RESUMEN
This study was conducted to determine the time course of gene expression associated with specific signaling pathways in normal human bronchial epithelial (NHBE) cells after exposure to 2 concentrations of 2R4F tobacco mainstream smoke (MSS). Expression of 84 genes representing 18 signal transduction pathways was quantitated in MSS- and air-exposed cultures using real-time polymerase chain reaction (PCR) arrays at 1, 4, and 24 hours following exposure. A confidence score, calculated based on statistical analysis of the degree and reproducibility of expression changes, was used to identify potential biologically significant changes in gene expression. Stimulation of NIAP, an apoptosis inhibitor, suppression of NFKB1 and MYC, representing pro-apoptotic activity, and down-regulation of TCF7 and up-regulation of KLK2, representing anti-/pro-inflammatory responses, were altered 1 hour after exposure to the high concentration of MSS. At the 4-hour time point, the pattern had changed such that 10 different genes were now up-regulated and an additional gene was now down-regulated. Significant changes included genes involved in inflammatory response (LTA, SELPLG, and IL8), repair and wound-healing activity (MMP10), and growth activity (GREB1, EGR1), suggesting repair in this period. By 24 hours, the only up-regulated genes in common with the 4-hour profile were SELPLG and IL8, suggesting continued inflammatory signaling. These results suggest that identification of specific gene expression-based biomarkers of MSS toxicity is promising for investigating specific mechanisms of cellular damage. As expected, the expressed signals were dependent on the concentration of MSS and the postexposure times.
Asunto(s)
Bronquios/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Nicotiana , Mucosa Respiratoria/efectos de los fármacos , Humo/efectos adversos , Bronquios/metabolismo , Bronquios/patología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Reproducibilidad de los Resultados , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Transducción de Señal/genética , Factores de TiempoRESUMEN
Bronchial epithelium is frequently exposed to air pollutants, and it is hypothesized that these cells elicit inflammatory responses as early elements in pulmonary defense. Our purpose was to evaluate changes in messenger RNA levels of 84 genes representing cytokines and receptors over a repetitive-exposure time course to further define the inflammatory responses associated with mainstream cigarette smoke (MSS) exposure in an in vitro lung model. Normal human bronchial epithelial cells were treated with mainstream cigarette smoke condensate (CSC) prepared from Kentucky 2R4F cigarettes (60 microg total particulate matter/mL media, 0.2% dimethylsulfoxide), and examined by quantitative real-time polymerase chain reaction. Applications of CSC were designed in seven groups to test immediate, early, intermediate, and late responses evaluated at the end of alternating exposure/recovery periods. Three predominant gene expression responses were observed: adaptive (return to baseline), sustained (maintained expression during treatment), and chronic (maintained expression posttreatment). Overall, 25 genes exhibited statistically significant changes: 14 genes exclusively elevated, 10 genes exclusively depressed, and 1, interleukin-8 (IL8), exhibiting both up- and downregulation in the seven groups. The most responsive genes were osteopontin (34-fold upregulation) and CXCL14 (23-fold downregulation). Our observations suggest that specific genes involved in inflammatory pathways respond to CSC in chronic, sustained, or adaptive patterns with the chronic pattern as the predominant behavior.
Asunto(s)
Bronquios/inmunología , Quimiocinas/biosíntesis , Interleucinas/biosíntesis , Nicotiana/efectos adversos , Mucosa Respiratoria/inmunología , Humo/efectos adversos , Breas/toxicidad , Adulto , Bronquios/efectos de los fármacos , Células Cultivadas , Quimiocinas CXC/biosíntesis , Regulación hacia Abajo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Perfilación de la Expresión Génica , Humanos , Interleucina-8/biosíntesis , Masculino , Osteopontina/inmunología , Mucosa Respiratoria/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Atherosclerosis is generally considered an inflammatory disease characterized by the accumulation of lipid in large and medium elastic arteries. Individuals who smoke are at increased risk for developing atherosclerosis and the clinical events associated with this disease. Underlying the mechanisms involved in atherosclerotic lesion development exists a complex pattern of signaling, involving molecules (cytokines and chemokines) that mediate the progression of arterial lesions. The unique nature of exposure to tobacco-related toxicants during the process of smoking prompted our investigation of the time-dependent responses of two critical cell types to cigarette smoke condensate exposure. In this study, we examined the kinetic responses, using suspension array technology and RT-PCR of 17 cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17 GM-CSF, G-CSF, INF-gamma, TNF-alpha, MCP-1 and MIP-1beta) in human aortic endothelial cells (HAECs) and THP-1 monocyte macrophages following exposure to cigarette smoke condensate (CSC) for 24h. In HAECs, IL-8 and IL-4 were rapidly stimulated by CSC exposure while, surprisingly, MCP-1 expression was downregulated. In THP-1 macrophages, IL-6, MIP-1beta, MCP-1 and IL-1beta protein expression were suppressed upon CSC exposure. All other measurable cytokines in THP-1 cells exposed to CSC had levels of protein and mRNA similar to controls. Depending on cell type, CSC uniquely influences the expression of cytokines. The complex interplay of these signaling molecules within the framework of atherosclerosis points to the ability of cigarette smoke components to alter such signaling following acute exposure, and by this mechanism may alter the course of both atherogenesis initiation and progression.
Asunto(s)
Citocinas/metabolismo , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Monocitos/efectos de los fármacos , Nicotiana , Humo , Aorta , Línea Celular , Células Cultivadas , Citocinas/biosíntesis , Citocinas/genética , Células Endoteliales/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Cigarettes that burn tobacco produce a complex mixture of chemicals, including mutagens and carcinogens. Cigarettes that primarily heat tobacco produce smoke with marked reductions in the amount of mutagens and carcinogens and demonstrate reduced mutagenicity and carcinogenicity in a battery of toxicological assays. Chemically induced oxidative stress, DNA damage, and inflammation may alter cell cycle regulation and are important biological events in the carcinogenic process. The objective of this study was to characterize and compare the effects of smoke condensates from cigarettes that burn tobacco and those that primarily heat tobacco on gene expression in NHBE cells. For this comparison, we used quantitative RT/PCR and further evaluated the effects on cell cycling using flow cytometry. Cigarette smoke condensates (CSCs) were prepared from Kentucky 1R4F cigarettes (a tobacco-burning product designed to represent the average full-flavor, low "tar" cigarette in the US market) and Eclipse (a cigarette that primarily heats tobacco) using FTC machine smoking conditions. The CSC from 1R4F cigarettes induced statistically significant increases in the mRNA levels of genes responsive to DNA damage (GADD45) and involved in cell cycle regulation (p21;WAF1/CIP1), compared to the CSC from Eclipse cigarettes. In addition, genes coding for cyclooxygenase-2 (COX-2) and interleukin 8 (IL-8), which are associated with oxidative stress and inflammation, respectively, were increased statistically significantly more by CSC from 1R4F than by that from Eclipse. Furthermore, a dose-dependent increase in IL-8 protein secretion into cell culture media was stimulated by 1R4F exposure, whereas minimal IL-8 protein was secreted after Eclipse treatment. The biological relevance of the differential effect on gene expression was reflected in differential cell cycle regulation, as cells exposed to 1R4F CSC exhibited more significant S phase and G2 phase accumulation than cells exposed to Eclipse CSC. These data indicate that the simplified smoke chemistry of the tobacco-heating Eclipse cigarette yields statistically significant reductions in the expression of key genes involved in DNA damage, oxidative stress, inflammatory response, and cell cycle regulation in normal human bronchial epithelial cells compared to a representative tobacco-burning cigarette.
Asunto(s)
Bronquios/metabolismo , Expresión Génica , Nicotiana , Humo , Secuencia de Bases , Bronquios/citología , Ciclo Celular , Células Cultivadas , Cartilla de ADN , Células Epiteliales/metabolismo , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Bronchial epithelial cells are often exposed to airborne mutagens that have the potential to induce genetic changes involved in the development of lung cancer. Although lung tumors often display alterations in the expression of oncogenes and/or tumor suppressor genes, the role of specific chemicals and/or metabolites in causing these alterations is not well defined. The polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P), a by-product of combustion, is a prevalent airborne environmental mutagen and a constituent of cigarette smoke. The primary objective of this study was to compare the effect of B[a]P and two of its reactive metabolites, benzo[a]pyrene diol epoxide (BPDE or bay region epoxide) and benzo[a]pyrene-4,5-dihydroepoxide (BPE or K-region epoxide), on expression of the proto-oncogene c-myc in normal human bronchial epithelial (NHBE) cells using a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. Changes in c-myc gene expression were compared with DNA adduct formation, growth inhibition, and cell-cycle progression as determined by (32)P-postlabelling, neutral red (NR), and flow cytometric analyses, respectively. None of the three test compounds altered the levels of 18S ribosomal RNA or beta-actin at the concentrations evaluated for c-myc expression, indicating that nonspecific changes in gene expression induced by cytotoxicity, for example, were not present at the concentrations evaluated. Cells exposed to B[a]P exhibited a dose-dependent increase in c-myc expression; conversely, a dose-dependent decrease in c-myc expression was observed following BPDE exposure. A marginal but concentration-dependent increase in c-myc mRNA levels was observed following exposure to the K-region epoxide. Our results demonstrated that, although B[a]P and its metabolites alter c-myc expression, the parent compound and its metabolites produce unequal and contrasting effects on the expression of this gene.
Asunto(s)
7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/toxicidad , Benzo(a)pireno/toxicidad , Benzopirenos/toxicidad , Bronquios/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Contaminantes Atmosféricos/toxicidad , Bronquios/citología , Bronquios/fisiología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , División Celular/efectos de los fármacos , División Celular/genética , Células Cultivadas , Colorantes/farmacocinética , Aductos de ADN/efectos de los fármacos , Aductos de ADN/metabolismo , Células Epiteliales/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mutágenos/toxicidad , Rojo Neutro/farmacocinética , Radioisótopos de Fósforo , Hidrocarburos Policíclicos Aromáticos/toxicidad , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/efectos de los fármacos , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Cigarette smoking has been associated with an increase in the severity and prevalence of atherosclerosis in the abdominal aorta. To begin our investigation of this finding, we used an integrated approach combining gene expression profiling, protein analysis, cytokine measurements, and cytotoxicity determinations to examine molecular responses of cultured human aortic and coronary endothelial cells exposed to cigarette smoke condensate (CSC) and nicotine. Exposure of endothelial cells to CSC (30 and 60 microg/mL TPM) for 24 h resulted in minimal cytotoxicity, and the upregulation of genes involved in matrix degradation (MMP-1, MMP-8, and MMP-9), xenobiotic metabolism (HO-1 and CYP1A2), and downregulation of genes involved in cell cycle regulation (including TOP2A, CCNB1, CCNA, CDKN3). Exposure of cells to a high physiological concentration of nicotine resulted in few differentially expressed genes. Immunoblot analysis of proteins selected from genes shown to be differentially regulated by microarray analysis revealed similar responses. Finally, a number of inflammatory cytokines measured in culture media were elevated in response to CSC. Together, these results describe a complex proinflammatory response, possibly mediating the recruitment of leukocytes through cytokine signaling. Additionally, fibrous cap destabilization may be facilitated by matrix metalloproteinase upregulation.