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.