Role of glutathione in the regulation of hepatic cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme of bile acid biosynthesis

The effect of in vivo variation of hepatic glutathione (using diethyl maleate and L-cysteine) on in vitro cholesterol 7 alpha-hydroxylase activity was studied in male Sprague-Dawley rats. Cholesterol 7 alpha-hydroxylase activity in glutathione-depleted rats (ca. 10% of control glutathione) was significantly reduced compared to that in vehicle-injected controls. While L-cysteine treatment of glutathione-depleted animals increased glutathione levels somewhat (ca. 20% of control glutathione), they were still significantly less than control levels. Similarly, cholesterol 7 alpha-hydroxylase activity in the partially glutathione replete animals was approximately 50% greater than that in the glutathione-depleted animals, but still significantly less than that in the controls. The rate of 7 alpha-hydroxylation of cholesterol was found to be dependent on liver glutathione content. The calculated maximal rate was 34.4 picomoles/mg/min with a half maximal activity at 1.89 mumoles glutathione/gm liver. These results suggest that hepatic glutathione may be an important modulator of in vivo activity of cholesterol 7 alpha-hydroxylase.     

Antioxidant activity of carotenoid lutein in vitro and in vivo

Carotenoid lutein was evaluated for its antioxidant potential both in vitro and in vivo. Lutein was found to scavenge superoxide radicals, hydroxyl radicals and inhibited in vitro lipid peroxidation. Concentrations needed for 50% inhibition (IC50) were 21, 1.75 and 2.2 microg/mL respectively. It scavenged 2,2-diphenyl-1-picryl hydrazyl (IC50 35 microg/mL) and nitric oxide radicals (IC50 3.8 microg/mL) while 2,2-azobis-3-ethylbenzthiozoline-6-sulfonic acid radicals were inhibited at higher concentration. Ferric reducing power (50%) of lutein was found to be equal 0.3 micromols/mL of FeSO4.7H2O. Its oral administration inhibited superoxide generation in macrophages in vivo by 34.18, 64.32 and 70.22% at doses of 50, 100 and 250 mg/kg body weight. The oral administration of lutein in mice for 1 month significantly increased the activity of catalase, superoxide dismutase, glutathione reductase and glutathione in blood and liver while the activity of glutathione peroxidase and glutathione-S-transferase were found to be increased in the liver tissue. Implication of these results in terms of its role in reducing degenerative diseases is discussed.     

Reduced glutathione inhibits the alkylation by N-nitrosodimethylamine of liver DNA in vivo and microsomal fraction in vitro

The transfer of radioactivity from N-nitroso-[14C]dimethylamine to trichloroacetic acid precipitable macromolecules in the microsomal fraction of rat liver was investigated. This transfer was found to depend on N-nitrosodimethylamine being metabolized. Cytosolic fraction and cytosol enriched with reduced glutathione inhibited the binding of radioactivity to acid insoluble proteins. Depletion of glutathione in rat liver with diethylmaleate prior to i.v. administration of 10 mg N-nitroso-[14C]dimethylamine/kg led to an increase in O6-methylguanine and N-7-methylguanine in DNA. If rats were fed disulfiram for 6 days (2 g/kg feed), glutathione and glutathione S-transferase were enhanced, and the degree of methylation of guanine by N-nitrosodimethylamine was greatly reduced, as was the metabolism of N-nitrosodimethylamine in the intact animal. Fasting rats for 24 h did not change the N-nitrosodimethylamine-demethylase activity in vitro but greatly enhanced the methylation of guanine in vivo, while the glutathione content and glutathione S-transferase activity were not changed compared to fed animals.     

Perfluorodecanoic acid decreases the enzyme activity and the amount of glutathione S-transferases proteins and mRNAs in vivo

The effects of the anti-wetting agent perfluoro-n-decanoic acid (PFDA) on various glutathione S-transferase (GST) enzyme activities were studied in vitro and in vivo. In addition the effects of PFDA treatment on the amount of some glutathione S-transferase subunits and their corresponding translatable mRNA were studied in vivo. PFDA like some other peroxisome proliferators was a non-competitive inhibitor of several GST enzyme activities in vitro. In vivo PFDA reduced the enzyme activity towards substrates which are indicative for the Ya, Yb1 and Yb2 subunits of GSTs to a larger extent than the enzyme activity towards the substrate indicative for the Yc subunit. Whereas the reduction of GST enzyme activities by other peroxisome proliferators was shown to be caused by an inhibition of the relevant enzymes in vivo, PFDA was found to decrease the GST enzyme activities at least in part by lowering the amount of the various GST subunits in vivo due to a lowered concentration of translatable mRNA coding for these enzymes. In addition PFDA abolished the inducibility of GST mRNAs by phenobarbital. Thus PFDA might be an interesting tool for mechanistic studies of the control of GST expression in the liver.     

Glutathione reductase is not required for maintenance of reduced glutathione in Escherichia coli K-12.

Seven independently isolated glutathione reductase-deficient (gor) Escherichia coli mutants were found to have an in vivo glutathione redox state that did not significantly differ from that of the parental strain, 98 to 99% reduced. Strains containing both a gor mutation and either a trxA mutation (thioredoxin deficient) or a trxB mutation (thioredoxin reductase deficient) were able to maintain a 94 to 96% reduced glutathione pool, suggesting that glutathione can be reduced independently of glutathione reductase and thioredoxin reductase.     

A major fraction of endoplasmic reticulum-located glutathione is present as mixed disulfides with protein

The tripeptide glutathione is the most abundant thiol/disulfide component of the eukaryotic cell and is known to be present in the endoplasmic reticulum lumen. Accordingly, the thiol/disulfide redox status of the endoplasmic reticulum lumen is defined by the status of glutathione, and it has been assumed that reduced and oxidized glutathione form the principal redox buffer. We have determined the distribution of glutathione between different chemical states in rat liver microsomes by labeling with the thiol-specific label monobromobimane and subsequent separation by reversed phase high performance liquid chromatography. More than half of the microsomal glutathione was found to be present in mixed disulfides with protein, the remainder being distributed between the reduced and oxidized forms of glutathione in the ratio of 3:1. The high proportion of the total population of glutathione that was found to be in mixed disulfides with protein has significant implications for the redox state and buffering capacity of the endoplasmic reticulum and, hence, for the formation of disulfide bonds in vivo.     

The role of oxidant injury in tumor cell sensitivity to recombinant human tumor necrosis factor in vivo. Implications for mechanisms of action

The intracellular glutathione levels of two human tumor lines and seven murine tumor lines were determined in order to investigate the role of oxidant injury in tumor cell sensitivity to human rTNF (rhTNF). Correlations were found between high intracellular glutathione levels and in vivo tumor resistance to rhTNF, and on the other hand, low glutathione levels and rhTNF sensitivity. The transplantable murine fibrosarcoma, Meth A, a TNF-sensitive line in vivo, was less sensitive to rhTNF and host toxicity was reduced when the hosts were pretreated with uric acid, a major reactive oxygen scavenger in humans and certain other primates. Conversely, pretreatment of the tumor-bearing hosts with DL-buthionine-(S,R)-sulfoximine, an inhibitor of GSH biosynthesis, resulted in an increased sensitivity of Meth A to rhTNF. This effect was not limited to tumor-bearing mice, as rats pretreated with diethyl maleate, a compound which irreversibly binds glutathione, were more sensitive to rhTNF toxicity than control rats. On the other hand, pretreatment with N-acetyl cysteine, an oxidant scavenger, reduced the toxicity of rhTNF treatment in rats. The data are consistent with the hypothesis that tumor cell sensitivity to rhTNF in vivo is dependent on its capacity to buffer oxidative attack. In addition, host toxicity is also related to the production of reactive oxygen species. Activated effector cells such as granulocytes and macrophages are hypothesized to produce most of this damage by their respiratory burst and oxidant release, although the direct action of rhTNF may also contribute to oxidative injury in vivo.     

The function of gamma-glutamyl transpeptidase as a determinant in cell sensitivity to azaserine toxicity

The enzyme gamma-glutamyl transpeptidase (GGT) is characteristically present at high levels in mammalian cells that are vulnerable in vivo to the selectively toxic and carcinogenic effects of the naturally occurring diazo amino acid L-azaserine. The possible role of GGT as a determinant of cellular sensitivity to azaserine toxicity was investigated. No correlation was found between GGT activity and the abilities of different cell lines or GGT-deficient cell strains of TuWi, a human nephroblastoma-derived line high in GGT, to accumulate azaserine. However, the thiols glutathione and cysteine were found to inhibit the toxicity of azaserine in cultures of TuWi. In addition, maleate lowered both intracellular and extracellular glutathione levels and enhanced sensitivity of TuWi cells to azaserine, while serine-borate, a potent inhibitor of GGT, increased extracellular glutathione levels and inhibited azaserine toxicity. Since extracellular glutathione accumulation, which may reflect the rate of cellular glutathione turnover, is increased in cultures of azaserine-resistant, GGT-deficient strains of TuWi, we propose that GGT enhances cellular sensitivity to azaserine primarily by increasing the rate of glutathione turnover, thus removing the glutathione from detoxification pathways.     

Extracellular metabolism of glutathione accounts for its disappearance from the basolateral circulation of the kidney

Glutathione labeled in each of its amino acid residues, the corresponding free amino acids, and gamma-glutamyl-amino acids were used to evaluate their renal basolateral transport and metabolism at physiological levels of glutathione. Recovery of label in the venous outflow was compared to that of co-administered inulin after a single-pass in vivo infusion of rat kidney. Metabolites of glutathione and of its constituent amino acids were determined. No net basolateral transport of glutathione was detected; instead there was extensive breakdown of glutathione by the actions of basolateral gamma-glutamyl transpeptidase and dipeptidase. Glutamate and 5-oxoproline showed net basolateral uptake. Recoveries of 35S greater than those of inulin were found after perfusion of [35S]cysteine and [35S]glutathione suggesting rapid net tubular reabsorption of cyst(e)ine. Recovery of label from perfused [U-14C]glycine was equivalent to that of inulin consistent with little or no net flux. Co-administration of large amounts of unlabeled metabolites together with the labeled glutathiones led to label recoveries closer to those of inulin, consistent with competitive inhibition of labeled metabolite transport. Treatment of rats with an inhibitor of gamma-glutamyl transpeptidase decreased basolateral glutathione metabolism and thus indirectly decreased transport of labeled metabolites. No net basolateral transport of gamma-glutamyl-amino acids was detected. Significant amounts of label perfused as [Glu-U-14C]glutathione appeared in the gamma-glutamyl-amino acid fraction of the renal venous outflows, providing direct evidence that glutathione is used in vivo for the formation of gamma-glutamyl-amino acids.     

Enzymatic Protection Against Peroxidative Damage in Isolated Brain Capillaries

The content of polyunsaturated fatty acids, the activities of superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, and catalase, and the concentration of reduced glutathione were measured in cerebral microvessels isolated from rat brain. Polyunsaturated fatty acids, mainly arachidonic, linoleic, and docosahexaenoic acids, accounted for 32% of total fatty acids in cerebral microvessels. Whereas total SOD activity in the microvessels was slightly lower than that found in cerebrum and cerebellum, glutathione peroxidase and glutathione reductase activities were twice as high and catalase activity was four times higher. Glutathione peroxidase in microvessels is active on both hydrogen peroxide and cumen hydroperoxide, and it is strongly inhibited by mercaptosuccinate. After several hours of preparation, the concentration of reduced glutathione in isolated microvessels was 0.7 mumol/mg of protein, which corresponds to a concentration of approximately 3.5 mM. Our results indicate that the blood-brain barrier contains large amounts of peroxide-detoxifying enzymes, which may act, in vivo, to protect its highly polyunsaturated membranes against oxidative alterations.     

Inhibition of rat liver glutathione S-transferase isoenzymes by acrolein

The in vitro effect of the toxin and teratogen, acrolein, on the fetal rat liver glutathione S-transferase isoenzyme, YcYfetus, was investigated and compared with acrolein's effect on some of the adult rat liver glutathione S-transferase isoenzymes. Acrolein was found to inhibit all the isoenzymes investigated and double-reciprocal plots suggest that inhibition is either noncompetitive or mixed-type noncompetitive. It is therefore attractive to suggest that should a similar situation arise in vivo, it may provide one mechanism for the teratogenicity of acrolein.     

Effect of low level zinc pretreatment on zinc-mediated toxicity in different lung cell lines

Reduced toxicity of high zinc exposure was observed after pretreatment of various lung cells with nonlethal zinc concentrations. This effect became significant when various parameters of cytotoxicity were assessed (e.g., inhibition of protein synthesis, depletion of reduced glutathione [GSH], increase of oxidized glutathione [GSSG], release of lactate dehydrogenase [LDH]). Similar protective effects by zinc have already been shown by several investigators for a variety of toxicity studies dealing with cadmium, in vitro and in vivo. Zinc-induced toxicity has been linked to glutathione metabolism and cellular GSH contents. Activity of glutathione reductase (GR) and rates of glutathione synthesis were identified as determinants of zinc (cyto)toxicity. However, these variables were virtually unaffected in our adapted cells. Consequently, another variable appears to be crucial for modulating cellular suscepticibility in zinc pretreated cells. Protection in our cells was achieved by pretreatment with 80–120 μmol/L zinc chloride for 24–72 h, roughly 10-fold more zinc in the medium than is normally found in human plasma. Protection was not observed when the cells were concomitantly exposed to cycloheximide, an inhibitor of protein synthesis, or actinomycin D, an inhibitor of RNA synthesis, but it was found in the presence of amanitin, an inhibitor of mRNA synthesis. It is therefore concluded that the altered zinc tolerance of pretreated cells is not attributable to the induction of metallothionein.     

Resistance to Fas-induced apoptosis in hepatocytes: role of GSH depletion by cell isolation and culture

The involvement of reduction/oxidation (redox) state in cell sensitivity to apoptosis has been suggested by several studies in which induction of apoptosis was shown to require oxidative stress or GSH extrusion. On the other hand, biochemical studies of caspases revealed that their activation necessitates a reduced cysteine in their active site. This is ensured by maintaining intact intracellular glutathione status during apoptotic induction as reported by in vivo studies. Therefore, we investigated the relationship between intracellular glutathione levels and the sensitivity of mouse hepatocytes in culture to Fas-induced apoptosis as well as potential mechanisms responsible for this sensitivity. We found that total and reduced glutathione levels are decreased by one-half after cell isolation procedure and further decline by 25% during cell culture for 2 h in normal Williams' E medium. Cell culture in medium supplemented with cysteine and methionine maintains glutathione at a level similar to that measured just after cell isolation. Results show that the capacity of Fas to activate caspase-8 and to induce apoptosis requires important intracellular glutathione levels and high GSH/total glutathione ratio. In conclusion, the present study shows that intracellular glutathione plays an important role in maintaining the apoptotic machinery functional and is thus capable of transmitting the apoptotic signal.     

Glutathione utilization by Candida albicans requires a functional glutathione degradation (DUG) pathway and OPT7, an unusual member of the oligopeptide transporter family

Candida albicans lacks the ability to survive within its mammalian host in the absence of endogenous glutathione biosynthesis. To examine the ability of this yeast to utilize exogenous glutathione, we exploited the organic sulfur auxotrophy of C. albicans met15Δ strains. We observed that glutathione is utilized efficiently by the alternative pathway of glutathione degradation (DUG pathway). The major oligopeptide transporters OPT1-OPT5 of C. albicans that were most similar to the known yeast glutathione transporters were not found to contribute to glutathione transport to any significant extent. A genomic library approach to identify the glutathione transporter of C. albicans yielded OPT7 as the primary glutathione transporter. Biochemical studies on OPT7 using radiolabeled GSH uptake revealed a K(m) of 205 μm, indicating that it was a high affinity glutathione transporter. OPT7 is unusual in several aspects. It is the most remote member to known yeast glutathione transporters, lacks the two highly conserved cysteines in the family that are known to be crucial in trafficking, and also has the ability to take up tripeptides. The transporter was regulated by sulfur sources in the medium. OPT7 orthologues were prevalent among many pathogenic yeasts and fungi and formed a distinct cluster quite remote from the Saccharomyces cerevisiae HGT1 glutathione transporter cluster. In vivo experiments using a systemic model of candidiasis failed to detect expression of OPT7 in vivo, and strains disrupted either in the degradation (dug3Δ) or transport (opt7Δ) of glutathione failed to show a defect in virulence.     

Redox regulation of 3'-phosphoadenylylsulfate reductase from Escherichia coli by glutathione and glutaredoxins

Inorganic sulfate (SO42-, S+VI) is reduced in vivo to sulfite (SO32-, S+IV) via phosphoadenylylsulfate (PAPS) reductase. Escherichia coli lacking glutathione reductase and glutaredoxins (gor-grxA-grxB-grxC-) barely grows on sulfate. We found that incubation of PAPS reductase with oxidized glutathione leads to enzyme inactivation with simultaneous formation of a mixed disulfide between glutathione and the active site Cys-239. A newly developed method based on thiol-specific fluorescent alkylation and gel electrophoresis showed that glutathionylated PAPS reductase is reduced by glutaredoxins via a monothiol mechanism. This glutathionylated species was also observed in poorly growing gor-grxA-grxB-grxC- cells expressing inactive glutaredoxin 2 (Grx2) C9S/C12S. However, it was absent in better growing cells expressing monothiol Grx2 C12S or wild type Grx2. Reversible glutathionylation may thus regulate the activity of PAPS reductase in vivo.     

Non-invasive Monitoring of L-2-Oxothiazolidine-4-Carboxylate Metabolism in the Rat Brain by In vivo 13C Magnetic Resonance Spectroscopy

The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5-(13)C]-thiazolidine-4-carboxylate, (13)C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by (13)C magnetic resonance spectroscopy. Although uptake and clearance of (13)C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of (13)C-OTZ over 20 h, however, resulted in (13)C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.     

Glutathione status in retinopathy of prematurity.

This study examines the glutathione status of red blood cells in patients with retinopathy of prematurity (ROP) both in vivo and after an in vitro oxidative challenge. Fifty ROP patients of different ages (between 6 weeks and 6 years), born prematurely (gestational age: 28.7 +/- 1.3 weeks; birth weight: 1210 +/- 313 g; mean +/- SD) suffering either from active ROP (<3 months old; n = 12) or from a visual handicap due to preceding ROP (3 months-6 years; n = 38) as well as control patients of similar age and maturity (n = 56) were included. Infants with active disease have the lowest levels of reduced glutathione (GSH), the highest levels of oxidized form (GSSG), the highest GSSG/GSH ratios and the greatest fall in GSH after an in vitro oxidative challenge. After an in vitro oxidative stress, defective glutathione recycling was found in patients with preceding ROP and was suggested as a factor predisposing to oxidative hemolysis. The glutathione redox ratio was warranted as a biochemical screen for active ROP in premature infants.     

Vitamin C is an essential antioxidant that enhances survival of oxidatively stressed human vascular endothelial cells in the presence of a vast molar excess of glutathione

Cellular glutathione levels may exceed vitamin C levels by 10-fold, generating the question about the real antioxidant role that low intracellular concentrations of vitamin C can play in the presence of a vast molar excess of glutathione. We characterized the metabolism of vitamin C and its relationship with glutathione in primary cultures of human endothelial cells oxidatively challenged by treatment with hydrogen peroxide or with activated cells undergoing the respiratory burst, and analyzed the manner in which vitamin C interacts with glutathione to increase the antioxidant capacity of cells. Our data indicate that: (i) endothelial cells express transporters for reduced and oxidized vitamin C and accumulate ascorbic acid with participation of glutathione-dependent dehydroascorbic acid reductases, (ii) although increased intracellular levels of vitamin C or glutathione caused augmented resistance to oxidative stress, 10-times more glutathione than vitamin C was required, (iii) full antioxidant protection required the simultaneous presence of intracellular and extracellular vitamin C at concentrations normally found in vivo, and (iv) intracellular vitamin C cooperated in enhancing glutathione recovery after oxidative challenge thus providing cells with enhanced survival potential, while extracellular vitamin C was recycled through a mechanism involving the simultaneous neutralization of oxidant species. Therefore, in endothelial cells under oxidative challenge, vitamin C functions as an essential cellular antioxidant even in the presence of a vast molar excess of glutathione.     

Enzymatic reaction of chlorotrifluoroethene with glutathione: 19F NMR evidence for stereochemical control of the reaction

Chlorotrifluoroethene, a potent nephrotoxin, is a substrate for the glutathione S-transferases present in the cytosolic and microsomal fractions of rat liver. The glutathione conjugate formed by both subcellular fractions has been identified as S-(2-chloro-1,1,2-trifluoroethyl)glutathione by 1H and 19F NMR and by secondary ion mass spectrometry. The conjugate formed by the cytosolic fraction is an equimolar mixture of two diastereomers, whereas the conjugate formed by the microsomal fraction is predominantly one diastereomer, as judged by the 19F NMR spectra. No evidence for the formation of S-(trihalovinyl)glutathione derivatives by an addition/elimination reaction was found. High-performance liquid chromatography was employed to measure the rates of glutathione conjugate formation in vitro. The rates of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation were 75-107 nmol min-1 (mg of protein)-1 and 151-200 nmol min-1 (mg of protein)-1 catalyzed by the cytosolic and microsomal fractions, respectively (measured at pH 7.4, 37 degrees C, with 5 mM glutathione). These results suggest that glutathione conjugation occurs at high rates in vivo to produce the highly nephrotoxic S-(2-chloro-1,1,2-trifluoroethyl)glutathione.     

Cystine Uptake and Glutathione Level in Fetal Brain Cells in Primary Culture and in Suspension

Abstract— The glutathione level and the factors affecting this level were investigated in fetal rat brain cells in a primary culture. Early in the culture, the glutathione level of the brain cells decreased, but after 5 h it began to increase. This increase was not observed in a cystine-free medium and was prevented by excess glutamate. Cystine was taken up in freshly isolated brain cell suspensions, and its rate increased during the culture. The cystine uptake was mediated by a Na⁺-independent, glutamate-sensitive route previously found in various types of cells and designated as system x⁻_c. The uptake of cystine is a crucial factor in maintaining the glutathione level of the cells under culture, because it provides cysteine for the cells for glutathione synthesis. Cysteine was undetectable in the medium before the culture, but it appeared, though at a very low level, when the brain cells were cultured there. The source of this cysteine was the cystine in the medium. Presumably the decrease in the glutathione level of the cells in the early stage of the culture resulted from the fact that the medium did not contain cysteine. The enhancement of the cystine uptake during culture may constitute a protective mechanism against the oxidative stress to which the cultured cells are exposed. Regulation of the glutathione level in fetal brain cells in vivo by the transport of cystine and cysteine is discussed.