RESUMO
Chlorobenzene is a volatile organic compound (VOC) that is widely used as a solvent, degreasing agent and chemical intermediate in many industrial settings. Occupational studies have shown that acute and chronic exposure to chlorobenzene can cause irritation of the mucosa of the upper respiratory tract and eyes. Using in vitro assays, we have shown in a previous study that human bronchial epithelial cells release inflammatory mediators such as the cytokine monocyte chemoattractant protein-1 (MCP-1) in response to chlorobenzene. This response is mediated through the NF-kappaB signaling pathway. Here, we investigated the effects of monochlorobenzene on human lung cells, with emphasis on potential alterations of the redox equilibrium to clarify whether the chlorobenzene-induced inflammatory response in lung epithelial cells is caused via an oxidative stress-dependent mechanism. We found that expression of cellular markers for oxidative stress, such as heme oxygenase 1 (HO-1), glutathione S-transferase pi1 (GSTP1), superoxide dismutase 1 (SOD1), prostaglandin-endoperoxide synthase 2 (PTGS2) and dual specificity phosphatase 1 (DUSP1), were elevated in the presence of monochlorobenzene. Likewise, intracellular reactive oxygen species (ROS) were increased in response to exposure. However, in the presence of the antioxidants N-(2-mercaptopropionyl)-glycine (MPG) or bucillamine, chlorobenzene-induced upregulation of marker proteins and release of the inflammatory mediator MCP-1 are suppressed. These results complement our previous findings and point to an oxidative stress-mediated inflammatory response following chlorobenzene exposure.
Assuntos
Clorobenzenos/toxicidade , Células Epiteliais/efeitos dos fármacos , Pulmão/citologia , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/farmacologia , Western Blotting , Linhagem Celular , Quimiocina CCL2/biossíntese , Quimiocina CCL2/genética , Glutationa/metabolismo , Glutationa S-Transferase pi/biossíntese , Glutationa S-Transferase pi/genética , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/genética , Humanos , Pulmão/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/efeitos dos fármacosRESUMO
Chlorobenzene is a volatile organic compound that is used as a solvent in many industrial settings and has been shown to be related with irritations of the respiratory tract. Exposure to chlorobenzene induces the release of monocyte chemoattractant protein 1 (MCP-1) by lung epithelial cells, a chemokine involved in inflammatory reactions. To characterize the underlying mechanisms we investigated the influence of chlorobenzene on the activation of two intracellular signalling pathways: the nuclear factor-kappa B (NF-kappa B) and the p38 mitogen-activated protein kinase (MAPK) pathways. Human lung epithelial cells (A549) were stimulated with tumor necrosis factor (TNF)-alpha in the presence or absence of specific inhibitors of NF-kappaB or the p38 MAP kinase and exposed to chlorobenzene using an air-liquid cell culture system. Exposure of lung epithelial cells to chlorobenzene resulted in an activation of NF-kappa B and p38 MAP kinase and a release of the chemokine MCP-1. In the presence of IKK-NBD, a specific NF-kappa B inhibitor, or the inhibitors of the p38 MAP kinase SB 203580 and SB 202190, the chlorobenzene-related MCP-1 release was suppressed, suggesting an involvement of both pathways in the chlorobenzene induced expression of MCP-1. Our data show that the release of MCP-1 following chlorobenzene exposure is dependent on the NF-kappa B and MAPK pathways.
Assuntos
Clorobenzenos/toxicidade , NF-kappa B/metabolismo , Alvéolos Pulmonares/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular , Quimiocina CCL2/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Inibidores Enzimáticos/farmacologia , Formazans/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Sais de Tetrazólio/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Styrene is a volatile organic compound (VOC) that is widely used as a solvent in many industrial settings. Chronic exposure to styrene can result in irritation of the mucosa of the upper respiratory tract. Contact of styrene with epithelial cells stimulates the expression of a variety of inflammatory mediators, including the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). To characterise the underlying mechanisms of the induction of inflammatory signals by styrene, we investigated the influence of this compound on the induction of oxidative stress and the activation of the nuclear factor-kappa B (NF-kappaB) signalling pathway in human lung epithelial cells (A549). The results demonstrate that styrene-induced MCP-1 expression, as well as the expression of the oxidative stress marker glutathione S-transferase (GST), is associated with a concentration dependent pattern of NF-kappaB activity. An inhibitor of NF-kappaB, IKK-NBD, and the anti-inflammatory antioxidant N-acetylcysteine (NAC) were both effective in suppressing styrene-induced MCP-1 secretion. In addition, NAC was capable of inhibiting the upregulation of GST expression. Our findings suggest that the activation of the NF-kappaB signalling pathway by styrene is mediated via a redox-sensitive mechanism.
Assuntos
Pulmão/efeitos dos fármacos , NF-kappa B/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Solventes/toxicidade , Estireno/toxicidade , Linhagem Celular , Quimiocina CCL2/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/efeitos dos fármacos , Glutationa Transferase/metabolismo , Humanos , Pulmão/metabolismo , NF-kappa B/metabolismo , Oxirredução , Transdução de Sinais/efeitos dos fármacos , Solventes/administração & dosagem , Estireno/administração & dosagemRESUMO
An in vitro cell culture system based on an air/liquid culture technique was developed which allows a direct exposure of cells to volatile chemicals without medium coverage. For the establishment of the experimental system, chlorobenzene was used as a model compound. Chlorobenzene is a volatile organic compound which is mainly used as a solvent. Beside other adverse health effects, chlorobenzene exposure has been shown to be associated with respiratory tract irritations, Th2 differentiation, and allergic sensitizations. Human peripheral blood mononuclear cells (PBMC) and lung epithelial cells (A549) were exposed to chlorobenzene via gas phase for 20 h. Additionally, PBMC were incubated with culture supernatants from exposed lung epithelial cells. High chlorobenzene concentrations (100 g/m(3)) induced IL-8 production in A549 cells, whereby lower concentrations (10 microg/m(3)-1 g/m(3)) stimulated the secretion of the monocyte chemoattractant protein-1 (MCP-1). A direct effect of chlorobenzene on the cytokine secretion of PBMC was not found. However, if PBMC were incubated with culture supernatants of exposed lung cells, an enhanced production of the Th2 cytokine IL-13 was observed. This induction was prevented in the presence of an anti-MCP-1 antibody. Our data suggest that chlorobenzene induces the production of inflammatory mediators in lung cells. The primary chlorobenzene caused release of MCP-1 in lung epithelial cells may secondarily result in a Th2 differentiation in T lymphocytes. These findings may contribute to the understanding of how chlorobenzene mediates the development of inflammatory reactions in the airways and contributes to the development of an allergic reactivity.