upstream stimulatory factor bound to a common palindromic E-box element in the distal promoter region. All three response elements were shown to be essential for constitutive expression of CYP2A5 in murine hepatocytes. ARNT appeared to control Cyp2a5 transcription without a heterodimerization partner suggesting active involvement of the ARNT homodimer in mammalian gene regulation. Aryl hydrocarbon receptor (AHR) ligands were shown to induce Cyp2a5 transcriptionally by an AHR-dependent mechanism, and established Cyp2a5 as a novel AHR-regulated gene. The AHR response element and the E-box, identified in these studies, were located near to each other and close to a separately defined nuclear factor (erythroid-derived 2)-like 2 binding site in the distal region of the Cyp2a5 promoter, suggesting cooperation between these elements. Peroxisome proliferator-activated receptor gamma coactivator-1α was shown to up-regulate Cyp2a5 transcription through coactivation of HNF-4α. This indicates that xenobiotic metabolism can be regulated by modification of co-activation. The present results show that CYP2A5 is regulated by several different cross-regulatory pathways. The regulatory mechanisms involved in the transcription of the Cyp2a5 gene may also control other CYP genes, especially the human ortholog CYP2A6, and may explain some of the individual variations in the metabolism of xenobiotics.

Introdução: A artrite reumatóide (AR) é uma artropatia autoimune, de caráter inflamatório, com prevalência em torno de 1% da população. O tabagismo é considerado o principal fator de risco para o desenvolvimento da AR. O receptor de hidrocarbonetos de arila (AhR), um fator de transcrição intracelular ativado por hidrocarbonetos aromáticos componentes da fumaça do cigarro, foi identificado como alvo de regulação da diferenciação de células Th17. Objetivos: Avaliar se os polimorfismos genéticos do AhR estariam associados ao desenvolvimento da AR , e se este receptor estaria mais expresso em pacientes com AR. Pacientes e Métodos: Nós analisamos sete polimorfismos genéticos por mudança de única base (SNP) por PCR em tempo real utilizando sondas TaqMan em 138 pacientes com AR e 129 indivíduos saudáveis. A quantificação da expressão do mRNA do AhR em células mononucleares isoladas de pacientes com AR e indivíduos saudáveis foi realizada por PCR em tempo real. Resultados: Identificamos que haplótipos formados por SNPs no AhR estariam associados com desenvolvimento da AR, podendo ser fator protetor ou de risco para a doença. Em adição, os pacientes com haplótipos de risco apresentavam doença com índice de atividade elevado, principalmente quando o tabagismo estava presente. De fato, pacientes com AR apresentaram aumento na expressão de AhR (mRNA) em relação a indivíduos saudáveis. Conclusões: Em conjunto estes resultados sugerem que o AhR possui um papel importante para o desenvolvimento da artrite reumatóide. Possivelmente mutações neste receptor podem estar relacionadas com alterações na sua atividade e conseqüentemente na diferenciação de células Th17 e a susceptibilidade a AR. / Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune arthropaty with inflammatory characteristics and prevalence around 1% in the population. Tabagism is the main risk factor to RA development. The aryl hydrocarbon receptor (AhR) is an intracellular transcription factor activated by aromatic hydrocarbons present in smoking, whichwas identified to be a target of regulation of Th17 differentiation. Purpose: Study the relationship of genetic polymorphisms in AhR with RA development, and if this receptor expression is upregulated in RA patients. Patients and Methods: We analyzed seven genetic single nucleotide polymorphisms by Real-Time PCR using TaqMan probes in 138 patients with Rheumatoid Arthritis and 129 healthy controls. The AhR mRNA quantization in mononuclear cells isolated from AR patients and healthy controls has been done by Realt-Time PCR. Results: We identified that AhR haplotypes were associated with RA development and that they could be protector or risk factors to disease. In addition, patients with risk haplotypes showed higher disease activity index, mainly when smoking was present. Indeed, patients with RA showed upregulation in the AhR expression (mRNA) when compared with healthy controls. Conclusions: These results suggest that AhR has an important role in AR development. Probably, mutations in this receptor could be related with alterations in its activity and consequently in the differentiation of Th17 cells and RA susceptibility.

Le récepteur aux hydrocarbures aromatiques (AhR) est un facteur de transcription activé par de nombreux xénobiotiques (molécules étrangères à l’organisme) qui régule l’expression d’enzymes et transporteurs permettant le métabolisme et l’élimination de ces ligands. Cette protéine exprimée dans toutes les cellules chez les vertébrés, joue un rôle majeur dans la détoxication et la protection des organismes vis à vis des molécules toxiques. Des orthologues de celle-ci ont été identifiés chez les invertébrés mais ne semblent pas jouer le même rôle; ils sont exprimés principalement dans des neurones et ne sont pas activés par des polluants. L’absence du AhR chez ces organismes entraîne au niveau cellulaire, des défauts de morphologie dendritique et sur le plan comportemental, des anomalies dans le comportement de nutrition. Malgré ces découvertes, peu de recherches ont été entreprises sur les conséquences d’une invalidation du AhR sur le fonctionnement du système nerveux central chez les vertébrés. Au cours de ma thèse, j’ai étudié ces conséquences au niveau moléculaire, cellulaire et comportemental: les souris AhR KO développent un nystagmus pendulaire horizontal dont l’origine est en partie liée à des défauts structuraux de la gaine de myéline. Au niveau moléculaire, nous avons mis en évidence un changement de la composition lipidique, de l’expression des gènes de la myéline et de l’inflammation, défauts qui sont retrouvés en partie chez des souris dont le AhR a été invalidé spécifiquement dans l’oligodendrocyte, la cellule responsable de la formation de la gaine de myéline. J’ai donc réalisé des études en parallèle sur la lignée murine d’oligodendrocyte, 158N, et montré que l’invalidation du AhR dans cette lignée ainsi que in vivo, modifiait l’expression du gène MAG (Myelin Associated Glycoprotein). Compte tenu du rôle du AhR en tant que récepteur de polluants, nous avons également exposé ou traité nos modèles avec de la TCDD (dioxine de Seveso) et montré que celle-ci modifiait également l’expression du gène MAG. Mes travaux démontrent donc que le AhR joue un rôle au niveau oligodendrocytaire dans la formation de la gaine de myéline. Son rôle connu en tant que récepteur de polluants laisse supposer que certaines contaminations environnementales pourraient jouer un rôle dans l’incidence de pathologies au niveau du système nerveux central, ce qui soulève de nombreuses questions en terme de santé publique. / The aryl hydrocarbon receptor (AhR) is a transcription factor activated by many xenobiotics (foreign molecules) that regulates the expression of enzymes and transporters which allow the metabolism and elimination of these ligands. This protein expressed in all cells in vertebrates, plays a major role in detoxication and protection of the organisms against toxic molecules. Some orthologs have been identified in invertebrates but do not seem to play the same role; they are expressed mainly in neurons and are not activated by pollutants. The absence of the AhR in these organisms causes at the cellular level, defects of the dendritic morphology and behaviourally, abnormalities in the feeding behavior. Despite these findings, little research has been conducted on the consequences of the AhR invalidation in the central nervous system of vertebrates. During my PhD, I studied these consequences at the molecular, cellular and behavioral : the AhR knockout mice develop a horizontal pendular nystagmus whose origin is partly related to structural defects in the myelin sheath. At the molecular level, we have shown modifications in the lipid composition, myelin and inflammation gene expression, defects that are found partly in mice whose AhR was invalidated specifically in the oligodendrocytes, the cells involved in myelin sheath formation. I therefore made parallel studies on the murine oligodendrocyte lineage, 158N, and showed that the invalidation of the AhR in this cell line and in vivo, altered MAG (Myelin Associated Glycoprotein) gene expression. Given the role of the AhR as a receptor of pollutants, we have also exposed or treated our models with TCDD (dioxin of Seveso) and showed that it also changed the expression of MAG gene. My works show that the AhR is involved in oligodendrocyte level in the formation of the myelin sheath. As the AhR is also a receptor of pollutants, some environmental contaminants may play a role in the incidence of diseases in the central nervous system, which raises many issues in terms of public health.

Introdução: A artrite reumatóide (AR) é uma artropatia autoimune, de caráter inflamatório, com prevalência em torno de 1% da população. O tabagismo é considerado o principal fator de risco para o desenvolvimento da AR. O receptor de hidrocarbonetos de arila (AhR), um fator de transcrição intracelular ativado por hidrocarbonetos aromáticos componentes da fumaça do cigarro, foi identificado como alvo de regulação da diferenciação de células Th17. Objetivos: Avaliar se os polimorfismos genéticos do AhR estariam associados ao desenvolvimento da AR , e se este receptor estaria mais expresso em pacientes com AR. Pacientes e Métodos: Nós analisamos sete polimorfismos genéticos por mudança de única base (SNP) por PCR em tempo real utilizando sondas TaqMan em 138 pacientes com AR e 129 indivíduos saudáveis. A quantificação da expressão do mRNA do AhR em células mononucleares isoladas de pacientes com AR e indivíduos saudáveis foi realizada por PCR em tempo real. Resultados: Identificamos que haplótipos formados por SNPs no AhR estariam associados com desenvolvimento da AR, podendo ser fator protetor ou de risco para a doença. Em adição, os pacientes com haplótipos de risco apresentavam doença com índice de atividade elevado, principalmente quando o tabagismo estava presente. De fato, pacientes com AR apresentaram aumento na expressão de AhR (mRNA) em relação a indivíduos saudáveis. Conclusões: Em conjunto estes resultados sugerem que o AhR possui um papel importante para o desenvolvimento da artrite reumatóide. Possivelmente mutações neste receptor podem estar relacionadas com alterações na sua atividade e conseqüentemente na diferenciação de células Th17 e a susceptibilidade a AR. / Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune arthropaty with inflammatory characteristics and prevalence around 1% in the population. Tabagism is the main risk factor to RA development. The aryl hydrocarbon receptor (AhR) is an intracellular transcription factor activated by aromatic hydrocarbons present in smoking, whichwas identified to be a target of regulation of Th17 differentiation. Purpose: Study the relationship of genetic polymorphisms in AhR with RA development, and if this receptor expression is upregulated in RA patients. Patients and Methods: We analyzed seven genetic single nucleotide polymorphisms by Real-Time PCR using TaqMan probes in 138 patients with Rheumatoid Arthritis and 129 healthy controls. The AhR mRNA quantization in mononuclear cells isolated from AR patients and healthy controls has been done by Realt-Time PCR. Results: We identified that AhR haplotypes were associated with RA development and that they could be protector or risk factors to disease. In addition, patients with risk haplotypes showed higher disease activity index, mainly when smoking was present. Indeed, patients with RA showed upregulation in the AhR expression (mRNA) when compared with healthy controls. Conclusions: These results suggest that AhR has an important role in AR development. Probably, mutations in this receptor could be related with alterations in its activity and consequently in the differentiation of Th17 cells and RA susceptibility.

The aryl hydrocarbon receptor (AhR) binds synthetic and chemoprotective phytochemicals, and research in this laboratory has developed selective AhR modulators (SAhRMs) for treatment of breast cancer. Activation of the AhR through agonists such as TCDD inhibits hormone activation of several E2-responsive genes in breast cancer cell lines. In this study, inhibition of E2-induced proliferation and gene expression by TCDD has been investigated in the uterus of wildtype, ERKO and AhRKO mice. Cyclin D1, DNA polymerase ?, and VEGF mRNA levels are induced by E2 through ER? in the uterus as determined by in situ hybridization studies. TCDD down-regulated E2-induced cyclin D1 and DNA polymerase ? expression, but not E2-induced VEGF expression, in wild-type mice, but not AhRKO mice, confirming the role of the AhR. Furthermore, protein synthesis was not necessary for induction of cyclin D1 or DNA polymerase ?gene expression by E2 or inhibition of these responses by TCDD. Therefore, AhR-ER? crosstalk directly regulates the expression of genes involved in cell proliferation in vivo. AhR agonists induce down-regulation of ErbB family receptors in multiple tissues/organs suggesting possible inhibitory interactions with chemotherapeutic potential. Recently, it has been reported that the SAhRM 1,1??,2,2??-tetramethyldiindolylmethane inhibited DMBA-induced mammary tumor growth in rats and also inhibited MAPK and PI3-K pathways in human breast cancer cells. BT-474 and MDA-MB-453 cell lines are ErbB2-overexpressing breast cancer cells that express functional AhR and exhibit constitutive activation of MAPK and PI3-K pathways. Therefore, 1,1??,2,2??-tetramethyldiindolylmethane-induced inhibition of ErbB2 signaling was investigated in these cells lines and in the MMTV-c-neu mouse mammary tumor model, which overexpresses ErbB2 in the mammary gland. The growth of ErbB2 overexpressing cell lines and mammary tumors was inhibited by 1,1??,2,2??-tetramethyldiindolylmethane; however, modulation of MAPK or PI3-K pathways and cell cycle proteins nor induction of apoptosis by 1,1',2,2'-tetramethyldiindolylmethane was observed in the ErbB2overexpressing cell lines. Current studies are investigating mitochondrial effects of 1,1??,2,2??-tetramethyldiindolylmethane in the ErbB2-overexpressing cell lines, as well as continuing studies on gene expression profiles in the mammary glands of MMTV-c-neu mice to better understand and identify critical genes that are responsible for ErbB2-mediated transformation and growth of cancer cells/tumors.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Feb. 19, 2010). Includes bibliographical references.

Alkylphenanthrenes such as retene (7-isopropyl-1-methylphenanthrene) are aquatic contaminants commonly found in anthropogenically-, industrially-, and petroleum-contaminated environments, and have been implicated in crude oil toxicity. In the early life stages (ELS) of fish, exposures to alkylphenanthrenes produce signs of toxicity typical of those observed in exposures to halogenated aromatic hydrocarbons, particularly to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD, the most toxic congener, serves as the basis of the current mechanism-based risk assessment model. The model assumes that congeners that produce TCDD-like toxicity share a common mode of action and act additively. The mechanism of TCDD-like toxicity is assumed to be mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor involved in the xenobiotic response (e.g., induction of cytochrome P450 1A enzymes; CYP1A) and in normal development. CYP1A enzymes are not involved in the mechanism of TCDD toxicity. Alkylphenanthrenes toxic to the ELS of fish are AhR ligands, but in contrast to TCDD, are readily metabolized by CYP1A enzymes. The byproducts of CYP1A metabolism have been implicated in retene toxicity. However, the target tissue of retene and the direct roles of AhR and CYP1A in retene toxicity are unknown, but are expected to be similar to those of TCDD. The results presented in this thesis suggest that in the ELS of fish: (1) the primary target of retene is the cardiovascular system (Chapters 2 & 5); (2) retene toxicity is stage-specific (Chapter 2); (3) the mechanism of retene toxicity is mediated by AhR2, and is independent of CYP1A enzymes (Chapter 5); (4) multiple CYP1A-independent toxicities can result from exposures to different mixtures of CYP1A inducing (retene) and CYP1A inhibiting (alpha-naphthoflavone or 2-aminoanthracene) PAHs (Chapters 3 & 4); and (5) multiple concentration-dependent mechanisms of toxicity (i.e., synergism and response addition) can occur in co-exposures of a CYP1A inducer (retene) with a range of CYP1A inhibitor (alpha-naphthoflavone) concentrations (Chapter 3). Thus, retene toxicity is mechanistically similar to that of TCDD toxicity, suggesting alkylphenanthrenes can be included in the current risk assessment model. However, the observed variable mixture toxicities and species differences in retene toxicity raise questions about the effectiveness of this model. / Thesis (Ph.D, Biology) -- Queen's University, 2009-01-13 12:10:31.373

Aryl hydrocarbon receptor (AHR) is a sensor protein, activated by aromatic chemical species for transcriptionally regulating xenobiotic metabolizing enzymes. AHR is also known to be involved in a variety of pathogenesis such as cancer, diabetes mellitus, cirrhosis, asthma, etc. The AHR signaling induced by xenobiotics has been intensively studied whereas its physiological role in the absence of xenobiotics is poorly understood. Despite a number of ligands of AHR have been reported thus far, further applications are still hampered by the lack of specificity and/or the partially agonistic activity. Thus, a pure AHR antagonist is needed for deciphering the AHR cryptic as well as potential therapeutic agent. The Proteolysis Targeting Chimera (PROTAC) is a bi-functional small molecule containing a ligand and proteolysis inducer. PROTAC recruits the target protein to proteolysis machinery and elicits proteolysis. Thus far, a number of PROTAC have been prepared and demonstrated to effectively induce the degradation of targeted protein in cultured cells, validating PROTAC as a useful research tool. In the present study, PROTACs based on apigenin was prepared and demonstrated to induce the degradation of AHR, providing the proof of concept. To improve activity, a synthetic structure, CH-223191, was optimized for antagonistic activity by positional scanning identifying several AHR antagonists. PROTACs based on the optimal structure were prepared and assessed their biological activity. The products and synthetic scheme described hereby will be helpful for the further understanding on AHR biology as well as for developing therapeutic agents targeting AHR.

Persistent organic pollutants (POPs) and adipose tissue hypoxia have been shown to independently affect adipocyte functions. The goals of this study were to (1) determine the effect of PCB-77, PCB-153, and DDE on the differentiation of human preadipocytes, and (2) investigate the cross-talk between PCB-77 and hypoxia in differentiated human adipocytes. First, human preadipocytes were exposed to PCB-77, PCB-153, or DDE during the entire 14-day differentiation period. We found no effect of low POP levels on lipid accumulation. Second, differentiated human adipocytes were exposed to a combination of PCB-77 and hypoxia. We demonstrated gene-specific cross-talk between PCB-77 and hypoxia, showing an additive effect of PCB-77 on VEGF, MCP-1, and adiponectin, as well as an inhibition of PCB-77-induced expression of CYP1A1 by hypoxia. This work has expanded our understanding of the role of POPs and hypoxia in differentiated human adipocytes.

Challenged by a huge and diverse antigenic stimulus, the intestinal mucosa has developed a unique immune system that mainly functions to maintain tolerance to innocuous antigens while retaining the ability to respond swiftly to pathogenic threats. Central to this specialised immune system are the Intraepithelial Lymphocytes (IELs). These cells are uniquely located between Intestinal Epithelial Cells (IECs) ready to respond to exogenous antigens in the intestinal lumen. The intestinal immune system is constantly influenced, not only by the commensal microbiota, but also by the nutritional status of the host and the availability of certain essential micronutrients that are derived from a healthy-balanced diet. Additionally, age has a significant impact on the efficiency of gut immunity in responding to infectious pathogens, as reflected by the increased burden of gastrointestinal infections at the extremes of age. In this thesis, using the Murine Norovirus (MNV) oral infection model, I aimed to characterize intestinal mucosal antiviral-responses with specific focus on the role of IELs, the impact of aging and the influence of certain micronutrients whose effects are mediated through the Aryl Hydrocarbon Receptor (AhR). Employing different knock-out and adoptive transfer experiments, I concluded that, at least in our experimental conditions and in a viral strain-specific manner, the activated IELs are not essential and may play a minor role in the protective response against MNV infection. This work also demonstrated that various MNV virus strains activate IELs differentially and for the first time (to our knowledge) revealed distinct abilities of these different Norovirus variants to infect IECs. Recognising an impaired response in old (2-year) mice, we were also able to identify a specific defect in the IFN-Lambda response of aged IECs. Furthermore, using the model of MNV infection to investigate the role of AhR signalling, the data I generated suggested a direct link between constitutive AhR signalling and innate interferon-mediated responses. These findings have uncovered a potential preventive/therapeutic targets for enhancing anti-viral responses.