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1 From the Departments of Biochemistry and 2 Ophthalmology, Program in Nutritional Health Sciences, School of Medicine, Emory University, Atlanta, Georgia; and the 3 Department of Environmental Health, University of Washington Seattle.
Abstract
PURPOSE. To determine the effect of dimethylfumarate (DMF), an inducer of glutathione (GSH)-dependent detoxification, on intracellular GSH levels in cultured human retinal pigment epithelium (hRPE) cells, its mechanism of action, and its effect on hRPE cells subjected to oxidative injury.
METHODS. Established hRPE cell lines were treated with DMF and assayed by
high-pressure liquid chromatography for intracellular and extracellular
GSH levels. Quantification of 
-glutamylcysteine synthetase (GLCL)
was determined through northern and western blot analyses, and activity
was measured. Effects of pretreatment with DMF on GSH redox status of
hRPE cells was determined. Sensitivity of hRPE cells to oxidative
stress was determined using tert-butylhydroperoxide as the
oxidative agent.
RESULTS. Dimethylfumarate caused a transient decrease followed by a significant increase in intracellular GSH. Glutathione increased maximally at 24 hours with 100 to 200 µM DMF. The initial decrease could be accounted for by the formation of a DMF-GSH conjugate. Dimethylfumarate treatment increased the steady state mRNA expression of the regulatory subunit of GLCL, but no increase was seen for the catalytic subunit. However, protein levels were increased for both, and the catalytic activity of GLCL was also increased. Whereas the initial decrease in GSH made hRPE cells more susceptible to oxidative damage, pretreatment with DMF under conditions that increased intracellular GSH protected hRPE cells against oxidative damage.
CONCLUSIONS. These results suggest a means by which the antioxidant capability of hRPE may be augmented without direct antioxidant supplementation. Specifically, a dietary compound that conjugates with GSH can induce GSH synthesis, increase GSH concentration, and improve protection by GSH-dependent detoxification pathways in hRPE. However, the early depletion of GSH before stimulated synthesis necessitates caution in prevention strategies using dietary inducers.
Age-related macular degeneration (ARMD) is the leading cause of blindness in elderly Americans.1 2 Although a causal link between oxidative injury and ARMD has not been established, studies suggest that the primary source of dysfunction in ARMD may be linked to oxidative injury of the retinal pigment epithelium.3 In vitro studies have shown that the antioxidant glutathione (GSH) protects human retinal pigment epithelial (hRPE) cells from peroxide-induced injury.4 It has also been shown that consumption of a diet of antioxidant-rich fruits and vegetables is associated with higher levels of the reduced form of GSH.5 These findings suggest that nutritional or therapeutic means to increase GSH levels in the hRPE may provide approaches for reducing oxidative injury, thus affecting onset or progression of ARMD.
A broad class of naturally occurring and synthetic compounds is known to induce synthesis of GSH and/or GSH-dependent detoxification systems in mammalian cells. Dimethylfumarate (DMF) is a nonnutritive compound found in some fruits that has been shown to increase activity of GSH-dependent detoxification systems in vivo in various murine tissues6 7 and to increase GSH levels in human U1 monocytoid cells.8 It has relatively low toxicity, exemplified by its use as a food additive. This is further supported by chronic toxicologic studies of the parent compound, fumaric acid,9 10 11 and the treatment of psoriasis with its derivatives.12 13 14 Thus, we selected DMF as a model compound to determine whether dietary inducers increase GSH concentration in human retinal pigment epithelial cells and whether this increase can protect against oxidative injury.
Methods
Methods of securing human tissues complied with the Emory University Human Investigation Review Board guidelines and the Declaration of Helsinki.
Cell Isolation and Culture Conditions
hRPE cell cultures were established from donor eyes obtained
through the Georgia Eye Bank as previously described.15
Appropriate approval for their use was obtained from the Eye Bank and
the Emory University Human Investigation Review Board. Primary cell
cultures were established in 6-well tissue culture dishes with
low-glucose (1 mg/ml) medium supplemented with fetal bovine serum
(20%), amphotericin B (3 µg/ml), and gentamicin sulfate (50
µg/ml). Cells were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) with 10% fetal bovine serum at 37°C under 95% air-5%
CO2, and the medium was changed every 2 to 3
days. Cells were passaged in 75-cm2 flasks every
7 to 14 days, and experiments were performed on hRPE cell cultures
between the 3rd and 10th passages.
Incubations were performed in DMEM with 10% fetal bovine serum at 37°C under 95% air-5% CO2. The DMF (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO, Sigma) and then added to the medium, resulting in a final DMSO concentration of 0.2%. The controls were treated with DMSO only. Experiments were performed on cell lines at 75% to 90% confluence. Data from these replicate tissue culture dishes within the same experiment were averaged before the calculation of mean ± SEM.
GSH Analysis
For GSH studies, media were aspirated, and plates were washed with
phosphate-buffered saline (PBS, pH 7.4) before addition of 1 ml
perchloric acid (5% vol/vol) containing 0.2 M boric acid and 5 or 10
µM -glutamylglutamate as an internal standard. Contents were
scraped and stored in 1.5-ml microcentrifuge tubes at -80°C before
derivatization.16
Supernatant (300 µl) was mixed with 60
µl iodoacetic acid (7.4 mg/ml H2O) and adjusted
to pH 9.0 ± 0.2 with a KOH/tetraborate solution (1 M KOH in
saturated
K2B4O7).
After 20 minutes at room temperature, 300 µl of a dansyl chloride
solution (20 mg/ml acetone) was added. Samples were mixed and placed in
the dark at room temperature for 24 hours. Five hundred microliters
chloroform was then added to each, and samples were stored in the
presence of both the perchlorate precipitate and the chloroform layer
at 0°C to 4°C until analysis through high-pressure liquid
chromatography (HPLC).
Samples were centrifuged, and aliquots of the aqueous layer were
collected for analysis. Separation was achieved on a 3-aminopropyl
column (Custom LC, Houston, TX), with initial conditions of 80%
solvent A (80% methanol) and 20% solvent B (4 M sodium acetate [pH
4.6] containing 64% methanol) run at 1 ml/min. After 10 minutes, a
linear gradient of 20% solvent A and 80% solvent B was run during the
next 20 minutes. From 30 to 46 minutes the conditions were maintained
at 20% A and 80% B and returned to 80% A and 20% B in 2 minutes.
Equilibration time for the next run was 12 minutes. Detection was
obtained by fluorescence monitoring with a band-pass filter (305 to 395
nm excitation and 510 to 650 nm emission; Gilson Medical Electronics,
Middleton, WI). Quantitation of glutathione was obtained by integration
relative to the internal standard, -glutamylglutamate.
Isolation of Total RNA and Northern Blot Analysis
Total RNA was isolated by addition of TRIzol (Life Technologies,
Gaithersburg, MD) to the plates. Cells were scraped, placed in 1.5-ml
microcentrifuge tubes (Molecular Research Center Inc., Cincinnati, OH)
and frozen in liquid nitrogen until resuspension in Formazol.
Total RNA (20 µg/well) was separated by electrophoresis on a 37%
formaldehyde-1.5% agarose gel. RNA was transferred to a positively
charged nylon membrane (Schleicher and Schuell, Keene, NH) and
cross-linked to the membrane by UV irradiation. The full length cDNAs
for the mouse glutamate-cysteine ligase catalytic and regulatory
subunits (GLCLc and GLCLr, respectively) were labeled with
[
-32P] dATP (Dupont, Boston, MA) using a random prime
labeling kit (Stratagene, La Jolla, CA), purified with NucTrap columns
(Stratagene) and used at 8 x 105 cpm/ml of
hybridization solution. Hybridizations were performed overnight at
60°C in a solution of 10% dextran sulfate, 2% sodium dodecyl
sulfate (SDS), and 1 M NaCl, washed with 2x SSC-0.1% SDS for 15
minutes at room temperature, then for 50 minutes at 60°C with 0.1x
SSC-0.1% SDS. The membranes were exposed to film with an intensifying
screen (Biomax; Kodak, Rochester, NY) at -80°C for 24 hours.
Membranes were stripped by boiling twice in 0.1% SDS for 15 minutes.
Glyceraldehyde 3-phosphate dehydrogenase (G3PDH; Clonetech, Palo Alto,
CA) was used as an internal standard. Autoradiographs were quantified
by densitometric analysis and expressed as percentage of G3PDH mRNA
expression levels.
Isolation of GLCL Protein and Western Blot Analysis
Plates of hRPE cells were washed twice with PBS (pH 7.4) scraped
in PBS and frozen at -80°C. On thawing, samples were homogenized in
the PBS plus peptidase inhibitors (100 µg/ml Pefabloc, 10 µg/ml
TLCK, 1 µg/ml pepstatin A, 1 µg/ml aprotonin, and 1 µg/ml
leupeptin [Boehringer Mannheim, Indianapolis, IN]) and centrifuged
for 10 minutes at 16,000g at 4°C. Protein levels in the
supernatants were assayed by the Bradford method (Bio-Rad, Hercules,
CA), and a total of 70 µg protein from each homogenate per lane was
separated by SDS-polyacrylamide gel electrophoresis. The proteins were
transferred to polyvinylidene difluoride membrane (Millipore, Bedford,
MA), and blots were incubated with blocking solution (3% bovine serum
albumin, 3% nonfat dry milk, 1% ovalbumin, 1% normal goat serum,
0.1% Tween-20, and 0.1% NaN3). Blots were
stained with rabbit polyclonal antisera raised against peptides from
GLCLc and GLCLr. The secondary antibody was horseradish
peroxidase–conjugated goat anti-rabbit IgG (Boehringer Mannheim) with
binding detected by enhanced chemiluminescence (Amersham, Arlington
Heights, IL). The autoradiographs were scanned and densitometry
performed using the GEL-DOC system with commercial software (Molecular
Analyst; Bio-Rad).
Reactive Oxygen Species Assay
The method used is based on that described by LeBel and
Bondy.17
It involves the use of the nonfluorescent probe
2',7'-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene,
OR). DCFH-DA is a nonfluorescent compound that readily crosses cell
membranes. Intracellularly, DCFH-DA is hydrolyzed to
2',7'-dichlorofluorescein (DCFH), a nonfluorescent product that cannot
cross cell membranes. If reactive oxygen species (ROS) are present,
DCFH is oxidized by ROS to the highly fluorescent product
2'7'-dichlorofluorescein (DCF).
The media were aspirated from cultured hRPE cells, and the cells were washed twice in DMEM. Cells were then incubated in Hanks’ balanced salt solution (HBSS, Life Technologies) containing 40 µM DCFH-DA for 30 minutes at 30°C in 5% CO2. Cells were washed with DMEM twice and incubated in 100 µM DMF in DMEM for 1 and 3 hours at 37°C. After incubation, cells were washed with DMEM twice. Fluorescence measurements (FACScan; Becton Dickinson, Braintree, MA) were then read, using an excitation wavelength of 488 nm and an emission wavelength of 588 nm. Controls consisted of hRPE cells incubated in DMEM without DMF and hRPE cells incubated in DMEM containing 300 µM H2O2 or 500 µM tert-butylhydroperoxide (tBH).
Assay of GLCL Activity
The method was a modification of that of Fernandez–Checa and
Kaplowitz,18
which we modified to perform using a 96-well
plate. Monochlorobimane (mBCl) forms a fluorescent adduct with GSH more
specifically than other bimanes and preferentially over other thiols
present in solution. Cultured hRPE cells were treated with
diethylmaleate in DMEM with 10% fetal bovine serum for 1 hour. Cells
were then washed twice with PBS and scraped into a reaction buffer
containing 100 mM Tris-HCl, 150 mM KCl, 20 mM
MgCl2, and 2 mM EDTA (pH 7.3). Cells were lysed
by repeated aspiration through a 25-gauge needle, and 50 µl cytosol
(0.03 mg protein) was added to 200 µl of the reaction buffer that
contained 100 µM glutamate, 10 mM glycine, 3 mM adenosine
triphosphate, 0.2 mM cysteine, 0.1 mM dithiothreitol, and 100 µM mBCl
(from a stock of 100 mM in ethanol). The increase in fluorescence with
time was used to quantify the enzymatic activity of GLCL. Fluorescence
increase was converted to nanomoles GSH using standardized solutions of
GSH and measuring the fluorescence increase after addition of 100 µM
mBCl plus purified glutathione-S-transferase (GST).
LDH Assay
To measure viability of cells exposed to tBH, a lactate
dehydrogenase (LDH) assay was used. PBS (0.9 ml) was added to a
cuvette. Forty microliters of a 3-mg/ml reduced nicotinamide adenine
dinucleotide (NADH) solution and 40 µl of a 3-mg/ml sodium pyruvate
solution were added to the PBS. Forty microliters of growth medium (4
ml/60 cm2 plate) was added to the reaction
mixture, and the change in absorbance at 340 nm with time was
immediately measured. Duplicates were measured for each plate. The
cells were lysed by the addition of 100 µl 10% (vol/vol) Triton
X-100 to the growth medium, and the rate of NADH oxidation was again
measured. The ratios of the rates before and after lysing provide the
percentage of LDH leakage and were used as a measure of cell viability,
with 100% viability taken as the ratio for untreated control cells.
Results
To determine whether DMF increased GSH in hRPE, cells were treated with increasing concentrations of DMF (0, 50, 100, 200, and 500 µM), and intracellular GSH concentrations were analyzed after 24 hours. The GSH content for hRPE cells under control conditions was 12.5 ± 1.5 nanomoles/mg protein. Glutathione concentration increased as a function of DMF concentration, with a maximal GSH level 2.5 times more than obtained in control samples at 200 µM DMF (Fig. 1) . At 500 µM DMF, GSH was substantially decreased, and examination of cell viability with trypan blue showed 100% cell death after 24 hours (data not shown). Examination of cells cultured at 100 and 200 µM DMF did not reveal signs of toxicity (e.g., blebbing, lifting off the plates, or loss of trypan blue exclusion). Thus, the results show that treatment of hRPE cells with DMF caused an increase in intracellular GSH levels. The maximum increase was seen after treatment with 200 µM DMF, with higher DMF concentrations proving toxic.
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-glutamyl-transpeptidase, an enzyme that degrades GSH conjugates,
before derivatization and HPLC. Thus, the results indicate that a
DMF-GSH conjugate was formed in hRPE cells and that this formation may
account for the loss of GSH after DMF treatment.
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To determine whether the increased GSH was associated with an increase in GSH-dependent detoxification activity, we measured the activity of GST after DMF treatment. GST acts as a detoxifier within the cell by coupling GSH to a wide variety of toxic molecules. In cells treated with DMF for 24 hours, GST activity was 28.9% ± 4.3% (SEM) greater than the activity observed in cells treated with DMSO only (data are representative of four experiments performed using established cell lines from four different donor eyes). Thus, the increase in GSH concentration caused by DMF exposure was also associated with increased activity of this GSH-dependent detoxification system.
To determine whether decreased intracellular GSH caused by a 3-hour pretreatment with DMF altered the sensitivity of the hRPE cells to oxidative stress, three different cell lines were incubated with tBH, a model oxidant that has been used extensively with cultured hRPE cells .4 19 Because the cell lines differ in their sensitivity to tBH, we initially exposed the cell lines to increasing concentrations of tBH for 24 hours to establish the highest tBH concentration at which the cells exhibited a 0% to 10% loss of cell viability, measured by trypan blue exclusion (Fig. 9 A, concentration identified as b). These tBH concentrations ranged from 250 to 300 µM. Each cell line was then incubated with 100 µM DMF for 3 hours followed by the aforementioned concentration of tBH for 24 hours. After 24 hours, viability was measured by LDH release into the media. Although this release is traditionally used to measure necrosis, our recent studies of oxidant-induced cell death in hRPE cells indicate that LDH release probably represents necrosis that occurs in vitro secondary to apoptosis.19 When compared with untreated control cells, cells pretreated with DMF showed a 98% reduction in viability at a tBH concentration that caused a 0% to 2% reduction in viability of cells treated with DMSO only (Fig. 9B) . Thus, cells were more sensitive to oxidative stress during the initial decrease of GSH after DMF exposure. It is likely this increased sensitivity was caused by lower GSH levels during oxidative stress, because low levels of GSH have been associated with oxidative cell death in previous studies.28 29
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Discussion
Epidemiologic data and animal studies have provided evidence that oxidative mechanisms may contribute to the progression of ARMD,30 31 32 33 and a large-scale interventional study is currently under way to assess whether supplementation with antioxidants and/or zinc protects against onset or progression of ARMD.34 Our present study was devised to gain information on an alternate strategy, namely, whether a nonnutritive compound that increases GSH-dependent detoxification enzymes can be used to enhance cellular defenses against oxidative injury.
Our experiments were performed with established normal cell lines of human RPE. Histologic studies show that RPE cells are affected early in the progression of macular disease,35 36 37 and animal studies show that these cells are vulnerable to light-induced injury.38 39 In vivo, the RPE cells are exposed to high partial pressures of oxygen, and the cells in the macula are exposed to focused light. Thus, although the evidence remains circumstantial, it seems possible that oxidative injury contributes to macular disease development and/or progression. Although RPE cell cultures used in the present study retain morphology characteristic of RPE, they differ from in vivo RPE because they have a decreased content of pigment granules and have acquired the capacity to proliferate. But these cultured cells remain sensitive to light-induced toxicity40 and chemical-induced oxidative injury.41 Thus, these cell cultures provide a useful in vitro model for study of potential mechanisms to protect the RPE from oxidative damage.
Our previous studies showed that cultured hRPE cells have the capacity to synthesize the antioxidant GSH.42 Moreover, the sensitivity of these cells to oxidative injury was decreased by supplying amino acid precursors of GSH, and this sensitivity was increased by inhibiting GSH synthesis.3 Thus, hRPE cells are sensitive to oxidative stress, and enhanced synthesis of GSH protects against oxidative stress in these cells. The present studies show that cellular GSH also can be increased by a nonnutritive dietary compound and that pretreatment with this compound also protects against oxidative injury. Thus, the results support the principle that antioxidant defenses can be enhanced in the hRPE by nutritionally or therapeutically increasing GSH synthesis.
Although DMF ultimately caused a large increase in intracellular GSH concentrations, it immediately depletes the cells of GSH. Our evidence suggests that this depletion was not caused by increased efflux of GSH from the cells or by an oxidative insult. The results suggest that DMF formed a conjugate with GSH that was exported and/or metabolized by the cell. A significant increase in extracellular GSH and CySSG concentration at 16 to 24 hours after DMF addition (Fig. 3) provides evidence that the efflux of GSH increased only after intracellular concentrations were increased. Therefore, our evidence supports the conclusion that a DMF-GSH conjugate, or a decrease in GSH per se, acts to induce the synthesis of glutathione. If induction is stimulated by a GSH conjugate, then it may be possible to stimulate synthesis with a more potent conjugate under conditions that do not deplete GSH.
The increase in GSH after treatment with DMF is consistent with previous studies showing transcriptional regulation of GLCL.21 22 23 24 However, in contrast to results showing increased expression of catalytic subunit only, DMF appeared to induce activity in hRPE cells in a previously unrecognized manner, by increasing the expression of the mRNA and the resultant protein for the regulatory subunit. An increase in the amount of regulatory subunit may result in an increased half-life of the catalytic subunit or alter its kinetic properties to account for the twofold increase in activity of GLCL.
Despite these very promising observations concerning increased GSH synthesis, it is unclear whether the initial GSH decrease would pose a health risk, whether GSH increases in the RPE can be achieved in vivo, whether such increases can be sustained over long periods, and whether such increases can protect against oxidative processes that may contribute to ARMD. The initial decrease in GSH concentration with DMF suggests that intermittent therapy may pose a risk. However, if long-term exposure allows a sustained elevation of GSH, the use of DMF as a therapeutic agent for the prevention and treatment of ARMD may nonetheless be feasible. Alternatively, other inducers that increase intracellular GSH levels without causing a transient decrease, such as oltipraz,43 may be effective and more useful therapeutically.
The present finding of an increase of GSH in cultured hRPE cells caused by DMF treatment suggests a possible mechanism that could contribute to epidemiologic findings of an association of fruit and vegetable consumption and reduced risk of ARMD.44 45 Plants contain a variety of compounds that induce detoxication enzymes, including the rate-limiting enzyme of GSH synthesis, GLCL. People who consume diets high in fruits and vegetables may have higher GSH concentration in their RPE cells as a consequence of this induction and therefore may have enhanced antioxidant defenses. In support of this hypothesis, a previous demographic study showed increased plasma GSH in subjects who reported higher fruit and vegetable consumption on a food frequency questionnaire.5
In conclusion, our results show that a nonnutritive dietary compound results in increased intracellular GSH in hRPE cells and consequent protection against oxidative injury. These results suggest that consumption of diets high in such compounds, or therapeutic administration of such compounds, could enhance antioxidant defenses and protect against ARMD. However, early depletion of GSH by DMF also suggests that experimental use of such a strategy must be undertaken cautiously to determine whether the transient decrease in GSH could contribute to increased susceptibility to oxidative injury.
Footnotes
Reprint requests: Paul Sternberg, Jr, Emory University School of Medicine, Department of Ophthalmology, 1365B Clifton Road, NE, Atlanta, GA 30322.
Supported by National Institutes of Health Grants EY07892 and EY06360 and Research to Prevent Blindness.
Submitted for publication November 11, 1998; revised March 12, 1999; accepted April 8, 1999.
Proprietary interest category: N.
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K. A. Rezaei, Y. Chen, J. Cai, and P. Sternberg Modulation of Nrf2-Dependent Antioxidant Functions in the RPE by Zip2, a Zinc Transporter Protein Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1665 - 1670. [Abstract] [Full Text] [PDF]
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L. Wang, Y. Chen, P. Sternberg, and J. Cai Essential Roles of the PI3 Kinase/Akt Pathway in Regulating Nrf2-Dependent Antioxidant Functions in the RPE Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1671 - 1678. [Abstract] [Full Text] [PDF]
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L. A. Voloboueva, J. Liu, J. H. Suh, B. N. Ames, and S. S. Miller (R)-{alpha}-Lipoic Acid Protects Retinal Pigment Epithelial Cells from Oxidative Damage Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 4302 - 4310. [Abstract] [Full Text] [PDF]
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