The Detoxification of Cumene Hydroperoxide by the Glutathione System of Cultured Astroglial Cells Hinges on Hexose Availability for the Regeneration of NADPH

Authors


  • Abbreviations used : buthionine sulfoximine ; CHP, cumene hydroperoxide ; GPx, glutathione peroxidase ; GSx, amount of GSH plus two times amount of GSSG ; IB, incubation buffer containing no glucose ; IB+, incubation buffer containing 5 mM glucose ; MM, minimal medium ; MS, mercaptosuccinate ; PPP, pentose phosphate pathway ; tBHP, tertbutyl hydroperoxide.

Address correspondence and reprint requests to Dr. R. Dringen at Physiologisch-chemisches Institut der Universität, Hoppe-Seyler-Str. 4, D-72076 Tübingen, Germany.

Abstract

Abstract : The ability of astroglia-rich primary cultures derived from the brains of newborn rats to detoxify exogenously applied cumene hydroperoxide (CHP) was analyzed as a model to study glutathione-mediated peroxide detoxification by astrocytes. Under the conditions used, 200 μM CHP disappeared from the incubation buffer with a half-time of ~ 10 min. The half-time of CHP in the incubation buffer was found strongly elevated (a) in cultures depleted of glutathione by a preincubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, (b) in the presence of mercaptosuccinae, an inhibitor of glutathione peroxidase, and (c) in the absence of glucose, a precursor for the regeneration of NADPH. The involvement of glutathione peroxidase in the clearance of CHP was confirmed by the rapid increase in the level of GSSG after application of CHP. The restoration of the initial high ratio of GSH to GSSG depended on the presence of glucose during the incubation. The high capacity of astroglial cells to clear CHP and to restore the initial ratio of GSH to GSSG was fully maintained when glucose was replaced by mannose. In addition, fructose and galactose at least partially substituted for glucose, whereas exogenous isocirate and malate were at best marginally able to replace glucose during peroxide detoxification and regeneration of GSH. These results demonstrate that CHP is detoxified rapidly by astroglial cells via the glutathione system. This metabolic process strongly depends on the availability of glucose or mannose as hydride donors for the regeneration of the NADPH that is required for the reduction of GSSG by glutathione reductase.

Peroxides are generated continuously in cells and have to be reduced to the corresponding alcohols to prevent oxidative damage. In the brain, the peroxide generated in largest quantities is H2O2 (Sinet et al., 1980 ; Hyslop et al., 1995), which is the product of the reactions catalyzed by superoxide dismutases and monoamine oxidases (Berry et al., 1994 ; Fridovich, 1995). In addition, soluble organic hydroperoxides are generated by brain cells during eicosanoid metabolism (Murphy et al., 1988 ; Bishai and Coceani, 1992 ; Piomelli, 1994). Moreover, lipid hydroperoxides are generated by nonenzymatic lipid peroxidation (Halliwell, 1992). All these peroxides are reduced to the corresponding alcohols by isoforms of glutathione peroxidase (GPx ; EC 1.11.1.9), enzymes that use GSH as a donor of reduction equivalents (Flohé, 1989 ; Ursini et al., 1995). An alternative pathway exists only for the detoxification of H2O2, the reaction catalyzed by catalase. Catalase is a diffusion-controlled enzyme (Aebi, 1984) and, therefore, is especially important if the clearance of high concentrations of H2O2 is necessary.

GPx activity has been found in brain (De Marchena et al., 1974), as well as in cultured astroglial cells (Copin et al., 1992 ; Huang and Philbert, 1995 ; Desagher et al., 1996 ; Dringen and Hamprecht, 1997). Recently, it has been reported that after application of peroxides to cultured astroglial cells glutathione is quickly oxidized, a process that is blocked by mercaptosuccinate (MS) (Dringen and Hamprecht, 1997 ; Dringen et al., 1998a), an inhibitor of GPx (Tappel, 1984). These results indicate that GPx is involved in the detoxification of peroxides by these cells. The GSSG produced in vivo during the reaction catalyzed by GPx is reduced by glutathione reductase (EC 1.6.4.2), an enzyme that needs NADPH as cosubstrate. Therefore, the detoxification of peroxides is linked directly to the availability and the regeneration of NADPH. As in other cells and tissues, the pentose phosphate pathway (PPP) appears to be the predominant source in brain cells for regeneration of NADPH (Hotta, 1962 ; Hotta and Seventko, 1968 ; Baquer et al., 1988). For cultured neural cells, this view has been supported recently by the strong activation of the PPP after application of H2O2 to astroglial and neuronal cultures (Ben-Yoseph et al., 1994, 1996).

However, besides the PPP enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), additional enzymes could also participate in the NADP+ reduction in astroglial cells, as has been suggested for the retina (Winkler et al., 1986). For cultured astroglial cells, the presence of the cytosolic isoform of malic enzyme (EC 1.1.1.40) has been demonstrated (Kurz et al., 1993 ; Vogel et al., 1998). In addition, both the mitochondrial and the cytosolic isoforms of isocitrate dehydrogenase (EC 1.1.1.42) have been reported for astroglial cultures (Juurlink, 1993).

As the detoxification of peroxides by GPx is directly coupled to the regeneration of GSH by glutathione reductase and consequently to the availability of NADPH, the velocity of peroxide clearance is an indicator for the availability and the regeneration of endogenous NADPH. Astroglial cells detoxify H2O2 partially via catalase (Dringen and Hamprecht, 1997). Therefore, the rate of clearance of H2O2 does not correlate with the demand of the glutathione system for the cofactor NADPH during peroxide clearance. In contrast, tert-butyl hydroperoxide (tBHP) is detoxified by astroglial cells exclusively via the glutathione system (Dringen et al., 1998a, b). Therefore, to study pathways of NADPH regeneration in asroglial cells during peroxide disposal, cumene hydroperoxide (CHP) was chosen, an organic hydroperoxide that is detoxified by astroglial cells and is stable under the incubation conditions applied (Dringen et al., 1998b).

Here we demonstrate that the glutathione system is exclusively esponsible for the rapid clearance of CHP by astroglial cultures and that this process depends on the availability of substrates for regenerating the NADPH in these cultures.

MATERIALS AND METHODS

Materials

Dulbecco’s modified Eagle’s medium was obtained from Life Technologies (Eggenstein, Germany). Fetal calf serum, GSH, GSSG, glutathione reductase from yeast, and NADH were from Boehringer (Mannheim, Germany). NADPH was purchased from Applichem (Darmstadt, Germany). 3-Aminotriazole, bovine serum albumin, buthionine sulfoximine (BSO), 5,5′ -dithio-bis(2-nitrobenzoic acid), fructose, malate, MS, 5-sulfosalicylic acid, and xylenol orange were obtained from Sigma (Deisenhofen, Germany). 2-Vinylpyridine was from Aldrich (Steinheim, Germany). CHP and monopotassium 1R, 2S-isocitrate were obtained from Fluka (Deisenhofen, Germany), Streptomycin sulfate, penicillin G, and Triton X-100 were from Serva (Heidelberg, Germany). All other chemicals, of the highest purity available, were obtained from E. Merck (Darmstadt, Germany). Cell culture dishes and 96-well microtiter plates were purchased from Nunc (Wiesbaden, Germany).

Cell culture

Astroglia-rich primary cultures derived from the brains of newborn Wistar rats were prepared, cultivated, and maintained as described (Hamprecht and Löffler, 1985). The cells were seeded in plastic culture dishes (50 mm in diameter) and incubated in culture medium (90% Dulbecco’s modified Eagle’s medium, 10% fetal cal serum, 20 units/ml penicillin G, and 20 μg/ml streptomycin sulfate). These cultures are widely used for the analysis of metabolic functions of astroglial cells (for overview, see Hamprecht and Dringen, 1995). The results were obtained with 15-21-day-old cultures and, at least in this range, did not depend on the age of the culture.

Experimental incubation

If not stated otherwise in the figure legends, the culture medium was removed and the cells were washed twice with 3 ml of a minimal medium (MM ; 44 mM NaHCO3, 110 mM NaCl, 1.8 mM CaCl2, 5.4 mM KCl, 0.8 mM MgSO4, 0.92 mM NaH2PO4, adjusted with CO2 to pH 7.4) and preincubated for 2 h with 3 ml of MM in a Heraeus cell incubator containing a humidified atmosphere of 10% CO2/90% air. To allow the cells to adapt to the buffer used in the main incubation, a second preincubation (37°C, 5 min) was performed on a water bath with 3 ml of incubation buffer [20 mM HEPES, 145 mM NaCl, 1.8 mM CaCl2, 5.4 mM KCl, 1 mM MgCl2, 0.8 mM Na2HPO4, pH 7.4, containing no glucose (IB) or 5 mM glucose (IB+)]. The subsequent main incubation was carried out in the absence or presence of glucose (5 mM) in 3 ml of IB containing 200 μM CHP.

CHP assay

CHP was determined using a modification (Dringen et al., 1998b) of the assay described by Jiang et al. (1990). In brief, 10 μl of the CHP-containing IB, collected after gentle swirling of the dish at the time points indicated, was added to 190 μl of 25 mM H2SO4 in a well of a microtiter plate. After the further addition of 200 μl of reaction mixture [0.5 mM (NH4)2Fe(SO4)2, 200 mM sorbitol, 200 μM xylenol orange, in 25 mM H2SO4] and incubation for 45 min, the absorbance at 550 nm was determined using a microtiter plate reader (Titertek Plus 212, ISN Biomedicals, Meckenheim, Germany) and compared with the absorbance read at known standard concentrations of CHP. The increase in absorption of the complex generated is proportional to the CHP conent in the range of 0-2.5 nmol of CHP per well of the microtiter plate or a concentration of 0-250 μM in the IB (Dringen et al., 1998b).

GSH regeneration assay

After removal of the culture medium, the cells were washed twice with 3 ml of MM and preincubated with 3 ml of MM to deplete the cells of endogenous hydride donors for NADPH regeneration. After a 2-h preincubation, the cells were washed with 3 ml of IB and incubated for 2.5 min with IB containing CHP (200 μM) and the indicated putative exogenous substrates for endogenous NADP+ reduction. Subsequently, the peroxide-containing buffer was removed and the cells were washed with 3 ml of IB and incubated for 60 s in IB containing the putative substrates to allow regeneration of GSH. Finally, the cells were washed twice with 3 ml of phosphate-buffered saline (10 mM potassium phosphate buffer, 150 mM NaCl, pH 7.4) and subsequently lysed with 1 ml of sulfosalicylic acid (1%, wt/vol). The lysate was used for determining the contents of total glutathione (GSx ; amount of GSH plus two times amount of GSSG) and of GSSG.

Determination of glutathione

The contents of GSx and GSSG (expressed as GSx) in cell lysates were determined as described (Dringen and Hamprecht, 1996) using a modification (Baker et al., 1990) of the assay developed by Tietze (1969). GSSG was used as a standard for the determination of GSx and GSSG in a range of 0-500 pmol/10 μl.

Determination of cell viability and of protein content

Cell viability was analyzed by determining the activity of lactate dehydrogenase in the incubation medium as described previously (Dringen et al., 1998b). Under all conditions used, the viability of the cells after incubation for 45 min with the peroxide was >90% (data not shown). Protein content of cultured cells was determined according to the method of Lowry et al. (1951) using bovine serum albumin as a standard.

Presentation of data

Each experiment was carried out on at least two independently prepared cultures with comparable results. If not stated otherwise, the results shown in the figures and the tables are those of one representative experiment, and the data represent means of triplicate values ± SD obtained from replica plates. In the figures, the vertical bars correspond to the SD and have been omitted if they are smaller than the symbols representing the mean values. Statistical analysis was performed by ANOVA followed by Bonferroni post hoc test.

RESULTS

After application of CHP at a concentration of 200 μM to astroglia-rich primary cultures, the peroxide disappeared from the IB with a half-time of ~10 min if glucose was present (Fig. 1). In contrast, glucose-deprived cells disposed of the peroxide more slowly (Fig. 1A). At least for the first 8 min of the incubation period, the disappearance of CHP from the IB followed firstorder kinetics (Fig. 1B). Therefore, half-times could be calculated for the initial period of incubation giving a measure for the rate of initial detoxification of CHP. In the absence of cells, but under otherwise identical conditions, the concentration of CHP was not reduced during an incubation for up to 30 min (Fig. 1A).

Figure 1.

A : Concentration of CHP in IB as a function of the time of incubation in the absence (open circles) or the presence (filled symbols) of astroglia-rich primary cultures. After a 2-h preincubation of the cells in MM and a 5-min preincubation in IB containing 0 mM (filled circles) or 5 mM glucose (filled triangles), the cells were incubated in IB containing CHP (200 μM) in the absence (filled circles) or the presence (filled triangles) of glucose (5 mM). The 18-day-old culture contained 0.9 mg of protein per dish. B : Semilogarithmic representation of the data obtained during the initial period of 8 min.

FIG. 1.

Glutathione is the essential substrate for hydroperoxide detoxification by GPx. Therefore, a reduction of the glutathione level should influence the rate of detoxification of CHP by astroglial cells. The intracellular glutathione content was reduced by preincubation with BSO, an irreversible inhibitor of γ-glutamylcysteine synthetase (Griffith and Meister, 1979). Preincubation of astroglial cells with BSO caused an increase in the half-times for the cell-dependent clearance of CHP from the IB from 10.6 min to 58.7 min (Table 1).

Table 1. Effects of BSO and MS on the half-times for CHP during incubation with astroglia-rich primary culturesThe cells were preincubated for 24 h in culture medium containing no BSO or 0.5 mM BSO followed by a subsequent 2-h preincubation in MM containing glucose (5 mM) in the presence or the absence of MS (10 mM). After a further 5-min preincubation of the cells in IB+ in the absence or the presence of MS, the cells were incubated with IB+ containing 200 μM CHP in addition to the components present during the 5-min preincubation. The half-times for CHP were calculated from the slopes of the regression lines in semilogarithmic representations of the data obtained for the initial 8 min. With the exception of the data obtained for cells treated with MS, the data represent the means ± SD (n = 6) of two sets of three dishes, each set representing an independently prepared culture. For MS-treated cells, the individual half-times are given for each of the two experiments performed (mean ± SD) on sets of three dishes of independent cultures, corresponding to no decrease (infinitive half-time) or to a decrease in the concentration of CHP from 200 μM to 185 μM (half-time : 72.3 min) within the initial 8 min of incubation.
TreatmentHalf-time (min)
  1. ap < 0.001, compared with the control.

Control10.6 ± 2.0
BSO58.7 ± 17.9 a
MS72.3 ± 8.1 a [unk]

TABLE 1.

MS, an inhibitor of GPx (Tappel, 1984 ; Dringen and Hamprecht, 1997), was used to demonstrate the involvement of GPx in the detoxification of CHP by astroglial cells. In the presence of glucose, the rapid disappearance of CHP from the IB was reduced drastically or even prevented by MS at a concentration of 10 mM (Table 1). If unstarved astroglial cultures were incubated with CHP in a glucose-free buffer, the peroxide disappeared with a half-time of ~10 min (Fig. 2). This indicates that endogenous sources of substrates for NADP+-reducing enzymes are available in concentrations sufficient to allow maximal detoxification of CHP. To use the rate of CHP clearance as an indicator for the utilization of exogenous substrates for astroglial NADP+ reduction, the availability of endogenous substrates had to be reduced first. This was achieved by a preincubation of the cells in MM before the clearance of CHP was determined. Astroglial cultures had to be preincubated for at least 30 min to increases the half-time for CHP significantly. Half-times four to five times that of untreated cells were calculated for astroglial cultures preincubated for up to 2 h in MM (Fig. 2). For further experiments, a preincubation for 2 h was used to deprive the cells of endogenous substrates for NADP+-reducing enzymes.

Figure 2.

Relationship between the half-time for CHP in the IB of astroglia-rich primary cultures and the time of preincubation of the cells in glucose-free medium. After the preincubation period indicated, the cells were incubated in glucose-free IB containing CHP (200 μM). The values are presented as the means ± SD (n = 6-14) of data obtained from dishes of two to four separate cultures. ***p < 0.001, compared with the half-time calculated for cells that were not preincubated.

FIG. 2.

If astroglial cultures were starved for 2 h in MM and subsequently incubated with CHP in the presence or the absence of glucose, the half-times for CHP were ~10 and ~60 min, respectively (Table 2). The presence of mannose, substituting for glucose, enabled astroglial cells to dispose of CHP with the maximal rate found in the presence of glucose. Also, fructose and galactose were at least partially able to replace glucose during CHP detoxification, as indicated by the significantly smaller half-time compared with that of cells incubated in the absence of glucose. For the effectiveness of the presence of hexoses to improve astroglial detoxification of CHP, the significance ranking glucose = mannose > fructose > galactose was found. In contrast to the hexoses, exogenous malate or isocitrate was unable to improve significantly the rate of CHP clearance observed in the absence of glucose (Table 2).

Table 2. Half-times for CHP in astroglia-rich primary cultures in the presence or the absence of putative exogenous substrates for NADPH regenerationThe cells were preincubated for 2 h in MM to deplete the cells of endogenous substrates. After a second preincubation (5 min) in IB containing the compounds indicated (5 mM), the cells were incubated with IB containing CHP (200 μM) in addition to the components indicated (5 mM). The half-times for CHP were calculated from the slopes of the regression lines in a semilogarithmic representation of the data obtained for the initial 15 min. The data represent the means ± SD of n dishes obtained from two to five independently prepared cultures.
SubstratesHalf-time (min)n
  1. ap < 0.001,

  2. bp < 0.05, compared with the half-times obtained for cells incubated in the absence of exogenous substrates (none).

None58.9 ± 12.015
Glucose10.2 ± 3.1 a15
Mannose7.8 ± 0.5 a6
Fructose23.1 ± 3.1 a6
Galactose40.6 ± 3.6 b6
Malate46.3 ± 15.911
Isocitrate52.3 ± 14.89

TABLE 2.

To confirm further the involvement of glutathione in the detoxification of CHP, the amount of GSx and GSSG in astroglia-rich primary cultures was determined after application of CHP at a concentration of 200 μM. The content of GSSG accounts for <2% of GSx in untreated astroglial cultures (Dringen and Hamprecht, 1997) and was not increased by preincubation in the absence of glucose (Fig. 3). In the absence of glucose, the proportion of GSSG rose to 45% of GSx within 1 min after application of CHP, and the cells were unable to restore completely the original high ratio of the levels of GSx and GSSG within 60 min (Fig. 3A). In contrast, if glucose was present, the rise was less dramatic and within 30 min the original ratio of the contents of GSx and GSSG was completely reestablished, because no GSSG was detectable anymore (Fig. 3B). During incubation with the peroxide, a loss of GSx was noticed that accounted during the first 2.5 min of the incubation for 36 and 28% of the original GSx content of cells incubated in the absence and the presence of glucose, respectively. After 2.5 min of incubation in the presence of glucose, the GSx content remained almost constant (Fig. 3B). In contrast, the GSx content of glucose-deprived astroglial cultures declined further to 17% of the initial level after 60 min of incubation (Fig. 3A).

Figure 3.

Changes of the contents of GSx (circles) and GSSG (squares, expressed as GSx) in cells of astroglia-rich primary cultures during exposure to CHP. After preincubation for a period of 2 h in MM and a second preincubation (5 min) in IB lacking (A) or containing glucose (5 mM ; B), the cells were exposed to CHP (200 μM) in IB in the absence (A) or the presence (B) of 5 mM glucose. The 20-day-old culture contained 1.2 mg of protein per dish.

FIG. 3.

After incubation of starved astroglial cultures with CHP (200 μM) for 2.5 min, a maximal GSSG content was found in the cells (Fig. 3). Removal of the peroxide after such an incubation with CHP caused a rapid decline in the level of GSSG, although the GSx content remained almost constant (Fig. 4). The rate of GSSG disappearance depended strongly on the presence of glucose. After a 1-min incubation in the presence and the absence of glucose, 14 and 58%, respectively, of the GSx content were accounted for by GSSG (Fig. 4).

Figure 4.

Contents of GSx (circles) and GSSG (squares) in astroglia-rich primary cultures after removal of CHP. After a preincubation for 2 h in MM and a second preincubation for 2.5 min in IB containing CHP (200 μM), the cells were incubated in IB in the absence (open symbols) or presence of 5 mM glucose (filled symbols). The 19-day-old cultures contained 1.3 mg of protein per dish.

FIG. 4.

If starved astroglial cultures were incubated for 2.5 min with CHP (200 μM) in the absence of exogenous substrates, GSSG levels of up to 80% of GSx were determined (Fig. 5A). This level was significantly lower if glucose or mannose was present during the incubation with the peroxide (Fig. 5A, open columns). Within 1 min after removal of the peroxide, the level of GSSG declined by ~25% in the absence of substrates. This disappearance of GSSG was improved significantly in the presence of fructose, galactose, and malate, and was almost complete within 1 min in the presence of glucose or mannose (Fig. 5A, hatched columns). The significance of the improving effects of glucose, mannose, fructose, galactose, and malate on the ability of astroglial cultures to reduce endogenous GSSG became also evident from the relative amounts of GSSG reduction during the 1-min incubation after removal of CHP (Fig. 5B). In contrast to the other substrates, isocitrate was unable to improve the GSSG reduction of peroxide-treated astroglial cultures (Fig. 5B).

Figure 5.

Content of GSSG in astroglia-rich primary cultures before and after removal of CHP. The cells were preincubated for 2 h in MM to deprive the cells of endogenous substrates for the reduction of NADP+. A second preincubation was performed for 2.5 min in IB containing 200 μM CHP and the substrates (5 mM) indicated. Subsequently, the peroxide was removed and the cells were incubated for 1 min in IB containing the substrates (5 mM) indicated. A : GSSG levels in the cells before (open columns) and after (hatched columns) the 1-min incubation. B : GSSG content after 1 min of recovery from the exposure to CHP expressed as a percentage of the GSSG amount before regeneration. The data represent the means ± SD (n = 8-12) of dishes obtained from three or four independently prepared cultures. Glc, glucose ; Man, mannose ; Frc, fructose ; Gal, galactose ; Mal, malate ; Icit, isocitrate. **p <0.01, ***p 0.001, compared with the data obtained for the incubation in the absence of substrate (none).

FIG. 5.

DISCUSSION

To investigate the ability of astroglial cells to detoxify CHP, this peroxide was applied to astroglia-rich primary cultures. The cells of these cultures disposed of CHP rapidly from the IB. Several lines of evidence demonstrate that the glutathione system is responsible for the rapid decrease in the peroxide concentration of the IB.

Immediately after administration of CHP to astroglial cells, GSH was found oxidized to GSSG. Such a rapid oxidation of GSH has been demonstrated after application of peroxides to erythrocytes (Srivastava et al., 1974), hepatocytes (Eklöw et al., 1984), and astroglial cells (Dringen and Hamprecht, 1997 ; Dringen et al., 1998a), indicating the involvement of GPx in the detoxification of CHP by astroglial cultures. This hypothesis is strongly supported by the inhibition of CHP clearance in the presence of the GPx inhibitor MS. In contrast to MS, 3-aminotriazole, an inhibitor of catalase (Aebi, 1984), did not affect the clearance of CHP from the IB (data not shown), excluding an involvement of catalase in the detoxification of CHP by astroglial cells. The rate of detoxification of CHP by the cells was strongly affected by the content of glutathione in the cells. During preincubation with BSO, the glutathione content of astroglial cells falls with a half-time of 5 h (Devesa et al., 1993), and after 24 h of incubation astroglial cells contain only 14% of the glutathione present in untreated cells (Dringen et al., 1998a). Apparently, a high level of glutathione is a prerequisite for fast detoxification of organic hydroperoxides, such as tBHP (Dringen et al., 1998a) or CHP by astroglial cells. This can most likely be explained by a reduction in the activity of cellular GPx due to a diminished saturation with its substrate GSH. Taking these pieces of evidence together, the glutathione system appears to be able and to be sufficient to completely detoxify organic peroxides applied to cultured astroglial cells.

Under all conditions used, the disappearance of CHP followed first-order kinetics for several minutes. This indicates that during this period the rate of clearance of CHP is always proportional to the actual concentration of this compound and demonstrates the ability of the astroglial glutathione system to detoxify hydroperoxides over a considerable range of concentrations. First-order kinetics were found as well for the clearance of H2O2 and tBHP by cultured fibroblasts (Makino et al., 1994, 1995) and astroglial cells (Dringen and Hamprecht, 1997 ; Dringen et al., 1998a).

If glucose-fed astroglial cultures were incubated with CHP in glucose-free IB, the peroxide was cleared with maximal rate. Therefore, astroglial cultures contain at the onset of the incubation with CHP, besides GSH and NADPH, also substrates needed for NADPH regeneration in concentrations sufficient to allow a maximal rate of detoxification of the peroxide. Substrates for NADPH regeneration could be all compounds that can be converted to glucose-6-phosphate, malate, or isocitrate, because the NADP+-reducing enzymes of the PPP (Ben-Yoseph et al., 1994), the cytosolic malic enzyme (Kurz et al., 1993 ; Vogel et al., 1998), as well as cytosolic and mitochondrial isoforms of isocitrate dehydrogenase (Juurlink, 1993) are present in cultured astroglial cells. Starvation of astroglial cultures for >15 min led to a significant increase in the half-time for CHP disappearance, indicating a reduced availability of endogenous substrates for NADPH regeneration. The endogenous compound most likely to provide substrates for NADP+ reduction is glycogen, because astroglial cultures contain ~100 nmol of glucosyl residues per culture dish (50 mm in diameter) stored as glycogen (Dringen and Hamprecht, 1992). The shuttling of all glycosyl residues derived from glycogen would generate 200 nmol of NADPH via the oxidative part of the PPP. As upon the onset of starvation the glycogen in astroglial cultures declines with a half-time between 7 and 15 min (Dringen and Hamprecht, 1992, 1993), this rapid mobilization could be responsible for the high capacity of astroglial cultures to detoxify CHP after removal of extracellular glucose.

After 2 h of starvation, both the clearance of CHP from the IB and the ratio of GSH to GSSG after application of CHP depended strongly on the presence of exogenous glucose. This indicates that under such conditions most likely the PPP is predominately responsible for the supply of reduction equivalents in the form of NADPH, which is required for the regeneration of GSH. This hypothesis is supported by the strong activation of the astroglial PPP on application of H2O2 (Ben-Yoseph et al., 1994).

Besides glucose, astroglial cells are able to metabolize other sugars. Especially mannose and fructose are good substrates for astroglial metabolism (Wiesinger et al., 1997). These sugars can be used by astroglial cultures as substrate for glycogen synthesis and lactate production (Dringen and Hamprecht, 1993 ; Dringen et al., 1994 ; Bergbauer et al., 1996). Therefore, it was not surprising that mannose was fully and fructose was at least partially able to substitute for glucose as an exogenous substrate for endogenous NADPH regeneration. As both sugars are metabolized in astroglial cells to fructose 6-phosphate, which can be converted to glucose 6-phosphate (Wiesinger et al., 1997), the NADPH required for GSSG reduction is most likely regenerated via the PPP. In contrast to mannose and fructose, the metabolism of galactose in astroglial cultures is almost unknown. No net synthesis of astroglial glycogen and only marginal production of lactate were reported for astroglial cultures incubated with galactose (Dringen and Hamprecht, 1993). However, the presence of galactose significantly improved the ability of starved astroglial cultures to dispose of CHP and to regenerate GSH, indicating that galactose is used by cultured astroglial cells and serves as a hydride donor for the reduction of NADP+.

During incubation with CHP, a loss of GSx from the cells was observed, which has been reported previously for peroxide-treated astroglial cells (O’Connor et al., 1995 ; Peuchen et al., 1996 ; Dringen and Hamprecht, 1997 ; Dringen et al., 1998a). A reason for this loss could be a release of GSSG, which has been reported for several cell types as a response to oxidative stress (Akerboom and Sies, 1990). Indeed, in preliminary experiments, a release of GSSG from astroglial cultures exposed to peroxides was found (data not shown).

The results presented here confirm the idea that astroglial cells possess a prominent PPP that is likely to be responsible for the fast supply of NADPH, at least in the cytosol. At best, weak evidence has been obtained for a function of malic enzyme and no evidence for an involvement of isocitrate dehydrogenases in the supply of NADPH for GSH regeneration during peroxide detoxification by astroglial cells. However, it has to be stressed that insufficient uptake of exogenous malate and/or isocitrate might strongly limit the intracellular availability of these substrates. In that case, the assay systems used in the present study would not be suited for an investigation of the contribution of malic enzyme or isocitrate dehydrogenases in NADPH regeneration.

In conclusion, astroglial cells in culture are highly effective in detoxifying exogenously applied CHP via their glutathione system. As the clearance of CHP is directly coupled to the availability of NADPH for the regeneration of GSH, the disposal of CHP and the regeneration of GSH can be used as tools to investigate the capability of astroglial cells to take up and use exogenous compounds as precursors for the reduction of NADP+.

Acknowledgements

The authors wish to thank Dr. Heinrich Wiesinger for critically reading the manuscript.

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