CHAPTER 17 Antioxidants and Horse Health
During the last two decades, the physiologic and pathophysiologic roles of oxidants and antioxidants have been intensively investigated in humans, laboratory rodents, and horses. Oxidants, which were initially considered potentially harmful metabolic byproducts, are now recognized as important cellular mediators and signaling molecules. Nevertheless, if the pro-oxidant burden overwhelms the antioxidant defense systems, oxidant-antioxidant disequilibrium or oxidative stress can develop. Biomarkers can be detected in body fluids, enabling assessment of oxidative stress in various physiologic and pathologic conditions. Antioxidants are recommended for maintaining optimal oxidant-antioxidant equilibrium as a means to prevent or treat oxidative stress-associated diseases, but the paucity of systematic research in this field limits the recommendations for therapeutic antioxidant use in equine medicine.
Oxidants or reactive oxygen species (ROS) are oxygen-containing molecules that are more reactive than the oxygen molecules that comprise the air, and include free radicals as well as reactive compounds without unpaired electrons in their outer orbit. The reactive nitrogen species (RNS) have been defined as a subgroup of oxidants derived from nitric oxide (NO°), and the specific term nitrosative stress has been introduced. In this chapter, the term oxidants will encompass ROS and RNS.
Organisms are constantly exposed to exogenous and endogenous oxidants. Exposure to exogenous oxidants is of particular importance for the airways and lungs, which are exposed to inhaled substances such as ozone, ultrafine particles, or endotoxins. The antioxidant defense system of the respiratory tract is particularly well developed, but pulmonary oxidative stress is observed in numerous respiratory tract diseases and is believed to play a role in their pathogenesis.
Endogenous oxidants can be divided into three groups, depending on their origin. The major source of endogenous oxidants is the electron transfer chain of the mitochondria, where 1% to 3% of oxygen reduced into water may form superoxide (O2°–). This pathway of oxidant formation is particularly important during exercise, when oxygen consumption increases up to 40 times in horses. Second contributors to endogenous oxidant generation are enzymes such as xanthine oxidase, membrane oxidases, and nitric oxide synthases, which produce oxidants. The relative contribution of this pathway to the pro-oxidant burden depends on the metabolic activity in each horse. The third source of oxidants is the respiratory burst of inflammatory cells, which generates superoxide anions (O2°–) via NADPH oxidase. This pathway of oxidant generation is strongly increased during inflammatory processes. Production and liberation of oxidants by neutrophils and macrophages form part of the main arm of the nonspecific immune response against invading microorganisms.
Oxidants play an important role by inactivating and destroying microorganisms through peroxidation and destabilization of lipid membranes, oxidation and inactivation of microbial protein receptors or enzymes, and oxidation of nuclear material. Oxidant generation can be further enhanced in presence of pro-oxidant elements, which transform oxidants into more reactive forms. These elements include enzymes such as myeloperoxidase liberated by polymorphonuclear neutrophils and unbound trace elements, such as iron or copper, favoring oxidant generation.
Initially oxidants were considered to be potentially harmful byproducts of cellular metabolism and part of the immune system. However, during the last two decades, oxidants have been identified as important messengers in numerous intracellular pathways. The implication of so-called redox signaling in activation of transcription factors and in mitotic and apoptotic processes indicates that oxidants are indispensable signaling molecules. The expression of inflammatory genes is also dependent on oxidation-reduction reactions, which makes oxidants potential pro-inflammatory stimuli (Figure 17-1).
Figure 17-1 The mitochondrial respiratory chain, enzymatic processes, and phagocytes respiratory burst are the primary sources of oxidants. Oxidants play a role of cellular messengers and pro-inflammatory signals, thereby allowing autoactivation of the respiratory burst during inflammatory processes. Enzymatic antioxidants and their catalyzing trace elements and nonenzymatic antioxidants counterbalance the oxidant burden and may prevent oxidative stress. Pro-oxidant elements favor oxidative stress, leading to irreversible modifications of cellular components that can be evidenced by detecting oxidation products. The oxidant-antioxidant equilibrium is characterized on the basis of measurement of antioxidant systems, pro-oxidant elements, and oxidation products.
Exposure to oxidants from various sources has led organisms to develop a series of defense mechanisms, including preventative mechanisms, antioxidant defenses, and repair mechanisms. Prevention of oxidant generation occurs mainly within the mitochondrial respiratory chain, where enzymatic complexes strongly limit electron leakage. Furthermore, proteins that bind free iron and copper (which promote oxidative processes), such as transferrin, ferritin, ceruloplasmin, and albumin, further decrease the cellular capacity for oxidant generation.
Antioxidants are implicated in the inactivation or transformation of oxidants, which can either be transformed by antioxidant enzymes into less reactive forms or react with antioxidant molecules that are chemically stable. The most important antioxidant enzymes are superoxide dismutase (SOD), catalase (CAT), and glutathione-peroxidase (GPx). The catalytic activity of these enzymes allows transformation of superoxide anion into hydrogen peroxide (H2O2) and water, thereby inactivating important amounts of oxidants. Trace elements, such as selenium (Se), zinc (Zn), copper (Cu), and manganese (Mn), play an important catalytic role in the enzymatic activity of GPx (Se) and SOD (Zn, Mn, Cu). Antioxidant molecules of reduced molecular weight are the most numerous endogenous antioxidants. They can be divided into hydrophobic and hydrophilic antioxidants.
Hydrophobic antioxidants include mainly α-tocopherol (vitamin E), β-carotene (vitamin A), flavonoids (genistein, quercetin, resveratrol, lycopene), ubiquinol, bilirubin, and melatonin and act to protect lipids from chain reactions of peroxidation. Hydrophilic antioxidants include glutathione, uric acid, ascorbic acid (vitamin C), thiols, proteoglycans, and hyaluronic acid; these molecules protect against lipid peroxidation and against oxidation of protein, carbohydrate, and nuclear material. Repair mechanisms induce lysis or repair of oxidized proteins, lipids, or nuclear material that have undergone oxidation, thereby limiting the functional repercussions of oxidative damage.
Oxidative stress is defined as a disturbance of the equilibrium between antioxidants and oxidants in favor of oxidants. Oxidative stress might develop when the antioxidant defense system is overwhelmed by an increased oxidant burden or reduced antioxidant supply. If exogenous or endogenous oxidant exposure increases or is insufficiently counterbalanced by antioxidants, oxidative damage occurs in the form of oxidized DNA, proteins, lipids, or carbohydrates. Although virtually all cellular components may undergo oxidation in the presence of high concentrations of oxidants, the intracellular origin and reactivity of the oxidant and the location and biochemical properties of the target molecule play a determining role for the “pecking order” of oxidants.
Molecules that have undergone oxidation can be detected and are used as oxidation markers. Their presence can disturb cell function, especially with oxidative modifications to DNA, and can lead to mutations. Oxidation of proteins contributes to enzyme dysfunction and cell membrane lipid peroxidation, initiating chain reactions that can compromise cell integrity. Such cellular perturbations appear to be of particular importance in inflammatory conditions in which increased oxidant production can further enhance the inflammatory process through a positive feedback mechanism (see Figure 17-1).