Free radicals are unstable molecules that can damage cells. They are formed through normal metabolic processes but also due to environmental toxins and radiation. The body has antioxidant defenses against free radicals but an excess can lead to oxidative stress and disease. Endogenous free radicals include reactive oxygen species like superoxide, hydrogen peroxide, and hydroxyl radicals produced during metabolism. Exogenous sources include tobacco smoke, drugs, radiation, and air pollution. Free radical damage accumulates with age and is linked to many age-related diseases.

1. FREE RADICALS AND ANTIOXIDANTS
IN HEALTH AND DISEASE
By
Dr. Madhumita Sen

2. What are free radicals?
• Free radicals are ionised particles in
the human body.
• Free radicals can be caused by
environmental toxins, stress, food
additives and cooking among others.
• Free radicals are also formed when
oxygen is used by the body during
normal metabolism.
• But they are not all bad.
• Free radicals actually help the body to
fight viruses, bacteria, waste and
toxins.

3. How Free Radicals Act
• The human body is composed of
many different types of cells.
• Cells are composed of many different
types of molecules and atoms
consisting of protons, neutrons and
electrons.
• Electrons are involved in chemical
reactions and are the substance that
bonds atoms together to form
molecules.

4. • Electrons surround, or "orbit" an atom in one or
more shells.
• The innermost shell is full when it has two electrons.
When the first shell is full, electrons begin to fill the
second shell. When the second shell has eight
electrons, it is full, and so on.
• The most important structural feature of an atom for
determining its chemical behaviour is the number of
electrons in its outer shell.
• A substance that has a full outer shell tends not to
enter in chemical reactions (an inert substance).

5. • Because atoms seek to reach a state of
maximum stability, an atom will try to fill
it’s outer shell by:
1. Gaining or losing electrons to either
fill or empty its outer shell
2. Sharing its electrons by bonding
together with other atoms in order to
complete its outer shell
• Atoms often complete their outer shells
by sharing electrons with other atoms.
By sharing electrons, the atoms are
bound together and satisfy the
conditions of maximum stability for the
molecule.

6. How Free Radicals are Formed
• Normally, bonds don’t split in a way that
leaves a molecule with an odd, unpaired
electron.
• But when weak bonds split, free radicals
are formed.
• Free radicals are very unstable and react
quickly with other compounds, trying to
capture the needed electron to gain
stability.

8. • Free radicals are highly reactive due to the
presence of unpaired electron(s).
• Any free radical involving oxygen can be
referred to as reactive oxygen species
(ROS).
• Oxygen centred free radicals contain two
unpaired electrons in the outer shell.

9. • In turn the newly formed radical then looks to return to
its ground state by stealing electrons from cellular
structures or molecules.
• Thus the chain reaction continues and can be "thousand
of events long."

10. • It's when the body has too many free radicals that
damage can occur.
• Free radicals can affect tissues, lipids, proteins and DNA.
• Cells can be damaged leading to many diseases and early
aging.
• The effects of free radical damage can increase over time
and lead to many age-related diseases.

11. Functions of Free Radicals
• Some free radicals arise normally during
metabolism.
• Sometimes the body’s immune system’s cells
purposefully create them to neutralize viruses
and bacteria.
• Normally, the body can handle free radicals, but if
antioxidants are unavailable, or if the free-radical
production becomes excessive, damage can
occur.
• Of particular importance is that free radical
damage accumulates with age.

12. Types: Endogenous and Exogenous
Free Radicals

13. TYPES OF ENDOGENOUS
FREE RADICALS
• The most important free radicals in the body are the radical
derivatives of oxygen better known as reactive oxygen
species. These include
1. Oxygen in its triplet state (3O2) or singlet state
(O2), hydroxyl radical (OH), nitric oxide (NO), hypochlorous
acid (HOCl), hydrogen peroxide (H2O2) and the superoxide
radical (O2_).
2. Others are carbon-centered free radicals that arise from
the attack of an oxidizing radical on an organic molecule.
3. Hydrogen centered radicals result from attack of the H
atom (H).
4. Another form is the sulfur-centered radical produced in the
oxidation of glutathione resulting in the thiyl radical.

14. Superoxide
• The superoxide free radical anion is formed
when oxygen is reduced by the transfer of a
single electron to its outer shells.
• The major source of superoxide is from the
electron transfer chain of the mitochondria.
• On its own it isn't particularly damaging.

15. Hydrogen peroxide
• Hydrogen peroxide(H2O2) is not a free radical but
falls in the category of reactive oxygen species.
• It is an oxidising agent that is not particularly
reactive but its main significance lies in that it is
the main source of hydroxyl (OH) radicals.
• It is also involved in the production of HOCl by
neutrophils.

16. • In biological systems hydrogen peroxide is
generated by the production of superoxide: two
superoxide molecules can react together to form
hydrogen peroxide and oxygen:
• 2O2⁻+ 2H⁺ = H2O2 + O2
• The above reaction is called a dismutation
reaction as the radical reactants produce non-
radical products.

17. Hydroxyl radical
• The hydroxyl radical is an extremely reactive
oxidising radical that will react with most
biomolecules.
• The hydroxyl free radical is important in radio-
biological damage.
• The hydroxyl radical can damage virtually all types
of macromolecules: carbohydrates, nucleic acids
(mutations), lipids, and amino acids.

18. Mechanisms for scavenging hydroxyl radicals for the
protection of cellular structures includes:
1. endogenous antioxidants such as melatonin and
glutathione, and
2. dietary antioxidants such as phytochemicals and
vitamin E.

19. Singlet oxygen
• Singlet oxygen (1O2) is an electronically excited
and mutagenic form of oxygen. It is similar to
normal oxygen but there is one difference… it has
an extra electron.
• It is generated by input of energy like radiation or
sunlight.
• This free radical is involved in joint diseases (like
arthritis) and eye diseases.
• Carotenoids, such as Vitamin A and lycopene, can
tame the singlet oxygen and so can Vitamin E.

20. Nitric Oxide
• It is a common gaseous free radical.
• It is now recognised to play a role in vascular
physiology and is also known as endothelium derived
relaxing factor.
• Vascular endothelium produces nitric oxide from arginine
using the enzyme nitric oxide synthetase (in brain, blood
vessels and immune cells).
• This event can be stimulated by cytokines, tumour
necrosis factor, or interleukins and exercise.
• Inhibition of production is known to reduce microbicidal
and tumouricidal activities of macrophages.

21. Actions of Nitric Oxide free radical
• Nitric Oxide is a key
messenger in the
cardiovascular
system.
• Nitric Oxide stimulates
growth hormone
production.
• It stimulates immune
cells.
• It improves memory
and nerve cell
plasticity.

22. Peroxy-nitrite
• It is produced by the reaction of nitric oxide with
superoxide.
• Because of its oxidizing properties, peroxy-nitrite can
damage a wide array of molecules in cells, including
DNA and proteins and results in cell apoptosis.

23. Hypochlorous acid
• Activated polymorphonuclear cells
produce HOCl as a major
bactericidal agent.
• This reaction occurs in the
neutrophils phagocytic lysosomal
vesicles.
• Hypochlorous acid can cross cell
membranes.
• It may contribute to tissue
damage during the inflammatory
process.

24. Transition metals ions
• Iron and copper play a major role in the generation of free
radical injury and the facilitation of lipid peroxidation.
• Transition metal ions generate OH from O2⁻ and H2O2.
H2O2 + Fe2⁺ = OH + OH⁻ + Fe3⁺
• The reaction accelerates the non-enzymatic oxidation of
molecules such as epinephrine and glutathione that
generates O2- and H2O2 and subsequently OH.

25. EXOGENOUS FREE RADICALS
• Drugs:
• A number of drugs can increase the
production of free radicals.
• These drugs include antibiotics that
depend on bound metals for activity
(nitrofurantoin), antineoplastic agents
as bleomycin, anthracyclines
(adriamycin) and methotrexate.
• In addition radicals derived from
penicillamine, phenylbutazone and
sulphasalazine might inactivate
protease and deplete ascorbic acid
accelerating lipid peroxidation.

26. • Radiation:
• Radiotherapy may cause tissue injury that is caused by
free radicals.
• Electromagnetic radiation (X rays, gamma rays) and
particulate radiation (electrons, photons, neutrons, alpha
and beta particles) generate primary radicals by
transferring their energy to cellular components such as
water.
• These primary radicals can undergo secondary reactions
with dissolved oxygen or with cellular solutes.

27. • Tobacco smoking:
• It has been estimated that each puff of smoke has an
enormous amount of oxidant materials.
• These include aldehydes, epoxides, peroxides, and other free
radicals that may be sufficiently long lived as to survive till
they cause damage to the alveoli.
• In addition it also contains other relatively stable radicals in
the tar phase.
• It was also found that smokers have elevated amounts of
neutrophils in the lower respiratory tract that could
contribute to a further elevation of the
concentration of free radicals.

28. • Inorganic particles:
• Inhalation of inorganic particles also known as mineral dust
(e.g. asbestos, quartz, silica) can lead to lung injury that
seems at least in part to be mediated by free radical
production.
• Asbestos inhalation has been linked to an increased risk of
developing pulmonary fibrosis (asbestosis), mesothelioma
and bronchogenic carcinoma.
• Silica particles as well as asbestos are phagocytosed by
pulmonary macrophages.
• These cells then rupture, releasing proteolytic enzymes and
chemotactic mediators that leads to increased production of
free radicals and other reactive oxygen species.

29. • Gases:
• Ozone is not a free radical but a very powerful oxidising
agent. Ozone (O3) contains two unpaired electrons and
degrades under physiological conditions to OH, suggesting
that free radicals are formed when ozone reacts with
biological substrates.
• Ozone can generate lipid peroxidation.
• Others:
• Fever, excess glucocorticoid therapy and hyperthyroidism
increase metabolism. This leads to the increased
generation of oxygen-derived radicals.
• In addition, a wide variety of environmental agents
including chemical air pollutants, pesticides, solvents,
anaesthetics, exhaust fumes, also cause free radical
damage to cells.

31. Free radicals: Our enemies or friends?
• Although essentially cancer and degenerative diseases are
to major extent caused by damage done to our DNA by
them, free radicals also play an important role in cell
metabolism.
• The immune system is the main body system that
utilizes free radicals.
• Foreign invaders or damaged tissue is marked with free
radicals by the immune system.
• This allows for determination of which tissue need to be
removed from the body.
• Because of this some question the need for antioxidant
supplementation, as they believe supplementation can
actually decrease the effectiveness of the immune system.

33. ANTIOXIDANTS, NATURE AND
CHEMISTRY
• In the aerobic environment, the most dangerous
by product are the species of reactive oxygen.
• The role of antioxidants is to detoxify reactive
oxygen intermediates (ROI) in the body.
• Over the past several years, nutritional
antioxidants have attracted considerable interest
as potential treatment for a wide variety of
disease states, including
cancer, atherosclerosis, chronic inflammatory
diseases and aging.

34. Definition
• An antioxidant is a substance that when present in low
concentrations relative to the oxidizable substrate
significantly delays or reduces oxidation of the substrate
(Halliwell, 1995).
• Antioxidants get their name because they combat
oxidation.
• They are substances that protect other chemicals of the
body from damaging oxidation reactions by reacting with
free radicals and other reactive oxygen species within the
body, hence hindering the process of oxidation.

35. • During this reaction the antioxidant sacrifices itself by
becoming oxidized.
• However, antioxidant supply is not unlimited as one
antioxidant molecule can only react with a single free
radical.
• Therefore, there is a constant need to replenish
antioxidant resources, whether endogenously or through
supplementation in diet and exercise.

36. Antioxidant System
• The body has developed several
endogenous antioxidant systems
to deal with the production of ROI.
The enzymatic antioxidants include
1. superoxide dismutase
(SOD), which catalyses the
conversion of O2⁻ to H2O2 and H2O;
2. catalase, which then converts
H2O2 to H2O and O2;and
3. glutathione peroxidase, which
reduces H2O2 to H2O.

37. • The glutathione redox cycle is a
central mechanism for reduction
of intracellular hydroperoxides.
• Source and Nature:
• It is a tetrameric protein and has
4 atoms of selenium (Se) that
confers the catalytic activity.
• Glutathione peroxidase reduces
H2O2 to H2O by oxidizing
glutathione.
• Re-reduction of the oxidized form
of glutathione (GSSG) is then
catalysed by glutathione
reductase.

38. These enzymes also require trace metal cofactors
for maximal efficiency, including selenium for
glutathione peroxidase; copper, zinc, or
manganese for SOD; and iron for catalase.

39. The nonenzymatic/Exogenous
antioxidants
This includes
1. the lipid-soluble vitamins, vitamin E and vitamin A or
provitamin A (beta-carotene),
2. the water-soluble vitamin C and
3. Glutathione (GSH), a tripeptide molecule.
• The enzymatic and nonenzymatic antioxidant systems are
intimately linked to one another and appear to interact with one
another.

40. Classification of major antioxidants
Antioxidant Role Remarks
Superoxide Dismutates O2⁻ to Contains Manganese
dismutase (SOD) (Mn.SOD)
Mitochondrial
H2O2 Contains Copper & Zinc
Cytoplasmic (CuZnSOD)
ENZY Extracellular Contains Copper (CuSOD)
MES
Catalase Dismutates Tetrameric hemoprotein
H2O2 to H2O present in peroxisomes
Glutathione Removes Selenoproteins (contains
peroxidase H2O2 and lipid Se2+)
(GSH.Px) peroxides Primarily in the cytosol also
mitochondria
Uses GSH

41. Alpha Breaks lipid peroxidation Fat soluble
tocopherol Lipid peroxide and O2⁻and OH vitamin
(Vit E) scavenger
VITA Beta Scavenges OH, O2⁻ and peroxy Fat soluble
MINS carotene radicals vitamin
Prevents oxidation of vitamin A
Binds to transition metals
Ascorbic Directly scavenges O2⁻, OH, and Water soluble
acid H2O2 vitamin
Neutralizes oxidants from
stimulated neutrophils
Contributes to regeneration of
vitamin E

42. Antioxidant Vitamins
• Vitamin E is more appropriately described as an
antioxidant than a vitamin. This is because, unlike most
vitamins, it does not act as a co-factor for enzymatic
reactions.
• Also, deficiency of vitamin E does not produce a disease
with rapidly developing symptoms such as scurvy or
beriberi.
• Overt symptoms due to vitamin E deficiency occur only in
cases involving fat malabsorption syndromes, premature
infants and patients on total parenteral nutrition.

43. • The effects of inadequate vitamin E intake usually develop
over a long time, typically decades, and have been linked to
chronic diseases such as cancer and atherosclerosis.
• Its main function is to prevent the peroxidation of membrane
phospholipids, and avoid cell membrane damage through its
antioxidant action.

44. • How It Acts:
• Tocopherol-OH can transfer a hydrogen atom with a single
electron to a free radical, thus removing the radical before it
can interact with cell membrane, proteins or generate lipid
peroxidation.
• When tocopherol-OH combines with the free radical, it
becomes tocopherol-O, itself a radical.
• When ascorbic acid (Vitamin C) is available, tocopherol-O
plus ascorbate yields semi-dehydro-ascorbate plus
tocopherol-OH.
• By this process, an aggressive ROI is eliminated and a
weak ROI (dehydroascorbate) is formed, and tocopherol-
OH is regenerated.

45. • Vitamin E also stimulates the immune response.
Some studies have shown lower incidence of
infections when vitamin E levels are high, and
vitamin E may inhibit cancer initiation through
enhanced immuno-competence.
• Vitamin E inhibits the conversion of nitrites in
smoked, pickled and cured foods to nitrosamines in
the stomach. Nitrosamines are strong tumour
promoters.
• {Alpha-tocopherol has been shown to be capable of
reducing ferric iron to ferrous iron, i.e. act as a pro-
oxidant}.

46. Beta Carotene
• Source and Nature:
• Carotenoids are pigmented micronutrients present in fruits
and vegetables.
• Carotenoids are precursors of vitamin A and also have
antioxidant effects.
• While over 600 carotenoids have been found in the food
supply, the most common forms are alpha-carotene, beta-
carotene, lycopene, crocetin, canthaxanthin, and
zeaxanthin.
• Beta-carotene is the most widely studied. It is composed
of two molecules of vitamin A (retinol) joined together.
• Dietary beta-carotene is converted to retinol at the level of
the intestinal mucosa.

47. • Mechanisms of Action:
• The antioxidant function of beta-carotene is due
to its ability to quench singlet oxygen, scavenge
free radicals and protect the cell membrane lipids
from the harmful effects of oxidative degradation.
• The ability of beta-carotene and other
carotenoids to quench excited
oxygen, however, is limited, because the
carotenoid itself can be oxidized during the
process (autoxidation).

48. • Carotenoids also have been
reported to have a number of
other biologic actions, including
• immuno-enhancement;
• inhibition of mutagenesis and
transformation; and
• regression of premalignant
lesions.

49. Ascorbic acid (vitamin C)
• Source and Nature:
• Ascorbic acid (vitamin C) is a water-soluble, antioxidant
present in citrus fruits, potatoes, tomatoes and green leafy
vegetables.
• Mechanism of Action:
• The chemopreventive action of vitamin C is attributed to
two of its functions.
1. It is a water-soluble, chain breaking antioxidant. As an
antioxidant, it scavenges free radicals and reactive
oxygen molecules, which are produced during metabolic
pathways of detoxification.
2. It also prevents formation of carcinogens from precursor
compounds.

50. • It has also been shown that ascorbate is more potent
than a-tocopherol in inhibiting the oxidation of LDL.
• Vitamin C also contributes to the regeneration of
membrane bound oxidized vitamin E. It will react with
the a -tocopheroxyl radical, resulting in the generation of
tocopherol in this process itself being oxidized to
dehydroascorbic acid. Vitamin C supplementation leads
to increased plasma and tissue levels of vitamin E.

51. Other antioxidants
• Glutathione (GSH):
• GSH is synthesized intra-cellularly from
cysteine, glycine, and glutamate.
• In addition to its role as a substrate in GSH redox
cycle, GSH is also a scavenger of hydroxyl radicals and
singlet oxygen.
• The majority of GSH is synthesized in the liver, and
approximately 40% is secreted in the bile.
• The biologic role of GSH in bile is believed to be defence
against dietary xenobiotics and protection of the intestinal
epithelium from oxygen radical attack.

52. • CoQ10:
• CoQ10 (Coenzyme Q10) is also known as ubiquinone. It is
found in almost every living cell and is essential to energy
production by the mitochondria.
• Far beyond producing energy, CoQ10 can protect the body
from destructive free radicals and enhance immune defences.
• Albumin:
• Albumin scavenges several free radicals and thus can be
considered as one of the primary extracellular defence
systems.
• Plasma proteins
• Namely ceruloplasmin and transferrin have also shown
antioxidant activity.

53. Melatonin
• Melatonin is a powerful antioxidant.
• Melatonin easily crosses cell
membranes and the blood-brain
barrier.
• Unlike other antioxidants, melatonin
does not undergo redox
cycling, which is the ability of a
molecule to undergo
repeated reduction and oxidation.
• Melatonin, once oxidized, cannot be
reduced to its former state.
Therefore, it has been referred to as a
terminal (or suicidal) antioxidant.

54. Uric acid:
• Acts as an endogenous radical scavenger and antioxidant.
It is present in about 0.5 mmol/L in body's fluids and is the
end product of purine metabolism.
• Uric acid is a powerful scavenger of singlet oxygen, peroxyl
radical (ROO) and OH radical.
• Concerns over elevated UA's contribution to gout must be
considered as one of its many risk factors.
• The effects of uric acid in conditions such as
atherosclerosis, ischemic stroke, and heart attacks are still
not well understood.
• This might be because high levels of uric acid act as a pro-
oxidant.

55. Drugs:
Several pharmaceutical agents have been found to exert
an antioxidant effect:
1. Xanthine oxidase inhibitors: e.g. allopurinol, folic acid.
2. NADPH inhibitors: e.g. adenosine, calcium channel
blockers.
3. Albumin.
4. Inhibitors of iron redox cycling: deferoxamine,
apotransferrin and ceruloplasmin
5. Statins, the anti-cholesterol drug

56. Dietary sources of antioxidants

57. POTENTIAL OF ANTIOXIDANT SUPPLEMENTS TO
DAMAGE HEALTH
• Oxidation reactions can produce free radicals. In turn,
these radicals can start chain reactions. When the chain
reaction occurs in a cell, it can cause damage or death to
the cell.
• An antioxidant is a molecule that inhibits the oxidation of
other molecules.
• Antioxidants terminate these chain reactions by removing
free radical intermediates, and inhibit other oxidation
reactions.
• Antioxidants are widely used in dietary supplements and
have been investigated for the prevention of diseases such
as cancer, coronary heart disease and even altitude
sickness.

58. • 1. Although initial studies suggested that antioxidant
supplements might promote health, later large clinical
trials with a limited number of antioxidants detected no benefit
and even suggested that excess supplementation with certain
antioxidants may be harmful.
• Antioxidants that are reducing agents can also act as
pro-oxidants.
• For example, vitamin C has antioxidant activity when it
reduces oxidizing substances such as hydrogen
peroxide, however, it will also reduce metal ions that generate
free radicals.
• That is, paradoxically, agents which are normally considered
antioxidants can act as conditional pro-oxidants and actually
increase oxidative stress.

59. • 2. Free radicals induce an endogenous response
which protects against exogenous radicals (and
possibly other toxic compounds).
• Recent experimental evidence strongly suggests that
such induction of endogenous free radical production can
extend the life span.
• Mito Hormesis: Most importantly, this induction of life
span is prevented by antioxidants, providing direct
evidence that toxic radicals may mito-hormetically exert
life extending and health promoting effects.

60. HORMESIS
• Hormesis is the term for generally favourable biological
responses to low exposures to toxins and other stressors.
• Examples in normal life are exercise, acute stress and
uric acid, all of which in small amounts increase
lifespan, immunity and health, but in larger amounts are
harmful.
• The biochemical mechanisms by which hormesis works
are not well understood.
• It is conjectured that low doses of toxins or other
stressors might activate the repair mechanisms of the
body.
• Free radicals may also help by causing apoptosis of
damaged cells and thus reducing aging and cancer
causing cells in the body.

61. • 3. Another fact:
• Antioxidant supplements have no clear effect on the risk
of chronic diseases such as cancer and heart disease in
the long run.
• This suggests that the health benefits of fruits and
vegetables come from other substances in fruits and
vegetables or come from a complex mix of compounds.
• For example, the antioxidant effect of flavonoid-rich foods
seems to be due to fructose-induced increases in the
synthesis of the antioxidant uric acid and not only to
dietary antioxidants.

62. Important points
• Diet plays an important role in reducing oxidative damage
to the body.
• The role of dietary antioxidants is not limited to its
antioxidant chemicals, minerals and vitamins alone, but to
an as yet unknown combination of all these.
• Supplementation pills of individual antioxidants have not
been shown to prolong life or improve health.

64. Homework
Explain how Mithormesis works to prolong life.

65. Any Questions?