LOO HARIYANTO RAHARJO,dr.,MSi.

 O2 is both essential to human life and

toxic.
 We are dependent on O2 for oxidation
reactions in the pathways of adenosine
triphosphate (ATP) generation,
detoxification, and biosynthesis.
 However, when O2 accepts single
electrons, it is transformed into highly
reactive oxygen radicals that damage
cellular lipids, proteins, and DNA.
 A radical is a molecule that has a
single unpaired electron in an orbital.
 A free radical is a radical capable of
independent existence.
 Radicals are highly reactive and
initiate chain reactions by extracting
an electron from a neighboring
molecule to complete their own
orbitals.
 The oxygen atom is a biradical,
which means it has two single
electrons in different orbitals.
 O2 is capable of accepting a total
of four electrons, which reduces it
to water (H2 O)
 Reactive oxygen species (ROS) are oxygen-containing compounds that are highly reactive free
radicals, or compounds readily converted to these oxygen free radicals in the cell.
 The hydroxyl radical is probably the most
potent of the ROS.
 It initiates chain reactions that form lipid
peroxides and organic radicals and adds
directly to compounds.
 The superoxide anion is also highly reactive,
but has limited lipid solubility and cannot
diffuse far.
 However, it can generate the more reactive
hydroxyl and hydroperoxy radicals by
reacting nonenzymatically with hydrogen
peroxide (H2O2) in the Haber–Weiss
reaction.
 Hydrogen peroxide (H2O2), although
not actually a radical, is a weak
oxidizing agent that is classified as an
ROS because it can generate the
hydroxyl radical (OH•).
 Transition metals, such as Fe 2+
+
(Ferrous) or Cu , catalyze formation of
the hydroxyl radical (OH•).
 from hydrogen peroxide in the
nonenzymatic Fenton reaction.
(SUPEROXIDE REDUCTION)

(Fenton reaction)

(Haber–Weiss reaction)
 Because hydrogen peroxide is lipid
soluble, it can diffuse through
membranes and generate OH• at
2+ +
localized Fe or Cu containing sites,
such as the mitochondria.
 Hydrogen peroxide is also the
precursor of hypochlorous acid
(HOCl), a powerful oxidizing agent
that is produced endogenously and
enzymatically by phagocytic cells
(=Respiratory Burst).
 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 (Complex III):
 Q + H + e QH
+ - . (CoQ semiquinone)
 QH . + H+ + e-  QH2 (reduced CoQ)
 Generation of O2.- occurs when O2
react directly with QH .

 QH + O2  Q + O2
. .- + H+
 O2 can further processed
.-
.
yielding H2O2 and OH As
follows:
 2O2.- +2H+  H2O2
 O2.- + H2O2  O2 + OH +
-
OH .

 Increased production of O2.-

can occur when further
transfer of electrons is
hindered for instance due to
damage to complex III or IV.
 Cytochrome P450 enzymes are a major
source of free radicals “leaked” from
reactions.
 Because these enzymes catalyze reactions in
which single electrons are transferred to O2
and an organic substrate, the possibility of
accidentally generating and releasing free
radical intermediates is high.
 Induction of P450 enzymes by alcohol,
drugs, or chemical toxicants leads to
increased cellular injury.
 Monoamine oxidase, which oxidatively degrades the
neurotransmitter dopamine, generates H2O2 at the
mitochondrial membrane of certain neurons.
 Peroxisomal fatty acid oxidase generates H2O2
rather than FAD(2H) during the oxidation of very-
long-chain fatty acids.
 Xanthine oxidase, an enzyme of purine
degradation that can reduce O2 to O2 or H2O2 in
.-
the cytosol, is thought to be a major contributor to
ischemia–reperfusion injury, especially in intestinal
mucosal and endothelial cells.
 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 cause,
at old age, the activity of
antioxidant enzymes is
known to decrease especially
catalase and Glutathion
peroxidase.
 Oxidants are electron acceptors
 Example: Fe 3+ +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. + OH .

H atom Hydroxyl radical

(Free radical) (Free radical)
 Term Definition

Oxidation Gain in oxygen

Loss of hydrogen
Loss of electrons
Reduction Loss of oxygen
Gain of hydrogen
Gain of electrons
Oxidant Oxidizes another chemical by
taking electrons, hydrogen, or by
adding oxygen
Reductant Reduces another chemical by
supplying electrons, hydrogen, or
by removing oxygen
 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) + R2.
radical Non-radical Non-radical New radical

The newly formed radical can then attack other molecules

 Oxidants are electron acceptors, i.e. It also attract
electrons: Fe +e  Fe
3+ - 2+

 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
 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 half-life.
 The relative stability of certain radicals is due to a physico-
chemical phenomenon called electron delocalization
(“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.
 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
 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

 TERMINation:
 R1. + R1.  R1-R1
 R1. + R2.  R1-R2
 R2. + R3.  R2-R3
 etc
 Are more damaging than non-radical oxidants because
of their:
 Higher reactivity
 Tendency to trigger chain reaction
 Are endogenous oxidants derived from oxygen
 Superoxide anion : O2.-

 Peroxyl radical : OOH .

 Hydrogen peroxide: H2O2

 Hydroxyl radical: OH 
. dangerous : high reactivity
 Hypochlorite anion: ClO.

 Singlet oxygen: 1O
2
 The reduction of O2 to H2O involves the transfer of 4
electrons: O2 + 4H + 4e 2 H2O
+ -
 All ROS except Cl and O2 , can be considered as being
- 1
the result of incomplete oxygen reduction:
 1 e- transfer: O2 + e-  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

 ClO.

1O
H2O2 2

O2
OOH.
OH.

O2.-

O2.- & H2O2 are primary ROS from which all, others are derived
 •OH Attack phospholipids (PUFA) causing a chain
reaction called lipid peroxidation.
 Initiation :L-H + •OH  L• + H2O
(lipid
radical)
 Propagation: L• + O2  LOO• + H2O

(peroxylipid radical)
L-H + LOO•  L• + LOOH
(lipid
 Membran lipids
ROS
Lipid peroxidation

Cross- Cleavage w3 & w6 PUFA chain


linking FA Toxic products:
chain MDA
8-OH nonenal
Ethane (C2H6)
Pentane (C5H10)

Membran cell damage

Disturbed osmotic balance

Water enters cell

Cell swelling Cell lysis
 Cysteine: Cys-SH + OH 
. Cys-S . + H2O
2Cys-S 
. Cys-S-S-Cys (cystine)
Cysteine is source of glutathione

Histidine is source of ketoglutarate

1. Hydroxilation DNA Cleavage of
of Pu & Py bases
phosphodiester
2. Ring opening of
backbone
Pu & Py bases

Repaired Not repaired

Not repaired

Mutation on No effect
proto- or Chromosome If severe
antioncogenes aberrations
Cell dies

Cancer Other effects

 Proteins

Cysteine residues Other amino

acids residues
Formation of
Disulfide (S-S) bonds Modified side chain

Intra- or
interchain
cross-linking

Loss of biological function

(e.g. Enzymes, peptide hormones, receptors, channel protein, etc
 In its original definition in chemistry antioxidants are
electron donors.
Example: Cu  Cu + e
+ 2+ -

 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
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)
 Accumulation of free & Cu+ Fe2+ ions are prevented by
transition metal binding proteins:
 Fe 2+
: Transferrin & Ferritin
 Cu : Ceruloplasmin & Albumin
+

 Accumulation of O2.- is prevented by a reaction catalyzed

by superoxide dismutase (SOD):
 2O2.- + 2H O
+
2 + H2O
 Mammalian cells contain & species of SOD one containing
Cu & Zn (CuZnSOD) and another containing Mn (MnSOD).
 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:
 OH . Once generated can still be inactivated by
glutathione (GSH) or Cysteine (Cys-SH)
GSH : GSH + OH  GS + H2O
. .

2GS  GSSG
.

Cys-SH: Cys-SH + OH  Cys + H2O

. .

2Cys  Cys SS Cys

.

(cystine)
 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.
 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
 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.