Moderate exercise is an antioxidant: upregulation of antioxidant genes by training

Exercise causes oxidative stress only when exhaustive. Strenuous exercise causes oxidation of glutathione, release of cytosolic enzymes, and other signs of cell damage. However, there is increasing evidence that reactive oxygen species (ROS) not only are toxic but also play an important role in cell signaling and in the regulation of gene expression. Xanthine oxidase is involved in the generation of superoxide associated with exhaustive exercise. Allopurinol (an inhibitor of this enzyme) prevents muscle damage after exhaustive exercise, but also modifies cell signaling pathways associated with both moderate and exhaustive exercise in rats and humans. In gastrocnemius muscle from rats, exercise caused an activation of MAP kinases. This in turn activated the NF-kappaB pathway and consequently the expression of important enzymes associated with defense against ROS (superoxide dismutase) and adaptation to exercise (eNOS and iNOS). All these changes were abolished when ROS production was prevented by allopurinol. Thus ROS act as signals in exercise because decreasing their formation prevents activation of important signaling pathways that cause useful adaptations in cells. Because these signals result in an upregulation of powerful antioxidant enzymes, exercise itself can be considered an antioxidant. We have found that interfering with free radical metabolism with antioxidants may hamper useful adaptations to training.     

Reassessment of antioxidant activity of arbutin: Multifaceted evaluation using five antioxidant assay systems

Abstract

Arbutin, a practically used skin-lightening agent, has been reported to possess a weak antioxidant activity compared to that of its precursor, hydroquinone. However, its antioxidant activity has not been systematically evaluated. Hence, this study reassessed its activity using five assay systems. Assays were first performed using model radicals, DPPH radical and ABTS^?+. Arbutin showed weak DPPH radical-scavenging activity compared to that of hydroquinone, but showed strong ABTS^?+-scavenging activity. Its activity by ORAC assay was then evaluated using a physiologically relevant peroxyl radical. Arbutin exerted weak but long-lasting radical-scavenging activity and showed totally the same antioxidant activity as that of hydroquinone. Finally, it was shown that, in two cell-based antioxidant assays using erythrocytes and skin fibroblasts, arbutin exerted strong antioxidant activity comparable or even superior to that of hydroquinone. These findings indicate that the antioxidant activity of arbutin may have been under-estimated and suggest that it acts as a potent antioxidant in the skin.     

Spectrophotometric determination of antioxidant activity

Summary

The spectrophotometric technique for total antioxidant activity (TAA)^1,2 measures the relative abilities of antioxidants to scavenge the 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) radical cation (ABTS^?+) in comparison with the antioxidant potency of standard amounts of Trolox, the water-soluble vitamin E analogue. This method is based on the progressive consumption of antioxidant activity by ABTS^?+ as it is generated in the reaction cuvette and can be automated with a spectrophotometric analyzer. Several different analytical strategies are possible using the same reagents, enabling the assay system to be used to determine the antioxidant activity of plasma, saliva, lipoprotein fractions, foods and beverages. To determine the activity of pure antioxidant substances, a hydrogen peroxide concentration of 75 μM is used, together with a 6 min measuring time. For biological samples with endogenous peroxidase activity the hydrogen peroxide concentration is increased fivefold and the measuring time shortened to 3.25 min. Assays with improved sensitivity are described for low-density lipoprotein (LDL) preparations and saliva. Use of a spectrophotometric endpoint makes the assay simple to carry out without special laboratory equipment. Measurement at 734 nm avoids a range of potential interfering factors, such as sample turbidity and non-specific absorbance by sample constituents. Current applications of the ABTS antioxidant assay are described and discussed.     

Enzymatic antioxidant system of endotheliocytes

It is shown that endothelial cells from human umbilical vein have a reduced activity and gene expression of the “classic” antioxidant enzymes (Cu,Zn-superoxide dismutase, catalase, and Se-containing glutathione peroxidase). At the same time, a high expression level of peroxiredoxin genes was identified in the same endothelial cells, which obviously indicates the predominant involvement of these enzymes in protecting the endothelium from the damaging effect of free radical peroxidation.     

Ozone-induced inactivation of antioxidant enzymes

Ozone is an air pollutant that damages a variety of biomolecules. We investigated ozone-induced inactivation of three major antioxidant enzymes. Cu/Zn superoxide dismutase was inactivated by ozone in a concentration-dependent manner. The concentration of ozone for 50% inactivation was approximately 45 microM when 10 microM Cu/Zn superoxide dismutase was incubated for 30 min in the presence of ozone. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was randomly fragmented. Both ascorbate and glutathione were very effective in protecting Cu/Zn superoxide dismutase from ozone-induced inactivation. The other two enzymes, catalase and glutathione peroxidase, were much more resistant to ozone than Cu/Zn superoxide dismutase. The ozone concentrations for 50% inactivation of 10 microM catalase and glutathione peroxidase were 500 and 240 microM, respectively. SDS-PAGE demonstrated that ozone caused formation of high molecular weight aggregates in catalase and dimerization in glutathione peroxidase. Glutathione protected catalase and glutathione peroxidase from ozone but the effective concentrations were much higher than that for Cu/Zn superoxide dismutase. Ascorbate was almost ineffective. The result suggests that, among the three antioxidant enzymes, Cu/Zn superoxide dismutase is a major target for ozone-induced inactivation and both glutathione and ascorbate are very effective in protecting the enzyme from ozone.     

The antioxidant activity of cyclohistidylproline

A cyclohistidyl-proline, cyclopeptide possessing a hormonal and neurotrophic activity is shown to be an inhibitor of the (Fe + ascorbic acid)-induced peroxidation of membrane lipids, its effect being dependent on its concentration. Inhibition of the malondialdehyde formation by cyclohistidil-proline is accompanied by protection of the membrane bound Ca-pump. In the test of the free radical cumole oxidation antioxidative effect of cyclohistidyl-proline is 4 times higher than that of the hydrophilic antioxidant carnosine. After peritoneal injection of cyclohistidil-proline (15 mg/kg of body weight) to rats the stationary level of thiobarbituric acid reactive products in rat brain or serum is pronouncedly decreased, this effect being in progress up to 6 h after injection. Antioxidative action of cyclohistidyl-proline suggests to be on the basis of a variety of its biological effects.     

Regulation of antioxidant enzymes.

Free radicals generated by a partial reduction of O2 pose a serious hazard to tissues and vital organs, especially membrane lipids, connective tissues, and the nucleic acids of cells. For protection, enzymes have evolved that specifically attack O2-, hydrogen, and organic peroxides, and repair any damage incurred to DNA. With few exceptions, antioxidant enzymes are found in all aerobic and aerotolerant anaerobic organisms. Logic assumes that a basal level of antioxidant enzyme activity is maintained at all times. This may be true. Yet cells must have ways to amplify antioxidant enzyme activity to counter sudden increases in oxygen metabolites. The full details of that regulation are slowly coming to light. Bacteria possess a series of elaborate and interacting genes that can sense specific increases in intracellular H2O2 and O2-. In higher organisms, hormones and metal ion cofactors impose pre- and posttranslational control over the genetic expression of antioxidant enzymes. Furthermore, aging, cellular differentiation, and organ specificity must also be factored into the final equation in higher organisms. This review will discuss some of the more recent findings relevant to antioxidant enzyme regulation in bacteria and higher organisms.     

Therapeutic uses of antioxidant liposomes

This review will focus on the therapeutic uses of antioxidant liposomes. Antioxidant liposomes have a unique ability to deliver both lipid- and water-soluble antioxidants to tissues. This review will detail the varieties of antioxidants which have been incorporated into liposomes, their modes of administration, and the clinical conditions in which antioxidant liposomes could play an important therapeutic role. Antioxidant liposomes should be particularly useful for treating diseases or conditions in which oxidative stress plays a significant pathophysiological role because this technology has been shown to suppress oxidative stress. These diseases and conditions include cancer, trauma, irradiation, retinotherapy or prematurity, respiratory distress syndrome, chemical weapon exposure, and pulmonary infections.     

Oxidative activation of antioxidant defence

Living cells maintain a delicate balance between oxidizing and reducing species, and many disorders such as rheumatoid arthritis and Alzheimer's disease have been associated with a disturbed intracellular 'redox equilibrium'. The past few years have witnessed accelerated research into how natural redox responses and antioxidant defence systems are activated and how they restore a healthy redox balance. To function properly, many of these processes rely on a powerful sulfur redox chemistry, which is best exemplified by the complex, newly emerging cysteine-based redox regulation of the glutathione and thioredoxin pathways. Other redox systems based on oxidatively activated amino acid side chains in proteins are also becoming increasingly important, but are still barely understood or explored.     

The influence of pH on antioxidant properties and the mechanism of antioxidant action of hydroxyflavones

The effect of the pH on antioxidant properties of a series of hydroxyflavones was investigated. The pKa of the individual hydroxyl moieties in the hydroxyflavones was compared to computer-calculated deprotonation energies. This resulted in a quantitative structure activity relationship (QSAR), which enables the estimation of pKa values of individual hydroxyl moieties, also in hydroxyflavones for which these pKa values are not available. Comparison of the pKa values to the pH-dependent antioxidant profiles, determined by the TEAC assay, reveals that for various hydroxyflavones the pH-dependent behavior is related to hydroxyl moiety deprotonation, resulting in an increase of the antioxidant potential upon formation of the deprotonated forms. Comparison of these experimental results to computer calculated O-H bond dissociation energies (BDE) and ionization potentials (IP) of the nondeprotonated and the deprotonated forms of the various hydroxyflavones indicates that especially the parameter reflecting the ease of electron donation, i.e., the IP, and not the BDE, is greatly influenced by the deprotonation. Based on these results it is concluded that upon deprotonation the TEAC value increases (radical scavenging capacity increases) because electron-, not H*-, donation becomes easier. Taking into account that the mechanism of radical scavenging antioxidant activity of the neutral form of the hydroxyflavones is generally considered to be hydrogen atom donation, this implies than not only the ease of radical scavenging, but also the mechanism of antioxidant action changes upon hydroxyflavone deprotonation.     

Dietary intake of antioxidant supplements modulate antioxidant status and heat shock protein 70 synthesis

The physiological effects and efficacy of dietary intake of antioxidant supplements in humans remains controversial. Experiments involving dietary, often high, intake of a single antioxidant or vitamin may be seriously flawed given the interactive nature of antioxidants in vivo. The present studies were conducted on individuals (35-60 years of age) taking a commercial antioxidant mixture in a double-blind, placebo-controlled, cross-over study. Intake was two capsules per day, for 4 weeks, with a 4-week washout period in between active dose or placebo. Intake of antioxidants was associated with little change in superoxide dismutase activity, but an increase in glutathione peroxidase was noted. Haemolysis of red blood cells (erythrocytes) induced by the free radical generator AAPH was significantly reduced in individuals on antioxidant supplements. In lymphocytes isolated from individuals taking supplements, there was a marked increase, as compared with individuals on placebo, in the synthesis of heat shock protein 70 (hsp70) following heat shock from 37 degrees C to 42.5 degrees C. We conclude that dietary intake of a mixed antioxidant supplement leads to modulation of cellular redox status resulting in decreased oxidative stress and increased ability of lymphocytes to mount a stress response.     

Creatine as an antioxidant

Creatine monohydrate (Cr), the most diffuse supplement in the sports industry, is receiving greater attention because of its beneficial effects in a wide number of human degenerative diseases and conditions. These effects can be barely explained on the basis of the sole ergogenic role of the Cr/CrP system. Indeed, a wide number of research articles indicate that Cr is capable of exerting multiple, non-energy related, effects on diverse and relevant cellular targets. Among these effects, the antioxidant activity of Cr emerges as an additional mechanism which is likely to play a supportive role in the Cr-cytoprotection paradigm.     

Mucolytics and antioxidant activity

We investigated effects of the mucolytics ambroxol and N-acetylcysteine on airways reactivity evoked by histamine in guinea pigs exposed to toluene vapors. We did not find significant changes in reactivity of tracheal smooth muscle from animals treated with mucolytics compared to the control group. However, the administration of ambroxol and N-acetylcysteine caused a significant decrease in lung tissue reactivity. The effect of ambroxol was more pronounced after intraperitoneal injection than after inhalation, while N-acetylcysteine was only effective after inhalation. The protective effects of mucolytics in the lung tissue may be due to their antioxidant activity together with other mechanisms.     

The antioxidant activity of phloretin: the disclosure of a new antioxidant pharmacophore in flavonoids

Phloretin is a dihydrochalcone flavonoid that displays a potent antioxidant activity in peroxynitrite scavenging and the inhibition of lipid peroxidation. Comparison with structurally related compounds revealed that the antioxidant pharmacophore of phloretin is 2,6-dihydroxyacetophenone. The potent activity of 2,6-dihydroxyacetophenone is due to stabilisation of its radical via tautomerisation. The antioxidant pharmacophore in the dihydrochalcone phloretin, i.e., the 2,6-dihydroxyacetophenone group, is different from the antioxidant pharmacophores previously reported in flavonoids.     

Mechanism of antioxidant action of carnosine

The comparative study of the antiradical activity of carnosine and vitamin C was carried out by the means of the evaluation of quenching of ESR signals of 2,2-diphenyl-1-picrylhydrazyl (DFPH) and semiquinone radical of alpha-tocopherol. It was shown that carnosine is not able to quench the ESR signals of the stable radical of DFPH and semiquinone radical of alpha-tocopherol. It permits to conclude that: a) carnosine does not interact directly with highly active free radicals; b) carnosine is unable to regenerate the radical of alpha-tocopherol to form the antiradical synergistic couple. The data obtained are consistent with the idea that there is a difference between on the antioxidant mechanism action of vitamin C and carnosine due to the difference in the antiradical activity of these compounds.     

Queuine promotes antioxidant defence system by activating cellular antioxidant enzyme activities in cancer

Constant generation of Reactive oxygen species (ROS) during normal cellular metabolism of an organism is generally balanced by similar rate of consumption by antioxidants. Imbalance between ROS production and antioxidant defense results in increased level of ROS causing oxidative stress which leads to promotion of malignancy. Queuine is a hyper modified base analogue of guanine, found at first anti-codon position of Q- family of tRNAs. These tRNAs are completely modified with respect to queuosine in terminally differentiated somatic cells, however hypomodification of Q-tRNAs is close association with cell proliferation. Q-tRNA modification is essential for normal development, differentiation and cellular functions. Queuine is a nutrient factor to eukaryotes. It is found to promote cellular antioxidant defense system and inhibit tumorigenesis. The activities of antioxidant enzymes like catalase, SOD, glutathione peroxidase and glutathione reductase are found to be low in Dalton's lymphoma ascites transplanted (DLAT) mouse liver compared to normal. However, exogenous administration of queuine to DLAT mouse improves the activities of antioxidant enzymes. The results suggest that queuine promotes antioxidant defense system by increasing antioxidant enzyme activities and in turn inhibits oxidative stress and tumorigenesis.     

Oxidative inactivation of paraoxonase1, an antioxidant protein and its effect on antioxidant action

Paraoxonase1 (PON1), one of antioxidant proteins to protect low density lipoprotein (LDL) from the oxidation, is known to lose its activity in the oxidative environment. Here, we attempted to elucidate the possible mechanisms for the oxidative inactivation of PON1, and to examine the capability of hydroxyl radicals-inactivated PON1 to prevent against LDL oxidation. Of various oxidative systems, the ascorbate/Cu₂₊ system was the most potent in inactivating the purified PON1 (PON1) as well as HDL-bound PON1 (HDL-PON1). In contrast to a limited inactivation by Fe₂₊ (2.0 μM), the inclusion of Cu₂₊ (0.1-1.0 μM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.5 mM). A similar result was also obtained with the inactivation of HDL-PON1. The inactivation of PON1 by ascorbate/Cu₂₊ was pevented by catalase, but not general hydroxyl radical scavengers, supporting Cu₂₊-catalyzed oxidative inactivation. In addition, Cu₂₊ alone inactivated PON1, either soluble or HDL-bound, by different mechanisms, concentration-dependent. Separately, there was a reverse relationship between the inactivation of PON1 and its preventive action against LDL oxidation during Cu₂₊-induced oxidation of LDL. Noteworthy, ascorbate/Cu₂₊-inactivated PON1, which was charaterized by the partial loss of histidine residues, expressed a lower protection against Cu₂₊-induced LDL oxidation, compared to native PON1. Based on these results, it is proposed that metal-catalyzed oxidation may be a primary factor to cause the decrease of HDL-associated PON1 activity under oxidative stress, and radicals-induced inactivation of PON1 may lead to the decrease in its antioxidant action against LDL oxidation.     

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.     

Genomics of berry fruits antioxidant components

Reactive oxygen and nitrogen metabolites, which are side products of cell metabolism, can produce a lot of damage in biological macromolecules and tissues, producing a number of chronic illnesses. On the other hand, antioxidant metabolites usually accumulated in fruits and vegetables can provide an effective protection by neutralizing these reactive molecules. Among comestible vegetables, berry fruits are considered one of the richest sources of antioxidant metabolites; hence, they represent a good model for molecular and biochemical investigations about the biosynthesis and the functional role of antioxidants in plants. This review illustrates how recent developments in the fields of genomics and bioinformatics can provide powerful tools to better understand the molecular mechanisms that trigger biosynthesis and accumulation of antioxidant metabolites in berries.