Free radicals are unstable molecules that contain unpaired electrons. This instability causes free radicals to be highly reactive and seek to pair their unpaired electrons by interacting with other molecules. This reactivity can damage cells and lead to diseases. Specifically: - Free radicals become more stable by taking electrons from other atoms, which can cause cellular damage and diseases or signs of aging over time. - Common diseases associated with free radicals include atherosclerosis, heart disease, arthritis, stroke, respiratory diseases, Parkinson's and Alzheimer's disease. - Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species/reactive nitrogen species like free radicals and a body's antioxidant defenses. This stress can damage biom

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Q. 1 Why free radical is an unstable molecule?
 Free radicals are the products of normal cellular metabolism. A free radical can be
defined as an atom or molecule containing one or more unpaired electrons in valency
shell or outer orbit and is capable of independent existence. Free radicals include:
 Hydroxyl (OH•
), Superoxide (O2
•–
), Nitric oxide (NO•)
, Nitrogen dioxide (NO2•
),
Neroxyl (ROO•
), Lipid peroxyl (LOO•
)
 The odd number of electron(s) of a free radical makes it unstable, short lived and
highly reactive. Because of their high reactivity, they can abstract electrons from other
compounds to attain stability. Thus the attacked molecule loses its electron and
becomes a free radical itself, beginning a chain reaction cascade which finally damages
the living cell.

FIGURE: Chemical reactions that generate reactive oxygen species. (A) Superoxide is
generated in the mitochondria when electrons leak out of the electron transport chain
and reduce singlet oxygen. (B) Superoxide can also be generated in the cell when
enzymes catalyze the transfer of an electron from NADPH to singlet oxygen, often
during metabolism reactions. (C) Two superoxide molecules can be converted to
hydrogen peroxide and oxygen by the superoxide dismutase enzymes. (D)
Myeloperoxidase catalyzes the conversion of hydrogen peroxide and a chloride anion
to hypochlorous acid which acts as a potent oxidizer in the respiratory burst. (E) When
hydrogen peroxide encounters free ferrous iron within the cell, the Fenton reaction
occurs, producing a hydroxyl radical.
Q. 2 Diseases caused by free radicals
 Free radicals are unsteady molecules that the body produces as a reaction to
environmental and other pressures, as the body processes food and reacts to the
environment. Therefore, it is totally normal to have some free radicals in your body.
Occasionally, however, the proportion of free radicals in the body may increase
significantly and, due to their high activity, will instead begin to attack the body’s
own cells, damaging the cells. The damaged cells, in turn, produce new free radicals.

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 Both ROS and RNS collectively constitute the free radicals and other non radical
reactive species. The ROS/RNS play a twofold job as both beneficial and toxic
compounds to the living system. At moderate or low levels ROS/RNS have beneficial
effects and involve in various physiological functions such as in immune function (i.e.
defense against pathogenic microorganisms), in a number of cellular signaling
pathways, in mitogenic response and in redox regulation. But at higher concentration,
both ROS as well as RNS generate oxidative stress and nitrosative stress, respectively,
causing potential damage to the biomolecules. The oxidative stress and nitrosative
stress are developed when there is an excess production of ROS/RNS on one side and
a deficiency of enzymatic and
non enzymatic antioxidants on
the other side. Most importantly,
the excess ROS can damage the
integrity of various biomolecules
including lipids, proteins and
DNA leading to increased
oxidative stress in various human
diseases such as diabetes
mellitus, neurodegenerative
diseases, rheumatoid arthritis,
cataracts, cardiovascular
diseases, respiratory diseases as
well as in aging process.
 Health Conditions Caused by
Free Radicals and Oxidative Stress
 Atherosclerosis.
 Vision loss – deterioration of the eye lens, which contributes to blindness.
 Heart disease – increased risk of coronary heart disease, since free radicals encourage
low-density lipoprotein (LDL) cholesterol to stick to artery walls.
 Arthritis – inflammation of the joints.
 Stroke.
 Respiratory diseases.
 Immune deficiency.
 Emphysema.
 Parkinson’s disease – damage to nerve cells in the brain, which contributes to this
condition.
 Alzheimer’s disease – also damage to nerve cells in the brain.
 Cancer – certain cancers are triggered by damaged cell DNA.
 Obesity.
 Hair loss.
 Fast aging – acceleration of the ageing process.
 An excessive release of free iron or copper ions.
 A disruption of electron transport chains.
 An increase in enzymes that generate free radicals.
 Inflammatory joint disease.

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 Asthma.
 Diabetes.
 Senile dementia.
 In addition, other inflammatory or ischemic conditions.
 Free radicals are unstable atoms. To become more stable, they take electrons from other
atoms. This may cause diseases or signs of aging.
 It is thought that the free radicals cause changes in the cells that lead to these and
possibly also other conditions. However, antioxidants help to neutralize free radicals in
our bodies, which also boosts our overall health.
 Oxidative stress can occur when there is an imbalance of free radicals and antioxidants
in the body. Several factors contribute to oxidative stress and excess free radical
production. These factors can include:
 Diet, lifestyle, certain conditions and environmental factors such as pollution and
radiation
 Antioxidants are substances that neutralize or remove free radicals by donating an
electron. The neutralizing effect of antioxidants helps protect the body from oxidative
stress. Examples of antioxidants include vitamins A, C, and E.
 Effects of oxidative stress The effects of oxidative stress vary and are not always
harmful. For example, oxidative stress that results from physical activity may have
beneficial, regulatory effects on the body.
 However, long-term oxidative stress damages the body’s cells, proteins, and DNA. This
can contribute to aging and may play an important role in the development of a range
of conditions.
 Some of these conditions are: 1. Chronic inflammation: Oxidative stress can cause
chronic inflammation. Infections and injuries trigger the body’s immune response.
Immune cells called macrophages produce free radicals while fighting off invading
germs. These free radicals can damage healthy cells, leading to inflammation.
 However, oxidative stress can also trigger the inflammatory response, which, in turn,
produces more free radicals that can lead to further oxidative stress, creating a cycle.
 Chronic inflammation due to oxidative stress may lead to several conditions,
including diabetes, cardiovascular disease, and arthritis.
 2. Neurodegenerative diseases: The effects of oxidative stress may contribute to
several neurodegenerative conditions, such as Alzheimer’s disease and Parkinson’s
disease. The brain is particularly vulnerable to oxidative stress because brain cells
require a substantial amount of oxygen.
 During oxidative stress, excess free radicals can damage structures inside brain cells
and even cause cell death, which may increase the risk of Parkinson’s disease.
 Conditions linked to oxidative stress: Oxidative stress may play a role in the
development of a range of conditions, including: cancer, Alzheimer’s disease,
Parkinson’s disease, diabetes, cardiovascular conditions such as high blood pressure,
atherosclerosis, and stroke, inflammatory disorders, chronic fatigue syndrome, asthma
male infertility
 Factors that may increase a person’s risk of long-term oxidative stress include:
 obesity
 diets high in fat, sugar, and processed foods

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 exposure to radiation
 smoking cigarettes or other tobacco products
 alcohol consumption
 certain medications
 pollution
 exposure to pesticides or industrial chemicals
Q.3 Discuss Life span and hydroxyl radical
 Hydroxyl radical is the most reactive free radical and can be formed from ·O2– and
H2O2 in the presence of metal ions such as copper or iron. Hydroxyl radicals have the
highest 1-electron reduction potential and are primarily responsible for the cytotoxic
effect in aerobic organism. Hydroxyl radicals react with lipids, polypeptides, proteins,
and nucleic acids, especially thiamine and guanosine. They also add readily to
unsaturated compounds. When a hydroxyl radical reacts with aromatic compounds, it
can add on across a double bond, resulting in hydroxycyclohexadienyl radical. The
resulting radical can undergo further reactions, such as reaction with oxygen, to give
peroxyl radical, or decompose by water elimination to phenoxyl type radicals.
 In living organisms there are two major reactive oxygen species, superoxide radical and
hydroxyl radical that are being continuously formed in a process of reduction of oxygen
to water.
 The hydroxyl radical has a very short in vivo half-life of approximately 10−9
seconds
and a high reactivity. This makes it a very dangerous compound to the organism.
 In a biological body, hydroxyl radicals attack the cell membrane, causing membrane
damage and destroying sugar groups and DNA base sequences, inducing the
disintegration of the double-helix structure, even causing cell death and mutations.
 The hydroxyl radical (⋅OH), the quintessential reactive oxygen species, is the mediator
of much of the DNA damage caused by ionizing radiation. This damage includes strand
breaks, which are initiated by abstraction of a deoxyribose hydrogen atom by the
hydroxyl radical.
 Body's cells have developed many enzymatic and non-enzymatic mechanisms to
inactivate these radicals, the so-called antioxidant mechanisms. Specifically, the skin
and mucous membranes have become barriers capable of neutralizing radicals, so they
will not affect body tissues.
Q. 4 What is spin trapping
 Spin trapping is an analytical technique employed in chemistry and biology for the
detection and identification of short-lived free radicals through the use of electron
paramagnetic resonance (EPR) spectroscopy.
 EPR spectroscopy detects paramagnetic species such as the unpaired electrons of free
radicals. However, when the half-life of radicals is too short to detect with EPR,
compounds known as spin traps are used to react covalently with the radical products
and form more stable adduct that will also have paramagnetic resonance spectra
detectable by EPR spectroscopy.
 The use of radical-addition reactions to detect short-lived radicals was developed by
several independent groups by 1968.

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 The most commonly used spin traps are alpha-phenyl N-tertiary-butyl nitrone (PBN)
and 5,5-dimethyl-pyrroline N-oxide (DMPO). More rarely, C-nitroso spin traps such as
3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) can be used: often additional
hyperfine information is derived, but at a cost of specificity (due to facile non-radical
addition of many compounds to C-nitroso species, and subsequent oxidation of the
resulting hydroxylamine).
 5-Diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DIPPMPO) spin trapping
has been used in measuring superoxide production in mitochondria.
 A common method for spin-trapping involves the addition of radical to a nitrone spin
trap resulting in the formation of a spin adduct, a nitroxide-based persistent radical, that
can be detected using EPR. The spin adduct usually yields a distinctive EPR spectrum
characteristic of a particular free radical that is trapped.
 The identity of the radical can be inferred based on the EPR spectral profile of their
respective spin adducts such as the g value, but most importantly, the hyperfine-
coupling constants of relevant nuclei.
 Unambiguous assignments of the identity of the trapped radical can often be made by
using stable isotope substitution of the radicals parent compound, so that further
hyperfine couplings are introduced or altered.
 It is worth noting that the radical adduct (or products such as the hydroxylamine) can
often be stable enough to allow non-EPR detection techniques. The groups of London,
and Berliner & Khramtsov have used NMR to study such adducts and Timmins and
co-workers used charge changes upon DBNBS trapping to isolate protein adducts for
study.
Q. 5 What is Nitric oxide signaling
 Nitric oxide (NO) is an essential molecule involved in several physiological and
pathological processes within the mammalian body. NO is synthesized by nitric oxide
synthase (NOS) which oxidizes a guanidine nitrogen of L-arginine releasing nitric
oxide in the form of a free radical and citrulline.
 Three isoforms of the NOS have been identified: endothelial (eNOS or NOS-3),
neuronal (nNOS or NOS-1), and inducible (iNOS or NOS-2) - each with separate
functions. The neuronal enzyme (NOS-1) and the endothelial isoform (NOS-3) are
calcium-dependent and produce low levels of gas as a cell signaling molecule. The
inducible isoform (NOS-2) is calcium independent and produces large amounts of gas
which can be cytotoxic.
 Nitric oxide thus generated acts as a messenger in diverse functions including
vasodilation neurotransmission, anti-tumor and anti-pathogenic activities. Sufficient
levels of NO production are necessary in protecting an organ such as the liver from
ischemic damage. However, sustained levels of NO production result in direct tissue
toxicity and contribute to the vascular collapse associated with septic shock, whereas
chronic expression of NO is associated with various carcinomas and inflammatory
conditions including juvenile diabetes, multiple sclerosis, arthritis, and ulcerative
colitis.

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What Is Nitric Oxide?
Nitric oxide (NO), a small molecule with a simple structure, is an extremely unstable gaseous
free radical. It is soluble in both aqueous and lipid media so that it can rapidly diffuse through
biofilms. And it has a short half-life of only a few seconds in vivo.
NO is widespread in various tissues especially in nervous tissues in mammals. And NO is one
of the productions of the oxidation of L-arginine by nitric oxide synthase (NOS). It is a novel
biological messenger molecule and plays an important role in the regulation of cardiovascular
＆ cerebrovascular, nerve, and immunity. And it was selected as a "star molecule" by Science
magazine in 1992.
What Is Nitric Oxide Signaling?
Nitric oxide signaling mainly refers to the processes by which NO exerts multiple biological
functions through autocrine and paracrine signaling pathways.
The Function of Nitric Oxide Signaling
Nitric oxide signaling mediates multiple biological processes by which NO acts as a signal
transducer to exert diverse functions including immune responses, the regulation of vascular
tone and neurotransmission, anti-tumor and anti-pathogenic.
Nitric Oxide Signaling pathway
As a small inorganic molecule with hydrophilic and lipophilic properties, NO is an important
player in many physiological processes. At present, the NO signaling pathway in the
cardiovascular system has been best studied.
When Ach stimulates vascular endothelial cells, the surface Ach receptor (GPCR) is activated.
GPCR activation activates phospholipase C (PLC), which catalyzes the production of IP3. IP3
diffuses into the cytoplasm and acts on the IP3-gated Ca2+ channel in the endoplasmic
reticulum, promoting the release of Ca2+ in the endoplasmic reticulum. The released Ca2+
binds to calmodulin (CaM) to form Ca2+-CaM complex, which reactivates NO synthase
(eNOS) in endothelial cells. At the catalytic action of NOS, a substantial of NO is made. NO
diffuses into vascular smooth muscle cells adjacent to the endothelial cells where it binds to
and activates soluble guanylate cyclase (sGC). Active sGC catalyzes the dephosphorylation of
GTP to cGMP. cGMP further activates cGMP-dependent kinase G (PKG). Active PKG
phosphorylates myosin light chain phosphatase (MLCP), which dephosphorylates the light
chain of myosin, leading to smooth muscle relaxation.

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PKG plays a central regulatory role in the signal pathway above. Except for direct stimulation
for eNOS to produce NO, PKG also activates CaM by inhibiting Ca2+ entry. Active CaM
evokes eNOS. Feedback regulation of sGC and activation of PDE5 by PKG increase the
hydrolysis of cGMP, reducing the concentration of cGMP. In addition to its role in the
cardiovascular system, NO is involved in the regulation of many processes, most notably in the
nervous system. It may act as a reverse transmitter in long-term enhancement in terms of
learning and memory. Although the process of NO participation has been understood quite a
lot, many details are still not completely clear, and its role in many aspects is not clear, such as
immunity, nerve, vision and so on. Therefore, there is still much work to be done on the NO
signal network.

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Q6. Harmful effects of ROS and RNS (see Q. 2)
 Indeed, when ROS overwhelm the cellular antioxidant defense system, oxidative stress
occurs, which results in oxidative damage of nucleic acids, proteins, and lipids. This
potentially harmful effect of ROS has been implicated in carcinogenesis,
neurodegeneration, atherosclerosis, diabetes, and aging.

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 Reactive nitrogen species (RNS) include peroxynitrite and its reaction products, such
as NO2. High, sustained levels of NO and superoxide, precursors of peroxynitrite
(ONOO−), are associated with tissue toxicity, cancer, and inflammatory conditions,
such as arthritis, juvenile diabetes, and ulcerative colitis
 Free radical nitric oxide (NO) is a biological messenger with diverse functions in plant
physiology, including in stress physiology. Together with NO, related molecules called
reactive nitrogen species (RNS), e.g. peroxynitrite or S-nitrosothiols, are associated
with plant metabolism under both physiological and stress conditions.
Q. 7 Role of mitrochondria in oxidative stress generation
 Mitochondria are deeply involved in the production of reactive oxygen species through
one-electron carriers in the respiratory chain; mitochondrial structures are also very
susceptible to oxidative stress as evidenced by massive information on lipid
peroxidation, protein oxidation, and mitochondrial DNA (mtDNA) mutations.
Oxidative stress can induce apoptotic death, and mitochondria have a central role in
this and other types of apoptosis, since cytochrome c release in the cytoplasm and
opening of the permeability transition pore are important events in the apoptotic
cascade.
 Mitochondrial DNA represents a critical target for such oxidative damage. Once
damaged, mitochondrial DNA can amplify oxidative stress by decreased expression of
critical proteins important for electron transport, leading to a vicious cycle of ROS and
organelle dysregulation that eventually triggers apoptosis
 Mitochondria are a major source of intracellular ROS and are particularly vulnerable to
oxidative stress. Mitochondrial dysfunction is a prominent feature of neurodegenerative
diseases, such as PD, AD, and ALS. As mentioned above, ROS can induce
mitochondrial DNA mutations, and damage the mitochondrial respiratory chain,
membrane permeability, Ca2+ homeostasis and mitochondrial defense systems; all
these aspects are implicated in the development of neurodegenerative diseases, which
mediate or amplifying neuronal dysfunction during the course of neurodegeneration
 Mitochondria as sources of ROS: Sources of ROS in living cells are represented by
physiological enzymatic mechanisms; oxidative stress may ensue when ROS
production is excessive, due either to a particular metabolic situation, or to the presence
of xenobiotic compounds, or also to damage-mediated liberation of non-enzymatic
catalysts such as free metals, or when the cellular defences are lowered by the depletion
of physiological antioxidants.
 The respiratory chain is a powerful source of ROS, primarily the superoxide radical and
consequently hydrogen peroxide, either as a product of superoxide dismutase or by
spontaneous disproportionation
 There are two major respiratory chain regions where ROS are produced, one being
complex I (NADH coenzyme Q reductase) and the other complex III (ubiquinol
cytochrome c reductase)
 In complex III, antimycin is known not to completely inhibit electron flow from
ubiquinol to cytochrome c: the antimycin-insensitive reduction of cytochrome c is
mediated by superoxide radicals; the source of superoxide in the enzyme may be either

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cytochrome b566, or ubisemiquinone or Rieske’s iron-sulfur center. Ubisemiquinone is
relatively stable only when protein bound, therefore the coenzyme Q (CoQ) pool in the
lipid bilayer is no source of ROS.
 There is evidence that the one-electron donor to oxygen in complex I is a non-
physiological quinone reduction site different from the physiological site(s); the former,
hydrophilic, site reduces several quinones to the corresponding semiquinone forms,
which are unstable and can reduce oxygen to superoxide. This mechanism is shared by
several quinones, including such drugs as anthracyclines and the clinically employed
CoQ analog, idebenone
 Mitochondria contain antioxidant enzymes, including superoxide dismutase (Mn form)
and glutathione peroxidase, and lipid-soluble antioxidants such as vitamin E and
reduced CoQ. Ubiquinol may exert its antioxidant function indirectly by reducing α-
tocopheroxyl radical back to vitamin E or directly as a quencher of oxygen and lipid
peroxyl radicals.
 Mitochondria as targets of ROS Being major producers of ROS, mitochondrial
structures are exposed to high concentrations thereof and may therefore be particularly
susceptible to their attack. Evidence exists, however, that even ROS produced outside
the mitochondrion may damage mitochondrial components
 Damage by oxidative stress to mitochondrial components includes lipid peroxidation,
protein oxidation and mtDNA mutations.
 Lipid peroxidation might be particularly harmful in mitochondria, that contain
cardiolipin as a major component of the inner mitochondrial membrane, since this lipid
is required for the activity of cytochrome oxidas and of other mitochondrial proteins
 Modification of the redox state of vital sulfhydryl groups may be at the basis also in
mitochondria of important regulatory mechanisms, similar to those suggested to
modulate signal transduction cascades. Inactivation of Mn-superoxide dismutase in
transgenic mice enhances ROS production and results in animal death by dilated
cardiomyopathy, with partial inactivation of mitochondrial enzymes containing iron-
sulfur centers.
 Mitochondria, ROS, and cell death Cell death can occur by either necrosis or
apoptosis as a result of exogenous and endogenous insults. There seems to be no net
border between these phenomena, depending mainly on the extent of stress and on the
ATP levels; however, the mechanisms are rather different, since apoptosis involves a
well-defined chain of enzymatic events which are genetically programmed. Apoptosis
induced by oxidative stress has been well documented and appears to involve the same
steps in the commitment and execution stages as in the other causes of apoptosis.
Actually, apoptosis may be a mechanism to eliminate ROS-producing cells.
 Mitochondria and ageing The concept that mitochondria are primarily involved in
ageing derives from the theory of Harman, linking senescence to the injurious effect of
free radicals arising from the one-electron reduction of oxygen during metabolism. In
accordance with the free radical theory of ageing is the inverse relation existing between
auto-oxidation rate in different animal species and life expectancy of the same species;
the auto-oxidation rate on its hand is directly proportional to metabolic rate, so that the
duration of life seems to be inversely related to the rate of oxygen consumption. The
increased longevity obtained by caloric restriction in rodents, which is accompanied by
decreased state 4 respiration and decreased superoxide production, and the relation of

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lifespan in Drosophila with the simultaneous expression of the antioxidant enzymes
superoxide dismutase and catalase are corollaries of this proposal.
Q. 8 Describe the mechanism of Glutathione antioxidant
 Mechanism of action
 Production of GSH occurs by two mechanisms, de novo synthesis and recycling of
GSSG. De novo synthesis occurs in a two-step reaction catalyzed by two separate
enzymes, glutamine-cysteine ligase (GCL) and glutathione synthase (GS), as shown in
Figure 1. The first step in the reaction is catalyzed by GCL, a heterodimer made up of
a catalytic subunit (GCLC) that possesses the enzyme’s active site and performs the
actual amino acid linkage, and a modulating subunit (GCLM) that regulates the activity
of GCLC, as reviewed in Figure 1. This first step is rate limiting, with cysteine
availability being the rate-limiting component. In the final step of de novo GSH
synthesis, glycine is linked to the dimer formed in the previous step reaction by GS. De
novo synthesis of GSH is regulated by negative feedback.

 Figure 1. Production of GSH occurs by two mechanisms, de novo synthesis and
recycling of GSSG. De novo synthesis occurs in a two-step reaction catalyzed by two
separate enzymes, glutamine-cysteine ligase (GCL) and glutathione synthase (GS). The
enzyme glutathione reductase (GSR) catalyzes the reduction of GSSG back to GSH.
Glutathione peroxidase (GSH-PX) is a selenium-based enzyme that reduces hydrogen
peroxide (H2O2) to water.
 Gamma-glutamyltransferase 1 (GGT1) is a membrane-bound enzyme that catalyzes the
breakdown of extracellular GSH into glutamate and cysteinyl-glycine, providing raw
materials for the de novo of GSH
 Finally, GSH can be obtained by the reduction of GSSG via the glutathione reductase
(GSR) enzyme. This reaction requires NADPH and forms two GSH molecules from
one GSSG molecule. It is now known that a number of interactions may occur that
prevent the de novo formation of GSH. For example, it has been shown that glutathione
is decreased due to an inability to produce glutathione in the extracellular lung fluid of
children with chronic asthma and in the macrophages of adults with human
immunodeficiency virus (HIV)
 GSH protects cells by neutralising (reducing) reactive oxygen species. This conversion
is illustrated by the reduction of peroxides:

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 2 GSH + R2O2 → GSSG + 2 ROH (R = H, alkyl)
and with free radicals:
 GSH + R•
→ 1⁄2 GSSG + RH
 Regulation: Aside from deactivating radicals and reactive oxidants, glutathione
participates in thiol protection and redox regulation of cellular thiol proteins under
oxidative stress by protein S-glutathionylation, a redox-regulated post-translational
thiol modification. The general reaction involves formation of an unsymmetrical
disulfide from the protectable protein (RSH) and GSH
 RSH + GSH + [O] → GSSR + H2O
 Glutathione is also employed for the detoxification of methylglyoxal and
formaldehyde, toxic metabolites produced under oxidative stress. This detoxification
reaction is carried out by the glyoxalase system. Glyoxalase I (EC 4.4.1.5) catalyzes
the conversion of methylglyoxal and reduced glutathione to S-D-lactoylglutathione.
Glyoxalase II (EC 3.1.2.6) catalyzes the hydrolysis of S-D-lactoylglutathione to
glutathione and D-lactic acid.
OR
Mechanism of action
 Glutathione (GSH) participates in leukotriene synthesis and is a cofactor for the enzyme
glutathione peroxidase. It also plays a role in the hepatic biotransformation and
detoxification process; it acts as a hydrophilic molecule that is added to other lipophilic
toxins or wastes prior to entering biliary excretion. It participates in the detoxification
of methylglyoxal, a toxic by-product of metabolism, mediated by glyoxalase enzymes.
Glyoxalase I catalyzes the conversion of methylglyoxal and reduced glutathione to S-
D-Lactoyl-glutathione. Glyoxalase II catalyzes the conversion of S-D-Lactoyl
Glutathione to Reduced Glutathione and D-lactate. Glyoxalase I catalyzes the
conversion of methylglyoxal and reduced glutathione to S-D-Lactoyl-glutathione.
Glyoxalase II catalyzes the conversion of S-D-Lactoyl Glutathione to Reduced
Glutathione and D-lactate. GSH is a cofactor of conjugation and reduction reactions
that are catalyzed by glutathione S-transferase enzymes expressed in the cytosol,
microsomes, and mitochondria. However, it is capable of participating in non-
enzymatic conjugation with some chemicals, as it is hypothesized to do to a significant
extent with n-acetyl-p-benzoquinone imine (NAPQI), the reactive cytochrome P450
reactive metabolite formed by toxic overdose of acetaminophen. Glutathione in this
capacity binds to NAPQI as a suicide substrate and in the process detoxifies it, taking
the place of cellular protein sulfhydryl groups which would otherwise be toxically
adducted. The preferred medical treatment to an overdose of this nature, whose efficacy
has been consistently supported in literature, is the administration (usually in atomized
form) of N-acetylcysteine, which is used by cells to replace spent GSSG and allow a
usable GSH pool.
Q.9 what is the difference between GSH and GSSG
 Glutathione (GSH) is a tri-peptide (g-glutamylcysteinylglycine) that acts as an
endogenous antioxidant, a xenobiotic detoxifier, and is involved in metabolic
regulation. GSH is the most abundant antioxidant in aerobic cells, present in
micromolar (mM) concentrations in bodily fluids and in millimolar (mM)
concentrations in tissue. The central nervous system (CNS) has GSH concentrations

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ranging from 1 to 3 mM, depending on the region. The forebrain and cortex` have the
highest concentration of GSH, followed by the cerebellum, brain stem, and spinal cord.
With high oxygen consumption and rich poly-unsaturated fatty acid (PUFAs) content,
the brain is particularly susceptible to oxidative stress. GSH is critical for protecting the
brain from oxidative stress, acting as a free radical scavenger and inhibitor of lipid
peroxidation
 GSH is synthesized from l-glutamate, l-cysteine, and l-glycine in two ATP requiring
steps catalyzed by the enzymes g-glutamylcysteine ligase and glutathione synthetase.
The cysteine thiol moiety gives GSH its antioxidant properties. The thiol is oxidized by
cellular pro-oxidants, such as free radicals and reactive aldehydes, to form oxidized
GSH disulfide (GSSG). The reduction of GSSG back to GSH requires NADPH and is
catalyzed by the enzyme glutathione reductase, thus regenerating GSH for cellular
antioxidant defense. In addition to the reactions listed that produce and regenerate GSH,
GSH is degraded by g-glutamyl transpeptidase to form glutamate and cysteinyl glycine.
The reactions described are summarized in Fig. 1.
Glutathione disulfide (GSSG)
 In living cells, glutathione disulfide is reduced into two molecules of glutathione with
reducing equivalents from the coenzyme NADPH. This reaction is catalyzed by the
enzyme glutathione reductase.
 Antioxidant enzymes, such as glutathione peroxidases and peroxiredoxins, generate
glutathione disulfide during the reduction of peroxides such as hydrogen peroxide
(H2O2) and organic hydroperoxides (ROOH)
 2 GSH + ROOH → GSSG + ROH + H2O
 Other enzymes, such as glutaredoxins, generate glutathione disulfide through thiol-
disulfide exchange with protein disulfide bonds or other low molecular mass
compounds, such as coenzyme A disulfide or dehydroascorbic acid.

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 2 GSH + R-S-S-R → GSSG + 2 RSH
 The GSH:GSSG ratio is therefore an important bioindicator of cellular health, with a
higher ratio signifying less oxidative stress in the organism. A lower ratio may even be
indicative of neurodegenerative diseases, such as Parkinson's disease (PD) and
Alzheimer's disease.
 GSSG, along with glutathione and S-nitrosoglutathione (GSNO), have been found to
bind to the glutamate recognition site of the NMDA and AMPA receptors (via their γ-
glutamyl moieties), and may be endogenous neuromodulators. At millimolar
concentrations, they may also modulate the redox state of the NMDA receptor complex.
Q. 10 Discuss the mechanism of action of superoxide dismutase
 Superoxide dismutases (SODs) are universal enzymes of organisms that live in the
presence of oxygen. They catalyze the conversion of superoxide into oxygen and
hydrogen peroxide.

 SOD catalyzes the conversion of the superoxide anion free radical (•
O2−
) to hydrogen
peroxide (H2O2) and molecular oxygen O2 (Figure 1 A,B). Subsequently, H2O2 is
reduced to water by the catalase (CAT) enzyme, glutathione peroxidase (GPx), and/or
thioredoxin (Trx)-dependent peroxiredoxin (Prx) enzymes (Figure 1B). H2O2 may also
generate another reactive oxygen species (ROS), the hydroxide ion (•
HO) via the
Fenton reaction in the presence of Fe2+
(Figure 1B).
 The major cellular defense against O2•−
and peroxynitrite is a group of oxidoreductases
known as SODs, which catalyze the dismutation of O2•−
into oxygen and H2O2. In
mammals, there are three isoforms of SOD (SOD1 [CuZnSOD]; SOD2 [MnSOD];
SOD3 [ecSOD]), and each is a product of distinct genes and distinct subcellular
localization, but catalyzes the same reaction. This distinct subcellular location of these
SOD isoforms is particularly important for compartmentalized redox signaling. The
mechanism of dismutation of O2•−
to H2
O2
by SOD involves alternate reduction and
reoxidation of a redox active transition metal, such as copper (Cu) and manganese (Mn)
at the active site of the enzyme as shown in Figure. This indicates that SOD activity
requires a catalytic metal.
 FIGURE Common
mechanism of scavenging O2
•−
by
SODs. Enzymatic activity of SOD
involves alternate reduction and
reoxidation of catalytic metal (i.e.,
Cu or Mn) at the active site of the
enzyme. Thus, Cu or Mn will be a
key modulator of SOD activity of
SOD1/SOD3 or SOD2, respectively.

15. ©Lekhan
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
 Figure 1. Superoxide dismutase enzymes. (A) Superoxide dismutases (SODs) are
metalloenzymes constitutively expressed in eukaryotes: SOD1 is a Cu, Zn-SOD and is
present in the cytosol and the mitochondrial intermembrane; SOD2 is a Mn-SOD
localized in the matrix and inner membrane of mitochondria; SOD3 is a Cu, Zn-SOD
expressed in the extracellular compartment. Nevertheless, all three forms catalyze the
conversion of the superoxide anion free radical (•
O2−
) into hydrogen peroxide (H2O2).
(B) In detail, SOD converts the •
O2−
, generated in several cellular insults/metabolism,
into H2O2 and molecular oxygen (O2). The resulting H2O2 may undergo reduction to
water via catalase (CAT), glutathione peroxidases (GPx), or thioredoxin (Trx)-
dependent peroxiredoxin (Prx). Otherwise, H2O2 originates •
OH via the Fenton reaction

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in the presence of Fe2+
. •
O2−
may also react with •
NO originating the oxidant and
nitrating agent peroxynitrite (ONOO−
), which further contributes to oxidative-stress
damage. GSH = glutathione; GSSG = glutathione disulfide; TrxSH2 = reduced
thioredoxin; TrxS2 = oxidized thioredoxin
Q. 11 Describe the Process of Oxidative stress in aging
 Reactive oxygen and nitrogen species (RONS) are produced by several endogenous and
exogenous processes, and their negative effects are neutralized by antioxidant defenses.
Oxidative stress occurs from the imbalance between RONS production and these
antioxidant defenses.
 Aging is a process characterized by the progressive loss of tissue and organ function.
The oxidative stress theory of aging is based on the hypothesis that age-associated
functional losses are due to the accumulation of RONS-induced damages. At the same
time, oxidative stress is involved in several age-related conditions (ie, cardiovascular
diseases [CVDs], chronic obstructive pulmonary disease, chronic kidney disease,
neurodegenerative diseases, and cancer), including sarcopenia and frailty.
 Pathophysiology of oxidative stress
 Free radicals are highly reactive atoms or molecules with one or more unpaired
electron(s) in their external shell and can be formed when oxygen interacts with certain
molecules. These radicals can be produced in cells by losing or accepting a single
electron, therefore, behaving as oxidants or reductants.
 The terms reactive oxygen species (ROS) and reactive nitrogen species (RNS) refer to
reactive radical and non-radical derivatives of oxygen and nitrogen, respectively.
 Reactive oxygen and nitrogen species (RONS) are produced by all aerobic cells and
play an important role in aging as well as in age-related diseases.4 RONS generation is
not only limited to determine deleterious effects but also involved in the extraction of
energy from organic molecules, in immune defense, and in the signaling process.
 There are endogenous and exogenous sources of RONS:
 Endogenous sources of RONS include nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase, myeloperoxidase (MPO), lipoxygenase, and angiotensin II.
NADPH oxidase is the prevalent source of the radical superoxide anion (O2
•
) which is
formed by the one-electron reduction of molecular oxygen, with electrons supplied by
NADPH, during cellular respiration. Most of the O2
•
is dismutated into the hydrogen
peroxide (H2O2) by superoxide dismutase (SOD). H2O2 is not a free radical because it
has no unpaired electrons, but it is able to form the highly reactive ROS hydroxyl ion
(OH•
) through the Fenton or Haber–Weiss reaction. Hydroxyl radicals are extremely
reactive and react especially with phospholipids in cell membranes and proteins. In
neutrophils, H2O2 in the presence of chloride and MPO can be converted to
hypochlorous acid, an ROS particularly damaging cellular proteins. Nitric oxide (NO)
is produced from l-arginine by three main isoforms of nitric oxide synthase (NOS):
epithelial NOS, related to vasodilation and vascular regulation, neuronal NOS, linked
to intracellular signaling, and inducible NOS, activated in response to various
endotoxin or cytokine signals. Finally, O2 may react with NO to form another relatively
reactive molecule, peroxynitrite (ONOO−
).
 Exogenous sources of RONS are air and water pollution, tobacco, alcohol, heavy or
transition metals, drugs (eg, cyclosporine, tacrolimus, gentamycin, and bleomycin),

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industrial solvents, cooking (eg, smoked meat, waste oil, and fat), and radiation, which
inside the body are metabolized into free radicals.
 Antioxidant defense protects biological systems from free radical toxicity and includes
both endogenous and exogenous molecules.
 Endogenous antioxidants include enzymatic and non-enzymatic pathways.
 The primary antioxidant enzymes are SOD, catalase (CAT), and glutathione peroxidase
(GSH-Px). As mentioned above, O2 is converted by SOD to H2O2, which is
decomposed to water and oxygen by CAT, preventing hydroxyl radicals production.
Additionally, GSH-Px converts peroxides and hydroxyl radicals into nontoxic forms by
the oxidation of reduced glutathione (GSH) into glutathione disulfide and then reduced
to GSH by glutathione reductase. Other antioxidant enzymes are glutathione-S-
transferase and glucose-6-phosphate dehydrogenase.
 The non-enzymatic antioxidants are molecules that interact with RONS and terminate
the free radical chain reactions: bilirubin, α-tocopherol (vitamin E), and β-carotene are
present in blood while albumin and uric acid account for 85% of antioxidant capacity
in plasma
 Exogenous antioxidants include ascorbic acid (vitamin C), which scavenges hydroxyl
and superoxide radical anion, α-tocopherol (vitamin E), which is involved against lipid
peroxidation of cell membranes, and phenolic antioxidants, which include stilbene
derivatives (resveratrol, phenolic acids, and flavonoids), oil lecitinas, selenium, zinc,
and drugs such as acetylcysteine. Oxidative stress occurs when there is an imbalance
between the formation and the removal of RONS because of an overproduction and/or
an impaired ability to neutralize them or to repair the resulting damage
 Oxidative stress and theory of aging
 Aging is the progressive loss of tissue and organ function over time. The free radical
theory of aging, later termed as oxidative stress theory of aging, is based on the
structural damage-based hypothesis that age-associated functional losses are due to the
accumulation of oxidative damage to macromolecules (lipids, DNA, and proteins) by
RONS.
 The exact mechanism of oxidative stress-induced aging is still not clear, but probably
increased RONS levels lead to cellular senescence, a physiological mechanism that
stops cellular proliferation in response to damages that occur during replication.
Senescent cells acquire an irreversible senescence-associated secretory phenotype
(SASP) involving secretion of soluble factors (interleukins, chemokines, and growth
factors), degradative enzymes like matrix metalloproteases (MMPs), and insoluble
proteins/extracellular matrix (ECM) components. RONS induce cellular senescence
acting on various components of SASP:
 regulation of mammalian target of rapamycin complexes’ functions;1
 production of IL-1α leading to a proinflammatory state, which increases nuclear factor
kappa-B (NFκB) activity and epithelial–mesenchymal transition and tumor metastatic
progression;1
 induction of MMPs expression, which is associated with age-related and chronic
diseases such as cancer, Alzheimer’s, atherosclerosis, osteoarthritis, and lung
emphysema;1
 inhibition of FOXO (Forkhead box) proteins activity, which is involved in
insulin/insulin-like growth factor-1-mediated protection from oxidative stress;

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 reduction of sarco/endoplasmic reticulum Ca2+-ATPase activity leading to cardiac
senescence;
 inhibition of sirtuins activity leading to an increased production of RONS by SOD
inhibition, a proinflammatory state by preventing their inhibition of tumor necrosis
factor alpha (TNFα) and NFκB, and tumorigenic effect by preventing their inhibitory
effect on c-Jun and c-Myc;20
 regulation of p16INK4a/pRB and p53/p21 pathways leading to senescence.
 Oxidative stress and age-related diseases
 xidative stress, cellular senescence, and consequently, SASP factors are involved in
several acute and chronic pathological processes, such as CVDs, acute and chronic
kidney disease (CKD), neurodegenerative diseases (NDs), macular degeneration (MD),
biliary diseases, and cancer. Cardiovascular (CV) risk factors (ie, obesity, diabetes,
hypertension, and atherosclerosis) are associated with the inflammatory pathway
mediated by IL-1α, IL-6, IL-8, and increased cellular senescence.1 Moreover, vascular
calcification is linked to an SASP-driven osteoblastic transdifferentiation of senescent
smooth muscle cells. In many neurodegenerative conditions, including Alzheimer’s
disease (AD), brain tissue biopsies show increased levels of p16, MMP, and IL-6.21
Chronic obstructive pulmonary disease, biliary cirrhosis, cholangitis, and osteoarthritis
share several damaging SASP profiles including IL-6, IL-8, and MMP.1 The induction
of epithelial to mesenchymal transition mediated by RONS promotes cancer
metastasis.22 In synthesis, given the close relationship between oxidative stress,
inflammation, and aging, the oxidation-inflammatory theory of aging or oxi-inflamm-
aging has been proposed: aging is a loss of homeostasis due to a chronic oxidative stress
that affects especially the regulatory systems, such as nervous, endocrine, and immune
systems. The consequent activation of the immune system induces an inflammatory
state that creates a vicious circle in which chronic oxidative stress and inflammation
feed each other, and consequently, increases the age-related morbidity and mortality
Abbreviations: 4-HNE, trans-4-
hydroxy-2-nonenal; 8oxodG, 7,8-
dihydro-8-oxo-2′-
deoxyguanosine; 8oxoGuo, 7,8-
dihydro-8-oxoguanosine; ADMA,
asymmetric dimethyl L-arginine;
AGEs, advanced glycation end
products; CKD, chronic kidney
disease; CVDs, cardiovascular
diseases; F2-IsoPs, F2-
isoprostanes; MDA,
malondialdehyde; MPO,
myeloperoxidase; NDs,
neurodegenerative diseases; NT,
nitrotyrosine; oxLDL, oxidized
low-density lipoprotein; PC, protein carbonyl; Prx, peroxiredoxins; P-VASP, phosphorylated
vasodilator-stimulated phosphoprotein; Trx, thioredoxin.