A seminar on

Oxygen free radicals and their Scavengers

ByHardik G. DaveIst year M. PharmHSKCOP, Bagalkot

Introduction

It is believed that life has originated from basic

chemicals by free radical reaction, largely

initialled by ionising radiation from sun.

Paradoxically the same reactions creating life

may also be responsible for many diseases,

ageing and death.

Free radical - what is it?• Free radical is an atom or group of atoms with

at least one unpaired electron.• Example :

• In the body it is usually an oxygen molecule that has lost an electron and will stabilize itself by stealing an electron from a nearby molecule.

• In the body free radicals are high-energy particles that ricochet wildly and damage cells.

Hydroxyl radical ( OH)O

H

Radicals can be formed by…

1. The LOSS of a single electron from a non-radical, or by the GAIN of a single electron by a non-radical. And

2. The breakage of covalent bond ‘homolytic fission’

AB A + B

• Example H2O H + OH

• These are highly reactive chemical entities that have a single unpaired electron in their outer most orbit.

• Under certain conditions can be highly toxic to the cells.• Generally unstable and try to become stable, either by

accepting or donating an electron.• Therefore if two free radical react, they neutralise each

other.• However, if the free radical react with stable molecules,

there is generation of more free radicals.• This character enables the FRs to participate in auto

catalytic chain reactions, – Molecules with which they react are themselves converted to

free radicals to propagate the chain of damages.

• Any free radical involving oxygen can be referred to as reactive oxygen species (ROS).

• Oxygen centered free radicals contain two unpaired electrons in the outer shell.

• When free radicals steal an electron from a surrounding compound or molecule, a new free radical is formed in its place.

• In turn the newly formed radical then looks to return to its ground state by stealing electrons with antiparallel spins from cellular structures or molecules.

• These results in damaging the cell membranes. It also responsible for more than 100 human diseases like cancer, heart attacks, stroke and arthritis

• TYPES OF FREE RADICALS :• The most common ROS include: i. the superoxide anion (O2-), ii. the hydroxyl radical (OH ·), iii. singlet oxygen (1O2 ), and iv. hydrogen peroxide (H2O2)

1. SUPEROXIDE ANION (O2-) :• Superoxide anions are formed when oxygen (O2) acquires an

additional electron, leaving the molecule with only one unpaired electron. Within the mitochondria.

• O2- ·is continuously being formed.2. HYDROXYL RADICAL (OH- .) :• Hydroxyl radicals are short-lived, but the most damaging radicals

within the body.• This type of free radical can be formed from O2- and H2O2 via

the Harber-Weiss reaction.• The interaction of copper or iron and H2O2 also produce OH · as

first observed by Fenton. 3. HYDROGEN PEROXIDE (H2O2) :• Hydrogen peroxide is produced in vivo by many reactions. • Hydrogen peroxide is unique in that it can be converted to the

highly damaging hydroxyl radical or be catalyzed and excreted harmlessly as water.

• Glutathione peroxidase is essential for the conversion of glutathione to oxidized glutathione, during which H2O2 is converted to water (2).

• If H2O2 is not converted into water 1O2 (singlet oxygen) is formed.

4. SINGLET OXYGEN (1O2) :• Singlet oxygen is not a free radical, but can be formed during

radical reactions and also cause further reactions.• When oxygen is energetically excited one of the electrons can

jump to empty orbital creating unpaired electrons.• Singlet oxygen can then transfer the energy to a new molecule

and act as a catalyst for free radical formation.• The molecule can also interact with other molecules leading to

the formation of a new free radical.

PHYSIOLOGICAL EFFECTS :• Under normal conditions (at rest) the antioxidant defense system

within the body can easily handle free radicals that are produced. • During times of increased oxygen flux (i.e. exercise) free radical

production may exceed that of removal ultimately resulting in lipid peroxidation.

• Free radicals have been implicated as playing a role in the - etiology of cardiovascular disease, cancer, Alzheimer's disease, and Parkinson's disease.

THE ROLE OF OXYGEN FREE RADICALS :

• Our bodies use oxygen to convert food items such as fat and sugar into energy, in this process, oxygen is converted to water, and each water molecule normally takes up four electrons.

• However some oxygen may escape before the conversion is complete, and this results in about 2% of the oxygen having an electron deficit.

• They may also be formed in the human body by air pollution, smoking and exposure to radiation.

• Free oxygen radicals are extremely reactive and can cause damage to body proteins and fats, and also to the hereditary material of cells, known as DNA.

• Fortunately our bodies have some protective mechanisms against the harmful effects of free oxygen radicals These are enzymes and antioxidants.

• In mitochondria:- generation of energy - ATP- glucose, fatty acids, amino acids- O2 2H2O

4e-

- leakage of O2-. (superoxide)

H2O2 (hydrogen peroxide)

• In Smooth Endoplasmic Reticulum (micro some)- detoxification (cytochrome P-450s)- toxins, drugs and xenobiotics- O2 + RH R-OH and H2O

- leakage of O2-.

- metabolic activation - X.

Leaking’ of electron (to oxygen) during electron transport leads to the formation of O2

- (O2 + e- O2-)

Superoxide Production from Mitochondrial Electron Transport Chain

• • • •

• NADPH

• NADP+

• O2

• O2-

• NADPH

• NADP+

• O2

• O2-

• outside• inside

• .• .• .• .

• Phagocytic vacuole (phagosome)

Electron is transferred from NADPH to O2, resulting in the formation of O2-

•Formation of NADPH oxidase complex

Oxygen burst (respiratory burst) during phagocytosis

• In Peroxisomes- containing oxidases for degradation of various substrates- glucose, amino acids, xanthine, etc.- requires O2

- by product is H2O2

• In Cytoplasm- nitric oxide (NO.) production from Arginine- functions as a biological messenger- in brain, vascular endothelial cells, and macrophages

- NO. + O2-. ONOO. (peroxynitrite)

• Other source of free radical– Ionizing radiation, Ultraviolet radiation ,Ultrasound

– Chemicals, tobacco smoke, etc

Roles of Free Radicals in Biological Systems

• Enzyme-catalyzed reactions• Electron transport in mitochondria• Signal transduction & gene expression• Activation of nuclear transcription factors• Oxidative damages of molecules, cells, tissues• Antimicrobial actions• Aging & diseases

1.Oxidative stress :• Damages caused by free radicals/reactive oxygen species• Cellular damages at different levels (membrane, proteins,

DNA, etc) lead to cell death, tissue injury, cellular toxicity, etc• Reduction of antioxidants

2. Free Radical Toxicity :• Causes of free radical toxicity

– Increase production of free radicals– Decrease level of defense system (e.g.,antioxidants)

• Lipid peroxidation• DNA damage• Protein oxidation

Lipid Peroxidation1. Initiation of first-chain reaction

• Abstraction of H+ by ROS (•OH)• Formation of lipid radical (LH•)• Formation of peroxyl radical (LOO•, ROO•)

2. Propagation• H+ abstraction by lipid peroxyl radical (LOO•)

3. Termination • Radical interaction non-radical product

•

Molecular rearrangement

Conjugated diene

Lipid hydroperoxide

Cyclic peroxide

Cyclic endoperoxide

••-H

Hydrogen abstractionI

•

O

O

O

O

H

•H

O2 Oxygen uptake

Peroxy radical: abstractH• from another fatty acidcausing an autocatalytic chain reactions

P

I

P

Initiation

Propagation

(LH•)

(LOO•)

Oxidative DNA Damage• Correlation with cancers and diseases• Oxidative DNA lesions by

-Direct attack

-Indirect activation of endonuclease enzymes

• Oxidative modification of bases – mutation• Oxidative modification of sugar moieties –

DNA strand

break

• Abstraction of H+ atom from carbon atoms of sugar molecules• Disproportionations and rearrangement lead to C-C bond fragmentation and DNA strand break

A computer image depicts a hydroxyl radical attacking the sugar on the back bone of a DNA molecule

Protein Oxidation

• Protein targets– Receptors, transport proteins, enzymes, etc– Secondary damage – autoimmunity

• Protein oxidation products– Protein carbonyl group, 3-nitrotyrosine, other

oxidized amino acids• Most susceptible amino acids

– Tyrosine, histidine, cysteine, methionine

Antioxidant/Scavengers

Damage(Pro-oxidants)

Defense(Antioxidants)

Oxidant-Antioxidant Balance

Damage(Pro-oxidants)

Defense(Antioxidants)

Decrease of antioxidant defense system

Oxidative damage

Cellular Defense Mechanisms

• Isolation of generation sites of reactive oxygen species

• Inhibition of propagation phase of reactive oxygen species

• Scavenging of reactive oxygen species• Repair of the damage caused by reactive

oxygen species

Protection Against ROS Damage

• Direct protection against ROS– Superoxide dismutase, Glutathione peroxidase, Catalase

• Non-specific reduction system– Glutathione, Vitamin C

• Protection against lipid peroxidation– Glutathione peroxidase, Vitamin E, -Carotene

• Sequestration of metals– Transferrin, Lactoferrin, Ferritin, Metalothionein

• Repair systems– DNA repair enzymes, Macroxyproteinases, Glutathione transferase

Superoxide Dismutase (SOD)2O2

. - + 2H+ H2O2 + O2

• SOD - is present in all oxygen-metabolizing cells, different cofactors (metals).

• An inducible in case of superoxide overproduction.

1. CuZn-SOD – Cytoplasm, nucleus, lysosomes

2. Mn-SOD – Mitochondrial matrix

3. EC (CuZn) – Plasma membrane, extracellular

4. EC Mn-SOD – Plasma membrane

Glutathione/Glutathione Peroxidase

Catalase

2 H2O2 2 H2O + O2

• High affinity to H2O2 : peroxisomes hepatocytes mitochondria, cytoplasm of erytrocytes.

• Tetramer with Fe, needs NADPH.• Prevents lipid peroxidation and protein oxidation.

Summary

Melatonin• Highly selective & electroactive endogenous indoleamine.• Present in good amounts in the nervous system.• Sacrificed & irreversibly oxidized.• Inhibit NOS• Stimulate brain GPX activity• Increases mRNA levels for Mn & Cu-Zn SOD

Ascorbic AcidAntioxidant Function• Donate 1 e- semidehydroascorbate (ascorbyl radical)

Relatively unreactive Tocopherol• Scavenges peroxyl radical• Inhibits chain reaction of lipid peroxidation• Chain-breaking antioxidant

REFERENCE

• Text book of Pharmacotherapy and physiological approach , JOSHEP T DIPIRO, 5th edition, pg no – 390 and 1085.• Harpers Biochemistry 26th edition.• Lehninger's Principles of Biochemistry 4th Edition - D L

Nelson, Cox Lehninger - W H Freeman 2004.• Free-Radical-Induced DNA Damage and Its Repair - A

Chemical Perspective (Springer, 2006)• Rang and Dale’s Pharmacology , 6th edition • www.google.com