Determination of S-nitrosoglutathione in human and rat plasma by high-performance liquid chromatography with fluorescence and ultraviolet absorbance detection after precolumn derivatization with o-phthalaldehyde.

An analytical method is described for the quantification of S-nitrosoglutathione (GSNO), a potent physiological vasodilator and inhibitor of platelet aggregation, in the presence of a high excess of reduced glutathione (GSH). The method is based on the quantitative elimination of GSH by N-ethylmaleimide, the conversion of GSNO by 2-mercaptoethanol to GSH, its reaction with o-phthalaldehyde (OPA) to form a highly fluorescent and UV-absorbing tricyclic isoindole derivative, and subsequent high-performance liquid chromatographic (HPLC) separation with fluorescence and/or UV absorbance detection. The OPA derivatives of GSH and GSNO obtained by this method were found to be identical by mass spectrometry. GSH (up to 50 microM) did not interfere with the analysis of GSNO (up to 1000 nM). The limits of detection of the method for buffered aqueous solutions of GSNO were determined as 3 nM using fluorescence and 70 nM using UV absorbance detection. Isolation of GSNO by HPLC analysis (pH 7.0) of plasma ultrafiltrate samples (200 microl) prior to derivatization allows specific and artifact-free quantification of GSNO in human and rat plasma. Reduced and oxidized glutathione, nitrite, and cysteine did not interfere with the measurement of GSNO in human and rat plasma. The limit of quantitation (LOQ) of the combined method was determined as 100 nM of GSNO in human plasma ultrafiltrate using fluorescence detection. No endogenous GSNO could be detected in ultrafiltrate samples of plasma of 10 healthy humans at concentrations exceeding the LOQ of the method. After iv infusion of GSNO (125 micromol/kg body wt) in a rat for 20 min GSNO and GSH were detected in rat plasma at 60 and 130 microM, respectively. The method should be useful to investigate formation, metabolism, and reactions of GSNO in vitro and in vivo at physiologically relevant concentrations.     

The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. II. Activation by the isolated perfused rat liver.

In this investigation Salmonella typhimurium strain TA 1530 and TA 1535 were combined with isolated perfused rat liver. Samples of perfusate and bile produced were tested for mutagenicity after treatment with 1,2-dichloroethane (DCE), 1,2-dibromoethane (DBE) or 2-chloroethanol. The results are in good agreement with our previous experiments which indicate that both DEC and DBE are activated through conjugation with glutathione (GSH). Most GSH conjugates are normally excreted in bile. Following liver perfusion the bile was highly mutagenic after DCE and DBE treatments, while 2-chloroethanol did not have this effect. The highest mutagenic effect was seen 15--30 min after the addition of DCE or DBE. The production of mutagenic bile also occurred in mice treated in vivo with DCE. One possible metabolic endproduct of a GSH conjugate is the corresponding mercapturic acid. Thus synthetic N-acetyl-S-(2-chloroethyl)-L-cysteine was tested on TA 1535 and found to be as mutagenic as S-(2-chloroethyl)-L-cysteine in the concentration range 0.2--0.6 mumol/plate. Differences and similarities in the metabolism of DCE and vinyl chloride are discussed on the basis of these results.     

The redox pathway of S-nitrosoglutathione, glutathione and nitric oxide in cell to neuron communications

Recent results demonstrated that S-nitrosoglutathione (GSNO) and nitric oxide (*NO) protect brain dopamine neurons from hydroxyl radical (*OH)-induced oxidative stress in vivo because they are potent antioxidants. GSNO and *NO terminate oxidant stress in the brain by (i) inhibiting iron-stimulated hydroxyl radicals formation or the Fenton reaction, (ii) terminating lipid peroxidation, (iii) augmenting the antioxidative potency of glutathione (GSH), (iv) mediating neuroprotective action of brain-derived neurotrophin (BDNF), and (v) inhibiting cysteinyl proteases. In fact, GSNO--S-nitrosylated GSH--is approximately 100 times more potent than the classical antioxidant GSH. In addition, S-nitrosylation of cysteine residues by GSNO inactivates caspase-3 and HIV-1 protease, and prevents apoptosis and neurotoxicity. GSNO-induced antiplatelet aggregation is also mediated by S-nitrosylation of clotting factor XIII. Thus the elucidation of chemical reactions involved in this GSNO pathway (GSH GS* + *NO-->[GSNO]-->GSSG + *NO-->GSH) is necessary for understanding the biology of *NO, especially its beneficial antioxidative and neuroprotective effects in the CNS. GSNO is most likely generated in the endothelial and astroglial cells during oxidative stress because these cells contain mM GSH and nitric oxide synthase. Furthermore, the transfer of GSH and *NO to neurons via this GSNO pathway may facilitate cell to neuron communications, including not only the activation of guanylyl cyclase, but also the nitrosylation of iron complexes, iron containing enzymes, and cysteinyl proteases. GSNO annihilates free radicals and promotes neuroprotection via its c-GMP-independent nitrosylation actions. This putative pathway of GSNO/GSH/*NO may provide new molecular insights for the redox cycling of GSH and GSSG in the CNS.     

Influence of glutathione and its derivatives on fibrin polymerization

A complex relationship exists between reduced, oxidized, and nitrosated glutathione (GSH, GSSG, and GSNO, respectively). Although previous studies have demonstrated S-nitrosoglutathione (GSNO) has potent antiplatelet efficacy, little work has examined the role of GSNO and related species on subsequent aspects of coagulation (e.g., fibrin polymerization). Herein, the effects of GSH, GSSG, and GSNO on the entire process of fibrin polymerization are described. Relative to normal fibrinogen, the addition of GSH, GSSG, or GSNO leads to prolonged lag times, slower rates of protofibril lateral aggregation and the formation of clots with lower final turbidities. Dose-dependent studies indicate the influence of GSH on fibrin formation is a function of both GSH and fibrinogen concentration. Studies with Aalpha251 recombinant fibrinogen (lacking alphaC regions) showed GSH had no influence on its polymerization, suggesting the glutathione species interact within the alphaC region of fibrinogen.     

Improved antimicrobial efficacy with nitric oxide releasing nanoparticle generated S-nitrosoglutathione

Nitric oxide (NO) plays a vital role in mammalian host defense through a variety of mechanisms. In particular, NO can oxidize to form reactive nitrogen species or interact with protein thiols and metal centers, blocking essential microbial processes. S-nitrosoglutathione (GSNO), a potent NO donor formed by the interaction of NO with intracellular glutathione (GSH), is a major factor in this pathway and is considered one of the strongest naturally occurring nitrosating agent. We previously described the broad-spectrum antimicrobial activity of a nanoparticulate platform capable of controlled and sustained release of NO (NO-np). Interestingly, in vivo efficacy of the NO-np surpassed in vitro data generated. We hypothesized that the enhanced activity was in part achieved via the interaction between the generated NO and available GSH, forming GSNO. In the current study, we investigated the efficiency of NO-np to form GSNO in the presence of GSH was evaluated, and assessed the antimicrobial activity of the formed GSNO against methicillin resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. When GSH was combined with NO-np, GSNO was rapidly produced and significant concentrations of GSNO were maintained for >24h. The GSNO generated was more effective compared to NO-np alone against all bacterial strains examined, with P. aeruginosa being the most sensitive and K. pneumoniae the most resistant. We conclude that the combination of NO-np with GSH is an effective means of generating GSNO, and presents a novel approach to potent antimicrobial therapy.     

Nitric oxide inactivates glyoxalase I in cooperation with glutathione

We previously found that glyoxalase I (Glo I) is inactivated upon exposure of human endothelial cells to extracellular nitric oxide (NO), and this event correlates with an increase in its pI on two-dimensional gels. In this study, we demonstrate that NO can modulate Glo I activity in cooperation with cellular glutathione (GSH). Severe depletion of intracellular GSH prevents the inactivation of Glo I in response to NO, although such depletion enhances the inactivation of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a well-known enzyme susceptible to NO-induced oxidation. S-Nitrosoglutathione (GSNO), an adduct of GSH and NO, lowers the activity of purified human Glo I, while S-nitrosocysteine (CysNO) inactivates the enzyme only in the presence of GSH. This indicates that a dysfunction in Glo I would require the formation of GSNO in situ. Competitive inhibitors of Glo I, S-(4-bromobenzyl)glutathione and its membrane-permeating form, completely abolish the NO action in vitro and inside cells, respectively. Taken together, these results reveal that Glo I can interact directly with GSNO, and that the interaction converts Glo I into an inactive form. Moreover, the data suggest that the substrate recognition site of Glo I might be involved in the interaction with GSNO.     

Production of Hydroxyl Free Radical in the Xanthine Oxidase System with Addition of 1-methyl-3-nitro-1-nitrosoguanidine

Recent results demonstrated that S-nitrosoglutathione (GSNO) and nitric oxide (^·NO) protect brain dopamine neurons from hydroxyl radical (^·OH)-induced oxidative stress in vivo because they are potent antioxidants. GSNO and ^·NO terminate oxidant stress in the brain by (i) inhibiting iron-stimulated hydroxyl radicals formation or the Fenton reaction, (ii) terminating lipid peroxidation, (iii) augmenting the antioxidative potency of glutathione (GSH), (iv) mediating neuroprotective action of brain-derived neurotrophin (BDNF), and (v) inhibiting cysteinyl proteases. In fact, GSNO — S-nitrosylated GSH — is approximately 100 times more potent than the classical antioxidant GSH. In addition, S-nitrosylation of cysteine residues by GSNO inactivates caspase-3 and HIV-1 protease, and prevents apoptosis and neurotoxicity. GSNO-induced antiplatelet aggregation is also mediated by S-nitrosylation of clotting factor XIII. Thus the elucidation of chemical reactions involved in this GSNO pathway (GSH → GS^· + ^·NO → [GSNO] → GSSG + ^·NO → GSH) is necessary for understanding the biology of ^·NO, especially its beneficial antioxidative and neuroprotective effects in the CNS. GSNO is most likely generated in the endothelial and astroglial cells during oxidative stress because these cells contain mM GSH and nitric oxide synthase. Furthermore, the transfer of GSH and ^·NO to neurons via this GSNO pathway may facilitate cell to neuron communications, including not only the activation of guanylyl cyclase, but also the nitrosylation of iron complexes, iron containing enzymes, and cysteinyl proteases. GSNO annihilates free radicals and promotes neuroprotection via its c-GMP-independent nitrosylation actions. This putative pathway of GSNO/GSH/^·NO may provide new molecular insights for the redox cycling of GSH and GSSG in the CNS.     

Influence of glutathione on the mutagenicity of 2-chloroethylnitrosoureas. Mutagenic potential of glutathione derivatives formed from 2-chloroethylnitrosoureas and glutathione

2-Chloroethylnitrosoureas (CNU) are antineoplastic agents whose therapeutic dose is limited by toxic and carcinogenic side effect. The clinically used drugs, bis-(2-chloroethyl)nitrosourea (BCNU) and 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea (HECNU) and their analogue N-(2-chloroethyl)-N-nitrosocarbamoyl-glycinamide (CNC-GA) were tested for mutagenicity and toxicity in the Salmonella typhimurium tester strain TA1535 in the presence and absence of glutathione (GSH). All 3 compounds proved to be potent mutagens. The cytotoxicity of these CNUs, however, varied depending on their carbamoylating activity. These cytotoxic effects were decreased considerably by the addition of GSH. It has been shown that the isocyanate decomposition product of the 2-chloroethylnitrosoureas reacts with GSH yielding S-carbamoylated GSH derivatives. The adducts resulting from coincubation of BCNU or HECNU with GSH, 2-chloroethyl-S-carbamoyl-GSH and 2-hydroxy-S-carbamoyl-GSH, were also tested for their mutagenic activity. While the hydroxyethylated compound exhibited no effects, 2-chloroethyl-S-carbamoyl-GSH and its cysteine analogue, 2-chloroethyl-S-carbamoyl-GSH, were strong mutagens. Further experiments with 3-chloropropyl-S-carbamoyl-GSH and t-butyl-S-carbamoyl-GSH indicate that a chlorine substituent in the beta position is necessary for the induction of a potent mutagenic response.     

DNA damage in tissues and organs of mice treated with diphenyl diselenide

Diphenyl diselenide (DPDS) is an organoselenium compound with interesting pharmacological activities and various toxic effects. In previous reports, we demonstrated the pro-oxidant action and the mutagenic properties of this molecule in bacteria, yeast and cultured mammalian cells. This study investigated the genotoxic effects of DPDS in multiple organs (brain, kidney, liver, spleen, testes and urinary bladder) and tissues (bone marrow, lymphocytes) of mice using in vivo comet assay, in order to determine the threshold of dose at which it has beneficial or toxic effects. We assessed the mechanism underlying the genotoxicity through the measurement of GSH content and thiobarbituric acid reactive species, two oxidative stress biomarkers. Male CF-1 mice were given 0.2-200 micromol/kg BW DPDS intraperitonially. DPDS induced DNA damage in brain, liver, kidney and testes in a dose response manner, in a broad dose range at 75-200 micromol/kg with the brain showing the highest level of damage. Overall, our analysis demonstrated a high correlation among decreased levels of GSH content and an increase in lipid peroxidation and DNA damage. This finding establishes an interrelationship between pro-oxidant and genotoxic effects. In addition, DPDS was not genotoxic and did not increase lipid peroxidation levels in any organs at doses < 50 micromol/kg. Finally, pre-treatment with N-acetyl-cysteine completely prevented DPDS-induced oxidative damage by the maintenance of cellular GSH levels, reinforcing the positive relationship of DPDS-induced GSH depletion and DNA damage. In summary, DPDS induces systemic genotoxicity in mammals as it causes DNA damage in vital organs like brain, liver, kidney and testes.     

Putative denitrosylase activity of Cu,Zn-superoxide dismutase

The Cu,Zn-superoxide dismutase (SOD1) has been reported to exert an S-nitrosylated glutathione (GSNO) denitrosylase activity that was augmented by a familial amyotrophic lateral sclerosis (FALS)-associated mutation in this enzyme. This putative enzymatic activity as well as the spontaneous decomposition of GSNO has been reexamined. The spontaneous decomposition of GSNO exhibited several peculiarities, such as a lag phase followed by an accelerating rate plus a marked dependence on GSNO concentration, suggestive of autocatalysis, and a greater rate in polypropylene than in glass vessels. Dimedone caused a rapid increase in absorbance likely due to reaction with GSNO, followed by a slower increase possibly due to reaction with an intermediate such as glutathione sulfenic acid. SOD1 weakly increased the rate of decomposition of GSNO, but did so only when GSH was present; and FALS-associated mutant forms of SOD1 were not more active in this regard than was the wild type. Decomposed GSNO, when added to fresh GSNO, hastened its decomposition, in accord with autocatalysis, and when added to GSH, generated GSNO in accord with the presence of nitrite. A mechanism is proposed that is in accord with these observations.     

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.     

Glutathione and other low-molecular-weight thiols relax guinea pig trachea ex vivo: interactions with nitric oxide?

The aim of this study was to determine the effects of glutathione (GSH) on trachea smooth muscle tension in view of previously reported interactions between GSH and nitric oxide (NO) (Gaston B. Biochim Biophys Acta 1411: 323-333, 1999; Kelm M. Biochim Biophys Acta 1411: 273-289, 1999; and Kharitonov VG, Sundquist AR, and Sharma VS. J Biol Chem 270: 28158-28164, 1995) and the high (millimolar) concentrations of GSH in trachea epithelium (Rahman I, Li XY, Donaldson K, Harrison DJ, and MacNee W. Am J Physiol Lung Cell Mol Physiol 269: L285-L292, 1995). GSH and other thiols (1.0-10 mM) dose dependently decreased the tension in isolated guinea pig tracheas. Relaxations by GSH were paralleled with sevenfold increased nitrite levels (P < 0.05) in the tracheal effluent, suggesting an interaction between GSH and NO. However, preincubation with a NO scavenger did not reduce the relaxations by GSH or its NO adduct, S-nitrosoglutathione (GSNO). Inhibition of guanylyl cyclase inhibited the relaxations induced by GSNO, but not by GSH. Blocking potassium channels, however, completely abolished the relaxing effects of GSH (P < 0.05). Preincubation of tracheas with GSH significantly (P < 0.05) suppressed hyperreactivity to histamine as caused by removal of tracheal epithelium. These data indicate that GSH plays a role in maintaining tracheal tone. The mechanism is probably an antioxidative action of GSH itself rather than an action of NO or GSNO.     

Leishmanicidal activity of primary S-nitrosothiols against Leishmania major and Leishmania amazonensis: implications for the treatment of cutaneous leishmaniasis.

Nitric oxide (NO) is considered a key molecule in the defense against intracellular pathogens, particularly Leishmania. The expression of inducible nitric oxide synthase and consequent production of NO by infected macrophages has been shown to correlate with leishmaniasis resistance in the murine model as well as in human patients. Nitric oxide donors have been used successfully in the treatment of cutaneous leishmaniasis in humans, although their mechanisms of action are not fully understood. In the present work, the dose-dependent cytotoxic effects of the NO-donors S-nitroso-N-acetyl-l-cysteine (SNAC) and S-nitrosoglutathione (GSNO) against Leishmania were evaluated. GSNO inhibited the growth of Leishmania major and Leishmania amazonensis with in vitro 50% inhibitory concentrations (IC(50)) of 68.8+/-22.86 and 68.9+/-7.9 micromol L(-1), respectively. The IC(50) for SNAC against L. major and L. amazonensis were, respectively, 54.6+/-8.3 and 181.6+/-12.5 micromol L(-1). The leishmanicidal activity of GSNO, but not of SNAC, was reversed by ascorbic acid (AA) and dithiothreitol (DTT), suggesting that the mechanism of action of GSNO is related to the transnitrosation of parasite proteins. These results demonstrate that SNAC and GSNO have leishmanicidal activity, and are thus potential therapeutic agents against cutaneous leishmaniasis.     

Oxidation and nitrosation of thiols at low micromolar exposure to nitric oxide. Evidence for a free radical mechanism

Although the nitric oxide (.NO)-mediated nitrosation of thiol-containing molecules is increasingly recognized as an important post-translational modification in cell signaling and pathology, little is known about the factors that govern this process in vivo. In the present study, we examined the chemical pathways of nitrosothiol (RSNO) production at low micromolar concentrations of .NO. Our results indicate that, in addition to nitrosation by the .NO derivative dinitrogen trioxide (N2O3), RSNOs may be formed via intermediate one-electron oxidation of thiols, possibly mediated by nitrogen dioxide (.NO2), and the subsequent reaction of thiyl radicals with .NO. In vitro, the formation of S-nitrosoglutathione (GSNO) from .NO and excess glutathione (GSH) was accompanied by the formation of glutathione disulfide, which could not be ascribed to the secondary reaction of GSH with GSNO. Superoxide dismutase significantly increased GSNO yields and the thiyl radical trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), inhibited by 45 and 98% the formation of GSNO and GSSG, respectively. Maximum nitrosation yields were obtained at an oxygen concentration of 3%, whereas higher oxygen tensions decreased GSNO and increased GSSG formation. When murine fibroblasts were exposed to exogenous .NO, RSNO formation was sensitive to DMPO and oxygen tension in a manner similar to that observed with GSH alone. Our data indicate that RSNO formation is favored at oxygen concentrations that typically occur in tissues. Nitrosothiol formation in vivo depends not only on the availability of .NO and O2 but also on the degree of oxidative stress by affecting the steady-state concentration of thiyl radicals.     

The effect of oxidative stress on endothelium-dependent and nitric oxide donor-induced relaxation: implications for nitrate tolerance.

Increased inactivation of nitric oxide (NO) by superoxide has been implicated in nitrate tolerance. Here, we set out to compare the inhibitory effect of superoxide on endothelium-dependent, acetylcholine (ACh)-mediated vascular relaxation with that on the endothelium-independent effects of glyceryl trinitrate (GTN) and another NO donor drug, S-nitrosoglutathione (GSNO). Rings of thoracic aorta from adult male Wistar rats (350-450 g) were precontracted with phenylephrine (approximately EC(90)) prior to cumulative additions (10 nM/L-10 microM/L) of GTN, GSNO, or ACh. Rings were then treated with the superoxide generator pyrogallol (300 micromol/L) alone or following pretreatment with the Cu/Zn superoxide dismutase inhibitor diethyldithiocarbamate (DETCA; 100 micromol/L), and cumulative additions of the vasodilators were repeated. All experiments were conducted in the presence of catalase (3000 U/ml) to prevent accumulation of hydrogen peroxide. Relaxation to ACh was abolished by pyrogallol-derived superoxide. Relaxation to GSNO was significantly inhibited by superoxide (P < 0.05, n = 8) and was more pronounced at lower GSNO concentrations. However, GTN was relatively resistant to inhibition by superoxide with modest inhibition only occurring in rings pretreated with DETCA prior to pyrogallol (P < 0.05; n = 8). In contrast to GSNO, the inhibitory effect was more pronounced with high concentrations of GTN, suggesting that the mechanism underlying superoxide-mediated inhibition is different for the two NO donor drugs. Further experiments showed that vascular responses to ACh were not inhibited (P > 0.05, n = 6) in aortic rings made tolerant to GTN (10 micromol/L, 2-h incubation) and that treatment of vessels with the antioxidant vitamin C (1 mmol/L) successfully prevented the development of tolerance. Taken together, these results suggest that superoxide is not a major factor in tolerance in vitro and imply that the protective actions of vitamin C are unrelated to its antioxidant activity in this setting.     

Free radical scavenging abilities of flavonoids as mechanism of protection against mutagenicity induced by tert-butyl hydroperoxide or cumene hydroperoxide in Salmonella typhimurium TA102

Mutagenicity induced by tert-butyl hydroperoxide (BHP) or cumene hydroperoxide (CHP) in Salmonella typhimurium TA102 was effectively reduced by flavonols with 3',4'-hydroxyl groups such as fisetin, quercetin, rutin, isoquercitrin, hyperoxide, myricetin, myricitrin, robinetin, and to a lesser extent also by morin and kaempferol (ID50=0.25-1.05 micromol per plate). With the exception of isorhamnetin, rhamnetin, morin, and kaempferol, closely similar results were obtained with both peroxides. Hydrogenation of the double bond between carbons 2 and 3 (dihydroquercetin, dihydrorobinetin) as well as the additional elimination of the carbonyl function at carbon 4 (catechins) resulted in a loss of antimutagenicity with the notable exception of catechin itself. Again, all flavones and flavanones tested were inactive except luteolin, luteolin-7-glucoside, diosmetin, and naringenin. The typical radical scavenger butylated hydroxytoluene also showed strong antimutagenicity against CHP (ID50=5.4 micromol per plate) and BHP (ID50=11.4 micromol per plate). Other lipophilic scavengers such as alpha-tocopherol and N,N'-diphenyl-1,4-phenylenediamine exerted only moderate effects, the hydrophilic scavenger trolox was inactive. The metal chelating agent 1,10-phenanthroline strongly reduced mutagenicities induced by CHP and BHP (ID50=2.75 and 2.5 micromol per plate) at low concentrations but induced mutagenic activities at higher concentrations. The iron chelator deferoxamine mesylate, however, was less effective in both respects. The copper chelator neocuproine effectively inhibited mutagenicity induced by BHP (ID50=39.7 micromol per plate) and CHP (ID50=25.9 micrommol per plate), the iron chelator 2,2'-dipyridyl was less potent (ID50=6.25 mmol per plate against BHP, 0.42 mmol per plate against CHP). In the absence of BHP and CHP, yet not in the presence of these hydroperoxides, quercetin, rutin, catechin, epicatechin, and naringenin induced strong mutagenic activities in S. typhimurium TA102. Radical scavenging activities of flavonoids against peroxyl radicals generated from 2,2'-azo-bis(2-amidinopropane)dihydrochloride (AAPH) as measured in the haemolysis test, confirmed that in general flavonoids with di- or trihydroxy hydroxyl functions especially in positions 3', 4', 5' are effective radical scavengers. In this test system, however, luteolin was the most potent compound, followed by epicatechin and eriodictyol. Again, isorhamnetin was a potent inhibitor of lysis of red blood cells despite the presence of a 3'-OCH3 function. Radical scavenging activities of flavonoids against the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) in principle obeyed the rules outlined above. Flavanones, tamarixetin, and rhamnetin, however, were only weakly active against DPPH, while isorhamnetin was again a potent compound. From these results we conclude that in the Salmonella/reversion assay with strain TA102 antimutagenic activities of flavonoids against the peroxide mutagens CHP and BHP are mainly caused by radical scavenging effects.     

Detection and quantification of S-nitrosoglutathione (GSNO) in pepper (Capsicum annuum L.) plant organs by LC-ES/MS

Glutathione (GSH) is one of the major, soluble, low molecular weight antioxidants, as well as the major non-protein thiol in plant cells. However, the relevance of this molecule could be even greater considering that it can react with nitric oxide (NO) to generate S-nitrosoglutathione (GSNO) which is considered to function as a mobile reservoir of NO bioactivity in plants. Although this NO-derived molecule has an increased physiological and phytopathological relevance in plants cells, its identification and quantification in plant tissues have not be reported so far. Using liquid chromatography-electrospray/mass spectrometry (LC-ES/MS), a method was set up to detect and quantify simultaneously GSNO as well reduced and oxidized glutathione (GSH and GSSG, respectively) in different pepper plant organs including roots, stems and leaves, and in Arabidopsis leaves. The analysis of NO and GSNO reductase (GSNOR) activity in these pepper organs showed that the content of GSNO was directly related to the content of NO in each organ and oppositely related to the GSNOR activity. This approach opens up new analytical possibilities to understand the relevance of GSNO in plant cells under physiological and stress conditions.     

Evaluation of hexamethylphosphoramide for gene mutations in Salmonella typhimurium using plate incorporation, preincubation, and suspension assays

Hexamethylphosphoramide (HMPA), a potent rat nasal carcinogen by inhalation, and three of its metabolites, pentamethylphosphoramide (PMPA), trimethylphosphoramide (TriMPA), and formaldehyde (HCHO), were assessed in Salmonella typhimurium gene mutation assays using various protocols, including plate incorporation, preincubation and suspension assays. HMPA (tested up to 15 000 μg/plate) was not mutagenic in plate incorporation or preincubation assays with or without metabolic activation. HCHO was mutagenic in the plate incorporation and preincubation assays (tested up to 150 μg/plate). In suspension assays, however, HMPA (tested up to 40 mg/ml), PMPA (up to 44 mg/ml) and HCHO (up to 45 μg/ml), but not TriMPA (up to 29 mg/ml), were mutagenic. HMPA and PMPA were positive only with activation. HMPA's mutagenicity was optimized using a relatively high level of rat liver S9 protein (3.5 mg/plate) in the metabolic activation mixture. Semicarbazide, an HCHO trapping agent, added at concentrations up to 167 μg/ml, markedly inhibited the mutagenic activities of HMPA and PMPA suggesting that HCHO generation may play a role in their mutagenicity. These studies show that HMPA is mutagenic in a modified Salmonella typhimurium reverse mutation assay with metabolic activation. Successive N-demethylation of HMPA eventually eliminates the mutagenic activity which further suggests that HMPA's mutagenic activity is related to the release of HCHO.     

Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors

GSNO (S-nitrosoglutathione) is emerging as a key regulator in NO signalling as it is in equilibrium with S-nitrosated proteins. Accordingly, it is of great interest to investigate GSNO metabolism in terms of competitive pathways and redox state. The present study explored ADH3 (alcohol dehydrogenase 3) in its dual function as GSNOR (GSNO reductase) and glutathione-dependent formaldehyde dehydrogenase. The glutathione adduct of formaldehyde, HMGSH (S-hydroxymethylglutathione), was oxidized with a k(cat)/K(m) value approx. 10 times the k(cat)/K(m) value of GSNO reduction, as determined by fluorescence spectroscopy. HMGSH oxidation in vitro was greatly accelerated in the presence of GSNO, which was concurrently reduced under cofactor recycling. Hence, considering the high cytosolic NAD(+)/NADH ratio, formaldehyde probably triggers ADH3-mediated GSNO reduction by enzyme-bound cofactor recycling and might result in a decrease in cellular S-NO (S-nitrosothiol) content in vivo. Formaldehyde exposure affected S-NO content in cultured cells with a trend towards decreased levels at concentrations of 1-5 mM, in agreement with the proposed mechanism. Product formation after GSNO reduction to the intermediate semimercaptal responded to GSH/GSNO ratios; ratios up to 2-fold allowed the spontaneous rearrangement to glutathione sulfinamide, whereas 5-fold excess of GSH favoured the interception of the intermediate to form glutathione disulfide. The sulfinamide and its hydrolysis product, glutathione sulfinic acid, inhibited GST (glutathione transferase) activity. Taken together, the findings of the present study provide indirect evidence for formaldehyde as a physiological trigger of GSNO depletion and show that GSNO reduction can result in the formation of GST inhibitors, which, however, is prevented under normal cellular redox conditions.     

Effect of S-nitrosoglutathione on renal mitochondrial function: a new mechanism for reversible regulation of manganese superoxide dismutase activity?

Mitochondria are at the heart of all cellular processes as they provide the majority of the energy needed for various metabolic processes. Nitric oxide has been shown to have numerous roles in the regulation of mitochondrial function. Mitochondria have enormous pools of glutathione (GSH≈5–10 mM). Nitric oxide can react with glutathione to generate a physiological molecule, S-nitrosoglutathione (GSNO). The impact GSNO has on mitochondrial function has been intensively studied in recent years, and several mitochondrial electron transport chain complex proteins have been shown to be targeted by GSNO. In this study we investigated the effect of GSNO on mitochondrial function using normal rat proximal tubular kidney cells (NRK cells). GSNO treatment of NRK cells led to mitochondrial membrane depolarization and significant reduction in activities of mitochondrial complex IV and manganese superoxide dismutase enzyme (MnSOD). MnSOD is a critical endogenous antioxidant enzyme that scavenges excess superoxide radicals in the mitochondria. The decrease in MnSOD activity was not associated with a reduction in its protein levels and treatment of NRK cell lysate with dithiothreitol (a strong sulfhydryl-group-reducing agent) restored MnSOD activity to control values. GSNO is known to cause both S-nitrosylation and S-glutathionylation, which involve the addition of NO and GS groups, respectively, to protein sulfhydryl (SH) groups of cysteine residues. Endogenous GSH is an essential mediator in S-glutathionylation of cellular proteins, and the current studies revealed that GSH is required for MnSOD inactivation after GSNO or diamide treatment in rat kidney cells as well as in isolated kidneys. Further studies showed that GSNO led to glutathionylation of MnSOD; however, glutathionylated recombinant MnSOD was not inactivated. This suggests that a more complex pathway, possibly involving the participation of multiple proteins, leads to MnSOD inactivation after GSNO treatment. The major highlight of these studies is the fact that dithiothreitol can restore MnSOD activity after GSNO treatment. To our knowledge, this is the first study showing that MnSOD activity can be reversibly regulated in vivo, through a mechanism involving thiol residues.