Antioxidants and Ocular Cell Type Differences in Cytoprotection from Formic Acid Toxicity in Vitro

Jaime L. Treichel*, Michele M. Henry{dagger}, Christine M. B. Skumatz{dagger}, Janis T. Eells* and Janice M. Burke*,{dagger},1

* Departments of Pharmacology & Toxicology and {dagger} Ophthalmology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Received April 2, 2004; accepted August 10, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinal photoreceptors and retinal pigment epithelial (RPE) cells are among the cell types that are sensitive to poisoning with methanol and its toxic metabolite formic acid. When exposed to formic acid in vitro, cultured cell lines from photoreceptors (661W) and the RPE (ARPE-19) were previously shown to accumulate similar levels of formate, but cytotoxic effects are greater in 661W cells. Here catalase and glutathione were analyzed in the two retinal cell lines to determine whether differences in these antioxidant systems contributed to cell-type-specific differences in cytotoxicity. Cells were exposed to formic acid (pH 6.8) in the culture medium in the presence or absence of a catalase activity inhibitor, 3-amino-1,2,4-triazole (AT), or a glutathione synthesis inhibitor, buthionine L-sulfoximine (BSO). Catalase protein, catalase enzyme activity, glutathione, glutathione peroxidase activity, cellular ATP, and cytotoxicity were analyzed. Compared to ARPE-19, 661W cells show lower antioxidant levels: 50% less glutathione, glutathione peroxidase and catalase protein, and 90% less catalase enzyme activity. In both cell types, formic acid treatment produced decreases in glutathione and glutathione peroxidase, and glutathione synthesis inhibition with BSO produced greater ATP depletion and cytotoxicity than formic acid treatment alone. In contrast, formate exposure produced decreases in catalase protein and activity in 661W cells, but increases in activity in ARPE-19. Treatment with the catalase inhibitor AT increased the formate sensitivity only of the ARPE-19 cells. ARPE-19 cells, therefore, may be less susceptible to formate toxicity due to higher levels of antioxidants, especially catalase, which increases on formate treatment and which has a significant cytoprotective effect for the RPE cell line.

Key Words: formate; methanol; photoreceptors; glutathione; catalase.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Methanol is recognized as a serious neurotoxin capable of producing severe visual impairment or blindness (Benton and Calhoun, 1952Go; Eells, 1992Go; Roe, 1955Go). Methanol is metabolized in the liver by sequential oxidative steps to formic acid, formaldehyde, and carbon dioxide (Roe, 1955Go). Formic acid is the toxic methanol metabolite, responsible for both the metabolic acidosis and the ocular toxicity characterizing human methanol poisoning (Hayreh et al., 1980Go; Tephly and McMartin, 1984Go).

Formic acid disrupts mitochondrial electron transport and energy production (Hayreh et al., 1980Go; Sharpe et al., 1982Go) by inhibiting cytochrome oxidase activity, the terminal electron acceptor of the electron transport chain (Nicholls, 1975Go, 1976Go). Cell death from cytochrome oxidase inhibition by formate is believed to result partly from depletion of ATP, reducing energy levels so that essential cell functions cannot be maintained. Evidence for ATP depletion by formate comes partly from studies showing decreased ATP synthesis in isolated mitochondria and decreased cellular ATP content in cultured neuronal cells exposed to formate (Dorman et al., 1993Go; Eells et al., 1997Go). Since ATP is required for fundamental cellular functions such as operating energy-requiring ion pumps, ATP depletion may reduce cell viability by loss of ionic and volume regulatory controls (Buja et al., 1993Go).

Inhibition of cytochrome oxidase by formate may also cause cell death by increased production of cytotoxic reactive oxygen species (ROS) secondary to the blockade of the electron transport chain (Richter et al., 1995Go). Although cells contain antioxidant defenses to guard against ROS damage, antioxidant systems may be depleted when oxidative stress is elevated, increasing the susceptibility of cells to the cytotoxic effects of ROS-induced damage to DNA and other molecules, notably membrane lipids (Richter et al., 1995Go).

Although mitochondria are ubiquitous, only certain tissues are affected in methanol/formate toxicity. In the eye, the optic nerve (Baumbach et al., 1977Go) and the retina (Murray et al., 1991Go; Roe, 1948Go) are sensitive tissues. In methanol-intoxicated rats, retinal ATP and ADP are depleted (Seme et al., 2001Go), and photoreceptors and the adjacent retinal pigment epithelium (RPE) show functional and/or structural changes. With methanol exposure of the animals, functional decrements are detected in photoreceptor cells by electroretinography (Seme et al., 1999Go), and both photoreceptors and RPE cells show morphologic changes (Murray et al., 1991Go). These changes tend to be regional or patchy and to include vacuolation of the cytoplasm and swelling or severe disruption of mitochondrial cristae. Recent analysis of a methanol poisoning case showed similar morphologic changes in both photoreceptor and RPE cells, indicating a retinotoxic effect on these tissues in humans as well (Treichel et al., 2004Go).

Since retinal photoreceptors and the RPE have a close structural and functional association, damage to one cell type can affect the viability of the other, making it difficult to discriminate in studies of whole organisms which retinal cell type(s) is sensitive to formate. We have therefore been developing a cell culture model to separately analyze formate effects on the retinal cells and have found greater formate-induced cytotoxicity in a photoreceptor (661W) as compared to an RPE (ARPE-19) cell line (Treichel et al., 2003Go). Differences in cytotoxicity were not due to differences in formate accumulation, which was similar for both cell types. To explain the observed differences in formate sensitivity between 661W and ARPE-19 cells, here we examined whether the cell types differ in antioxidant levels. This question is particularly relevant for photoreceptors and RPE, because in situ both cell types are at a high risk for oxidative damage. Photoreceptors are highly aerobically active, oxygen levels are high in the outer retina (Linsenmeier, 1986Go), and both tissues are exposed to high levels of light irradiation. Further, photoreceptor outer segment membranes are rich in polyunsaturated fatty acids (PUFAs), which are susceptible to lipid peroxidation (Handelman and Dratz, 1986Go), and outer segments are shed and then phagocytized by the RPE, increasing the oxidative burden for these cells (Miceli et al., 1994Go).

Perhaps because the retina is at high risk for oxidative damage, tissues of the outer retina have a rich complement of antioxidants, including catalase and glutathione. Catalase has not been specifically measured in photoreceptors, but catalase activity is the highest in the RPE of all ocular tissues where it appears to suppress the generation of lipid peroxides (Liles et al., 1991Go; Miceli et al., 1994Go). Glutathione is found in photoreceptor outer segments and in the RPE (Beatty et al., 2000Go; Newsome et al., 1994Go). Further, RPE cells have higher activity levels of glutathione peroxidase, an enzyme that uses glutathione as an electron donor to reduce hydroperoxides, than many other tissues including photoreceptors (Handelman and Dratz, 1986Go; Naash and Anderson, 1989Go).

Here we analyzed catalase and glutathione/glutathione peroxidase in 661W photoreceptor and ARPE-19 cell lines and compared the cell type sensitivity to formate when antioxidant systems were inhibited. We show that the two ocular cell types differ in endogenous levels of both antioxidants. Further, we demonstrate cell type differences in how catalase levels are affected with formate exposure, and in the dependence upon catalase for cytoprotection from the toxic effects of formate.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures. The spontaneously arising human RPE cell line ARPE-19 (Dunn et al., 1996Go) (American Type Culture Collection, Rockville, MD) was propagated in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum and antibiotics. Cultures were fed biweekly and maintained in a gas environment of 95% air/5% CO2. The SV-40 transformed mouse photoreceptor cell line 661W (a generous gift of Dr. Al-Ubaidi, University of Oklahoma) was grown similarly except using Dulbecco's modified Eagle's Medium supplemented with 5% fetal bovine serum (Al-Ubaidi et al., 1992Go; Tan et al., 2004Go). Cells were fed 24 h before each experiment and plated in replicate wells for each test or control group, using the same number of cells of both types, in 24-well plates or 12-well plates (for Western blot analysis). Experiments were initiated when cultures were at 90% confluency as estimated by phase contrast microscopy.

Experimental cultures were treated for intervals to 48 h with 30 mM formic acid, pH 6.8. The treatment parameters, which were chosen to simulate the formate concentration and blood pH found in human methanol poisoning cases (Hayreh et al., 1980Go), were previously shown to produce differential cytotoxic effects on the ocular cells in vitro (Treichel et al., 2003Go). Paired control cultures were in medium containing 30 mM HCl, pH 6.8 (acid control). In some experiments, cells were pretreated for 2 h with 20 mM 3-amino-1,2,4-triazole (AT) (Sigma, St. Louis, MO) (Lord-Fontaine and Averill, 1999Go) to inhibit catalase activity, or for 24 h with 1.0 mM L-buthionine-(S,R)-sulfoximine (BSO) (Sigma, St. Louis, MO) (Lord-Fontaine and Averill, 1999Go) to inhibit gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione synthesis (Griffith and Meister, 1979Go). At harvest for each time point, a sample of the culture medium was taken for measurement of released lactate dehydrogenase (LDH) as described below. Cultures were then placed on ice and rinsed with phosphate-buffered saline (PBS), followed by retrieving cell samples for measurements of formate, ATP concentration, and total cell protein, also as described below. Samples were frozen at –80°C prior to assay.

Assay methods. LDH release into the culture medium from damaged cells was used as a measure of cytotoxicity and quantified by a colorimetric method using the Cytotoxicity Detection Kit (Roche Molecular Biochemicals). According to the assay, NADH that is produced from the LDH-catalyzed conversion of lactate to pyruvate is used to reduce tetrazolium to the colored product formazan. ATP was extracted from cells and assayed by a luciferin–luciferase assay using the Sigma kit and the Strehler method (Strehler, 1968Go). Formate was extracted from cells with 0.1 M sodium hydroxide and assayed by the fluorimetric method of Makar and Tephly (1982)Go. LDH, ATP, and formate measurements were standardized to sample protein determined by the Bradford assay (Bradford, 1976Go) from paired, identically treated culture wells. Measurements were expressed as follows: percent LDH release above baseline pretreatment levels (at time 0 h), nmoles ATP/mg protein, or µmoles formate/mg protein.

Antioxidant Measurements
Catalase protein and catalase enzyme activity. To measure catalase protein in 661W and ARPE-19 cells, quantitative Western blotting was performed as previously described (Cao and Burke, 1997Go). Briefly, cells in 12-well plates were harvested from replicate wells by rinsing with cold PBS; then cells were extracted with a 1:1 mixture of Laemmli's (Laemmli, 1970Go) electrophoresis buffer and the following Triton detergent buffer: 10 mM Tris–HCl, pH 7.5, 120 mM NaCl, 25 mM KCl, 2 mM EGTA, 2 mM EDTA, 0.5% (vol/vol) Triton X-100, and a cocktail of protease inhibitors. Aliquots were taken for protein measurements; then ß-mercaptoethanol (5 mM) was added to the remainder of the sample. Samples were boiled, and proteins were separated by electrophoresis using 10% separating SDS gels. Blots were prepared by standard methods, probed with a polyclonal antibody to catalase (Abcam), and visualized by the ECL detection system capturing the signal on film (Amersham). Blotting signals were quantified in four replicate lanes by densitometry, using the computer-assisted NIH Image program, and expressed in arbitrary densitometric units. Preliminary experiments were performed to identify antibody dilution (1:2500 was used here), time of incubation, and protein loading to achieve a specific, linear blotting signal. Equivalent protein loading was confirmed by inspection of band density on Coomassie Blue stained gels (not shown). Due to higher catalase in ARPE-19 cells (see Results), to compare catalase protein by densitometric analysis required a lower protein loading for ARPE-19 (15 µg/lane) than for 661W cells (30 µg/lane).

Catalase enzyme activity was measured using the Amplex Red Catalase Assay Kit (Molecular Probes). Following the reaction of catalase with hydrogen peroxide, the Amplex Red reagent reacts with unreacted hydrogen peroxide and produces a fluorescent product, which was quantified using a 96-well fluorometric plate reader with excitation and emission wavelengths of 560 and 590 nm, respectively. Measurements were standardized to sample protein and expressed in units/mg protein.

Because catalase is markedly higher in ARPE-19 than in 661W cells (see Results), for demonstration purposes in the figures, different y-axis scales are used for the two cell types in the graphs showing catalase protein and catalase activity (Fig. 1).



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FIG. 1. Catalase in 661W (A, C) and ARPE-19 cells (B, D) at intervals after addition of formic acid to the culture medium as described in the Methods. A and B show catalase Western blots (formic acid treatment only) and analyses of band density (in arbitrary densitometric units) for catalase protein; C and D show catalase enzyme activity (in units per mg protein). Data are means (± SD) of four replicate culture wells from representative experiments. Note that the y-axis scales differ for the two cell types due to the markedly higher catalase values for ARPE-19 cells. Asterisks (*) indicate significant differences (p values are provided) between acid control (HCl) and formic acid (FA) treatment at the same time interval (ANOVA followed by Bonferroni's).

 
Glutathione and glutathione peroxidase activity. Reduced glutathione (GSH) was measured using the Glutathione (GSH) Detection Kit (Chemicon Internation, Inc.). The dye monochlorobimane binds with high affinity to thiol groups on glutathione, causing its conversion to a fluorescent product that was quantified using a 96-well fluorometric plate reader with excitation and emission wavelengths of 380 and 460 nm, respectively.

Glutathione peroxidase was measured by a modification of the method of Ohrloff et al. (1980)Go, using a 3-ml total reaction volume containing 0.3 ml sample extracted from culture wells with phosphate buffered saline, and the following reaction mixture: 60 mM potassium phosphate buffer (pH 7.6), 1 mM EDTA, 0.33 mM NADPH, 4 U glutathione reductase, 4 mM reduced glutathione, and 0.4 mM t-butylhydroperoxide. The decrease in extinction was measured for 4 min at 366 nm in a spectrophotometer. Measurements from a sample-free blank were subtracted.

Measures of both glutathione and glutathione peroxidase were standardized to sample protein and expressed in nmoles/mg protein (for glutathione) or mU/mg protein (for glutathione peroxidase).

Statistical analysis. Results are expressed as the mean ± SD of replicate culture wells within an experiment. Statistical analysis of group means was made using a group Student's t-test if only one comparison was made between two groups. In all cases in which several comparisons were required, a one-way ANOVA with repeated measures was performed. This was followed by a Bonferroni's test procedure for multiple comparisons with a control. For data presented graphically, the p value is reported in the figures; the minimum level of significance was taken as p < 0.05.

For each protocol that was performed and each assay method, a minimum of four independent experiments was conducted on each cell type. Representative data are shown.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Catalase
661W cells have significantly less catalase than ARPE-19 cells (Student's t-test, p < 0.05), with 661W cells containing half the amount of catalase protein and only 14% the amount of catalase activity as ARPE-19 cells (Table 1). When 661W cells are treated with formic acid, catalase declines even further (Fig. 1). Catalase protein decreases earlier than catalase activity, by 6 h of formic acid treatment, ultimately declining significantly, to 44% below the same time interval control level by the termination of the experiment at 48 h (Fig. 1A). Initially, catalase catalytic activity is maintained in 661W cells even with declines in catalase protein. However, by 48 h of formic acid treatment, catalase activity also decreases significantly, to 27% below the same time interval control level (Fig. 1C).


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TABLE 1 Antioxidant Comparison in 661W and ARPE-19 Cells

 
ARPE-19 cells have higher baseline levels of catalase protein and activity than 661W cells (Table 1). Additionally, treatment of ARPE-19 with formic acid results at 48 h in higher, not lower, levels of catalase activity. Formic acid treatment of ARPE-19 cells produces an initial (at 6 h) significant increase in catalase protein and activity relative to the same time interval control. This is followed by a transient decrease (at 24 h) and then a second increase (at 48 h) (Figs. 1B and 1D), a pattern that was reproducible among experiments. The increase in catalase protein occurring between 24 and 48 h returns protein to baseline levels, which are the same as the matched time interval control (Fig. 1B). Catalase activity, however, is significantly greater at 48 h (42% above the baseline pretreatment level) (Fig. 1D).

Inhibition of Catalase Activity
The catalase inhibitor AT significantly inhibits baseline catalase activity below untreated cultures (Student's t-test, p < 0.05), and the inhibition was equivalent (approximately 90%) in both 661W and ARPE-19 cells (Table 1). Exposure to AT alone has no effect on baseline ATP or LDH release in either cell type, nor does AT affect formate accumulation (not shown). Formic acid exposure produced greater ATP depletion and LDH release than exposure to acid control medium in both cell types (Fig. 2), as we have previously observed (Treichel et al., 2003Go). The addition of AT to acid control medium did not affect ATP or LDH measurements in either cell type (Fig. 2), but AT differentially affected these measurements in the two retinal cell lines when it was added to medium containing formic acid. For 661W cells, the effects of formic acid exposure were unchanged by the addition of AT. Regardless of whether AT was present, 48 h after formic acid treatment ATP levels were significantly (72%) below the paired acid control (Fig. 2A), and LDH release was significantly (53%) above the paired acid control (Fig. 2C).



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FIG. 2. ATP levels in 661W (A) and ARPE-19 (B) and lactate dehydrogenase (LDH) release into culture medium by 661W (C) and ARPE-19 (D) cells exposed to acid control (HCl) or formic acid-containing (FA) medium, with or without 20 mM AT pretreatment. Data are means (± SD) of four replicate culture wells from a representative experiment. Symbols indicating significant differences (ANOVA followed by Bonferroni's test, with p values provided) are as follows: Asterisks (*): FA versus HCl at the same time interval; daggers ({dagger}): FA in the presence of AT versus HCl in the presence of AT; double daggers ({ddagger}): FA in the presence of AT versus FA.

 
In contrast to 661W cells, AT augmented the effects of formic acid on ARPE-19 cells. In the absence of AT, formic acid significantly depleted ATP in ARPE-19 cells, less than for the more sensitive 661 W cells but nonetheless 46% below the paired acid control at 48 h (Fig. 2B) (which was similar to the 6 and 24 h time points). When AT was added, ATP depletion was significantly greater, dropping to 72% below the paired acid control at 48 h; a similar level of ATP depletion occurred across the time course. LDH release after 48 h of formic acid treatment was 25% above the paired acid control (Fig. 2D) and similar to the 6 and 24 h time points, but again less than that for the more sensitive 661W cells. But when AT was added, LDH release from ARPE-19 cells was greater, increasing significantly to 60% above the paired acid control at 48 h. Further, LDH release at 48 h with AT treatment was significantly higher than the release seen in the AT treatment groups at 6 and 24 h. Inhibition of catalase activity therefore increases the sensitivity of ARPE-19 cells to formic acid exposure, resulting in an ATP depletion and an LDH release that are similar to the results for the more sensitive 661W cells.

Glutathione and Glutathione Peroxidase
661W cells have significantly lower amounts of glutathione or glutathione peroxidase than ARPE-19 cells (Student's t-test, p < 0.05) (Table 1). In this way, glutathione/glutathione peroxidase is similar to catalase: endogenous levels of both antioxidants are higher in the ARPE-19 cell line.

For both cell lines, exposure to formic acid produces a decrease in cellular levels of glutathione and glutathione peroxidase. For 661W cells, formic acid treatment for 48 h induces significant decreases in glutathione and glutathione peroxidase, to 38% (Fig. 3A) and 22% (Fig. 3C), respectively, below the same time interval controls. ARPE-19 cells show similar significant declines at 48 h in glutathione and glutathione peroxidase, to 42% (Fig. 3B) and 26% (Fig. 3D), respectively, below the same time interval controls.



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FIG. 3. Glutathione (A and B) and glutathione peroxidase activity (C and D) in 661W (A and C) and ARPE-19 cells (B and D). Cells were exposed to formic acid- or HCl-containing medium for intervals to 48 h. Data are means (±SD) of four replicate culture wells from representative experiments. Asterisks (*) indicate significant differences (p values are provided) between acid control (HCl) and formic acid (FA) treatment at the same time interval (ANOVA followed by Bonferroni's).

 
Glutathione Synthesis Inhibition
Similar to the catalase inhibitor, the glutathione synthesis inhibitor BSO has significant (Student's t-test, p < 0.05) and equivalent inhibitory effects (approximately 80% inhibition) on baseline glutathione activity in both 661W and ARPE-19 cells (Table 1). Also like the catalase inhibitor, the glutathione synthesis inhibitor alone does not affect baseline ATP, LDH release, or formate accumulation (not shown), or the response to treatment with acid control medium (Fig. 4) in either cell type.



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FIG. 4. ATP levels in 661W (A) and ARPE-19 (B) cells and lactate dehydrogenase (LDH) release into culture medium by 661W (C) and ARPE-19 (D) exposed to acid control (HCl) or formic acid-containing (FA) medium, with or without 1 mM BSO pretreatment. Data are means (±SD) of four replicate culture wells from a representative experiment. Symbols indicating significant differences (ANOVA followed by Bonferroni's test, with p values provided) are as follows: Asterisks (*): FA versus HCl at the same time interval; daggers ({dagger}): FA in the presence of BSO versus HCl in the presence of BSO; double daggers ({ddagger}): FA in the presence of BSO versus FA.

 
In contrast to the catalase inhibitor, however, which augmented the toxic effects of formic acid only in ARPE-19 cells, the glutathione synthesis inhibitor BSO augmented the toxic effects for both cell types (Fig. 4). For 661W cells, ATP depletion by formic acid at 48 h was 62% below paired acid controls in the absence of BSO, increasing to 83% below controls in the presence of BSO. LDH release was 47% above paired acid controls in the absence of the inhibitor, increasing to 63% above controls in its presence. For ARPE-19 cells, comparable values at 48 h for ATP were 50% (without BSO) and 79% (with BSO) below paired acid controls and, for LDH, 22% (without BSO) and 56% (with BSO) above acid controls. With formic acid treatment alone ATP depletion and LDH release were similar across the time course, but in the presence of BSO there was an increasing effect over time for both measures of cytotoxicity in both cell types. Therefore, both retinal cell lines show dependence upon glutathione for protection from the cytotoxic effects of formic acid.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In methanol poisoning, the metabolite formic acid acts as a mitochondrial toxin. For reasons that are unclear, only certain tissues such as photoreceptors and RPE cells of the outer retina (Murray et al., 1991Go; Seme et al., 1999Go, 2001Go, Treichel et al., 2004Go) show cytotoxic effects with methanol/formic acid exposure. To analyze in more detail how these ocular cells respond to formic acid treatment and to determine whether the effects differ between cell types, we are evaluating an in vitro model that employs 661W photoreceptor and ARPE-19 cell lines. In previous studies of formic acid addition to the culture medium (Treichel et al., 2003Go), the two ocular cell lines accumulated similar levels of formate, but 661W cells showed greater cytotoxicity, which was manifest as greater ATP depletion and LDH release (see also Figs. 2 and 4 herein). Since generation of cytotoxic ROS is considered one mechanism of formic acid-induced cell damage (Richter et al., 1995Go), we examined here the role of antioxidants in cytoprotection. We find a higher complement of glutathione/glutathione peroxidase and (especially) catalase in ARPE-19 than in 661W cells, suggesting that antioxidant status contributes to the greater resistance of ARPE-19 cells to formic acid-induced cytotoxicity.

Glutathione has been previously demonstrated in both photoreceptors (Beatty et al., 2000Go) and the RPE (Newsome et al., 1994Go), although the measurements were made in separate studies, so comparative levels in the two tissues were not obtained. Here, using cell lines derived from the two cell types, ARPE-19 cells showed more than two-fold higher levels of glutathione and of glutathione peroxidase activity than 661W cells. From other investigations, RPE cells are known to have notably high levels of glutathione peroxidase activity compared to other ocular tissues, including photoreceptors, suggesting that the differences in culture reflect tissue-specific differences. For example, relative to extracts of the entire retina, glutathione peroxidase enzyme activity is reportedly somewhat higher (1.5 times) in the outer segments of rod photoreceptors, but higher still (5 times) in the RPE (Naash and Anderson, 1989Go).

Both 661W and ARPE-19 cells appear to depend upon glutathione for protection against formic acid-induced toxicity, since inhibition of glutathione synthesis by BSO increases ATP depletion and LDH release in both cell lines. This phenomenon of increased sensitivity to oxidative injury when treated with BSO occurs in many cultured cell types (Andreoli et al., 1986). With formic acid treatment, both retinal cells types examined here show decreases in glutathione and glutathione peroxidase. Similar decreases occur in situ in methanol-intoxicated rats in which retinal glutathione (Seme et al., 2001Go) and liver glutathione peroxidase (Skrzydlewska and Farbiszewski, 1997Go) are depleted. Depletion of glutathione with formic acid treatment could result from ATP depletion, since glutathione synthesis is ATP dependent, or from increased ROS generation, both of which occur as a consequence of inhibition of the mitochondrial electron transport chain (Meister, 1995Go). Regardless of the mechanism, 661W and ARPE-19 cells treated with formic acid show a similar depletion of glutathione, as well as a similar dependence on glutathione as a protective antioxidant. Perhaps the higher baseline levels of glutathione/glutathione peroxidase in ARPE-19 cells contribute to the greater cytoprotection of these cells when exposed to formic acid, but the contribution may be relatively small.

Catalase appears to serve as a greater discriminator than glutathione/glutathione peroxidase of the selective sensitivity of the two ocular cell lines to formic acid-induced toxicity. ARPE-19 cells have markedly higher amounts of catalase protein and of catalase activity than 661W cells. Catalase levels have not specifically been compared in extracts of photoreceptor and RPE cells, but the high catalase in the ARPE-19 cell line observed here is consistent with reports indicating that the RPE has the highest levels of catalase activity of several ocular tissues (retina, iris, or vitreous [Liles et al., 1991Go]). Not only do ARPE-19 cells have higher baseline levels of catalase than 661W cells, but with formic acid exposure, catalase activity decreases in 661W cells and increases in ARPE-19 cells. Increases in catalase activity have been observed in liver homogenates of methanol-treated rats (Skrzydlewska and Farbiszewski, 1997Go) and in cultured RPE cells subjected to other forms of oxidative stress resulting from treatment with H2O2 or rod outer segments (Miceli et al., 1994Go; Tate et al., 1995Go).

In the latter investigations where increases in RPE catalase were found, activity was measured at a single posttreatment interval without concomitant catalase protein measurements. Here, however, changes in both catalase protein and activity were examined at multiple intervals after formic treatment (Fig. 1), which revealed a complex pattern, especially in the ARPE-19 cells. For 661W cells, in which catalase protein declines within 6 h of formic acid treatment, activity levels are nonetheless initially maintained, suggesting that not all of the catalase is maximally functional, and that a ‘reserve activity’ can be invoked during formic acid exposure. A similar observation suggesting submaximal activity of catalase in tissues was made by Makar and Mannering (1968), who reported that only one-fifth of the total catalase activity available in rodent liver homogenates is used during methanol oxidation. For 661W cells, catalase activity is not sustained after longer periods of formate treatment, perhaps because the catalase protein declines to levels that are too low to maintain activity. In ARPE-19 cells after formate treatment there is a reproducible oscillation in amounts of catalase protein, which increases at 6 h, drops below baseline at 24 h, then returns to baseline at 48 h. At the 24 h time period, when catalase protein levels are lower than at the baseline pretreatment interval, catalase activity is nonetheless maintained, again suggesting that not all of the catalase is maximally functional and a reserve activity may be invoked after formate treatment. At the latest treatment interval in ARPE-19 cells, catalase activity levels are significantly increased. It appears, therefore, that formic acid treatment triggers an increase in the enzyme activity of catalase only in the ARPE-19 cells.

The mechanism(s) whereby formic acid treatment induces increased catalase activity in the cultured ARPE-19 cells is unknown. One possibility is an increase in the concentration of the enzyme's substrate hydrogen peroxide, which is likely produced during formate exposure and which has been speculated to regulate catalase activity (Makar and Mannering, 1968). Formate treatment may also trigger posttranslational modifications of catalase, such as phosphorylation, which could regulate its activity. However, no such mechanisms of catalase regulation have been identified.

Regardless of the mechanism of catalase upregulation, as a consequence of the higher baseline levels of catalase in ARPE-19 cells and the increases in catalase activity induced by formic acid treatment, ARPE-19 cells may be better protected from the cytotoxic effects of formic acid than 661W cells. Indeed ARPE-19 cells appear more dependent than 661W cells upon catalase for cytoprotection, since catalase inhibition by AT increases the formic acid-induced ATP depletion and LDH release only in ARPE-19 cells.

We conclude that treatment with the methanol-derived toxin formic acid produces lower toxicity in ARPE-19 cells than 661W cells, due at least in part to the high antioxidant levels in ARPE-19. This is especially true for catalase, which is markedly higher than in 661W cells, which increases further on formate exposure, and on which ARPE-19 cells disproportionately depend for cytoprotection. Other antioxidant systems, or other differences between the cell lines, may also contribute to differential cytotoxicity, but the antioxidants analyzed here, catalase and glutathione, are major antioxidants of these ocular cells. We cannot conclude with certainty that the results obtained here using cultured cell lines reflect properties of their tissues of origin. However, the ARPE-19 and 661W cell lines do express several tissue-specific features (Al-Ubaidi et al., 1992Go; Dunn et al., 1996Go; Tan et al., 2004Go), which has led to their use in studies of the cellular response to other agents that induce oxidative stress (Gao et al., 2001Go; Krishnamoorthy et al., 1999Go). One might add to the list of cell-type-specific properties retained by the ARPE-19 cells the expression of high levels of glutathione/glutathione peroxidase and catalase; high levels of these molecules are known to occur in the RPE in situ (Handelman and Dratz, 1986Go; Liles et al., 1991Go; Miceli et al., 1994Go; Naash and Anderson, 1989Go). Since the photoreceptors of the outer retina and the RPE monolayer are highly interdependent in situ, it would be difficult to advance the understanding of cell-type-specific effects of toxins such as formate without using cell culture models in which the cells can be separated. Primary cultures might seem preferable to cell lines, but adequate numbers of photoreceptors free of contaminating cell types are difficult to obtain and short lived, and RPE cultures grown from donor eyes are structurally and functionally heterogeneous, offering a cellular background that is too variable for cytotoxicity studies. The 661W and ARPE-19 ocular cell lines therefore offer a useful model for investigating cell type differences in the effects of formic acid, and perhaps also for probing broader questions of why some cells are more sensitive to mitochondrial toxins than others.


    ACKNOWLEDGMENTS
 
This work was supported by NIH grants R01 EY13722 (J.M.B.), R01 ES06648 (J.T.E.) and a Core Grant for Vision Research P30 EY01931 from the National Institutes of Health. Support was also provided by the Posner Foundation, and the David and Ruth Coleman Charitable Foundation (both Milwaukee, WI), and by an unrestricted grant from Research to Prevent Blindness, Inc.


    NOTES
 

1 To whom correspondence should be addressed at Eye Institute, 925 N. 87th Street, Milwaukee, WI 53226. Fax: (414) 456-6304. E-mail: jburke{at}mcw.edu.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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