1 ESPECIES REACTIVAS DE OXIGENO (ROS) Y DE NITROGENORADICALES LIBRES ESPECIES REACTIVAS DE OXIGENO (ROS) Y DE NITROGENO

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3 ROS

4 ¿Dónde se produce normalmente ROS?

5 ROS production - I 90% ROS are generated here Membrane potential and ROS production 70% Japanese centanarians have mutation in complex 1 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

6 ROS production - II Immune Response CVD, autoimmune diseaseNeutrophils ‘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

7 Efectos de los ROS sobre las moléculas biológicas

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9 Radical Mediated Cleavage of Peptide BondsInstead 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

10 Deamidation, Racemization and Isomerization of Protein ResiduesBesides 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 D-isomers. Asymmetric side chains of Thr and Ile residues convert from the L-isomer to the D-isomer. Spontaneous prolyl cis-trans isomerization occurs.

11 Modified Proteins Which Are Not DegradedThe 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.

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15 Hydroperoxides - SourcesHydrogen peroxide: 1. Redox - Free radical reactions 2. Enzymatic MAOI, Aminoacid oxidase, Glyclate oxidase, Fatty acid oxidase (in peroxisomes + catalase) SOD - Leukocytes Lipid Hydroperoxides (LOOH): 1. Redox - Lipid peroxidation From Arachidonate - Cyclo / lipoxygenase Cyclic endoperoxides - PGG2 /PGH2 Hydroperoxy eicosotetraenoic acids (HPETEs)

16 Fate of Hydrogen Peroxide1. Low Steady State Levels - GPx (Se) H2O2 + 2GSH = GSSG + 2H2O 2. High Concentrations - Catalase 2H2O2 ==> O2 + 2H2O 3. In presence of Transition Metals (TM) Fenton H2O2 + Fe 2+ ==> Fe 3+ + OH- + OH * 4. In presence of TM and Superoxide Haber Weiss H2O2 + O Fe 2+ ==> Fe 3+ + O2 + OH- + OH. *

17 Hydroperoxides & Cellular Oxidative DamageOxidized SH Inflammation Shock H2O2 ATP decrease Lipid Peroxidation PGI2 Thromboxane Release DNA Damage

18 Mecanismos anti-oxidantes

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20 Reaccion de la Superoxido DismutasaO2- + O2- + 2H+ -> H2O2 + O2

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24 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.

25 Trypanothione metabolism in trypanosomatids

26 Defense against ROS TR: trypanothione reductase TPN: tryparedoxinTPX: tryparedoxin peroxidase PDX: peroxyredoxin SOD: superoxide dismutase

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33 BIOLOGICAL ANTI-OXIDANT SYSTEMS1. 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

34 Organizational Hierarchy in Consumption of Plasma Antioxidants1. vs aqueous peroxyl radicals Plasma Ascorbic acid > Protein Thiols > Bilirubin > Uric Acid > a-tocopherol [Stocker et al, Frei et al, ] 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]

35 Oxidative Stress SIGMA-ALDRICH

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37 Radicales de nitrogeno

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39 Nitric Oxide MetabolismSIGMA-ALDRICH

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43 El estrés oxidativo y su relaciòn con el envejecimiento

44 La Hipòtesis de la Tasa de Vida“La tasa metabòlica de una especie determina su expectativa de vida”

45 Relaciòn entre metabolismo y envejecimientoEn 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

46 Los oxidantes contribuyen al desarrollo del fenotipo de senescenciaFibroblastos 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

47 Mitochondrial respiratory Chainincreased 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- increases adhesion molecules and PMN infiltration Lipoxygenase/cycloxygenase Activated by cytokines, hormones and toxins

48 Source of Free Radicals in Skeletal MuscleWith 2 mM pyruvate and 2 mM malate as mitochondrial respiration substrates Replace pyr- malate wiith 1.7 mM ADP, 0.1 mM NADPH and Fe+3 Ji & Bejma J.A.P. (1999) An acute bout of exercise in rats increases ROS production 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.

49 p53 puede presentar un loop de retroalimentacion pro-apoptotica

50 Control + Peroxido de hidrògeno

51 Antioxidant activity vs Lipid (LDL) Peroxidation1. 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

52 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

53 “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

54 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”

55 1. All antioxidants may be prooxidants 21 All antioxidants may be prooxidants 2 Regulated antioxidant system - Redox 3 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

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