RESUMO
CYP1A1 bioactivates several procarcinogens and detoxifies several xenobiotic compounds. Transcription of CYP1A1 is highly induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the aryl hydrocarbon receptor. We recently described an RNAi high throughput screening performed in the Hepa-1 mouse hepatoma cell line, which revealed that SIN3A is necessary for the induction of CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) enzymatic activity by TCDD. In the current studies, we sought to provide insight into the role of SIN3A in this process, particularly because studies on SIN3A have usually focused on its repressive activity on transcription. We report that ectopic expression of human SIN3A in Hepa-1 cells enhanced EROD induction by TCDD and efficiently rescued TCDD induction of EROD activity in cells treated with an siRNA to mouse SIN3A, thus validating a role for SIN3A in CYP1A1 induction. We demonstrate that SIN3A is required for TCDD induction of the CYP1A1 protein in Hepa-1 cells but not for expression of the aryl hydrocarbon receptor protein. In addition, siRNAs for SIN3A decreased TCDD-mediated induction of CYP1A1 mRNA and EROD activity in human hepatoma cell line Hep3B. We establish that TCDD treatment of Hepa-1 cells rapidly increases the degree of SIN3A binding to both the proximal promoter and enhancer of the Cyp1a1 gene and demonstrate that increased binding to the promoter also occurs in human Hep3B, HepG2, and MCF-7 cells. These studies establish that SIN3A physically interacts with the CYP1A1 gene and extends the transcriptional role of SIN3A to a gene that is very rapidly and dramatically induced.
Assuntos
Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica/fisiologia , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Camundongos , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/genética , Proteínas Repressoras/genética , Complexo Correpressor Histona Desacetilase e Sin3 , Teratogênicos/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) adversely affects many mammalian organs and tissues. These effects are mediated by the aryl hydrocarbon receptor (AHR). CYP1A1, CYP1A2 and CYP1B1 are upregulated by the liganded AHR. These (and other) cytochromes P450 can metabolize arachidonic acid into a variety of bioactive eicosanoids. Towards investigating a potential role of eicosanoids in TCDD toxicity, arachidonic acid, two other unsaturated long-chain fatty acids, and up to twenty-five eicosanoids were measured in five organs/tissues of male and female wild-type and Ahr null mice treated or untreated with TCDD. TCDD generally increased the levels of the four dihydroxyeicosatrienoic acids (DHETs) and (where measured) 5,6-epoxyeicosatrienoic acid and 18-, 19- and 20-hydroxyeicosatrienoic acids (HETEs) in the serum, liver, spleen and lungs, but not the heart, of both sexes, and increased the levels in the serum, liver and spleen of several metabolites that are usually considered products of lipoxygenase activity, but which may also be generated by cytochromes P450. TCDD also increased the levels of the esterified forms of these eicosanoids in the liver in parallel with the corresponding free forms. The levels of prostanoids were generally not affected by TCDD. The above changes did not occur in Ahr null mice, and are therefore mediated by the AHR. TCDD increased the mRNA levels of Cyp1a1, Cyp1a2, Cyp1b1 and the Pla2g12a form of phospholipase A(2) to varying degrees in the different organs, and these increases correlated with some but not all the changes in eicosanoids levels in the organs, suggesting that other enzymes may also be involved.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Eicosanoides/metabolismo , Poluentes Ambientais/toxicidade , Dibenzodioxinas Policloradas/análogos & derivados , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Eicosanoides/sangue , Feminino , Coração/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Dibenzodioxinas Policloradas/toxicidade , RNA/química , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Organismos Livres de Patógenos Específicos , Baço/efeitos dos fármacos , Baço/metabolismo , Espectrometria de Massas em TandemRESUMO
We have tested the entire Keio collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to one of seven different antibiotics (ciprofloxacin, rifampin, vancomycin, ampicillin, sulfamethoxazole, gentamicin, or metronidazole). We used high-throughput screening of several subinhibitory concentrations of each antibiotic and reduced more than 65,000 data points to a set of 140 strains that display significantly increased sensitivities to at least one of the antibiotics, determining the MIC in each case. These data provide targets for the design of "codrugs" that can potentiate existing antibiotics. We have made a number of double mutants with greatly increased sensitivity to ciprofloxacin, and these overcome the resistance generated by certain gyrA mutations. Many of the gene knockouts in E. coli are hypersensitive to more than one antibiotic. Together, all of these data allow us to outline the cell's "intrinsic resistome," which provides innate resistance to antibiotics.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Genes Bacterianos/genética , Mutação , Ampicilina/farmacologia , Ciprofloxacina/farmacologia , Escherichia coli/genética , Gentamicinas/farmacologia , Metronidazol/farmacologia , Testes de Sensibilidade Microbiana , Rifampina/farmacologia , Sulfametoxazol/farmacologia , Vancomicina/farmacologiaRESUMO
Exposure to ambient particulate matter (PM) has been associated with the onset of neurodevelopmental and neurodegenerative disorders, but the mechanism of toxicity remains unclear. To gain insight into this neurotoxicity, this study sought to examine global gene expression changes caused by exposure to ambient ultrafine PM. Microarray analysis was performed on primary human neurons derived from fetal brain tissue after a 24h exposure to 20µg/mL of ambient ultrafine particles. We found a majority of the changes in noncoding RNAs, which are involved in epigenetic regulation of gene expression, and thereby could impact the expression of several other protein coding gene targets. Although neurons from biologically different lot numbers were used, we found a significant increase in the expression of metallothionein 1A and 1F in all samples after exposure to particulate matter as confirmed by quantitative PCR. These metallothionein 1 proteins are responsible for neuroprotection after exposure to environmental insult but prolonged induction can be toxic. Epidemiological studies have reported that in utero exposure to ultrafine PM not only leads to neurodevelopmental and behavioral abnormalities, but may also predispose the progeny to neurodegenerative disease later in life by genetic imprinting. Our results pinpoint some of the PM-induced genetic changes that may underlie these findings.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Metalotioneína/metabolismo , Neurônios/efeitos dos fármacos , Material Particulado/farmacologia , Trifosfato de Adenosina , Encéfalo/citologia , Células Cultivadas , Feto , Humanos , Metalotioneína/genética , Análise em Microsséries , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Exposure to particulate matter (PM), a component of urban air pollution, may cause adverse effects in the brain. Although the exact mechanisms involved are unknown, both oxidative and inflammatory responses have been reported. Since the main route of exposure to particulate matter is through inhalation, there is a potential for compounds to directly enter the brain and alter normal cellular function. Enhancement in both oxidative stress and neuroinflammatory markers has been observed in neurodegenerative disorders and PM-induced potentiation of these events may accelerate the disease process. The objective of this pilot study was to use normal human brain cells, a model system which has not been previously used, to assess cell-type-specific responses after exposure to ultrafine particles (UFP). Human microglia, neurons, and astrocytes were grown separately or as co-cultures and then exposed to aqueous UFP suspensions. Reactive Oxygen Species (ROS) formation and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) were measured as markers of oxidative stress or inflammation respectively. Our results revealed that after exposure to 2 µg/ml of particles, normal human neurons exhibit a decrease in ROS formation and an increase in TNF-α. The observed decrease in ROS formation persisted in the presence of glial cells, which contrasts previous studies done in rodent cells reporting that PM-induced microglial activation modulates neuronal responses. Our study indicates that human CNS cells may respond differently compared to rodent cells and that their use may be more predictive in risk assessment.
Assuntos
Poluentes Atmosféricos/toxicidade , Astrócitos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Material Particulado/toxicidade , Astrócitos/metabolismo , Encéfalo/citologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Humanos , Microglia/metabolismo , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The aryl hydrocarbon receptor (AHR) has a plethora of physiological roles, and upon dysregulation, carcinogenesis can occur. One target gene of AHR encodes the xenobiotic and drug-metabolizing enzyme CYP1A1, which is inducible by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the AHR. An siRNA library targeted against over 5600 gene candidates in the druggable genome was used to transfect mouse Hepa-1 cells, which were then treated with TCDD, and subsequently assayed for CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) activity. Following redundant siRNA activity (RSA) statistical analysis, we identified 93 hits that reduced EROD activity with a p value ≤ .005 and substantiated 39 of these as positive hits in a secondary screening using endoribonuclease-prepared siRNAs (esiRNAs). Twelve of the corresponding gene products were subsequently confirmed to be necessary for the induction of CYP1A1 messenger RNA by TCDD. None of the candidates were deficient in aryl hydrocarbon nuclear translocator expression. However 6 gene products including UBE2i, RAB40C, CRYGD, DCTN4, RBM5, and RAD50 are required for the expression of AHR as well as for induction of CYP1A1. We also found 2 gene products, ARMC8 and TCF20, to be required for the induction of CYP1A1, but our data are ambiguous as to whether they are required for the expression of AHR. In contrast, SIN3A, PDC, TMEM5, and CD9 are not required for AHR expression but are required for the induction of CYP1A1, implicating a direct role in Cyp1a1 transcription. Our methods, although applied to Cyp1a1, could be modified for identifying proteins that regulate other inducible genes.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Citocromo P-450 CYP1A1/biossíntese , Hepatócitos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Dibenzodioxinas Policloradas/toxicidade , Interferência de RNA , Receptores de Hidrocarboneto Arílico/agonistas , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/genética , Indução Enzimática , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Reguladores de Proteínas de Ligação ao GTP/genética , Reguladores de Proteínas de Ligação ao GTP/metabolismo , Genes Reporter , Estudo de Associação Genômica Ampla , Hepatócitos/enzimologia , Camundongos , Oxazinas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Complexo Correpressor Histona Desacetilase e Sin3 , Especificidade por Substrato , Tetraspanina 29/genética , Tetraspanina 29/metabolismo , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
We previously reported that 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increased the levels of several cytochrome P450 metabolites of the omega-6 polyunsaturated fatty acids (PUFAs), arachidonic acid (ARA) and linoleic acid in the serum, liver, lung and spleen of C57BL/6 mice in an aryl hydrocarbon receptor (AHR)-dependent fashion. These increases correlated with increased levels of CYP1A1, CYP1A2 and/or CYP1B1. In the current study, we measured 77 oxylipins, including 59 that we had not measured previously, and demonstrate that TCDD also markedly increases the levels of many epoxide and diol metabolites of the omega-3 PUFAs, α-linolenic acid, eicosapentaenoic acid (EPA) and docasahexaenoic acid (DHA) in these mice. Since these epoxide metabolites have been reported to have opposite effects on angiogenesis, tumor growth and tumor metastasis compared with the equivalent metabolites of omega-6 PUFA, these observations have important implications with regard to the potential involvement of the cytochrome P450 metabolites of PUFAs in mediating the biological effects of TCDD and other agonists of AHR.
Assuntos
Fígado/metabolismo , Pulmão/metabolismo , Oxilipinas/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Animais , Sistema Enzimático do Citocromo P-450 , Ácidos Graxos Ômega-3/metabolismo , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Dibenzodioxinas Policloradas/administração & dosagem , Receptores de Hidrocarboneto Arílico/agonistasRESUMO
Cytochrome P450s are monooxygenase proteins involved in the metabolism of both exogenous and endogenous compounds. CYP2S1 can metabolize eicosanoids in the absence of both NADPH and NADPH cytochrome P450 reductase, and can also activate the anticancer agent 1 AQ4N [1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxy anthracene-9,10-dione]. CYP2S1 is mainly expressed in extrahepatic tissues such as the trachea, lung, stomach, small intestine, spleen, skin, breast, kidney and placenta. Furthermore, increased expression of CYP2S1 occurs in several tumors of epithelial origin, making the characterization of CYP2S1 regulation relevant to the treatment of disease. We report that the synthetic glucocorticoid receptor ligand dexamethasone (DEX) represses CYP2S1 expression. The ED(50) is between 1 nM and 3 nM and maximal repression is reached by 48 h. Other corticosteroids are also effective at repressing CYP2S1. We show that repression by DEX is mediated by the glucocorticoid receptor and requires histone deacetylase activity.
Assuntos
Corticosteroides/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/biossíntese , Corticosteroides/antagonistas & inibidores , Western Blotting , Linhagem Celular , Dactinomicina/farmacologia , Dexametasona/antagonistas & inibidores , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/enzimologia , Inibidores da Síntese de Proteínas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Glucocorticoides/efeitos dos fármacosRESUMO
Titanium dioxide (TiO(2)) nanoparticles are manufactured worldwide in large quantities for use in a wide range of applications including pigment and cosmetic manufacturing. Although TiO(2) is chemically inert, TiO(2) nanoparticles can cause negative health effects, such as respiratory tract cancer in rats. However, the mechanisms involved in TiO(2)-induced genotoxicity and carcinogenicity have not been clearly defined and are poorly studied in vivo. The present study investigates TiO(2) nanoparticles-induced genotoxicity, oxidative DNA damage, and inflammation in a mice model. We treated wild-type mice with TiO(2) nanoparticles in drinking water and determined the extent of DNA damage using the comet assay, the micronuclei assay, and the gamma-H2AX immunostaining assay and by measuring 8-hydroxy-2'-deoxyguanosine levels and, as a genetic instability endpoint, DNA deletions. We also determined mRNA levels of inflammatory cytokines in the peripheral blood. Our results show that TiO(2) nanoparticles induced 8-hydroxy-2'-deoxyguanosine, gamma-H2AX foci, micronuclei, and DNA deletions. The formation of gamma-H2AX foci, indicative of DNA double-strand breaks, was the most sensitive parameter. Inflammation was also present as characterized by a moderate inflammatory response. Together, these results describe the first comprehensive study of TiO(2) nanoparticles-induced genotoxicity in vivo in mice possibly caused by a secondary genotoxic mechanism associated with inflammation and/or oxidative stress. Given the growing use of TiO(2) nanoparticles, these findings raise concern about potential health hazards associated with TiO(2) nanoparticles exposure.