The Glutathione System of Peroxide ... - Wiley Online Library

Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry

The Glutathione System of Peroxide Detoxification Is Less Efficient in Neurons than in Astroglial Cells Ralf Dringen, Lothar Kussmaul, Jan Mirko Gutterer, Johannes Hirrlinger, and Bernd Hamprecht Physiologisch-chemisches Institut der Universita¨t, Tu¨bingen, Germany

Abstract: The ability of neurons to detoxify exogenously applied peroxides was analyzed using neuron-rich primary cultures derived from embryonic rat brain. Incubation of neurons with H2O2 at an initial concentration of 100 ␮M (300 nmol/3 ml) led to a decrease in the concentration of the peroxide, which depended strongly on the seeding density of the neurons. When 3 ⫻ 106 viable cells were seeded per dish, the half-time for the clearance by neurons of H2O2 from the incubation buffer was 15.1 min. Immediately after application of 100 ␮M H2O2 to neurons, glutathione was quickly oxidized. After incubation for 2.5 min, GSSG accounted for 48% of the total glutathione. Subsequent removal of H2O2 caused an almost complete regeneration of the original ratio of GSH to GSSG within 2.5 min. Compared with confluent astroglial cultures, neuron-rich cultures cleared H2O2 more slowly from the incubation buffer. However, if the differences in protein content were taken into consideration, the ability of the cells to dispose of H2O2 was identical in the two culture types. The clearance rate by neurons for H2O2 was strongly reduced in the presence of the catalase inhibitor 3-aminotriazol, a situation contrasting with that in astroglial cultures. This indicates that for the rapid clearance of H2O2 by neurons, both glutathione peroxidase and catalase are essential and that the glutathione system cannot functionally compensate for the loss of the catalase reaction. In addition, the protein-normalized ability of neuronal cultures to detoxify exogenous cumene hydroperoxide, an alkyl hydroperoxide that is reduced exclusively via the glutathione system, was lower than that of astroglial cells by a factor of 3. These results demonstrate that the glutathione system of peroxide detoxification in neurons is less efficient than that of astroglial cells. Key Words: Astrocytes—Cumene hydroperoxide —Glutathione —Hydrogen peroxide —Neurons—Oxidative stress. J. Neurochem. 72, 2523–2530 (1999).

especially important for the brain, because oxidative stress generated by these compounds has been connected with neurodegenerative diseases, i.e., Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (Halliwell, 1992; Weber, 1994; Bowling and Beal, 1995; Bains and Shaw, 1997; Cadet and Brannock, 1998). Evidence is growing that glutathione plays an important role in the detoxification of reactive oxygen species in brain. Glutathione deficiency induced in newborn rats by application of an inhibitor of ␥-glutamylcysteine synthetase, buthionine sulfoximine, led to mitochondrial damage in brain (Jain et al., 1991). Furthermore, reduction of the brain glutathione content by buthionine sulfoximine enhanced the toxic effects of insults that are suggested to act by generation of reactive oxygen species, e.g., ischemia (Mizui et al., 1992) or treatment with MPP⫹ (Wu¨llner et al., 1996) or 6-hydroxydopamine (Pileblad et al., 1989). H2O2 is generated continuously in cells and, therefore, has to be detoxified continuously. Generation of H2O2 has been reported for brain (Sinet et al., 1980; Hyslop et al., 1995). At least under experimental conditions, extracellular H2O2 concentrations of up to 100 ␮M have been measured in brain by microdialysis (Hyslop et al., 1995). The predominant biochemical sources for H2O2 in brain cells appear to be the reactions catalyzed by superoxide dismutases and by monoamine oxidases (Halliwell, 1992; Berry et al., 1994; Fridovich, 1995). H2O2 and organic hydroperoxides are reduced by GPx, an enzyme that uses GSH as donor of reduction equivalents. GPx activity has been found in brain (De Marchena et al., 1974), as well as in cultured neurons (Huang and Philbert, 1995; Desagher et al., 1996). The Received December 23, 1998; revised manuscript received February 8, 1999; accepted February 8, 1999. Address correspondence and reprint requests to Dr. R. Dringen at Physiologisch-chemisches Institut der Universita¨t, Hoppe-Seyler-Str. 4, D-72076 Tu¨bingen, Germany. Abbreviations used: 3AT, 3-aminotriazol; CHP, cumene hydroperoxide; D, specific detoxification rate constant; DMEM, Dulbecco’s modified Eagle’s medium; GPx, glutathione peroxidase; GR, glutathione reductase; GSx, amount of GSH plus two times amount of GSSG; PPP, pentose phosphate pathway; PS, penicillin/streptomycin; tBHP, tert-butyl hydroperoxide.

In mammalian cells, reactive oxygen species are generated continuously during aerobic metabolism. To prevent toxic effects caused by radicals and peroxides, these reactive compounds have to be eliminated. Enzymes involved in such processes are superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), glutathione peroxidase (GPx; EC 1.11.1.9), and glutathione reductase (GR; EC 1.6.4.2). Detoxification of reactive oxygen species is 2523

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GSSG produced during the GPx reaction is reduced by GR, which uses NADPH as electron donor. Therefore, the detoxification of peroxides is linked to the availability and the generation of NADPH. As in other cells and tissues, the pentose phosphate pathway (PPP) appears to be the predominant source in brain cells for generation of NADPH, which subsequently is necessary for the regeneration of GSH (Hotta, 1962; Hotta and Seventko, 1968; Baquer et al., 1988). For neuronal cultures, it has been demonstrated that during the detoxification of H2O2 the PPP was activated (Ben-Yoseph et al., 1994, 1996). This indicates that the glutathione system exerts a key function in the detoxification of H2O2 by neurons. Neurons in culture have been reported to be more vulnerable than cultured astroglial cells against damaging compounds such as H2O2, tert-butyl hydroperoxide (tBHP), or peroxynitrite (Ben-Yoseph et al., 1994; Bolanõs et al., 1995; Abe and Saito, 1998). One reason for this vulnerability might be that neurons in culture contain glutathione at a lower concentration than astroglial cells (Raps et al., 1989; Bolanõs et al., 1995). This hypothesis has been supported by data showing that glutathionedeprived astroglial cells are no longer resistant against peroxynitrite (Barker et al., 1996). The importance of the glutathione system for the detoxification of peroxides by brain cells has been described recently for astroglial cells in culture. On administration of H2O2 or tBHP to astroglial cells, glutathione is oxidized immediately (Dringen and Hamprecht, 1997; Dringen et al., 1998a). Besides GPx, catalase also is involved in the detoxification of H2O2 by astroglial cells (Dringen and Hamprecht, 1997). In contrast, for the clearance of tBHP by astroglial cells, the glutathione system is essential and sufficient (Dringen et al., 1998a). Like astroglial cells (Desagher et al., 1996; Dringen and Hamprecht, 1997), cultured neurons also are able to rid themselves of H2O2 albeit less effectively than astroglial cells (Desagher et al., 1996). To investigate the involvement of the glutathione system in the neuronal detoxification of H2O2, the ratio of GSH to GSSG in the cells and the clearance of H2O2 from the incubation buffer were measured. In addition, the detoxification of an organic hydroperoxide by neurons was investigated, a metabolic pathway performed exclusively by the glutathione system. MATERIALS AND METHODS Materials Dulbecco’s modified Eagle’s medium (DMEM) and horse serum were obtained from Life Technologies (Eggenstein, Germany). Fetal calf serum, GSH, GSSG, GR from yeast, insulin, and NADH were purchased from Boehringer (Mannheim, Germany). NADPH was from Applichem (Darmstadt, Germany). 3-Amino-1,2,4-triazole (3AT), bovine serum albumin, cytosine arabinoside, 4⬘,6-diamidino-2-phenylindole dihydrochloride, 5,5⬘-dithiobis(2-nitrobenzoic acid), poly-D-lysine, progesterone, putrescine, transferrin, 5-sulfosalicylic acid, and xylenol orange were obtained from Sigma (Deisenhofen, Germany). Cumene hydroperoxide (CHP), sodium pyruvate, and sodium

J. Neurochem., Vol. 72, No. 6, 1999

selenite were purchased from Fluka (Neu-Ulm, 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 (50 mm in diameter) and 96-well microtiter plates were from Nunc (Wiesbaden, Germany).

Primary cultures Neuron-rich primary cultures were prepared from the brains of embryonal Wistar rats as described (Lo¨ffler et al., 1986). If not stated otherwise, 3 ⫻ 106 viable cells were seeded into poly-D-lysine-coated plastic culture dishes (50 mm in diameter) in 3 ml of 90% DMEM/10% horse serum containing penicillin and streptomycin (PS; 20 units/ml penicillin G and 20 ␮g/ml streptomycin sulfate). The cultures were maintained as previously described (Lo¨ffler et al., 1986) and used at a culture age of 5–7 days. These cultures contain minor contaminations of astroglial cells, but no oligodendroglial or ependymal cells (Lo¨ffler et al., 1986). The average contribution in these cultures of cells positive for the astroglial marker protein glial fibrillary acidic protein was 5.3 ⫾ 1.9% (n ⫽ 3 cultures) determined by immunocytochemical staining as previously described (Reinhart et al., 1990) and subsequent staining of the cells with the DNA dye 4⬘,6-diamidino-2-phenylindole (1 ␮g/ml) for 5 min to determine the total number of cells (Russel et al., 1975). Astroglia-rich primary cultures derived from the brains of newborn Wistar rats were prepared, cultivated, and maintained as described (Hamprecht and Lo¨ffler, 1985). Viable cells (3 ⫻ 106) were seeded in plastic culture dishes (50 mm in diameter) and incubated in 90% DMEM containing 10% fetal calf serum and PS. These cultures are widely used for the analysis of metabolic functions of astroglial cells (for review, see Hamprecht and Dringen, 1995). The results presented here were obtained with 14 –21-day-old cultures.

Experimental incubation After removal of the culture medium, the cells were washed twice 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, 5 mM glucose, pH 7.4) and incubated in incubation buffer containing peroxides (100 ␮M), the culture plates being placed on an aluminum grid touching the surface of the water of a 37°C water bath for the time periods indicated.

Peroxide assay The concentration of peroxides in the incubation buffer was determined using a modification (Dringen et al., 1998b) of the assay described by Jiang et al. (1990). In brief, 10 ␮l of peroxide-containing incubation buffer, collected after gentle swirling of the dish at the time points indicated, was added to 190 ␮l of 25 mM H2SO4 in a well of a microtiter plate. Forty-five minutes after the further addition of 200 ␮l of reaction mixture [0.5 mM (NH4)2Fe(SO4)2, 200 mM sorbitol, and 200 ␮M xylenol orange in 25 mM H2SO4], the absorbance at 550 nm was determined using a microtiter plate reader (Titertek Plus MS212, ISN Biomedicals, Meckenheim, Germany) and compared with the absorbance read at known standard concentrations of the peroxide investigated. The increase in absorbance of the complex generated is proportional to the peroxide content in the range of 0 –2.5 nmol of peroxide per well of the microtiter plate (Dringen et al., 1998b).

Determination of glutathione The contents of GSx (amount of GSH plus two times amount of GSSG) and GSSG in cell lysates were determined as described (Dringen and Hamprecht, 1996) using a modification

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Student’s t test. For multiple comparison, data were analyzed by ANOVA followed by the Bonferroni post hoc test. A p value of ⬎0.05 was considered as statistically insignificant.

RESULTS

FIG. 1. Time course of the concentration of H2O2 in incubation buffer in the absence of cells (open triangles) or in the presence of astroglia-rich (open circles) and neuron-rich (filled symbols) primary cultures. For the generation of neuron-rich primary cultures of different cell densities, 6 ⫻ 106 (filled circles), 3 ⫻ 106 (filled triangles), or 2 ⫻ 106 (filled squares) viable cells were seeded per dish. The neuronal and astroglial cultures were used at ages of 6 and 16 days, respectively. The cells were incubated in incubation buffer containing 100 ␮M H2O2. The content of cellular protein was determined for each individual dish of the cultures. The mean value of protein per dish was 1.18 ⫾ 0.05 mg for astroglial cultures. The corresponding data for the neuronal cultures were 607 ⫾ 47, 298 ⫾ 27, and 156 ⫾ 7 ␮g for cultures seeded at 6 ⫻ 106, 3 ⫻ 106, and 2 ⫻ 106 viable cells per dish, respectively. Inset: Semilogarithmic representation of the data obtained during the initial period of 10 min.

(Baker et al., 1990) of the assay described first by Tietze (1969). GSSG was used as a standard for the determination of GSx and GSSG in a range of 0 –500 pmol/10 ␮l.

Determination of enzyme activities The activities of GPx and catalase were determined according to the methods described by Flohe´ and Gu¨nzler (1984) and Aebi (1984), respectively, as described previously (Dringen and Hamprecht, 1997).

Determination of cell viability and of protein content Cell viability was analyzed by determining the activity of lactate dehydrogenase in the incubation medium by a modification (Dringen et al., 1998b) of the lactate dehydrogenase assay described by Vassault (1983). Under all conditions used, the viability of the cells 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 independent cultures with comparable results. In the figures, the data are presented as means of triplicate values ⫾ SD obtained from replicate plates of one representative experiment. The bars have been omitted when they were smaller than the symbols representing the mean values. In the tables, the data presented were from n dishes derived from two or more independently prepared cultures. In the tables, the significance of differences between two groups of data was analyzed using the unpaired

The ability of neuron-rich primary cultures to detoxify H2O2 was investigated by monitoring the time course of the concentration of the peroxide in incubation buffer. In the absence of cells, no reduction of the peroxide concentration was found (Fig. 1). In contrast, in the presence of neurons or of astroglial cells, H2O2 disappeared from the incubation buffer. The clearance rate of H2O2 by neurons depended on the seeding density of the neurons and increased strongly with the cell density (Fig. 1). However, even neurons seeded at a density as high as 6 ⫻ 106 viable cells were unable to detoxify H2O2 with the rate found for confluent astroglial cultures (Fig. 1, the lower curves in both the main figure and the inset). For all seeding densities of neurons, H2O2 disappeared at least for the first 10 min of incubation from the incubation buffer following first-order kinetics (Fig. 1, inset). There were no obvious changes in cell morphologies and no decline in cell viability during the experiments (data not shown). Both the protein content per dish and the half-time of the peroxide depended strongly, but inversely, on the seeding density of the cultures (Table 1). To compare the ability of cell cultures to detoxify peroxides, the protein content per dish has to be taken into consideration. If one assumes that the rate constant k for the first-order reaction is proportional to the amount p of cellular protein per dish, then it can be derived from the known relationship in first-order kinetics between k and the half-time t1/2 that p is inversely proportional to t1/2. In the equation t1/2⫺1 ⫽ Dp, the proportionality constant D represents the specific detoxification rate constant, which is characteristic for a certain experimental cell culture paradigm

TABLE 1. Protein content per dish, half-times of H2O2, and specific detoxification rate constant D for the clearance of H2O2 by neuron-rich primary cultures of different cell densities Seeding density 2 ⫻ 106 3 ⫻ 106 6 ⫻ 106

Protein content (␮g/dish)

Half-time (min)

D (min ⫻ mg of protein)⫺1

170 ⫾ 16a 319 ⫾ 30 658 ⫾ 68a

23.2 ⫾ 2.8a 14.5 ⫾ 0.7 7.6 ⫾ 0.3a

0.258 ⫾ 0.014b 0.217 ⫾ 0.018 0.202 ⫾ 0.024

The cultures were generated by seeding the given number of viable cells per dish and maintaining the cultures as described (Lo¨ffler et al., 1986). At a culture age of 6 days, the cultures were incubated in incubation buffer containing 100 ␮M H2O2. The half-times for H2O2 were calculated from the slopes of the straight lines obtained in the semilogarithmic representations of the data obtained for the first 10 min of incubation. The data represent means ⫾ SD (n ⫽ 6) of two sets of three dishes, each set representing independently prepared cultures. a p ⬍ 0.001, b p ⬍ 0.01 compared with the values obtained from cultures generated by seeding 3 ⫻ 106 viable cells per dish (ANOVA followed by the Bonferroni post hoc test).


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TABLE 2. Half-times and specific detoxification rate constants D for the clearance of H2O2 and CHP: comparison of neuron-rich and astroglia-rich primary cultures Neuron-rich primary cultures Peroxide H2O2 CHP

Astroglia-rich primary cultures

Half-time (min)


n

Half-time (min)


n

15.1 ⫾ 1.4a,c 43.9 ⫾ 33.5b

0.250 ⫾ 0.046c 0.094 ⫾ 0.039a

24 12

2.8 ⫾ 0.9d 4.4 ⫾ 1.1

0.291 ⫾ 0.061c 0.186 ⫾ 0.020

9 9

The cultures were incubated in incubation buffer containing a 100 ␮M concentration of the peroxides indicated. The half-times for the peroxides were calculated from the slopes of the semilogarithmic presentations of the data obtained for the first 10 min of incubation. The data represent means ⫾ SD of n dishes derived from three to five independently prepared cultures. The data were analyzed by ANOVA followed by the Bonferroni post hoc test. a p ⬍ 0.001, b p ⬍ 0.01 compared with the detoxification of the same peroxide by the other culture type; c p ⬍ 0.001, d p ⬍ 0.01 compared with the detoxification of the other peroxide by the same culture type.

(D ⫽ p⫺1 t1/2⫺1). Compared with neuronal cultures generated by seeding 3 ⫻ 106 cells per dish, the D values of neuron-rich cultures obtained by seeding 2 ⫻ 106 or 6 ⫻ 106 cells differed by ⬍16% (Table 1). With a half-time of 15.1 min, the detoxification of H2O2 by cells of a neuron-rich culture obtained after seeding 3 ⫻ 106 viable cells was slower by a factor of 5 than that by astroglial cultures. Nevertheless, due to the five-times higher protein content of astroglial cultures, identical D values of neuronal and astroglial cultures regarding the detoxification of H2O2 were obtained (Table 2). Before application of H2O2 to cultured neurons, the cells contained only marginal amounts of GSSG (Fig. 2). In contrast, after application of H2O2, GSSG levels were strongly elevated (Fig. 2). A transient maximum in the amount of GSSG (48% of total glutathione) was found 2.5 min after application of H2O2. During the incubation for up to 60 min, the amount of GSSG remained ⬎15% of the total glutathione. In contrast, when the peroxide was removed from the cells, the original ratio of GSSG to GSx was reestablished within 2.5 min (Fig. 2). To test for an involvement of catalase in the detoxification of H2O2 by neurons, the catalase inhibitor 3AT (Aebi, 1984) was applied. In the presence of 3AT, the disappearance of H2O2 from the incubation buffer was slowed down (Fig. 3A). Both the half-time for H2O2 and the D value were elevated threefold in 3AT-treated neurons compared with neurons incubated in the absence of 3AT (Table 3). Besides H2O2, the organic hydroperoxide CHP also was detoxified by cultured neurons (Fig. 3B). However, the clearance rate was significantly slower than that for H2O2 (Fig. 3B). The increased half-time for CHP led to an elevated D value, which was 2.7-fold that of H2O2. In contrast, astroglial cells detoxify CHP only somewhat more slowly than H2O2 (Dringen et al., 1998b; Table 2), leading to a 56% elevated D value for the detoxification of CHP by astroglial cells (Table 2). To elucidate why the glutathione system in neurons is less efficient than that in astroglial cultures, glutathione levels and specific activities of enzymes involved in peroxide detoxification were measured. Compared with astroglial cells, the specific glutathione content and the J. Neurochem., Vol. 72, No. 6, 1999

specific activity of GPx in neurons were 26 and 38% lower, respectively. In contrast, no significant differences between neurons and astroglial cultures were found in the specific activities of catalase (Table 4). DISCUSSION To investigate the ability of neurons to detoxify peroxides, H2O2 was applied to neuron-rich primary cultures. Immediately after administration of H2O2, 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 (Eklo¨w et al., 1984), or astroglial cells (Dringen and Hamprecht, 1997; Dringen et al., 1998a), indicating the involvement of GPx in the detoxification of H2O2. The rapid regeneration of the original ratio of GSH to GSSG after removal of H2O2 demonstrates that neurons in culture have a high capacity of GR-mediated reduction of GSSG. In the cytosol, the NADPH essential for this reaction is presumably provided by the PPP confined to this compartment. This hypothesis is supported by the

FIG. 2. Changes of the contents of GSx (circles) and GSSG (squares) in cells of neuron-rich primary cultures during exposure to H2O2. After addition of H2O2 (100 ␮M), the cells were incubated with the peroxide for the time periods indicated (open symbols). Alternatively, the cells were washed with incubation buffer after a 2.5-min incubation with H2O2 (100 ␮M in incubation buffer) followed by an incubation in peroxide-free incubation buffer (filled symbols). Inset: The content of GSSG expressed as percentages of GSx content.

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TABLE 4. Glutathione content and specific activities of GPx and catalase in neuron-rich and astroglia-rich primary cultures Specific activity of

FIG. 3. Clearance of peroxides from the incubation buffer of neuron-rich primary cultures. A: Neurons were preincubated for 2 h in DMEM/PS in the absence or the presence of 3AT (10 mM). The main incubation was performed in incubation buffer containing H2O2 (100 ␮M) in the absence (open circles) or the presence (filled circles) of 3AT (10 mM). B: Neurons were incubated with incubation buffer containing 100 ␮M H2O2 (open circles) or of CHP (filled squares). The neuron cultures were used at culture ages of 6 (A) and 5 days (B). The content of cellular protein was in the range between 250 and 300 ␮g of protein per dish.

known activation of the PPP after application of H2O2 to cultured neurons (Ben-Yoseph et al., 1994, 1996). In addition, alternative sources of NADPH production must be invoked, especially the mitochondrial isoform of malic enzyme that has been demonstrated to occur in neurons (Vogel et al., 1998). Neuronal cultures have been reported to be less effective in detoxifying H2O2 than astroglial cultures (Desagher et al., 1996). However, it has to be considered that such cultures are not confluent and contain less peroxidedetoxifying cells per dish than confluent astroglial cultures. Therefore, the reduced ability of cultured neurons to detoxify H2O2 might be due to a lower number of cells TABLE 3. Protein content per dish, half-times for H2O2, and specific detoxification rate constants D for the clearance of H2O2 by neuron-rich primary cultures in the presence or the absence of the catalase inhibitor 3AT

Control 3AT

Protein content (␮g/dish)

Half-time (min)


286 ⫾ 21 297 ⫾ 12

15.1 ⫾ 0.7 47.7 ⫾ 8.2a

0.232 ⫾ 0.014 0.073 ⫾ 0.015a

After a preincubation (2 h) in DMEM/PS containing 0 mM (control) or 10 mM 3AT, the cultures were incubated in incubation buffer containing H2O2 (100 ␮M) in the presence or the absence of 3AT (10 mM). The half-times for H2O2 were calculated from the slopes of the semilogarithmic representations of the data obtained for the first 10 min of incubation. The data represent means ⫾ SD (n ⫽ 6) of two sets of three dishes, each set representing independently prepared cultures. a p ⬍ 0.001 with respect to the control values (Student’s t test).

Primary culture

Glutathione content (nmol/mg)

GPx (nmol/min/mg)

Catalase k (1/min/mg)

Neuron-rich Astroglia-rich

23.7 ⫾ 6.0a 32.1 ⫾ 5.4b

112.5 ⫾ 37.5 182.6 ⫾ 22.7b

0.79 ⫾ 0.05 0.78 ⫾ 0.11

The data represent means ⫾ SD of the glutathione content measured in 27 experiments on neuron cultures and in 20 experiments on astroglial cultures. The specific activities of the enzymes were measured in homogenates obtained from the cells of culture dishes (n ⫽ 11) derived from three independently prepared cultures. The activities of GPx and catalase were normalized on the content of soluble proteins in the respective homogenate and on the total protein content of replicate plates, respectively. The catalase activity is expressed as first-order rate constant as recommended by Aebi (1984). a Reference values published by Dringen et al. (1999). b The differences in the specific glutathione content and in the specific activity of GPx between neurons and astroglial cells are significant ( p ⬍ 0.001, Student’s t test).

per dish. Indeed, with increasing seeding density, the ability of the neuronal cultures to clear H2O2 from the incubation buffer was improved. Nevertheless, even neuronal cultures obtained by seeding 6 ⫻ 106 viable cells did not detoxify H2O2 with the rate encountered in confluent astroglial cultures. Under all conditions chosen, the disappearance of H2O2 and CHP followed first-order kinetics for several minutes. This indicates that during this period the rate of clearance of a peroxide is always proportional to the concentration of this compound. First-order kinetics were found as well for the clearance of H2O2 or tBHP by cultured fibroblasts (Makino et al., 1994, 1995) and astroglial cells (Dringen and Hamprecht, 1997; Dringen et al., 1998a). The protein content per dish of a neuron-rich culture was almost proportional to the number of neurons seeded. Therefore, the detoxification rate of a cell culture dish has to be normalized to the protein content per dish to compare cells of different cell densities regarding their capacity for peroxide detoxification. The specific detoxification rate constant D equals the inversed product of the half-time of a peroxide (as indicator for the detoxification by the cells present on one dish) with the protein content (as indicator for the amount of cells responsible for peroxide detoxification). D is proportional to the capacity of a cell type to detoxify a peroxide. The higher the D value, the better the ability of the cultured cells to dispose of a peroxide. The D values for neuronal cultures obtained from different seeding densities varied at best marginally compared with the half-times for H2O2 and the protein content of these cultures, demonstrating that indeed the D value could be a valuable factor for the comparison of peroxide detoxification between cultures of different cell densities. The D values of neurons and astroglial cultures J. Neurochem., Vol. 72, No. 6, 1999

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regarding H2O2 detoxification were not significantly different, although the half-times calculated for the clearance of H2O2 differed significantly between these types of cultures. These results suggest that neuron-rich cultures are as capable as astroglial cultures to detoxify H2O2, if the different protein contents per dish of the two culture types are taken into consideration. Inhibition of catalase by 3AT reduced strongly the clearance rate of neurons for H2O2. This finding contrasts with the situation in astroglial cells, where the inhibition by 3AT of catalase during peroxide clearance is functionally buffered by the glutathione system (Dringen and Hamprecht, 1997). Therefore, it has to be concluded that the capacity of the glutathione system in neurons is insufficient to compensate for the loss of catalase activity during H2O2 detoxification. To confirm a reduced efficacy of the neuronal glutathione system of peroxide disposal, the detoxification of an organic hydroperoxide by neurons was investigated. Under the conditions used, such compounds do not serve as substrates for catalase (Aebi, 1984; Dringen et al., 1998a), and, therefore, only the glutathione system is responsible for the clearance of organic hydroperoxides. Consequently, if the glutathione system of neurons would be less effective in peroxide detoxification than that of astroglial cells, a reduction in the clearance rate for CHP should be found in neurons. That was indeed the case. Both the half-times and the D value for CHP were elevated approximately threefold compared with the values calculated for the clearance of H2O2 by neurons. Thus, if the glutathione system is made responsible for the clearance of a peroxide, either by inhibition of catalase to study the detoxification of H2O2 or by application of an organic hydroperoxide, astroglial cells can dispose of the peroxide three times faster than neurons. Therefore, only the participation of both GPx and catalase enables neurons to detoxify H2O2 with a D value identical to that of astroglial cells. CHP was used as a tool to investigate the glutathionedependent peroxide clearance by neuronal cultures. However, the disposal of CHP can also be considered as a model for the reduction to the corresponding alcohols of soluble organic hydroperoxides, such as the products of the cyclooxygenase and lipoxygenase reactions. Both neurons and astroglial cells have been reported to synthesize prostaglandins and leukotrienes (Murphy et al., 1988; Bishai and Coceani, 1992; Piomelli, 1994). Also, in neural cells, GPx might be involved in the generation of prostaglandin H2 and 12-hydroxyeicosatetraenoic acid, as has been reported for GPx from other tissues (Hong et al., 1989; Jahn and Hansch, 1990). Consequently, differences in the capacity of the glutathione system in astrocytes and neurons might reflect different rates in the metabolism of eicosanoids in these brain cell types. Neurons are less effective than astroglial cells in detoxifying peroxides via the glutathione system. A reason for this situation could be the lower specific activity of GPx and the lower glutathione content of neurons comJ. Neurochem., Vol. 72, No. 6, 1999

pared with astroglial cultures, which confirms reported data for murine cultures (Raps et al., 1989; Bolanõs et al., 1995; Huang and Philbert, 1995; Desagher et al., 1996). If for the cultured neural cells the intracellular concentration of GSH and the Km value of GPx for GSH are in the low millimolar range, as has been reported for brain (De Marchena et al., 1974; Cooper, 1997), both lower GSH content and lower GPx activity will lead to a reduced rate of peroxide detoxification by GPx. Oxidative stress has been connected with several neurodegenerative diseases (Halliwell, 1992; Weber, 1994; Bowling and Beal, 1995; Bains and Shaw, 1997; Cadet and Brannock, 1998). A reduced ability of the neuronal glutathione system to detoxify peroxides might contribute to an increased oxidative stress in neurons compared with other brain cell types. That might be especially the case for neurons in brain areas such as the pars compacta of the substantia nigra, where a 40 –50% loss in glutathione levels has been reported for patients who suffered from Parkinson’s disease (Sofic et al., 1992; Sian et al., 1994). In conclusion, neurons in culture are able to detoxify exogenously applied H2O2 with approximately the same rate as astroglial cells. However, for this fast detoxification of H2O2 in neurons, the involvement of catalase is essential. The compromised detoxification of H2O2 in 3AT-treated neurons and the lower clearance rate of CHP indicate that the glutathione system of peroxide detoxification in neurons is less efficient than in astroglial cells. These data support the hypothesis that brain neurons are more vulnerable to oxidative stress than astrocytes due to an insufficient detoxification of reactive oxygen species via their glutathione system. REFERENCES Abe K. and Saito H. (1998) Characterization of t-butyl hydroperoxide toxicity in cultured rat cortical neurones and astrocytes. Pharmacol. Toxicol. 83, 40 – 46. Aebi H. E. (1984) Catalase, in Methods of Enzymatic Analysis, Vol. 3 (Bergmeyer H. U., ed), pp. 273–286. Verlag Chemie, Weinheim, Germany. Bains J. S. and Shaw C. A. (1997) Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Res. Rev. 25, 335–358. Baker M. A., Cerniglia G. J., and Zaman A. (1990) Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples. Anal. Biochem. 190, 360 –365. Baquer N. Z., Hothersall J. S., and McLean P. (1988) Function and regulation of the pentose phosphate pathway in brain. Curr. Top. Cell. Regul. 29, 265–289. Barker J. E., Bolanõs J. P., Land J. M., Clark J. B., and Heales S. J. R. (1996) Glutathione protects astrocytes from peroxynitrite-mediated mitochondrial damage: implication for neuronal/astrocytic trafficking and neurodegeneration. Dev. Neurosci. 18, 391–396. Ben-Yoseph O., Boxer P. A., and Ross B. D. (1994) Oxidative stress in the central nervous system: monitoring the metabolic response using the pentose phosphate pathway. Dev. Neurosci. 16, 328 – 336. Ben-Yoseph O., Boxer P. A., and Ross B. D. (1996) Assessment of the role of the glutathione and pentose phosphate pathways in the protection of primary cerebrocortical cultures from oxidative stress. J. Neurochem. 66, 2329 –2337.

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