Electrophile and antioxidant regulation of enzymes that detoxify carcinogens

Detoxication (phase 2) enzymes, such as glutathione S-transferases (GSTs), NAD(P)H:(quinone-acceptor) oxidoreductase (QR), and UDP-glucuronsyltransferase, are induced in animal cells exposed to a variety of electrophilic compounds and phenolic antioxidants. Induction protects against the toxic and neoplastic effects of carcinogens and is mediated by activation of upstream electrophile-responsive/antioxidant-responsive elements (EpRE/ARE). The mechanism of activation of these enhancers was analyzed by transient gene expression of growth hormone reporter constructs containing a 41-bp region derived from the mouse GST Ya gene 5'-upstream region that contains the EpRE/ARE element and of constructs in which this element was replaced with either one or two consensus phorbol 12-tetradecanoate 13-acetate (TPA)-responsive elements (TREs). When these three constructs were compared in Hep G2 (human) and Hepa 1c1c7 (murine) hepatoma cells, the wild-type sequence was highly activated by diverse inducers, including tert-butylhydroquinone, Michael reaction acceptors, 1,2-dithiole-3-thione, sulforaphane,2,3-dimercapto-1-propanol, HgCl2, sodium arsenite, and phenylarsine oxide. In contrast, constructs with consensus TRE sites were not induced significantly. TPA in combination with these compounds led to additive or synergistic inductions of the EpRE/ARE construct, but induction of the TRE construct was similar to that induced by TPA alone. Transfection of the EpRE/ARE reporter construct into F9 cells, which lack endogenous TRE-binding proteins, produced large inductions by the same compounds, which also induced QR activity in these cells. We conclude that activation of the EpRE/ARE by electrophile and antioxidant inducers is mediated by EpRE/ARE-specific proteins.     

Differential distribution of purine metabolizing enzymes between glia and neurons

Previous studies showed that in cultured chick ciliary ganglion neurons and CNS glia, adenosine can be synthesized by hydrolysis of 5'-AMP and that the accumulation of the adenosine degradative products inosine and hypoxanthine was significantly greater in glial than in neuronal cultures. Furthermore, previous immunochemical and histochemical studies in brain showed that adenosine deaminase and nucleoside phosphorylase are localized in endothelial and glial cells but are absent in neurons; however, adenosine deaminase may be found in a few neurons in discrete brain regions. These results suggested that adenosine degradative pathways may be more active in glia. Thus, we have determined if there is a differential distribution of adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase enzyme fluxes in glia, comparing primary cultures of central and ciliary ganglion neurons and glial cells from chick embryos. Hypoxanthine-guanine phosphoribosyltransferase and production of adenosine by S-adenosylhomocysteine hydrolase activity were also examined. Our results show that there is a distinct profile of purine metabolizing enzymes for glia and neurons in culture. Both cell types have an S-adenosylhomocysteine hydrolase, but it was more active in neurons than in glia. In contrast, in glia the enzymatic activities of xanthine oxidase (443 +/- 61 pmol/min/10(7) cells), nucleoside phosphorylase (187 +/- 8 pmol/min/10(7) cells), and adenosine deaminase (233 +/- 32 pmol/min/10(7) cells) were more active at least 100, 20, and five times, respectively, than in ciliary ganglion neurons and 100, 100, and nine times, respectively, than in central neurons.     

Disruption of c-Fos leads to increased expression of NAD(P)H:quinone oxidoreductase1 and glutathione S-transferase

Regulation of the basal and induced expression of detoxifying enzymes such as NAD(P)H:quinone oxidoreductasel (NQO1) and glutathione S-transferase (GST) by the antioxidant response element (ARE) is important for cellular protection against oxidative stress. The ARE contains AP1 and AP1-like elements and is known to bind to several leucine zipper proteins including c-Fos. Previous studies (Venugopal, R., and Jaiswal, A.K. (1996) Proc. NatL Acad. Sci. USA 93, 14960-14965) have shown that overexpression of c-Fos in transfected cells leads to repression of ARE-mediated gene expression. In the present report, we used c-Fos-/- mice and investigated the physiological (in vivo) role of c-Fos in repression of the NQO1 and GST genes expression. The analysis of enzyme activity levels showed significant increases in NQO1 and GST activities in several tissues of c-Fos-/- mice, as compared with wild type (c-Fos+/+) mice. The increases in enzyme activities were supported by Wetern analysis of respective proteins. Western analyses showed significant increases in the expression of NQO1 in kidney, liver and skin tissues of c-Fos-/- mice, as compared with wild type (c-Fos+/+) controls. Western analyses also demonstrated an increased expression of the GST Ya gene in kidney and liver tissues of the c-Fos-/-mice. These results confirm a negative (repressive) role for c-Fos in the expression of NQO1, GST Ya, and other detoxifying enzyme genes.     

Ability of retinal Müller glial cells to protect neurons against excitotoxicity in vitro depends upon maturation and neuron-glial interactions

Glutamate is the most abundant excitatory amino acid in the central nervous system. It has also been described as a potent toxin when present in high concentrations because excessive stimulation of its receptors leads to neuronal death. Glial influence on neuronal survival has already been shown in the central nervous system, but the mechanisms underlying glial neuroprotection are only partly known. When cells isolated from newborn rat retina were maintained in culture as enriched neuronal populations, 80% of the cells were destroyed by application of excitotoxic concentrations of glutamate. Massive neuronal death was also observed in newborn retinal cultures containing large numbers of glia, or when neurons were seeded onto feeder layers of purified cells prepared from immature (postnatal 8 day) rat retina. When newborn retinal neurons were seeded onto feeder layers of purified glial cells prepared from adult retinas, application of excitotoxic amino acids no longer led to neuronal death. Furthermore, neuronal death was not observed in mixed neuron/glial cultures prepared from adult retina. However, in all cases (newborn and adult) application of kainate led to amacrine cell-specific death. Activity of glutamine synthetase, a key glial enzyme involved in glutamate detoxification, was assayed in these cultures in the presence or absence of exogenous glutamate. Whereas pure glial cultures alone (from young or adult retina) showed low activity that was not stimulated by glutamate addition, mixed or co-cultured neurons and adult glia exhibited up to threefold higher levels of activity following glutamate treatment. These data indicate that two conditions must be satisfied to observe glial neuroprotection: maturation of glutamine synthetase expression, and neuron-glial signalling through glutamate-elicited responses.     

Olfactory epithelial organotypic slice cultures: a useful tool for investigating olfactory neural development

An in vitro slice culture was established for investigating olfactory neural development. The olfactory epithelium was dissected from embryonic day 13 rats; 400 microns slices were cultured for 5 days in serum-free medium on Millicell-CM membranes coated with different substrates. The slices were grown in the absence of their appropriate target, the olfactory bulb, or CNS derived glia. The cultures mimic many features of in vivo development. Cells in the olfactory epithelium slices differentiate into neurons that express olfactory marker protein (OMP). OMP-positive cells have the characteristic morphology of olfactory receptor neurons: a short dendrite and a single thin axon. The slices support robust axon outgrowth. In single-label experiments, many axons expressed neural specific tubulin, growth-associated protein 43 and OMP. Axons appeared to grow equally well on membranes coated with type I rat tail collagen, laminin or fibronectin. The cultures exhibit organotypic polarity with an apical side rich in olfactory neurons and a basal side supporting axon outgrowth. Numerous cells migrate out of the slices, of which a small minority was identified as neurons based on the expression of neural specific tubulin and HuD, a nuclear antigen, expressed exclusively in differentiated neurons. Most of the migrating cells, however, were positive for glial fibrillary acidic protein and S-100, indicating that they are differentiated glia. A subpopulation of these glial cells also expressed low-affinity nerve growth factor receptors, indicating that they are olfactory Schwann cells. Both migrating neurons and glia were frequently associated with axons growing out of the slice. In some cases, axons extended in advance of migrating cells. This suggests that olfactory receptor neurons in organotypic cultures require neither a pre-established glial/neuronal cellular terrain nor any target tissue for successful axon outgrowth. Organotypic olfactory epithelial slice cultures may be useful for investigating cellular and molecular mechanisms that regulate early olfactory development and function.     

Age-dependent differences in the effects of GDNF and NT-3 on the development of neurons and glia from neural crest-derived precursors immunoselected from the fetal rat gut: expression of GFRalpha-1 in vitro and in vivo

No enteric neurons or glia develop in the gut below the rostral foregut in mice lacking glial cell line-derived neurotrophic factor (GDNF) or Ret. We analyzed the nature and age dependence of the effects of GDNF and, for comparison, those of NT-3, on the in vitro development of the precursors of enteric neurons and glia. Positive and negative immunoselection with antibodies to p75(NTR) were used to isolate crest-derived and crest-depleted populations of cells from the fetal rat bowel at E12, 14, and 16. Cells were typed immunocytochemically. GDNF stimulated the proliferation of nestin-expressing precursor cells isolated at E12, but not at E14-16. GDNF promoted the development of peripherin-expressing neurons (E12 > E14-16) and expression of TrkC. GDNF inhibited expression of S-100-expressing glia at E14-16. NT-3 did not affect cells isolated at E12, never stimulated precursors to proliferate, and promoted glial as well as neuronal development at E14-16. GFRalpha-1 was expressed both by crest- and non-crest-derived cells, although only crest-derived cells anchored GFRalpha-1 and GFRalpha-2 (GFRalpha-1 > GFRalpha-2). GDNF increased the number of neurons anchoring GFRalpha-1. GFRalpha-1 is immunocytochemically detectable in neurons of the E13 intestine and persists in adult neurons of both plexuses. We suggest that GDNF stimulates the proliferation of an early (E12) NT-3-insensitive precursor common to enteric neurons and glia; by E14, this common precursor is replaced by specified NT-3-responsive neuronal and glial progenitors. GDNF exerts a neurotrophic, but not a mitogenic, effect on the neuronal progenitor. The glial progenitor is not maintained by GDNF.     

The redox-sensitive human antioxidant responsive element induces gene expression under low oxygen conditions

Transient transfection studies of human HepG2 and mouse Hepa hepatocarcinoma cells with a reporter gene construct regulated by a human antioxidant responsive element (ARE) from the NQO1 gene demonstrated that the element is responsive to low oxygen conditions. The antioxidant N-acetyl L-cysteine (NAC) strongly inhibited basal aerobic reporter gene activity in HepG2 cells without obviously affecting the hypoxic induction, as is consistent with ARE sensitivity to oxidative stress in aerobic cultures. Electrophoretic mobility shift (EMS) assays of nuclear extracts of HepG2 and Hepa cells lysed under aerobic or hypoxic conditions or after exposure to the phenolic compound 3-(2)-tert-butyl-4-hydroxyanisole (BHA), showed specific and constitutive protein binding to the ARE under all of these conditions. Taken together, these findings show that the ARE can mediate gene expression in response to low oxygen conditions. Co-ordinately regulated expression of ARE-dependent genes, such as phase II detoxification enzymes, may be an important phenotype of solid tumors containing significant regions of pathophysiological hypoxia.     

Responses of Bergmann glia and granule neurons in situ to N-methyl-D-aspartate, norepinephrine, and high potassium

To gain insight into neuronal-glial signaling in brain, cerebellar Bergmann glia and granule neurons were studied in acutely isolated slices with the aid of laser scanning confocal microscopy. Both Bergmann glia and granule neurons responded to N-methyl-D-aspartate (NMDA) with a rise in [Ca2+]i. However, the glial NMDA response was frequently inhibited by tetrodotoxin, suggesting that the response depended on neuronal action potentials, rather than on direct activation of NMDA receptors on the Bergmann glia. Further experiments demonstrated that the NMDA response in Bergmann glia was not inhibited by a combination of non-NMDA glutamate receptor blockers 6-cyano-7-nitroquinoxaline-2,3-dione and alpha-methyl-4-carboxyphenylglycine. Bergmann glia also responded to norepinephrine and high K+, and the responses were not inhibited by tetrodotoxin. The glial norepinephrine response was blocked by phentolamine but not by the removal of external Ca2+, indicating a direct activation of alpha1-adrenergic receptors that mediated release of Ca2+ from intracellular stores. The KCl-induced response in both neurons and glia was dependent on external Ca2+ and was blocked by verapamil or nifedipine. In summary, our data indicate that Bergmann glia in situ recognize a signal(s) released from neurons during neuronal activity.     

Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons

Sodium-dependent transport into astrocytes is critical for maintaining the extracellular concentrations of glutamate below toxic levels in the central nervous system. In this study, the expression of the glial glutamate transporters GLT-1 and GLAST was studied in primary cultures derived from cortical tissue. In primary astrocytes, GLAST protein levels were approximately one half of those observed in cortical tissue, but GLT-1 protein was present at very low levels compared with cortical tissue. Maintenance of these astrocytes in medium supplemented with dibutyryl-cAMP (dbcAMP) caused a dramatic change in cell morphology, increased GLT-1 and GLAST mRNA levels approximately 5-fold, increased GLAST protein approximately 2-fold, and increased GLT-1 protein >/=8-20-fold. These increases in protein expression were accompanied by 2-fold increases in the Vmax and Km values for Na+-dependent L-[3H]glutamate transport activity. Although GLT-1 is sensitive to inhibition by dihydrokainate in heterologous expression systems, no dihydrokainate sensitivity was observed in astrocyte cultures that expressed GLT-1. Biotinylation with a membrane-impermeant reagent, separation of the biotinylated/cell surface proteins, and subsequent Western blotting demonstrated that both GLT-1 and GLAST were present at the cell surface. Coculturing of astrocytes with neurons also induced expression of GLT-1, which colocalized with the glial specific marker, glial fibrillary acidic protein. Neurons induced a small increase in GLAST protein. Several studies were performed to examine the mechanism by which neurons regulate expression of the glial transporters. Three different protein kinase A (PKA) antagonists did not block the effect of neurons on glial expression of GLT-1 protein, but the addition of dbcAMP to mixed cultures of neurons and astrocytes did not cause GLT-1 protein to increase further. This suggests that neurons do not regulate GLT-1 by activation of PKA but that neurons and dbcAMP regulate GLT-1 protein through convergent pathways. As was observed with GLT-1, the increases in GLAST protein observed in cocultures were not blocked by PKA antagonists, but unlike GLT-1, the addition of dbcAMP to mixed cultures of neurons and astrocytes caused GLAST protein to increase approximately 2-fold. Neurons separated from astrocytes with a semipermeable membrane increased GLT-1 protein, indicating that the effect of neurons was mediated by a diffusible molecule. Treatment of cocultures with high concentrations of either N-methyl-D-aspartate or glutamate killed the neurons, caused GLT-1 protein to decrease, and caused GLAST protein to increase. These studies suggest that GLT-1 and GLAST protein are regulated independently in astrocyte cultures and that a diffusible molecule secreted by neurons induces expression of GLT-1 in astrocytes.     

Developmentally regulated and spatially restricted antigens of radial glial cells

Radial glial cells, present in many parts of the embryonic vertebrate central nervous system (CNS), have been implicated in the guidance of neuroblasts from the ventricular zone to their laminar destinations. Moreover, radial glial cells may be progenitors of some CNS neurons and glia. To gain new insight into the structure and development of these cells, we have generated and characterized a panel of monoclonal antibodies that recognize radial glial cells of the chick optic tectum. Mice were immunized with homogenates of embryonic day (E) 10 tectum, and antibodies were analyzed by immunofluorescence and immunoblotting. We describe here three pairs of antibodies. 1) H5 and a previously generated antibody, R5 (Dr?ger et al., J. Neurosci. 4:2025, 1984), stain the whole extent of the radial glial cell from E7 to E20. In cultures prepared from E10 tecta, both stain a filamentous meshwork in glial cells but not in neurons. On immunoblots, both recognize a protein of approximately 52 kD that is closely related (or identical) to vimentin. 2) H28 and H29 stain radial glia between E7 and E14, but not later. Moreover, H28 and H29 staining is markedly more intense in the ventricular and intermediate zones than in the laminae of the tectal plate. Both of these antibodies recognize an intracellular epitope in cultured glial cells and a protein of approximately 35 kD on immunoblots. 3) H2 and H27 recognize antigens concentrated in the most superficial processes and endfeet of radial glia in late (E16-E20) embryos. They stain distinct structures in cultured glia, suggesting that they recognize distinct antigens. H27 recognizes a protein of approximately 29 kD on immunoblots. Thus antibodies H5 and R5 are good markers of radial glial cells at all stages, whereas the others define antigens that are developmentally regulated and localized to discrete domains. Together, these antibodies can be used to study temporal and spatial specializations of radial glia.     

Activation of protein kinase C induces neurofilament fragmentation, hyperphosphorylation of perikaryal neurofilaments and proximal dendritic swellings in cultured motor neurons

Characteristic responses of motor neurons to injury include an apparent increase in the phosphorylation of C-terminal domains of neurofilament proteins in the perikaryal and dendritic compartments. This change was induced in dissociated cultures of embryonic spinal cord by activation of protein kinase C (PKC). PKC was activated by: (a) exposure of cultures to 10 nM 12-o-tetradecanoyl phorbol 13-acetate (TPA); (b) microinjection of 1 mM dioctanoylglycerol (diC8) directly into perikaryal of motor neurons;(c) addition of 10 microM diC8 to culture medium. Activation of PKC led to different immediate and long term effects on neurofilaments of motor neurons. After 30 minutes (min), fragmentation of the neurofilament network was observed by labeling with antibodies to low and high molecular weight neurofilaments proteins; glial filaments were disassembled after 10 min and reassembled by 1 hour (h). From 4 to 24 h, motor neuron were observed with extensions of perikaryal cytoplasm or massive enlargements of proximal dendritic processes, both containing intact neurofilament networks. Over 1 to 12 days, there was a gradual increase in the number of motor neuronal perikarya immunoreactive with antibodies to neurofilament proteins phosphorylated at KSP sites on the C-terminal domains (SMI31, SMI34). It is proposed that activation of PKc secondary to other injurious events may contribute to the changes in neurofilaments observed in motor neuron diseases.     

Metanil yellow: a bifunctional inducer of hepatic phase I and phase II xenobiotic-metabolizing enzymes

Metanil yellow, a non-permitted food colour, has been found in various foodstuffs. The induction potential of metanil yellow on hepatic microsomal cytochrome P-450 (P-450)-dependent monooxygenases and cytosolic detoxification enzymes, namely, glutathione S-transferase (GST) and quinone reductase (QR), was investigated. Oral administration of metanil yellow (430 mg/kg body weight) to four animals for seven days caused significant induction of hepatic P-450 (48%) and its dependent aryl hydrocarbon hydroxylase (100%) activity and cytosolic GST (136%) and QR (92%) activities. Parenteral administration of metanil yellow (80 mg/kg body weight) to another set of four animals for 3 days resulted in higher induction of ethoxyresorufin-O-deethylase (228%) as compared to other monooxygenases (64-92%), while GST and QR were also found to be induced (59-95%). Spectra of metanil yellow-induced microsomes showed an increase in P-450 with a shift of 2.2 nm in the soret region. The results suggest that metanil yellow acts as a bifunctional inducer of specific isozymes of P-450 and cytosolic enzymes and thus may involve the cytosolic aryl hydrocarbon (Ah) receptor for this type of induction.     

Glia modulate NMDA-mediated signaling in primary cultures of cerebellar granule cells

Excessive activation of N-methyl-D-aspartate (NMDA) receptor channels (NRs) is a major cause of neuronal death associated with stroke and ischemia. Cerebellar granule neurons in vivo, but not in culture, are relatively resistant to toxicity, possibly owing to protective effects of glia. To evaluate whether NR-mediated signaling is modulated when developing neurons are cocultured with glia, the neurotoxic responses of rat cerebellar granule cells to applied NMDA or glutamate were compared in astrocyte-rich and astrocyte-poor cultures. In astrocyte-poor cultures, significant neurotoxicity was observed in response to NMDA or glutamate and was inhibited by an NR antagonist. Astrocyte-rich neuronal cultures demonstrated three significant differences, compared with astrocyte-poor cultures: (a) Neuronal viability was increased; (b) glutamate-mediated neurotoxicity was decreased, consistent with the presence of a sodium-coupled glutamate transport system in astrocytes; and (c) NMDA- but not kainate-mediated neurotoxicity was decreased, in a manner that depended on the relative abundance of glia in the culture. Because glia do not express NRs or an NMDA transport system, the mechanism of protection is distinct from that observed in response to glutamate. No differences in NR subunit composition (evaluated using RT-PCR assays for NR1 and NR2 subunit mRNAs), NR sensitivity (evaluated by measuring NR-mediated changes in intracellular Ca2+ levels), or glycine availability as a coagonist (evaluated in the presence and absence of exogenous glycine) were observed between astrocyte-rich and astrocyte-poor cultures, suggesting that glia do not directly modulate NR composition or function. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked NMDA-mediated toxicity in astrocyte-poor cultures, raising the possibility that glia effectively reduce the accumulation of highly diffusible and toxic arachidonic acid metabolites in neurons. Alternatively, glia may alter neuronal development/phenotype in a manner that selectively reduces susceptibility to NR-mediated toxicity.     

Rosemary extract and carnosol stimulate rat liver glutathione-S-transferase and quinone reductase activities

The effects of dietary intake and intraperitoneal (i.p.) administration of an extract of the spice rosemary and of the rosemary constituent carnosol on the liver activities of glutathione-S-transferase (GST) and NAD(P)H-quinone reductase (QR) in the female rat were evaluated. Rosemary extract at concentrations from 0.25 to 1.0% (by wt.) in the diet resulted in a significant 3.5- to 4.5-fold increase in liver GST and a 3.3- to 4.0-fold increase in liver QR activities compared to controls. Carnosol supplemented in the diet at levels from 0.01 to 1.0% did not enhance GST activity. When rosemary extract and carnosol were administered i.p. there was a significant increase in liver GST and QR activities. The injection of rosemary extract (200 mg/kg) was associated with 1.5-fold and 3.2-fold increases in GST and QR activities, respectively, compared to controls. The injection of carnosol at doses from 100 to 400 mg/kg was associated with 1.6- to 1.9-fold increases in GST activity and 3.1- to 4.8-fold increases in QR activity, compared to controls. These data indicate that rosemary extract in the diet or injected i.p. and carnosol administered i.p. are effective enhancers of the in vivo activity of liver GST and QR in the female rat.     

Protein kinase C expression and activity after global incomplete cerebral ischemia in dogs

BACKGROUND AND PURPOSE: Studies suggest that protein kinase C (PKC) activation during ischemia plays an important role in glutamate neurotoxicity and that PKC inhibition may be neuroprotective. We tested the hypothesis that elevations in the biochemical activity and protein expression of Ca2+-dependent PKC isoforms occur in hippocampus and cerebellum during the period of delayed neurodegeneration after mild brain ischemia. METHODS: We used a dog model of 20 minutes of global incomplete ischemia followed by either 6 hours, 1 day, or 7 days of recovery. Changes in PKC expression (Western blotting and immunocytochemistry) and biochemical activity were compared with neuropathology (percent ischemically damaged neurons) by means of hematoxylin and eosin staining. RESULTS: The percentage of ischemically damaged neurons increased from 13+/-4% to 52+/-10% in CA1 and 24+/-11% to 69+/-6% in cerebellar Purkinje cells from 1 to 7 days, respectively. The occurrence of neuronal injury was accompanied by sustained increases in PKC activity (240% and 211% of control in hippocampus and cerebellum, respectively) and increased protein phosphorylation as detected by proteins containing phosphoserine residues. By Western blotting, the membrane-enriched fraction showed postischemic changes in protein expression with increases of 146+/-64% of control in hippocampal PKCalpha and increases of 138+/-38% of control in cerebellar PKCalpha, but no changes in PKCbeta and PKCgamma were observed. By immunocytochemistry, the neuropil of CA1 and CA4 in hippocampus and the radial glia in the molecular layer of cerebellum showed increased PKCalpha expression after ischemia. CONCLUSIONS: This study shows that during the period of progressive ischemic neurodegeneration there are regionally specific increases in PKC activity, isoform-specific increases in membrane-associated PKC, and elevated protein phosphorylation at serine sites.     

Cytokine-mediated neuronal apoptosis

Cytokines have been reported to induce neuronal injury via the free radical nitric oxide (NO); however, the precise mechanism underlying cytokine-mediated neurotoxicity is unclear. We investigated the hypothesis that cytokine-mediated neurotoxicity in primary cultures of human fetal neurons occurs via an apoptotic mechanism triggered by NO. Treatment of mixed neuronal/glial cell cultures with interferon (IFN)-gamma plus interleukin (IL)-1 beta for 13 days induced a high output of NO accompanied by marked neuronal loss. The NO synthase inhibitor N-monomethyl-L-arginine (NMMA) significantly attenuated cytokine-induced neuronal loss, confirming the involvement of NO. Cytokine-mediated neuronal injury was accompanied by morphologic changes and a DNA fragmentation pattern consistent with apoptosis. Treatment of neuronal cell cultures with NMMA protected against cytokine-mediated apoptotic death. These findings, using primary human neuronal cell cultures, support the hypothesis that cytokine-mediated neurotoxicity involving NO proceeds via an apoptotic mechanism. These findings could lead to the development of new therapies for neurodegenerative diseases involving glia, cytokines, and NO.