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A seminar onOxygen free radicals and their Scavengers By Hardik G. Dave Ist year M. Pharm HSKCOP, Bagalkot
IntroductionIt is believed that life has originated from basicchemicals by free radical reaction, largelyinitialled by ionising radiation from sun.Paradoxically the same reactions creating lifemay also be responsible for manydiseases, ageing and death.
Free radical - what is it?• Free radical is an atom or group of atoms with at least one unpaired electron.• Example : O H Hydroxyl radical ( OH)• In the body it is usually an oxygen molecule that has lost an electron and will stabilize itself by stealing an electron from a nearby molecule.• In the body free radicals are high-energy particles that ricochet wildly and damage cells.
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. And2. The breakage of covalent bond ‘homolytic fission’ A B A +B• Example H2O H + OH
• These are highly reactive chemical entities that have a single unpaired electron in their outer most orbit.• Under certain conditions can be highly toxic to the cells.• Generally unstable and try to become stable, either by accepting or donating an electron.• Therefore if two free radical react, they neutralise each other.• However, if the free radical react with stable molecules, there is generation of more free radicals.• This character enables the FRs to participate in auto catalytic chain reactions, – Molecules with which they react are themselves converted to free radicals to propagate the chain of damages.
• Any free radical involving oxygen can be referred to as reactive oxygen species (ROS).• Oxygen centered free radicals contain two unpaired electrons in the outer shell.• When free radicals steal an electron from a surrounding compound or molecule, a new free radical is formed in its place.• In turn the newly formed radical then looks to return to its ground state by stealing electrons with antiparallel spins from cellular structures or molecules.• These results in damaging the cell membranes. It also responsible for more than 100 human diseases like cancer, heart attacks, stroke and arthritis
• TYPES OF FREE RADICALS :• The most common ROS include:i. the superoxide anion (O2-),ii. the hydroxyl radical (OH ·),iii. singlet oxygen (1O2 ), andiv. hydrogen peroxide (H2O2)
1. SUPEROXIDE ANION (O2-) :• Superoxide anions are formed when oxygen (O2) acquires an additional electron, leaving the molecule with only one unpaired electron. Within the mitochondria.• O2- ·is continuously being formed.2. HYDROXYL RADICAL (OH- .) :• Hydroxyl radicals are short-lived, but the most damaging radicals within the body.• This type of free radical can be formed from O2- and H2O2 via the Harber-Weiss reaction.• The interaction of copper or iron and H2O2 also produce OH · as first observed by Fenton.3. HYDROGEN PEROXIDE (H2O2) :• Hydrogen peroxide is produced in vivo by many reactions.• Hydrogen peroxide is unique in that it can be converted to the highly damaging hydroxyl radical or be catalyzed and excreted harmlessly as water.
• Glutathione peroxidase is essential for the conversion of glutathione to oxidized glutathione, during which H2O2 is converted to water (2).• If H2O2 is not converted into water 1O2 (singlet oxygen) is formed.4. SINGLET OXYGEN (1O2) :• Singlet oxygen is not a free radical, but can be formed during radical reactions and also cause further reactions.• When oxygen is energetically excited one of the electrons can jump to empty orbital creating unpaired electrons.• Singlet oxygen can then transfer the energy to a new molecule and act as a catalyst for free radical formation.• The molecule can also interact with other molecules leading to the formation of a new free radical.
PHYSIOLOGICAL EFFECTS :• Under normal conditions (at rest) the antioxidant defense system within the body can easily handle free radicals that are produced.• During times of increased oxygen flux (i.e. exercise) free radical production may exceed that of removal ultimately resulting in lipid peroxidation.• Free radicals have been implicated as playing a role in the - etiology of cardiovascular disease, cancer, Alzheimers disease, and Parkinsons disease.
THE ROLE OF OXYGEN FREE RADICALS :• Our bodies use oxygen to convert food items such as fat and sugar into energy, in this process, oxygen is converted to water, and each water molecule normally takes up four electrons.• However some oxygen may escape before the conversion is complete, and this results in about 2% of the oxygen having an electron deficit.• They may also be formed in the human body by air pollution, smoking and exposure to radiation.• Free oxygen radicals are extremely reactive and can cause damage to body proteins and fats, and also to the hereditary material of cells, known as DNA.• Fortunately our bodies have some protective mechanisms against the harmful effects of free oxygen radicals These are enzymes and antioxidants.
• In mitochondria: - generation of energy - ATP - glucose, fatty acids, amino acids - O2 2H2O 4e- - leakage of O2-. (superoxide) H2O2 (hydrogen peroxide)• In Smooth Endoplasmic Reticulum (micro some) - detoxification (cytochrome P-450s) - toxins, drugs and xenobiotics - O2 + RH R-OH and H2O - leakage of O2-. - metabolic activation - X.
Superoxide Production from MitochondrialElectron Transport Chain Leaking’ of electron (to oxygen) during electron transport leads to the formation of O2 - (O2 + e- O 2 -)
Oxygen burst (respiratory burst) during phagocytosis•Formation of NADPH oxidase complexElectron is transferred from NADPH to O2, resulting in the formation of O2 - NADPH O2 NADPH . O2 • ... NADP+ • O2 - - NADP+ O2outside inside Phagocytic vacuole (phagosome)
• In Peroxisomes - containing oxidases for degradation of various substrates - glucose, amino acids, xanthine, etc. - requires O2 - by product is H2O2• In Cytoplasm - nitric oxide (NO.) production from Arginine - functions as a biological messenger - in brain, vascular endothelial cells, and macrophages - NO . + O -. ONOO . (peroxynitrite) 2
• Other source of free radical – Ionizing radiation, Ultraviolet radiation ,Ultrasound – Chemicals, tobacco smoke, etc
Roles of Free Radicals in Biological Systems• Enzyme-catalyzed reactions• Electron transport in mitochondria• Signal transduction & gene expression• Activation of nuclear transcription factors• Oxidative damages of molecules, cells, tissues• Antimicrobial actions• Aging & diseases
1.Oxidative stress :• Damages caused by free radicals/reactive oxygen species• Cellular damages at different levels (membrane, proteins, DNA, etc) lead to cell death, tissue injury, cellular toxicity, etc• Reduction of antioxidants2. Free Radical Toxicity :• Causes of free radical toxicity – Increase production of free radicals – Decrease level of defense system (e.g.,antioxidants)• Lipid peroxidation• DNA damage• Protein oxidation
Lipid Peroxidation1. Initiation of first-chain reaction • Abstraction of H+ by ROS ( OH) • • Formation of lipid radical (LH ) • • Formation of peroxyl radical (LOO , ROO ) • •2. Propagation • H+ abstraction by lipid peroxyl radical (LOO ) •3. Termination • Radical interaction non-radical product
I Hydrogen abstraction -H • • (LH•) Molecular rearrangement Conjugated diene • O2 Oxygen uptake Peroxy radical: abstractP H• from another fatty acid (LOO•) causing an autocatalytic O chain reactions O H• • Lipid hydroperoxide O Cyclic peroxide O I Initiation H Cyclic endoperoxide P Propagation
Oxidative DNA Damage• Correlation with cancers and diseases• Oxidative DNA lesions by -Direct attack -Indirect activation of endonuclease enzymes• Oxidative modification of bases – mutation• Oxidative modification of sugar moieties –DNA strand break
• Abstraction of H+ atom fromcarbon atoms of sugarmolecules• Disproportionations andrearrangement lead to C-Cbond fragmentation and DNAstrand break A computer image depicts a hydroxyl radical attacking the sugar on the back bone of a DNA molecule
Protein Oxidation• Protein targets – Receptors, transport proteins, enzymes, etc – Secondary damage – autoimmunity• Protein oxidation products – Protein carbonyl group, 3-nitrotyrosine, other oxidized amino acids• Most susceptible amino acids – Tyrosine, histidine, cysteine, methionine
Oxidant-Antioxidant Balance Defense Damage (Antioxidants) (Pro-oxidants)Decrease of antioxidant defense system Oxidative damage
Cellular Defense Mechanisms• Isolation of generation sites of reactive oxygen species• Inhibition of propagation phase of reactive oxygen species• Scavenging of reactive oxygen species• Repair of the damage caused by reactive oxygen species
Protection Against ROS Damage• Direct protection against ROS – Superoxide dismutase, Glutathione peroxidase, Catalase• Non-specific reduction system – Glutathione, Vitamin C• Protection against lipid peroxidation – Glutathione peroxidase, Vitamin E, -Carotene• Sequestration of metals – Transferrin, Lactoferrin, Ferritin, Metalothionein• Repair systems – DNA repair enzymes, Macroxyproteinases, Glutathione transferase
Superoxide Dismutase (SOD) 2O2. - + 2H+ H2O2 + O2• SOD - is present in all oxygen-metabolizing cells, different cofactors (metals).• An inducible in case of superoxide overproduction.1. CuZn-SOD – Cytoplasm, nucleus, lysosomes2. Mn-SOD – Mitochondrial matrix3. EC (CuZn) – Plasma membrane, extracellular4. EC Mn-SOD – Plasma membrane
Catalase 2 H2O2 2 H2O + O2• High affinity to H2O2 : peroxisomes hepatocytes mitochondria, cytoplasm of erytrocytes.• Tetramer with Fe, needs NADPH.• Prevents lipid peroxidation and protein oxidation.
REFERENCE• Text book of Pharmacotherapy and physiological approach , JOSHEP T DIPIRO, 5th edition, pg no – 390 and 1085.• Harpers Biochemistry 26th edition.• Lehningers Principles of Biochemistry 4th Edition - D L Nelson, Cox Lehninger - W H Freeman 2004.• Free-Radical-Induced DNA Damage and Its Repair - A Chemical Perspective (Springer, 2006)• Rang and Dale’s Pharmacology , 6th edition• www.google.com