Role of a partially purified glutathione S-transferase from rat liver nuclei in the inhibition of nuclear lipid peroxidation

Glutathione protects isolated rat liver nuclei against lipid peroxidation by inducing a lag period prior to the onset of peroxidation. This GSH-dependent protection was abolished by exposing isolated nuclei to the glutathione S-transferase inhibitor S-octylglutathione. In incubations containing 0.2 mM S-octylglutathione, the GSH-induced lag period was reduced from 30 to 5 min. S-Octylglutathione (0.2 mM) also completely inhibited nuclear glutathione S-transferase activity and reduced glutathione peroxidase activity by 85%. About 70% of the glutathione S-transferase activity associated with isolated nuclei was solubilized with 0.3% Triton X-100. This solubilized glutathione S-transferase activity was partially purified by utilizing a S-hexylglutathione affinity column. The partially purified nuclear glutathione S-transferase exhibited glutathione peroxidase activity towards lipid hydroperoxides in solution. The data from the present study indicate that a glutathione S-transferase associated with the nucleus may contribute to glutathione-dependent protection of isolated nuclei against lipid peroxidation. Evidence was obtained which indicates that this enzyme is distinct from the microsomal glutathione S-transferase.     

The effect of age and sex on glutathione reductase and glutathione peroxidase activities and on aerobic glutathione oxidation in rat liver homogenates

1. Changes in liver glutathione reductase and glutathione peroxidase activities in relation to age and sex of rats were measured. Oxidation of GSH was correlated with glutathione peroxidase activity. 2. Glutathione reductase activity in foetal rat liver was about 65% of the adult value. It increased to a value slightly higher than the adult one at about 2-3 days, decreased until about 16 days and then rose after weaning to a maximum at about 31 days, finally reaching adult values at about 45 days old. 3. Weaning rats on to an artificial rat-milk diet prevented the rise in glutathione reductase activity associated with weaning on to the usual diet high in carbohydrate. 4. In male rats glutathione peroxidase activity in the liver increased steadily up to adult values. There were no differences between male and female rats until sexual maturity, when, in females, the activity increased abruptly to an adult value that was about 80% higher than that in males. 5. The rate of GSH oxidation in rat liver homogenates increased steadily from 3 days until maturity, when the rate of oxidation was about 50% higher in female than in male liver. 6. In the liver a positive correlation between glutathione peroxidase activity and GSH oxidation was found. 7. It is suggested that the coupled oxidation-reduction through glutathione reductase and glutathione peroxidase is important for determining the redox state of glutathione and of NADP, and also for controlling the degradation of hydroperoxides. 8. Changes in glutathione reductase and glutathione peroxidase activities are discussed in relation to the redox state of glutathione and NADP and to their effects on the concentration of free CoA in rat liver and its possible action on ketogenesis and lipogenesis.     

Adaptive response of antioxidant enzymes to catalase inhibition by aminotriazole in goldfish liver and kidney

This study was undertaken to clarify the physiological role of catalase in the maintenance of pro/antioxidant balance in goldfish tissues by inhibiting the enzyme in vivo with 3-amino 1,2,4-triazole. Intraperitoneal injection of aminotriazole (0.5 mg/g wet mass) caused a decrease in liver catalase activity by 83% after 24 h that was sustained after 168 h post-injection. In kidney catalase activity was reduced by approximately 50% and 70% at the two time points, respectively. Levels of protein carbonyls were unchanged in liver but rose by 2-fold in kidney after 168 h. Levels of thiobarbituric acid-reactive substances were elevated in both tissues after 24 h but were reversed by 168 h. Glutathione peroxidase and glutathione-S-transferase activities increased in kidney after aminotriazole treatment whereas activities of glutathione peroxidase and glutathione reductase in liver decreased after 24 h but rebounded by 168 h. Liver glucose-6-phosphate dehydrogenase activity was reduced at both time points. Activities of these three enzymes in liver correlated inversely with the levels of lipid damage products (R2=0.65-0.81) suggesting that they may have been oxidatively inactivated. Glutathione-S-transferase activity also correlated inversely with catalase (R2=0.86). Hence, the response to catalase depletion involves compensatory changes in the activities of enzymes of glutathione metabolism.     

A comparative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P

Glutathione peroxidase catalyzes the reduction of hydrogen peroxide and organic hydroperoxide by glutathione and functions in the protection of cells against oxidative damage. Glutathione peroxidase exists in several forms that differ in their primary structure and localization. We have also shown that selenoprotein P exhibits a glutathione peroxidase-like activity (Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K. (1999) J. Biol. Chem. 274, 2866-2871). To understand the physiological significance of the diversity among these enzymes, a comparative study on the peroxide substrate specificity of three types of ubiquitous glutathione peroxidase (cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, and extracellular glutathione peroxidase) and of selenoprotein P purified from human origins was done. The specific activities and kinetic parameters against two hydroperoxides (hydrogen peroxide and phosphatidylcholine hydroperoxide) were determined. We next examined the thiol specificity and found that thioredoxin is the preferred electron donor for selenoprotein P. These four enzymes exhibit different peroxide and thiol specificities and collaborate to protect biological molecules from oxidative stress both inside and outside the cells.     

Subcellular distribution of selenium-containing proteins in the rat

The subcellular distribution of selenium in rat tissues was studied by measuring 75Se in animals provided for 5 months with [75Se]selenite as the main dietary source of selenium. Equilibration of the animals to a constant specific activity allowed the measurement of 75Se to be used as a specific elemental assay for selenium. Of the whole-body selenium, 51% was in the soluble fractions and 48% was bound to the particulate fractions as follows: 21% in plasma membranes, 11% in microsomes, and 16% in mitochondria. Glutathione peroxidase was primarily a soluble enzyme, but part of the activity was associated with plasma membrane in liver, mitochondria in liver and kidney, and microsomes in testes. Selenium in glutathione peroxidase accounted for about one-third of the particulate-associated selenium. These results indicate that other selenium-containing proteins besides glutathione peroxidase are present in membranes.     

Development of Mechanisms of Protection Against Oxidative Stress in Doxorubicin-Resistant Rat Tumoral Cells in Culture

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene.

Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H₂O₂ in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.     

Glutathione level and its relation to radiation therapy in patients with cancer of uterine cervix

Glutathione functions as an important antioxidant in the destruction of hydrogen peroxide and lipid peroxides by providing substrate for the glutathione peroxidase and also promotes the ascorbic acid. Glutathione plays a vital role in detoxification of xenobiotics, carcinogens, free radicals and maintenance of immune functions. The study was aimed to determine plasma glutathione as well as erythrocyte glutathione and glutathione peroxidase in patients with invasive cervical carcinoma (n = 30) before initiation and after completion of radiotherapy and subsequently, at the time of first three monthly follow-up visit. The levels of plasma glutathione, erythrocyte glutathione and glutathione peroxidase activity were found to be lower in all cervical cancer patients as compared to age matched normal control women. The study indicates a change in antioxidant status in relation with the glutathione system among patients with invasive carcinoma of the uterine cervix. This study also demonstrates the effect of radiation therapy on this antioxidant system.     

Immunolocalization of antioxidant enzymes in adult hamster kidney

Summary Immunoperoxidase and immunogold techniques were used to localize the following antioxidant enzyme systems in the adult hamster kidney at the light and ultrastructural levels: superoxide dismutases, catalases, peroxidases and glutathione S-transferases. Each cell type in the kidney showed specific patterns of labelling of these enzymes. For example, proximal and distal tubular and transitional epithelial cells showed significant staining for all of these enzymes, while glomerular cells and cells of the thin loop of Henle did not show significant staining at the light microscope level. In addition, high levels of glutathione peroxidase were found in smooth muscle cells of renal arteries. At the ultrastructural level, each enzyme was found in a specific subcellular location. Manganese superoxide dismutase was found in mitochondria, catalase was localized in peroxisomes, while copper, zinc superoxide dismutase and glutathione S-transferase (liver and placental forms) were found in both the nucleus and cytoplasm. Glutathione peroxidase was found to have a broad intracellular distribution, with localization in mitochondria, peroxisomes, nucleus, and cytoplasm. Microvilli of tubular cells were labelled by antibodies to catalase, copper, zinc superoxide dismutase, glutathione peroxidase, and glutathione S-transferases. Cell types that were negative by light microscopy immunoperoxidase studies showed definite labelling with immunogold post-embedding ultrastructural techniques (glomerular cells and cells of the loop of Henle), demonstrating the greater sensitivity of the latter technique. These observations demonstrate that there are large variations in the levels of antioxidant enzymes in different cell types, and that even within a distinct cell type, the levels of these enzymes vary in different subcellular locations. Our results demonstrate for the first time the overall antioxidant enzyme status of individual kidney cell types, thereby explaining why different cell types have differing susceptibilities to oxidant stress. Possible physiological and pathological consequences of these findings are discussed.     

Species-related variations in tissue antioxidant status--I. Differences in antioxidant enzyme profiles.

1. Antioxidant enzyme activity profiles in red cells of man, rabbit, quail, pig and rat have been investigated and found to exhibit striking differences. 2. No direct correlations between activities of "functionally coupled" enzymes (superoxide dismutase/catalase and glutathione peroxidase/glutathione reductase) were apparent, suggesting their independent regulation. 3. However, activities of red cell catalase and glutathione peroxidase in the various species studied were inversely correlated. 4. This was most evident in quail red cells, which showed negligible catalase activity but the highest levels of glutathione peroxidase of all the species examined. 5. A significant positive correlation between catalase and glutathione reductase activities was also demonstrated. 6. This may be relevant to the suggestion that the binding of NADPH to catalase may serve to decrease the intracellular inactivation of this reducing cofactor which may be limiting in the glutathione reductase reaction. 7. Basal levels of glutathione, which have been claimed to be limiting for the glutathione peroxidase reaction, were found to correlate positively with the activity of this enzyme in red cells. 8. Myocardial tissues also exhibited species-related differences in antioxidant enzyme profiles but these did not bear any obvious relationship to patterns observed in the corresponding red cells.     

Phospholipid hydroperoxide glutathione peroxidase: specific activity in tissues of rats of different age and comparison with other glutathione peroxidases

The tissue distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPX) was studied in rats of different ages. In the same samples the activities of Se-dependent glutathione peroxidase (GPX), and non-Se-dependent glutathione peroxidase (non Se-GPX) were also determined using specific substrates for each enzyme. Enzymatically generated phospholipid hydroperoxides were used as substrate for PHGPX, hydrogen peroxide for GPX, and cumene hydroperoxide for non-Se-GPX (after correction for the activity of GPX on this substrate). PHGPX specific activity in different organs is as follows: liver = kidney greater than heart = lung = brain greater than muscle. Furthermore, this activity is reasonably constant in different age groups, with a lower specific activity observed only in kidney and liver of young animals. GPX activity is expressed as follows: liver greater than kidney greater than heart greater than lung greater than brain = muscle, and substantial age-dependent differences have been observed (adult greater than old greater than young). Non-Se-GPX activity was present in significant amount only in liver greater than lung greater than heart and only in adult animals. These results suggest a tissue- and age-specific expression of different peroxidases.     

Glutathione peroxidase is neither required nor kinetically competent for conversion of 5-HPETE to 5-HETE in rat PMN lysates

In the 5-lipoxygenase pathway for arachidonic acid metabolism, reduction of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to 5-hydroxyeicosatetraenoic acid (5-HETE) is catalyzed by an activity different from glutathione peroxidase. Glutathione peroxidase here refers to the nonspecific peroxidase that catalyzes the reduction by glutathione of cumene hydroperoxide and a variety of other peroxides including 5-HPETE. This enzyme is inhibited by mercaptosuccinic acid. Preparations of the 15,000xg supernatant from lysed rat peritoneal polymorphonuclear leukocytes were the source of these activities. Thus, when glutathione peroxidase is inhibited to less than 0.5% of its normal activity by mercaptosuccinic acid, 5-HPETE is reduced as efficiently as in the absence of mercaptosuccinate. In lysate preparations from which endogenous glutathione has been removed, reduction of 5-HPETE is still observed but only in the presence of added reducing agents, e.g., 0.2 mM glutathione. When endogenous glutathione peroxidase is not inhibited, reduction of 5-HPETE occurs at a rate greater than 15-fold faster than can be accounted for by this activity. We conclude, therefore, that the glutathione peroxidase in rat PMNs is not kinetically competent to account for reduction of 5-HPETE. There is a distinct peroxidase that catalyzes this reaction. The 5-HPETE peroxidase can utilize glutathione as reducing agent but is not inhibited by mercaptosuccinate, and additional results indicate that it is inactivated during turnover.     

Role of Glutathione-Dependent Peroxidase in Regulation of Lipoperoxide Utilization in Malignant Tumors

Glutathione content, the activity of glutathione-dependent enzymes (glutathione reductase, glutathione peroxidase, and glutathione S-transferase), and also SOD (superoxide dismutase) and catalase were studied in human malignant tumors (uterus, breast, and ovaries) and normal tissues. Glutathione level and the activity of glutathione-dependent enzymes were 2-3 times higher in the malignant tumors than in normal tissues. A negative correlation between the level of glutathione and glutathione-dependent enzymes (glutathione peroxidase and glutathione S-transferase) in tumors and the efficacy of postoperative chemotherapy may characterize the degree of tumor resistance to chemotherapy and therefore may have prognostic value. Low SOD and catalase activity and high activity of glutathione-dependent enzymes in tumors suggest that glutathione peroxidase and glutathione S-transferase play a major role in peroxide utilization in malignant tumors.     

gamma-Butyrobetaine hydroxylase and the protective role of glutathione peroxidase

The selenoenzyme glutathione peroxidase in the presence of GSH effectively replaced catalase in the in vitro assay for gamma-butyrobetaine hydroxylase. Quantitatively, glutathione peroxidase was an order of magnitude more efficient than catalase, with maximal activity at less than 0.1 microM glutathione peroxidase in a standard reaction. Glutathione peroxidase prevented the loss of gamma-butyrobetaine hydroxylase during preliminary incubation with ferrous ions but without other substrates as well as in the course of the reaction. Regardless of whether glutathione peroxidase or catalase was present in the assay, the ascorbate concentrations needed to achieve half-maximal rates were similar (about 1 mM). Phosphate stimulated the rate of L-carnitine synthesis. Ferrous ion saturation indicated a pronounced effect of phosphate on the maximal velocity of the enzyme-catalyzed reaction, but its mechanism of action remains to be elucidated. Based on the subcellular distribution of gamma-butyrobetaine hydroxylase, catalase, and glutathione peroxidase, the role of glutathione peroxidase assumes importance. However, initial studies indicated that the assayable activity of liver gamma-butyrobetaine hydroxylase and L-carnitine concentrations in liver, blood plasma, and muscle were not significantly altered in selenium-deficient rats.     

Subcellular localization of adrenal cortical glutathione peroxidase and protective role of the mitochondrial enzyme against lipid peroxidative damage

In view of the physiological importance of adrenocortical lipid peroxidation, we have carried out subcellular fractionation to determine the location of glutathione peroxidase, an enzyme which protects against lipid peroxidation. Glutathione peroxidase is present in both cytosolic (92%) and mitochondrial (8%) fractions. The small activity in mitochondria is not due to contamination by the cytosolic activity as evidenced by several rigorous approaches. The mitochondrial enzyme is located in the matrix and appears to be effective in protection from NADPH-dependent lipid peroxidative damage of cytochrome P-450 and succinic dehydrogenase, which are located exclusively in the inner membrane.     

The effect of melaxen and valdoxan on the activity of the glutathione antioxidant system in rats with experimental hyperthyroidism

The activity of the glutathione system and conjugated diene content (CD) have been investigated in the liver and blood serum of rats with experimental hyperthyroidism treated with melaxen and valdoxan. The study of glutathione reductase (GR), glutathione peroxidase (GP) and glutathione transferase (GST) activities increased under this pathology has shown that administration of these compounds decreased these activities towards control levels. Melaxen and valdoxan administration increased reduced glutathione (GSH) content as compared with untreated hyperthyroid rats. This increase may be associated with its decreased utilization for detoxification of toxic products of free radical oxidation (FRO). Administration of the melatonin correcting drugs also tended to normalize the CD level increased in the liver and blood serum of hyperthyroid rats. Results of this study indicate that melaxen and valdoxan exhibit positive effect on free radical homeostasis. This appears to be accompanied by a decrease in the load of the glutathione antioxidant system in comparison with the examined pathology.     

Postmortem Stability of Enzymes Detoxifying Peroxide In Brain

Abstract: Glutathione peroxidase, glutathione reductase, and catalase activities were measured to 48 h after death in mouse brains held at temperatures replicating the cooling occurring in human cadaver brain. Glutathione peroxidase was stable for 48 h; catalase was stable for 24 h and then declined 20% in activity. Glutathione reductase was stable for 4 h and then decreased to 55% of its initial activity by 48 h. Perfusion of mouse brain with 0.9% (wt/vol) NaCl did not decrease enzyme activities, indicating that erythrocyte contamination has little effect on measured brain activities. The results suggest that glutathione peroxidase would not be affected by moderate time delays in obtaining human postmortem brains but catalase activity may be affected if brains are not promptly removed. Glutathione reductase is not stable and measurements would require controls carefully matched for postmortem conditions.     

Development of Mechanisms of Protection Against Oxidative Stress in Doxorubicin-Resistant Rat Tumoral Cells in Culture

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene.

Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H₂O₂ in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.     

Glutathione peroxidase is neither required nor kinetically competent for conversion of 5-HPETE to 5-HETE in rat PMN lysates

In the 5-lipoxygenase pathway for arachidonic acid metabolism, reduction of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to 5-hydroxyeicosatetraenoic acid (5-HETE) is catalyzed by an activity different from glutathione peroxidase. Glutathione peroxidase here refers to the nonspecific peroxidase that catalyzes the reduction by glutathione of cumeme hydroperoxide and a variety of other peroxides including 5-HPETE. This enzyme is inhibited by mercaptosuccinic acid. Preparations of the 15,000xg supernatant from lysed rat peritoneal polymorphonuclear leukocytes were the source of these activities. Thus, when glutathione peroxidase is inhibited to less than 0.5% of its normal activity by mercaptosuccinic acid, 5-HPETE is reduced as efficiently as in the absence of mercaptosuccinate. In lysate preparations from which endogenous glutathione has been removed, reduction of 5-HPETE is still observed but only in the presence of added reducing agents, e.g., 0.2 mM glutathione. When endogenous glutahione peroxidase is not inhibited, reduction of 5-HPETE occurs at a rate >15-fold faster than can be accounted for by this activity. We conclude, therefore, that the glutathione peroxidase in rat PMNs is not kinetically competent to account for reduction of 5-HPETE. There is a distinct peroxidase that catalyzes this reaction. The 5-HPETE peroxidase can utilize glutathione as reducing agent but is not inhibited by mercaptosuccinate, and additional results indicate that it is inactivated during turnover.     

Hydroperoxide metabolism in cyanobacteria

The enzymes involved in antioxidative activity and the cellular content of the antioxidants glutathione and ascorbate in the cyanobacteria Nostoc muscorum 7119 and Synechococcus 6311 have been examined for their roles in hydroperoxide removal. High activities of ascorbate peroxidase and catalase were found in vegetative cells of both species and in the heterocysts of N. muscorum. The affinity of ascorbate peroxidase for H2O2 was 15- to 25-fold higher than that of catalase. Increased activity of ascorbate peroxidase was observed in N. muscorum when H2O2 production was enhanced by photorespiration. Catalase activity was decreased in dilute cultures whereas ascorbate peroxidase activity increased. Ascorbate peroxidase activity also increased when the CO2 concentration was reduced. Ascorbate peroxidase appears to be a key enzyme in a cascade of reactions regenerating antioxidants. Dehydroascorbate reductase was found to regenerate ascorbate, and glutathione reductase recycled glutathione. In vegetative cells glutathione was present in high amounts (2-4 mM) whereas the ascorbate content was almost 100-fold lower (20-100 microM). Glutathione peroxidase was not detected in either cyanobacterium. It is concluded from the high activity of ascorbate peroxidase activity and the levels of antioxidants found that this enzyme can effectively remove low concentrations of peroxides. Catalase may remove H2O2 produced under photooxidative conditions where the peroxide concentration is higher.