Antioxidant defence mechanisms: From the beginning to the end (of the beginning)

When life first evolved on Earth, there was little oxygen in the atmosphere. Evolution of antioxidant defences must have been closely associated with the evolution of photosynthesis and of O₂-dependent electron transport mechanisms. Studies with mice lacking antioxidant defences confirm the important roles of MnSOD and transferrin in maintaining health, but show that glutathione peroxidase (GPX) and CuZnSOD are not essential for everyday life (at least in mice). Superoxide can be cytotoxic by several mechanisms: one is the formation of hydroxyl radicals. There is good evidence that OH^· formation occurs in vivo. Other important antioxidants may include thioredoxin, and selenoproteins other than GPX. Nitric oxide may be an important antioxidant in the vascular system. Diet-derived antioxidants are important in maintaining human health, but recent studies employing “biomarkers” of oxidative DNA damage are questioning the “antioxidant” roles of β-carotene and ascorbate. An important area of future research will be elucidation of the reasons why levels of steady-state oxidative damage to DNA and lipids vary so much between individuals, and their predictive value for the later development of human disease.     

Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence

Multiple reactive oxygen/nitrogen species induce oxidative stress. Mammals have evolved with an elaborate defense network against oxidative stress, in which multiple antioxidant compounds and enzymes with different functions exert their respective roles. Radical scavenging is one of the essential roles of antioxidants and vitamin E is the most abundant and important lipophilic radical-scavenging antioxidant in vivo. The kinetic data and physiological molar ratio of vitamin E to substrates show that the peroxyl radicals are the only radicals that vitamin E can scavenge to break chain propagation efficiently and that vitamin E is unable to act as a potent scavenger of hydroxyl, alkoxyl, nitrogen dioxide, and thiyl radicals in vivo. The preventive effect of vitamin E against the oxidation mediated by nonradical oxidants such as hypochlorite, singlet oxygen, ozone, and enzymes may be limited in vivo. The synergistic interaction of vitamin E and vitamin C is effective for enhancing the antioxidant capacity of vitamin E. The in vitro and in vivo evidence of the function of vitamin E as a peroxyl radical-scavenging antioxidant and inhibitor of lipid peroxidation is presented.     

Reactive Oxygen Species and the Central Nervous System

Radicals are species containing one or more unpaired electrons, such as nitric oxide (NO.). The oxygen radical superoxide (O2.-) and the nonradical hydrogen peroxide (H2O2) are produced during normal metabolism and perform several useful functions. Excessive production of O2.- and H2O2 can result in tissue damage, which often involves generation of highly reactive hydroxyl radical (.OH) and other oxidants in the presence of "catalytic" iron or copper ions. An important form of antioxidant defense is the storage and transport of iron and copper ions in forms that will not catalyze formation of reactive radicals. Tissue injury, e.g., by ischemia or trauma, can cause increased metal ion availability and accelerate free radical reactions. This may be especially important in the brain because areas of this organ are rich in iron and CSF cannot bind released iron ions. Oxidative stress on nervous tissue can produce damage by several interacting mechanisms, including increases in intracellular free Ca2+ and, possibly, release of excitatory amino acids. Recent suggestions that free radical reactions are involved in the neurotoxicity of aluminum and in damage to the substantia nigra in patients with Parkinson's disease are reviewed. Finally, the nature of antioxidants is discussed, it being suggested that antioxidant enzymes and chelators of transition metal ions may be more generally useful protective agents than chain-breaking antioxidants. Careful precautions must be used in the design of antioxidants for therapeutic use.     

A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

Various catalytic reaction models have been proposed as the reaction mechanisms of glycosidases, but a reasonable and unitary model capable of interpreting both “inverting” and “retaining” glycosidase reactions remains to be established. As for the models proposed to date, the nucleophilic displacement mechanism and the oxocarbenium ion intermediate mechanism are widely known, but recently the former is widely accepted, and so the general tendency of world opinion appears to favor it. This reaction model, however, is considered to comprise some inconsistencies that cannot be neglected from the viewpoint of reactivity in organic chemistry. While the nucleophilic displacement mechanism is often applied to reactions of glycosidases, it appears unlikely that such reactions actually occur. This review argues that the oxocarbenium ion intermediate reaction mechanism is more rational than the nucleophilic displacement reaction mechanism, as the action mode of glycosidases and related enzymes.     

Carotenoids in evolutionary ecology: re‐evaluating the antioxidant role

The antioxidant role of carotenoids in the living organism was proposed as a possible basis for the honesty of carotenoid‐based signals. However, recent studies have questioned the relevance of carotenoids as powerful antioxidants in vivo. Current evidence does not seem to support the “antioxidant role” hypothesis, but it does not allow us to reject it either. This paper proposes some steps to solve this controversy, such as taking a dynamic approach to antioxidant responses, designing protocols that expose individuals to oxidative challenges, analyzing tissues other than blood, and obtaining measures of antioxidant capacity and oxidative damage simultaneously. However, it should be considered that, irrespective of their antioxidant potential, carotenoids might still give information on oxidative stress levels if they are particularly sensitive to free radicals. Finally, lumping together the immunostimulatory and antioxidant roles of carotenoids should be avoided as these functions are not necessarily associated.     

抗氧化系统研究新进展

氧化还原系统主要由活性氧、自由基、活性氧生成系统和抗氧化系统组成。大量的研究表明,氧化还原系统在机体多种生物学功能中发挥关键的调节作用。抗氧化系统主要包括酶类抗氧化剂和非酶类抗氧化剂。抗氧化系统一方面可以通过调节活性氧的水平影响各种生物学功能,另一方面各种酶类抗氧化剂和非酶类抗氧化剂本身也可以参与多种生化反应,调节机体功能。近年来的研究表明,机体内除了典型的抗氧化酶,如超氧化物歧化酶和过氧化氢酶等,还存在多种抗氧化新型抗氧化酶,如硫氧还蛋白、谷氧还蛋白和金属基质蛋白酶等。在本文中,我们将回顾近年来的一些文献,综述抗氧化系统的研究新进展,旨在为抗氧化系统的深入研究提供理论基础。     

Antioxidant activity of carotenoids

Carotenoids are pigments which play a major role in the protection of plants against photooxidative processes. They are efficient antioxidants scavenging singlet molecular oxygen and peroxyl radicals. In the human organism, carotenoids are part of the antioxidant defense system. They interact synergistically with other antioxidants; mixtures of carotenoids are more effective than single compounds. According to their structure most carotenoids exhibit absorption maxima at around 450 nm. Filtering of blue light has been proposed as a mechanism protecting the macula lutea against photooxidative damage. There is increasing evidence from human studies that carotenoids protect the skin against photooxidative damage.     

Effects of antioxidants on X-ray- or hyperthermia-induced apoptosis in human lymphoma U937 cells

Hydroxyl radicals (.OH) and superoxide anion radicals (O2.-) are known to play cardinal roles in cell killing and various types of cell damage. In order to elucidate the mechanism of the involvement of both free radicals on apoptosis, the correlation between anti-apoptotic effects and free radical scavenging abilities of anti-oxidants was studied. As an indicator of anti-apoptotic effects, C1/2 (antioxidant concentration to inhibit DNA fragmentation by 50%) was evaluated in human lymphoma cell line U937 cells 6 hr after X-ray (10 Gy) or hyperthermia (44 degrees C, 30 min) treatment. Rate constants of the reactions between antioxidants and .OH or O2.- were calculated as the scavenging ability of the antioxidants with graded concentration estimated by EPR spectroscopy. No apparent correlation between C1/2 obtained in apoptosis induced by X-rays or hyperthermia and the rate constants of antioxidants for .OH or O2.- was observed. On the other hand, the partition coefficients in 1-octanol/water of the antioxidants, an indicator of hydrophobicity, revealed a correlation with the C1/2 of the agents with hyperthermia, but not with X-ray irradiation. These results indicate that the prevention of apoptosis by an antioxidant is not simply associated with its scavenging ability for .OH or O2.-. The hydrophobicity of the antioxidant, among other possible factors, is involved in the inhibition of hyperthermia- induced apoptosis.     

Influence of vitamin C and vitamin E on redox signaling: Implications for exercise adaptations

The exogenous antioxidants vitamin C (ascorbate) and vitamin E (α-tocopherol) often blunt favorable cell signaling responses to exercise, suggesting that redox signaling contributes to exercise adaptations. Current theories posit that this antioxidant paradigm interferes with redox signaling by attenuating exercise-induced reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. The well-documented in vitro antioxidant actions of ascorbate and α-tocopherol and characterization of the type and source of the ROS/RNS produced during exercise theoretically enable identification of redox-dependent mechanisms responsible for the blunting of favorable cell signaling responses to exercise. This review aimed to apply this reasoning to determine how the aforementioned antioxidants might attenuate exercise-induced ROS/RNS production. The principal outcomes of this analysis are (1) neither antioxidant is likely to attenuate nitric oxide signaling either directly (reaction with nitric oxide) or indirectly (reaction with derivatives, e.g., peroxynitrite); (2) neither antioxidant reacts appreciably with hydrogen peroxide, a key effector of redox signaling; (3) ascorbate but not α-tocopherol has the capacity to attenuate exercise-induced superoxide generation; and (4) alternate mechanisms, namely pro-oxidant side reactions and/or reduction of bioactive oxidized macromolecule adducts, are unlikely to interfere with exercise-induced redox signaling. Out of all the possibilities considered, ascorbate-mediated suppression of superoxide generation with attendant implications for hydrogen peroxide signaling is arguably the most cogent explanation for blunting of favorable cell signaling responses to exercise. However, this mechanism is dependent on ascorbate accumulating at sites rich in NADPH oxidases, principal contributors to contraction-mediated superoxide generation, and outcompeting nitric oxide and superoxide dismutase isoforms. The major conclusions of this review are: (1) direct evidence for interference of ascorbate and α-tocopherol with exercise-induced ROS/RNS production is lacking; (2) theoretical analysis reveals that both antioxidants are unlikely to have a major impact on exercise-induced redox signaling; and (3) it is worth considering alternate redox-independent mechanisms.     

Antioxidant lipoate and tissue antioxidants in aged rats

Oxidative metabolism produces free radicals that must be removed from the cellular environment for the cell to survive. The levels of nonenzymic antioxidants involved in the elimination of free radicals were investigated in an attempt to correlate any changes in the levels of enzymic antioxidants during aging with changes in free radical mediated cellular damage. Antioxidants were measured in liver and kidney of young and aged rats with respect to DL-alpha-lipoic acid supplemented rats. In both organs lipid peroxidation damage (a marker of free radical mediated damage) increased with age, and a significant decrease in antioxidant systems was observed. Moreover, DL-alpha-lipoic acid treated aged rats showed a decrease in the level of lipid peroxides and an increase in the antioxidant status. The results of this study provide evidence that DL-alpha-lipoic acid treatment can improve antioxidants during aging and minimize the age-associated disorders in which free radicals are the major cause.     

Relative importance of the antioxidant and anesthetic properties of propylene phenoxetol in its action as a “preservative” for living holothurians

The local name “sea pudding” for the Bermudan holothurian, Stichopus badionotus, might stem from a certain resemblance to a black pudding but it also now appropriately suggests the softening and degeneration of the body wall that occurs in response to local trauma. In mock cloacal dissections, an antioxidant, propylene phenoxetol, provided effective protection against local degeneration. This observation led to a series of experiments, with standardized local trauma, in which local degeneration of the body wall was retarded by the following antioxidants: ethyl gallate, propylene phenoxetol, propyl gallate, and butylated hydroxyanisole (BHA). Of eight local anesthetics tested, only ethyl ether retarded local degeneration. Nevertheless, it may be thought that the mechanism of local degeneration is susceptible both to antioxidants and to local anesthetics.     

A modulating role for antioxidants in desiccation tolerance

Most organisms depend on the availability of water. However, some life-forms, among them plants and fungi, but very few animals, can survive in the desiccated state. Here we discuss biochemical mechanisms that confer tolerance to desiccation in photosynthetic and non-photosynthetic organisms. We first consider damage caused by water removal and point out that free radicals are a major cause of death in intolerant tissue. Free radicals impair metabolism and necessitate protection and repair during desiccation and rehydration, respectively. As a consequence, desiccation tolerance and prolonged longevity in the desiccated state depend on the ability to scavenge free radicals, using antioxidants such as glutathione, ascorbate, tocopherols and free radical-processing enzymes. Some 'classic' antioxidants may be absent in lower plants and fungi. On the other hand, lichens and seeds often contain secondary phenolic products with antioxidant properties. The major intracellular antioxidant consistently found in all life forms is glutathione, making it essential to survive desiccation. We finally discuss the role of glutathione to act as a signal that initiates programmed cell death. The failure of the antioxidant system during long-term desiccation appears to trigger programmed cell death, causing ageing and eventual death of the organism. In turn, this suggests that a potent antioxidant machinery is one of the underlying mechanisms of desiccation tolerance.     

Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems

Free radicals derived from oxygen, nitrogen and sulphur molecules in the biological system are highly active to react with other molecules due to their unpaired electrons. These radicals are important part of groups of molecules called reactive oxygen/nitrogen species (ROS/RNS), which are produced during cellular metabolism and functional activities and have important roles in cell signalling, apoptosis, gene expression and ion transportation. However, excessive ROS attack bases in nucleic acids, amino acid side chains in proteins and double bonds in unsaturated fatty acids, and cause oxidative stress, which can damage DNA, RNA, proteins and lipids resulting in an increased risk for cardiovascular disease, cancer, autism and other diseases. Intracellular antioxidant enzymes and intake of dietary antioxidants may help to maintain an adequate antioxidant status in the body. In the past decades, new molecular techniques, cell cultures and animal models have been established to study the effects and mechanisms of antioxidants on ROS. The chemical and molecular approaches have been used to study the mechanism and kinetics of antioxidants and to identify new potent antioxidants. Antioxidants can decrease the oxidative damage directly via reacting with free radicals or indirectly by inhibiting the activity or expression of free radical generating enzymes or enhancing the activity or expression of intracellular antioxidant enzymes. The new chemical and cell-free biological system has been applied in dissecting the molecular action of antioxidants. This review focuses on the research approaches that have been used to study oxidative stress and antioxidants in lipid peroxidation, DNA damage, protein modification as well as enzyme activity, with emphasis on the chemical and cell-free biological system.     

Synergistic inhibition of LDL oxidation by phytoestrogens and ascorbic acid

Increasing evidence indicates that oxidative modification of low-density lipoprotein (LDL) is an important determinant in atherogenesis, and following menopause, the incidence of coronary heart disease is as prevalent in women as it is in men. Estrogen has been demonstrated to inhibit the susceptibility of LDL to be oxidized, and more recently the use of phytoestrogens has been considered for estrogen replacement therapy. In this study the antioxidant activity of the three major phytoestrogens: genistein, daidzein, and equol were measured in terms of LDL oxidative susceptibility. Increasing levels of genistein, daidzein, and equol inhibited LDL oxidation, and this inhibitory effect was further enhanced in the presence of ascorbic acid. The synergism exhibited by these compounds is of clinical importance to phytoestrogen therapy since the efficacy of phytoestrogens as effective antioxidants is evident at concentration well within the range found in the plasma of subjects consuming soy products. However, this synergism, combined with the low reactivity of the phytoestrogens with peroxyl radicals, suggests that an antioxidant mechanism other then free radical scavenging reactions account for the phytoestrogen antioxidant effect. A structural basis for inhibition of LDL oxidation involving interaction of the phytoestrogens with apoB-100 is postulated.     

Protection of Cells against Oxidative Stress by Nanomolar Levels of Hydroxyflavones Indicates a New Type of Intracellular Antioxidant Mechanism

Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 µM cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals.     

Can nitroxides evoke the Keap1–Nrf2–ARE pathway in skin?

Nitroxides are stable cyclic radicals of diverse size, charge, and lipophilicity. They are cell-permeative, which effectively protects cells, tissues, isolated organs, and laboratory animals from radical-induced damage. The mechanisms of activity through which nitroxides operate are diverse, including superoxide dismutase-mimetic activity, oxidation of semiquinone radicals, oxidation of reduced metal ions, procatalase-mimetic activity, interruption of radical chain reactions, and indirect modulation of NO levels. Nitroxides possess both a nucleophilic (reducing properties) and an electrophilic (oxidizing properties) nature and, therefore, they may affect different cellular pathways. In the current study, a novel mechanism of action by which nitroxides provide skin protection based on their electrophilic nature is suggested. This study shows that nitroxides may act as electrophiles, directly or indirectly, capable of activating the Keap1–Nrf2–ARE pathway in human keratinocytes (HaCaT) and in human skin (human organ culture model). The high potency of oxoammonium cations versus hydroxylamines in activating the system is demonstrated. The mechanism of action by which nitroxides activate the Keap1–Nrf2–ARE pathway is discussed. Understanding the mechanism of activity may expand the usage of nitroxides as a skin protection strategy against oxidative stress-related conditions.     

Effects of intracellular Mn on the radiation resistance of the halophilic archaeon Halobacterium salinarum

Ionizing radiation (IR) is of particular interest in biology because its exposure results in severe oxidative stress to the cell’s macromolecules. Our recent work with extremophiles supports the idea that IR resistance is most likely achieved by a metabolic route, effected by manganese (Mn) antioxidants. Biochemical analysis of “super-IR resistant” mutants of H. salinarum, evolved over multiple cycles of exposure to high doses of IR, confirmed the key role for Mn antioxidants in the IR resistance of this organism. Analysis of the proteome of H. salinarum “super-IR resistant” mutants revealed increased expression for proteins involved in energy metabolism, replenishing the cell with reducing equivalents depleted by the oxidative stress inflicted by IR. Maintenance of redox homeostasis was also activated by the over-expression of coenzyme biosynthesis pathways involved in redox reactions. We propose that in H. salinarum, increased tolerance to IR is a combination of metabolic regulatory adjustments and the accumulation of Mn-antioxidant complexes.     

The use of cycloamylose to probe the “charge-relay” system

The effect of the combination of imidazolyl and carboxyl groups on the cleavage of m-t-butylphenyl acetate in the presence of α-cyclodextrin was examined to shed light on the role of the “charge-relay” system in serine esterases. 2-Benzimidazole-acetic acid, which has both the imidazolyl and carboxyl groups in the same molecule, accelerates the cleavage of m-t-butylphenyl acetate in the presence of α-cyclodextrin. On the other hand, neither benzimidazole (which has only an imidazolyl group) nor 2-naphthaleneacetic acid (which has only a carboxyl group) exhibited measurable acceleration. The cleavage of m-t-butylphenyl acetate by the α-cyclodextrin-2-benzimidazolecetic acid system takes place through inclusion complex formation between m-t-butylphenyl acetate and α-cyclodextrin, followed by catalysis associated with the combination of the carboxyl anion, the neutral imidazolyl group, and the alkoxide anion. The most probable explanation for the combination of the three groups in the catalysis involves nucleophilic attack by the imidazolyl group, assisted by the carboxyl and alkoxide anions. The mechanism of the combination of the imidazolyl, carboxyl, and hydroxyl groups is apparently different from those shown by the “charge-relay” system in enzymatic reactions.     

The reaction of tertiary amino alcohols with active esters: I. Acylation and deacylation steps

The reactions of triethanolamine and four other tertiary amino alcohols with six active ester substrates were studied in the pH range 6–10 at 30°C. The reaction products were in all cases the respective O-acyl-amino alcohols. Analysis of the effects of substituents in the leaving group as well as in the acyl moiety of the substrates showed that the ester product was formed by direct attack of the nucleophilic hydroxyl group. Comparison with reactions of tertiary amines with the same substrates supports this conclusion. The reactions of tertiary amino alcohols were also compared with those of zwitterionic quaternary amino alcohols and 3-quinuclidinol, a “rigid” tertiary amino alcohol. On the basis of these comparisons, it is proposed that one of the pathways for the predominant effect of the neutral species of tertiary amino alcohols involves intramolecular general base assistance by the tertiary amino group to the nucleophilic attack of the hydroxylic oxygen on the substrate. The contribution of this pathway to the rate of reaction is evaluated.In several systems the first product of the reaction, an O-acyl-amino alcohol, undergoes relatively rapid deacylation, the overall reaction being thus hydrolysis of active esters, catalyzed by the amino alcohol via an acylation-deacylation mechanism.