Kafoury R M, Pryor W A, Squadrito G L, Salgo M G, Zou X, Friedman M
Department of Environmental Health Sciences, Tulane University Medical Center, New Orleans, Louisiana 70112, USA.
Toxicol Appl Pharmacol. 1998 Jun;150(2):338-49. doi: 10.1006/taap.1998.8418.
Ozone exposure, in vitro, has been shown to activate phospholipases A2 (PLA2), 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 microM) produced from the ozonation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and measured the activity of PLA2, PLC, and PLD. The PLA2 isoform responsible for arachidonic acid release (AA) in stimulated cultures was also characterized. Activation of PLA2, PLC, and PLD by three oxidants, hydrogen peroxide (H2O2), 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 PLA2 in a dose-dependent fashion. Cytosolic PLA2 (cPLA2) 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 H2O2 alone did not stimulate PLC; however, in the presence of the aldehyde, nonanal, a 62 +/- 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 PLA2 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 PLA2, 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.
体外实验表明，臭氧可激活气道上皮细胞中的磷脂酶A2（PLA2）、C（PLC）和D（PLD）。然而，由于其高反应活性，臭氧无法深入穿透气/肺组织界面。有人提出，臭氧与上皮衬液（ELF）和细胞膜中的不饱和脂肪酸（UFA）反应，生成一系列脂质臭氧化产物（LOP），介导臭氧诱导的毒性。为验证这一假设，我们将培养的人支气管上皮细胞（BEAS-2B）暴露于由1-棕榈酰-2-油酰-sn-甘油-3-磷脂酰胆碱（POPC）臭氧化产生的LOP（1-100 microM）中，并测量PLA2、PLC和PLD的活性。还对刺激培养物中负责花生四烯酸释放（AA）的PLA2同工型进行了表征。还测量了三种氧化剂过氧化氢（H2O2）、叔丁基过氧化氢（t-BOOH）和2,2'-偶氮二（2-脒基丙烷）二盐酸盐（AAPH）对PLA2、PLC和PLD的激活作用，并与LOP的激活作用进行比较。sn-2位臭氧化POPC的衍生物9-氧代壬酰基（PC-ALD）、9-羟基-9-氢过氧壬酰基（PC-HHP）和8-（-5-辛基-1,2,4-三氧杂环戊烷-3-基-）辛酰基（POPC-OZ）以剂量依赖方式选择性激活PLA2。在刺激细胞裂解物的胞质部分中测得的胞质PLA2（cPLA2）是负责AA释放的主要同工型。PLC激活仅由羟基氢过氧化物衍生物诱导。PC-HHP和9-碳羟基氢过氧化物（HHP-C9）增加了PLC活性。PLD活性也由POPC产生的LOP诱导。单独用H2O2孵育培养物不会刺激PLC；然而，在醛壬醛存在下，发现PLC活性增加了62±2%，这表明活性增加是由于中间产物HHP-C9的形成。在与LOP相同的暴露条件下，t-BOOH和AAPH也未能诱导PLA2激活，但确实激活了PLC。只有t-BOOH激活了PLD。这些结果表明，生物学相关浓度的LOP可激活气道上皮细胞中的PLA2、PLC和PLD，气道上皮细胞是臭氧暴露的主要靶标。这些磷脂酶的激活可能在臭氧暴露期间肺部炎症的发展中起作用。
