cGPx Activity Research Articles

Effect of Selenium Supplementation on Glutathione Peroxidase and Catalase Activities in Senescent Cultured Human Fibroblasts

Aims: To investigate the effect of senescence and selenium supplementation on glutathione peroxidase (cGPx) and catalase (CAT) activities, and concurrent hydrogen peroxide (H₂O₂) generation in subcultured human fibroblasts. Methods: cGPx and CAT activities and H₂O₂ levels were assayed in presenescent passage 5 and 10 cells, and in senescent passage 20, 25, 30 and 35 cells cultured in routine medium (MEM1) and supplemented media MEM2 and MEM3 containing normal and triple human plasma levels of Se, respectively. Senescent cells were identified by studying their growth and replication states, and by monitoring their activity of key glucose and glycogen degradative enzymes. Results: cGPx activity showed moderate increases in senescent cells at passages 20–35 subcultured in MEM1 or MEM2. This activity underwent highly significant progressive increases in the same senescent cells subcultured in MEM3. In contrast, CAT activity showed progressive, highly significant increases in senescent cells at passages 20–35 regardless of the culture medium type. Concurrent H₂O₂ generation was significantly increased in passage 15–25 cells and peaked to higher levels in passage 30 and 35 cells cultured in MEM1 or MEM2. These rates, however, were significantly reduced in senescent passage 20–35 cells cultured in MEM3. Conclusions: The highest cGPx activity and coupled lower H₂O₂ generation were achieved in senescent cells cultured in MEM3.

Measurement of ascorbic acid concentration and glutathione peroxidase activity in biological samples collected from horses with recurrent airway obstruction

To measure the ascorbic acid (AA) concentration in bronchoalveolar lavage fluid (BALF) and cellular glutathione peroxidase (cGPx) activity in RBCs and WBCs from peripherally obtained blood and in cells from BALF to determine whether differences existed between the 2 major redox systems in recurrent airway obstruction (RAO)-affected and -nonaffected (control) horses and between systemic and local pulmonary responses in the glutathione redox system. 16 adult horses in pairs: 8 healthy (control) and 8 RAO-affected horses. Physical examination data and biological samples were collected from horses before (remission), during, and after (recovery) environmental challenge with dusty straw and hay. At each stage, BALF cell AA concentration and RBC, WBC, and BALF cell cGPx activity were measured. Compared with control horses, RAO-affected horses had significantly higher cGPx activity in RBCs at all points and in WBCs during remission and challenge. The BALF cell cGPx activity was higher in RAO-affected horses during recovery than during remission The BALF cell AA concentration did not differ significantly in control horses at any point, but total and free AA concentrations were significantly lower in RAO-affected horses during the challenge period than during remission and recovery periods. High cGPx activity suggested this redox system was upregulated during exposure to dusty straw and hay to combat oxidative stress, as AA was depleted in RAO-affected horses. The relative delay and lack of comparative increase in cGPx activity within the local environment (represented by BALF cells), compared with that in RBCs and WBCs, might contribute to disease in RAO-affected horses.

Measurement of ascorbic acid and glutathione peroxidase activity in biological samples collected from recurrent airway obstruction affected horses and their controls.

Multiple biomarkers of oxidative stress have been measured and utilized in human medicine to assess the redox status of the lung. The purpose of the study was to determine the relationships between ascorbic acid concentration in bronchoalveolar lavage fluid (BALF), and the activity of cellular glutathione peroxidase (cGPx) in red blood cells (RBC), white blood cells (WBC), and cells of BALF, to distinguish recurrent airway obstruction (RAO)-affected horses from their matched controls. Sixteen horses in matched pairs were used: eight non-RAO-affected (controls) and eight RAO-affected horses. Samples from all horses were collected at remission (S1), during environmental challenge (S2) and at recovery (S3). In RAO-affected horses, RBC cGPx were significantly higher at all sample times (p = 0.0313 at S1, p = 0.0208 at S2, p = 0.0207 at S3). WBC cGPx activity was also greater in these horses during remission (p = 0.0128 at S1) and challenge (p = 0.0087 at S2). In the BALF, RAO-affected horses had significantly higher cGPx activity during recovery compared to remission (p = 0.0077). BALF ascorbic acid concentration (total, free and oxidized) was not significantly different in control horses, but total (p = 0.0009) and free (p = 0.0021) forms were significantly reduced during challenge in RAO-affected horses. The elevation in cGPx concentration suggests an up-regulation of this redox system to combat oxidative stress as the ascorbic acid system is depleted during challenge in RAO-affected horses. This difference supports further research into antioxidant interventions that may prevent or minimize clinical signs of RAO.

Effects of selenium status and supplementary seleno-chemical sources on mouse T-cell mitogenesis

Although selenium is thought to be essential for various immune responses, the excess supplementation may have an adverse effect on certain immunological functions. The present study was designed to determine the effective chemical forms of selenium and their optimal levels on T-cell mitogenesis with splenic cells from mice given a selenium-deficient diet for 8 weeks to avoid effects of cellular selenium sources. Although selenium in tissues, except for spleen and thymus, was almost depleted by feeding selenium-deficient diet, the lymphoid organs still contained low levels of selenium. Both activities of cellular glutathione peroxidase (cGPx) and thioredoxin reductase (TR) in liver and splenic cells showed a tendency to decrease by selenium deficiency. However, splenic cells were tolerant against decrease of the selenoenzyme activities, and TR was also more tolerant than cGPx. T-cell proliferation of the selenium-insufficient splenic cells induced by concanavalin A was increased by addition of Na 2SeO 3, Na 2SeO 4, Na 2Se, seleno- dl-cystine, seleno- l-methionine and selenocystamine. Their promoting action was observed at levels lower than 0.1 μmol/L and was completely suppressed at the highest concentration (1 μmol/L), except for selenocystamine. Na 2SeO 3 was one of the efficient selenocompounds for the mitogenesis, which was concomitant with the significant induction of cGPx and TR. However, recovery of cGPx activity in the selenium-insufficient cells by supplementary Na 2SeO 3 was only partial, while TR activity was readily recovered from selenium deficiency. These results therefore indicate that only low levels of selenium is essential for T-cell mitogenesis even in selenium-insufficient splenic cells, and TR, which is readily recovered by Na 2SeO 3, may be the critical enzyme.

Lower Erythrocyte Glutathione Peroxidase Activity in Bronchopulmonary Dysplasia in the First Week of Neonatal Life

Background: Bronchopulmonary dysplasia (BPD) is a major pulmonary complication in premature infants and is considered a free radical disorder. Erythrocyte catalase (CAT) and cellular glutathione peroxidase (c-GPx) are antioxidant enzymes that detoxify peroxides generated from dismutation of superoxide anion. The study goal was to measure enzyme activity for preterm infants during the first week of life. Methods: Blood samples were collected from umbilical arterial lines or the radial artery of 44 preterm infants (gestational age range: 25–30 weeks) on days 0, 2, and 5 after delivery. Erythrocyte CAT and c-GPx activities were evaluated, while clinical data were obtained and the correlations of enzyme activity and BPD status were examined. Results: There was no correlation between enzyme activity and gestational age or birth weight. Packed red cell transfusion did not affect enzyme activity and there was no difference in CAT activity between the patient groups. C-GPx activity from preterm infants with BPD was significantly lower than those of preterm infants without BPD on day 5. Conclusions: Erythrocyte c-GPx may play an important role in the development of BPD. Lower c-GPx activity in early post-natal life might be a risk factor of BPD.

Mechanism for proliferation inhibition by various selenium compounds and selenium-enriched broccoli extract in rat glial cells

The objective of this study was to investigate the differential effects of various selenium (Se) compounds and Se-enriched broccoli extracts on cell proliferation and the possible mechanism responsible for the Se-induced growth inhibition. C6 rat glial cells were incubated with graded concentrations up to 1000 nM of selenite, selenate, selenomethionine (SeM), Se-methyl-selenocysteine (SeMCys), high-Se broccoli (H-SeB) extract or low-Se broccoli (L-SeB) extract for 24 and 48 h. MTT results indicated that all Se sources and levels examined inhibited C6 cell proliferation at 48 h. The results from cell cycle progression and apoptosis analysis indicated that SeM, SeMCys, H-SeB or L-SeB treatments at the concentration of 1000 nM reduced the cell population in G(0)/G(1) phase, but induced G(2)/M phase arrest and increased apoptosis and secondary necrosis in C6 cells at 24 h. The populations of apoptotic cells and secondary necrotic cells were increased by all Se sources examined. The COMET assay indicated that there was no significant DNA single-strand break found for all Se treatments in C6 cells for 48 h. In addition, the Se-induced proliferation inhibition may involve a hydrogen peroxide (H(2)O(2))-dependent mechanism with elevated cellular glutathione peroxidase (cGPX) activity. Both H-SeB and L-SeB inhibited C6 cell proliferation but H-SeB was less inhibitory than L-SeB. The proliferation inhibition by H-SeB in C6 cells is apparently related to the increased H(2)O(2) with the elevated cGPX activity, but the inhibition by L-SeB was H(2)O(2)-independent without change in cGPX activity.

Antioxidants do not explain the disparate longevity between mice and the longest-living rodent, the naked mole-rat

The maximum lifespan of naked mole-rats (NMRs; Heterocephalus glaber) is greater than that of any other rodent. These hystricognaths survive in captivity >28 years, eight-times longer than similar-sized mice. The present study tested if NMRs possess superior antioxidant defenses compared to mice and if age-related interspecies changes in antioxidants were evident. Activities of Cu/Zn superoxide dismutase (Cu/Zn, SOD), Mn SOD, catalase and cellular glutathione peroxidase (cGPx) were measured in livers of physiologically equivalent age-matched NMRs (30, 75 and 130 months) and CB6F1 mice (4, 12 and 18 months). In mice, Mn SOD activity increased with age, while the activity of catalase and cGPx declined. None of the antioxidants changed with age in mole-rats. cGPx activity of NMRs was 70-times lower ( p < 0.0001) than in mice, and resembled that of cGPx knock-out animals. NMRs may partially compensate for the lower cGPx when compared to mice, by having moderately higher activities of the other antioxidants. It is nonetheless unlikely that antioxidant defenses are responsible for the eight-fold longevity difference between these two species. Maintenance of constant antioxidant defenses with age in NMRs concurs with previous physiological data, suggesting delayed aging in this species.

Renal tubular cell damage and oxidative stress in renal stone patients and the effect of potassium citrate treatment

Our objective was to evaluate the oxidative stress and renal tubular cell damage in patients who have renal stones compared to normal subjects. The patients were re-evaluated after 1-months supplementation with potassium citrate. We recruited 30 patients (11 males and 19 females) diagnosed with kidney stones and scheduled for surgical stone removal the following month, and 30 healthy non-stone formers (14 males and 16 females). Two 24-h urine samples and one heparinized blood sample were collected from each subject. Plasma was separated from the erythrocytes and assayed for creatinine, potassium, sodium, calcium, magnesium, phosphate, malondialdehyde (MDA, a lipid peroxidation product) (P-MDA), protein thiol as an indicator of protein oxidation, and vitamin E. Erythrocytes were analysed for MDA (E-MDA), reduced glutathione (GSH) and cellular glutathione peroxidase (cGPx) activity. The urine was analyzed for pH, creatinine, potassium, sodium, calcium, magnesium, phosphate, oxalate, citrate, MDA (U-MDA), total protein (U-protein) and N-acetyl-beta-glucosaminidase (NAG) activity. For the stone patients, urine and blood samples were re-evaluated after supplementation with potassium citrate (60 mEq/day) for 1 month. Renal stone patients had higher plasma creatinine and lower plasma potassium, urinary pH, potassium, magnesium, phosphate and citrate than the controls. The patients had higher P-MDA, E-MDA, U-MDA, U-protein and NAG activity, but lower GSH, cGPx activity, protein thiol and vitamin E, when compared with controls. After potassium citrate supplementation, P-MDA and E-MDA decreased while plasma vitamin E, urinary NAG activity and citrate increased. Renal stone disease is associated with high oxidative stress and damage to renal tubular cells. These abnormalities are coincident with an increase in blood lipid peroxidation products and a decrease in antioxidant status. Although supplementation with potassium citrate improved urinary citrate levels and oxidative stress, it neither reduced urinary lipid peroxidation products nor remedied the damage to renal tubular cells, probably due to the existence of kidney stones.

Superoxide release and cellular gluthatione peroxidase activity in leukocytes from children with persistent asthma

Asthma is an inflammatory condition characterized by the involvement of several mediators, including reactive oxygen species. The aim of the present study was to investigate the superoxide release and cellular glutathione peroxidase (cGPx) activity in peripheral blood granulocytes and monocytes from children and adolescents with atopic asthma. Forty-four patients were selected and classified as having intermittent or persistent asthma (mild, moderate or severe). The spontaneous or phorbol myristate acetate (PMA, 30 nM)-induced superoxide release by granulocytes and monocytes was determined at 0, 5, 15, and 25 min. cGPx activity was assayed spectrophotometrically. The spontaneous superoxide release by granulocytes from patients with mild (N = 15), moderate (N = 12) or severe (N = 6) asthma was higher at 25 min compared to healthy individuals (N = 28, P < 0.05, Duncan test). The PMA-induced superoxide release by granulocytes from patients with moderate (N = 12) or severe (N = 6) asthma was higher at 15 and 25 min compared to healthy individuals (N = 28, P < 0.05 in both times of incubation, Duncan test). The spontaneous or PMA-induced superoxide release by monocytes from asthmatic patients was similar to healthy individuals (P > 0.05 in all times of incubation, Duncan test). cGPx activity of granulocytes and monocytes from patients with persistent asthma (N = 20) was also similar to healthy individuals (N = 10, P > 0.05, Kruskal-Wallis test). We conclude that, under specific circumstances, granulocytes from children with persistent asthma present a higher respiratory burst activity compared to healthy individuals. These findings indicate a risk of oxidative stress, phagocyte auto-oxidation, and the subsequent release of intracellular toxic oxidants and enzymes, leading to additional inflammation and lung damage in asthmatic children.

Ability of GDNF to diminish free radical production leads to protection against kainate-induced excitotoxicity in hippocampus.

The primary aim of this study is to explore the protective mechanisms of glial-derived neurotrophic factor (GDNF) during excitotoxicity by kainate in the hippocampus. After a 15-min microinjection with kainate, excitotoxicity was induced in the rat hippocampus. The protective effect of GDNF in the hippocampus was evaluated by administering GDNF 14 min after injection of kainate. The resulting hydroxyl free radicals were quantified by microdialysis of the hippocampus. The results show that GDNF can effectively suppress the production of kainate-induced hydroxyl free radical production. In addition, histological observation indicated the ability of GDNF to decrease the damage level of pyramidal neurons in the CA3 and CA4 areas of the hippocampus. Superoxide dismutase (SOD) activity in the hippocampus was elevated significantly at 30 min and 7 days after kainate induction, while glutathione peroxidase (cGPx) activity did not increase significantly until the seventh day. With GDNF treatment, SOD and cGPx activity in the hippocampus was elevated significantly 7 days after kainate induction. We suggest that mechanisms including a decrease in free radical generation and scavenging of free radicals might be involved in GDNF protection against kainate-induced excitotoxicity.

Zinc and Selenium Requirements for Glutathione Peroxidase Activity and Cell Survival in Chinese Hamster Ovary Cells Overexpressing Metallothionein

Many defined cell culture media were formulated over 30 years ago and may be deficient in certain micronutrients whose essentiality has only subsequently been recognised. The objective of this study was to evaluate whether alpha-minimal essential medium (MEM) supplemented with 10% foetal bovine serum contained sufficient selenium for optimal activity of the selenium containing enzymes cytosolic glutathione peroxidase (cGPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in cultured Chinese hamster ovary (CHO) cells. Additionally, the effect of zinc deficiency and metallothionein (MT) overexpression on cGPx and PHGPx activity was studied. The addition of 100 nM of selenous acid to the culture medium increased cGPx expression by 10-fold and PHGPx by about 2-fold in both wild-type CHO-K1 cells and CHO-K1 cells overexpressing mouse MT-1. Zinc deficiency had no significant effect on enzyme activity, but cells overexpressing mouse MT-1 had higher levels of cGPx activity. Zinc deficiency decreased cell survival but overexpression of MT-1 was partially protective, probably because its presence in quantity favoured the uptake, sequestration and cellular retention of any remaining zinc. This study demonstrates that selenium in complete alpha-MEM is insufficient for optimal cGPx and PHGPx activity and may compromise the cellular response to oxidative stress.

Oxidative Stress in Erythrocytes from Premature and Full-term Infants During their First 72 h of Life

Objective : The aim of this study was to evaluate the extent of lipid peroxidation and the response of the enzymatic and non-enzymatic antioxidant defence system in erythrocytes from full-term and premature infants at birth, after 3 and after 72 h of life. Study design: Twenty infants were selected and divided in two groups according to their gestational age. Blood samples were taken at birth, at 3 and at 72 h of life, erythrocytes were isolated and the following parameters were measured: fatty-acid profile, coenzyme Q, f -tocopherol, hydroperoxides and the activity of the antioxidant enzymes catalase, superoxide dismutase (SOD) and cytosolic glutathione peroxidase (cGPx). Results: For the three studied periods, several differences between full-term and premature infants were found. Premature children showed a higher concentration of hydroperoxides, a lower level of f -tocopherol and lower SOD and cGPx activity (except for cGPx at birth). Moreover, n-3 polyunsaturated fatty-acids percentages (essential for good neonatal development) were higher in full term children throughout all the study. Conclusion: Results suggest a strong imbalance between oxidants and antioxidants in premature infants during their first 72 h of life, a situation which could lead to several pathologies. Therefore, further research is needed, including possible nutritional intervention (with antioxidant therapy, supplementation of essential fatty acids and other dietary constituents) before and after birth.

Differential influences of various arsenic compounds on glutathione redox status and antioxidative enzymes in porcine endothelial cells.

The cellular response and detoxification mechanisms in porcine endothelial cells (PAECs) to arsenic trioxide (As2O3), sodium arsenite (NaAsO2) and sodium arsenate (Na2HAsO4) were investigated. NaAsO2 at 20 microM for 72 h increased Cu/Zn superoxide dismutase activity resulting in elevated intracellular hydrogen peroxide levels, but As2O3 and Na2HAsO4 did not. Trivalent arsenic compounds increased intracellular oxidized glutathione (GSSG) and total glutathione (GSH) and cellular glutathione peroxidase (cGPX) and glutathione S-transferase (GST) activity, but not glutathione reductase activity. The increased cGPX activity resulted in an elevated cellular GSSG content. Na2HAsO4 increased the cellular GSSG level at 72 h compared to controls. These results imply that the increased GSH content responding to the oxidative stress by trivalent arsenic compounds may be mainly related to the regulation of GSH turnover. The increased GST activity implies that the elevated intracellular GSH level responding to the oxidative stress may be used to conjugate arsenic in PAECs and facilitate arsenic efflux.

Dual regulation of glutathione peroxidase by docosahexaenoic acid in endothelial cells depending on concentration and vascular bed origin

Docosahexaenoic acid (DHA) has been reported to elicit oxidative stress, which in turn can induce antioxidant enzymes. Glutathione peroxidase (GPx) has received particular attention in this respect, as this enzyme is specifically required for the degradation of lipid hydroperoxides. Because we previously found that DHA could protect against oxidative stress when used in low amounts, we have compared the effect of a low (10 μM) versus high (100 μM) concentration of DHA on oxidant/antioxidant balance in bovine retinal and bovine aortic endothelial cells (BREC and BAEC). At 100 μM, DHA elicited a marked oxidative stress, as evidenced by high malondialdehyde levels and decreased plasmalogen phosphatidylethanolamine in both cells, and for BAEC only, a decrease of α-tocopherol. At 10 μM, DHA induced a slight increase of malondialdehyde in both cells, but did not affect α-tocopherol levels, which is indicative of a mild oxidative stress. In BREC, 10 μM DHA slightly but significantly decreased cytosolic GPx (cGPx) activity whereas 100 μM had no effect. In contrast, in BAEC, DHA 10 μM did not affect cGPx activity, whereas 100 μM increased it. The decreased cGPx activity in BREC was associated with a lower level of protein, suggesting a transcriptional and/or posttranscriptional effect. Phospholipid hydroperoxide GPx (PHGPx) activity was not modified by DHA at either concentration in BREC, whereas it was increased in BAEC when using 100 μM. Our results confirm that large amounts of DHA lead to oxidative stress, but do no support an antioxidant action of DHA at low concentration, in endothelium. Nevertheless, we showed that DHA can exert opposite effects on GPx regulation in endothelial cells, with regard to its concentration and to vascular bed origin.

Differential selenium-dependent expression of type I 5'-deiodinase and glutathione peroxidase in the porcine epithelial kidney cell line LLC-PK1.

The Se-dependent expression of two selenoproteins, cytosolic glutathione peroxidase (cGPx) and type I iodothyronine-5'-deiodinase (5'DI), was investigated in the porcine epithelial kidney cell line LLC-PK1 in serum-free medium. The selenite-dependent expression of cGPx and 5'DI was revealed by enzyme-activity measurements, affinity labelling of 5'DI, metabolic labelling of proteins with 75Se and steady-state mRNA analysis. The expression of the two enzymes strongly depended on selenite concentrations of the culture medium. cGPx required 2-fold higher selenite levels than 5'DI to reach half-maximal activity. The Se-dependent enzyme activities were approximately paralleled by the corresponding steady-state mRNA levels. The response of the two enzymes to Se supply was further characterized by kinetic Se-depletion and -repletion experiments. Upon removal of medium selenite, cGPx activity decreased exponentially, whereas after an initial decrease over 1-2 days, 5'DI levels completely recovered during a further 2 days. These data indicate a differential Se-dependent regulation of the two selenoproteins, with 5'DI being preferentially supplied with the trace element Se, thus ensuring a continuous cellular capacity for thyroid-hormone activation, even under Se-deficient conditions. The abundant cGPx in cells with sufficient Se supply might serve as a cellular Se store which can be mobilized for the synthesis of more vital selenoproteins such as 5'DI under shortage conditions. Thus, a cellular hierarchy of selenoprotein expression, reflected by different individual regulation mechanisms at the transcriptional and post-transcriptional level, adds to the previously recognized tissue-specific hierarchy of Se retention.

Dehydroepiandrosterone and a β-agonist, energy transducers, alter antioxidant enzyme systems: Influence of chronic training and acute exercise in rats

We examined the influence of dehydroepiandrosterone (DHEA), a β-agonist and exercise training on enzymes that detoxify toxic oxygen species. Feeding 0.4% DHEA decreased hepatic cytosolic (c) selenium-dependent glutathione peroxidase (GPX), (−26%, P < 0.0001) and increased hepatic mitochondrial (m) Mn superoxide dismutase (SOD), (+38%, P < 0.001). DHEA decreased myocardial c-GPX (−21%, P < 0.05) when compared to a β-agonist (βA; L644969 Merck and Co.) fed at 5 ppm but neither differed from the Control (C). In contrast, the βA increased hepatic m-GPX (+25%, P < 0.05). In skeletal muscle, DHEA and βA decreased muscle c-GPX by 20 and 12%, respectively ( P < 0.0009). DHEA increased both muscle (+20%, P < 0.01) and myocardial (+20%, P < 0.05) c-glutathione S-transferase (GST) over βA (+20%, P < 0.01) but neither was significantly different from C. Similar to DHEA, chronic training (Tr) (1 h/day, 5 days/week at 27 m/min, 15% grade on treadmill) decreased hepatic c-GPX (−16%, P < 0.003). Tr elevates muscle c-GPX (+36%, P < 0.05) in C. Tr increased myocardial c-GPX by 28% in the βA-treated rats, whereas Tr decreased myocardial c-GPX by 22% in the C ( P < 0.05, interaction). One hour of acute exercise (Ex) (70% VO 2 max relative work load) decreased hepatic homogenate catalase (−12%, P < 0.02) and increased hepatic m-Mn SOD (+28%, P < 0.03). Ex decreased myocardial c-GST ( P < 0.05) only in the DHEA-treated rats. DHEA and Tr may improve efficiency of oxygen utilization at the tissue level with lower antioxidant enzyme activity in liver and locally protective up-regulation in muscle. βA stresses oxygen utilization systems and liver responds by up-regulation of antioxidant enzymes. The increase in myocardial c-GPX activity in the βA-treated group may be a protective effect against indirect catecholamine-induced myocardial necrosis which results from free radical generation.