LIPID OZONATION PRODUCTS ACTIVATE PHOSPHOLIPASES A(2), C, AND D

Ozone exposure, in vitro, has been shown to activate phospholipases A( 2)(PLA(2)), C (PLC), and D (PLD) in airway epithelial cells. However, because of its high reactivity, ozone cannot penetrate far into the ai r/lung tissue interface. It has been proposed that ozone reacts with u nsaturated 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 expo sed cultured human bronchial epithelial cells (BEAS-2B) to LOP (1-100 mu M) produced from the ozonation of almitoyl-2-oleoyl-sn-glycero-3-ph osphatidylcholine (POPC) and measured the activity of PLA,, PLC, and P LD. The PLA, isoform responsible for arachidonic acid release (AA) in stimulated cultures was also characterized. Activation of PLA,, PLC, a nd PLD by three oxidants, hydrogen peroxide (H2O2), tert-butyl hydrope roxide (t-BOOH) and 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH ) also was measured and compared to that of LOP. The derivatives of oz onized POPC at the sn-2 residue, 9-oxononanoyl (PC-ALD), 9-hydroxy-9-h ydroperoxynonanoyl (PC-HHP), and 8-(-5-octyl-1,2,4-trioxolan-3-yl-) oc tanoyl (POPC-OZ) selectively activated PLA, in a dose-dependent fashio n. Cytosolic PLA, (cPLA(2)) measured in the cytosolic fraction of stim ulated cell lysates was found to be the predominant isoform responsibl e for AA release. PLC activation was exclusively induced by the hydrox yhydroperoxide derivatives. PC-HHP and the 9-carbon hydroxyhydroperoxi de (HHP-C9) increased PLC activity. PLD activity also was induced by L OP generated from POPC. Incubation of cultures with H2O2 alone did not stimulate PLC; however, in the presence of the aldehyde, nonanal, a 6 2 +/- 2% increase in PLC activity was found, suggesting that the incre ase in activity was due to the formation of the intermediate HHP-C9. t -BOOH, and AAPH also failed to induce PLA, activation, but did activat e PLC, under conditions of exposure identical to that of LOP. Only t-B OOH activated PLD. These results suggest that biologically relevant co ncentrations of LOP activate PLA,, PLC, and PLD in the airway epitheli al cell, a primary target to ozone exposure. The activation of these p hospholipases may play a role in the development of lung inflammation during ozone exposure. (C) 1998 Academic Press.