Effects of arsenite on glutamate metabolism in primary cultured astrocytes

The aim of this study was to explore the mechanisms that contribute to neurotoxicity induced by arsenite exposure focusing on the alteration of glutamate metabolism in primary cultured astrocytes. The cells were exposed to 0-30μM arsenite for 24h, and then cell viability, intracellular nonprotein sulfhydryl (NPSH) levels, mitochondrial membrane potential, activity of Na(+)/K(+)-ATPase, glutamine synthetase (GS) and glutamate transporter (GLAST and GLT-1), and protein expression of GS, GLAST and GLT-1 were examined. Compared with those in control, exposure to arsenite resulted in damages of astrocytes in a concentration dependent manner, which were shown by cell viabilities, and supported by morphological observation, mitochondrial membrane potential and intracellular NPSH levels. On the other hand, activities and protein expression of GS, GLAST and GLT-1 were significantly inhibited by arsenite exposure. Moreover, protein expression of GLAST and activities of GS were much more sensitive to arsenite. However, activities of Na(+)/K(+)-ATPase were not influenced obviously by arsenite exposure. In conclusion, findings from this study indicated that exposure to arsenite could inhibit glutamate metabolism in astrocytes, which might be related to arsenic-induced neurotoxicity.     

Microcystin-LR and nodularin induce intracellular glutathione alteration, reactive oxygen species production and lipid peroxidation in primary cultured rat hepatocytes

Microcystin-LR (MCYST-LR) and nodularin (NOD) produced by cyanobacteria are potent specific hepatotoxins. However, the mechanisms of their hepatotoxicity have not been fully elucidated. In the present study the effect of non cytotoxic low concentrations of MCYST-LR and NOD on intracellular reduced glutathione (GSH) alteration, reactive oxygen species (ROS) production and lipid peroxidation was investigated in primary cultured rat hepatocytes. Cell viability was determined by the methylthiazoltetrazolium (MTT) dye assay, reduced GSH was evaluated by enzymatic methods, ROS were evaluated by the dichlorofluorescein diacetate (H2DCF-DA) fluorescent probe and lipid peroxidation by dosing malondialdehyde (MDA) by the thiobarbituric acid method. The 24 h LC50 values of MCYST-LR and NOD were 48 and 62 ng/ml, respectively. Exposure of freshly isolated rat hepatocytes to MCYST-LR or NOD at non cytotoxic low concentrations (2, 10 ng/ml) for 3, 24 and 48 h periods resulted in a significant rise of GSH levels and production of ROS. NOD significantly induced in a time- and concentration-dependent lipid peroxidation. However, MCYST-LR treatment did result in a significant decrease in MDA levels compared with controls. Although MCYST-LR and NOD are closely related in terms of structure and inhibition of protein phosphatases, they induce differently the oxidative stress at non cytotoxic low concentrations. Therefore, the results indicate that oxidative stress mediated by reactive intermediates may be a mechanism by which these cyanotoxins induce their hepatotoxic effect.     

Lanthanum chloride promotes mitochondrial apoptotic pathway in primary cultured rat astrocytes

Population surveys and animal experiments have shown that rare earth elements (REEs) cause neurological defects. However, the detailed mechanisms underlying these effects are still unclear. Given that lanthanum is commonly used for investigating into REEs‐induced neurological defects, this study chose lanthanum chloride (LaCl₃) to show that LaCl₃ promotes mitochondrial apoptotic pathway in primary cultured rat astrocytes by regulating expression of Bcl‐2 family proteins. The main findings of this study are (1) LaCl₃ treatment (0.25, 0.5, and 1.0 mM for 12–48 h) induced the astrocytes damages with a concentration‐dependent manner, which were confirmed with methyl thiazolyl tetrazolium and lactate dehydrogenase release assays, and morphological examination. (2) A 24 h treatment of LaCl₃ concentration‐dependently decreased mitochondrial membrane potential, increased cytochrome c release from mitochondria into cytosol, elevated caspase 9 and 3 expression, and promoted astrocyte apoptosis. (3) LaCl₃ treatment increased the ratio of pro‐apoptotic Bax and antiapoptotic Bcl‐2 proteins, which in turn broke the balance among pro‐apoptotic and antiapoptotic Bcl‐2 family proteins, leading to astrocyte apoptosis. Our results indicate that LaCl₃ alters Bcl‐2 family protein expressions, which in turn promote mitochondrial apoptotic pathway, and thus astrocytic damage. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 489–497, 2013.     

Lead toxicity in primary cultured cerebral astrocytes and cerebellar granular neurons

Neurons are more sensitive than astrocytes to lead toxicity in vivo. In order to understand the bases for the differences in brain cell responses to lead, the effects of lead acetate on cell morphology and on aerobic energy metabolism were studied in rat primary cultured neurons and astrocytes. By transmission electron microscopy, neuronal cell damage was seen with exposure to lead concentrations which were much lower than those required for similar changes in the astrocyte. As previously described in our studies of in vivo lead exposure, astrocytes in primary culture concentrated lead in nuclear, cytoplasmic, and lysosomal inclusions while neurons showed lead densities only in lysosomes. With acute lead exposures, inhibition of maximal respiratory capacity was greater and occurred at lower lead concentrations in neurons than in astrocytes. Similarly, respiratory rates were inhibited at lower lead concentrations in cerebral cortical slices from 8-day-old rat pups compared to those from adults. We conclude that primary cultured brain cells are appropriate in vitro systems for studying the in vivo cellular responses to lead. As in vivo, neurons are more sensitive than astrocytes to lead toxicity. In both cells, inhibition of aerobic energy metabolism appears to be closely associated with cell damage. The capacity of the astrocyte to sequester lead in nonmitochondrial intracellular sites may be critical in resistance to lead toxicity in vitro and in the mature brain.     

Cytodifferentiation enhances Erk activation induced by endothelin-1 in primary cultured astrocytes

We have previously demonstrated that endothelin-1 (ET-1)-induced extracellular signal-regulated kinase (Erk) activity via the ETB receptor (EDNRB) is mediated through two independent pathways, a protein kinase C-dependent pathway and a pertussis toxin (PTX)-sensitive pathway, in astrocytes. In this study, we showed that the molar potency of ET-1 to induce Erk activation was two orders of magnitude higher in dibutyryl cAMP (DBcAMP)-treated astrocytes than in quiescent astrocytes. This DBcAMP-enhanced molar potency of ET-1 in Erk activation was selectively inhibited by pretreatment of astrocytes with PTX. The expression level of EDNRB in astrocytes was markedly upregulated by DBcAMP-induced cytodifferentiation. However, this up-regulation was simply attributed to the high expression of low-affinity sites. The molar potency of ET-1 to induce both stimulation of inositol trisphosphate production and activation of protein kinase C in DBcAMP-treated astrocytes was similar to that in quiescent astrocytes. On the contrary, the molar potency of ET-1 to induce accumulation of Ras-GTP was two orders of magnitude higher in DBcAMP-treated astrocytes than in quiescent astrocytes, which was consistent with the case of ET-1-induced Erk activation. Moreover, the ET-1-induced Ras activation was PTX sensitive. These results suggest that cytodifferentiation selectively enhances the PTXsensitive Ras/Erk pathway induced by ET-1 in astrocytes, and that cytodifferentiation-induced EDNRB up-regulation might not contribute to this selective potentiation of ET-1 signaling.     

Uptake and toxicity of copper oxide nanoparticles in cultured primary brain astrocytes

To test for consequences of an exposure of brain cells to copper oxide nanoparticles (CuO-NPs), we synthesised and characterised dimercaptosuccinate-coated CuO-NPs. These particles had a diameter of around 5 nm as determined by transmission electron microscopy, while their average hydrodynamic diameter in aqueous dispersion was 136 ± 4 nm. Dispersion in cell-culture medium containing 10% fetal calf serum increased the hydrodynamic diameter to 178 ± 12 nm and shifted the zeta potential of the particles from –49 ± 7 mV (in water) to –10 ± 3 mV. Exposure of cultured primary brain astrocytes to CuO-NPs increased the cellular copper levels and compromised the cell viability in a time-, concentration- and temperature-dependent manner. Application of CuO-NPs in concentrations above 100 µM copper (6.4 µg/ml) severely compromised the viability of the cells, as demonstrated by a lowered 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction capacity, a lowered cellular lactate dehydrogenase activity and an increased membrane permeability for the fluorescent dye propidium iodide. Copper internalisation as well as cell toxicity of astrocytes exposed to CuO-NPs were similar to that observed for cells that had been incubated with copper salts. The CuO-NP-induced toxicity was accompanied by an increase in the generation of reactive oxygen species (ROS) in the cells. Both, ROS formation and cell toxicity in CuO-NP-treated astrocytes, were lowered in the presence of the cell-permeable copper chelator tetrathiomolybdate. These data demonstrate that CuO-NPs are taken up by cultured astrocytes and suggest that excess of internalised CuO-NPs cause cell toxicity by accelerating the formation of ROS.     

Ethanol and acetaldehyde disturb TNF-alpha and IL-6 production in cultured astrocytes

Ethanol disturbs astroglial growth and differentiation and causes functional alterations. Furthermore, many signalling molecules produced by astrocytes contribute to these processes. The aim of the present study was to investigate the influence of ethanol and its primary metabolite, acetaldehyde, on TNF-alpha and IL-6 production in a rat cortical astrocyte primary culture. We are the first to report that both ethanol and acetaldehyde can modulate TNF-alpha and IL-6 secretion from cultured astrocytes. Long-term exposure (7 days) to ethanol and acetaldehyde was more toxic than an acute (24 hours) exposure. However, both compounds showed a biphasic, hormestic effect on the IL-6 secretion after the acute as well as the long-term exposure, and the maximum stimulation was reached for 50-mM ethanol and 1-mM acetaldehyde after 7-day exposure. In contrast, both compounds reduced the TNF-alpha secretion, where the effect was concentration-dependent. The catalase inhibitor 2-amino-1,2,4 triazole significantly reduced the ethanol toxicity in the cultured astrocytes after the acute as well as the long-term exposure. In conclusion, both ethanol and acetaldehyde affect the production of IL-6 and TNF-alpha in cultured astrocytes. The effect depends on the concentration of the compounds and the duration of the exposure. Acetaldehyde is a more potent toxin than ethanol, and ethanol's toxicity in the brain is at least partially due to its primary metabolite, acetaldehyde.     

6-Hydroxydopamine-induced aggression in cats: effects of various drugs

The effects of intracerebroventricular injections (ICV) in the unanesthetized cat of antimuscarinic drugs, ganglionic blocking agents, alpha and beta adrenergic blocking substances, dopamine and 5-hydroxytryptamine (5-HT) antagonists, and an antihistamine on aggressive behavior produced by 6-hydroxydopamine injected similarly was investigated. It was found that atropine, hyoscine, hexamethonium, mecamylamine, yohimbine, phenoxybenzamine, propranolol, practolol, chlorpromazine, haloperidol, antazoline and methysergide exerted virtually no effect on the pattern of aggressive responses evoked by ICV 6-hydroxydopamine. It is thus concluded that the aggressive behavior induced by 6-hydroxydopamine is not related to the release of acetylcholine, norepinephrine, dopamine, histamine or 5-hydroxytryptamine from endogenous storage sites in the brain.     

谷氨酸和γ-氨基丁酸对原代培养星形胶质细胞神经甾体合成释放的影响

目的研究氨基酸类神经递质对原代培养大鼠大脑皮质星形胶质细胞神经甾体合成释放的影响。方法采用原代培养的大鼠大脑皮质星形胶质细胞,分别加入不同浓度的谷氨酸和γ-氨基丁酸处理48h;采用固相萃取结合高效液相色谱质-谱联用分析方法提取分离和测定细胞培养液中游离型(脱氢表雄酮,DHEA;孕烯醇酮,PREG;别孕烯醇酮,AP)及结合型神经甾体(脱氢表雄酮硫酸酯,DHEAS;孕烯醇酮硫酸酯,PREGS)。结果与生理盐水对照组比较,谷氨酸处理使PREG和PREGS水平明显下降,DHEAS水平明显升高;γ-氨基丁酸处理使PREG水平明显降低,AP水平增加。结论谷氨酸和γ-氨基丁酸两种神经递质对原代培养的星形胶质细胞PREG合成释放均呈抑制作用;谷氨酸对DHE-AS、γ-氨基丁酸对AP的合成释放分别呈现明显促进作用;高剂量的谷氨酸还可以抑制PRGES的合成和释放。     

KATP channel openers facilitate glutamate uptake by GluTs in rat primary cultured astrocytes.

Increasing evidence, including from our laboratory, has revealed that opening of ATP sensitive potassium channels (KATP channels) plays the neuronal protective roles both in vivo and in vitro. Thus KATP channel openers (KCOs) have been proposed as potential neuroprotectants. Our previous studies demonstrated that KATP channels could regulate glutamate uptake activity in PC12 cells as well as in synaptosomes of rats. Since glutamate transporters (GluTs) of astrocytes play crucial roles in glutamate uptake and KATP channels are also expressed in astrocytes, the present study showed whether and how KATP channels regulated the function of GluTs in primary cultured astrocytes. The results showed that nonselective KCO pinacidil, selective mitochondrial KCO diazoxide, novel, and blood-brain barrier permeable KCO iptakalim could enhance glutamate uptake, except for the sarcolemmal KCO P1075. Moreover pinacidil, diazoxide, and iptakalim reversed the inhibition of glutamate uptake induced by 1-methyl-4-phenylpyridinium (MPP+). These potentiated effects were completely abolished by mitochondrial KATP blocker 5-hydroxydecanoate. Furthermore, either diazoxide or iptakalim could inhibit MPP+-induced elevation of reactive oxygen species (ROS) and phosphorylation of protein kinases C (PKC). These findings are the first to demonstrate that activation of KATP channel, especially mitochondrial KATP channel, improves the function of GluTs in astrocytes due to reducing ROS production and downregulating PKC phosphorylation. Therefore, the present study not only reveals a novel pharmacological profile of KCOs as regulators of GluTs, but also provides a new strategy for neuroprotection.     

Alteration in mitochondrial function and glutamate metabolism affected by 2-chloroethanol in primary cultured astrocytes

The aim of this study was to explore the mechanisms that contribute to 1,2-dichloroethane (1,2-DCE) induced brain edema by focusing on alteration of mitochondrial function and glutamate metabolism in primary cultured astrocytes induced by 2-chloroethanol (2-CE), a metabolite of 1,2-DCE in vivo. The cells were exposed to different levels of 2-CE in the media for 24 h. Mitochondrial function was evaluated by its membrane potential and intracellular contents of ATP, lactic acid and reactive oxygen species (ROS). Glutamate metabolism was indicated by expression of glutamine synthase (GS), glutamate–aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) at both protein and gene levels. Compared to the control group, exposure to 2-CE could cause a dose dependent damage in astrocytes, indicated by decreased cell viability and morphological changes, and supported by decreased levels of nonprotein sulfhydryl (NPSH) and inhibited activities of Na⁺/K⁺-ATPase and Ca^2 +-ATPase in the cells. The present study also revealed both mitochondrial function and glutamate metabolism in astrocytes were significantly disturbed by 2-CE. Of which, mitochondrial function was much vulnerable to the effects of 2-CE. In conclusion, our findings suggested that mitochondrial dysfunction and glutamate metabolism disorder could contribute to 2-CE-induced cytotoxicity in astrocytes, which might be related to 1,2-DCE-induced brain edema.     

心脑舒通胶囊及有效组分对原代培养大鼠星形胶质细胞的保护作用

<正>星形胶质细胞(astrocytes,AS)是中枢神经系统的重要组成成分,在神经保护方面发挥重要作用。在缺血/再灌注等病理损伤下,AS形态和功能会发生相应变化:一方面,通过分泌炎症因子等,加重脑损伤;另一方面,激活的AS会释放大量神经营养因子,如脑源性神经营养因子(brain derived neurotrophic factor,BDNF)、胶质细胞源性神经营养因子(glial cell-derived neurotrophic factor,GDNF)、神经生长因子(nerve growth factor,NGF)等,其大量表达能支持和营养受损神经元,从而发挥神经保护作用[1]。心脑舒通胶囊是由蒺藜地上全草部分经干燥提取后制成的胶囊剂,具有活血化瘀、     

The nitric oxide donor NOC12 protects cultured astrocytes against apoptosis via a cGMP-dependent mechanism

We examined the effect of 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC12), a nitric oxide (NO) donor, on apoptosis in cultured astrocytes. Reperfusion after hydrogen peroxide (H2O2) exposure caused a decrease in cell viability, loss of mitochondrial membrane potential, caspase-3 activation, DNA ladder formation, and nuclear condensation. NOC12 at 10-100 microM significantly attenuated these apoptotic changes, while the NO donor at 1 mM caused cell injury and exacerbated the H202-induced cell injury. NOC12 increased intracellular cGMP levels in a dose dependent manner with the maximal effect at 100 microM. The protective effect of NOC12 was mimicked by the NO-independent guanylate cyclase activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole, and was attenuated by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and the cGMP-dependent protein kinase inhibitor KT5823. ODQ and KT5823 did not block but rather exacerbated the cytotoxic effect of NOC12 at 1 mM. These findings demonstrate that lower concentrations of NOC12 inhibit the H2O2-induced apoptosis of astrocytes in a cGMP-dependent way, but higher concentrations of NOC12 show a toxic effect on astrocytes in a cGMP-independent way.     

钩藤碱对缺血再灌注诱导大鼠星形胶质细胞损伤的作用

目的:以原代培养的大鼠大脑星形胶质细胞进行缺血再灌注损伤处理,观察钩藤碱(Rsy)对星形胶质细胞损伤的保护作用.方法:1～3 d龄SD大鼠乳鼠大脑星形胶质细胞原代培养,含连二亚硫酸钠的无糖Earle's液进行缺血缺氧处理造模,MTT法测定细胞存活率,Hoechst 33258荧光显微镜观察细胞形态学变化,流式细胞仪检测细胞坏死、凋亡率,用LDH试剂盒测定LDH漏出率.结果:与模型组比较,钩藤碱0.02、0.2 mg·mL-1均能显著提高细胞存活率,显著降低细胞坏死率和细胞凋亡率,也能显著降低细胞LDH漏出率.结论:钩藤碱对缺血再灌注损伤后星形胶质细胞的坏死凋亡具有显著抑制作用,提示钩藤碱可能通过抑制星形胶质细胞凋亡和坏死而对脑缺血损伤产生保护作用.     

Benomyl affects the microtubule cytoskeleton and the glutathione level of mammalian primary cultured hepatocytes

Rat primary hepatocyte cultures have been used to study the effect of Benomyl alone or in combination with Pirimiphos-methyl. The results presented demonstrate that Benomyl alone is responsible for the microtubular disorganization in both a time- and dose-dependent manner, that the effect is reversible after the agent is removed, and that Benomyl is a potent glutathione-depleting agent. Pirimiphos-methyl, alone or combined with Benomyl had no effect on microtubule organization, but reinforced the decrease in glutathione.     

碱性成纤维细胞生长因子上调原代培养鼠星形胶质细胞中血管内皮生长因子的表达(英文)

目的:观察碱性成纤维细胞生长因子(bFGF)对鼠星形胶质细胞中血管内皮生长因子(VEGF)表达的影响。方法:用半定量聚合酶链反应(PCR)法和免疫组织化学法分别考察bFGF对VEGF信使RNA(mRNA)水平和蛋白质水平的影响。对不同剂量bFGF(1,10,100 ug·L~(-1))和不同孵育时间(3,6,12,24 h)的作用均进行了分析。 结果:bFGF可以剂量依赖性升高VEGF的mRNA水平。bFGF 10ug·L~(-1)在孵育3 h后即可上调VEGF mRNA水平,孵育24 h后细胞内VEGF mRNA水平显著高于对照组。免疫细胞化学分析结果表明bFGF也能升高细胞的VEGF蛋白质水平。结论:碱性成纤维细胞生长因子可以上调鼠星形胶质细胞中血管内皮生长因子的表达。     

Ebselen attenuates oxidative stress in ischemic astrocytes depleted of glutathione. Comparison with glutathione precursors

In this study, we investigated the protective effect of ebselen, a seleno-organic compound with antioxidant activity, towards astrocyte degeneration caused by exposure to simulated in vitro ischemic conditions and simultaneous depletion of glutathione (GSH). Depletion of GSH was induced by 24 h pretreatment with L-buthionine-(S,R)-sulfoximine (BSO). In this experimental paradigm, we examined the effects of ebselen (1-40 microM) on apoptosis, mitochondrial function, reactive oxygen species (ROS) production, intracellular GSH level and mitochondrial transmembrane potential (MTP). In addition, we also compared the antioxidant potential of ebselen with cystine and methionine as precursors of GSH synthesis as well as with GSH ethyl ester. Our study demonstrated that toxicity of simulated ischemia conditions was enhanced when intracellular GSH was depleted. Treatment with ebselen, especially at concentrations of 20 and 40 microM prevented ischemia-induced cytotoxicity. Our study has shown that antiapoptotic effect of ebselen is associated with its strong antioxidant properties, preservation of MTP and possibly conservation of mitochondrial GSH during cytoplasmatic GSH depletion caused by oxidative damage. Also, promoting GSH synthesis by the delivery of its substrates, like cystine or inhibition of the efflux by methionine may be a powerful strategy to minimize cell damage in the nervous tissue after ischemia.     

Efflux of 45calcium from cultured primary astrocytes: effects of glutamate receptor agonists and antagonists

The effects of high-K, glutamate and glutamate receptor agonists on the efflux of Ca2+ were studied in cultured primary astrocytes, prepared from the brains of newborn rats. An increase in efflux of 45Ca2+ of produced by a large extracellular concentration of K+ was effectively inhibited by verapamil, a blocker of voltage-gated Ca2+ channels, suggesting that these cells have functional voltage-sensitive Ca2+ channels. Glutamate and its agonists kainate, quisqualate and N-methyl-D-aspartate (NMDA) stimulated the efflux of preloaded 45Ca2+, in a dose-dependent manner. The most effective agonist was quisqualate followed by glutamate, whereas kainate and NMDA were less potent. In the Mg-free medium, the response to NMDA was significantly increased. The quisqualate receptor agonist (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) also stimulated the efflux, about equally effectively as quisqualate. The glutamate-, quisqualate- and kainate-induced efflux of 45Ca2+ was significantly inhibited by L-glutamatediethylester (GDEE) and that of NMDA by DL-2-amino-5-phosphonopentanoic acid (AP5). The kainate-induced efflux was totally inhibited by verapamil, but that of glutamate only partially. No effect of verapamil was observed on the quisqualate-induced efflux of 45Ca2+. The results imply that, in cultured astrocytes, Ca2+ fluxes induced by glutamate agonists, occur partially through voltage-dependent Ca2+ channels. The extensive release of 45Ca2+ caused by quisqualate was mainly due to a release from internal stores.     

Assay system for synapse formation between primary cultured neurons

Synapse plasticity, in particular, formation of new synapses, plays crucial roles in learning and memory. We have developed a convenient assay system for measuring the number of newly formed synapses between cultured rat cerebrocortical neurons using the multisite fluorometry system of intracellular calcium. We found that cultured neurons exhibited spontaneous oscillatory changes in intracellular calcium levels and that the frequency of the oscillation was strongly correlated with synaptic density. Combined with immunohistochemical studies, this assay system enables us to study the molecular mechanism of synapse formation, in particular, the involvement of ecto-protein kinase. Other applications of the assay system are discussed here.     

Manganese induces oxidative impairment in cultured rat astrocytes.

Excessive free radical formation has been implicated as a causative factor in neurotoxic damage associated with exposures to a variety of metals, including manganese (Mn). It is well established that Mn accumulates in astrocytes, affecting their ability to indirectly induce and/or exacerbate neuronal dysfunction. The present study examined the effects of Mn treatment on the following endpoints in primary astrocyte cultures: (1) oxidative injury, (2) alterations in high-energy phosphate (adenosine 5'-triphosphate, ATP) levels, (3) mitochondrial inner membrane potential, and (4) glutamine uptake and the expression of glutamine transporters. We quantified astrocyte cerebral oxidative damage by measuring F(2)-isoprostanes (F(2)-IsoPs) using stable isotope dilution methods followed by gas chromatography-mass spectrometry with selective ion monitoring. Our data showed a significant (p < 0.01) elevation in F(2)-IsoPs levels at 2 h following exposure to Mn (100 microM, 500 microM, or 1 mM). Consistent with this observation, Mn induced a concentration-dependent reduction in ATP and the inner mitochondrial membrane potential (DeltaPsi(m)), measured by the high pressure liquid chromatography method and the potentiometric dye, tetramethyl rhodamine ethyl ester, respectively. Moreover, 30 min of pretreatment with Mn (100 microM, 500 microM, or 1 mM) inhibited the net uptake of glutamine (GLN) ((3)H-glutamine) measured at 1 and 5 min. Expression of the messenger RNA coding the GLN transporters, SNAT3/SN1 and SNAT1, was inhibited after 100 and 500 microM Mn treatment for 24 h. Our results demonstrate that induction of oxidative stress, associated mitochondrial dysfunction, and alterations in GLN/glutamate cycling in astrocytes represent key mechanisms by which Mn exerts its neurotoxicity.