radicales libres especies reactivas de oxigeno (ros) y de nitrogeno
TRANSCRIPT
RADICALES LIBRES
ESPECIES REACTIVAS DE OXIGENO (ROS) Y DE
NITROGENO
ROS
¿Dónde se produce normalmente ROS?
ROS production - I
Mitochondria ATP generating organellesE.T.C. system common to all lifeelectron leak - birds, bats, other mammalsState 3 and 4Turtles, ischemia/reperfusion
ROS production - II
time
ROS
Neutrophils ‘oxidative burst’
T-cells
Immune Response CVD, autoimmune disease
Cell signalling linked to redox state of cellMany receptors insulin, vegfMany 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
Alpha amidation
Diamide
Glutamateoxidation
Prolineoxidation
-CONH2 RCOCONH- NH3, RCOCOOH
-CONHC(R)O O C N- CO2, RCOOH, NH3
-CONH2CH3COCONH- CH3COCOOH, NH3,
NOC
O HOCONH- H2NCH2(CH2)COOH, CO2
HOOC-COOH
Type of Cleavage
C-terminalgroup of N-terminal fragment
N-terminal group of C-terminal fragment
Hydrolysis products
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 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.
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 - SourcesHydrogen peroxide:1. Redox - Free radical reactions2. Enzymatic
MAOI, Aminoacid oxidase, Glyclate oxidase, Fatty acid oxidase (in peroxisomes + catalase)SOD - Leukocytes
Lipid Hydroperoxides (LOOH):1. Redox - Lipid peroxidation2. Enzymatic
From Arachidonate - Cyclo / lipoxygenase Cyclic endoperoxides - PGG2 /PGH2
Hydroperoxy eicosotetraenoic acids (HPETEs)
Fate of Hydrogen Peroxide1. Low Steady State Levels - GPx (Se)
H2O2 + 2GSH = GSSG + 2H2O
2. High Concentrations - Catalase2H2O2 ==> 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 + O2 .- + Fe 2+ ==> Fe 3+ + O2 + OH- + OH. *
Hydroperoxides & Cellular Oxidative Damage
H2O2 Lipid Peroxidation
DNA Damage
ATP decrease
Oxidized SH
ThromboxaneRelease
PGI2
Shock Inflammation
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.
H2O2 + O2 GSSG
superoxidedismutase
glutathioneperoxidase
2O2
GSH + H2O2 H2O + O2 + GSSG
NH3 NH
HN
O-
O-O
O
O
SH
GSH
NH3 NH
HN
O-
O-O
O
O
S
GSSG
2
Trypanothione metabolism in trypanosomatids
Defense against ROS
NADP+
NADPH + H+
TR
T[SH]2
TS2
TPNox
TPNred
TPXred
TPXox
H2O2
H2O
ROOH
ROH
PDXred
PDXox2O2-
O2
2H+
SODlocalized to glycosomes sequence identified PTS2 identified PTS1 identified
TR: trypanothione reductaseTPN: tryparedoxinTPX: tryparedoxin peroxidasePDX: peroxyredoxinSOD: superoxide dismutase
1. INTRACELLULAR1. INTRACELLULAR CatalaseCatalase SODSOD PeroxidasePeroxidase Glutathione Glutathione SeleniumSelenium DNA DNA (Repair)(Repair)
2. MEMBRANE2. MEMBRANE Vitamin EVitamin E ß Caroteneß Carotene UbiquinoneUbiquinone (Chain Breaking)(Chain Breaking)
3. EXTRACELLULAR (PLASMA)3. EXTRACELLULAR (PLASMA)Metal-Binding ProteinsMetal-Binding Proteins (Preventive)(Preventive)
Caeruloplasmin, TransferrinCaeruloplasmin, TransferrinAlbuminAlbumin Uric acidUric acidVitamin EVitamin E Vitamin CVitamin C
BIOLOGICAL ANTI-OXIDANT SYSTEMSBIOLOGICAL ANTI-OXIDANT SYSTEMS
Organizational Hierarchy in Consumption Organizational Hierarchy in Consumption of Plasma Antioxidantsof Plasma Antioxidants
11.. vs aqueous peroxyl radicals Plasma vs aqueous peroxyl radicals Plasma Ascorbic acid > Protein Thiols > BilirubinAscorbic acid > Protein Thiols > Bilirubin
> Uric Acid > > Uric Acid > -tocopherol-tocopherol [ [Stocker et al, Frei et al, 1988-9] Stocker et al, Frei et al, 1988-9]
2.2. vs lipid-soluble radical generator [vs lipid-soluble radical generator [Frei et al, 1989]Frei et al, 1989]
-tocopherol > Ascorbic acid > Alb-Bilirubin-tocopherol > Ascorbic acid > Alb-Bilirubin
3.3. vs singlet oxygen vs singlet oxygen - Lycopene, Bilirubin- Lycopene, Bilirubin
4.4. LDL [LDL [Esterbauer et al, 1987,1989]Esterbauer et al, 1987,1989]
-tocopherol / Ubiquinol > -tocopherol / Ubiquinol > -tocopherol > -tocopherol > Lycopene >[Uric acid / Ascorbic acid]Lycopene >[Uric acid / Ascorbic acid] > > -carotene-carotene
5.5. Phorbol myristate-activated PMn [Phorbol myristate-activated PMn [Frei et al 1988]Frei et al 1988]
Ascorbic acid = Protein Thiols = Bilirubin > Ascorbic acid = Protein Thiols = Bilirubin > Uric Acid Uric Acid [vit E neg][vit E neg]
Oxidative Stress
SIGMA-ALDRICH
Radicales de nitrogeno
Nitric Oxide MetabolismSIGMA-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 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 oxidaseIL-1, IL-6 and TNF- increases adhesion molecules and PMN infiltration
• Lipoxygenase/cycloxygenaseActivated by cytokines, hormones and toxins
0
20
40
60
80
8 mo 25 mo
pm
ol D
CF
x m
in-1 x
mg
-10
40
80
120
160
8 mo 25 mo
pm
ol D
CF x
min-1
x m
g-1 Rested
Exercised
Source of Free Radicals in Skeletal Muscle
• 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.
With 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)
p53 puede presentar un loop de retroalimentacion pro-apoptotica
Control + Peroxido de hidrògeno
Antioxidant activity vsLipid (LDL) Peroxidation
1. Remove Oxygen, or decrease its concentration1. Remove Oxygen, or decrease its concentration
2. Remove transition metal catalytic ions2. Remove transition metal catalytic ions
3. Remove ROS (reactive O3. Remove ROS (reactive O22 species) - O species) - O22--, H, H22OO22
4. Scavenging initiating radicals - OH*, RO*, ROO*4. Scavenging initiating radicals - OH*, RO*, ROO*
5. Chain breakers: Vitamin E5. Chain breakers: Vitamin E
6. Quenching singlet oxygen: beta carotene6. Quenching singlet oxygen: beta carotene
ROS manipulationDietary 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 damageMetabolic rate unaltered Mitochondria characteristics – lipid membrane, lessROS with same membrane potential
Exercise (up to 10% LS extension)Acute can lead to immune response and damageDepletion of Vitamin ETraining generally beneficial with more mitochondriaproduced
“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 Cantioxidant or pro-oxidant ?
Pro-oxidant with transition metals ==> Lipid PeroxidationWills 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 peroxidationWefers & Sies. Eur J Biochem. 174: 353, 1988
Conclusion: “You can tell an antioxidant’s activity
by the company it keeps”
1 All antioxidants may be prooxidants2 Regulated antioxidant system - Redox3 Other natural agents – OVERDOSES?
Carotene:Carotene: Increased Increased Carcinoma of Lung in SmokersCarcinoma of Lung in SmokersVitamin CVitamin C Low dose: antioxidantLow dose: antioxidant High dose: pro-oxidant - interaction with FeHigh dose: pro-oxidant - interaction with FeVitamin EVitamin E Interfere with phagocyte functionInterfere with phagocyte function Cytochrome P450Cytochrome P450SODSOD must work with catalase; otherwise formsmust work with catalase; otherwise forms dangerous Hdangerous H22OO22