Transcript Oxidative stress - The language of Biochemistry

Oxidative stress
Anju .M.P
BCH.10.05.12
• One of the paradoxes of life on this planet is that
the molecule that sustains aerobic life, oxygen, is
not only fundamentally essential for energy
metabolism and respiration, but it has been
implicated in many diseases and degenerative
conditions
• A common element in such diverse human
disorders as ageing, arthritis, cancer, Lou Gehrig's
disease and many others is the involvement of
partially reduced forms of oxygen.
• In the sequential univalent process by which O2
undergoes reduction, several reactive intermediates
are formed, such as superoxide (O2 – ), hydrogen
peroxide (H2O2), and the extremely reactive
hydroxyl radical (.OH): collectively termed as the
reactive oxygen species (ROS).
O2 +e- → O2¯+e- → H2O2+e-→ .OH+e-→ H2O
• O2 can behave like a radical (a diradical) owing to
the presence of two unpaired electrons of parallel
spin, it does not exhibit extreme reactivity due to
quantum-mechanical restrictions.
• Its electronic structure result in formation of water
by reduction with four electrons, i.e:
O2 +4H +4e →2H O.
• Atm oxygen in its ground-state is a biradical, or it
has two unpaired electrons.
• This makes oxygen paramagnetic; it also makes
oxygen very unlikely to participate in reactions with
organic molecules unless it is "activated".
• The requirement for activation occurs because the
two unpaired electrons in oxygen have parallel
spins.
• According to Pauli's exclusion principle, this
precludes reactions with a divalent reductant,
unless this reductant also has two unpaired
electrons with parallel spin opposite to that of the
oxygen, which is a very rare occurrence.
• Hence, oxygen is usually non-reactive to organic
molecules which have paired electrons with
opposite spins.
• This spin restriction means that the most common
mechanisms of oxygen reduction in biochemical
reactions are those involving transfer of only a
single electron (monovalent reduction).
Activation of oxygen may occur by two different
mechanisms:
1. absorption of sufficient energy to reverse the spin on one
of the unpaired electrons, or monovalent reduction
• If triplet oxygen absorbs sufficient energy to reverse
the spin of one of its unpaired electrons, it will form
the singlet state, in which the two electrons have
opposite spins
• This activation overcomes the spin restriction and
singlet oxygen can consequently participate in
reactions involving the simultaneous transfer of two
electrons (divalent reduction).
• Since paired electrons are common in organic
molecules, singlet oxygen is much more reactive
towards organic molecules than its triplet
counterpart.
2. by the stepwise monovalent reduction of oxygen to
form superoxide ,hydrogen, hydroxyl radical and
finally water
• The first step in the reduction of oxygen forming
superoxide is endothermic but subsequent
reductions are exothermic
REACTIONS THAT LEAD TO THE FORMATION OF SOME ROS
In the presence of trace amounts of iron, the reaction
of superoxide and hydrogen peroxide will form the
destructive hydroxyl radical and initiate the oxidation
of organic substrates
What is oxidative stress?
• our body constantly reacts with oxygen during
breathing and our cells produce energy. As a
consequence of this activity, highly reactive
molecules are produced known as free radicals.
Free radicals interact with other molecules within
cells. This can cause oxidative damage to proteins,
membranes and genes.
Oxidative damage has been implicated in the cause of
many diseases, such as cancer and Alzheimer's and
has an impact on the body's aging process.
External factors, such as pollution, sunlight and
smoking, also trigger the production of free radicals.
BIOLOGICAL REACTIONS OF OXYGEN RADICALS
• The reactions of activated oxygen in biological
systems there are even more complications due to
the surface properties of membranes, electrical
charges, binding properties of macromolecules, and
compartmentalisation of enzymes, substrates and
catalysts
1. OXIDATIVE DAMAGE TO LIPIDS
• The lipid bilayer membrane is composed of a
mixture of phospholipids and glycolipids that have
fatty acid chains
• Initiation-the production of R‘/PUFA radical/ROO‘
by the interaction of PUFA with free radicals
generated by other means
R‘ andROO‘ are degraded to malon dialdehyde—an
indicator of fatty acid break down by free radicals
• Propagation-one free radical generates another free
radical in the neighbouring molecules→chain
reaction→destruction of fine architechture &
integrity of the membranes
• Termination- reactions in membrane lipids are
terminated when the carbon or peroxy radicals
cross-link to form conjugated products that are not
radicals
• Lipid peroxy radicals react with other lipids,
proteins, and nucleic acids; propagating thereby the
transfer of electrons and bringing about the
oxidation of substrates.
• Cell membranes, which are structurally made up of
large amounts of PUFA, are highly susceptible to
oxidative attack and, consequently, changes in
membrane fluidity, permeability, and cellular
metabolic functions result.
2.OXIDATIVE DAMAGE TO PROTEINS
• Oxidative attack on proteins results in site-specific amino
acid modifications, fragmentation of the peptide chain,
aggregation of cross-linked reaction products, altered
electrical charge and increased susceptibility to proteolysis
• Sulphur containing amino acids, and thiol groups
specifically, are very susceptible sites
• the oxidation of iron-sulphur centres by superoxide
destroys enzymatic function
• Thus it destroys the structure,functions of essential
proterins and enzymes and whole cell metabolism is
blocked
• In the process of cataractogenesis, oxidative
modification plays a significant role in cross-linking
of crystalline lens protein,leading to high-molecularweight aggregates, loss of solubility, and lens
opacity. Lipofuscin – an aggregate of peroxidized
lipid andproteins – accumulates in lysosomesof
aged cells, Alzheimer’s disease brain cells, and iron
overloaded hepatocytes.
3.OXIDATIVE DAMAGE TO DNA
• Activated oxygen and agents that generate oxygen
free radicals, such as ionizing radiation, induce
numerous lesions in DNA that cause deletions,
mutations and other lethal genetic effects
• Degradation of the base will produce numerous
products, including 8-hydroxyguanine,
hydroxymethyl urea, urea, thymine glycol, thymine
and adenine ring-opened and -saturated products.
• Characterizations of this damage to DNA has
indicated that both the sugar and the base moieties
are susceptible to oxidation, causing base
degradation, single strand breakage, and crosslinking to protein .
• Mutation arising from selective modification of G : C
sites specially indicates oxidative attack on DNA by
ROS.
• The action of 8-oxodeoxy- guanosine as a
promutagen, as well as in altering the binding of
methylase to the oligomer so as to inhibit
methylation of adjacent cytosine has been reported
in cases of cancer development
• ROS have also been shown to activate mutations
inhuman C-Ha-ras-1 protooncogene, and to induce
mutation in the p53 tumour-suppressor gene4
• ROS may interfere with normal cell signalling, resulting
thereby in alteration of the gene expression, and
development of cancer by redox regulation of
transcriptional factors/activator and/or by oxidatively
modulating the protein kinase cascades.
• ROS also induce various early response or stressresponse genes like c-fos, c-jun, jun-B, jun-D, c-myc,erg1, and heme oxygenase-1.
• The oxidative damage of mitochondrial DNA also
involves base modification and strand breaks, which
leads to formation of abnormal componentsof the ETC
• This results in the generation ofmore ROS through
increased leakage of electrons, and cell damage.
• Oxidative damage to mitochondrial DNA may promote
cancer and aging
• Mitochondria are a primary site of production of
free radicals. While more than 98% of the molecular
oxygen taken up by cells is fully utilized by
cytochrome c oxidase to form water, this enzyme
can release partly reduced species. Other enzymes
of the respiratory chain, and in particular complexes
I and III, also produce partly reduced oxygen species
including superoxide.
• Various oxidative processes, including oxidation,
hydroxylations, dealkylations, deaminations,
dehalogenation and desaturation, occur on the SER.
• Mixed function oxygenases that contain a heme
moiety add an oxygen atom into an organic
substrate using NAD(P)H as the electron donor
• The generalised reaction catalysed by cytochrome
P450
• The best characterised cytochrome P450 in plants is
cinnamate-4-hydroxylase which functions in
flavonoid and lignin biosynthesis
Cytochrome P450 reacts first with its organic substrate, RH. The
complex is reduced by a flavoprotein to form a radical intermediate
that can readily react with triplet oxygen because each has one
unpaired electron. This oxygenated complex may be reduced by
cytochrome b or occasionally the comples may decompose releasing
superoxide
• The plant NAD(P)H oxidase have an analogous function
to the animal enzyme.
• Leucocytes contain an NADH oxidase on the outer
membrane surface which is activated in response to a
foreign agent, generating superoxide that initiates
oxidative reactions that destroy the potential pathogen
• In plants, fungal elicitors cause a similar formation of
superoxide that has been linked to the hypersensitive
response to some pathogenic fungi .
• Wounding, heat shock and xenobiotics transiently
activate this superoxide generating reaction, and
consequently, it has been proposed that these
superoxide generating reactions may serve as a signal
in plant cells to elicit responses to biological, physical or
chemical stress
Reactive oxygen species and aging
• Eversince Harman104 first proposed the free-radical
theory of aging, as early as 1956, the molecular
basis of aging and the role of ROS
• aging and age-related diseases result from ROSmediated oxidative damage of lipid, protein, and
nuclear and mitochondrial DNA molecules.
• The concentration of oxidatively damaged proteins,
lipids, and DNA has been reported to increase with
age
• The hydroxyl and peroxy radicals cause extensive
damage of proteins resulting in aging and age
related degenerative diseases
• mutation in mitochondrial DNA also leads to the
formation of defective respiratory enzymes which
not only result in decreased ATP synthesis but also
generate more ROS to cause further oxidative damage
• This vicious cycle is mainly responsible for aging and
age-related disorders.
• Melatonin, having antioxidant property, declines
significantly with the increase in age.
• This decline in melatonin coincides with the
increased oxidative damage and pathogenesis.
• ROS have also been implicated in the regulation of
at least two well-defined transcription factors, AP-1
and NFkB
• These transcription factors bind at the promoter
regions of a large variety of genes and play a very
significant role in the expression of various proteins
such as TNFa, interleukin-1 and -2, collagenase,
matrix metalloproteinase, etc. which are involved in
inflammatory responses and tissue injury
• Blocking the expression of these genes by suitable
antioxidants should be one of the approaches for
controlling the ROS-mediated pathogenesis
Enhanced oxidative stress is now well
documented to occur in a number of
degenerative diseases including
Parkinson's disease, Alzheimer's and
diabetes
DEFENCE MECHANISMS
1.SUPEROXIDE DISMUTASE
• was first isolated by Mann and Keilis and thought to
be a copper storage protein. SOD is now known to
catalyse the dismutation of superoxide to hydrogen
peroxide and oxygen
2.CATALASE
• Catalase is a heme-containing enzyme that catalyses
the dismutation of hydrogen peroxide into water and
oxygen.
• found in all aerobic eukaryotes and is important in the
removal of hydrogen peroxide generated in
peroxisomes (microbodies) by oxidases involved in ßoxidation of fatty acids, the glyoxylate cycle and purine
catabolism
• Careful examination of the structure of beef liver
catalase has shown four NADPH binding sites per
catalase tetramer , but these sites were not in close
association with the hydrogen peroxide reaction centre.
Instead, NADPH functions in animal catalase to protect
against inactivation by hydrogen peroxide
3.GLUTATHIONE
• Glutathione (GSH) is a tripeptide (Glu-Cys-Gly)
whose antioxidant function is facilitated by the
sulphydryl group of cysteine
• is found in most tissues, cells and subcellular
compartments of higher plants
• can react chemically with singlet oxygen,
superoxide and hydroxyl radicals and therefore
function directly as a free radical scavenger
• stabilise membrane structure by removing acyl
peroxides formed by lipid peroxidation reactions
• The reduction of GSSG to GSH is catalysed by the
enzyme glutathione reductase (GR) in presence
NADPH(HMP)
• GR is associated mainly with the chloroplast but
significant activity is also found in the cytosol and a
lesser amount in the mitochondria
GLUTATIONE PEROXIDASE
• Glutathione peroxidase catalyses the reaction of
hydroperoxides with reduced glutathione (GSH) to
form glutathione disulphide (GSSG) and the
reduction product of the hydroperoxide
• This enzyme is specific for its hydrogen donor, GSH,
andnonspecific for the hydroperoxides ranging from
H2O2 to organic hydroperoxides.
• It is a seleno-enzyme; two-third of which (inliver) is
present in the cytosol and one-third in the
mitochondria2.
Antioxidants and oxidative stress
• To counteract oxidative stress, the body produces an
armoury of antioxidants to defend itself. It's the job
of antioxidants to neutralise or 'mop up' free
radicals that can harm our cells.
• our body's ability to produce antioxidants (its
metabolic process) is controlled by our genetic
makeup and influenced by our exposure to
environmental factors, such as diet and smoking.
• wecan help your body to defend itself by increasing
our dietary intake of antioxidants.
Ascorbic acid
• L-ascorbic acid (vitamin C) is an important vitamin
in the human diet and is abundant in plant tissues.
• Ascorbate functions as a reductant for many free
radicals, thereby minimising the damage caused by
oxidative stress
Structure of ascorbic acid and its metabolites
• can directly scavenge oxygen free radicals with and
without enzyme catalysts and can indirectly scavenge them
by recycling tocopherol to the reduced form.
• By reacting with activated oxygen more readily than any
other aqueous component, ascorbate protects critical
macromolecules from oxidative damage
Tocopherol
• The tocopherol (vitamin E), have been studied
extensively in mammalian research as membrane
stabilisers and multifaceted antioxidants, that
scavenge oxygen free radicals, lipid peroxy radicals,
and singlet oxygen
• The active oxygen of the a-tocopherol is located
near the surface of the bilayer and it readily
diffuses laterally in the plane of the bilayer,
tocopherol can react with peroxyl radicals formed in
the bilayer as they diffuse to the aqueous phase.
• The antioxidant properties of tocopherol are the
result of its ability to quench both singlet oxygen
and peroxides
• it may function to break carbon radical chain
reactions by trapping peroxyl radicals:
ROO + tocopherol ROOH + tocopherol
Carotenoids
• Carotenoids are C40 isoprenoids and tetraterpenes
that are located in the plastids of both
photosynthetic and non-photosynthetic plant
tissues.
• In chloroplasts, the carotenoids function act as
accessory pigments in light harvesting,
• more important role is their ability to detoxify
various forms of activated oxygen and triplet
chlorophyll that are produced as a result of
excitation of the photosynthetic complexes by light.
β-car + ROO β-car + ROOH
β-car + ROO inactive products
Structure of two common carotenoids found in plants, β-carotene and
zeaxanthinin.
Foods and antioxidants
Tomatoes
• Tomatoes contain a pigment called lycopene that
is responsible for their red colour but is also a
powerful antioxidant.
• Tomatoes in all their forms are a major source of
lycopene, including tomato products like canned
tomatoes, tomato soup, tomato juice
• Lycopene is also highly concentrated in
watermelon
Citrus fruits
• Oranges, grapefruit, lemons and limes possess
many natural substances that appear to be
important in disease protection, such as
carotenoids, flavonoids, terpenes, limonoids and
coumarins.
• It's always better to eat the fruit whole in its
natural form, because some of the potency is lost
when the juice is extracted
Tea
• Black tea, green tea and oolong teas have antioxidant
properties. All three varieties come from the plant
Camellia sinenis.
• Common brands of black tea do contain antioxidants,
but by far the most potent source is green tea
(jasmine tea) which contains the antioxidant catechin.
• Black tea has only 10 per cent as many antioxidants as
green tea.
• Oolong tea has 40 per cent as many antioxidants as
green tea.
• This because some of the catechins are destroyed
when green tea is processed (baked and fermented) to
make black tea.
Carrots
• Beta-carotene is an orange pigment that was
isolated from carrots 150 years ago.
• It is found concentrated in deep orange and green
vegetables (the green chlorophyll covers up the
orange pigment).
• Beta-carotene is an antioxidant that has been
much discussed in connection with lung cancer
rates.