OXIDATIVE STRESS AND

ANTIOXIDANTS
in health and diseases

MUHAMMAD IQBAL CHOUDHARY

Dr. Panjwani Center for Molecular Medicine and Drug Research

International Center for Chemical and Biological Sciences
University of Karachi, Karachi-75270
 Oxidation and Human Health

One of the paradoxes of life on this planet is

that the molecule that sustain aerobic life,
oxygen, is not only fundamentally essential for
energy metabolism and respiration, but
implicated in many diseases and degenerative
conditions.

Marx, Science, 235, 529-531 (1985).

 Learning Objectives
Understanding the relationship between oxidative
stress, and health and diseases…

• What are oxidants or ROS, types, sources, and activities?

• Their role in normal physiological process
• Their detrimental role in the onset, and progression of diseases,
chemical basis of oxidative damage!
• Biomarkers of oxidative damage to the vital biomolecules, and
their analysis
• What are antioxidants, types, sources, and activities?
• Perceived role of antioxidants in the preservation of health, and
prevention of diseases
• Anti-oxidant drug discovery and development- challenges and
opportunities
 Video Lecture to be used
• https://www.youtube.com/watch?v=mMl9eH
C4kyA

• Oxygen free radicals & cellular injury - causes,

symptoms & pathology Published on 15 Jan 2016 by
Osmosis
 CONTENT
What is oxidation?
• Oxidation in biological system
• What are free radicals?
• Sources of free radicals
• Harmful effects of free radicals
• Damage to proteins and associated diseases
• Damage to DNA and associated diseases
• Damage to lipids and associated diseases
• Damage to carbohydrates and associated diseases
• What are antioxidants?
• Nature’s antioxidants system
• Dietary sources of antioxidants
• Oxidative stress- Imbalance between oxidation and anti-oxidation
• Types of antioxidants
• Mechanism of anti-oxidation
• Bioassays used in the discovery of new antioxidants
 Why This Topic?

If you search for Antioxidants, Oxidative

Stress, ROS…

• Sci-Finder 108,762 Articles (Feb. 2018)

• Pubmed Over 800,000 Hits (Feb. 2018)
• Google Search Over 85,000,000 Web pages (Feb.
2018)
• Chemical Abstracts 131,961 Publications (2003-2013)
• Global Business US $ 3.25 billion
 What is Oxidation?
• Combination of substrate with oxygen.
• Reactions which involve the loss of the
hydrogen.
• Reaction in which some atom in a compound
lose electrons.
• Any compound, including oxygen, that can
accept electrons is an oxidant or oxidizing
agent (pro-oxidant), whereas a substance that
donates electrons is a reductant or a reducing
agent (antioxidant).
 Oxidation in Biological System
• We live in an aerobic environment
• Oxygen is the life sustaining element
• We consume approximately 3.5 kilograms of
oxygen every day
• 2.8 percent of the oxygen is not properly used
and forms free radicals
• Several kilograms of peroxides (harmful
oxidized lipids) are produced in our body
every year
 What are Free Radicals?
• Free radical is an atom that has lost an electron, and as a
result, has a net positive (+) charge.

• Free radicals are chemically reactive species, looking to

steal an electron from another molecule, thus generating
another free radical.

• This lead to initiation of chain reaction, in which more and

more free radicals are formed.

• If not controlled, free radical can cause major damage to

biological system (oxidative injury), such as injury to cell
membrane.
 What are Free Radicals?
• Free radicals (pro-oxidants) are any chemical
species, capable of independent (although
extremely short) existence with one or more
unpaired electrons
• Highly unstable and reactive
• Most common among them are Reactive Oxygen
Species (ROS)
• Reactive Nitrogen Species (RNS) (NO., ONOO-, etc) or
Reactive Sulfur Species (RSS) also exist
• Collectively called RONS (Reactive Oxygen and
Nitrogen Species)
 What are Reactive Oxygen Species
(ROS)?

• ROS are:
a. oxygen derived free radicals (O2.-, .OH, ROO.,
1O , RO.)
2
b. oxygen-derived non-radical reactive species (O2,
H2O2, O3, ROOH, HOCl)
• They are now considered as major players in
biochemical reactions, cellular response, and
clinical outcome.
Triplet Oxygen (Ground state) O O

Singlet Oxygen O O
Superoxide O O
Perhydroxyl Radical O O H

Hydrogen Peroxide H O O H

Hydroxyl Radical H O
.
Hydroxyl Ion O H
.
Hydrogen Peroxide H O O H
 Common Free Radicals
Oxidant Description
•O2-, superoxide One-electron reduced state of O2, formed in many
anion autoxidation reactions and by the electron transport
2+
chain in cell. Rather unreactive but can release Fe
from iron-sulfur proteins and ferritin. Undergoes
dismutation to form H2O2 spontaneously or by
enzymatic catalysis, and is a precursor for metal-
catalyzed •OH formation.
H2O2, hydrogen Two-electron reduction state, formed by
peroxide dismutation of •O2- or by direct reduction of O2.
Lipid soluble, and thus able to diffuse across
membranes.
•OH, hydroxyl Three-electron reduction state, formed by Fenton
radical reaction and decomposition of peroxynitrite.
Extremely reactive, will attack most cellular
components
 Common Free Radicals
Oxidant Description

ROOH, organic Formed by radical reactions with cellular

hydroperoxide components, such as lipids and nucleobases.

RO• alkoxy, and Oxygen centered organic radicals. Lipid forms

ROO• peroxy participate in lipid peroxidation reactions. Produced
radicals in the presence of oxygen by radical addition to
double bonds or hydrogen abstraction.
ONOO-, Formed in a rapid reaction between •O2- and NO•.
Peroxynitrite Lipid soluble and similar in reactivity to
hypochlorous acid. Protonation forms peroxynitrous
acid, which can undergo homolytic cleavage to form
hydroxyl radical and nitrogen dioxide.
Source= Wikipedia
 History: Harmful Effect of Free
Radicals
• 1775 Priestly- Toxicity of oxygen to the
organism similar to burning of candle
• 1954 Gilbert and Gersham- Free radicals are
important player in biological environment
and responsible for deleterious process in the
cell
• 1969 Mc Cord and Fridovich- Superoxide
theory of toxicity
 What Free Radical does?
Free radicals are cellular renegades; they wreak havoc
by damaging DNA, altering biochemical compounds,
corroding cell membranes, and killing cells outright.
Such molecular mayhem, scientists increasingly believe,
plays a major role in the development of ailments like
cancer, heart or lung diseases, and cataracts. Many
researchers are convinced that the cumulative effects
of free radicals also underlie the gradual deterioration
that is the hallmark of aging in all individuals, healthy
as well as sick.

TIME, April 6, 1992 publications

 Internal Sources of Free Radicals
• Mitochondria
• Phagocytes (macrophages)
• Xanthine oxidase
• Reaction involving iron and other transition metals
• Arachidonate pathways
• Exercise
• Inflammation
• Ischaemia/Reperfusion
 External Sources of Free Radicals
• Cigarette smoke
• Stress
• Unhealthy diet
• Environmental pollutants
• Radiations including ultraviolet radiations
• Ozone
• Certain drugs, pesticides, anesthetics and
industrial solvents
 Paradox of Oxidation
• Physiological levels of RONS (Reactive Oxygen
and Nitrogen Species) are crucial for a proper
cell functions, such as intracellular signalling,
inflammation, and immune functions.

• Problem arise when RONS levels exceed their

physiological concentrations, this leads to
oxidative stress.
 Useful Functions of Free Radicals
• Necessary in the maturation processes of cellular
structures
• Necessary in the antibacterial activity- White blood
cells (phagocytes) releases free radicals to destroy
invading pathogenic microbes as part of the body’s
defense mechanism
• Necessary in the immune system
• Necessary in the prostaglandin biosynthesis
• Some of them play an important role in cell
signaling, such as NO. (Nitric oxide)
 Anti-oxidative Defence Mechanisms
• Protection at cellular level is mainly carried
out by enzymes (superoxide dismutase,
catalase, glutathione peroxidase, glutathione
reductase, etc), and most importantly
glutathione (GSH).

• In plasma, various small molecules (a-

tocopherol, b-carotene, vitamin C, GSH, urate,
and plyphenols) (non enzymatic) play a major
role.
 Harmful Effects of Free
Radicals- Perception or
Reality
• Free radicals can damage all cellular
macromolecules, including proteins,
carbohydrates, lipids and nucleic acids.

• Destructive effect play a role in the onset and

progression of different diseases and in aging.
 Harmful Effect of Free Radicals-
Reality
• Experimental evidences has shown that
plasma TAC (Total antioxidant capacity) of
patients affected by different chronic diseases,
such as diabetes, AIDS, ulcerative colites,
Crohn’s disease, meningitis, CVDs, and
colorectal, lung and breast cancers is lower as
compared to healthy controls, suggesting the
deep impairment of the antioxidant network
during the development of these pathologies.
Diseases Related to Oxidative Damage
Ageing is one of the major consequences
of oxidative damage

1956 D. Harman- Free Radical Theory of Ageing

 Free Radical Theory of Ageing
• First proposed by Harman (1956).

• Ageing is due to accumulation of oxidative damages to

tissues and organs caused by free radicals.

• This is one of the major theories providing a testable

hypothesis.

Chen et al., Biology of Ageing and Role of Dietary Antioxidants, BioMed Research
International, 2014.
 Human Ailments Associated with
Oxidative Damage
Neurological
Alzheimer’s Disease
Parkinson‘s Disease
Endocrine
Diabetes
Gastrointestinal
Acute Pancreatitis
 Human Ailments Associated with
Oxidative Damage

Others Conditions

 Obesity
• Loss of catalytic functions of proteins
• Toxicity
• Chronic Inflammation and arthritis
• Cancers
 Exogenous v/s Endogenous
Sources of Free Radicals
• Exogenous ROS are in extremely high amount.
• Exposure to endogenous oxidants is much more
important and extensive, because it is a continued
process during the entire life span
• Mitochondria play an extremely important role in
endogenous ROS production
• Presence of metals (iron, copper, chromium, cobalt,
vanadium), in un-complexed forms, significantly
increases the level of oxidative stress.
 Damage to Lipids
• Lipids are highly prone to get oxidized.
• Polyunsaturated fatty acid (PUFA)- major part
of the low-density lipoproteins (LDL) in blood.
 Damage to Lipids
• Lipid peroxidation, if not terminated rapidly, can cause damage to
cell membranes.
• Removal of lipid peroxides is essential for mammalian life
(Glutathione peroxidase IV knock-out mouse doesn’t survive
beyond embryonic state).
• Malondialdehyde (MDA) is an important biomarker of oxidative
stress. It reacts with DNA bases to for DNA-adduct
End Products of Lipid Peroxidation
These end products are the
markers for lipid
peroxidation
determination. For example
malondialdehyde (MDA) is
detected in TBARS
(Thiobarb-
ituric Acid Reactive
Substance)
assay, specific for lipid
peroxidation determination.

13-HPODE= 13-
Hydroperxy-
9Z,11E-octdecdienoic acid

4-HNE= Hydroxynonenal
Damage to Lipids- Associated
Diseases
• Alterations in the structures of lipid molecules lead
to change in their physical properties, such as
permeability, surface adhesion, etc.

• It causes damage to the cell membrane, made up of

mainly lipids.

• Risk of cardiovascular diseases (CVD), including

atherosclerosis.
 Free Radical Damage to Cell
Membrane
• Cell membrane is composed of
polyunsaturated based lipid bilayers
• Free radicals, and other ROS cause sustainable
damage to cell membrane.
Damage to Lipids- Associated
Diseases
• End products of lipid peroxidation can also cause
mutagenesis and carcinogenesis. For example
malondialdehyde reacts with deoxyadenosine and
deoxyguanosine in DNA to form a variety of DNA
adducts.
• Body has evolved a range of molecules, such as
Vitamin E, and enzymes such as SOD, catalase, and
peroxidase to control lipid peroxidation.
• Knockout animals (which can not produce anti-
oxidant enzymes), generally do not survive.
 Atherosclerosis and Oxidative
Stress
• Compelling evidence points oxidative stress as an important
trigger in the complex chain of events leading to atherosclerosis.
• It involves accumulation of macrophages in the arterial wall.
Which then promptly incorporate oxidized LDL to form foam
cells.
• ROS can lead to platelet activation and thrombosis formation.
• Probucol has shown reduced progression of carotid
atherosclerosis in clinical trials.
 Oxidative Damage to
Proteins
• .OH and RO. and cause damage to proteins
• Direct damage include peroxidation, damage
to specific amino acid residues, change in
tertiary structures, degradation and
fragmentation
• No efficient mechanism of repair of protein
damage exists
• Proteolytic enzymes play an important role in
the removal of damaged proteins
Free Radical Damage to Proteins
 Protein Oxidation Products
• Aldehydes, keto, and other carbonyl compounds

• 3-Nitrotyrosine, produced by interaction of tyrosine

and ONOO-, is a useful biomarker of oxidative
protein damage

• Ortho- and meta-tyrosines from phenylalanine.

• Other damaged products include hydroxyproline,

glutamyl semialdehyde, etc

• Crossed linked proteins

 Damage to Proteins-
Associated Problems
• Modified oxidized proteins are susceptible to many
changes in their functions

• This include chemical fragmentation, inactivation, and

increased proteolytic degradation

• Oxidative changes in the structures of catalytic proteins

lead to loss of enzyme activity
 Damage to Proteins-
Associated Problems
•
• Altered cellular functions such as energy production,
interference with the creation of membrane potential
and change in the type and level of cellular proteins
• Non- enzymatic glycation of proteins lead to multiple
proteopathic disorders

• Serum protein carbonyl concentration is directly related

to muscle dysfunctions.
 Mechanism of Glycation of Protein- Role of
ROS
Glucose + Protein Schiff Amadori product
base

Protein Enediol

O2 Mn

O2 O2 M (n -1)
Mn M (n -1)
Protein dicabonyl
OH H2O2

Advanced
Glycation
Endproducts
Catalyzed by transition metals (M) and the superoxide radical generated are converted to
the hydroxyl radical via the Fenton reaction.
 Oxidative Damage to DNA

• DNA is stable, well protected molecules

• ROS, specially .OH, can interact with it and
cause several types of damage
• This includes modification of DNA bases, single-
and double-helical breaks, loss of purines,
damage to deoxyribose sugar, DNA-protein
cross linkage, and damage to DNA repair system
• Out of four bases, guanine is the most easily
oxidizable nucleic acid base
 Oxidative Damage to DNA

•
 Oxidative Damage to DNA
• Oxidative products of guanosine serve as
biomarkers of damage to DNA molecule.
 Oxidative Damage to DNA

• ROS in the cells lead to DNA damage, cause stable

DNA lesions which are mutagenic, if un-repaired
• Damaged DNA provide the wrong genetic code
leading to unregulated protein synthesis and/or cell
growth which results in cancer.
• Presence of 8-oxo-2-deoxyguanosine (oxo8dG) in
DNA is an important indicator of oxidative damage
to DNA
• Oxidative damage to DNA accumulate with ageing,
increasing the possibilities of cancers, and other
disorders
 Damage to DNA- Associated
Problems
• Number of oxidative hits to DNA per cell per day is
about 100,000 in the rat and about 10,000 in the
human (Reason????)
• There is an inherent mechanism (specific repair
glycosylases, etc.) to repair most of the DNA damage
caused by ROS
• Oxidative lesions in DNA accumulate with age and
eventually lead to serious health challenges (well
established relationship between onset of cancers
and age)
 Oxidative Stress Markers
Oxidative stress end products detection

Lipoperoxidation markers:
malondialdehyde (MDA), conjugated dienes,
isoprostanes
•Oxidative damage to protein markers :
protein hydroperoxides
•Oxidative damage to DNA :
modified nucleosides
 Potentialities of oxidative/nitrosative
stress-related biomarkers

Acta Medica Okayama, 61 (4), 181-189, 2007

 What are Antioxidants?

• Antioxidants (reductants or reducing agents)

are compounds capable of preventing the pro-
oxidation process or biological oxidative
damage by scavenging or stabilizing reactive
oxidative species.
An antioxidant is a molecule stable enough to donate an
electron to a rampaging free radical and neutralize it, thus
reducing its capacity to damage..
 What are Antioxidants?
• Antioxidants are produced during normal metabolism,
include glutathione, ubiquinol, and uric acid
• Antioxidant enzymes include glutathione peroxidases,
superoxide dismutases, catalase, and glutathione
reductase
• Antioxidants from dietary sources, such as Vitamins E
and C and carotenoids
• Antioxidants from non-dietary sources include
phenolic or polyphenolic compounds as well as
selenium
What Antioxidant Do???
 WHY ARE ANTIOXIDANTS
IMPORTANT ?
 They inhibit the conversion of nitrites to nitrosamines
(which are tumor promoters) and enhance the immune
response.
 Vitamins E, and C, ubiquinones, etc. remove free
radicals from the epidermis of the skin and counteract
their potentially damaging effect.
 They terminate free radical- induced cellular
damage and functional degeneration (aging).
 They trap and neutralize free radicals and protect our
body tissues from environmental pollutants.
 Sources of Antioxidants
• More than 4,000 antioxidants are known
• Endogenous- Antioxidant enzymes, include
glutatione peroxidases, superoxide
dismutases and catalase
• Antioxidants from dietary sources, such as
Vitamin E, Vitamin C and carotenoids
• Antioxidants from non-dietary sources include
phenolic or polyphenolic compounds
 Antioxidant Enzymes

• Glutathione peroxidases (Seleno proteins) catalyze

the reduction of lipid hydroperoxides to their
corresponding alcohols
• Superoxide dismutases, a family of metal-containing
enzymes (Mn, Fe, Zn, Cu), catalyze the dismutation
of superoxide into oxygen and hydrogen peroxide
• Catalases catalyze the decomposition of hydrogen
peroxide to water and oxygen
 In Vivo Anti-oxidant System –
Enzyme Based
Step 1 SOD
.O2- - +. O2- + 2H+ O 2 + H 2O 2

CAT
Step 2 H 2O 2 + H 2O 2 2H2O + O2

GPx
Step 3 H2O2 + 2GSH 2H2O + GSSG

GR

What we will do with .OH (Hydroxyl radical)?

 In Vivo Antioxidants- Small
Molecule
• Ubiquinol (Co-enzyme Q10) a 2,3-dimethoxy-
5-methyl-6-poly prenyl-1,4-benzoquinol, with
a 9-10 carbons long polyprenylated side-chain
in mammals.

• Uric Acid

• Glutathione
 Major Antioxidants-
Vitamin E
• Vitamin E, fat-soluble vitamin which exists in
eight different forms. a-Tocopherol is the
most active form in humans.
• Vitamin E protect cells through its ability to
limit production of free radicals
• Dietary sources include wheat, almonds,
sunflower seeds, etc.
 Major Antioxidants- Vitamin C
• Vitamin C, or L-ascorbate is an essential
nutrient, water-soluble
• Vitamin C scavenge aqueous peroxyl radicals
 Major Antioxidants- Carotenoids

• Organic pigments naturally occur in plants

• 600 known carotenoids, and tetraterpenoids exist in
nature
• Most common are lycopene and vitamin A precursor b-
carotene
• They quench singlet oxygen primarily by a physical
mechanism in which the excess energy of the singlet
oxygen is transferred to the carotenoids electron rich
structure
Lycopene
 Major Antioxidants-
Plant Phenolics

• A large number of plant phenolics, such as

flavanoids, and catechins act as free radical
scavengers
• Tea is the richest source of plant phenolics
• French paradox – Flavanoids in red wine
 Major Antioxidants-
Plant Phenolics

Apigenin

Apigenin Catechin
 Antioxidants at a Glance
Nutrient RDI Dietary Sources Evidence

Vitamin E 30 IU Vegetable oils (soy, corn, olive, 100-800 IU may lower

cotton-seed, safflower, sunflower), heart disease risk by
nuts, sunflower seed, wheat germ 30%-40%

Vitamin C 60 mg Citrus, strawberries, tomatoes, no evidence

that RDI
cantaloupe, broccoli, asparagus, supplement form can
peppers, spinach, potatoes prevent CHD or
cancer

ß-Carotene NA Dark green, yellow, and orange may protect against

vegetables: spinach, collard green CHD and macular
broccoli, carrots, peppers, sweet degeneration
potatoes; yellow fruits: peaches

Selenium 70 ug Egg yolks, tuna, seafood, chicken, 150-200 ug may lower

55 ug liver, whole grains, plant grown in prostate cancer risk
selenium-rich soil.
 Balance Between Oxidation
and Antioxidation- Key to
Health
• Balance between pro-oxidants and oxidants is tightly
regulated and extremely important for maintaining
vital cellular and biological functions

• The objective of the antioxidant therapy is not to

eliminate all the ROS, but to strike a healthy balance.
 Oxidative and reductive stress

Oxidant Reductant

Oxidant Reductant
Reductive stress
Oxidative stress
 What is Oxidative Stress?

• If there are too many free radicals produced and too

few anti-oxidants, a condition of “oxidative stress”
develop which may cause chronic diseases.
• An imbalance between the production of various
reactive species and the ability of the organism’s
natural protective mechanism to cope with these
reactive compounds and prevent adverse effects.
• It has been proven to be related to degenerative
diseases such as cancers, diabetes, premature ageing,
Alzheimer’s disease, arthritis, etc.
• Difficult to measure.
 Oxidative Stress Markers
Direct detection of free radicals
• Very difficult to analyze because of chemical
and physical properties e.g. short half life

Oxidative stress end products detection (foot

print measurement)
• more simple, a wide range of techniques
available
Some Common In Vitro Antioxidant
Assays

• NO· ( Nitric Oxide) Radical Inhibition

• Assay for DPPH Radical Scavenging Activity
• Assay for superoxide anion scavenging Activity
a. Enzymatic generation of superoxide anion (through xanthine
oxidase)

b. Non-Enzymatic generation of superoxide anion (through

NADH/Phenazine methosulphate system)

• Assay for xanthine oxidase inhibition Activity

Some Common In Vivo Antioxidant Assays

• In vivo CCl4 (Carbon tetrachloride) induced hepatoxicity

assay.

• Antioxidant testing kits are now available to detects free

radical activity in body.

• TAC (Total antioxidant capacity) of plasma can also be

measured by TAC kits.
 DILEMA OF REDOX IN
LIVING SYSTEM
• Difficult to quantify the oxidizing stress
• Difficult to extrapolate in vitro and in
vivo situations
• Bioavailability is an issue
• Interaction with other molecules
• Whether antioxidants do help in the
prevention of diseases?
• More questions than answers!!!!
 Antioxidant Drug Development-
Challenges and Opportunities

• Clinical trials nightmare

• Non-conclusive results
• Requires new type of clinical studies than
conventional double blind placebo trials.
• Preventive v/s therapeutic
• Raise questions to old believes?
THANK YOU VERY MUCH