REACTIVE OXGEN AND NITROGEN SPECIES AND THEIR BIOLOGICAL EFFECTSPeter Kaplán

Reactive oxygen and nitrogen species ROS/RNSFree radical – each molecule or its fragment, which can exists independently And contains one or two unpaired electronsReactive oxygen species - species, which contain one or more oxygen atoms and are much more reactive than molecular oxygen

ROS/RNSFree radicals

superoxide radical

hydroperoxyl radical

hydroxyl radical

nitric oxide

hydrogen peroxide

Some characteristics of ROS ROS Symbol Half-

lifeProperties

Superoxide radical

O2•- 10-6 s poor oxidant

Hydroperoxyl radical

HO2• stronger oxidant than O2

•-

Hydrogen peroxide

H2O2 minúty oxidant, diffuses across membranes

Hydroxyl radical OH• 10-9 s extremely reactive, diffuses only to very low distance

Alkoxyl radical LO• 10-6 s less reactive than OH•, but more than ROO•

Peroxyl radical LOO• 10-2 s weak oxidant, high diffusability

Singlet oxygen 1O2 10-6 s powerful oxidizing agent

Sources of ROS, RNS and other oxidants

H 2O O H + H

ioniz ing radiation

h

Ionizing radiation:homolytic break of covalent bondsin water, DNA and other biomolecules

Cellular sources of ROS

xanthine oxidasehemoglobinriboflavincatecholamines

Cytochrome P450

electron transport chain

lipid peroxidationNADPH oxidase (oxidative burst: phagocytes)

oxidasesflavoproteins

myeloperoxidase (oxidative burst: phagocytes)

transient metals

Cellular sources of ROS - examples

O 2 O 2- H 2O 2 H 2O + O H H 2O

e- e- + 2H+ e- + H+ e- + H+

Hb(Fe2+)-O 2 metHb(Fe 3+) + O 2-

Fe2+ + H 2O 2 Fe 3+ + HO + O H -

Electron transport system:

Autooxidation of hemoglobin:

Fenton reaction:

Cellular sources of RNSO

O

NHNH2

NH2

OH

NH

O

NHNH2

NH2

OH

NO synthase

NADPH NADP+

O2, biopterin

+ NO

Formation of nitric oxide

From arginine:

NO + O2- ONOO- ONOOH

H+

ONOOH HO + NO2

NO and superoxide radical combine to form strong oxidizing agent peroxynitrite:

Peroxynitrous acid breaks down to form additional ROS/RNS (hydroxyl radical and nitrogen dioxide):

Three tissue-specific NO synthases: 1) neuronal (nNOS)2) inducible (iNOS) –in cells of the immune system, macrophages – responsible for the overproduction of NO 3) endothelial (eNOS)

Formation of ROS and peroxynitrous acid in phagocytic vacuole of phagocytes

SOD – superoxid dismutaseMPO - myeloperoxidase

LH + HO L + H2O

L + O2 LOO

LH + LOO L + LOOH

Formation of lipid (alkyl) radical initiatedby ROS:

Alkyl radical react with O2 to produceperoxyl radical:

Peroxyl radical attacks another poly-unsturated FA to produce new alkyl radical and lipid peroxide:

Oxidative damage to lipids - Lipid peroxidation (LPO)

ROS

O

O

NH2

NH2

Proteín

NH

N

Proteín

+

NH Proteín

N ProteínNH2 Proteín

+

NH2 Proteín

O

O

O O

OCH3

OH

malondialdehyde

4-hydroxynonenal

HNE – highly toxic aldehyd, which reacts with free –SH and -NH2 groups of proteins and -NH2 groups of DNA

MDA - toxic aldehyde, which reacts with free -NH2 groups of proteins and DNA

Loss of biological activityDNA mutation

Oxidative damage to proteins and DNA - Modification by LPO products. Formation of intra- and intermolecular cross-links.

Oxidative modification of proteins

OH OH

R R

dityrosine

OH

NO2

R

3-nitrotyrosine

dityrosine and 3-nitrotyrosine –markers of protein damage by ROS and RNS

Oxidative damage to DNA

Formation of thymine dimers

Modification of purines – 8-hydroxy-deoxyguanosine Marker of the oxidative damage to DNA

Oxidative damage to DNA – DNA mutation

Glucose and cell oxidative injury

Hyperglycemia

Glucose autooxidation

Oxidative stress

Formation of AGE products Polyol pathway

Angiopathy Nefropathy Neuropathy Retinopathy

Glucose and oxidative damageFormation of AGE products

AGE – advanced glycation end-products (pentosidine and others)

C

C

C

C

C

H2C OH

O

H OH

OH H

H OH

H OH

HC

C

C

C

C

H2C OH

N

H OH

OH H

H OH

H OH

HH2C

C

C

C

C

H2C OH

OH H

H OH

H OH

NH

OH2N protein

protein protein

ROS H2N protein

glucose Schiff base Amadori products pentosidine glycated proteins AGE

N

N

NH

NH Arg +

Lys protein

protein

Mechanisms of cell injury mediated by ROS and RNS

ROS a RNS

Modification of aa, fragmentation and

aggregation of proteins

Lipid peroxidation DNA damage

Membrane damage

Loss of membrane integrity

Damage to Ca2+ and other ion transport systems

Inability to maintain normal ion gradients

Activation/deactivation of various enzymes

Altered gene expression

Depletion of ATP

Lipids Proteins DNA

Cell injury/ Cell death

Antioxidants and secondary defense systems

Prevent ROS formation Eliminate radicals by formation of nonradicals or less reactive radicals Repair dameged molecules and cell structures Expression of genes coding for antioxidant enzymes

Antioxidants and secondary defense systems

Enzyme antioxidants

Nonenzymatic antioxidants

Chelating agents Enzymes of repair and de

novo synthesis of damaged molecules

Water-solubleLipid-soluble

EndogenousPresent in diet

Antioxidant enzymes

ENZYME TISSUE SITE ______ Superoxide dismutase

Cu/Zn SOD primarily cytosol, nucleus

Mn SOD mitochondria

EC SOD extracellular fluid

Catalase peroxisomes

Glutathione peroxidase GPx cytosol, mitochondria

Glutathione reductase GRed cytosol, mitochondria _________________________________________________

Antioxidant enzymes

O 2- + O 2

- + 2H + H 2O 2 + O 2 SOD

SOD scavenges superoxide radical:

2H 2O 2 2H 2O + O 2

Catalase decomposes hydrogen peroxid in peroxisomes :

Glutathione peroxidase (GPx) decomposes H2O2 and lipid peroxides in

cytosol and mitochondria by help of GSH, NADPH and glutathionereductase

(GRed):

GPx GRed

H2O + LOH GSSG NADPH

LOOH 2GSH NADP+

Nonenzymatic antioxidants

Endogenous antioxidants - Synthesized in the bodybilirubín glutathione and other thiocompounds (thioredoxin)uric acidcoenzyme Q (Ubichinon-10/Ubichinol-10)lipooic acidmelatoninsex hormones 2-oxoacids (pyruvate, 2-oxoglutarate)dipeptides containig His (carnosine, anserine)albumin (-SH groups)

Dietary antioxidantsascorbic acidvitamine Ecarotenoidsflavonoids – plant phenols (catechin, quercetin etc)

Synthetic antioxidants N-acetylcystein (scavenger of ROS), deferoxamine (chelator), alopurinol (inhibitor of XO), acetyl salicylic acid (feritine synthesis)

Nonenzymatic antioxidants – regeneration of α-tocopherol by ascorbic acid

dehydroascorbic acid

ascorbic acid

Chelating agents and secondary defenses_________________________________________________

CHELATING AGENTS Transferin sequestration of Fe3+ Ferritin sequestration of Fe3+ Lactoferin sequestration of Fe3+ Ceruloplasmin sequestration of Cu2+ Albumin sequestration of Cu2+, weakly Fe3+ ENZYMES OF REPAIR AND DE NOVO SYNTHESIS OF DAMAGED

MOLECULES Fosfolipases Proteinases Enzymes of DNA repair ADAPTATION Increased synthesis of antioxidant enzymes _________________________________________________

ROS, RNS and signal transmission – redox signaling

ROS/RNS

ionic channels and pumps

thioredoxin GSH

transcription factors

protein kinasesprotein phosphatases

Enzymes of CTCrespiratory chain

cell growth/apoptosis/survival

Molecular mechanisms:

modification of the redox state of protein (protein thiol groups)

nitrosylation of protein

NO – signaling molecule, which difuses through water and lipid membranes

At low concentrations serves as a second messenger and neurotrasmitter and participates in:

smooth muscle relaxation lowering blood pressure inhibition of pletelet aggregation inflammatory and immune responses

Pro-oxidants, antioxidants and oxidative stress

Oxidative stress – imbalance of pro-oxidants and antioxidants in favor of the former

Oxidative stress – disruption of redox signaling and control

Major diseases/disorders linked to ROS/RNS Respiratory system

Inhalation of oxidants (SO2, NO2, O3)

Smoking

BrainAlzheimer`s diseaseParkinson`s diseaseAmyotrophic lateral sclerosisDown syndromeTraumatic injury

Cardiovascular systemAtherosclerosisIschemia-reperfusion injuryMyocardial infarction and heart

failureSelenium deficiency (Keshan

disease)

SkinIonizing radiationThermal injuryPorphyriasPhotosenzitizers and other

reagents

MuscleExerciseMuscular dystrophy

OthersAgingCancerCataractsDiabetes mellitusInflamatory and autoimmune

diseasesLiver damage by endotoxins

or halogen derivatives of hydrocarbons

Kidney diseases/disordersAIDS

Formation of ROS during ischemia and reperfusion

ATP

ADP

AMP

adenosine

inosine

hypoxanthine

Xanthine dehydrogenase

Xanthine oxidase

Xanthine + •O2- + H2O2

I S C

H E

M I

A

R E P E R F USION

Ca2+ proteolysis