Free Radicals and Antioxidants PDF

Free Radicals and Antioxidants Biochem 474 By Dr. Mai Madkour Lecturer of Biochemistry Faculty of Science, Mansoura University Basics of redox chemistry Oxidation ▪ Gain in oxygen ▪ Loss of hydrogen or electrons Reduction ▪...

Free Radicals and Antioxidants Biochem 474 By Dr. Mai Madkour Lecturer of Biochemistry Faculty of Science, Mansoura University Basics of redox chemistry Oxidation ▪ Gain in oxygen ▪ Loss of hydrogen or electrons Reduction ▪ Loss of oxygen ▪ Gain of hydrogen or electrons Oxidant ▪ Oxidize another chemical compound by accepting electrons, hydrogen Reductant ▪ Reduces another chemical compound by donating electrons, hydrogen  Free radicals ▪ A free radical is defined as an atom or molecule that contains one or more unpaired electrons in its outer orbital rather than the usual paired electrons that spin in opposite directions ▪ It is an electron-deficient species ▪ It is represented by a superscript dot to the right R. ▪ Examples: - Hydroxyl radical (HO·), a molecule that has one unpaired electron on the oxygen atom Free radical nomenclature A free radical is donated by a superscript dot to the the oxygen or carbon e.g.,.OH, NO.,. CH3 If a free radical is a charged species, the dot is put and then the charge e.g., O2-. Characteristics of Free radicals Free radicals are ▪ Highly reactive and react quickly with other compounds ▪ Unstable and try to become stable ▪ Short life span (short lived ) as they tend to catch electron from other molecules ▪ Generation of new free radicals by chain reaction ▪ Damage to various tissues Radicals can be formed by 1. The loss of a single electron from a non-radical, or by the gain of a single electron by a non-radical 2. The breakage of covalent bond “homolytic fission’ - Covalent bond breakage in which the shared electrons is split evenly between the products - In the presence of heat/light ▪ Therefore if two free radicals react, they neutralize each other. ▪ However, if the free radicals react with stable molecules, there is generation of more free radials. ▪ This character enables the free radicals to participate in autocatalytic chain reactions. - Molecules are themselves converted to free radicals to propagate the chain of damages. Function of free radical ▪ Some of free radicals arise normally during metabolism ▪ Sometimes the body’s immune system create them to neutralize viruses and bacteria - Respiratory burst in WBC - NO signaling ▪ Some free radicals at low levels are signaling molecules, i.e. they are responsible for turning on and off of genes. ▪ Some free radicals kill cancer cells. ▪ Normally, the body can handle free radicals, but if antioxidants are unavailable, or if the free radicals production becomes excessive, damage can occur Non-radicals ▪ Species that have strong oxidizing potential ▪ These nonradicals molecules can produce oxidation “per se” or can also be converted into free radicals. Hydrogen peroxide ▪ Examples - Hydrogen peroxide (H₂O₂) Hypochlrous acid - Hypochlorus acid (HClO) Ozone - Ozone (O₃) - Singlet oxygen (1O2 ) Singlet oxygen - Peroxynitrite (ONOO−) - Transition metals Peroxynitrite Ex: manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu) Source of free radicals Exogenous source of free radical Ionizing radiation Ultraviolet radiation Chemicals, smoking Pollution Diet Endogenous source of free radical ▪ Free radical formation occurs continuously in the cells as a consequence of both enzymatic and nonenzymatic reactions ▪ Enzymatic reactions, which serve as source of free radicals, include those involved in the respiratory chain, phagocytosis, prostaglandin synthesis, and in the cytochrome P-450 system ▪ Free radicals can also be formed in nonenzymatic reactions of oxygen with organic compounds as well as those initiated by ionizing reactions (x-ray and UV) which lyse water leading to formation of hydroxyl radical ▪ Transition metal ions including Cu +2, Co +2, Ni +2 and Fe +2 Can react nonenzymatically with oxygen ▪ or react with hydrogen peroxide leading to the formation of hydroxyl radicals Examples ▪ Cells (neutrophil, eosinophil,……) ▪ Inflammation ▪ Phagocytosis ▪ Enzymes (Nitric oxide synthase, xanthine oxidase, NADPH oxidase) ▪ Electron transport chain in mitochondria ▪ Cytochrome P450 in endoplasmic reticulum  Source of free radical Endogenous ▪ Endogenous free radical ▪ Exogenous free radicals Transitiom metals E.g. ionizing radiation Types of free radicals Reactive oxygen species (ROS): - Hydrogen peroxide (H2O2) - Hypochlorous acid (HOCl) - Singlet oxygen ( 1O2 ) - Superoxide anion (O2-.) - Hydroxyl radical (.OH) Reactive nitrogen species (RNS): - Nitric oxide ( NO. ) - Peroxy nitrite (ONOO-) - Peroxy nitrate (O NOO-) 2 Other reactive species - Lipid peroxyl radical (LOO.) - Lipid hydroperoxide (LOOH)  Activation of oxygen ▪ Atmospheric oxygen in its ground-state is distinctive among the gaseous elements because it is a biradical, or in other words it has two unpaired electrons. This feature makes oxygen paramagnetic ▪ The high reactivity of atmospheric oxygen is due to its biradical state (the two unpaired electrons in oxygen has parallel spins) Activation of oxygen may occur by two different mechanisms: ▪ Absorption of sufficient energy to reverse the spin on one of the unpaired electrons, or monovalent reduction. ▪ singlet oxygen is much more reactive towards organic molecules than its triplet oxygen A singlet state refers to a system in which all the electrons are paired. Whereas, the triplet state of a system describes that the system has two unpaired electrons. ▪ The second mechanism of activation is by the stepwise monovalent reduction of oxygen to form superoxide (O-2), hydrogen peroxide (H2O2), hydroxyl radical (.OH) and finally water ▪ The first step in the reduction of oxygen forming superoxide is endothermic but subsequent reductions are exothermic. The activation states of oxygen  Oxidative stress ▪ Oxidative stress represents an imbalance between the production of reactive oxygen species or decrease in the antioxidant status in the body ▪ Disturbance in the normal redox state of tissue can cause toxic effects ▪ Free radicals damage all components of the cell, including protein, lipids and DNA ▪ Free radical may cause mutation, cancer, and autoimmune disease Under normal physiologic conditions, the production of oxygen free radicals and peroxides is balanced by an efficient system of antioxidants, which are molecules capable of "scavenging" ROS, thereby preventing oxidative damage Superoxide radical (O2-.) ▪ Formed chemically by addition of an extra electron to O2 molecule ▪ The major source of superoxide is from the electron transport chain of the mitochondria ▪ This reaction occur due to two reasons: 1) Electron leak from their carriers within the respiratory chain of mitochondria 2) phagocyte produce superoxide as an antibacterial agent ▪ The enzymes that can produce superoxide include - xanthine oxidase - Lipoxygenase - Cyclooxygenase - NADPH dependent oxidase - Mitochondria electron transport chain Reactivity of Superoxide radical O2-. ▪ The superoxide radical (O2-.) can function both: As an oxidant in which case is gains an electron and produces H2O2, or as a reductant in which case it loses its electrons and is oxidized to oxygen. + e- - e- ▪ Dismutation Reaction - Two superoxide radicals can interact that ONE anion is reduced and the other oxidized with resulting formation of H2O2 + O2 ▪ This superoxide can spontaneously dismutase in aqueous solution to form hydrogen peroxide & singlet oxygen - The dismutation reaction (with superoxide dismutase) (SOD) well is greatly affected by the pH, the spontaneous production of H2O2 from O2-. occur at a significant rate in an acidic medium. - In contrast, at neutral pH, the dismutation reaction is much slower because of the electrostatic repulsion between the two-superoxide anion. ▪ Superoxide forms the highly toxic hydroxyl radical ( OH) non enzymatically by reacting with hydrogen peroxide (Haber-Weiss reaction) ▪ Under physiological pH the most occurring form is superoxide. It can act as reducing agent and it reduces iron complexes such as cytochrome-c and ferric-ethylene diaminetetraacetic acid (Fe+3 -EDTA), in which Fe+3 is reduced to Fe+2. ▪ The univalent reduction of superoxide produces hydrogen peroxide which is not a free radical because all of its electrons are paired Estimation of superoxide anion (O2-. ): ▪ O2-. can both reduce Ferricytochrome C and catalyze the oxidation of epinephrine. ▪ The reaction involves the transfer of only a single electron and the product of Ferricytochrome C is stable during assay ▪ Cytochrome c peroxidase, is a water-soluble heme-containing enzyme that takes reducing equivalents from cytochrome c and reduces hydrogen peroxide to water Ferrocytochrome C Cytochrome c peroxidase Ferricytochrome C ▪ The second method depend on conversion of Epinephrine to Adrenochrome. ▪ The reaction involve a Four-electron for oxidation and a chain reaction is variable length and the production of Adrenochrome ▪ the chemical conversion of adrenaline is coupled with the formation of O2-. Hydrogen Peroxide (H2O2) ▪ It is not a free radical but falls in the category of reactive oxygen species ▪ A molecule of hydrogen peroxide will break into stable water and oxygen ▪ The extra oxygen atom makes hydrogen peroxide more reactive than water ▪ Hydrogen peroxide (H2O2) itself is not especially toxic unless it is present in high concentrations within cells ▪ H2O2 produced, probably mainly via O2 from phagocytic cells in mitochondria and chloroplast ▪ several oxidases enzymes can produce H2O2 such as urate oxidase and xanthine oxidase ▪ Hydrogen peroxide is formed in vivo in a dismutation reaction catalyzed by the enzyme superoxide dismutase (SOD) ▪ H2O2 is converted to innocuous products by the actions of two important antioxidant enzymes - Catalase - Selenium-dependent glutathione peroxidase (GPx) Formation of ROS by the Fenton and Haber– Weiss reactions. (A) Fenton first described the oxidizing power of solutions of Fe+2 and. This reaction generates the strong oxidant OH. Cu+ catalyzes the same reaction. (B) The Haber–Weiss reaction describes the production of OH from H2O2. (C) Under physiologic conditions, the Haber– Weiss reaction is catalyzed by redox-active metal ions. Toxicity of H2O2 ▪ It is not a free radical but it can cause damage to the cell at relatively low concentration (10 μM) ▪ It can easily penetrate the biological membranes. H2O2 has no direct effect on DNA but can damage DNA by producing hydroxyl radical (OH−) in the presence of transition metal ions ▪ Its most vital property is the ability to cross cell membranes freely, which superoxide generally can not do ▪ Hence, hydrogen peroxide generated in one location might diffuse a considerable distance before decomposing to yield the highly reactive hydroxyl radical ▪ H2O2 generated within the cells, and may be removed by the addition of catalase outside the cells, due to the H2O2 leave the cell through plasma membrane. Hydroxyl Radicals (HO.) ▪ Most reactive radical ▪ Formation of hydroxyl radicals in biological systems by: 1) Ionizing radiation -It can be produced when water is exposed to ionizing radiation, this leads to fragmenting of O – H bond, leaving a single electron on H and one on O atom 2) Formation of hydroxyl radical from ozone Ozonide radical (O3.- ) 3) Haber-Weiss reaction - The superoxide forms the highly toxic hydroxyl radical (OH) non enzymatically by reacting with hydrogen peroxide 4) Fenton reaction - Hydroxyl radical can be formed by hydrogen peroxide in the presence of Fe2+ or Cu+2 5) Also, reaction of nitric oxide (.NO) with superoxide anion (O2.-) to produce HO. ▪ Nitric oxide (NO) reacts rapidly with superoxide, producing peroxynitrite. ▪ Peroxynitrous acid decomposes via homolysis producing a pair of discrete OH and NO2 radicals Peroxynitrous acid ▪ Reactions of hydroxyl radicals - Hydrogen atom abstraction - Addition - Electron transfer Singlet Oxygen 1O2 SINGLET OXYGEN ▪ Singlet oxygen is an electronically excited and mutagenic form of oxygen. ▪ Not a true radical as it does not contain an unpaired electron ▪ It is a highly reactive toxic reactive oxygen species ▪ 1O2 is not a diradical as molecular oxygen, and it is highly reactive toward most olefins; thus, it can subtract H+ from a PUFA to initiate lipid peroxidation. 1O exist in two states: 2 - Delta state (1Δg O2), both electrons are paired with opposite spin and exist in one orbital leaving the other empty.( Lifetime=2x10-6sec) - The delta state singlet oxygen is a non-radical - Sigma State ( 1∑g O2 ), both electrons occupy different orbitals as in the ground state, but are of opposite spins.( Lifetime=10-11sec) -The sigma state singlet oxygen is a free radical ▪ Formation of singlet oxygen in biological systems by: - It is produced in vivo by the activation of neutrophils and eosinophils HOCl + H2O2 → 1O2 + H2O + Cl− - It is also formed by some of the enzymatic reactions catalyzed by enzymes such as lipoxygenases, dioxygenases, and lactoperoxidase. - It may be generated from ozone - It is a highly potent oxidizing agent that can cause DNA damage and tissue damage Nitric Oxide NITRIC OXIDE (free radical) ▪ Endothelium-derived relaxing factor (EDRF) ▪ It can be produced by macrophage ▪ It generated from the catalysis of L-arginine by nitric oxide synthase (NOS) enzymes, which are in three forms 1) Type I NOS- brain enzyme (bNOS) 2) Type II NOS- inducible enzyme found in macrophage (iNOS) 3) Type III NOS- endothelial cell enzyme (eNOS) Peroxynitrite (OONO−) PEROXY NITRITE ▪ Macrophage derived nitric oxide when released simultaneously with superoxide, forms new reactive nitrogen species, peroxynitrite anion (ONOO−) ▪ It is formed by the reaction between O2-. and.NO. NO + O -. OONO − 2 ▪ The ONOOH further undergo homolysis to form both OH and.NO2 or rearrange to form nitrate (NO3). ▪ OONO- can oxidize lipids, oxidize methionine and tyrosine residues in proteins and oxidizes DNA Lipid free radicals ▪ The interaction of oxygen free radicals with polyunsaturated fatty acids in the phospholipids of cell membrane leads to the formation of lipid free radicals ▪ Types: - Fatty acid radical (L.) - Lipide peroxide (LOO.) Endogenous Sources of ROS and RNS 1. Mitochondria (electron transport chain) 2. Cytochrome P450 3. Peroxisome 4. Phagocytosis (NADPH oxidase, myeloperoxidase) 5. Nitric oxide synthase 6. Xanthine oxidase 7. Non enzymatic (transition metals) 1- Electron transport chain ▪ Inner mitochondrial membrane ▪ The final stage of aerobic respiration is known as oxidative phosphorylation ▪ Electron transport chain is the transfer of electrons from NADH and FADH2 to oxygen via multiple carriers ▪ The reduction of O2 to water by the mitochondrial electron transport chain ▪ This process require - oxygen ( To accept the electrons and hydrogen at the end) - Reduced NAD+ and FAD which are carrying hydrogen - Electron carriers ▪ The electrons that transferred from NADH and FADH2 to the ETC involves 4 multi-subunit large enzymes complexes and 2 mobile electron carriers Components of ETC 1. Enzyme complex I, NADH dehydrogenase 2. Enzyme complex II, succinate dehydrogenase 3. Enzyme complex III, cytochrome bc1 reductase 4. Enzyme complex IV, cytochrome c oxidase - ATP synthase :- synthesis of ATP from ADP Two mobile carrier ▪ These are connected by two mobile carrier coenzyme Q and cytochrome c - Coenzyme Q (ubiquinone) connects between either Complex I or Complex II to Complex III - Cytochrome c connects complex III and IV ▪ Electron flow from more electronegative to electropositive components ▪ NADH passes its electron on to complex 1 – NADH dehydrogenase ▪ FADH2 passes its electron to complex 2- succinate dehydrogenase ▪ In complex IV, when a total of four electrons are transferred to oxygen, two water molecules are formed ▪ Some electrons can “escape” the electron transport chain and combine with oxygen to form a very unstable form of oxygen called a superoxide radical (O2 -) ▪ The electrons derived from NADH and FADH2 combine with O2 , resulting in the generation of reactive oxygen species. ▪ Mitochondrial electron transport chain complex I, II, III take part in O2-. ▪ Leakage of electrons at complex I and complex III from electron transport chains leads to partial reduction of oxygen to form superoxide. Subsequently, superoxide is quickly dismutated to hydrogen peroxide by SOD1 and SOD2 ▪ Products of partial reduction of oxygen are highly reactive and damage the living tissue - sometimes the levels of superoxide rise, for example after alcohol exposure (which generates a lot of NADH). - Thus, more hydrogen peroxide is formed and can’t be detoxified by the limited amount of catalase. - Instead hydrogen peroxide becomes reduced by iron (Fe2+), which donates an electron to produce the hydroxyl radical ( OH) - It is extremely reactive and a great oxidizing agent. 2- Cytochrome P450 ▪ Cytochrome P450 is a superfamily of heme enzyme can catalyze different reaction types, mainly hydroxylation. (P450 = absorbs a very characteristic wavelength (450 nm) of UV light when it is exposed to carbon monoxide) Located in the smooth endoplasmic reticulum of all major organs and tissues especially liver ▪ Use NADPH as a source of reducing equivalent ▪ Acting on both xenobiotics and endogenous compounds methene ▪ CYP enzymes catalyze the oxygenation of an organic substrate and the simultaneous reduction of molecular oxygen. ▪ If the transfer of oxygen to a substrate is not tightly controlled, uncoupling occurs and leads to the formation of reactive oxygen species ▪ In this oxidation – reduction process, two microsomal enzymes play a key role. ▪ The first enzyme:-A heme protein known as cytochrome p450, it is a terminal oxidase and plays the important role of transferring an oxygen atom to the substrate RH and convert it to ROH ▪ The second enzyme, the flavoprotein known as cytochrome p450 reductase which is Fe+3 NADPH dependent. It function as an electron carrier catalyzing the reduction of cytochrome p450 to the ferrous form by cytochrome p450 reductase transferring an electron from NADPH Cinnamate 4-hydroxylase It is a cytochrome P450 monooxygenase associated externally with the endoplasmic reticulum of plant cells. The enzyme uses NADPH-cytochrome P450 reductase as a donor of electrons and hydroxylates cinnamic acid to form 4-coumaric acid p-Coumaric acid serves as a precursor of organic compounds that are essential for plant metabolism including flavonoids and lignin cinnamic acid 4-coumaric acid ▪ Superoxide is produced by microsomal NAD(P)H dependent electron transport involving cytochrome P450 1. Cytochrome P450 reacts first with its organic substrate, RH 2. The complex is oxidized by a flavoprotein to form a radical intermediate 3. They can readily react with triplet oxygen because each has one unpaired electron. 4. This oxygenated complex may be reduced by cytochrome b or occasionally the complex may decompose releasing superoxide 3- Electron transport system with Microbodies ▪ A microbody is a type of organelles that is found in the cells of plants and animals ▪ They are two types of microbodies namely: 1. Peroxisome 2. Glyoxysome ▪ Peroxisomes and glyoxysomes are organelles with a single membrane that compartmentalizes enzymes involved in the ß-oxidation of fatty acids, and the glyoxylate acid cycle (TCA cycle) ▪ Glycolate oxidase produces H2O2 in a two-electron transfer from glycolate to oxygen ▪ Xanthine oxidase, urate oxidase and NADPH oxidase generate superoxide because of the oxidation of their substrates Peroxisome ▪ These also called microbodies are organelles found in all eukaryotic cells called peroxisome because of their ability to produce or utilize hydrogen peroxide ▪ They are small, oval or spherical in shape. They have a fine network of tubules in their matrix ▪ About 50 enzymes have been identified Glyoxysome ▪ These are found in plant ▪ They convert stored lipid into carbohydrates so they can be used for plant growth. ▪ In glyoxysome, the fatty acids are hydrolyzed to acetyl-coA by oxidation enzymes. ▪ One of the reactions catalyzed by using the coenzyme FMN involves the enzyme glycolate oxidase. ▪ Glycolate oxidase is a peroxisomal enzyme. ▪ It catalyzes the oxidation of alpha-hydroxy acids. It is one of the key enzymes in photorespiration, where it oxidizes glycolate to glyoxylate. ▪ This reaction can be divided into to main steps. First, glycolate is oxidized to glyoxylate by FMN in a two-electron transfer. Then, the reduced FMN is reoxidized by oxygen and peroxide is formed ▪ Xanthine oxidase (XO) is a form of xanthine oxidoreductase (XOR) catalyzes oxidative hydroxylation of hypoxanthine to xanthine to uric acid, accompanying the production of reactive oxygen species (ROS) x x ▪ NADPH oxidase catalyze the transfer of electron to O2 generating superoxide or H2O2 using NADPH as an electron donor. NADPH oxidase NADPH + 2O2 NADP+ + H+ + 2O2 − 4- During inflammation ▪ The tissue damage produced free radicals has to contribute to the injury process ▪ O2 − is generated by one-electron reduction of O2 through enzymatic catalysis by NADPH oxidase or xanthine oxidase (XO) ▪ H2O2 can change to highly reactive HOCl at the inflammatory sites by an enzyme known as myeloperoxidase (MPO), which is abundantly expressed in neutrophils ▪ H2O2 can also change to the highly toxic OH in presence of Fe2+ by Fenton's reaction Reaction ▪ In addition, metal containing protein (Hb) released from lysed erythrocyte at sites of inflammation. Metal containing in these proteins can react with free radical and ROS through a series of reactions called Haber – Weiss ▪ In the presence of the transition metal ion, O2 − and H2O2, in turn, generate the highly reactive OH− and OH (Haber–Weiss reaction) ▪ In the first step of this reaction, O2 − reacts with Fe3+ to form Fe2+ and O2. ▪ The second step of this reaction is also known as Fenton's reaction and occurs under the biological conditions in which Fe2+ reacts with H2O2 to form both OH and OH− 5- Ischemia ▪ Ischemia is defined as a condition of inadequate blood supply to an area of tissue ▪ It occurs during myocardial infarction and transplantation of organs ▪ As a result of oxygen deprivation ATP is metabolized to Xanthine and Hypoxanthine, while the enzyme Xanthine dehydrogenase is proteolytically converted into Xanthine Oxidase ▪ Upon reperfusion and restoration of O2 , Xanthine Oxidase metabolites Hypoxanthine and Xanthine to Uric acid, but in doing so produces O2-. ▪ The production of OFRs plays a major part in generating the tissues damage seen in Ischemia/reperfusion injury 1. NAD + is used in the metabolism of hypoxanthine by xanthine dehydrogenase in aerobic condition In ischemia (anaerobic condition) 1. Low ATP pump activity prevent the transport of Na+2 and Ca+2 ions to the outside of the cell Ca +2 2. The intracellular concentrations of Na+2 and Ca+2 ions increase 3. Calcium ion activate protease enzyme, which convert xanthine dehydrogenase to xanthine oxidase 4. The accumulated hypoxanthine cannot be transformed into xanthine until reperfusion occurs Mechanism of xanthine oxidase- mediated free radical injury 6- Phagocyte-Derived Free Radicals ▪ Phagocytes (neutrophils, macrophages, monocytes) release free radicals to destroy invading pathogenic microbes as part of the body’s defense mechanism against disease ▪ The process of phagocytosis follows five main steps: 1) Recognition & Adherence 2) Ingestion and formation of phagosome 3) Formation of phagolysosome 4) Intracellular killing 5) Elimination or exocytosis ▪ Oxygen dependent killing ▪ Activated macrophage produces ROS and RNS Respiratory burst ▪ During phagocytosis inflammatory cells (macrophage, neutrophil) produce superoxide anion by the action of NADPH oxidase ▪ The superoxide is converted to H2O2 and hypochlorous acid (HCLO) by the action of SOD and myeloperoxidase (MPO) which have bactericidal action ▪ This is the deliberate production of free radicals by the body for defence ▪ GSH is crucial for the detoxification of H2O2 that has diffused into the cytosol. G6P: glucose-6-phosphate 6PG: 6-phosphgluconate 1. The O2-. dismutes to H2O2, which can induce chloride secretion in ileum and colon, increase mucosal permeability, and increases resting tension of smooth muscle 2. In addition, the H2O2 can react with low molecular chelates of iron (e.g., ferritin that has reacted with superoxide) or copper, yielding the damaging hydroxyl radical 3. Superoxide also may interact with nitric oxide, secreted by endothelium and macrophages, to generate ONOO., which decomposes into ONOOH and, finally, the hydroxyl radical In addition to oxygen-free radicals, activated neutrophils and monocytes secrete myeloperoxidase extracellularly hypochlorous acid (HOCL), which is 100 to 1000 times more toxic than superoxide or H2O2 7- Cytokines and growth factor signaling ▪ Cytokines act by binding to specific membrane receptors, which then signal the cell via second messenger, often tyrosine kinase, to alter its behavior (gene expression) ▪ Growth factor receptors are tyrosine kinases (RTKs) that play a key role in the transmission of information from outside the cell into the cytoplasm and the nucleus. ▪ The information is transmitted via the activation of mitogen-activated protein kinases (MAPKs) signaling pathways. ROS production because of activated growth factor receptor signaling includes ▪ Tyrosine kinase receptor such as - epidermal growth factor (EGF) receptor - Platelet derived growth factor (PDGF) receptor - vascular endothelial growth factor (VEGF). ▪ cytokine receptors such as tumor necrosis factor (TNF-α) and interferon-8 (IFN-8) ▪ Interleukin receptors (IL-1β). - It is generally accepted that ROS generated by these ligand/receptor initiated pathways can function as true second messengers and mediate important cellular functions such as proliferation and programmed cell death 8- Metals, toxicity and oxidative stress ▪ Common mechanisms involving the Fenton reaction, generation of the superoxide radical and the hydroxyl radical appear to be involved for iron, copper, chromium, vanadium and cobalt primarily associated with mitochondria, microsomes and peroxisomes ▪ Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing malondialdehyde, 4-hydroxynonenal OH Fe2+ LH LOOH Lipid peroxidation Lipid hydroperoxides (Malondialdehyde , 4 hydroxynonenal) ▪ Reactions of heavy metals (mercury, cadmium, and nickel (Ni)) with sulfhydryl groups of protein ▪ Arsenic (As) is thought to bind directly to critical thiols ▪ the carcinogenic effect of metals has been related to mainly redox-sensitive transcription factors, involving AP-1 (activator protein) and p53 (This protein acts as a tumor suppressor). ▪ Experimental results have also shown a link between vanadium and oxidative stress in the etiology of diabetes. ▪ Neurodegenerative role of zinc (and copper) in the etiology of Alzheimer's disease ▪ The impact of zinc (Zn) on the immune system, the ability of zinc to act as an antioxidant to reduce oxidative stress and the neuroprotective Exogenous source of oxygen free radical 1- Ionizing Radiation ▪ Radiotherapy may cause tissue injury that is caused by free radicals ▪ Electromagnetic radiation (x ray, gamma ray) and particulate radiation (electrons, photons, neutrons, alpha and beta particles) generate primary radicals by transferring their energy to cellular component such as water ▪ the decrease in the protection ozone layer with it the increased risk of skin cancer ▪ Hydroxyl radicals are generated by ionizing radiation either directly by oxidation of water, or indirectly by the formation of secondary partially ROS ▪ Secondary radiation injury is therefore influenced by the cellular antioxidant status 2- Food ▪ All food will eventually oxidized on storage, even at temperature as low as -70oC ▪ Natural foods such as meat, fish and vegetable contain antioxidants, these can gradually be consumed or overwhelmed by oxidation process ▪ Processed foods, because of antioxidants depletion during production often have large amounts of synthetic antioxidants added to them such as: Butylated Hydroxy Toluene (BHT). Oxidative damage of ROS ▪ Oxidative stress is a state that occurs when there is an excess of free radicals in the body's cells. ▪ In biological systems there are even more complications due to the surface properties of membranes, electrical charges, binding properties of macromolecules, enzymes, substrates and catalysts. ▪ Oxidative damage in biological system - Oxidative damage to lipids - Oxidative damage to proteins - Oxidative damage to DNA Oxidative damage to lipids ▪ The deleterious effects are considered to be caused by free radicals produced during peroxide formation from fatty acids containing double bond ▪ The lipid bilayer membrane is composed of a mixture of phospholipids and glycolipids that have fatty acid chains attached to carbon 1 and 2 of the glycerol backbone by an ester linkage ▪ The peroxidation reactions differ among these fatty acids depending on the number and position of the double bonds on the acyl chain Lipid peroxidation ▪ Lipid peroxidation refers to the oxidative degradation of lipids ▪ It is the process whereby free radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage. This process proceeds by a free radical chain reaction mechanism ▪ Lipid peroxidation most often affects polyunsaturated fatty acids, because they contain multiple double bonds in between which lie methylene -CH2- groups that possess especially reactive hydrogens. ▪ The reaction consists of three major steps - Initiation - propagation - termination Initiation phase - Fatty acid radical is produced - The most initiators in living cell are reactive oxygen species (ROS), such as hydroxyl radical (OH.) - This step involves the removal of hydrogen atom (H) from polyunsaturated fatty acids (RH) to make a fatty acid radical and water RH + OH. R. + H2O Propagation phase ▪ The fatty acid radical is not a very stable molecule, so it reacts readily with molecular oxygen, thereby creating a peroxyl-fatty acid radical. ▪ This too is an unstable species that reacts with another free fatty acids, producing a different fatty acid radical and a lipid hydroperoxide (ROOH) or cyclic peroxide if it had reacted with itself. ▪ This cycle continues, as the new fatty acid radical reacts in the same way R. + O2 ROO. ROO. + RH ROOH + R. ▪ The hydroperoxides are capable of further stimulating lipid peroxidation as they can form alkoxyl (RO.) & peroxyl (ROO.)radicals 2 ROOH RO. + RO2. Termination phase ▪ The radical reaction stops when two radicals react and produce a non-radical species ▪ Living organisms have different molecules that speed up termination by neutralizing free radicals and, therefore, protecting the cell membrane. Antioxidants such as vitamin C and vitamin E may inhibit lipid peroxidation. R.+ R. R-R R. + ROO. ROOR ROO. + ROO. ROOR + O2 Process of lipid peroxidation Example: Oxidation of linoleate fatty acid which is common in cell membrane Initiation ▪ The initiation reaction between linoleate and the hydroxyl radical involves the abstraction of an H atom from the methylene group on the fatty acid (occurs at carbon 11) ▪ The remaining carbon centered radical, forms a resonance structure sharing this unpaired electron among carbons 9 to 13. ▪ The role of the hydroxyl radical is analogous to a "spark" that starts a fire ▪ At very low concentrations it initiates a chain reaction involving triplet oxygen Propagation ▪ Triplet oxygen that has two unpaired electrons may attach to this structure at either carbon -9 or -13 forming a peroxy radical ▪ The peroxy radical then abstracts an H atom from a second fatty acid forming a lipid hydroperoxide and leaving another carbon centered free radical that can participate in a second H abstraction ▪ The lipid hydroperoxide (ROOH) is unstable in the presence of Fe2+, or other metal catalysts because ROOH will participate in a Fenton reaction leading to the formation of reactive alkoxy radicals: ▪ Therefore, in the presence of Fe2+, the chain reactions are not only propagated but amplified. ▪ Among the degradation products of ROOH are - aldehydes, such as Malondialdehyde - Hydrocarbons, such as ethane and ethylene, - 4-hydroxy-2-nonenal (4-HNE) :- Major membrane lipid peroxidation product (That are commonly measured end products of lipid peroxidation) Termination ▪ The peroxidation reactions in membrane lipids are terminated when the carbon or peroxy radicals cross-link to form conjugated products that are not radical ▪ When a radical reacts with a non-radical, it always produces another radical, which is why the process is called “a chain reaction mechanism” ▪ The radical reaction stops when two radicals react and produce a non-radical species ▪ Lipid peroxidation proceeds as a chain reaction until the PUFA gets oxidized Oxidative damage to proteins ▪ Protein oxidation: modification of side chain residue in a protein or effect on the protein backbone due to oxidative stress ▪ Oxidative attack on proteins results in site-specific amino acid modifications, fragmentation of the peptide chain, aggregation of cross-linked reaction products, altered electrical charge and increased susceptibility to proteolysis ▪ Oxidative damage to protein depend on : 1- The amino acids in a peptide differ in their susceptibility to attack 2- The various forms of activated oxygen differ in their potential reactivity 3- Primary, secondary, and tertiary protein structures alter the relative susceptibility of certain amino acids. ▪ Protein oxidation results from the reaction of reactive oxygen species and reactive nitrogen species with both amino acid side chains and peptide backbone ▪ Sulphur containing amino acids and thiol groups specifically, are very susceptible sites ▪ Activated oxygen can abstract an H atom from cysteine residues to form a thiyl radical that will cross-link to a second thiyl radical to form disulphide bridges ▪ Oxygen can add to a methionine residue to form methionine sulphoxide derivatives. ▪ Methionine oxidation denatures proteins and converts the hydrophobic properties of Met into hydrophilic properties, resulting in structural alterations ▪ A protein-methionine-S-oxide reductase (MSRA) or methionine sulfoxide reductase has been measured in pea chloroplasts. This enzyme reduces the methionyl sulfoxide back to methionyl residues in the presence of thioredoxin. ▪ Many amino acids undergo specific irreversible modifications when a protein is oxidized. ▪ Irreversible oxidation products of other amino acids are most frequently - Hydroxylated and carbonylated amino acid derivatives - The oxidation of iron-sulphur centres by superoxide destroys enzymatic function. ▪ Tyrosine is readily cross-linked to form dityrosine products (side chain oxidation) ▪ Protein carbonylation 1) ROS directly attack the protein producing highly reactive carbonyl derivatives by oxidation of the side chains of histidine, lysine, proline, arginine, and serine residues. 2) The addition of reactive aldehyde or ketone groups to the side chains of amino acids -Protein carbonylation could be mediated by reactive aldehydes (4-hydroxy nonenal, malondialdehyde) ▪ The oxidative degradation of protein is enhanced in the presence of metal cofactors that are capable of redox cycling, such as Fe. ▪ In these cases, the metal binds to a divalent cation binding site on the protein. ▪ The metal then reacts with hydrogen peroxide in a Fenton reaction to form a hydroxyl radical that rapidly oxidizes an amino acid residue at or near the cation binding site of the protein. ▪ This site-specific alteration of an amino acid usually inactivates the enzyme by destruction of the cation binding site. ▪ The accumulation of such inactive proteins could functionally compete with their active counterparts. ▪ Accumulation of such protein aggregates may result in cell death. ▪ Oxidative modification of specific amino acids is one mechanism of marking a protein for proteolysis. ▪ In E. coli there are specific proteases that degrade oxidized proteins ▪ One of the most effective ones is the hydrolysis based on specific for these compartment proteases like cysteine, serine, aspartate proteases and metalloproteases Oxidative damage to DNA ▪ Activated oxygen and agents that generate oxygen free radicals, such as ionizing radiation, induce numerous lesions in DNA that cause deletions, mutations, and other lethal genetic effects. ▪ Characterization of this damage to DNA has indicated that both the sugar and the base moieties are susceptible to oxidation ▪ Can be recognized by enzyme ▪ Types of DNA damage: - Base degradation - Single or double strand breakage - Cross linking to protein Base degradation ▪ Degradation of the base will produce numerous products, including 8-hydroxyguanine, hydroxymethyl urea, urea, thymine glycol, thymine and adenine ring-opened. ▪ The highly mutagenic guanine residue 7,8-dihydro-8-oxoguanine, which is by far the most common DNA lesion formed as a result of oxidative stress ▪ It is formed by oxidation, yielding a guanine with an extra oxygen at the C8 position. ▪ The highly reactive hydroxyl radical ( OH) reacts with DNA by addition to double bonds of DNA bases and by abstraction of an H atom from the methyl group of methyl cytosine Single or double strand breakage ▪ The principal cause of single strand breaks is oxidation of the sugar moiety by the hydroxyl radical. ▪ Oxidative stress by hydroxyl radical also causes DNA damage, mainly by strand cleavage and oxidation of pyrimidine and purine bases. ▪ DNA, because of the negative charge of its phosphate groups, acts as an anion and is therefore capable of binding many cations, including those required for Fenton chemistry, like Fe2+ and Cu+. Cross-linking of DNA to protein ▪ Hydroxyl radical attack on either DNA or its associated proteins. ▪ Treatment with ionizing radiation or other hydroxyl radical generating agents causes covalent linkage such as guanine-lysine adducts, between DNA and protein ▪ Crosslinks are particularly hazardous, as they can effectively block gene transcription and DNA replication. Reaction scheme for DPC formation involving the ε- amino group of lysine (-NH2 in black) in protein and the amino group of DNA bases (-NH2 in purple). DNA-protein crosslink ▪ DNA is an obvious weak link in a cell's ability to tolerate oxygen free radical attack ▪ First, it seems that DNA is effective in binding metals that are involved in Fenton reactions ▪ secondly less damage can be tolerated in DNA than other macromolecules. As a consequence, the cell has a number of DNA repair enzymes ▪ One reason why eukaryotic organisms have compartmentalized DNA in the nucleus, away from sites of redox cycling that are high in NAD(P)H and other reductants, may be to avoid oxidative damage. Free radical damage to DNA ▪ Oxidative damage mainly occurs by formation of free radical ▪ 70 % damage by.OH ▪ Radiolysis of H2O produce peroxide ▪ Formation of. OH requires metal near to the DNA ▪ Fe+2 mainly form.OH by fenton reaction Two additional possible mechanisms by which oxidant stress may participate in tissue injury: First Mechanism a) The presence of low concentrations of H2O2 has been shown to activate the transcription factor, NF-kappa B, which can then enter the nucleus of cells, bind to DNA control elements, and induce synthesis of specific mRNAs b) This transcription factor is not only a marker of oxidant stress but also has a role in inducing viral replication, particularly HIV, and inducing the synthesis of interferons and other cytokines Second mechanism ▪ Involve the regulation of collagen synthesis by fibroblasts and lipocytes ▪ Lipid peroxide products, such as malondialdehyde, have been shown to increase transcription and synthesis of collagen by these two cell types in iron overloads other models of hepatic oxidant injury TGF-β1 (Transforming growth factor beta 1) Oxidative stress and its role in cancer ▪ It is well known that species derived from oxygen are cytotoxic and are involved in the etiology of cancer ▪ Several carcinogens during metabolism exert their effect by producing ROS, it plays a vital role in the process of carcinogenesis ▪ Free radicals linked damage of protein and DNA has been suggested to play a major role in the development of diseases such as cancer ▪ Cancer cell exhibits a higher oxidative stress level compared to normal cells ▪ This focuses on 8-hydroxy-2-deoxyguanosine (8- OHdG) and antioxidative enzymes as biomarkers for measurement of oxidative stress in different types of cancer ▪ Also deals with the product of lipid peroxidation, malondialdehyde (MDA), and across a variety of cancers ▪ These damage can result mutations that are heritable change in DNA that can yield cancer in somatic cell ▪ In general, levels of antioxidative enzymes are mostly lower in cancer patients, while 8-OHdG and MDA are higher. ▪ ROS activation of AP-1 (activator protein) and NF-kappaB (nuclear factor kappa B) signal transduction pathways, which in turn lead to the transcription of genes involved in cell growth regulatory pathways. ▪ The "two-faced" character of ROS is substantiated by growing body of evidence ▪ ROS within cells act as secondary messengers in intracellular signaling cascades, which induce and maintain the oncogenic phenotype of cancer cells ▪ ROS can also induce aging and apoptosis and can therefore function as anti- tumourigenic species. Defenses against ROS ▪ Antioxidants that are reducing agents can also act as pro-oxidants. ▪ An antioxidant is a molecule capable of inhibiting the oxidation of other molecules ▪ Oxidation reaction can form free radicals and these start chain reactions that damage cells ▪ Antioxidants terminate these chain reaction by removing free radical intermediate and inhibit other oxidation reactions Types of antioxidant There are two types: i) Enzymatic antioxidants -Superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione transferase II) Non-enzymatic antioxidant - Nutrient: carotenoid, tocopherol, vitamin c, selenium, methionine - Metabolic: glutathione, heme protein, coenzyme Q, bilirubin, urates Antioxidant enzymes 1- Superoxide dismutase (SOD) ▪ catalyze the breakdown of the superoxide anion into oxygen and hydrogen peroxide ▪ It is the major antioxidant defense system against O2 − ▪ Superoxide dismutase enzymes contain metal ion cofactors that, depending on the isozyme ▪ There are three isoforms of SOD in mammals: - The cytoplasmic Cu/ZnSOD (SOD1) - The mitochondrial MnSOD (SOD2) - The extracellular Cu/ZnSOD (SOD3) ▪ In plants, SOD isozymes are present in the cytosol and mitochondria, with an iron SOD found in chloroplasts that is absent from vertebrates and yeast 2- Catalase ▪ Catalase is a heme-containing enzyme ▪ This antioxidant enzyme can catalyze the conversion of hydrogen peroxide into water and oxygen, using either an iron or manganese cofactor ▪ All forms of the enzyme are tetramers in excess of 220,000 molecular weight. ▪ It is localized in the matrix of peroxisomes in mammalian cells. ▪ It’s cofactor is oxidized by one molecule of hydrogen peroxide and then regenerated by transferring the bound oxygen to a second molecule of substrate Two-stage mechanism of catalase action 1) The reaction cycle of the catalase begins with the ferric (III ) state, which reacts with peroxide molecule to form compound I intermediate 2) In the next step, a second hydrogen peroxide molecule is used as a reductant to regenerate the enzyme, producing water and oxygen. II) Non-enzymatic antioxidant Ascorbic acid (vitamin c) ▪ Vitamin c also known as ascorbic acid or ascorbate ▪ It is a dibasic acid with an enediol group built into a five-membered heterocyclic lactone ring ▪ It is a hydrophilic antioxidant (water soluble vitamin) ▪ Antioxidants that are reducing agents can also act as pro-oxidants. ▪ Ascorbic acid and dehydroascorbic acid. The oxidized form, dehydroascorbic acid, can be reduced back to ascorbic acid by glutathione (GSH). Function ▪ Neutralization of hydrogen peroxide ▪ Vitamin C also enhances iron absorption by reducing Fe3+ to Fe2+ ▪ Growth and repair of tissues in all parts of the body ▪ Maintain healthy collagen in skin , repair damaged tissue, healthy teeth and bones ▪ Ascorbate can directly scavenge oxygen free radicals with and without enzyme catalysts and can indirectly scavenge them by recycling tocopherol to the reduced form Synthesis ▪ Most plants and animals synthesize ascorbic acid for their own requirement. However, humans can not synthesize ascorbic acid due to lack of an enzyme gulonolactone oxidase ▪ Synthesis of ascorbate occurs in the cytosol ▪ L-ascorbic acid is synthesized from hexose sugars (d-glucose) ▪ The pathway involves the oxidation of carbon-1 of D-glucose and enediol formation between carbons 2 and 3. Mechanism of anti-oxidant activity of ascorbic acid ▪ Ascorbate has been found in the chloroplast, cytosol 1- ascorbate will react with superoxide, hydrogen peroxide or the tocopheroxyl radical to form monodehydroascorbic acid and/or dehydroascorbic acid 2- The oxidized forms are recycled back to ascorbic acid by monodehydroascorbate reductase and dehydroascorbate reductase using reducing equivalents from NAD(P)H or glutathione, respectively 3- Dehydroascorbate may decompose into tartrate and oxalate. Direct role of ascorbate The reaction with superoxide may serve a physiologically similar role to SOD 2 O2.- + 2H+ + ascorbate = 2H2O2 + dehydroascorbate ▪ The reaction with hydrogen peroxide is catalyzed by ascorbate peroxidase H2O2 + 2 ascorbate = 2H2O + 2 monodehydroascorbate Indirect role of ascorbate ▪ The indirect role of ascorbate as an antioxidant is to regenerate membrane-bound antioxidants, such as tocopherol, that scavenge peroxyl radicals and singlet oxygen, respectively: tocopheroxyl radical + ascorbate = tocopherol + monodehydroascorbate ▪ The reactions indicate that their are two different products of ascorbate oxidation, monodehydroascorbate and dehydroascorbate, that represent one and two electron transfers, respectively ▪ The monodehydroascorbate can either spontaneously dismutate or is reduced to ascorbate by NAD(P)H monodehydroascorbate reductase : 2 monodehydroascorbate = ascorbate + dehydroascorbate monodehydroascorbate + NAD(P)H = ascorbate + NAD(P)+ ▪ The dehydroascorbate is unstable at pH greater than 6 decomposing into tartrate and oxalate. ▪ To prevent this, dehydroascorbate is rapidly reduced to ascorbate by dehydroascorbate reductase using reducing equivalents from glutathione (GSH): 2 GSH + dehydroascorbate = GSSG + ascorbate reduced form Oxidized form Prooxidant effect of vitamin C ▪ The reducing agents, antioxidants, can also act as prooxidants. Vitamin C is also known to act as a prooxidant in vitro. Antioxidant vitamin C reduces oxidizing substances, such as hydrogen peroxide; however, it also reduces metal ions that generate free radicals through the Fenton reaction ▪ This is a potentially dangerous prooxidative compound; thus, vitamin C supplements are not recommended in people with high iron levels Fe3+ + ascorbate = Fe2+ + monodehydroascorbate Fe2+ + O2 = Fe3+ + O2.- Fe2+ + H2O2 = Fe3+ + OH. + OH- ▪ The cell wall is also an important site of ascorbate metabolism because it contains mM concentrations of ascorbate. ▪ The cell wall does not contain ascorbate peroxidase, but contains ascorbate oxidase. This enzyme contains 8-12 copper molecules per enzyme and catalyzes the reaction: ▪ The enzymes to recycle oxidized forms of ascorbate are not present in the cell wall, it has been proposed that the plasma membrane may have an ascorbate translocator to shuttle oxidized and reduced forms between the cytosol and cell wall. ▪ Ascorbate has been found in the chloroplast, cytosol, and extra-cellular compartments of the cell. ▪ Plant chloroplast normally exhibit relatively high concentration of ascorbate ▪ This pathway is to prevent H2O2 react with O2.- producing hydroxyl radical + H+ The redox cycling of ascorbate + alpha tocopherol (vitamin E) ▪ It is a lipid soluble and among them α-tocopherol is biologically active ▪ Most important antioxidant in the body due to chromane ring ▪ Prevent lipid peroxidation ▪ This can break the covalent links that ROS have formed between fatty acid side chains in membrane lipids. ▪ It directly acts on oxyradicals and severe important chain breaking antioxidant ▪ Vitamin E is used as treatment for many chronic diseases including Alzeimer’s disease chromane ring Antioxidant activity of tocopherol It terminates free radicals by donating a hydrogen from the hydroxyl group on its chromane ring The presence of other antioxidant such as vitamin c, is required to regenerate the antioxidant capacity of tocopherol Glutathione ▪ It is a tripeptide made of cysteine, glutamic acid and glycine ▪ It is a hydrophilic antioxidant found in every cell in human body ▪ It is the most important antioxidant due to the fact that it is directly found in cells and breakdown free radicals within in the cell ▪ GSH concentration is highest in the chloroplast ▪ Two forms of glutathione -GSH (glutathione) called reduced glutathione -GSSG (glutathione disulphide) called Oxidized glutathione ▪ Glutathione is the most abundant non-protein thiol in the cell (1 to 10 Mm depending on cell type) ▪ Glutathione is a tripeptide that has gamma linkage between the first amino acids (instead of typical alpha linkage), which resists degradation by intercellular peptidase ▪ On oxidation, the sulphur forms a thiyl radical that reacts with a second oxidized glutathione forming a disulphide bond (GSSG). Antioxidant activity of glutathione ▪ Reactive oxygen species (ROS) oxidize GSH to disulfide (GSSG) by glutathione peroxidase ▪ The reduction of GGSH to GSH is catalyzed by glutathione reductase in presence of NADH ▪ Glutathione is associated mainly with chloroplast but significantly activity is also found in the cytosol and lesser amount in the mitochondria Functions of GSH 1- It can react chemically with singlet oxygen, superoxide and hydroxyl radicals and therefore function directly as a free radical scavenge. 2- GSH may stabilize membrane structure by removing acyl peroxides formed by lipid peroxidation reactions 3- GSH is the reducing agent that recycles ascorbic acid from its oxidized to its reduced form by the enzyme dehydroascorbate reductase. GSH can also reduce dehydroascorbate by a non-enzymatic mechanism at pH > 7 4- There are alternative functions for GSH in cellular metabolism independent of its antioxidant properties 5- GSH also participates in the detoxification of xenobiotics as a substrate for the enzyme glutathione-S-transferase 6- GSH is also the precursor of the phytochelatins that act as heavy metal binding peptides in plants. Synthesis of glutathione Glutathione is synthesized in two ATP-dependent steps: ▪ First, gamma-glutamylcysteine is synthesized from L-glutamate and cysteine via the enzyme gamma-glutamylcyteine synthetase. This reaction is the rate limiting step in glutahthione synthesis ▪ Second, glycine is added to the c-terminal of gamma glutamylcysteine via the enzyme glutathione synthetase ▪ In the legumes that accumulate hGSH, the addition of alanine by the enzyme homoglutathione synthetase Glu + Cys = Glu-Cys Glu-Cys + Gly = Glu-Cys-Gly Glu-Cys + Ala = Glu-Cys-Ala  ▪ The degradation of GSH involves first the cleavage of the bond between glutamate and cysteine by glutamyl transpeptidase ▪ the transfer of the glutamate residues to an acceptor amino acid ▪ Subsequently the Cys-Gly dipeptide is degraded by dipeptidases and Glu-aa by glutamylcyclotransferase Glu-Cys-Gly + aa = Glu-aa + Cys-Gly Cys-Gly = Cys + Gly Glu-aa = 5-oxoproline + aa Nitric oxide ▪ NO is paramagnetic and is a radical ▪ NO is less reactive than many free radicals in that it cannot react with itself ▪ It is uncharged molecule having an unpaired electron ▪ Diffuse freely within and between cells across membrane ▪ NO is an important cellular signaling molecule and involved in physiological processes such as neurotransmission and the control of vascular tone ▪ On the other hand can be effective in causing cell death Synthesis of NO ▪ Nitric oxide synthase catalyzes transport of electrons for reactions between molecular oxygen and L-arginine. ▪ Nitric oxide synthases produce NO by catalyzing a five-electron oxidation of L-arginine (L-Arg). Oxidation of L-Arg to L-citrulline occurs via two step process producing Nω- hydroxy-L-arginine as an intermediate. ▪ 2 mol of O2 and 1.5 mol of NADPH are consumed per mole of Nitric oxide formed ▪ NOSs are unusual in that they require five cofactors. ▪ The electron flow in the NO synthase reaction is: NADPH → FAD → FMN → heme → O2 Isoforms of NOS ▪ There are three isoforms of nitric oxide synthase (NOS) named according to their activity or the tissue type - NOS I (neuronal NOS, nNOS) - NOS II (inducible NOS, iNOS) - NOS III (endothelial NOS, eNOS) NO release i) Constitutive NO release ▪ NOS I (nNOS) & NOS III (eNOS) ▪ They are found in cytosol and membrane ▪ Synthesize small amount of NO on demand ▪ dependent on Ca2+ and calmodulin (Synthesis NO in response to increases in intracellular calcium levels) ▪ Act on target cell e.g. vascular smooth muscles, platelets ii) Induced NO release ▪ NOS II (iNOS) ▪ Synthesis large amount of NO ▪ They are synthesized in cells after induction by bacterial endotoxins or cytokine ▪ Ca2+ and calmodulin independent (iNOS activity doesn't respond to changes in calcium levels in the cell) ▪ Acts as a killer molecule (act as immune defense mechanism)  Mechanism of action of Nitric oxide ▪ Constitutive NOS acts via calcium-calmodulin mechanism to release NO which acts on smooth muscle endothelium ▪ NO has been shown to interact with the haem group on soluble guanylate cyclase ▪ NO is the most potent activator of soluble guanylate cyclase ▪ This interaction produces a conformational change that activates the enzyme leading to increased intracellular levels of cGMP & thus muscle relaxation ▪ The role of nitric oxide as the endothelium-derived relaxing factor Properties and targets of NO 1- NO reacts with molecular oxygen to produce nitrogen dioxide 2- Nitric oxide (NO) reacts rapidly with superoxide, producing peroxynitrite - In high concentrations, NO can inhibit a variety of metabolic processes and can also cause direct damage to DNA. 3- Nitric oxide reacts with iron, present in proteins as haem or Fe-S complex - Nitric oxide may provide an intracellular signal for the regulation of iron homeostasis ▪ The main target sites for NO within the cell are thiol containing proteins ▪ In addition to thiol proteins, there are high concentrations of thiols, such as glutathione and cysteine, within the cell which form a pool that can protect the cell against oxidant stress. ▪ It can interact with thiols to produce nitrosothiols ▪ This lead to inactivate NO ▪ The half-life of nitrosothiols is considerably greater than that of free. (S-nitrosothiol) Free Radicals and antioxidants in Human Disease 1. Age-Related Macular Degeneration (AMD) ▪ AMD is eye disease may be slow or progressive due to disturbances of pigmentation of retinal pigment epithelium (RPE) ▪ Retinal metabolism results in the production of several highly reactive species of oxygen, a process which might be speeded up by exposure to light. ▪ The high content of polyunsaturated fatty acids (PUFA) in the photoreceptor outer segment membranes makes photoreceptors more susceptible to free radical damage. ▪ Antioxidant enzymes such as catalase, glutathione peroxidase, glutathione reductase and superoxide dismutase protect against oxidative damage by catalyzing decomposition of reactive oxygen species. 2. Insulin dependent diabetes mellitus (IDDM) also known as type 1 ▪ It characterized by the selective destruction of the insulin-producing β-cells in the pancreas. ▪ The levels of the vitamins and antioxidant enzymes such as SOD, CAT and GPx decreased ▪ Uric acid comprises 30-65% of the peroxyl radical-scavenging capacity of blood plasma and the reduced level of this important antioxidant in the IDDM patients may contribute to a situation of oxidative stress in vivo. ▪ Higher levels of the oxidized purine base 8-hydroxy-deoxyguanosine, a recognized biomarker of DNA damage. 3. Respiratory Diseases ▪ A type of disease that affects the lungs and other parts of the respiratory system. ▪ The fluids which line the respiratory tract form a first line of defense against the potential adverse effects of inhaled oxidants ▪ Various antioxidants are present in epithelial lining fluid (ELF), including : - Low molecular weight chain-breaking antioxidants, antioxidant enzymes and transition metal binding proteins Antioxidants in respiratory diseases ▪ Uric acid is the major low-molecular-weight antioxidant - urate is the most potent scavenger of ozone. - urate can also chelate transition metal ▪ Ascorbate is the aqueous phase chain-breaking antioxidant in plasma and makes a major contribution to antioxidant protection in the lung ▪ Glutathione is the third low-molecular-weight antioxidant ▪ Tocopherol has been measured in only low concentrations in ELF in comparison with the above antioxidants. ▪ Transferrin, ceruloplasmin, catalase and superoxide dismutase (SOD). - In the presence of lung inflammation, metals may be released from damaged cells, and transition metal binding proteins will then serve an important antioxidant function. ▪ Albumin has copper-binding activity, but will also serve as a antioxidant by its rich content of sulphydryl groups (-SH) ▪ The aim of such treatment would be to increase antioxidant levels in ELF ▪ There are two potential routes for the administration: either systemically or via inhalation of an aerosol - Glutathione cannot be administered orally, due to lack of bioavailability. However, oral or intravenous administration of N-acetylcysteine (NAC) is an effective means of increasing glutathione in plasma 4. Cystic Fibrosis (CF) ▪ CF is an inherited condition that causes sticky mucus to build up in the lungs and pancreas ▪ CF patients suffer from diminished pancreatic function, resulting in the inadequate breakdown and absorption of fat-soluble vitamins. This leads to deficiencies in α- tocopherol and β-carotene ▪ In addition, CF patients have a 1000-fold increase in lung neutrophil numbers Antioxidants in cystic fibrosis ▪ Glutathione (GSH), as it is the antioxidant in epithelial lining fluid (ELF). ▪ Mucus may play an important antioxidant role in CF - Several components in mucus, such as carbohydrates, are powerful scavengers of ROS, including OH ▪ The other major antioxidants, such as vitamin E, vitamin C, β-carotene and the GSH-Px and superoxide dismutase (SOD) enzymes, are also believed to play a role in CF Free Radical Production due to Inflammation in CF ▪ During this‘ respiratory burst’, the neutrophils have released high concentrations of the ROS. The bacterium ingested by the phagolysosome has been exposed to this high concentration of free radicals, leading to its destruction. Some of these free radicals, however, leak into the cell. ▪ Macrophages release the cytokine interleukin-8 (IL-8), which is also a neutrophil chemoattractant. ▪ Other neutrophil chemoattractants found in CF include leukotriene B4 (LTB4), and other cytokines including interleukin-1 and -6 and tumour necrosis factor-α (TNF ). ▪ During periods of inflammation or infection, neutrophil elastase (NE) is released by neutrophils. - NE is a highly destructive protease, capable of hydrolyzing all the major connective tissue proteins that form the lung matrix Free radical production due to increased metabolic rate in CF ▪ More than 95% of the oxygen we breathe is reduced by four electrons to produce water, catalyzed by the cytochrome c oxidase enzyme in the mitochondrial electron transport chain. Thus, oxygen becomes a sink for electrons. ▪ About 1.5% of the consumed oxygen is not reduced to water, but produces ROS ▪ In CF patients, an elevated resting metabolic rate is observed. Free radical production due to exogenous oxidants in CF ▪ Exogenous oxidants such as cigarette smoke (containing NO, NO2, ONOO-) and air pollution (containing NO2, O3, SO2, CC14) are metabolized by cytochrome P450 enzymes. Reduced antioxidant protection due to malabsorption in CF ▪ The pancreas produces thick secretions that often block the pancreatic ducts, preventing the digestive enzymes from reaching the intestines ▪ This amplifies oxidative injury due to severe deficiencies in fat-soluble antioxidants, such as vitamin E and carotenoids, resulting in impaired antioxidant protection. ▪ Oxidative stress markers ▪ Increased lipid peroxidation (higher plasma levels of malondialdehyde and free fatty acid hydroperoxides) ▪ Elevated protein peroxidation however, was not observed, suggesting that CF patients are more susceptible to lipid oxidative damage ▪ Reduced levels of antioxidant enzymes ▪ Lower selenium concentrations and increased levels of free iron ▪ Oxidative damage to DNA in CF patients by measuring a urinary marker, 8- hydroxydeoxyguanosine ▪ An increase in myeloperoxidase leads to increased oxidative stress. ▪ The proinflammatory cytokines tumor necrosis factor-α (TNF ) and interleukins 1, 6 and 8 (IL-1 , IL-6 and IL-8), which are produced by macrophages in response to bacteria, promote the destruction of lung tissue by encouraging production of ROS. ▪ Increased levels of plasma 8-iso-prostaglandin F2α. It is an isoprostane that is produced by the non-enzymatic peroxidation of arachidonic acid in membrane phospholipids. 5. Schizophrenia ▪ It is a brain disorder with a broad range of behavioral and biological manifestations ▪ It is more likely that the membrane abnormalities are sublethal and lead to neuronal dysfunction. ▪ Neuronal membrane phospholipids contain high proportions of essential polyunsaturated fatty acids (PUFA), primarily arachidonic acid (AA; n-6) and docosahexaenoic acid (DHA; n-3) ▪ Primarily concentrations of membrane phospholipid and essential fatty acids (EFA), may be associated with clinical features of the illness ▪ Increased blood malondialdehyde levels have been reported in schizophrenic patients 6. Alzheimer’s Disease (AD) ▪ Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by loss of memory and cognition. ▪ The most common neurological disorder in old age ▪ A hallmark of Alzheimer's disease is the build-up of toxic proteins in the brain. One of these proteins, known as amyloid beta, ▪ Amyloid beta (Aβ) causes membrane damage by lipid peroxidation ▪ One consequence of lipid peroxidation is the production of 4-hydroxynonenal, an aldehyde that covalently binds to membrane proteins involved in the regulation of ion homeostasis and impairs their function. These proteins include the Na+ /K+ -ATPase, the Ca2+-ATPase, and the glutamate and glucose transporters 7. Pregnancy ▪ Pregnancy is characterized by dynamic changes in multiple body systems resulting in increased basal oxygen consumption and in changes in energy substrate use by different organs including the fetoplacental unit. ▪ The placenta (major source of free radical) ▪ During pregnancy, the placenta is a site of active oxygen metabolism that continuously generates oxidative stress ▪ Placental macrophages in the presence of infection are a source of nitric oxide (NO), and other cytokines that induce mitochondrial alterations and production of free radicals. ▪ S-nitrosothiols, which are important circulating reservoirs of NO, increase in pregnancy. 8. Ageing ▪ Ageing is a process of becoming older in which cells accumulate free radical damage over time. ▪ Free radicals cause the progressive oxidation of protein and lipid components in the cellular membranes and also activate phospholipases, proteases and endonucleases. ▪ Increased lipid peroxidation has been implicated in the ageing process. ▪ Levels of antioxidants such as glutathione peroxidase are reported to decrease with age