Biochemistry of free radicals

Joël de Leiris
Laboratoire Stress cardiovasculaire et Pathologies associées,
Faculté de Médecine et Pharmacie, Université Joseph Fourier, Grenoble, France
Correspondence : Dr Joël de Leiris, SCPA, Bâtiment Jean Roget,
Domaine de la Merci, UJF, 38706 La Tronche, France Tel : + 33 (0) 4 76 63 71 17, fax : + 33 (0) 4 76 63 71 52, e-mail : joel.deleiris@ujf-grenoble.fr

Introduction
In the mid 1950s, a small number of scientists first postulated the role of free radicals in the pathophysiological mechanisms of various types of human disease [3]. However, the importance of free radicals in biological systems was not recognized until the early 1970s. Indeed these reactive chemical species are short-lived species and their detection requires the use of sophisticated techniques. Prior to 1975, owing to the technical difficulty of measuring free radical amounts and of quantitating oxidative damage, it was very difficult to prove that free radicals could contribute to cell pathology. In the early 80s, tools such as EPR spectroscopy or fluorescent probes allowed to study free radical biochemistry and to get useful informations about the nature and consequences of free radical-induced protein and lipid oxidation in vitro as well as in vivo.

Reactive oxygen, free radicals and pathology
Today, it is widely accepted that molecular oxygen, although essential for maintaining cell viability in living organisms, may also act as one of the primary factors involved in the pathogenesis of several forms of tissue injury, for instance that occuring when ischemic
tissues are reperfused (‘reperfusion injury’). Nowadays, overproduction of free radicals is considered as a common feature of a large broad of diseases and oxidative stress is generally thought to make a significant contribution to ischemic diseases, drug-induced cardiomyopathy, heart failure, hypertension, inflammatory diseases, cancer, adult respiratory distress syndrome, organ transplantation, neurologic diseases, smoking-related diseases, and AIDS, as well as many others [9]. Moreover oxidative damage is considered to be a major factor in the progressive decline of tissue functions associated with aging [10].
However, in 1987, the discovery of nitric oxide (a nitrogen-derived free radical) and its identification as the endothelium-derived relaxing factor led to the concept that, beyond their toxic effects, free radicals may also play a useful role as second messengers in cellular signal transduction. As such, they are involved in the maintenance of cellular homeostasis and in the communication of the cell with its external milieu [7].
In 1973, the pioneering work of David Hearse and colleagues [4] clearly demonstrated that the reintroduction of oxygen after a period of severe hypoxia in an isolated, buffer-perfused heart preparation, is associated with the occurrence of ‘oxygen paradox’. Oxygen paradox is characterized by the development of a sudden alteration in the integrity of cardiac cells and a definitive loss of myocardial contractile function, associated with severe and irreversible ultrastructural damage. The demonstration that molecular oxygen is involved in the development of such reoxygenation injury (or reperfusion injury) was provided by the observation that if reperfusion of the anoxic heart with an oxygenated buffer enhanced cellular injury, anoxic reperfusion did not produce any extent of tissue injury [5].
In clinical conditions of myocardial ischemia, early reperfusion is imperative for salvage of the remaining viable myocardial cells within the jeopardized area. However, post-ischemic reperfusion is often associated with some specific alterations brought about by oxidative stress upon reflow, such as life-threatening ventricular arrhythmias and persistent contractile dysfunction (myocardial stunning).

Biochemistry of free radicals
The way by which oxygen may exert such deleterious effects is through its reactive forms (reactive oxygen species: ROS, or reactive oxygen metabolites: ROM), including oxygen-derived free radicals as well as nonradical reactive species. Chemically, a free radical is any chemical species (atom, ion, or molecule) containing a single, unpaired electron in its outer orbitals. This single electron confers to the radical a very high reactivity. Oxygen-derived free radicals, including superoxide radical (°O2–) and hydroxyl radical (°OH), or nonradical ROS (hydrogen peroxide H2O2, singlet oxygen 1O2) are partially reduced forms of molecular oxygen (Figure 1).

Figure 1. Univalent reduction of oxygen leading to the formation of ROS.

The reduction of superoxide anion (°O2–) can occur spontaneously or can be catalysed by superoxide dismutase (SOD) or diverse organic SOD-like compounds [1, 11].
The resulting hydrogen peroxide (H2O2) can be: i) divalently reduced by catalase (CAT) or glutathione peroxidase (GSH-Px), ii) divalently reduced by diverse peroxidases producing singlet oxygen (1O2), iii) monovalently reduced to produce hydroxyl radical (°OH) through the Fenton reaction catalysed by transition metals (M) such as iron or copper, or iv) monovalently reduced by reaction with °O2– to produce 1O2.

They are highly unstable and extremely reactive. All cellular components can be attacked by free radicals. Thus, oxygen free radicals react with membrane lipids, especially those containing unsaturated double bonds, leading to the formation of lipid peroxides or hydroperoxides, and aldehydes. Membrane proteins, especially those containing sulfhydryl groups, are also important targets for oxyradicals, leading to marked alterations in cellular ionic homeostasis. Finally, several enzymes are inactivated by ROS, including catalase, glyceraldehyde-3-phosphate dehydrogenase, glutathione peroxidase,adenylate cyclase, myofibrillar ATPase, and creatine kinase [9]. The one-electron reduction product of molecular oxygen is the superoxide radical (superoxide anion °O2–), the half-life of which is 10–9-10–11 sec (10–15 sec in presence of superoxide dismutase) (Table I). Superoxide radical is a requirement for the production of hydroxyl radical °OH. It can be dismutated into hydrogen peroxide H2O2, a nonradical oxygen species (half-life : 10–3 sec ; 10–8 sec in presence of catalase), either spontaneously, or, at a much higher rate, under the catalyzing effect of superoxide dismutases SOD (Figure 1). In mammalian cells, there are two different SODs. These enzymes are metallo-proteins and their metal center is essential for their catalytic activity. Manganese-centered SOD (Mn-SOD) is located in mitochondria, and copper-zinc-centered SOD (Cu/Zn-SOD) in the cytosol. Hydrogen peroxide is a strong oxidant which is able to interact slowly with most organic substrates. In presence of transition metals such as iron or copper (Table I), hydrogen peroxide can oxidize superoxide radical at rapid rates (Fenton reaction) to produce hydroxyl radical °OH, the most highly reactive oxidant, which, unlike superoxide radical or hydrogen peroxide, is reactive with most biological substrates.

Table I. Formation of oxygen-derived free radicals and reactive species.
(Production and dismutation of superoxide radical, Fenton and Haber-Weiss reactions).

Due to its high reactivity, hydroxyl radical immediately reacts with surrounding target molecules at the site where it is generated. The Fenton reaction is the major source of hydroxyl radicals under physiological conditions. Alternatively, °OH can be generated through the Haber-Weiss reaction (Table I) when a superoxide radical and a H2O2 molecule spontaneously combine to form molecular oxygen and two hydroxyl radicals [2]. Moreover, °OH can be generated through a metal-independent pathway involving the interaction of superoxide radical and nitric oxide.
Singlet oxygen (1O2) is formed if one of the unpaired electrons of molecular oxygen absorbs energy to undergo spin inversion and orbital transition. Its half-life is approximately 2.10–6 sec. Intracellular generation of 1O2 may take place as a reaction product of hypochlorite and H2O2, dismutation of °O2–, or Haber-Weiss reaction. Altough there is currently no satisfying way to provide direct evidence of 1O2 formation in vivo, recent experimental studies have suggested that singlet oxygen might play an important role in myocardial ischemia-reperfusion injury [13].

Sources of oxygen radicals
In normal aerobic tissue, there is a continuous generation of oxygen radicals, which are detoxified by the endogenous antioxidants. Approximately 2% to 5% of the electron flow through the electron transport chain during normal mitochondrial respiration is subjected to partial univalent reduction of molecular oxygen, producing ROS including superoxide radical. In such conditions, Fenton reaction appears to be the major source of hydroxyl radical [2]. In various pathological conditions, for instance upon reperfusion of the ischemic myocardium, the activity of cellular antioxidant enzymes is considerably reduced, whereas the rate of oxyradical production is greatly increased, due to reintroduction of molecular oxygen [11]. Such an imbalance between production of ROS and antioxidant defence can result in oxidative stress. Under these conditions, the most important sources of oxyradical generation are mitochondrial electron transport system (univalent reduction of molecular oxygen, NADH dehydrogenase complex) (Figure 1), endothelial cells (xanthine oxidase reaction), inflammatory cells (myeloperoxidase, NADPH oxidase), catecholamine oxidation, and metabolism of arachidonic acid (Table II) [11].

Table II. Formation of oxygen-derived free radicals and reactive species through reactions catalyzed by xanthine oxidase, myeloperoxidase, and NADPH oxidase.

Conclusion
During the last decade, the terms ‘free radicals’, ‘oxidative stress’, and ‘antioxidants’ have become commonly used to discuss the cellular mechanisms of an increasing number of human diseases even though there are still many gaps in our understanding of the role of free radicals in the pathogenesis of such diseases. Available evidence from animal and human studies illustrate that antioxidant reserve might well be an important factor in promoting tissue protection against oxidative stress-mediated injury. Antioxidant enzyme mimics (mostly SOD mimics) or catalytic drugs hold much promise for treating conditions in which the damaging oxidant molecule is continuously overproduced following an insult such as myocardial ischemia followed by reperfusion [1, 6, 12]. Recently, the research in the area of SOD mimics has been particularly fascinating because the discovery of these new catalysts has evolved in parallel to greater understanding of the biological role of ROS in general and superoxide radical in particular. It is clear that antioxidants are not a panacea for treating or preventing aging and diseases, but the importance of certain dietary antioxidants in preventing life-threatening diseases, such as heart disease and certain types of cancer, is still debated. Finally, if antioxidant therapy may be of some interest for limiting the damage caused by ROS, an important question now arises regarding their physiological role in biological signaling.

References
1. Gershman R, Gilbert DL, Nye SW, Dwyer P, Fenn WO. Oxygen poisoning and X-irradiation: a mechanism in common. Science. 1954;119:623–626.

 
The evolution of free radicals and oxidative stress.

McCord JM.

Webb-Waring Institute, University of Colorado Health Sciences Center, Denver, Colorado, USA.

The superoxide free radical has come to occupy an amazingly central role in a wide variety of diseases. Our metabolic focus on aerobic energy metabolism in all cell types, coupled with some chemical peculiarities of the oxygen molecule itself, contribute to the phenomenon. Superoxide is not, as we once thought, just a toxic but unavoidable byproduct of oxygen metabolism. Rather it appears to be a carefully regulated metabolite capable of signaling and communicating important information to the cell's genetic machinery. Redox regulation of gene expression by superoxide and other related oxidants and antioxidants is beginning to unfold as a vital mechanism in health and disease.

Publication Types:

• Review
• Review, Tutorial

PMID: 10856414 [PubMed - indexed for MEDLINE]

 
Reactive oxygen species and protein oxidation in aging: a look back, a look ahead.

Hensley K, Floyd RA.

Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA. Kenneth-Hensley@omrf.ouhsc.edu

The existence of free radicals, as chemical entities, was inferred 100 years ago but not universally accepted for some 30-40 years. The existence and importance of free radicals in biological systems was not recognized until the mid 1950s, by a small number of visionary scientists who can be credited with founding the field of reactive oxygen biochemistry. For most of the remaining 20th century, reactive oxygen species (ROS) were considered a type of biochemical "rusting agent" that caused stochastic tissue damage and disease. As we enter the 21st century, reactive oxygen biochemistry is maturing as a discipline and establishing its importance among the biomedical sciences. It is now recognized that virtually every disease state involves some degree of oxidative stress. Moreover, we are now beginning to recognize that ROS are produced in a well-regulated manner to help maintain homeostasis on the cellular level in normal, healthy tissue. This review summarizes the history of reactive oxygen biochemistry, outlining major paradigm shifts that the field has undergone and continues to experience. The contributions of Earl Stadtman to the recent history of the field (1980-present) are especially highlighted. The role of ROS in signal transduction is presented in some detail as central to the latest paradigm shift. Emerging technologies, particularly proteomic technologies, are discussed that will facilitate further evolution in the field of reactive oxygen biochemistry. (c)2001 Elsevier Science.

Publication Types:

• Review
• Review, Tutorial

PMID: 11795897 [PubMed - indexed for MEDLINE]

 
Effect of antioxidant trace elements on the response of cardiac tissue to oxidative stress.

Barandier C, Tanguy S, Pucheu S, Boucher F, De Leiris J.

Groupe de Physiopathologie Cellulaire Cardiaque, Universite Joseph Fourier, Grenoble, France.

It is now well established that several trace elements, because of their involvement in the catalytic activity and spatial conformation of antioxidant enzymes, may contribute to the prevention of oxidative stress such as occurs upon reperfusion of ischemic tissue. The aim of this paper is (1) to review the role of these trace elements (Cu, Mn, Se, and Zn) in antioxidant cellular defenses in the course of post-ischemic reperfusion of cardiac tissue, (2) to provide experimental data suggesting that variations in trace element dietary intake may modulate the vulnerability of cardiac tissue to ischemia-reperfusion, and (3) to discuss in more detail the effect of Mn ions, which seem to play a special protective role against reperfusion injury. Some results obtained from experiments in animal models of myocardial reperfusion have shown that the dietary intake of such trace elements can modulate cardiac activity of antioxidant enzymes and, consequently, the degree of reperfusion damage. In addition, experimental data on the protective effects of an acute treatment with Mn are presented. Finally, experimental evidence on the protective role of salen-Mn complexes, which exhibit catalytic SOD- and CAT-like activities against reperfusion injury, are described. These complexes should be of considerable interest in clinical conditions.

Publication Types:

• Review
• Review, Tutorial

PMID: 10415528 [PubMed - indexed for MEDLINE]

 9. Lucchesi BR. Free radicals and tissue injury. Dialogues Cardiovasc Med. 1998;3:3–22.

 
Cardiac toxicity of singlet oxygen: implication in reperfusion injury.

Toufektsian MC, Boucher FR, Tanguy S, Morel S, de Leiris JG.

Laboratoire Stress Cardiovasculaires et Pathologies Associees, Universite Joseph Fourier, Batiment Jean Roget, Domaine de la Merci, Grenoble, France.

Oxygen-derived free radicals (O2.-, H2O2, and .OH) that are produced during postischemic reperfusion are currently suspected to be involved in the pathogenesis of tissue injury. Another reactive oxygen species, the electronically excited molecular oxygen (1O2), is of increasing interest in the area of experimental research in cardiology. In this review are discussed the main potential sources of singlet oxygen in the organism, particularly in the myocardium, the various cardiovascular cytotoxic effects induced by this reactive oxygen intermediate, and the growing evidence of its involvement in ischemia/reperfusion injury.

Publication Types:

• Review
• Review, Tutorial

PMID: 11291599 [PubMed - indexed for MEDLINE]

12. Henke SL. Superoxide dismutase mimics as future therapeutics. Exp Opin Ther Patents. 1999;9:169–180.

 
Cytoprotection against oxidative stress in rat isolated cardiomyocytes: effect of EUK 8, a nonprotein catalytic antioxidant.

Tanguy S, Boucher F, Besse S, de Leiris J.

Publication Types:

• Letter

PMID: 9825180 [PubMed - indexed for MEDLINE]