Effects of antioxidants and MAPK inhibitors on cell death and reactive oxygen species levels in H2O2-treated human pulmonary fibroblasts

H2O2-induced cytotoxicity in normal human pulmonary fibroblasts (HPFs) is of interest in toxicological research since HPFs are involved in lung inflammation, fibrosis and cancer. The present study investigated the cytotoxic effects of H2O2 on normal HPFs in relation to reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) using the well-known antioxidants N-acetyl cysteine (NAC) and propyl gallate (PG), as well as MAPK inhibitors. Treatment with 50 μM H2O2 inhibited the growth of the HPFs by ∼45% in 24 h. H2O2 induced cell death via apoptosis and triggered the loss of mitochondrial membrane potential (MMP; Δψm) in the HPFs. H2O2 also increased the ROS levels, including O2•−, in the HPFs and induced glutathione (GSH) depletion. NAC and PG attenuated the death of the HPFs and the loss of MMP (Δψm) through the use of H2O2. NAC decreased the ROS levels in the H2O2-treated HPFs and PG markedly prevented an increase in O2•− levels in these cells. However, PG alone induced cell death in the HPF control cells and increased the ROS levels in these cells. None of the MAPK (MEK, JNK and p38) inhibitors affected cell growth inhibition or cell death by H2O2. In addition, these inhibitors did not significantly affect the ROS levels and GSH depletion in the H2O2-treated HPFs. In conclusion, H2O2 induced growth inhibition and cell death in the HPFs via GSH depletion. NAC and PG attenuated H2O2-induced HPF cell death but each regulated the ROS levels in a different manner. Treatment with MAPK inhibitors did not affect cell death or the ROS levels in the H2O2-treated HPFs.


Introduction
Reactive oxygen species (ROS) are mainly comprised of hydrogen peroxide (H 2 O 2 ), superoxide anions (O 2 •-) and hydroxyl radicals ( • OH), which affect numerous cellular processes, including metabolism, differentiation and cell proliferation and death, by regulating critical signaling pathways (1,2). Unlike other ROS, H 2 O 2 is capable of freely diffusing through biological membranes the width of several cells prior to reacting with specific molecular targets. ROS are mostly generated during the process of mitochondrial respiration and by specific oxidases, including nicotine adenine diphosphate (NADPH) oxidase and xanthine oxidase (3). The main metabolic pathways use superoxide dismutases, which metabolize O 2 •to H 2 O 2 (4). Further metabolism by catalase or glutathione (GSH) peroxidase yields O 2 and H 2 O (5). Oxidative stress occurs through an increase in ROS levels and/or a decrease in cellular antioxidants, and leads to cell death (6)(7)(8). Exogenous H 2 O 2 is frequently used as a representative ROS in modeling and inducing oxidative stress.
Three major groups of mitogen-activated protein kinases (MAPKs) exist: extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 (9). MAPKs are involved in crucial signaling pathways in cell proliferation, differentiation and cell death in response to various signals produced by growth factors, hormones and cytokines, as well as genotoxic and oxidative stressors (9,10). Each MAPK pathway has comparatively varied upstream activators and unambiguous substrates (11). Abundant evidence has demonstrated that JNK and p38 are activated by ROS or mild oxidative shifts in the intracellular thiol/disulfide redox state, initiating processes associated with apoptosis (12,13). ROS provoke ERK phosphorylation and also stimulate the ERK pathway (14). In the majority of instances, ERK activation has a pro-survival effect rather than a pro-apoptotic effect (15). In addition, MAPK pathways are also activated by the direct inhibition of MAPK phosphatases by the ROS. Since the differing and opposing effects on MAPKs are caused by various ROS in the cells, the correlation between ROS and MAPKs requires further clarification, particularly with regard to the signaling associated with cell survival and death.
Cultured normal human cells are invaluable biological models for mechanistic studies of oxidative stress. H 2 O 2 -induced cytotoxicity in normal fibroblast cells in vitro may be of interest in toxicological research with regard to the toxic potential of exogenous H 2 O 2 in human pulmonary fibroblasts (HPFs) since HPFs are closely involved in lung inflammation, fibrosis and cancer. However, the toxicological mechanism of the effects of exogenous H 2 O 2 on normal HPFs remains unknown with regard to MAPKs. The present study investigated the effects of the well-known antioxidants N-acetyl cysteine (NAC) and propyl gallate (PG), as well as the MAPK inhibitors, on H 2 O 2 -treated HPFs in relation to cell growth and death and the ROS and GSH levels.

Materials and methods
Cell culture. HPFs purchased from PromoCell GmbH (Heidelberg, Germany) were maintained in a humidified incubator at 37˚C with 5% CO 2 . The HPFs were cultured in RPMI-1640 supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (GIBCO BRL, Grand Island, NY, USA). The HPFs were grown in 100-mm plastic tissue culture dishes (Nunc, Roskilde, Denmark) and harvested with trypsin-EDTA solution while in the logarithmic growth phase. The HPFs between passages four and eight were used. The study was approved by the Ethics Committee of Chonbuk National University, Jeonju, Republic of Korea.
Reagents. H 2 O 2 , NAC and PG were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). The NAC was dissolved in buffer [20 mM HEPES (pH 7.0)], while the PG was dissolved in ethanol at 200 mM as a stock solution. JNK inhibitors (SP600125), MEK inhibitors (PD98059) and p38 inhibitors (SB203580) were purchased from Calbiochem (San Diego, CA, USA). All the inhibitors were dissolved in DMSO at 10 mM as stock solutions. The HPFs were pretreated with 2 mM NAC, 400 µM PG or 10 µM MAPK inhibitors for 1 h prior to treatment with H 2 O 2 . Ethanol (0.2%) and DMSO (0.2%) were used as control vehicles and did not affect cell growth or death.
Cell growth and cell number assays. The changes in cell growth in the HPFs were indirectly determined by measuring the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich Chemical Company) dye absorbance. In brief, 4x10 4 cells per well were seeded in 96-well microtiter plates (Nunc). Following exposure to 50 µM H 2 O 2 , with or without 2 mM NAC, 400 µM PG or 10 µM MAPK inhibitors for 24 h, 20 µl MTT solution (2 mg/ml in PBS) was added to each well of the 96-well plates. The plates were incubated for an additional 4 h at 37˚C. The media in the plates were withdrawn by pipetting and 200 µl DMSO was added to each well to solubilize the formazan crystals. The optical density was measured at 570 nm using a microplate reader (Synergy™ 2; BioTek Instruments Inc., Winooski, VT, USA).
Annexin V-fluorescein isothiocyanate (FITC) staining for cell death detection. Apoptosis was determined by staining cells with annexin (Invitrogen Corporation, Camarillo, CA, USA; Ex/Em = 488 nm/519 nm). In brief, 1x10 6 cells were incubated in a 60-mm culture dish (Nunc) with 50 µM H 2 O 2 , with or without 2 mM NAC, 400 µM PG or 10 µM MAPK inhibitors for 24 h. The cells were washed twice with cold PBS, then resuspended in 500 µl binding buffer (10 mM HEPES/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM CaCl 2 ) at a concentration of 1x10 6 cells/ml. Annexin V-FITC (5 µl) was then added to the cells, which were analyzed with a FACStar flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).  •levels were expressed as the mean fluorescence intensity (MFI), which was calculated by CellQuest software (Becton Dickinson).

Measurement of mitochondrial membrane potential (MMP
Detection of the intracellular GSH. The cellular GSH levels were analyzed using a 5-chloromethylfluorescein diacetate dye (CMFDA, Ex/Em = 522 nm/595 nm; Invitrogen Molecular Probes). In brief, 1x10 6 cells were incubated in a 60-mm culture dish (Nunc) with 50 µM H 2 O 2 , with or without 2 mM NAC, 400 µM PG or 10 µM MAPK inhibitors for 24 h. The cells were then incubated with 5 µM CMFDA at 37˚C for 30 min. The CMF fluorescence intensity was determined using a FACStar flow cytometer (Becton Dickinson). CMF-negative (GSH-depleted) cells were expressed as the percent of CMFcells.
Statistical analysis. The results represent the mean of at least two independent experiments (mean ± SD). The data were analyzed using Instat software (GraphPad Prism4; GraphPad Software, San Diego, CA, USA). The Student's t-test and a one-way analysis of variance (ANOVA) with post hoc analysis, using Tukey's multiple comparison, were applied to the parametric data. P<0.05 was considered to indicate a statistically significant difference.

Effects of NAC and PG on cell growth and death and MMP (∆ψ m ) levels in H 2 O 2 -treated
HPFs. The effects of NAC and PG on cell growth and death and MMP (∆ψ m ) levels were investigated in H 2 O 2 -treated HPFs at 24 h using MTT assays. A concentration of 50 µM H 2 O 2 was used as an optimal dose in this experiment; this inhibited the growth of the HPFs by ~45% in 24 h (Fig. 1A). NAC and PG significantly reduced the growth inhibition caused by H 2 O 2 , with PG showing a more marked effect (Fig. 1A). H 2 O 2 increased the percentage of annexin V-FITC stained cells among the HPFs, indirectly indicating that the HPF cell death caused by H 2 O 2 occurred via apoptosis (Fig. 1B). NAC and PG significantly reduced the number of annexin V-FITC-positive cells in the H 2 O 2 -treated HPFs, while PG completely prevented the HPF cell death caused by H 2 O 2 (Fig. 1B). Notably, PG alone increased the number of annexin V-FITC-positive cells among the control HPFs (Fig. 1B). Since cell death is closely associated with the collapse of MMP (∆ψ m ) (16), the effect of H 2 O 2 on MMP (∆ψ m ) in the HPFs was assessed using a rhodamine 123 dye. Treatment with 50 µM H 2 O 2 significantly induced the loss of MMP (∆ψ m ) in the HPFs (Fig. 1C). NAC and PG attenuated the loss of MMP (∆ψ m ) caused by H 2 O 2 , while PG totally prevented this loss (Fig. 1C). Similar to the number of annexin V-FITC-positive cells, PG also increased the number of cells that lost MMP (∆ψ m ) among the control HPFs (Fig. 1C).  (Fig. 2A). Moreover, PG alone markedly increased the ROS (DCF) levels in the control HPFs ( Fig. 2A). When the intracellular O 2

Effects of NAC and PG on intracellular ROS and GSH levels
•levels were assessed in the H 2 O 2 -treated HPFs, the level of DHE fluorescence dye, which  •level in the H 2 O 2 -treated HPFs, PG entirely attenuated the increase in these cells (Fig. 2B). However, PG alone increased the O 2 •level in the control HPFs (Fig. 2B). When the intracellular GSH levels were measured in the H 2 O 2 -treated HPFs using a CMFDA dye, 50 µM H 2 O 2 was shown to increase the number of GSH-depleted cells in the HPFs at 24 h (Fig. 2C). NAC and PG significantly reduced the number of GSH-depleted cells in the H 2 O 2 -treated HPFs, while PG completely prevented the GSH depletion (Fig. 2C).

Effects of MAPK inhibitors on cell growth and death and MMP (∆ψ m ) levels in H 2 O 2 -treated HPFs.
The effect of the MAPK inhibitors on cell growth and death and MMP (∆ψ m ) levels in the H 2 O 2 -treated HPFs was evaluated. Based on previous studies (17,18), 10 µM of each MAPK inhibitor was used as an optimal dose in the present study. None of the MAPK inhibitors affected the growth inhibition caused by H 2 O 2 (Fig. 3A). p38 inhibitor alone increased the growth of control HPFs (Fig. 3A). Additionally, none of the MAPK inhibitors affected the number of annexin V-stained cells among the H 2 O 2 -treated or -untreated HPFs (Fig. 3B). All the MAPK inhibitors appeared to enhance the loss of MMP (∆ψ m ) in the H 2 O 2 -treated HPFs (Fig. 3C).

Effects of MAPK inhibitors on intracellular ROS and GSH levels in H 2 O 2 -treated HPFs.
The changes in intracellular ROS levels in the H 2 O 2 and/or each MAPK inhibitor-treated HPF were assessed. As shown in Fig. 4A, the MEK and JNK inhibitors only marginally enhanced the ROS levels in the H 2 O 2 -treated HPFs, whereas the p38 inhibitor appeared to decrease the ROS levels (Fig. 4A). In addition, all the MAPK inhibitors marginally intensified the O 2 •level increase caused by H 2 O 2 (Fig. 4B). The p38 inhibitor alone increased the ROS levels, including the O 2 •level, in the HPF control cells (Fig. 4A and B). Moreover, none of the MAPK inhibitors affected the number of GSH-depleted cells in the H 2 O 2 -treated or untreated HPFs (Fig. 4C).

Discussion
HPFs are pathophysiologically involved in lung inflammation, fibrosis and cancer since these cells synthesize extracellular matrix and collagen to maintain the structural and functional integrity of the lung. The present study focused on elucidating   PG, as a synthetic antioxidant, exerts a variety of effects on tissues and cells. For example, PG is an efficient protector of liver cells against lipid peroxidation by oxygen radicals (20). By contrast, PG has pro-oxidant properties (21,22). The antioxidative and cytoprotective properties of PG may change to pro-oxidative, cytotoxic and genotoxic properties in the presence of Cu(II) (23). The present results demonstrated that PG alone marginally inhibited the growth of the HPFs and induced cell death accompanied by the loss of MMP (∆ψ m ). In addition, PG increased the ROS levels, including those of O 2 •-, in the HPFs. Thus, it is possible that PG, as a pro-oxidant, is able to directly generate mitochondrial O 2 •in the HPFs by impairing mitochondrial function, consequently leading to HPF cell death via oxidative stress. Similarly, it has been reported that PG causes cytotoxic effects in isolated rat hepatocytes by causing mitochondrial damage (24) and that it also increases mitochondrial O 2 •levels in HeLa cells (25). Notably, PG markedly attenuated the growth inhibition and cell death in the H 2 O 2 -treated HPFs and also prevented MMP (∆ψ m ) loss in these cells. Moreover, PG completely abrogated the O 2 •-(DHE) level increase caused by H 2 O 2 . Therefore, PG appeared to protect the HPFs against exogenous H 2 O 2 by protecting the mitochondria. However, PG increased the ROS (DCF) levels in the H 2 O 2 -treated HPFs. Numerous studies, including the present study, support the hypothesis that PG has a role as an antioxidant (20,26,27) or as a pro-oxidant (21,22), depending on various conditions, such as the cell culture media, the co-treated drugs and the cell types. PG is likely to have differing effects on the levels of the different ROS in the cells. Further studies are required to elucidate the exact roles of the types of ROS in PG-treated HPFs.
In the present study, the MEK inhibitor, which is likely to inactivate ERK, did not affect growth inhibition or cell death in the H 2 O 2 -treated HPFs. Thus, H 2 O 2 did not directly regulate the signaling associated with ERK in the HPFs to induce their growth inhibition and death. In addition, the JNK and p38 MAPKs, which are generally associated with cell death (12,13), were not likely to be affected by H 2 O 2 in the HPFs since none of the inhibitors affected the growth inhibition and cell death caused by H 2 O 2 . The p38 inhibitor alone increased the growth of the control HPFs, suggesting that p38 signaling is involved in the basal level of HPF growth. With regard to MMP (∆ψ m ), all the MAPK inhibitors marginally increased the loss of MMP (∆ψ m ) in the H 2 O 2 -treated HPFs, indicating that the dysregulation of these MAPK signalings enhanced the loss in these cells. Moreover, the MAPK inhibitors marginally, but not significantly, affected the ROS levels, including that of O 2 GSH is a key cellular non-protein antioxidant, which reduces H 2 O 2 to H 2 O using GSH peroxidase (28). The intracellular GSH content has a significant effect on anticancer drug-induced apoptosis, indicating that apoptotic effects are inversely proportional to the GSH content (29,30). Similarly, in the present study, H 2 O 2 increased the number of GSH-depleted cells in the HPFs. NAC and PG demonstrated anti-apoptotic effects on the H 2 O 2 -treated HPFs, significantly suppressing the GSH depletion in these cells. In addition, none of the MAPK inhibitors affected the GSH depletion in the H 2 O 2 -treated HPFs. Therefore, the intracellular GSH content appears to be a decisive factor in HPF cell death. However, PG alone induced cell death in the HPF control cells but it did not significantly induce GSH depletion,, suggesting that PG-induced HPF cell death is not highly associated with changes in the GSH level.
In conclusion, H 2 O 2 induced growth inhibition and death in the HPFs via GSH depletion. NAC and PG attenuated H 2 O 2 -induced HPF cell growth inhibition and death, but each antioxidant affected the ROS levels, including that of O 2 •-, differently in the H 2 O 2 -treated and -untreated HPFs. Treatment with MAPK inhibitors did not affect cell death or the ROS levels in the H 2 O 2 -treated HPFs. The present data provide useful information concerning the toxicological effect of exogenous H 2 O 2 on normal HPFs with regard to ROS and MAPKs.