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RADICALES LIBRES ESPECIES REACTIVAS DE OXIGENO (ROS) Y DE NITROGENO ROS ¿Dónde se produce normalmente ROS? ROS production - I Mitochondria ATP generating organelles E.T.C. system common to all life electron leak - birds, bats, other mammals State 3 and 4 Turtles, ischemia/reperfusion ROS production - II Immune Response CVD, autoimmune disease Neutrophils ‘oxidative burst’ ROS T-cells time Cell signalling linked to redox state of cell Many receptors insulin, vegf Many transcription factors NF-Kb, AP-1 Efectos de los ROS sobre las moléculas biológicas Radical Mediated Cleavage of Peptide Bonds Instead of forming carbonyl adduct products, ROS can directly cleave and oxidize the peptide bond. Table 1 illustrates the four most common types of radical mediated cleavages and the corresponding products. Table 1 Type of Cleavage C-terminal group of Nterminal fragment Alpha amidation -CONH2 Diamide -CONHC(R)O Glutamate oxidation -CONH2 Proline oxidation N-terminal group of Cterminal fragment RCOCONH- O C N- CH3COCONH- Hydrolysis products NH3, RCOCOOH CO2, RCOOH, NH3 CH3COCOOH, NH3, HOOC-COOH O NOC HOCONH- H2NCH2(CH2)COOH, CO2 Deamidation, Racemization and Isomerization of Protein Residues Besides introducing carbonyl groups into the protein, ROS are also responsible for deamidation, racemization and isomerization of residues. Gln and Asn residues deamidate and racemize about their C alpha atoms to the Disomers. Asymmetric side chains of Thr and Ile residues convert from the L-isomer to the Disomer. Spontaneous prolyl cis-trans isomerization occurs. Modified Proteins Which Are Not Degraded The previous slides dealt with chemical modifications which lead to protein degradation, but not all aberrant proteins are recognized by degradation systems in the cells. For example, modified proteins in eye lens are not recognized. Therefore, modified lens proteins accumulate over a lifetime with deleterious effects to vision. Chemically modified lens proteins lead to the formation of cataracts. Hydroperoxides - Sources Hydrogen peroxide: 1. 2. Redox - Free radical reactions Enzymatic MAOI, Aminoacid oxidase, Glyclate oxidase, Fatty acid oxidase (in peroxisomes + catalase) SOD - Leukocytes Lipid Hydroperoxides (LOOH): 1. 2. Redox - Lipid peroxidation Enzymatic From Arachidonate - Cyclo / lipoxygenase Cyclic endoperoxides - PGG2 /PGH2 Hydroperoxy eicosotetraenoic acids (HPETEs) Fate of Hydrogen Peroxide 1. Low Steady State Levels - GPx (Se) H2O2 + 2GSH = GSSG + 2H2O 2. 3. High Concentrations - Catalase 2H2O2 ==> O2 + 2H2O In presence of Transition Metals (TM) Fenton H2O2 + Fe 2+ ==> Fe 3+ + OH- + OH * 4. In presence of TM and Superoxide Haber Weiss H2O2 + O2 .- + Fe 2+ ==> Fe 3+ + O2 + OH- + OH. * Hydroperoxides & Cellular Oxidative Damage Oxidized SH Inflammation Shock ATP decrease Thromboxane Release H 2O 2 DNA Damage Lipid Peroxidation PGI2 Mecanismos anti-oxidantes Reaccion de la Superoxido Dismutasa O2- + O2- + 2H+ -> H2O2 + O2 Cellular Defense Mechanisms to Prevent ROS Buildup. Due to the oxygen rich environment in which proteins exist, reactions with ROS are unavoidable. Superoxide dismutase and glutathione peroxidase are natural antioxidants present in organisms which eliminate some ROS. Glutathione peroxidase catalyzes the reduction of peroxide by oxidizing glutathione (GSH) to GSSG. O O- superoxide dismutase 2O2 H2O2 + O2 GSH + H2O2 O SH glutathione peroxidase GSSG H2O + O2 + GSSG O- S 2 O O H N H N NH3 O- N H NH3 O GSH O- N H O GSSG Trypanothione metabolism in trypanosomatids Defense against ROS NADP+ T[SH]2 TPNox TPXred ROOH TR NADPH + H + TS2 TPNred TPXox PDXred 2H+ 2O2 - H2O 2 SOD PDXox O2 H2O TR: TPN: TPX: PDX: SOD: trypanothione reductase tryparedoxin tryparedoxin peroxidase peroxyredoxin superoxide dismutase localized to glycosomes sequence identified PTS2 identified PTS1 identified ROH BIOLOGICAL ANTI-OXIDANT SYSTEMS 1. INTRACELLULAR Catalase SOD Peroxidase Glutathione Selenium DNA (Repair) 2. MEMBRANE Vitamin E ß Carotene Ubiquinone (Chain Breaking) 3. EXTRACELLULAR (PLASMA) Metal-Binding Proteins (Preventive) Caeruloplasmin, Transferrin Albumin Uric acid Vitamin E Vitamin C Organizational Hierarchy in Consumption of Plasma Antioxidants 1. vs aqueous peroxyl radicals Plasma Ascorbic acid > Protein Thiols > Bilirubin > Uric Acid > a-tocopherol [Stocker et al, Frei et al, 1988-9] 2. vs lipid-soluble radical generator [Frei et al, 1989] a -tocopherol > Ascorbic acid > Alb-Bilirubin 3. vs singlet oxygen - Lycopene, Bilirubin 4. LDL [Esterbauer et al, 1987,1989] a -tocopherol / Ubiquinol > g -tocopherol > Lycopene >[Uric acid / Ascorbic acid] > b-carotene 5. Phorbol myristate-activated PMn [Frei et al 1988] Ascorbic acid = Protein Thiols = Bilirubin > Uric Acid [vit E neg] Oxidative Stress SIGMA-ALDRICH Radicales de nitrogeno Nitric Oxide Metabolism SIGMA-ALDRICH 1885 1955 El estrés oxidativo y su relaciòn con el envejecimiento La Hipòtesis de la Tasa de Vida “La tasa metabòlica de una especie determina su expectativa de vida” Relaciòn entre metabolismo y envejecimiento • En 1957 Denham Harman propone la teorìa de envejecimiento por radicales libres • En 1969 se identifica la superoxido dismutasa (SOD) • Se unifica empiricamente el concepto de “a mayor tasa metabòlica, mayor producciòn de ROS, menor tiempo de vida” • Se corrige y se simplifica la correlaciòn ROS y longevidad Los oxidantes contribuyen al desarrollo del fenotipo de senescencia • Fibroblastos crecidos en baja tensiòn de O2 viven mas tiempo • Fibroblastos crecidos en baja tensiòn de O2 reducen su tiempo de vida y presentan acortamiento de telomeros mas ràpido • H2O2 detienen el crecimiento celular y muestran senescencia • Efecto de Ras puede ser revertido por anti oxidantes permeables • • • • Mitochondrial respiratory Chain increased oxygen consumption produces more O2.- and H2O2. Xanthine oxidase Insufficient blood flow (hypoxia) leads to degradation of ATP to hypoxanthine producing .O2 and H2O2 . Neutrophil (PMN) Respiratory burst by NADPH oxidase IL-1, IL-6 and TNF-a increases adhesion molecules and PMN infiltration Lipoxygenase/cycloxy genase Activated by cytokines, hormones and toxins Source of Free Radicals in Skeletal Muscle • • Aged rats generates more ROS at rest and during exercise (15 m/min, 0%) at the same relative workload as young rats (25 m/min, 10%). Both mitochondria and NADPH oxidase are sources of ROS in young muscle during exercise. For aged muscle, mitochondria seem to be the main source. ROS generation is also increased in the heart. pm ol DCF x m in -1 x m g-1 • An acute bout of exercise in rats increases ROS production in skeletal muscle. Rested 120 With 2 mM pyruvate and 2 mM malate as mitochondrial respiration substrates Exercised 80 40 0 8 mo 25 mo 80 pmol DCF x min-1 x mg -1 • 160 60 Replace pyrmalate wiith 1.7 mM ADP, 0.1 mM NADPH and Fe+3 40 Ji & Bejma J.A.P. (1999) 20 0 8 mo 25 m o p53 puede presentar un loop de retroalimentacion pro-apoptotica Control + Peroxido de hidrògeno Antioxidant activity vs Lipid (LDL) Peroxidation 1. Remove Oxygen, or decrease its concentration 2. Remove transition metal catalytic ions 3. Remove ROS (reactive O2 species) - O2-, H2O2 4. Scavenging initiating radicals - OH*, RO*, ROO* 5. Chain breakers: Vitamin E 6. Quenching singlet oxygen: beta carotene ROS manipulation Dietary supplementation Very mixed results except in particular cases such as Vitamin E and ischemea/reperfusion Dietary Restriction (up to 50% LS extension) Less evidence of oxidative damage Metabolic rate unaltered Mitochondria characteristics – lipid membrane, less ROS with same membrane potential Exercise (up to 10% LS extension) Acute can lead to immune response and damage Depletion of Vitamin E Training generally beneficial with more mitochondria produced “Es casi un milagro que los mètodos modernos de enseñanza no hayan estrangulado aùn enteramente la sagrada curiosidad de la investigaciòn; para lo cual èsta pequeña planta, necesita mas que nada, ademàs de estimulaciòn, libertad The problem with vitamin C antioxidant or pro-oxidant ? Pro-oxidant with transition metals ==> Lipid Peroxidation Wills ED, Biochem Pharmacol 21: 239, 1972 Ascorbate and Glutathione protect against microsomal peroxidation only in the presence of vitamin E. In Vit E-deficient microsomes, enhanced peroxidation Wefers & Sies. Eur J Biochem. 174: 353, 1988 Conclusion: “You can tell an antioxidant’s activity by the company it keeps” 1 2 3 All antioxidants may be prooxidants Regulated antioxidant system - Redox Other natural agents – OVERDOSES? Carotene: Increased Carcinoma of Lung in Smokers Vitamin C Low dose: antioxidant High dose: pro-oxidant - interaction with Fe Vitamin E Interfere with phagocyte function Cytochrome P450 SOD must work with catalase; otherwise forms dangerous H2O2