Lipid ozonation products activate phospholipases A sub(2), C, and D

Ozone exposure, in vitro, has been shown to activate phospholipases A sub(2) (PLA sub(2)), C (PLC), and D (PLD) in airway epithelial cells. However, because of its high reactivity, ozone cannot penetrate far into the air/lung tissue interface. It has been proposed that ozone reacts with unsaturated fatty acids (UFA) in the epithelial lining fluid (ELF) and cell membranes to generate a cascade of lipid ozonation products (LOP) that mediate ozone-induced toxicity. To test this hypothesis, we exposed cultured human bronchial epithelial cells (BEAS-2B) to LOP (1-100 mu M) produced from the ozonation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and measured the activity of PLA sub(2), PLC, and PLD. The PLA sub(2) isoform responsible for arachidonic acid release (AA) in stimulated cultures was also characterized. Activation of PLA sub(2), PLC, and PLD by three oxidants, hydrogen peroxide (H sub(2)O sub(2)), tert-butyl hydroperoxide (t-BOOH) and 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) also was measured and compared to that of LOP. The derivatives of ozonized POPC at the sn-2 residue, 9-oxononanoyl (PC-ALD), 9-hydroxy-9-hydroperoxynonanoyl (PC-HHP), and 8-(-5-octyl-1,2,4-trioxolan-3-yl-) octanoyl (POPC-OZ) selectively activated PLA sub(2) in a dose-dependent fashion. Cytosolic PLA sub(2) (cPLA sub(2)) measured in the cytosolic fraction of stimulated cell lysates was found to be the predominant isoform responsible for AA release. PLC activation was exclusively induced by the hydroxyhydroperoxide derivatives. PC-HHP and the 9-carbon hydroxyhydroperoxide (HHP-C9) increased PLC activity. PLD activity also was induced by LOP generated from POPC. Incubation of cultures with H sub(2)O sub(2) alone did not stimulate PLC; however, in the presence of the aldehyde, nonanal, a 62 plus or minus 2% increase in PLC activity was found, suggesting that the increase in activity was due to the formation of the intermediate HHP-C9. t-BOOH, and AAPH also failed to induce PLA sub(2) activation, but did activate PLC, under conditions of exposure identical to that of LOP. Only t-BOOH activated PLD. These results suggest that biologically relevant concentrations of LOP activate PLA sub(2), PLC, and PLD in the airway epithelial cell, a primary target to ozone exposure. The activation of these phospholipases may play a role in the development of lung inflammation during ozone exposure.