Altered gene expression in the human airways during oxidative stress

Sammanfattning: The atmospheric accumulation of pollutant gases, such as N02 and ozone, has been associated with an increased airways hyper-reactivity within the healthy population. Moreover, those with conditions such as asthma and chronic obstructive pulmonary diseases generally experience an exacerbation of their symptoms upon exposure to these noxious gases. Cells lining the respiratory tract can be considered as the primary target for the potential toxic effects of airborne chemicals, but the molecular mechanisms behind the injury occurring in these cells upon gas inhalation remain to be fully elucidated. It has been hypothesised that the toxicity of these gases relies on their reactivity towards macromolecules in the lining fluid of the lung and the formation of reactive secondary products, which cause a situation of oxidative stress. Cells posses a well-developed defence against reactive species, but when this defence is overwhelmed damage can occur to important cellular components, including DNA and proteins. The major aim of this thesis was to study the effects of oxidative stress on the redox-sensitive machinery of gene expression in human lung cells. Cells are able to sense oxidative damage, as well as changes in the cellular redox status and adapt their gene expression profiles in a manner either promoting protection against the insult, or taking the decision to induce cell death to prevent propagation of damaged genetic templates. The pulmonary defence against inhaled antigens is based on the activation of immunological events, most of them dependent on cytokine signalling. Analysis of human alveolar macrophages exposed to N02 in vitro revealed inhibition of transcription and release of some inflammationmodulating cytokines. The inhibition was more pronounced in macrophages from habitual smokers. The effects of ozone on mRNA profiles were studied in human alveolar macrophages exposed to lowconcentration of ozone in vivo. Large-scale screening of these events resulted in the detection of a discrete number of ozone-responsive genes belonging to disparate biological pathways, including nucleic acids synthesis and repair, inter- and intra-cellular signal transduction, cytoskeletal organisation, inflammation and protein modification. These findings also report on considerable biochemical effects of ozone in the lung at levels close to the safety limits recommended by international air quality guidelines and standards. Exposure of cells to the thiol- oxidising agent diamide in vitro, lead to increased mRNA levels and expression of stress responsive- genes and to the oxidation of GSH with the concomitant S-glutathionylation of cellular proteins. In contrast, in vitro exposure of cells to hydrogen peroxide failed to induce many of the diamide- responsive genes, significant oxidation of GSH or formation of protein-GSH mixed disulphides. Thus, redox-sensitive alteration of gene expression could be correlated to the oxidation of GSH and the concomitant formation of mixed protein-GSH disulphides. In addition, induced expression of protein and DNA chaperones, including heat shock proteins, by diamide exposure, resulted in cytoprotection against both heath shock and the DNA-damaging pro-oxidant potassium bromate. Exposure of the human A549 lung epithelial cell line to sub-toxic levels of hydrogen peroxide caused sustained arrest in cell cycle progression and activation of apoptotic events, including activation of caspase-3 and the augmentation of TRAIL-dependent caspase-3 activation. These effects may be in consequence of the DNA damage caused by the oxidant. Hydrogen peroxide treatment lead also to the alteration in mRNA profiles for a variety of genes, many of them known to be p53-dependent and other not previously associated with changes in cellular redox states. These alterations could be functionally related to cell cycle arrest, apoptosis and DNA damage. In summary, airborne pollutants and their secondary oxidant products can damage important components of lung cells and alter their gene expression profiles. The results of these studies contribute to our understanding of the mechanisms of oxidative stress in human lung cells, as well as provide evidence for co-ordination in the regulation of the redox-sensitive machinery of gene expression.