Mercuric chloride, but not methylmercury, inhibits glutamine synthetase activity in primary cultures of cortical astrocytes

Brain Research 891 (2001) 148–157 www.elsevier.com / locate / bres

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Mercuric chloride, but not methylmercury, inhibits glutamine synthetase activity in primary cultures of cortical astrocytes Jeffrey W. Allen, Lysette A. Mutkus, Michael Aschner* Department of Physiology and Pharmacology, Interdisciplinary Program in Neuroscience, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 -1083, USA Accepted 31 October 2000

Abstract Methylmercury (MeHg) is highly neurotoxic with an apparent dose-related latency period between time of exposure and the appearance of symptoms. Astrocytes are known targets for MeHg toxicity and a site of mercury localization within the central nervous system (CNS). Glutamine synthetase (GS) is an enzyme localized predominately within astrocytes. GS converts two potentially toxic molecules, glutamate and ammonia, to the relatively non-toxic amino acid, glutamine. During prolonged exposure to MeHg, inorganic mercury (I-Hg) accumulates within the brain, suggesting in situ demethylation of MeHg to I-Hg. To determine if speciation of mercurials would differentially alter GS activity and expression, neonatal rat primary astrocyte cultures were exposed to MeHg or mercuric chloride (HgCl 2 ) for 1 or 6 h. MeHg produced no changes in GS activity, protein, or mRNA at any time or dose tested. In contrast, HgCl 2 produced a dose dependent decrease in astrocytic GS activity at both 1 and 6 h. There were no changes in GS protein or mRNA levels following HgCl 2 exposure. Additional studies were carried out to determine GS activity in cell lysates incubated with HgCl 2 or MeHg. In cell lysates, HgCl 2 was three-times more potent than MeHg in inhibiting GS activity. The inhibition of GS activity in cell lysates by HgCl 2 was reversed by the addition of dithiothreitol (DTT), while DTT did not restore GS activity following MeHg. These data suggest that astrocytic GS activity is not inhibited by physiologically relevant concentrations of MeHg, but is inhibited by I-Hg, which is present in CNS following chronic MeHg exposure.  2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the Nervous System Topic: Neurotoxicology Keywords: Glutamine synthetase; Mercury; Methylmercury; Astrocytes

1. Introduction Methylmercury (MeHg) is a highly neurotoxic compound producing neuronal death that is partially mediated by glutamate [17,36,80] and over production of reactive oxygen species (ROS) [5,42,59,60,70,81]. Although MeHg produces neuronal death [15,32,75] and alters neuronal function [6,11,18,66,67,82], mercurials localize predominantly within astrocytes [22,32,44,72]. MeHg produces astrocytic swelling both in cultured astrocytes [8] and in vivo [21,23,26,37], stimulates the efflux of excitatory amino acids (EAA) from astrocytes, and inhibits the uptake of EAA [8,10,25,51], suggesting MeHg-induced *Corresponding author. Tel.: 11-336-716-8530; fax: 11-336-7168501. E-mail address: [email protected] (M. Aschner).

neuronal toxicity may be mediated by changes in astrocytic functions [9]. Astrocytes are vital in maintaining homeostasis of the central nervous system (CNS). Up to 80% of the potentially neurotoxic amino acid, glutamate, is removed by two astrocytic Na 1 -dependent excitatory amino acid transporters, EAAT1 and EAAT2 [57]. Once localized in astrocytes, glutamate is combined with ammonia to form glutamine by the enzyme glutamine synthetase (GS, glutamate-ammonia ligase, E.C. 6.3.1.2) [48]. Within the CNS, GS is localized almost exclusively in astrocytes [47]; however, oligodendrocytes possess some GS activity [19,73]. Glutamine produced by astrocytes via the action of GS is released from astrocytes and taken up by neighboring glutamatergic or GABAergic neurons as precursors for neurotransmitter synthesis as part of the glutamate–glutamine cycle [14,61,62,69]. In addition to

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03185-1

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providing glutamine to neurons, other glutamate derived metabolites, such as the tricarboxcylic acid (TCA) cycle precursors lactate, malate and citrate are utilized by neurons for energy production [46,63,68]. Thus, inhibition of GS activity may result in decreased neuronal energy levels [24], decreased synthesis of glutamate and GABA [56,61,62], and the inability to detoxify ammonia within the CNS [3,14,33]. Decreases in GS activity might be mediated by multiple mechanisms. The GS macromolecule is susceptible to oxidative modification resulting in decreased synthetic activity in vitro [43,45,52] and in vivo [54]. In addition to direct inactivation by ROS, the GS protein is ‘marked’ following oxidative modification for rapid degradation by intracellular proteases leading to decreases in both activity and protein levels [55,71]. GS activity is also inhibited by the neurotoxic heavy metal lead [27,64,65,74]. Lead inhibits GS activity in cellular lysates [27], cultured astrocytes [65,74], and in vivo [64]. In cellular lysates, the inhibitory actions of lead on GS activity are reversed by the addition of dithiothreitol (DTT) or ethylenediaminetetraacetic acid (EDTA), suggesting direct interactions of metals with protein thiols [27]. Thus, GS activity is inhibited by multiple mechanisms, including direct protein interactions with ROS and thiol reactive metals, and increased degradation following oxidative ‘marking’. MeHg exposure in vivo and in cultured cells results in overproduction of ROS [5,36,42,59,60,81], which may cause oxidatively-mediated inactivation and degradation of the GS protein. In addition, MeHg has an extremely high affinity for protein thiols [20,35,78] and therefore may inactivate GS via direct interactions with protein thiols. MeHg readily crosses the blood–brain barrier (BBB) [7]. However, following MeHg exposure in vivo, there is a characteristic latency period between initial exposure and the onset of clinical symptoms [12]. Following chronic exposure to MeHg, there is a time-dependent increase in I-Hg levels within the CNS. Given that I-Hg is poorly transported across the BBB, it has been suggested that the in situ demethylation of MeHg to I-Hg accounts for the increased levels of I-Hg [22,26,30,50,75]. Following chronic exposure to MeHg in humans [26] and non-human primates [75], I-Hg accounts for greater than 80% of mercury within the CNS. Given that I-Hg is generally more potent than MeHg in disrupting cellular functions [1,6,16], the actions of I-Hg must be considered as a potential toxic species following chronic MeHg exposure in the CNS. In the present study, we assessed GS activity, mRNA, and protein levels in primary cultures of astrocytes following 1-h or 6-h exposure to MeHg or HgCl 2 . In addition, the ability of MeHg or HgCl 2 to inhibit GS activity when added directly to the cellular lysates of control cells (in vitro) was also assessed. GS activity is inhibited both by direct inactivation of the GS macromolecule, and also by protein degradation, thus concurrent evaluation of GS

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activity and protein levels allowed determination of potential mechanisms of mercurial-induced GS inhibition.

2. Methods

2.1. Cell culture Primary cultures of neonatal rat cortical astrocytes were prepared as described previously [4,29]. In brief, the cerebral cortices were removed from 1-day-old Sprague– Dawley rat pups and the meninges were carefully dissected away from the tissue. The tissue was digested with a bacterial neutral protease (dispase) and astrocytes are recovered by repeated removal of dissociated cells from non-dissociated sedimenting tissue. Astrocytes were maintained in Minimal Essential Media with Earl’s salts containing 10% heat-inactivated horse serum, 100 U / ml penicillin, 100 mg / ml streptomycin, and 0.25 mg / ml Fungizone  (all from Gibco Life Technologies, Gaithersburg, MD, USA). Cells were grown in 100 mm culture dishes for 3–5 weeks in 95% air–5% CO 2 , 95% relative humidity atmosphere until confluent monolayers were formed. Cultures are routinely .95% positive for the astrocytic marker glial fibrillary acidic protein (GFAP), attesting to the purity of the cultures.

2.2. Exposure of astrocyte cultures to HgCl2 or MeHg Astrocytes were washed 23 with 10 ml of buffer consisting of 122 mM NaCl, 3.3 mM KCl, 0.4 mM MgSO 4 , 1.3 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 10 mM glucose, and 25 mM N-2-hydroxy-ethylpiperazine-N9-2ethansolfonic acid (HEPES) adjusted to pH 7.4 with 10 M NaOH. Cells were exposed to HgCl 2 (100 nM, 1 mM, or 5 mM) or MeHg (100 nM, 1 mM, or 10 mM) in HEPES buffer for 1 or 6 h at 378C. The highest concentrations used for HgCl 2 and MeHg were different due to unsatisfactory levels of toxicity produced by 10 mM HgCl 2 after 6 h of exposure.

2.3. Glutamine synthetase assay Following exposure to HgCl 2 or MeHg, GS activity was assessed in astrocytes using the g-glutamyl transferase assay [58]. Astrocytes were washed 33 with 48C phosphate buffered saline (PBS) and lysed with 750 ml of imidazole lysis buffer (1 mM imidazole, 0.1% TritonX100, pH 6.5) for 60 min at 48C. Cells were transferred to a 1.5-ml microcentrifuge tubes and sonicated twice for 30 s. Cell lysates were centrifuged for 10 min at 10,0003g to remove non-soluble cellular debris, and the protein content of the resultant supernatant was determined with the bicinchoninic acid method (Pierce Chemical, Rockford, IL, USA). Protein (150 mg) was added to a fresh microcentrifuge tube on ice and imidazole lysis buffer was

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added to a final volume of 400 ml. A 23 reaction buffer was prepared fresh daily to provide a final assay concentration of 120 mM L-glutamine, 10 mM sodium arsenate, 50 mM imidazole, 30 mM hydroxylamine, pH 6.5. Immediately prior to use, MnCl 2 (30 mM final concentration) and ATP (200 mM final concentration) were added to the 23 reaction buffer. A 400-ml aliquot of the 23 reaction buffer was added to each sample of cellular lysate on ice. Tubes were gently vortexed and incubated at 378C for 60 min. Standards of L-glutamic acid g-monohydroxamate (g-glutamyl hydroxamate) were prepared in imidazole lysis buffer and incubated in parallel with the experimental samples. Following incubation, 200 ml of FeCl 3 buffer [FeCl 3 ?7H 2 O (15%, w / v), trichloroacetic acid (25%, v / v), hydrochloric acid (2.5 N)] was added and proteins were allowed to precipitate for 60 min at 48C, after which time samples were centrifuged at 10,0003g for 10 min. The absorbance of the supernatant was measured at 490 nm with a Molecular Devices Vmax kinetic microplate reader (Sunnyvale, CA, USA). The direct effects of mercurials on GS activity were assessed in cellular lysates obtained from control cells. Aliquots of 5–50 mM MeHg or HgCl 2 were added to the cellular lysates immediately prior to initiation of the GS assay. To assess the reversibility of the in vitro inhibition of GS activity by mercurials, lysates were co-incubated with DTT (1 mM) and HgCl 2 (20 mM) or MeHg (50 mM). DTT (1 mM) had no effect on GS activity in control cellular lysates (data not shown).

2.4. GS western blotting An aliquot of the cellular lysates used for GS assays was also used for western blotting to determine GS protein levels. Proteins (100 mg) were concentrated from the imidazole lysis buffer by organic extraction [77]. Sample volumes were brought up to 400 ml with water and an equal volume of methanol (400 ml) was added, followed by 100 ml of chloroform. Samples were vortexed for 20 s and centrifuged at 14,0003g for 3 min. The upper layer was removed and discarded. An additional 300 ml of methanol was added to the samples, and they were again vortexed and centrifuged. The supernatant was removed and the pellet was allowed to dry. Each pellet was dissolved in 25 ml of 2% sodium dodecyl sulfate (SDS) heated to 658C. A 5-ml sample of 53 loading buffer (50% glycerol; 10% SDS; 0.25 M Tris, pH 6.8) and DTT (final concentration 100 mM) were added to extracted proteins, and samples were boiled for 10 min. Bromophenol blue [1 ml of a 50% (w / v) solution] was added and proteins were separated by denaturing SDS–PAGE using 5% stacking and 12% resolving acrylamide gels in 0.1% SDS, 25 mM Tris, 192 mM glycine buffer. Following fractionation, proteins were electrophoretically transferred to a nitrocellulose membrane (Protran, BA83, Schleicher and

Schuell, Keene, NH, USA) in 20% methanol, 0.1% SDS, 25 mM Tris, and 192 mM glycine for 3 h at 60 V. Membranes were blocked with 5% low-fat powered milk in Tris-buffered saline with Tween (TBST, 0.1% Tween 20; 150 mM NaCl; 20 mM Tris) containing 0.1% gelatin (type B, from bovine skin, Sigma, St. Louis, MO, USA). GS proteins were detected with a monoclonal antibody (Chemicon, Temecula, CA, USA) diluted 1:2000 followed by incubation with a horse radish peroxidase conjugated secondary antibody diluted to 1:2000 (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA), both in TBST and 5% milk for 1 h. Protein bands were visualized with the Renaissance enhanced chemiluminescence system (New England Nuclear, Boston, MA, USA) followed by exposure to X-ray film (BioMax MR, Eastman Kodak, Rochester, NY, USA). Films were digitized and band density was determined using the Tina v2.09e computer program (Raytest USA, Inc., Wilmington, NC, USA).

2.5. GS northern analysis Following a 6-h exposure to MeHg or HgCl 2 , the total RNA was isolated from astrocyte cultures with a monophase phenol and guanidine isothiocyanate solution (RNAStat-60, Tel-Test Laboratories, Friendswood, TX, USA). RNA (5 mg) was separated by denaturing agarose electrophoresis and transferred to nitrocellulose (Nytran SuPerCharge, Schleicher and Schuell, Keene, NH, USA) by capillary action in 103 SSC overnight. Membranes were cross-linked with an ultraviolet cross-linker and prehybridized at 508C for at least 1 h in 50% de-ionized formamide, 53 Denhardt’s solution, 10% dextran sulfate, 0.1% SDS, 43 SSC, 100 mg / ml denatured salmon sperm DNA, 20 mM Tris, pH 8.0. An 800 base pair sequence of rat GS cDNA (a generous gift from Dr. Steve Abcouwer, Harvard Medical School, Boston, MA, USA) was used as a template to create a a- 32 P-dCTP labeled probe by random priming (RadPrime DNA Labeling System, Life Technologies, Gaithersburg, MD, USA). Unincorporated nucleotides were removed by Spehadex-50 column chromatography (Nick Spin Columns, Amersham, Boston, MA, USA) and 1310 6 CPM / ml hybridization buffer was added and allowed to hybridize overnight. Blots were washed and exposed to X-ray film (BioMax MR, Eastman Kodak, Rochester, NY, USA). To correct for total RNA levels, blots were stripped in boiling 0.13 SSC–0.1% SDS–40 mM Tris buffer and reprobed for ribosomal 28S as described by Barbu and Dautry [13] using the conditions listed above. Films were digitized and band density was determined using the Tina v2.09e computer program (Raytest USA, Inc., Wilmington, NC, USA).

2.6. Statistical analysis All experiments were conducted in 3–6 individual

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astrocytes preparations. To determine statistical significant one-way analysis of variance (ANOVA) was used, and when overall significance resulted in rejection of the null hypothesis (P,0.05), the source of the variance was determined with the Student-Newman-Keuls post-test. All analyses were performed using GraphPad InStat version 3.02 for Windows, (GraphPad Software, San Diego, CA, USA).

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mM for 1 h, also produced no changes in protein levels as seen in Fig. 3. Despite significant changes in GS activity following a 6-h exposure to 1 and 5 mM HgCl 2 (Fig. 2), astrocytic GS protein levels were unchanged, as seen in Fig. 4. There were no significant changes in GS protein levels following exposure of up to 10 mM MeHg for 6 h.

3.3. GS mRNA levels 3. Results

3.1. GS activity following MeHg or HgCl2 treatment As shown in Fig. 1, 1-h exposure of astrocytes to 5 mM HgCl 2 decreased GS activity to 57.568.2% of control (mean6S.E.M., P,0.001). Treatment of astrocytes with MeHg for 1 h produced a decrease in GS activity at a concentration of 10 mM that approached, but did not reach, statistical significance (P50.07). Following 6 h of exposure (Fig. 2), astrocytic GS activity was decreased to 74.368.0% of control by 1 mM HgCl 2 (P,0.05) and 59.165.1% of control by 5 mM HgCl 2 (P,0.01). Astrocytic GS activity was not statistically different from controls following 6 h of treatment with up to 10 mM MeHg (P50.065).

3.2. GS protein levels As assessed by western blotting, 1 h exposure of astrocytes to concentrations of HgCl 2 up to 5 mM produced no changes in GS protein levels. MeHg, up to 10

Treatment of astrocytes with concentrations of up to 5 mM HgCl 2 or 10 mM MeHg for 6 h did not alter steadystate levels of GS mRNA as assessed by northern blotting (Fig. 5).

3.4. In vitro GS activity As shown in Fig. 6, addition of HgCl 2 to astrocytic cellular lysates inhibited GS activity in a dose-dependent manner. GS activity was significantly inhibited as compared to controls by 10 mM (7263%, P,0.001), 20 mM (6.362.3%, P,0.001), and 50 mM (161%, P,0.001) of HgCl 2 . MeHg also inhibited GS in a dose-dependent fashion, but its effect was not as pronounced and significant inhibition of GS activity occurred only at 50 mM (32619%, P,0.001). Co-incubation of 20 mM HgCl 2 with 1 mM DTT completely reversed inhibition of GS activity induced by HgCl 2 (9262.9% of control) as seen in Fig. 7. In contrast, the inhibition of GS activity induced by 50 mM MeHg was not reversed by co-incubation with 1 mM DTT (34616%). Incubation of control cell lysates with 1 mM DTT alone did not alter GS activity (data not shown).

Fig. 1. GS activity in astrocytes following exposure to HgCl 2 or MeHg for 1 h. GS activity was calculated as mmol of g-glutamylhydroxamate formed / h / 150 mg protein then expressed as percent control. GS activity in astrocytes exposed to 5 mM HgCl 2 for 1 h was significantly decreased as compared to control astrocytes (*P,0.001, N55–7). Exposure to concentrations of up to 10 mM MeHg produced no significant effects on GS activity (P50.07, N56).

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Fig. 2. GS activity in astrocytes following exposure to HgCl 2 or MeHg for 6 h. GS activity was expressed as mmol of g-glutamylhydroxamate formed / h / 150 mg protein then expressed as percent control. GS activity in astrocytes expososed to 1 mM HgCl 2 or 5 mM HgCl 2 for 6 h significantly decreased GS activity as compared to control astrocytes (*P,0.05, N56–9). Exposure to concentrations of up to 10 mM MeHg produced no significant effects on GS activity (P50.065, N54).

4. Discussion Rats intracerebrally injected with MeHg or HgCl 2 show similar structural alterations [31] suggesting both species are capable of producing neurotoxicity. The ability of MeHg or HgCl 2 to alter GS activity or expression has not been previously investigated in primary cultures of astrocytes, the main site of GS activity [47] and mercurial localization [15,21,23] in the brain. The present study demonstrated that exposure to HgCl 2 for 1 and 6 h decreased GS activity in cultured astrocytes (Figs. 1 and 2). Immunoreactive GS protein levels were unchanged (Figs. 3 and 4) suggesting HgCl 2 mediated inhibition of

GS activity is due to direct interactions with the GS macromolecule rather than increased protein degradation. Inhibition of GS activity by HgCl 2 produced no changes in GS mRNA levels (Fig. 5). The lack of changes in mRNA expression despite changes in GS activity, but not GS protein levels, are in agreement with studies in lung tissue suggesting GS mRNA expression is regulated by protein stability [41]. Previous studies have shown that HgCl 2 inhibits GS activity when added directly to cellular lysates of astrocytes [27], or in aggregate brain cultures exposed to 1 mM HgCl 2 for 10 days [49]. GS protein levels were not analyzed in these aggregate cultures preventing inves-

Fig. 3. Western blotting of astrocytic GS protein following exposure to HgCl 2 or MeHg for 1 h. Immunoreactive GS protein levels were not significantly altered following 1 h of exposure to either HgCl 2 or MeHg (N53–4).

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Fig. 4. Western blotting of astrocytic GS protein following exposure to HgCl 2 or MeHg for 6 h. Immunoreactive GS protein levels were not significantly altered following 6 h of exposure to either HgCl 2 or MeHg (N53–4).

tigation of possible inhibitory mechanisms of HgCl 2 . In the present study, in vitro HgCl 2 inhibited GS activity in astrocyte cellular lysates as demonstrated previously [27]. The inhibition of GS by HgCl 2 was reversed by addition of DTT, replicating the effect seen with lead [27], suggesting direct interactions between these neurotoxic metals and the GS macromolecules [27,74]. In contrast to the inhibitory effects of HgCl 2 on GS activity, MeHg produced no significant changes in GS activity in cultured astrocytes following 1 or 6 h of exposure, although GS activity following exposure to 10 mM MeHg approached statistical significance (Figs. 1 and 2). As seen with HgCl 2 , there were no changes in either GS protein levels (Figs. 3 and 4) or mRNA levels (Fig. 5).

Exposure of aggregate brain cell to 1 mM MeHg for 10 days produced a 40% decrease in GS activity [49] suggesting prolonged exposure to MeHg may be necessary to alter GS activity. No demethylation of MeHg to I-Hg was seen in the aggregate cultures; therefore, the inhibitory effects on GS activity were not due to I-Hg [49]. In vitro studies, which mercurials were added directly to cellular lysates of astrocytes, MeHg was less potent than HgCl 2 in decreasing GS activity with inhibition occurring only at 50 mM (Fig. 6). This concentration of MeHg produces acute toxicity in cultured astrocytes (data not shown) and is approximately two-fold higher than mercury concentrations found in human brains of Minamata Bay victims, in which total mercury were estimated to range

Fig. 5. GS mRNA levels in cultured neonatal rat astrocytes following exposure to HgCl 2 or MeHg for 6 h. Steady-state GS mRNA levels were determined by northern blotting. Relative levels of GS expression were corrected for total RNA levels in each lane as determined by ribosomal 28S levels. Exposure to HgCl 2 or MeHg produced no alterations in GS mRNA levels (N53–4).

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Fig. 6. In vitro inhibition of astrocytic GS activity by mercurials. HgCl 2 (5–50 mM) or MeHg (5–50 mM) was added to cellular lysates of control astrocytes immediately prior to addition of the GS activity assay buffer. Addition of 10–50 mM HgCl 2 in vitro significantly decreased GS activity (*P,0.001, N54–7) with concentrations of 20 mM and above decreasing activity to ,7% of control levels. In vitro MeHg also significantly decreased GS activity as compared to control levels, but only at 50 mM (*P,0.001, N54–7).

from 2 to 26 mM [30]. Therefore, the 50 mM concentration that was used in the in vitro cell lysate studies probably has little physiological relevance. The concentrations of mercurials used for DTT reversibility studies were chosen because they were the lowest concentrations tested to produce at least 50% inhibition of GS activity. The approximate IC 50 for HgCl 2 on GS activity on cellular lysates was 12 mM, while the IC 50 for MeHg was approximately 38 mM. Unlike the reversibility seen with HgCl 2 , 1 mM DTT was unable to restore GS activity suggesting possibly permanent modification of the GS molecule (Fig. 7). In rat

brain homogenates, in vitro exposure to 100 mM MeHg produced no inhibition of GS activity [39]. In rats exposed to doses of MeHg that caused a lack of weight gain compared to controls, but no clinical signs of MeHg toxicity (3.36 mg / kg, p.o. for 14 days), no changes in brain GS activity were reported. In rats receiving intoxicating doses of MeHg producing weight loss and partial hind-limb paralysis (7.05 mg / kg, p.o. for 7 days), GS activity was increased in the cerebellum suggesting astrocyte proliferation [40]. The lack of convergence of the effects of MeHg on GS activity studied in vitro, in cultured cells, and in vivo suggests that further studies are necessary to elucidate the interaction between MeHg and GS The identification of the toxic species in MeHg poisonings has been questioned given the latency period between exposure and development of toxicity [12,15,53]. In cultured cells, HgCl 2 is generally more toxic than MeHg [1,16] suggesting in situ demethylation to the more toxic HgCl 2 species may in part underlie the delayed toxicity of MeHg. Studies in non-human primates have shown timedependent increases in the percentages of brain I-Hg ranging from 10 to nearly 80% of total mercury [22,75]. Vahter et al. [75] treated primates with MeHg for 6, 12, or 18 months by mouth or with HgCl 2 by intravenous infusion for 3 months at sub clinical doses (50 mg / kg / day MeHg or 200 mg / kg / day HgCl 2 ). These studies found MeHg levels in brain reached a steady state at approximately 12 months but I-Hg levels increased during the entire 18 months. An additional group of primates was exposed to MeHg for 12 months and was allowed a 6-month washout period. In this group, MeHg levels dramatically decreased with a half-time of around 36 days, but I-Hg levels remained essentially unchanged with an estimated half-time of years,

Fig. 7. DTT reverses HgCl 2 -mediated, but not MeHg-mediated inhibition of astrocytic GS activity in vitro. The in vitro inhibition of GS activity by 20 mM HgCl 2 (*P,0.001, N53) was completely restored to control levels by 60 min co-incubation of 1 mM DTT. In contrast, in vitro inhibition of GS activity by MeHg (50 mM) was not reversed by co-incubation with 1 mM DTT (*P,0.001, N53).

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which is similar to the half-time in humans exposed to Hg 0 vapor [38]. Because of the long half-time, at the end of the 6 month washout period 82% of the mercurials were in the inorganic form. In monkeys exposed to HgCl 2 , brain levels of I-Hg were only 15–35% of those reached in MeHg treated monkeys, despite intravenous administration of an eight-fold larger dose of HgCl 2 suggesting very little transfer of I-Hg across the blood–brain barrier. These data, and others, suggest accumulation of I-Hg with the CNS is due to in situ demethylation and not transport of peripherally derived I-Hg across the BBB [30,50,75]. Although no studies have directly examined the consequences of mercurial-induced inhibition of GS activity, inhibition of GS with methionine-L-sulfoxamine (MSO) [76] in rats [56] demonstrated that tissue glutamate levels were unchanged; however, Ca 12 -dependent, potassiumstimulated neuronal glutamate release from striatal tissue was decreased by 50%. Total glutamine levels and GS activity were also decreased by 50%. The lack of effect on total glutamate levels, with a decrease in neuronal glutamate release, suggests a shift in glutamate levels from neurons to either the extracellular space or astrocytes. Following MSO treatment, levels of glutamate in cultured astrocytes are increased approximately four-fold over that of control cells [28] suggesting the localization of glutamate within astrocytes [34,79]. Following HgCl 2 treatment, astrocytes have reduced intracellular glutamine levels and reduced glutamine efflux [17] suggesting decreased GS activity. In addition to the direct inhibition of GS activity demonstrated in this study and by others [17,49], MeHg and HgCl 2 have dramatic effects of glutamate homeostasis in the CNS by inhibiting the uptake of EAA in astrocytes and promoting astrocytic EAA efflux [2,8,10,51]. The mercurial induced inhibition of glutamate transport may limit the function of GS beyond that induced by direct interaction with the GS protein due to decreased intracellular astrocytic glutamate. Approximately 70% of the glutamate used by GS to synthesize glutamine and detoxify ammonia is derived from extracellular glutamate [28], thus mercurials will decrease glutamine levels [17]. This suggests that following mercurial exposure, there may be decreases in neuronal glutamate and GABA levels due to decreases in their precursor glutamine similar to that seen following MSO treatment [56]. This decrease may be due to inhibition of GS activity via direct protein interactions by mercurials, either by mercurial-mediated inhibition of EAA transport, or a combination of both processes. Mercurial-induced alterations in astrocytic function, such as inhibition of excitatory amino transport, have been previously demonstrated to occur within 60 min [2,10,16,25] and thus acute exposure of astrocyte to MeHg or HgCl 2 represents a valid method to assess mercurial toxicity. The concentrations of mercurials used in the present study are similar to those used in prior studies examining GS activity in cultured astrocytes following

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exposure to lead [65] and in mixed aggregating cultures [40]. In summary, for the first time we have demonstrated HgCl 2 is more potent than MeHg in inhibiting GS activity in cultured astrocytes. This inhibition is likely due to direct protein interactions, because the in vitro inhibitory effects of HgCl 2 on GS activity was fully reversed by addition of DTT. Given the complex interactions of mercurials with multiple sites in the glutamate–glutamine cycle, additional metabolic studies on GS and other enzymes are warranted, as they are likely to shed new light on the mechanisms of mercurial neurotoxicity.

Acknowledgements This study was supported by Public Health Service grant ES07331 to M.A., and J.W.A. was supported by a NIAAA pre-doctoral trainee fellowship (T32 AA 07565).

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