Antioxidant System in

body
Molecular Biology team

Agents that Damage DNA

Certain wavelengths of radiation

ionizing radiation such as gamma rays and x-rays
ultraviolet rays, especially the UV-C rays (~260 nm)
that are absorbed strongly by DNA but also the
longer-wavelength UV-B that penetrates the
ozone shield .
Highly-reactive oxygen radicals produced during
normal cellular respiration as well as by other
biochemical pathways.

Who is responsible?
Major cause
Reactive Oxygen Species (ROS)
Reactive Nitrogen Species (RNS)
Oxidants are also generated by different types of
radiation, with X-irradiation generating the hydroxyl
radical and irradiation with ultraviolet light generating
electronically excited states
with subsequent radical formation. Ultrasound and
microwave radiation can also generate reactive oxygen
species. Even shear stress, e.g. in homogenization, is
known to generate radicals.

Free radicals
Superoxide

O2-

Hydroxyl

HO

Hydroperoxyl

HO2

Hydrogen peroxide

H2O2

Lipid peroxide

LO2H

Reactive nitrogen species

Thiyl
Molecular oxygen can be reduced to water. The intermediate
steps of' oxygen reduction are the formation of- the superoxide
anion radical, hydrogen peroxide and the hydroxyl radical,
corresponding to the steps of' reduction by one, two and three
electrons, respectively.

Types of DNA Damage

All four of the bases in DNA (A, T, C, G) can
be covalently modified at various positions.
One of the most frequent is the loss of an
amino group ("deamination") resulting, for
example, in a C being converted to a U.
Mismatches of the normal bases because of a
failure of proofreading during DNA
replication.
Common example: incorporation of the
pyrimidine U (normally found only in RNA)
instead of T.

http://www.benbest.com/lifeext/aging.html#radical

Other damages
Breaks in the backbone.
Can be limited to one of the two strands (a singlestranded break, SSB) or
on both strands (a double-stranded break (DSB).
Ionizing radiation is a frequent cause, but some
chemicals produce breaks as well.
Crosslinks Covalent linkages can be formed between
bases
on the same DNA strand ("intrastrand") or
on the opposite strand ("interstrand").
Several chemotherapeutic drugs used against
cancers crosslink DNA [Link].

How?
Mutagenesis by ROS/RON contribute to cancer
due to
1.Cause structural changes e.g. base pair
modification, rearrangement, deletion, insertion
and sequence amplifications
2.Affect cytoplasmic and nuclear signal
transduction
3.Modulate the activity of the proteins and genes
that respond to stress and which act to regulate
the genes that are related to cell proliferation,
differentiation and apoptosis

Fig. 1. Reactive oxygen species (ROS) can play a role in cell signaling.
Oxidative stress can activate numerous intracellular signaling pathways via
ROS-mediated modulation of various enzymes and critical transcription
factors. In one scenario, transcription factors activated in response to an
increase in ROS or oxidative damage travel from the cytoplasm to the
nucleus within a cell and bind to promoter regions of particular genes. As a
result, these stress-activated pathways can have a significant impact on
gene expression, which will ultimately affect the fate of a cell (e.g.,
apoptosis, proliferation, cytokines). The balance between ROS production,
cellular antioxidant defenses, activation of stress-related signaling
pathways, and the production of various gene products, as well as the
effect of aging on these processes, will determine whether a cell exposed
to an increase in ROS will be destined for survival or death.

Where?
Mitochondria more than
nuclear DNA
Intracelluar source is
Mitochondiral electron
transport may generate
radicals ROS
Prevented by low
calorie intake and free
radical inhibitors
Fe and Cu are
associated with ROS

Membranes

Enzymes

Scientific American, Dec. 1992

Why different types of antioxidants?

The half-lives of the major reactive oxygen species are
vastly different, underscoring the necessity for different
types of defense mechanisms
Highest rate constants for the reaction with target
molecules are found for the hydroxyl radical; its reactions
are diffusion limited, i.e. they take place practically at the
site of generation.
In contrast, some peroxyl radicals are relatively stable, with
half-lives in the range of seconds. Such molecules
may diffuse away from their site of generation and thus
transport the radical or oxidant function to other target
sites.

Oxidative Stress
An imbalance between oxidants and
antioxidants in favor of the oxidants, potentially
leading to damage, is termed 'oxidative stress
Antioxidant defense involves several
strategies,

enzymatic and

non-enzymatic.
.

Natures Strategy
Prevention, Interception and Repair
Prevention
Cytochrome oxidase, which carries outmost of the
cellular oxygen reduction, does not release
superoxide or other radicals, even though it contains
iron and copper ions. Likewise, the three-dimensional
structure of the enzyme

Ribonucleotide reductase keeps the radical character

of the tyrosyl function in subunit B from spreading to
the environment by forming an appropriate 'cage'.
Metal chelation is a major means of controlling lipid
peroxidation and DNA fragmentation. Thus, the metalbinding proteins ferritin, transferrin, coeruloplasmin
and others, e.g. metallothionein, are of central
importance in the control

Prevention (contd. )

Protection of cells from incident radiation may occur

through specialized pigments, e.g. the melanins for
ultraviolet radiation or the carotenoids for
electronically excited states such as singlet oxygen.
However, these and other strategies are not
completely preventative, because they operate by
decreasing the yield of a given challenging agent with
less than 100 % efficiency.
the intestinal mucosal cells. These cells are exposed
to a variety of reactive intermediates and xenobiotics,
and the rate of accumulation of products of oxidative
damage in these cells is high. The turnover and
elimination of whole cells prevents further spread of
the challenging species.

2. Interception
Antioxidant defense involves several strategies,
enzymatic and
non-enzymatic.
In general, this means transferring the oxidizing equivalents
from the hydrophobic phases into the aqueous phases, e.g.
from the membrane to the cytosol or from lipoproteins to the
aqueous phase of the plasma.
Such intercepting chain-breaking antioxidants are often
phenolic compounds. (R,R,R)-a-Tocopherol is probably the
most efficient compound in the lipid phase
A prerequisite for efficient interception by the phenolic
antioxidants is that the lifetime of the radical to be intercepted
must not be too short.

Non-enzymatic
In the lipid phase, tocopherols and
carotenes as well as oxy-carotenoids are
of' interest, (as are vitamin A and
ubiquinols.
In the aqueous phase, there are
ascorbate, glutathione and other
compounds.
In addition to the cytosol, the nuclear and
mitochondrial matrices and extracellular
fluids are protected.

Supplements
Antioxidants from our diet appear to be of great
importance in controlling damage by free
radicals.
it is not clear if supplements should be taken
and, if so, how much. Once thought to be
harmless, we now know that consuming megadoses of antioxidants can be harmful due to their
potential toxicity and interactions with
medications. Remember -- antioxidants
themselves may act as pro-oxidants at high
levels.

Enzymatic
Overall, these low molecular mass
antioxidant molecules add
significantly to the defense provided
by the enzymes
superoxide dismutase,
catalase and
glutathione peroxidases

Antioxidant Enzymes
The antioxidant enzymes are proteins with
antioxidant properties. There are three known
classes of antioxidant enzymes:

Superoxide dismutases
Catalases
Glutathione peroxidases
There are many forms of each class of protein. In
general, cancer cells have low levels of these enzymes,
when compared to an appropriate normal cell control.

Primary Antioxidant Enzyme

System

SOD
FeSOD which contains an iron ion
and is generally found in some
prokaryotes, and CuZnSOD,
which is active in the cytoplasm of
eukaryotic cells.
CuZnSOD occurs as a dimer of
identical 16 KDa subunits. Each
subunit is 151 amino acids long,
and the total protein weighs 32
KDa
Absence of Zn

Alzheimer Disease
SOD act as pro-oxidant than
Antioxidant

SOD
Superoxide dismutase is categorized as
an oxidoreductase class of enzyme, and
has the Enzyme Commission identifier of
EC1.15.1.1. SOD is a metalloenzyme,
meaning that in addition to amino acids, it
contains metal ions.
Cu2+ + O2- Cu+ + O2
Cu+ + O2- + 2H+ Cu2+ + H2O2
2 O2- + 2H+ H2O2 + O2

Repair
Since prevention and interception
processes are not completely effective,
products of damage are continuously
formed in low yields and hence may
accumulate. there are multiple enzyme
systems involved in
DNA repair and lipolytic as well as
proteolytic enzymes capable of serving the
functions of restitution or replenishment.

Peroxynitrile (RON)
which is formed from nitric oxide and
superoxide. It was observed that a selenoorganic compound reacts very efficiently
with peroxynitrile

Biomarkers-1
Lipid Peroxidation:
The appearance of 8-epi-prostaglandin PGF2a (8-epiPGF2a) in plasma or urine has been suggested by a
number of investigators as a reliable index of in vivo free
radical generation and oxidative lipid formation. There is
very strong evidence from animal studies that 8-epiPGF2a increase in plasma and urine as a result of
oxidative stress, and in human, this product is elevated
in smokers. Comparison with other measures of lipid
peroxidation, 8-epi-PGF2a is specific product of lipid
peroxidation, and is very stable. In addition, its formation
is modulated by antioxidant status, and its level is not
affected by lipid content of the diet.

Biomarkers-2
Protein Oxidation:
Oxidative damage can affect proteins giving rise
among others to protein carbonyl derivatives, via
a variety of mechanisms that include
fragmentation and amino acid oxidation. Protein
oxidation has major deleterious effects on
normal functioning of organism. 2-Oxohistine
and nitrotyrosine are thought to be an indicator
of protein oxidation induced by peroxyl radical
and peroxynitrite, respectively.

Biomarkers-3
DNA damage:
One of the major products of oxygen
radical attack is 8-hydroxy-2'deoxyguanosine. Numerous publications
reported that there is an age-dependent
increase in the level of this adduct in
human brain tissue. Brunswick
Laboratories has developed a LC/MS
method to measure 8-hydroxy-2'deoxyguanosine in urine.