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