RESUMO
This study tested the hypothesis that gene expression profiling can reveal indicators of subtle injury to the liver induced by a low dose of a substance that does not cause overt toxicity as defined by conventional criteria of toxicology (e.g., abnormal clinical chemistry and histopathology). For the purpose of this study we defined this low dose as subtoxic, i.e., a dose that elicits effects which are below the detection of conventional toxicological parameters. Acetaminophen (APAP) was selected as a model hepatotoxicant because (1) considerable information exists concerning the mechanism of APAP hepatotoxicity that can occur following high doses, (2) intoxication with APAP is the leading cause of emergency room visits involving acute liver failure within the United States, and (3) conventional clinical markers have poor predictive value. Rats treated with a single dose of 0, 50, 150, or 1500 mg/kg APAP were examined at 6, 24, or 48 h after exposure for conventional toxicological parameters and for gene expression alterations. Patterns of gene expression were found which indicated cellular energy loss as a consequence of APAP toxicity. Elements of these patterns were apparent even after exposure to subtoxic doses. With increasing dose, the magnitude of changes increased and additional members of the same biological pathways were differentially expressed. The energy loss suggested by gene expression changes was confirmed at the 1500 mg/kg dose exposure by measuring ATP levels. Only by ultrastructural examination could any indication of toxicity be identified after exposure to a subtoxic dose of APAP and that was occasional mitochondrial damage. In conclusion, this study provides evidence that supports the hypothesis that gene expression profiling may be a sensitive means of identifying indicators of potential adverse effects in the absence of the occurrence of overt toxicity.
Assuntos
Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Acetaminofen/administração & dosagem , Trifosfato de Adenosina/metabolismo , Administração Oral , Analgésicos não Narcóticos/administração & dosagem , Animais , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Fígado/metabolismo , Fígado/patologia , Masculino , Análise em Microsséries , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/ultraestrutura , Ratos , Ratos Endogâmicos F344 , Testes de Toxicidade/métodosRESUMO
The International Agency for Research on Cancer (IARC) convened a working Group of experts in March 1999 to evaluate the cancer preventive potential of nine retinoids and to compile the fourth volume of the IARC Handbooks of Cancer Prevention. The handbook provides a comprehensive review of the relevant information in the published scientific literature through March 1999 on the potential role of all-trans-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans N-(4-hydroxyphenyl)retinamide, etretinate, acitretin, N-ethylretinamide, targretin and LGD 1550 in cancer prevention. Of these, the data suggest that all-trans-retinoic acid, 13-cis-retinoic acid and N-ethylretinamide are not suitable for chemoprevention of cancer in humans either because they are too toxic, may enhance cancer occurrence or are ineffective. In contrast, 9-cis-retinoic acid, etretinate and acitretin show some promise, but more data are required, while all- trans N-(4-hydroxyphenyl)retinamide is quite promising. Targretin and LGD 1550 are of interest, based on theoretical grounds, but there are no significant human and little experimental data as yet.
RESUMO
A novel computational method based on a genetic algorithm was developed to study composite structure of promoters of coexpressed genes. Our method enabled an identification of combinations of multiple transcription factor binding sites regulating the concerted expression of genes. In this article, we study genes whose expression is regulated by a ligand-activated transcription factor, aryl hydrocarbon receptor (AhR), that mediates responses to a variety of toxins. AhR-mediated change in expression of AhR target genes was measured by oligonucleotide microarrays and by reverse transcription-polymerase chain reaction in human and rat hepatocytes. Promoters and long-distance regulatory regions (>10 kb) of AhR-responsive genes were analyzed by the genetic algorithm and a variety of other computational methods. Rules were established on the local oligonucleotide context in the flanks of the AhR binding sites, on the occurrence of clusters of AhR recognition elements, and on the presence in the promoters of specific combinations of multiple binding sites for the transcription factors cooperating in the AhR regulatory network. Our rules were applied to search for yet unknown Ah-receptor target genes. Experimental evidence is presented to demonstrate high fidelity of this novel in silico approach.
Assuntos
Receptores de Hidrocarboneto Arílico/genética , Fatores de Transcrição/metabolismo , Algoritmos , Animais , Sequência de Bases , Sítios de Ligação , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Masculino , Camundongos , Modelos Teóricos , Dados de Sequência Molecular , Rede Nervosa , Filogenia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
Proinflammatory cytokines, lipopolysaccharide (LPS), and neutrophil elastase (NE) have been implicated in the induction of hypersecretion of respiratory mucus. In this study, we demonstrated that interleukin-1beta (IL-1beta) increased MUC2 and MUC5AC mRNA levels 2- to 3-fold in a time- and dose-dependent manner in NCI-H292 cells. In contrast, MUC5B mRNA was not significantly changed. A transcription inhibitor blocked the stimulation of MUC2 and MUC5AC gene expression by IL-1beta. A translation inhibitor did not interfere with the induction of MUC2 mRNA expression, whereas stimulation of MUC5AC mRNA was blocked, suggesting de novo protein synthesis is required for the stimulation of MUC5AC mRNA. We previously reported that induction of MUC2, MUC5AC, and MUC5B gene expressions by retinoic acid is mediated by the retinoic acid receptor (RARalpha), and inhibited by the specific RARalpha antagonist Ro 41-5253. Here, we demonstrate that the RARalpha antagonist can effectively inhibit IL-1beta-induced MUC2 and MUC5AC gene expression and reduce intracellular MUC5AC protein. Further investigation showed that the RARalpha antagonist also inhibited the stimulation of MUC2 and MUC5AC mRNA expression by tumor necrosis factor-alpha, LPS, and NE.
Assuntos
Benzoatos/farmacologia , Cromanos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucina-1/farmacologia , Elastase de Leucócito/farmacologia , Lipopolissacarídeos/farmacologia , Mucinas/genética , Receptores do Ácido Retinoico/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Humanos , Interleucina-1/antagonistas & inibidores , Elastase de Leucócito/antagonistas & inibidores , Lipopolissacarídeos/antagonistas & inibidores , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mucina-5AC , Mucina-2 , Mucinas/antagonistas & inibidores , Mucinas/biossíntese , Proteínas de Neoplasias/genética , Biossíntese de Proteínas/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptor alfa de Ácido Retinoico , Retinoides/farmacologia , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
We reported recently that interleukin (IL)-1beta exposure resulted in a prolonged increase in MUC5AC mucin production in normal, well differentiated, human tracheobronchial epithelial (NHTBE) cell cultures, without significantly increasing MUC5AC mRNA (Am J Physiol 286:L320-L330, 2004). The goal of the present study was to elucidate the signaling pathways involved in IL-1beta-induced MUC5AC production. We found that IL-1beta increased cyclooxygenase-2 (COX-2) mRNA expression and prostaglandin (PG) E(2) production and that the COX-2 inhibitor celecoxib suppressed IL-1beta-induced MUC5AC production. Addition of exogenous PGE(2) to NHTBE cultures also increased MUC5AC production and IL-1beta-induced Muc5ac hypersecretion in tracheas from wild-type but not from COX-2-/- mice. NHTBE cells expressed all four E-prostanoid (EP) receptor subtypes and misoprostol, an EP2 and EP4 agonist, increased MUC5AC production, whereas sulprostone, an EP1 and EP3 agonist, did not. Furthermore, specific protein kinase A (PKA) inhibitors blocked IL-1beta and PGE(2)-induced MUC5AC production. However, neither inhibition of epidermal growth factor receptor (EGFR) activation with the tyrosine kinase inhibitor 4-(3-chloroanilino)-6,7-dimethoxyquinazoline HCl (AG-1478) or EGFR blocking antibody nor inhibition of extracellular signal-regulated kinase/P-38 mitogen activated protein kinases with specific inhibitors blocked IL-1beta stimulation of MUC5AC mucin production. We also observed that tumor necrosis factor (TNF)-alpha, platelet activating factor (PAF), and lipopolysaccharide (LPS) induced COX-2 and increased MUC5AC production that was blocked by celecoxib, suggesting a common signaling pathway of inflammatory mediator-induced MUC5AC production in NHTBE cells. We conclude that the induction of MUC5AC by IL-1beta, TNF-alpha, PAF, and LPS involves COX-2- generated PGE(2), activation of EP2 and/or EP4 receptor(s), and cAMP-PKA-mediated signaling.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Epitélio/efeitos dos fármacos , Interleucina-1/farmacologia , Isoenzimas/metabolismo , Mucinas/biossíntese , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores de Prostaglandina E/metabolismo , Transdução de Sinais/fisiologia , Brônquios/citologia , Células Cultivadas , AMP Cíclico/metabolismo , Ciclo-Oxigenase 2 , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Epitélio/metabolismo , Receptores ErbB/metabolismo , Humanos , Isoenzimas/genética , Lipopolissacarídeos/farmacologia , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/genética , Receptores de Prostaglandina E Subtipo EP1 , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP3 , Receptores de Prostaglandina E Subtipo EP4 , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Mucociliary transport in the airways significantly depends on the liquid and mucin components of the airway surface liquid (ASL). The regulation of ASL water and mucin content during pathological conditions is not well understood. We hypothesized that airway epithelial mucin production and liquid transport are regulated in response to inflammatory stimuli and tested this hypothesis by investigating the effects of the pleiotropic, early-response cytokine, IL-1beta, on cultured primary human bronchial epithelial and second-passage, normal human tracheo-bronchial epithelial (NHTBE) cell cultures. Fully differentiated NHTBE cultures secreted two major airway mucins, MUC5AC and MUC5B. IL-1beta, in a dose- and time-dependent manner, increased the secretion of MUC5AC, but not MUC5B. MUC5AC mRNA levels were only transiently increased at 1 and 4 h after the start of IL-1beta treatment and returned to control levels thereafter, even though MUC5AC mucin production remained elevated for at least 72 h. Synchronous with elevated MUC5AC secretion, ASL volume increased, its percentage of solid was reduced, and the pH/[HCO(3)(-)] of the ASL was elevated. ASL volume changes reflected altered ion transport, including an upregulation of Cl(-) secretory currents (via CFTR and Ca(2+)-activated Cl(-) conductance) and an inhibition of epithelial sodium channel (ENaC)-mediated absorptive Na(+) currents. IL-1beta increased CFTR mRNA levels without affecting those for ENaC subunits. The synchronous regulation of ASL mucin and liquid metabolism triggered by IL-1beta may be an important defense mechanism of the airway epithelium to enhance mucociliary clearance during airway inflammation.