15-deoxy-delta 12, 14-Prostaglandin J2 prevents reactive oxygen species generation and mitochondrial membrane depolarization induced by oxidative stress

doi:10.1186/1471-2210-4-6

BMC Pharmacol. 2004; 4: 6.

Published online 2004 May 18. doi: 10.1186/1471-2210-4-6

PMCID: PMC446193

Copyright © 2004 Garg and Chang; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

15-deoxy-delta 12, 14-Prostaglandin J₂prevents reactive oxygen species generation and mitochondrial membrane depolarization induced by oxidative stress

Tarun K Garg¹ and Jason Y Chang^1,²

¹Department of Neurobiology & Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

²Department of Ophthalmology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

Corresponding author.

Tarun K Garg: gargtarunk/at/uams.edu; Jason Y Chang: changjasony/at/uams.edu

Received December 26, 2003; Accepted May 18, 2004.

This article has been cited by other articles in PMC.

Abstract

Background

With the use of cultured human retinal pigment epithelial cells, we have previously described a number of cellular responses to oxidative stress caused by H₂O₂. We also demonstrated that the cytotoxicity caused by H₂O₂could be prevented by the prostaglandin derivative, 15-deoxy-delta 12, 14-Prostaglandin J₂(15d-PGJ₂).

Results

Further characterization of the experimental system indicated that the half-life of H₂O₂in cultures was ~1 hour. At a fixed H₂O₂concentration, the cytotoxicity was dependent on the volume of H₂O₂solution used in the culture, such that higher volume caused more cytotoxicity. Most cells were committed to die if the culture was treated for 2 hours with a cytotoxic concentration of H₂O₂. The prostaglandin derivative, 15d-PGJ₂, could prevent oxidative damage caused by t-butyl hydroperoxide, in addition to H₂O₂. Further studies indicated that both H₂O₂and tBH caused an increase in reactive oxygen species and depolarization of mitochondrial membrane potential. Pretreatment of cells with 1 μM 15d-PGJ₂led to a modest decrease in reactive oxygen species generation, and a significant restoration of mitochondrial membrane potential.

Conclusion

This agent may be used in the future as a pharmacological tool for preventing cellular damage caused by oxidative stress.

Keywords: 15d-PGJ₂, oxidative stress, ROS (reactive oxygen species), MMP (mitochondrial membrane potential), RPE (retinal pigment epithelium).

Background

Oxidative stress on retinal pigment epithelial cells (RPE) has been suggested as a cause of age-related macular degeneration [1,2]. With the use of primary cultures of human RPE and the ARPE-19 cell line, we reported earlier that oxidative stress caused a number of structural and biochemical changes [3]. For example, cytoskeletal changes were observed shortly after RPE cells were treated with H₂O₂. This was accompanied by activation of the ERK (Extracellular signal-regulated Kinase) MAP kinase, and formation of lipid peroxidation products (4-hydroxynonanel protein adducts). Nuclear condensation was observed 12 hours after H₂O₂treatment, and cells died in a manner consistent with apoptosis.

In an effort to identify pharmacological agents capable of preventing oxidative stress-induced cytotoxicity in RPE, we found the prostaglandin derivative, 15-deoxy-delta 12, 14-Prostaglandin J₂(15d-PGJ₂), could prevent H₂O₂-induced cytotoxicity both in primary human RPE cultures and in ARPE-19 cells [3]. Even though 15d-PGJ₂is a ligand for peroxisome proliferator-activated receptors (PPARs) [4,5], further studies indicated that the saving effect of 15d-PGJ₂was not shared by other agonists of PPARs. It was thus concluded that this agent saved these cells in a way not involving PPAR activation. In other experimental systems, 15d-PGJ₂has been shown to have a potent inhibitory effect on immune cell functions [6,7] and is effective in preventing disease progression of the Experimental Autoimmune Encephalomyelitis, an animal model of the autoimmune disease multiple sclerosis [8].

A series of intracellular events occur as a result of oxidative stress, which may eventually lead to apoptosis. For example, an intracellular increase in reactive oxygen species (ROS) is accompanied by depolarization of the mitochondrial membrane potential (MMP, ΔΨm) [9]. This is followed by the release of cytochrome c from mitochondria, caspase activation and other events that lead to apoptosis [10]. A study of mitochondrial alterations is an important aspect of apoptosis, and is a subject of recent reviews [11,12]. Oxidative stress-induced mitochondrial DNA damage in RPE has been suggested to be a cause of age-related macular degeneration [13,14]. The current study was designed to test the hypothesis that 15d-PGJ₂prevented oxidative stress-induced RPE cell death by preventing early events caused by oxidative stress, such as the generation of intracellular ROS and the depolarization of mitochondrial membrane potential. While we used H₂O₂as the primary source of oxidative stress in most experiments, we also used t-butyl hydroperoxide (tBH) [15] in this study to test the validity of results obtained with H₂O₂.

Results

H₂O₂degradation and its effect on cell viability

We have previously shown that H₂O₂caused a dose- and time-dependent decrease in cellular viability in ARPE-19 cells. Furthermore, the cytotoxicity of H₂O₂was dependent on the cell density in each well [3]. If ARPE-19 cells were plated at 20,000 cells/well in a 96-well plate for two days, 2 mM H₂O₂(100 μl/well) decreased cell viability to ~15% of controls after a 1-day treatment while 1 mM H₂O₂had little effect on cell viability under identical experimental conditions.

As a further analysis of the experimental system, experiments were performed to determine the degradation of H₂O₂in the cultures, and its effect on cell viability. Cells were treated with 2 mM H₂O₂for various periods of time to assess the H₂O₂concentration that remained in each well. Results (Fig. (Fig.1A,1A, open squares) indicated that the readings were so close to background that H₂O₂degradation could not be adequately assessed. Subsequent experiments using 5 mM or 10 mM H₂O₂indicated that H₂O₂degraded in a time-dependent manner (Fig. (Fig.1A),1A), with a half-life of ~1 hour (Fig. (Fig.1B1B).

Figure 1

Degradation of H₂O₂and its effect on cellular viability. (A): Degradation of H₂O₂in cell cultures. ARPE-19 cells were plated in a 24-well plate (140,000 cells/well) for a day, fed with serum-free medium for a day, then treated with 700 μl of (more ...)

The decrease in H₂O₂concentration was inversely related to the volume of H₂O₂solution in each well, such that cultures with more H₂O₂solution maintained H₂O₂concentration better (Fig. (Fig.1C).1C). For example, the OD readings in cells treated with 2,100 μl, 1,400 μl or 700 μl (10 mM H₂O₂) per well were 0.30, 0.23 or 0.15, respectively after 1 hour (open diamonds), and 0.21, 0.15 or 0.07, respectively, after 2 hours (open triangles). Without cells, the H₂O₂concentrations remained almost constant regardless of the volume added to each well (Fig. (Fig.1C,1C, solid diamonds).

Based on these results, it was hypothesized that at a fixed H₂O₂concentration and a fixed cell density, the volume of H₂O₂solution added to each well should affect the cytotoxicity of H₂O₂. To test this, a set of cells grown in 96-well plates were treated with H₂O₂of various volumes for one day, then the viability of each well was determined by the MTT assay. Results indicated that while 100 μl of 2 mM H₂O₂decreased the viability to ~20% of controls, 50 μl of 2 mM H₂O₂had little effect on the viability (Fig. (Fig.1D,1D, solid squares). Similar volume-dependent cytotoxicity was observed with 1.5 mM and 1 mM H₂O₂. At 1.5 mM H₂O₂, it took 100 μl to decrease the viability to ~50% of controls (Fig. (Fig.1D,1D, open circles).

In view of these results, in addition to maintaining a constant cell density, the volume of H₂O₂solution used in this study was adjusted proportionally throughout according to the surface area of the culture wells. For example, while 100 μl/well was used in a 96-well plate, 700 μl/well was used in a 24-well plate.

Temporal commitment point of oxidative stress induced cytotoxicity

Results from Fig. Fig.1D1D indicated that if cells were treated with 100 μl of 2 mM H₂O₂, the majority of cells would die by 24 hours after treatment. The length of time required for cells to commit to death under these conditions was determined. Cultures were treated with 2 mM H₂O₂at time zero, and then H₂O₂was replaced with fresh medium from a set of cells at 30-minute intervals. Viability was then determined 24 hours later. Results (Fig. (Fig.2,2, solid circles) indicated that if cells were treated with 2 mM H₂O₂for 1 hour followed by fresh medium (without H₂O₂) for 23 hours, the viability decreased to ~75% of control. If the cells were treated with H₂O₂for 2 hours, then with fresh medium for 22 hours, the viability decreased to ~10% of control. It was thus concluded that with 2 mM H₂O₂treatment, most cells were committed to die 2 hours after treatment.

Figure 2

Temporal commitment point of H₂O₂and tBH. Cells plated in 96-well plates for 2 days were treated with 2 mM H₂O₂(solid circles) or 600 μM tBH (open circles) for various duration as indicated, then with fresh medium (without the testing agent) (more ...)

In a set of analogous experiments, the commitment point of cells after tBH treatment was determined. Approximately 80% of cells survived 600 μM tBH treatment if this agent was removed within 2 hours of treatment (Fig. (Fig.2,2, open circles). On the other hand, most cells were committed to die if the treatment continued for 4 hours (see below for more experiments involving tBH).

Effect of 15d-PGJ₂on H₂O₂-induced ROS generation

We have previously shown that pretreatment of ARPE-19 cells with 15d-PGJ₂greatly reduced the cytotoxicity caused by H₂O₂[3]. For example, while H₂O₂at 1.3 mM, 1.4 mM or 1.5 mM reduced the viability to 64%, 46% or 28% of controls, pretreatment of these cells with 1 μM 15d-PGJ₂raised the viability to 95%, 84% or 68% of control, respectively. Similar saving effects were also observed in primary cultures of human RPE. Since H₂O₂could induce intracellular generation of ROS, experiments were performed to determine whether 15d-PGJ₂could reduce intracellular ROS levels after H₂O₂treatment, thereby reducing the H₂O₂-induced cytotoxicity.

Initial experiments indicated that H₂O₂treatment of cells caused a dose-dependent increase in ROS levels in ARPE-19 cultures, as indicated by the conversion of H₂DCF-DA to its oxidized form, DCF-DA. The maximal ROS generation was caused by 1 mM H₂O₂, which reached ~3× of basal levels (Fig. (Fig.3A).3A). It is of interest to note that total DCF-DA generated in each well was ~2× as much as that associated with cells. For example, while H₂O₂at 250, 500 or 1000 μM generated ~55 FU, 67 FU or 70 FU of DCF-DA in each well (Fig. (Fig.3A,3A, solid circles), the "cell associated" DCF-DA was ~28 FU, 33 FU or 35 FU, respectively (Fig. (Fig.3A,3A, open circles). DCF-DA formation appears to occur intracellularly, because without cells, H₂O₂did not cause conversion of H₂DCF-DA to DCF-DA (data not shown). Together, these results indicate that approximately half of the DCF-DA generated inside the cells diffused out of the cells.

Figure 3

Effect of 15d-PGJ₂on H₂O₂-induced ROS generation. (A): ROS generation by H₂O₂. Cells were treated with 10 μM H₂DCF-DA for 20 min, followed by various concentrations of H₂O₂for 20 min, then the "total" and "cell-associated" ROS generated was (more ...)

15d-PGJ₂was tested to see if it could prevent H₂O₂induced ROS elevation. Cells were pretreated with 1 μM 15d-PGJ₂overnight, challenged with 1 mM H₂O₂the next day for 20 minutes, then ROS in each well was determined. Results (Fig. (Fig.3B)3B) indicated that while 1 mM H₂O₂generated ~132 FU "total" ROS in this set of experiments, 15d-PGJ₂pretreatment reduced ROS to ~108 FU (p < 0.005). Similarly, the "cell associated" ROS was reduced by 15d-PGJ₂from ~64 to ~54 FU (p < 0.005). The decrease in ROS levels caused by 15d-PGJ₂may be partially responsible for its ability to prevent H₂O₂-induced cytotoxicity.

H₂O₂-induced mitochondrial membrane depolarization in ARPE-19 cells

Other than ROS generation, H₂O₂treatment of cells leads to depolarization of mitochondrial membrane potential, which can be measured by the JC-1 dye [12]. Depolarization of mitochondrial membrane causes a shift in the emission spectrum from red to green color, which can be quantified by a fluorescence plate reader. Initial experiments indicated that the emission intensity of the green peak and the red peak could be determined optimally at ~545 nm and ~595 nm, respectively (see Fig. Fig.5A).5A). Treatment of cells with H₂O₂led to an alteration of the relative intensity of these two peaks (See Fig. Fig.5C).5C). The ratio of readings at 545 nm and 595 nm was thus used to assess mitochondrial membrane potential. A higher 545/595 emission intensity ratio suggests more mitochondrial depolarization.

Figure 5

Prevention of H₂O₂-induced mitochondrial membrane depolarization by 15d-PGJ₂. A-D: The JC-1 emission spectra between 520 nm to 620 nm were determined for cells under various conditions. (A): Untreated cells; (B): Cells treated with 1 μM 15d-PGJ (more ...)

The relative intensity of the 545/595 peaks as a function of H₂O₂concentration and duration of exposure was determined. Results indicated that H₂O₂treatment for 30 min caused a slight dose-dependent increase of both peaks (Fig. (Fig.4A).4A). Similar observations were made in cells treated with various concentrations of H₂O₂for 1 hour (Fig. (Fig.4B).4B). There was little change in 545/595 emission intensity ratio (not shown).

Figure 4

H₂O₂caused depolarization of mitochondrial membrane potential. Cells were treated with various concentrations of H₂O₂for 0.5 hour (A), 1 hour (B), 2 hours (C) or 4 hours (D), then processed for the JC-1 assay as described in the Materials and Methods. (more ...)

A change of the relative intensities of the 545 nm peak and 595 nm peak occurred when cells were treated with higher concentrations of H₂O₂for 2 hours. Untreated cells at this time had an emission reading of 173 FU at 545 nm (Fig. (Fig.4C,4C, open square at 0 mM H₂O₂), and 377 FU at 595 nm (Fig. (Fig.4C,4C, solid circle at 0 mM H₂O₂). The 545/595 ratio was ~0.46 (i.e., 173/377). Cells treated with 0.5 mM or 1 mM H₂O₂had emission readings at 545 nm or 595 nm slightly higher than those of controls. The 545/595 emission intensity ratio was similar to that in untreated cells (Fig. (Fig.4C).4C). However, cells treated with 1.5 mM H₂O₂had an increased emission reading at 545 nm and a decreased reading at 595 nm, such that these two readings were almost identical (Fig. (Fig.4C4C at 1.5 mM). With 2 mM H₂O₂treatment, the reading at 545 nm was higher than that of 595 nm (Fig. (Fig.4C4C).

At 4 hours after treatment, there was a further decrease in the emission readings at 595 nm in cells treated with 1.5 mM or 2 mM H₂O₂(as compared to untreated cells, see Fig. Fig.4D,4D, open circles), and a corresponding increase in the emission readings at 545 nm (Fig. (Fig.4D,4D, solid circles).

Results from Fig. 4A,4B,4C,4D were re-plotted to show the change of 545/595 emission intensity ratio as a function of H₂O₂concentrations and duration of treatment. There was little change of the ratio when cells were treated with 1 mM H₂O₂up to 4 hours (Fig. (Fig.4E,4E, open circles). Most cells survived this concentration of H₂O₂after 1-day treatment (Fig. (Fig.1D,1D, solid circle at 100 μl). In contrast, 2 mM H₂O₂treatment led to a time-dependent increase in the 545/595 emission intensity ratio with a significant increase (over untreated cells) occurring at 2 hours after treatment (Fig. (Fig.4E,4E, solid circle at 2-hour treatment point), and a further increase at 4 hours after treatment. Most of the cells were committed to die at this time (see Fig. Fig.2,2, solid circle at 2-hour time point).

The mitochondrial uncoupler, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) is commonly used as a positive control that causes mitochondrial depolarization [9]. This agent caused a dose-dependent increase of the 545/595 emission intensity ratio under the same experimental conditions. Cells treated with 0, 5, 10, 20 or 40 μM FCCP for 4 hours had a 545/595 emission intensity ratio of 0.8, 1.3, 2.1, 2.9 or 3.1, respectively (Fig. (Fig.4F4F).

Effect of 15d-PGJ₂on H₂O₂-induced mitochondrial membrane depolarization

We determined whether 15d-PGJ₂pretreatment could prevent H₂O₂-induced mitochondrial membrane potential depolarization. Cells were pretreated with 1 μM 15d-PGJ₂overnight, challenged with 1.5 mM H₂O₂for 2 hours, then the emission spectrum of each treatment was determined. Results indicated that untreated cells showed two peaks at 545 nm and 595 nm (Fig. (Fig.5A).5A). Similar peaks were observed in cells treated with 15d-PGJ₂overnight (Fig. (Fig.5B).5B). Treatment of cells with H₂O₂caused a shift in the relative intensity of these two peaks (Fig. (Fig.5C).5C). However, overnight pretreatment of cells with 15d-PGJ₂restored the relative intensity to that close to the untreated cells (Fig. (Fig.5D5D).

This set of experiments was performed 4 times and the results (Fig. (Fig.5E)5E) indicated that H₂O₂led to an increase of 545/595 emission intensity ratio from 0.62 ± 0.06 (control) to 1.77 ± 0.11. Pretreatment of cells with 15d-PGJ₂had little effect on the 545/595 emission intensity ratio (0.65 ± 0.05, p > 0.05 between control and 15d-PGJ₂), but the pretreatment greatly reduced the 545/595 emission ratio of H₂O₂-treated cells to 0.85 ± 0.06. Similar results were obtained when H₂O₂challenge was extended to 4 hours (Fig. (Fig.5F).5F). The 545/595 emission intensity ratios for control, H₂O₂-treated, 15d-PGJ₂-treated or 15d-PGJ₂/H₂O₂-treated cells were 0.82 ± 0.07, 2.74 ± 0.22, 0.85 ± 0.05 or 1.46 ± 0.09, respectively. The difference between H₂O₂-treated cells and 15d-PGJ₂+H₂O₂-treated cells was statistically significant (p < 0.001). These results suggested that the inhibition of mitochondrial membrane depolarization by 15d-PGJ₂might be partially responsible for its ability to prevent H₂O₂-induced cytotoxicity.

We reported earlier that ciglitazone (a PPARγ agonist, tested between 1–10 μM) and WY14643 (a PPARα agonist, tested between 10–40 μM) had no saving effect in this experimental setting [3]. Subsequent experiments indicated that these two agents could not restore H₂O₂-induced mitochondrial membrane depolarization (see Additional file: 1).

Prevention of tBH-induced cell death by 15d-PGJ₂

Given the saving effect of 15d-PGJ₂against H₂O₂-induced oxidative stress, we determined whether 15d-PGJ₂could prevent the oxidative stress caused by tBH. Results indicated that tBH at 400 μM or less had little cytotoxicity for ARPE-19 cells; however, 600 μM tBH killed most of the cells (Fig. (Fig.6,6, solid circles). Overnight pre-treatment of cells with 1 μM 15d-PGJ₂greatly prevented the cytotoxicity caused by tBH (Fig. (Fig.6A,6A, open circles). For example, while only ~21% of the cells remained viable after 600 μM tBH treatment, 15d-PGJ₂pre-treatment raised the viability to ~78%.

Figure 6

Prevention of tBH-induced cytotoxicity by 15d-PGJ₂. (A): Cells plated in 96-well plates were pre-treated with serum-free medium (solid circles) or 1 μM 15d-PGJ₂(prepared in serum-free medium, open circles) overnight, then with various concentrations (more ...)

Experiments were performed to determine the dose-dependent saving effect of 15d-PGJ₂. In this set of experiments, 1-day treatment of cells with 450 μM tBH decreased cell viability to ~17% of control. Pre-treatment of cells with 0.25, 0.5 or 1 μM 15d-PGJ₂raised the viability to ~43%, 64% or 75%, respectively (Fig. (Fig.6B).6B). This saving effect was consistent with morphological observations with a microscope (Fig. (Fig.6C6C).

Effect of 15d-PGJ₂on tBH-induced ROS generation and mitochondrial membrane depolarization

Treatment of cells with tBH caused a dose-dependent increase of ROS generation. At 1000 μM tBH, the "total" ROS was ~3× control (Fig. (Fig.7A,7A, solid squares). There was also a parallel increase of "cell-associated" ROS (Fig. (Fig.7A,7A, open squares). To test whether 15d-PGJ₂pre-treatment could reduce tBH-induced ROS, cells were pre-treated with 1 μM 15d-PGJ₂overnight followed by 1000 μM tBH challenge, then the "total" and "cell-associated" ROS were determined. Results (Fig. (Fig.7B)7B) indicated that tBH generated ~190 FU of "total" ROS, which was decreased to ~149 FU by 15d-PGJ₂pre-treatment (p < 0.001). Similarly, the "cell-associated" ROS was reduced from ~139 FU (tBU alone) to ~100 FU (with 15d-PGJ₂pre-treatment, p < 0.001).

Figure 7

Effect of 15d-PGJ₂on tBH-induced ROS generation. (A): ROS generation by tBH. Cells were treated with H₂DCF-DA for 20 min, followed by tBH for 20 min, and then the "total" (solid squares) and "cell-associated" (open squares) ROS generation was determined. (more ...)

We next determined whether tBH caused mitochondrial membrane depolarization and if 15d-PGJ₂could prevent the depolarization. Cells were treated with 400 μM, 500 μM or 600 μM tBH for 4 hours, and then processed for the JC-1 assay. Results indicated that tBH at these concentrations increased 545/595 emission intensity ratio from 0.76 ± 0.17 (control) to 1.03 ± 0.11, 1.23 ± 0.14 and 1.42 ± 0.19, respectively (Fig. (Fig.8,8, solid circles). Pretreatment of cells with 1 μM 15d-PGJ₂greatly decreased the 545/595 emission intensity ratio resulting from the tBH challenge (Fig. (Fig.8,8, open circles). The inhibition of mitochondrial membrane depolarization by 15d-PGJ₂may be partially responsible for its ability to prevent tBH-induced cytotoxicity.

Figure 8

Prevention of tBH-induced mitochondrial membrane depolarization by 15d-PGJ₂. Cells were pretreated with serum-free medium (control) or with 1 μM 15d-PGJ₂(prepared in serum-free medium) overnight, then with 400 μM, 500 μM or 600 (more ...)

Discussion

H₂O₂cytotoxicity as a function of volume

There are several important factors that can determine the cytotoxicity of H₂O₂in cultured cells. In our previous studies with 96-well plates, we noticed that the degree of H₂O₂cytotoxicity at a fixed concentration was very much dependent on cell density [3]. If we performed experiments 2 days after plating 20,000 cells per well in 96-well plates, the cytotoxicity of H₂O₂(100 μl/well) could be observed between 1–2 mM. H₂O₂at concentrations lower than 1 mM had no apparent cytotoxicty. Cells were more sensitive to H₂O₂if they were plated at a lower density. As a result, we paid much attention to cell density in subsequent experiments when we intended to use culture plates other than 96-well plates. While using a 6-well plate, we plated down 30× as many cells per well as that used in each well of a 96-well plate in order to maintain the appropriate density. With this density issue in mind, we were puzzled to see that results of a series of experiments in 6-well plates using 2 ml/well H₂O₂solution yielded much less cytotoxicity than we expected. Comparable cytotoxicity could only be observed when we used 3 ml/well H₂O₂solution regarding surface area versus volume ratio.

These observations prompted us to determine H₂O₂degradation in the culture wells, and analyze its effect on cytotoxicity. Results (Fig. (Fig.1A1A and and1B)1B) suggested that H₂O₂(either 5 mM or 10 mM, fixed volume) degraded at a rate that reached ~50% of the original concentrations in one hour. Further experiments (Fig. (Fig.1C)1C) indicated that the rate of degradation (at a fixed concentration) was dependent on the volume of H₂O₂used in the culture well. It appeared that cells (at a fixed density) would consume a fixed mass amount of H₂O₂molecules such that wells containing more volume (thus more H₂O₂molecules) would have more remaining H₂O₂molecules after a given time period. As a result, wells containing a higher volume maintained the concentration better than those with a lower volume. The resulting higher concentration led to a higher cytotoxicity, as illustrated by Fig. Fig.1D1D.

These results indicated that it is very important to control the volume of H₂O₂solution used in the experiments. The volume used in this study was adjusted proportionally according to the surface area in order to obtain a consistent H₂O₂cytotoxicity. As a result, other than control for uniformed cell density, we used 100 μl/well in a 96-well plate, 700 μl/well in a 24-well plate and 3 ml/well in a 6-well plate for all of our experiments. Even though these results were obtained using H₂O₂as the cytotoxic agent, there is a possibility that similar considerations should be made while testing other cytotoxic agents.

Temporal commitment point: H₂O₂vs. tBH

H₂O₂and tBH have been used in a number of studies testing the responses of RPE toward oxidative stress. For example, it was shown that these two agents could alter gene expression in a specific manner [15]. Results from the current study indicate that 2 mM of H₂O₂killed most of the cells after a 1-day treatment (Fig. (Fig.1D,1D, see the solid square at 100 μl/well) but it took only 600 μM tBH to achieve similar cytotoxicity (Fig. (Fig.6A,6A, solid circles). Based on this comparison, it appeared that tBH was more potent than H₂O₂. It was reported that H₂O₂could be metabolized ~3 times as fast as tBH [16], which may partially explain the observation that it took ~3× concentrations of H₂O₂to achieve similar cytotoxicity as tBH.

It is generally believed that ROS are short-lived and damage to cellular structures occurs rapidly. With this in mind, experiments were performed to test how soon cells were committed to die following treatment with 2 mM H₂O₂or 600 μM tBH, concentrations that killed most of the cells in 24 hours. Results indicated that tBH, even with its apparent higher potency, appeared to have a slower action. In fact, if it was removed within 2 hours, ~80% of the cells had survived the treatment one day later. It took 4 hours for most cells to commit to die following tBH treatment (Fig. (Fig.2,2, open circles). In contrast, H₂O₂appeared to damage cells more rapidly (Fig. (Fig.2,2, solid circles). Some cell damage should have occurred during the first hour of treatment based on the viability curve. The damage appeared to increase in a time-dependent manner such that most of the cells committed to die if the cultures were treated with H₂O₂for 2 hours.

DCF-DA generation caused by H₂O₂vs. tBH: membrane leakiness

One possible explanation for the finding that cells treated with H₂O₂had an earlier commitment point is that H₂O₂treatment led to a faster or higher rise in intracellular ROS. Results from this study indicated that both H₂O₂(Fig. (Fig.3A)3A) and tBH (Fig. (Fig.7A)7A) treatment led to an increase in cellular ROS within 20 min and the maximal "total" ROS levels were ~3× of control levels in both cases. It is of interest to note that the "cell-associated" ROS was ~50% of total ROS in H₂O₂-treated cells, while it was about ~80% in tBH-treated cells. These results suggest that H₂O₂treatment made the cell membrane more leaky, and more of the intracellular DCF-DA generated by H₂O₂treatment leaked out into the extracellular compartment. Significant plasma membrane injury caused by H₂O₂has been reported earlier by others [17]. The membrane leakiness caused by H₂O₂might be so severe that it partially contributed to the rapid commitment of cell death observed in Fig. Fig.22 (solid circles).

These results also suggest that experiments designed to assess ROS generation should consider whether the agent of interest causes membrane leakiness. If it does, repeated washing of the cultures after treatment could remove much of the DCF-DA made. This would lead to an under-estimation of ROS generation in the experimental system.

Disparity between ROS curves and viability curves

It should be noted that the ROS generation caused by H₂O₂did not fit its cytotoxicity profiles. ROS generation reached a plateau at 500–1000 μM (Fig. (Fig.3A),3A), however, the cell death caused by H₂O₂occurred at 1–2 mM (Fig. (Fig.1D,1D, see 100 μl/well for each concentration). It is possible that the stability of H₂O₂in the culture partially accounted for this observation. Even though H₂O₂at 500 μM could generate large quantity of ROS, it might be short-lived and fell under the threshold of cytotoxicity as H₂O₂degraded in the culture. In contrast, while H₂O₂at 1 mM or higher made similar amount of ROS, a higher concentration of H₂O₂in the culture could ensure that intracellular ROS remained elevated and continued to cause cellular damage. This idea is consistent with the results from Fig. Fig.1,1, which suggested that more cytotoxicity was observed if H₂O₂concentrations remained high for a longer period of time.

Similar observations regarding ROS generation and cytotoxicity occurred in cultures treated with tBH. This agent at 400 μM or less exhibited little cytotoxicity (Fig. (Fig.6,6, solid circles). However, short-term treatment of cells with 400 μM tBH generated large amount of ROS (Fig. (Fig.7A).7A). Analogous to the discussion for H₂O₂above, sustained intracellular ROS generation caused by high concentrations of tBH may partially account for these observations.

Temporal commitment point vs. 545/595 emission intensity ratio

The temporal commitment of cell death after H₂O₂treatment correlated well with a change in mitochondrial membrane potential. Treatment of cells with 2 mM H₂O₂for less than one hour had little effect on 545/595 emission intensity ratio. A significant increase in the ratio, signaling depolarization of mitochondrial membrane potential, occurred at 2 hours after treatment, and continued to increase at 4-hour after treatment (Fig. (Fig.4E,4E, solid circles). This transition could be observed clearly in the 545 nm and 595 nm emission intensity plots in Fig. 4A,4B,4C,4D. A decrease of the 595 nm readings and an increase of the 545 nm readings began at 2 hours after 2 mM H₂O₂treatment. Cells treated with 1 mM H₂O₂maintained the mitochondrial membrane potential well up to 4 hours after treatment (Fig. (Fig.4E,4E, open circles), and remained mostly viable one day later (Fig. (Fig.1,1, solid circle at 100 μl/well). Taken together, these results suggest that changes in mitochondrial membrane potential soon after H₂O₂treatment may be a predictor of cellular viability assessed 24 hours later.

Even though oxidative stress led to mitochondrial membrane depolarization in this study, it should be noted that RPE apoptosis is not always preceded by depolarization of mitochondria membrane potential. For example, Kim et al. reported that RPE apoptosis caused by proteosome inhibition was preceded by a sustained hyperpolarization of mitochondrial membrane potential [18].

15d-PGJ₂prevented oxidative-stress induced cytotoxicity, ROS generation and mitochondrial membrane depolarization

We previously reported that 15d-PGJ₂could prevent H₂O₂-induced cytotoxicity. Results from this study further indicated that 15d-PGJ₂blocked tBH-induced cytotoxicity (Fig. (Fig.6).6). Pre-treatment of cells with 15d-PGJ₂caused a modest decrease of ROS levels after either H₂O₂(Fig, (Fig,3B)3B) or tBH (Fig. (Fig.7B)7B) treatment. This inhibition may be partially responsible for its saving effect.

In this respect, it is important to mention that 15d-PGJ₂at higher concentrations (5–10 μM) was shown to induce intracellular ROS formation and cell death in human hepatic myofibroblasts [19] and in human neuroblastoma SH-SY5Y cells [20]. In our experimental system, 10 μM 15d-PGJ₂treatment for several days could also lead to cell death. Therefore, the effect of this agent can differ according to the concentration used. Since in this study we focused on the protective effect of 15d-PGJ₂, we used this agent at 1 μM or lower concentrations. In this concentration range 15d-PGJ₂did not increase intracellular ROS in ARPE-19 cells after either short-term (20 min) or long-term (overnight) treatment (results not shown).

In order to further determine the mechanism(s) responsible for the saving effect of 15d-PGJ₂against H₂O₂-and tBH-induced cytotoxicity, we tested the hypothesis that 15d-PGJ₂could block mitochondrial membrane depolarization caused by those agents. Results indicated that 15d-PGJ₂prevented mitochondrial membrane depolarization caused by either H₂O₂(Fig. (Fig.5)5) or tBH (Fig. (Fig.88).

Given the observations that cells treated with H₂O₂or tBH showed ROS generation within 20 min of treatment (Fig. (Fig.33 and Fig. Fig.7)7) but detectable mitochondrial depolarization occurred at 2–4 hours after treatment (Fig. (Fig.44 and Fig. Fig.8),8), there is a possibility that ROS generation could lead to mitochondrial damage and membrane depolarization. 15d-PGJ₂appeared to prevent both events, but with a more prominent inhibitory effect on mitochondrial membrane depolarization.

It was reported that thiazolidinedione activation of PPARγ could enhance mitochondrial membrane potential and promote cell survival [21]. Even though 15d-PGJ₂can activate PPARγ, we have previously reported that the saving effect of this agent in this experimental system does not appear to involve PPARγ activation [3]. For example, ciglitazone (a thiazolidinedione PPARγ agonist) did not prevent H₂O₂-induced cytotoxicity. Consistent with our previous findings, the results from this study indicated that ciglitazone did not prevent H₂O₂-induced depolarization of mitochondrial membrane potential. WY14643, a PPARα agonist that had no saving effect in this experimental system [3], also did not prevent mitochondrial membrane depolarization.

Conclusion

This study documented ROS generation and alteration of mitochondrial membrane potential resulting from retinal pigment epithelial cells exposed to H₂O₂and tBH. The prostaglandin derivative, 15d-PGJ₂, inhibited ROS generation and prevented mitochondrial membrane depolarization, which may contribute to the prevention of cell death caused by oxidative agents. In an earlier report we also demonstrated that 15d-PGJ₂prevented cell death caused by oxysterols, toxic oxidative products of cholesterol [22]. Thus, 15d-PGJ₂may be used as a pharmacological tool to protect cells against oxidative stress or toxic products of oxidative stress.

Methods

Materials

15d-PGJ₂was purchased from Cayman (Ann Arbor, MI). FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), hydrogen peroxide, t-butyl hydroperoxide, Hank's Balanced Salt Solution (HBSS) and other general biochemical reagents were purchased from Sigma (St. Louis, MO) unless otherwise stated. H₂DCF-DA (2', 7'-dichlorodihydrofluorescein diacetate) and JC-1 (5,5', 6,6'-tetrachloro-1, 1', 3,3'-tetraethylbenzimidazolocarbocyanine iodide) were from Molecular Probes (Eugene, OR).

Cell cultures

ARPE-19 cells [23] were obtained from the American Type Culture Collection and grown in DMEM/F12 containing 10% newborn calf serum and 2.5 mM glutamine. In order to maintain a comparable growth condition with regard to cell density and medium volume, cells were plated at 20,000 cells/well in 96-well plates (100 μl medium per well) or 140,000 cells/well in 24-well plates (700 μl medium per well).

H₂O₂degradation

Cells were plated in 24-well plates for 2 days, rinsed with HBSS, then treated with 700 μl H₂O₂solution (prepared in HBSS). Solutions from a set of 3 wells were removed at various periods of time after treatment, then 100 μl of solution were transferred to a quartz 96-well plate, and the concentration of H₂O₂was estimated by measuring the optical density (OD) at 240 nm [24,25] using a plate reader (Spectra Max 190, Molecular Devices, Sunnyvale, CA). "Blank" readings, obtained from cultures treated with HBSS only, were subtracted before the final results were plotted. Experiments were performed 3 times with 3 replicates per treatment.

Cell viability

As a general protocol, cells were plated in 96-well plates (20,000 cells/well) overnight, then fed with serum-free medium or treated with a testing agent (prepared in serum-free medium) the next day. Cultures were subjected to oxidative stress (H₂O₂or tBH) for a day, and then the viability from each treatment was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [3,22]. The OD of each well was measured by a plate reader (Spectra Max 190, Molecular Devices, Sunnyvale, CA) with a filter setting at 570 nm (reference filter setting was 630 nm). See figure legends for specific descriptions regarding each experiment. Cells were plated in 24-well plates (140,000 cells/well) for microscopic observations. After treatment, cells were washed twice with phosphate-buffered saline (PBS), then fixed for 10 minutes with 3.7% buffered formaldehyde, examined under a phase contrast microscope Nikon Eclipse TE 200 (Nikon Instruments Inc., Melville, NY). Cells were photographed digitally at a fixed exposure time by using a Photometrics CoolSNAP fx camera (Roper Scientific, Inc., Tucson, AZ) and the software MetaVue, Meta Imaging Series version 4.6r9 (Universal Imaging Corporation, Downingtown, PA).

Measurement of reactive oxygen species

Cells were plated in 96-well plates (20,000 cells/well) overnight, fed with serum-free medium or treated with a testing agent (prepared in serum-free medium) the next day then the assay was performed the following day. Medium from each well was removed and each culture was treated with 10 μM H₂DCF-DA (100 μl/well prepared in HBSS) for 20 minutes at 37°C. After removal of H₂DCF-DA, cells were treated with H₂O₂(or tBH, 100 μl/well prepared in HBSS) for 20 minutes at 37°C. The fluorescence of each well was detected by a fluorescence plate reader (Spectra Max Gemini XS, Molecular Devices, Sunnyvale, CA) with the following settings: excitation 485 nm, emission 535 nm and cutoff 530 nm. This first reading was designated as "total" fluorescence. The H₂O₂(or tBH) solution was then replaced with 100 μl HBSS, and the fluorescence from each well was determined again. This second reading was designated as "cell associated" fluorescence. A set of cells without H₂DCF-DA treatment was used as "blank" in each experiment. The reading of blank was subtracted before the results were plotted, expressed as net fluorescence units (FU).

Measurement of mitochondrial membrane potential (MMP, ΔΨm)

Cells were plated in 24-well plates (140,000 cells/well) overnight, fed with serum-free medium or treated with a testing agent (prepared in serum-free medium) the next day. The assay was performed the following day. Each culture was treated with H₂O₂or tBH (700 μl/well prepared in serum-free medium) for a period of time (indicated in each experiment) at 37°C. After H₂O₂treatment, cells were washed once with 500 μl HBSS then incubated with 5 μM JC-1 (300 μl/well) for 15 minutes at 37°C. After this incubation period, cells from each well were rinsed with 500 μl HBSS, then scraped into 300 μl HBSS, transferred into a 96-well tissue culture plate. The fluorescence of each well was detected by a fluorescence plate reader with the following settings: excitation 485 nm, emission 545 nm and 595 nm, cutoff 530 nm. In some experiments, the emission spectra between 520–620 nm were obtained. To prepare JC-1, stock solution (5 mM prepared in DMSO) was diluted (1:4) into 5% BSA, then further diluted (1:199) into HBSS. After proper mixing, this solution was filtered through a 0.2 μm filter, shielded from light until use.

Statistical analysis

Unless otherwise stated, results were pooled from 12 replicate samples derived from 3 independent experiments (cell viability and ROS) or 4 independent experiments (mitochondrial membrane potential), and expressed as mean ± SEM. Statistical analyses were performed by unpaired, two-tailed t test or by analysis of variance (one-way ANOVA) followed by the Bonferroni test to determine the significance of difference among means.

Abbreviations

15d-PGJ₂, 15-deoxy-delta 12, 14-Prostaglandin J₂; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FU, fluorescence units; H₂DCF-DA, 2', 7'-dichlorodihydrofluorescein diacetate; HBSS, Hank's Balanced Salt Solution; JC-1, 5,5', 6,6'-tetrachloro-1, 1', 3,3'-tetraethylbenzimidazolocarbocyanine iodide; MMP, mitochondrial membrane potential (ΔΨm), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); OD, optical density; PPARs, peroxisome proliferator-activated receptors; ROS, reactive oxygen species; RPE, retinal pigment epithelium; tBH, t-butyl hydroperoxide

Authors' contributions

TKG and JYC were both involved in the experimental design and data collection. Both authors read and approved the final manuscript.

Additional file 1

The PPARγ agonist Ciglitazone and the PPARα agonist WY14643 failed to prevent H₂O₂-induced mitochondrial membrane depolarization.

This set of experiments was performed under conditions as those described in Fig. Fig.5.5. (A): Untreated cells; (B): Cells treated with 1.5 mM H₂O₂for 2 hours; (C): Cells treated with 10 μM Ciglitazone overnight, then with 1.5 mM H₂O₂(without 15d-PGJ₂) for 2 hours. (D): Cells treated with 40 μM WY14643 overnight, then with 1.5 mM H₂O₂(without 15d-PGJ₂) for 2 hours. Note H₂O₂caused a shift of the relative intensity of the peaks, indicating mitochondrial depolarization occurred. Pre-treatment of cells with either Ciglitazone or WY14643 failed to prevent H₂O₂-induced mitochondrial membrane depolarization.

Supplementary Material

Additional file 1

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Acknowledgements

This work was supported by funds from Research to Prevent Blindness. Support by the Jones Eye Institute and the Pat & Willard Walker Eye Research Center is greatly appreciated. Suggestions provided by Dr. Joel Weinberg (University of Michigan) and Dr. Matt Whiteman (National University of Singapore) in design of the JC-1 assay, and Dr. John M. Ong (Cedars-Sinai Medical Center) in design of the ROS assay are highly appreciated. We also appreciate the helpful discussions provided by the research group led by Dr. Robert Reis (University of Arkansas for Medical Sciences).

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