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Cadmium-induced apoptotic death of human retinal pigment epithelial cells is mediated by MAPK pathway
Experimental Eye Research 89 (2009) 494–502
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Cadmium-induced apoptotic death of human retinal pigment epithelial cells is mediated by MAPK pathway Nilesh M. Kalariya a, Nancy K. Wills b, Kota V. Ramana c, Satish K. Srivastava c, Frederik J.G.M. van Kuijk a, * a
Department of Ophthalmology & Visual Sciences, University of Texas Medical Branch, Galveston, TX 77555, USA Department of Neuroscience & Cell Biology, University of Texas Medical Branch, Galveston, TX 77555, USA c Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 April 2009 Accepted in revised form 12 May 2009 Available online 12 June 2009
Cadmium (Cd), released from cigarette smoke and metal industrial activities, is known to accumulate in human body organs including retina and is particularly higher in retinal tissues of age-related macular degeneration (AMD) eyes compared to non-AMD eyes. We have determined the cytotoxic effects of Cd on human retinal pigment epithelial (RPE) cells. Upon Cd treatment, there was a dose- and time-dependent decline in ARPE-19 cell viability as well as early apoptotic changes such as altered mitochondrial membrane potential (MMP) and Cytochrome C release in cytosol. Depletion of GSH by buthionine-[S,R]sulfoximine (BSO) resulted in increased Cd toxicity in ARPE-19 cells. Cadmium also caused reactive oxygen species (ROS) generation and activation of mitogen-activated protein kinases (MAPKs) pathway including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (Erk1/2), and p38 in ARPE-19 cells. Antioxidants such as N-acetylcysteine (NAC) significantly reduced Cd-induced toxicity. These results indicate that elevated ROS-induced activation of the MAPK signaling pathway could be associated with Cd-induced RPE cell apoptosis, one of the major contributing factors in AMD. The toxic effects of Cd on ARPE-19 cells indicate that environmental heavy metals such as Cd could be important potential factors in RPE cells death associated retinal diseases particularly related to smoking. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: cadmium retinal pigment epithelium oxidative stress apoptosis MAPK AMD
1. Introduction Cadmium (Cd), a heavy metal, has increased in the environment due to industrial activities such as mining and manufacturing. It is also a contaminant in fertilizers and as a result absorbed and gradually accumulated by plants (Nordberg et al., 2007). The uptake of Cd in human body occurs through food intake as well as smoking (Satarug and Moore, 2004; Satarug et al., 2004). The half-life of Cd in the human body is approximately 10–30 years. Although the systemic circulation does not retain Cd, vital organs like liver, kidney, lung, and bone are known to accumulate Cd with age (Baker et al., 2005; Friberg, 1983). Cadmium accumulation in human body organs has been reported to contribute to a variety of pathological conditions such as carcinogenesis, neurodegeneration, lung dysfunction, renal dysfunction, vascular disease, hypertension, coronary heart disease, myocardial infarction, anemia, osteoporosis, Parkinson’s disease, and Alzheimer’s disease (Arisawa et al., 2007; Gallagher
* Corresponding author at: Department of Ophthalmology & Visual Sciences, Room 2.100, 700 University Blvd, University of Texas Medical Branch, Galveston, TX 77555, USA. Tel.: þ1 409 747 5411; fax: þ1 409 747 5402. E-mail address: [email protected] (F.J.G.M. van Kuijk). 0014-4835/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2009.05.011
et al., 2008; Horiguchi, 2007; Houston, 2007; Johnson, 2001; Kim et al., 2005; Lopez et al., 2003; Mannino et al., 2004; Okuda et al., 1997; Singh et al., 2009). Recent reports have demonstrated Cd accumulation in human retina and implicated Cd in development as well as progression of smoking-related age-related macular degeneration (AMD) (Erie et al., 2005; Wills et al., 2008a,b, 2009). The reported Cd levels in the retinal pigment epithelium (RPE)/choroid tissues are approximately double (4000 mg/g tissue) in smokers compared to nonsmokers (1650 mg/g tissue) (Erie et al., 2005). In addition, higher urinary Cd levels, indicating a higher total body burden of Cd, have also been reported in smokers who had AMD compared to smokers who did not have AMD (Erie et al., 2007). We have demonstrated that human retinal tissues accumulate Cd during aging (Wills et al., 2008a). We have also observed gender difference in terms of Cd accumulation. Significantly higher Cd levels have been found in the RPE and choroid from female donor eyes compared to male donor eyes (Wills et al., 2008b). Higher Cd levels have also been found in the neural retina and RPE of AMD eyes (90 and 220 pmol/mg protein, respectively) compared to non-AMD eyes (40 and 120 pmol/mg protein, respectively) (Wills et al., 2009). The accumulation as well as possible toxic effects of Cd in RPE cells could be prevented by manganese (Mn) and zinc (Zn) (Satarug et al., 2008).
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Since Cd and Mn exhibit equal affinity for metal transporter proteins such as ZIP8 and ZIP14, Cd uptake as well as accumulation could be reduced when the cells are treated with Cd and Mn (He et al., 2006). Moreover, Mn could cause induction of heme oxygenase-1 (HO-1) in ARPE-19 cells thereby reducing the toxic effects of Cd (Satarug et al., 2008). These findings have raised the possibility that cadmium exposure might play a role in tobacco-related AMD. Cadmium has been demonstrated to have toxic effects on many cell types, including neurons, lung cells and renal tubules (Kukner et al., 2007; Lei et al., 2008; Lopez et al., 2003; Prozialeck et al., 2007; Thijssen et al., 2007). Cadmium-induced damage in most cells is indirectly caused in part by increased levels of reactive oxygen species (ROS) (Bertin and Averbeck, 2006). Retinal cells and the RPE routinely cope with ROS generated by metabolic processes and light. However, during aging, decreased levels of specific antioxidant enzymes in the retina (Liles et al., 1991) diminish the ability of cells to survive oxidative stress. In view of this fact, it is important to determine whether cadmium accumulation in retina contributes to ROS generation and its possible consequences on RPE cells. In this study we observed that Cd induces ROS production and leads to dose- and time-dependent retinal cell apoptosis. We have also delineated the Cd-induced mechanism of apoptosis in ARPE-19 cells. We found that Cd activates MAPK members JNK, Erk1/2, and p38 in ARPE-19 cells. Antioxidants such as N-acetylcysteine (NAC) prevented ROS generation induced by Cd. Thus activation of MAPK pathway and subsequent cell death could be attenuated. These findings indicate that Cd-induced RPE cell apoptosis could be associated with the activation of the MAPK signaling pathways which could play an important role in smokingrelated AMD. 2. Materials and methods 2.1. Materials Human retinal pigment epithelial cells (ARPE-19) were purchased from American Type Culture Collection (ATCC). Cadmium chloride (CdCl2) and buthionine-[S,R]-sulfoximine (BSO) were purchased from Sigma, USA. JC-1, Dihydroethidium (hydroethidine) dye, TACSÔ Annexin V-FITC Apoptosis detection kit were purchased from Molecular Probes, USA. CellTiter 96Ò AQueous one solution cell proliferation assay kit was purchased from Promega. USA. RIPA cell lysis buffer was purchased from Santa Cruz biotechnology, USA. Protein assay reagent was obtained from Bio-Rad, USA. Fetal bovine serum, trypsin/EDTA, antibiotics, N-acetylcysteine (NAC), 20 , 70 dichlorofluorescin diacetate (DCF-DA) and all other chemicals were obtained from Sigma. Antibodies against the MAPK cascade proteins such as phospho-JNK, JNK, phospho-c jun, c-jun, phosphoErk1/2, Erk1/2, phospho-p38 and p38, were obtained from Cell Signaling Inc. Cytochrome C antibodies were purchased from Santa Cruz, CA, USA. Mouse anti-rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were obtained from Research Diagnostics Inc. 2.2. Cell culture and treatment The ARPE-19 cells were grown to confluency in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 C in a humidified atmosphere of 5% CO2. Sub-confluent cells were growth-arrested in 0.1% FBS medium. Sub culturing was done using trypsin/EDTA solution and the cells were sub cultivated with a split ratio of 1:4. The CdCl2 treatment was done in 0.1% serum medium. Whenever BSO and NAC were used, cells were preincubated with it for 24 and 1 h respectively.
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2.3. Cell viability assay The ARPE-19 cells were plated (5000 cells/well) in a 96-well plate. After 24 h, cells were serum starved in 0.1% FBS medium for 24 h and CdCl2 was added to the media after pretreating the cells with BSO or NAC for 24 or 1 h respectively. Cells incubated without CdCl2 and NAC served as control. Cell viability was determined by CellTiter 96 AQueous one solution cell proliferation assay kit. CellTiter reagent was added to culture well, incubated for 3 h and absorbance recorded at 490 nm using a 96-well micro plate reader (BioTek, USA). 2.4. Determination of apoptosis Apoptosis was determined by using the TACS Annexin V-FITC Apoptosis kit (Molecular Probes) which detects cell surface changes that occur early in the apoptotic process. The assay was performed according to manufacturer’s instructions and cell death was detected by flow cytometry. After growth and starvation the cells were washed with PBS and incubated with CdCl2 and H2O2 (positive control) for 12 h without or with NAC. Pellets of approximately 1 106 cells were suspended in 100 ml of Annexin binding buffer containing Annexin-V and Propidium Iodide (PI) dye solution. After incubation at 37 C for 30 min, 400 ml of Annexin binding buffer was added and solution was subjected to flow cytometry. 2.5. Measurement of mitochondrial membrane potential (MMP) Flow cytometry was performed using the cationic dye JC-1 that exhibits its membrane potential-dependent accumulation in the mitochondria as determined by a fluorescence emission shift from green (525 nm) to red (590 nm). After growth and starvation the cells were washed with PBS and incubated with CdCl2 and H2O2 (positive control) for 5 h without or with NAC. Pellets of approximately 1 106 cells were suspended in 100 ml of PBS containing 5 ml of JC-1 solution (20 mg/ml) was added. After incubation at 37 C for 30 min, cells were washed with PBS, resuspended in 500 ml PBS, and subjected to flow cytometry. 2.6. ROS determination The intracellular ROS levels were measured using a fluorescent dye, DCF-DA. The cells were plated (5000 cells/well) in a 96-well plate. After 24 h, cells were serum starved for 24 h with BSO and/or NAC treatment, washed with PBS and incubated with 10 mM DCFDA at 37 C for 30 min in media without phenol red. Cells were washed again to remove excess DCF-DA and treated with CdCl2 in media without FBS for 30 min. At the end of the treatment, cells were washed twice with PBS. Media without phenol red was added and absorbance determined after 1 h at 485 nm excitation and 538 nm emission wavelengths. Relative ROS production was expressed as a change in fluorescence compared to fluorescence of the appropriate control. The intracellular ROS were also detected using a fluorescent dye, Dihydroethidium. The cells were plated (100,000 cells/Chamber) in a 2-chamber slide. After 24 h, cells were serum starved for 24 h without or with NAC treatment, washed with PBS and incubated with 5 mM Dihydroethidium at RT for 30 min in PBS. Cells were washed again to remove excess Dihydroethidium and treated with CdCl2 for 1 h. At the end of the treatment, cells were washed twice with PBS mounted with Fluorosave (Molecular Probes), covered with the coverslip and the coverslip borders were sealed with nail polish. The cells were visualized under a Nikon Eclipse 800 epifluorescence microscope equipped with a xenon arc lamp using a Texas Red filter set for red fluorescence. Photographs were taken
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using a Roper Scientific CoolSNAP Fx monochrome cooled CCD 12bit digital camera.
3. Results 3.1. Cd causes decreased cell viability
2.7. Western blot analysis After treatment, ARPE-19 cells were washed twice with ice-cold PBS and lysed in ice-cold RIPA lysis buffer for 15 min with occasional vortexing at maximum speed at 4 C. The crude lysates were cleared by centrifugation at 12,000g for 10 min at 4 C. Aliquots of the lysates were diluted with 2 SDS sample buffer and boiled for 5 min. The lysates were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore, Bedford, MA). The membranes were then incubated in blocking solution containing 5% wt/vol dried fat-free milk and 0.1% vol/vol Tween-20 in Tris-buffered saline. Subsequently, the membranes were incubated with appropriate primary antibodies. The membranes were then probed with horseradish peroxidase– conjugated secondary antibody (GE Healthcare, Piscataway, NJ) and visualized by chemiluminescence (Pierce Biotechnology, Rockford, IL). For Cytochrome C analysis, cytoplasmic fractions were obtained by method mentioned elsewhere (Kalariya et al., 2008) 2.8. Statistical analysis Results are expressed as mean S.D. and the p values were determined by Student’s t-test using Microsoft Excel software.
In order to investigate the effects of Cd on cell viability, ARPE-19 cells were incubated with Cd for 24 h. A concentration-dependent (0–80 mM Cd) decrease in cell viability was observed (Fig. 1A). The LC50 for Cd was approximately 20 mM. To elucidate the timedependent effect of Cd, ARPE-19 cells were incubated with 20 mM Cd for various time intervals. A 15% decline in cell viability occurred in the first 6 h and approximately 21% and 40% by 12 h and 24 h, respectively (Fig. 1B). We observed, a significant (p < 0.001) decline in cell viability by 12 h. The RPE cells are known to have high degree of oxidative stress and depleted antioxidants like GSH due to aging as well as pathological states such as AMD. Therefore, we investigated the effect of Cd on cell viability in ARPE-19 cells that were pretreated with BSO to reduce total cellular GSH levels. As shown in Fig. 1C, effects of Cd toxicity increased substantially in cells treated with BSO. The decline in cell viability was highly significant (p < 0.001) even at lower concentration (10 mM) in cells treated with BSO compared to cells without BSO treatment. Cadmium at 10 mM caused 15% or 40% decline in viability of ARPE-19 cells pretreated without or with BSO, respectively (Fig. 1C). These results indicate that the LC50 for Cd and degree of oxidative stress in retinal cells could have an inverse relationship.
Fig. 1. Effect of Cadmium (Cd) on the viability of ARPE-19 cells. (A) Cd caused concentration-dependent decrease in ARPE-19 cell viability. Cells were exposed to various concentrations of Cd (0–80 mM) and after 24 h cell viability was determined. Data represents the mean SD of three experiments (*p < 0.01, **p < 0.001 vs Control). (B) Timedependent effect of Cd on ARPE-19 cell viability. Cells were exposed to 20 mM Cd for 3 h, 6 h, 12 h and 24 h and cell viability was determined. Data represents the mean SD of three experiments (*p < 0.01, **p < 0.001 vs Control). (C) Cd caused substantial decline in ARPE-19 cell viability under reduced or depleted GSH levels by BSO. Cells pretreated with BSO (1 mM) were exposed to various concentrations of Cd (10, 20, and 40 mM) and after 24 h cell viability was determined. Data represents the mean SD of three experiments (*p < 0.01, **p < 0.001 vs Control, #p < 0.001 vs 10, 20, 40 mM Cd without BSO respectively). (D) NAC prevented Cd-induced decline in ARPE-19 cell viability. Cells pretreated with BSO (1 mM) were exposed to NAC (1 mM) followed by Cd (20 mM) and after 24 h cell viability was determined. Data represents the mean SD of three experiments (*p < 0.001 vs Control, **p < 0.001 vs Cd, #p < 0.001 vs Cd, ##p < 0.001 vs Cd þ BSO).
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Since the toxic effect of Cd seemed dependent on the degree of cellular GSH levels, use of antioxidants could be a useful tool to prevent Cd toxicity in retinal cells. Therefore, we used NAC as preventive measure in cells treated without or with BSO. As shown in Fig. 1D, Cd-induced decline in cell viability was significantly (p < 0.001) prevented by NAC in cells treated without or with BSO. These results indicate that status of antioxidant defense could be the determining factor in Cd-induced toxicity in RPE cells. 3.2. Increase in ROS levels by Cd Since our preliminary results on retinal cell viability indicated that Cd-induced toxicity is associated with cellular GSH levels, we investigated if Cd could generate ROS in retinal cells. Incubation of ARPE-19 cells with Cd resulted in a dose-dependent (0–80 mM) increase in ROS levels (Fig. 2A). Cadmium at lower concentrations (5 & 10 mM) did not have a marked effect on ROS generation, but at higher concentration 20 mM, significant increase (p < 0.001) in ROS levels was observed. These results indicate that Cd can be a potent oxidant at 20 mM concentration and could lead to increase in ROS levels and eventual toxicity in retinal cells. This observation was further strengthened by using ROS scavenger, NAC, to ameliorate Cd-induced increase in ROS levels and toxicity. Pretreatment of cells with NAC significantly (p < 0.001) attenuated Cd-induced ROS increase in ARPE-19 cells (Fig. 2A and B). Fluorescence microscopy was also carried out to visualize Cd-induced ROS increase as well as scavenging effect of NAC (Fig. 2B). Cadmium treated cells showed red fluorescence indicating an increase in intracellular ROS levels (Fig. 2B III). The increase in ROS levels was prevented by pretreating the cells with NAC (Fig. 2B IV). Cells in control and NAC-treated groups did not exhibit increase in ROS levels (Fig. 2B I & II). The levels of ROS increased significantly in cells with compromised GSH levels by treatment with BSO compared to cells without BSO treatment. As shown in Fig. 2C, the increase in ROS in BSO þ Cd treated cells was highly significant (p < 0.001) compared to cells without BSO treatment. Moreover, NAC scavenged (p < 0.001) Cdinduced ROS increase in cells pretreated without or with BSO. 3.3. Cd-induced apoptosis in RPE cells A growing body of evidence suggests that increased oxidative stress due to elevated ROS levels in RPE cells could cause apoptotic cell death which is a known pathogenic event in AMD. Therefore, ARPE-19 cells were incubated with Cd (20 mM) or H2O2 (200 mM) without or with NAC (1 mM) for 12 h. An increase in apoptosis was observed when cells were treated with Cd or H2O2 (200 mM), a positive control (Fig. 3C & D, respectively). The Cd- as well as H2O2-induced apoptosis was 32% and 23%, respectively and NAC significantly (p < 0.001) prevented it (Fig. 3E and F). 3.4. Effects of Cd on mitochondrial membrane potential (MMP) The alteration in MMP has been a marker for an early stage of apoptosis. Therefore, the effect of Cd as well as H2O2 on the early stages of apoptosis was also assessed by measuring MMP. ARPE-19 cells were exposed to Cd (20 mM) or H2O2 (200 mM) for 5 h without or with pretreatment with NAC, followed by incubation with JC-1 dye. Flow cytometry analysis revealed that Cd as well as H2O2 treatment caused alteration of MMP in approximately 38% and 27% of the cells, respectively (Fig. 4 C & D) and pretreatment with NAC attenuated Cd-induced change in MMP (Fig. 4 E & F). Fig. 4 A & B represent control and NAC-treated groups, respectively. It is also known that Cytochrome C, which plays a crucial role in apoptosis, is released from mitochondria due to change in MMP. We
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also determined the release of Cytochrome C in cytosol upon Cd treatment. As shown in Fig. 4 G, western blot analysis demonstrates the release of Cytochrome C in Cd treated group. Pretreatment with NAC reduced the release of Cytochrome C from mitochondria. 3.5. Cd-induced activation of MAPK pathway Since we observed that Cd induces ROS generation as well as eventual retinal cell apoptosis, we determined the role of MAPK signaling pathway. In order to determine the role of MAPK members such as JNK, Erk1/2 and p38 in Cd-induced retinal cell apoptosis, ARPE-19 cells were exposed to various concentration of Cd (0–20 mM) for 12 h. As shown in Fig. 5 A, there was robust phosphorylation of JNK, Erk1/2, and p38 with >5 mM Cd. Moreover, when ARPE-19 cells were treated with 20 mM Cd for various time intervals, the robust phosphorylation of JNK, Erk1/2, and p38 was initiated approximately at 4 h and sustained until 12 h (Fig. 5B). The activation of MAPK pathway along with ROS generation in retinal cells suggests that antioxidants could attenuate Cd-induced upstream apoptotic signals in RPE cells. Therefore, we treated ARPE19 cells with 20 mM Cd without or with 1 mM NAC for 10 h. As shown in Fig. 5C, NAC suppressed the phosphorylation of JNK, Erk1/2, and p38. These results suggest that Cd-induced ROS generation could activate the MAPK pathway and trigger apoptosis of the RPE cells. 4. Discussion Smoking has been associated with higher incidence and progression of AMD (Klein et al., 2008; Zarbin, 2004). However, there is little understanding in molecular mechanisms by which smoking could contribute to AMD. Chronic cigarette smoke has been shown to cause oxidative mitochondrial DNA damage, RPE apoptosis, and ultrastructural degeneration of the RPE and Bruch’s membrane in mice (Fujihara et al., 2008; Wang et al., 2009). The toxic elements of smoke, such as Benzo(a)Pyrene (B(a)P), has been shown to cause extensive mitochondrial DNA damage as well as increased lysosomal activity and exocytotic activity in RPE cells as well as in mice (Wang et al., 2009). The B(a)P could also increase complement pathway components surrounding Bruch’s membrane in mice (Wang et al., 2009). Most of the structural abnormalities as well as biochemical markers observed in in vitro and in vivo studies are consistent with findings in AMD patients (Wang et al., 2009). Furthermore, genetic studies have also identified susceptibility locus, i.e. LOC387715, for AMD and that polymorphism of this locus and smoking could confer a higher risk for AMD than either factor alone (Rivera et al., 2005; Fritsche et al., 2008; Schmidt et al., 2006). Thus, smoking could be a potential contributor to AMD and merits further investigation. In this study, we have shown that Cd which increases with smoking could induce cell death in RPE cells in a dose- and timedependent manner. These data are consistent with that observed in neuronal cells (Chen et al., 2008a). The observations that Cd could activate MAPK cascade in ARPE-19 cells is consistent with previous studies which demonstrated that Cd can cause apoptosis in various cell types through activation of MAPK cascade (Chen et al., 2008a,b; Chuang et al., 2000; Jung et al., 2008; Kim et al., 2005; Lag et al., 2005; Rockwell et al., 2004). Further, oxidative stress has been known to play a crucial role in various pathological conditions through activation of apoptotic mechanism. Induction of ROS is a profound feature of many degenerative diseases such as AMD, Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis (Hollyfield et al., 2008; Johnson, 2001; Li et al., 2004; Okuda et al., 1997). Cadmium is a potent oxidative stress and inflammatory agent
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Fig. 2. Effect of Cadmium (Cd) on ROS levels in ARPE-19 cells. (A) Cd generated concentration-dependent elevation in ROS levels in ARPE-19 cells. The cells were incubated with DCF-DA (10 mM) dye for 30 min. After washing with PBS, cells were treated with Cd (0–80 mM) for 1 h without or with NAC (1 mM). Data represents the mean SD of three experiments (*p < 0.05, **p < 0.001 vs Control; #p < 0.05, ##p < 0.001 vs Cd treated group). (B) The serum-starved ARPE-19 cells without or with NAC (1 mM) were treated with the ROS-sensitive dye dihydroethidium (5 mM) for 30 min. Subsequently, the cells were exposed to Cd (20 mM) without or with NAC (1 mM) for 1 h and fluorescence was measured with a Nikon epifluorescence microscope. Panels: (I) Control, (II) 1 mM NAC, (III) 20 mM Cd, (IV) 20 mM Cd þ 1 mM NAC. Picture is a representative of three different sets of experiments. (C) Cd-induced elevation in ROS levels in ARPE-19 cells with depleted GSH levels by BSO. The cells were incubated with BSO (1 mM) for 24 h and/or NAC (1 mM) for 1 h. The cells were washed with PBS and incubated with DCF-DA (10 mM) dye for 30 mints. After washing with PBS, cells were treated with Cd (20 mM) for 1 h. Data represents the mean SD of three experiments (*p < 0.05 vs Control, **p < 0.05 vs Cd, #p < 0.001 vs Cd, ##p < 0.001 vs Cd þ BSO).
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Fig. 3. Cadmium (Cd)-induced apoptosis in ARPE-19 cell. ARPE-19 cells (1 106) were incubated with Cd (20 mM) or H2O2 (200 mM) without or with NAC (1 mM) and harvested after 12 h. Apoptosis was detected by Annexin V-FITC as described in the text. Panels: (A) Control, (B) 1 mM NAC, (C) 20 mM Cd and (D) 200 mM H2O2, (E) 20 mM Cd þ 1 mM NAC, (F) 200 mM H2O2 þ 1 mM NAC. Picture is a representative of three different sets of experiments.
(Bhattacharyya et al., 2000) and there is growing a growing body of evidence suggests that under pathological conditions, Cd-induced excessive ROS generation could alter structure and function of proteins, lipids, and DNA besides activating various signaling pathways which collectively cause apoptosis (Chen et al., 2008b; Kim et al., 2005; Monroe and Halvorsen, 2006). Cadmium toxicity has been related to induction of ROS levels (Chen et al., 2008b; Monroe and Halvorsen, 2006). However, the role of Cd in causing oxidative stress in RPE cells has not been known so far. Our demonstration that Cd can increase ROS levels in ARPE-19 cells is
consistent with many previous studies which have shown Cdinduced ROS generation in many cell types (Chen et al., 2008b; Monroe and Halvorsen, 2006). When intracellular oxidative stress is elevated, GSH is oxidized to GSSG which is transported out of cells (Srivastava and Beutler, 1969; Meister et al., 1986; Ramana et al., 2006). As a result cellular GSH levels are significantly depleted. Therefore, cells are vulnerable to ROS-induced toxicity but supplementing the cells with GSH protects against ROS-induced cytotoxicity (Yadav et al., 2008; Cao et al., 2003). In this study, we have depleted GSH levels in
Fig. 4. Cadmium (Cd)-induced changes in ARPE-19 cell MMP represent an early apoptotic change. ARPE-19 cells (0.5 106) were exposed to Cd (20 mM) or H2O2 (200 mM) for 5 h without or with pretreatment with NAC (1 mM) for 1 h. At the end of experiment, the cells were harvested, washed with PBS, incubated with JC-1 dye for 30 min, followed by 3 wash with PBS, suspended in PBS and subjected to flow cytometry. Panels: (A) Control, (B) 1 mM NAC, (C) 20 mM Cd and (D) 200 mM H2O2, (E) 20 mM Cd þ 1 mM NAC, (F) 200 mM H2O2 þ 1 mM NAC. Picture is a representative of three different sets of experiments. (G) ARPE-19 cells treated with Cd (20 mM) for 5 h without or with NAC were harvested and cytoplasmic fraction were subjected to western blot analysis using antibodies against the Cytochrome C which released into cytoplasm from Mitochondria due to change in MMP. The blots were probed with GAPDH antibodies as a loading control. Similar results were observed in at least three independent experiments.
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Fig. 5. Activation of mitogen-activated protein kinase (MAPK) cascade in ARPE-19 cells by Cadmium (Cd). ARPE-19 cells treated with Cd (0–20 mM) for 12 h, or with Cd (20 mM) for 0–12 h, or with Cd (20 mM) for 10 h without or with NAC (1 mM) were harvested and total lysates were subjected to western blot analysis using antibodies against the indicated proteins. The blots were probed with GAPDH antibodies as a loading control. Similar results were observed in at least three independent experiments. Cd activated phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (Erk1/2), and p38 in a concentration-dependent (A) and time-dependent manner (B). Effects of NAC on Cd-induced phosphorylation of JNK, Erk1/2, and p38 (C).
ARPE-19 cells by pretreating the cells with BSO. Our observations that Cd-induced ROS generation in ARPE-19 cells with BSO treatment increased significantly compared to cells without BSO treatment indicates that with decrease in GSH levels, Cd could cause greater increase in ROS and related pathologies. This fact was strengthened by the observation that NAC prevented the Cdinduced ROS generation significantly in cells with depleted or decreased GSH levels. Furthermore, when intracellular antioxidant defenses are compromised by aging, repeated infections or chemically such as by BSO, Cd would increase toxicity even at low concentrations. In the present study we found that the LC50 for Cd is 20 mM in normal conditions. However, when the ARPE-19 cells were pretreated with BSO to decrease cellular GSH pool, Cd induced significant toxicity even at 10 mM. These results indicate that Cd toxicity is not only dependent on Cd concentration but also on the status of cellular antioxidant defense system including GSH levels in RPE cells. The MAPK pathway is reported to play a role in ROS-induced apoptosis (Chen et al., 2008b). Moreover, recent studies have demonstrated that Cd-induced activation of the MAPK pathway leads to apoptosis in various cell types (Chen et al., 2008a,b; Chuang et al., 2000; Kim et al., 2005; Lag et al., 2005). It is also demonstrated that Cd-induced activation of JNK, Erk1/2, and p38 depends on cell type as well as Cd concentration (Chen et al., 2008b; Kim et al., 2005; Martin et al., 2006). Further, elevated Cd can activate JNK, Erk1/2, and p38 in neuronal PC12 cells and cause apoptosis (Chen et al., 2008a,b). In contrast, Kim et al. (2005) demonstrated Cd-induced activation of JNK and p38 but not Erk1/2 in SH-SY5Y cells. Since in ARPE-19 cells, Cd increases ROS levels which would activate the MAPK pathway. We observed a dose- and time-dependent phosphorylation of JNK, Erk1/2, and p38 in Cd treated ARPE-19 cells (Fig. 5A & B). However, when the cells were
pretreated with NAC, the phosphorylation of JNK, Erk1/2, and p38 could be suppressed (Fig. 5C). These data indicate that Cd-induced activation of MAPK pathway could be mediated by ROS in ARPE-19 cells. This finding is consistent with the results of previous studies (Chen et al., 2008a,b). Glotin et al. (2006) reported a role for Erk1/2 but not JNK and p38 in t-BHP-induced apoptotic signaling in ARPE19 cells. However, Ho et al. (2006) found evidence for JNK and p38 but not Erk1/2 involvement in the mediation of H2O2-induced ARPE-19 cell apoptosis. In view of this paradox, more studies are needed to delineate the role of each member of MAPK signaling pathway in RPE cell apoptosis. Our data indicate that Cd-induced apoptosis can be in part prevented by using antioxidants like NAC. Further work is in progress to define the role of MAPK members using specific inhibitors of this pathway (SP600125 -JNK inhibitor, U0126 -Erk1/2 inhibitor, and SB203580 -p38 MAPK inhibitor) to prevent apoptosis in RPE cells. Besides, MAPK pathway, other signaling pathways such as mTOR has also been implicated in neuronal cell death (Chen et al., 2008a,b). Therefore, detailed signaling studies should be undertaken to understand the mechanism of Cd cytotoxicity and its relationship to smoke-related AMD. In conclusion, we have identified that Cd induces apoptosis of RPE cells by activation of JNK, Erk1/2, and p38. Our findings support the notion that inhibitors of the MAPK pathways may be exploited for prevention of Cd-induced degenerative changes in retina. Acknowledgments This work was supported by National Institutes of Health (NIH) Grants GM71036 (to K.V.R.), DK36118 (to S.K.S.), and Philip Morris Fund (N.W.), Wilkins AMD Fund as well as Research to Prevent Blindness (to FJGMvK).
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References Arisawa, K., Uemura, H., Hiyoshi, M., Takeda, H., Saito, H., Soda, M., 2007. Cadmiuminduced renal dysfunction and mortality in two cohorts: disappearance of the association in a generation born later. Toxicol. Lett. 169 (3), 214–221. Baker, J.R., Edwards, R.J., Lasker, J.M., Moore, M.R., Satarug, S., 2005. Renal and hepatic accumulation of cadmium and lead in the expression of CYP4F2 and CYP2E1. Toxicol. Lett. 159, 182–191. Bertin, G., Averbeck, D., 2006. Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences. Biochimie 88 (11),1549–1559. Bhattacharyya, M.H., Wilson, A.K., Rajan, S.S., Jonah, M., 2000. Biochemical pathways in cadmium toxicity. In: Zalups, R.R., Koropatrick, J. (Eds.), Molecular Biology and Toxicology of Metals. Taylor and Francis, London 2000. Cao, Z., Hardej, D., Trombetta, L.D., Li, Y., 2003. The role of chemically induced glutathione and glutathione S-transferase in protecting against 4-hydroxy-2nonenal-mediated cytotoxicity in vascular smooth muscle cells. Cardiovasc. Toxicol. 3 (2), 165–177. Chen, L., Liu, L., Luo, Y., Huang, S., 2008a. MAPK and mTOR pathways are involved in cadmium-induced neuronal apoptosis. J. Neurochem. 105, 251–261. Chen, L., Liu, L., Huang, S., 2008b. Cadmium activates the mitogen-activated protein kinase (MAPK) pathway via induction of reactive oxygen species and inhibition of protein phosphatases 2A and 5. Free Radic. Biol. Med. 45, 1035–1044. Chuang, S.M., Wang, I.C., Yang, J.L., 2000. Roles of JNK, p38 and ERK mitogen-activated protein kinases in the growth inhibition and apoptosis induced by cadmium. Carcinogenesis 21, 1423–1432. Erie, J.C., Butz, J.A., Good, J.A., Erie, E.A., Burritt, M.F., Cameron, J.D., 2005. Heavy metal concentrations in human eyes. Am. J. Ophthalmol. 139 (5), 888–893. Erie, J.C., Good, J.A., Butz, J., Hodge, D.O., Pulido, J.S., 2007. Urinary cadmium and age related macular degeneration. Am. J. Ophthalmol. 144 (3), 414–418. Friberg, L., 1983. Cadmium. Annu. Rev. Public Health 4, 367–373. Fritsche, L.G., Loenhardt, T., Janssen, A., Fisher, S.A., Rivera, A., et al., 2008. Agerelated macular degeneration is associated with an unstable ARMS2 (LOC387715) mRNA. Nat. Genet. 40, 892–896. Fujihara, M., Nagai, N., Sussan, T.E., Biswal, S., Handa, J.T., 2008. Chronic cigarette smoke causes oxidative damage and apoptosis to retinal pigmented epithelial cells in mice. PLoS ONE 3, e3119. Gallagher, C.M., Kovach, J.S., Meliker, J.R., 2008. Urinary cadmium and osteoporosis in U.S. Women > or ¼ 50 years of age: NHANES. Environ. Health Perspect. 116 (10), 1338–1343. Glotin, A., Calipel, A., Brossas, J., Faussat, A., Treton, J., Mascarelli, F., 2006. Sustained versus transient Erk1/2 signalling underlies the anti- and proapoptotic effects of oxidative stress in human RPE cells. Invest. Ophthalmol. Vis. Sci. 47 (10), 4614–4623. He, L., Girijashanker, K., Dalton, T.P., Reed, J., Li, H., Soleimani, M., Nebert, D.W., 2006. ZIP8, member of the solute-carrier-39 (SLC39) metal-transporter family: characterization of transporter properties. Mol. Pharmacol. 70, 171–180. Ho, T.C., Yang, Y.C., Cheng, H.C., Wu, A.C., Chen, S.L., Chen, H.K., Tsao, Y.P., 2006. Activation of mitogen-activated protein kinases is essential for hydrogen peroxideinduced apoptosis in retinal pigment epithelial cells. Apoptosis 11, 1899–1908. Hollyfield, J.G., Bonilha, V.L., Rayborn, M.E., Yang, X., Shadrach, K.G., Lu, L., Ufret, R.L., Salomon, R.G., Perez, V.L., 2008. Oxidative damage-induced inflammation initiates age related macular degeneration. Nat. Med. 14 (2), 194–198. Horiguchi, H., 2007. Anemia induced by cadmium intoxication. Nippon Eiseigaku Zasshi 62 (3), 888–904. Houston, M.C., 2007. The role of mercury and cadmium heavy metals in vascular disease, hypertension, coronary heart disease, and myocardial infarction. Altern. Ther. Health Med. 13 (2), 128–133. Johnson, S., 2001. Gradual micronutrient accumulation and depletion in Alzheimer’s disease. Med. Hypotheses 56, 595–597. Jung, Y.S., Jeong, E.M., Park, E.K., Kim, Y.M., Sohn, S., Lee, S.H., Baik, E.J., Moon, C.H., 2008. Cadmium induces apoptotic cell death through p38 MAPK in brain microvessel endothelial cells. Eur. J. Pharmacol. 578 (1), 11–18. Kalariya, N.M., Ramana, K.V., Srivastava, S.K., van Kuijk, F.J., 2008. Carotenoid derived aldehydes-induced oxidative stress causes apoptotic cell death in human retinal pigment epithelial cells. Exp. Eye Res. 86 (1), 70–80. Kim, S., Moon, C., Eun, S., Ryu, P., Jo, S., 2005. Identification of ASK1, MKK4, JNK, cJun, and caspase-3 as a signaling cascade involved in cadmium-induced neuronal cell apoptosis. Biochem. Biophys. Res. Commun. 328, 326–334. Klein, R., Knudtson, M.D., Cruickshanks, K.J., Klein, B.E., 2008. Further observations on the association between smoking and the long-term incidence and progression of age-related macular degeneration: the Beaver Dam Eye Study. Arch. Ophthalmol. 126 (1), 115–121. Kukner, A., Colakoglu, N., Kara, H., Oner, H., Ozogul, C., Ozan, E., 2007. Ultrastructural changes in the kidney of rats with acute exposure to cadmium and effects of exogenous metallothionein. Biol. Trace Elem. Res. 119 (2), 137–146. Lag, M., Refsnes, M., Lilleaas, E.M., Holme, J.A., Becher, R., Schwarze, P.E., 2005. Role of mitogen activated protein kinases and protein kinase C in cadmium-induced apoptosis of primary epithelial lung cells. Toxicology 211 (3), 253–264. Lei, Y.X., Wei, L., Wang, M., Wu, G.R., Li, M., 2008. Malignant transformation and abnormal expression of eukaryotic initiation factor in bronchial epithelial cells induced by cadmium chloride. Biomed. Environ. Sci. 21 (4), 332–338.
Li, Z., Arnaud, L., Rockwell, P., Figueiredo-Pereira, M.E., 2004. A single amino acid substitution in a proteasome subunit triggers aggregation of ubiquitinated proteins in stressed neuronal cells. J. Neurochem. 90, 19–28. Liles, M.R., Newsome, D.A., Oliver, P.D., 1991. Antioxidant enzymes in the aging human retinal pigment epithelium. Arch. Ophthalmol. 109 (9), 1285–1288. Lopez, E., Figueroa, S., Oset-Gasque, M., Gonzalez, M.P., 2003. Apoptosis and necrosis: two distinct events induced by cadmium in cortical neurons in culture. Br. J. Pharmacol. 138, 901–911. Mannino, D.M., Holguin, F., Greves, H.M., Savage-Brown, A., Stock, A.L., Jones, R.L., 2004. Urinary cadmium levels predict lower lung function in current and former smokers: data from the Third National Health and Nutrition Examination Survey. Thorax 59 (3), 194–198. Martin, P., Poggi, M.C., Chambard, J.C., Boulukos, K.E., Pognonec, P., 2006. Low dose cadmium poisoning results in sustained ERK phosphorylation and caspase activation. Biochem. Biophys. Res. Commun. 350 (3), 803–807. Meister, A., Anderson, M.E., Hwang, O., 1986. Intracellular cysteine and glutathione delivery systems. J. Am. Coll. Nutr. 5, 137–151. Monroe, R.K., Halvorsen, S.W., 2006. Cadmium blocks receptor-mediated Jak/STAT signaling in neurons by oxidative stress. Free Radic. Biol. Med. 41, 493–502. Nordberg, G.F., Nogawa, K., Nordberg, M., Friberg, L.T., 2007. Cadmium. In: Aitio, A., Andersson, I., Fowler, B.A., Friberg, L.T., Nordberg, G.F., Nordberg, M., Part, P., Skerfving, S. (Eds.), Handbook on the Toxicology of Metals. Elsevier, Amsterdam, pp. 445–486. 2007. Okuda, B., Iwamoto, Y., Tachibana, H., Sugita, M., 1997. Parkinsonism after acute cadmium poisoning. Clin. Neurol. Neurosurg. 99, 263–265. Prozialeck, W.C., Vaidya, V.S., Liu, J., Waalkes, M.P., Edwards, J.R., Lamar, P.C., Bernard, A.M., Dumont, X., Bonventre, J.V., 2007. Kidney injury molecule-1 is an early biomarker of cadmium nephrotoxicity. Kidney Int. 72 (8), 985–993. Ramana, K.V., Bhatnagar, A., Srivastava, S., Yadav, U.C., Awasthi, S., Awasthi, Y.C., Srivastava, S.K., 2006. Mitogenic responses of vascular smooth muscle cells to lipid peroxidation-derived aldehyde 4-hydroxy-trans-2-nonenal (HNE): role of aldose reductase-catalyzed reduction of the HNE-glutathione conjugates in regulating cell growth. J. Biol. Chem. 281 (26), 17652–17660. Rivera, A., Fisher, S.A., Fritsche, L.G., Keilhauer, C.N., Lichtner, P., et al., 2005. Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk. Hum. Mol. Genet. 14, 3227–3236. Rockwell, P., Martinez, J., Papa, L., Gomes, E., 2004. Redox regulates COX-2 upregulation and cell death in the neuronal response to cadmium. Cell. Signal. 16, 343–353. Satarug, S., Moore, M.R., 2004. Adverse health effects of chronic exposure to lowlevel cadmium in foodstuffs and cigarette smoke. Environ. Health Perspect. 112 (10), 1099–1103. Satarug, S., Ujjin, P., Vanavanitkun, Y., Baker, J.R., Moore, M.R., 2004. Influence of body iron store status and cigarette smoking on cadmium body burden of healthy Thai women and men. Toxicol. Lett. 148 (3), 177–185. Satarug, S., Kikuchi, M., Wisedpanichkij, R., Li, B., Takeda, K., Na-Bangchang, K., Moore, M.R., Hirayama, K., Shibahara, S., 2008. Prevention of cadmium accumulation in retinal pigment epithelium with manganese and zinc. Exp. Eye Res. 87 (6), 587–593. Schmidt, S., Hauser, M.A., Scott, W.K., Postel, E.A., Agarwal, A., et al., 2006. Cigarette smoking strongly modifies the association of LOC387715 and age-related macular degeneration. Am. J. Hum. Genet. 78, 852–864. Singh, K.P., Kumari, R., Pevey, C., Jackson, D., Dumond, J.W., 2009. Long duration exposure to cadmium leads to increased cell survival, decreased DNA repair capacity, and genomic instability in mouse testicular Leydig cells. Cancer Lett. 279 (1), 84–92. Srivastava, S.K., Beutler, E., 1969. The transport of oxidized glutathione from the erythrocytes of various species in the presence of chromate. Biochem. J. 114, 833–837. Thijssen, S., Lambrichts, I., Maringwa, J., Van Kerkhove, E., 2007. Changes in expression of fibrotic markers and histopathological alterations in kidneys of mice chronically exposed to low and high Cd doses. Toxicology 238 (2–3), 200–210. Wang, A.L., Lukas, T.J., Yuan, M., Du, N., Handa, J.T., Neufeld, A.H., 2009. Changes in retinal pigment epithelium related to cigarette smoke: possible relevance to smoking as a risk factor for age-related macular degeneration. PLoS ONE 4 (4), e5304. Wills, N.K., Sadagopa Ramanujam, V.M., Chang-Strepka, J., Kalariya, N., Lewis, J.R., van Kuijk, F.J., 2008a. Cadmium accumulation in the aging human retina. Exp. Eye Res. 86 (1), 41–51. Wills, N.K., Sadagopa Ramanujam, V.M., Kalariya, N., Lewis, J.R., van Kuijk, F.J.M., 2008b. Copper and zinc distribution in the human retina: relationship to cadmium accumulation, age, and gender. Exp. Eye Res. 87 (2), 80–88. Wills, N.K., Kalariya, N., Sadagopa Ramanujam, V.M, Lewis, J.R., Haji Abdollahi, S., Husain, A., van Kuijk, F.J., 2009. Human retinal cadmium accumulation as a factor in etiology of age-related macular degeneration. Exp. Eye Res. 89 (1), 79–87. Yadav, U.C., Ramana, K.V., Awasthi, Y.C., Srivastava, S.K., 2008. Glutathione level regulates HNE-induced genotoxicity in human erythroleukemia cells. Toxicol. Appl. Pharmacol. 227 (2), 257–264. Zarbin, M.A., 2004. Current concepts in the pathogenesis of age-related macular degeneration. Arch. Ophthalmol. 122 (4), 598–614.
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Cadmium-induced apoptotic death of human retinal pigment epithelial cells is mediated by MAPK pathway Nilesh M. Kalariya a, Nancy K. Wills b, Kota V. Ramana c, Satish K. Srivastava c, Frederik J.G.M. van Kuijk a, * a
Department of Ophthalmology & Visual Sciences, University of Texas Medical Branch, Galveston, TX 77555, USA Department of Neuroscience & Cell Biology, University of Texas Medical Branch, Galveston, TX 77555, USA c Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 April 2009 Accepted in revised form 12 May 2009 Available online 12 June 2009
Cadmium (Cd), released from cigarette smoke and metal industrial activities, is known to accumulate in human body organs including retina and is particularly higher in retinal tissues of age-related macular degeneration (AMD) eyes compared to non-AMD eyes. We have determined the cytotoxic effects of Cd on human retinal pigment epithelial (RPE) cells. Upon Cd treatment, there was a dose- and time-dependent decline in ARPE-19 cell viability as well as early apoptotic changes such as altered mitochondrial membrane potential (MMP) and Cytochrome C release in cytosol. Depletion of GSH by buthionine-[S,R]sulfoximine (BSO) resulted in increased Cd toxicity in ARPE-19 cells. Cadmium also caused reactive oxygen species (ROS) generation and activation of mitogen-activated protein kinases (MAPKs) pathway including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (Erk1/2), and p38 in ARPE-19 cells. Antioxidants such as N-acetylcysteine (NAC) significantly reduced Cd-induced toxicity. These results indicate that elevated ROS-induced activation of the MAPK signaling pathway could be associated with Cd-induced RPE cell apoptosis, one of the major contributing factors in AMD. The toxic effects of Cd on ARPE-19 cells indicate that environmental heavy metals such as Cd could be important potential factors in RPE cells death associated retinal diseases particularly related to smoking. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: cadmium retinal pigment epithelium oxidative stress apoptosis MAPK AMD
1. Introduction Cadmium (Cd), a heavy metal, has increased in the environment due to industrial activities such as mining and manufacturing. It is also a contaminant in fertilizers and as a result absorbed and gradually accumulated by plants (Nordberg et al., 2007). The uptake of Cd in human body occurs through food intake as well as smoking (Satarug and Moore, 2004; Satarug et al., 2004). The half-life of Cd in the human body is approximately 10–30 years. Although the systemic circulation does not retain Cd, vital organs like liver, kidney, lung, and bone are known to accumulate Cd with age (Baker et al., 2005; Friberg, 1983). Cadmium accumulation in human body organs has been reported to contribute to a variety of pathological conditions such as carcinogenesis, neurodegeneration, lung dysfunction, renal dysfunction, vascular disease, hypertension, coronary heart disease, myocardial infarction, anemia, osteoporosis, Parkinson’s disease, and Alzheimer’s disease (Arisawa et al., 2007; Gallagher
* Corresponding author at: Department of Ophthalmology & Visual Sciences, Room 2.100, 700 University Blvd, University of Texas Medical Branch, Galveston, TX 77555, USA. Tel.: þ1 409 747 5411; fax: þ1 409 747 5402. E-mail address: [email protected] (F.J.G.M. van Kuijk). 0014-4835/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2009.05.011
et al., 2008; Horiguchi, 2007; Houston, 2007; Johnson, 2001; Kim et al., 2005; Lopez et al., 2003; Mannino et al., 2004; Okuda et al., 1997; Singh et al., 2009). Recent reports have demonstrated Cd accumulation in human retina and implicated Cd in development as well as progression of smoking-related age-related macular degeneration (AMD) (Erie et al., 2005; Wills et al., 2008a,b, 2009). The reported Cd levels in the retinal pigment epithelium (RPE)/choroid tissues are approximately double (4000 mg/g tissue) in smokers compared to nonsmokers (1650 mg/g tissue) (Erie et al., 2005). In addition, higher urinary Cd levels, indicating a higher total body burden of Cd, have also been reported in smokers who had AMD compared to smokers who did not have AMD (Erie et al., 2007). We have demonstrated that human retinal tissues accumulate Cd during aging (Wills et al., 2008a). We have also observed gender difference in terms of Cd accumulation. Significantly higher Cd levels have been found in the RPE and choroid from female donor eyes compared to male donor eyes (Wills et al., 2008b). Higher Cd levels have also been found in the neural retina and RPE of AMD eyes (90 and 220 pmol/mg protein, respectively) compared to non-AMD eyes (40 and 120 pmol/mg protein, respectively) (Wills et al., 2009). The accumulation as well as possible toxic effects of Cd in RPE cells could be prevented by manganese (Mn) and zinc (Zn) (Satarug et al., 2008).
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Since Cd and Mn exhibit equal affinity for metal transporter proteins such as ZIP8 and ZIP14, Cd uptake as well as accumulation could be reduced when the cells are treated with Cd and Mn (He et al., 2006). Moreover, Mn could cause induction of heme oxygenase-1 (HO-1) in ARPE-19 cells thereby reducing the toxic effects of Cd (Satarug et al., 2008). These findings have raised the possibility that cadmium exposure might play a role in tobacco-related AMD. Cadmium has been demonstrated to have toxic effects on many cell types, including neurons, lung cells and renal tubules (Kukner et al., 2007; Lei et al., 2008; Lopez et al., 2003; Prozialeck et al., 2007; Thijssen et al., 2007). Cadmium-induced damage in most cells is indirectly caused in part by increased levels of reactive oxygen species (ROS) (Bertin and Averbeck, 2006). Retinal cells and the RPE routinely cope with ROS generated by metabolic processes and light. However, during aging, decreased levels of specific antioxidant enzymes in the retina (Liles et al., 1991) diminish the ability of cells to survive oxidative stress. In view of this fact, it is important to determine whether cadmium accumulation in retina contributes to ROS generation and its possible consequences on RPE cells. In this study we observed that Cd induces ROS production and leads to dose- and time-dependent retinal cell apoptosis. We have also delineated the Cd-induced mechanism of apoptosis in ARPE-19 cells. We found that Cd activates MAPK members JNK, Erk1/2, and p38 in ARPE-19 cells. Antioxidants such as N-acetylcysteine (NAC) prevented ROS generation induced by Cd. Thus activation of MAPK pathway and subsequent cell death could be attenuated. These findings indicate that Cd-induced RPE cell apoptosis could be associated with the activation of the MAPK signaling pathways which could play an important role in smokingrelated AMD. 2. Materials and methods 2.1. Materials Human retinal pigment epithelial cells (ARPE-19) were purchased from American Type Culture Collection (ATCC). Cadmium chloride (CdCl2) and buthionine-[S,R]-sulfoximine (BSO) were purchased from Sigma, USA. JC-1, Dihydroethidium (hydroethidine) dye, TACSÔ Annexin V-FITC Apoptosis detection kit were purchased from Molecular Probes, USA. CellTiter 96Ò AQueous one solution cell proliferation assay kit was purchased from Promega. USA. RIPA cell lysis buffer was purchased from Santa Cruz biotechnology, USA. Protein assay reagent was obtained from Bio-Rad, USA. Fetal bovine serum, trypsin/EDTA, antibiotics, N-acetylcysteine (NAC), 20 , 70 dichlorofluorescin diacetate (DCF-DA) and all other chemicals were obtained from Sigma. Antibodies against the MAPK cascade proteins such as phospho-JNK, JNK, phospho-c jun, c-jun, phosphoErk1/2, Erk1/2, phospho-p38 and p38, were obtained from Cell Signaling Inc. Cytochrome C antibodies were purchased from Santa Cruz, CA, USA. Mouse anti-rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were obtained from Research Diagnostics Inc. 2.2. Cell culture and treatment The ARPE-19 cells were grown to confluency in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 C in a humidified atmosphere of 5% CO2. Sub-confluent cells were growth-arrested in 0.1% FBS medium. Sub culturing was done using trypsin/EDTA solution and the cells were sub cultivated with a split ratio of 1:4. The CdCl2 treatment was done in 0.1% serum medium. Whenever BSO and NAC were used, cells were preincubated with it for 24 and 1 h respectively.
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2.3. Cell viability assay The ARPE-19 cells were plated (5000 cells/well) in a 96-well plate. After 24 h, cells were serum starved in 0.1% FBS medium for 24 h and CdCl2 was added to the media after pretreating the cells with BSO or NAC for 24 or 1 h respectively. Cells incubated without CdCl2 and NAC served as control. Cell viability was determined by CellTiter 96 AQueous one solution cell proliferation assay kit. CellTiter reagent was added to culture well, incubated for 3 h and absorbance recorded at 490 nm using a 96-well micro plate reader (BioTek, USA). 2.4. Determination of apoptosis Apoptosis was determined by using the TACS Annexin V-FITC Apoptosis kit (Molecular Probes) which detects cell surface changes that occur early in the apoptotic process. The assay was performed according to manufacturer’s instructions and cell death was detected by flow cytometry. After growth and starvation the cells were washed with PBS and incubated with CdCl2 and H2O2 (positive control) for 12 h without or with NAC. Pellets of approximately 1 106 cells were suspended in 100 ml of Annexin binding buffer containing Annexin-V and Propidium Iodide (PI) dye solution. After incubation at 37 C for 30 min, 400 ml of Annexin binding buffer was added and solution was subjected to flow cytometry. 2.5. Measurement of mitochondrial membrane potential (MMP) Flow cytometry was performed using the cationic dye JC-1 that exhibits its membrane potential-dependent accumulation in the mitochondria as determined by a fluorescence emission shift from green (525 nm) to red (590 nm). After growth and starvation the cells were washed with PBS and incubated with CdCl2 and H2O2 (positive control) for 5 h without or with NAC. Pellets of approximately 1 106 cells were suspended in 100 ml of PBS containing 5 ml of JC-1 solution (20 mg/ml) was added. After incubation at 37 C for 30 min, cells were washed with PBS, resuspended in 500 ml PBS, and subjected to flow cytometry. 2.6. ROS determination The intracellular ROS levels were measured using a fluorescent dye, DCF-DA. The cells were plated (5000 cells/well) in a 96-well plate. After 24 h, cells were serum starved for 24 h with BSO and/or NAC treatment, washed with PBS and incubated with 10 mM DCFDA at 37 C for 30 min in media without phenol red. Cells were washed again to remove excess DCF-DA and treated with CdCl2 in media without FBS for 30 min. At the end of the treatment, cells were washed twice with PBS. Media without phenol red was added and absorbance determined after 1 h at 485 nm excitation and 538 nm emission wavelengths. Relative ROS production was expressed as a change in fluorescence compared to fluorescence of the appropriate control. The intracellular ROS were also detected using a fluorescent dye, Dihydroethidium. The cells were plated (100,000 cells/Chamber) in a 2-chamber slide. After 24 h, cells were serum starved for 24 h without or with NAC treatment, washed with PBS and incubated with 5 mM Dihydroethidium at RT for 30 min in PBS. Cells were washed again to remove excess Dihydroethidium and treated with CdCl2 for 1 h. At the end of the treatment, cells were washed twice with PBS mounted with Fluorosave (Molecular Probes), covered with the coverslip and the coverslip borders were sealed with nail polish. The cells were visualized under a Nikon Eclipse 800 epifluorescence microscope equipped with a xenon arc lamp using a Texas Red filter set for red fluorescence. Photographs were taken
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using a Roper Scientific CoolSNAP Fx monochrome cooled CCD 12bit digital camera.
3. Results 3.1. Cd causes decreased cell viability
2.7. Western blot analysis After treatment, ARPE-19 cells were washed twice with ice-cold PBS and lysed in ice-cold RIPA lysis buffer for 15 min with occasional vortexing at maximum speed at 4 C. The crude lysates were cleared by centrifugation at 12,000g for 10 min at 4 C. Aliquots of the lysates were diluted with 2 SDS sample buffer and boiled for 5 min. The lysates were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore, Bedford, MA). The membranes were then incubated in blocking solution containing 5% wt/vol dried fat-free milk and 0.1% vol/vol Tween-20 in Tris-buffered saline. Subsequently, the membranes were incubated with appropriate primary antibodies. The membranes were then probed with horseradish peroxidase– conjugated secondary antibody (GE Healthcare, Piscataway, NJ) and visualized by chemiluminescence (Pierce Biotechnology, Rockford, IL). For Cytochrome C analysis, cytoplasmic fractions were obtained by method mentioned elsewhere (Kalariya et al., 2008) 2.8. Statistical analysis Results are expressed as mean S.D. and the p values were determined by Student’s t-test using Microsoft Excel software.
In order to investigate the effects of Cd on cell viability, ARPE-19 cells were incubated with Cd for 24 h. A concentration-dependent (0–80 mM Cd) decrease in cell viability was observed (Fig. 1A). The LC50 for Cd was approximately 20 mM. To elucidate the timedependent effect of Cd, ARPE-19 cells were incubated with 20 mM Cd for various time intervals. A 15% decline in cell viability occurred in the first 6 h and approximately 21% and 40% by 12 h and 24 h, respectively (Fig. 1B). We observed, a significant (p < 0.001) decline in cell viability by 12 h. The RPE cells are known to have high degree of oxidative stress and depleted antioxidants like GSH due to aging as well as pathological states such as AMD. Therefore, we investigated the effect of Cd on cell viability in ARPE-19 cells that were pretreated with BSO to reduce total cellular GSH levels. As shown in Fig. 1C, effects of Cd toxicity increased substantially in cells treated with BSO. The decline in cell viability was highly significant (p < 0.001) even at lower concentration (10 mM) in cells treated with BSO compared to cells without BSO treatment. Cadmium at 10 mM caused 15% or 40% decline in viability of ARPE-19 cells pretreated without or with BSO, respectively (Fig. 1C). These results indicate that the LC50 for Cd and degree of oxidative stress in retinal cells could have an inverse relationship.
Fig. 1. Effect of Cadmium (Cd) on the viability of ARPE-19 cells. (A) Cd caused concentration-dependent decrease in ARPE-19 cell viability. Cells were exposed to various concentrations of Cd (0–80 mM) and after 24 h cell viability was determined. Data represents the mean SD of three experiments (*p < 0.01, **p < 0.001 vs Control). (B) Timedependent effect of Cd on ARPE-19 cell viability. Cells were exposed to 20 mM Cd for 3 h, 6 h, 12 h and 24 h and cell viability was determined. Data represents the mean SD of three experiments (*p < 0.01, **p < 0.001 vs Control). (C) Cd caused substantial decline in ARPE-19 cell viability under reduced or depleted GSH levels by BSO. Cells pretreated with BSO (1 mM) were exposed to various concentrations of Cd (10, 20, and 40 mM) and after 24 h cell viability was determined. Data represents the mean SD of three experiments (*p < 0.01, **p < 0.001 vs Control, #p < 0.001 vs 10, 20, 40 mM Cd without BSO respectively). (D) NAC prevented Cd-induced decline in ARPE-19 cell viability. Cells pretreated with BSO (1 mM) were exposed to NAC (1 mM) followed by Cd (20 mM) and after 24 h cell viability was determined. Data represents the mean SD of three experiments (*p < 0.001 vs Control, **p < 0.001 vs Cd, #p < 0.001 vs Cd, ##p < 0.001 vs Cd þ BSO).
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Since the toxic effect of Cd seemed dependent on the degree of cellular GSH levels, use of antioxidants could be a useful tool to prevent Cd toxicity in retinal cells. Therefore, we used NAC as preventive measure in cells treated without or with BSO. As shown in Fig. 1D, Cd-induced decline in cell viability was significantly (p < 0.001) prevented by NAC in cells treated without or with BSO. These results indicate that status of antioxidant defense could be the determining factor in Cd-induced toxicity in RPE cells. 3.2. Increase in ROS levels by Cd Since our preliminary results on retinal cell viability indicated that Cd-induced toxicity is associated with cellular GSH levels, we investigated if Cd could generate ROS in retinal cells. Incubation of ARPE-19 cells with Cd resulted in a dose-dependent (0–80 mM) increase in ROS levels (Fig. 2A). Cadmium at lower concentrations (5 & 10 mM) did not have a marked effect on ROS generation, but at higher concentration 20 mM, significant increase (p < 0.001) in ROS levels was observed. These results indicate that Cd can be a potent oxidant at 20 mM concentration and could lead to increase in ROS levels and eventual toxicity in retinal cells. This observation was further strengthened by using ROS scavenger, NAC, to ameliorate Cd-induced increase in ROS levels and toxicity. Pretreatment of cells with NAC significantly (p < 0.001) attenuated Cd-induced ROS increase in ARPE-19 cells (Fig. 2A and B). Fluorescence microscopy was also carried out to visualize Cd-induced ROS increase as well as scavenging effect of NAC (Fig. 2B). Cadmium treated cells showed red fluorescence indicating an increase in intracellular ROS levels (Fig. 2B III). The increase in ROS levels was prevented by pretreating the cells with NAC (Fig. 2B IV). Cells in control and NAC-treated groups did not exhibit increase in ROS levels (Fig. 2B I & II). The levels of ROS increased significantly in cells with compromised GSH levels by treatment with BSO compared to cells without BSO treatment. As shown in Fig. 2C, the increase in ROS in BSO þ Cd treated cells was highly significant (p < 0.001) compared to cells without BSO treatment. Moreover, NAC scavenged (p < 0.001) Cdinduced ROS increase in cells pretreated without or with BSO. 3.3. Cd-induced apoptosis in RPE cells A growing body of evidence suggests that increased oxidative stress due to elevated ROS levels in RPE cells could cause apoptotic cell death which is a known pathogenic event in AMD. Therefore, ARPE-19 cells were incubated with Cd (20 mM) or H2O2 (200 mM) without or with NAC (1 mM) for 12 h. An increase in apoptosis was observed when cells were treated with Cd or H2O2 (200 mM), a positive control (Fig. 3C & D, respectively). The Cd- as well as H2O2-induced apoptosis was 32% and 23%, respectively and NAC significantly (p < 0.001) prevented it (Fig. 3E and F). 3.4. Effects of Cd on mitochondrial membrane potential (MMP) The alteration in MMP has been a marker for an early stage of apoptosis. Therefore, the effect of Cd as well as H2O2 on the early stages of apoptosis was also assessed by measuring MMP. ARPE-19 cells were exposed to Cd (20 mM) or H2O2 (200 mM) for 5 h without or with pretreatment with NAC, followed by incubation with JC-1 dye. Flow cytometry analysis revealed that Cd as well as H2O2 treatment caused alteration of MMP in approximately 38% and 27% of the cells, respectively (Fig. 4 C & D) and pretreatment with NAC attenuated Cd-induced change in MMP (Fig. 4 E & F). Fig. 4 A & B represent control and NAC-treated groups, respectively. It is also known that Cytochrome C, which plays a crucial role in apoptosis, is released from mitochondria due to change in MMP. We
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also determined the release of Cytochrome C in cytosol upon Cd treatment. As shown in Fig. 4 G, western blot analysis demonstrates the release of Cytochrome C in Cd treated group. Pretreatment with NAC reduced the release of Cytochrome C from mitochondria. 3.5. Cd-induced activation of MAPK pathway Since we observed that Cd induces ROS generation as well as eventual retinal cell apoptosis, we determined the role of MAPK signaling pathway. In order to determine the role of MAPK members such as JNK, Erk1/2 and p38 in Cd-induced retinal cell apoptosis, ARPE-19 cells were exposed to various concentration of Cd (0–20 mM) for 12 h. As shown in Fig. 5 A, there was robust phosphorylation of JNK, Erk1/2, and p38 with >5 mM Cd. Moreover, when ARPE-19 cells were treated with 20 mM Cd for various time intervals, the robust phosphorylation of JNK, Erk1/2, and p38 was initiated approximately at 4 h and sustained until 12 h (Fig. 5B). The activation of MAPK pathway along with ROS generation in retinal cells suggests that antioxidants could attenuate Cd-induced upstream apoptotic signals in RPE cells. Therefore, we treated ARPE19 cells with 20 mM Cd without or with 1 mM NAC for 10 h. As shown in Fig. 5C, NAC suppressed the phosphorylation of JNK, Erk1/2, and p38. These results suggest that Cd-induced ROS generation could activate the MAPK pathway and trigger apoptosis of the RPE cells. 4. Discussion Smoking has been associated with higher incidence and progression of AMD (Klein et al., 2008; Zarbin, 2004). However, there is little understanding in molecular mechanisms by which smoking could contribute to AMD. Chronic cigarette smoke has been shown to cause oxidative mitochondrial DNA damage, RPE apoptosis, and ultrastructural degeneration of the RPE and Bruch’s membrane in mice (Fujihara et al., 2008; Wang et al., 2009). The toxic elements of smoke, such as Benzo(a)Pyrene (B(a)P), has been shown to cause extensive mitochondrial DNA damage as well as increased lysosomal activity and exocytotic activity in RPE cells as well as in mice (Wang et al., 2009). The B(a)P could also increase complement pathway components surrounding Bruch’s membrane in mice (Wang et al., 2009). Most of the structural abnormalities as well as biochemical markers observed in in vitro and in vivo studies are consistent with findings in AMD patients (Wang et al., 2009). Furthermore, genetic studies have also identified susceptibility locus, i.e. LOC387715, for AMD and that polymorphism of this locus and smoking could confer a higher risk for AMD than either factor alone (Rivera et al., 2005; Fritsche et al., 2008; Schmidt et al., 2006). Thus, smoking could be a potential contributor to AMD and merits further investigation. In this study, we have shown that Cd which increases with smoking could induce cell death in RPE cells in a dose- and timedependent manner. These data are consistent with that observed in neuronal cells (Chen et al., 2008a). The observations that Cd could activate MAPK cascade in ARPE-19 cells is consistent with previous studies which demonstrated that Cd can cause apoptosis in various cell types through activation of MAPK cascade (Chen et al., 2008a,b; Chuang et al., 2000; Jung et al., 2008; Kim et al., 2005; Lag et al., 2005; Rockwell et al., 2004). Further, oxidative stress has been known to play a crucial role in various pathological conditions through activation of apoptotic mechanism. Induction of ROS is a profound feature of many degenerative diseases such as AMD, Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis (Hollyfield et al., 2008; Johnson, 2001; Li et al., 2004; Okuda et al., 1997). Cadmium is a potent oxidative stress and inflammatory agent
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Fig. 2. Effect of Cadmium (Cd) on ROS levels in ARPE-19 cells. (A) Cd generated concentration-dependent elevation in ROS levels in ARPE-19 cells. The cells were incubated with DCF-DA (10 mM) dye for 30 min. After washing with PBS, cells were treated with Cd (0–80 mM) for 1 h without or with NAC (1 mM). Data represents the mean SD of three experiments (*p < 0.05, **p < 0.001 vs Control; #p < 0.05, ##p < 0.001 vs Cd treated group). (B) The serum-starved ARPE-19 cells without or with NAC (1 mM) were treated with the ROS-sensitive dye dihydroethidium (5 mM) for 30 min. Subsequently, the cells were exposed to Cd (20 mM) without or with NAC (1 mM) for 1 h and fluorescence was measured with a Nikon epifluorescence microscope. Panels: (I) Control, (II) 1 mM NAC, (III) 20 mM Cd, (IV) 20 mM Cd þ 1 mM NAC. Picture is a representative of three different sets of experiments. (C) Cd-induced elevation in ROS levels in ARPE-19 cells with depleted GSH levels by BSO. The cells were incubated with BSO (1 mM) for 24 h and/or NAC (1 mM) for 1 h. The cells were washed with PBS and incubated with DCF-DA (10 mM) dye for 30 mints. After washing with PBS, cells were treated with Cd (20 mM) for 1 h. Data represents the mean SD of three experiments (*p < 0.05 vs Control, **p < 0.05 vs Cd, #p < 0.001 vs Cd, ##p < 0.001 vs Cd þ BSO).
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Fig. 3. Cadmium (Cd)-induced apoptosis in ARPE-19 cell. ARPE-19 cells (1 106) were incubated with Cd (20 mM) or H2O2 (200 mM) without or with NAC (1 mM) and harvested after 12 h. Apoptosis was detected by Annexin V-FITC as described in the text. Panels: (A) Control, (B) 1 mM NAC, (C) 20 mM Cd and (D) 200 mM H2O2, (E) 20 mM Cd þ 1 mM NAC, (F) 200 mM H2O2 þ 1 mM NAC. Picture is a representative of three different sets of experiments.
(Bhattacharyya et al., 2000) and there is growing a growing body of evidence suggests that under pathological conditions, Cd-induced excessive ROS generation could alter structure and function of proteins, lipids, and DNA besides activating various signaling pathways which collectively cause apoptosis (Chen et al., 2008b; Kim et al., 2005; Monroe and Halvorsen, 2006). Cadmium toxicity has been related to induction of ROS levels (Chen et al., 2008b; Monroe and Halvorsen, 2006). However, the role of Cd in causing oxidative stress in RPE cells has not been known so far. Our demonstration that Cd can increase ROS levels in ARPE-19 cells is
consistent with many previous studies which have shown Cdinduced ROS generation in many cell types (Chen et al., 2008b; Monroe and Halvorsen, 2006). When intracellular oxidative stress is elevated, GSH is oxidized to GSSG which is transported out of cells (Srivastava and Beutler, 1969; Meister et al., 1986; Ramana et al., 2006). As a result cellular GSH levels are significantly depleted. Therefore, cells are vulnerable to ROS-induced toxicity but supplementing the cells with GSH protects against ROS-induced cytotoxicity (Yadav et al., 2008; Cao et al., 2003). In this study, we have depleted GSH levels in
Fig. 4. Cadmium (Cd)-induced changes in ARPE-19 cell MMP represent an early apoptotic change. ARPE-19 cells (0.5 106) were exposed to Cd (20 mM) or H2O2 (200 mM) for 5 h without or with pretreatment with NAC (1 mM) for 1 h. At the end of experiment, the cells were harvested, washed with PBS, incubated with JC-1 dye for 30 min, followed by 3 wash with PBS, suspended in PBS and subjected to flow cytometry. Panels: (A) Control, (B) 1 mM NAC, (C) 20 mM Cd and (D) 200 mM H2O2, (E) 20 mM Cd þ 1 mM NAC, (F) 200 mM H2O2 þ 1 mM NAC. Picture is a representative of three different sets of experiments. (G) ARPE-19 cells treated with Cd (20 mM) for 5 h without or with NAC were harvested and cytoplasmic fraction were subjected to western blot analysis using antibodies against the Cytochrome C which released into cytoplasm from Mitochondria due to change in MMP. The blots were probed with GAPDH antibodies as a loading control. Similar results were observed in at least three independent experiments.
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Fig. 5. Activation of mitogen-activated protein kinase (MAPK) cascade in ARPE-19 cells by Cadmium (Cd). ARPE-19 cells treated with Cd (0–20 mM) for 12 h, or with Cd (20 mM) for 0–12 h, or with Cd (20 mM) for 10 h without or with NAC (1 mM) were harvested and total lysates were subjected to western blot analysis using antibodies against the indicated proteins. The blots were probed with GAPDH antibodies as a loading control. Similar results were observed in at least three independent experiments. Cd activated phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (Erk1/2), and p38 in a concentration-dependent (A) and time-dependent manner (B). Effects of NAC on Cd-induced phosphorylation of JNK, Erk1/2, and p38 (C).
ARPE-19 cells by pretreating the cells with BSO. Our observations that Cd-induced ROS generation in ARPE-19 cells with BSO treatment increased significantly compared to cells without BSO treatment indicates that with decrease in GSH levels, Cd could cause greater increase in ROS and related pathologies. This fact was strengthened by the observation that NAC prevented the Cdinduced ROS generation significantly in cells with depleted or decreased GSH levels. Furthermore, when intracellular antioxidant defenses are compromised by aging, repeated infections or chemically such as by BSO, Cd would increase toxicity even at low concentrations. In the present study we found that the LC50 for Cd is 20 mM in normal conditions. However, when the ARPE-19 cells were pretreated with BSO to decrease cellular GSH pool, Cd induced significant toxicity even at 10 mM. These results indicate that Cd toxicity is not only dependent on Cd concentration but also on the status of cellular antioxidant defense system including GSH levels in RPE cells. The MAPK pathway is reported to play a role in ROS-induced apoptosis (Chen et al., 2008b). Moreover, recent studies have demonstrated that Cd-induced activation of the MAPK pathway leads to apoptosis in various cell types (Chen et al., 2008a,b; Chuang et al., 2000; Kim et al., 2005; Lag et al., 2005). It is also demonstrated that Cd-induced activation of JNK, Erk1/2, and p38 depends on cell type as well as Cd concentration (Chen et al., 2008b; Kim et al., 2005; Martin et al., 2006). Further, elevated Cd can activate JNK, Erk1/2, and p38 in neuronal PC12 cells and cause apoptosis (Chen et al., 2008a,b). In contrast, Kim et al. (2005) demonstrated Cd-induced activation of JNK and p38 but not Erk1/2 in SH-SY5Y cells. Since in ARPE-19 cells, Cd increases ROS levels which would activate the MAPK pathway. We observed a dose- and time-dependent phosphorylation of JNK, Erk1/2, and p38 in Cd treated ARPE-19 cells (Fig. 5A & B). However, when the cells were
pretreated with NAC, the phosphorylation of JNK, Erk1/2, and p38 could be suppressed (Fig. 5C). These data indicate that Cd-induced activation of MAPK pathway could be mediated by ROS in ARPE-19 cells. This finding is consistent with the results of previous studies (Chen et al., 2008a,b). Glotin et al. (2006) reported a role for Erk1/2 but not JNK and p38 in t-BHP-induced apoptotic signaling in ARPE19 cells. However, Ho et al. (2006) found evidence for JNK and p38 but not Erk1/2 involvement in the mediation of H2O2-induced ARPE-19 cell apoptosis. In view of this paradox, more studies are needed to delineate the role of each member of MAPK signaling pathway in RPE cell apoptosis. Our data indicate that Cd-induced apoptosis can be in part prevented by using antioxidants like NAC. Further work is in progress to define the role of MAPK members using specific inhibitors of this pathway (SP600125 -JNK inhibitor, U0126 -Erk1/2 inhibitor, and SB203580 -p38 MAPK inhibitor) to prevent apoptosis in RPE cells. Besides, MAPK pathway, other signaling pathways such as mTOR has also been implicated in neuronal cell death (Chen et al., 2008a,b). Therefore, detailed signaling studies should be undertaken to understand the mechanism of Cd cytotoxicity and its relationship to smoke-related AMD. In conclusion, we have identified that Cd induces apoptosis of RPE cells by activation of JNK, Erk1/2, and p38. Our findings support the notion that inhibitors of the MAPK pathways may be exploited for prevention of Cd-induced degenerative changes in retina. Acknowledgments This work was supported by National Institutes of Health (NIH) Grants GM71036 (to K.V.R.), DK36118 (to S.K.S.), and Philip Morris Fund (N.W.), Wilkins AMD Fund as well as Research to Prevent Blindness (to FJGMvK).
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References Arisawa, K., Uemura, H., Hiyoshi, M., Takeda, H., Saito, H., Soda, M., 2007. Cadmiuminduced renal dysfunction and mortality in two cohorts: disappearance of the association in a generation born later. Toxicol. Lett. 169 (3), 214–221. Baker, J.R., Edwards, R.J., Lasker, J.M., Moore, M.R., Satarug, S., 2005. Renal and hepatic accumulation of cadmium and lead in the expression of CYP4F2 and CYP2E1. Toxicol. Lett. 159, 182–191. Bertin, G., Averbeck, D., 2006. Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences. Biochimie 88 (11),1549–1559. Bhattacharyya, M.H., Wilson, A.K., Rajan, S.S., Jonah, M., 2000. Biochemical pathways in cadmium toxicity. In: Zalups, R.R., Koropatrick, J. (Eds.), Molecular Biology and Toxicology of Metals. Taylor and Francis, London 2000. Cao, Z., Hardej, D., Trombetta, L.D., Li, Y., 2003. The role of chemically induced glutathione and glutathione S-transferase in protecting against 4-hydroxy-2nonenal-mediated cytotoxicity in vascular smooth muscle cells. Cardiovasc. Toxicol. 3 (2), 165–177. Chen, L., Liu, L., Luo, Y., Huang, S., 2008a. MAPK and mTOR pathways are involved in cadmium-induced neuronal apoptosis. J. Neurochem. 105, 251–261. Chen, L., Liu, L., Huang, S., 2008b. Cadmium activates the mitogen-activated protein kinase (MAPK) pathway via induction of reactive oxygen species and inhibition of protein phosphatases 2A and 5. Free Radic. Biol. Med. 45, 1035–1044. Chuang, S.M., Wang, I.C., Yang, J.L., 2000. Roles of JNK, p38 and ERK mitogen-activated protein kinases in the growth inhibition and apoptosis induced by cadmium. Carcinogenesis 21, 1423–1432. Erie, J.C., Butz, J.A., Good, J.A., Erie, E.A., Burritt, M.F., Cameron, J.D., 2005. Heavy metal concentrations in human eyes. Am. J. Ophthalmol. 139 (5), 888–893. Erie, J.C., Good, J.A., Butz, J., Hodge, D.O., Pulido, J.S., 2007. Urinary cadmium and age related macular degeneration. Am. J. Ophthalmol. 144 (3), 414–418. Friberg, L., 1983. Cadmium. Annu. Rev. Public Health 4, 367–373. Fritsche, L.G., Loenhardt, T., Janssen, A., Fisher, S.A., Rivera, A., et al., 2008. Agerelated macular degeneration is associated with an unstable ARMS2 (LOC387715) mRNA. Nat. Genet. 40, 892–896. Fujihara, M., Nagai, N., Sussan, T.E., Biswal, S., Handa, J.T., 2008. Chronic cigarette smoke causes oxidative damage and apoptosis to retinal pigmented epithelial cells in mice. PLoS ONE 3, e3119. Gallagher, C.M., Kovach, J.S., Meliker, J.R., 2008. Urinary cadmium and osteoporosis in U.S. Women > or ¼ 50 years of age: NHANES. Environ. Health Perspect. 116 (10), 1338–1343. Glotin, A., Calipel, A., Brossas, J., Faussat, A., Treton, J., Mascarelli, F., 2006. Sustained versus transient Erk1/2 signalling underlies the anti- and proapoptotic effects of oxidative stress in human RPE cells. Invest. Ophthalmol. Vis. Sci. 47 (10), 4614–4623. He, L., Girijashanker, K., Dalton, T.P., Reed, J., Li, H., Soleimani, M., Nebert, D.W., 2006. ZIP8, member of the solute-carrier-39 (SLC39) metal-transporter family: characterization of transporter properties. Mol. Pharmacol. 70, 171–180. Ho, T.C., Yang, Y.C., Cheng, H.C., Wu, A.C., Chen, S.L., Chen, H.K., Tsao, Y.P., 2006. Activation of mitogen-activated protein kinases is essential for hydrogen peroxideinduced apoptosis in retinal pigment epithelial cells. Apoptosis 11, 1899–1908. Hollyfield, J.G., Bonilha, V.L., Rayborn, M.E., Yang, X., Shadrach, K.G., Lu, L., Ufret, R.L., Salomon, R.G., Perez, V.L., 2008. Oxidative damage-induced inflammation initiates age related macular degeneration. Nat. Med. 14 (2), 194–198. Horiguchi, H., 2007. Anemia induced by cadmium intoxication. Nippon Eiseigaku Zasshi 62 (3), 888–904. Houston, M.C., 2007. The role of mercury and cadmium heavy metals in vascular disease, hypertension, coronary heart disease, and myocardial infarction. Altern. Ther. Health Med. 13 (2), 128–133. Johnson, S., 2001. Gradual micronutrient accumulation and depletion in Alzheimer’s disease. Med. Hypotheses 56, 595–597. Jung, Y.S., Jeong, E.M., Park, E.K., Kim, Y.M., Sohn, S., Lee, S.H., Baik, E.J., Moon, C.H., 2008. Cadmium induces apoptotic cell death through p38 MAPK in brain microvessel endothelial cells. Eur. J. Pharmacol. 578 (1), 11–18. Kalariya, N.M., Ramana, K.V., Srivastava, S.K., van Kuijk, F.J., 2008. Carotenoid derived aldehydes-induced oxidative stress causes apoptotic cell death in human retinal pigment epithelial cells. Exp. Eye Res. 86 (1), 70–80. Kim, S., Moon, C., Eun, S., Ryu, P., Jo, S., 2005. Identification of ASK1, MKK4, JNK, cJun, and caspase-3 as a signaling cascade involved in cadmium-induced neuronal cell apoptosis. Biochem. Biophys. Res. Commun. 328, 326–334. Klein, R., Knudtson, M.D., Cruickshanks, K.J., Klein, B.E., 2008. Further observations on the association between smoking and the long-term incidence and progression of age-related macular degeneration: the Beaver Dam Eye Study. Arch. Ophthalmol. 126 (1), 115–121. Kukner, A., Colakoglu, N., Kara, H., Oner, H., Ozogul, C., Ozan, E., 2007. Ultrastructural changes in the kidney of rats with acute exposure to cadmium and effects of exogenous metallothionein. Biol. Trace Elem. Res. 119 (2), 137–146. Lag, M., Refsnes, M., Lilleaas, E.M., Holme, J.A., Becher, R., Schwarze, P.E., 2005. Role of mitogen activated protein kinases and protein kinase C in cadmium-induced apoptosis of primary epithelial lung cells. Toxicology 211 (3), 253–264. Lei, Y.X., Wei, L., Wang, M., Wu, G.R., Li, M., 2008. Malignant transformation and abnormal expression of eukaryotic initiation factor in bronchial epithelial cells induced by cadmium chloride. Biomed. Environ. Sci. 21 (4), 332–338.
Li, Z., Arnaud, L., Rockwell, P., Figueiredo-Pereira, M.E., 2004. A single amino acid substitution in a proteasome subunit triggers aggregation of ubiquitinated proteins in stressed neuronal cells. J. Neurochem. 90, 19–28. Liles, M.R., Newsome, D.A., Oliver, P.D., 1991. Antioxidant enzymes in the aging human retinal pigment epithelium. Arch. Ophthalmol. 109 (9), 1285–1288. Lopez, E., Figueroa, S., Oset-Gasque, M., Gonzalez, M.P., 2003. Apoptosis and necrosis: two distinct events induced by cadmium in cortical neurons in culture. Br. J. Pharmacol. 138, 901–911. Mannino, D.M., Holguin, F., Greves, H.M., Savage-Brown, A., Stock, A.L., Jones, R.L., 2004. Urinary cadmium levels predict lower lung function in current and former smokers: data from the Third National Health and Nutrition Examination Survey. Thorax 59 (3), 194–198. Martin, P., Poggi, M.C., Chambard, J.C., Boulukos, K.E., Pognonec, P., 2006. Low dose cadmium poisoning results in sustained ERK phosphorylation and caspase activation. Biochem. Biophys. Res. Commun. 350 (3), 803–807. Meister, A., Anderson, M.E., Hwang, O., 1986. Intracellular cysteine and glutathione delivery systems. J. Am. Coll. Nutr. 5, 137–151. Monroe, R.K., Halvorsen, S.W., 2006. Cadmium blocks receptor-mediated Jak/STAT signaling in neurons by oxidative stress. Free Radic. Biol. Med. 41, 493–502. Nordberg, G.F., Nogawa, K., Nordberg, M., Friberg, L.T., 2007. Cadmium. In: Aitio, A., Andersson, I., Fowler, B.A., Friberg, L.T., Nordberg, G.F., Nordberg, M., Part, P., Skerfving, S. (Eds.), Handbook on the Toxicology of Metals. Elsevier, Amsterdam, pp. 445–486. 2007. Okuda, B., Iwamoto, Y., Tachibana, H., Sugita, M., 1997. Parkinsonism after acute cadmium poisoning. Clin. Neurol. Neurosurg. 99, 263–265. Prozialeck, W.C., Vaidya, V.S., Liu, J., Waalkes, M.P., Edwards, J.R., Lamar, P.C., Bernard, A.M., Dumont, X., Bonventre, J.V., 2007. Kidney injury molecule-1 is an early biomarker of cadmium nephrotoxicity. Kidney Int. 72 (8), 985–993. Ramana, K.V., Bhatnagar, A., Srivastava, S., Yadav, U.C., Awasthi, S., Awasthi, Y.C., Srivastava, S.K., 2006. Mitogenic responses of vascular smooth muscle cells to lipid peroxidation-derived aldehyde 4-hydroxy-trans-2-nonenal (HNE): role of aldose reductase-catalyzed reduction of the HNE-glutathione conjugates in regulating cell growth. J. Biol. Chem. 281 (26), 17652–17660. Rivera, A., Fisher, S.A., Fritsche, L.G., Keilhauer, C.N., Lichtner, P., et al., 2005. Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk. Hum. Mol. Genet. 14, 3227–3236. Rockwell, P., Martinez, J., Papa, L., Gomes, E., 2004. Redox regulates COX-2 upregulation and cell death in the neuronal response to cadmium. Cell. Signal. 16, 343–353. Satarug, S., Moore, M.R., 2004. Adverse health effects of chronic exposure to lowlevel cadmium in foodstuffs and cigarette smoke. Environ. Health Perspect. 112 (10), 1099–1103. Satarug, S., Ujjin, P., Vanavanitkun, Y., Baker, J.R., Moore, M.R., 2004. Influence of body iron store status and cigarette smoking on cadmium body burden of healthy Thai women and men. Toxicol. Lett. 148 (3), 177–185. Satarug, S., Kikuchi, M., Wisedpanichkij, R., Li, B., Takeda, K., Na-Bangchang, K., Moore, M.R., Hirayama, K., Shibahara, S., 2008. Prevention of cadmium accumulation in retinal pigment epithelium with manganese and zinc. Exp. Eye Res. 87 (6), 587–593. Schmidt, S., Hauser, M.A., Scott, W.K., Postel, E.A., Agarwal, A., et al., 2006. Cigarette smoking strongly modifies the association of LOC387715 and age-related macular degeneration. Am. J. Hum. Genet. 78, 852–864. Singh, K.P., Kumari, R., Pevey, C., Jackson, D., Dumond, J.W., 2009. Long duration exposure to cadmium leads to increased cell survival, decreased DNA repair capacity, and genomic instability in mouse testicular Leydig cells. Cancer Lett. 279 (1), 84–92. Srivastava, S.K., Beutler, E., 1969. The transport of oxidized glutathione from the erythrocytes of various species in the presence of chromate. Biochem. J. 114, 833–837. Thijssen, S., Lambrichts, I., Maringwa, J., Van Kerkhove, E., 2007. Changes in expression of fibrotic markers and histopathological alterations in kidneys of mice chronically exposed to low and high Cd doses. Toxicology 238 (2–3), 200–210. Wang, A.L., Lukas, T.J., Yuan, M., Du, N., Handa, J.T., Neufeld, A.H., 2009. Changes in retinal pigment epithelium related to cigarette smoke: possible relevance to smoking as a risk factor for age-related macular degeneration. PLoS ONE 4 (4), e5304. Wills, N.K., Sadagopa Ramanujam, V.M., Chang-Strepka, J., Kalariya, N., Lewis, J.R., van Kuijk, F.J., 2008a. Cadmium accumulation in the aging human retina. Exp. Eye Res. 86 (1), 41–51. Wills, N.K., Sadagopa Ramanujam, V.M., Kalariya, N., Lewis, J.R., van Kuijk, F.J.M., 2008b. Copper and zinc distribution in the human retina: relationship to cadmium accumulation, age, and gender. Exp. Eye Res. 87 (2), 80–88. Wills, N.K., Kalariya, N., Sadagopa Ramanujam, V.M, Lewis, J.R., Haji Abdollahi, S., Husain, A., van Kuijk, F.J., 2009. Human retinal cadmium accumulation as a factor in etiology of age-related macular degeneration. Exp. Eye Res. 89 (1), 79–87. Yadav, U.C., Ramana, K.V., Awasthi, Y.C., Srivastava, S.K., 2008. Glutathione level regulates HNE-induced genotoxicity in human erythroleukemia cells. Toxicol. Appl. Pharmacol. 227 (2), 257–264. Zarbin, M.A., 2004. Current concepts in the pathogenesis of age-related macular degeneration. Arch. Ophthalmol. 122 (4), 598–614.