Uploaded byNoel Martin Bautista
A Student's Prayer
The document discusses oxygen metabolism and toxicity. It begins by describing the properties of oxygen and how it can form reactive oxygen species (ROS) through one-electron reductions. The major ROS - superoxide, hydrogen peroxide, and hydroxyl radical - are introduced along with their sources within the cell, such as from the electron transport chain, respiratory burst in phagocytes, and various oxidases and oxygenases. Exogenous sources of ROS like radiation, drugs, smoking, and pollutants are also noted. The document concludes by outlining how ROS can damage important cellular components through radical reactions and cause diseases.
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A Student's Prayer
- 1. A Student’s Prayer Father, I have knowledge, so I pray you'll show me now How to use it wisely and find a way somehow To make the world I live in a little better place, And make life, with its problems, a bit easier to face... Grant me faith and courage and put purpose in my days, And show me how to serve Thee in the most effective ways So all my education, my knowledge and my skill, May find their true fulfillment as I learn to do Thy will... And may I ever be aware in everything I do That knowledge comes from learning - And wisdom comes from You. 1
- 2. Oxygen Metabolism and OxygenToxicity NOEL MARTIN S. BAUTISTA, MD, DPPS, MBAH Department of Biochemistry, Molecular Biology and Nutrition
- 3. Oxygen Metabolism and Toxicity Properties of Oxygen / O2 Properties of ROS Major Sources of ROS in the Cell Oxygen Radical Reactions with Cellular Components Cellular Defenses Against Oxygen Toxicity 3
- 4. Oxygen: The Elementof Life one of the most abundant elements on this planet earth's crust (46.6% by weight), oceans (86% by weight), atmosphere (21% by volume) comes from the Greek stems oxys, "acid," and gennan, "to form or generate, " literally means “acid former” term introduced by Lavoisier, who noticed that compounds rich in oxygen (eg, SO2) when dissolved in water generate acids 4
- 5. Oxygen: Chemistry colorless, odorless diatomic molecule with the formula O2 two oxygen atoms are bonded bond has a bond order of two, and is thus often simplified in description as a double bond 5
- 6. Oxygen: Chemistry chemical element with the chemical symbol O atomic number 8 valency of 2 8 neutrons, 8 protons, 8 electrons 6
- 7. Oxygen: Chemistry electron configuration of the molecule has two unpaired electrons occupying two molecular orbitals orbitals are classified as anti- bonding, so the diatomic oxygen bond is weaker than the diatomic nitrogen bond, where all bonding molecular orbitals are filled unpaired electrons are commonly associated with high reactivity in chemical compounds 7
- 8. Properties of Oxygen biradical molecule 2 single electrons in different orbitals with parallel spins high tendency to form toxic reactive oxygen species (ROS) 8
- 9. Oxygen: Toxicity Most of the damaging effects of oxygen can be explained by oxygen free radicals - Gershman and Gilbert, 1954 9
- 10. O2: Radical Nature radicals - molecules that possess a single unpaired electron in an orbital highly reactive and can initiate chain reactions Paired Electrons Unpaired Electron 10 Stable Molecule Free Radical
- 11. O2: Radical Nature Free radical low chemical specificity “steals” electrons from nearest stable molecule free radical chain reaction Cell damage 11
- 12. O2: Reduction Products O2 is capable of accepting 4 electrons, reducing it to water 4-electron reduction steps for O2 progressively generate superoxide, hydrogen peroxide, and the hydroxyl radical plus 12 water
- 13.
- 14. ROS: Properties major oxygen metabolites produced by one-electron reduction of oxygen react indiscriminately by extracting electrons from other molecules include oxygen ions, free radicals and peroxides levels can increase dramatically with environmental stress resulting to significant damage to cell structures (oxidative stress) 14
- 15. Reactive Oxygen Species O2- superoxide radical H2O2 hydrogen peroxide OH• hydroxyl radical R•, RO•, R-S• organic radicals RCOO• organic peroxide radical HOCL hypochlorous acid 1O singlet oxygen 2 NO nitric oxide ONOO- peroxynitrite 15
- 16. ROS: Superoxide Anion O2- can be formed from free O2 by donation of an electron to another free radical highly reactive but has limited lipid solubility and cannot diffuse far from site of origin contains one additional unpaired electron 16
- 17. ROS: Superoxide Anion reacts non- enzymatically with hydrogen peroxide in the Haber Weiss reaction to generate other ROS (hydroxyl and hydroperoxy radicals) 17
- 18. ROS: Superoxide Anion Sources: produced by the ETC other sites 18
- 19. ROS: Hydrogen Peroxide H 2O 2 contains two additional paired electrons formed by two- electron reduction of oxygen not a free radical, but a weak oxidizing agent 19
- 20. ROS: Hydrogen Peroxide classified as ROS because it can generate the hydroxyl free radical by reaction with a transition metal (Fe2+) in the non-enzymatic Fenton Reaction 20
- 21. ROS: Hydrogen Peroxide lipid soluble and thus can diffuse into and through cell membranes dismutation Reaction 2O2- + 2H+ H2O2 + O2 precursor of the powerful oxidizing agent, hypochlorous acid (HOCl) 21
- 22. ROS: Hydroxyl Radical OH• most reactive species in attacking biological molecules produced by H2O2 in the presence of Fe++ or Cu+ (Fenton Reaction) or via the Haber-Weiss reaction one of its damaging immediate effects is the initiation of lipid peroxidation 22
- 23. ROS: Organic Radicals R•, RO•, R-S• organic free radical produced from RH by superoxide or •OH attack by extracting electrons RH can be the carbon or a double bond in fatty acid (resulting in –C• =C-) or RSH (resulting in R-S•) 23
- 24. ROS: Organic Peroxide Radical RCOO• organic peroxyl radicals, such as occurs during lipid degradation (also denoted as LOO•) important reaction because the primary molecules that undergo this chemistry are the PUFAs Allylic carbonyl radicals are generated; organic peroxyl radical participates in a chain reaction of lipid oxidations cell membrane damage and death 24
- 25. ROS: Hypochlorous Acid (Hypochlorite) HOCl produced in neutrophils (respiratory burst) to destroy invading organisms; toxicity via halogenation and oxidation reactions generated by myeloperoxidase on Cl- ions in the presence of H2O2 H2O2 + Cl- HOCl + OH- can lead to formation of more toxic ROS (OH•) HOCl + O2- •OH + Cl- + O2 HOCl + Fe2+ •OH + Cl- + Fe3+ 25
- 26. ROS: Singlet Oxygen high energy species of oxygen molecule with anti-parallel spins no unpaired electrons, but one orbital is completely empty highly reactive can react with organic conjugated double bonds to form endoperoxides, dioxetanes and hydroperoxides produced at high-oxygen tensions from absorption of UV light Decays rapidly, not significant 26
- 27. ROS: Nitric Oxide NO free radical produced endogenously by nitric oxide synthase endothelium derived relaxing factor synthesized from arginine via action of nitric oxide synthetase binds to metal ions combines with O2 or other oxygen-containing radicals to produce additional RNOS example: peroxynitrite (strong oxidizing agent) O2-• + NO• ONOO- 27
- 28. Sources of ROSin the Cell
- 29. A. Coenzyme Q major source of superoxide ETC “leaks” free radicals at CoQ The one-electron reduced form of CoQ (CoQH•) is free within the membrane and can accidentally transfer an electron to dissolved O2, thereby forming the superoxide 29
- 30. B. Respiratory Burst process by which phagocytic cells consume large amounts of oxygen during phagocytosis and release ROS major source of superoxide anion, hydrogen peroxide, hydroxyl radical, and hypochlorite (HOCl), nitric oxide 30 (NO) and other free
- 31.
- 32. B. Respiratory Burst 1. NADPH Oxidase catalyzes the transfer of an electron from NADPH to O2 to form superoxide activation of NADPH oxidase initiates the respiratory burst at the cell membrane superoxide 32
- 33. B. Respiratory Burst 2. Superoxide Dismutase (SOD) H2O2 4. Fenton Reaction hydroxyl free radical 33
- 34. B. Respiratory Burst 3. Myeloperoxidase formation of hypochlorous acid from H2O2 is catalyzed by myeloperoxidase hypochlorous acid is a powerful toxin that destroys bacteria within seconds through halogenation and oxidation reactions 34
- 35. B. Respiratory Burst 5. Nitric Oxide Synthase generates NO NO reacts rapidly with superoxide to generate peroxynitrite, whi ch forms additional RNOS 35
- 36. C. Oxidases, Oxygenases and Peroxidases oxidases, peroxidases and oxygenases in the cell bind O2 and transfer single electrons to it via a metal free radical intermediates of these reactions may be accidentally released hydrogen peroxide and lipid peroxides are generated enzymatically as major reaction products by a number of oxidases present in peroxisomes, mitochondria and the endoplasmic reticulum 36
- 37. C. Oxidases, Oxygenases and Peroxidases Examples: Cytochrome P450 enzymes – major source of free radicals “leaked” from reactions Monoamine oxidase oxidatively degrades the neurotransmitter dopamine and generates H2O2 Peroxisomal fatty acid oxidase generates H2O2 rather than FAD (2H) during the oxidation of very long chain fatty acids Xanthine oxidase, an enzyme of purine degradation that can reduce O2 to O2- or H2O2 in the cytosol; major contributor to ischemia-reperfusion injury Lipid peroxides are formed enzymatically as intermediates in the synthesis of many eicosanoids 37
- 38. D. Exogenous Sources ionizing radiation drugs tobacco smoking alcohol consumption inorganic substances gases (ozone) 38
- 39. Exogenous Sources: Ionizing Radiation electromagnetic radiation generate primary radicals by transferring their energy to cellular components such as water its high energy level can split water into hydroxyl and hydrogen radicals radiation damage to skin, mutations, cancer and cell death may generate organic radicals through direct collision with organic cellular components 39
- 40. Exogenous Sources: Drugs appear to increase free radical production in the presence of increased oxygen tensions (hyperoxia) antibiotics (quinoid groups or bound metals) antineoplastics (bleomycin, anthracyclines, methotrexate) 40
- 41. Exogenous Sources: Tobacco Smoking tobacco smoke contains enormous amount of oxidant material (aldehydes, epoxides, peroxides, NO and semiquinones) that may cause damage to the alveoli micro-hemorrhages causes iron deposition in the smokers’ lung tissue formation of the lethal hydroxyl radical from H2O2 (Fenton reaction) elevated amounts of neutrophils found in the lower respiratory tract of smokers increased formation of free radicals smoke oxidants deplete intracellular antioxidants 41
- 42.
- 43. Exogenous Sources: Alcohol Consumption excessive alcohol ingestion induce oxidative reactions in the liver 43
- 44. Exogenous Sources: Inorganic Particles asbestos, quartz, silica (mineral dust) Leads to cell rupture lung injury ↑ production of ROS 44
- 45. Exogenous Sources: Inorganic Particles Phagocytosis by pulmonary macrophages Release proteolytic enzymes and chemotactic factors inflammation Increased free radical and ROS production 45
- 46. Exogenous Sources: Gases (Ozone) not a free radical; highly potent oxidizing agent contains two unpaired electrons and degrades under physiological conditions to hydroxyl radicals photo-dissociation of chlorofluorocarbons (aerosol sprays) can lead to chlorine radicals Cl• 46
- 47. Oxygen Radical Reactions withCellular Components
- 48. Free Radical Mediated Cellular Injury 48
- 49. Diseases Associated with Free Radical Injury Atherogenesis Cerebrovascular disorders Emphysema bronchitis Ischemia/reperfusion injury Duchenne-type muscular Neurodegenerative disorders dystrophy Pregnancy/pre-eclampsia Amyotrophic lateral sclerosis Cervical cancer Alzheimer’s disease Alcohol-induced liver disease Down’s syndrome Hemodialysis Ischemia/reperfusion injury following stroke Diabetes Mitochondrial DNA disorders Acute renal failure Multiple sclerosis Aging Parkinson’s disease Retrolental fibroplasia 49
- 50. A. Membrane Attack formation of lipid and lipid peroxy radicals free radical auto-oxidation requires an initiator (e.g., hydroxyl radical from Fenton reaction) to begin the chain reaction 50
- 51. Lipid Peroxidation: 1.Initiation initiated by a hydroxyl or other radical that extracts a hydrogen atom from polyunsaturated lipid (LH), thereby forming a lipid radical (L•) 51
- 52. Lipid Peroxidation: 2. Propagation free radical chain reaction is propagated by reaction with O2, forming the lipid peroxy radical (LOO•) and lipid peroxide (LOOH) 52
- 53. Lipid Peroxidation: 3. Degradation rearrangements of the single electron result in degradation of the lipid malondialdehyde, one of the compounds formed, is soluble and appears in blood and urine (marker of free radical damage) 53
- 54. Lipid Peroxidation: 4. Termination chain reaction can be terminated by Vitamin E and other lipid-soluble antioxidants that donate single electrons. two subsequent reduction steps form a stable, oxidized antioxidant 54
- 55. Lipid Peroxidation: Effects invariably changes or damages lipid molecular structure the aldehydes that are formed can cross- link with proteins disrupts the cohesive lipid bilayer arrangement and stable structural organization disruption of mitochondrial membrane integrity may result in further free radical production 55
- 56. B. Proteins andPeptides proline, histidine, arginine, cysteine and methionine: most susceptible to OH• attack and oxidative damage protein may fragment or residues may cross-link damaged cysteine residues cross-link and form aggregates that prevent their degradation oxidative damage increases the susceptibility of other proteins to proteolytic digestion free radical attack and oxidation of the cysteine sulfhydryl residues of glutathione increases oxidative damage throughout the cell 56
- 57. C. DNA Damage oxygen-derived free radicals are a major source of DNA damage non-specific binding of Fe2+ to DNA facilitates localized production of the hydroxyl radical (Fenton reaction) causes strand breaks and base alterations in the DNA 57
- 58. Cellular Defenses Against OxygenToxicity
- 59. Oxidative Stress occurs when the rate of ROS production over-balances the rate of their removal by cellular defense mechanisms 59
- 60. Cellular Defense Systems: Components antioxidant scavenging enzymes non-enzymatic antioxidants (free radical scavengers) endogenous antioxidants metal chelators other cellular defenses compartmentation repair mechanisms 60
- 61. Cellular Defense Systems: Overview 61
- 62. A. Anti-Oxidant Scavenging Enzymes 2O2- Superoxide dismutase H2O2 OH . 2H+ O2 NADP+ 2GSH GSH GSH H2O2 peroxidase reductase GSSG NADPH Catalase + H+ 2H2O + O2 2H2O 62
- 63. A. Anti-Oxidant Scavenging Enzymes Superoxide Dismutase primary defense against oxidative stress speeds up dismutation of O2- H2O2 and O2 Exists as three isoenzyme forms Cu+-Zn2+ – cytosol Mn2+ – mitochondria Cu+-Zn2+ – extracellular activity of Cu+-Zn2+ SOD is by chemicals or conditions (such as hyperbaric oxygen) that increase the production of superoxide 63
- 64. A. Anti-Oxidant Scavenging Enzymes Catalase heme-containing enzyme catalyzing dismutation of hydrogen peroxide into water and oxygen found principally in the peroxisomes and cytosol and microsomal fraction of the cell highest activities are found in tissues with a high peroxisomal content (kidney and liver) in the immune system, catalase serves to protect the cell against its own respiratory burst 64
- 65. A. Anti-Oxidant Scavenging Enzymes Glutathione Peroxidase and Glutathione Reductase 65
- 66. Glutathione -glutamylcysteinyl- glycine one of body’s principal substances against oxidative damage sulfhydryl group oxidized to a disulfide, transferring electrons to H2O2 to produce water 66
- 67. A. Anti-Oxidant Scavenging Enzymes Glutathione Peroxidase one of principal means of protection against oxidative damage catalyzes the reduction of H2O2 and lipid peroxides (LOOH) by glutathione reactive sulfhydryl groups reduce H2O2 to water and lipid peroxides to non-toxic alcohols two glutathione molecules are oxidized to form a single molecule, glutathione disulfide (GSSG) 67
- 68. A. Anti-Oxidant Scavenging Enzymes Glutathione Peroxidase sulfhydryl reactions are also oxidized in non-enzymatic chain terminating reactions with organic radicals within cells, found principally in the cytosol and mitochondria, and are a major means for removing H2O2 produced outside of peroxisomes contribute to our dietary requirement for selenium and account for the protective effect of selenium in the prevention of free radical injury 68
- 69. A. Anti-Oxidant Scavenging Enzymes Glutathione Reductase reduces oxidized glutathione (GSSG) back to the reduced form contains an FAD and catalyzes transfer of electrons from NADPH to the disulfide bond of GSSG NADPH is essential for protection against free radical injury; major source is the pentose phosphate pathway (HMP) 69
- 70. B. Nonenzymatic Antioxidants (Free Radical Scavengers) convert free radicals to a nonradical nontoxic form in nonenzymatic reactions mostly are antioxidants compounds that neutralize free radicals by donating a hydrogen atom (with its one electron) to the radical reduce free radicals but themselves are oxidized 70
- 71. B. Nonenzymatic Antioxidants (Free Radical Scavengers) common structural feature: conjugated double bond system a system of atoms covalently bonded with alternating single and multiple bonds (e.g., C=C-C=C-C) enables the electrons to be delocalized over the whole system and so be shared by many atoms delocalized electrons may move around the whole system 71
- 72. B. Nonenzymatic Antioxidants (Free Radical Scavengers) common structural feature: conjugated double bond system may also be an aromatic ring example: phenol (C6H5OH, benzene with hydroxyl group) 72
- 73. B. Nonenzymatic Antioxidants (Free Radical Scavengers) Vitamin E -tocopherol lipid soluble efficient antioxidant and terminator of free radical chain reactions; radical trap little pro-oxidant activity; most widely distributed; most potent anitoxidant 73
- 74. B. Nonenzymatic Antioxidants (Free Radical Scavengers) Vitamin E primarily to protect against lipid peroxidation donates single electrons to lipid peroxyl radicals (LOO•) to form stable lipid peroxide (LOOH) 74
- 75. B. Nonenzymatic Antioxidants (Free Radical Scavengers) Vitamin C oxidation-reduction coenzyme (collagen synthesis, etc) water soluble circulates unbound in blood, extracellular fluid important role in free radical defense – regenerate reduced Vitamin E 75
- 76. B. Nonenzymatic Antioxidants (Free Radical Scavengers) L-ascorbate donates single electrons to free radicals / disulfides in 2 steps reacts also with superoxide, H2O2, hypochlorite, hydroxyl and peroxyl radicals and NO2 76
- 77. B. Nonenzymatic Antioxidants (Free Radical Scavengers) Carotenoids -carotene compounds with functional oxygen- containing substituents on the rings “chain-breaking” antioxidants “quench” singlet O2 “health benefits” of diets high in fruits / vegetables 77
- 78. B. Nonenzymatic Antioxidants (Free Radical Scavengers) Carotenoids 78
- 79. Carotenoids Lycopene
- 80.
- 81. B. Nonenzymatic Antioxidants (Free Radical Scavengers) Flavonoids found in red wine, green tea, chocolates, other plant-derived foods group of structurally similar compounds with 2 spatially separate aromatic rings 81
- 82. B. Nonenzymatic Antioxidants (Free Radical Scavengers) Flavonoids exhibit the same ring structure differ in ring substituents (=O, - OH and OCH3) eg. Quercetin fruit skins antioxidant activity 82
- 83. B. Nonenzymatic Antioxidants (Free Radical Scavengers) Flavonoids contribute to free radical defenses in various ways: free radical scavengers by donating electrons to superoxide or lipid peroxy radicals stabilize free radicals by complex-formation inhibit enzymes responsible for superoxide anion production regeneration of reduced Vitamin E efficient chelators of Fe and Cu (Fenton reaction) e.g. Quercetin – effective in Fe chelation 83
- 84.
- 85. Time to TakeFive NATIONAL INSTITUTES OF HEALTH National Cancer Institute Department of Health and Human Services Public Health Service 85
- 86. Count 'Em Up! What's a serving of fruits and vegetables? A serving is: 1 medium fruit or 1/2 cup of small or cut-up fruit 3/4 cup of 100% fruit juice 1/4 cup dried fruit 1/4 cup raw or cooked vegetables 1 cup raw leafy vegetables (such as lettuce, spinach) 1/2 cup cooked beans or peas (such as lentils, pinto beans, kidney beans) 86
- 87. C. Endogenous Antioxidants Uric Acid formed from degradation of purines and is released into the extracellular fluids, including blood, saliva and lung-lining fluid with protein thiols, accounts for the major free radical trapping capacity of plasma directly scavenge hydroxyl radicals, oxyheme oxidants formed between the reaction of hemoglobin and peroxy radicals and peroxy radicals themselves 87
- 88. C. Endogenous Antioxidants Melatonin nonenzymatic free radical scavenger that donates an electron to “neutralize” free radicals reacts with ROS and RNOS to form addition products (“suicidal transformations”) hydrophilic/hydrophobic; can pass through membranes and the blood brain barrier 88
- 89. D. Metal Chelators bind Fe and Cu disable them from participating in Fenton reaction ↓ OH• production ferritin – multi-subunit protein shell surrounding a Fe+3 core transferrin – binds Fe+3 ceruloplasmin – converts Fe+2 to Fe+3 albumin – binds Cu+2 tightly and Fe+2 weakly 89
- 90. E. Compartmentation various defenses against ROS are found in different subcellular compartments location of free radical defense enzymes matches the type and amount of ROS generated in each subcellular compartment Separation of species and sites involved in ROS generation from the rest of the cell Fe being tightly bound to the storage protein, ferritin, cannot react with ROS. enzymes that produce H2O2 are sequestered in peroxisomes 90
- 91.
- 92. F. Repair Mechanisms Repair mechanisms for: DNA removal of oxidized fatty acids from membrane lipids oxidized amino acids through protein degradation and re- synthesis of new proteins 92
- 93. Thank you verymuch! NOEL MARTIN S. BAUTISTA, MD, DPPS, MBAH Department of Biochemistry, Molecular Biology and Nutrition