Molecular mechanisms of nitrogen dioxide induced lung damage

Nitrogen dioxide (NO2) is an oxidant gas which contaminates ambient air in many urban andindustrial locations, and indoor air in homes with combustion appliances. The EnvironmentalProtection Agency (USA) regulates N02 in ambient air as a "criteria" pollutant. In spite of decades of laboratory, clinical, and epidemiological research, the mechanisms of the well-known adverse effects of NO2 exposure on the lung, the most important target for this gas, are not well understood. Of particular interest are the molecular mechanisms behind the airways inflammation induced by this gas which are far from clear. The general aims of this study: 1. To establish cell culture models and an invitro gas exposure model for mechanistic studies of factors underlying the pulmonary toxicity of theNO2 and, eventually, 03. 2. To define the cytoprotective roles of major airway antioxidants in thealveolar structure against NO2. 3. To study the molecular mechanisms of NO2-induced airwayinflammation.We developed a new, optimal in vitro gas exposure modd for the exposure of inverted cell culturesto NO2, which present several advantages. Firstly, the cell monolayer can be directly exposed toNO2 in the gas phase for up to 1 hour, without the interposition of a thick aqueous layer. Secondly,the chamber system allows the simple and precise control of the gas concentration during theexposure. Finally, the system allows the simultaneous exposure of large numbers of cells understerile conditions, facilitating further culture of the cells after the exposure.By using this gas exposure model, the relative importance of water-soluble nonenzymatic antioxidantcomponents in airway epithelial lining fluids, ascorbate and glutathione (GSH), in protecting humancells against NO2-induced cytotoxicity has been evaluated. From the results we conclude that bothascorbate and GSH play important roles in defending human cells from the toxicity of NO2 underdirect exposure conditions and that both extracdlular and intracdlular ascorbate can very efficiently play a protective role. The results also strengthen the premise that ascorbate and GSH co-operate in such antioxidative protection. Chemical oxidation assays illustrated that NO2 directly reacts with ascorbate and GSH being itself reduced to nitrite.Considering alveolar macrophages (AMs) may be direct cellular target of NO2 in vivo andconsidering their important potential roles of the cells in airway inflammation due to their capacity of releasing many kind of inflammatory mediators including proinflammatory cytokines andchemokines, a murine macrophage cell line IC-21 and human primary alveolar macrophages weredirectly exposed to NO2 using our modd. Results indicated that NO2 does not stimulate the mRNAexpression and protein release of TNF-ac, IL-l~, IL,8 and MIP-l from these cells.Our knowledge of understanding the involvement of endothdial and epithelial cell metabolism inairway inflammation, due partially to their capacity to produce and release proinflammatory cytokines and chemokines, has been increasing rapidly. Toxicological data has indicated that airway epithelial and alveolar endothelial cells may be damaged directly and/or indirectly by inhaled N02. Therefore, we further studied the effects of NO2 on expression of proinflammatory cytokines, chemokines and infammatory cell adhesion molecules in human endothelial cdls, airway epithdial cells and bronchial biopsies. The results from RT-PCR, semi-quantitative RT-PCR and ELISA show that NO2 does notinduce or stimulate the expression of inflammatory cytokines, chemokines or cell adhesion moleculesin any of the probed cells/tissues under the respective condition of exposure.In summary, the in vitro gaseous exposure model is a suitable system for the study of, the biologicaleffects of oxidative gaseous pollutants. Both ascorbate and GSH dependently and independentlyprotect human cells against the NO2-induced cytotoxicity. More sensitive tools and more quantitativestudies are required for understanding the involvement of proinflammatory cytokines, chemokinesand inflammatory cell adhesion molecules as well as other mediator, from different cell types inairways, in NO2-induced airways inflammation.Doctoral Thesis1995 Ba TuISBN 91-628-1837-