Paraquat Induces Lung Alveolar Epithelial Cell Apoptosis via Nrf-2-Regulated Mitochondrial Dysfunction and ER stress
Ya-Wen Chen, Yuan-Ting Yang, Dong-Zong Hung, Chin-Chuan Su, Kuo-Liang Chen
Y. W. Chen:
Department of Physiology, and Graduate Institute of Basic Medical Science, College of Medicine,China Medical University, No.91 Hsueh-Shih Road, Taichung, 40402
Taiwan. Y. T. Yang:
Graduate Institute of Drug Safety, College of Pharmacy, China Medical University, No.91 Hsueh-Shih Road, Taichung, 40402 Taiwan.
D. Z. Hung:
Division of Toxicology, Trauma and Emergency Center, China Medical University Hospital, Taichung 40402, Taiwan.
C. C. Su:
Department of Otorhinolaryngology, Head and Neck Surgery, Changhua Christian Hospital, Changhua, 500 Taiwan
K. L. Chen:
Department of Urology, China Medical University Hospital, Taichung, 40402 Taiwan.
To e-mail address, telephone, and fax numbers of corresponding author and first author:
E-mail: [email protected] (Y. W. Chen) Tel.: +886 4 22052121 ext. 7728 (Y. W. Chen) Fax: + 886 4 22333641(Y. W. Chen)
Abstract
Paraquat (1,1’-dimethyl-4,4’-bipyridinium chloride; PQ) is widely and commonly used as a herbicides in the world. PQ has been reported to be a major hazard because it causes lung injury. However, the molecular mechanisms underlying PQ-induced lung toxicity still need to be elucidated. Here, we found that PQ significantly decreases cell viability, increases sub-G1 hypodiploids DNA contents and caspase 3/7 activity in lung alveolar epithelial cell-derived L2 cells, which also caused mitochondrial dysfunction and decreased the mRNA expression of Bcl-2 and increased that of Bax, Bak, and p53. Moreover, the protein expressions of Bax and Bak were increased in PQ-treated cells. In addition, when PQ was exposed to L2 cells, the expressions of ER stress-related signaling genes (including Grp78, CHOP and caspase-12 mRNA) and proteins (including phospho-eIF-2, CHOP, Grp78, calpain-I and -II, and caspase-12) were significantly increased. PQ also decreased the protein expressions of procaspase-9/7/3. Next, we investigated the role of Nrf-2 in PQ-induced alveolar epithelial cell toxicity. In L2 cells, paraquat induced Nrf-2 translocation from the cytosol to the nucleus. Cells transfected with Nrf-2 siRNA significantly reversed the PQ-induced toxicity, including depolarization of MMP, increased the Bax, Bak, p53 mRNAs expression, decreased the Bcl-2 mRNA expression, increased the caspase 3/7 activity, increased the Grp78, CHOP and caspase-12 mRNAs expression, increased the expression of proteins Grp78 and CHOP, and decreased that of pro-caspase-3. Taken together, these results suggest that Nrf-2-regulated mitochondria and ER stress-related pathways are
involved in the PQ-induced alveolar epithelial cell injury.
dysfunction, ER stress Introduction
Paraquat (1,1’-dimethyl-4,4’-bipyridinium chloride; PQ) is a bipyridyl compound, which is widely and commonly used herbicides worldwide. In 1962, there were thousand of human deaths due to occupational, accidental or voluntary ingestion of PQ (Liu et al., 2011). In Jamaica, paraquat was the most common herbicide to cause fatal poisoning, which was found in the coroner’s autopsies at West Indies from 1980 to 1999 (Escoffery and Shirley, 2004). A study in 375 cases of paraquat poisoning has shown that 49 have evidence of renal toxicity, 41 receive haemodialysis or charcoal haemoperfusion, 61 develop pulmonary sequelae, and 44 have lesions in the upper gastrointestinal tract (Jones et al., 1999). PQ induced pathological changes, involving fibroblast proliferation and augmentation of collagen synthesis in multiple tissues, such as the liver, kidney, and heart, and in arteries, and the nervous system. The amount of PQ accumulated in the lungs is 6~10 times more than that in the plasma; this pulmonary effect might be due to the participation of the polyamine transport system abundantly expressed in the membrane of type I and II alveolar cells and Clara cells (Dinis-Oliveira et al., 2008). Rose and colleagues have also demonstrated that only lung slices were accumulated with paraquat and its concentration was higher than the medium when incubation of [14C]paraquat to tissue slices (Rose et al., 1974; Rose et al., 1976). Thus, these
results demonstrated that paraquat was preferential accumulation in the lung.
Importantly, this results in irreversible lung damage, which causes death (Bismuth et al., 1990; Mitsopoulos and Suntres, 2010; Moran et al., 2010; Park et al., 2010). Besides, it has been shown that PQ-induced lung injury causes pulmonary edema, infiltration of inflammatory cells, hemorrhage, and blockade in the alveolar
epithelium, leading to the progression of lung fibrosis (Suntres, 2002). PQ shows selective localization and primarily causes injury in the lung epithelial cells (Smith et al., 1990; Hoet and Nemery, 2000; Suntres, 2002; Mitsopoulos and Suntres, 2010). It has been suggested that the toxicity of PQ is based on the extent of its induction of redox cycling, which results in the oxidation of NADPH to NADP+
(Park et al., 2010). Several studies have also shown that NADPH-cytochrome P450 oxidoreductase mediates electron transfer from NADPH to cytochrome P450 enzyme (Enoch and Strittmatter, 1979; Han et al., 2006). NADPH-cytochrome P450 oxidoreductase has been reported to be the major enzyme to catalyze the one-electron reduction in paraquat-initiated redox cycling (Fussell et al., 2011). These effects increase the oxygen utilization, generation of ROS, and induction of oxidative stress in cells (Saito et al., 1985; Fussell et al., 2011). Thus, PQ induced the generation of superoxide anions, nitric oxide (NO), hydroxyl radicals, and oxidant peroxynitrite and led to oxidative stress damage-related cell death and inflammation (Liu et al., 2011). These free radicals can react with DNA, proteins, and lipids in the cells, and lead to protein inactivation, DNA breakage, and lipid peroxidation (Halliwell and Gutteridge, 1992; Berisha et al., 1994). Glutathione (GSH) plays a protective role in the ROS generation in many xenobiotics, including paraquat-induced oxidative stress. A study has demonstrated that administration of paraquat to rat, the GSH levels are decreased and GSH/GSSG ratio is diminished in oxidative injury of liver (Adams et al., 1983; Konstantinova and Russanov, 1999).
However, oxidative stress inhibition as a treatment strategy against PQ-induced toxicity is not ideal (Turrens et al., 1984; Smith et al., 1992; Suntres and Shek, 1995;
Liu et al., 2011).
basic leucine zipper containing the basic region and heptad repeats of leucine, and an N-terminal acidic domain (Moi et al., 1994; Lee and Johnson, 2004). Recently, it was discussed that Nrf-2 is involved in the cell damage cascade. A previous study showed that Nrf-2 acts as a novel caspase substrate. Overexpression of the C-terminal cleavage fragment containing the DNA binding domain and leucine-zipper domain of Nrf-2 induced apoptosis in HeLa cells (Ohtsubo et al., 1999). In one study, it was found that Nrf-2 translocation to the nucleus enhanced oxidative stress and increased the sub-G1 DNA content; this in turn led to cell damage in human bronchial epithelial cells (Eom and Choi, 2009). Another study demonstrated that ambient urban particulate matters induced Nrf-2 protein expression and subsequently caused apoptosis (Choi et al., 2004). Thus, Nrf-2 may play an important role in apoptosis.
Apoptosis is characterized by widespread chromatin condensation and blebbing of plasma membrane and nuclear envelope (Bellomo et al., 1992). To change the calcium homeostasis in cells may alter cell growth, cell differentiation and sensitivity to activation apoptosis (Nicotera et al., 1997; Nicotera and Orrenius, 1998). Paraquat has been demonstrated to induce oxidation-reduction reaction, which leads to generation free radicals, lipid peroxidation, and produces oxidative stress in the lung (Dinis-Oliveira et al., 2008; Huang et al., 2011). Paraquat could also lead to rupture, hemorrhage, edema, and induced marked apoptosis in lung epithelial cells (Huang et al., 2011). Lung epithelial cells are easily affected by stress and induced apoptosis (Franek et al., 2001). The mitochondrial apoptotic pathway is induced by cellular stress or damage, which leads to depolarization of the mitochondrial transmembrane potential (Son et al., 2010). Stress also induced the expression of pro-apoptotic proteins, including Bax and Bak, which play a key role
in the loss of MMP (Selimovic et al., 2011). Loss of MMP led to the release of cytochrome c, apoptosis-inducing factor (AIF), and endo-G from the mitochondria. Subsequently, cytochrome c worked together with Apaf-1 (apoptotic protease activating factor-1) and activated caspase-9 to initiate downstream effector caspases,
resulting in apoptosis (Liang and Sundberg, 2011; Lin et al., 2011).
Recently, many studies demonstrated that the endoplasmic reticulum (ER) plays an important role in chemical toxicant-induced apoptosis. It has been reported that ER stress-mediated apoptosis is involved in the pathogenesis of several diseases (Shang et al., 2011; Wang et al., 2011). ER is an organelle that maintains the intracellular calcium homeostasis, protein synthesis, post-translational modifications, and proper protein folding. If toxicants altered the calcium homeostasis and increased the amount of unfolded protein, then it would cause ER stress (Selimovic et al., 2011). A previous study reported that stress led to an increase in the expression of Bax and Bak, which in turn brought about the release of calcium from the ER lumen into the cytoplasm and subsequent calpain activation, and thus triggered ER stress-mediated apoptosis (Zong et al., 2003; Selimovic et al., 2011).
PQ is accumulated in the lung, and preferentially in alveolar epithelial cells (Mitsopoulos and Suntres, 2010). However, the distinct mechanisms underlying PQ-induced lung alveolar epithelial cell damage have not been clarified. Hence, in this study, we investigated the mechanisms underlying PQ toxicity in alveolar epithelial cells, and the role of Nrf-2 in PQ-induced apoptosis.
Materials and Methods
L2 cell culture
The rat lung epithelial derived cell lines, L2 cell were purchased from ATCC (CCL-149). Cells were cultured in a humidified chamber with 5% CO2 and 95% air mixture at 37 ◦C and maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Gibco BRL, Life Technologies).
Cell viability
L2 cells were washed with phosphate-buffered saline (PBS) twice from 10 cm2 dishes and cultured in 24-well plate (2x105 cells/well). After, cells were exposed PQ for 24 h. After, cells were washed with twice of PBS and replaced with culture medium containing 30 (2 mg/ml) 3-(4,5-dimethyl thiazol-2-yl-)-2,5-diphenyl tetrazolium bromide for 4 h. After, the medium was removed and added 1 ml dimethyl sulfoxide to dissolved blue formazan crystal. The enzyme-linked immunosorbent assay reader (Thermo Fisher Scientific, Waltham, MA, USA) was
used for fluorescence detection at a wavelength of 570 nm.
Cells were washed twice with PBS and detached. After, cells were fixed with 70% (v/v) ethanol, 4℃, for 24 h. Then cells (1x106 cells/ml) washed with PBS and stained with propidium iodide (50 g/mL) for 30 min. Quantification of Sub G1 DNA content after PI staining was carried out using flow cytometric analysis (FACScalibur, Becton Dickinson).
Preperation of cytosolic and nuclear fractions
The method was according to the previous study (Schreiber et al., 1990; Kang et al., 2002). Briefly, cells were treated with PQ for various time points. After, cells were washed twice of ice-cold PBS and transferred to microtube. Cells were swelling by adding 100 L of hypotonic buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride) and incubated for 30 min in ice. After, cells were centrifuged at 7,200 x g for 5 min, and supernatant was used as cytosilic fraction. Besides, pellets were resuspended with 50 L of extraction buffer (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 10 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride) and incubated for 30 min in ice. Samples were centrifuged at 15,800 x g for 10 min, and supernatant was used as nuclear fraction in
Nrf-2 translocation assay.
Western blot analysis
The method of Western blot analysis was performed as we previously study (Chen et al., 2010). After cells treated with indicated drugs, cells were washed with twice of PBS and lysed. After, samples were centrifuged at 14,000 x rpm for 20 min at
4℃ Each sample of 50 g protein was used to electrophoresis on 10% (w/v) SDS-. polyacrylamide gels and transferred to polyvinylidine difluoride (PVDF) membrane. After, membranes were blocked in in PBST (PBS and 0.05% Tween 20) containing 5% nonfat dry milk for 1 h. After blocking, the membrane was incubated with antibodies against Nrf-2, PCNA, -tubulin, AIF, cytochrome c, Bax, Bak, phospho-eIF-2, CHOP, Grp78, procaspase-12, calpain I, calpain II, procaspase-9, procaspase-7, procaspase-3 for 1 h. after, membranes were washed with 0.1% PBST and incubated with secondary antibodies conjugated to horseradish peroxidase for 45 min. The antibody-reactive bands were revealed using enhanced chemiluminescence reagents (Amersham Biosciences, Sweden) and exposed to radiographic film (Kodak, Rochester, NY, USA).
Determination of mitochondrial transmembrane potential (MMP)
The flow cytometry was used to detect the MMP in cells. After cells were treated indicated drugs, cells were harvested and washed twice with PBS. Then cells were treated with 40 nM DiOC6 for 30 min and analyzed in a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Detection of caspase 3/7 activity
To determine the apoptotic effects on paraquat-induced toxicity in L2 cells. The caspase 3/7 activity was assayed by FLICA DEVD-FMK caspase 3/7 assay kit (Immunohistochemistry Technologies, LCC.). Briefly, cells were treated with or without paraquat for 24 h. After, cells were collected and centrifuged at 200 x g for 5 min at 4℃. Then cells were washed twice with PBS and stained with fluorescence
probe for 10 min, at room temperature in a dark. Caspase 3/7 activity was determined by measuring the fluorescence intensity of the cells by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) analysis. More than 10,000 individual
cells were collected for each group.
Quantitative real-time PCR analysis
This method was performed as previously described (Chen et al., 2010). Total RNA was extracted after cells were treated with PQ. Total RNA (5 g) was heated to 90°C for 5 min to remove any secondary structures and then rapidly placed on ice. Samples were then reverse transcribed into cDNA using AMV RTase (Promega, Madison, WI, USA) at 42 °C in reaction buffer containing 2.5 mM dNTPs, 40U/L RNasin (Promega, Madison, WI, USA), 100 nmol random-hexamer primers, 1 RTase buffer; 30U AMV RTase in nuclease-free water at a final volume of 20 L. The mixture was incubated at 42 °C for 60 min. Samples were then denatured at 95 °C for 10 min and placed on ice. Total cDNA (100 ng) was added per 25-μl reaction with sequence-specific primers and real-time Sybr Green PCR reagent (Invitrogen, Carlsbad, CA, USA). The amplification of sample was performed using an ABI Prism 7900HT real-time thermal cycler (Applied Biosystems, Carlsbad, CA, USA). The fold difference in mRNA expression between treatment groups was determined using the relative quantification method utilizing real-time PCR efficiencies and normalized to the β-actin gene, thus comparing relative CT changes between control and experimental samples. Prior to conducting statistical analyses, the fold change from the mean of the control group was calculated for each individual sample, including individual control samples to assess variability within the group.
Measurement of calpain activity
Calpain activity was measured with a calpain fluorometric assay kit according to the manufacturer’s protocol (Biomol). Cells were cultured in 24 well plates (5×104 cells/well) and treated with PQ for 1, 3, 6, 16, 24 h. Untreated controls were incubated with media for 24 h. The 5µl of calpain substrate (Suc-Leu-Leu-Val-Tyr-AMC) were added in each well. After incubated at 37℃ for 60 min, cells were washed twice with PBS, and then added with 70 µl of cold RIPA buffer. After centrifugation at 14,000×g for 5 min, supernatant were aliquot into a 96-well plate and analyzed the 7-amino-4-methylcoumarin (AMC) fluorescence by a
spectrofluorometer (Spectramax, Molecular devices).
The siRNA transfection
The siRNA against Nrf-2 were purchased commercially from Thermo, Biotechnology. Cells were transfected with Nrf-2 siRNA (100 nM) by using lipofectamine 2000 (Invitrogen Life Technology) according to the manufacturer's instructions.
Statistical analysis
These values given as the means ± S.D. Statistical significance between two groups was assessed using the paired Student’s t test (Sigma Plot 10.0; Systat Software, San Jose, CA, USA). One-way ANOVA was used for multiple groups’ analysis, and Duncans’s post hoc test was applied to identify group differences. A P value of less
than 0.05 was considered significant. The statistical package SPSS 11.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis.
Results
Effects of paraquat on cell viability, sub-G1 DNA contents and caspase 3/7 activity in lung epithelial cell-derived L2 cells
To investigate the extent of paraquat toxicity on lung epithelial cells, we performed an MTT assay that would detect the level of cytotoxicity in paraquat-treated L2 cells. The results showed that cell viability was significantly reduced to about 50% after the cells were treated with paraquat (300 M) for 24 hours (Figure 1A). In order to further investigate paraquat-induced cell death, flow cytometry was used to explore paraquat toxicity in sub-G1 DNA content. Results found that paraquat (300 M) significantly increased genomic DNA fragmentation (sub-G1 DNA content) for 24 hours in L2 cells (Figure 1B). Besides, paraquat (300 M) significantly increased the caspase 3/7 activity, which is a cell death indicator in cells (Figure 1C). These results showed that paraquat was cytotoxic to L2 cells, and these effects may be
harmful for cell viability.
Effects of paraquat on mitochondrial transmembrane potential (MMP) and mitochondrial apoptosis-related protein expression in lung epithelial cell-derived L2
cells
We next examined if paraquat-induced cytotoxicity through the mitochondrial-apoptosis pathway. Cells were treated with or without paraquat (300 M) for 6, 16, and 24 h. The mitochondrial transmembrane potential (MMP) was detected via flow cytometry, and DiOC6 fluorescence was an indicator of MMP. The results showed that paraquat significantly resulted in MMP depolarization (Figure 2A). Besides, cytosolic fraction of apoptosis-inducing factor (AIF) and cytochrome c expression levels were significantly increased (Figure 2B). We also found that apoptosis-related signaling involving the induction of Bax, Bak, and p53 mRNA expression was significantly increased, and the expression of pro-apoptotic Bcl-2 was decreased after cells were treated with paraquat (300 M) for 24 h (Figure 3A). Besides, paraquat increased the expression of Bax and Bak proteins in L2 cells (Figure 3B). These results indicated that paraquat disrupted mitochondrial function and promoted
the apoptosis pathway.
Effects of paraquat on endoplasmic reticulum (ER) stress-related signaling in lung epithelial cell-derived L2 cells
To examine whether ER stress-related signals are involved in paraquat-induced lung epithelial cell damage, we performed quantitative real-time PCR and detected mRNAs generated under ER stress in paraquat-treated L2 cells. The results showed that the mRNA expression of Grp78, CHOP and caspase-12 was significantly increased after paraquat treatment (Figure 4A). Besides, ER stress-related signaling involving expression of phospho-eIF-2, CHOP, Grp78, and pro-caspase-12 proteins was significantly increased (Figure 4B). To further investigate ER stress
signaling in paraquat-treated L2 cells, we determined the effects of paraquat on calpain protein expression and calpain activity. The results showed that paraquat (300 M) significantly increased calpain I and calpain II protein expression (Figure 4C) and calpain activity (Figure 4D) in cells. These results indicate that
paraquat-induced cytotoxicity may act through the ER stress pathway.
Effects of paraquat on caspase family proteins expression in lung epithelial cell-derived L2 cells
To evaluate whether paraquat-induced cytotoxicity which through mitochondrial and ER-stress signals promoted apoptotic progression, we investigated the expression of proteins caspase-9, caspase-7, and caspase-3 in paraquat-treated L2 cells for 6, 18, and 24 h. The results showed that paraquat significantly decreased procaspase-9, procaspase-7, and procaspase-3 protein expression in the cells (Figure 5). These results indicated the paraquat-induced apoptosis in L2 cells.
The role of Nrf-2 in paraquat-induced apoptosis of lung epithelial cell-derived L2 cells
To investigate the role of Nrf-2 in paraquat-treated L2 cells, we performed a Western blotting and detected Nrf-2 protein expression in the cytosol and nucleus. The results showed that the nuclear fraction of the Nrf-2 protein was significantly increased and the cytosol fraction of the Nrf-2 protein was decreased after L2 cells were treated with paraquat (300 M) for 6 h (Figure 6). Next, we investigated the role of Nrf-2 in the apoptosis of paraquat-induced L2 cells. The results showed that paraquat significantly reduced MMP, which could be reversed by Nrf-2 siRNA
transfection (Figure 7A). Besides, Nrf-2 siRNA reversed the paraquat-induced Bax, Bak and p53 mRNAs increased, and Bcl-2 mRNA decreased (Figure 7B). Further, Nrf-2 siRNA reversed the paraquat-induced caspase 3/7 activity increased (Figure 7C). We further investigated whether Nrf-2 regulated ER stress and apoptosis signals. The results showed that paraquat-induced Grp78, CHOP and caspase-12 mRNAs expression could be reduced by Nrf-2 siRNA transfection (Figure 7D). Besides, paraquat-induced expression of Grp78 and CHOP proteins could also be reduced by Nrf-2 siRNA transfection (Figure 7E). Further, paraquat decreased procaspase-3 protein expression, which could be reversed by Nrf-2 siRNA transfection (Figure 7E). These results indicated that paraquat-induced apoptosis through ER stress and mitochondrial pathway was regulated by Nrf-2.
Discussion
It has been reported that paraquat accumulated and induced lung alveolar epithelial cell damage, which in turn disrupted the antioxidant system (Suntres, 2002; Park et al., 2010; Liu et al., 2011). Production of ROS leads to oxidative stress and cell damage in paraquat-treated alveolar epithelial cells (Liu et al., 2011). However, although many reports have reported damage of paraquat-induced alveolar epithelial cells, the distinct mechanisms underlying this have not been clarified (Smith et al., 1990; Hoet and Nemery, 2000; Suntres, 2002; Mitsopoulos and Suntres, 2010). In this study, we investigated mitochondrial and ER stress signaling, and the role of Nrf-2 in the apoptosis of paraquat-induced alveolar epithelial cells.
Paraquat is mainly accumulated in the lung. When exposed to paraquat, the amount of paraquat in the lung is 6~10 times more than that in the plasma. It has been reported that oxidative stress plays a key role in paraquat-induced cellular injury (Dinis-Oliveira et al., 2008). A previous study has demonstrated that paraquat caused loss of alveolar epithelial cells and subsequently led to apoptosis (Kliment et al., 2009; Lu et al., 2010). In the results of the present study, we found that paraquat induced significant cytotoxicity in the alveolar epithelial cell line, L2 cells. Besides, paraquat increased the sub-G1 hypodiploid DNA content and caspase 3/7 activity in paraquat-treated L2 cells. These results indicated that paraquat indeed induced cell damage in alveolar epithelial cells.
It has been demonstrated that apoptosis is related to MMP depolarization. In the human corneal endothelium, paraquat induced MMP depolarization and caused apoptosis (Cheng et al., 2007). A previous study has shown that permeabilization of the mitochondrial outer membrane is a critical step in apoptosis. Loss of MMP resulted in the release of cytochrome c from the mitochondria to the cytosol; this activates pro-apoptotic enzymes and leads to progression of the apoptotic cascade (Woo et al., 2011). Bcl-2, which belongs to the anti-apoptotic family, can bind to Bax or Bak and prevent permeabilization of the mitochondrial outer membrane (Fei and Ethell, 2008). However, the activation of Bid/Bim, which are pro-apoptotic BH-2 proteins, induced Bax or Bak activation and led to a change in mitochondrial outer membrane permeabilization (Fei and Ethell, 2008). Furthermore, Bid-mediated cytochrome c release triggered caspase-9 and caspase-3 activation during apoptosis
(Gogada et al., 2011). Then AIF and endo-G were released from the mitochondria, and these cleaved specific substrates leading to the progression of apoptosis (Lin et al., 2011). Recently, it has been shown that p53 activates the apoptotic pathway through the effect of p53 on members of the Bcl-2 family (Fei and Ethell, 2008). In a previous study, when p53 siRNA was used to block p53 expression, cell apoptosis was inhibited with an increase in Bcl-2 expression. p53/Bcl-2-mediated signaling is required for apoptosis (Son et al., 2010). In our results, we found that paraquat decreased MMP and increased the release of cytosolic AIF and cytochrome c. Besides, paraquat decreased the mRNA expression of Bcl-2 and increased that of Bax, Bak, and p53. Furthermore, Bax and Bak protein levels were increased after cells were treated with paraquat. These results suggested that paraquat induced mitochondrial apoptotic signaling and progression of cell death.
ER stress-induced apoptosis. It has been reported that biochemical and physiological stimuli triggered cellular dysfunction in ER stress (Zhao et al., 2011). In ER-stress, ATF-6 activation resulted in the up-regulation of chaperone proteins such as glucose-related protein (Grps) and Bip. Subsequently, XBP1 expression was increased and then PERK was activated; this led to the phosphorylation of eukaryotic initiation factor 2α (p-eIF2α) (Lawson et al., 2011). CHOP is a transcription factor downstream of PERK/ATF4. It has been reported that CHOP can decrease Bcl-2 transcription and promote ROS production (McCullough et al., 2001). Calpains are proteins that belong to the family of calcium-dependent intracellular cysteine proteases. The activation of I (-calpain) and calpain-II (m-calpain) proteases has been demonstrated to result in the cleavage of caspase-12 and ER stress (Huang and Wang, 2001; Lu et al., 2011). In the present study, the results showed that paraquat increased the expression of phosoho-eIF2α, CHOP, and Grp78 and decreased that of pro-caspase-12. Besides, paraquat induced calpain I and calpain II protein expression, and increased calpain activity in paraquat-treated L2 cells. These results implicated the involvement of ER stress in paraquat-induced apoptosis.
Nrf-2 is a member of the NF-E2 family of basic region leucine-zipper transcription factors (Ohtsubo et al., 1999). It has been reported that Nrf-2 binds to the antioxidant responsive element (ARE) and then initiates detoxification. Nrf-2 activation protects cells against oxidative stress (Itoh et al., 1997; Kobayashi and Yamamoto, 2005; Johnson et al., 2008). Many studies have shown that Nrf-2 decreases oxidative stress, which is associated with neuronal cell death, and helps manage neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (Johnson et al.,
2008). However, it has been found that Nrf-2 acts as a caspase-3-like substrate, and is thus involved in apoptotic signals (Ohtsubo et al., 1999). Another study has also found that the activation of Nrf-2 increased cell damage in human bronchial epithelial cells (Eom and Choi, 2009). Hence, although many studies have reported that Nrf-2 prevents oxidative stress, Nrf-2 may still play an important role in cell damage and apoptosis progression. To understand the mechanism underlying the observed paraquat-induced oxidative stress, the role of Nrf-2 was investigated. From our results, we found that paraquat increased Nrf-2 translocation from the cytosol to the nucleus in alveolar epithelial cells. Hence, Nrf-2 activation may be associated with paraquat-induced alveolar cell damage.
Many studies reported that paraquat-induced cell apoptosis is associated with oxidative stress. However, because of different antioxidant properties, the effect of paraquat toxicity cannot be perfectly reversed. A previous study has shown that glutathione (GSH) is quickly cleared from the lung, and is thus ineffective in oxidant-induced lung injury. The effects of superoxide dismutase and catalase are limited because of their inability to traverse the biological membrane. Other antioxidants such as -tocopherol are extremely insoluble and offer limited protection against paraquat toxicity (Turrens et al., 1984; Smith et al., 1992; Suntres and Shek, 1995; Liu et al., 2011). Hence, we want to find a suitable and effective treatment for paraquat-induced alveolar epithelial cell apoptosis. In our results, we found that transfection of Nrf-2 siRNA inhibited paraquat-induced MMP depolarization and reversed paraquat-induced Bax, Bak, p53 mRNAs decrease and Bcl-2 increase in paraquat-treated cells. Besides, transfection of Nrf-2 si-RNA inhibited paraquat-induced caspase 3/7 activity increase. In ER stress related signals, we also found that Nrf-2 siRNA decreased the Grp78, CHOP and caspase-12
mRNAs expression in paraquat-treated cells. Besides, transfection of Nrf-2 siRNA resulted in a decrease in Grp78 and CHOP protein expression. Furthermore, Nrf-2 siRNA reversed paraquat-induced pro-caspase 3 cleavage. These results suggested that Nrf-2 has an important role in the regulation of paraquat-induced alveolar epithelial cell apoptosis.
Conclusion
In conclusion, the results of this study provide evidence that paraquat caused alveolar lung epithelial cell injury leading to the progression of apoptosis. More importantly, the mechanism of paraquat-induced alveolar epithelial cell apoptosis was through Nrf-2 regulated mitochondrial and ER stress pathways (Figure 8). Inhibition of Nrf-2 may be an effective treatment strategy against paraquat-induced alveolar epithelial cell toxicity.
Conflict of interest statement
Acknowledgments
This study was supported by research grants from the Changhua Christian Hospital, Changhua, Taiwan (BSC-100-CCH-IRP-53), and also supported in part by
Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH101-TD-B-111-004).
References
Adams JD, Jr Lauterburg BH, Mitchell JR, (1983) Plasma glutathione and glutathione disulfide in the rat: regulation and response to oxidative stress. J Pharmacol Exp Ther 227: 749-54
Bellomo G, Perotti M, Taddei F, Mirabelli F, Finardi G, Nicotera P, Orrenius S, (1992) Tumor necrosis factor alpha induces apoptosis in mammary adenocarcinoma cells by an increase in intranuclear free Ca2+ concentration and
DNA fragmentation. Cancer Res 52: 1342-6
Berisha HI, Pakbaz H, Absood A, Said SI, (1994) Nitric oxide as a mediator of oxidant lung injury due to paraquat. Proc Natl Acad Sci U S A 91: 7445-9 Bismuth C, Garnier R, Baud FJ, Muszynski J, Keyes C, (1990) Paraquat poisoning.
An overview of the current status. Drug Saf 5: 243-51
Chen YW, Chen KL, Chen CH, Wu HC, Su CC, Wu CC, Way TD, Hung DZ, Yen CC, Yang YT, Lu TH, (2010) Pyrrolidine dithiocarbamate (PDTC)/Cu complex induces lung epithelial cell apoptosis through mitochondria and ER-stress pathways. Toxicol Lett 199: 333-40
Cheng Q, Nguyen T, Song H, Bonanno J, (2007) Hypoxia protects human corneal endothelium from tertiary butyl hydroperoxide and paraquat-induced cell death
in vitro. Exp Biol Med (Maywood) 232: 445-53
study of PM2.5 - and PM10 - induced oxidative stress in rat lung epithelial cells. J Vet Sci 5: 11-8
Dinis-Oliveira RJ, Duarte JA, Sanchez-Navarro A, Remiao F, Bastos ML, Carvalho F, (2008) Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment. Crit Rev Toxicol 38: 13-71
Enoch HG, Strittmatter P, (1979) Cytochrome b5 reduction by NADPH-cytochrome P-450 reductase. J Biol Chem 254: 8976-81
Eom HJ, Choi J, (2009) Oxidative stress of silica nanoparticles in human bronchial epithelial cell, Beas-2B. Toxicol In Vitro 23: 1326-32
Escoffery CT, Shirley SE, (2004) Fatal poisoning in Jamaica: a coroner's autopsy study from the University Hospital of the West Indies. Med Sci Law 44: 116-20
Fei Q, Ethell DW, (2008) Maneb potentiates paraquat neurotoxicity by inducing key Bcl-2 family members. J Neurochem 105: 2091-7
Franek WR, Horowitz S, Stansberry L, Kazzaz JA, Koo HC, Li Y, Arita Y, Davis JM, Mantell AS, Scott W, Mantell LL, (2001) Hyperoxia inhibits oxidant-induced apoptosis in lung epithelial cells. J Biol Chem 276: 569-75
Fussell KC, Udasin RG, Gray JP, Mishin V, Smith PJ, Heck DE, Laskin JD, (2011) Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing
NADPH-cytochrome P450 reductase. Free Radic Biol Med 50: 874-82
Gogada R, Prabhu V, Amadori M, Scott R, Hashmi S, Chandra D, (2011) Resveratrol Induces p53-Independent, XIAP-Mediated Bax Oligomerization on Mitochondria to Initiate Cytochrome c Release and Caspase Activation. J Biol Chem286: 28749-60
Halliwell B, Gutteridge JM, (1992) Biologically relevant metal ion-dependent hydroxyl radical generation. An update. FEBS Lett 307: 108-12
Han JF, Wang SL, He XY, Liu CY, Hong JY, (2006) Effect of genetic variation on human cytochrome p450 reductase-mediated paraquat cytotoxicity. Toxicol Sci
91: 42-8
Hoet PH, Nemery B, (2000) Polyamines in the lung: polyamine uptake and polyamine-linked pathological or toxicological conditions. Am J Physiol Lung Cell Mol Physiol 278: L417-33
Huang WD, Wang JZ, Lu YQ, Di YM, Jiang JK, Zhang Q, (2011) Lysine acetylsalicylate ameliorates lung injury in rats acutely exposed to paraquat. Chin Med J (Engl) 124: 2496-501
Huang Y, Wang KK, (2001) The calpain family and human disease. Trends Mol Med 7: 355-62
Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, Yamamoto M, Nabeshima Y, (1997) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun 236: 313-22
Johnson JA, Johnson DA, Kraft AD, Calkins MJ, Jakel RJ, Vargas MR, Chen PC, (2008) The Nrf2-ARE pathway: an indicator and modulator of oxidative stress
in neurodegeneration. Ann N Y Acad Sci 1147: 61-9
Jones AL, Elton R, Flanagan R, (1999) Multiple logistic regression analysis of plasma paraquat concentrations as a predictor of outcome in 375 cases of paraquat poisoning. QJM 92: 573-8
Kang KW, Lee SJ, Park JW, Kim SG, (2002) Phosphatidylinositol 3-kinase regulates nuclear translocation of NF-E2-related factor 2 through actin rearrangement in response to oxidative stress. Mol Pharmacol 62: 1001-10 Kliment CR, Englert JM, Gochuico BR, Yu G, Kaminski N, Rosas I, Oury TD,
(2009) Oxidative stress alters syndecan-1 distribution in lungs with pulmonary fibrosis. J Biol Chem 284: 3537-45
Kobayashi M, Yamamoto M, (2005) Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation. Antioxid Redox Signal 7: 385-94
Konstantinova SG, Russanov EM, (1999) Studies on paraquat-induced oxidative stress in rat liver. Acta Physiol Pharmacol Bulg 24: 107-11
Lawson WE, Cheng DS, Degryse AL, Tanjore H, Polosukhin VV, Xu XC, Newcomb DC, Jones BR, Roldan J, Lane KB, Morrisey EE, Beers MF, Yull FE, Blackwell TS, (2011) Endoplasmic reticulum stress enhances fibrotic
remodeling in the lungs. Proc Natl Acad Sci U S A 108: 10562-7
Lee JM, Johnson JA, (2004) An important role of Nrf2-ARE pathway in the cellular defense mechanism. J Biochem Mol Biol 37: 139-43
Liang Y, Sundberg JP, (2011) SHARPIN regulates mitochondria-dependent apoptosis in keratinocytes. J Dermatol Sci63: 148-53
Lin CC, Kuo CL, Lee MH, Lai KC, Lin JP, Yang JS, Yu CS, Lu CC, Chiang JH, Chueh FS, Chung JG, (2011) Wogonin triggers apoptosis in human osteosarcoma U-2 OS cells through the endoplasmic reticulum stress, mitochondrial dysfunction and caspase-3-dependent signaling pathways. Int J Oncol 39: 217-24
Liu S, Liu K, Sun Q, Liu W, Xu W, Denoble P, Tao H, Sun X, (2011) Consumption of hydrogen water reduces paraquat-induced acute lung injury in rats. J Biomed
Biotechnol 2011: 305086
Lu T H, Chen C H, Lee MJ, Ho TJ, Leung YM, Hung DZ, Yen CC, He TY , Chen YW, (2010) Methylmercury chloride induces alveolar type II epithelial cell damage through an oxidative stress-related mitochondrial cell death pathway.
Toxicol Lett 194: 70-8
Lu TH, Su CC, Chen YW, Yang CY, Wu CC, Hung DZ, Chen CH, Cheng PW, Liu SH, Huang CF, (2011) Arsenic induces pancreatic beta-cell apoptosis via the oxidative stress-regulated mitochondria-dependent and endoplasmic reticulum stress-triggered signaling pathways. Toxicol Lett 201: 15-26
McCullough KD, Martindale JL, Klotz LO, Aw TY, Holbrook NJ, (2001) Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state. Mol Cell Biol 21: 1249-59
Mitsopoulos P, Suntres ZE, (2010) Cytotoxicity and gene array analysis of alveolar epithelial A549 cells exposed to paraquat. Chem Biol Interact 188: 427-36 Moi P, Chan K, Asunis I, Cao A, Kan YW, (1994) Isolation of NF-E2-related factor
2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region. Proc Natl Acad Sci U S A 91: 9926-30
Moran JM, Ortiz-Ortiz MA, Ruiz-Mesa LM, Fuentes JM, (2010) Nitric oxide in paraquat-mediated toxicity: A review. J Biochem Mol Toxicol 24: 402-9
Nicotera P, Ankarcrona M, Bonfoco E, Orrenius S, Lipton SA, (1997) Neuronal necrosis and apoptosis: two distinct events induced by exposure to glutamate or
oxidative stress. Adv Neurol 72: 95-101
Nicotera P, Orrenius S, (1998) The role of calcium in apoptosis. Cell Calcium 23: 173-80
Ohtsubo T, Kamada S, Mikami T, Murakami H, Tsujimoto Y, (1999) Identification of NRF2, a member of the NF-E2 family of transcription factors, as a substrate
for caspase-3(-like) proteases. Cell Death Differ 6: 865-72
Park HK, Kim SJ, Kwon do Y, Park JH, Kim YC, (2010) Protective effect of quercetin against paraquat-induced lung injury in rats. Life Sci 87: 181-6
Rose MS, Lock EA, Smith LL, Wyatt I, (1976) Paraquat accumulation: tissue and species specificity. Biochem Pharmacol 25: 419-23
Rose MS, Smith LL, Wyatt I, (1974) Evidence for energy-dependent accumulation of paraquat into rat lung. Nature 252: 314-5
Saito M, Thomas CE, Aust SD, (1985) Paraquat and ferritin-dependent lipid peroxidation. J Free Radic Biol Med 1: 179-85
Schreiber E, Harshman K, Kemler I, Malipiero U, Schaffner W, Fontana A, (1990) Astrocytes and glioblastoma cells express novel octamer-DNA binding proteins distinct from the ubiquitous Oct-1 and B cell type Oct-2 proteins. Nucleic Acids Res 18: 5495-503
Selimovic D, Ahmad M, El-Khattouti A, Hannig M, Haikel Y, Hassan M, (2011) Apoptosis related protein-2 triggers melanoma cell death by a mechanism including both endoplasmic reticulum stress and mitochondrial dysregulation. Carcinogenesis 32: 1268-78
Shang Y, Wang F, Bai C, Huang Y, Zhao LJ, Yao XP, Li Q, Sun SH, (2011) Dexamethasone protects airway epithelial cell line NCI-H292 against lipopolysaccharide induced endoplasmic reticulum stress and apoptosis. Chin Med J (Engl) 124: 38-44
Smith LJ, Anderson J, Shamsuddin M, (1992) Glutathione localization and distribution after intratracheal instillation. Implications for treatment. Am Rev Respir Dis 145: 153-9
Smith LL, Lewis CP, Wyatt I, Cohen GM, (1990) The importance of epithelial uptake systems in lung toxicity. Environ. Health Perspect 85: 25-30
Son YO, Lee JC, Hitron JA, Pan J, Zhang Z, Shi X, (2010) Cadmium induces intracellular Ca2+- and H2O2-dependent apoptosis through JNK- and p53-mediated pathways in skin epidermal cell line. Toxicol Sci 113: 127-37
Suntres ZE, (2002) Role of antioxidants in paraquat toxicity. Toxicology 180: 65-77 Suntres ZE, Shek PN, (1995) Liposomal alpha-tocopherol alleviates the progression
of paraquat-induced lung damage. J Drug Target 2: 493-500
Turrens JF, Crapo JD, Freeman BA, (1984) Protection against oxygen toxicity by intravenous injection of liposome-entrapped catalase and superoxide dismutase.
J Clin Invest 73: 87-95
Wang Y, Xiao J, Zhou H, Yang S, Wu X, Jiang C, Zhao Y, Liang D, Li X, Liang G, (2011) A Novel Monocarbonyl Analogue of Curcumin, (1E,4E)-1,5-Bis(2,3-dimethoxyphenyl)penta-1,4-dien-3-one, Induced Cancer Cell H460 Apoptosis via Activation of Endoplasmic Reticulum Stress Signaling Pathway. J Med Chem 54: 3768-78
Woo IS, Jin H, Kang ES, Kim HJ, Lee JH, Chang KC, Park JY, Choi WS, Seo HG, (2011) TMEM14A inhibits N-(4-hydroxyphenyl)retinamide-induced apoptosis through the stabilization of mitochondrial membrane potential. Cancer Lett
309: 190-8
Zhao Y, Zhu C, Li X, Zhang Z, Yuan Y, Ni Y, Liu T, Deng S, Zhao J, Wang Y, (2011) Asterosaponin 1 induces endoplasmic reticulum stress-associated apoptosis in A549 human lung cancer cells. Oncol Rep 26: 919-24
Zong WX, Li C, Hatzivassiliou G, Lindsten T, Yu QC, Yuan J, Thompson CB, (2003) Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis. J Cell Biol 162: 59-69
Figure Legends
Figure 1. Effects of paraquat (PQ) on cell viability, sub-G1 DNA contents and caspase 3/7 activity in lung epithelial cell-derived L2 cell. (A) Cells were exposed with or without PQ (0, 50, 100, 300 and 500, 900 M) for 24h. The cell viability was by MTT assay. (B) Cells were treated with or without PQ (300 M) for 24 h. The genomic DNA fragmentation (sub-G1 DNA content) was assayed via flow cytometery. (C) The caspase 3/7 activity was assayed after cells were treated with PQ (300 M) for 24 h via flow cytometery. Data are presented as means ± S.D. for four independent experiments with triplicate determinations. *P<0.05 as compared
with control.
Figure 2. Effects of paraquat (PQ) on mitochondrial transmembrane potential (MMP), cytosolic AIF and cytochrome c protein expression in lung epithelial cell-derived L2 cell. (A) Cells were treated with or without PQ (300 M) for 24 h. Then
the DiOC6 fluorescence was used to detect MMP alteration by flow cytometery. (B) Cells were treated as described above. The cytosolic fractions of AIF and cytochrome c proteins expression were detected by Western blot analysis. Data in (A) are presented as means ± S.D. for four independent experiments with triplicate determinations. *P<0.05 as compared with control. Data in (B) are representative of
four independent experiments.
Figure 3. Effects of paraquat (PQ) on Bcl-2, Bax, Bak, p53 mRNAs expression, and Bax, Bak proteins expression in lung epithelial cell-derived L2 cell. (A) Cells were exposed with or without PQ (300 M) for 24 h. The mRNAs of Bcl-2, Bax, Bak, and p53 were determined by quantitative real-time PCR. (B) Cells were treated as described above. The proteins expression of Bax and Bak were analyzed by Western blot. Data are representative of three independent experiments. Data in (A) are presented as means ± S.D. for four independent experiments with triplicate determinations. *P<0.05 is increased as compared with control. #P<0.05 is decreased as compared with control. Data in (B) are representative of four independent
experiments.
Figure 4. Effects of paraquat (PQ) on Grp78, CHOP and caspase-12 mRNAs expression, and phospho-eIF-2, CHOP, Grp78, pro-caspase-12, calpain I, calpain II proteins expression, and calpain activity, in lung epithelial cell-derived L2 cell. (A) Cells were treated with or without PQ (300 M) for 24 h. The mRNAs of Grp78, CHOP and caspase-12 were determined by quantitative real-time PCR. (B) Cells were exposed with or without PQ (300 μM) for 6, 16 and 24 h. The proteins expression of phospho-eIF-2, CHOP, Grp78, pro-caspase-12 were analyzed by Western blot. (C) The calpain I and calpain II protein expression was analysis by
Western blot, and (D) calpain activity was determined in L2 cells. Data in (A) and (D), data are presented as means ± S.D. for four independent experiments with triplicate determinations. *P<0.05 as compared with control. Data in (B) and (C), protein expression data are representative of three independent experiments.
Figure 5. Effects of paraquat (PQ) on 9, 7 and pro-caspase-3 proteins expression in lung epithelial cell-derived L2 cell. Cells were exposed with or without PQ (300 M) for 6, 18, 24 h. The proteins expression of pro-caspase-9, pro-caspase-7 and pro-caspase-3 were analyzed by Western blot. Data are representative of three independent experiments.
Figure 6. Effects of paraquat (PQ) on Nrf-2 protein expression in lung epithelial cell-derived L2 cell. Cells were exposed with or without PQ (300 μM) for 6, 16, 24 h. The cytosolic and nuclear fractions of Nrf-2 protein expression were determined by Western blot analysis. Results shown are representative of four independent
experiments.
Figure 7. Effects of Nrf-2 siRNA on paraquat (PQ)-induced mitochondrial toxicity in lung epithelial cell-derived L2 cell. Cells were transfected with or without Nrf-2 siRNA, and treated with or without PQ (300 M) for 24 h. The (A) DiOC6 fluorescence of mitochondrial transmembrane potential (MMP) and (C) caspase 3/7 activity were detected via flow cytometery. (B) The Bcl-2, Bax, Bak and p53 mRNA level was detected after cells were transfected with or without Nrf-2 siRNA and treated with or without PQ (300 M) for 24 h. (D) The Grp78, CHOP and caspase-12 mRNAs expression were determined by quantitative real-time PCR. (E) The Grp78, CHOP, pro-caspase-3 protein expression was detected by Western blot. Data
in (A), (B), (C) and (D) are presented as means ± S.D. for four independent experiments with triplicate determinations. *P<0.05 as compared with control, which is increased. #P<0.05 as compared with control, which is decreased. Data in
(E) are representative of three independent experiments.
Figure 8. The schematic representation of proposed mechanisms according to paraquat (PQ) induced apoptosis in lung alveolar epithelial cells.
