Transcript Diapositiva 1

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
NH3
H
N
O-
N
H
NH3
O
GSH
O-
N
H
O
GSSG
Trypanothione metabolism in
trypanosomatids
Defense against ROS
NADP +
T[SH]2
TPNox
TPXr ed
ROOH
TR
NADPH + H +
TS2
TPNr ed
TPXox
PDX r ed
2H+
2O2 -
H2O 2
SOD
PDX ox
O2
H2O
TR:
TPN:
TPX:
PDX:
SOD:
trypanothione reductase
tryparedoxin
tryparedoxin peroxidase
peroxyredoxin
superoxide dismutase
localized to glycos om e s
s e que nce ide ntifie d
PTS2 ide ntifie d
PTS1 ide ntifie d
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