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1.
Cell Physiol Biochem ; 54(1): 126-141, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32017483

RESUMO

BACKGROUND/AIMS: Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter and hormone with important physiological functions in many organs, including the intestine. We have previously shown that 5-HT activates the aryl hydrocarbon receptor (AhR) in intestinal epithelial cells (IECs) via a serotonin transporter (SERT)-dependent mechanism. AhR is a nuclear receptor that binds a variety of molecules including tryptophan (TRP) metabolites to regulate physiological processes in the intestine including xenobiotic detoxification and immune modulation. We hypothesized that 5-HT activates AhR indirectly by interfering with metabolic clearance of AhR ligands by cytochrome P450 1A1 (CYP1A1). METHODS: Inhibition of CYP1A1 activity by 5-HT was assessed in the human intestinal epithelial cell line Caco-2 and recombinant CYP1A1 microsomes using both luciferase and LC-MS/MS. Degradation of 5-HT by recombinant CYP1A1 was measured by LC-MS/MS. For in vitro studies, CYP1A1 and CYP1B1 mRNA expression levels were measured by RT-PCR and CYP1A1 activity was measured by ethoxyresorufin-O-deethylase (EROD) assays. For in vivo studies, AhR ligands were administered to SERT KO mice and WT littermates and intestinal mucosa CYP1A1 mRNA was measured. RESULTS: We show that 5-HT inhibits metabolism of both the pro-luciferin CYP1A1 substrate Luc-CEE as well as the high affinity AhR ligand 6-formylindolo[3,2-b] carbazole (FICZ). Recombinant CYP1A1 assays revealed that 5-HT is metabolized by CYP1A1 in an NADPH dependent manner. Treatment with 5-HT in TRP-free medium, which is devoid of trace AhR ligands, showed that 5-HT requires the presence of AhR ligands to activate AhR. Cotreatment with 5-HT and FICZ confirmed that 5-HT potentiates induction of AhR target genes by AhR ligands. However, this was only true for ligands which are CYP1A1 substrates such as FICZ. Administration of ß-napthoflavone by gavage or indole-3-carbinol via diet to SERT KO mice revealed that lack of SERT impairs intestinal AhR activation. CONCLUSION: Our studies provide novel evidence of crosstalk between serotonergic and AhR signaling where 5-HT can influence the ability of AhR ligands to activate the receptor in the intestine.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Serotonina/farmacologia , Transcrição Genética/efeitos dos fármacos , Animais , Células CACO-2 , Carbazóis/farmacologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Humanos , Ligantes , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/deficiência , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , beta-Naftoflavona/administração & dosagem
2.
Chemosphere ; 239: 124747, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31514003

RESUMO

BACKGROUNDS: Polychlorinated biphenyls are persistent environmental pollutants associated with the onset of non-alcoholic fatty liver disease in humans, but there is limited information on the underlying mechanism. In the present study, we investigated the alterations in gene expression profiles in normal human liver cells L-02 following exposure to 2, 3, 3', 4, 4', 5 - hexachlorobiphenyl (PCB 156), a potent compound that may induce non-alcoholic fatty liver disease. METHODS: The L-02 cells were exposed to PCB 156 for 72 h and the contents of intracellular triacylglyceride and total cholesterol were subsequently measured. Microarray analysis of mRNAs and long non-coding RNAs (lncRNAs) in the cells was also performed after 3.4 µM PCB 156 treatment. RESULTS: Exposure to PCB 156 (3.4 µM, 72 h) resulted in significant increases of triacylglyceride and total cholesterol concentrations in L-02 cells. Microarray analysis identified 222 differentially expressed mRNAs and 628 differentially expressed lncRNAs. Gene Ontology and pathway analyses associated the differentially expressed mRNAs with metabolic and inflammatory processes. Moreover, lncRNA-mRNA co-expression network revealed 36 network pairs comprising 10 differentially expressed mRNAs and 34 dysregulated lncRNAs. The results of bioinformatics analysis further indicated that dysregulated lncRNA NONHSAT174696, lncRNA NONHSAT179219, and lncRNA NONHSAT161887, as the regulators of EDAR, CYP1B1, and ALDH3A1 respectively, played an important role in the PCB 156-induced lipid metabolism disorder. CONCLUSION: Our findings provide an overview of differentially expressed mRNAs and lncRNAs in L-02 cells exposed to PCB 156, and contribute to the field of polychlorinated biphenyl-induced non-alcoholic fatty liver disease.


Assuntos
Fígado/efeitos dos fármacos , Bifenilos Policlorados/toxicidade , Transcriptoma/efeitos dos fármacos , Aldeído Desidrogenase/genética , Linhagem Celular , Colesterol/metabolismo , Citocromo P-450 CYP1B1/genética , Receptor Edar/genética , Perfilação da Expressão Gênica , Humanos , Fígado/citologia , Fígado/fisiologia , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Longo não Codificante , RNA Mensageiro/metabolismo , Testes de Toxicidade , Triglicerídeos/metabolismo
3.
Chemosphere ; 237: 124467, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31549677

RESUMO

In the present study, the carcinogenic effects of the wastewater sample collected from the Dongming Canal in Shijiazhuang city were first detected by the rat medium-term liver bioassay. The experiment contained five groups: a negative control group, a DEN-alone group, 25% wastewater, 50% wastewater, and 100% wastewater. The body weight of rats decreased significantly as the dose increased. Morphologically, we also found that the damage of the hepatic lobule was more serious and the proliferation of liver cells was more obvious as the dose increased. In addition, we observed a significantly increased liver organ coefficient in rat. With the increase in dose, the damage of the hepatocytes was more serious, which was manifested in significantly elevated of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gammaglutamyl transfer peptide enzyme (γ-GT). And, the irrigative wastewater significantly increased GST-p in the liver of rats at both the transcriptional and translational levels dose-dependently, eventually causing precancerous lesions in the liver tissues. CYP1A1 and CYP1B1 expressions in the rat liver cells at the level of transcription and translation were also significantly increased dose-dependently. Our data clearly demonstrated that the irrigative wastewater had a carcinogenetic effect that was associated with CYP1A1 and CYP1B1. The risk of carcinogenic potential to human health might be due to joint action and accumulative effects over a long period of exposure. We can also concluded that the medium-term liver bioassay could be used as an effective method for evaluating the carcinogenicity of complex water mixtures such as irrigative wastewater.


Assuntos
Irrigação Agrícola , Citocromo P-450 CYP1A1/metabolismo , Exposição Ambiental/estatística & dados numéricos , Lesões Pré-Cancerosas , Eliminação de Resíduos Líquidos , Águas Residuárias/toxicidade , Animais , Carcinogênese , Carcinógenos/metabolismo , China , Citocromo P-450 CYP1B1 , Hepatócitos/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos
4.
Chem Biol Interact ; 312: 108816, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31505164

RESUMO

Indirubins E804 (indirubin-3'-(2,3 dihydroxypropyl)-oximether) and 7BIO (7-Bromoindirubin-3'-oxime) are synthetic derivatives of natural indirubin, the active compound in Danggui Longhui Wan, a traditional Chinese remedy for cancer and inflammation. Herein, we explore E804 and 7BIO for their potential to modulate key pro-inflammatory genes and cytokines in LN-18 and T98G glioblastoma cells. High grade gliomas typically secrete large amounts of inflammatory cytokines and growth factors that promote tumor growth in an autocrine fashion. Inflammation is emerging as a key concern in the success of new treatment modalities for glioblastomas. Studies indicate that select indirubin derivatives bind and activate signaling of the AHR pathway, as well as inhibit cyclin-dependent kinases and STAT3 signaling. AHR signaling is involved in hematopoiesis, immune function, cell cycling, and inflammation, and thus may be a possible target for glioma treatment. To determine the significance of the AHR pathway in LN-18 and T98G glioma inflammatory profiles, and on the effects of E804 and 7BIO on these profiles, we used 6,2',4'-trimethoxyflavone (TMF), a putative selective AHR antagonist. It was confirmed that E804 and 7BIO activates the AHR leading to cyp1b1 expression, and that TMF antagonizes expression. We then employed a commercial cancer inflammation and immunity crosstalk qRT-PCR array to screen for anti-inflammatory related properties. TMF alone inhibited expression of ifng, ptsg2, il12b, tnfa, il10, il13, the balance between pd1 and pdl1, and even expression of mhc1a/b. E804 was very potent in suppressing many pro-inflammatory genes, including il1a, il1b, il12a, ptgs2, tlr4, and others. E804 also affected expression of il6, vegfa, and stat3. Conversely, 7BIO induced cox2, but suppressed a different selection of pro-inflammatory genes including nos2, tnfa, and igf1. Secretion of IL-6 protein, an iconic inflammatory cytokine, was decreased by E804. VEGF (vascular endothelial growth factor) protein secretion was upregulated by 7BIO, yet downregulated by E804 and E804 plus TMF. Thus, E804 is both an AHR ligand and regulator of important pro-inflammatory cytokines such as IL-6 and oncogene STAT3, among others. Our results point to the use of E804 and TMF in combination as a promising new treatment for glioblastoma.


Assuntos
Indóis/farmacologia , Oximas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Citocinas/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Indóis/química , Oximas/química , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
BMC Med Genet ; 20(1): 148, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477036

RESUMO

BACKGROUND: Single nucleotide polymorphisms (SNPs) in several CYP genes have been associated with altered breast cancer (BC) risk in different populations. Despite this, there is a dearth of information on the roles of these SNPs in Jordanian BC patients. Therefore, this study aims to determine if there is any single nucleotide polymorphism (SNP) within CYP19A1, CYP2C19, CYP2C9, CYP1B1, CYP3A4, and CYP1A2 genes associated with BC in the Jordanian population. In addition, this work investigates the association between selected BC prognostic factors and variants of the aforementioned CYP candidate genes. METHODS: Blood samples were withdrawn from 221 BC patients and 218 healthy volunteers recruited from the Jordanian population. Genomic DNA was withdrawn and, after quantification and quality control, was genotyped using the Sequenom MassARRAY® system (iPLEX GOLD). Statistical analysis was then carried out to assess allelic and genotypic frequencies as well as genetic association between cases and controls. RESULTS: The CYP19A1 SNP rs7176005 (p < 0.0045) and the CYP1A2 SNP rs762551 (p = 0.004) were significantly associated with BC risk. However, no such association was found for the screened SNPs of the CYP2C9, CYP1B1, CYP2C19 and CYP3A4 genes. Regarding the prognostic factors of BC, several of the screened SNPs were associated with different pathological and clinical features. CONCLUSIONS: Certain CYP genes, particularly CYP19A1 and CYP1A2, were associated with BC risk and development in the Jordanian population.


Assuntos
Neoplasias da Mama/genética , Ciclofilinas/genética , Estudos de Associação Genética , Predisposição Genética para Doença/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Aromatase/genética , Estudos de Coortes , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP3A/genética , Feminino , Genótipo , Humanos , Jordânia , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Prognóstico , Risco , Adulto Jovem
6.
J Exp Clin Cancer Res ; 38(1): 335, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370872

RESUMO

BACKGROUND: The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity. METHODS: Molecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA. RESULTS: We first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC. CONCLUSIONS: In the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Metilcolantreno/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metilcolantreno/química , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Transporte Proteico , Receptores de Hidrocarboneto Arílico/química , Receptores Estrogênicos/química , Receptores Acoplados a Proteínas-G/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
7.
Int J Mol Sci ; 20(16)2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31434333

RESUMO

Left ventricular hypertrophy (LVH) can be adaptive, as arising from exercise, or pathological, most commonly when driven by hypertension. The pathophysiology of LVH is consistently associated with an increase in cytochrome P450 (CYP)1B1 and mitogen-activated protein kinases (MAPKs) and a decrease in sirtuins and mitochondria functioning. Treatment is usually targeted to hypertension management, although it is widely accepted that treatment outcomes could be improved with cardiomyocyte hypertrophy targeted interventions. The current article reviews the wide, but disparate, bodies of data pertaining to LVH pathoetiology and pathophysiology, proposing a significant role for variations in the N-acetylserotonin (NAS)/melatonin ratio within mitochondria in driving the biological underpinnings of LVH. Heightened levels of mitochondria CYP1B1 drive the 'backward' conversion of melatonin to NAS, resulting in a loss of the co-operative interactions of melatonin and sirtuin-3 within mitochondria. NAS activates the brain-derived neurotrophic factor receptor, TrkB, leading to raised trophic signalling via cyclic adenosine 3',5'-monophosphate (cAMP)-response element binding protein (CREB) and the MAPKs, which are significantly increased in LVH. The gut microbiome may be intimately linked to how stress and depression associate with LVH and hypertension, with gut microbiome derived butyrate, and other histone deacetylase inhibitors, significant modulators of the melatonergic pathways and LVH more generally. This provides a model of LVH that has significant treatment and research implications.


Assuntos
Citocromo P-450 CYP1B1/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Animais , Citocromo P-450 CYP1B1/genética , Microbioma Gastrointestinal/fisiologia , Humanos , Hipertrofia Ventricular Esquerda/fisiopatologia , Melatonina/metabolismo , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Sirtuínas/metabolismo
8.
Artigo em Inglês | MEDLINE | ID: mdl-31373329

RESUMO

E-cigarette aerosol contains lower levels of most known carcinogens than tobacco smoke, but many users of e-cigarettes are also smokers, and these individuals may be vulnerable to possible promoting and/or cocarcinogenic effects of e-cigarettes. We investigated the possibility that a condensate of e-cigarette aerosol (EAC) enhances the metabolism of the tobacco carcinogen, benzo(a)pyrene (BaP), to genotoxic products in a human oral keratinocyte cell line. Cells were pretreated with EAC from two popular e-cigs and then with BaP. Metabolism to its ultimate carcinogenic metabolite, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro B[a]P (BPDE), was assayed by measuring isomers of its spontaneous hydrolysis products, BaP tetrols. The pretreatment of cells with EAC enhanced the rate of BaP tetrol formation several fold. Pretreatment with the e-liquid resulted in a smaller enhancement. The treatment of cells with EAC induced CYP1A1/1B1 mRNA and protein. The enhancement of BaP tetrol formation was inhibited by the aryl hydrocarbon receptor (AhR) inhibitor, α-napthoflavone, indicating EAC likely induces CYP1A1/1B1 and enhances BaP metabolism by activating the AhR. To our knowledge, this is first report demonstrating that e-cigarettes can potentiate the genotoxic effects of a tobacco smoke carcinogen.


Assuntos
Aerossóis/efeitos adversos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Benzo(a)pireno/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Mutagênicos/efeitos adversos , Receptores de Hidrocarboneto Arílico/genética , Fumaça/efeitos adversos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinógenos/toxicidade , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Sistemas Eletrônicos de Liberação de Nicotina , Humanos , Receptores de Hidrocarboneto Arílico/metabolismo
9.
Int J Mol Sci ; 20(11)2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31181746

RESUMO

Exposure to ultrafine particles (UFPs) leads to adverse effects on health caused by an unbalanced ratio between UFPs deposition and clearance efficacy. Since air pollution toxicity is first direct to cardiorespiratory system, we compared the acute and sub-acute effects of diesel exhaust particles (DEP) and biomass burning-derived particles (BB) on bronchoalveolar Lavage Fluid (BALf), lung and heart parenchyma. Markers of cytotoxicity, oxidative stress and inflammation were analysed in male BALB/c mice submitted to single and repeated intra-tracheal instillations of 50 µg UFPs. This in-vivo study showed the activation of inflammatory response (COX-2 and MPO) after exposure to UFPs, both in respiratory and cardiovascular systems. Exposure to DEP results also in pro- and anti-oxidant (HO-1, iNOS, Cyp1b1, Hsp70) protein levels increase, although, stress persist only in cardiac tissue under repeated instillations. Statistical correlations suggest that stress marker variation was probably due to soluble components and/or mediators translocation of from first deposition site. This mechanism, appears more important after repeated instillations, since inflammation and oxidative stress endure only in heart. In summary, chemical composition of UFPs influenced the activation of different responses mediated by their components or pro-inflammatory and pro-oxidative molecules, indicating DEP as the most damaging pollutant in the comparison.


Assuntos
Exposição por Inalação/efeitos adversos , Material Particulado/toxicidade , Emissões de Veículos/toxicidade , Animais , Biomarcadores/análise , Líquido da Lavagem Broncoalveolar/química , Ciclo-Oxigenase 2/análise , Citocromo P-450 CYP1B1/análise , Proteínas de Choque Térmico HSP70/análise , Heme Oxigenase-1/análise , Inflamação/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/análise
10.
Toxicol In Vitro ; 60: 203-211, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31154061

RESUMO

The development and normal function of prostate tissue depends on signalling interactions between stromal and epithelial compartments. Development of a prostate microtissue composed of these two components can help identify substance exposures that could cause adverse effects in humans as part of a non-animal risk assessment. In this study, prostate microtissues composed of human derived stromal (WPMY-1) and epithelial (RWPE-1) cell lines grown in scaffold-free hydrogels were developed and characterized using immunohistochemistry, light microscopy, and qRT-PCR. Within 5 days after seeding, the microtissues self-organized into spheroids consisting of a core of stromal WPMY-1 cells surrounded by epithelial RWPE-1 cells. The RWPE-1 layer is reflective of intermediate prostatic epithelium, expressing both characteristics of the luminal (high expression of PSA) and basal (high expression of cytokeratins 5/6 and 14) epithelial cells. The response of the microtissues to an androgen (dihydrotestosterone, DHT) and an anti-androgen (flutamide) was also investigated. Treatment with DHT, flutamide or a mixture of DHT and flutamide indicated that the morphology and self-organization of the microtissues is androgen dependent. qRT-PCR data showed that a saturating concentration of DHT increased the expression of genes coding for the estrogen receptors (ESR1 and ESR2) and decreased the expression of CYP1B1 without affecting the expression of the androgen receptor. With further development and optimization RWPE-1/WPMY-1 microtissues can play an important role in non-animal risk assessments.


Assuntos
Alternativas aos Testes com Animais , Próstata , Antagonistas de Androgênios/farmacologia , Androgênios/farmacologia , Linhagem Celular , Técnicas de Cocultura , Citocromo P-450 CYP1B1/genética , Di-Hidrotestosterona/farmacologia , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Flutamida/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrogéis , Masculino , Receptores Androgênicos/genética
11.
Chem Biol Interact ; 309: 108700, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31176714

RESUMO

Aryl hydrocarbon receptor (AhR) is a highly conserved ligand-activated transcription factor with high affinity to aromatic planar compounds, such as ß-naphthoflavone (BNF), benzo[a]pyrene (BaP) or dioxin (TCDD). After binding the ligand, AhR triggers induction of the expression of phase I and phase II drug-metabolizing genes, together with numerous other genes that are not directly involved in the metabolism of xenobiotics. Several studies have shown that AhR plays a role in tumor initiation, promotion and progression, but the molecular mechanisms involved in these processes are not fully understood. A previous study from our laboratory indicated that the SERPINB2 gene is presumably regulated by AhR. To prove that such induction is really AhR-dependent, in the present study we knocked down the expression of AhR by stable transfection of a laryngeal squamous cell carcinoma cell line (UT-SCC-34) with shRNA, resulting in 92% reduction of BNF-induced expression of SERPINB2. However, in silico analysis did not reveal AhR-dependent responsive elements in the promoter of the SERPINB2 gene. Therefore, to address this problem, we have used cycloheximide, an inhibitor of translation, and our results clearly indicate that an additional, newly synthesized protein is involved in AhR-dependent induction of SERPINB2 expression by BNF. So, to exclude that AhR binds to the putative xenobiotic-responsive elements (XREs) localized upstream of the SERPINB2 gene, we performed chromatin immunoprecipitation assays. As expected, we found no direct binding of AhR to its responsive elements in the vicinity of the SERPINB2 gene, further demonstrating the indirect SERPINB2 induction by AhR. However, the further analysis demonstrated that the expression of the enhancer RNA encoded by the region of DNA 20 kbp upstream from the SERPINB2 gene was AhR-dependent. Although AhR-mediated SERPINB2 induction clearly requires the synthesis of an additional protein, the kinetics of SERPINB2 induction is as fast as the kinetics of CYP1A1 and CYP1B1 induction (both genes directly regulated by AhR). Therefore, given previous studies regarding the induction of SERPINB2 expression by bacterial lipopolysaccharides (LPS), we think that, similarly, the interaction with pause-release proteins may be responsible for AhR-dependent regulation of SERPINB2 expression.


Assuntos
Receptores de Hidrocarboneto Arílico/metabolismo , Serpinas/metabolismo , Benzo(a)pireno/farmacologia , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Serpinas/genética , beta-Naftoflavona/farmacologia
12.
J Ethnopharmacol ; 240: 111951, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31085226

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Teucrum chamaedrys L. is one of the known medicinal plants, useful for treatment of various health problems, especially digestive. In this study, we investigated methanol, ethyl-acetate and acetone extracts of T. chamaedrys in respect to their anticancer properties in SW480 colorectal cancer cells. MATERIALS AND METHODS: Cytotoxicity and proapoptotic potential were assessed by MTT cell viability assay and AO/EB double staining. Molecular mechanisms of induced apoptosis were determined by monitoring Fas receptor protein expression through immunofluorescence, Caspase 8 and 9 activity, as well as concentrations of O2.- spectrophotometrically. Additionally, mRNA expression of biotransformation enzymes (CYP1A1, CYP1B1, GSTP1) and membrane transporters (MRP1 and MRP2) involved in drug resistance were investigated by qPCR method. Qualitative analysis of individual phenolic compounds was performed by reversed phase HPLC-MS analysis. RESULTS: Methanol extract shows the best cytotoxicity and selectivity compared to ethyl-acetate and acetone extracts, mainly causing apoptosis of SW480 cells, without affecting normal HaCaT keratinocytes. The increased expression of Fas receptor protein and caspase 8 activity indicate that the death receptor-mediated pathway plays a crucial role in the observed apoptosis. The increased caspase 9 activity and O2.- concentration suggest that mitochondria are also involved in the apoptosis. T. chamaedrys methanol extract inhibits mRNA expression of CYP1A1, CYP1B1, GSTP1, MRP1 and MRP2 in SW480 cells. CONCLUSIONS: Induction of apoptosis and inhibition of CYP1A1, CYP1B1, GSTP1, MRP1 and MRP2 mRNA expression implies that T. chamaedrys can serve as a valuable source of bioactive compounds as dietary supplements or selective anticancer agents, with the ability to induce apoptosis and modulate drug resistance in colorectal cancer cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Extratos Vegetais/farmacologia , Teucrium , Apoptose/efeitos dos fármacos , Biotransformação/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Flores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutationa S-Transferase pi/genética , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética
13.
Environ Toxicol ; 34(9): 992-1000, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31087746

RESUMO

Polycyclic aromatic hydrocarbons (PAHs) comprise a large family of toxic compounds that come from natural and anthropogenic sources. Chrysene is a PAH with multiple effects, but the toxic potentials of mono-methylchrysenes are less characterized. A comparison of chrysene and six mono-methylchrysenes was performed using assays for cytotoxicity, human aryl hydrocarbon receptor (AhR) reporter gene signaling, and AhR-regulated target gene and protein expression. Sulforhodamine B and trypan blue dye binding assays revealed these chrysenes to be similar in their cytotoxic effects on HepG2 cells. A yeast-based reporter assay detecting human AhR-mediated gene expression identified 4-methylchrysene as being six times more potent and 5-methylchrysene about one-third as potent as chrysene. Other methylchrysenes were more similar to chrysene in the ability to act as AhR ligands. The mono-methylchrysenes all strongly induced CYP1A1 mRNA and protein and moderately induced CYP1B1 expression in HepG2 cells. Levels of CYP1A2 mRNA were induced at higher concentrations of the chrysenes, but protein expression was not significantly altered. The PCR-based gene expression and immunoblotting analyses indicated induced expression differences across the chrysene members were similar to each other. Overall, the effects of methylated chrysenes were comparable to unsubstituted chrysene, suggesting members of this group may be considered approximately equivalent in their effects. © 2019 Wiley Periodicals, Inc.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Crisenos/toxicidade , Expressão Gênica/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1B1/genética , Genes Reporter/efeitos dos fármacos , Células Hep G2 , Humanos , Receptores de Hidrocarboneto Arílico/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos
14.
Mol Biol Rep ; 46(3): 3299-3306, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30989560

RESUMO

Uterine leiomyomas (UL) are prevalent benign tumors, especially among women of African ancestry. The disease also has genetic liability and is influenced by risk factors such as hormones and obesity. This study investigates the haplotypes of the Cytochrome P450 1B1 gene (CYP1B1) related to hormones and coiled-coil domain containing 57 gene (CCDC57) related to obesity in Afro-Caribbean females. Each haplotype was constructed from unphased sequence data using PHASE v.2.1 software and Haploview v.4.2 was used for linkage disequilibrium (LD) studies. There were contrasting LD observed among the single nucleotide polymorphisms of CYP1B1 and CCDC5. Accordingly, the GTA haplotype of CYP1B1 was significantly associated with UL risk (P = 0.02) while there was no association between CCDC57 haplotypes and UL (P = 0.2) for the ATG haplotype. As such, our findings suggest that the Asp449Asp polymorphism and GTA haplotype of CYP1B1 may contribute to UL susceptibility in women of Afro-Caribbean ancestry in this population.


Assuntos
Citocromo P-450 CYP1B1/genética , Leiomioma/genética , Adulto , Grupo com Ancestrais do Continente Africano/genética , Alelos , Região do Caribe , Estudos de Casos e Controles , Citocromo P-450 CYP1B1/metabolismo , Grupos Étnicos , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença , Haplótipos/genética , Humanos , Desequilíbrio de Ligação/genética , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Neoplasias Uterinas/genética
15.
Phytomedicine ; 59: 152898, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30986715

RESUMO

BACKGROUND: Previous evidence show foods and beverages rich in polyphenolic compounds to have favourable effects on the cardiovascular system. HYPOTHESIS: The current study assessed the modulation of oxidative stress and associated inflammation induced by diesel exhaust particles (DEP - SRM 2975) by pre-treatment of human umbilical vein endothelial cells (HUVECs) with aqueous extracts of rooibos [fermented (FR) as well as green form (GR)] and honeybush [fermented form (FH)]. STUDY DESIGN: HUVEC are either exposed to DEP (10 µg/ml) for 4 h or pre-treated with 40 and 60 µg/ml of FR or GH or FR, or 50 µg/ml orientin (OR) for 6 h prior to DEP exposure. METHODS: In vitro antioxidant capacity of the extracts was assessed and the polyphenol contents were also assessed by HPLC. ROS, cell viability, lactate dehydrogenase leakage, lipid peroxidation, GSH:GSSG ratios, conjugated diene and protein carbonyl levels were determined as indices of oxidative stress and cytotoxicity. RT-qPCR and western blot were used to assess inflammatory cytokines and antioxidant genes expression. RESULTS: DEP caused a dose and time-dependent increase in ROS production, significant (p < 0.001) increase in protein carbonyl (PC) formation, thiobarbituric acid reactive substances and conjugated dienes levels (p < 0.01) and a significant reduction in glutathione (GSH) redox status. Pre-incubation with either the herbal extracts or orientin attenuated these effects. The significant increase in IL-1α, IL-6, IL-8, VCAM-1 and ATF4 gene expression caused by DEP (10 µg/ml) were also attenuated by the presence of the FR, GR and FH extracts, and OR . Pre-treatment with the rooibos extracts or flavone orientin enhanced cell viability, reduced LDH leakage, enhanced mRNA expression of NQO1 and Nrf2, but repressed CYP1B1 mRNA induced by DEP. Western blot showed both the herbal tea extracts and orientin to enhance NQO1 and γGSC protein induction by DEP. CONCLUSION: Taken together, the herbal extracts offer protection against DEP-induced oxidative stress and inflammatory response.


Assuntos
Fabaceae/química , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Emissões de Veículos/toxicidade , Antioxidantes/metabolismo , Aspalathus/química , Citocromo P-450 CYP1B1/genética , Flavonoides/farmacologia , Glucosídeos/farmacologia , Glutationa/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Extratos Vegetais/química , Polifenóis/análise , Substâncias Protetoras/farmacologia
16.
Nat Commun ; 10(1): 1150, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850646

RESUMO

Frontal fibrosing alopecia (FFA) is a recently described inflammatory and scarring type of hair loss affecting almost exclusively women. Despite a dramatic recent increase in incidence the aetiopathogenesis of FFA remains unknown. We undertake genome-wide association studies in females from a UK cohort, comprising 844 cases and 3,760 controls, a Spanish cohort of 172 cases and 385 controls, and perform statistical meta-analysis. We observe genome-wide significant association with FFA at four genomic loci: 2p22.2, 6p21.1, 8q24.22 and 15q2.1. Within the 6p21.1 locus, fine-mapping indicates that the association is driven by the HLA-B*07:02 allele. At 2p22.1, we implicate a putative causal missense variant in CYP1B1, encoding the homonymous xenobiotic- and hormone-processing enzyme. Transcriptomic analysis of affected scalp tissue highlights overrepresentation of transcripts encoding components of innate and adaptive immune response pathways. These findings provide insight into disease pathogenesis and characterise FFA as a genetically predisposed immuno-inflammatory disorder driven by HLA-B*07:02.


Assuntos
Alopecia/congênito , Loci Gênicos , Predisposição Genética para Doença , Antígeno HLA-B7/genética , Transcriptoma/imunologia , Imunidade Adaptativa , Alopecia/diagnóstico , Alopecia/genética , Alopecia/fisiopatologia , Estudos de Casos e Controles , Estudos de Coortes , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/imunologia , Feminino , Expressão Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Antígeno HLA-B7/imunologia , Humanos , Imunidade Inata , Polimorfismo de Nucleotídeo Único
17.
Mol Vis ; 25: 144-154, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820150

RESUMO

Purpose: Primary congenital glaucoma (PCG) is a clinically and genetically heterogeneous disease. The present study was undertaken to find the genetic causes of PCG segregating in 36 large consanguineous Pakistani families. Methods: Ophthalmic examination including fundoscopy, or slit-lamp microscopy was performed to clinically characterize the PCG phenotype. Genomic nucleotide sequences of the CYP1B1 and LTBP2 genes were analyzed with either Sanger or whole exome sequencing. In silico prediction programs were used to assess the pathogenicity of identified alleles. ClustalW alignments were performed to determine evolutionary conservation, and three-dimensional (3D) modeling was performed using HOPE and Phyre2 software. Results: Among the known loci, mutations in CYP1B1 and LTBP2 are the common causes of PCG. Therefore, we analyzed the genomic nucleotide sequences of CYP1B1 and LTBP2, and detected probable pathogenic variants cosegregating with PCG in 14 families. These included the three novel (c.542T>A, c.1436A>G, and c.1325delC) and five known (c.868dupC, c.1168C>T, c.1169G>A, c.1209InsTCATGCCACC, and c.1310C>T) variants in CYP1B1. Two of the novel variants are missense substitutions [p.(Leu181Gln), p.(Gln479Arg)], which replaced evolutionary conserved amino acids, and are predicted to be pathogenic by various in silico programs, while the third variant (c.1325delC) is predicted to cause reading frameshift and premature truncation of the protein. A single mutation, p.(Arg390His), causes PCG in six (~43%) of the 14 CYP1B1 mutations harboring families, and thus, is the most common variant in this cohort. Surprisingly, we did not find any LTBP2 pathogenic variants in the families, which further supports the genetic heterogeneity of PCG in the Pakistani population. Conclusions: In conclusion, results of the present study enhance our understanding of the genetic basis of PCG, support the notion of a genetic modifier of CYP1B1, and contribute to the development of genetic testing protocols and genetic counseling for PCG in Pakistani families.


Assuntos
Citocromo P-450 CYP1B1/genética , Heterogeneidade Genética , Glaucoma/genética , Mutação , Adolescente , Adulto , Idoso , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Criança , Pré-Escolar , Consanguinidade , Análise Mutacional de DNA , Feminino , Expressão Gênica , Frequência do Gene , Glaucoma/congênito , Glaucoma/patologia , Glaucoma/cirurgia , Humanos , Lactente , Proteínas de Ligação a TGF-beta Latente/genética , Masculino , Pessoa de Meia-Idade , Paquistão , Linhagem , Alinhamento de Sequência , Trabeculectomia/métodos
18.
Int J Biochem Cell Biol ; 110: 111-121, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30858141

RESUMO

The extrahepatic CYP enzymes, CYP1B1 and CYP2U1, have been predominantly found in both astrocytes and brain microvessels. We investigated the alteration in the production of hydroxyeicosatetraenoic acids (HETEs) from arachidonic acid (AA) mainly via CYP1B1 and CYP2U1 by glutamate. CYP1B1 and CYP2U1 mRNA levels were dose-dependently induced by glutamate in human U251 glioma cells and hCMEC/D3 blood-brain barrier cells. The increases in the CYP1B1 and CYP2U1 mRNA levels and the binding of CREB to CYP1B1 and CYP2U1 promoters following glutamate treatment were attenuated by mGlu5 receptor antagonist. The mRNA levels of CYP1B1 and CYP2U1 were increased in the cortex, hippocampus, and cerebellum from adult rats that received a subcutaneous injection of monosodium l-glutamate at 1, 3, 5, and 7 days of age; meanwhile, the protein levels of CYP1B1 and CYP2U1 in the astrocytes were induced by glutamate. Glutamate treatment significantly increased the production of 5-HETE, 8-HETE, 11-HETE, and 20-HETE in the cortex and cerebellum. These data suggested that the neuron-astrocyte reciprocal signaling can change the CYP-mediated AA metabolism (e.g. EETs and HETEs) in astrocytes via its specific receptor.


Assuntos
Ácido Araquidônico/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Família 2 do Citocromo P450/metabolismo , Ácido Glutâmico/farmacologia , Receptor de Glutamato Metabotrópico 5/metabolismo , Animais , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxieicosatetraenoicos/biossíntese , Hidroxilação/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
19.
Artigo em Inglês | MEDLINE | ID: mdl-30744811

RESUMO

We investigated the mechanism responsible for the oxidative stress and pro-inflammatory response triggered by PM2.5 collected from Nanjing of China. Two human bronchial epithelia cell lines (HBE and BEAS-2B) were used. Human gene expression profile microarray was performed to investigate the alteration of gene expression in PM2.5-treated HBE cells. The results of ROS assay and ELISA indicated that PM2.5 (150 µg/ml) increased the level of cellular reactive oxygen species (ROS) and promoted the release of interleukin-6 (IL-6) in HBE cells. CYP1A1 and CYP1B1 were the top two up-regulated genes by PM2.5 (150 µg/ml, 48 h of exposure) in HBE cells. Co-knockdown of CYP1A1/1B1 by siRNA substantially inhibited PM2.5-induced ROS generation, IL-6/IL-8 secretion and STAT3/P-STAT3 expression. Similarly, the knockdown of STAT3 also effectively inhibited PM2.5-induced rise in ROS level and IL-6/IL-8 secretion. In summary, PM2.5 mediated oxidative stress and pro-inflammatory response via up-regulating the expression of CYP1A1/1B1 in two human bronchial epithelial cell lines.


Assuntos
Brônquios/patologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Células Epiteliais/patologia , Mediadores da Inflamação/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Material Particulado/farmacologia , Poluentes Atmosféricos/farmacologia , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Brônquios/metabolismo , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Espécies Reativas de Oxigênio
20.
Environ Mol Mutagen ; 60(5): 428-442, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30706525

RESUMO

Mexico City's Metropolitan Area (MCMA) includes Mexico City and 60 municipalities of the neighbor states. Inhabitants are exposed to emissions from over five million vehicles and stationary sources of air pollutants such as particulate matter (PM) and ozone. MCMA PM contains elemental carbon and organic carbon (OC). OCs include polycyclic aromatic hydrocarbons (PAHs), many of which induce mutagenic and carcinogenic DNA adducts. Gestational exposure to air pollution has been associated with increased risk of intrauterine growth restriction, preterm birth or low birth weight risk, and PAH-DNA adducts. These effects also depend on the presence of risk alleles. We investigated the presence of bulky PAH-DNA adducts, plasma 8-iso-PGF2α (8-iso-prostaglandin F2α ) and risk allele variants in neonates cord blood and their non-smoking mothers' leucocytes from families that were living in a highly polluted area during 2014-2015. The presence of adducts was significantly associated with both PM2.5 and PM10 levels, mainly during the last trimester of gestation in both neonates and mothers, while the last month of pregnancy was significant for the association between ozone levels and maternal plasma 8-iso-PGF2α . Fetal CYP1B1*3 risk allele was associated with increased adduct levels in neonates while the presence of the maternal allele significantly reduced the levels of fetal adducts. Maternal NQO1*2 was associated with lower maternal levels of adducts. Our findings suggest the need to reduce actual PM limits in MCMA. We did not observe a clear association between PM and/or adduct levels and neonate weight, length, body mass index, Apgar or Capurro score. Environ. Mol. Mutagen. 60:428-442, 2019. © 2019 Wiley Periodicals, Inc.


Assuntos
Adutos de DNA/análise , Exposição Materna , Troca Materno-Fetal/fisiologia , Ozônio/toxicidade , Material Particulado/toxicidade , Hidrocarbonetos Policíclicos Aromáticos/análise , Efeitos Tardios da Exposição Pré-Natal/patologia , Adulto , Poluição do Ar/análise , Citocromo P-450 CYP1B1/genética , Adutos de DNA/genética , Feminino , Sangue Fetal/química , Humanos , Recém-Nascido , Isoprostanos/sangue , México , NAD(P)H Desidrogenase (Quinona)/genética , Gravidez , Emissões de Veículos/análise , Adulto Jovem
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