Differential Expression of Glutathione Reductase and Cytosolic Glutathione Peroxidase,GPX1, in Developing Rat Lungs and Kidneys

A mutant rat GPX1 (a cytosolic predominant form), in which the selenocysteine residue in the catalytic center was replaced by cysteine, was prepared and an antibody against the mutant enzyme was raised. The resultant antibody specifically reacted with rat GPX1 and was, together with the Glutathione reductase (GR) antibody, used in a Western blot analysis and immunohistochemistry experiments. To elucidate the physiological coupling of these enzymes under oxidative stress which accompanies the birth, developmental changes of the protein levels and enzymatic activities of GR and GPX1 were examined for lungs and kidneys from prenatal fetus to adult rats. The expression of GR was already evident at the prenatal stage and remained high in lungs at all stages. However, GR activity in kidneys gradually increased after birth reaching maximal levels at adulthood. An immunohistochemical study showed that GR was strongly bound to the bronchial epithelia in lungs and the epithelial cells of renal tubes. GPX1 was expressed in the renal tube epithelial cells and its level gradually increased after birth in a manner similar to that of GR. The expression of GPX1 in the lungs was, on the other hand, variable and occurred in some alveolar cells and bronchial epithelia only at restricted periods. It preferentially localized in nuclei at a late stage of development. Thus, the expression of the two functionally coupled enzymes via GSH did not appear to coordinate with development, tissue localization or under oxidative stress. Since many gene products show GSH-dependent preoxidase activity, other peroxidase(s) may be induced to compensate for the low GPX1 levels at stages with high GR expression.     

溴化1-己基-3-甲基咪唑对斜生栅藻谷胱甘肽及其代谢酶的影响

研究了浓度为0、1、5、10、15、20 mg/L的新兴离子液体溴化1-己基-3-甲基咪唑([C6mim]Br)在24h、48h、72h和96h对斜生栅藻还原型谷胱甘肽（GSH）及其代谢酶-谷胱甘肽过氧化物酶（GPX）、谷胱甘肽转硫酶（GST）和谷胱甘肽还原酶（GR）的影响。结果表明：GSH含量在24h、48h和72h时,在最低处理浓度下不变,其他处理浓度下随胁迫浓度增加而降低,96h时则与对照无差异或较小;GPX和GST的活性在72h之前明显升高（最高浓度组的GST活性有波动）,96h时均降低至对照水平;GR活性在24h时,[C6mim]Br=1 mg/L时升高,之后降低,在48h增高至对照水平,72h时,[C6mim]Br≥10 mg/L的处理组高于对照水平,96h时,除最低处理组外,均降至对照水平以下。GR是GSH系统中的限速酶,GST则是该系统中活性和灵敏性最高的酶,可作为[C6mim]Br胁迫时的敏感的生物标志物。1 mg/L的[C6mim]Br可引起藻细胞的氧化胁迫,具有环境毒性。     

Immunohistochemical study of glutathione reductase in rat ocular tissues at different developmental stages

Glutathione, which is found in high levels in eye tissues, is involved in multiple functions, including serving as an antioxidant and as an electron donor for peroxidases. Although the activities of enzymes related to glutathione metabolism have been reported in the eye, the issue of which cells produce these proteins, where they are produced and at what levels is an important one. Glutathione reductase, an enzyme which recycles oxidized glutathione by transferring electrons from NADPH, was localized immunohistochemically in adult rat eye in this study. The reductase was distributed in the corneal and conjunctival epithelia, corneal keratocytes and endothelium, iridial and ciliary epithelia, neural retina, and retinal pigment epithelium. In addition, it was highly expressed in ganglion cells, which are responsible for transmitting photophysiological signals from the retina to the higher visual centres. To clarify the correlation of glutathione reductase expression and oxidative stress, the enzymatic activity and the level of protein expression at the pre- and postnatal stages was examined. Expression of the enzyme was detected first in the ganglion cell layer of a late prenatal stage, and appeared in the inner plexyform layer after birth. Along with an increasing differentiation between the inner nuclear and outer nuclear layers, glutathione reductase expression became detectable in the outer plexyform layer. Pigment epithelial cells were positively stained only after birth. Expression was also detected in the lens epithelium from the prenatal to early postnatal stages although its level was low in the adult lens. Collectively, these data, except for lens epithelia, suggest the pivotal role of glutathione reductase in recycling oxidized glutathione for the protection of the tissues against oxidative stress, which is caused by eye opening accompanied by the initiation of various ocular processes, such as accession of light and transduction of the photochemical signal.     

A trifunctional enzyme with glutathione S-transferase,glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.     

Effects of Se-depletion on glutathione peroxidase and selenoprotein W gene expression in the colon

Selenium (Se)-containing proteins have important roles in protecting cells from oxidative damage. This work investigated the effects of Se-depletion on the expression of the genes encoding selenoproteins in colonic mucosa from rats fed diets of different Se content and in human intestinal Caco-2 cells grown in Se-adequate or Se-depleted culture medium. Se-depletion produced statistically significant (P<0.05) falls in glutathione peroxidase (GPX) 1 mRNA (60-83%) and selenoprotein W mRNA (73%) levels, a small but significant fall in GPX4 mRNA (17-25%) but no significant change in GPX2. The data show that SelW expression in the colon is highly sensitive to Se-depletion.     

The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesis.

Glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) using an NADPH as the electron source. The function of GR in the male genital tract of the rat was examined by measuring its enzymatic activity and examining the gene expression and localization of the protein. Levels of GR activity, the protein, and the corresponding mRNA were the highest in epididymis among testes, vas deferens, seminal vesicle, and prostate gland. The localization of GR, as evidenced by immunohistochemical techniques, reveals that it exists at high levels in the epithelia of the genital tract. In testis, GR is mainly localized in Sertoli cells. The enzymatic activity and protein expression of GR in primary cultured testicular cells confirmed its predominant expression in Sertoli cells. Intracellular GSH levels, expressed as mol per mg protein, was higher in spermatogenic cells than in Sertoli cells. As a result of these findings, the effects of buthionine sulfoximine (BSO), an inhibitor for GSH synthesis, and 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU), an inhibitor for GR, on cultured testicular cells were examined. Sertoli cells were prone to die as the result of BCNU, but not BSO treatment, although intracellular levels of GSH declined more severely with BSO treatment. Spermatogenic cells were less sensitive to these agents than Sertoli cells, which indicates that the contribution of these enzymes is less significant in spermatogenic cells. The results herein suggest that the GR system in Sertoli cells is involved in the supplementation of GSH to spermatogenic cells in which high levels of cysteine are required for protamine synthesis. In turn, the genital tract, the epithelia of which are rich in GR, functions in an antioxidative manner to protect sulfhydryl groups and unsaturated fatty acids in spermatozoa from oxidation during the maturation process and storage.     

Alteration of glutathione reductase expression in the female reproductive organs during the estrous cycle.

The enzyme glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) in an NADPH-dependent manner. A specific antibody raised against recombinant rat GR was used to localize the protein in the female reproductive organs during the estrous cycle in the rat. In the ovary, the strongest reactivity to the antibody was observed in oocytes, followed by granulosa cells, corpus luteum, and interstitial cells. A strongly positive reaction was also observed mainly in the oviduct epithelia, uterine epithelia, and endometrial gland in the reproductive tract. Oviducts contained the highest GR activity. The GR activity of uterus during metestrus was about twice as high as that for other stages of the cycle. The levels of GR proteins in the tissues roughly matched the activities. The expression of the GR mRNA was highest during metestrus. Because GSH is known to increase gamete viability and the efficiency of fertility, GR, which is expressed in these tissues, is predicted to play a pivotal role in the reproduction process as a source of GSH.     

Changes in expression and activity of glutathione S-transferase in different organs of schistosoma haematobium-infected hamster

Schistosomiasis is a major health problem in many subtropical developing countries, causing a number of serious pathologies, including bladder cancer. Most of the toxic compounds formed as a result of these infestations are derived either exogenously or formed endogenously and can be conjugated with glutathione (GSH) via glutathione S-transferase (GST). The present study investigates the effect of Schistosma haematobium infection on the activity of GST and glutathione reductase (GR) and levels of glutathione and free radicals (measured as thiobarbituric acid reactive substances) in different organs of the male hamster. The total activity of GST was increased in several organs; in kidney by 50 and 46% at 6 and 10 weeks postinfection, respectively, and in bladder tissues by 169, 23, and 130% at 2, 4, and 6 weeks postinfection, respectively. In support of this, the expression of GST isozymes was also induced in kidney and bladder tissues at early stages (2, 4, and 6 weeks) and reduced at the later stages of infection (8 and 10 weeks). In contrast, the expression of these isozymes was decreased in the spleen and liver at 2, 4, 6, 8, and 10 weeks postinfection. Also, such activity was decreased in lungs by 74 and 78% and in bladders by 65 and 72% at 8 and 10 weeks postinfection, respectively. GSH levels increased in lungs by 95, 40, and 56% at 2, 4, and 6 weeks and in spleen by 26 and 74% at 4 and 6 weeks, respectively, but decreased at later stages of S. haematobium infection in these organs. The depletion of GSH levels also occurred in bladders by 72 and 54% at 8 and 10 weeks postinfection, respectively. The activity of GR was increased in the livers, lungs, and kidneys of the S. haematobium-infected hamster. TBARS also increased in the lung by 14, 65, 53, 828, and 624% and in the kidney by 64, 29, 87, 190, and 111%, and in the bladder by 216, 23, 1468, 528, and 1025% at 2, 4, 6, 8, and 10 weeks postinfection, respectively. This study indicates that low GST expression and high levels of free radicals could provide new evidence for damage to the bladder and other organs as a result of S. haematobium infection.     

Reduction of selenium-binding protein 1 sensitizes cancer cells to selenite via elevating extracellular glutathione: A novel mechanism of cancer-specific cytotoxicity of selenite

Selenium is an essential trace element and has been extensively studied for preventive effects on cancers. Recent emerging evidence has also shown that selenium at supranutritional dosage has a preferential cytotoxicity in cancer cells and chemotherapeutic drug-resistant cells, but the underlying mechanisms remain largely unknown. This study was to investigate the roles of two distinct representatives of selenium-containing proteins, selenium-binding protein 1 (SBP1) and glutathione peroxidase 1 (GPX1), in selenite-mediated cancer-specific cytotoxicity. We found that there was a significantly inverse correlation between SBP1 and GPX1 protein level in human breast cancers and adjacent matched nontumor tissues (Pearson r=–0.4347, P=0.0338). Ectopic expression of GPX1 enhanced selenite cytotoxicity through down-regulation of SBP1, and SBP1 was likely to be a crucial determinant for selenite-mediated cytotoxicity. Reduction of SBP1 in cancer cells and epirubicin-resistant cells on selenite exposure resulted in a dramatic increase in the generation of hydrogen peroxide and superoxide anion, which in turn caused oxidative stress and triggered apoptosis. Furthermore, knockdown SBP1 by small interfering RNA increased selenite sensitivity by elevating extracellular glutathione (GSH), which spontaneously reacted with selenite and led to the rapid depletion of selenium (IV) in growth medium and the high-affinity uptake of selenite. In conclusion, these findings would improve our understanding of the roles of selenium-containing proteins in selenite-mediated cytotoxicity, and revealed a potent mechanism of the selective cytotoxicity of selenite in cancer cells and drug-resistant cells, in which SBP1 was likely to play an important role in modulating the extracellular microenvironment by regulating the levels of extracellular GSH.     

A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.     

Molecular characterization and mRNA expression of glutathione peroxidase and glutathione S-transferase during osmotic stress in olive flounder (Paralichthys olivaceus)

Glutathione peroxidase (GPX) and glutathione S-transferase (GST) are key enzymes of cellular detoxification systems that defend cells against reactive oxygen species (ROS). In this study, we isolated the GPX and GST full-length cDNA and investigated the expression of these mRNAs from livers of olive flounder during salinity changes (35, 17.5, 8.75, 4 and 0 psu) by quantitative PCR (QPCR). GPX cDNA consists of 429 base pairs (bp) and encodes a protein of 142 amino acids. GST cDNA consists of 663 bp and encodes a protein of 220 amino acids. Both of GPX and GST mRNA expressions were the highest in 4 psu and then decreased in 0 psu. Also, the levels of Na(+) and Cl(-) decreased, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased during the experimental period. These findings provide molecular characterization of GPX and GST in olive flounder and suggest that GPX and GST play important roles in detoxification of ROS, thereby these maybe indicators of oxidative stress responses by salinity changes in olive flounder.     

Genetic analysis of glutathione peroxidase in oxidative stress response of Saccharomyces cerevisiae.

Three glutathione peroxidase homologs (YKL026C, YBR244W, and YIR037W/HYR1) were found in the Saccharomyces Genome Database. We named them GPX1, GPX2, and GPX3, respectively, and we investigated the function of each gene product. The gpx3Delta mutant was hypersensitive to peroxides, whereas null mutants of the GPX1 and GPX2 did not show any obvious phenotypes. Glutathione peroxidase activity decreased approximately 57 and 93% in the gpx3Delta and gpx1Delta/gpx2Delta/gpx3Delta mutants, respectively, compared with that of wild type. Expression of the GPX3 gene was not induced by any stresses tested, whereas that of the GPX1 gene was induced by glucose starvation. The GPX2 gene expression was induced by oxidative stress, which was dependent upon the Yap1p. The TSA1 (thiol-specific antioxidant) gene encodes thioredoxin peroxidase that can reduce peroxides by using thioredoxin as a reducing power. Disruption of the TSA1 gene enhanced the basal expression level of the Yap1p target genes such as GSH1, GLR1, and GPX2 and that resulted in increases of total glutathione level and activities of glutathione reductase and glutathione peroxidase. However, expression of the TSA1 gene did not increase in the gpx1Delta/gpx2Delta/gpx3Delta mutant. Therefore, de novo synthesis and recycling of glutathione were increased in the tsa1Delta mutant to maintain the catalytic cycle of glutathione peroxidase reaction efficiently as a backup system for thioredoxin peroxidase.     

Saccharomyces cerevisiae expresses three phospholipid hydroperoxide glutathione peroxidases.

The GPX1, GPX2, and GPX3 genes of Saccharomyces cerevisiae have been reported previously to encode glutathione peroxidases (GPxs). We re-examined the sequence alignments of these proteins with GPxs from higher eukaryotes. Sequence identities, particularly with phospholipid hydroperoxide glutathione peroxidases (PHGPxs), were enhanced markedly by introduction to the yeast sequences of gaps that are characteristic of PHGPxs. PHGPx-like activity was detectable in extracts from wild-type S. cerevisiae and was diminished in extracts from gpx1 Delta, gpx2 Delta, and gpx3 Delta deletion mutants; PHGPx activity was almost absent in a gpx1 Delta/gpx2 Delta/gpx3 Delta triple mutant. Studies with cloned GPX1, GPX2, and GPX3 expressed heterologously in Escherichia coli confirmed that these genes encode proteins with PHGPx activity. An S. cerevisiae gpx1 Delta/gpx2 Delta/gpx3 Delta mutant was defective for growth in medium supplemented with the oxidation-sensitive polyunsaturated fatty acid linolenate (18:3). This sensitivity to 18:3 was more marked than sensitivity to H(2)O(2). Unlike H(2)O(2) toxicity, delayed toxicity of 18:3 toward gpx1 Delta/gpx2 Delta/gpx3 Delta cells was correlated with the gradual incorporation of 18:3 into S. cerevisiae membrane lipids and was suppressible with alpha-tocopherol, an inhibitor of lipid peroxidation. The results show that the GPX genes of S. cerevisiae, previously reported to encode GPxs, encode PHGPxs (PHGPx1, PHGPx2, and PHGPx3) and that these enzymes protect yeast against phospholipid hydroperoxides as well as nonphospholipid peroxides during oxidative stress. This is the first report of an organism that expresses PHGPx from more than one gene and produces PHGPx in the absence of a GPx.     

Glutathione peroxidase-1 gene knockout on body antioxidant defense in mice

To determine the in vivo role of cellular glutathione peroxidase (E.C.1.11.1.9, GPX1), we challenged the GPX1 knockout [GPX1(-/-)], the GPX1 overexpressing [GPX1(+)], and their respective wild-type (WT) mice of different Se and vitamin E status with acute oxidative stress. After these mice were injected with pro-oxidants paraquat or diquat at 12 to 125 mg/kg of body weight, their survival rate and time were a function of their GPX1 activity levels. The GPX1 protection was associated with attenuation of NADPH and NADH oxidation, protein carbonyl and F(2)-isoprostanes formation, and alanine transaminase release in various tissues, and was irreplaceable by high levels of dietary vitamin E or other selenoproteins. The GPX1 expression was also protective against moderate oxidative stress induced by low levels of paraquat or diquat, particularly in the Se-deficient mice. Alteration of GPX1 expression showed no impact on the expression of other selenoproteins and antioxidant enzymes in unstressed mice. Total Se content in liver of the Se-adequate GPX1(-/-) mice was reduced by 60% the WT controls. In conclusion, normal expression of GPX1 is essential and overexpression of GPX1 is beneficial to protect mice against acute oxidative stress.     

Post-translational activation of non-selenium glutathione peroxidase of Chlamydomonas reinhardtii by specific incorporation of selenium

Glutathione peroxidase (GPX) plays a pivotal role in the protection of cells against oxidative damage. The green alga Chlamydomonas reinhardtii expresses both selenocysteine-containing GPX and the non-selenium GPX homolog (GPXH). We previously reported that supplementation of selenium to algal culture induces GPXH to exhibit GPX activity. Here we investigated the incorporation of selenium into GPXH and its causal relationship with the upregulation of the enzymatic activity. GPXH was purified from algal cells grown with selenium and proteolytically digested into four fragments. Selenium content analysis for these proteolytic fragments confirmed that GPXH-incorporated selenium is predominantly enriched in a fragment that carries the putative catalytic residue Cys-38. We next constructed three kinds of engineered GPXH proteins by substituting Ser for one of three Cys residues in native GPXH, Cys-38, -66, and -84, using a bacterial overexpression system, resulting in Cys38Ser, Cys66Ser, and Cys84Ser derivatives, respectively. Of these, the Cys66Ser and Cys84Ser derivatives exhibited the same level of selenium-dependent GPX activity as the normal recombinant GPXH, whereas the Cys38Ser mutant GPXH not only lost its activity completely but also demonstrated severely impaired incorporation of selenium. These findings strongly suggest that selenium is post-translationally assimilated into the Cys-38 of the GPXH protein, thereby enhancing its enzymatic activity.     

Induction of glutathione peroxidase in response to inactivation by nitric oxide

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 μg/ml) and tumor necrosis factor-α (TNF-α, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-α exerted a transient effect on the GPX activity. The treatment with SNAP (200 μM) or sodium nitroprusside (200 μM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-α, and aminoguanidine blocked the effect. In cells metabolically labeled with ⁷⁵Se, LPS + TNF-α accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.     

Age-related changes in antioxidant and glutathione S-transferase enzyme activities in the Asian clam

Aging is accompanied by increased production of free oxygen radicals and impairment of normal cellular functions. The aim of this work was to provide preliminary data on age-related differences in the activities of antioxidant enzymes and phase II biotransformation enzyme glutathione S-transferase (GST) in a wild population of the Asian clam Corbicula fluminea. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and GST were assessed in visceral mass of four age classes (0+-, 1+-, 2+-, and 3+-year-old) of C. fluminea clams. Age-related changes were seen in antioxidant enzyme status: levels of total SOD (totSOD) (P < 0.05), MnSOD, and CuZnSOD (P < 0.05) activities increased progressively during aging from younger to older clams. Changes in CAT and GR activities with advancing age were found, the levels being the highest in age class II, then being lower in age classes III and IV (P < 0.05). Activities of GPX and GST were lower in the senescent individuals (2+- and 3+-year-old clams) compared with young individuals (0+- and 1+-year-old clams). Overall, the decline of glutathione-dependent enzyme activities, coupled with higher and lower activities of totSOD and CAT, respectively, as the individual grows older, may render the older animals more susceptible to oxidative stress. Data reported here are not intended to be exhaustive since they concern only age/size structure of the population at one locality, so more detailed studies on both the developmental stages and levels of antioxidant enzymes of this new alien species in Serbian rivers are required.     

Characterization of UPF peptides,members of the glutathione analogues library,on the basis of their effects on oxidative stress-related enzymes

Previously the authors have designed and synthesized a library of antioxidative glutathione analogues called UPF peptides which are superior to glutathione in hydroxyl radical elimination. This paper is a follow-up study which investigated the effects of the most promising members of the library (UPF1 and UPF17) on oxidative stress-related enzymes. At concentrations used in vivo experiments neither UPF peptide influenced the activity of glutathione peroxidase (GPx) when purified enzyme or erythrocyte lysate was used. At higher concentrations they inhibited GPx activity. UPF peptides had no effect on glutathione reductase (GR) activity. Also they, as well as glutathione itself, slightly increased MnSOD activity in human brain mitochondria and inhibited oxidative burst caused by neutrophil NAD(P)H oxidase. RT-PCR measurements showed that UPF1 and UPF17 have no effect on GPx and MnSOD expression level in human blood mononuclear cells. The results of this study confirm that investigated UPF peptides do not interfere with the enzymatic mechanisms of antioxidative defence and can be used as themselves or as a lead for the protector molecule design against excessive oxidative stress.