In vitro inhibition of topoisomerase IIα by reduced glutathione

In most cells, the major intracellular redox buffer is glutathione (GSH) and its disulfide-oxidized (GSSG) form. The GSH/GSSG system maintains the intracellular redox balance and the essential thiol status of proteins by thiol disulfide exchange. Topoisomerases are thiol proteins and are a target of thiol-reactive substances. In this study, the inhibitory effect of physiological concentration of GSH and GSSG on topoisomerase IIα activity in vitro was investigated. GSH (0-10 mM) inhibited topoisomerase IIα in a concentration-dependent manner while GSSG (1-100 μM) had no significant effect. These findings suggest that the GSH/GSSG system could have a potential in vivo role in regulating topoisomerase IIα activity.     

Differential oxidation of thioredoxin-1, thioredoxin-2, and glutathione by metal ions

Metal toxicity often includes the generation of reactive oxygen species (ROS) and subsequent oxidative stress, but whether metals have different effects on the major thiol antioxidant systems is unknown. Here, we examine the effects of arsenic, cadmium, cesium, copper, iron, mercury, nickel, and zinc on glutathione (GSH), cytoplasmic thioredoxin-1 (Trx1), and mitochondrial thioredoxin-2 (Trx2) redox states. GSH/GSSG redox states were determined by HPLC, and Trx1 and Trx2 redox states were determined by Redox Western blot methods. Copper, iron, and nickel showed significant oxidation of GSH but relatively little oxidation of either Trx1 or Trx2. Arsenic, cadmium, and mercury showed little oxidation of GSH but significantly oxidized both Trx1 and Trx2. The magnitude of effects of arsenic, cadmium, and mercury was greater for the mitochondrial Trx2 (>60 mV) compared to the cytoplasmic Trx1 (20 to 40 mV). Apoptosis signal-regulating kinase 1 (ASK1) may be activated by two different pathways, one dependent upon GSH and glutaredoxin and the other independent of GSH and dependent upon thioredoxin. ASK1 activation and cell death were observed with metals that oxidized thioredoxins but not with metals that oxidized GSH. These findings show that metals have differential oxidative effects on the major thiol antioxidant systems and that activation of apoptosis may be associated with metal ions that oxidize thioredoxin and activate ASK1. The differential oxidation of the major thiol antioxidant systems by metal ions suggest that the distinct thiol/disulfide redox couples represented by GSH/GSSG and the thioredoxins may convey different levels of control in apoptotic and toxic signaling pathways.     

Glutathione and homoglutathione synthesis in legume root nodules

High-performance liquid chromatography (HPLC) with fluorescence detection was used to study thiol metabolism in legume nodules. Glutathione (GSH) was the major non-protein thiol in all indeterminate nodules examined, as well as in the determinate nodules of cowpea (Vigna unguiculata), whereas homoglutathione (hGSH) predominated in soybean (Glycine max), bean (Phaseolus vulgaris), and mungbean (Vigna radiata) nodules. All nodules had greater thiol concentrations than the leaves and roots of the same plants because of active thiol synthesis in nodule tissue. The correlation between thiol tripeptides and the activities of glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) in the nodules of eight legumes, and the contrasting thiol contents and activities in alfalfa (Medicago sativa) leaves (98% hGSH, 100% hGSHS) and nodules (72% GSH, 80% GSHS) indicated that the distribution of GSH and hGSH is determined by specific synthetases. Thiol contents and synthesis decreased with both natural and induced nodule senescence, and were also reduced in the senescent zone of indeterminate nodules. Thiols and GSHS were especially abundant in the meristematic and infected zones of pea (Pisum sativum) nodules. Thiols and gamma-glutamylcysteinyl synthetase were also more abundant in the infected zone of bean nodules, but hGSHS was predominant in the cortex. Isolation of full-length cDNA sequences coding for gamma-glutamylcysteinyl synthetase from legume nodules revealed that they are highly homologous to those from other higher plants.     

Effect of cysteine on stability and toxicity to aphids of a cyclic hydroxamic acid from gramineae

Toxicity of DIMBOA, the major cyclic hydroxamic acid in maize extracts, to the aphid Schizaphis graminum, was decreased by addition of cysteine to the insect diet. The ld₅₀ for DIMBOA on aphids was, after 24 hr, 2.1 and 0.9 mM in diets with and without added cysteine, respectively. DIMBOA decomposed 1.5 times faster in diets or buffer with added cysteine. Decomposition products of DIMBOA (4 mM) in insect diets with or without added cysteine were not toxic. It is suggested that the observed variations in toxicity of DIMBOA are a consequence of differences in its rate of disappearance from the diet.     

Thiol reducing compounds prevent human amylin-evoked cytotoxicity

Human amylin (hA) is a small fibrillogenic protein that is the major constituent of pancreatic islet amyloid, which occurs in most subjects with type-2 diabetes mellitus (T2Dm). There is growing evidence that hA toxicity towards islet beta-cells is responsible for their gradual loss of function in T2Dm. Preventing hA-mediated cytotoxicity has been proposed as a route to halt the progression of this disease, although this has not yet been demonstrated in vivo. The aim of our studies, in which we show that a small number of hA-treated cells exhibit intracellular accumulation of reactive oxygen species (ROS), was to evaluate the role of oxidative stress in the mechanism of hA-mediated cytotoxicity. Here we report that catalase and n-propyl gallate, antioxidants that are thought to act mainly as free radical scavengers, afford RINm5F cells only limited protection against hA-mediated toxicity. By contrast, the thiol antioxidants, N-acetyl-L-cysteine (NAC), GSH and dithiothreitol, which not only react with ROS, but also modulate the cellular redox potential by increasing intracellular levels of GSH and/or by acting as thiol reducing agents, afford almost complete protection and inhibit the progression of hA-evoked apoptosis. We also show that hA treatment is not associated with changes in intracellular GSH levels and that inhibition of GSH biosynthesis has no effect on either hA-mediated cytotoxicity or NAC-mediated protection. These results indicate that, in addition to the induction of oxidative stress, hA appears to mediate cytotoxicity through signalling pathways that are sensitive to the actions of thiol antioxidants.     

Ascorbate-dependent recycling of the vitamin E homologue Trolox by dihydrolipoate and glutathione in murine skin homogenates

In the redox antioxidant network, dihydrolipoate can synergistically enhance the ascorbate-dependent recycling of vitamin E. Since the major endogenous thiol antioxidant in biological systems is glutathione (GSH) it was of interest to compare the effects of dihydrolipoate with GSH on ascorbate-dependent recycling of the water-soluble homologue of vitamin E, Trolox, by electron spin resonance (ESR). Trolox phenoxyl radicals were generated by a horseradish peroxidase (HRP)-hydrogen peroxide (H2O2) oxidation system. In the presence of dihydrolipoate, Trolox radicals were suppressed until both dihydrolipoate and endogenous levels of ascorbate in skin homogenates were consumed. Similar experiments made in the presence of GSH revealed that Trolox radicals reappeared immediately after ascorbate was depleted and that GSH was not able to drive the ascorbate-dependent Trolox recycling reaction. However, at higher concentrations GSH was able to increase ascorbate-mediated Trolox regeneration from the Trolox radical. ESR and spectrophotometric measurements demonstrated the ability of dihydrolipoate or GSH to react with dehydroascorbate, the two-electron oxidation product of ascorbate in this system. Dihydrolipoate regenerated greater amounts of ascorbate at a much faster rate than equivalent concentrations of GSH. Thus the marked difference between the rate and efficiency of ascorbate generation by dihydrolipoate as compared with GSH appears to account for the different kinetics by which these thiol antioxidants influence ascorbate-dependent Trolox recycling.     

Glutathione synthesis and its role in redox signaling

Glutathione (GSH) is the most abundant antioxidant and a major detoxification agent in cells. It is synthesized through two-enzyme reaction catalyzed by glutamate cysteine ligase and glutathione synthetase, and its level is well regulated in response to redox change. Accumulating evidence suggests that GSH may play important roles in cell signaling. This review will focus on the biosynthesis of GSH, the reaction of S-glutathionylation (the conjugation of GSH with thiol residue on proteins), GSNO, and their roles in redox signaling.     

Metabolic activation and hepatotoxicity. Metabolism of bromobenzene in isolated hypatocytes.

Rat liver microsomes and isolated rat hepatocytes metabolized bromobenzene to watersoluble and protein-bound metabolites. The latter fraction—which normally accounted for 2–5% of the total products—was slightly increased when 1,2-epoxy-3,3,3-trichloropropane, an inhibitor of microsomal epoxide hydrase, was added to the microsomal incubate. The presence of reduced glutathione (GSH), on the other hand, caused an almost complete inhibition of the formation of protein-bound metabolites from bromobenzene in microsomes. The rates of bromobenzene metabolism were similar in liver microsomes and hepatocytes, and increased severalfold after phenobarbital pretreatment of the rats. Metyrapone and SKF 525-A were inhibitory in both systems. Bromobenzene metabolism in hepatocytes isolated from phenobarbital-treated rats was associated with a rapid and marked decrease in the level of intracellular GSH. When the cells were incubated in a complete medium, however, the decrease in GSH leveled off at about 40% of the original concentration and there was no evidence of any accelerated rate of cell death even when the incubation with bromobenzene was prolonged to 10 h. This was most probably due to resynthesis of GSH by the hepatocytes, which partly compensated for the loss of this thiol associated with bromobenzene metabolism. Accordingly, in a deficient medium (lacking amino acids), the cytotoxic effect of bromobenzene metabolism was pronounced—less than 5% of the zerotime level of GSH and only 25% cell viability remaining after 5 h of incubation. It is concluded that the intracellular level of GSH is of major importance in regard to the cytotoxic effect of bromobenzene metabolism and that hepatocytes incubated in a complete medium are protected against toxicity by their ability to resynthesize this thiol.     

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, an Inhibitor from Zea mays with Differential Activity against Soft Rotting Erwinia Species

[2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] DIMBOA was extracted with ethyl acetate from acidified water homogenates of corn (Zea mays L.) seedlings. Pure DIMBOA or ethyl acetate extracts of corn tissue were added to bacterial growth medium at five concentrations (measured as hydroxamates). DIMBOA and corn extracts were more inhibitory to soft rot bacteria (Erwinia spp.) that are nonpathogenic to corn than to soft rot bacteria that are corn pathogens. The inhibitory activity of DIMBOA was similar to that of the ethyl acetate extracts. Both corn extracts and DIMBOA prolonged the lag phase of bacterial growth without significantly changing log phase growth rates. At various concentrations of the inhibitor, 50 to 100% of the activity of corn extracts inhibitory to different bacterial isolates was attributable to DIMBOA. Extracts of DIMBOA-deficient plants (genotype bxbx) were not inhibitory to Erwinia spp. It was concluded that DIMBOA is the major active component in those corn extracts which are inhibitory to soft rot Erwinia species.     

Exploration of in vitro pro-drug activation and futile cycling by glutathione S-transferases: thiol ester hydrolysis and inhibitor maturation

In addition to glutathione (GSH) conjugating activity, glutathione S-transferases (GSTs) catalyze "reverse" reactions, such as the hydrolysis of GSH thiol esters. Reverse reactions are of interest as potential tumor-directed pro-drug activation strategies and as mechanisms for tissue redistribution of carboxylate-containing drugs. However, the mechanism and specificity of GST-mediated GSH thiol ester hydrolysis are uncharacterized. Here, the GSH thiol esters of ethacrynic acid (E-SG) and several nonsteroidal antiinflammatory agents have been tested as substrates with human GSTs. The catalytic hydrolysis of these thiol esters appears to be a general property of GSTs. The hydrolysis of the thiol ester of E-SG was studied further with GSTA1-1 and GSTP1-1, as a model pro-drug with several possible fates for the hydrolysis products: competitive inhibition, covalent enzyme adduction, and sequential metabolism. In contrast to hydrolysis rates, significant isoform-dependent differences in the subsequent fate of the products ethacrynic acid and GSH were observed. At low [E-SG], only the GSTP1-1 efficiently catalyzed sequential metabolism, via a dissociative mechanism.     

Regulation of sulphate assimilation by glutathione in poplars (Populus tremula x P. alba) of wild type and overexpressing gamma-glutamylcysteine synthetase in the cytosol

Glutathione (GSH) is the major low molecular weight thiol in plants with different functions in stress defence and the transport and storage of sulphur. Its synthesis is dependent on the supply of its constituent amino acids cysteine, glutamate, and glycine. GSH is a feedback inhibitor of the sulphate assimilation pathway, the primary source of cysteine synthesis. Sulphate assimilation has been analysed in transgenic poplars (Populus tremula x P. alba) overexpressing gamma-glutamylcysteine synthetase, the key enzyme of GSH synthesis, and the results compared with the effects of exogenously added GSH. Although foliar GSH levels were 3-4-fold increased in the transgenic plants, the activities of enzymes of sulphate assimilation, namely ATP sulphurylase, adenosine 5'-phosphosulphate reductase (APR), sulphite reductase, serine acetyltransferase, and O-acetylserine (thiol)lyase were not affected in three transgenic lines compared with the wild type. Also the mRNA levels of these enzymes were not altered by the increased GSH levels. By contrast, an increase in GSH content due to exogenously supplied GSH resulted in a strong reduction in APR activity and mRNA accumulation. This feedback regulation was reverted by simultaneous addition of O-acetylserine (OAS). However, OAS measurements revealed that OAS cannot be the only signal responsible for the lack of feedback regulation of APR by GSH in the transgenic poplars.     

Radiation response of cells during altered protein thiol redox

The major focus of this work was to investigate how altered protein thiol redox homeostasis affects radiation-induced cell death. We used the cells of wild-type CHO cell line K1, the CHO cell line E89, which is null for G6PD activity, and a radiation-sensitive CHO cell line, XRS5. The protein-thiol redox status of cells was altered with cell-permeable disulfides, hydroxyethyldisulfide (HEDS) or lipoate. HEDS is primarily reduced by thioltransferase (glutaredoxin), with GSH as the electron donor. In contrast, lipoate is reduced by thioredoxin reductase. HEDS was reduced at a greater rate than lipoate by G6PD-containing K1 (wild-type) cells. Reduction of disulfides by G6PD-deficient cells was significantly slower with HEDS as substrate and was nearly absent with lipoate. The rate of reduction of HEDS by E89 cells decelerated to near zero by 30 min, whereas the reduction continued at nearly the same rate during the entire measurement period for K1 cells. HEDS treatment decreased the GSH and protein thiol (PSH) content more in G6PD-deficient cells than in G6PD-containing cells. On the other hand, lipoate did not significantly alter the protein thiol, but it increased the GSH in K1 cells. Acute depletion of GSH by l-buthionine-sulfoximine (l-BSO) in combination with dimethylfumarate significantly decreased the rate of reduction of HEDS by K1 cells close to that of G6PD-deficient cells. Prior GSH depletion by l-BSO alone significantly decreased the PSH in glucose-depleted E89 cells exposed to HEDS, but this did not occur with K1 cells. The radiation response of G6PD-deficient cells was significantly sensitized by HEDS, but HEDS did not have this effect on K1 cells. The DNA repair-deficient XRS5 CHO cells displayed the same capacity as K1 cells for HEDS reduction, and like K1 cells the XRS5 cells were not sensitized to radiation by HEDS treatment. Deprivation of glucose, which provides the substrate for G6PD in the oxidative pentose phosphate cycle, decreased the rate of bioreduction of HEDS and lipoate in G6PD-containing cells to the level in G6PD-deficient cells. In the absence of glucose, HEDS treatment diminished non-protein thiol and protein thiol to the same level as those in G6PD-deficient cells and sensitized the K1 cells to HEDS treatment. However, depletion of glucose did not alter the sensitivity of XRS5 cells in either the presence or absence of HEDS. Overall the results suggest a major role for pentose cycle control of protein redox state coupled to the activities of the thioltransferase and thioredoxin systems. The results also show that protein thiol status is a critical factor in cell survival after irradiation.     

The physiological consequences of glutathione variations.

The major low molecular weight thiol inside cells, the tripeptide glutathione (GSH), is of importance for protection of the cell against oxidative challenge, for thiol homeostasis required to guarantee basic functions, and for defence mechanisms against xenobiotics. Since the pathophysiological significance of a perturbed GSH status in human disease is less clear, this review evaluates the consequences of in vivo variations of GSH. Owing to intracellular GSH concentrations above 2 mM depletion of GSH as such has little metabolic consequences unless an additional stress is superimposed. The kinetic properties of GSH-dependent enzymes imply that loss of up to 90% of intracellular GSH may still be compatible with cellular integrity. Mitochondrial GSH, which accounts for about 10% of total cellular GSH, may define the threshold beyond that toxicity commences. Thus, in cases of severe GSH-depletion a substitution of GSH as a therapeutic measure seems justified. Such a severe depletion of GSH has been described for some diseases such as liver dysfunction, AIDS or pulmonary fibrosis.     

Characterization of normal, glutathione-deficient and arginase-deficient sheep erythrocytes by 1H-NMR spectroscopy

Normal sheep erythrocytes as well as glutathione- (GSH-) deficient and arginase-deficient sheep erythrocytes have been characterized by 1H nuclear magnetic resonance spectroscopy. The GSH deficiency is a result of defective amino acid transport (lesion 1), diminished gamma-glutamylcysteine synthetase activity (lesion 2), or both (lesions (1 + 2)). 1H-NMR spectra of normal sheep erythrocytes are similar to those for human erythrocytes, and consist of resonances from a number of small intracellular molecules, including GSH. In contrast, the resonances for GSH in the GSH-deficient erythrocytes are much weaker, and strong resonances are observed for lysine, threonine and ornithine or arginine, depending on the arginase activity, in erythrocytes with lesion 1 and lesions (1 + 2). A comparison of the intensity of GSH resonances in spectra for normal and GSH-deficient erythrocytes with GSH levels determined spectrophotometrically following reaction with the nonspecific thiol reagent 5,5'-dithiobis(2-nitrobenzoate) (DTNB) indicates that either not all of the GSH determined with Ellman's reagent is free and observable by 1H-NMR or that not all of the thiol determined by Ellman's reagent is GSH. If the latter is the case, the GSH levels determined with Ellman's reagent for erythrocytes with lesions (1 + 2) are most affected, which might account for their high susceptibility to oxidative stress.     

Homoglutathione: isolation, quantification and occurrence in legumes

Homoglutathione (hGSH: γ-glutamyl-eysteinyl-β-alanine) was purified from seeds of Phaseolus coccineus L. cv. Preisgewinner, using anion-exchange chromatography and Cu₂O precipitation. Quantitative and specific determination of this thiol is possible by high-performance liquid chromatography (HPLC) after monobromobimane derivatization. The enzymatic recycling assay based on yeast glutathione reductase (EC 1.6.4.2) can also be applied, but only to samples containing either hGSH or glutathione (GSH), since enzyme reaction with hGSH is 2.7 times faster than with GSH. Using the very sensitive HPLC method, the thiol content of leaves, roots and seeds of several legumes was investigated. Although GSH and hGSH were found in all plants analysed, the GSH/hGSH ratio varied greatly within the different tribes as well as within the different organs of plants of one species. In seeds and leaves of Vicieae, only traces of hGSH were found beside the main thiol GSH, whereas in roots the hGSH content exceeded the GSH content. The Trifolieae contained both tripeptides and in the tribe Phaseoleae, hGSH predominated by far.     

Downregulation of glutaredoxin but not glutathione loss leads to mitochondrial dysfunction in female mice CNS: implications in excitotoxicity

Oxidative stress, excitotoxicity and mitochondrial dysfunction play synergistic roles in neurodegeneration. Maintenance of thiol homeostasis is important for normal mitochondrial function and dysregulation of protein thiol homeostasis by oxidative stress leads to mitochondrial dysfunction and neurodegeneration. We examined the critical roles played by the antioxidant, non-protein thiol, glutathione and related enzyme, glutaredoxin in maintaining mitochondrial function during excitotoxicity caused by beta-N-oxalyl amino-L-alanine (L-BOAA), the causative factor of neurolathyrism, a motor neuron disease involving the pyramidal system. L-BOAA causes loss of GSH and inhibition of mitochondrial complex I in lumbosacral cord of male mice through oxidation of thiol groups, while female mice are resistant. Reducing GSH levels in female mice CNS by pretreatment with diethyl maleate or L-propargyl glycine did not result in inhibition of complex I activity, unlike male mice. Further, treatment of female mice depleted of GSH with L-BOAA did not induce inhibition of complex I indicating that GSH levels were not critical for maintaining complex I activity in female mice unlike their male counterpart. Glutaredoxin, a thiol disulfide oxidoreductase helps maintain redox status of proteins and downregulation of glutaredoxin results in loss of mitochondrial complex I activity. Female mice express higher levels of glutaredoxin in certain CNS regions and downregulation of glutaredoxin using antisense oligonucleotides sensitizes them to L-BOAA toxicity seen as mitochondrial complex I loss. Ovariectomy downregulates glutaredoxin and renders female mice vulnerable to L-BOAA toxicity as evidenced by activation of AP1, loss of GSH and complex I activity indicating the important role of glutaredoxin in neuroprotection. Estrogen protects against mitochondrial dysfunction caused by excitotoxicity by maintaining cellular redox status through higher constitutive expression of glutaredoxin in the CNS. Therapeutic interventions designed to upregulate glutaredoxin may offer neuroprotection against excitotoxicity in motor neurons.     

The role of thiols in cellular response to radiation and drugs

Cellular nonprotein thiols (NPSH) consist of glutathione (GSH) and other low molecular weight species such as cysteine, cysteamine, and coenzyme A. GSH is usually less than the total cellular NPSH, and with thiol reactive agents, such as diethyl maleate (DEM), its rate of depletion is in part dependent upon the cellular capacity for its resynthesis. If resynthesis is blocked by buthionine-S,R-sulfoximine(BSO), the NPSH, including GSH, is depleted more rapidly, Cellular thiol depletion by diamide, N-ethylmaleimide, and BSO may render oxygenated cells more sensitive to radiation. These cells may or may not show a reduction in the oxygen enhancement ratio (OER). Human A549 lung carcinoma cells depleted of their NPSH either by prolonged culture or by BSO treatment do not show a reduced OER but do show increased aerobic responses to radiation. Some nitroheterocyclic radiosensitizing drugs also deplete cellular thiols under aerobic conditions. Such reactivity may be the reason that they show anomalous radiation sensitization (i.e., better than predicted on the basis of electron affinity). Other nitrocompounds, such as misonidazole, are activated under hypoxic conditions to radical intermediates. When cellular thiols are depleted peroxide is formed. Under hypoxic conditions thiols are depleted because metabolically reduced intermediates react with GSH instead of oxygen. Thiol depletion, under hypoxic conditions, may be the reason that misonidazole and other nitrocompounds show an extra enhancement ratio with hypoxic cells. Thiol depletion by DEM or BSO alters the radiation response of hypoxic cells to misonidazole. In conclusion, we propose an altered thiol model which includes a mechanism for thiol involvement in the aerobic radiation response of cells. This mechanism involves both thiol-linked hydrogen donation to oxygen radical adducts to produce hydroperoxides followed by a GSH peroxidase-catalyzed reduction of the hydroperoxides to intermediates entering into metabolic pathways to produce the original molecule.     

Identification of glutathione modifications by cigarette smoke

Although it has been recognized for decades that cigarette smoke (CS) is toxic to respiratory tract tissues, and that glutathione (GSH) and other thiols are able to ameliorate some of the adverse effects of CS, the precise interactions between thiols and critical CS components are only partially characterized. In the present study, we used HPLC and MALDI-MS approaches to more rigorously characterize the products of CS reactions with GSH, the major cellular thiol and an important antioxidant constituent in respiratory tract lining fluids, in an attempt to increase our understanding of mechanisms of CS respiratory tract toxicity. Exposure of solutions of GSH to gas phase CS resulted in its rapid depletion, and about 50% of this depletion could be accounted for by reaction with acrolein and crotonaldehyde, the two major alpha, beta-unsaturated aldehydes in CS. Similar aldehyde adducts with GSH could also be detected in cells exposed to CS, although the relative yields were limited, presumably because of further reactions of these adducts and/or their excretion. Further characterization of in vivo thiol-aldehyde formation in respiratory tract cells can be expected to provide significant insights into the mechanisms of CS toxicity.     

Inhibition of protein carbonyl formation and lipid peroxidation by glutathione in rat liver microsomes.

The peroxidation of rat liver microsomal lipids is stimulated in the presence of iron by the addition of NADPH or ascorbate and is inhibited by the addition of glutathione (GSH). The fate of GSH and the oxidative modification of proteins under these conditions have not been well studied. Rat liver microsomes were incubated at 37 degrees C under 95% O2:5% CO2 in the presence of 10 microM ferric chloride, 400 microM ADP, and either 450 microM ascorbic acid or 400 microM NADPH. Lipid peroxidation was assessed in the presence 0, 0.2, 0.5, 1, or 5 mM GSH by measuring thiobarbituric acid reactive substance (TBARS) and oxidative modification of proteins by measuring protein thiol and carbonyl groups. GSH inhibited TBARS and protein carbonyl group formation in both ascorbate and NADPH systems in a dose-dependent manner. Heat denaturing of microsomes or treatment with trypsin resulted in the loss of this protection. The formation of protein carbonyl groups could be duplicated by incubating microsomes with 4-hydroxynonenal. Ascorbate-dependent peroxidation caused a loss of protein thiol groups which was diminished by GSH only in fresh microsomes. Both boiling and trypsin treatment significantly decreased the basal protein thiol content of microsomes and enhanced ascorbate-stimulated lipid peroxidation. Protection against protein carbonyl group formation by GSH correlated with the inhibition of lipid peroxidation and appeared not to be due to the formation of the GSH conjugate of 4-hydroxynonenal as only trace amounts of this conjugate were detected. Ninety percent of the GSH lost after 60 min of peroxidation was recoverable as borohydride reducible material in the supernatant fraction. The remaining 10% could be accounted for as GSH-bound protein mixed disulfides. However, only 75% of the GSH lost during peroxidation appeared as glutathione disulfide, suggesting that some was converted to other soluble borohydride reducible forms. These data support a role for protein thiol groups in the GSH-mediated protection of microsomes against lipid peroxidation.     

A flow cytometric assay for intracellular nonprotein thiols using mercury orange

The level of nonprotein thiols was assayed in individual mammalian cells using flow cytometry. Previous determinations of glutathione (GSH, the most abundant nonprotein thiol in most cells) by flow cytometry were based on UV laser excitation of fluorochromes. Because of several shortcomings of UV excitation, an assay for GSH using visible light is of interest. Selective staining of nonprotein thiols with mercury orange (a mercurial compound that binds stoichiometrically to sulfhydryl groups) was obtained by restricting the staining time. By using various drugs that affect GSH levels and overall thiol levels in cells, it was shown that GSH is the primary thiol group being stained. Thus a quick, specific technique using mercury orange has been developed for the flow cytometric determination of nonprotein thiols and preferentially for GSH in individual mammalian cells.