Oxidative Stress and Oxidative Stress and AtherosclerosisAtherosclerosis

ObjectivesObjectives

What is “free radicalfree radical” ? Reactive oxygen and nitrogen species (RONSRONS) Are the RONS always dangerous? Well known term “oxidative stressoxidative stress” - what is it? AntioxidantsAntioxidants - types DisordersDisorders Associated with Oxidative stress Oxidative stress and atherosclerosisatherosclerosis

TermTerm DefinitionDefinition

OxidationOxidation Gain in oxygenLoss of hydrogenLoss of electrons

ReductionReduction Loss of oxygenGain of hydrogenGain of electrons

OxidantOxidant Oxidizes another chemical by takingelectrons hydrogen or by adding oxygen

ReductantReductant Reduces another chemical by supplying electrons, hydrogen or by removing oxygen

Basics of Redox ChemistryBasics of Redox Chemistry

Free RadicalsFree Radicals A molecule with an unpaired electron in an

outer valence shell• Tend to reach equilibrium, plucks an electron plucks an electron

from the nearest intact molecule.• Most of biomoleculs are not radicals• Radicals are highly reactive species

R3C. Carbon-centered

R3N. Nitrogen-centered

R-O. Oxygen-centered

R-S. Sulfur-centered

Reactive SpeciesReactive Species

Non RadicalsNon Radicals Species that have strong oxidizing potentialoxidizing potential Species that favor the formation favor the formation of strong

oxidants (e.g. transition metals)

H2O2 Hydrogen peroxide

HOCl- Hypochlorous acid

O3 Ozone1O2 Singlet oxygen

ONOO- Peroxynitrite

Men+ Transition metals

Reactive Oxygen Species (ROS)Reactive Oxygen Species (ROS)

RadicalsRadicals

O2.- Superoxide

OH .- Hydroxyl

RO2. Peroxyl

RO. Alkoxyl

HO2. Hydroperoxyl

Non-RadicalsNon-Radicals

H2O2 Hydrogen peroxide

HOCl- Hypochlorous acid

Reactive Nitrogen Species (RNS)Reactive Nitrogen Species (RNS)

Radicals as:

NO. Nitric Oxide

NO2. Nitrogen dioxide

Non-Radicals as:Peroxynitrite

“An An imbalanceimbalance favoring (pro) oxidants and/or disfavoring favoring (pro) oxidants and/or disfavoring antioxidants, potentially leading to antioxidants, potentially leading to damagedamage” ”

AntioxidantsAntioxidants

OxidantsOxidants

Oxidative StressOxidative Stress

Oxidative StressOxidative Stress

• It is a state in the cells in which there is increased concentration of reactive species which is not counterbalanced by increased levels of antioxidants.

• This imbalance was implicated in production of different diseases as atherosclerosis

Endogenous sources of ROS & RNSEndogenous sources of ROS & RNS

MitochondriaMitochondria

LysosomesLysosomes

PeroxisomesPeroxisomes

Endoplasmic ReticulumEndoplasmic Reticulum

CytoplasmCytoplasm

Microsomal Oxidation, Microsomal Oxidation, Flavoproteins, CYP enzymesFlavoproteins, CYP enzymes MyeloperoxidaseMyeloperoxidase

(phagocytes) (phagocytes)

Electron transportElectron transport

Oxidases,Oxidases,FlavoproteinsFlavoproteins

Plasma MembranePlasma MembraneLipoxygenases,Lipoxygenases,

Prostaglandin synthaseProstaglandin synthaseNADPH oxidase

Xanthine Oxidase,Xanthine Oxidase,NOS isoformsNOS isoforms

FeCu

Transition Transition metalsmetals

Reactive Oxygen Species (ROS)Reactive Oxygen Species (ROS)

ROS are:ROS are:Free radicalsFree radicals: : e.g. Superoxide & hydroxyl radicalse.g. Superoxide & hydroxyl radicalsNon radicalsNon radicals: Hydrogen peroxide: Hydrogen peroxide

ROSROS are formed continuously by partial reduction of molecular oxygen in electron transport chain in mitochondria

For example:For example:Oxygen dependent pathway for microbial killingOxygen dependent pathway for microbial killing

In phagocytic cells (neutrophils & monocytes), In phagocytic cells (neutrophils & monocytes), microorganisms are phagocytosedmicroorganisms are phagocytosed

NADPH oxidase in these cells convertsNADPH oxidase in these cells convertsmolecular oxygen into superoxide (Respiratory molecular oxygen into superoxide (Respiratory Burst). Burst).

Superoxide + chloride ions are converted into Superoxide + chloride ions are converted into hypochlorous acid that kills bacteriahypochlorous acid that kills bacteria(by help of the lysosomal enzyme myeloperoxidsase(by help of the lysosomal enzyme myeloperoxidsaseMPO)MPO)

Sometimes, ROS is useful for humansSometimes, ROS is useful for humans

AntioxidantsAntioxidants

Enzymes that catalyze antioxidant Enzymes that catalyze antioxidant reactionsreactions::1- Superoxide dismutase 2- Catalase3- Glutathione system (glutathione, NADPH, reductase, peroxidase & selenium).

VitaminsVitamins1- Vitamin A & β-carotenes2- Vitamin C (ascorbic acid)3- Vitamin E

Trace elementsTrace elementsSelenium

Enzymatic Antioxidant MechanismsEnzymatic Antioxidant Mechanisms

Glutathione SystemGlutathione System

SeleniumSelenium

Glutathione system Glutathione system in RBCSin RBCS

Nitric Oxide (NO)Nitric Oxide (NO)Nitric oxide (NO):Nitric oxide (NO):Free radical gasVery short half-life (seconds)Metabolized into nitrates & nitrites & perooxynitrite

SynthesisSynthesis

Enzyme: NO synthasePrecursor: L-Arginine

EffectsEffectsRelaxes vascular smooth musclePrevents platelet aggregationBactricidal & tumoricidal effectsNeurotransmitter in brain

NNitric itric OOxide xide SSynthase ynthase (NOS)(NOS)

Two constitiutive NOSTwo constitiutive NOSs s

in endothelium = eNOSeNOS neural = nNOSnNOSconstantlyconstantly produce lowlow level of NOcalcium-calmodulin dependent

One inducible (iNOS)One inducible (iNOS)

Can be expressed in many cells including hepatocytes, macrophages & neutrophils.InducersInducers include bacterial toxins, tumor-necrosis factor & inflammatory cytokinesIt can produce largelarge amounts of NO over hours or even daysCalcium independent

Endotheilal Nitric Oxide Endotheilal Nitric Oxide Synthase (eNOS)Synthase (eNOS)

NO productionNO production

Diffuses to smooth muscles

Activates guanylate cyclase

production of cGMP from GTP

Activation of protein kinase G

Phosphorylstes myosin light-chain kinase (becomes inactive)

Decrease smooth muscular contraction

(RELAXATION)

NO effect on vascular endotheliumNO effect on vascular endothelium

Oxidative Stress: Role of Nitric Oxide (NO)

NO produced by endothelial NOS (eNOS) improving

vascular dilation and perfusion (i.e. beneficial).

Vasodilators such as nitroglycerin is metabolized into NO and

causes vasodilatation

Increased iNOS activity is generally associated with

inflammatory processes

LipidsLipids Proteins Proteins DNA DNA Oxidation of

vitamin E

Lipid peroxidationLipid peroxidation

Membrane damageMembrane damage

Thiol oxidationThiol oxidationCarbonyl formationCarbonyl formation

Damage to CaDamage to Ca2+2+ and andother ion transportother ion transport

systemssystems

DNA damageDNA damage

Cell InjuryCell Injury

Disruption of normalDisruption of normalion gradientsion gradients

Activation/deactivationActivation/deactivationof various enzyme systemsof various enzyme systems

MutationsMutations

Depletion of ATPand NAD(P)H

Adapted from: Kehrer JP, 1993

Cellular Damage Cellular Damage caused by Oxidative Stresscaused by Oxidative Stress

PathologicalPathological conditions that conditions that involve involve

oxidative stressoxidative stress•InflammationInflammation•AtherosclerosisAtherosclerosis•Ischemia/reperfusion injuryIschemia/reperfusion injury•CancerCancer•AgingAging•ObesityObesity

Oxidation of the Oxidation of the lipid components & apo B in LDLlipid components & apo B in LDL

Oxidized LDL Oxidized LDL

Injury of endothelium (inner lining of blood vessels)Injury of endothelium (inner lining of blood vessels)

Monocytes adhere to endothelial cells Monocytes adhere to endothelial cells

Move to (subendothelium) intimaMove to (subendothelium) intima

Monocytes are converted to Macrophages Monocytes are converted to Macrophages

Macrophages uptakes oxidized LDL through scavenger receptor Macrophages uptakes oxidized LDL through scavenger receptor class A (SR-A)class A (SR-A)

Macrophages are converted to Foam cells Macrophages are converted to Foam cells

Release cytokines Release cytokines & participates in formation of & participates in formation of

ATHEROSCLEROTIC PLAQUESATHEROSCLEROTIC PLAQUES

Oxidative stress & Oxidative stress & AtherosclerosisAtherosclerosis

Athersclerotic Plaque FormationAthersclerotic Plaque Formation