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Chemicals bearing hydrosulfide groups are normally detected by a reaction based on the formation of poorly soluble mercaptides of silver, mercury, and lead. Thiols, disulfides, and thioethers are identified by [...], A novel method is developed for the detection cysteine, reduced glutathione, using organoselenium compound diacetophenonyl selenide as a reagent, and the optimal conditions of performing the reaction are found. The method is applicable to the detection of diacetophenonyl selenide by its reaction with cysteine as well. Keywords: cysteine, reduced glutathione, diacetophenonyl selenide, elementary selenium, thin-layer chromatography DOI: 10.1134/S1061934813110105

This study investigated the mechanisms responsible for the disrupted homeostasis of reduced glutathione (GSH) in aging muscles with stress (14 days of hind-limb unloading [HU]). Adult and old rats were randomized into four groups: weight bearing and 3, 7, and 14 days of HU. Soleus muscles were harvested to investigate the activity or content of enzymes involved in GSH metabolism (utilization and synthesis). The activities of glutathione S transferase, glutathione reductase, [gamma]-glutamyl transpeptidase, and glutamate cysteine ligase (GCL) were determined. The protein content of the two subunits of GCL, catalytic subunit (GCLC) and modifier subunit (GCLM), were evaluated. The major results, failure to maintain the accelerated GCLC production and GCL activity, are associated with the GSH depletion in aging muscles with 14 days of HU. The results suggest that the regulation of GCL, especially the catalytic subunit, with stress may be compromised in aging muscles. Key Words: GCL--GCLC--Hind-limb unloading. doi: 10.1093/gerona/glp194

Oxidative stress plays an important role in obesity-related metabolic diseases. Glutathione peroxidase (GPX) is an antioxidant enzyme downregulated in adipose tissue of obese mice. However, the role of GPX in adipocytes remains elusive. The objective of this study was to clarify the pathophysiological changes in GPX activity and glutathione metabolism and their roles in the pathogenesis of insulin resistance in adipocytes. To achieve this goal, we measured cellular GPX activity, glutathione (GSH) contents, GSH/GSSG ratio, and mRNA expression of [gamma]-glutamylcysteine synthetase ([gamma]-GCS), a rate-limiting enzyme for de novo GSH synthesis, in adipose tissue of control and ob/ob mice and in 3T3-L1 adipocytes treated with insulin, [H.sub.2][O.sub.2], free fatty acid (FFA), or TNF[alpha]. Furthermore, we investigated the effects of GPX inhibition with a specific GPX inhibitor or RNA interference against GPX, [H.sub.2][O.sub.2], and reduced GSH on insulin signaling in 3T3-L1 adipocytes, ob/ob Mice showed not only a decrease in cellular activity of GPXs (GPX1, -4, and -7) but also an increase in [gamma]-GCS expression, resulting in increased GSH contents in adipose tissue. These alterations in glutathione metabolism were also observed during differentiation of 3T3-L1 cells and their exposure to insulin, FFA ,or [H.sub.2][O.sub.2]. Inhibition of GPX activity, addition of GSH, and [H.sub.2][O.sub.2] resulted in impaired insulin signaling in 3T3-L1 adipocytes. These results suggest that decreased GPX activity and increased [gamma]-GCS expression lead to overaccumulation of GSH, which might be involved in the pathogenesis of insulin resistance in obesity. glutathione peroxidase; [gamma]-glutamylcysteine synthetase; oxidative stress; insulin resistance

Reactive oxygen species (ROS), such as hydrogen peroxide ([H.sub.2][O.sub.2]) and nitric oxide (NO), have been shown to differentially alter the [Ca.sup.2+] sensitivity of the contractile apparatus of fast-twitch skeletal muscle, leading to the proposal that normal muscle function is controlled by perturbations in the amounts of these two groups of molecules (28). However, no previous studies have examined whether these opposing actions are retained when the contractile apparatus is subjected to both molecule types. Using mechanically skinned fast- and slow-twitch skeletal muscle fibers of the rat, we compared the effects of sequential addition of nitrosoglutathione (GSNO), a NO donor, and [H.sub.2][O.sub.2] on the [Ca.sup.2+] sensitivity of the contractile apparatus. As expected from previous reports in fast-twitch fibers, when added separately, GSNO (1 mM) reduced the [Ca.sup.2+] sensitivity of the contractile apparatus, whereas [H.sub.2][O.sub.2] (10 mM; added during contractions) increased the [Ca.sup.2+] sensitivity of the contractile apparatus. When added sequentially to the same fiber, such that the oxidation by one molecule (e.g., GSNO) preceded the oxidation by the other (e.g., [H.sub.2][O.sub.2]), and vice versa, the individual effects of both molecules on the [Ca.sup.2+] sensitivity were retained. Interestingly, neither molecule had any effect on the [Ca.sup.2+] sensitivity of slow-twitch skeletal muscle. The data show that [H.sub.2][O.sub.2] and GSNO retain the capacity to independently affect the contractile apparatus to modulate force. Furthermore, the absence of effects in slow-twitch muscle may further explain why this fiber type is relatively insensitive to fatigue. nitric oxide; reactive oxygen species, nitrosoglutathione; hydrogen peroxide

Glutathione (GSH) and bilirubin are prominent endogenous antioxidant cytoprotectants. Despite tissue levels that are thousands of times lower than GSH, bilirubin is effective because of the biosynthetic cycle wherein it is generated from biliverdin by biliverdin reductase (BVR). When bilirubin acts as an antioxidant, it is oxidized to biliverdin, which is immediately reduced by BVR to bilirubin. Why does the body employ both of these 2 distinct antioxidant systems? We show that the water-soluble GSH primarily protects water soluble proteins, whereas the lipophilic bilirubin protects lipids from oxidation. Mice with deletion of heine oxygenase-2, which generates biliverdin, display greater lipid than protein oxidation, while the reverse holds for GSH depletion. RNA interference depletion of BVR increases oxidation of lipids more than protein. Depletion of BVR or GSH augments cell death in an oxidant-specific fashion. apoptosis | biliverdin | cell death | heme oxygenase | neuroprotection

Small coronary arteries (SCA) from diabetic rats exhibit enhanced peroxynitrite ([ONOO.sup.-]) formation and concurrent impairment of voltage-dependent potassium ([K.sub.v]) channel function. However, it is unclear whether [ONOO.sup.-] plays a causative role in this impairment. We hypothesized that functional loss of [K.sub.v] channels in coronary smooth muscle cells (SMC) in diabetes is due to ONOO with subsequent tyrosine nitration of [K.sub.v] channel proteins. Diabetic rats and nondiabetic controls were treated with or without ebselen (Eb) for 4 wk. SCA were prepared for immunohistochemistry (IHC), immunoprecipitation (IP) followed by Western blot (WB), videomicroscopy, and patch-clamp analysis. IHC revealed excess [ONOO.sup.-] in SCA from diabetic rats. IP and WB revealed elevated nitration of the [K.sub.v]1.2 [alpha]-subunit and reduced [K.sub.v]l.2 protein expression in diabetic rats. Each of these changes was improved in Eb-treated rats. Protein nitration and [K.sub.v]1.5 expression were unchanged in SCA from diabetic rats. Forskolin, a direct cAMP activator that induces [K.sub.v]1 channel activity, dilated SCA from nondiabetic rats in a correolide (Cor; a selective [K.sub.v]1 channel blocker)-sensitive fashion. Cor did not alter the reduced dilation to forskolin in diabetic rats; however, Eb partially restored the Cor-sensitive component of dilation. Basal [K.sub.v] current density and response to forskolin were improved in smooth muscle cells from Eb-treated DM rats. We conclude that enhanced nitrosative stress in diabetes mellitus contributes to [K.sub.v]1 channel dysfunction in the coronary microcirculation. Eb may be beneficial for the therapeutic treatment of vascular complications in diabetes mellitus. diabetes; peroxynitrite; oxidative stress; coronary circulation

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is the only known apical glutathione (GSH) transporter in the lung. The purpose of these studies was to determine whether oral GSH or glutathione disulfide (GSSG) treatment could increase lung epithelial lining fluid (ELF) GSH levels and whether CFTR plays a role in this process. The pharmacokinetic profile of an oral bolus dose of GSH (300 mg/kg) was determined in mice. Plasma, ELF, bronchoalveolar lavage (BAL) cells, and lung tissue were analyzed for GSH content. There was a rapid elevation in the GSH levels that peaked at 30 min in the plasma and 60 min in the lung, ELF, and BAL cells alter oral GSH dosing. Oral GSH treatment produced a selective increase in the reduced and active form of GSH in all lung compartments examined. Oral GSSG treatment (300 mg/kg) resulted in a smaller increase of GSH levels. To evaluate the role of CFTR in this process, Cftr knockout (KO) mice and gut-corrected Cftr KO-transgenic (Tg) mice were given an oral bolus dose of GSH (300 mg/kg) and compared with wild-type mice for changes in GSH levels in plasma, lung, ELF, and BAL cells. There was a twofold increase in plasma, a twofold increase in lung, a fivefold increase in ELF, and a threefold increase in BAL cell GSH levels at 60 min in wild-type mice; however, GSH levels only increased by 40% in the plasma, 60% in the lung, 50% in the ELF, and twofold in the BAL cells within the gut-corrected Cftr KO-Tg mice. No change in GSH levels was observed in the uncorrected Cftr KO mice. These studies suggest that CFTR plays an important role in GSH uptake from the diet and transport processes in the lung. nutrition; transport; high-performance liquid chromatography; mice; cystic fibrosis; antioxidant; cystic fibrosis transmembrane conductance regulator doi: 10.1152/ajplung.00365.2006

Higher requirements for disulfide bond formation in professional secretory cells may affect intracellular redox homeostasis, particularly during an endoplasmic reticulum (ER) stress response. To assess this hypothesis, we investigated the effects of the ER stress response on the major redox couple (GSH/GSSG), endogenous ROS production, expression of genes involved in ER oxidative protein folding, general antioxidant defense, and thiol metabolism by use of the well-validated MIN6 [beta]-cell as a model and mouse islets. The data revealed that glucose concentration-dependent decreases in the GSH/GSSG ratio were further decreased significantly by ER-derived oxidative stress induced by inhibiting ER-associated degradation with the specific proteasome inhibitor lactacystin (10 [micro]M) in mouse islets. Notably, minimal cell death was observed during 12-h treatments. This was likely attributed to the upregulation of genes encoding the rate limiting enzyme for glutathione synthesis ([gamma]-glutamylcysteine ligase), as well as genes involved in antioxidant defense (glutathione peroxidase, peroxiredoxin-1) and ER protein folding (Grp78/BiP, PDI, Erol). Gene expression and reporter assays with a NO synthase inhibitor ([N.sup.[omega]]-nitro-L-arginine methyl ester, 1-10 mM) indicated that endogenous NO production was essential for the upregulation of several ER stress-responsive genes. Specifically, gel shift analyses demonstrate NO-independent binding of the transcription factor NF-E2-related factor to the antioxidant response element Gclc-ARE4 in MIN6 cells. However, endogenous NO production was necessary for activation of Gclc-ARE4-driven reporter gene expression. Together, these data reveal a distinct protective role for NO during the ER stress response, which helps to dissipate ROS and promote [beta]-cell survival. endoplasmic reticulum-associated degradation; glutathione; proteasome doi: 10.1152/ajpendo.00620.2006

A study was conducted to identify the specific cysteine residues involved in the protein S-glutathiolation of complex I at both the 51 kDa and 75 kDa subunits of NQR with a combination of immunoblotting and mass spectrometry. The results implies that S-glutathiolation of the 75 kDa subunit might play a role in protecting the 4Fe-4S cluster of the 51 kDa subunit from redox modification when complex 1 is exposed to redox change in the GSH pool.

We have previously demonstrated that chronic alcohol exposure decreases glutathione in the alveolar space. Although alcohol use is associated with decreased alveolar macrophage function, the mechanism by which alcohol impairs macrophage phagocytosis is unknown. In the current study, we examined the possibility that ethanol-induced alveolar macrophage dysfunction was secondary to decreased glutathione and subsequent chronic oxidative stress in the alveolar space. After 6 wk of ethanol ingestion, oxidant stress in the alveolar macrophages was evidenced by a 30-mV oxidation of the GSH/GSSG redox potential (P [less than or equal to] 0.05). For control macrophages, ~80% internalized fluorescent Staphylococcus aureus were added in vitro. In contrast, only 20% of the macrophages from the ethanol-fed rats were able to bind and internalize fluorescent S. aureus. This ethanol-induced decreased capacity for phagocytosis was paralleled by increased apoptosis. When added to the ethanol diet, the glutathione precursors procysteine or N-acetyl cysteine normalized glutathione and oxidant stress in the epithelial lining fluid as well as the alveolar macrophages to control values. This attenuation of oxidant stress was associated with normalization of macrophage phagocytosis and viability. These results suggested that decreased glutathione availability in the alcoholic lung contribute to alveolar macrophage dysfunction via oxidative stress, resulting in not only decreased function but decreased viability. oxidative stress; apoptosis; lung; antioxidants

The cause of 95% of Parkinson's disease (PD) cases is unknown. It is hypothesized that PD arises from an interaction of free-radicalgenerating agents with an underlying genetic susceptibility to these compounds. Here we use the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of parkinsonism to examine the role of a dual function protein, GST [pi], in dopaminergic neuron death. GST [pi] is the only GST family member expressed in substantia nigra neurons. GST [pi] reduction by pharmacological blockade, RNA inhibition, and gene targeting increases sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, suggesting that differential expression of GST [pi] contributes to the sensitivity to xenobiotics in the substantia nigra and may influence the pathogenesis of reactive oxygen species-induced neurological disorders including PD. glutathione | oxidative stress | Parkinson's disease | substantia nigra | detoxification

Glutathione (GSH) concentration affects cell proliferation and apoptosis in intestinal and other cell lines in vitro. However, in vivo data on gut mucosal GSH redox status and cell turnover are limited. We investigated the effect of altered GSH redox status on the ileal mucosa in a rat model of short bowel syndrome following massive small bowel resection (SBR). Rats underwent 80% mid-jejunoileal resection (RX) or small bowel transection (TX; as operative controls), with administration of either saline or D, L-buthionine-sulfoximine (BSO), a specific inhibitor of cellular GSH synthesis. Ileal mucosal redox, morphology, and indices of cell proliferation and apoptosis were determined at different days after surgery. Ileal GSH redox status was assessed by GSH and GSH disulfide (GSSG) concentrations and the redox potential of GSH/GSSG ([E.sub.h]). Ileal lipid peroxidation [free malondialdehyde (MDA)] was measured as an index of lipid peroxidation. BSO markedly decreased ileal mucosal GSH, oxidized GSH/GSSG [E.sub.h], and increased MDA content without inducing morphological damage as assessed by light or electron microscopy. As expected, SBR stimulated adaptive growth of ileal villus height and total mucosal height at 7 d after surgery, but this response was unaffected by BSO treatment despite a modest increase in crypt cell apoptosis. Ileal cell proliferation (crypt cell bromodeoxyuridine incorporation) increased at 2 d after SBR but was unaffected by BSO. Collectively, our in vivo data show that marked depletion of ileal GSH and oxidation of the GSH redox pool does not alter indices of ileal epithelial proliferation or SBR-induced ileal mucosal adaptive growth.

Oxidative stress during cardiac arrest may inactivate myocardial enzymes and thereby exacerbate ischemic derangements of myocardial metabolism. This study examined the impact of cardiac arrest on left ventricular enzymes. Beagles were subjected to 5 min of cardiac arrest and 5 min of open-chest cardiac compressions (OCCC) before epicardial direct current countershocks were applied to restore sinus rhythm. Glutathione/glutathione disulfide redox state (GSH/GSSG) and a panel of enzyme activities were measured in snap-frozen left ventricle. To test whether oxidative stress during arrest inactivated the enzymes, metabolic (pyruvate) or pharmacological (N-acetyl-L-cysteine) antioxidants were infused intravenously for 30 min before arrest. During cardiac arrest, activities of phosphofructokinase, citrate synthase, aconitase, malate dehydrogenase, creatine kinase, glucose-6-phosphate dehydrogenase, and glutathione reductase fell by 56, 81, 55, 34, 42, 55, and 45%, respectively, coincident with 50% decline in GSH/GSSG. OCCC effected full recovery of glutathione reductase and partial recovery of citrate synthase and aconitase, in parallel with GSH/GSSG. Phosphofructokinase, malate dehydrogenase, creatine kinase, and glucose-6-phosphate dehydrogenase recovered only after cardioversion. Antioxidant pretreatments augmented phosphofructokinase, aconitase, and malate dehydrogenase activities before arrest and enhanced these activities, as well as those of citrate synthase and glucose-6-phosphate dehydrogenase, during arrest. In conclusion, cardiac arrest reversibly inactivates several important myocardial metabolic enzymes. Antioxidant protection of these enzymes implicates oxidative stress as a principal mechanism of enzyme inactivation during arrest. glutathione; pyruvate; N-acetyl-L-cysteine; phosphofructokinase; creatine kinase; citrate synthase

Peroxiredoxin 6 (Prdx6) is a novel peroxidase enzyme belonging to the Prdx family, which in mammals contains five more peroxiredoxins (Prdx1-Prdx5). Like glutathione peroxidase (GSHPx) and catalase, Prdx6 possesses [H.sub.2][O.sub.2]-scavenging activities, and, like the former, it also removes hydroperoxides. Since significant amounts of catalase and GSHPx are present in the heart contributing toward the attenuation of [H.sub.2][O.sub.2] and hydroperoxides formed during ischemia-reperfusion injury and thereby providing cardioprotection, we asked whether Prdx6 also has any role in this process. In the present study we used Prdx[6.sup.-/-] mice to assess the role of Prdx6 in ischemic injury. Western blot analysis revealed the absence of any Prdx activity in the Prdx[6.sup.-/-] mouse heart, while the GSHPx-1 and catalase levels remained unchanged. Randomly selected hearts from Prdx[6.sup.-/-] mice and wild-type mice were subjected to 30 min of global ischemia followed by 120 min of reperfusion at normothermia. The hearts from the Prdx[6.sup.-/-] mice were more susceptible to ischemic reperfusion injury as evidenced by reduced recovery of left ventricular function, increased myocardial infarct size, and higher amount of apoptotic cardiomyocytes compared with wild-type mouse hearts. These Prdx[6.sup.-/-] hearts were also subjected to a higher amount of oxidative stress as evidenced by the presence of higher amount of malondialdehyde. The present study thus indicates a nonredundant role of Prdx6 in myocardial ischemic reperfusion injury as catalase, and GSHPx could not make up for the deficiency of Prdx6 activities. redox signaling; reactive oxygen species; glutathione; catalase; glutathione peroxidase; peroxiredoxin gene knockout mice

Abstract: Formation of the shoot apical meristem (SAM) has been extensively investigated in zygotic embryos of flowering plants, where it follows a prolonged and dynamic developmental pattern underlined by precise [...]

The nucleophilic addition of the aminothiols homocysteine (HCY), cysteine (CYS), and glutathione (GSH) to the electrogenerated quinone of fluorone black (1) via the ECE mechanism is reported. It is demonstrated that 1 selectively reacts with GSH to form the bis-GSH adduct, 1-[(GSH).sub.2], while only the monothiol adducts were generated in the presence of HCY and CYS (1-HCY and 1-CYS, respectively). The more anodic [E.sub.pa] of 1-[(GSH).sub.2] relative to 1 and 1-GSH ([DELTA][E.sub.pa] ~ +0.14) is the voltammetric signature that allows the discrimination of GSH from HCY and CYS. It is also shown that the presence of structurally similar aminothiols--HCY and CYS--posed no interference to the signature voltammetric response of 1-[(GSH).sub.2].

The precise mechanisms underlying skeletal muscle damage in Duchenne muscular dystrophy (DMD) remain ill-defined. Functional ischemia during muscle activation, with subsequent reperfusion during rest, has been documented. Therefore, one possibility is the presence of increased oxidative stress. We applied a model of acute hindlimb ischemia/reperfusion (I/R) in mdx mice (genetic homolog of DMD) to evaluate dynamic in vivo responses of dystrophic muscles to this form of oxidative stress. Before the application of I/R, mdx muscles showed: 1) decreased levels of total glutathione (GSH) with an increased oxidized (GSSG)-to-reduced (GSH) glutathione ratio; 2) greater activity of the GSH-metabolizing enzymes glutathione peroxidase (GPx) and glutathione reductase; and 3) lower activity levels of NADP-linked isocitrate dehydrogenase (ICDH) and aconitase, two metabolic enzymes that are sensitive to inactivation by oxidative stress and also implicated in GSH regeneration. Interestingly, nondystrophic muscles subjected to UR exhibited similar changes in total glutathione, GSSG/GSH, GPx, ICDH, and aconitase. In contrast, all of the above remained stable in mdx muscles subjected to I/R. Taken together, these results suggest that mdx muscles are chronically subjected to increased oxidative stress, leading to adaptive changes that attempt to protect (although only in part) the dystrophic muscles from acute I/R-induced oxidative stress. In addition, mdx muscles show significant impairment of the redox-sensitive metabolic enzymes ICDH and aconitase, which may further contribute to contractile dysfunction in dystrophic muscles. reduced glutathione; glutathione reductase; Duchenne muscular dystrophy doi:10.1152/ajpregu.00031.2006

The development of menadione chemistry had led to choice of 6-[2'-(3'-methyl)-1',4'-naphthoquinolyl]hexanoic acid, called [M.sub.5] as a potent reversible and uncompetitive inhibitor of both human and Plasmodium falciparum glutathione reductases. The synthesis and kinetic characterization of a fluoromethyl-[M.sub.5] analogue that acts as a mechanism-based inhibitor of both enzymes is described.

Glutathione (GSH)-conjugated xenobiotics and GSH-conjugated metabolites (e.g. the cysteinyl leukotriene [C.sub.4]) must be exported from the cells in which they are formed before they can be eliminated from the body or act on their cellular targets. This efflux is often mediated by the multidrug resistance protein 1 (MRP1) transporter, which also confers drug resistance to tumour cells and can protect normal cells from toxic insults. In addition to drugs and GSH conjugates, MRP1 exports GSH and GSH disulfide, and might thus have a role in cellular responses to oxidative stress. The transport of several drugs and conjugated organic anions by MRP1 requires the presence of GSH, but it is not well understood how GSH (and its analogues) enhances transport. Site-directed mutagenesis studies and biophysical analyses have provided important insights into the structural determinants of MRP1 that influence GSH and GSH conjugate binding and transport.

The mammalian liver tightly regulates its free cysteine pool, and intracellular cysteine in rat liver is maintained between 20 and 100 nmol/g even when sulfur amino acid intakes are deficient or excessive. By keeping cysteine levels within a narrow range and by regulating the synthesis of glutathione, which serves as a reservoir of cysteine, the liver addresses both the need to have adequate cysteine to support normal metabolism and the need to keep cysteine levels below the threshold of toxicity. Cysteine catabolism is tightly regulated via regulation of cysteine dioxygenase (CDO) levels in the liver, with the turnover of CDO protein being dramatically decreased when intracellular cysteine levels increase. This occurs in response to changes in the intracellular cysteine concentration via changes in the rate of CDO ubiquitination and degradation. Glutathione synthesis also increases when intracellular cysteine levels increase as a result of increased saturation of glutamate-cysteine ligase (GCL) with cysteine, and this contributes to removal of excess cysteine. When cysteine levels drop, GCL activity increases, and the increased capacity for glutathione synthesis facilitates conservation of cysteine in the form of glutathione (although the absolute rate of glutathione synthesis still decreases because of the lack of substrate). This increase in GCL activity is dependent on up-regulation of expression of both the catalytic and modifier subunits of GCL, resulting in an increase in total catalytic subunit plus an increase in the catalytic efficiency of the enzyme. An important role of cysteine utilization for coenzyme A synthesis in maintaining cellular cysteine levels in some tissues, and a possible connection between the necessity of controlling cellular cysteine levels to regulate the rate of hydrogen sulfide production, have been suggested by recent literature and are areas that deserve further study. KEY WORDS: * cysteine * cysteine dioxygenase * glutathione * glutamate-cysteine ligase * hypotaurine * taurine * coenzyme A * cysteamine * hydrogen sulfide

Redox-induced regulation of the Na-K-ATPase was studied in dispersed rat cerebellar granule cells. Intracellular thiol redox state was modulated using glutathione (GSH)-conjugating agents and membrane-permeable ethyl ester of GSH (et-GSH) and Na-K-ATPase transport and hydrolytic activity monitored as a function of intracellular reduced thiol concentration. Depletion of cytosolic and mitochondrial GSH pools caused an increase in free radical production in mitochondria and rapid ATP deprivation with a subsequent decrease in transport but not hydrolytic activity of the Na-K-ATPase. Selective conjugation of cytosolic GSH did not affect free radical production and Na-KATPase function. Unexpectedly, overloading of cerebellar granule cells with GSH triggered global free radical burst originating most probably from GSH autooxidation. The latter was not followed by ATP depletion but resulted in suppression of active [K.sup.+] influx and a modest increase in mortality. Suppression of transport activity of the Na-K-ATPase was observed in granule cells exposed to both permeable et-GSH and impermeable GSH, with inhibitory effects of external and cytosolic GSH being additive. The obtained data indicate that redox state is a potent regulator of the Na-K-ATPase function. Shifts from an 'optimal redox potential range' to higher or lower levels cause suppression of the Na-K pump activity. glutathione; free radicals; redox state

The multidrug resistance protein ABCC4 (MRP4), a member of the ATP-binding cassette superfamily, mediates ATP-dependent unidirectional efflux of organic anions out of cells. Previous studies showed that human ABCC4 is localized to the sinusoidal membrane of hepatocytes and mediates, among other substrates, the cotransport of reduced glutathione (GSH) with bile acids. In the present study, using inside-out membrane vesicles, we demonstrated that human ABCC4 in the presence of physiological concentrations of GSH has a high affinity for the taurine and glycine conjugates of the common natural bile acids as well as the unconjugated bile acid cholate. Chenodeoxycholyltaurine and chenodeoxycholylglycine were the GSH cosubstrates with the highest affinities for ABCC4, with [K.sub.m] values of 3.6 and 5.9 [micro]M, respectively. Ursodeoxycholyltaurine and ursodeoxycholylglycine were cotransported together with GSH by ABCC4 with [K.sub.m] values of 7.8 and 12.5 [micro]M, respectively, but no transport of ursodeoxycholate and deoxycholate was observed. The simultaneous transport of labeled GSH and cholyltaurine or cholylglycine was demonstrated in double-labeled cotransport experiments with a bile acid-to-GSH ratio of ~1:22. [K.sub.m] values of the bile acids for ABCC4 were in a range similar to those reported for the canalicular bile salt export pump ABCB 11. Under physiological conditions, the sinusoidal ABCC4 may compete with canalicular ABCB11 for bile acids and thereby play a key role in determining the hepatocyte concentration of bile acids. In cholestatic conditions, ABCC4 may become a key pathway for efflux of bile acids from hepatocytes into blood. liver; multidrug resistance protein; sinusoidal transporter

Enteropathogenic Escherichia coli (EPEC) secretes many Esps (E. coli-secreted proteins) and effectors via the type III secretion (TTS) system. We previously identified a novel needle complex (NC) composed of a basal body and a needle structure containing an expandable EspA sheath-like structure as a central part of the EPEC TTS apparatus. To further investigate the structure and protein components of the EPEC NC, we purified it in successive centrifugal steps. Finally, NCs with long EspA sheath-like structures could be separated from those with short needle structures on the basis of their densities. Although the highly purified NC appeared to lack an inner ring in the basal body, its core structure, composed of an outer ring and a central rod, was observed by transmission electron microscopy. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, Western blot, and immunoelectron microscopic analyses revealed that EscC was a major protein component of the outer ring in the core basal body. To investigate the mechanisms of assembly of the basal body, interactions between the presumed components of the EPEC TTS apparatus were analyzed by a glutathione S-transferase pulldown assay. The EscC outer ring protein was associated with both the EscF needle protein and EscD, a presumed inner membrane protein. EscF was also associated with Esc J, a presumed inner ring protein. Furthermore, escC, escD, and escJ mutant strains were unable to produce the TTS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTS apparatus.

The mechanisms through which thiol oxidation and cellular redox influence the regulation of soluble guanylate cyclase (sGC) are poorly understood. This study investigated whether promoting thiol oxidation via inhibition of NADPH generation by the pentose phosphate pathway (PPP) with 1 mM 6-aminonicotinamide (6-AN) or the thiol oxidant diamide (1 mM) alters sGC activity and cGMP-associated relaxation to nitric oxide (NO) donors [S-nitroso-N-acetylpenicillamine (SNAP) and spermine-NONOate]. Diamide and 6-AN inhibited NO-elicited relaxation of endothelium-denuded bovine pulmonary arteries (BPA) and stimulation of sGC activity in BPA homogenates. Treatment of BPA with the thiol reductant DTT (1 mM) reversed inhibition of NO-mediated relaxation and sGC stimulation by 6-AN. The increase in cGMP protein kinase-associated phosphorylation of vasodilator-stimulated phosphoprotein on [Ser.sup.239] elicited by 10 [micro]M SNAP was also inhibited by diamide. Activation of sGC by SNAP was attenuated by low micromolar concentrations of GSSG in concentrated, but not dilute, homogenates of BPA, suggesting that an enzymatic process contributes to the actions of GSSG. Relaxation to agents that function through cAMP (forskolin and isoproterenol) was not altered by inhibition of the pentose phosphate pathway or diamide. Thus a thiol oxidation mechanism controlled by the regulation of thiol redox by NADPH generated via the pentose phosphate pathway appears to inhibit sGC activation and cGMP-mediated relaxation by NO in a manner consistent with its function as an important physiological redox-mediated regulator of vascular function. cGMP; glutathione redox; NADPH redox; nitrovasodilators

Reactive oxygen species (ROS) enhance myocardial ischemia-reperfusion (I/R) injury. Ischemic preconditioning (PC) provides potent cardio-protective effects in I/R. However, it has not been elucidated whether PC diminishes ROS stress in UR and whether PC protects the myocardium from ROS stress transmurally and homogeneously. Isolated rabbit hearts perfused with Krebs-Henseleit buffer underwent 30 min of ischemia and 60 min of reperfusion. Hemodynamic changes and myocardial damage extent were analyzed in four groups. The control group underwent I/R alone. The [H.sub.2][O.sub.2] group underwent I/R with [H.sub.2][O.sub.2] infusion (50 M) in the first minute of reperfusion to enhance oxidative stress. The PC and [H.sub.2][O.sub.2]+PC groups underwent 5 min of PC before control and [H.sub.2][O.sub.2] protocols, respectively. Extracted myocardial DNA was analyzed for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative DNA damage, with the use of the HPLC-electrochemical detection method. Glutathione peroxidase (GPX) activity and the reduced form of GSH were measured by spectrophotometric assays. The myocardial infarct size was significantly reduced in the PC group (19 [+ or -] 2%) compared with the control group (37 [+ or -] 4%; P < 0.05), particularly in the subendocardium. [H.sub.2][O.sub.2] transmurally increased the infarct size by 59 [+ or -] 4% (P < 0.05), which was significantly diminished in the [H.sub.2][O.sub.2]+PC group (31 [+ or -] 4%; P < 0.01). The GSH levels, but not GPX activity, were well preserved transmurally in protocols with PC. The 8-OHdG levels were significantly decreased in PC and were significantly enhanced in H202 (P < 0.01). These changes in oxidative DNA damage were effectively diminished by PC. In conclusion, PC enhanced the scavenging activity of GSH against ROS transmurally, reduced myocardial damage, particularly in the subendocardium, and diminished the transmural difference in myocardial infarct size. reperfusion injury; 8-hydroxydeoxyguanosine; glutathione

Glutathione is a tripeptide and antioxidant, synthesized at high levels by cells during the production of reactive oxygen and nitrogen intermediates. Glutathione also serves as a carrier molecule for nitric oxide in the form of S-nitrosoglutathione. Previous studies from this laboratory have shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. Glutathione is not transported into the cells as a tripeptide. Extracellular glutathione is converted to a dipeptide due to the action of transpeptidase, and the dipeptide is then transported into the bacterial cells. The processing of glutathione and S-nitrosoglutathione is brought about by the action of the enzyme [gamma]-glutamyl transpeptidase. The function of [gamma]-glutamyl transpeptidase is to cleave glutathione and S-nitrosoglutathione to the dipeptide (Cys-Gly), which is then transported into the bacterium by the multicomponent ABC transporter dipeptide permease. We have created a mutant strain of Mycobacterium tuberculosis lacking this metabolic enzyme. We investigated the sensitivity of this strain to glutathione and S-nitrosoglutathione compared to that of the wild-type bacteria. In addition, we examined the role of glutathione and/or S-nitrosoglutathione in controlling the growth of intracellular M. tuberculosis inside mouse macrophages.

Cysteine synthesis from homocysteine is catalyzed by two pyridoxal 5'-phosphate (PLP)-dependent enzymes. This suggests that vitamin B-6 status might affect cysteine and glutathione homeostasis, but it is unclear whether this occurs in humans. We assessed the effects of vitamin B-6 status on static and kinetic parameters of cysteine and glutathione metabolism in healthy female (n = 5) and male (n = 4) volunteers (20-30 y) before and after 4 wk of dietary vitamin B-6 restriction ( KEY WORDS: * cysteine * glutathione * human * transsulfuration * vitamin B-6

Glutathione S-transferase pie (GST pie) is shown to reactivate oxidized 1-cysteine peroxiredoxin (1-cys Prx, Prx VI, Prdx6 and AOP2). A heterodimer complex is formed where 1-Cys Prx is fully active towards either H2O2 or phospholipid hydroperoxide, while the GST pie activity is -25% of that of the GST pie homodimer.

Phytochelatin synthase (PCS) is a key enzyme for heavy-metal detoxification in plants. PCS catalyzes the production of glutathione (GSH)-derived peptides (called phytochelatins or PCs) that bind heavy-metal ions before vacuolar sequestration. The enzyme can also hydrolyze GSH and GS-conjugated xenobiotics. In the cyanobacterium Nostoc, the enzyme (NsPCS) contains only the catalytic domain of the eukaryotic synthase and can act as a GSH hydrolase and weakly as a peptide ligase. The crystal structure of NsPCS in its native form solved at a 2.0-[Angstrom] resolution shows that NsPCS is a dimer that belongs to the papain superfamily of cysteine proteases, with a conserved catalytic machinery. Moreover, the structure of the protein solved as a complex with GSH at a 1.4-[Angstrom] resolution reveals a [gamma]-glutamyl cysteine acyl-enzyme intermediate stabilized in a cavity of the protein adjacent to a second putative GSH binding site. GSH hydrolase and PCS activities of the enzyme are discussed in the light of both structures. cysteine protease | heavy-metal detoxification | phytochelatin synthase

Reactive [alpha],[beta]-unsaturated aldehydes are major components of common environmental pollutants and are products of lipid oxidation. Although these aldehydes have been demonstrated to induce apoptotic cell death in various cell types, we recently observed that the [alpha],[beta]- unsaturated aldehyde acrolein (ACR) can inhibit constitutive apoptosis of polymorphonuclear neutrophils and thus potentially contribute to chronic inflammation. The present study was designed to investigate the biochemical mechanisms by which two representative [alpha],[beta]- unsaturated aldehydes, ACR and 4-hydroxynonenal (HNE), regulate neutrophil apoptosis. Whereas low concentrations of either aldehyde ( inflammation; reduced glutathione; caspase-3; adenosine 5'-triphosphate; cytochrome c

The bacterium Caulobacter crescentus and related stalk bacterial species are known for their distinctive ability to live in low-nutrient environments, a characteristic of most heavy metal-contaminated sites. Caulobacter crescentus is a model organism for studying cell cycle regulation with well-developed genetics. We have identified the pathways responding to heavy-metal toxicity in C. crescentus to provide insights for the possible application of Caulobacter to environmental restoration. We exposed C. crescentus cells to four heavy metals (chromium, cadmium, selenium, and uranium) and analyzed genome-wide transcriptional activities postexposure using an Affymetrix GeneChip microarray. C. crescentus showed surprisingly high tolerance to uranium, a possible mechanism for which may be the formation of extracellular calcium-uranium-phosphate precipitates. The principal response to these metals was protection against oxidative stress (up-regulation of manganese-dependent superoxide dismutase sodA). Glutathione S-transferase, thioredoxin, glutaredoxins, and DNA repair enzymes responded most strongly to cadmium and chromate. The cadmium and chromium stress response also focused on reducing the intracellular metal concentration, with multiple efflux pumps employed to remove cadmium, while a sulfate transporter was down-regulated to reduce nonspecific uptake of chromium. Membrane proteins were also up-regulated in response to most of the metals tested. A two-component signal transduction system involved in the uranium response was identified. Several differentially regulated transcripts from regions previously not known to encode proteins were identified, demonstrating the advantage of evaluating the transcriptome by using whole-genome microarrays.

During growth on ethanolamine, Salmonella enterica synthesizes a multimolecular structure that mimics the carboxysome used by some photosynthetic bacteria to fix C[O.sub.2]. In S. enterica, this carboxysome-like structure (hereafter referred to as the ethanolamine metabolosome) is thought to contain the enzymatic machinery needed to metabolize ethanolamine into acetyl coenzyme A (acetyl-CoA). Analysis of the growth behavior of mutant strains of S. enterica lacking specific functions encoded by the 17-gene ethanolamine utilization (eut) operon established the minimal biochemical functions needed by this bacterium to use ethanolamine as a source of carbon and energy. The data obtained support the conclusion that the ethanolamine ammnonia-lyase (EAL) enzyme (encoded by the eutBC genes) and coenzyme [B.sub.12] are necessary and sufficient to grow on ethanolamine. We propose that the EutD phosphotransacetylase and EutG alcohol dehydrogenase are important to maintain metabolic balance. Glutathione (GSH) had a strong positive effect that compensated for the lack of the EAL reactivase EutA protein under aerobic growth on ethanolamine. Neither GSH nor EutA was needed during growth on ethanolamine under reduced-oxygen conditions. GSH also stimulated growth of a strain lacking the acetaldehyde dehydrogenase (EutE) enzyme. The role of GSH in ethanolamine catabolism is complex and requires further investigation. Our data show that the ethanolamine metabolosome is not involved in the biochemistry of ethanolamine catabolism. We propose the metabolosome is needed to concentrate low levels of ethanolamine catabolic enzymes, to keep the level of toxic acetaldehyde low, to generate enough acetyl-CoA to support cell growth, and to maintain a pool of free CoA.

Selenium has been shown to reduce cancer incidence in animal models and more recent data indicate that it may be protective in humans as well. However, little is known about the mechanism by which selenium prevents cancer. Cytosolic glutathione peroxidase (GPX1), a selenium-containing antioxidant enzyme, has been implicated in the development of cancer of the head and neck, lung, and breast, in part because of allelic loss at the GPX1 locus. The study of allelic loss at the GPX1 locus in colon cancer was investigated by examining loss of heterozygosity (LOH) in DNA extracted from both tumor and adjacent histopathologically normal tissue obtained by laser capture microdissection. Tissue samples were obtained from 53 colon cancer patients. Two highly polymorphic markers, alanine codon repeats and a proline-leucine polymorphism (198P/L) present in the GPX1 gene, were used to examine LOH at this locus. Analysis of both polymorphisms identified LOH at GPX1 in a significant percentage of colorectal cancer (42%). These results indicated that LOH at the GPX1 locus is a common event in cancer development and that GPX1 or other tightly linked genes may be involved in the etiology of this disease. KEY WORDS: * selenium * selenoproteins * glutathione peroxidase * cancer

Ti[O.sub.2]/ITO modified electrodes were developed to quantitatively photooxidize adsorbed ds-DNA and to study the effect of antioxidants as ds-DNA protecting agents. Ti[O.sub.2] films are used for efficient ds-DNA immobilization, for ds-DNA oxidation through photogenerated hydroxyl radicals, and as electrodes for amperometric sensing. The films, prepared by a sol--gel process, are deposited on ITO glass electrodes. Damages occurring after ds-DNA oxidation by ROS are detected by adding MB as an intercalant probe and by monitoring the electrochemical reduction current of the intercalated redox probe. The MB electrochemical signal is found to be sensitive enough to monitor ds-DNA structure changes, and the electrochemical sensor has been applied to the evaluation of the antioxidant properties of glutathione and gallic acid.

Both biotic and abiotic stress activate the oxylipin pathway in plants. As reactive electrophile species (RES), some oxylipins are expected to bind cellular nucleophiles in a Michael-type addition reaction. Using the HPLC-tandem mass spectrometry techniques, we have established the analytical basis for the investigation of oxylipin conjugation to glutathione (GSH) in plant extracts. The GSH adducts to the four keto fatty acid isomers issued from both linoleic and linolenic acids were first produced and their mass spectrometric features analyzed in the positive electrospray ionization mode. In all cases, the main fragmentation ([MS.sup.2] mode) of the pseudomolecular ion leads to the neutral loss of a glutamyl moiety (-129 Da), affording an ion that gives structural information upon an additional fragmentation ([MS.sup.3] mode). The glutamyl loss was confirmed by the analysis of other GSH adducts to oxylipin RES and appeared as being characteristic of GSH adducts. It is thus proposed to search GSH adducts in plant extracts by HPLC MS/MS, using initially the neural loss mode and then the MS2 mode to further characterize the identified compounds. This methodology was successfully applied to the analysis of GSH adducts upon infiltration into leaves of the four previous keto fatty acids at 5 mM, a concentration inducing cell death. The production of GSH adducts to oxylipin RES was observed for the first time in plant tissues. Furthermore, the levels of adduct production explain in part the observed GSH depletion. These results support the role of RES in altering protein activities and cellular redox balance of plant cells, via addition reactions to cellular nucleophiles.

Thioredoxin f (TRXf) is a key factor in the redox regulation of chloroplastic carbon fixation enzymes, whereas glutathione is an important thiol buffer whose status is modulated by stress conditions. Here, we report specific glutathionylation of TRXf. A conserved cysteine is present in the TRXf primary sequence, in addition to its two active-site cysteines. The additional cysteine becomes glutathionylated when TRXf is exposed to oxidized glutathione or to reduced glutathione plus oxidants. No other chloroplastic TRX, from either Arabidopsis or Chlamydomonas, is glutathionylated under these conditions. Glutathionylation decreases the ability of TRXf to be reduced by ferredoxin-thioredoxin reductase and results in impaired light activation of target enzymes in a reconstituted thylakoid system. Although several mammalian proteins undergoing glutathionylation have already been identified, TRXf is among the first plant proteins found to undergo this posttranslational modification. This report suggests that a crosstalk between the TRX and glutathione systems mediates a previously uncharacterized form of redox signaling in plants in stress conditions. Chlamydomonas | Arabidopsis | Calvin cycle | enzyme light activation | thiol

Changes in intracellular redox couples and redox reactive molecules have been implicated in the regulation of a variety of cellular processes, including cell proliferation and growth arrest by contact inhibition. However, the magnitude, direction, and temporal relationship of redox changes to cellular responses are incompletely defined. The present work sought to characterize redox and metabolic changes associated with proliferative stages to contact inhibition of growth in rat IEC-6 intestinal epithelial cells. From the first day of culture until 1 day before confluence, an increase in GSH concentrations and a significant reduction in the redox potential of the GSSG/2GSH couple were observed. These changes were accompanied by a decrease in relative reactive oxygen species (ROS) and nitric oxide (NO) concentrations and oxidation of the redox potential of the NAD[P.sup.+]/reduced NADP and NA[D.sup.+]/NADH couples. Postconfluent cells exhibited a significant decrease in GSH concentrations and a significant oxidation of the GSSG/2GSH couple. When cell proliferation decreased, relative ROS concentrations increased (P < 0.01), whereas NO concentrations remained unchanged, and the NA[D.sup.+]/NADH couple became more reduced. Together, these data indicate that the redox potential of distinct couples varies differentially in both magnitude and direction during successive stages of IEC-6 growth. This finding points out the difficulty of defining intracellular redox status at particular stages of cell growth by examining only one redox species. In addition, the data provide a numerical framework for future research of regulatory mechanisms governed by distinct intracellular redox couples. cell proliferation; contact inhibition; glutathione

The health consequences of mild copper excess in humans are unknown. In a previous study, 2 mo of supplementation with up to 6 mg Cu/L in drinking water did not induce detectable changes. Here we assessed a copper supplement at the upper level of dietary recommendations for 'healthy' adults. The study was a prospective controlled trial; participants (women and men, 18-50 y old), represented the upper and lower 5% of the ceruloplasmin distribution curve obtained from a community-based sample of 800 healthy adults (n = 41/ group, each ~50% men). Individuals received a single daily dose of 10 mg Cu for 60 d. Before and after supplementation, blood [copper, ceruloplasmin protein, homocysteine, liver aminotranferases, Cu-Zn -superoxide dismutase activity in erythrocytes (eSOD), and glutathione in peripheral mononuclear cells] and urine [copper excretion after a 5-h administration of a chelator 2,3-dimercapto-1-propano-sodium sulfonate (DMPS)] analyses were performed. After 2 mo, liver enzyme activities remained below the clinical cutoff value used to diagnose liver dysfunction, but had increased significantly in both groups and genders. These increases were no longer present 12 mo after the copper loading period was completed. Glutathione in mononuclear cells (mmol/g of protein) also increased after the 2-mo copper loading in both groups and in both genders (P = 0.01). eSOD activity, serum homocysteine concentration, and urinary copper excretion 5 h after DMPS administration were not affected. We conclude that copper administered as described induced a transient, mild, but significant elevation of aminotransferases. KEY WORDS: * copper * liver aminotransferases * super oxide dismutase * glutathione * humans

Cardioplegic arrest for bypass surgery imposes global ischemia on the myocardium, which generates oxyradicals and depletes myocardial high-energy phosphates. The glycolytic metabolite pyruvate, but not its reduced congener lactate, increases phosphorylation potential and detoxifies oxyradicals in ischemic and postischemic myocardium. This study tested the hypothesis that pyruvate mitigates oxidative stress and preserves the energy state in cardioplegically arrested myocardium. In situ swine hearts were arrested for 60 min with a 4:1 mixture of blood and crystalloid cardioplegia solution containing 188 mM glucose alone (control) or with additional 23.8 mM lactate or 23.8 mM pyruvate and then reperfused for 3 min with cardioplegiafree blood. Glutathione (GSH), glutathione disulfide (GSSG), and energy metabolites [phosphocreatine (PCr), creatine (Cr), [P.sub.i]] were measured in myocardium, which was snap frozen at 45 min arrest and 3 min reperfusion to determine antioxidant GSH redox state (GSH/ GSSG) and PCr phosphorylation potential {[PCrl/([Cr][[P.sub.i]])}. Coronary sinus 8-isoprostane indexed oxidative stress. Pyruvate cardioplegia lowered 8-isoprostane release ~40% during arrest versus control and lactate cardioplegia. Lactate and pyruvate cardioplegia dampened (P < 0.05 vs. control) the surge of 8-isoprostane release following reperfusion. Pyruvate doubled GSH/GSSG versus lactate cardioplegia during arrest, but GSH/GSSG fell in all three groups after reperfusion. Myocardial [PCr]/([Cr] [[P.sub.i]]) was maintained in all three groups during arrest. Pyruvate cardioplegia doubled [PCr]/([Cr][[P.sub.i]]) versus control and lactate cardioplegia after reperfusion. Pyruvate cardioplegia mitigates oxidative stress during cardioplegic arrest and enhances myocardial energy state on reperfusion. glutathione; reactive oxygen species; cardioplegia

Glutathione (GSH) is the most abundant non-protein thiol in eukaryotic cells and acts as reducing equivalent in many cellular processes. We investigated the role of glutathione in Dictyostelium development by disruption of [gamma]-glutamylcysteine synthetase (GCS), an essential enzyme in glutathione biosynthesis. GCS-null strain showed glutathione auxotrophy and could not grow in medium containing other thiol compounds. The developmental progress of GCS-null strain was determined by GSH concentration contained in preincubated media before development. GCS-null strain preincubated with 0.2 mM GSH was arrested at mound stage or formed bent stalk-like structure during development. GCS-null strain preincubated with more than 0.5 mM GSH formed fruiting body with spores, but spore viability was significantly reduced. In GCS-null strain precultured with 0.2 mM GSH, prestalk-specific gene expression was delayed, while prespore-specific gene and spore-specific gene expressions were not detected. In addition, GCS-null strain precultured with 0.2 mM GSH showed prestalk tendency and extended G1 phase of cell cycle. Since G1 phase cells at starvation differentiate into prestalk cells, developmental defect of GCS-null strain precultured with 0.2 mM GSH may result from altered cell cycle. These results suggest that glutathione itself is essential for growth and differentiation to prespore in Dictyostelium. Keywords: Glutathione; [gamma]-Glutamylcysteine synthetase; Cell fate; Dictyostelium discoideum

In mammals, >100 genes regulate pigmentation by means of a wide variety of developmental, cellular, and enzymatic mechanisms. Nevertheless, genes that directly regulate pheomelanin production have not been described. Here, we demonstrate that the subtle gray (sut) mouse pigmentation mutant arose by means of a mutation in the Slc7a11 gene, encoding the plasma membrane cystine/glutamate exchanger xCT [Kanai, Y. & Endou, H. (2001) Curr. Drug Metab. 2, 339-354]. A resulting low rate of extracellular cystine transport into sut melanocytes reduces pheomelanin production. We show that Slc7a11 is a major genetic regulator of pheomelanin pigment in hair and melanocytes, with minimal or no effects on eumelanin. Furthermore, transport of cystine by xCT is critical for normal proliferation, glutathione production, and protection from oxidative stress in cultured cells. Thus, we have found that the Slc7a11 gene controls the production of pheomelanin pigment directly. Cells from sut mice provide a model for oxidative stress-related diseases and their therapies. glutathione | melanin | pigmentation | cystine | melanocyte