Dehydroepiandrosterone inhibits the death of immunostimulated rat C6 glioma cells deprived of glucose

Brain Research 922 (2001) 267–275 www.elsevier.com / locate / bres

Research report

Dehydroepiandrosterone inhibits the death of immunostimulated rat C6 glioma cells deprived of glucose Chan Young Shin a ,1 , Ji-Woong Choi a ,1 , Eun Sook Jang a , Chung Ju b , Won-Ki Kim b , c d a, Hyoung-Chun Kim , Chang-Rak Choi , Kwang Ho Ko * a

Department of Pharmacology, College of Pharmacy, Seoul National University, San 56 -1, Shillim-Dong, Kwanak-Gu, Seoul 151 -742, South Korea b Department of Pharmacology, College of Medicine, Ewha Institute of Neuroscience, Ewha Women’ s University, Seoul 158 -710, South Korea c College of Pharmacy, Kang Won National University, Chunchon 200 -701, South Korea d Department of Neurosurgery, St. Mary’ s Hospital, The Catholic University of Korea, Seoul 150 -713, South Korea Accepted 25 September 2001

Abstract Pretreatment of interferon-g and lipopolysaccharides made C6 glioma cells highly vulnerable to glucose deprivation. Neither 12 h of glucose deprivation nor 2-day treatment with interferon-g (100 U / ml) and lipopolysaccharides (1 mg / ml) altered the viability of C6 glioma cells. However, significant death of immunostimulated C6 glioma cells was observed after 5 h of glucose deprivation. The augmented death was prevented by dehydroepiandrosterone (DHEA) treatment during immunostimulation, but not by DHEA treatment during glucose deprivation. DHEA reduced the rise in nitrotyrosine immunoreactivity, a marker of peroxynitrite, and superoxide production in glucose-deprived immunostimulated C6 glioma cells. DHEA, however, did not protect glucose-deprived C6 glioma cells from the exogenously produced peroxynitrite by 3-morpholinosydnonimine. Further, DHEA did not alter the production of total reactive oxygen species and nitric oxide in immunostimulated C6 glioma cells. Superoxide dismutase (SOD) and the synthetic SOD mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin inhibited the death of glucose-deprived immunostimulated C6 glioma cells. In addition, a superoxide anion generator paraquat reversed the protective effect of DHEA on the augmented death. The data indicate that DHEA prevents the glucose deprivation-evoked augmented death by inhibiting the production of superoxide anion in immunostimulated C6 glioma cells.  2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Dehydroepiandrosterone; Peroxynitrite; C6 glioma; Immunostimulation; Glucose deprivation

1. Introduction Following various brain insults including seizures and ischemia, glial cells are stimulated by locally or systemically produced cytokines [6,43]. Immunostimulated glial cells then produce cytokines and chemokines along with nitric oxide (NO) and reactive oxygen species (ROS), which greatly contribute to the neuronal toxicity observed in the aforementioned pathophysiological conditions [13,19,20]. In the cerebral ischemia, progressive metabolic deterio*Corresponding author. Tel.: 182-2-880-7848; fax: 182-2-885-8211. E-mail address: [email protected] (K. Ho Ko). 1 These authors equally contributed to this work.

ration has been reported to eventually lead to pan-necrosis of both glial and neuronal cells [8]. Recently, we reported that immunostimulation made murine astrocytes highly vulnerable to glucose deprivation-induced cell death [9– 11]. Endogenously produced peroxynitrite appeared to be mainly involved in the augmented vulnerability of immunostimulated astrocytes to glucose deprivation [9–11]. Because astrocytes support normal neuronal functions by tightly regulating the extracellular environment and providing energy substrates such as glucose, dysfunction or loss of astrocytes will lead to neuronal death [29,33,40]. Therefore, protection of astrocytes should be a new neuroprotective strategy in the post-ischemic brain. Dehydroepiandrosterone (DHEA) and its active metabolite dehydroepiandrosterone sulfate (DHEAS) have been

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03185-7

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reported to have various actions in brain such as neuroprotection, blockade of calcium ion channels, or antagonism of the GABAA receptor [14,15,32,38]. DHEA was shown to protect hippocampal neurons against excitatory amino acid- or glucocorticoid-induced neurotoxicity [25,26] and oxidative stress-induced damage by H 2 O 2 / FeSO 4 complex [4]. However, no data have been reported of the inhibitory effect of DHEA on the susceptibility of immunostimulated glial cells to oxidative stress. In the present study, therefore, we investigated the effects of DHEA on the potentiated death in immunostimulated rat C6 glioma cells deprived of glucose.

2. Materials and methods

2.1. Materials Lipopolysaccharide (LPS, serotype 026:B6 from Escherichia coli) and DHEA were purchased from Sigma– Aldrich Co. (St. Louis, MO). Recombinant rat interferon-g (IFN-g), glucose-free Dulbecco’s modified Eagle’s medium (DMEM), DMEM / F12 and fetal bovine serum (FBS) were from Gibco BRL (Grand Island, NY). N G nitro-L-arginine (NNA) was obtained from Research Biochemical International (Natick, MA). 3-Morpholinosydnonimine (SIN-1) was obtained from Calbiochem (La Jolla, CA). Monoclonal anti-nitrotyrosine antibody was purchased from Upstate Biotechnology (Lake Placid, NY). All other chemicals including N v -nitro-L-arginine methyl ester ( L-NAME) and Cu / Zn-SOD, were purchased from Sigma.

product of NO, as described previously [18]. In brief, nitrite levels were determined by adding the Greiss reagent (mixing equal volumes of 0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid) to the medium. After 10 min, the absorbance at 550 nm was determined using a UV spectrophotometer (Beckman DU-650, Fullerton, CA).

2.4. Measurement of superoxide anion production C6 glioma cells were seeded into poly-L-lysine-coated plastic cuvettes and cultured overnight followed by 24–48 h of immunostimulation. The culture medium was replaced with DMEM containing 10 mM lucigenin (Molecular Probes, Eugene, OR) and then chemiluminescence was monitored (Autolumat LB953, E.G.&G. Berthold, Germany) at 378C for 1 h.

2.5. Measurement of total reactive oxygen species ( ROS) production The production of ROS was assessed by using the ROS-sensitive fluorescence indicator 29,79-dichlorodihydrofluoroscein diacetate (H 2 DCF-DA; Molecular Probes). Cells were loaded with H 2 DCF-DA (5 mg / ml) in PBS for 30 min and then rinsed with the same solution. The fluorescence of DCF was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a spectrofluorophotometer (Tecan, Austria). Fluorescence intensities were measured only once due to the potential oxidation of the dye by the excitation light and were corrected for autofluorescence (i.e. fluorescence of cells not loaded with DCF-DA).

2.2. C6 glioma cell culture 2.6. Measurement of cell death C6 glioma cells were derived from American Type Culture collection (ATCC) and cultured in DMEM / F12 supplemented with 10% heat-inactivated FBS and 100 U / ml penicillin / streptomycin at 378C in 5% CO 2 . Confluent cultures of C6 glioma cells were harvested by brief trypsinization and seeded at a density of 10 5 cells / ml on 12-well culture plates. Cells were adapted to serum-free condition for 48 h after seeding. During the adaptation period, cell mortality was less than 5% as evidenced by trypan blue exclusion. C6 glioma cells were immunostimulated for 36–48 h with IFN-g (100 U / ml) and LPS (1 mg / ml). After immunostimulation, glucose deprivation was achieved by repeated rinsing and incubation in glucose-free DMEM that was not supplemented with serum, which interfered with the lactate dehydrogenase (LDH) assay.

2.3. Measurement of NO NO production from immunostimulated C6 glioma cells was determined by measuring nitrite, a stable oxidation

Cell death was assessed by measuring LDH release into the medium at various time points after starting glucose deprivation, as described previously [11]. The LDH amount (total LDH release) corresponding to complete glial death was measured in sister cultures treated with 0.1% Triton X-100 for 30 min at 378C. Basal LDH levels (generally less than 10% of total LDH release) were determined in sister cultures subjected to sham wash with 5 mM glucose containing DMEM and subtracted from the levels in experimental conditions to yield the LDH signal specific to experimental injury.

2.7. Immunocytochemical staining of nitrotyrosine production C6 glioma cells were fixed with 4% paraformaldehyde for 30 min. After thorough washing with PBS, the cells were incubated with polyclonal anti-nitrotyrosine antibody (rabbit immunoaffinity purified IgG, Upstate Biotechnology, Lake Placid, NY) diluted 1:200 in PBS containing

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0.1% Triton X-100 for 1 h at room temperature. After washing, the cells were incubated with biotin-labeled goat anti-rabbit IgG for 1 h at room temperature. After washing, the cells were incubated for 30 min in Vectastain Elite ABC reagent (Vector, Burlingame, CA). After washing, the cells were then reacted with 400 mg / ml 3,39-diaminobenzidine tetrahydrochloride and 0.02% hydrogen peroxide in 50 mM Tris–HCl, pH 7.4 for 5–10 min.

2.8. Statistical analysis Data are expressed as the mean6S.E.M. and analyzed for statistical significance by using one-way analysis of variance followed by Newman–Keuls test as a post hoc test, and a P value of less than 0.01 was considered significant.

3. Results NO production, measured as nitrite formation, increased in C6 glioma cells treated with LPS and IFN-g, and was inhibited by co-treatment of nitric oxide synthase (NOS) inhibitors NNA and L-NAME (Fig. 1A). Glucose deprivation markedly evoked LDH release in immunostimulated C6 glioma cells (Fig. 1B). However, neither 12 h glucose deprivation nor 48 h immunostimulation caused LDH releases from C6 glioma cells. As reported previously, dying astrocytes displayed a morphological and biochemical features consistent with necrotic rather than apoptotic cell death [9,10]. Near complete death of immunostimulated C6 glioma cells was observed 8 h after glucose deprivation. Thus, we routinely used this time point in LDH determination for the rest of the experiments. Just pretreatment with NNA or L-NAME during the immunostimulation period (prior to glucose deprivation) inhibited LDH release from immunostimulated C6 glioma cells during the subsequent glucose deprivation period (Fig. 1B). Like NOS inhibitors, pretreatment with 10–100 mM DHEA during immunostimulation suppressed LDH release caused by glucose deprivation in immunostimulated C6 glioma cells (Fig. 2). Pretreatment with DHEA for at least 12 h prior to glucose deprivation (i.e. 36 h after immunostimulation) was required to significantly block the death of glucose-deprived immunostimulated C6 glioma cells (Fig. 3). Co-treatment with DHEA during glucose deprivation did not block LDH release in glucose-deprived immunostimulated C6 glioma cells (data not shown). In this study, DHEA did not increase proliferation of C6 glioma cells (data not shown). Previously, we showed that endogenously produced peroxynitrite plays a crucial role in the synergistic injury and death of immunostimulated glial cells exposed to glucose deprivation [9–11]. Therefore, we further investigated whether the protective effect of DHEA was due to

Fig. 1. (A) Production of NO from C6 glioma cells. C6 glioma cells were stimulated for 48 h with IFN-g (100 U / ml) / LPS (1 mg / ml) in the absence and presence of NOS inhibitors (NNA, L-NAME). Culture medium was analyzed for nitrite formation by Greiss reaction as described in Section 2. Each bar indicates the mean6S.D. (n54). *P, 0.01, significantly different from untreated control. (B) Augmented death of immunostimulated C6 glioma cells during glucose deprivation (GD). Immunostimulated cells were further incubated in the absence and presence of glucose. The amounts of LDH released into the medium from C6 glioma cells were determined at indicated times. Data are the mean6S.E.M. (n54). *P,0.01, significantly different from immunostimulated C6 glioma cells.

the decreased production of peroxynitrite. Nitrotyrosine immunoreactivity (a marker for peroxynitrite) was markedly increased in glucose-deprived immunostimulated C6 glioma cells (Fig. 4). The enhanced nitrotyrosine immunoreactivity was reduced by DHEA (Fig. 4) as well as by NNA or L-NAME (data not shown). As shown previously in primary astrocyte cultures [9], addition of an exogenous peroxynitrite generator SIN-1 during glucose deprivation period, concentration-dependently increased the death of glucose-deprived C6 glioma cells (Fig. 5A). Interestingly, however, 48 h pretreatment with DHEA did not block the SIN-1-induced death of glucose-deprived C6 glioma cells, indicating that DHEA might not directly scavenge peroxynitrite (Fig. 5B). Further, DHEA did not inhibit the

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termined by DCF (data not shown). SOD and MnTMPyP almost completely inhibited LDH release in glucose-deprived immunostimulated C6 glioma cells (Fig. 8). Addition of paraquat, a superoxide generator, into glucosedeprived immunostimulated C6 glioma cells reversed the effect of 48 h pretreatment of DHEA on their LDH release (Fig. 8). Despite the suggestive blockade of superoxide anion production by DHEA in immunostimulated C6 glioma cells, DHEA did not directly scavenge the superoxide anion. As determined by lucigenin, DHEA did not alter the production of superoxide anion from 200 mM SIN-1: control, 922668; DHEA-treated, 898654 arbitrary luminescence units (data not shown).

4. Discussion Fig. 2. Concentration-dependent protective effect of DHEA. C6 glioma cells were immunostimulated for 48 h in the presence of various concentrations of DHEA. The amounts of LDH released into the medium were determined 8 h after starting glucose deprivation. Data are the mean6S.E.M. (n54). *P,0.01, significantly different from C6 glioma cells immunostimulated in the absence of DHEA.

production of NO (Fig. 6A) and total oxygen free radicals (Fig. 6B) in immunostimulated C6 glioma cells. In contrast, 48 h pretreatment of DHEA completely blocked the increase in superoxide anion production in immunostimulated C6 glioma cells (Fig. 7). Similar to DHEA, superoxide dismutase (SOD) and a synthetic SOD mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin (MnTMPyP) did not alter the production of NO and total oxygen free radicals in immunostimulated C6 glioma cells, as de-

Fig. 3. Time-dependent protective effect of DHEA. C6 glioma cells were immunostimulated for 48 h during which DHEA (100 mM) was added at indicated times after starting IFN-g/ LPS treatment and were then incubated in glucose-free DMEM. The amounts of LDH released into the medium were determined at 8 h after starting glucose deprivation. Data are the mean6S.E.M. (n54). *P,0.01, significantly different from LDH release from C6 glioma cells without DHEA treatment during immunostimulation (i.e. 48 h).

DHEA and its active metabolite DHEAS are the most abundant steroids in the blood of young adult human [34]. Aging and stress cause the reduction of DHEA level in blood [17,21,22,39]. Several researchers have reported neuroprotective effect of DHEA. DHEA protected hippocampal neurons against either excitatory amino acid- or glucocorticoid-induced toxicity [25,26]. DHEA also protected mixed hippocampal neurons from H 2 O 2 or sodium nitroprusside (SNP)-induced toxicity [4]. However, the exact mechanism for neuroprotection by DHEA is still uncertain.Previously, we showed that overproduction of peroxynitrite in rat astrocytes resulted in mitochondrial dysfunction and subsequent cell death [11,24]. In the present study the cytoprotective effect of DHEA appeared to involve the suppression of peroxynitrite production in glucose-deprived immunostimulated C6 glioma cells. However, DHEA did not inhibit the cytotoxicity induced by peroxynitrite exogenously produced from SIN-1. This finding implies that DHEA neither scavenges peroxynitrite nor interferes with the downstream pathway of peroxynitrite-evoked cytotoxicity. In the present study DHEA was not found to affect the production of NO and total ROS in immunostimulated C6 glioma cells. Thus, a question arises how DHEA decreases the production of peroxynitrite in immunostimulated C6 glioma cells. Peroxynitrite is produced from NO and superoxide. Elimination of either species may result in the decreased peroxynitrite production. Several observations indicate that the cytoprotective effect of DHEA may be associated with inhibition of production of superoxide anion from immunostimulated C6 glioma cells. In the present study, SOD and the cell membrane-permeable SOD mimetic MnTMPyP inhibited the death of glucose-deprived immunostimulated C6 glioma cells. Previously, DHEA was reported to inhibit spontaneous or 12-O-tetra-decanoylphorbol-13-acetatestimulated release of superoxide anion [36,45]. Similar to DHEA, SOD and MnTMPyP caused little changes in DCF fluorescence (total intracellular ROS marker) while superoxide production is significantly reduced, as deter-

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Fig. 4. DHEA inhibits nitrotyrosine immunoreactivity in immunostimulated C6 glioma cells. (A) C6 glioma cells were immunostimulated for 48 h with LPS (b) and IFN-g (c) alone or in combination (d). DHEA (100 mM) was added during 48 h IFN-g/ LPS treatment (e). After immunostimulation, cells were three times washed and deprived of glucose for 4 h and the nitrotyrosine content was visualized by immunocytochemistry as described in Section 2. Cells were counterstained with hematoxylin for comparison. Micrographs are representative of three independent experiments. (B) Quantification of nitrotyrosine immunoreactivity. After immunostaining, nitrotyrosine positive and negative cells were counted by manual inspection. Nitrotyrosine immunoreactivity was expressed as percentage of positive cells against total cell number. Data are the mean6S.E.M. (n53). *P,0.01, significantly different from untreated C6 glioma cells. ¶ P,0.01, significantly different from LPS1IFN-g-stimulated C6 glioma cells.

mined by lucigenin. These results suggest that superoxide anion produced from immunostimulated C6 glioma cells constitute small portion of total ROS detected with DCF fluorescence. Many researchers reported that DCF may be used to detect hydrogen peroxide, but not superoxide [30,35]. Furthermore, we observed that immunostimulation-induced increase in DCF fluorescence was

inhibited by catalase (data not shown), which suggest that DCF-sensitive ROS is mainly hydrogen peroxide and related species. DHEA-mediated inhibition of superoxide anion production in immunostimulated C6 glioma cells was further supported by the reversal of the inhibitory effect of DHEA by paraquat, a superoxide generator. Paraquat was used for the generation of intracellular

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Fig. 5. Potentiated LDH release by SIN-1 in glucose-deprived C6 glioma cells. (A) C6 glioma cells were exposed to various concentrations of SIN-1 in the absence and presence of glucose. *P,0.01, significantly different from C6 glioma cells treated with various concentrations of SIN-1 in the presence of glucose. (B) C6 glioma cells were pretreated with DHEA (5–100 mM) for 48 h, and then exposed to 50 mM SIN-1 in the absence of glucose. The amounts of LDH released into the medium were determined 8 h after starting glucose deprivation. Data are the mean6S.E.M. (n54).

superoxide radicals in several works [12,27]. Here we showed that continuous supply of superoxide radicals by paraquat block the protective effect of DHEA against cell death induced by glucose deprivation in immunostimulated C6 cells (Fig. 8). These results suggest that DHEA-induced inhibition of superoxide production mediates decreased peroxynitrite production and contributes to the inhibition of cell death under glucose deprivation in immunostimulated C6 cells. Despite the suggested blockade of superoxide anion production by DHEA in immunostimulated C6 glioma cells, DHEA did not directly scavenge the superoxide anion. Thus, DHEA neither caused any changes in O 2 2 production from 200 mM SIN-1 nor prevented cell death caused by SIN-1. In addition,

Fig. 6. (A) DHEA does not alter NO production in immunostimulated C6 glioma cells. C6 glioma cells were immunostimulated for 48 h in the presence of various concentrations of DHEA. Data are the mean6S.E.M. (n54). (B) DHEA does not alter ROS generation in immunostimulated C6 glioma cells. C6 glioma cells were immunostimulated for 48 h in the absence and presence of DHEA (100 mM). ROS generation was measured using H 2 DCF-DA, as described in Section 2. Data are the mean6S.E.M. (n54). *P,0.01, significantly different from untreated control.

DHEA treatment only during glucose deprivation period did not protect cell death in immunostimulated astrocytes. Moreover, DHEA did not block nitration of tyrosine residues of bovine serum albumin induced by SIN-1 in vitro (data not shown). The data suggest that DHEA decreases the superoxide anion generation and consequent peroxynitrite production in cells rather than directly scavenging superoxide anion. DHEA has been widely used as an inhibitor of glucose6-phosphate dehydrogenase (G6PD) activity [16,41,42]. It has been reported that DHEA prevent rat tracheal epithelial cells from oxidant-induced toxicity by inhibiting G6PD [31]. This inhibitory action may result in the decrease in NADPH level, a substrate necessary for membrane bound flavoprotein NADPH oxidase to generate superoxide anion [31,37]. The decreased substrate availability may diminish superoxide production, which can lead to the inhibition of peroxynitrite generation. However, G6PD also plays a role in cell survival and it is possible that complete inhibition

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Fig. 7. DHEA markedly suppresses superoxide generation in immunostimulated C6 glioma cells. Cells were immunostimulated in the presence of SOD (50 U / ml) or DHEA (100 mM) for 24 or 48 h, respectively. The amounts of superoxide anion were determined using lucigenin, as described in Section 2. Data are the mean6S.E.M. (n54). *P,0.01, significantly different from C6 glioma cells untreated with IFN-g and LPS. ¶ P,0.01, significantly different from IFN-g/ LPS-treated group.

Fig. 8. Effects of SOD, MnTMPyP and paraquat on the LDH release from immunostimulated C6 glioma cells. C6 glioma cells were immunostimulated in the presence of SOD (50 U / ml) or DHEA (100 mM) for 24 or 48 h, respectively, and then subjected to glucose deprivation. MnTMPyP was added in immunostimulated C6 glioma cells only during glucose deprivation period. Paraquat (100 mM) was added in control or DHEA-treated C6 glioma cells during glucose deprivation. The amounts of LDH released into the medium were determined 8 h after starting glucose deprivation. Data are the mean6S.E.M. (n54). *P,0.01, significantly different from untreated control. ¶ P,0.01, significantly different from DHEA-treated group.

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of G6PD may induce cell toxicity. It was reported that 100 mM of DHEA in the presence of glucose (25 mM) slightly inhibited cell viability in BV-cells (25%), although lower doses did not inhibit cell viability [46]. However, in the present study, DHEA co-treatment with LPS and IFN-g for 48 h did not induce significant cell death as determined by morphological examination or by measurement of LDH release into the medium (data not shown). The factors underlying these differences are not clear yet. Different experimental conditions such as the method for the determination of cell death (MTT vs. LDH assay) and cell types (BV-cells vs. C6 glioma cells) may cause those differences. In addition, BV-cells are a murine microglial cell line and microglia is more susceptible than astrocytes to toxic insults including ROS [23]. To investigate whether the inhibition of G6PD results in the decreased superoxide production, the determination of intracellular NADPH level and activity of NADPH oxidase after DHEA treatment would be necessary. Astrocytes appear to be the most active steroidogenic cells in the brain and have the capacity to metabolize DHEA into sex steroid hormones, expressing for 17bhydroxysteroid dehydrogenase and cytochrome P450 aromatase [47]. Testosterone, as well as its metabolite 17b-estradiol, the product of aromatase, have been shown to protect hippocampal neurons from neurotoxin-induced death [3], and 17b-estradiol treatments also protect male brain in experimental stroke [44]. It should be determined in future studies whether those metabolites are involved in the protective effect of DHEA on the glucose deprivationinduced cell death in immunostimulated C6 cells. Previously, DHEA was shown to exert its cytoprotective effect by ameliorating lipid peroxidation by a mechanism involving inhibition of hydroxyl radical production [4]. This finding coincided with the reported in vivo inhibitory effect of DHEA on lipid peroxidation induced by copper (Cu 21 ) or by acute hyperglycemia in the rat brain [1,7]. Previously, however, we showed that the lipid-soluble vitamin E analogue trolox, a well-known antioxidant that protects against lipid peroxidation, did not alter the augmented death in glucose-deprived immunostimulated glial cultures [24]. Therefore, the cytoprotective effect of DHEA on lipid peroxidation might not be a critical factor in inhibiting the augmented death of glucose-deprived immunostimulated C6 glioma cells shown in the present study. In the cerebral ischemic penumbra, both glial and neuronal cells have been reported to undergo necrotic and / or apoptotic death [8]. Because of the supportive action of astrocytes in normal neuronal functions, dysfunction or loss of astrocytes leads to neuronal death [29,33,40]. As administered peripherally, DHEA rapidly crosses blood–brain barrier and protects brain from various insults [2,25,45]. The concentrations of DHEA used in the present study appeared to be somewhat higher than those occurring naturally in brain, which is 19.6 nmol / kg tissue

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in the human brain and 0.24 ng / g tissue in the rat brain [5,28]. Such a high concentration was also required for the protective effect of DHEA on oxidative stress-induced hippocampal neuronal toxicity [4]. However, the concentration of DHEA in human brain is 10 times higher than that in rat brain, suggesting the profound action of DHEA in human brain [5,28]. Considering the inhibitory effect on superoxide generation as well as cytoprotective effect of DHEA on glucose-deprived, immunostimulated glial cells, it would be interesting to investigate whether DHEA is also effective in the condition of hypoxia / ischemia. Also, it should be determined whether DHEA protection of astrocytes will necessarily protect neurons before expanding it as a new neuroprotective strategy in the post-ischemic brain.

[11]

[12]

[13]

[14]

[15]

[16]

Acknowledgements This work was supported in part by a grant of ‘The Good Health R & D Project (1999)’ and also supported in part by BK21 project from the Ministry of Education, Republic of Korea.

[17]

[18]

References [19] [1] M. Aragno, E. Brignardello, E. Tamagno, V. Gatto, O. Danni, G. Boccuzzi, Dehydroepiandrosterone administration prevents the oxidative damage induced by acute hyperglycemia in rats, J. Endocrinol. 155 (1997) 233–240. [2] M. Aragno, E. Tamagno, G. Boccuzzi, E. Brignardello, E. Chiarpotto, A. Pizzini, O. Danni, Dehydroepiandrosterone pretreatment protects rats against the pro-oxidant and necrogenic effects of carbon tetrachloride, Biochem. Pharmacol. 46 (1993) 1689–1694. [3] I. Azcoitia, A. Sierra, S. Veiga, S. Honda, N. Harada, L.M. GarciaSegura, Brain aromatase is neuroprotective, J. Neurobiol. 47 (2001) 318–329. [4] S. Bastianetto, C. Ramassamy, J. Poirier, R. Quirion, Dehydroepiandrosterone (DHEA) protects hippocampal cells from oxidative stress-induced damage, Brain Res. Mol. Brain Res. 66 (1999) 35–41. [5] E.E. Baulieu, P. Robel, Dehydroepiandrosterone and dehydroepiandrosterone sulfate as neuroactive neurosteroids, J. Endocrinol. 150 (Suppl.) (1996) S221–S239. [6] B. Becher, A. Prat, J.P. Antel, Brain-immune connection: immunoregulatory properties of CNS-resident cells, Glia 29 (2000) 293– 304. [7] G. Boccuzzi, M. Aragno, M. Seccia, E. Brignardello, E. Tamagno, E. Albano, O. Danni, G. Bellomo, Protective effect of dehydroepiandrosterone against copper-induced lipid peroxidation in the rat, Free Radic. Biol. Med. 22 (1997) 1289–1294. [8] J.B. Brierly, D.I. Graham, Hypoxia and vascular disorders of the nervous system, in: J.H. Adams, J.A.N. Corsellis, L.W. Duchen (Eds.), Greenfield’s Neuropathology, 4th Edition, Wiley, New York, 1984, pp. 125–207. [9] J.J. Choi, W.K. Kim, Potentiated glucose deprivation-induced death of astrocytes after induction of iNOS, J. Neurosci. Res. 54 (1998) 870–875. [10] J.J. Choi, W.K. Kim, Proliferation-dependent vulnerability of

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

glucose-deprived astrocytes to nitric oxide-induced cytotoxicity, Neurosci. Lett. 256 (1998) 109–112. I.Y. Choi, S.J. Lee, C. Ju, W. Nam, H.C. Kim, K.H. Ko, W.K. Kim, Protection by a manganese porphyrin of endogenous peroxynitriteinduced death of glial cells via inhibition of mitochondrial transmembrane potential decrease, Glia 31 (2000) 155–164. J.C. Copin, Y. Gasche, Y. Li, P.H. Chan, Prolonged hypoxia during cell development protects mature manganese superoxide dismutasedeficient astrocytes from damage by oxidative stress, FASEB J. 15 (2001) 525–534. M. Downen, T.D. Amaral, L.L. Hua, M.L. Zhao, S.C. Lee, Neuronal death in cytokine-activated primary human brain cell culture: role of tumor necrosis factor-alpha, Glia 28 (1999) 114–127. J.M. Ffrench-Mullen, K.T. Spence, Neurosteroids block Ca 21 channel current in freshly isolated hippocampal CA1 neurons, Eur. J. Pharmacol. 202 (1991) 269–272. J.F. Flood, J.E. Morley, E. Roberts, Memory-enhancing effects in male mice of pregnenolone and steroids metabolically derived from it, Proc. Natl. Acad. Sci. USA 89 (1992) 1567–1571. P. Garcia-Nogales, A. Almeida, E. Fernandez, J.M. Medina, J.P. Bolanos, Induction of glucose-6-phosphate dehydrogenase by lipopolysaccharide contributes to preventing nitric oxide-mediated glutathione depletion in cultured rat astrocytes, J. Neurochem. 72 (1999) 1750–1758. I.M. Goodyer, J. Herbert, P.M. Altham, J. Pearson, S.M. Secher, H.M. Shiers, Adrenal secretion during major depression in 8- to 16-year-olds. I. Altered diurnal rhythms in salivary cortisol and dehydroepiandrosterone (DHEA) at presentation, Psychol. Med. 26 (1996) 245–256. S.J. Green, M.S. Meltzer, J.S. Hibbs Jr., C.A. Nacy, Immunostimulated macrophages destroy intracellular Leishmania major amastigotes by an L-arginine-dependent killing mechanism, J. Immunol. 144 (1990) 278–283. B. Halliwell, Reactive oxygen species and the central nervous system, J. Neurochem. 59 (1992) 1609–1623. S.J. Heales, J.P. Bolanos, V.C. Stewart, P.S. Brookes, J.M. Land, J.B. Clark, Nitric oxide, mitochondria and neurological disease, Biochim. Biophys. Acta 1410 (1999) 215–228. M. Hedman, E. Nilsson, B. de la Torre, Low blood and synovial fluid levels of sulpho-conjugated steroids in rheumatoid arthritis, Clin. Exp. Rheumatol. 10 (1992) 25–30. J. Herbert, I.M. Goodyer, P.M. Altham, J. Pearson, S.M. Secher, H.M. Shiers, Adrenal secretion and major depression in 8- to 16-year-olds. II. Influence of co-morbidity at presentation, Psychol. Med. 26 (1996) 257–263. S.B. Hollensworth, C. Shen, J.E. Sim, D.R. Spitz, G.L. Wilson, S.P. LeDoux, Glial cell type-specific responses to menadione-induced oxidative stress, Free Radic. Biol. Med. 28 (2000) 1161–1174. C. Ju, K.N. Yoon, Y.K. Oh, H.C. Kim, C.Y. Shin, J.R. Ryu, K.H. Ko, W.K. Kim, Synergistic depletion of astrocytic glutathione by glucose deprivation and peroxynitrite: Correlation with mitochondrial dysfunction and subsequent cell death, J. Neurochem. 74 (2000) 1989–1998. V.G. Kimonides, N.H. Khatibi, C.N. Svendsen, M.V. Sofroniew, J. Herbert, Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) protect hippocampal neurons against excitatory amino acid-induced neurotoxicity, Proc. Natl. Acad. Sci. USA 95 (1998) 1852–1857. V.G. Kimonides, M.G. Spillantini, M.V. Sofroniew, J.W. Fawcett, J. Herbert, Dehydroepiandrosterone antagonizes the neurotoxic effects of corticosterone and translocation of stress-activated protein kinase 3 in hippocampal primary cultures, Neuroscience 89 (1999) 429– 436. W.Y. Kuo, T.K. Tang, Effects of G6PD overexpression in NIH3T3 cells treated with tert-butyl hydroperoxide or paraquat, Free Radic. Biol. Med. 24 (1998) 1130–1138. C. Lacroix, J. Fiet, J.P. Benais, B. Gueux, R. Bonete, J.M. Villette,

C. Young Shin et al. / Brain Research 922 (2001) 267 – 275

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

B. Gourmel, C. Dreux, Simultaneous radioimmunoassay of progesterone, androst-4-enedione, pregnenolone, dehydroepiandrosterone and 17-hydroxyprogesterone in specific regions of human brain, J. Steroid Biochem. 28 (1987) 317–325. C. Largo, P. Cuevas, G.G. Somjen, R. Martin, O. Herreras, The effect of depressing glial function in rat brain in situ on ion homeostasis, synaptic transmission, and neuron survival, J. Neurosci. 16 (1996) 1219–1229. C.P. LeBel, H. Ischiropoulos, S.C. Bondy, Evaluation of the probe 29,79-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress, Chem. Res. Toxicol. 5 (1992) 227–231. T.C. Lee, G.J. Lai, S.L. Kao, I.C. Ho, C.W. Wu, Protection of a rat tracheal epithelial cell line from paraquat toxicity by inhibition of glucose-6-phosphate dehydrogenase, Biochem. Pharmacol. 45 (1993) 1143–1147. M.D. Majewska, S. Demirgoren, C.E. Spivak, E.D. London, The neurosteroid dehydroepiandrosterone sulfate is an allosteric antagonist of the GABAA receptor, Brain Res. 526 (1990) 143–146. E.K. O’Malley, B.A. Sieber, I.B. Black, C.F. Dreyfus, Mesencephalic type astrocytes mediate the survival of substantia nigra dopaminergic neurons in culture, Brain Res. 582 (1992) 65–70. N. Orentreich, J.L. Brind, J.H. Vogelman, R. Andres, H. Baldwin, Long-term longitudinal measurements of plasma dehydroepiandrosterone sulfate in normal men, J. Clin. Endocrinol. Metab. 75 (1992) 1002–1004. Y. Oyama, A. Hayashi, T. Ueha, K. Maekawa K, Characterization of 29,79-dichlorofluorescin fluorescence in dissociated mammalian brain neurons: estimation on intracellular content of hydrogen peroxide, Brain Res. 635 (1994) 113–117. W.N. Rom, T. Harkin, Dehydroepiandrosterone inhibits the spontaneous release of superoxide radical by alveolar macrophages in vitro in asbestosis, Environ. Res. 55 (1991) 145–156. S. Sankarapandi, J.L. Zweier, G. Mukherjee, M.T. Quinn, D.L. Huso, Measurement and characterization of superoxide generation in

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

275

microglial cells: evidence for an NADPH oxidase-dependent pathway, Arch. Biochem. Biophys. 353 (1998) 312–321. M.L. Schlegel, J.F. Spetz, P. Robel, M. Haug, Studies on the effects of dehydroepiandrosterone and its metabolites on attack by castrated mice on lactating intruders, Physiol. Behav. 34 (1985) 867–870. D.I. Spratt, C. Longcope, P.M. Cox, S.T. Bigos, C. Wilbur-Welling, Differential changes in serum concentrations of androgens and estrogens (in relation with cortisol) in postmenopausal women with acute illness, J. Clin. Endocrinol. Metab. 76 (1993) 1542–1547. T. Takeshima, J.M. Johnston, J.W. Commissiong, Mesencephalic type 1 astrocytes rescue dopaminergic neurons from death induced by serum deprivation, J. Neurosci. 14 (1994) 4769–4779. W.N. Tian, L.D. Braunstein, J. Pang, K.M. Stuhlmeier, Q.C. Xi, X. Tian, R.C. Stanton, Importance of glucose-6-phosphate dehydrogenase activity for cell growth, J. Biol. Chem. 273 (1998) 10609– 10617. W.N. Tian, L.D. Braunstein, K. Apse, J. Pang, M. Rose, X. Tian, R.C. Stanton, Importance of glucose-6-phosphate dehydrogenase activity in cell death, Am. J. Physiol. 276 (1999) C1121–C1131. S. Toulmond, P. Parnet, A.C. Linthorst, When cytokines get on your nerves: cytokine networks and CNS pathologies, Trends Neurosci. 19 (1996) 409–410. T.J. Toung, R.J. Traystman, P.D. Hurn, Estrogen-mediated neuroprotection after experimental stroke in male rats, Stroke 29 (1998) 1666–1670. J.M. Whitcomb, A.G. Schwartz, Dehydroepiandrosterone and 16 alpha-Br-epiandrosterone inhibit 12-O-tetradecanoylphorbol-13-acetate stimulation of superoxide radical production by human polymorphonuclear leukocytes, Carcinogenesis 6 (1985) 333–335. N.C. Yang, K.C. Jeng, W.M. Ho, S.J. Chou, M.L. Hu, DHEA inhibits cell growth and induces apoptosis in BV-2 cells and the effects are inversely associated with glucose concentration in the medium, J. Steroid. Biochem. Mol. Biol. 75 (2000) 159–166. I.H. Zwain, S.S. Yen, Dehydroepiandrosterone: biosynthesis and metabolism in the brain, Endocrinology 140 (1999) 880–887.