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 dialdehydean indicator of fatty acid break down by free radicals

 Propagation-one free radical generates another free radical in the neighbouring moleculeschain reactiondestruction 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, Alzheimers 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 oxidation 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.

-car + ROO -car + ROOH -car + ROO inactive products

Structure of two common carotenoids found in plants, -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.