Free radicals and antioxidants in human health: current status and future prospects

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Content: Review Article free radicals and Antioxidants in Human Health: Current Status and Future Prospects TPA Devasagayam*, JC Tilak*, KK Boloor*+, Ketaki S Sane**, Saroj S Ghaskadbi**, RD Lele***
Abstract Free radicals and related species have attracted a great deal of attention in recent years. They are mainly derived from oxygen (reactive oxygen species/ROS) and nitrogen (reactive nitrogen species/RNS), and are generated in our body by various endogenous systems, exposure to different physicochemical conditions or pathophysiological states. Free radicals can adversely alter lipids, proteins and DNA and have been implicated in aging and a number of human diseases. Lipids are highly prone to free radical damage resulting in lipid peroxidation that can lead to adverse alterations. Free radical damage to protein can result in loss of enzyme activity. Damage caused to DNA, can result in mutagenesis and carcinogenesis. Redox signaling is a major area of free radical research that is attracting attention. Nature has endowed us with protective antioxidant mechanisms- superoxide dismutase (SOD), catalase, glutathione, glutathione peroxidases and reductase, vitamin E (tocopherols and tocotrienols), vitamin C etc., apart from many dietary components. There are epidemiological evidences correlating higher intake of components/ foods with antioxidant abilities to lower incidence of various human morbidities or mortalities. current research reveals the different potential applications of antioxidant/free radical manipulations in prevention or control of disease. Natural products from dietary components such as Indian spices and medicinal plants are known to possess antioxidant activity. Newer and future approaches include gene therapy to produce more antioxidants in the body, genetically engineered plant products with higher level of antioxidants, synthetic antioxidant enzymes (SOD mimics), novel biomolecules and the use of functional foods enriched with antioxidants. ©
INTRODUCTION AND BASICS OF FREE RADICAL RESEARCH In recent years there is an upsurge in the areas related to newer developments in prevention of disease especially the role of free radicals and antioxidants. So it will be pertinent to examine the possible role of `free radicals' in disease and `antioxidants' in its prevention, especially the current status of the subject matter and future prospects, in this review. Free Radicals- Friends or Foes? The events of World War II (1939-1945) led directly to the birth of free radical biochemistry. The two atom bombs (6th August 1945, Hiroshima and 9th August 1945, Nagasaki) led *Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai - 400 085, +Present address: Wimta Laboratories, Hyderabad; **Department of Zoology, University of Pune, Ganeshkhind, Pune 411 007; ***Honorary Chief Physician and Director of Nuclear Medicine/Director of Research, Jaslok Hospital and Research Centre, Mumbai 400 026. Received : 2.4.2004; Revised : 2.8.2004; Accepted : 16.7.2004
to massive deaths to entire population, and the survivors had shortened life-span. In 1954, Gershman and Gilbert speculated that the lethal effects of ionizing radiation might be ascribed to formation of reactive oxygen species (ROS). Since then free radicals (atoms with an unpaired electron) such as ROS and reactive nitrogen species (RNS) have gained notoriety. (Gilbert et al, 1981).1 In popular scientific/biomedical literature the term `free radical' is used in a broad sense and also includes related reactive species such as `excited states' that lead to free radical generation or those species that results from free radical reactions. In general, free radicals are very short lived, with half-lives in milli-, micro- or nanoseconds. Details about some of the biologically important reactive species are presented as Table 1. Free radicals have been implicated in the etiology of several human diseases as well as ageing (Harman, 1958; Halliwell and Gutteridge, 1997).2,3 But it has to be emphasized that ROS and RNS are both produced in a well regulated manner to help maintain homeostasis at the cellular level in the normal healthy tissues and play an important role as signaling molecules. Most cells can produce superoxide (O2·-), hydrogen peroxide (H2O2) and
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Table 1: Reactive oxygen and nitrogen species of biological interest
Reactive species
Symbol Half life Reactivity / Remarks (in sec)
Reactive oxygen species :
Superoxide
O2 - 10-6 s
Hydroxyl radical
OH 10-9 s
Hydrogen peroxide
H2O2 stable
Peroxyl radical ROO
s
Organic
ROOH
hydroperoxide
Singlet oxygen 1O2
stable 10-6 s
Ozone
O3
s
Reactive nitrogen species:
Nitric oxide
NO
s
Peroxynitrite ONOO- 10-3 s
Peroxynitrous acid Nitrogen dioxide
ONOOH NO2
fairly stable s
Generated in mitochondria, in cardiovascular system and others Very highly reactive, generated during iron overload and such conditions in our body Formed in our body by large number of reactions and yields potent species like OH Reactive and formed from lipids, proteins, DNA, sugars etc. during oxidative damage Reacts with transient metal ions to yield reactive species Highly reactive, formed during photosensitization and chemical reactions Present as an atmospheric pollutant, can react with various molecules, yielding 1O2 Neurotransmitter and blood pressure regulator, can yield potent oxidants during pathological states Formed from NO. and superoxide, highly reactive Protonated form of ONOO- Formed during atmospheric pollution
nitric oxide (NO) on demand. Hence, it is worth emphasizing the important beneficial role of free radicals. 1. Generation of ATP (universal energy currency) from ADP in the mitochondria: oxidative phosphorylation 2. Detoxification of xenobiotics by Cytochrome P450 (oxidizing enzymes) 3. Apoptosis of effete or defective cells 4. Killing of micro-organisms and cancer cells by macrophages and cytotoxic lymphocytes 5. Oxygenases (eg. COX: cyclo-oxygenases, LOX: lipoxygenase) for the generation of prostaglandins and leukotrienes, which have many regulatory functions. In recent years, it has become increasingly clear the ROS, such as O2·- and H2O2 may act as second messengers. Observations made some twenty years ago had suggested that ROS may play a role in modulating cellular function. Studies done then revealed that exogenous H2O2 could mimic the action of the insulin growth factor. The discovery of redoxsensitive transcription factors and that NO·, a free radical produced enzymatically, plays a physiological role in vasodialation and neurotransmission through activation of soluble guanylated cyclase further supported the concept that ROS and RNS can act as second messengers to modulate signaling pathways. This led to the renaissance of the field
of redox signaling and with the accumulation of data in various systems, a clearer picture is emerging of the signaling pathways and specific targets affected by ROS/RNS (Yoshikawa et al, 2000).4
Other sources of free radicals include redox cycling of
xenobiotics, exposure to physicochemical agents like ionizing
radiations such as X-rays and -rays besides visible light or
UV in the presence of oxygen and an endogenous compound
or a drug that act as photosensitizer. Most of the damage
induced by ionizing radiations in biological systems is indirect
and is mediated by products of radiolysis of water including
hydrogen radical (·H), ·OH, hydrated electron (e -), H O ,
aq
22
peroxyl radical (ROO·), O2·-, 1O2 etc. (Von Sonntag, 1987;
Devasagayam and Kesavan, 1996).5,6 Cigarette smoke contains
a large amount of reactive species (Devasagayam and Kamat,
2002).7 Cigarette tar contains quinone-hydroquinone-
semiquinone system which reduces O to form O ·-, H O and
2
2
22
·OH, while cigarette smoke contains small oxygen- and
carbon-centered radicals as well as active oxidants such as
NO· and nitrogen dioxide (NO ). Recent studies by Wentworth 2 et al. (2003)8 showed that antibodies, regardless of origin or
antigenic specificity, could convert 1O2 into H2O2 via a process that they have postulated to involve dihydrogen trioxide
(H2O3). During ischemia-reperfusion, oxidants like O2·-, ·OH and H O are produced. This occurs during non-fatal 22 myocardial infarction, surgeries, stroke, transplantation,
blockage of arteries under pathological conditions, etc.
During ischemia in the heart (in myocyte mitochondria)
conversion of ATP to adenosine causes the generation of
O ·-, while in the blood vessels (endothelium) the pathway 2 involved is the transition from xanthine to uric acid (Yoshikawa
et al 2000).4
Antioxidants
`Antioxidants' are substances that neutralize free radicals or their actions (Sies, 1996).9 Nature has endowed each cell with adequate protective mechanisms against any harmful effects of free radicals: superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, thioredoxin, thiols and disulfide bonding are buffering systems in every cell. -Tocopherol (vitamin E) is an essential nutrient which functions as a chain-breaking antioxidant which prevents the propagation of free radical reactions in all cell membranes in the human body. Ascorbic acid (vitamin C) is also part of the normal protecting mechanism. Other non-enzymatic antioxidants include carotenoids, flavonoids and related polyphenols, -lipoic acid, glutathione etc.
Levels of Antioxidant Action
Antioxidants, capable of neutralizing free radicals or their actions, act at different stages. They act at the levels of prevention, interception and repair (see Figure 1 for details). Preventive antioxidants attempt to stop the formation of ROS. These include superoxide dismutase (SOD) that catalyses the dismutation of superoxide to H2O2 and catalase that breaks it down to water (Sies, 1996; Cadenas and Packer, 1996).9,10 Interception of free radicals is mainly by radical scavenging, while at the secondary level scavenging of
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peroxyl radicals are effected. The effectors include various antioxidants like vitamins C and E, glutathione, other thiol compounds, carotenoids, flavonoids, etc. At the repair and reconstitution level, mainly repair enzymes are involved (Sies, 1996; Cadenas and Packer, 1996; Halliwell and Aruoma, 1993).9-11
Concept of Oxidative Stress
The relation between free radicals and disease can be explained by the concept of `oxidative stress' elaborated by Sies (1986).12 In a normal healthy human body, the generation of pro-oxidants in the form of ROS and RNS are effectively kept in check by the various levels of antioxidant defense. However, when it gets exposed to adverse physicochemical, environmental or pathological agents such as atmospheric pollutants, cigarette smoking, ultraviolet rays, radiation, toxic chemicals, overnutrition and advanced glycation end products (AGEs) in diabetes, this delicately maintained balance is shifted in favor of pro-oxidants resulting in `oxidative stress'. It has been implicated in the etiology of several (>100) of human diseases and in the process of ageing.
Molecular damage induced by free radicals
All the biological molecules present in our body are at risk of being attacked by free radicals. Such damaged molecules can impair cell functions and even lead to cell death eventually resulting in diseased states.
Lipids and Lipid Peroxidation
Membrane lipids present in subcellular organelles are highly susceptible to free radical damage. Lipids when reacted with free radicals can undergo the highly damaging chain reaction of lipid peroxidation (LP) leading to both direct and indirect effects. During LP a large number of toxic byproducts are also formed that can have effects at a site away from the area of generation, behaving as `second messengers'. The damage caused by LP is highly detrimental to the functioning of the cell (Devasagayam et al 2003).13
Lipid peroxidation is a free radical mediated process. Initiation of a peroxidative sequence is due to the attack by any species, which can abstract a hydrogen atom from a methylene group (CH2), leaving behind an unpaired electron on the carbon atom (·CH). The resultant carbon radical is stabilized by molecular rearrangement to produce a conjugated diene, which then can react with an oxygen molecule to give a lipid peroxyl radical (LOO·). These radicals can further abstract hydrogen atoms from other lipid molecules to form lipid hydroperoxides (LOOH) and at the same time propagate LP further. The peroxidation reaction can be terminated by a number of reactions. The major one involves the reaction of LOO· or lipid radical (L·) with a molecule of antioxidant such as vitamin E or -tocopherol (-TOH) forming more stable tocopherol phenoxyl radical that is not involved in further chain reactions. This can be `recycled' by other cellular antioxidants such as vitamin C or GSH.
LH + ·OH
L· + H2O
L· + O2
LOO·
LOO· + LH
L· + LOOH
LOO· + -TOH LOOH + -TO·
The process of LP, gives rise to many products of toxicological interest like malondialdehyde (MDA), 4hydroxynonenal (4-HNE) and various 2-alkenals. Isoprostanes are unique products of lipid peroxidation of arachidonic acid and recently tests such as Mass Spectrometry and ELISA-assay kits are available to detect isoprostanes (Yoshikawa et al. 2000).4
Carbohydrates
Free radicals such as ·OH react with carbohydrates by randomly abstracting a hydrogen atom from one of the carbon atoms, producing a carbon-centered radical. This leads to chain breaks in important molecules like hyaluronic acid. In the synovial fluid surrounding joints, an accumulation and activation of neutrophils during inflammation produces significant amounts of oxyradicals, that is also being implicated in rheumatoid arthritis.
DNA
Oxidative damage to DNA is a result of interaction of DNA with ROS or RNS. Free radicals such as ·OH, eaq- and H· react with DNA by addition to bases or abstractions of hydrogen atoms from the sugar moiety. The C4-C5 double bond of pyrimidine is particularly sensitive to attack by ·OH, generating a spectrum of oxidative pyrimidine damage products, including thymine glycol, uracil glycol, urea residue, 5-hydroxydeoxyuridine, 5-hydroxydeoxycytidine, hydantoin and others. Similarly, interaction of ·OH with purines will generate 8-hydroxydeoxyguanosine (8-OHdG), 8hydroxydeoxyadenosine, formamidopyrimidines and other less characterized purine oxidative products. Several repair pathways repair DNA damage (Halliwell and Aruoma, 1993).11 8-OHdG has been implicated in carcinogenesis and is considered a reliable marker for oxidative DNA damage.
Proteins
Oxidation of proteins by ROS/RNS can generate a range of stable as well as reactive products such as protein hydroperoxides that can generate additional radicals particularly upon interaction with transition metal ions. Although most oxidised proteins that are functionally inactive are rapidly removed, some can gradually accumulate with time and thereby contribute to the damage associated with ageing as well as various diseases. Lipofuscin, an aggregate of peroxidized lipids and proteins accumulates in lysosomes of aged cells and brain cells of patients with Alzheimer's disease (Stadtman, 1992).14
Significance of antioxidants in relation to disease
Antioxidants may prevent and/or improve different diseased states (Knight, 2000).15 Zinc is an essential trace element, being a co-factor for about 200 human enzymes, including the cytoplasmic antioxidant Cu-Zn SOD, isoenzyme of SOD mainly present in cytosol. Selenium is also an essential trace element and a co-factor for glutathione peroxidase. Vitamin E and tocotrienols (such as those from palm oil) are
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efficient lipid soluble antioxidants that function as a `chain breaker' during lipid peroxidation in cell membranes and various lipid particles including LDL (Packer and Ong, 1998; Kagan et al. 2002).16,17 Vitamin E is considered as the `standard antioxidant' to which other compounds with antioxidant activities are compared, especially in terms of its biological activity and clinical relevance. The daily dietary allowance varies between 400 IU to 800 IU. Vitamin C (ascorbic acid) is a water-soluble free radical scavenger. The daily recommended dietary allowance is 60 mg. Apart from these carotenoids such as beta-carotene, lycopene, lutein and other carotenoids function as important antioxidants and they quench 1O2 and ROO·. Flavonoids, mainly present as colouring pigments in plants also function as potent antioxidants at various levels (Sies, 1996; Cadenas and Packer, 1996; Kagan et al. 2002).9,10,17 Antioxidants and protection against human disease There are a number of epidemiological studies that have shown inverse correlation between the levels of established antioxidants/phytonutrients present in tissue/blood samples and occurrence of cardiovascular disease, cancer or mortality due to these diseases. However, some recent metaanalysis show that supplementation with mainly single antioxidants may not be that effective (Vivekananthan et al. 2003),18 a view that contrasts with those of preclinical and epidemiological studies on consumption of antioxidant-rich foods. Based on the majority of epidemiological and casecontrol studies recommendations were made for the daily dietary intake of some established antioxidants like vitamin E and C as well as others. Requirement for antioxidants in Indian conditions differ from that of industrialized western countries due to the nutritional differences. There are also a number of dietary supplements rich in antioxidants tested for their efficacy. There are many laboratories from India working on the antioxidant effect of plant compounds, mainly derived from natural sources that are capable of protecting against such damage. Such studies show that compounds with potent antioxidant activity include carotenoids, curcumin from turmeric, flavonoids, caffeine present in coffee, tea, etc., orientin, vicenin, glabridin, glycyrrhizin, emblicanin, punigluconin, pedunculagin, 2-hydroxy-4-methoxy benzoic acid, dehydrozingerone, picroliv, withaferin, yakuchinone, gingerol, chlorogenic acid, vanillin (food flavouring agent) and chlorophyllin (a water-soluble analogue of chlorophyll). (For more details see Table 2). Newer therapeutic approaches using antioxidants Antioxidant-based drugs/formulations for prevention and treatment of complex diseases like atherosclerosis, stroke, diabetes, Alzheimer's disease (AD), Parkinson's disease, cancer, etc. appeared over the past three decades. Free radical theory has greatly stimulated interest in the role of dietary antioxidants in preventing many human diseases, including cancer, atherosclerosis, stroke, rheumatoid arthritis, neurodegeneration and diabetes.
Dietary antioxidants may have promising therapeutic potential in delaying the onset as well as in preventing the ageing population with AD and its related complications. Two neuroprotective clinical trials are available with antioxidants: Deprenyl and tocopherol antioxidant therapy of Parkinson's study. By fusing ancient wisdom and modern science, India can create world-class products. Therefore, it has embarked on a fast track programme to discover new drugs by building on traditional medicines and screening the diverse plants and microbial sources of the country. In terms of its size, diversity and access to talent and resources this programme is not only the world's largest project of its kind, but is also unique (Jayaraman, 2003).19 Ayurveda, antioxidants and therapeutics Employing a unique holistic approach, Ayurvedic medicines are usually customized to an individual constitution. Ayurvedic Indian and traditional Chinese systems are living `great traditions' and have important roles in bioprospecting of new medicines from medicinal plants, which are also rich sources of antioxiodants. Current estimate indicates that about 80% of people in developing countries still rely on traditional medicine-based largely on various species of plants and animals for their primary healthcare. Ayurveda remains one of the most ancient and yet living traditions practiced widely in India. Sources of antioxidants, phytonutrients and functional foods Natural compounds, especially derived from dietary sources provide a large number of antioxidants (Table 3). Some beverages such as tea are also rich sources of antioxidants. A growing body of evidence suggests that moderate consumption of tea may protect against several forms of cancer, cardiovascular diseases, the formation of kidney stones, bacterial infections, and dental cavities. Tea is particularly rich in catechins, of which epigallocatechin gallate (EGCG) is the most abundant. Indian Medicinal Plants Apart from the dietary sources, Indian medicinal plants also provide antioxidants and these include: (with common/ ayurvedic names in brackets) Aegle marmelos (Bengal quince, Bel), Allium cepa (Onion), Allium sativum (Garlic, Lahsuna), Aloe vera (Indian aloe, Ghritkumari), Amomum subulatum (Greater cardamom, Bari elachi), Andrographis paniculata (The creat, Kiryat), Asparagus racemosus (Shatavari), Azadirachta indica (Neem, Nimba), Bacopa monniera (Brahmi), Camellia sinensis (Green tea), Cinnamomum verum (Cinnamon), Cinnamomum tamala (Tejpat), Curcuma longa (Turmeric, Haridra), Emblica officinalis (Indian gooseberry, Amlaki), Glycyrrhiza glabra (Yashtimadhu), Hemidesmus indicus (Indian Sarasparilla, Anantamul), Momordica charantia (Bitter gourd), Nigella sativa (Black cumin), Ocimum sanctum (Holy basil, Tulsi), Picrorrhiza kurroa (Katuka), Plumbago zeylanica (Chitrak), Syzigium cumini (Jamun), Terminalia bellarica (Behda), Tinospora cordifolia
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Table 2 : Epidemiological studies on antioxidants in humans
Disease
Antioxidant
Parameter
Effect
Cancer Gastric cancer Lung cancer in smokers prostate cancer Lung cancer in workers exposed to asbestos Lung cancer Cardiovascular diseases Myocardial infarction Coronary heart disease Atherosclerosis Stroke and myocardial infarction Cardiovascular disease Coronary heart disease Hypertension Heart failure Neurodegenerative diseases Parkinson's disease Alzheimer's disease Others Diabetes/hyperglycemia type 2 diabetes Renal dysfunction Subarachnoid hemorrhage in mice Pre-eclampsia
Vit E, -carotene, selenium Vit E, -carotene and both together Vit E -carotene + Vit A ,-carotene, lutein, lycopene and -crypto-xanthine in diet for 10 years Aspirin -carotene Vit E Vit E N-3 PUFA , Vit E And both together Catechin, Quercetin Vit C Vit C Carvedilol (25 mg bid) Metoprolol (50 mg bid) for 4, 8, 12 weeks. Vit E. (2000 UI/day), Deprenyl (10 mg/day) and in combination for 14 months Selegiline(10mg/day), Vit E (2000 UI/day) and in combination for 2 yrs Vit C (24 mg/min) intra arterially for 10 min Vit E Acetylcysteine (600 mg bid) i.v. Transgenic Cu-Zn SOD (22.7 U/mg protein) as compared to 7.9 in non transgenic mice Vit C (1000 mg) + Vit E (400 mg) during pregnancy
Stenosis Collagen-induced platelet aggregation Improved vascular flow Peripheral vascular resistance
In non-smokers heart rate and exercise tolerance
Death, institutional-

ization, severe dementia
Blood flow, Impaired endothelium-dependent vasodilatation Age, body-mass index, fasting plasma glucose and insulin, vit E and TBARS Serum creatinine and blood urea nitrogen levels iNOS levels
Restores levels of iNOS
Plasminogen activator inhibitor in PA-I (PA-I), a marker
reduced effect; no significant effect; increased effect
(Heart-leaved moonseed, Guduchi), Trigonella foenumgraecum (Fenugreek), Withania somnifera (Winter cherry, Ashwagandha) and Zingiber officinalis (Ginger). There are also a number of ayurvedic formulations containing ingredients from medicinal plants that show antioxidant activities (Tilak et al, 2001).20 In respect of the above we would like to give some data on our studies with Terminalia arjuna (Tilak et al, in press).21 Since possible antioxidant properties have been correlated with cardioprotective effects (Yoshikawa et al),4 we examined the antioxidant properties of different preparations from the bark of T. arjuna that has been extensively studied for its cardioprotective effects (Tilak et al, in press-b).21 One of its active ingredient, baicalein, also was subjected to these studies. The assays employed pertain to different levels of antioxidant protection such as radical formation, radical scavenging and membrane damage. Our studies have shown that the extracts of T. arjuna bark and baicalein possessed significant antioxidant properties. We also studied the possible uptake of components from T. arjuna and baicalain
by feeding rats and by using the ex vivo model of `inverted rat intestine loop'. Our studies indicate that about 25% baicalein is taken up and other components are being absorbed. These components may confer protective properties by their antioxidant effects. Further studies are needed to characterize and to estimate their metabolism of such potentially useful components. Importance of phytonutrients The idea of growing crops for health rather than for food or fiber is slowly changing plant biotechnology and medicine. Rediscovery of the connection between plants and health is responsible for launching a new generation of botanical therapeutics that include plant-derived pharmaceuticals, multicomponent botanical drugs, dietary supplements, functional foods and plant-produced recombinant proteins. Among polyphenols, flavonoids constitute the most important single group, including more than 5000 compounds that have been thus far identified. Apart from nutrient components such as -carotene, vitamins C and E, and selenium, compounds such as phenols,
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Table 3 : Major group of antioxidant compounds and their dietary sources
Polyphenolic compounds Flavonoids with antioxidant effect: Anthocyanidins, Aurones, Chalcones, Flavanones (Naringenin), Flavanols (Procyanidin), Flavan-3-ol (Epicatechin, Catechin), Flavones (Apigenin, Luteolin), Flavonols (Isorhamnetin, Kaempferol, Myricetin, Quercetin, Quercetin glycosides, Rutin), Isoflavonoids (Anisole, Cumestrol, Daidzein, Genistein) Other Polyphenols: Cinnamic acid, Coumarin, Condensed tannins, Hydroxybenzoic acid (Gallic aid, Protocatechuic acid, Vanillic acid), Hydroxycinnamic acid (Caffeic acid, Caftaric acid, Chlorogenic acid, Coumaric acid, , Ferulic acid, Sinapic acid), Proanthocyanidins, Carotenoids with antioxidant effects: Astaxanthin, Bixin, Canthaxanthin, Capsorubin, -Carotene, -Carotene, -Carotene, Crocin, -Cryptoxanthin, Lutein, Lycopene, Zeaxanthin, Vitamins: Vitamin C, Vitamin E ( Tocopherols, Tocotreinols), Nicotinamide. Other compounds: Curcumin, Caffeine, Chlorophyllin, Sesaminol, Zingerone Functional Foods
Dietary sources Fruits: Apples, Blackberries, Blueberries, citrus fruits, Grapes, Pears, Pomegranate, Raspberries, Strawberries Vegetables: Beetroot, Brinjal, Broccoli, Celery, Endives, Leek, Lettuce, Onion (white and red), Pepper, Spinach, Tomatoes Legumes: Horsegram, Greengram, Lupin Peas, Soy beans, White and Black Beans Spices: Cardamom, Cinnamon, Cloves, Coriander, Cumin Beverages: Cocoa, Tea, Wine (Red and White Wines, Sherry) Oil:Olive oil Chocolates Fruits: Apples, Apricot, Banana, Blackberries, Blueberries, Cherries, Grapefruits, Grapes, Jack fruit, Kiwi fruit, Lemon, Mango, Melon, Orange, Papaya, Peach, Pears, Pineapple, Plum, Strawberries, Watermelon. Vegetables: Amaranthus, Asparagus, Beet Beetroot, Brinjal, Broccoli, Brussels sprouts, Cabbage, Cauliflower, Cucumber Carrots, Celery, Lettuce, Mushroom, Onion Pepper, Tomatoes, Potatoes, Pumpkin, Spinach, Spring greens, Spring onions. Cereals: Sweetcorn/Corn. Legumes: Beans (Broad, Green, Runner, Kidney), Bean sprouts, Peas. Spices: Chillies, Saffron Oil: Red palm oil Dairy products: Butter, Cheese, Margarine, Milk, Yogurt. Eggs: Whole and yolk, Mayonnaise Amla (Indian gooseberry), Lemon, Oranges, Oil: Groundnut oil, Olive oil, Palm oil, Cashew nuts, Germinated pulses, Rasins Coffee, Cocoa, Colas, Green vegetables, Tea, Turmeric, Zinger, Food colorants Soy protein, fish oil fatty acids, bengal gram, amla, bitter gourd, til, winged bean, guar, Spirulina (blue green algae), sunflower, cowpea, linseed, ground nut, kenaf, safflower, rape seed, horse gram, rice bran, pearl millet, wheat grass, sorghum, soybean, Amaranthus, ivy guard, cabbage, cassava, sweet potato and yams
flavonoids, isoflavones, isothiocyanates, diterpenes, methylxanthines, dithiols, and coumarins appear to be important in cancer prevention through their role on the inhibition of tumor production. (Krishnaswami, 1996).22 Functional foods The very concept of food is changing from a past emphasis on health maintenance to the promising use of foods to promote better health to prevent chronic illnesses. `Functional foods' are those that provide more than simple nutrition; they supply additional physiological benefit to the consumer. Because dietary habits are specific to populations and vary widely, it is necessary to study the disease-preventive potential of functional micronutrients in the regional diets. Indian food constituents such as spices as well as medicinal plants with increased levels of essential vitamins and nutrients (eg. vitamin E, lycopene, vitamin C, bioflavonoids, thioredoxin etc.) provide a rich source of compounds like antioxidants that can be used in functional foods (Devasagayam et al. In Press).23 Pro-oxidant effect of antioxidant under certain conditions Antioxidants also have the potential to act as prooxidants
under certain conditions. For example, ascorbate, in the presence of high concentration of ferric iron, is a potent potentiator of lipid peroxidation. Recent studies suggest that ascorbate sometimes increase DNA damage in humans. Recent mechanistic studies on the early stage of LDL oxidation show that the role of vitamin E is not simply that of a classical antioxidant. Unless additional compounds are present, vitamin E can have antioxidant, neutral or prooxidant activity. Beta-carotene also can behave as a prooxidant in the lungs of smokers. The paradoxical role (pro-oxidant effect) of antioxidants is also directly related to the recently described `redox signaling' of the antioxidants. The functional role of many antioxidants depends on redox cycling. For example, the best-described intracellular antioxidant vitamin E supplementation in the face of infarcted myocardium exerted prooxidant effects resulting in the rupture of the plaques. When a cell is attacked by environmental stress, the cell's defense is lowered because of massive generation of ROS. The cell immediately responds to this stress by upregulating its antioxidant defense. During the induction process ROS function as signaling molecules. It should be easily understood that in these pathophysiological conditions even though the antioxidants
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are lowered and supplementation of the antioxidants are warranted, the antioxidants should be harmful because they will prevent the function of the ROS to perform signal transduction to induce intracellular antioxidants. CURRENT TOPICS OF INTEREST AND STUDIES IN FREE RADICAL RESEARCH The field of free radical research is undergoing a tremendous advancement in recent years. Here we present some of the interesting areas of current research and some novel observations pertaining to free radicals and antioxidants in relation to human health. Cardiovascular disease In the vascular endothelium, the free radical NO· is produced from arginine by nitric oxide synthase (NOS), converting the substrate L-arginine to L-citrulline. L-Arg + O2 + NADPH NOS NO· + citrulline The reaction requires calmodulin, NADPH and tetrahydrobiopterin (BH4) as cofactors. Under normal conditions, NO· is protective against adhesion of platelets and leucocytes, anti-inflammatory, anti-proliferative and regulates the expression and synthesis of extracellular matrix proteins. It inhibits redox sensitive transcription factor, NFKappa expression through its binding with IKappa, linked to a number of diseases which shorten life. Endothelial dysfunction can be caused due to altered gene expression as a product of interaction of genes with the environment such as maternal malnutrition in fetus. The gene responsible for this is endothelial nitric oxide synthase (eNOS): NOS3, which on polymorphism can also cause hypertension. Oxidative damage to cholesterol component
of the low-density lipoprotein (LDL) leads to oxidised LDL by a series of consecutive events. This induces endothelial dysfunction, which promotes inflammation during atherosclerosis. Oxidative stress involved in other clinically recognised conditions such as smoking, advanced glycation end-products (AGEs) in diabetes mellitus, shear stress etc. intensify endothelial dysfunction. Endothelial NOS can constitutively produce both NO· and O ·-. However, due to above-mentioned reasons, the ROS 2 produced increase Ca2+ concentration in endothelial cells. In patients having hypertension, there is increased angiotensin II which mediates O ·- production through oxidation of 2 membrane NADP/NADPH oxidases and increase lipid peroxidation as shown by elevated levels of F2 isoprostanes. Excessive O2·- produced scavenges NO· to form highly damaging reactive species, peroxynitrite. This can be attenuated by SOD bound to the outer endothelial cell surface, which protect NO· from O2·-. SOD mimic tempol has been shown to restore vasodilation in afferent arterioles in experimental diabetic nephropathy. Depletion of BH4 has been associated with endothelial dysfunction in hypertension, diabetes mellitus and atherosclerosis. It plays a key role in controlling production of NO· and O2·- in vivo. In insulin resistance, abnormal biopterin metabolism causes impaired vasorelaxation because insulin stimulates synthesis of BH4. Thus endothelial dysfunction and insulin resistance are basis of hypertension, type II diabetes and atherosclerosis in the Indian population (Lele, 2003; 2004).24,25 Oxidised LDL acts as a trigger to initiate endothelial inflammation leading to atherosclerosis and vascular thrombosis (heart attack and stroke) (Figure 2). LDL oxidation and atherogenesis can be inhibited by nutritional antioxidants. There are also epidemiological evidences and interventional studies to correlate higher level of antioxidant-rich food uptake with lower incidence of coronary heart disease (Chopra et al, 1996).26
Level of Antioxidant Action Non-enzymatic, enzymatic and ancillary enzymes & defense systems in vivo against oxidative damage Fig. 1: Schematic diagram showing the five different levels of antioxidant action
Fig. 2 : Diagram showing mechanism by which the oxidation of LDL may contribute to atherosclerosis. The figure shows oxidative modification of low-density lipoprotein (LDL) caused by reactive oxygen species and then formation of foam cells which is the initial lesion of atherogenesis.
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Neurodegenerative Disorders Nervous tissue including brain is highly susceptible for free radical damage due to high content of lipids especially polyunsaturated fatty acids. In Alzheimer's disease (AD) biochemical and histological studies have provided evidence for increased levels of oxidative stress and membrane LP. Alterations in levels of antioxidant enzymes such as catalase and CuZn- and Mn-SOD in neurons in AD patients are consistent with their being under increased stress. Increased protein oxidation, protein nitration and LP occur in neurofibrillary tangles and neuritic plaques. Lipid peroxidation is quite extensive as indicated by increased levels of peroxidation products such as 4-hydroxynonenal (4-HNE) in the cerebrospinal fluid of AD patients. Iron (Fe2+) likely contributes to increased LP in AD. Lipid peroxidation may promote neuronal death in AD by multiple mechanisms that include impairment of the function of membrane ion-motive ATPases (Na+/K+-ATPase and Ca2+-ATPase), glucose transporters and glutamate transporters. Lipid peroxidation leads to production of the aldehyde 4-HNE that appears to play a central role in the neurotoxic actions of amyloid peptide (Yoshikawa et al. 2000).4 Free Radical Damage to DNA and Cancer DNA is a major target of free radical damage. The types of damages induced are many and include strand breaks (single or double strand breaks), various forms of base damage yielding products such as 8-hydroxyguanosine, thymine glycol or abasic sites, damage to deoxyribose sugar as well as DNA protein cross links. These damages can result in mutations that are heritable change in the DNA that can yield cancer in somatic cells or foetal malformations in the germ cells. The involvement of free radicals with tumor suppressor genes and proto-oncogenes suggest their role in the development of different human cancers (Halliwell and Aruoma, 1993).11 Cancer develops through an accumulation of genetic changes. Initiating agents can be tobacco smoking and chewing, UV rays of sunlight, radiation, viruses, chemical pollutants, etc. Promoting agents include hormones (androgens for prostate cancer, estrogens for breast cancer and ovarian cancer). Inflammation induces iNOS (inducible nitric oxide synthase) as well as COX and LOX. These can initiate carcinogenesis. Experimental as well as epidemiological data indicate that a variety of nutritional factors can act as antioxidants and inhibit the process of cancer development and reduce cancer risk. Some of these include vitamins A, C, E, beta-catotene, and micronutrients such as antioxidants and anticarcinogens. (Croce, 2001).27 Surh (2003)28 recently reviewed mechanisms behind anticancer effects of dietary phytochemicals. Chemopreventive phytochemicals can block initiation or reverse the promotion stage of multistep carcinogenesis. They can also halt or retard the progression of precancerous cells into the malignant ones. Many molecular alterations associated with carcinogenesis occur in cell-signalling pathways that regulate cell proliferation and differentiation. One of the central components of the intracellular- signalling
network that maintains homeostasis is the family of mitogenactivated protein kinases (MAPKs). Numerous intracellular signal-transduction pathways converge with the activation of the transcription factors NF-B and AP1. As these factors mediate pleiotropic effects of both external and internal stimuli in the cellular- signalling cascades, they are prime targets of diverse classes of chemopreventive phytochemicals (Surh, 2003).28 The active principle of Curcuma longa (Turmeric, Haldi) curcumin, down-regulates the expression of COX2, LOX, iNOS, MMP-9, TNF, chemokines and other cell-surface adhesion molecules and cyclin D1. Human clinical trials have shown safety at doses upto 10 g/day curcumin, which can suppress tumour initiation, promotion and metastasis. Many Ayurvedic herbal drugs have anti-inflammatory, antioxidant and immunomodulatory activity and can be used for chemoprevention. Validation of the concept necessitates long-term prospective CLINICAL STUDIES. Free Radicals, Diabetes and AGEs Experimental evidences suggest the involvement of free radicals in the onset of diabetes and more importantly in the development of diabetic complications (Lipinsky, 2001).29 Scavengers of free radicals are effective in preventing experimental diabetes in animal models and in type 1 (IDDM) and type 2 (NIDDM) patients as well as reducing severity of diabetic complications. Persistent hyperglycemia in the diabetic patients leads to generation of oxidative stress due to a) autooxidation of glucose; b) non-enzymatic glycosylation and c) polyol pathway. Auto-oxidation of glucose involves spontaneous reduction of molecular oxygen to superoxide and hydroxyl radicals, which are highly reactive and interact with all biomolecules. They also accelerate formation of advanced glycation end products (AGEs). AGEs such as pyrroles and imidazoles tend to accumulate in the tissue. Crosslinking AGE-protein with other macromolecules in tissues results in abnormalities in the cell and tissue function. Polyol pathway is the third mechanism by which free radicals are generated in the tissues (Glugliano et al, 1995).30 Lot of NADPH is deleted during this pathway, hence it impairs generation of antioxidants such as glutathione. Due to protein glycation capacity of antioxidant enzymes is also reduced. Free radicals generated also react with nitric oxide in endothelial cells leading to loss of vasodilation activity. Long-lived structural proteins, collagen and elastin, undergo continual non-enzymatic crosslinking during ageing and in diabetic individuals (Vasan et al. 2003).31 This abnormal protein crosslinking is mediated by AGEs generated by nonenzymatic glycosylation of proteins by glucose. Free radicals and ageing Mitochondrial ROS production and oxidative damage to mitochondrial DNA results in ageing. Further increased lipid peroxidation in cellular membranes due to oxidative stress leads to fatty acid unsaturation. The most recent review on `free radicals and ageing' by Barja (2004)32 emphasizes that caloric restriction (CR) is the only known experimental manipulation that decreases rate of mammalian ageing, and it
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has many beneficial effects on the brains of rodents and possibly of humans. Calorie-restricted mitochondria, similar to those of long-lived animal species, avoid generation of ROS efficiently at complex I with pyruvate and malate. The mitochondrial oxygen consumption remains unchanged, but the free radical leak from electron transport chain is decreased in CR. Many investigators realized that increasing the level of defense mechanisms against oxidative stress could extend an organism's health span. Arking's research group's work on artificial selection in flies also produced organisms with a much higher level of oxidative stress resistance and more efficient mitochondria. In fact, the lower level of oxidative damage and delayed onset of senescence in those flies arose from decreased production and increased destruction of ROS. However, using genetic engineering techniques to insert some extra copies of these oxidative stress-resistance genes into mice has not yet resulted in extending longevity (Lane et al 2002).33 Mitochondria, oxidative protein damage and proteomics The rapid advance of proteomic methodologies and their application to large scale studies of protein-protein interactions and protein expression profiles suggest that these methods are well suited to provide the molecular details needed to fully understand oxidative injury induced by free radicals (Gibson, 2004).34 Over the last two decades, considerable progress has been made in identifying individual proteins that are localized to the mitochondria. In particular the 100 or so subunits that constitute the five complexes of the electron transport chain (ETC). Recently, using modern mass spectrometry (MS)-based proteomic strategies, several groups have begun to tackle the larger job of determining the composition of entire mitochondrial proteomes from a number of important model systems as well as from human tissues. Using mitochondria isolated from human heart, Gibson and coworkers have identified 684 unique proteins from the combined peptide data obtained from over 100,000 mass spectra generated by MALDI-MS and high performance liquid chromatography (HPLC) MS/MS analyses. These data are now part of `MitoProteome', a publicly accessible database for the human heart mitochondrial proteome. NEWER AND NOVEL APPROACHES TO REDUCE FREE RADICAL DAMAGE AND FUTURE PROSPECTS There are several novel approaches in the study of free radicals/antioxidants for the improvement of human health. A number of neuronal and behavioral changes occur with ageing, even in the absence of degenerative disease. A decline in cognitive function is one of the manifestations of changes that occur in neuronal functions with age. Several recent studies have found associations between the decline of memory performance and lower status of dietary antioxidants. The totality of evidence from experimental, clinical, and
epidemiological studies support the notion that consumption of foods obtaining high levels of dietary antioxidants, in addition to exerting several health benefits, may prevent or reduce the risk of cognitive deterioration. Recently a new class of SOD mimetic drugs, like tempol, was also developed to alleviate acute and chronic pain. These drugs substantially reduced tissue damage by inflammation and reperfusion. Unlike the naturally derived SOD enzymes the mimetic is well suited for use as a drug because it has a much lower molecular weight, is more stable and appears not to elicit an immune response in the body. SOD mimics also have ability to increase antitumour effects of interleukins, besides being efficient radioprotectors. Development of genetically engineered plants, to yield vegetables with higher level of certain compounds is another approach to increase antioxidant availability. Tomatoes with upto 3 times lycopene concentration as well as with longer shelf life were developed. `Orange cauliflower' is found to be rich in (-carotene. One way of checking the antioxidant ability of vegetables and fruits is measuring its ORAC value or oxygen radical absorbance capacity. Some fruits/vegetables with their ORAC values/100 g in (brackets) are raisins (2830), black berries (2036), strawberries (1540), oranges (750), grapes (739), cherries (670), spinach (1260), beets (840), onion (450) and eggplant (390). Intake of fruits and vegetables with ORAC values between 3000 and 5000 per day is recommended to have significant impact of the beneficial effect of antioxidants (Lachnicht, 2000).35 Given their inherently nutritious composition, most fruit and vegetable breeding programmes have tended to focus on improving aspects like appearance, taste, texture, shape and shelf-life. But more and more research energy is being channeled into making fruits and vegetables even healthier. The Vegetable and Fruit Improvement Cente at Texas A and M University is a leading research hub in the area and created one of the world's first `super-vegetables' about a decade ago - a purple carrot breed that had 40% more beta-carotene than usual. The carrot, `Betasweet', now available throughout the US, was also bred to have a higher sugar content to improve flavour, as well as a crispy texture to make it more palatable for children. Other similar work being conducted include: developing the anti-carcinogen properties of citrus fruits; increasing the carotenoid content of watermelons and cantaloupes; developing milder and sweeter onions with increased quercetin and anthocyanin levels; increasing the quercetin levels of peppers and making them sweeter; increasing anthocyanin levels in stone fruits; developing lycopene-enriched tomatoes. Among the different products delivering essential nutrients to the body, an egg arguably has a special place, being a rich and balanced source of essential amino and fatty acids as well some minerals and vitamins. The advantages of simultaneous enrichment of eggs with vitamin E, carotenoids, selenium and DHA include better stability of polyunsaturated fatty acids during egg storage and cooking, high availability of such nutrients as vitamin E and carotenoids, absence of
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off-taste and an improved anti-oxidant and n-3status of people consuming these eggs. Designer eggs can be considered as a new type of functional food. Indian traditional systems of medicine and home remedies have identified many forms of health foods. Mainly whole foods are consumed as functional foods rather than supplements or processed foods. It is a time, after a period of flouring research on oxidants and antioxidants, to critically reflect on the fields. Speculation that many (if not all) diseases are related to radical damage needs to be supported by more secure data. The hope that antioxidants can prevent or cure a number of pathological situations also required reconsideration. The relatively new nothion that molecules with strong antioxidant activity in vitro may have `non-antioxidant' effects in cells and tissues should stimulate, rather than discourage, important research in this field. Finally, the discrepancies in the outcome of intervention studies may be understood if, instead of considering the simple paradigm of bad oxidants and good antioxidants, scientists will start talk about the real molecular function of such compounds in each particular situation (Azzi et al, 2004).36 CONCLUSION Free radicals have been implicated in the etiology of large number of major diseases. They can adversely alter many crucial biological molecules leading to loss of form and function. Such undesirable changes in the body can lead to diseased conditions. Antioxidants can protect against the damage induced by free radicals acting at various levels. Dietary and other components of plants form major sources of antioxidants. The relation between free radicals, antioxidants and functioning of various organs and organ systems is highly complex and the discovery of `redox signaling' is a milestone in this crucial relationship. Recent research centers around various strategies to protect crucial tissues and organs against oxidative damage induced by free radicals. Many novel approaches are made and significant findings have come to light in the last few years. The traditional Indian diet, spices and medicinal plants are rich sources of natural antioxidants. Higher intake of foods with functional attributes including high level of antioxidants in functional foods is one strategy that is gaining importance in advanced countries and is making its appearance in our country. Coordinated research involving bioMedical Scientists, nutritionists and physicians can make significant difference to human health in the coming decades. Research on free radicals and antioxidants involving these is one such effort in the right direction. REFERENCES 1. Gilbert DL 1981, Oxygen and living processes: an interdisciplinary approach, Springer, NY. 2. Harman D. Ageing: a theory based on free radical and radiation chemistry. J Gerontol 1956;11:298-300. 3. Halliwell B, Gutteridge JMC, (eds), Free Radicals in Biology and Medicine, Oxford University Press, Oxford, 1997.
4. Yoshikawa T, Toyokuni S, Yamamoto Y and Naito Y, (eds) Free Radicals in Chemistry Biology and Medicine, OICA International, London, 2000. 5. Von Sonntag C, (ed) In: The Chemical Basis of Radiation Biology, Taylor and Francis, London, 1987. 6. Devasagayam TPA and Kesavan PC, Radioprotective and antioxidant action of caffeine: Mechanistic considerations. Indian J Expt Biol 1996;34, 291-7. 7. Devasagayam TPA and Kamat JP, Biological significance of singlet oxygen. Ind J Expt Biol 2002;40, 680-92. 8. Wentworth P, Wentworth AD, Zhy X, et al. Evidence for the production of trioxygen species during antibody-catalyzed chemical modification of antigens. Proc Natl Ac Sc USA 2003;100:1490-93. 9. Sies H. (ed.) Antioxidants in Disease, Mechanisms and Therapy, Academic Press, New York, 1996. 10. Cadenas E and Packer L, (eds) Hand Book of Antioxidants. Plenum Publishers, New York, 1996. 11. Halliwell B and Aruoma OI. (eds) DNA and Free Radicals, Boca Raton Press, 1993. 12. Sies H, Biochemistry of Oxidative Stress. Angew Chem Internat Ed Eng 1986;25,1058-71. 13. Devasagayam TPA, Boloor KK and Ramsarma T. Methods for estimating lipid peroxidation: Analysis of merits and demerits (minireview). Indian J Bioche Biophys 2003;40:300-8. 14. Stadtman ER. Protein oxidation and aging. Science 1992;257:1220-25. 15. Knight JA. Review: Free radicals, antioxidants and the Immune System. Ann Clin Lab Sci 2000;30:145-58. 16. Packer L and Ong ASH. (eds) Biological Oxidants and Antioxidants:Molecular Mechanisms and Health Effects, AOCS Press, Champaign, 1998. 17. Kagan VE, Kisin ER, Kawai K, et al. Towards mechanismbased antioxidant interventions. Ann N Y Acad Sci 2002;959:188-98. 18. Vivekananthan DP, Penn MS, Sapp SK, et al. Use of antioxidant vitamins for the prevention of cardiovascular disease:metaanalysis of randomized trials. The Lancet 2003;361:2017-23. 19. Jayaraman KS. Technology, tradition unite India's drug discovery scheme. Nat Med 2003;9:982. 20. Tilak JC, Devasagayam TPA and Lele RD. Antioxidant activities from Indian medicinal plants: A review - Current status and future prospects. International Conference on Natural Antioxidants and Free Radicals in Human Health and Radiation Biology (NFHR). July 21-24, 2001. Mumbai, India. (Abstract book, page no. 5). 21. Tilak JC, Devasagayam TPA and Lele RD. Cardioprotective Properties of Some Indian Medicinal Plants: A Review and Possible Mechanisms. Biochem Pharmacol (in press). 22. Krishnaswami K. Indian functional food: role in prevention of cancer. Nutr rev 1996;54:S127-31. 23. Devasagayam TPA, Tilak JC and Singhal R. Functional foods in India; history and scope in Angiogenesis, functional and medicinal foods (Eds Losso JN, Shahidi F, Bagchi D), Marcel Dekker Inc. New York. (In Press). 24. Lele RD. The human genome project: its implications in clinical medicine. J Assoc Physicians India 2003,51:373-80.
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25. Lele RD. Hypertension: Molecular approach. J Assoc Physicians India 2004,52:53-62. 26. Chopra MU, McLoone M, O'Neill N, and Williams DI. Fruit and vegetable supplementation- effect on ex-vivo LDL oxidation in humans, In Natural Antioxidant and food quality in Atherosclerosis and Cancer Prevention, (eds Kumpulaine, JT, Salonel, JP). The Royal Society of Chemistry. Cambridge, UK 1996, pp151-55. 27. Croce CM. How can we prevent cancer? Proc Natl Acad Sci USA 2001;98:10986-88. 28. Surh YJ. Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 2003;3:768-80. 29. Lipinski B. Pathophysiology of oxidative stress in diabetes mellitus. J Diabetes Complications 2001;15:203-10. 30. Glugliano D, Cerriello A and Paolisso G. Diabetes mellitus, hypertension and cardiovascular disease: which role for
oxidative stress. Metabolism 1995;44:363-8. 31. Vasan S, Foiles P and Founds H, Therapeutic potential of breakers of advanced glycation end products-protein crosslinks. Arch Biochem Biophys 2003;419:89-96. 32. Barja G. Free radicals and ageing. Trends Neurosci 2004;257:16. 33. Lane MA, Ingram DK and Roth GS, The serious search for an anti-ageing pill, Scientific American 2002;24-9. 34. Gibson BW. Exploiting proteomics in the discovery of drugs that target oxidative damage. Science 2004;304:176-7. 35. Lachnicht D, Brevard PB, Wagner TL, DeMars CE. Dietary oxygen radical absorbance capacity as a predictor of bone mineral density. Nutr Res 2002;22:1389-99. 36. Azzi A, Davies KJA, and Kelly F. Free radical biologyterminology and critical thinking (minireview). FEBS Letters 2004;558:3-6.
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