Mechanisms of superoxide signaling in epigenetic processes: relation to aging and cancer.

Superoxide is a precursor of many free radicals and reactive oxygen species (ROS) in biological systems. It has been shown that superoxide regulates major epigenetic processes of DNA methylation, histone methylation, and histone acetylation. We suggested that superoxide, being a radical anion and a strong nucleophile, could participate in DNA methylation and histone methylation and acetylation through mechanism of nucleophilic substitution and free radical abstraction. In nucleophilic reactions superoxide is able to neutralize positive charges of methyl donors S-adenosyl-L-methionine (SAM) and acetyl-coenzyme A (AcCoA) enhancing their nucleophilic capacity or to deprotonate cytosine. In the reversed free radical reactions of demethylation and deacetylation superoxide is formed catalytically by the (Tet) family of dioxygenates and converted into the iron form of hydroxyl radical with subsequent oxidation and final eradication of methyl substituents. Double role of superoxide in these epigenetic processes might be of importance for understanding of ROS effects under physiological and pathological conditions including cancer and aging.

New nucleophilic mechanisms of ros-dependent epigenetic modifications: comparison of aging and cancer.

It has been shown that ROS (reactive oxygen species, superoxide and hydrogen peroxide) regulate major epigenetic processes, DNA methylation and histone acetylation, although the mechanism of ROS action (ROS signaling) is still unknown. Both DNA methylation and histone acetylation are nucleophilic processes and therefore ROS signaling through typical free radical processes, for example hydrogen atom abstraction is impossible. However, being "super-nucleophile" superoxide can participate in these reactions. Now we propose new nucleophilic mechanisms of DNA methylation and histone modification. During DNA methylation superoxide can deprotonate the cytosine molecule at C-5 position and by this accelerate the reaction of DNA with the positive-charged intermediate S-adenosyl-L-methionine (SAM). Superoxide can also deprotonate histone N-terminal tail lysines and accelerate the formation of their complexes with acetyl-coenzyme A (AcCoA), the supplier of acetyl groups. In cancer cells ROS enhance DNA methylation causing the silencing of tumor suppressor and antioxidant genes and enhancing the proliferation of cancer cells under condition of oxidative stress. ROS signaling in senescent cells probably causes DNA hypomethylation although there are insufficient data for such proposal.

ROS and RNS signaling in heart disorders: could antioxidant treatment be successful?

There is not too much success in the antioxidant treatment of heart deceases in humans. However a new approach is now developed that suggests that depending on their structures and concentrations antioxidants can exhibit much more complicated functions in many pathological disorders. It is now well established that physiological free radicals superoxide and nitric oxide together with their derivatives hydrogen peroxide and peroxynitrite (all are named reactive oxygen species (ROS) and reactive nitrogen species (RNS)) play a more important role in heart diseases through their signaling functions. Correspondingly this work is dedicated to the consideration of damaging signaling by ROS and RNS in various heart and vascular disorders: heart failure (congestive heart failure or CHF), left ventricular hypertrophy (LVH), coronary heart disease, cardiac arrhythmias, and so forth. It will be demonstrated that ROS overproduction (oxidative stress) is a main origin of the transformation of normal physiological signaling processes into the damaging ones. Furthermore the favorable effects of low/moderate oxidative stress through preconditioning mechanisms in ischemia/reperfusion will be considered. And in the last part we will discuss the possibility of efficient application of antioxidants and enzyme/gene inhibitors for the regulation of damaging ROS signaling in heart disorders.

Reactive oxygen species signaling in cancer: comparison with aging.

This work considers reactive oxygen species (ROS) signaling in solid tumors. Most (probably all) cancer cells are characterized by ROS overproduction that is they exist under conditions of incessant oxidative stress. For example ROS overproduction has been shown in prostate, pancreatic, melanoma, and glioma cells. ROS overproduction has been also demonstrated in breast, liver, bladder, colon, and ovarian cancers. Although these examples probably do not incorporate all the described data concerning ROS overproduction in cancer cells, they clearly support a proposal about enhanced oxidative stress in these cells. Therefore the mechanisms of ROS signaling in the survival and death of cancer cells and comparison with ROS signaling in senescent cells ought to be considered. It might be suggested that ROS overproduction in cancer cells is a major origin of their survival and resistance to anticancer treatment while the enhanced oxidative stress responsible for aging development. However it is of particular interest that additional ROS production by prooxidants can induce apoptosis in cancer cells. We suggest that moderate oxidative stress can stimulate proliferation and survival of cancer sells by conditioning mechanism while the enhancement of ROS overproduction by prooxidants under severe oxidative stress results in apoptosis and cell death. Aging development is always characterized by harmful ROS overproduction although the moderate increase in ROS formation in senescent cells might be not dangerous. Similar double-edged sword effects of ROS might be observed during the development of other pathologies for example diabetes mellitus.

Reactive oxygen species and age-related genes p66shc, Sirtuin, FOX03 and Klotho in senescence.

Reactive oxygen species (ROS) superoxide and hydrogen peroxide perform important signaling functions in many physiological and pathophysiological processes. Cell senescence and organismal age are not exemptions.Aging-regulating genes p66shc, Sirtuin, FOXO3a and Klotho are new important factors which are stimulated by ROS signaling. It has been shown that ROS participate in initiation and prolongation of gene-dependent aging development.ROS also participate in the activation of protein kinases Akt/PKB and extracellular signal-regulated kinase ERK, which by themselves or through gene activation stimulates or retards cell senescence.Different retarding/stimulating effects of ROS might depend on the nature of signaling species-superoxide or hydrogen peroxide. Importance of radical anion superoxide as a signaling molecule with"super-nucleophilic" properties points to the possibility of the use of superoxide scavengers (SOD mimetics, ubiquinones and flavonoids) for retarding the development of aging.

Signaling by reactive oxygen and nitrogen species in skin diseases.

For many years the formation of reactive oxygen and nitrogen species (ROS) and (RNS) in living organisms has been considered to be dangerous phenomenon due to their damaging action on biomolecules. However, present studies demonstrated another important activity of ROS and RNS: their signaling functions in physiological and pathological processes. In this work we discuss the new data concerning a role of ROS and RNS in many enzymatic/gene cascades causing damaging changes during the development of skin diseases and pathological disorders (skin cancer, the toxic effects of irradiation on the skin, and skin wounding). It has been suggested that the enhancement of ROS formation in tumor cells through the inactivation of mitochondrial MnSOD or the activation of NADPH oxidase leads to apoptosis and might be applied for developing a new cancer therapy. On the other hand ROS overproduction might stimulate malignant transformation of melanoma. Role of ROS signaling is also considered in the damaging action of UVA, UVB, and IRA irradiation on the skin and the processes of wound healing. In the last part of review the possibility of the right choice of antioxidants and free radical scavengers for the treatment of skin disease is discussed.

Signaling and Damaging Functions of Free Radicals in Aging-Free Radical Theory, Hormesis, and TOR.

Harman's Free Radical Theory of Aging has been considered as a major theory of aging for more than 50 years. In 1956 Dr. Harman proposed that the accumulation of free radicals with the age causes the damage of biomolecules by these reactive species and the development of pathological disorders resulting in cell senescence and organismal aging. His hypothesis was supported by numerous experimental studies demonstrated an increase in free radical levels in cells and living organisms with aging. In subsequent years important discoveries of new physiological free radicals superoxide and nitric oxide have been made that led to understanding of other important functions of free radicals. It has been shown that superoxide and nitric oxide together with their diamagnetic reaction products hydrogen peroxide and peroxynitrite (all are now named reactive oxygen and nitrogen species, ROS and RNS) function as signaling species in many physiological enzymatic/gene processes. Furthermore, the disturbance of ROS and RNS physiological signaling can be an origin of various pathologies and aging. These discoveries demanded to widen original free radical theory of aging and to consider the damaging ROS signaling as an important, maybe major route to cell senescence and organismal aging. However, some experimental findings such as the extension of lifespan by calorie restriction of yeast, flies, worms, and mice, and favorable effects of physical exercises stimulated criticism of free radical theory because the expansion of lifespan accompanied in some cases by increasing oxidative stress. On these grounds such theories as Hormesis and Target of rapamycin (mTOR) theories refute the role of ROS and oxidative stress in aging. Accordingly, a major purpose of this review to show that ROS signaling is probably the most important enzyme/gene pathway responsible for the development of cell senescence and organismal aging and that ROS signaling might be considered as further development of free radical theory of aging. In spite of apparent contradictions the Hormesis or TOR theories are also describing processes of aging development regulated by ROS signaling.

Signaling of reactive oxygen and nitrogen species in Diabetes mellitus.

Disorder of physiological signaling functions of reactive oxygen species (ROS) superoxide and hydrogen peroxide and reactive nitrogen species (RNS) nitric oxide and peroxynitrite is an important feature of diabetes mellitus type 1 and type 2. It is now known that hyperglycemic conditions of cells are associated with the enhanced levels of ROS mainly generated by mitochondria and NADPH oxidase. It has been established that ROS stimulate many enzymatic cascades under normal physiological conditions, but hyperglycemia causes ROS overproduction and the deregulation of ROS signaling pathways initiating the development of diabetes mellitus. On the other hand the deregulation of RNS signaling leads basically to a decrease in NO formation with subsequent damaging disorders. In the present work we will consider the pathological changes of ROS and RNS signaling in enzyme/gene regulated processes catalyzed by protein kinases C and B (Akt/B), phosphatidylinositol 3'-kinase (PI3-kinase), extracellular signal-regulated kinase 1/2 (ERK1/2), and some others. Furthermore we will discuss a particularly important role of several ROS-regulated genes and adapter proteins such as the p66shc, FOXO3a, and Sirt2. The effects of low and high ROS levels in diabetes will be also considered. Thus the regulation of damaging ROS levels in diabetes by antioxidants and free radical scavengers must be one of promising treatment of this disease, however, because of the inability of traditional antioxidative vitamin E and C to interact with superoxide and hydrogen peroxide, new free radical scavengers such as flavonoids, quinones and synthetic mimetics of superoxide dismutase (SOD) should be intensively studied.

Superoxide and nitric oxide in senescence and aging.

In this review some aspects of free radical theory of aging are discussed. Many new and interesting findings concerning the role of physiological free radicals superoxide and nitric oxide in senescence and aging development are considered and the mechanisms of processes mediated by these radicals are discussed. It has been known for a long time that being themselves mostly harmless species, superoxide and NO are precursors of really reactive species hydroxyl radicals and peroxynitrite, the initiators of aging and various pathologies. However, contemporary studies demonstrate the other maybe more important ways of damaging activity of physiological free radicals. Numerous studies show that lessening of NO production and its bioavailability could be a starting point of aging development. It results in a decrease in NO inhibition of mitochondrial cytochrome c oxidase and an increase in dioxygen consumption. That in its turn leads to an increase in the production of superoxide and the other reactive oxygen and nitrogen species and initiation of apoptosis, In conclusion the possibilities of pharmacological intervention with antioxidants and other antiradical procedures to suppress aging and senescence or even to expand the life span of animals are considered.

Signaling functions of free radicals superoxide & nitric oxide under physiological & pathological conditions.

Superoxide and nitric oxide are ubiquitous physiological free radicals that are responsible for many pathological disorders. Both radicals by themselves are relatively harmless but are the precursors of many toxic species such as peroxy and hydroxyl radicals, hydrogen peroxide, and peroxynitrite. However, it has been shown now that both superoxide and nitric oxide are also able to perform important signaling functions in physiological and pathophysiological processes. Wrongly named "superoxide," the radical anion of dioxygen is not a super-oxidant but the strong super-nucleophile, an efficient catalyst of heterogenic nucleophilic reaction. Due to this, superoxide plays an important role in many enzymatic processes such as the phosphorylation and activation of numerous protein kinases. On the other hand, superoxide inhibits the activation of phosphatases, the enzymes catalyzed by dephosphorylation of protein kinases. We suggest that superoxide catalyzes these enzymatic processes as a result of its nucleophilic properties. Another important physiological function of superoxide and nitric oxide is their competition for the interaction with mitochondrial cytochrome c oxidase. Disturbance of superoxide/nitric oxide balance leads to the dysfunction of mitochondria and the enhancement of apoptosis and oxidative stress, which are primary causes of various pathological disorders and aging. In conclusion, interplay between superoxide and nitric oxide, one of major factors of aging development, is considered.

Interplay between superoxide and nitric oxide in thalassemia and Fanconi's anemia.

Thalassemia (thal) and Fanconi's Anemia (FA) are genetic disorders associated with iron-catalyzed free radical damage. Therefore, the contemporary and most successful treatment of thalassemic patients depends on the application of iron (Fe) chelators. However, there is another pathway of free radical-mediated damaging processes in these pathologies, depending on the interplay between physiological free radicals superoxide and nitric oxide (NO). In the present study, we have considered the major routes of superoxide damaging effects in mitochondria: the initiation of apoptosis through the reduction of cytochrome c, the activation of uncoupled proteins by superoxide, and the mitochondrial damage due to the competition between superoxide and nitric oxide at the Complex IV site (cytochrome oxidase). The application of the effective scavengers superoxide dismutases and flavonoids for the treatment of thalassemic and FA patients, is discussed.

Free radical mechanisms of aging processes under physiological conditions.

Free radical theory of aging predicts crucial role for free radicals produced by external factors (environmental contamination, irradiation, etc.) or pathological disorders (hereditary diseases or infections) in the initiation of aging. Does it mean that under hypothetical completely physiological conditions aging processes could be fully suppressed? To answer this question, we will consider the possible mechanisms of free radical formation in such hypothetical state. There are two major mechanisms, which are responsible for free radical-mediated damage in a living organism: superoxide overproduction by mitochondria and nonenzymatic lipid peroxidation. Superoxide overproduction causes the inhibition of nitric oxide formation and bioavailability, one of principal characteristics of aging, while nonenzymatic lipid peroxidation, which is already demonstrated at physiological conditions, produces toxic isoprostanes. We suggest that major initiators of free radical-mediated damaging processes leading to aging at physiological state are oxidizable components of diet. The possibility of inhibition of aging processes by supplementation of nontoxic antioxidants and calorie restriction is discussed. Scheme demonstrating the potential mechanisms of starting the free radical-mediated aging processes is presented, which are discussed on the grounds of known literature data.

Interplay between superoxide and nitric oxide in aging and diseases.

Free radicals, superoxide and nitric oxide, are important signaling molecules, which mediate numerous physiological functions (phagocytosis, vasorelaxation, etc.). However, regulation errors may lead to free-radical-mediated damaging processes in cells and tissues. In this work, the effects of an interplay between superoxide and NO, which may be responsible for the development of aging and diseases, are considered. We are suggesting that the superoxide-mediated proton leak leading to the inhibition of oxidative phosphorylation and the competition between NO and O2*- in their reactions with cytochrome oxidase can be a cause of mitochondrial aging.

Detection of superoxide in vascular tissue.

During the past decade, it has become apparent that reactive oxygen species play a critical role in the genesis of many vascular diseases. The superoxide anion is among the most important of these, not only because of its rapid reaction with NO but also because it serves as a progenitor for many other reactive oxygen species. Although there are many approaches to detecting and quantifying superoxide in chemical systems, its detection in intact tissues is more difficult. The validity of the most popular and frequently used assay for this purpose, lucigenin-enhanced chemiluminescence, has been recently questioned. It has been suggested that lucigenin itself, especially at high concentrations (>50 micromol/L), may act as a source for superoxide via redox cycling. Lower lucigenin concentrations (5 micromol/L) do not participate in redox cycling to an important extent in intact tissues and, therefore, provide an accurate assessment of the rate of superoxide production in such samples. Other useful assays for superoxide include those using the fluorescent dye dihydroethidine, 2-methyl-6-phenyl-3,7-dihydroimidazo(1,2-alpha)pyrazin-3-one (CLA), and 2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl) 8-benzylimidazo[1,2-alpha]pyrazin-3-one (coelenterazine). The chemiluminescent compound 5-amino-2,3-dihydroxy-1,4-phthalayineidone (luminol) may also be used to detect various reactive oxygen species and may be made specific for various oxidants, such as hydrogen peroxide, superoxide, and peroxynitrite, by altering the experimental conditions. Although each of these methods may be associated with potential artifacts, the use of > or =2 different techniques that yield similar results provides a reliable approach for the study of reactive oxygen species in intact vascular tissues.