Free Radicals

&
Antioxidants

Dr.Dr Prasad DKVasad

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 RADICALS

“-any species capable of independent existence

that contains one or more unpaired electrons”

Example : 
 O  H Hydroxyl radical (OH)


Free Radicals can be formed by…

 The LOSS of a single electron from a non-radical, or
by the GAIN of a single electron by a non-radical
 Due to leaks in ETC in mitochondria
FREE RADICAL NOMENCLATURE

 A free radical is denoted by a superscript

dot to the oxygen (or carbon)
– e.g., HO , NO ,  CH3

 If a free radical is a charged species, the

dot is put and then the charge
– e.g., O2 -
Formation of free radicals
Univalent reduction of molecular
oxygen in tissues gives rise to the
production of ‘Superoxide radical’
.-
(O ), Hydrogen peroxide (H O )
2 2 2

.
and Hydroxyl radical (OH )
TYPES OF FREE RADICALS

 Oxygen-centered radicals
 Singlet oxygen, superoxide, hydroxyl radicals
 Sulfur-centered radicals
 Thiyl radical (RS )
•

 Carbon-centered radicals
 CCl3, CH2 CHOH (Ethenol radical)
• •

 Nitrogen-centered radicals
 NO , R2NO ( Aminoxyl radical)
• •
 FREE RADICALS

.
Superoxide anion radical (O - )
2

 Hydroperoxyl radical (HOO • )

 Hydrogen peroxide (H2O2)
 Hydroxyl radical (OH • )
 Lipid peroxide radical (ROO•)
 Singlet oxygen ( ’O2)
 Nitric oxide (NO • )
 Peroxy nitrite (ONOO- • )
When oxygen molecule takes up one
electron, by univalent reduction, it
forms ‘Superoxide’ anion, (O2-).
O2+ e- (O2-)

This superoxide anion is highly reactive

and toxic to cell membranes
OTHER FREE RADICALS IN THE BODY ARE-

By sequence of reactions that occur to

form other free radicals
1. Superoxide anion can capture further
electrons to form “hydrogen peroxide”
(H2O2), which is also toxic and injurious.

2e-,2H+
(O2 -) e-. 2H+ H2O2 2H2O
 Hydrogen Peroxide

 Not a free radical but ROS (reactive

oxygen species)
 Formed in the cell by dismutation
reactions by
– Spontaneously during respiratory burst
– By enzymes Eg: Superoxide dismutase
 H2O2 can further react with Superoxide
anion , in the presence of Fe+2
(Ferrous ion) to form “Hydroxyl”
radical, and “Singlet oxygen”
.
H2O2 + O2 - OH + ‘O2 + OH-
Fe++
Free Singlet
hydroxyl oxygen
radical

 This is Haber’s reaction or Haber-Weiss-

Fenten’s reaction
FENTON REACTION

• Discovered by Fenton (1894)

• “A mixture of hydrogen peroxide and an iron(II) salts
causes the formation of hydroxyl radical”

Fe2+ + H2O2 intermediate complex Fe3+ + OH- + HO

- +
Fe3+ + H2O2 intermediate complex Fe2+ + O2 + 2H

• Haber-Weiss reaction

Fe2+ + H O Fe3+ + OH- + HO

2 2


Fe3+ + O 2 - Fe2+ + O 2 metal
O2 + HO + OH-
catalyst
 Role of Ceruloplasmin

 It acts as Ferroxidase, can serve as

antioxidant
 It can convert Fe++ to Fe+++ and thus
it can halt the “Haber’s reaction”.,
preventing further formation of highly
reactive “hydroxyl” free radicals.
Superoxide anion can accept a H+
and form “Hydroperoxy” radical.

H+ HOO’
O2- HOO’ H2O2 + O2
Nitric oxide produced in the body
from Arginine (by the action of
(NOS), 3-4 sec half- life
It reacts with Oxygen and Superoxide
(O2-) to form “Peroxynitrite” radical
(ONOO-).
On decomposition, it forms highly
.
reactive OH radical.
CHARACTERISTICS of Free Radicals
 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 and cell membranes.
 Generally unstable and try to become stable,
either by accepting or donating an electron.
 Short life span
 Generation of new ROS by chain reaction
  
O   O Oxygen (O2)
 
Reactive Oxygen Species

• Radicals – Hydroxyl radical  O  H Hydroxyl radical (OH)


H
 
• Molecules – Hydrogen  O O 
   Hydrogen peroxide (H2O2)
peroxide  
H


 -
 O H Hydroxy anion (OH-)


• Ions – Hypochlorite ion   -

 O  Cl 
   Hypochlorite anion (OCl-)

• Superoxide anion – which is   -

both ion and radical  O  O Superoxide anion (O )
-
  2
FORMATION OF SUPEROXIDE ANION
IN METABOLIC PATHWAYS

 Xanthine oxidase (enzymatic reduction)

 Mitochondrial electron transport chain
 During phagocytosis (Respiratory burst)
 During hydroxylations of steroids, drugs, and
xenobiotics
 Aldehyde dehydrogenase
 Univalent oxidations with O2
 During methaemoglobin formation
 Exposed in ionizing radiations
Mitochondrial
Electron Transport Chain

• The most important source of O2- in vivo is

most aerobic cells
BIOLOGICAL GENERATION OF
SUPEROXIDE
• Enzymatic reduction of oxygen

Xanthine/hypoxanthine XOD Uric acid

[XOD = xanthine oxidase]
O2 O2
-

• Redox cycling : Paraquat (Xenobiotic)

Oxidized
cytochomre +
NADPH
P450
H3C N • N CH3 O2
reductase

Reduced
+ +
cytochomre N
NADP
P450
H3C N CH3 O2  -
reductase
e-
Paraquat
Superoxide Production from
Mitochondrial Electron Transport Chain

‘Leaking’ of electron (to oxygen) during electron transport leads to

the formation of O2- (O2 + e- O2- )
• Passage of electrons from Complex I to
QH2 involving the radical Q· as an
intermediate.
 Respiratory Burst

NADPH oxidase complex

• Cytoplasmic proteins
(p47, p67, gp91, p22)
 NADPH NADP+ + H+
 Electron is transferred
from NADPH to O2,
resulting in the formation
of O2-
[NADPH : Reduced Nicotinamide Adenine Dinucleotide Phosphate]
 Hypochlo-
rous acid

GENERATION OF ROS in MACROPHAGES and PMNs

Respiratory Burst

• Myeloperoxidase
– Oxidizes Cl- to hypochlorous acid
– Chronic granulomatous disease

• NADPH oxidase enzyme

NADPH
O2 NADPH
.. . O2
• • . -
O2
NADP+
O- NADP+
outside inside 2

Phagocytic vacuole (phagosome)

Nitrogen-Centered Radicals
• Nitric oxide (NO)
– Endothelial derived-relaxing factor (EDRF)
– Generated from the catalysis of L-arginine by
nitric oxide synthase (NOS) enzymes
– Functions
• Vascular function, platelet aggregation, immune
response, neurotransmitter, signal transduction
• cytotoxicity
– NO + O2- ONOO- (highly toxic)
 BH4
L-arginine L-citrulline + NO
NOS

• In the absence of L-arginine and BH4(tetrahydrobiopterin),

the activation of molecular by NOS results in a divalent
reduction of O2 to yield superoxide anions and hydrogen
peroxide.
SOURCES OF FREE
RADICALS

• Endogenous sources of free radicals

– Oxidative metabolic transformation
• Mitochondrial respiratory chain
• Oxygen burst (respiratory burst) during
phagocytosis
• Eicosanoid synthesis
• Enzymatic reactions (oxygenases, oxidases)
– Xenobiotic metabolism (redox cycling)
SOURCES OF FREE
RADICALS

• Exogenous sources
of free radicals
– Ionizing radiation
– Ultraviolet radiation
– Ultrasound
– Chemicals, tobacco
smoke, etc
SCAVENGERS OF FREE RADICALS

 Superoxide dismutase (SOD)- O2-.

 Catalase (CAT)- H2O2
 Glutathione Peroxidase (GPx)- H2O2

 Ferricytochrome- O2-.
 Endogenous ceruloplasmin- H2O2
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
OXIDATIVE
STRESS
O 2

Non-enzymatic sources
Mitochondrial respiratory chain  OH
Glucose autoxidation
Enzymatic sources
NADPH oxidase Fenton reaction
Xanthine oxidase (Fe or Cu)
Cyclooxygenase

SOD
O2 H2O2
GSH
NO
GPx Catalase
GSSG

ONOO-
H2O + O2
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
 EFFECT OF FREE RADICALS ON
BIOMEMBRANES
 Free radicals can initiate chain reaction and brings
about lipid peroxidation. Hydroxyl radical is most
reactive and also mutagenic.
 The oxidants can oxidize
 –SH groups to S-S groups
 Lipids and Poly unsaturated FA to lipid
peroxides and lipoxides. This will affect the
fluidity of membrane causing membranopathy
Lipid Peroxidation

1. Initiation of first-chain reaction

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

RH + OH . .
R + H O --------1A
2

Metal ion
ROOH ROO
. + H+ ------------------------------1B
2. Propagation
H+ abstraction by lipid peroxyl radical
(LOO )
•

Under aerobic conditions, the fatty acid

.
radical (R ) takes up oxygen to form
peroxy radical (ROO-) which can attack
another PUFA.
The latter, in turn, can remove H-atom from
another PUFA (RH) to form lipid
hydroperoxide (ROOH).
.
R +O ROO-
2

ROO- + RH ROOH + R.
The hydroperoxides are capable of further
stimulating lipid peroxidation as they can
form alkoxy (RO-) & peroxyl (ROO-)radicals.

2ROOH RO- + ROO- + H2O

ROOH RO- + ROO- + aldehydes
3. Termination
Lipid peroxidation proceeds as a chain
reaction until the PUFA gets oxidized.

.
ROO + ROO
. ROOR+ O2
.
ROO + R
. ROOR

.
R +R
. RR
The products of lipid peroxidation are unstable
e.g. carbonyls, esters, alkanes, alkenes, 2-
alkenal ,2 ,4- alkadienal, MDA.

Malondialdehyde (-CHO-CH2-CHO-) is a marker

for the assessment of lipid peroxidation.
MDA & other aldehydes react with
thiobarbituric acid & produce red-coloured
products - thiobarbituric acid reactive
substances (TBARS) which can be measured
colorimetrically.
I Hydrogen abstraction

-H •
(LH•)
•
Molecular rearrangement
Conjugated diene
•
O2 Oxygen uptake
Peroxy radical: abstract
P (LOO•) H• rom another fatty acid
causing an autocatalytic
O chain reactions
O H•
• Lipid hydroperoxide

O Cyclic peroxide
O I Initiation
H Cyclic endoperoxide
P Propagation
Products of Lipid Peroxidation

Reactive Oxygen Species

Lipid peroxides
Aldehyde
Alkanes products

Conjugated dienes ,-unsaturated

aldehydes
Malondialdehyde
(MDA) n-aldehydes
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
 A computer image depicts a
hydroxyl radical attacking the
sugar on the back bone of a
DNA molecule
Oxidative DNA Damage

• Abstraction of H+ atom from

carbon atoms of sugar molecules
• Disproportionations and
rearrangement lead to C-C bond
fragmentation and DNA strand
break
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
Protein Oxidation

Oxidative protein degradations

Modifications of Modifications of
amino acid chain prosthetic group
of enzymes

Protein aggregation
Protein fragmentation
Activations of protease enzymes
Free Radical Toxicity
Free Radicals and Diseases
• Cancer
• Inflammation/Infection
• Ischemia-reperfusion injury
• Neurodegenerative diseases
• Cardiovascular diseases
• Aging
• Others (e.g., drug/chemical-induced toxicity,
etc)
Ischemia-Reperfusion Injury

• Ischemic – reoxygenation
• Tissue damages caused by excessive
production of free radicals
• Cells lose ability to pump sodium outward
& develop intracellular edema
• Organs function well after resuscitation but
deteriorate in ensuing hours
Reasons for IRI
• Associated with the generation of ROS
• Leukocyte sequestration & activation
associated with generation of many
inflammatory mediators such as TNF, PAF,
& various proteases.
• Disseminated intravascular coagulation
Ischemia-Reperfusion Injury
ATP Xanthine
i CO2 dehydrogenase
s TNF Ca2+-dependent
c AMP protease
h
IL1 TISSUE
e pH C5
Xanthine INJURY
m
i Adenosine oxidase
a

Hypoxanthine/Purine

REPERFUSION Fe2+
O2 O2 + H2O2 OH- +

XOD
Xanthine/Hypoxanthine O2.- + uric acid
O2
Sources for reperfusion injury
• The catabolism of ATP to hypoxanthine
• The activation of neutrophils
• Ferrous form of iron catalyzing the
conversion of SOR and H2o2 to the
hydroxyl radical
Sequelae
• Lipid peroxidation of cell & organelle membranes
• Oxidize sulfhydryl groups
• Activate or inactivate enzyme systems
– Impaired calcium transport
– Decreased phosphocreatinine
– Activated collagenases degrade basement membranes
– Activated hyaluronidases degrade interstitial matrix
– Myocardial contractility is impaired
• Cause DNA & RNA depolymerisation
Measurement of Oxidative Stress
• Oxygen consumption
• Oxidative markers “footprints”
– Lipid peroxidation products (TBARs, lipid hydroperoxides, etc)
– DNA hydroxylation products (8-OHGua,
– Protein hydroxylation products (nitrosation products)
• Free radical detection
– Single photon counting
– Chemiluminescence
– Fluorescent probe
– Electron paramagnetic resonance spectroscopy (EPR)
ANTIOXIDANTS
Contents
• Oxidant-Antioxidant balance
• Biological actions of antioxidant defense
system
• Antioxidant defense system
– Superoxide dismutase (SOD)
– Catalase
– Glutathione cycle/Glutathione peroxidase
– Diet-derived antioxidants & Low molecular weight
antioxidants
• Roles in the cellular protection against oxidative
stress & oxidative stress-related diseases
 Oxidant-Antioxidant Balance

Damage Defense
(Pro-oxidants) (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
Antioxidant Defense System
• Antioxidant Enzymes
– Superoxide dismutase (SOD)
– Catalase (CAT)
– Glutathione peroxidase (GPx)
• Endogenous non-enzymatic
antioxidants
– GSH, bilirubin
Antioxidant Defense System
• Exogenous antioxidant molecules
– -Tocopherol -- prevents oxidation of fatty
acids
– Carotenoids (-carotene, leutin, lycopene,
etc) -- destroy a particularly damaging form of
singlet oxygen
– Ascorbic acid -- radical scavenging, recycling
of vitamin E
– Bioflavonoids -- potent antioxidant activity
SUPEROXIDE DISMUTASE
(SOD)
Function
2O2•- + 2H+ H2O2 + O2

k ~ 2-4 x 109 M-1s-1

• Only enzyme known to react with free radical

• The presence of SOD implies O2.- produced in
cell during normal metabolism
• * SOD is a primary antioxidant enzyme
Intracellular Location of SOD
• CuZn-SOD
– Cytoplasm, nucleus, lysosomes
• Mn-SOD
– Mitochondrial matrix
• EC (CuZn)
– Plasma membrane, extracellular
• EC Mn-SOD
– Plasma membrane
Structure and Properties of SOD
• CuZn-SOD
– One of the most stable protein
– Inactivated by guanidine HCl, CN-,
diethyldithiocarbamate (DETC)
• EC-SOD
– Inhibited by CN-, azide, H2O2, SDS
– Located in extracellular fluids
– Suppresses inflammation
• Fe/Mn-SOD
– Not stable
CATALASE (CAT)

Function : Removes H2O2

2 H2O2 2 H2O + O2

Prevents lipid peroxidation

and protein oxidation
Glutathione Cycle
Glutathione ~ Glu-Cys-Gly
Reduced glutathione (GSH)
Oxidized glutathione (GSSG)

Function : gets rid of H2O2 or ROOH (hydroperoxide)

ROOH 2 GSH NADPH

Glutathione Glutathione
peroxidase reductase

ROH + H2O GSSG NADP

GLUTATHIONE PEROXIDASE
(GPX)
Function : Removes H2O2 & ROOH
(lipid hydroperoxide)
H2O2 + 2 GSH Se GSSG + 2H2O

 Deficiency in GPX leads to oxidative

hemolysis.

 Protects against lipid peroxidation

 Selenium containing enzyme

Low Molecular Mass Agents

• Compounds synthesized in vivo

– bilirubin, melatonin, lipoic acid, uric acid, etc.

• Compounds derived from the diet

– Ascorbic acid
– Vitamin E
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-ZnSOD
Ascorbic Acid

Antioxidant Function
Donate 1 e- semidehydroascorbate (ascorbyl radical)

Relatively unreactive
Tocopherol

“Chain-breaking antioxidant”
Scavenges peroxyl radical
Inhibits chain reaction of lipid peroxidation

Eight naturally-occurring substances

d--, d--, d--tocopherols
d--, d--, d--tocotrienols
Biological Properties of Natural
Antioxidants
• Natural antioxidants
– Polyphenols (phenolic, flavonoids), carotenoids,
lycopene, etc
• Electron donor property
– Ability of antioxidant to donate an
electron to a species (free radical) –
reducing property
– Antioxidant remains stable
CLINICAL SIGNIFICANCE
 Role in ageing
 Neonatal oxygen radical diseases
 Rheumatoid arthritis
 Atherosclerosis
 Chronic granulomatous diseases
 Role in phagocytosis
 Role in malaria
 Role in cancer
 Role in DM
 Role in respiratory diseases
 SUMMARY

• Characteristics of free radicals/reactive

oxygen species
• Endogenous/Exogenous formation of free
radicals
• Oxidative cell damage (lipids, DNA, proteins)
• Oxidative damage-related carcinogenesis
• Antioxidants (types, functions)
• Roles in the preventions against oxidative
damage
BIBLIOGRAPHY

• Reactive Oxygen Species in Biological Systems-An

Interdisciplinary Approach (Gilbert,2002).
• Free-Radical-Induced DNA Damage and Its Repair - A
Chemical Perspective (Springer, 2006)
• Clinical Biochemistry of Domestic Animals (Sixth
Edition)
• Harpers Biochemistry 26th ed
• Lehninger's Principles of Biochemistry 4th Edition - D L
Nelson, Cox Lehninger - W H Freeman 2004
• Textbook of Small Animal Surgery (Slatter)
Thank You ALL…