Fenton, more than hundred years ago, described an oxidizing potential of hydrogen peroxide mixed with ferrous salts (Fenton, 1894; 1899).
Oxidative stress and Alzheimer's disease
OXIDATIVE STRESS AND
The formation of free radicals or oxidants is a well-established
physiological event in aerobic cells, which convene enzymic
and nonenzymic resources, known as antioxidant defenses,
to remove these oxidizing species.
An imbalance between oxidants and antioxidants known as
oxidative stress, and the consequent damage to cell
molecules constitutes the basic tenet of several
pathophysiological states, such as neurodegeneration, cancer,
mutagenesis, cardiovascular diseases, and aging.
OXYGEN FREE RADICALS
Oxygen is a relatively unreactive compound that can be
metabolized in vivo to form highly reactive oxidants known as
oxygen free radicals.
A free radical is defined as any species that contains one or more
unpaired electron occupying an atomic or molecular orbital by
Mechanisms of Formation of Oxygen Free
Oxygen radicals or reactive oxygen species may be generated
Superoxide anion radical
Triplet carbonyl compounds
Lipid alkoxyl and peroxyl
Formation of Oxidants by Electron Transfer
Univalent reduction of oxygen to water with formation of
different intermediates: superoxide anion radical (O2.–),
hydrogen peroxide (H2O2), and hydroxyl radical (HO.):
HOW REACTIVE ARE OXYGEN
Reactivity of Superoxide Anion
The reactivity of superoxide radical is dependent on the cellular
environment. Two reactions are important in a cellular setting,
which change the chemical reactivity of superoxide anion (O2.–):
Reactivity of superoxide anion with itself
O2. – + O2. – + 2H + → H2O2 + O2
Protonation of superoxide anion
O2.– + H+ → HO2.
Reactivity of Hydrogen Peroxide
Hydrogen peroxide (H2O2) is not a free radical, but it may be considered
as an oxidant. Per se, hydrogen peroxide (H2O2) is little reactive. Its
reactivity in biological systems depends on two properties:
It can diffuse long distances crossing membranes
It reacts with transition metals by a homolytic cleavage yielding the
highly reactive hydroxyl radical (HO.).
Reactivity of Hydroxyl radical
The chemical reactivity of hydroxyl radical (HO.) may
be assumed to encompass two main reactions:
HOW ARE OXYGEN RADICALS GENERATED
IN THE CELL?
Sources of Superoxide Radical
- xanthine oxidase
- NADH oxidase
Present in leukocytes: bactericidal activity
- NADPH-cytochrome P450 reductase
• Cellular sources
- leukocytes and macrophages
- mitochondrial electron transfer
- microsomal monooxygenase
• Environmental factors
- X rays
- toxic chemicals
- aromatic hydroxylamines
- aromatic nitro compounds
- insecticides, such as paraquat
- chemotherapeutic agents, such as quinone
Mitochondria are major cellular sources of reactive oxygen species.
Mitochondria consume oxygen associated with the process of oxidative
Under normal conditions, approximately 95-97% of the oxygen is reduced to
water; a small fraction of the oxygen consumed (3-5%) is reduced univalently
to superoxide anion (O2.–).
Coenzyme Q or ubiquinone is a mobile electron carrier in the respiratory
chain and it collects electrons from complex I and complex II. The coenzyme Q
pool faces both the intermembrane space and the mitochondrial matrix.
Coenzyme Q or ubiquinone is reduced by Complex I and Complex II and
donates electrons to complex III (the bc1 segment). Because of these redox
transitions, ubiquinone exists as a quinone (fully oxidized), semiquinone, and
hydroquinone (fully reduced):
Electron leakage, accounting for about 3-5% of the total oxygen consumed by
mitochondria, is associated with the generation of oxygen radicals:
Ubisemiquinone donates one electron to molecular oxygen yielding superoxide
anion and ubiquinone; this is known as autoxidation of ubisemiquinone.
Sources of Hydrogen Peroxide
Hydrogen peroxide (H2O2) is generated within
the cell by two distinct processes:
• Nonradical or enzymic generation
The following enzymes do generate hydrogen peroxide (H2O2) upon
reduction of their co-substrate, molecular oxygen:
glycolate oxidase, D-amino acid oxidase, urate oxidase, acetyl-CoA
oxidase ,NADH oxidase and monoamine oxidase
The latter enzyme, monoamine oxidase (MAO) occurs in two forms
A and B and it catalyzes the oxidative deamination of biogenic
amines. It is present in the outer mitochondrial membrane.
• Radical generation or from superoxide anion disproportionation
This is achieved upon dismutation or disproportionation of
superoxide anion (O2.–), according to the reaction mentioned before:
O2. – + O2. – + H+ → H2O2 + O2
Sources of Hydroxyl Radical
Most of the hydroxyl radical (HO.) generated in vivo, except
for that during excessive exposure to ionizing radiation,
originates from the breakdown of hydrogen peroxide (H2O2)
via a Fenton reaction.
The Fenton reaction entails a metal-dependent reduction of
hydrogen peroxide (H2O2) to hydroxyl radical (HO.).
Transition metals, such as copper (Cu), iron (Fe), and cobalt
(Co), in their reduced form catalyze this reaction:
Fe++ + H2O2 → Fe+++ + HO– + HO.
HOW DO OXIDANTS MEDIATE CELLULAR
HOW DO CELLS PROTECT
THEMSELVES AGAINST OXIDANTS?
The cell convenes specific enzymic defenses against oxygen radical attack,
which can be considered preventive antioxidants. On the other hand, there exist
small antioxidant molecules, which can react with a variety of free radicals and
that may be considered as chain-breaking antioxidants.
Specific enzymic defenses or preventive antioxidants
Mammalian cells contain specific enzymes, which remove either superoxide
anion or hydrogen peroxide, the two required precursors of hydroxyl radical
Removal of Superoxide Anion: Superoxide Dismutases
Superoxide anion radical is formed by different nonenzymic and enzymic
reactions within the cell. Superoxide dismutases (abbreviated SOD) catalyze the
rapid dismutation of superoxide radical to hydrogen peroxide and oxygen. The
rate of this reaction is 10,000-fold higher than that of the spontaneous
+ O2.– + 2H+ H2O2 +
O2.– + O2.– + 2H+ H2O2 + O2
Removal of Hydrogen
Catalase: This enzyme is located in the peroxisomes and catalyses the following
H2O2 + H2O2 → 2H2O + O2
Glutathione Peroxidase: The enzyme occurs in cytosol and the mitochondrial
matrix and it requires glutathione, a tripeptide present in high concentrations in
most mammalian cells. During this reaction hydrogen peroxide (H2O2) is
reduced to water and glutathione (GSH) is oxidized to glutathione disulfide
H2O2 + 2GSH → H2O + GSSG
Increasing evidence suggests that the generation of
these oxygen free radicals plays an important role
in the pathophysiology of at least three disease
states: ischemia reperfusion injury, phagocytedependent
neurodegenerative disorders as well as aging.
Alzheimer’s disease (AD) is a neurodegenerative disorder associated with a
decline in cognitive impairments, progressive neurodegeneration and
formation of amyloid-β (Aβ) containing plaques and neurofibrillary tangles.
(Nain et al., 2011)
Alzheimer's disease (AD) is a slowly progressive disease of the brain that is
characterized by impairment of memory and eventually by disturbances in
reasoning, planning, language, and perception.
of amyloid precursor protein or presenilin genes or
apolipoprotein E gene polymorphism appear to affect amyloid formation,
which in turn causes neuronal death via a number of possible mechanisms,
including Ca2+ homeostasis disruption, oxidative stress, excitotoxicity,
energy depletion, neuro- inflammation and apoptosis.
Oxidative stress in AD??
Oxidative stress occurs when there is an imbalance between the
production and quenching of free radicals from oxygen species. These
reactive oxygen species (ROS) play a role in many chronic diseases
including mitochondrial diseases, neurodegenerative diseases, renal
disease, arteriosclerosis, diabetes , cancer and SLE .
The process of aging is also associated with increased oxidative stress.
Through pathological redox reactions ROS can denature biomolecules
such as proteins, lipids and nucleic acids. This can initiate tissue damage
via apoptosis and necrosis.
Oxidative stress plays a central role in the pathogenesis of AD leading to
neuronal dysfunction and cell death.
Peripheral markers of oxidative stress are elevated in AD indicating that
the damage is not brain-limited.
One study suggested that the level of oxidative markers is directly related
to the severity of cognitive impairment.
The increased level of oxidative stress in the AD brain is reflected
increased protein and DNA oxidation,
decreased level of cytochrome c oxidase and advanced
glycosylation end products.
enhanced lipid peroxidation,
Lipid peroxidation can weaken cell membranes causes ion
imbalance and impair metabolism.
Oxidative stress can influence DNA methylation which regulates
Internalized beta-amyloid may play a role in this process.
Mitochondrial dysfunction, which is associated with an
accumulation of ROS, appears to play a role in the early events of
A recent study, using mouse models, showed that mitochondria targeted
antioxidant catalase helps prevent abnormal beta-amyloid processing
decreasing plaque burden. There is also evidence that beta-amyloid
deposits lead to more mitochondrial damage.
Beta-amyloid peptide has been shown to inhibit cytochrome oxidase
leading to disruption of the electron transport chain and production of
ROS. Thus a viscous cycle may be initiated that culminates in
Under stressful conditions and in aging, the electron transport system
can increase ROS formation considerably. Thus, the mitochondria are
both a source and a target of toxic ROS. Mitochondrial dysfunction and
oxidative metabolism may play an important role in the pathogenesis of
AD and other neurodegenerative diseases.
Oxidative Stress Response
e.g. Neurotrophic factors,
Neurogenesis, DNA repair etc
Failure to adapt
Oxidation of proteins,
lipids and DNA
Oxidative radicals – mechanisms of oxygen radicals
generation in Alzheimer’s disease
A dismutation reaction of superoxide radical leading to the formation of
hydrogen peroxide and oxygen can occur spontaneously or is catalysed by
the enzyme superoxide dismutase (SOD).
There are three distinct types of SOD classified on the basis of the metal
the copper/zinc (Cu/Zn–SOD, cytoplasmic),
the manganese (Mn–SOD, mitochondrial) and
the iron (Fe–SOD) isozymes (Bannister et al, 1987).
Superoxide can act as either an oxidant or a reductant.
It can oxidize sulphur, ascorbic acid or NADPH and it can reduce
cytochrome c and metal ions. Superoxide forms the perhydroxyl radical
(.OOH), which is a powerful oxidant (Gebicki and Bielski, 1981), but its
biological relevance is probably minor because of its low concentration at
Numerous enzymes (peroxidases) use hydrogen peroxide as a
oxidation reactions involving the synthesis of complex
The well-known reactivity of hydrogen peroxide is not due to its
reactivity per se, but requires the presence of a metal reductant to form
the highly reactive hydroxyl radical, which is the strongest oxidizing
agent known and reacts with organic molecules at diffusion-limited
The reaction of Fe2++ with H2O2 produces the highly reactive
hydroxyl radical (.OH) via Fenton reaction.
In biological systems the availability of ferrous ions limits the rate of
reaction, but the recycling of iron from the ferric to the ferrous form
by a reducing agent can maintain an ongoing Fenton reaction leading
to the generation of hydroxyl radicals. Haber and Weiss (1934)
identified reaction resulting into .OH formation through an interaction
between O2.– and H2O2 in the presence of Fe2+ or Fe3+.
Increased lipid peroxidation occurs in the brain in AD and is most
prominent where degenerative changes are most pronounced.
Brain membrane phospholipids are composed of polyunsaturated fatty
acids, which are especially vulnerable to free radical attack because
their double bonds allow easy removal of hydrogen ions. Decreases in
docosahexaenoic acids, accompany lipid peroxidation in AD.
Oxidation of polyunsaturated fatty acids produces aldehydes, one of
the most important of which is 4-hydroxynonenal (HNE), a highly
reactive cytotoxic substance capable of inhibiting glycolysis, nucleic
acid and protein synthesis, and degrading proteins.
Glutathione transferases, a group of enzymes that inactivate the toxic
products of oxygen metabolism including 4-hydroxyalkenals such as
HNE, are markedly diminished in multiple brain regions and in the
CSF in subjects with AD, suggesting a loss of protection against HNE.
The oxidation of proteins by free radicals may also play a meaningful
role in AD.
Hydrazide-reactive protein carbonyl is a general assay of oxidative
damage to protein.
Several studies demonstrate an increase in protein carbonyls in
multiple brain regions in subjects with AD and in their NFTs. The
oxidation of brain proteins may be at the expense of enzymes critical
to neuron and glial function.
Two enzymes that are especially sensitive to oxidative modification
are glutamine synthetase and creatine kinase, both of which are
markedly diminished in the brains of subjects with AD.
Oxidative alterations in glutamine synthetase could cause alteration of
glutamate concentrations and enhance excitotoxicity, whereas
oxidative impairment of creatine kinase could cause diminished
energy metabolism in AD.
Four-hydroxynonenal causes degeneration and death of cultured hippocampal
neurons by impairing ion motive adenosine triphosphatase activity and
disrupting calcium homeostasis.6 Exposure of cultured hippocampal neurons
to amyloid (A) peptide causes a significant increase in levels of free and
protein-bound HNE and increases ROS. Four-hydroxynonenal impairs
glucose and glutamate transport and is capable of inducing apoptosis in
cultured neurons. Administration of HNE into the basal forebrain of rats
causes damage to cholinergic neurons, diminished choline acetyltransferase,
and impaired visuospatial memory.
Biological systems have evolved with endogenous defense mechanisms to
help protect against free radical induced cell damage.
Glutathione peroxidase, catalase and superoxide dismutases are
antioxidant enzymes, which metabolize toxic oxidative intermediates.
They require micronutrient as cofactors such as selenium, iron, copper,
zinc, and manganese for optimum catalytic activity and effective
antioxidant defence mechanisms.
SOD, catalase, and glutathione peroxidase are three primary enzymes,
involved in direct elimination of active oxygen species (hydroxyl radical,
superoxide radical, hydrogen peroxide) whereas glutathione reductase,
glucose-6-phosphate dehydrogenase, and cytosolic GST are secondary
enzymes, which help in the detoxification of ROS by decreasing peroxide
levels or maintaining a steady supply of metabolic intermediates like
glutathione and NADPH necessary for optimum functioning of the
primary antioxidant enzymes.
The most widely studied dietary antioxidants are vitamin C,
vitamin E, and beta-carotene.
Vitamin C is considered the most important water-soluble
antioxidant in extracellular fluids, as it is capable of neutralising
ROS in the aqueous phase before lipid peroxidation is initiated.
Vitamin E is a major lipid-soluble antioxidant, and is the most
effective chain-breaking antioxidant within the cell membrane
where it protects membrane fatty acids from lipid peroxidation.
Beta-carotene and other carotenoids also provide antioxidant
protection to lipid rich tissues.
Fruits and vegetables are major sources of vitamin C and
carotenoids, while whole grains, i.e., cereals and high quality
vegetable oils are major sources of vitamin E.
Drugs for AD with
Common Adverse effects
• Nausea, diarrhea, insomnia, vomiting, muscle
cramps, fatigue, and anorexia
• Nausea, vomiting, loss of appetite, dyspepsia,
asthenia, and weight loss
(Morrison et al.,2005)
PLANTS USED IN
MANAGEMENT OF AD
(Raghavendra et al., 2013, Kapoor et al., 2011, Sandhya et al., 2010)
Filipcik P, Cente M, Ferencik M, Hulin, Novak: The role of oxidative
stress in the pathogenesis of Alzheimer’s disease. 2006; 107 (9–10):
Ravindra Pratap Singh, Shashwat Sharad, Suman Kapur: Free Radicals
and Oxidative Stress in Neurodegenerative Diseases: Relevance of
Dietary Antioxidants. JIACM 2004; 5(3): 218-25
FREE RADICALS, OXIDATIVE STRESS, AND DISEASES.
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