Enzyme activities and level of SH groups in breast carcinomas

• 1.1. The carcinoma showed higher enzyme activities than the normal mammary tissue.
• 2.2. The ratios of glutamate dehydrogenase, glutathione reductase and catalase to lactate dehydrogenase were lower in carcinomas than in normal tissues. Similarly, the ratios of glutamate dehydrogenase, glutathione reductase and catalase to glucose-6-phosphate dehydrogenase were also significantly lower in carcinomas.
• 3.3. There were no significant differences in enzyme activities between stages I and II of disease, however in the metastatic tissues, there were significant differences between stages I and II.
• 4.4. SH groups were higher in the tissues of cancer patients than in normal tissues. The levels of thiols groups were higher in carcinomas at stage III of disease.

     

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.     

Temperature increase results in oxidative stress in goldfish tissues. 2. Antioxidant and associated enzymes

Activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glucose-6-phophate dehydrogenase (G6PDH) were measured in four tissues of goldfish, Carassius auratus L., over 1-12 h of high temperature (35 degrees C) exposure followed by 4 or 24 h of lower temperature (21 degrees C) recovery. SOD activity was strongly affected by heat shock, increasing 4-fold in brain, liver, and kidney, but was mainly reversed at recovery. In some tissues, activities of SOD, catalase, GPx, and G6PDH decreased significantly after 1 h heat shock exposure suggesting that thermal inactivation possibly occurred, but were renewed at further exposure. In many cases, 4 h of return to the initial temperature decreased enzyme activities. High correlation coefficients between SOD activities and levels of lipid peroxidation products suggest that these products might be involved in up-regulation of antioxidant defense. Several enzymes (SOD, GST, GR) responded to stress in coordinated manner.     

High content of endogenous cytokinins stimulates activity of enzymes and proteins involved in stress response in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

Transgenic Pssu-ipt tobacco with elevated content of endogenous cytokinins grown under in vitro conditions exhibited elevated activities of antioxidant enzymes (i.e. catalase, ascorbate peroxidase, guaiacol and syringaldazine peroxidase, glutathione reductase) and some of enzymes involved in anaplerotic pathways such as glucose-6-phosphate dehydrogenase, glycolate oxidase, NADP-malic enzyme, NADP-isocitrate dehydrogenase, and glutamate dehydrogenase compared to control non-transgenic SR1 tobacco. Higher activities of peroxidases, NADP-malic enzyme, and glutamate dehydrogenase were maintained in transgenic grafts after several weeks of the growth under ex vitro conditions, while transgenic rooted plants showed only the increase in activity of glycolate oxidase compared to control non-transformed tobacco. Total activities of superoxide dismutase were lower in both types of Pssu-ipt tobacco contrary to controls under both growth conditions. The presence of PR-1 protein and proteins with elevated activities of chitinase was proved in the extracellular fluid in both transgenic types under both in vitro and ex vitro conditions.     

Oxidant-antioxidant imbalance in the erythrocytes of sporadic amyotrophic lateral sclerosis patients correlates with the progression of disease

Free radicals are implicated in numerous disease processes including motor neuron degeneration (MND). Antioxidant defense enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) in the erythrocytes are capable of detoxifying reactive oxygen species produced endogenously or exogenously. In the present study, the extent of lipid peroxidation (LPO) and antioxidant defenses were evaluated in the erythrocytes of 20 sporadic amyotrophic lateral sclerosis (ALS) patients and 20 controls. We observed that lipid peroxidation in the erythrocytes of amyotrophic lateral sclerosis patients significantly increased with respect to controls (P<0.001). On the other hand, catalase activity was found to be significantly lower (P<0.001). The activities of glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione levels were also found to be significantly reduced in ALS patients compared to healthy subjects (P<0.001, P<0.01 and P<0.01, respectively). It was further observed that lipid peroxidation started to increase and catalase, glutathione reductase, glucose-6-phosphate dehydrogenase enzyme activities and glutathione levels started to decrease as amyotrophic lateral sclerosis progressed from 6 to 24 months, suggesting a correlation between these parameters and duration of amyotrophic lateral sclerosis. This study confirms the involvement of oxidative stress during the progression of amyotrophic lateral sclerosis and the need to develop specific peripheral biomarkers.     

Developmental Aspects of Detoxifying Enzymes in Fish (Salmo Iridaeus)

The activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione transferase and glyoxalase I have been studied during the embryologic development of rainbow trout (Salmo iridaeus) and in several other trout tissues to investigate the protective development metabolism.

A gradual increase of superoxide dismutase, catalase, glutathione reductase, glyoxalase I and glutathione transferase activities was noted throughout embryo development.

In all trout tissues investigated glutathione peroxidase was found to be extremely low compared to catalase activity. The highest activity of superoxide dismutase, glyoxalase I and glutathione reductase was found in liver followed by kidney.

No change in the number of GST subunits was noted with the transition from the embryonic to the adult stages of life according to the SDS/PAGE and HPLC analyses performed on the GSH-affinity purified fractions.     

Comparative study of antioxidant enzymes in tissues surrounding implant in rabbits

There is a possible role of reactive oxygen species (SROS) in the complication of implants although there is presently little information. The aim of this study was to investigate the alterations in lipid peroxidation (LP) and antioxidant enzyme activities in tissues surrounding implants in rabbits. Thirty New Zealand albino male rabbits were used. They were randomly divided into five groups. The first group (I) was used as control. Groups II, III, IV and V were implanted with stainless steel, ceramic, titanium and polyethylene, respectively. One month after the administration of implant, the tissues surrounding the implant were carefully removed for antioxidant enzyme analysis. Glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase (GR), superoxide dismutases (SOD), glutathione peroxidase (GPx), catalase (CAT) in tissues surrounding the implants in the groups II, III and IV were significantly (p<0.05-p<0.001) lower than in the control group although glutathione S-transferase (GST) activities and LP values were increased. CAT activity and LP level did not decrease in group V. In conclusion, these data demonstrate that there is an increase in lipid peroxidation in the tissues surrounding ceramic and titanium implants of animals whereas there is a decrease in antioxidant enzymes. Oxidative stress plays a very important role in the complications of ceramic and titanium implants. The polyethylene implant seems to be the best of the four implant materials tested.     

Hyperoxia results in transient oxidative stress and an adaptive response by antioxidant enzymes in goldfish tissues

The effects of hyperoxia on the status of antioxidant defenses and markers of oxidative damage were evaluated in goldfish tissues. The levels of lipid peroxides, thiobarbituric acid reactive substances, carbonyl proteins and the activities of some antioxidant enzymes were measured in brain, liver, kidney and skeletal muscle of goldfish, Carassius auratus L., over a time course of 3-12 h of hyperoxia exposure followed by 12 or 36 h of normoxic recovery. Exposure to high oxygen resulted in an accumulation of protein carbonyls in tissues throughout hyperoxia and recovery whereas lipid peroxides and thiobarbituric acid reactive substances accumulated transiently under short-term hyperoxia stress (3-6 h) but were then strongly reduced. This suggests that hyperoxia stimulated an enhancement of defenses against lipid peroxidation or mechanisms for enhancing the catabolism of peroxidation products. The activities of principal antioxidant enzymes, superoxide dismutase and catalase, were not altered under hyperoxia but catalase increased during normoxic recovery; activities may rise in anticipation of further hyperoxic excursions. In most tissues, the activities of glutathione-utilizing enzymes (glutathione peroxidase, glutathione-S-transferase, glutathione reductase) as well as glucose-6-phosphate dehydrogenase, were not affected under hyperoxia but increased sharply during normoxic recovery. Correlations between some enzyme activities and oxidative stress markers were found, for example, an inverse correlation was seen between levels of thiobarbituric acid reactive substances and glutathione-S-transferase activity in liver and catalase and glucose-6-phosphate dehydrogenase in kidney. The results suggest that liver glutathione-S-transferase plays an important role in detoxifying end products of lipid peroxidation accumulated under hyperoxia stress.     

Different effects of exercise tests on the antioxidant enzyme activities in lymphocytes and neutrophils

We have determined the effects of maximal and submaximal cycloergometer tests on the antioxidant enzyme defences of neutrophils and lymphocytes. We also compared the neutrophil and lymphocyte basal enzyme antioxidant activities. A total of 17 well-trained amateur athletes, runners, and cyclists participated in this study. Two tests were performed on an electromagnetic reduction cycloergometer: the maximal exercise test, and the submaximal prolonged exercise test. Blood samples were taken before and after the tests. Basal enzyme activity of superoxide dismutase was higher in lymphocytes but neutrophils presented higher activities of catalase and glutathione peroxidase. The maximal test increased the circulating number of lymphocytes and the activities of catalase and glutathione peroxidase. No changes were observed in lymphocyte number or in lymphocyte antioxidant enzyme activities after the submaximal test. The circulating number of neutrophils increased significantly after the submaximal test. Maximal and submaximal tests decreased the activities of neutrophil glutathione dependent antioxidant enzymes (glutathione peroxidase and glutathione reductase), but no changes were observed in catalase or superoxide dismutase activities after either test. Neither the maximal nor submaximal test produced increases in serum activities of lactate dehydrogenase and creatine kinase (CK).     

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.     

Redox imbalance in peripheral blood of type 1 myotonic dystrophy patients

Objectives: The aim of our study was to determine if redox imbalance caused by the activities of antioxidant enzymes existed in erythrocytes of type 1 myotonic dystrophy (DM1) patients.

Methods: The activities of erythrocyte superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were measured in 30 DM1 patients and 15 healthy controls (HCs). The obtained values were correlated with the Muscular Impairment Rating Scale (MIRS) score and creatine kinase (CK).

Results: Superoxide dismutase and catalase activities were lower in DM1 patients compared to HCs. A positive correlation was found between disease duration and MIRS score as well as with glutathione reductase activity. In DM1 patients, there were positive correlations between catalase, glutathione peroxidase, and glutathione reductase activities. After sub-dividing DM1 patients according to CK levels, superoxide dismutase activity was still statistically different from HCs. However, catalase activity was significantly lower only in DM1 patients with increased CK.

Discussion: Undesirable alterations in antioxidant enzyme activities during DM1 disease progression may result in conditions favoring oxidative stress and changes in metabolism which together could contribute to muscle wasting.     

The regulation of activity of the enzymes involved in the assimilation of nitrate by higher plants

1. Possible mechanisms regulating the activities of three enzymes involved in nitrate assimilation, nitrate reductase, nitrite reductase and glutamate dehydrogenase, were studied in radish cotyledons. 2. Nitrate-reductase and nitrite-reductase activities are low in nitrogen-deficient cotyledons, and are induced by their substrates. 3. Glutamate dehydrogenase is present regardless of the nitrogen status, and the enzyme can be increased only slightly by long-term growth on ammonia. 4. Although nitrate is the best inducer of nitrate reductase, lower levels of induction are also obtained with nitrite and ammonia. The experiments did not distinguish between direct or indirect induction by these two molecules. 5. Nitrite reductase is induced by nitrite and only indirectly by nitrate. 6. The induction of both nitrate reductase and nitrite reductase is prevented by the inhibitors actinomycin D, puromycin and cycloheximide, indicating a requirement for the synthesis of RNA and protein. 7. The decay of nitrate reductase, determined after inhibition of protein synthesis, is slower than the synthesis of the enzyme. Nitrite reductase is much more stable than nitrate reductase. 8. The synthesis of nitrate reductase is not repressed by ammonia, but is repressed by growth on a nitrite medium. 9. There is no inhibition of nitrate reductase, nitrite reductase or glutamate dehydrogenase by the normal end products of assimilation, but cyanate is a fairly specific inhibitor of nitrate reductase.     

Comparative study of nitrogen and oxygen metabolism enzymes in Yugoslav cultivars of alfalfa (Medicago sativa L.)

Four Yugoslav cultivars of alfalfa were investigated in order to determine nitrogen fixing (nitrogenase), nitrogen assimilation (nitrate reductase, glutamine synthetase, glutamate dehydrogenase) and antioxidant (Superoxide dismutase, catalase, peroxidase) enzymes activities. The level of lipid peroxidation and protein content were also investigated. On the basis of the results obtained a resistant cultivar with high nitrogen fixing and a cultivar with high nitrogen assimilation abilities were chosen. The cultivar with high nitrogen fixing ability had high activities of nitrogenase, Superoxide dismutase, peroxidase and catalase, and also a low level of lipid peroxidation. The cultivar with high assimilation ability had high activities of nitrate reductase, glutamine synthetase and glutamate dehydrogenase and high soluble protein content.     

Effect of chronic ethanol administration on testicular antioxidant system and steroidogenic enzyme activity in rats

In order to find out the effect of chronic ethanol administration on testicular antioxidant system and steroidogenic enzyme activity, male rats fed with ethanol 1.6g/kg body weight per day for four weeks were studied. Besides a drastic reduction in body and testis weight, there was decrease in ascorbic acid, reduced glutathione and activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase in the testicular tissue of the treated animals. Simultaneously, there was increase in lipid peroxidation and glutathione S-transferase activity. Activities of 3 beta-hydroxy steroid dehydrogenase and 17 beta-hydroxy steroid dehydrogenase were also found decreased in the treated animals. The results indicate that chronic ethanol administration resulted in increase in oxidative stress and decrease in the activities of steroidogenic enzymes in the rat testes.     

Effects of piperine on antioxidant pathways in tissues from normal and streptozotocin-induced diabetic rats

Using diabetes mellitus as a model of oxidative damage, this study investigated whether subacute treatment (10 mg/kg/day, intraperitoneally for 14 days) with the compound piperine would protect against diabetes-induced oxidative stress in 30-day streptozotocin-induced diabetic Sprague-Dawley rats. Liver, kidney, brain, and heart were assayed for degree of lipid peroxidation, reduced and oxidized glutathione (GSH and GSSG, respectively) content, and activities of the free-radical detoxifying enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase. Piperine treatment of normal rats enhanced hepatic GSSG concentration by 100% and decreased renal GSH concentration by 35% and renal glutathione reductase activity by 25% when compared to normal controls. All tissues from diabetic animals exhibited disturbances in antioxidant defense when compared with normal controls. Treatment with piperine reversed the diabetic effects on GSSG concentration in brain, on renal glutathione peroxidase and superoxide dismutase activities, and on cardiac glutathione reductase activity and lipid peroxidation. Piperine treatment did not reverse the effects of diabetes on hepatic GSH concentrations, lipid peroxidation, or glutathione peroxidase or catalase activities; on renal superoxide dismutase activity; or on cardiac glutathione peroxidase or catalase activities. These data indicate that subacute treatment with piperine for 14 days is only partially effective as an antioxidant therapy in diabetes.     

Riboflavin nutritional status and flavoprotein enzymes in streptozotocin-diabetic rats

Riboflavin nutritional status was assessed on the basis of activity coefficients of glutathione reductase in erythrocyte hemolysates of normal and streptozotocin-diabetic rats. Activity coefficient values higher than 1.3 were regarded as evidence of riboflavin deficiency. All diabetic animals were found to be riboflavin-deficient, with activity coefficient values of 1.47–2.11. Treatment of diabetic rats with either insulin or riboflavin returned their activity coefficients to normal. Rats fed a restricted diet had normal activity coefficient values. The erythrocyte glutathione reductase activity was significantly lower in diabetic rats, and the augmentation of enzyme activity in the presence of flavin-adenine dinucleotide (FAD) was 72% compared to 16% in normal rats. Hepatic activities of glutathione reductase and succinate dehydrogenase, both FAD-containing enzymes, were significantly lower in diabetic than in normal rats. Like activity coefficient values, all enzyme activities were normalized after insulin or riboflavin treatments. These data suggest that insulin and riboflavin enhance the synthesis of erythrocyte and hepatic FAD. The results of the present study suggest that experimental diabetes causes riboflavin deficiency, which in turn decreases erythrocyte and hepatic flavoprotein enzyme activities. These changes can be corrected for by either insulin or riboflavin. The pathogenesis of riboflavin deficiency in diabetes mellitus is not clearly understood. The data of the present study provide evidence in addition to the previous findings of an increased prevalence of riboflavin deficiency in genetically diabetic KK mice.     

Oxidative stress and antioxidative defense in cephalopods: a function of metabolic rate or age?

Activities of the antioxidative enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX) and glutathione reductase (GR) were measured in the cephalopods Sepia officinalis and Lolliguncula brevis. Maximal enzyme activities were higher in gill tissue than in the mantle musculature of both species. Activities were generally lower in tissues of L. brevis than in S. officinalis. Comparison with other ectothermic animals showed both cephalopod species to have a low enzymatic antioxidative status despite their high metabolic rate. Furthermore, changes in antioxidative enzyme activities were measured in the cuttlefish S. officinalis with increasing age. The concentrations of malondialdehyde (MDA) and lipofuscin were determined as indicators of lipid peroxidation. Investigated animals were between 1.5 months and over 12 months old. Changes of antioxidative enzyme activities with age were not uniform. SOD and GPX activities increased with age, while catalase activity declined. In contrast, GR activity remained almost unchanged in all age groups. The low level of antioxidative defense might allow for the significant age-induced rise in MDA levels in gills and mantle musculature and for the increase in lipofuscin levels in mantle and brain tissue. It might thereby contribute to increased oxidative damage and a short life span in these cephalopods.     

Evaluation of effect of some corticosteroids on glucose-6-phosphate dehydrogenase and comparative study of antioxidant enzyme activities

Corticosteroids are anti-inflammatory drugs that are similar to the natural corticosteroid hormones produced by the cortex of the adrenal glands. The objective of this study was to scrutinize effects of some corticosteroids on glucose-6-phosphate dehydrogenase (G6PD) and some antioxidant enzymes. Initially, G6PD was purified from human erythrocytes by using ammonium sulphate precipitation and affinity chromatography. The two drugs, dexamethasone phosphate and prednisolone, investigated on the purified enzyme inhibited the enzyme activity. Comparative in vivo studies were performed to determine the effects of dexamethasone phosphate on the antioxidant enzyme activities using Spraque-Dawley rats. G6PD and catalase (CAT) activities were found significantly lower than in the control, whereas glutathione peroxidase (GP) activity was significantly increased in the erythrocytes of rats the receiving drug; glutathione reductase (GR) activity was unaffected. The results imply that dexamethasone phosphate may affect oxidative stress by changing antioxidant enzyme activities.     

Activities of antioxidant and redox enzymes in human normal hepatic and hepatoma cell lines

The cellular defense system (including glutathione, glutathione-related enzymes, antioxidant and redox enzymes) plays a crucial role in cell survival and growth in aerobic organisms. To understand its physiological role in tumor cells, the glutathione content and related enzyme activities in the human normal hepatic cell line, Chang and human hepatoma cell line, HepG2, were systematically measured and compared. Superoxide dismutase, catalase, and glutathione peroxidase activities are 2.8-, 4.3-, and 2.9-fold higher in HepG2 cells than in Chang cells. Total glutathione content is also about 1.4-fold higher in HepG2, which is supported by significant increases in gamma-glutamylcysteine synthetase and glutathione synthetase activities. Two other glutathione-related enzymes, glutathione reductase and gamma-glutamyltranspeptidase, are upregulated in HepG2 cells. However, thioredoxin reductase and glutathione S-transferase activities are significantly lower in HepG2 cells. These results propose that defense-related enzymes are largely modulated in tumor cells, which might be linked to their growth and maintenance.     

Growth on ethanol results in co-ordinated Saccharomyces cerevisiae response to inactivation of genes encoding superoxide dismutases

Superoxide dismutase (SOD) is an essential enzyme protecting cells against oxidative stress. However, its specific role under different conditions is not clear. To study the possible role of SOD in the cell during respiration, Saccharomyces cerevisiae single and double mutants with inactivated SOD1 and/or SOD2 genes growing on ethanol as an energy and carbon source were used. Activities of antioxidant and associated enzymes as well as the level of protein carbonyls were measured. SOD activity was significantly higher in a Mn-SOD deficient strain than that in the wild-type parental strain, but significantly lower in a Cu, Zn-SOD mutant. A strong positive correlation between SOD and catalase activities (R(2) = 0.99) shows possible protection of catalase by SOD from inactivation in vivo and/or decrease in catalase activity because of lower H(2)O(2) formation in the mutant cells. SOD deficiency resulted in a malate dehydrogenase activity increase, whereas glucose-6-phosphate dehydrogenase (G6PDH) activity was lower in SOD-deficient strains. Linear and non-linear positive correlations between SOD and isocitrate dehydrogenase activities are discussed. No changes in the activity of glutathione reductase and protein carbonyl levels support the idea that SOD-deficient cells are not exposed to strong oxidative stress during exponential growth of yeast cultures on ethanol.