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Am J Physiol Cell Physiol 292: C2197-C2203, 2007. First published March 7, 2007; doi:10.1152/ajpcell.00620.2006
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CELLULAR METABOLISM

Upregulation of {gamma}-glutamate-cysteine ligase as part of the long-term adaptation process to iron accumulation in neuronal SH-SY5Y cells

Pabla Aguirre, Pamela Valdés, Paula Aracena-Parks, Victoria Tapia, and Marco T. Núñez

Department of Biology, Faculty of Sciences, and Cell Dynamics and Biotechnology Research Center, University of Chile, Santiago, Chile

Submitted 13 December 2006 ; accepted in final form 8 February 2007


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reactive iron is an important prooxidant factor, whereas GSH is a crucial component of a long-term adaptive system that allows cells to function during extended periods of high oxidative stress. In this work, the adaptive response of the GSH system to prolonged iron loads was characterized in human dopaminergic SH-SY5Y neuroblastoma cells. After the initial death of a substantial portion of the cell population, the surviving cells increased their GSH content by up to fivefold. This increase was traced to increased expression of the catalytic and modulatory subunits of {gamma}-glutamate-cysteine ligase. Under conditions of high iron load, cells maintained a low GSSG content through two mechanisms: 1) GSSG reductase-mediated recycling of GSSG to GSH and 2) multidrug resistant protein 1-mediated extrusion of GSSG. Increased GSH synthesis and low GSSG levels contributed to recover the cell reduction potential from –290 mV at the time of cell death to about –320 mV. These results highlight the fundamental role of GSH homeostasis in the antioxidant response to cellular iron accumulation and provide novel insights into the adaptive mechanisms of neurons subjected to increased iron loads, such as those observed in Parkinson's disease.

oxidative stress; glutathione; multidrug resistance protein 1; oxidixed glutathione reductase; neurodegenerative diseases

SOME NEURODEGENERATIVE PROCESSES such as Parkinson's and Alzheimer's disease have been associated with iron accumulation in redox-sensitive tissues such as the substantia nigra and hippocampal neurons (12, 28, 31, 37, 38). Postmortem studies have described higher levels of iron in the normal substantia nigra pars compacta (SNpc) than in other brain regions and increased iron accumulation in the SNpc from Parkinson's disease compared with non-Parkinson's disease individuals (10, 26, 38). Similarly, an increasing body of evidence indicates that neurons from Alzheimer's disease brains are subject to a high oxidative load related to iron and copper accumulation (5, 9, 22, 23, 29).

The mechanisms by which neurons and other cell types avert iron-mediated oxidative stress are fairly unknown. As most neuronal cells do not divide, their antioxidant defenses must be highly resilient to maintain neuronal function throughout human life. Neuronal antioxidant defenses rely mainly on cellular levels of GSH (7, 8). Decreased activity of antioxidant enzymes is observed in Alzheimer's disease brains (21), an indication that the normal handling of GSH may be altered. In conjunction, elevated GSH levels in the hippocampus and midbrain have been reported in Alzheimer's disease (1, 27, 39), which may reflect a long-term response to chronic oxidative stress. In contrast, GSH levels in Parkinson's disease are specifically decreased in the SNpc without a concomitant increase in the levels of GSSG (24, 26, 32). Thus, substantial evidence indicates profound changes in GSH metabolism in neurodegenerative processes.

In the adaptive response toward oxidative stress, cells increase their GSH content by activating its de novo synthesis (15, 33). {gamma}-Glutamate-cysteine ligase (GCL; EC 6.3.2.2 [EC] , also known as {gamma}-glutamyl-cysteine synthetase), the rate-limiting enzyme for GSH synthesis, is a heterodimer composed of a catalytic or heavy subunit (GCLC; molecular mass ~73 kDa) and a modulatory or light subunit (GCLM; molecular mass ~30 kDa), which are encoded by different genes (34, 35). In the case of human GCL, both genes contain upstream regions with antioxidant response elements (AREs; also known as electrophilic response elements) and with consensus binding regions for the nuclear transcription factors neuronal response factor 2 (Nrf2), NF-{kappa}B, and activator protein-1 (6, 15, 36).

A biphasic response of cellular GSH levels to progressive iron accumulation has been recently reported, with the decreasing of GSH being associated with death of ~50% of the cell population (20). The focus of the present work was to elucidate the strategies by which part of the cells survived iron loading, with the thought that knowledge of the survival strategies may be relevant to understand the progress of neurodegenerative diseases such as Parkinson's disease.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. FBS, minimal essential medium (MEM), F-12 culture medium, MEM nonessential amino acids, and the antibiotic-antimycotic mixture were purchased from Invitrogen-GIBCO Life Technologies (Carlsbad, CA). The GSSG reductase inhibitor 1,3-bis(2-chloroethyl)-2-nitrosourea (BCNU), N-acetyl-L-cysteine (NAC), 2,2'-dinitro-5,5'-di-thiobenzoic acid (DNTB), 2-vinylpyridine, protease inhibitors, buffers, and salts were purchased from Sigma Chemical (St. Louis, MO). 2,5-Di-(t-butyl)-1,4-hydroquinone (TBH) and (E)-3-{3-[2-(7-chloro-2-quinolinyl)ethenylphenyl]}-3-(dimethylamino-3-oxopropylthio)methylthiol propanoic acid, sodium salt (MK571) were purchased from Calbiochem (San Diego, CA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2',7'-dichlorodihydrofluorescin diacetate (DCDHF-DA) were obtained from Molecular Probes (Eugene, OR). Culture plasticware and Transwells were purchases from Corning-Costar (Cambridge, MA).

Cell culture and iron challenge. Human neuroblastoma SH-SY5Y cells (CRL-2266, American Type Culture Collection, Rockville, MD) were seeded at 1 x 105 cells in 2-cm2 plastic wells and cultured at 37°C in a 5% CO2 incubator in MEM-F-12 medium supplemented with 10% FBS, MEM nonessential amino acids, antibiotic-antimycotic mixture, and 20 mM HEPES buffer. The total iron concentration in this culture medium was 7.02 µM as determined by atomic absorption spectrometry. The medium was replaced every 2 days. Under these conditions, the doubling time was ~48 h. After 8 days, cultures reached a steady-state number of cells. At that time, cells were challenged with iron for 2–10 days by supplementing the culture medium with either deferrioxamine or the complex FeCl3-sodium nitrilotriacetate [1:2.2 (mol:mol)] to achieve the desired final iron concentration. Three iron concentrations were selected: 2 µM Fe (low-iron medium), 7 µM Fe (control medium), and 10–80 µM Fe (high-iron medium). Under all conditions, the medium was changed every 2 days.

Cell viability. Cell viability was quantified in 96-well microplates by the MTT assay (Molecular Probes) following the manufacturer's instructions. This assay determines the mitochondrial-dependent formation of a colored product (18).

Assessment of oxidative stress. Generation of reactive oxygen species was determined using the membrane-permeable fluorescent probe DCDHF-DA as previously described (20). The fluorescence emitted by dichlorofluorescein (DCF) directly reflects the overall oxidative status of a cell (25). Cells were grown in glass coverslips for 8 days, after which time they were challenged with 2, 7, 40, or 80 µM Fe, and the cultures were continued for 2, 4, 6, 8, or 10 days. Cells were then incubated with DCDHF-DA, and cell-associated DCF fluorescence was detected in a Zeiss Axiovert 200M epifluorescence microscope. High-magnification fields from each culture condition were imaged with an Axiocam HR camera attached to a personal computer equipped with Axiovision software. Shutters and a neutral- density filter were used to minimize photobleaching. Alternatively, SH-SY5Y cells were grown in 96-well plates as described above, and cell-associated DCF fluorescence was quantified in a Cytofluor II plate reader (Applied Biosystems, Foster City, CA).

GSH and GSSG determination. The amount of GSH plus two times the amount of GSSG (GSx) was quantified using the enzymatic recycling method (11) adapted to microplate readers according Baker et al. (3). Ten microliters of the cell lysates or GSSG standards (0–200 pmol of GSx/10 µl) were transferred into wells of microtiter plates and diluted with 90 µl water. After the addition of 100 µl of the reaction mixture [0.1 M sodium phosphate buffer (pH 7.5) containing 1 mM EDTA, 0.3 mM DNTB, 0.4 mM NADPH, and 1 U/ml GSSG reductase], the increase in extinction at 405 nm was followed in a microplate reader (Tecan Sunrise, Grödig/Salzburg, Austria) at 5-min intervals until a steady state was achieved (usually >60 min). Glutathione content was evaluated using a calibration curve established with standard samples. Glutathione disulfide was quantified after the derivatization of GSH with 2-vinylpyridine (11). In brief, 130 µl of the protein-free supernatant were mixed with 5 µl of 2-vinylpyridine and adjusted with 0.2 M Tris to pH 6–7 (11). Standard amounts of GSSG were treated the same way. After a 1-h incubation at room temperature, 10 µl of the 2-vinylpyridine-treated samples or standards were assayed as described above using a calibration curve established between 0 and 50 pmol GSSG/well. Determination was performed on freshly lysed cell extracts, since storage, even at –80°C, resulted in GSH oxidation and the consequent increase of GSSG content.

Effect of iron on GCL expression. Total SH-SY5Y cell RNA was isolated with TRIzol reagent (Invitrogen-GIBCO Life Technologies), and total cDNA was obtained by a standard reverse transcription reaction. The expression of GCLC (GenBank Accession No. bc039894) and GCLM (GenBank Accession No. NM_002061) was determined by real-time PCR using the following primers: GCLC, 5'-ATGGAGGTGCAATTAACAGAC-3' (sense) and 5'-ACTGCATTGCCACCTTTGCA-3' (antisense); and GCLM, 5'-GCTGTATCAGTGGGCACAG-3' (sense) and 5'-CGCTTGAATGTCAGGAATGC-3' (antisense). GAPDH (GenBank Accession No. NP_002037) mRNA was used as an internal control. The primers for GAPDH were 5'-TGGGTGTGAACCATGAGAAG-3' (sense) and 5'-CCATCACGACACAGTTTCC-3' (antisense). Quantitative PCR amplification was performed with a Lightcycler device (Roche Farma) and the SYBR Green DNA Master Kit (Roche Farma) following the manufacturer's instructions. After an initial denaturation at 95°C for 10 min, all genes were amplified with 45 cycles of the following protocol: 58°C for 5 s and 72°C for 8 s. This PCR amplification was followed by a melting-curve analysis (95°C for 5 s and 58°C for 20 s) and by a 0.2°C ramping from 58 to 95°C to control for specific amplification products. Data obtained for GCLC and GCLM were normalized by comparison with GAPDH expression. Protein levels of GCLC were determined in cell extracts by Western blot analysis as previously described (2). Blotted membranes were incubated overnight at 4°C with rabbit anti-human GCLC polyclonal antibody (Lab Vision, Fremont, CA) at a 1:1,500 dilution, rinsed, and incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody for 1 h at 25°C. Transferred proteins were detected with a peroxidase-based chemiluminiscence assay kit (SuperSignal, Pierce Chemical, Rockford, IL). Chemiluminiscence was detected using a Molecular Imager FX device (Bio-Rad, Hercules, CA).

Data analysis. Variables were tested in triplicate, and experiments were repeated at least twice. Variability among experiments was <20%. One-way ANOVA was used to test for significant differences among mean values, and Tukey's post hoc test was used for comparisons (InStat, GraphPad Software, San Diego, CA). Differences were considered significant if P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Iron induces cell death through oxidative stress. The capacity of SH-SY5Y neuroblastoma cells to survive for an extended period of time under an iron challenge was tested. To this end, cells were cultured for 8 days to achieve proliferative quiescence and then for up to 10 days with various concentrations of iron in the culture medium (Fig. 1A). Whereas ≥80% cells survived for up to 10 days with moderate (7–20 µM) iron concentrations, iron in the range of 60–80 µM produced massive cell death in a dose-dependent manner during the first 4 days of exposure. The surviving cells adapted to high iron conditions, as no further decline in cell number was evident from days 4–10 (Fig. 1A).


Figure 1
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  		Fig. 1. Long-term viability of SH-SY5Y cells cultured under various iron concentrations. A: SH-SY5Y cells grown in 96-well plates for 8 days were challenged with various concentrations of iron for 2, 4, 6, 8, or 10 days. Cell viability at the end of the second culture period was determined by the MTT method. Each individual point was tested in triplicate. Shown is 1 of 3 similar experiments. Decreased cell viability was apparent with increasing iron concentration and time of incubation. B: cells grown on glass coverslips for 8 days in the standard medium were challenged with 2, 7, or 80 µM Fe, and the culture was continued for 2, 4, or 10 days. The levels of reactive oxygen species were imaged in a Zeiss Axiovert 200M epifluorescence microscope equipped with an Axiocam HR camera. Shown are representative frames. C: dichlorofluorescein (DCF) fluorescence from each of the conditions shown in B was quantified in a microplate reader as described in MATERIALS AND METHODS. Values are means ± SE of 30-plicates of each condition. Significant differences (P < 0.001) were found between 2 µM Fe and 7 or 80 µM Fe at all days tested. Significant differences (P < 0.01) were also found between day 2 and days 4 or 10 in culture for all iron concentrations tested.

 
Because iron accumulation induces oxidative stress, it was of interest to determine the degree of oxidative stress in cells cultured for up to 10 days with increasing iron concentrations (Fig. 1, B and C). At all times tested, iron induced increased oxidative stress when its concentration was raised from 2 µM to either 7 or 80 µM. Significant increases in DCF fluorescence were observed between days 2 and 4, with a plateau generally seen at day 10. Nevertheless, a counteracting response was evident because no significant differences (P > 0.05) in DCF fluorescence were observed between 10-day cultures exposed to 7 or 80 µM Fe (Fig. 1C). These data are consistent with an adaptation process whereby cells neutralize increased oxidative stress with increased antioxidant defenses.

Expression of GCLC and GCLM. Critical to the adaptive response to oxidative stress is the transcriptional upregulation of several antioxidant enzymes, including GCLC and GCLM, the rate-limiting enzyme in GSH synthesis (15). Thus, we explored the possibility that iron induced GCLC or GCLM expression in SH-SY5Y cells (Fig. 2). As a positive control, we tested the effect of TBH, an inductor of both GCLC and GCLM in cultured rat hepatocytes (4). With 2 µM Fe in the culture medium, TBH strongly activated both GCLC and GCLM expression, and this activation was largely inhibited by the antioxidant NAC (Fig. 2A).


Figure 2
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  		Fig. 2. Expression analysis of the {gamma}-glutamate-cysteine ligase (GLC) catalytic subunit (GCLC) and the modulatory GLC subunit (GCLM). A: expression of GCLC and GCLM following a 16-h treatment with 2,5-di-(t-butyl)-1,4-hydroquinone (TBH) in the absence or presence of 10 mM N-acetyl-L-cysteine (NAC). Cells were cultured for 2 days in 2 µM Fe. Data are means ± SD of 3 determinations. aSignificant difference (P < 0.001) for both GCLC and GCLM between control and TBH conditions. bSignificant difference (P < 0.001) for both GCLC and GCLM between TBH and TBH + NAC conditions. B and C: mRNA expression of GCLC and GCLM was determined by real-time PCR of total RNA obtained from cells cultured for 2 or 10 days in medium with either 2, 7, or 80 µM Fe. GAPDH mRNA expression was used as an internal standard. cSignificant difference in GCLC expression (P < 0.001) were found between 2 µM values and 7 and 80 µM values for all days tested. dSignificant difference (P < 0.05–0.001) between days 2 and 10 for 7 and 80 µM Fe, but not for 2 µM Fe. e,f,gSignificant increases in GCLM expression were found for couples 2 µM Fe/2 days vs. 80 µM Fe/2 days (eP < 0.001), 2 µM Fe/2 days vs. 80 µM Fe/10 days (fP < 0.001), and 7 µM Fe/2days vs. 80 µM Fe/2 days (gP < 0.001). D: Western blot analysis of GCLC expression. Immunodetection (top) and quantification of band intensity normalized for actin (bottom) show a significant increase (hP < 0.01) in GCLC at 7 and 80 µM Fe compared with the basal 2 µM Fe condition after a 2-day culture period.

 
Significant differences in GCLC expression were found between the 2 µM values and the 7 and 80 µM values for all days tested (Fig. 2B). There were also significant increases between days 2 and 10 for 7 and 80 µM Fe (Fig. 2B). Thus, iron induced GCLC expression as a function of concentration and time of incubation. GCLM responded less markedly to iron induction. The only significant increases in GCLM expression were found between 2 and 80 µM iron, both at days 2 and 10 (Fig. 2C). We then evaluated the expression of GCLC protein to see if the increases in mRNA were observed at the protein level (Fig. 2D). Iron induced a significant increase in GCLC protein (P < 0.001), although the increase was not as marked as that observed for mRNA. Thus, iron activated the expression of GCLC and, to a lower extent, of GCLM as a function of both concentration and time in culture.

GSH accumulation over time. Increased GCL expression predicts increased GSH levels, but, at the same time, a sustained iron load should result in increased GSH consumption, since inhibition of GSH synthesis by a submaximal concentration of L-buthionine sulfoximine produces an accentuated decrease of GSH levels with iron (20). Thus, it was of interest to determine GSH levels in surviving cells. At all iron concentrations tested, SH-SY5Y cells markedly increased their GSH levels as a function of time in culture (Fig. 3). A robust fivefold increase in GSH content occurred from days 2 to 10 of culture with 80 µM Fe (Fig. 3). Smaller increases of 3-, 2.2-, and 2.7-fold were observed for 40, 7, and 2 µM Fe, respectively, by day 10. Thus, although attenuated, increases in GSH level with time correlated with increases in GCLC mRNA.


Figure 3
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  		Fig. 3. Variations in GSH and GSSG content under long-term culture conditions with various iron concentrations. SH-SY5Y cells were grown in 96-well plates for 8 days in the standard medium, after which time they were challenged with 2, 7, 40, or 80 µM Fe, and the cultures were continued for 2, 4, 6, 8, or 10 days. Cell content of GSH or GSSG was determined by a colorimetric assay in a microplate reader with each individual point tested in triplicate. Values represent means of 4 independent determinations. Variation between experiments was <20% of mean values. Statistic analysis showed significant differences (aP < 0.001 and bP < 0.01, between day 2 values and day 8 and 10 values. There was also a statistical difference between days 2 and 6 at 80 µM Fe. At day 10 in 80 µM Fe, cultures presented a 5-fold increase in GSH content compared with day 2 cultures, whereas no significant variations were observed in GSSG values.

 
No significant changes in GSSG concentration occurred between days 2 and 10 for all iron concentration tested (Fig. 3). The maintenance of low cellular GSSG concentrations allowed the surviving cells to maintain a reductant environment throughout the culture period, as determined by the GSH/GSSG half-cell reduction potential under high (80 µM) iron conditions (Fig. 4). Indeed, after the initial period of cell death by day 2 of treatment, which corresponded with an increase in the reduction potential from –337 mV at day 0 to –290 mV at day 2, surviving cells increased their reduction potential to about –320 mV at days 6–10. These data are in agreement with those of a previous report (20) that established that an increase in reduction potential to about –300 mV resulted in massive cell death in SH-SY5Y cells.


Figure 4
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  		Fig. 4. Calculated cellular redox potential. Data from Fig. 4 were used to calculate the GSH/GSSG-determined redox potential (ERedox) using the following equation: ERedox = –240 – (59.1/2) log [GSH]2/[GSSG]. The change in redox potential as a function of time for cells cultured in 80 µM Fe is shown. Under these conditions, about half the cell population died by day 2 (see Fig. 1).

 
Maintaining a low GSSG concentration is vital to the preservation of an adequate reduction potential in SH-SY5Y cells exposed to increased iron-induced oxidative stress. As shown in Fig. 5A, cell GSSG content is determined by a balance between GSSG production, GSSG recycling to GSH mediated by GSSG reductase, and GSSG exit from the cell via the multidrug resistance protein (MRP) pathway (14, 17). Treatment of SH-SY5Y cells with the GSSG reductase inhibitor BCNU greatly decreased GSH content but failed to produce GSSG accumulation (Fig. 5B), whereas treatment with MK571, a specific inhibitor of MRP1 (17), promoted a significant increase in GSSG (Fig. 5C). After 6 h of incubation with 50 µM MK571, a modest but significant decrease in GSH content was also observed. Calculation of the GSH/GSSG half-cell reduction potential under these conditions indicated that the inhibition of either GSSG reductase or MRP1 activity induced a more oxidative environment (Table 1). Since changes in reduction potential to values above –300 mV for a sustained period of time are deleterious for SH-SY5Y cell survival (20), both MRP1 and GSSG reductase appear to be crucial to maintain low GSSG levels.


Figure 5
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  		Fig. 5. GSSG processing. A: schematic diagram of GSSG processing showing that GSSG can be either recycled to GSH by GSSG reductase or extruded from the cell by multidrug resistance protein 1 (MRP1). GSSG reductase is inhibited by 1,3-bis(2-chloroethyl)-2-nitrosourea (BCNU), whereas MRP1 is inhibited by MK571. B and C: SH-SY5Y cells cultured for 2 days in 40 µM Fe medium were challenged with 50 µM of the GSSG reductase inhibitor BCNU (B) or with 50 µM of the MRP1 inhibitor MK571 (C) for 0, 2, 4, or 6 h. Cell content of GSH and GSSG was determined as described for Fig. 3. Values represent means of 3 independent experiments done in triplicates. Significant differences compared with values at time 0 are indicated as follows: aP < 0.001 and bP < 0.01.

 

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  		Table 1. GSH, GSSG, and GSH/GSSG half-cell reduction potential under oxidative conditions

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In studying the factors of cell adaptation to iron accumulation, we found that surviving cells underwent an adaptive process through the activation of the GSH synthesis machinery. We determined that cells greatly increased their GSH content over time, reaching two- to fivefold higher concentrations after 10 days of culture. It is possible that the increase of GSH in time is an adaptative response to increases in total cell iron content with time (2). This is the first time that such an adaptation strategy against iron accumulation has been reported. These findings are consistent with a strategy of dopaminergic cells to stabilize oxidative stress through increased GSH synthesis (1, 10, 26, 27, 32).

The increase in GSH was associated with increased mRNA levels of GCLC and, to a lesser degree, of GCLM. This disproportionate regulation of the two subunits is similar to the "oxidative stress" type of response described in HBE1 cells challenged with 4-hydroxy-2-nonenal-2,3-dimethoxy-1,4-naphthoquinone or t-butylhydroquinone (15) and different from the "sulfur amino acid starvation" response, which is characterized by a more pronounced increase in GCLM compared with GCLC expression (16). Thus, the pattern of activation of GCLC and GCLM expression caused by iron in SH-SY5Y cells corresponds to an oxidative stress type of response. In human hepatoblastoma cells, both Nrf2 and c-Jun are involved in the upregulation of the GCLC gene through its AREs (13), and hemin treatment of SH-SY5Y cells leads to Nrf2 translocation to the nuclei and to the upregulation of ARE-dependent oxidative stress-related enzymes (6, 19). Therefore, it is likely that oxidative stress induced by iron overload leads to activation of Nrf2 and increased expression of GCLC in SH-SY5Y cells.

No significant changes in GSSG content were noticed upon increased iron loading. We observed that these low and sustained levels of GSSG were increased by MK571, a specific inhibitor of MRP1, whereas BCNU, an inhibitor of GSSG reductase, largely decreased GSH levels. Thus, iron-produced GSSG seems to be a substrate for both GSSG reductase (thereby regenerating GSH) and MRP1 (which extrudes GSSG from the cells). The activity of these two proteins kept GSSG at basal levels, thus maintaining a controlled thiol redox potential. Because the reduction potential of neuronal cells depends mainly on the GSH/GSSG half-cell reduction potential (30), GSSG extrusion has a highly relevant role in maintaining a negative reduction potential that is suitable for cell function in the face of the high consumption of GSH induced by oxidative stimuli such as iron (20).

In summary, exposure of SH-SY5Y neuroblastoma cells to conditions of iron overload elicited a long-term increase in GSH synthesis through the upregulation of GCLC and GCLM. Because neurodegenerative conditions such as Parkinson's and Alzheimer's disease involve intracellular oxidative stress related to iron overload, our data support the idea that to ensure survival, the in vivo neuronal response includes the upregulation of GSH synthesis machinery.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a project from the Millennium Scientific Initiative, Santiago, Chile.


   ACKNOWLEDGMENTS
 
Present address of P. Aracena-Parks: Dept of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. T. Núñez, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile (e-mail: mnunez{at}uchile.cl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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