A

Singlet oxygen

NADPH NADP+

myeloperoxidase Fe2+ Fe3+

NADPH oxidase ^ _J SOD Hydrogen peroxide

Hydroxyl radical no l [> OONO c

Nitric Peroxynitrite oxide anion

Figure 7.3 Biological mechanisms for ROS formation.

Endogenous sources

Mitochondria Peroxisomes Lipoxygenases NADPH-oxidase Cytochrome P450

Antioxidant defenses

Catalase,SOD, Glutahione Peroxidase, Thioredoxin Reductase

Glutathione, Ascorbate, Tocopherol

Exogenous sources

Ultra violet Ionizing radiation Environmental toxin Chemotherapeutics Inflammatory cytokines

Decreased proliferative response Defective host defenses

Impaired physiological function

Reactive Oxygen Species (ROS) Reactive Nitrogen Species (RNS)

(Superoxide, peroxide, singlet oxygen, hydroxyl radical, NO, OONO)

Homeostasis

Normal growth and metabolism

Impaired physiological function

Random cellular damage

Specific signaling pathways

Ageing Disease Cell death

Figure 7.4 Reactive oxygen species mediated pathogenecity.

oxidant (56,57). The oxidants derived from NO are often referred to as reactive nitrogen species (RNS) (Figure 7.3).

Reactive oxygen species and reactive nitrogenspecies (RNS) are constantly produced in aerobic organisms both enzymatically and non-enzymatically. Many sources for the formation of ROS have been identified in cellular systems of living organisms (58,59). Superoxide is formed upon the one electron reduction of oxygen mediated by enzymes such as NADPH oxidase located on the cell membrane of polymorphonuclear cells, macrophages, and endothelial cells (60-62); from xanthine oxidase or from the respiratory chain (63). Superoxide radicals can also be formed from the electron cycling carried out by the cytochrome P450 dependent enzymes (64,65). Mitochondrial leakage of electrons or direct transfer of electron to oxygen via coenzymes or prosthetic groups such as flavins and Fe-S centers constitute another important source of ROS in cellular systems (58,59). The homolytic cleavage of water, or the breakdown of hydrogen peroxide in a high energy radiation (e.g., x-rays, UV) or metal catalyzed process, forms the hydroxyl radical which is the most reactive oxygen species (Figure 7.3, Figure 7.4). Another ROS, the hypochlorite ion, is formed by the macrophage myeloperoxidase, or related peroxidase activities, which catalyze the halide driven reduction of hydrogen peroxide to form the oxidant hypochlorous acid (HOCl) to kill invading microorganisms (63). Biological conversion of l-arginine to l-citrulline by nitric oxide synthase forms a cytotoxic oxygen species, nitric oxide (NO), which is involved in the cellular defense against malignant cells, invading fungi, and protozoa (56,57). Nitric oxide is also involved critically in signaling mechanisms in the vasculature controlling the vascular force in the blood vessels by vasodilation and inflammation (66).

Reactive oxygen species are ideally suited to be signaling molecules, as they are small, can diffuse small distances, and have several mechanisms of production that can be controlled and regulated. Low levels of ROS therefore have been implicated in many cellular processes, including intracellular signaling responsible for proliferation or apoptosis (67), for modulation of immune response (68), and for mounting a defense response against pathogens (69) (Figure 7.4). Even though the exact mechanism of action of ROS in effecting these physiological processes is not very well understood, there is growing evidence that ROS at some level are capable of activating or repressing many biological effector molecules (70). It has been shown to activate or repress transcription factors by directly activating them by oxidizing the sulphydryl groups present, or by modulating a complex array of kinases and phosphatases, which are important in signal transduction. Many ions that maintain the electrostatic balance of the cell required for many physiological processes such as cell growth and cell death are also now shown to be regulated by ROS. Another important mechanism by which ROS are now shown to be regulating the cell function is by altering the redox status of the cell, often by regulating the levels of oxidized and reduced glutathione (GSH/GSSG) (71).

7.2.2.1 Antioxidant Defense Systems

Redox regulated physiological processes are inevitably sensitive against excessive ROS production by any source. Such excessive levels of ROS may be generated either by overstimulation of the otherwise tightly regulated NAD(P)H oxidases or by other mechanisms that produce ROS in a nonregulated fashion, including the production of ROS by the mitochondrial electronic transport chain (ETC) or by xanthine oxidase. Diets rich in saturated fatty acids, and carbohydrates; and environmental factors such as exposure to high energy radiation, and ingestion of or exposure to toxins and carcinogens can also overstimulate metabolic systems resulting in the formation of ROS, which are beyond the cellular need for their regular functions (63-65) (Figure 7.4).

Several antioxidant systems are in place in the cell that can quickly remove the ROS from cellular systems. Biological defenses against ROS comprise a complex array of endogenous antioxidant enzymes, endogenous antioxidant factors including glutathione (GSH) and other tissue thiols, heme proteins, coenzyme Q, bilirubin and urates, and a variety of nutritional factors, primarily the antioxidant vitamins and phenolic phytochem-icals from diet (72-74) (Figure 7.4, Figure 7.5).

Glutathione is the most abundant intracellular reductant in all aerobic cells. Tissue GSH and other tissue thiols exist at millimolar concentrations and are important systems to protect the cell against oxidative stress and tissue injury (71). Glutathione acts as a redox sensor and as a sulfhydryl buffer in cellular systems. Upon generation of ROS, GSH preferentially reacts with ROS in an almost sacrificial manner to form oxidized glutathione (GSSH) to prevent the oxidation of other biological molecules. Enzymes such as glutathione peroxidases (GPX) reduce soluble peroxides and membrane bound peroxides to the corresponding alcohols, at the expense of GSH, which is oxidized to GSSG and ascorbic acid, which is then oxidized to dehydroascorbic acid (71-73). Another class of proteins, called glutaredoxins (GRx), are reduced by GSH, and are capable of reducing protein disulfides from oxidative stress (Figure 7.4, Figure 7.5). However, for this antioxidant response to continue, cellular systems have to regenerate GSH constantly (74). Tissue GSH/GSSH ratios are maintained in the reduced state by the concerted action of antioxi-dant vitamins such as ascorbate and tocopherols using many antioxidant enzyme systems

Oxidative Stress

Activation of antioxidant defenses

Repression of ROS producing systems

Induction of Antioxidant Genes

Catalase SOD

Glutahione peroxidase y-glutamylcystiene synthase Thioredoxin reductase

Down Regulation

CYP class of enzymes mitochondrial activity NADPH oxidase EPO

Figure 7.5 Biological adaptive responses to manage oxidative stress. (Morel, Y., R. Barouki, Biochem. J. 342(3): 481-496, 1999.)

involving several oxidoreductases (72-74). The cascade of endogenous antioxidant enzymes requires energy to maintain the living system in the reduced state. Glutathione reductase and ascorbate peroxidase maintain the tissue GSH in the reduced state at the expense of NADPH and FADH2 (72-74) (Figure 7.6). Enzymatic reactions of the thioredoxin system contain a set of oxidoreductases with wide range substrate specificity that reduce the active site disulfide in thioredoxin (Trx) and several other substrates, directly under the consumption of NADPH. Reduced Trx is highly efficient in reducing disulfides in proteins and peptides including glutathione disulfide (GSSG).

Another biological defense against superoxide free radicals is the superoxide dis-mutase (SOD) enzyme, which is often considered the most effective antioxidant system.

Superoxide dismutase is an enzyme that catalyzes dismutation of two superoxide anions into hydrogen peroxide and molecular oxygen

The importance of SOD is that it is a very efficient enzyme system in removing superoxide radicals from biological systems. The function of SOD is so imperative for the protection of cells that it represents a substantial proportion of the proteins manufactured by the body (72,73). One of the contributing factors for the high efficiency of SOD is that its dismutation is always coupled to another enzyme system called catalase (CAT). Catalase is involved in removing hydrogen peroxide molecules, which are byproducts of the reactions created by SOD (72,73) (Figure 7.6). Catalase is also abundantly present in the body and is integrated into all the cellular systems that operate in an oxidant environment. Red blood cells, which transport oxygen to different cellular systems, have high levels of CAT and help to remove hydrogen peroxide from tissues to prevent both cellular damage and propagation ah2 a

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