OXIDANT, FREE RADICALS AND ANTIOXIDANT

OXIDANTS
Oxidants are implicated in several disease processes such
as:
Cardiovascular disease
Respiratory disease
Reperfusion injury
Diabetes Mellitus
Impairment of the immune system
Carcinogenesis
Aging

OXIDANTS
Oxidants may affect cell`s integrity because they react with
and destroy cell`s component either structural (e.g. Cell
membrane & cytosceleton) or functional ( e.g. Enzymes &
DNA)
Oxidants maybe produced inside our body (endogenous) or
may come from source outside the body (exogenous)

ENDOGENOUS OXIDANTS
Are produced as a by product of normal physiological
processes such as:
ATP production (OxPhos) in mitochondria
Oxygenation of Hb in erythrocytes

Secreted by inflammatory cells.

Formed by the action of ionizing radiation.

GENERATION of FREE RADICALS by

MITOCHONDRIA

Under normal conditions 95%-98% of O2 is completeley

reduced to H2O, but:
2%-5% of O2 is reduced to O2 is by one electron transfer
During OxPhos, CoQ is reduced by a two steps reaction:
Q + + QH (CoQ semiquinone)
.
+
QH + H + e QH2 (reduced CoQ)
+
H

Generation of

O2 occurs

QH + O2 Q +
.

O2 +

+
H

when O2 react directly with QH

GENERATION of FREE RADICALS by

MITOCHONDRIA
O2 can

further processed yielding H2O2 and OH. As

follows:
2O2 +
H2O2
.
O2 + H2O2 O2 + OH + OH
-

+
2H

Increased production of O2
occur when further transfer
of electrons is hindered for instance due to damage to
complex III or IV.
- can

OXIDATIVE STRESS

OXIDATIVE STRESS
Oxidative stress can either be due to increased
production of ROS/free radicals or decreases activity
of antioxidant enzymes or both.
Due to a still unknown couse, at old age, the activity
of antioxidant enzymes is known to decrease
especially catalase and Glutathion peroxidase.

EXOGENOUS OXIDANTS
May come from several source such as:
Pollutants
Drugs
Food Additives
Chemicals used in industry
etc

EXOGENOUS SOURCE of FREE RADICALS

Radiation: uv light, x-rays, -rays
Chemicals that react to form peroxides:
Ozone and singlet oxygen

Chemicals that promote superoxide formation:

Quinones, nitroaromatics, bipyrimidiulium herbicides

Chemicals that are metabolized to radicals

e.g. Polyhalogenated alkanes, phenols, aminophenols

Chemicals that release iron

Ferritin

OXIDANTS & FREE RADICALS

Oxidants are electron acceptors
Example:

3+
Fe

+e

Fe

2+

Free radicals are atoms or molecules

possessing one or more unpaired
electron
Example: homolytic cleavage of water
due to ionizing radiation
H : O : H (H-O-H) H.

H atom
(Free radical)

OH .

Hydroxyl radical
(Free radical)

Definition

Oxidation

Gain in oxygen
Loss of hydrogen
Loss of electrons

Reduction

Loss of oxygen
Gain of hydrogen

Gain of electrons
Oxidant
Reductant

Oxidizes another chemical by

taking electrons, hydrogen, or by
adding oxygen
Reduces another chemical by
supplying electrons, hydrogen, or
by removing oxygen

BASICS of REDOX CHEMISTRY

Term

OXIDANTS & FREE RADICALS

Free radicals show a great tendency to attract electrons (e-) due to the
presence of unpaired electron.
Electron donor is another radical:
R1 + R2 R1 : R2 (R1 R2)
.

(non-radical)

Electron donor is a non-radical:

R1. + R2:H (R2-H) R1:H (R1-H) +
radical

Non-radical

Non-radical

R2.

New radical

The newly formed radical can then attack other molecules

OXIDANTS & FREE RADICALS

Oxidants are electron acceptors, i.e. It also attract
3+
2+
electrons: Fe +e Fe
Since free radicals also attract electrons, free radicals can
also be considered oxidants.
Free radicals are all oxidants but not all oxidants are free
radicals, example:
Hydrogen peroxide (H2O2) : oxidant, non radical
.
Hydroxyl radical (OH ) : oxidant, radical

OXIDANTS & FREE RADICALS

The tendency to attract electrons among free radicals may vary,
some are highly reactive, very unstable and have a short half-life
others are less reactive, relatively stable and have a longer halflife.
The relative stability of certain radicals is due to a physicochemical phenomenon called electron delocalisation
(wandering electron).
Non-radical oxidants, on the other hand, are stable compounds
and their oxidative reactivity are thus less than the most stable
free radical.

FREE RADICAL CAN TRIGGER CHAIN REACTION

When a free radical react with a non-radical the result with
.
.
be the formation of a new radical: R1 + R2-H R1-H + R2
The newly formed radical can again react with a non-radical
giving rise to another radical: R2. + R3-H R2-H + R3.
This process can be repeated again and again resulting in a chain reaction

Such a chain reaction will only stop when 2 radical meet.

R1. + R1. R1-R1
R1. + R2. R1-R2
etc

3 stages of a chain reaction

Initiation:
Fe2+ + H2O2 Fe3+ + OH- + OH.
R1-H + OH. R1. + H2O

Propagation:
R1. + R2-H R1-H + R2.
R2. + R3-H R2-H + R3.
etc

Propagation:

R1. + R1. R1-R1

R1. + R2. R1-R2
R2. + R3. R2-R3
etc

Free Radicals
Are more damaging than non-radical oxidants because of
their:
Higher reactivity
Tendency to trigger chain reaction

Reactive Oxygen Species (ROS)

Are endogenous oxidants derived from oxygen
. Superoxide anion : O2
.
Peroxyl radical : OOH
Hydrogen peroxide: H2O2
.
Hydroxyl radical: OH dangerous : high reactivity
Hypochlorite anion:
1
Singlet oxygen: O2

.
ClO

Reactive Oxygen Species (ROS)

The reduction of O2 to H2O involves the transfer of 4 electrons:
+
4H

O2 +
+
2 H2O
1
All ROS except Cl and O2 , can be considered as being the
result of incomplete oxygen reduction:
transfer: O2 +

+
.
O2 + e + H OOH
+
2 e transfer: O2 + 2e + 2H 2 H2O2
3 e- transfer: O2 + 3e- + 3H+ H2O +OH.
+
4 e transfer: O2 + 4e + 4H 2 H2O
1

4e

.O2

Genesis and relationship between ROS

ClO.
1O
2

H2O2
O2

OOH.
OH.
O2.-

O2.- & H2O2 are primary ROS from which all, others are derived

Effect of ROS on membrane lipids

.
OH

Attack phospholipids (PUFA) causing a chain reaction

called lipid peroxidation.
.
.
Initiation :L-H + OH L
+ H2O
.
L

LOO. +
H2O
(peroxylipid radical)
.
.
L-H + LOO
L
+ LOOH
(lipid peroxide)
.
.
Termination: L
+L
L-L

Propagation:

+ O2

(lipid radical)

Effect of ROS on membrane lipids

Membran lipids

ROS

Lipid peroxidation
Crosslinking FA
chain

Cleavage w3 & w6 PUFA chain

Toxic products:
MDA
8-OH nonenal
Ethane (C2H6)
Pentane (C5H10)

Membran cell damage

Disturbed osmotic balance
Water enters cell
Cell swelling

Cell lysis

Effect of ROS on amino acids

Cysteine: Cys-SH +

.
OH

Cys-S. + H2O

.
2Cys-S

Cys-S-S-Cys (cystine)

Cysteine is source of glutathione

Histidine is source of ketoglutarate

EFFECT ROS on DNA

1. Hydroxilation
of Pu & Py bases
2. Ring opening of
Pu & Py bases

DNA

Cleavage of
phosphodiester
backbone

Not repaired

Repaired

Mutation on
proto- or
antioncogenes

No effect

Not repaired

Chromosome
aberrations

If severe
Cell dies

Cancer

Other effects

EFFECT ROS on PROTEINS

Proteins

Cysteine residues
Formation of
Disulfide (S-S) bonds

Other amino
acids residues
Modified side chain

Intra- or
interchain
cross-linking

Loss of biological function

(e.g. Enzymes, peptide hormones, receptors, channel protein, etc

Antioxidants
In its original definition in chemistry antioxidants are
electron donors.
+
2+
Example: Cu Cu + e
Antioxidants can be classified according:
1. Its mode of action:
Preventive antioxidants prevent undue accumulation of oxidants
Chain breaking antioxidants prevent propagation of chain reactions
initiated by free radicals

Antioxidants
2. Its solubility:
Lipophilic antioxidants, hydrophobic, fat solluble molecules, act in cell
membranes:
Tocopherols (vitamin E)
-carotene (provitamin A)

Hydrophilic antioxidants, hydrophilic, water solluble molecules, act in

cytosol and extracellular fluid:

Ascorbic acid (vitamin C)

Glutathione
Cysteine
Others (e.g. Uric acid)

PREVENTIVE ANTIOXIDANTS
+
Cu

2+
Fe

Accumulation of free
&
transition metal binding proteins:

ions are prevented by

2+
Fe

: Transferrin & Ferritin

+
Cu : Coeruloplasmin & Albumin

Accumulation of is prevented by a reaction catalyzed by

superoxide dismutase (SOD):
.+
2O2 + 2H O2 + H2O
Mammalian cells contain & species of SOD one containing
Cu & Zn (CuZnSOD) and another containing Mn (MnSOD).

PREVENTIVE ANTIOXIDANTS
Accumulation of H2O2 prevented by the actions of enzymes called
Catalase and Peroxidases
Catalase : 2 H2O2 H2O + O2

Peroxidases are enzymes catalyzing the general reaction:

A + H2O2 AO + H2O
Among the peroxidases, the most important of which is Glutathione
peroxidase (GPx) a Se containing enzyme catalyzing the reaction:
2 GSH (Glutathione) + 2 H2O2 GSSG (Oxidized glutathione) + 2H2O

GSH is restored by the action of glutathione reductase:

+
+
GSSG + NADPH + H 2 GSH + NADP

PREVENTIVE ANTIOXIDANTS

.
OH

Once generated can still be inactivated by glutathione

(GSH) or Cysteine (Cys-SH)
GSH :

GSH +

GS + H2O
.

.
OH

2GS GSSG
.

Cys-SH: Cys-SH +

Cys

.
OH

+ H2O

2Cys Cys SS Cys (cystine)

.

CHAIN-BREAKING ANTIOXIDANTS
Lipid peroxidation is quantitatively the most important chain reaction
occuring in cells. This lipophilic antioxidants can stop this reaction
from progressing.
Tocopherols is major lipophilic antioxidants present in cell membranes
(and also in lipoproteins).
.
.
Although tocopherols (ToCH) can react with lipid radical (L ): L +
.
ToCH LH + ToC (Tocopheryl radical)
.
.
Its main action is probably on peroxylipid radicals (LOO ): LOO +
.
ToCH LOOH + ToC
.
Although ToC is relatively stable because of electron delocalisation,
it still remains to be inactivated.

CHAIN-BREAKING ANTIOXIDANTS
.

Inactivation ToC of can occur by several ways:

Intramolecular rearrangement can give rise to a non-radical called
tocoquinone (ToqQ).
Moving to the cell membrane surface, it reacts with ascorbic acid (Asc
H2):
.
ToC + Asc H2 ToCH + Asc .- + H+
Ascorbyl radical
The ascorbyl radical is then spontaneously inactivated by a dismutation
reaction:
+
.2 Asc + 2 H Asc H2 + DHAA

Dehydro-ascorbic acid

CHAIN-BREAKING ANTIOXIDANTS
Alternatively, ToC Can also react with cysteine (Cys-SH) or
glutathione (GSH), generating cystine (Cys SS Cys) or
oxidized glutathione (GSSG).
Tocopherols can only react at a relatively high PO2
.

At PO2 low , the role of tocopherols is replaced by carotene , whose radical (-carotenyl radical) is also
relatively stable due to electron delocalisation.