Pathological Roles of Reactive Oxygen Species and Their Defence Mechanisms

ion, hydrogen addition or electron transfer, while superoxide anion reacts via only hydrogen abstraction and electron transfer (Section 1.2) (5). Although, hydrogen peroxide and singlet oxygen are classified as ROS, they are not radicals and do not necessarily interact with tissue through radical reactions directly (6). Hence, they are not toxic per se but in presence of free radicals or catalysts (e.g. iron and copper) more reactive intermediates can be produced. Transition metals, such as iron and copper, which are found at the active site of many oxidase enzymes, e.g. cytochrome oxidase, have the ability to facilitate the transfer of single electrons to molecular oxygen to form free radicals (7). ROS cause damage to cellular macromolecules. This is known as oxidative stress or "oxygen toxicity" (for more details see Sections 2.3-2.6). 1.1. Formation of ROS from molecular oxygen: Reactive oxygen species are formed by the one or two electron reduction of molecular oxygen (8). The addition of one electron to molecular oxygen produces the superoxide anion while the addition of two electrons yields the peroxide ion (O2), which in biological systems is protonated to give hydrogen peroxide. Hydrogen peroxide can then react with superoxide anion and ferric or cupric ions in the Haber-Weiss reaction, to produce the highly reactive hydroxyl radical (Figure 1). In the first step of this reaction, superoxide anion reduces ferric iron (Fe III) to ferrous iron (Fe II) or cupric (Cu II) to cuprous (Cu I), which in turn reduces hydrogen peroxide to form a hydroxyl radical and hydroxyl ion (9). The reduced metal ion undergoes re-oxidation during the second stage. The hydroxyl radical is the most active ROS (10). This arises from a much higher reduction potential in comparison to other ROS. Its activity is minimized by removal of hydrogen peroxide and transition metals from the cell. As a result of its reactivity, the hydroxyl radical does not travel far and has a half-life of a few microseconds (10). However, hydrogen peroxide can cross cell membranes almost as readily as water while the PATHOLOGICAL ROLES OF REACTIVE OXYGEN SPECIES 3 Saudi Pharmaceutical Journal, Vol. 12, No. 1 January 2004 charged superoxide anion molecule can cross membranes only via transmembrane anion channels (11). Therefore, the presence of trace amounts of the transition metal ions Fe(III) or Cu(II) is a significant influence on the production of hydroxyl radicals in biological systems. 1.2. Mechanisms of ROS toxicity: When free radicals react with a non-radical, other free radicals can be formed. This enables free radicals to induce chain reactions that may be thousands of events long; e.g. hydroxyl radical induces lipid peroxidation of polyunsaturated fatty acids via hydrogen abstraction (12). In addition, the reaction of hydroxyl radicals with aromatic compounds, such as the purine base, guanine in DNA, is processed via hydrogen addition (9). Thus ROS can act as both oxidant and reducing agents. Although the initial free radical produces only local effects, secondary radicals and degradation products can have biological effects distant from the site where the first free radical was formed. However, when two free radicals react with each other, a stable molecule may be formed (13). This explains the eventual termination of free radical-induced chain reactions. The superoxide anion is potentially toxic. It may directly influence local homeostasis by oxidizing catecholamines, or it can be transformed into the hydroxyl radical via the Haber-Weiss reaction (Figure 1) (1). In contrast, hydrogen peroxide, per se, is not especially toxic to the cell macromolecules, but it can cross cellular membranes and this feature is potentially important because the extracellular environment possesses few antioxidant defense mechanisms. In the presence of low concentrations of transition metal ions, hydroxyl radicals are formed from hydrogen peroxide, via the Fenton reaction (Figure 2). Alternatively, hydrogen peroxide can interact with superoxide anion to produce the hydroxyl radical, by Haber-Weiss type reactions (Figure 1). Due to the charged nature of superoxide anion it is more concentrated in the intracellular compartment. As a result, hydroxyl radicals are produced predominantly from hydrogen peroxide by Haber-Weiss reactions in the intracellular compartment whereas the Fenton reaction is more important in extracellular compartments (14). 1.3. Sources of ROS: Reactive oxygen species can be produced by a number of enzymatic reactions including oxidases and cytochrome P450s (15, 16). However, they are also produced non-enzymatically, often through redox cycling (17). Redox cycling is the cyclic reduction-oxidation of drugs or other xenobiotics, which can generate ROS. Several xenobiotics, such as adriamycin (a quinone derivative), are capable of redox cycling (Figure 3) (5). As shown in figure 3, quinones can undergo a one-electron reduction reaction catalyzed by an enzyme such as the microsomal flavoprotein NADPH-cytochrome P450 reductase, resulting in the formation of the semiquinone free radical. This semiquinone metabolite is unstable in the presence of oxygen and is rapidly reoxidized to form the superoxide anion radical. In this process the parent quinone is regenerated leading to redox cycling of the quinone-semiquinone. The superoxide anion undergoes spontaneous or enzymatic dismutation to form hydrogen peroxide. Alternatively, in the presence of catalyst metals, excess superoxide anion can react with hydrogen peroxide to form hydroxyl radicals by a Haber-Weiss type reaction (Figure 1). Redox cycling also plays an important role in the toxicity of nitrofurantoin and bipyridyl compounds such as paraquat (4). In addition, redox cycling can induce lipid peroxidation and therefore, alterations in membrane permeability (12). Adriamycin and other anthracycline anticancer drugs can induce in vivo cardiac toxicity, which is associated with peroxidation of cardiac lipids (12). It has been proposed that this side effect is associated with hydroxyl radical formation, which is generated by redox cycling in the presence of catalyst metals (Figure 3b). Xanthine oxidase generates superoxide anion and hydrogen peroxide during the re-oxidation of the enzyme with molecular oxygen (18, 19). Consequently, this enzyme is widely used to generate ROS in vitro (20). However, it has been reported that human xanthine oxidase serves as an NAD dependent-dehydrogenase in vivo with minimal oxygen-reductase activity (21, 22). More recently, it has been shown that the dehydrogenase form may react slowly with oxygen to produce superoxide anion, although this activity is inhibited by NAD (23). Hence, the importance of xanthine dehydrogenase in vivo as a source of ROS is not