Pseudomonas aeruginosa causes severe acute nosocomial pneumonias and chronic lung infections in patients with cystic fibrosis (CF). Chronic lung injury is the primary cause of death in CF and is linked to co-existent P. aeruginosa infection. The mechanisms involved in P. aeruginosa-mediated lung damage remain uncertain. Pyocyanin (1-hydroxy-5-methylphenazine), one of several small phenazine compounds synthesized and secreted by most P. aeruginosa strains, has been linked to organism virulence. Pyocyanin undergoes eukaryotic cell-mediated aerobic redox cycling, resulting in formation of reactive oxygen species (ROS) such as superoxide (O2.-) and hydrogen peroxide (H2O2). How pyocyanin enters eukaryotic cells, the site(s), mechanism(s), and nature of the oxidants produced during cellular exposure to pyocyanin, the cellular targets modified and their functional consequences are each poorly understood. There is even less known about other related phenazines produced by P. aeruginosa, such as 1-hydroxyphenazine (1-HP) and phenazine-1-carboxylic acid (PCA). We are testing the hypothesis that during P. aeruginosa lung infection pyocyanin release leads to site-specific oxidant-mediated effects on airway epithelial cells that disrupt cellular energy generation, activate oxidant-sensitive signaling pathways, and promote inflammation in the lung. We have shown that mitochondria are a key site of pyocyanin-mediated ROS production and a target of cytotoxicity. We have also found that pyocyanin negatively affects two critical antioxidant defense mechanisms in epithelial cells, catalase and glutathione (GSH). It decreases steady state catalase MRNA, and protein and also directly inhibits the activity of the enzyme. It decreases cellular GSH levels in part by leading to GSH oxidation and export from the cell. Furthermore, paradoxically, GSH appears to be capable of accelerating the formation of ROS by pyocyanin by directly reducing pyocyanin. Furthermore, pyocyanin exposure alters a number of oxidant-sensitive transcription factors (e.g. NF-κB), as well as gene products influenced by them. We have also employed [14C]pyocyanin to assess the ability of pyocyanin to cross intact airway epithelial cell monolayers; Calu3 and primary human epithelial cells cultured on millicell filters. When [14C]pyocyanin and [3H]mannitol were placed in the upper chamber (apical side), steady movement of [14C]pyocyanin from the apical to basolateral fluid occurred over time. In contrast, <1% of the membrane impermeable [3H]mannitol was detected on the basolateral side of the monolayer, indicating that movement of pyocyanin did not occur via paracellular leak. When human fibroblasts were placed below the epithelial cell monolayer, pyocyanin that had traversed the monolayer was able to induce ROS production in the fibroblasts as assessed by oxidation of intracellular DCF. This suggests that luminal pyocyanin may be able to influence the function of cell types located beneath the epithelium. We also find that PCA has significant biologic effects on airway epithelial cells, the type, magnitude, or mechanism of which in some cases vary from those of pyocyanin. PCA increased epithelial cell IL-8 and ICAM-1 expression (4-5- and 3-fold, respectively) and decreased TNF-dependent RANTES and MCP-1 release 50-90% in a ROS-dependent process. In contrast to results with pyocyanin, PCA did not appreciably oxidize NADH or NADPH at pH 7 and oxidation was considerably slower than that caused by pyocyanin at pH 5. However, the addition of PCA (10-100 μM), like pyocyanin, increased ROS formation in epithelial cells. Interestingly, despite ROS production by both pyocyanin and PCA, we have observed significant differences in the ability of some antioxidants to inhibit their pro-inflammatory effects. We are seeking to develop systems to confirm the in vivo relevance of many of our in vitro observations. We find that when 50μl of HBSS, 50μM pyocyanin, or 50 μM PCA was injected intratracheally in mice, both pyocyanin and PCA markedly increased the number of PMNs in BAL over the ensuing 24h. Interestingly, the time course for PMN influx was different for pyocyanin (peak = 48 h) and for PCA (peak = 24 h). Moreover, increased influx of PMNs was accompanied by increased levels of PMNchemokines and of intercellular adhesion molecule-1 (ICAM-1). These findings support the hypothesis that pyocyanin and other P. aeruginosa-derived phenazines can participate in biologically important redox reactions that may significantly alter the function of airway epithelial cells during acute and chronic P. aeruginosa lung infections.