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Toxicology Letters 196 (2010) 168–174

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

ctivation of phospholipase D involved in both injury and survival in A549lveolar epithelial cells exposed to H2O2

ing Wua, Qi Wanga, Jiang-Yun Luod, Bo Jiangb, Xu-Yun Lic, Ru-Kun Chena, Yun-Bi Lud,∗

Department of Cardiothoracic Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, ChinaDepartment of Clinical Pharmacy, Second Affiliated Hospital, School of Medicine, Zhejiang University, ChinaDepartment of Physiology, School of Medicine, Zhejiang University, ChinaDepartment of Pharmacology, School of Medicine, Zhejiang University, Yu Hang Tang Road 388, Hangzhou, Zhejiang 310058, China

r t i c l e i n f o

rticle history:eceived 8 March 2010eceived in revised form 15 April 2010ccepted 16 April 2010vailable online 24 April 2010

a b s t r a c t

To determine the role of the phospholipase D (PLD) pathway in injury and survival of alveolar epithelialcells, A549 cells were exposed to H2O2 (500 �M) which resulted in time-dependent injury and bi-phasicincrease of PLD activity at 5 min and at 3 h, respectively. n-Butanol (0.5%) inhibited PLD activation,attenuated cell injury at 5 min of H2O2 exposure, but enhanced injury at 3 h of exposure. This activa-tion was inhibited by treatment with catalase (500 units/ml). Exogenous phosphatidic acid mimickedthe effects of PLD activation, and diphenyliodonium (NADPH oxidase inhibitor) reversed the decline in

eywords:ydrogen peroxidehospholipase Dhosphatidic acid549 cell

cell viability induced by H2O2 exposure. Propranolol (phosphatidic acid phospholydrolase inhibitor) andquinacrine (phospholipase A2 inhibitor) had weak effects on H2O2-induced PLD activation but reversedH2O2-induced injury. We speculate that PLD activation at the initiation of H2O2 exposure predominantlyresults in NAPDH oxidase activation, which mediates A549 cell injury, but turns to mediating cell survivalas the H2O2 attack continues, which might be mainly due to the accumulation of intracellular phosphatidicacid.

. Introduction

Acute lung injury and acute respiratory distress syndrome areharacterized by alveolar epithelial and alveolar-capillary dam-ge which result in nonhydrostatic pulmonary edema and severeypoxemia (Lucas et al., 2009; Manicone, 2009). Oxidative dam-ge plays an important role in the loss of integrity of the epithelialarrier which leads to the influx of protein-rich edema fluid andccumulation of neutrophils in the alveolar space (Lucas et al.,009; Manicone, 2009). In particular, a mass of reactive oxygenpecies (ROS) released by neutrophils in the alveolar space con-ribute greatly to alveolar tissue injury (Mendez and Hubmayr,005; Tsushima et al., 2009). However, a new paradigm of redoxignaling has emerged recently, whereby some oxidants are con-idered to function as intracellular signaling molecules which canontribute to either cell death or survival (Giorgio et al., 2007;

rachootham et al., 2008).

Phospholipase D (E.C.3.1.4.4; PLD) is ubiquitous in mammalianells and is activated by various extracellular stimuli, including2O2, in different cell types. H2O2 stimulates PLD activity by a

∗ Corresponding author. Tel.: +86 571 88208223; fax: +86 571 88208022.E-mail address: yunbi@zju.edu.cn (Y.-B. Lu).

378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2010.04.014

© 2010 Elsevier Ireland Ltd. All rights reserved.

poorly understood, probably direct or indirect signaling pathway(Min et al., 2001; Natarajan et al., 1993; Oh et al., 2000; Xiao etal., 2005). Two mammalian PLD isozymes, PLD1 and PLD2, havebeen identified, characterized and cloned (Cockcroft, 2001; Jenkinsand Frohman, 2005). PLD catalyzes the hydrolysis of phosphatidyl-choline and other membrane phospholipids to phosphatidic acid(PA) and choline. PA can be subsequently converted to lyso-PA byphospholipase A2 (PLA2) or to diacylglycerol (DAG) by PA phos-pholydrolase (PAP), where PA is considered to be the main effectorof the functions of PLD in cells (Cockcroft, 2001; Cockcroft andFrohman, 2009; Exton, 2002; Jenkins and Frohman, 2005). In thepresence of primary alcohols, PLD catalyzes a transphosphatidyla-tion reaction producing phosphtidylalcohols at the expense of PA;this feature provides a tool to implicate PLD in cellular responses(Exton, 2002).

PLD and its metabolites are involved in various cellular func-tions, such as activation of NADPH oxidase (oxidative burst),membrane trafficking, exocytosis (Cockcroft et al., 2002), phago-cytosis (Corrotte et al., 2006), cell adhesion and chemotaxis

(Gomez-Cambronero et al., 2007), cytoskeletal reorganization, cellproliferation, apoptosis, and survival (Cockcroft and Frohman,2009). In inflammatory cells or phagocytic cells, PLD plays a rolein stimulation of NADPH during the respiratory oxidative burst.PLD functions both directly, by generating PA, which binds to and

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value decreased, but remained higher than control at 60 min. At180 min, the value of PLD activity increased to 0.32 ± 0.04 units

M. Wu et al. / Toxicolog

timulates the p47 (phox) component of the NADPH oxidase com-lex (Usatyuk et al., 2009), and indirectly, by converting of somef the PA into DAG, which is required for NADPH activation (Paliczt al., 2001). Once NADPH oxidase is activated, it generates H2O2hich leads to further PLD activation (Xiao et al., 2005). These

eatures constitute a positive feedback cycle: exogenous H2O2 acti-ates PLD and PLD activation promotes NADPH oxidase activationhich leads to H2O2 production.

However, it is unclear that whether this positive feedback cyclexists in non-phagocytic cells, e.g., in the alveolar epithelial cell.oreover, the roles of PLD and its metabolites play in alveolar

pithelial cells during acute lung injury are unclear. Here, we reporthe involvement of the PLD pathway in H2O2-induced A549 alveo-ar epithelial cell injury and survival.

. Materials and methods

.1. Drugs

Phosphatidylcholine (C10:0), 4-aminoantipyrine, sodium oleate, 1-butanol,ropranolol, phosphatidic acid (C10:0), catalase (from bovine liver) (Sigma, St.ouis, MO, USA), phenol (ShengGong Biotechnology Co. Ltd., Shanghai, China),nd horseradish peroxidase (XiTang Biotechnology Co. Ltd., Shanghai, China),ere dissolved in Millipore water. Quinacrine, diphenyliodonium (DPI), and 2,7-ichlorofluorescein diacetate (Sigma) were dissolved in dimethyl sulfoxide (DMSO,igma) and diluted in Millipore water before use (the concentration of DMSO wasess than 0.001% after dilution). H2O2 (Sigma) was diluted in Earle’s solution (con-entrations in mM: NaCl, 117; KCl, 5.3; CaCl2, 1.8; NaHCO3, 26; MgSO4, 0.8; NaH2PO4,.0; glucose, 5.6; pH 7.4). Choline oxidase (Sigma) was dissolved in a solution con-aining (in mM) Tris–HCl, 10; EDTA, 2.0; KCl, 134; pH 8.0.

.2. Cell culture

A549 alveolar epithelial cells (Institute of Cell Biology, Chinese Academy of Sci-nce, Shanghai, China) were cultured in F-12 Kaighn’s nutrient mixture (Invitrogen,arlsbad, CA, USA) containing 10% fetal bovine serum (Invitrogen) in a culture flaskith 1 × 106 cells/75 cm2 and in 96-well plates with 4000 cells/well under air and

% CO2 at 37 ◦C. Before exposure to H2O2, the culture medium was switched to F-12aighn’s nutrient mixture containing 1% fetal bovine serum for 48 h and then theells were rinsed twice with Earle’s solution.

.3. Agent treatments

Catalase (500 units/ml), phosphatidic acid (0.1 �M), the PLD inhibitor 1-butanol0.5%), the NADPH oxidase inhibitor diphenyliodonium (1 nM), the PAP inhibitorropranolol (100 �M), and the PLA2 inhibitor quinacrine (1 �M) were used. Thegents were continuously applied from 30 min before H2O2 exposure to the end ofxposure. The control group received the same treatment except for H2O2 expo-ure.

.4. Assessment of cell viability and cell injury

At the end of H2O2 exposure, 3-(4,5-dimehythiazol-2-yl)-2,5-iphenyltetrazolium bromide (MTT, Sigma) was added to each well to a finaloncentration 0.5 mg/ml. After incubation for 4 h at 37 ◦C, the medium wasemoved and 100 �l DMSO was added to each well for 10 min at 37 ◦C. The plateas read at 570 nm using a microplate reader (Elx800, Bio-Tek Instruments,ermont, USA). Results are reported as percentages of control. In another series,

he cytotoxicity of H2O2 on A549 cells was quantified using lactate dehydrogenaseLDH) activity assays in the medium and the cell layer. The LDH activity assay wasarried out by a colorimetric method using an LDH assay kit (JianCheng Biotechnol-gy Co. Ltd., Nanjing, China). The net percentage of LDH release was calculated asollows: net percentage of LDH release = 100% × (stimulated release − spontaneouselease)/(total release − spontaneous release).

.5. Phospholipase D-catalyzed reaction

After treatment, the cells were trypsinized and rinsed with PBS (phosphateuffered saline, pH 7.4) 3 times at 4 ◦C. The cells were resuspend in lysis solutionKangCheng Biotechnology Co. Ltd., Shanghai, China), lysed by ultrasound in an iceath, and the lysate was centrifuged to eliminate nuclei and unbroken cells. All

ysates were tested for protein quantity. A PLD-catalyzed reaction was carried outn a 37 ◦C water bath for 90 min. The 360 �l reaction system (Vinggaard et al., 1996)

as composed of 25 mM HEPES (pH 7.4), 2 mM phosphatidylcholine, 6 mM oleiccid, 1.6 M (NH4)2SO4, 1 mM CaCl2 and 5 mM MgCl2. A549 cell lysate containing00–300 �g protein (in 95 �l) was added at the onset of the reaction. To terminatehe reaction, the tubes were placed in boiling water for 10 min. When cooled to room

rs 196 (2010) 168–174 169

temperature, each sample was mixed with 360 �l chloroform and vortexed for 1 minbefore centrifugation (4000 × g, 10 min). After centrifugation, the supernatant wasused for PLD activity assay.

2.6. PLD activity assay

As described previously (Lucas et al., 1995; Pedruzzi et al., 1998; Wu et al., 2001),the PLD activity assay was based on a reaction system containing 200 �l supernatantand 800 �l color reagent (Tris–HCl 45 mM, pH 8.0, peroxidase 5 units, choline oxi-dase 1 unit, 4-aminoantipyrine 0.3 mg, phenol 0.2 mg). The tube containing thisreaction was incubated in a 37 ◦C water bath for 90 min. The reaction was termi-nated by adding 0.5 ml ice-cold 50 mM Tris–HCl (pH 8.0). A standard curve wasconstructed each time with a fresh choline standard (10, 20, 40, 80, and 160 nmol).The PLD activity was quantified by calculation of produced choline using a standardcurve. One unit of PLD activity was defined as 1 nmol choline produced by 1 mg celllysate protein during 1 min at 37 ◦C.

2.7. Measurement of intracellular H2O2

The amount of intracellular H2O2 was assessed using the fluorescent probe2,7-dichlorofluorescein diacetate (DCF-DA). After diffusion into cells, the acetategroup of DCF-DA is cleaved by esterases, trapping it inside; subsequent oxidationyields a fluorescent adduct. A549 cells cultured on 96-well plates were rinsed withEarle’s solution, and incubated with 25 �M DCF-DA in Earle’s solution for 30 min at37 ◦C. Then the cells were exposed to H2O2 at indicated concentrations and dura-tions. At the end of H2O2 exposure, the cells were rinsed twice to remove excessprobe, and fluorescence was measured with a multi-well plate fluorescence reader(FLUOStar, BMG LABTECH GmbH, Offenburg, Germany) at excitation 485 nm andemission 520 nm. Results are expressed as percentage of control.

2.8. Statistical analysis

All data are expressed as mean ± SD. For comparison, unpaired t-test, one-wayANOVA and Dunnett’s test or Dunn’s test were performed using GraphPad Prism4.00 for Windows (GraphPad Software, San Diego, CA, USA). A P value of less than0.05 was considered statistically significant.

3. Results

3.1. H2O2-induced A549 cell viability changes and cell injury

We determined the effects of exposure to various concentra-tions of H2O2 at different durations on A549 cell viability by assayof MTT reduction, and on cell injury by assay of LDH release. Theresults showed that exposure to H2O2 induced a decline of cellviability and increment of LDH release in a concentration- and time-dependent manner (Fig. 1). After exposure to 500 �M H2O2, the cellviability gradually decreased by 8.6%, 9.2%, 9.7%, 26% and 40% ofcontrol at 5, 30, 60, 180 and 360 min, respectively (Fig. 1A). Whenthe H2O2 exposure was longer than 3 h, the LDH release increaseddramatically (Fig. 1B).

3.2. H2O2-induced changes of PLD activity in A549 cells

PLD activation was induced by H2O2 in a concentration-dependent and a time-dependent manner (Fig. 2). The basal activity(control level) of PLD in A549 cells was 0.16 units. When cellswere exposed to 0–1000 �M H2O2 for 30 min, the highest PLDactivity appeared when H2O2 concentration was 500 �M (Fig. 2A).Thus, in another series of experiments, cells were treated with500 �M H2O2 for 5–360 min (Fig. 2B). We found the highest PLDactivity appeared at an exposure of 5 min, when the value was0.41 ± 0.06 units (2.56-fold control; **P < 0.01, n = 5), and then the

but was lower than the peak value. The PLD activity decreased tocontrol at 6 h.

On the basis of these results, we assessed the responses of A549cells to H2O2 exposure at 500 �M for 5 min and 180 min in thesubsequent experiments.

170 M. Wu et al. / Toxicology Letters 196 (2010) 168–174

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ig. 1. Effects of H2O2 on viability and lactate dehydrogenase (LDH) release in A54DH assay kit. Data are mean ± SD; n = 8–12; *P < 0.05, **P < 0.01, compared with con

.3. Effects of enzyme inhibitors on H2O2-induced viabilityhanges and injury in A549 cells

To reveal the involvement of the PLD pathway, 1-butanol (aLD inhibitor, 0.5%), propranolol (a PAP inhibitor, 100 �M) and

uinacrine (a PLA2 inhibitor, 1 �M) were used (Fig. 3). None of thesehemicals administered alone had any effects on A549 cell viabilityt the durations and concentrations used (Fig. 3C).

After exposure to 500 �M H2O2 for 5 min, A549 cells showedecreased cell viability, which was reversed by 1-butanol (0.5%),

ig. 2. H2O2-induced phospholipase D (PLD) activation in a concentration- andime-dependent manner in A549 cells. (A) PLD activity in cells with or without H2O2

0–1000 �M) for 30 min. (B) PLD activity in cells with or without H2O2 (500 �M)or 5–360 min. Data are expressed as mean ± SD; n = 5; *P < 0.05, **P < 0.01, com-ared with control; #P < 0.05, ##P < 0.01, compared with H2O2 at 500 �M; $P < 0.05,

$P < 0.01, compared with exposure time 5 min; +P < 0.05, ++P < 0.01, compared withxposure time 180 min, one-way ANOVA.

s. (A) Cell viability measured using MTT assay; (B) LDH release measured using anne-way ANOVA.

propranolol (100 �M), and quinacrine (1 �M) (Fig. 3A). Whenthe cells were exposed to 500 �M H2O2 for 3 h, the cell viabil-ity decreased further, which was partly reversed by propranolol(100 �M) and quinacrine (1 �M); however, 0.5% 1-butanol aggra-vated the H2O2-induced decline in viability at this exposure time(Fig. 3C).

After exposure to 500 �M H2O2 for 5 min, A549 cells showedincreased LDH release, which was significantly attenuated by 1-butanol (0.5%), propranolol (100 �M), quinacrine (1 �M) (Fig. 3B).When the cells were treated with 500 �M H2O2 for 3 h, LDH releaseincreased further. The results suggested a tendency for the incre-ment of LDH release induced by 3 h of 500 �M H2O2 exposure to bereversed by 100 �M propranolol and 1 �M quinacrine, but aggra-vated by 1-butanol (0.5%), no statistical significance was shown(Fig. 3D).

Thus, the results suggested that PLD activation induced by 5 minof H2O2 exposure mediates a cell injury signal, while PLD activationinduced by 3 h of H2O2 exposure mediates a cell survival signal inA549 cells. And these results suggested that A549 cells maintainedbetter survival when the concentration of intracellular PA increasedas a result of inhibition of PA hydrolysis by PAP or PLA2.

3.4. Exogenous phosphatidic acid mimic the effects ofH2O2-induced A549 cells viability changes

To confirm that PA plays a key role in H2O2 exposure-inducedA549 cell injury and survival, exogenous PA was used to mimic PLDactivation. The results showed that treatment with 0.1 �M PA alonefor 30 min induced cell viability decline (91% ± 7.5% vs. control100% ± 5.2%, *P < 0.05, n = 7), while treatment with 0.1 �M PA alonefor 210 min induced a marked viability increment in A549 cells(125% ± 6.8% vs. control 100% ± 12%, ***P < 0.001, n = 7) (Fig. 4). Theresults were coincident with the effects of PLD activation inducedby 500 �M H2O2 exposure for 5 min and 3 h.

3.5. Increase of intracellular ROS production in A549 cells byexogenous H2O2

As the increase of PLD activity appeared to be bi-phasic after

H2O2 exposure in A549 cells, we carried out experiments todetermine whether changes of intracellular or extracellular ROSconcentration caused the phenomena.

First, we measured intracellular ROS production by detect-ing DCF-DA fluorescence in A549 cells after H2O2 exposure. The

M. Wu et al. / Toxicology Letters 196 (2010) 168–174 171

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DPI at 1 nM (Fig. 6B). After exposure to 500 �M H2O2 for 3 h, cells

ig. 3. Effects of enzyme inhibitors on H2O2-induced viability changes and lactatncubated with agents [1-butanol (0.5%), propranolol (100 �M), quinacrine (1 �M)]

TT assay; LDH release was measured using an LDH assay kit. Data are expressed as#P < 0.01, compared with H2O2 treatment alone, one-way ANOVA.

esults showed that the intracellular ROS production increasedfter 180 min of H2O2 exposure and the increased level decreasedfter 360 min of exposure (Fig. 5).

Then, to determine whether agents that had effects on theADPH oxidase pathway caused changes in intracellular H2O2 con-entration, the cells were treated with catalase (500 units/ml) or

PI (1 nM). As shown in Fig. 6A, the increment of intracellular H2O2

nduced by H2O2 exposure for 5 min was reversed by catalase toontrol level, but not by DPI. Moreover, treatment with catalase500 units/ml) or DPI (1 nM) significantly reversed the increment

ig. 4. Effects of exogenous phosphatidic acid on viability in A549 cells. A549 cellsrown on 96-well plate were incubated with phosphatidic acid (PA, 0.1 �M) for thendicated times. Cell viability was measured using MTT assay. Data are expressed as

ean ± SD; n = 7; *P < 0.05, ***P < 0.001, compared with control; unpaired t-test.

ydrogenase (LDH) release in A549 cells. A549 cells grown on 96-well plate wereor without 500 �M H2O2 for the indicated times. Cell viability was measured using± SD; n = 6–8; *P < 0.05, **P < 0.01, compared with control (without H2O2); #P < 0.05,

of intracellular H2O2 induced by exogenous H2O2 exposure for 3 h(Fig. 6C). These results were coincident with the protective effectsof catalase (500 units/ml) and DPI (1 nM) on A549 cells exposed toH2O2. A 5-min exposure to 500 �M H2O2 resulted in decreased cellviability, which was reversed by catalase at 500 units/ml, but not by

showed a further decrease in cell viability, which was reversed sig-nificantly by catalase (500 units/ml) and DPI (1 nM) to the controllevel (Fig. 6D).

Fig. 5. Increase of intracellular H2O2 level in A549 cells induced by exogenous500 �M H2O2. Cells were exposed to H2O2 for 5–360 min and intracellular H2O2

levels measured with DCF-DA fluorescent probe. Data are expressed as mean ± SD;n = 6–8; **P < 0.01 vs. control, one-way ANOVA.

172 M. Wu et al. / Toxicology Letters 196 (2010) 168–174

Fig. 6. Effects of catalase and diphenyliodonium on the increment of intracellular H2O2 (A and C) and on the changes of viability (B and D) induced by H2O2 exposure inA549 cells. Cells were incubated with agents [catalase (500 units/ml) or diphenyliodonium (DPI, 1 nM)] with or without H2O2 (500 �M) for 5 min (A and B) or 3 h (C and D).Intracellular H2O2 was assayed with the DCF-DA probe; cell viability was measured using MTT assay. Data are expressed as mean ± SD; n = 6–8; *P < 0.05, **P < 0.01 vs. control,##P < 0.01 vs. H2O2 treatment alone, one-way ANOVA.

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ig. 7. Effects of 1-butanol, catalase, propranolol and quinacrine on PLD activatioean ± SD; n = 5; *P < 0.05, **P < 0.01, compared with control; ##P < 0.01, compared

Finally, to determine whether the increase of PLD activity wasaused by H2O2, the cells were incubated with catalase. As shownn Fig. 7, treatment with catalase (500 units/ml) attenuated the up-egulation of PLD activity but not to control levels at 5 min exposureo H2O2, and treatment with catalase (500 units/ml) completelybolished PLD activation at an H2O2 exposure for 180 min.

.6. Effects of the enzyme inhibitors on H2O2-induced PLDctivation in A549 cells

After H2O2 exposure (500 �M) for 5 min or 3 h, treatment with-butanol (0.5%) completely inhibited PLD activation. PLD activa-

549 cells exposed to 500 �M H2O2 for 5 min (A) or 3 h (B). Data are expressed as2O2 treatment alone, one-way ANOVA.

tion was inhibited weakly and insignificantly by both propranolol(100 �M) and quinacrine (1 �M) (Fig. 7).

4. Discussion

The most important finding in the present study is the definitionof distinct roles for PLD at different durations of exposure to 500 �M

H2O2 in A549 cells. The results demonstrated that, H2O2 exposurecaused a bi-phasic increment of PLD activity and PLD activationinduced by 5 min of H2O2 exposure mediates a cell injury signal,while PLD activation induced by 3 h of H2O2 exposure mediates acell survival signal in A549 cells.

M. Wu et al. / Toxicology Letters 196 (2010) 168–174 173

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Fig. 8. Schematic of the effects of PLD activation induced by 500 �M H2O

H2O2 is a small, diffusible molecule and functions as a sec-nd messenger which contributes to either cell death or survivalAntunes and Cadenas, 2000; Bienert et al., 2006). Lines of evi-ences have shown that H2O2 is important for many signalingascades (Giorgio et al., 2007; Trachootham et al., 2008; Valkot al., 2007). One of these cascades involves H2O2 promotion ofLD activation (Min et al., 2001; Natarajan et al., 1993; Oh et al.,000; Xiao et al., 2005). When PLD is activated, PLD hydrolyzeshosphatidylcholine to produce PA, which stimulates the NADPHxidase complex to generate H2O2 (Stahelin et al., 2003). Thus,he relationship between H2O2, PLD, and NADPH oxidase consti-utes a positive feedback loop (Xiao et al., 2005). PLD activationnduced NADPH oxidase activation results in increased intracellularnd extracellular oxidants in non-phagocytic cells too (Thannickalnd Fanburg, 2000), which may enhance H2O2-induced cell dam-ge. Thus, we developed a schematic model to describe our findingsnd the potential mechanism underlying in this situation (Fig. 8).

Consistent with our observations, it was reported that PLD2ctivation is induced in PC12 cells exposed to 500 �M H2O2 formin, and this activation is involved in H2O2-mediated PC12 cellpoptosis (Kim et al., 2003). A study on PC12 cells (exposed to.2 mM H2O2 for 6 h) showed that PLD2 activity is specifically up-egulated by H2O2 and plays a suppressive role in H2O2-inducedpoptosis (Lee et al., 2000). PLD2 may be a crucial component forell survival against apoptosis induced by various stimuli. Oh etl. (2007) reported that PLD2 is involved in an anti-apoptotic sig-aling pathway via PA generation. Furthermore, overexpression ofLD isozymes results in inhibition of taxotere-induced apoptoticell death which is due to increased Bcl-2 expression by PA (Cho etl., 2008).

Our results also supported that PA was important for cell sur-ival. We found that the PAP inhibitor propranolol and the PLA2nhibitor quinacrine reversed the decline in A549 cell viabilitynduced by H2O2 exposure for 5 min or 3 h, while propranolol anduinacrine did not inhibit the activation of PLD under the same

onditions. These results suggested that A549 cells maintained bet-er survival when the concentration of intracellular PA increaseds a result of inhibition of PA hydrolysis by PAP or PLA2; in otherords, the results suggested that DAG (downstream factor of PAP)

nd lyso-PA (downstream factor of PLA2) might be unimportant

sure for 5 min or 3 h in A549 cells. : major effects, : minor effects.

for viability when A549 cells were treated with H2O2. Besides, theimportance of PA in cell survival was re-confirmed by the othertwo results in the study: (1) cell viability declined as intracellularPA accumulation decreased with the inhibition of PLD by 1-butanol,particularly when the cells were treated with H2O2 for 3 h; (2) treat-ment with exogenous PA over 3 h increased the viability of A549cells.

Several reports indicate that PA acts as a second messenger, andmany relevant targets, including phosphatidylinositol 4-phosphate5′-kinase, Bcl-2, and mammalian target of rapamycin (mTOR), aretargets for survival signals (Cho et al., 2008; Foster, 2009; Oh et al.,2007). Taking these findings and our results together, we suggestthat up-regulation of PLD activity induced by 3 h of exposure toH2O2 (500 �M) results in PA accumulation in A549 cells, and thisaccumulation triggers downstream signals which benefit cell sur-vival predominantly (Fig. 8). Meanwhile, our results showed thatthe NADPH oxidase inhibitor DPI treatment reversed the decline ofviability induced by H2O2 (500 �M) exposure for 3 h, it suggestedthat NADPH oxidase was activated and in part involved in injury toA549 cells exposed to H2O2 (500 �M) for 3 h (Fig. 8).

In A549 cells, PLD activation and cell injury induced by 5-min exposure to 500 �M H2O2 might also be due to the effectof H2O2 through an underlying mechanism partly involving PAactivating NADPH oxidase. Though 1 nM DPI did not reverse thedecline of A549 cell viability and the increment of intracellularH2O2 level induced by H2O2 (500 �M) exposure for 5 min, we foundthat treatment with exogenous PA for 30 min mimicked the cellinjury effects of PLD activation. It has been well confirmed thatPA activated superoxide-generating enzyme NADPH oxidase dur-ing respiratory burst in neutrophils (Palicz et al., 2001; Regier etal., 2000). Together with our findings, it was indicated that, whenA549 cells were exposed to H2O2 (500 �M) for 5 min, activationof NADPH oxidase-induced intracellular H2O2 increase enhancedextracellular H2O2-induced cell damage (Fig. 8).

Regarding the involvement of cellular antioxidant system, in the

situation of 500 �M H2O2 exposure for 5 min, the antioxidant sys-tem was mobilized for a short time, and thus the concentration ofH2O2 was still high enough to damage the cells; however, after 3 hof exposure, the antioxidant system was mobilized so adequatelythat the concentration of H2O2 was low enough to fall into the range

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74 M. Wu et al. / Toxicolog

f the physiological concentration where H2O2 triggered cellularurvival signals (Giorgio et al., 2007), i.e., ROS-mediated actions thatrotect cells against ROS-induced oxidative stress and re-establishredox homeostasis” (Trachootham et al., 2008). Under both condi-ions, PLD was activated by H2O2, as this study indicated, the firsthase of PLD activity increase was induced by the sudden incre-ent of extracellular H2O2 level (500 �M) and the second phase of

LD activity increase was due to the elevation of intracellular H2O2evel, while different downstream pathways of PLD/PA resulted inell injury or survival (Fig. 8).

Taking these considerations together (Fig. 8), we speculate thatLD activation at the initiation of H2O2 exposure predominantlyesults in NAPDH oxidase activation, which mediates A549 cellnjury. Then, PLD activation turns to mediating cell survival as the

2O2 attack continues, and might be mainly due to the accumula-ion of intracellular PA.

onflict of interest statement

None declared.

cknowledgements

We thank Dr. Iain C. Bruce (Department of Physiology, Schoolf Medicine, Zhejiang University) for critically reading and revis-ng the manuscript. This study was supported by grants from theational Natural Science Foundation of China (30500228), the Zhe-

iang Provincial Natural Science Foundation (Y2080105), and theey Research Program of Collaboration between the Ministry ofealth of China and the Department of Health of Zhejiang Province

wkj2009-2-026).

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