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International Journal of Research in Pharmacy and Biosciences Volume 3, Issue 6, 2016, PP 1-8 ISSN 2394-5885 (Print) & ISSN 2394-5893 (Online)

The Role of Reactive Species and in

Ozougwu, Jevas C. Ph.D and Biomedical Research Unit, Department of Biological Sciences, College of Basic and Applied Sciences, Rhema University Aba, Abia State, Nigeria

ABSTRACT Under normal conditions, the physiologically important intracellular levels of reactive oxygen species (ROS) are maintained at low levels by various systems in the body but at high concentrations, they produce adverse modifications to components such as lipids, and DNA. In humans, oxidative stress is involved in the development of cancer, parkinson's disease, alzheimer's disease, atherosclerosis, heart failure, , fragile X syndrome, sickle cell disease, lichen planus, vitiligo, autism, infection, chronic fatigue syndrome and depression. However, reactive oxygen species can be beneficial, as they are used by the as a way to attack and kill pathogens, also short-term oxidative stress is important in the prevention of aging by induction of mitohormesis. ROS can be from endogenous or exogenous sources and they are produced from molecular oxygen as a result of normal cellular , they are divided into free radicals and non-radicals. The major endogenous sources of ROS of physiological importance are anion, hydroxyl , , hypochlorous acid, peroxyl radical and radical while the exogenous sources of ROS include cigarette smoke, ozone exposure, , and heavy metal ions. Aerobic organisms have integrated antioxidant systems, which include enzymatic and non-enzymatic antioxidant that are usually effective in blocking harmful effects of reactive oxygen species. Enzymatic defense is a system of which includes peroxidases, superoxide dismutases and , which decrease the concentration of the most harmful ROS whereas non-enzymatic antioxidants include low molecular weight compounds such as vitamins C and E, β-carotene, uric acid and glutathione. Humans have a complex antioxidant protection system, which functions interactively and synergistically to neutralize free radicals and knowledge as presented in this review will be important in the management of oxidative stress and its associated diseases. Keywords: Role, Oxidative stress, Antioxidant, ROS

INTRODUCTION Oxidative stress is an imbalance between the systemic manifestation of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Reactive oxygen species are produced by living organisms as a result of normal cellular metabolism [1]. At low to moderate concentrations they function in physiological cell processes, but at high concentrations, they produce adverse modifications to cell components such as lipids, proteins and DNA [2, 3, 4]. Oxidative stress from oxidative metabolism causes base damage, as well as strand breaks in DNA. Base damage is mostly indirect and caused by reactive oxygen species generated, examples O2− (superoxide radical), OH () and H2O2 (hydrogen peroxide). Some reactive oxidative species act as cellular messengers in redox signaling. Thus, oxidative stress can cause disruptions in normal mechanisms of cellular signaling. In humans, oxidative stress is thought to be involved in the development of cancer, Parkinson's disease, Alzheimer's disease, [5, 6] atherosclerosis, heart failure,[7] myocardial infarction, [8,9] fragile X syndrome, [10] Sickle Cell Disease, [11] lichen planus, [12] vitiligo, [13] autism, [14] infection, [15] Chronic fatigue syndrome, [16] and Depression. However, reactive oxygen species can be beneficial, as they are used by the immune system as a way to attack and kill pathogens [17]. Short-term oxidative stress may also be important in prevention of aging by induction of mitohormesis [18]. Aerobic organisms have integrated antioxidant systems, which include enzymatic and non-enzymatic antioxidant that are usually effective in blocking harmful effects of reactive oxygen species. However, in pathological conditions, the antioxidant systems are overwhelmed by reactive oxygen species. In view of the central role of oxidative stress in aging and disease progression, the critical role of antioxidant in maintaining International Journal of Research in Pharmacy and Biosciences V3● I6● 2016 1 Ozougwu, Jevas C. “The Role of Reactive Oxygen Species and Antioxidants in Oxidative Stress” optimum health and well-being and the increased exposure to free radicals such as pollution, cigarette smoke, drug, stress, exercise etc this review becomes necessary to x-ray the role of ROS and antioxidant in oxidative stress. ROLE OF ROS IN OXIDATIVE STRESS Pro-oxidants are chemicals that induce oxidative stress, either by generating reactive oxygen species or by inhibiting antioxidant systems [19]. Oxidative stress produced by these chemicals can damage cells and tissues, for example an overdose of paracetamol (acetaminophen) damaged the liver of experimental , partly through its production of reactive oxygen species [20, 21]. Some of the conditions that make a substance serve as pro-oxidants are the concentration of the chemical and if oxygen or transition metals are present. Reactive oxygen species are produced from molecular oxygen as a result of normal cellular metabolism. They are divided into free radicals and non-radicals (Table 1). containing one or more unpaired and thus giving reactivity to the are called free radicals. When two free radicals share their unpaired electrons, non-radical forms are created. ROS can be from endogenous or exogenous sources. The major endogenous sources of ROS of physiological importance are superoxide anion, hydroxyl radical, hydrogen peroxide, hypochlorous acid, peroxyl radical and hydroperoxyl radical (Table 1). Superoxide anion is formed by the addition of one to the molecular oxygen [22]. Hydrogen peroxide is produced by , amino acid oxidase and NAD(P)H oxidase [23, 24] and in by consumption of molecular oxygen in metabolic reactions. Hydroxyl radical is the most reactive of reactive oxygen species and can damage proteins, lipids, carbohydrate and DNA. It can also start by taking an electron from polyunsaturated fatty acids [1]. Hypochlorous acid (HOCl) is highly oxidative and plays an important role in killing of the pathogens in the airways [25]. HOCl can also react with DNA and induce DNA interactions and produce pyrimidine oxidation products and add chloride to DNA bases [26, 27]. Other oxygen-derived free radicals are the peroxyl radicals (ROO-), the simplest form of these radicals is hydroperoxyl radical (HOO-) and has a role in fatty acid peroxidation. Free radicals can trigger lipid peroxidation chain reactions by abstracting a hydrogen atom from a side-chain methylene carbon. The lipid radical then reacts with oxygen to produce peroxyl radical. Peroxyl radical initiates a chain reaction and transforms polyunsaturated fatty acids into lipid hydroperoxides. Lipid hydroperoxide are very unstable and easily decompose to secondary products such as aldehydes and malondialdehydes. Peroxidation of lipids disturbs the integrity of cell membranes and lead to rearrangement of membrane structure. Exogenous sources of ROS includes cigarette smoke, ozone exposure, hyperoxia, ionizing radiation and heavy metal ions. Free radicals are electrically charged molecules that are unpaired electron, which causes them to seek out and capture electrons from other substances in order to neutralize themselves. The initial attack of free radicals cause the free radical to become neutralized, another free radical is formed in the process causing a chain reaction to occur [28], which continues until subsequent free radicals are deactivated, thousands of free radical reactions can occur within seconds of the initial reaction. Table1. Some Important Reactive Oxygen Species in Living Organisms FREE RADICALS Hydroxyl radical OH+ Superoxide radical O2+ Nitric oxide radical NO+ Lipid peroxyl radical LOO+ Hydroperoxyl radical HOO NON RADICALS Hydrogen peroxide H2O2 1O2 Hypochlorous acid HOCl ozone O3 All ROS are capable of reacting with membrane lipids, nucleic acids, proteins and enzymes and other small molecules resulting in cellular damage [28]. Reactive oxygen species are produced continuously in the human body as a consequence of normal metabolic processes. If free radicals are not inactivated, their chemical level can damage all types of cellular macromolecules, including proteins, carbohydrates, lipids and nucleic acids [29]. Free radicals and other reactive oxygen species in the human body are derived either from normal internal metabolic processes or from external sources as shown in Table 3.

2 International Journal of Research in Pharmacy and Biosciences V3● I6● 2016 Ozougwu, Jevas C. “The Role of Reactive Oxygen Species and Antioxidants in Oxidative Stress” Table3. Some Sources of Free Radicals Internally Generated Sources Mitochondria Xanthine oxidase Reactions involving and other transition metals Arachidonate pathways Peroxisomes Exercise /reperfusion Externally Generated Sources Cigarette smoke Environmental pollutants Ionizing radiation light Certain drugs, pesticides, anaesthetics and industrial solvent Ozone exposure Heavy metal ions Source: Langseth (1995). Free radicals are not always harmful, they serve useful purposes in the human body. For example, free radicals play a role in the destruction of disease-causing micro-organisms by specialized cells called phagocytes [29]. Reactive oxygen species are generated by a number of pathways. [28] reported that most of the ROS produced by cells occur as: i A consequence of normal aerobic metabolism and approximately 90% of the oxygen utilized by the cell is consumed by the mitochondrial electron transport system. ii Oxidative burst from phagocytes (white blood cells) as part of the mechanism by which bacteria and are killed and by which foreign proteins (antigens) are denatured. iii Xenobiotic metabolism, that is, detoxification of toxic substances. Consequently, vigorous exercise, which accelerates cellular metabolism, chronic inflammation, infections and other illnesses; exposure to allergens and the presence of “leaky gut” syndrome and exposure to drugs or toxins such as cigarette smoke, pollution, pesticides and insecticides may all contribute to an increase in the body’s oxidant load. ROLE OF ANTIOXIDANTS IN OXIDATIVE STRESS Antioxidants are substances which can delay or inhibit oxidation. Cell damage caused by free radicals is the major contributor to aging and to degenerative diseases of aging such as cancer, , cataracts, immune system decline and dysfunction [30]. Free radicals have been implicated in the pathogenesis of at least 50 diseases [31]. Fortunately, free radical formation is controlled naturally by various beneficial compounds known as antioxidant. It is when the availability of antioxidant becomes limited that damage caused by ROS can become cumulative and debilitating. Antioxidant are capable of stabilizing or deactivating free radicals before they attack cells which make them absolutely critical for maintaining optimal cellular and systemic health and well-being [28]. The human body has several mechanisms for defense against free radicals and other reactive oxygen species. The various defenses are complementary to one another because they act on different ROS or in different cellular compartments [29]. These defenses can be grouped into enzymatic and non- enzymatic antioxidant. Enzymatic defense is a system of enzymes which includes glutathione peroxidases, superoxide dismutases and catalase, which decrease the concentration of the most harmful ROS. Superoxide dismutases are a family of antioxidant enzymes which are important in the catalytic decomposition of the superoxide radical to hydrogen peroxide and oxygen [29]. Catalase specifically catalyzes the decomposition of hydrogen peroxide and glutathione peroxidases are a family of antioxidant enzymes containing selenium which are important in the reduction of hydro peroxides. Non-enzymatic antioxidants include low molecular weight compounds such as and E, β- carotene, uric acid and glutathione. To protect the cells and organ systems of the body against reactive oxygen species, humans have evolved a highly sophisticated and complex antioxidant protection system which functions interactively and synergistically to neutralize free radicals [32]. [28] reported that these components include: International Journal of Research in Pharmacy and Biosciences V3● I6● 2016 3 Ozougwu, Jevas C. “The Role of Reactive Oxygen Species and Antioxidants in Oxidative Stress” i Nutrient-derived antioxidant like ascorbic acid (vitamin C), and (), , and other low molecular weight compounds such as glutathione and lipoic acid. ii Antioxidant enzymes for example , and , which catalyze free radical quenching reactions. iii Metal binding proteins, such as , lactoferrin, albumin, and ceruloplasmin that sequester free iron and ions that are capable of catalyzing oxidative reactions. iv Numerous other antioxidant phytonutrients present in a wide variety of foods. Table4. Various ROS and Corresponding Neutralizing Antioxidant Reactive oxygen species (ROS) Neutralizing antioxidant Hydroxyl radical Vitamin C, glutathione, flavonoids, lipoic acid Superoxide radical Vitamin C, glutathione, flavonoids, SOD Hydrogen peroxide Vitamin C, glutathione, beta carotene, vitamin E, CoQ10 Flavonoids, lipoic acid Lipid peroxides Beta carotene, vitamin E, ubiquinone, flavonoids, glutathione peroxidase Source: Percival (1998). Dietary Antioxidant Vitamin C, vitamin E and Beta carotene are among the most widely studied dietary antioxidant. The food sources of antioxidant vitamins are shown in Table 5. Table5. Food Sources of Antioxidant Vitamins. Vitamin E Best sources: vegetable oils, cold pressed seed oils, wheat germ Other significant sources: vegetables, fruits, meat/poultry/ fish Vitamin C Fruits: especially citrus fruits, strawberries, cantaloupe melon Vegetables: especially tomatoes, leafy greens, cabbage family vegetables such as broccoli and cauliflower. Carotenoids β- carotene: yellow-orange vegetables and fruits, dark green vegetables α- carotene: carrots Lycopene: tomatoes Lutein and zeaxanthin: dark green leaf vegetables, broccoli Β- cryptoxanthin: citrus fruits Source: Langseth (1995). Vitamin C is considered the most important -soluble antioxidant in extracellular fluids [28]. It is capable of neutralizing ROS in the aqueous phase before lipid peroxidation is initiated. Vitamin E, a major lipid-soluble antioxidant, is the most effective chain-breaking antioxidant within the cell membrane where it protects membrane fatty acids from lipid peroxidation. Vitamin C has been reported as being capable of regenerating vitamin E [30]. Beta carotene and other carotenoids are also believed to provide antioxidant protection to lipid-rich tissues. Research suggests that beta carotene may work synergistically with vitamin E [32, 33]. A diet that is excessively low in fat may negatively affect beta carotene and vitamin E absorption, as well as other fat-soluble nutrients. Fruits and vegetables are major sources of vitamin C and carotenoids, while whole grains and high quality, properly extracted and protected vegetable oils are major sources of vitamin E [28]. A number of other dietary antioxidant substances exist beyond the traditional vitamins. Many plant-derived phytochemicals are becoming increasingly known for their antioxidant level. Phenolic compounds such as flavonoids are ubiquitous within the plant kingdom, approximately 3,000 flavonoid substances have been described [34]. In , flavonoids serve as protectors against a wide variety of environmental stresses while, in humans, flavonoids appear to function as biological response modifiers. Flavonoids have been demonstrated to have anti-inflammatory, antiallergenic, anti-viral, anti-aging and anti-carcinogenic level [35]. The broad therapeutic effects of flavonoids can be largely attributed to their antioxidant properties [28]. In addition to an antioxidant effect, flavonoid compounds may exert protection against heart disease through the inhibition of cyclooxygenase and lipoxygenase level in and macrophages [31]. The best way to ensure an adequate intake of phytonutrients is to eat a diet rich in a wide variety of fresh fruits and vegetables. 4 International Journal of Research in Pharmacy and Biosciences V3● I6● 2016 Ozougwu, Jevas C. “The Role of Reactive Oxygen Species and Antioxidants in Oxidative Stress” Endogenous Antioxidant The body relies on several endogenous defense mechanisms to help protect against free radical- induced cell damage. The antioxidant enzymes – glutathione peroxidase, catalase and superoxide dismutase (SOD) - metabolize oxidative toxic intermediates and require micronutrient cofactors such as selenium, iron, copper, zinc and for optimum catalytic level [28]. It has been suggested that an inadequate dietary intake of these trace minerals may compromise the effectiveness of these antioxidant defense mechanisms [37]. Research indicates that consumption and absorption of these important trace minerals may decrease with aging [37]. Glutathione, an important water-soluble antioxidant, is synthesized from the amino acids glycine, glutamate, and cysteine. Glutathione directly quenches ROS such as lipid peroxides and also plays a major role in xenobiotic metabolism [28]. Exposure of the liver to xenobiotic substances induces oxidative reactions through the up regulation of detoxification enzymes that is cytochrome P -450 mixed-function oxidase. When an individual is exposed to high levels of xenobiotics, more glutathione is utilized for conjugation making it less available to serve as an antioxidant. Research suggests that glutathione and vitamin C work interactively to quench free radicals and that they have a sparing effect upon each other. Lipoic acid, another important endogenous antioxidant, categorized as a thiol or biothiol, is a sulfur-containing molecule that is known for its involvement in the reaction that catalyzes the oxidative decarboxylation of alpha-keto acids, such as pyruvate and alphaketoglutarate, in the Krebs cycle. Lipoic acid and its reduced form, dihydrolipoic acid, are capable of quenching free radicals in both lipid and aqueous domains and as such has been called a universal antioxidant. Lipoic acid may also exert its antioxidant effect by chelating with pro-oxidant metals. Research reports indicate that lipoic acid has a sparing effect on other antioxidant [38]. Animal studies have demonstrated supplemental lipoic acid protect against the symptoms of vitamin E or vitamin C deficiency. Other physiological antioxidant are shown in Table 6. Table6. Antioxidant Protection System Endogenous antioxidant Bilirubin Thiols, eg glutathione,lipoic acid, N-acetyl cysteine NADPH and NADH Ubiquinone (coenzyme Q10) Uric acid Enzymes: copper/zinc and manganese-dependent superoxide dismutase (SOD), Iron –Dependent Catalase, selenium-dependent glutathione peroxidase. Dietary antioxidant Vitamin c Vitamin E Beta carotene and other carotenoids and oxycarotenoids, e.g lycopene and lutein. Polyphenols e.g flavonoids, flavones, flavonols and proanthocyanidins. Metal binding proteins Albumins (coopper) Ceruloplasmin (copper) Metallothionein (copper) Ferritin (iron) (iron) Transferring(iron) Source: Percival (1998). EFFECTS OF OXIDATIVE STRESS ON DNA, LIPIDS AND PROTEINS DNA modifications as a result of ROS occurs in several ways, which involves degradation of bases, single or double stranded DNA breaks, purine, pyrimidine or sugar bound modifications, mutations, deletions or translocations and cross-linking with proteins. Most of these DNA modifications are highly relevant to , aging, neurodegenerative, cardiovascular and autoimmune diseases. Lipid peroxidation can be induced by ROS which disrupts the membrane lipid bilayer arrangement that may inactivate membrane bound receptors and enzymes and increase permeability [39]. Products of lipid peroxidation, such as MDA and unsaturated aldehydes, are capable of inactivating many cellular proteins forming protein cross-linkage [40, 41, 42]. 4- Hydroxy-2-nonenal causes depletion of intracellular GSH and induces peroxide production [43, 44], activates epidermal growth International Journal of Research in Pharmacy and Biosciences V3● I6● 2016 5 Ozougwu, Jevas C. “The Role of Reactive Oxygen Species and Antioxidants in Oxidative Stress” factor receptor [45] and induces fibronectin production [46]. Lipid peroxidation products, such as isoprostanes and thiobarbituric acid reactive substances, have been used as indirect biomarkers of oxidative stress and increased levels were shown in the exhaled breath condensate or bronchoalveolar lavage fluid or lung of chronic obstructive pulmonary disease patients or smokers [47, 48, 49]. ROS can cause fragmentation of the peptide chain, alteration of electrical charge of proteins, cross-linking of proteins and oxidation of specific amino acids and therefore lead to increased susceptibility to proteolysis by degradation by specific proteases [50]. Cysteine and residues in proteins are particularly more susceptible to oxidation [51]. Oxidation of sulfhydryl groups or methionine residues of proteins cause conformational changes, protein unfolding and degradation [51, 52]. Enzymes that have metals on or close to their active sites are especially more sensitive to metal catalyzed oxidation. Oxidative modification of enzymes has been shown to inhibit their activities [53, 54]. CONCLUSIONS Damage to the body cells caused by ROS play central role in the aging process and in disease progression. Under normal conditions, the physiologically important intracellular levels of reactive oxygen species (ROS) are maintained at low levels by various antioxidant systems in the body but at high concentrations, they produce adverse modifications to cell components such as lipids, proteins and DNA. Reactive oxygen species can be beneficial, as they are used by the immune system as a way to attack and kill pathogens and short-term oxidative stress is important in prevention of aging by induction of mitohormesis. Humans have evolved a complex antioxidant protection system, which functions interactively and synergistically to neutralize free radicals and knowledge as presented in this review will be beneficial in the management of oxidative stress and its associated diseases. REFERENCES

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