NADPH oxidase activation and 4-hydroxy-2-nonenal/aquaporin-4 adducts as possible new players in oxidative neuronal damage presents in drug-resistant epilepsy.

A correlation between epilepsy and cellular redox imbalance has been suggested, although the mechanism by which oxidative stress (OS) can be implicated in this disorder is not clear. In the present study several oxidative stress markers and enzymes involved in OS have been determined. In particular, we examined the levels of 4-hydroxy-2-nonenal protein adducts (HNE-PA), a by-product of lipid peroxidation, and the activation of NADPH oxidase 2 (NOX2), as cellular source of superoxide (O(2)(-)), in surgically resected epileptic tissue from drug-resistant patients (N=50). In addition, we investigated whether oxidative-mediated protein damage can affect aquaporin-4 (AQP4), a water channel implicated in brain excitability and epilepsy. Results showed high levels of HNE-PA in epileptic hippocampus, in both neurons and glial cells and cytoplasmic positivity for p47(phox) and p67(phox) suggesting NOX2 activation. Interestingly, in epileptic tissue immunohistochemical localization of AQP4 was identified not only in perivascular astrocytic endfeet, but also in neurons. Nevertheless, negativity for AQP4 was observed in neurons in degeneration. Of note, HNE-mediated post-translational modifications of AQP4 were increased in epileptic tissues and double immunofluorescence clearly demonstrated co-localization of AQP4 and HNE-PA in epileptic hippocampal structures. The idea is that sudden, disorderly, and excessive neuronal discharges activates NOX2 with O(2)(-) production, leading to lipid peroxidation. The resulting generation of HNE targets AQP4, affecting water and ion balance. Therefore, we suggest that seizure induces oxidative damage as well as neuronal loss, thereby promoting neuronal hyperexcitability, also affecting water and ion balance by AQP4 modulation, and thus generating a vicious cycle.

Electrolytic univalent reduction of oxygen in aqueous solution demonstrated with superoxide dismutase.

The superoxide anion, generated electrolytically at a platinum electrode in buffered aqueous solution, was detected by its ability to cause the oxidation of epinephrine to adrenochrome. The rate of electrolytic oxidation of epinephrine varied with the applied potential in a manner reminiscent of an oxygen reduction half wave. This oxidation of epinephrine was dependent upon the presence of oxygen and was completely inhibitable by superoxide dismutase. It may be concluded that superoxide radicals, generated at the electrode, diffuse into the solution to an extent which allows reaction with small molecules such as epinephrine or with enzymes such as superoxide dismutase.

A dual role for calcium in regulation of superoxide generation by stimulated rat alveolar macrophages.

Superoxide production in alveolar macrophages is stimulated by agonists which act through Ca2+-mediated (concanavalin A) and/or protein kinase C (phorbol ester or diacylglycerol analogues) -mediated events. Simultaneous addition of saturating concentrations of concanavalin A and a protein kinase C activator (either phorbol 12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol) caused a supra-additive enhancement of the initial rate of O2-. production. This synergism closely correlated with the known time-course of Ca2+ mobilization induced by concanavalin A; however, it occurred under conditions in which protein kinase C activation is reportedly not Ca2+ dependent. Phorbol ester-induced O2-. production was partially inhibited by the Ca2+ ionophore, A23187. Although phorbol ester-stimulated O2-. production initially was enhanced by concanavalin A, the duration of this O2-. production was reduced in comparison to that induced by phorbol ester alone. These results suggest a dual role for intracellular Ca2+ in both stimulatory and inhibitory regulation of O2-. production.

Release of aldehydes from rat alveolar macrophages exposed in vitro to low concentrations of nitrogen dioxide.

This study demonstrated that aldehydes are released into the extracellular medium when alveolar macrophages (AM) are exposed to nitrogen dioxide (NO2) at concentrations that impair cell function but do not cause cell death. Butanal, glycolaldehyde, 4-hydroxynonenal, pentanal, pentenal, and hexanal were found. Dinitrophenylhydrazine (DNP) derivitization, thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry were used to identify the products. Some of the aldehydes have potential toxicity and may be responsible, in part, for altered AM function observed following NO2 exposure.

Inhibition of production of LTB4 and chemotactic agent from rat alveolar macrophages treated with t-butyl hydroperoxide is independent of ATP depletion.

In rat alveolar macrophages treated with 100 microM t-butyl hydroperoxide (tBOOH), leukotriene B4 (LTB4) synthesis was significantly lower than the basal level while levels of cyclooxygenase pathway products were increased. LTB4, 5,6-dihydroxyeicosatetraenoic acid (5,6-DiHETEs), and 5-hydroxyeicosatetraenoic acid (5-HETE) production in macrophages was significantly stimulated by 2 microM A23187, but this was suppressed 40% by simultaneous addition of 10 microM tBOOH and completely abolished by 100 microM tBOOH. Basal and A23187-stimulated macrophage production of chemotactic agents were similarly suppressed by addition of tBOOH; this effect paralleled depression of cellular LTB4 synthesis. In contrast to the significant depression of A23187-stimulated formation of 5-lipoxygenase products by 10 microM tBOOH, cellular adenosine triphosphate (ATP) was unchanged. Macrophages pretreated with KCN led to a 42% decline in ATP levels; however, LTB4, 5,6-DiHETEs, and 5-HETE production in response to A23187 was not suppressed. The results indicate that inhibition of 5-lipoxygenase pathway products in macrophages treated with tBOOH did not occur by depletion of cellular ATP levels.

Glutathione, stress responses, and redox signaling in lung inflammation.

Changes in the ratio of intracellular reduced and disulfide forms of glutathione (GSH/GSSG) can affect signaling pathways that participate in various physiological responses from cell proliferation to gene expression and apoptosis. It is also now known that many proteins have a highly conserved cysteine (sulfhydryl) sequence in their active/regulatory sites, which are primary targets of oxidative modifications and thus important components of redox signaling. However, the mechanism by which oxidants and GSH/protein-cysteine-thiols actually participate in redox signaling still remains to be elucidated. Initial studies involving the role of cysteine in various proteins have revealed that cysteine-SH may mediate redox signaling via reversible or irreversible oxidative modification to Cys-sulfenate or Cys-sulfinate and Cys-sulfonate species, respectively. Oxidative stress possibly via the modification of cysteine residues activates multiple stress kinase pathways and transcription factors nuclear factor-kappaB and activator protein-1, which differentially regulate the genes for proinflammatory cytokines as well as the protective antioxidant genes. Understanding the redox signaling mechanisms for differential gene regulation may allow for the development of novel pharmacological approaches that preferentially up-regulate key antioxidants genes, which, in turn, reduce or resolve inflammation and injury. This forum article features the current knowledge on the role of GSH in redox signaling, particularly the regulation of transcription factors and downstream signaling in lung inflammation.

Biphasic effects of 15-deoxy-delta(12,14)-prostaglandin J(2) on glutathione induction and apoptosis in human endothelial cells.

The lipid products derived from the cyclooxygenase pathway can have diverse and often contrasting effects on vascular cell function. Cyclopentenone prostaglandins (cyPGs), such as 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)), a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist, have been reported to cause endothelial cell apoptosis, yet in other cell types, cyPGs induce cytoprotective mediators, such as heat shock proteins, heme oxygenase-1, and glutathione (GSH). Herein, we show in human endothelial cells that low micromolar concentrations of 15d-PGJ(2) enhance GSH-dependent cytoprotection through the upregulation of glutamate-cysteine ligase, the rate-limiting enzyme of GSH synthesis, as well as GSH reductase. The effect of 15d-PGJ(2) on GSH synthesis is attributable to the cyPG structure but is independent of PPAR, inasmuch as the other cyPGs, but not PPARgamma or PPARalpha agonists, are able to increase GSH. The increase in cellular GSH is accompanied by abrogation of the proapoptotic effects of 4-hydroxynonenal, a product of lipid peroxidation present in atherosclerotic lesions. However, higher concentrations of 15d-PGJ(2) (10 micromol/L) cause endothelial cell apoptosis, which is further enhanced by depletion of cellular GSH by buthionine sulfoximine. We propose that the GSH-dependent cytoprotective pathways induced by 15d-PGJ(2) contribute to its antiatherogenic effects and that these pathways are distinct from those leading to apoptosis.

Redox signaling in macrophages.

Macrophages are phagocytic cells that produce and release reactive oxygen species (ROS) in response to phagocytosis or stimulation with various agents. The enzyme responsible for the production of superoxide and hydrogen peroxide is a multi-component NADPH oxidase that requires assembly at the plasma membrane to function as an oxidase. In addition to participating in bacterial killing, ROS, which have recently been shown to be produced enzymatically by non-phagocytic cells, have been implicated in inflammation and tissue injury. These toxic effects have been largely explored over the years and these studies have overshadowed initial observations supporting a role for ROS in modulating cellular function. In recent years, it has become increasingly evident that ROS can function as second messengers and, at low levels, can activate signaling pathways resulting in a broad array of physiological responses from cell proliferation to gene expression and apoptosis. Macrophages can also produce large amounts of nitric oxide (nitrogen monoxide, *NO). *NO was first identified as the endothelial-derived relaxing factor, EDRF and its role in the signaling pathway leading to its physiological effect was rapidly established. The ability of *NO to react with O(2)(*-) to produce peroxynitrite (ONOO(-)) was later recognized. As it is diffusion-limited, this reaction is more likely to occur in cells like macrophages that produce both ROS and RNS. In this review, we will summarize the current knowledge in redox signaling, and describe more specifically studies that are particular to macrophages.

Activation of NF kappa B by the respiratory burst of macrophages.

H2O2 and other reduced oxygen species have been proposed as activators of the transcription factor, NF Kappa B. Stimulated macrophages produce superoxide and H2O2 (the respiratory burst). We tested the hypothesis that production of these species could serve as part of the NF Kappa B activation pathway in rat alveolar macrophages and the J774A.1 mouse monocyte/macrophage cell line. Phorbol myristate acetate (PMA) and ADP, which stimulate the respiratory burst, caused NF Kappa B activation in both cells. Catalase abolished NF kappa B activation, while superoxide dismutase produced little inhibition. Thus, H2O2 was the principal agent of respiratory burst-associated NF kappa B activation. Abolition of NF kappa B activation by catalase also suggested that intermediate signaling pathways, such as protein kinase C activation or intracellular free calcium elevation must not be involved. Exogenous H2O2 added as a bolus > or = 50 microM (> or = 50 nmol/10(6) macrophages) also activated NF kappa B in macrophages. Nevertheless, the maximum endogenous production of H2O2 by stimulated alveolar macrophages during a 30-min incubation was < or = 1.3 nmol H2O2/10(6) cells for PMA stimulation and < or = 0.2 nmol H2O2/10(6) cells for ADP stimulation. Thus, relatively little endogenous H2O2 generation was required to produce NF kappa B activation compared to the required amount of exogenous H2O2. As H2O2 rapidly diffuses and is consumed, these results suggest that the site of action for endogenously generated H2O2 is probably close to its origin, the plasma membrane.

Adhering lung macrophages produce superoxide demonstrated with desferal-Mn(IV).

It has been shown that H2O2, the dismutation product of O2., is produced at cell-surface interfaces. Nevertheless, the relationships between the degree of attachment itself, type of surface, and O2. production are not clear. Superoxide production can be measured by the O2.-dependent reduction of nitroblue tetrazolium to an insoluble formazan. Superoxide dismutase (SOD) may be unable to scavenge O2. produced between alveolar macrophages (AM) and a surface. Desferal-Mn(IV) (Des-Mn), a low molecular weight mimic of SOD, is protective against paraquat toxicity in vivo, presumably because of specificity for O2-. Using that assumption, Des-Mn was used to measure O2. production that occurred during adherence of AM. AM suspensions were placed on fibronectin-coated glass coverslips or uncoated glass coverslips or non-stick tissue culture plates. Adherence to the surfaces varied with fibronectin greater than glass greater than non-stick and the percent formazan positive cells was 60, 24, and 4, respectively. With SOD present, the percentage of formazan positive cells were 40, 17, and 2; however, in the presence of Des-Mn the percent stained cells was 4, 4, and 0. When phorbol myristate acetate (PMA) was added during adherence, the percent of formazan positive cells was 82, 57, and 44, respectively. With PMA, Des-Mn was able to inhibit 88-100% of formazan staining whereas SOD inhibition decreased more markedly with increasing adherence. These results indicated that the degree of attachment correlated with both the degree of NBT reduction and the relative effectiveness of Des-Mn versus SOD to scavenge O2..

Oxidants as stimulators of signal transduction.

Redox (oxidation-reduction) reactions regulate signal transduction. Oxidants such as superoxide, hydrogen peroxide, hydroxyl radicals, and lipid hydroperoxides (i.e., reactive oxygen species) are now realized as signaling molecules under subtoxic conditions. Nitric oxide is also an example of a redox mediator. Reactive oxygen species induce various biological processes such as gene expression by stimulating signal transduction components such as Ca(2+)-signaling and protein phosphorylation. Various oxidants increase cytosolic Ca2+; however, the exact origin of Ca2+ is controversial. Ca2+ may be released from the endoplasmic reticulum, extracellular space, or mitochondria in response to oxidant-influence on Ca2+ pumps, channels, and transporters. Alternatively, oxidants may release Ca2+ from Ca2+ binding proteins. Various oxidants stimulate tyrosine as well as serine/threonine phosphorylation, and direct stimulation of protein kinases and inhibition of protein phosphatases by oxidants have been proposed as mechanisms. The oxidant-stimulation of the effector molecules such as phospholipase A2 as well as the activation of oxidative stress-responsive transcription factors may also depend on the oxidant-mediated activation of Ca(2+)-signaling and/or protein phosphorylation. In addition to the stimulation of signal transduction by oxidants, the observations that ligand-receptor interactions produce reactive oxygen species and that antioxidants block receptor-mediated signal transduction led to a proposal that reactive oxygen species may be second messengers for transcription factor activation, apoptosis, bone resorption, cell growth, and chemotaxis. Physiological significance of the role of biological oxidants in the regulation of signal transduction as well as the mechanisms of the oxidant-stimulation of signal transduction are discussed.

Transmembrane redox signaling activates NF-kappaB in macrophages.

In macrophages, NF-kappaB can be activated by H2O2 generated by the respiratory burst or added exogenously. The mechanism of H2O2 signaling may involve changes in the cellular redox state or a redox reaction at the plasma membrane; however, the site of H2O2 action cannot be readily ascertained because of its membrane permeability. Ferricyanide, a nonpermeable redox active anion, activated NF-kappaB in the macrophage cell line, J774A.1. In contrast with exogenous H2O2, activation by ferricyanide did not correlate with net oxidation of NAD(P)H or glutathione, suggesting that a transplasma membrane redox reaction itself was the first signaling process in NF-kappaB activation.

ADP stimulates the respiratory burst without activation of ERK and AKT in rat alveolar macrophages.

Alveolar macrophages (AM) are the first line of defense against infection in the lungs. We previously showed that the production of superoxide and hydrogen peroxide, i.e., the respiratory burst, is stimulated by adenine nucleotides (ADP >> ATP) in rat AM through signaling pathways involving calcium and protein kinase C. Here, we further show that ADP induces a rapid increase in the tyrosine phosphorylation of several proteins that was reduced by the tyrosine kinase inhibitor genistein, which also inhibited the respiratory burst. Interestingly, ADP did not trigger the activation of the mitogen-activated protein kinases ERK1 and ERK2, or that of protein kinase B/AKT, a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway. This is in contrast to another stimulus of the respiratory burst, zymosan-activated serum (ZAS), which activates both the ERK and PI3K pathways. Thus, this study demonstrates that the receptor for ADP in rat AM is not coupled to the ERK and AKT pathways and, that neither the ERK pathway nor AKT is essential to induce the activation of the NAPDH oxidase by ADP in rat AM while tyrosine kinases appeared to be required. The rate and amount of hydrogen peroxide released by the ADP-stimulated respiratory burst was similar to that produced by ZAS stimulation. The absence of ERK activation after ADP stimulation therefore suggests that hydrogen peroxide is not sufficient to activate the ERK pathway in rat AM. Nonetheless, as hydrogen peroxide was necessary for ERK activation by ZAS, this indicates that, in contrast to ADP, ZAS stimulates a pathway that is targeted by hydrogen peroxide and leads to ERK activation.

Extracellular glutathione and gamma-glutamyl transpeptidase prevent H2O2-induced injury by 2,3-dimethoxy-1,4-naphthoquinone.

Quinones are intracellular H2O2 generators that have been used extensively in models of oxidant injury; however, their toxicity is mediated partially through direct conjugation with glutathione (GSH). To focus upon the action of extracellular GSH in preventing H2O2-mediated toxicity, we used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which cannot conjugate with GSH but does continuously generate H2O2 through redox cycling. A eukaryotic cell line (3T3-GGT) stably overexpressing gamma-glutamyl transpeptidase (GGT) activity was used to study the role of GGT in utilizing extracellular GSH against DMNQ-induced oxidative stress. DMNQ (0 to 150 microM) caused a dose-dependent decrease of intracellular GSH and adenosine 5'-triphosphate (ATP) in both control and 3T3-GGT cells. The rate of H2O2 escape into the medium during DMNQ exposure was also the same in both cell lines. Administration of GSH helped to maintain intracellular GSH and supported resistance to ATP depletion caused by DMNQ in 3T3-GGT cells but not in control cells. The protective effect of extracellular GSH was completely prevented by acivicin, an inhibitor of GGT. Our results suggest that GGT-dependent breakdown of extracellular GSH for subsequent intracellular resynthesis helped to maintain cellular GSH levels and increased cellular resistance against DMNQ-induced oxidative injury.

Detecting and identifying volatile aldehydes as dinitrophenylhydrazones using gas chromatography mass spectrometry.

The detection of aldehydes has become an important measure of lipid oxidation in biological milieu. Aldehyde 2,4-dinitrophenylhydrazones are easily prepared and readily purified by HPLC and/or TLC and have proven useful for the detection of aldehydes. The lower limit of detection for dinitrophenylhydrazones was significantly reduced by using gas chromatography-mass spectrometric (GC-MS) techniques. Individual dinitrophenylhydrazones were readily separated by GC and detected by both positive and negative ion MS. The two major ions in negative ion spectra were the 182 m/z fragment ion and the molecular ion. Positive ion spectra showed strong ions corresponding to the protonated molecular ion and a protonated iminium ion. The greatest sensitivity was obtained with negative ion detection (10 pg per injection). However, more structural information was obtained from analysis of the positive ion spectra. Dinitrophenylhydrazones of hydroxyaldehydes, like 4-hydroxynonenal, were analyzed after converting the dinitrophenylhydrazones into trimethylsiloxylethers. GC-MS with negative ion detection was used to identify and quantitate the release of 4-hydroxynonenal by alveolar macrophages exposed to nitrogen dioxide.

Modulation of the alveolar macrophage respiratory burst by hydroperoxides.

Exposure of alveolar macrophages to hydroperoxides (ROOH) inhibits subsequent stimulation of O2.- production (the respiratory burst). Previous studies (under nonoxidant stress conditions) have shown that elevation of intracellular free calcium ([Ca2+]i) participates in both initiation and termination of O2.- production. In this investigation, the effects of sublethal ROOH exposure on [Ca2+]i and the respiratory burst of rat alveolar macrophages were compared. Exposure to a sublethal range of H2O2 or tert-butylhydroperoxide (10-100 pmol/10(6) cells; initially 10-100 microM under the experimental conditions) for 15 min resulted in dose-dependent effects on the respiratory burst stimulated by various agents, ADP, ATP, zymosan-activated serum, and phorbol myristate acetate. Low concentrations of the ROOH (10 or 25 pmol/10(6) cells) were found to enhance stimulation, whereas exposure to 75 or 100 pmol/10(6) cells resulted in significant inhibition for all of the stimuli. All concentrations of ROOH caused a rapid elevation in [Ca2+]i. For those concentrations of ROOH that produced enhancement of subsequent stimulation of the respiratory burst, [Ca2+]i returned to near baseline before the end of the 15-min preincubation. The temporal- and concentration-dependent effects of ROOH on [Ca2+]i correlate with subsequent enhancement or inhibition of stimulated O2.- production. Similarities between the ROOH-induced changes in [Ca2+]i and the effect of [Ca2+]i changes in physiological regulation of the respiratory burst suggest a potential relationship.

Dominant-negative Jun N-terminal protein kinase (JNK-1) inhibits metabolic oxidative stress during glucose deprivation in a human breast carcinoma cell line.

Signal transduction pathway involved in glucose deprivation-induced oxidative stress were investigated in human breast carcinoma cells (MCF-7/ADR). In MCF-7/ADR, glucose deprivation-induced prolonged activation of c-Jun N-terminal kinase (JNK1) as well as cytoxicity and the accumulation of oxidized glutathione. Glucose deprivation also caused significant increases in total glutathione, cysteine, gamma-glutamylcysteine, and immunoreactive proteins corresponding to the catalytic as well as regulatory subunits of gamma-glutamylcysteine, and immunoreactive proteins corresponding to the catalytic as well as regulatory subunits of gamma-glutamylcysteine synthetase, suggesting that the synthesis of glutathione increased as an adaptive response. Expression of a catalytically inactive dominant negative JNK1 in MCF-7/ADR inhibited glucose deprivation- induced cell death and the accumulation of oxidized glutathione as well as altered the duration of JNK activation from persistent (> 2 h) to transient (30 min). In addition, stimulation of glutathione synthesis during glucose deprivation was not observed in cells expressing the highest levels of dominant negative protein. Finally, a linear dose response suppression of oxidized glutathione accumulation was noted for clones expressing increasing levels of dominant negative JNK1 during glucose deprivation. These results show that expression of a dominant negative JNK1 protein was capable of suppressing persistent JNK activation as well as oxidative stress and cytotoxicity caused by glucose deprivation in MCF-7/ADR. These findings support the hypothesis that JNK signaling pathways may control the expression of proteins contributing to cell death mediated by metabolic oxidative stress during glucose deprivation. Finally, these results support the concept that JNK signaling-induced shifts in oxidative metabolism may provide a general mechanism for understanding the diverse biological effects seen during the activation of JNK signaling cascades.

The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells: a role for gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase.

Nitric oxide protects cells from oxidative stress through a number of direct scavenging reactions with free radicals but the effects of nitric oxide on the regulation of antioxidant enzymes are only now emerging. Using bovine aortic endothelial cells as a model, we show that nitric oxide, at physiological rates of production (1-3 nM/s), is capable of inducing the synthesis of glutathione through a mechanism involving gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase. This novel nitric oxide signalling pathway is cGMP-independent and we hypothesize that it makes an important contribution to the anti-atherosclerotic and antioxidant properties of nitric oxide.

Buthionine sulphoximine alone and in combination with melphalan (L-PAM) is highly cytotoxic for human neuroblastoma cell lines.

Buthionine sulphoximine (BSO) selectively inhibits glutathione (GSH) synthesis and may enhance the antineuroblastoma activity of melphalan (L-PAM). We determined the cytotoxicity of BSO (dose range 0-1000 microM) alone and in combination with L-PAM (dose range 0-0 microM) in a panel of 18 human neuroblastoma cell lines. BSO alone was highly cytotoxic with 16/18 neuroblastoma cell lines having IC90 values (range 2.1- > 1000 microM) below the clinically achievable steady-state plasma level of 500 microM BSO. Maximal cell killing correlated with GSH levels decreased to less than 10% baseline, and was partially reversed by the addition of exogenous anti-oxidants (GSH, vitamin E and ascorbate). Fluorocytometric analysis of DNA fragments by the Tunnel method detected 92% of a BSO sensitive cell line in apoptosis after a 48 h exposure to 500 microM BSO. The combination of L-PAM and BSO synergistically enhanced the cell killing of L-PAM alone by > 1-3 logs (combination index < 1). We conclude that BSO has significant single-agent cytotoxicity against neuroblastoma and enhances cell killing when combined with L-PAM.

Adaptation to oxidative stress: quinone-mediated protection of signaling in rat lung epithelial L2 cells.

Cells can respond to a sublethal oxidative stress by up-regulating their intracellular glutathione (GSH) pool. Such increased GSH concentration is likely to be protective against further oxidative challenge, and, in fact, pre-exposure to low levels of oxidants confers increased cellular resistance to subsequent greater oxidative stress. Previously, we have shown that pretreatment of rat lung epithelial L2 cells with sublethal concentrations of tert-butylhydroquinone (TBHQ) increases intracellular GSH concentration in a concentration- and time-dependent manner. This increase resulted from up-regulation of both gamma-glutamyltranspeptidase (GGT) and gamma-glutamylcysteine synthetase (GCS). Therefore, we investigated whether such increased GSH concentration protected these cells against a subtle loss in function caused by a subsequent challenge with sublethal concentrations of tert-butyl hydroperoxide (tBOOH) (< or = 200 microM), mimicking a physiological oxidative stress. Activation of L2 cell purinoreceptors with 100 microM ADP caused an elevation of intracellular Ca2+. This response was suppressed by a brief pre-exposure to tBOOH. The inhibition, however, was alleviated dramatically by a 16-hr pretreatment with 50 microM TBHQ. The same TBHQ pretreatment also protected the cells from ATP-depletion induced by tBOOH. L-Buthionine S,R-sulfoximine (BSO), an irreversible inhibitor of GCS, prevented the increase in intracellular GSH and also completely removed the protection by TBHQ in maintaining the ATP level. Thus, pre-exposure to a sublethal level of TBHQ results in protection of cell functions from hydroperoxide toxicity. This protection appears to depend on alteration of the intracellular GSH pool, the modulation of which constitutes an adaptive response to oxidative stress.