Blood glutathione redox status in gestational hypertension

Gestational hypertension during the third trimester reflects an exaggerated maternal inflammatory response to pregnancy. We hypothesized that oxidative stress present even in normal pregnancy becomes uncompensated in hypertensive patients. A glucose-6-phosphate dehydrogenase (G6PD) activity sufficient to meet the increased reductive equivalent need of the cells is indispensable for defense against oxidative stress. The erythrocyte glutathione redox system was studied, where G6PD is the only NADPH source. The glutathione (GSH) redox status was measured both in vivo and after an in vitro oxidative challenge in pregnant women with gestational hypertension (n = 19) vs. normotensive pregnant subjects (n = 18) and controls (n = 20). An erythrocyte GSH depletion with an increase in the oxidized form (GSSG) resulted in an elevated ratio GSSG/GSH (0.305 +/- 0.057; mean +/- SD) in hypertensive pregnant women vs. normotensive pregnant or control subjects (0.154 +/- 0.025; 0.168 +/- 0.073; p <.001). In hypertensive pregnant patients, a "GSH stability" decrease after an in vitro oxidative challenge suggested a reduced GSH recycling capacity resulting from an insufficient NADPH supply. The erythrocyte GSSG/GSH ratio may serve as an early and sensitive parameter of the oxidative imbalance and a relevant target for future clinical trials to control the effects of antioxidant treatment in women at increased risk of the pre-eclampsia syndrome.     

Genetic analysis of tissue glutathione concentrations and redox balance

Glutathione redox balance—defined as the ratio GSH/GSSG—is a critical regulator of cellular redox state, and declines in this ratio are closely associated with oxidative stress and disease. However, little is known about the impact of genetic variation on this trait. Previous mouse studies suggest that tissue GSH/GSSG is regulated by genetic background and is therefore heritable. In this study, we measured glutathione concentrations and GSH/GSSG in liver and kidney of 30 genetically diverse inbred mouse strains. Genetic background caused an approximately threefold difference in hepatic and renal GSH/GSSG between the most disparate strains. Haplotype association mapping determined the loci associated with hepatic and renal glutathione phenotypes. We narrowed the number of significant loci by focusing on those located within protein-coding genes, which we now consider to be candidate genes for glutathione homeostasis. No candidate genes were associated with both hepatic and renal GSH/GSSG, suggesting that genetic regulation of GSH/GSSG occurs predominantly in a tissue-specific manner. This is the first quantitative trait locus study to examine the genetic regulation of glutathione concentrations and redox balance in mammals. We identified novel candidate genes that have the potential to redefine our knowledge of redox biochemistry and its regulation and inform future therapeutic applications.     

Renal antioxidant enzymes and glutathione redox status in leptin-induced hypertension

Previously, we have demonstrated that leptin increases blood pressure (BP) in the rats through two oxidative stress-dependent mechanisms: stimulation of extracellular signal-regulated kinases (ERK) by H(2)O(2) and scavenging of nitric oxide (NO) by superoxide (O(2-.)). Herein, we examined if renal glutathione system and antioxidant enzymes determine the mechanism of prohypertensive effect of leptin. Leptin administered at 0.5 mg/kg/day for 4 or 8 days increased BP and renal Na(+),K(+)-ATPase activity and reduced fractional sodium excretion; these effects were prevented by NADPH oxidase inhibitor, apocynin. Superoxide scavenger, tempol, abolished the effect of leptin on BP and renal Na(+) pump in rats receiving leptin for 8 days, whereas ERK inhibitor, PD98059, was effective in animals treated with leptin for 4 days. Leptin administered for 4 days decreased glutathione (GSH) and increased glutathione disulfide (GSSG) in the kidney. In animals receiving leptin for 8 days GSH returned to normal level, which was accompanied by up-regulation of gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme of the GSH biosynthetic pathway. In addition, superoxide dismutase (SOD) activity was decreased, whereas glutathione peroxidase (GPx) was increased in rats receiving leptin for 8 days. Cotreatment with gamma-GCS inhibitor, buthionine sulfoximine (BSO), accelerated, whereas GSH precursor, N-acetylcysteine (NAC), attenuated leptin-induced changes in gamma-GCS, SOD, and GPx. In addition, coadministration of BSO changed the mechanism of BP elevation from H(2)O(2)-ERK to (O(2-.))-NO dependent in animals receiving leptin for 4 days, whereas NAC had the opposite effect in rats treated with leptin for 8 days. These results suggest that initial change in GSH redox status induces decrease in SOD/GPx ratio, which results in greater amount of (O)2-.)) versus H(2)O(2) in later phase of leptin treatment, thus shifting the mechanism of BP elevation from H(2)O(2)-ERK to (O(2-.))-NO dependent.     

Effect of alpha-tocopherol on superoxide dismutase and glutathione lipoperoxidase activities of mouse liver cytosol and mitochondria

The maximal radioactivity of mouse liver cytosol was observed 18 hours after intraperitoneal injection of 5-CH3-[3H] alpha-tocopherol emulsion with Tween 80. Intraperitoneal injections of Tween 80 (500 mg/kg) without alpha-tocopherol significantly decreased the glutathione lipoperoxidase and superoxide dismutase activities of liver cytosol. Vitamin E effect on the glutathione lipoperoxidase activity was weakly pronounced, while the superoxide dismutase activities of cytosol and liver mitochondria were markedly increased thereby. It was assumed that natural free radical scavengers can induce enzymatic utilization of superoxide radical anions, while synthetic free radical scavengers can trigger on enzymatic systems of lipoperoxide utilization.     

Effect of glutathione manipulation on prostaglandin synthesis in renal medullary homogenates.

1. The effect of glutathione (GSH) manipulation on arachidonic acid (AA) metabolism in renal medullary (RM) homogenates was investigated. 2. Diethyl maleate (DEM) depleted GSH initially by 50% (P less than 0.05) and produced a general suppression (P less than 0.05) of all PGs with the exception of TXB2. GSH was further depleted during homogenization and a 30-min incubation period (P less than 0.01). 3. Adding glutathione monoethyl ester (GSH-MEE) (0, 0.8, 1.6 or 3.2 mmol/ml) to RM homogenates increased GSH (P less than 0.01) and decreased RM homogenates' PGs-synthesizing capability (P less than 0.05), with the exception of PGE2 and TXB2 at the highest concentration. 4. The results indicate that homogenization has a significant impact (P less than 0.05) on GSH concentration of the media and alterations in GSH concentration affect the profile and quantity of AA metabolites in renal medullary homogenates.     

On the polydispersity of mitochondria in tissue homogenates and the determination of the average sedimentation coefficients of mixed populations of mitochondria

The sedimentation profile (sediterm) of subcellular particles in homogenous media depends on the average sedimentation coefficient ( value) and the size distribution. The present study has focused on the two common types of polydispersity, i.e., (i) a variable standard deviation in a normal (Gaussian) size distribution, and (ii) two populations of partieles with defined values and size distributions. Theoretical considerations and experimental data indicate that rat liver mitochondria have a normal size distribution, () with much smaller standard deviation than previously assumed (σ = 0.118 μm) based on isokinetic gradient centrifugation and electron microscopy. Sedimentation of a mixture of rat liver and guinea pig ileal mitochondria having the values 17,040 S and 5640 S, respectively, gave the expected profile (sediterm) of two populations of particles. Their values were estimated to be identical to those obtained when the individual mitochondrial populations were sedimented. The ratio between the populations (based on the assay of marker enzyme) was found to be identical to the expected value.     

An NADH-tetrazolium-coupled sensitive assay for malate dehydrogenase in mitochondria and crude tissue homogenates

A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 microg protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application.     

Effect of mitochondria on the redox reaction between oxyhemoglobin and ferricytochrome c

The effect of mitochondria on the redox reaction between oxymyoglobin (oxy-Mb) and ferricytochrome c was studied. The parameters of this reaction in the absence of mitochondria have been investigated earlier. It is shown that the course of oxidation of oxymyoglobin by cytochrome c in the presence of mitochondria differs from that without mitochondria: no reduced cytochrome c is observed; in addition, the order of this redox reaction and its dependence on pH and ionic strength change. The factors influencing the state of mitochondrial membrane and uncouples enhance markedly the reaction rate. The conclusion was drawn that mitochondria directly participate in the oxymyoglobin-cytochrome c redox reaction. The possibility of this reaction in vivo under extreme conditions and during pathological processes is discussed.     

Dietary supplementation with cysteine prodrugs selectively restores tissue glutathione levels and redox status in protein-malnourished mice(1)

Protein malnutrition (PM) is a major health problem in the world. PM compromises antioxidant defense in the body. In particular, PM decreases tissue glutathione (GSH) levels. A high protein diet was found to restore tissue GSH levels in animal studies, however it is not recommended for the early phase of PM rehabilitation. Therefore, using dietary supplementation to restore tissue GSH without giving a high protein diet may be an adjunct therapy that helps improve antioxidant status during the early rehabilitation of PM. In this study, we systematically compared the efficacy of dietary supplementation of four cysteine prodrugs: N-acetylcysteine, L-2-oxo-4-thiazolidine-carboxylate, methionine, and GSH, on tissue GSH in mice fed a protein-deficient (0.5%) diet. Results showed that dietary supplementation of cysteine prodrugs to PM mice restored GSH levels in liver, lung, heart and spleen, but not in colon. GSH and GSSG levels in brain and kidney were not affected by cysteine prodrug or PM. Supplementation also restored the redox status in liver and heart (based on GSH/GSSG), and in liver and spleen (based on GSSG/2GSH reduction potential). This suggests that the restoration of GSH levels and redox status by cysteine prodrugs are tissue-specific, and that the two indicators of redox status are not always interchangeable. However, all four prodrugs exhibited similar GSH-enhancing capacities, showing no prodrug-specificity as seen in cell culture studies. In conclusion, this study provided information that may be useful in a clinical setting where a short-term oral supplementation of cysteine prodrugs is necessary for the early rehabilitation of PM patients.     

Microinjected glutathione reductase crystals as indicators of the redox status in living cells

The flavoenzyme glutathione reductase catalyses electron transfer reactions between two major intracellular redox buffers, namely the NADPH/NADP+ couple and the 2 glutathione/glutathione disulfide couple. On this account, microcrystals of the enzyme were tested as redox probes of intracellular compartments. For introducing protein crystals into human fibroblasts, different methods (microinjection, particle bombardment and optical tweezers) were explored and compared. When glutathione reductase crystals are present in a cytosolic environment, the transition of the yellow Eox form to the orange-red 2-electron reduced charge transfer form, EH2, is observed. Taking into account the midpoint potential of the Eox/EH2 couple, the redox potential of the cytosol was found to be < -270 mV at pH 7.4 and 37 degrees C. As a general conclusion, competent proteins in crystalline--that is signal-amplifying--form are promising probes for studying intracellular events.     

Induction of lipid peroxidation and alteration of glutathione redox status by endosulfan

The oxidant stress-inducing effects of endosulfan, a chlorinated hydrocarbon insecticide of the cyclodiene group, have been examined following ig administration of single and repeated doses. A single dose of 30 mg/kg (∼30% LD₅₀) endosulfan significantly (p<0.001) increased the TBARS and, hence, the lipid peroxidation in cerebral and hepatic tissues of rats. Administration of endosulfan with doses of 10 or 15 mg/kg/d for 5 d has also induced lipid peroxidation significantly (p<0.05). The same doses caused a significant alteration in glutathione redox status of cerebral and hepatic tissues, where total glutathione and oxidized glutathione were measured by an enzymatic cycling procedure. Selenium levels were also determined and compared with controls. With repeated doses, oxidant stress was more pronounced in cerebral tissue, where endosulfan shows a GABA-antagonistic activity. The possible relationship between the neurotoxicity of endosulfan and its oxidant stress-inducing effect was discussed.     

Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells

Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.     

Effect of cold storage on tissue and cellular glutathione

One of the mechanisms thought to cause injury in preserved organs is the formation of oxygen free radicals. The cell is protected from oxidative stress by many defense mechanisms. A major defense mechanism involves glutathione and glutathione-dependent enzymes. During organ preservation by simple cold storage the loss of glutathione may sensitize the organ to free radical damage after transplantation. In this study we show that glutathione is depleted from the rabbit liver, kidney, and heart cold-stored (5 degrees C) for up to 72 h in the UW solution without glutathione. In the first 24 h kidney glutathione decreased to 84 +/- 3% of control values, liver glutathione decreased to 49 +/- 3% of control values, and heart glutathione decreased to 73 +/- 3% of control values. After 48 h of storage the kidney and liver lost an additional 30 and 20%, respectively, whereas heart glutathione changed very little. By 72 h all three organs had lost more than 50% of the glutathione found in freshly obtained tissue. To determine if glutathione added to the UW solution can effectively prevent this loss of glutathione during preservation, hepatocytes were cold-stored for up to 72 h in a preservation solution with and without glutathione. We found that adding glutathione to the preservation solution slowed the rate of loss of glutathione from the cells. These data suggest that at hypothermia the cell may be permeable to GSH. Methods to suppress the loss of glutathione during preservation of organs may be an important factor in suppressing oxygen free radical injury.     

Alterations of the glutathione redox cycle status in fumonisin B1-treated pig kidney cells

Fumonisin B₁ (FB₁) causes equine leukoencephalomalacia, porcine pulmonary edema, and liver tumors and chronic nephritis in rats. To investigate mechanisms by which FB₁ induces toxicity, effects of FB₁ on cellular glutathione (GSH) redox status and GSH depletion on FB₁ toxicity in pig kidney (LLC-PK₁) cells were studied. Treatment of LLC-PK₁ cells with 50 μM FB₁ for 24 hours significantly decreased cellular GSH contents from 56 ± 3.2 to 42.7 ± 4.4 nmol/mg protein (p < 0.05) and increased the activities of glutathione reductase (GR) from 25.7 ± 2.4 to 35.7 ± 5.0 μmol NADPH/mg protein (p < 0.05). The activities of glutathione peroxidase (GSHpx), catalase, and Cu,Zn-superoxide dismutase (SOD) were not changed by this treatment. Treatment of LLC-PK₁ cells for 12 hours with 0.1. mM buthionine sulfoximine (BSO), a selective inhibitor of the enzyme γ-glutamylcysteine synthetase that catalyzes the rate-limiting reaction in de novo GSH synthesis, decreased cellular GSH levels to about 20% of that found in the control cells. The cells pretreated with 0.1 mM BSO for 12 hours were significantly sensitized to the FB₁ cytotoxicity as determined by a long-term survival assay (p < 0.05). The results demonstrate that FB₁ changes GSH redox cycle status in LLC-PK₁ cells, and GSH may play a role in cytoprotection against FB₁ toxicity. © 1997 John Wiley & Sons, Inc.