Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake

Using the N18-RE-105 neuroblastoma X retina cell line, we previously described Ca2(+)-dependent quisqualate-type glutamate toxicity caused by the inhibition of high-affinity cystine uptake, leading to glutathione depletion and accumulation of cellular oxidants. We now demonstrate that primary cultures of rat cortical neurons (E17; 24-72 h in culture), but not glia, also degenerate when exposed to culture medium with reduced cystine or containing competitive inhibitors of cystine uptake, including glutamate. At this developmental stage, neurotoxicity did not occur as a consequence of continuous exposure to glutamate receptor subtype agonists, N-methyl-D-aspartate, kainate, or 2(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. However, those that inhibited neuronal cystine uptake--quisqualate, glutamate, homocysteate, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, and ibotenate--were neurotoxic. Toxicity related to quisqualate did not correlate with the development of quisqualate-stimulated phosphatidylinositol turnover. The toxic potencies of glutamate, quisqualate, and homocysteate were inversely proportional to the concentration of cystine in the medium, suggesting that they competitively inhibit cystine uptake. Autoradiographic analysis of the cellular localization of L-[35S]cystine uptake indicated that embryonic neurons have a high-affinity transport system that is sensitive to quisqualate, whereas non-neuronal cells in the same cultures have a low-affinity system that is insensitive to quisqualate but potently blocked by D-aspartate and glutamate. Exposure to glutamate or homocysteate resulted in a time-dependent depletion of the cellular antioxidant glutathione. The centrally acting antioxidant idebenone and alpha-tocopherol completely blocked the neurotoxicity resulting from glutamate exposure. We propose that competitive inhibition of cystine transport and reduction of extracellular cystine levels result in neuronal cell death due to accumulation of cellular oxidants.     

Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress

Glutamate binds to both excitatory neurotransmitter binding sites and a Cl(-)-dependent, quisqualate- and cystine-inhibited transport site on brain neurons. The neuroblastoma-primary retina hybrid cells (N18-RE-105) are susceptible to glutamate-induced cytotoxicity. The Cl(-)-dependent transport site to which glutamate and quisqualate (but not kainate or NMDA) bind has a higher affinity for cystine than for glutamate. Lowering cystine concentrations in the cell culture medium results in cytotoxicity similar to that induced by glutamate addition in its morphology, kinetics, and Ca2+ dependence. Glutamate-induced cytotoxicity is directly proportional to its ability to inhibit cystine uptake. Exposure to glutamate (or lowered cystine) causes a decrease in glutathione levels and an accumulation of intracellular peroxides. Like N18-RE-105 cells, primary rat hippocampal neurons (but not glia) in culture degenerate in medium with lowered cystine concentration. Thus, glutamate-induced cytotoxicity in N18-RE-105 cells is due to inhibition of cystine uptake, resulting in lowered glutathione levels leading to oxidative stress and cell death.     

Activation of Endogenous Antioxidant Defenses in Neuronal Cells Prevents Free Radical-Mediated Damage

Abstract: Dopamine (DA) is oxidized to the neurotoxic prooxidant species H₂O₂, OH^•, and DA quinones. We tested whether dimethyl fumarate (DMF), an electrophile shown to induce a pleiotropic antioxidant response in nonneuronal cells, could reduce the toxicity of DA metabolites in neural cells. Treatment of the N18-RE-105 neuroblastoma-retina hybridoma cell line with 30–150 µ M dopamine led to cell death within 24 h, which increased steeply with dose, decreased with higher plating density, and was blocked by the H₂O₂-metabolizing enzyme catalase. Pretreatment with DMF (30 µ M , 24 h) significantly attenuated DA and H₂O₂ toxicity (40–60%) but not that caused by the calcium ionophore ionomycin. DMF treatment also elevated total intracellular GSH and increased activities of the antioxidant enzymes quinone reductase (QR), glutathione S -transferase (GST), glutathione reductase, and the pentose phosphate enzyme glucose-6-phosphate dehydrogenase. To assess the protective efficacy of QR and GST, a stable cell line was constructed in which these enzymes were overexpressed. Cell death in the overexpressing line was not significantly different from that in a cell line expressing normal QR and GST activities, indicating that these two enzymes alone are insufficient for protection against DA toxicity. Although the relative importance of a single antioxidant enzyme such as QR or GST may be small, antioxidant inducers such as DMF may prove valuable as agents that elicit a broad-spectrum neuroprotective response.     

l-Glutamate Binding Site on N18-RE-105 Neuroblastoma Hybrid Cells Is Not Coupled to an Ion Channel

We studied the properties of the N18-RE-105 neuronal cell line to determine if its glutamate binding site represents a neurotransmitter receptor. In immunocytochemical experiments, these cells stained strongly for neurofilament, but not for glial fibrillary acidic protein. In whole-cell patch clamp experiments, cells exhibited voltage-dependent Na+, Ca2+, and K+ currents characteristic of neurons. However, perfusion with L-glutamate or other excitatory amino acids did not evoke the inward current expected of a receptor/channel complex. In binding studies, the maximum accumulation of L-[3H]glutamate by washed membrane vesicles at 37 degrees C was 69 pmol/mg protein, and half-maximal accumulation occurred at 0.64 microM. This accumulation was blocked completely by quisqualate, partially by DL-2-amino-4-phosphonobutyric acid and L-cystine, but not at all by 1 mM kainate or N-methylaspartate. L-[3H]Glutamate accumulation was stimulated by Cl-, but reduced by Na+, 0.01% digitonin, or hyperosmotic (400 mM glucose) assay medium. The release of L-[3H]glutamate from vesicles was much faster in the presence of 100 microM unlabelled glutamate than 100 microM unlabelled quisqualate or DL-2-amino-4-phosphonobutyric acid. Thus, although N18-RE-105 cells possess many neuronal properties, the results obtained are not those expected from reversible binding of L-glutamate to a receptor/channel complex, but are consistent with a Cl- -stimulated sequestration or exchange process.     

Potent antioxidative activity of cacalol, a sesquiterpene contained in Cacalia delphiniifolia Sleb et Zucc

The potent antioxidative sesquiterpene, cacalol, was isolated from Cacalia delphiniifolia Sleb et Zucc, and identified by an analysis of the MS and NMR spectral data. Cacalol showed potent antioxidative activity in a rat brain homogenate model (IC(50) of 40 nM). Cacalol also proved to be a potent neuroprotective substance which could protect neuronal hybridoma N18-RE-105 cells from L-glutamate toxicity.     

N18-RE-105 cells: differentiation and activation of p53 in response to glutamate and adriamycin is blocked by SV40 large T antigen tsA58

Process extension was induced in cells of the N18-RE-105 neuroblastoma-retinal hybrid line by toxic agents, including glutamate and the p53-inducing anticancer agents adriamycin and etoposide. Both adriamycin and glutamate activated p53 as measured by a plasmid transfection assay. It was therefore hypothesized that SV40 large T antigen, which binds p53, would interfere with cellular differentiation. To test this hypothesis, the temperature-sensitive form of SV40 large T was transduced into N18-RE-105 cells by retroviral infection. SV40 large T-infected cells became de-differentiated, grew in tightly-packed colonies, lost expression of neurofilament, and lost the ability to differentiate in response to glutamate and adriamycin. The de-differentiating effect of SV40 large T antigen may be due to binding and inactivation of cellular proteins, such as p53, p107, p130, p300, and retinoblastoma protein, which are important in cellular growth and differentiation. It is suggested that p53 may play a role in cellular differentiation, perhaps under unusual circumstances involving stress or cytotoxicity. Received: 29 April 1997 / Accepted: 18 June 1997     

Characterization of a glutamic acid neurotransmitter binding site on neuroblastoma hybrid cells

Glutamate is thought to be a major excitatory neurotransmitter in the central nervous system. To study the glutamate receptor and its regulation under carefully controlled conditions, the specific binding of [3H]glutamate was characterized in washed membranes isolated from a neuroblastoma X retina hybrid cell line, N18-RE-105. [3H]Glutamate bound in a saturable and reversible fashion with an apparent dissociation constant, KD, of 650 nM and a maximum binding capacity, Bmax, of 16 pmol/mg of protein. Pharmacologic characterization of the site indicates that it closely resembles the Na+-independent binding site for glutamate found on brain membranes and thought to be an excitatory amino acid neurotransmitter receptor. Thus, while kainate, N-methyl-DL-aspartate, and nonamino acid ligands did not displace [3H]glutamate, quisqualate and ibotenate were potent inhibitors of specific binding. Furthermore, this binding site is regulated by ions in a manner which resembles that described in the hippocampus (Baudry, M., and Lynch, G. (1979) Nature (Lond.) 282, 748-750). Calcium (10 mM) increased the number of binding sites 2.6-fold with no change in receptor-ligand affinity. Lanthanum (1 mM) was the only other cation added which enhanced (3-fold) the binding of [3H]glutamate. Monovalent cations resulted in a decrease in the number of glutamate binding sites. Incubation of membranes in the presence of chloride ions caused a marked increased in [3H] glutamate binding, an effect which was synergistic with that of calcium incubation. Thus, N18-RE-105 cells possess a binding site for [3H]glutamate pharmacologically similar to an excitatory neurotransmitter binding site in brain and which exhibits regulatory properties resembling those previously described in hippocampal membranes, providing an excellent model for mechanistic studies.     

Acidosis Potentiates Oxidative Neuronal Death by Multiple Mechanisms

Both acidosis and oxidative stress contribute to ischemic brain injury. The present study examines interactions between acidosis and oxidative stress in murine cortical cultures. Acidosis (pH 6.2) was found to potentiate markedly neuronal death induced by H2O2 exposure. To determine if this effect was mediated by decreased antioxidant capacity at low pH, the activities of several antioxidant enzymes were measured. Acidosis was found to reduce the activities of glutathione peroxidase and glutathione S-transferase by 50-60% (p < 0.001) and the activity of glutathione reductase by 20% (p < 0.01) in lysates of the cortical cultures. Like acidosis, direct inhibition of glutathione peroxidase with mercaptosuccinate also potentiated H2O2 toxicity. Because acidosis may accelerate hydroxyl radical production by the Fenton reaction, the effect of iron chelators was also examined. Both desferrioxamine and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, two structurally different iron chelators, significantly reduced H2O2-induced neuronal death under both pH 7.2 and pH 6.2 conditions. These results suggest that the increased cell death produced by severe acidosis during cerebral ischemia may result in part from exacerbation of oxidative injury. This exacerbation may result from both impaired antioxidant enzyme functions and increased intracellular free iron levels.     

Induction of renal oxidative stress and cell proliferation response by ferric nitrilotriacetate (Fe-NTA): diminution by soy isoflavones

Ferric nitrilotriacetate (Fe-NTA) is a known potent nephrotoxic agent. In this communication, we report the chemopreventive effect of soy isoflavones on renal oxidative stress, toxicity and cell proliferation response in Wistar rats. Fe-NTA (9 mg Fe/kg body weight, intraperitoneally) enhances gamma-glutamyl transpeptidase, renal lipid peroxidation, xanthine oxidase and hydrogen peroxide (H2O2) generation with reduction in renal glutathione content, antioxidant enzymes, viz., glutathione peroxidase, glutathione reductase, catalase, glucose-6-phosphate dehydrogenase and phase-II metabolising enzymes such as glutathione-S-transferase and quinone reductase. Fe-NTA treatment also induced tumor promotion markers, viz., ornithine decarboxylase (ODC) activity and thymidine [3H] incorporation into renal DNA. A sharp elevation in the levels of blood urea nitrogen and serum creatinine has also been observed. Treatment of rats orally with soy isoflavones (5 mg/kg body weight and 10 mg/kg body weight) resulted in significant decreases in gamma-glutamyl transpeptidase, lipid peroxidation, xanthine oxidase, H2O2 generation, blood urea nitrogen, serum creatinine, renal ODC activity and DNA synthesis (P < 0.001). Renal glutathione content (P < 0.01), glutathione metabolizing enzymes (P < 0.001) and antioxidant enzymes were also returned to normal levels (P < 0.001). Thus, our data suggest that soy isoflavones may be used as an effective chemopreventive agent against Fe-NTA-mediated renal oxidative stress, toxicity and cell proliferation response in Wistar rats.     

A549 subclones demonstrate heterogeneity in toxicological sensitivity and antioxidant profile

In A549 cell culture, significant variability was found in sensitivity to actinomycin D. Using limiting dilution, actinomycin D-susceptible (G4S) and -resistant (D3R) subclones were isolated. G4S cells were also susceptible to protein synthesis inhibitors, a redox cycling quinone, and an electrophile with concomitant activation of caspases 3 and 9. D3R cells were resistant to these agents without caspase activation. Antioxidant profiles revealed that D3R cells had significantly higher glutathione and glutathione reductase activity but markedly lower catalase, glutathione peroxidase, and aldehyde reductase activities than G4S cells. Thus A549 cells contain at least two distinct subpopulations with respect to predisposition to cell death and antioxidant profile. Because sensitivities to agents and the antioxidant profile were inconsistent, mechanisms independent of antioxidants, including the apparent inability to activate caspases in D3R cells, may play an important role. Regardless, the results suggest that antioxidant profiles of asymmetrical cell populations cannot predict sensitivity to oxidants and warn that the use of single subclones is advisable for mechanistic studies using A549 or other unstable cell lines.     

Cooperative action of glutamate transporters and cystine/glutamate antiporter system Xc- protects from oxidative glutamate toxicity

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress. In this paradigm, an excess of extracellular glutamate blocks the glutamate/cystine-antiporter system Xc-, depleting the cell of cysteine, a building block of the antioxidant glutathione. Loss of glutathione leads to the accumulation of reactive oxygen species and eventually cell death. We selected cells resistant to oxidative stress, which exhibit reduced glutamate-induced glutathione depletion mediated by an increase in the antiporter subunit xCT and system Xc- activity. Cystine uptake was less sensitive to inhibition by glutamate and we hypothesized that glutamate import via excitatory amino acid transporters and immediate re-export via system Xc- underlies this phenomenon. Inhibition of glutamate transporters by l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and DL-threo-beta-benzyloxyaspartic acid (TBOA) exacerbated glutamate-induced cell death. PDC decreased intracellular glutamate accumulation and exacerbated glutathione depletion in the presence of glutamate. Transient overexpression of xCT and the glutamate transporter EAAT3 cooperatively protected against glutamate. We conclude that EAATs support system Xc- to prevent glutathione depletion caused by high extracellular glutamate. This knowledge could be of use for the development of novel therapeutics aimed at diseases associated with depletion of glutathione like Parkinson's disease.     

Exacerbation of copper toxicity in primary neuronal cultures depleted of cellular glutathione

Perturbations to glutathione (GSH) metabolism may play an important role in neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases. A primary function of GSH is to prevent the toxic interaction between free radicals and reactive transition metals such as copper (Cu). Due to the potential role of Cu in neurodegeneration, we examined the effect of GSH depletion on Cu toxicity in murine primary neuronal cultures. Depletion of cellular GSH with L-buthionine-[S,R]-sulfoximine resulted in a dramatic potentiation of Cu toxicity in neurons without effect on iron (Fe) toxicity. Similarly, inhibition of glutathione reductase (GR) activity with 1,3-bis(2-chloroethyl)-1-nitrosurea also increased Cu toxicity in neurons. To determine if the Alzheimer's amyloid-beta (Abeta) peptide can affect neuronal resistance to transition metal toxicity, we exposed cultures to nontoxic concentrations of Abeta25-35 in the presence or absence of Cu or Fe. Abeta25-35 pretreatment was found to deplete neuronal GSH and increase GR activity, confirming the ability of Abeta to perturb neuronal GSH homeostasis. Abeta25-35 pretreatment potently increased Cu toxicity but had no effect on Fe toxicity. These studies demonstrate an important role for neuronal GSH homeostasis in selective protection against Cu toxicity, a finding with widespread implications for neurodegenerative disorders.     

FQR1, a novel primary auxin-response gene, encodes a flavin mononucleotide-binding quinone reductase

FQR1 is a novel primary auxin-response gene that codes for a flavin mononucleotide-binding flavodoxin-like quinone reductase. Accumulation of FQR1 mRNA begins within 10 min of indole-3-acetic acid application and reaches a maximum of approximately 10-fold induction 30 min after treatment. This increase in FQR1 mRNA abundance is not diminished by the protein synthesis inhibitor cycloheximide, demonstrating that FQR1 is a primary auxin-response gene. Sequence analysis reveals that FQR1 belongs to a family of flavin mononucleotide-binding quinone reductases. Partially purified His-tagged FQR1 isolated from Escherichia coli catalyzes the transfer of electrons from NADH and NADPH to several substrates and exhibits in vitro quinone reductase activity. Overexpression of FQR1 in plants leads to increased levels of FQR1 protein and quinone reductase activity, indicating that FQR1 functions as a quinone reductase in vivo. In mammalian systems, glutathione S-transferases and quinone reductases are classified as phase II detoxification enzymes. We hypothesize that the auxin-inducible glutathione S-transferases and quinone reductases found in plants also act as detoxification enzymes, possibly to protect against auxin-induced oxidative stress.     

Activation of stimulatory heterotrimeric G proteins increases glutathione and protects neuronal cells against oxidative stress

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress, where an excess of extracellular glutamate inhibits import of cystine, a building block of the antioxidant glutathione. The subsequent decrease in glutathione then leads to the accumulation of reactive oxygen species (ROS) and programmed cell death. We used pharmacological compounds known to interact with heterotrimeric G-protein signalling and studied their effects on cell survival, morphology, and intracellular events that ultimately lead to cell death. Cholera toxin and phorbol esters were most effective and prevented cell death through independent pathways. Treating HT22 cells with cholera toxin attenuated the glutamate-induced accumulation of ROS and calcium influx. This was, at least in part, caused by an increase in glutathione due to improved uptake of cystine mediated by the induction of the glutamate/cystine-antiporter subunit xCT or, additionally, by the up-regulation of the antiapoptotic protein Bcl-2. Gs activation also protected HT22 cells from hydrogen peroxide or inhibition of glutathione synthesis by buthionine sulfoximine, and immature cortical neurones from oxidative glutamate toxicity. Thus, this pathway might be more generally implicated in protection from neuronal death by oxidative stress.     

Role of NAD(P)H:quinone oxidoreductase in the progression of neuronal cell death in vitro and following cerebral ischaemia in vivo

A direct involvement of the antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO1) in neuroprotection has not yet been shown. The aim of this study was to examine changes, localization and role of NQO1 after different neuronal injury paradigms. In primary cultures of rat cortex the activity of NQO1 was measured after treatment with ethylcholine aziridinium (AF64A; 40 micro m), inducing mainly apoptotic cell death, or oxygen-glucose deprivation (OGD; 120 min), which combines features of apoptotic and necrotic cell death. After treatment with AF64A a significant NQO1 activation started after 24 h. Sixty minutes after OGD a significant early induction of the enzyme was observed, followed by a second increase 24 h later. Enzyme activity was preferentially localized in glial cells in control and injured cultures, however, expression also occurred in injured neuronal cells. Inhibition of the NQO1 activity by dicoumarol, cibacron blue or chrysin (1-100 nM) protected the cells both after exposure to AF64A or OGD as assessed by the decreased release of lactate dehydrogenase. Comparable results were obtained in vivo using a mouse model of focal cerebral ischaemia. Dicoumarol treatment (30 nmol intracerebroventricular) reduced the infarct volume by 29% (p = 0.005) 48 h after the insult. After chemical induction of NQO1 activity by t-butylhydroquinone in vitro neuronal damage was exaggerated. Our data suggest that the activity of NQO1 is a deteriorating rather than a protective factor in neuronal cell death.     

D-Glucose prevents glutathione oxidation and mitochondrial damage after glutamate receptor stimulation in rat cortical primary neurons

The possible neuroprotective effect of D-glucose against glutamate-mediated neurotoxicity was studied in rat cortical neurons in primary culture. Brief (5-min) exposure of neurons to glutamate (100 microM) increased delayed (24-h) necrosis and apoptosis by 3- and 1.8-fold, respectively. Glutamate-mediated neurotoxicity was accompanied by a D-(-)-2-amino-5-phosphonopentanoate (100 microM) and N(omega)-nitro-L-arginine methyl ester (1 mM)-inhibitable, time-dependent ATP depletion (55% at 24 h), confirming the involvement of NMDA receptor stimulation followed by nitric oxide synthesis in this process. Furthermore, the presence of D-glucose (20 mM), but not its inactive enantiomer, L-glucose, fully prevented glutamate-mediated delayed ATP depletion, necrosis, and apoptosis. Succinate- cytochrome c reductase activity, but not the activities of NADH-coenzyme Q(1) reductase or cytochrome c oxidase, was inhibited by 32% by glutamate treatment, an effect that was abolished by incubation with D-glucose. Lactate accumulation in the culture medium was unmodified by any of these treatments, ruling out the possible involvement of the glycolysis pathway in either glutamate neurotoxicity or D-glucose neuroprotection. In contrast, D-glucose, but not L-glucose, abolished glutamate-mediated glutathione oxidation and NADPH depletion. Our results suggest that NADPH production from D-glucose accounts for glutathione regeneration and protection from mitochondrial dysfunction. This supports the notion that the activity of the pentose phosphate pathway may be an important factor in protecting neurons against glutamate neurotoxicity.     

Neurotoxicity of Methylmercury in Isolated Astrocytes and Neurons: the Cytoskeleton as a Main Target

In the present work, we focused on mechanisms of methylmercury (MeHg) toxicity in primary astrocytes and neurons of rats. Cortical astrocytes and neurons exposed to 0.5–5 μM MeHg present a link among morphological alterations, glutathione (GSH) depletion, glutamate dyshomeostasis, and cell death. Disrupted neuronal cytoskeleton was assessed by decreased neurite length and neurite/neuron ratio. Astrocytes presented reorganization of actin and glial fibrillary acidic protein (GFAP) networks and reduced cytoplasmic area. Glutamate uptake and Na⁺K⁺ATPase activity in MeHg-treated astrocytes were preserved; however, downregulated EAAC1-mediated glutamate uptake was associated with impaired Na⁺K⁺ATPase activity in neurons. Oxidative imbalance was found in astrocytes and neurons through increased 2′7′-dichlorofluorescein (DCF) production and misregulated superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GPX) activities. Glutathione (GSH) levels were downregulated in both astrocytes and neurons. MeHg reduced neuronal viability and induced caspase 3-dependent apoptosis together with downregulated PI3K/Akt pathway. In astrocytes, necrotic death was associated with increased TNF-α and JNK/MAPK activities. Cytoskeletal remodeling and cell death were fully prevented in astrocytes and neurons by GSH, but not melatonin or Trolox supplementation. These findings support a role for depleted GSH in the cytotoxicity of MeHg leading to disruption of the cytoskeleton and cell death. Moreover, in neurons, glutamate antagonists also prevented cytoskeletal disruption and neuronal death. We propose that cytoskeleton is an end point in MeHg cytotoxicity. Oxidative imbalance and glutamate mechanisms mediate MeHg cytoskeletal disruption and apoptosis in neurons. Otherwise, redox imbalance and glutamate-independent mechanisms disrupted the cytoskeleton and induced necrosis in MeHg-exposed astrocyte.     

The toxicity of menadione (2-methyl-1,4-naphthoquinone) and two thioether conjugates studied with isolated renal epithelial cells

Menadione (2-methyl-1,4-naphthoquinone) was used as a model compound to test the hypothesis that thioether conjugates of quinones can be toxic to tissues associated with their elimination through a mechanism involving oxidative stress. Unlike menadione, the glutathione (2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone; MGNQ) and N-acetyl-L-cysteine (2-methyl-3-(N-acetylcysteine-S-yl)-1,4-naphthoquinone; M(NAC)NQ) thioether conjugates were not able to arylate protein thiols but were still able to redox cycle with cytochrome c reductase/NADH and rat kidney microsomes and mitochondria. Interestingly, menadione and M(NAC)NQ were equally toxic to isolated rat renal epithelial cells (IREC) while MGNQ was nontoxic. The toxicity of both menadione and M(NAC)NQ was preceded by a rapid depletion of soluble thiols and was associated with a depletion of soluble thiols and was associated with a depletion of protein thiols. Treatment of IREC with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1-nitrosourea, potentiated the thiol depletion and toxicity observed with menadione and M(NAC)NQ indicating the involvement of oxidative stress in this model of renal cell toxicity. The lack of MGNQ toxicity can be attributed to an intramolecular cyclization reaction which destroys the quinone nucleus and therefore eliminates its ability to redox cycle. These findings have important implications with regard to our understanding of the toxic potential of quinone thioether conjugates and of quinone toxicity in general.     

Geldanamycin Provides Posttreatment Protection Against Glutamate-Induced Oxidative Toxicity in a Mouse Hippocampal Cell Line

Abstract : The benzoquinoid ansamycin geldanamycin interferes with many cell signaling pathways and is currently being evaluated as an anticancer agent. The main intracellular target of geldanamycin is the 90-kDa heat shock protein, hsp90. In this report we demonstrate that geldanamycin is effective at preventing glutamate-induced oxidative toxicity in the HT22 mouse hippocampal cell line, even if given 4 h after glutamate treatment. Geldanamycin prevents glutamate-induced internucleosomal DNA cleavage in the HT22 cells but does not reverse the depletion of glutathione levels brought about by glutamate treatment. Both anabolic and catabolic effects are generated by geldanamycin treatment of HT22 cells, as evidenced by the induction of hsp70 expression and degradation of c-Raf-1 protein, respectively. Thus, geldanamycin may provide an effective strategy for manipulating signaling pathways in neuronal cells that use hsp90 as they proceed through a programmed cell death pathway in response to oxidative stress.