Oxidative stress - Université de Montpellier
Transcript of Oxidative stress - Université de Montpellier
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Oxidative stress :From toxicity to cell interactions
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Oxidative stress an umballancebetween oxidants and antioxidants
Antioxidants:
Oxidants:
Which place for oxidative stress biomarkers ?
Are biomarkers of oxidant production available ?
Is it possible to appreciate defense mechanism ?
Enzymatic or non enzymatic defenses
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Jonathan Himmelfarb, Peter Stenvinkel, T Alp Ikizler and Raymond M Hakim, Kidney International (2002) 62, 1524–1538
Exploring oxidative stress :a difficult chalenge
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I) Biomarkers of oxidative stress-related toxicity : « oxidative stress biomarkers »
II) Quantification of Oxidant production ?
III) Investigation of defense mechanisms ?
Exploring Oxidative Stress
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Proteins Glucides Lipids
AOPP AGEsIsoprostanes
TBARS
Oxidative stress : molecular targets
Membrane desorganisationLoss of protein fonctionsImpairement of genetic information
Nucleic acids
ModifiedBases
Oxidativestress
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Lipid peroxidation compounds as biomarkers of oxida tive stress
ROS
Lipidperoxidation
AldehydesMalonedialdehyde (MDA - TBARS)
4-hydroxynonénal (HNE)
F2-isoprostanes
anti-oxidized antibodies
PCOOH (oxydized phosphatidylcholine )
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Arachidonic Acid AAC20:4 ( n-6)
Docosahexaenoïc acid DHA(C22/6(n-3)
Oxidative stress
IsoprostanesNeuroprostanes
iPF2a-III(8-iso-PGF2a) iPF2a-IV
iPF2a-V F4-NPs
Isoprostanes : biomarkers of oxidative stress
CO2H
C18C15
C12
C9 C6
COOH
+ de 64 possibles isomers
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15-F2t-isoPPGs
- OTMS
Detection of Isoprostanes using GC-MS
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Isoprostane as a biomarker of oxidative stress in clinical situations
Monnier et al, JAMA, Avril 2006.
15 F2t- Isop pg/mg créatinine
0
200
400
600
800
DS II Contrôles
* p=0.0001
15-F2t-isoPs (pg/ml)
*
§
0
100
200
300
400
500
HV BPCD
F. Gouzi et al., J Appl Physiol. 2013;115(12):1796-805
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Oxidative stress Biomarkers:Nucleic acids
ROS
ModifiedNucleic Acid
8-hydroxy-2’deoxyguanosine (8-OHdG)Guanine oxidation
Test « COMET »Detection of breakdown of DNA usingelectrophoresis
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Increase in Oxydative stress in CKD
Tarng et coll. JASN, 2002Tarng et coll. JASN, 2002
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Protein oxidative products
ROS
ProteinOxidativeproducts
AOPP advanced oxydation protein products
Dityrosine, chlorotyrosine, nitrotyrosineOxyd. de la tyrosine
Carbonyl Proteins
AGEs, pentosidine, Carboxymethyl Lysine
Thiols oxidation : equilibrium GSH/GSSG
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Witko-Sarsat et al., Kidney Int. 1996 Wagner et al., Am J Kidney Dis. 2006
0
10
20
30
40
Controls HD patients
* *
Se
rum
CM
L co
nte
nt
(ng
/mg
pro
tein
)
Carboxymethyllysine and pentosidine : specific biomarkers
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AOPP : analytical limitations
Valli et al Clinica Chimica Acta 379 (2007) 87–94
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GSH/GSSG ratio a clear indicator of redox status� Valeurs sanguines
GSH libre: 800 – 1500µMGSSG: 1 – 10µM
� Méthode de dosage
Mesure du GSH sanguin difficile: peu stable
Variabilité intra-individuelle élevée
Important de maîtriser les étapes pré-analytiques
Robustesse de la méthode de dosage= LCMSMS
GSSG
GSH
Homocystine-d8= étalon interne
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I) Oxidative stress biomarkers : One lipid biomarker (IsoPs), 1 protein biomarker (C ML or pentosidine) or GSH/GSSG ratio, 1 nucleic acid biomarker : 8-OH-des oxyguanosine
II) Quantification of Oxidant production ?How are ROS produced?How to quantify ROS ?How to moduate ROS ?
III) Investigation of defense mechanisms ?
Exploring Oxidative Stress
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ROS are mainly producted as a coproduct of Energy supply or phagocyte activation
Chlorinated oxidants
ROS Nitrated oxidants
O2°-
Phagocyte activationCell metabolism
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Superoxide anion as a cofactorof mitochondria respiration
O2 ----------------------------------------> 2H2O
H+
e- e- e- e-
H+ H+ H+
ADP
ATP
O2°-
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ADP ATP
V
H+
β-oxidation
Krebs Cycle
ATP « energy fuels »
H+
O2 H2O
H+
Acyl-CoApyruvate
H+
O2.- and ROS
Desequiibrium between ATP
consumption and « energy fuel »
Electron flow
Complexes I et III : main source of ROS
Murphy MP 2009
ROS production by the
respiratory chain
(K. Begriche et al. 2006;
Wallace et al. 2010)
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The oxidative burst : an ehanced production of superoxide anion
Soluble Compounds:C5a, IL1, TNF, ProstanoïdesAgII, ET1
Insoluble compounds :Bacteria, LPS, IgG.
NADPH NADP+
2O2 2O2°-
2O22O2
°-
NADPH NADP+
NADPH Oxydase
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Inflammation and ROS poduction
InflammatoryMediators
H2O2
MPO
ClO-
Lactoferrine
OH°
Fe2+/ Fe3+
O2
O2°-
NADPH Oxydase
iNOS
L-Arginine
NO° ONOO-
O2
O2°- +
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NAD(P)H oxydase : a NOX superfamilly
Neurospora crassa
Humain
Bengtsson et al., 2005 Bedard. Physio Rev 2007 ; 87 : 245-313
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NOX 1, 3 and 4 stimuli and fonctions
Migdal. Médecine sciences 2011; 27 : 405 412
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Monocytes
Smooth Muscle Vascular cells
O2 O2°-
O2 O2°- PMNs
Endothelial cells
O2O2°-
NOX
NOX
NOX
NOX
SOD
SODSOD
SOD
H2O2
H2O2
MPO
MPO
O2O2°-
MPO
ClO-
ClO-
ClO-
ClO-
The synergistic action of NADPH oxidase and MPO
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Interactions between NADPH Oxydase -MPO - Mitochondria
Yokota T. et al., Am J Physiol Heart Circ Physiol 297:H1069-H1077, 2009
(Ray et Shah 2005)
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I) Oxidative stress biomarkers : One lipid biomarker (IsoPs), 1 protein biomarker (C ML or pentosidine) or GSH/GSSG ratio, 1 nucleic acid biomarker : 8-OH-des oxyguanosine
II) Quantification of Oxidant production ?How are ROS produced?How to quantify ROS ?How to moduate ROS ?
III) Investigation of defense mechanisms ?
Exploring Oxidative Stress
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DCFH-DA →DCFH2piégé à l'intérieur de la cellule
DCFH2 piégé subit l'oxydation → DCF fluorescent
ROS determination could be determined using DCFH -DA(dichlorodihydrofluorescein diacétate)
DCFH-DA DCFH2 DCF
Non fluorescent
Non fluorescent
fluorescent
ERO
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La production d’anion superoxyde par la lucigénine : principe
Li Y. The Journal of Biological Chemistry Vol. 273(4) : 2015–2023, 1998
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Activity and expression ofNADPH Oxydase : cell models
- Activité cellulaire : - Etude des sous unités :Lucigénine : anion superoxyde Western Blotting
time
0.6
0.5
0.4
0.3
0.2
0.1
p21 rac 2
actine
PMA PMA + simva
CTR
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I) Oxidative stress biomarkers : One lipid biomarker (IsoPs), 1 protein biomarker (C ML or pentosidine) or GSH/GSSG ratio, 1 nucleic acid biomarker : 8-OH-des oxyguanosine
II) Quantification of Oxidant production ?How are ROS produced?How to quantify ROS ?How to moduate ROS ?
III) Investigation of defense mechanisms ?
Exploring Oxidative Stress
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Cell components Transcriptionfactor
Oxidative stress and cell information
Oxidative stress biomarkersCell toxicity
de novo protein synthesis
Oxidativestress
Proinflammatory proteins
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CYTOKINES
LIPID NETWORK
FRO
+
+Cell
Stress or Injury
Amplification loops between inflammation and oxidative stress.
+NADPH Oxydase
PhenotypicModification
+
+
Mitochondria
Dysfonction
NFKB
AP-1+
Endothelial Cells monocytes/macrophages,
sMVC
AO
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Analyse UnivariéeHcy(p<0.01), CRP(p<0.01), Fibrinogène (p<0.01), α-1 glycoprotéine acide (p=0.03),
Albumine(p=0.01), TG(p=0.02), CT(p=0.05)
Analyse Multivariée : Déterminants de la production d’O2●-
Homocystéine (p<0.02), CRP (p<0.01)
(Ventura et al, Free Radical Biol Med, 2009)
Working hypothesis …A clinical proof in elderly
n = 478> 65 ans
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Analyse UnivariéeMDRD(p<0.004), Fibrinogène (p<0.02), HDL (p=0.03), PTH (p=0.04),
Hémoglobine (p=0.05), HTA (p=0.08)
Analyse Multivariée : Déterminants de la production d’O2●-
Fibrinogène (p<0.04), HDL (p<0.04), MDRD (p<0.04)
n = 136Stades 1-5
Morena et al., Free Radical Research, June 2011; 45(6): 735 –745
Working hypothesis …A clinical proof in CKD
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Oxidative stress, amplification loopsand aterosclerosis
Monocytes/Macrophages
Cellulesendothéliales Cellules musculaires lisses
Contractiles
Cellules spumeuses
VasorelaxationAntiagrégant –biocompatible
Dysfonction endothéliale :Vasoconstricton
AdhérenceProthrombotique
Sécrétoires
Ostéoblastiques
Inflammation Stress Oxydant
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Stress oxydant et transdifférentiation cellulaire
Monocytes/Macrophages
Cellulesendothéliales Cellules musculaires lisses
Contractiles
Cellules spumeuses
VasorelaxationAntiagrégant –biocompatible
Dysfonction endothéliale :Vasoconstricton
AdhérenceProthrombotique
Sécrétoires
Ostéoblastiques
Anti-oxidants
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Nutritional preventionof atherosclerosis:
CTR Aorta
Tota
l cho
lest
erol
(m
mol
/L)
0
4
8
12
Pro
duct
ion
supe
roxi
deC
ount
s / m
g pr
otei
n
0
4000
8000
12000
0
Blo
t int
ensi
ty
(arb
itrar
y un
its x
10-
3)
10
20
StandardAthérogène
Dépôts lipidiquesNutritionalprevention
Vegetal extract
(Sutra.T. et al., 2007)
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Les Polyphenols préviennent la dysfonction endothéliale chez les hamsters
Suh JH, et al., Food Funct. 2011
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I) Oxidative stress biomarkers : One lipid biomarker (IsoPs), 1 protein biomarker (C ML or pentosidine) or GSH/GSSG ratio, 1 nucleic acid biomarker : 8-OH-des oxyguanosine
II) Quantification of Oxidant production ?How are ROS produced?How to quantify ROS ?How to moduate ROS ?
III) Investigation of defense mechanisms ?How to measure the defense mechanismHow to interprete the defense mechanismHow to modulate defense mechanism
Exploring Oxidative Stress
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Defense mechanisms
H2O2O2°-
OH°ONOO-ClO-
Superoxyde anionproduction
Stress Oxydant
H2OSOD GPx
CatalaseTransitionMetal
chelation
Vitamines E/C
CaroténoïdesGSHMannitol...
Polyphénols
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�Les vitamines A, C et E plasmatiques sont dosées par HPLC-UV
�La vitamine E érythrocytaire est dosée par HPLC-Electrochimie
Conditions chromatographiques similaires
Colonne en phase inverse C18
Elution avec un gradient de solvant de polarité croissante
Seul le détecteur change:
�Le détecteur UVmesure l'absorption de la lumière par le produit à la sortie de la colonne.
Conditions: Il faut que le produit à détecter absorbe la lumière à une longueur d'onde accessible à l'appareil et que la phase mobile n'absorbe pas la lumière à la longueur d'onde choisie par l'opérateur
�Le détecteur électrochimiquemesure les réactions d’oxydoréductions qui produisent un courant proportionnel à la concentration du soluté.Meilleure sensibilité
Détection des vitamines : méthodes analytiques
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Limitations of antioxidant system determination
• Tissue variability :
• Different origin of antioxidant system :• Enzymatic system : synthesis and induction• Non enzymatic : strictly dependant of nutritional support
• Duration of the insult :• Consomtion• Induction
• Comorbidity and nutritional status:• age :• nutritional status…
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Enzymatic system : kinetics analysis
Cu/Zn SOD : rein de rat Mn SOD : rein de rat
Leach M., B Pharmacol J., 1998
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I) Oxidative stress biomarkers : One lipid biomarker (IsoPs), 1 protein biomarker (C ML or pentosidine) or GSH/GSSG ratio, 1 nucleic acid biomarker : 8-OH-des oxyguanosine
II) Quantification of Oxidant production ?How are ROS produced?How to quantify ROS ?How to moduate ROS ?
III) Investigation of defense mechanisms ?How to measure the defense mechanismHow to interprete the defense mechanismHow to modulate defense mechanism
Exploring Oxidative Stress
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Inducing enzymatic defense: the Nrf2 pathway
Keap1
Jaiswal AK, www.SABiosciences.com/support_literature .php
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Un équilibre dépendant de l’intensité du stress oxydant ?
L.M. Pedruzzi et al. / Biochimie xxx (2012) 1-6
EAO
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Lee I.K. Environmental toxicology and pharmacology 2 0 1 1
Les antioxydants (flavonoïdes) stimulent …Les enzymes antioxydantes …. via le Nrf 2 ?
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CYTOKINES
LIPID NETWORK
FRO
+
++NADPH
OxydaseModificationPhénotype
+
+
NFKB
AP-1
HIF
+
Prévention
-
Agression cellulaire
Redox system pro or antiinflammation
+-NrF-1
NrF-2
AO Nutrition
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Nutrition and COVID -19 mortality ?
Bousquet et al., Allergy. 2020
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CYTOKINES
LIPID NETWORK
Ang I
+NADPH Oxydase
NFKB
AP-1
HIF
+
Redox system and COVID infection
+- NrF-2
AOE
NutritionLactobacillusCabbageFermented vegetables …
Redox Status
High oxidative
stress
Maintained
Equilibrated
Redox status
FRO
production
Ang II
AT1R
HTA, Insulinorésistance, Obesity, Diabetes
ACE2Ang 1-7
Lung Injury
Endothelium damage
Death
Paucisymptomatic
infecition
SARS-CoV-2
-
Bousquet et al., Clin Transl Allergy. 2020