1.OXIDATIVE STRESS AND ALZHEIMERS DISEASEPresented by MANDEEP KAUR M.PHARM(2ND YR.)

2. INTRODUCTON The formation of free radicals or oxidants is a well-establishedphysiological event in aerobic cells, which convene enzymic and nonenzymic resources, known as antioxidant defenses, to remove these oxidizing species. An imbalance between oxidants and antioxidants known as oxidative stress, and the consequent damage to cell molecules constitutes the basic tenet of several pathophysiological states, such as neurodegeneration, cancer, mutagenesis, cardiovascular diseases, and aging. 3. OXYGEN FREE RADICALS Oxygen is a relatively unreactive compound that can bemetabolized in vivo to form highly reactive oxidants known as oxygen free radicals.Free Radicals??? A free radical is defined as any species that contains one or moreunpaired electron occupying an atomic or molecular orbital by itself 4. Mechanisms of Formation of Oxygen Free Radicals Oxygen radicals or reactive oxygen species may be generated by: electron-transfer reactions energy-transfer reactions ENERGYTRANSFER ELECTRONTRANSFER REACTIONS REACTIONS Singlet oxygen Superoxide anion radical Triplet carbonyl compounds Hydrogen peroxide Hydroxyl radicals Lipid alkoxyl and peroxyl radicals 5. Formation of Oxidants by Electron Transfer Reactions Univalent reduction of oxygen to water with formation ofdifferent intermediates: superoxide anion radical (O2.), hydrogen peroxide (H2O2), and hydroxyl radical (HO.):+1e-+2e-O2 O2. Molecular superoxide Oxygen anionH2O2 hydrogen peroxide+1e-HO. hydroxyl radicalH2O water 6. HOW REACTIVE ARE OXYGEN RADICALS?Reactivity of Superoxide Anion The reactivity of superoxide radical is dependent on the cellular environment. Two reactions are important in a cellular setting, which change the chemical reactivity of superoxide anion (O2.): Reactivity of superoxide anion with itself O2. + O2. + 2H + H2O2 + O2 Protonation of superoxide anion O2. + H+ HO2. 7. Reactivity of Hydrogen Peroxide Hydrogen peroxide (H2O2) is not a free radical, but it may be considered as an oxidant. Per se, hydrogen peroxide (H2O2) is little reactive. Its reactivity in biological systems depends on two properties: It can diffuse long distances crossing membranes It reacts with transition metals by a homolytic cleavage yielding the highly reactive hydroxyl radical (HO.).Reactivity of Hydroxyl radical The chemical reactivity of hydroxyl radical (HO.) may be assumed to encompass two main reactions: 8. Hydrogen abstractionRH + HO. R. + H2O Addition reactions 9. HOW ARE OXYGEN RADICALS GENERATED IN THE CELL? Sources of Superoxide Radical SourcePathophysiological SignificanceEnzymic reactions - xanthine oxidase Intestinal ischemia/reperfusion - NADH oxidase Present in leukocytes: bactericidal activity - NADPH-cytochrome P450 reductase Cellular sources - leukocytes and macrophages Bactericidal activity - mitochondrial electron transfer - microsomal monooxygenase Environmental factors ultraviolet light - X rays - toxic chemicals - aromatic hydroxylamines - aromatic nitro compounds - insecticides, such as paraquat - chemotherapeutic agents, such as quinone 10. CONTI Mitochondria are major cellular sources of reactive oxygen species. Mitochondria consume oxygen associated with the process of oxidative phosphorylation. Under normal conditions, approximately 95-97% of the oxygen is reduced to water; a small fraction of the oxygen consumed (3-5%) is reduced univalently to superoxide anion (O2.). Coenzyme Q or ubiquinone is a mobile electron carrier in the respiratory chain and it collects electrons from complex I and complex II. The coenzyme Q pool faces both the intermembrane space and the mitochondrial matrix. Coenzyme Q or ubiquinone is reduced by Complex I and Complex II and donates electrons to complex III (the bc1 segment). Because of these redox transitions, ubiquinone exists as a quinone (fully oxidized), semiquinone, and hydroquinone (fully reduced): Electron leakage, accounting for about 3-5% of the total oxygen consumed by mitochondria, is associated with the generation of oxygen radicals: Ubisemiquinone donates one electron to molecular oxygen yielding superoxide anion and ubiquinone; this is known as autoxidation of ubisemiquinone. 11. Sources of Hydrogen Peroxide Hydrogen peroxide (H2O2) is generated within the cell by two distinct processes: Nonradical or enzymic generation The following enzymes do generate hydrogen peroxide (H2O2) upon reduction of their co-substrate, molecular oxygen: glycolate oxidase, D-amino acid oxidase, urate oxidase, acetyl-CoA oxidase ,NADH oxidase and monoamine oxidase The latter enzyme, monoamine oxidase (MAO) occurs in two forms A and B and it catalyzes the oxidative deamination of biogenic amines. It is present in the outer mitochondrial membrane. Radical generation or from superoxide anion disproportionation This is achieved upon dismutation or disproportionation of superoxide anion (O2.), according to the reaction mentioned before: O2. + O2. + H+ H2O2 + O2 12. Sources of Hydroxyl Radical Most of the hydroxyl radical (HO.) generated in vivo, exceptfor that during excessive exposure to ionizing radiation, originates from the breakdown of hydrogen peroxide (H2O2) via a Fenton reaction. The Fenton reaction entails a metal-dependent reduction of hydrogen peroxide (H2O2) to hydroxyl radical (HO.). Transition metals, such as copper (Cu), iron (Fe), and cobalt (Co), in their reduced form catalyze this reaction: Fe++ + H2O2 Fe+++ + HO + HO. 13. Summary of Cellular Sources of Oxygen Radicals Cellular sources enzymic reactions leukocytes macrophages mitochondria microsomes Environmental factors UV light X rays toxic chemicalsO2O2.Cellular sources enzymic reactions leukocyte macrophages mitochondria H2O2No cellular sources Fenton reactionHO.DISMUTATIONMitochondrial Respiratory ChainH2O 14. HOW DO OXIDANTS MEDIATE CELLULAR DAMAGE? LIPID PEROXIDATIONDNA OXIDATION 15. HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? The cell convenes specific enzymic defenses against oxygen radical attack, which can be considered preventive antioxidants. On the other hand, there exist small antioxidant molecules, which can react with a variety of free radicals and that may be considered as chain-breaking antioxidants. Specific enzymic defenses or preventive antioxidants Mammalian cells contain specific enzymes, which remove either superoxide anion or hydrogen peroxide, the two required precursors of hydroxyl radical (HO.). Removal of Superoxide Anion: Superoxide Dismutases Superoxide anion radical is formed by different nonenzymic and enzymic reactions within the cell. Superoxide dismutases (abbreviated SOD) catalyze the rapid dismutation of superoxide radical to hydrogen peroxide and oxygen. The rate of this reaction is 10,000-fold higher than that of the spontaneous dismutation. 16. O2.+ O2. + 2H+ H2O2 + dismutation O2. + O2. + 2H+ H2O2 + O2Removal of Hydrogen Glutathione PeroxidasesO2spontaneous, nonezymic enzymic dismutationPeroxide:CatalaseandCatalase: This enzyme is located in the peroxisomes and catalyses the following reaction: H2O2 + H2O2 2H2O + O2 Glutathione Peroxidase: The enzyme occurs in cytosol and the mitochondrial matrix and it requires glutathione, a tripeptide present in high concentrations in most mammalian cells. During this reaction hydrogen peroxide (H2O2) is reduced to water and glutathione (GSH) is oxidized to glutathione disulfide (GSSG). H2O2 + 2GSH H2O + GSSG 17. catalase O2O2.H2O2HO.H 2Oglutathione peroxidasesuperoxide dismutase 2 GSHGSSG 18. Increasing evidence suggests that the generation ofthese oxygen free radicals plays an important role in the pathophysiology of at least three disease states: ischemia reperfusion injury, phagocytedependent inflammatory damage, and neurodegenerative disorders as well as aging. 19. Arteriosclerosis AsthmaAlzheimersOxidative stressDiabetesParkinsonsIschemiaCardiovascular diseasesArthritis Cancer 20. Alzheimers disease Alzheimers disease (AD) is a neurodegenerative disorder associated with adecline in cognitive impairments, progressive neurodegeneration and formation of amyloid- (A) containing plaques and neurofibrillary tangles. (Nain et al., 2011) Alzheimer's disease (AD) is a slowly progressive disease of the brain that ischaracterized by impairment of memory and eventually by disturbances in reasoning, planning, language, and perception. Mutationsof amyloid precursor protein or presenilin genes orapolipoprotein E gene polymorphism appear to affect amyloid formation,which in turn causes neuronal death via a number of possible mechanisms, including Ca2+ homeostasis disruption, oxidative stress, excitotoxicity, energy depletion, neuro- inflammation and apoptosis. 21. PATHOPHYSIOLOGY OF AD 22. Oxidative stress in AD?? Oxidative stress occurs when there is an imbalance between the production and quenching of free radicals from oxygen species. These reactive oxygen species (ROS) play a role in many chronic diseases including mitochondrial diseases, neurodegenerative diseases, renal disease, arteriosclerosis, diabetes , cancer and SLE . The process of aging is also associated with increased oxidative stress. Through pathological redox reactions ROS can denature biomolecules such as proteins, lipids and nucleic acids. This can initiate tissue damage via apoptosis and necrosis. Oxidative stress plays a central role in the pathogenesis of AD leading to neuronal dysfunction and cell death. Peripheral markers of oxidative stress are elevated in AD indicating that the damage is not brain-limited. One study suggested that the level of oxidative markers is directly related to the severity of cognitive impairment. 23. Conti The increased level of oxidative stress in the AD brain is reflectedby increased protein and DNA oxidation, decreased level of cytochrome c oxidase and advanced glycosylation end products. enhanced lipid peroxidation, Lipid peroxidation can weaken cell membranes causes ion imbalance and impair metabolism. Oxidative stress can influence DNA methylation which regulates gene expression. Internalized beta-amyloid may play a role in this process. Mitochondrial dysfunction, which is associated with an accumulation of ROS, appears to play a role in the early events of AD pathology. 24. Conti.... A recent study, using mouse models, showed that mitochondria targetedantioxidant catalase helps prevent abnormal beta-amyloid processing decreasing plaque burden. There is also evidence that beta-amyloid deposits lead to more mitochondrial damage. Beta-amyloid peptide has been shown to inhibit cytochrome oxidase leading to disruption of the electron transport chain and production of ROS. Thus a viscous cycle may be initiated that culminates in progressive disease. Under stressful conditions and in aging, the electron transport system can increase ROS formation considerably. Thus, the mitochondria are both a source and a target of toxic ROS. Mitochondrial dysfunction and oxidative metabolism may play an important role in the pathogenesis of AD and other neurodegenerative diseases. 25. Oxidative Stress Response e.g. Neurotrophic factors, Neurogenesis, DNA repair etcAdaptation Responses Failure to adapt ROS/RNS ApoptosisNecrosisOxidation of proteins, lipids and DNAOrganelle dysfunctionCalcium dysregulation 26. Oxidative radicals mechanisms of oxygen radicals generation in Alzheimers disease SUPEROXIDE RADICAL A dismutation reaction of superoxide radical leading to the formation ofhydrogen peroxide and oxygen can occur spontaneously or is catalysed by the enzyme superoxide dismutase (SOD). There are three distinct types of SOD classified on the basis of the metal cofactor: the copper/zinc (Cu/ZnSOD, cytoplasmic), the manganese (MnSOD, mitochondrial) and the iron (FeSOD) isozymes (Bannister et al, 1987). Superoxide can act as either an oxidant or a reductant. It can oxidize sulphur, ascorbic acid or NADPH and it can reduce cytochrome c and metal ions. Superoxide forms the perhydroxyl radical (.OOH), which is a powerful oxidant (Gebicki and Bielski, 1981), but its biological relevance is probably minor because of its low concentration at physiological pH. 27. Conti HYDROGEN PEROXIDE Numerous enzymes (peroxidases) use hydrogen peroxide as asubstrate in oxidation reactions involving the synthesis of complex organic molecules. The well-known reactivity of hydrogen peroxide is not due to its reactivity per se, but requires the presence of a metal reductant to form the highly reactive hydroxyl radical, which is the strongest oxidizing agent known and reacts with organic molecules at diffusion-limited rates. The reaction of Fe2++ with H2O2 produces the highly reactive hydroxyl radical (.OH) via Fenton reaction. In biological systems the availability of ferrous ions limits the rate of reaction, but the recycling of iron from the ferric to the ferrous form by a reducing agent can maintain an ongoing Fenton reaction leading to the generation of hydroxyl radicals. Haber and Weiss (1934) identified reaction resulting into .OH formation through an interaction between O2. and H2O2 in the presence of Fe2+ or Fe3+. 28. LIPID PEROXIDATION Increased lipid peroxidation occurs in the brain in AD and is mostprominent where degenerative changes are most pronounced. Brain membrane phospholipids are composed of polyunsaturated fatty acids, which are especially vulnerable to free radical attack because their double bonds allow easy removal of hydrogen ions. Decreases in polyunsaturated fatty acids, primarily arachidonic and docosahexaenoic acids, accompany lipid peroxidation in AD. Oxidation of polyunsaturated fatty acids produces aldehydes, one of the most important of which is 4-hydroxynonenal (HNE), a highly reactive cytotoxic substance capable of inhibiting glycolysis, nucleic acid and protein synthesis, and degrading proteins. Glutathione transferases, a group of enzymes that inactivate the toxic products of oxygen metabolism including 4-hydroxyalkenals such as HNE, are markedly diminished in multiple brain regions and in the CSF in subjects with AD, suggesting a loss of protection against HNE. 29. PROTEIN OXIDATION The oxidation of proteins by free radicals may also play a meaningful role in AD. Hydrazide-reactive protein carbonyl is a general assay of oxidative damage to protein. Several studies demonstrate an increase in protein carbonyls in multiple brain regions in subjects with AD and in their NFTs. The oxidation of brain proteins may be at the expense of enzymes critical to neuron and glial function. Two enzymes that are especially sensitive to oxidative modification are glutamine synthetase and creatine kinase, both of which are markedly diminished in the brains of subjects with AD. Oxidative alterations in glutamine synthetase could cause alteration of glutamate concentrations and enhance excitotoxicity, whereas oxidative impairment of creatine kinase could cause diminished energy metabolism in AD. 30. Four-hydroxynonenal causes degeneration and death of cultured hippocampalneurons by impairing ion motive adenosine triphosphatase activity and disrupting calcium homeostasis.6 Exposure of cultured hippocampal neurons to amyloid (A) peptide causes a significant increase in levels of free and protein-bound HNE and increases ROS. Four-hydroxynonenal impairs glucose and glutamate transport and is capable of inducing apoptosis in cultured neurons. Administration of HNE into the basal forebrain of rats causes damage to cholinergic neurons, diminished choline acetyltransferase, and impaired visuospatial memory. 31. Endogenous antioxidants Biological systems have evolved with endogenous defense mechanisms tohelp protect against free radical induced cell damage. Glutathione peroxidase, catalase and superoxide dismutases are antioxidant enzymes, which metabolize toxic oxidative intermediates. They require micronutrient as cofactors such as selenium, iron, copper, zinc, and manganese for optimum catalytic activity and effective antioxidant defence mechanisms. SOD, catalase, and glutathione peroxidase are three primary enzymes, involved in direct elimination of active oxygen species (hydroxyl radical, superoxide radical, hydrogen peroxide) whereas glutathione reductase, glucose-6-phosphate dehydrogenase, and cytosolic GST are secondary enzymes, which help in the detoxification of ROS by decreasing peroxide levels or maintaining a steady supply of metabolic intermediates like glutathione and NADPH necessary for optimum functioning of the primary antioxidant enzymes. 32. Exogenous antioxidants The most widely studied dietary antioxidants are vitamin C,vitamin E, and beta-carotene. Vitamin C is considered the most important water-soluble antioxidant in extracellular fluids, as it is capable of neutralising ROS in the aqueous phase before lipid peroxidation is initiated. Vitamin E is a major lipid-soluble antioxidant, and is the most effective chain-breaking antioxidant within the cell membrane where it protects membrane fatty acids from lipid peroxidation. Beta-carotene and other carotenoids also provide antioxidant protection to lipid rich tissues. Fruits and vegetables are major sources of vitamin C and carotenoids, while whole grains, i.e., cereals and high quality vegetable oils are major sources of vitamin E. 33. Drugs for AD with Common Adverse effects Nausea, diarrhea, insomnia, vomiting, muscle DonepezilRivastigminecramps, fatigue, and anorexia Nausea, vomiting, loss of appetite, dyspepsia, asthenia, and weight lossMemantine Confusion,dizziness,headache,andconstipationTacrine hepatotoxicity (Morrison et al.,2005) 34. PLANTS USED IN MANAGEMENT OF AD Melissa officinalisLycorus radiataRosemarinus officanalisCurcuma longaSalvia officanalisTinospora cordifoliaAllium sativumCentella asiaticaMacleaya cordataSecurinega suffruticosaGalanthus woronowiiCoptis chinensesAzadirachta indicaWithania somniferaGinkgo bilobaCatharanthus roseus(Raghavendra et al., 2013, Kapoor et al., 2011, Sandhya et al., 2010) 35. REFERENCES Filipcik P, Cente M, Ferencik M, Hulin, Novak: The role of oxidativestress in the pathogenesis of Alzheimers disease. 2006; 107 (910): 384394 Ravindra Pratap Singh, Shashwat Sharad, Suman Kapur: Free Radicals and Oxidative Stress in Neurodegenerative Diseases: Relevance of Dietary Antioxidants. JIACM 2004; 5(3): 218-25 FREE RADICALS, OXIDATIVE STRESS, AND DISEASES. ENRIQUE CADENAS, PSC 61