H2O2 degradation and its effect on cell viability
We have previously shown that H2
caused a dose- and time-dependent decrease in cellular viability in ARPE-19 cells. Furthermore, the cytotoxicity of H2
was 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 H2
(100 μl/well) decreased cell viability to ~15% of controls after a 1-day treatment while 1 mM H2
had 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 H2O2 in the cultures, and its effect on cell viability. Cells were treated with 2 mM H2O2 for various periods of time to assess the H2O2 concentration that remained in each well. Results (Fig. , open squares) indicated that the readings were so close to background that H2O2 degradation could not be adequately assessed. Subsequent experiments using 5 mM or 10 mM H2O2 indicated that H2O2 degraded in a time-dependent manner (Fig. ), with a half-life of ~1 hour (Fig. ).
Figure 1 Degradation of H2O2 and its effect on cellular viability. (A): Degradation of H2O2 in 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 H2O2 concentration was inversely related to the volume of H2O2 solution in each well, such that cultures with more H2O2 solution maintained H2O2 concentration better (Fig. ). 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 H2O2 concentrations remained almost constant regardless of the volume added to each well (Fig. , solid diamonds).
Based on these results, it was hypothesized that at a fixed H2O2 concentration and a fixed cell density, the volume of H2O2 solution 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 H2O2 of 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 H2O2 decreased the viability to ~20% of controls, 50 μl of 2 mM H2O2 had little effect on the viability (Fig. , 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. , open circles).
In view of these results, in addition to maintaining a constant cell density, the volume of H2O2 solution 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. 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 H2O2 at time zero, and then H2O2 was replaced with fresh medium from a set of cells at 30-minute intervals. Viability was then determined 24 hours later. Results (Fig. , solid circles) indicated that if cells were treated with 2 mM H2O2 for 1 hour followed by fresh medium (without H2O2) for 23 hours, the viability decreased to ~75% of control. If the cells were treated with H2O2 for 2 hours, then with fresh medium for 22 hours, the viability decreased to ~10% of control. It was thus concluded that with 2 mM H2O2 treatment, most cells were committed to die 2 hours after treatment.
Figure 2 Temporal commitment point of H2O2 and 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. , 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-PGJ2 on H2O2-induced ROS generation
We have previously shown that pretreatment of ARPE-19 cells with 15d-PGJ2
greatly reduced the cytotoxicity caused by H2
]. For example, while H2
at 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-PGJ2
raised the viability to 95%, 84% or 68% of control, respectively. Similar saving effects were also observed in primary cultures of human RPE. Since H2
could induce intracellular generation of ROS, experiments were performed to determine whether 15d-PGJ2
could reduce intracellular ROS levels after H2
treatment, thereby reducing the H2
Initial experiments indicated that H2O2 treatment 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. ). 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 H2O2 at 250, 500 or 1000 μM generated ~55 FU, 67 FU or 70 FU of DCF-DA in each well (Fig. , solid circles), the "cell associated" DCF-DA was ~28 FU, 33 FU or 35 FU, respectively (Fig. , open circles). DCF-DA formation appears to occur intracellularly, because without cells, H2O2 did 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-PGJ2 on 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 H2O2 for 20 min, then the "total" and "cell-associated" ROS generated was (more ...)
15d-PGJ2 was tested to see if it could prevent H2O2 induced ROS elevation. Cells were pretreated with 1 μM 15d-PGJ2 overnight, challenged with 1 mM H2O2 the next day for 20 minutes, then ROS in each well was determined. Results (Fig. ) indicated that while 1 mM H2O2 generated ~132 FU "total" ROS in this set of experiments, 15d-PGJ2 pretreatment reduced ROS to ~108 FU (p < 0.005). Similarly, the "cell associated" ROS was reduced by 15d-PGJ2 from ~64 to ~54 FU (p < 0.005). The decrease in ROS levels caused by 15d-PGJ2 may be partially responsible for its ability to prevent H2O2-induced cytotoxicity.
H2O2-induced mitochondrial membrane depolarization in ARPE-19 cells
Other than ROS generation, H2
treatment 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. ). Treatment of cells with H2
led to an alteration of the relative intensity of these two peaks (See Fig. ). 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 H2O2 concentration and duration of exposure was determined. Results indicated that H2O2 treatment for 30 min caused a slight dose-dependent increase of both peaks (Fig. ). Similar observations were made in cells treated with various concentrations of H2O2 for 1 hour (Fig. ). There was little change in 545/595 emission intensity ratio (not shown).
Figure 4 H2O2 caused depolarization of mitochondrial membrane potential. Cells were treated with various concentrations of H2O2 for 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 H2O2 for 2 hours. Untreated cells at this time had an emission reading of 173 FU at 545 nm (Fig. , open square at 0 mM H2O2), and 377 FU at 595 nm (Fig. , 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 H2O2 had 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. ). However, cells treated with 1.5 mM H2O2 had an increased emission reading at 545 nm and a decreased reading at 595 nm, such that these two readings were almost identical (Fig. at 1.5 mM). With 2 mM H2O2 treatment, the reading at 545 nm was higher than that of 595 nm (Fig. ).
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. , open circles), and a corresponding increase in the emission readings at 545 nm (Fig. , solid circles).
Results from Fig. were re-plotted to show the change of 545/595 emission intensity ratio as a function of H2O2 concentrations and duration of treatment. There was little change of the ratio when cells were treated with 1 mM H2O2 up to 4 hours (Fig. , open circles). Most cells survived this concentration of H2O2 after 1-day treatment (Fig. , solid circle at 100 μl). In contrast, 2 mM H2O2 treatment 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. , 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. , 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. ).
Effect of 15d-PGJ2 on H2O2-induced mitochondrial membrane depolarization
We determined whether 15d-PGJ2 pretreatment could prevent H2O2-induced mitochondrial membrane potential depolarization. Cells were pretreated with 1 μM 15d-PGJ2 overnight, challenged with 1.5 mM H2O2 for 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. ). Similar peaks were observed in cells treated with 15d-PGJ2 overnight (Fig. ). Treatment of cells with H2O2 caused a shift in the relative intensity of these two peaks (Fig. ). However, overnight pretreatment of cells with 15d-PGJ2 restored the relative intensity to that close to the untreated cells (Fig. ).
This set of experiments was performed 4 times and the results (Fig. ) indicated that H2O2 led 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-PGJ2 had 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 H2O2 challenge was extended to 4 hours (Fig. ). 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-PGJ2 might 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 H2
-induced mitochondrial membrane depolarization (see Additional file: 1
Prevention of tBH-induced cell death by 15d-PGJ2
Given the saving effect of 15d-PGJ2 against H2O2-induced oxidative stress, we determined whether 15d-PGJ2 could 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. , solid circles). Overnight pre-treatment of cells with 1 μM 15d-PGJ2 greatly prevented the cytotoxicity caused by tBH (Fig. , open circles). For example, while only ~21% of the cells remained viable after 600 μM tBH treatment, 15d-PGJ2 pre-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-PGJ2 raised the viability to ~43%, 64% or 75%, respectively (Fig. ). This saving effect was consistent with morphological observations with a microscope (Fig. ).
Effect of 15d-PGJ2 on 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. , solid squares). There was also a parallel increase of "cell-associated" ROS (Fig. , open squares). To test whether 15d-PGJ2 pre-treatment could reduce tBH-induced ROS, cells were pre-treated with 1 μM 15d-PGJ2 overnight followed by 1000 μM tBH challenge, then the "total" and "cell-associated" ROS were determined. Results (Fig. ) indicated that tBH generated ~190 FU of "total" ROS, which was decreased to ~149 FU by 15d-PGJ2 pre-treatment (p < 0.001). Similarly, the "cell-associated" ROS was reduced from ~139 FU (tBU alone) to ~100 FU (with 15d-PGJ2 pre-treatment, p < 0.001).
Figure 7 Effect of 15d-PGJ2 on 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-PGJ2 could 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. , solid circles). Pretreatment of cells with 1 μM 15d-PGJ2 greatly decreased the 545/595 emission intensity ratio resulting from the tBH challenge (Fig. , open circles). The inhibition of mitochondrial membrane depolarization by 15d-PGJ2 may 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 ...)