Clinica Chimica Acta 436 (2014) 332–347

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Clinica Chimica Acta

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Antioxidants and human diseases

Peramaiyan Rajendran a, Natarajan Nandakumar b, Thamaraiselvan Rengarajan a, Rajendran Palaniswami d,Edwinoliver Nesamony Gnanadhas e, Uppalapati Lakshminarasaiah f, Jacob Gopas b,c, Ikuo Nishigaki a,⁎a NPO-International Laboratory of Biochemistry, 1-166, Uchide, Nakagawa-ku, Nagoya 454-0926, Japanb Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Israelc Oncology Department Soroka University Medical Center, Be'er-Sheva 84105, Israeld Department of Applied Zoology and Biotechnology, Vivekananda College (A Gurukula Institute of Life Training), Affiliated to Madurai Kamaraj University, ThiruvedakamWest, Madurai 625234, Indiae Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israelf Department of Clinical Biochemistry and Pharmacology, Soroka University Medical Center, Ben-Gurion University of the Negev, Be'er-Sheva 84105, Israel

⁎ Corresponding author.E-mail address: nishigaki@se.starcat.ne.jp (I. Nishigaki

http://dx.doi.org/10.1016/j.cca.2014.06.0040009-8981/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 April 2014Received in revised form 4 June 2014Accepted 5 June 2014Available online 13 June 2014

Keywords:AntioxidantOxidative stressDiabetesCancer

Oxidative stress plays a pivotal role in the development of human diseases. Reactive oxygen species (ROS) thatincludes hydrogen peroxide, hyphochlorus acid, superoxide anion, singlet oxygen, lipid peroxides, hypochloriteandhydroxyl radical are involved in growth, differentiation, progression and death of the cell. They can reactwithmembrane lipids, nucleic acids, proteins, enzymes and other small molecules. Low concentrations of ROS has anindispensable role in intracellular signalling and defence against pathogens, while, higher amounts of ROS play arole in number of human diseases, including arthritis, cancer, diabetes, atherosclerosis, ischemia, failures in im-munity and endocrine functions. Antioxidants presumably act as safeguard against the accumulation of ROSand their elimination from the system. The aim of this review is to highlight advances in understanding of theROS and also to summarize the detailed impact and involvement of antioxidants in selected human diseases.

© 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3332. Measuring oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

2.1. Pro-oxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3333. Potential antioxidant biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

3.1. Superoxide dismutase (SOD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3343.2. Catalase (CAT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3343.3. Glutathione peroxidase (GPx) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3343.4. Thiol index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3343.5. Xanthine oxidase (XO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3343.6. NADPH Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3343.7. A biomarker of cardiovascular damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

4. Mechanism of antioxidant action and nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3355. Antioxidants vs human diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

5.1. Antioxidant vs arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3365.2. Anti oxidant vs cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3385.3. Antioxidant vs diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3395.4. Antioxidant vs arthrosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3415.5. Anti oxidant vs neurodegerative diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

).

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1. Introduction

The beneficial effect of antioxidants on themaintenance of health inhuman has become an important subject that has engaged manyscientists across theworld over the last decade. In the last few years, an-tioxidants have become the indispensable nutrients of the nutritionalworld. Antioxidants are important in terms of their ability to protectagainst oxidative cell damage that can lead to conditions, such asAlzheimer's disease, cancer, heart disease and also linked with chronicinflammation [1]. It is defined as the substances which at low concen-tration significantly inhibits or delay the oxidative process, while oftenbeing oxidized themselves. Recent reports suggest that several endoge-nous and exogenous antioxidants are used to neutralize free radicalsand protect the body from free radicals by maintaining redox balance[2,3]. Singh et al. (2010) [4] quoted that antioxidants have gone from“Miracle Molecules” to “Marvellous Molecules” and finally to “Physio-logical Molecules” that they play a vital role in metabolic pathwaysand protect cells. However recent conflicting evidence has forced thescientists to dig deeper in order to explore the role of antioxidants andpro-oxidants, since free radical reactions have been implicated inevery human pathological condition which includes neurodegenerativedisorders like Alzheimer's disease, Parkinson's disease, multiplesclerosis, amyotrophic lateral sclerosis, memory loss, depression andcardiovascular diseases such as atherosclerosis, ischemic heart disease,cardiac hypertrophy, hypertension, shock and trauma. Further, it alsoimplicated in pulmonary disorders which include inflammatory lungdiseases such as asthma and chronic obstructive pulmonary diseaseand additionally diseases associated with premature infants such asbronchopulmonary dysplasia, periventricular leukomalacia, intraven-tricular hemorrhage, retinopathy of prematurity and necrotizing en-terocolitis and in some autoimmune diseases like rheumatoid arthritisand also in several renal disorders such as glomerulonephritis,tubulointerstitial nephritis, chronic renal failure, proteinuria, uremiaand finally gastrointestinal diseases like peptic ulcer, inflammatorybowel disease and colitis, diabetes, tumors and cancers [5].

2. Measuring oxidative stress

As oxidative stress is indicative of an inequity between oxidants andantioxidants, methods for quantifying oxidative stress mostly includestraight or indirect measurement of oxidants and antioxidants [6,7]. Inthe following segment, some principles and commonly used methodsfor the measurement of oxidative stress and damage will be brieflyoutlined.

2.1. Pro-oxidants

The most abundant free radicals in biological systems are theoxygen-centered free radicals and their metabolites, usually referredto as ROS [7]. ROS are formed continuously as normal by-products ofcellular metabolism; and, in low concentrations, they are essential forseveral physiological processes, including protein phosphorylation,transcription factor activation, cell differentiation, apoptosis, oocytematuration, steroidogenesis, cell immunity, and cellular defense againstmicroorganisms [7]. However, when produced in excess, ROS can dam-age cell functionality as they can harm cellular lipids, proteins, and DNA[7,8]. The plasma level of ROMs is considered an indicator of free-radicalproduction [7]. ROMs is a collective term that includes not only oxygen-centered free radicals such as the superoxide anion and hydroxylradical, but also some non-radical derivatives of oxygen, such as hydro-gen peroxide (H2O2) and hypochlorous acid [9]. A ROMs kit has beendeveloped to assess oxidant levels in plasma and other biological fluids;and the ROMs test has been validated by electron spin resonance [10],which is considered the “gold standard” for measuring total oxidativestatus. However, electron spin resonance is not suitable for routine anal-ysis, as themethod is complex and requires specific technical assistance

not available in most laboratories. The utility of the ROMs assay inmonitoring oxidative stress in goats [11], sheep [12], and dairy cows[13] has been reported.

The concentrations of individual oxidant components can bemeasured separately in the laboratory; but such measurements aretime-consuming, labor intensive, and costly. Free-radical analyticalsystem 4 technology has been shown to offer a quick, simple, precise,and reliable method for assessing the oxidative status in dairy cows[14] and in horses [15]. Such technology is particularly useful in thefield, where it is not always practical or possible to get samples to alaboratory immediately. The possibility of assessing oxidative stressdirectly in blood samples provides veterinarians with a simple andreliable method for measuring oxidative stress in clinical situationssuch as the monitoring of therapy and in the antioxidant supplementa-tion of domestic animals. However, given the lack of reference valuesfor ROMs in ruminants, it is difficult to establish if and when these ani-mals are actually experiencing oxidative stress. Therefore, it is importantto calculate the specific referral ranges; because a correct biochemicalevaluation of oxidative status is an essential premise to prevent andeventually to treat the effects of oxidative stress in ruminant medicine.Advanced oxidation protein products (AOPPs) are terminal products ofproteins exposed to free radicals and arise from the reaction betweenplasma proteins and chlorinated oxidantsmediated bymyeloperoxidase,a neutrophil enzyme [16,17]. In humans, AOPPs have been linked toseveral diseases, such as chronic renal failure [18], diabetes mellitus[19], diabetic nephropathy [20], coronary artery diseases [21], andobesity [22]. Chronic accumulation of AOPPs has been demonstrated topromote inflammatory processes in the diabetic kidney [20] and inchronic renal failure [18], indicating that these products might be a by-product of neutrophil activation during infections. Studies in ruminantshave shown higher levels of AOPPs than normal in lambs [23] anddairy cows [24] supplemented with Yerba Mate (Ilex paraguanensis).More information about the role of protein oxidation in ruminants'health and about oxidated proteins could be obtained by a comparisonof AOPPs with other indicators of protein oxidation, such as advancedglycation end products (AGE). However, a correlation between AGEand inflammatory parameters is usually not found or is only weak, andthe induction of proinflammatory activities caused by AOPPs seems tobe more intense [25,26], suggesting that oxidative stress is more closelylinked to inflammation and acute-phase reactions than to the advancedglycation process and its end products. AOPPs could thus better describeacute inflammation, whereas AGE might serve more as a marker ofchronic long-lasting damage [25]. These observations are highly relevant,as increased levels of AOPPs could indicate the presence of inflammatoryprocesses that can potentially compromise the correct embryonicdevelopment in dairy cows [14,27]. Lipids, in particular those that arepolyunsaturated, are prone to oxidation. Lipids are one of the mostsusceptible substrates to damage by free radicals, and biomarkers oflipid peroxidation are considered the best indicators of oxidative stress[28]. Malondialdehyde (MDA) is one of several low-molecular-weightend products formed during radical-induced decomposition of polyun-saturated fatty acids [29]. MDA readily reacts with thiobarbituric acid,producing a red pigment that can be easilymeasured by spectrophotom-etry in the form of thiobarbituric acid-reactive substances (TBARS, [29]).It is worth noting that the MDA assay has been criticized for its lowspecificity and for artifact formation, since only a fraction of the MDAmeasured is actually generated in vivo. Furthermore, the TBARS assay,a common method used to measure MDA, is considered inaccurate,and returns results that differ according to the assay conditions used[30]. For example, studies on dairy cows have yielded contrasting results,with some reports failing to show any significant changes in plasmaMDA concentrations during the peripartum period [31]; whereas inother studies MDA or TBARS concentrations were shown to increasearound calving [8,16,32]. This apparent discrepancy could also havebeenmainly due to the great variation in individual MDA concentrationsmeasured in the studies by Castillo et al. [31]. Similarly, studies on

334 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347

transported cattle have failed to report a consistent change in their MDAconcentration. It seems that this discrepancy is partly due to the differentmethodologies employed to assess MDA levels. TBARS represent a widerange of lipid peroxidation products, and the thiobarbituric acid reactionis rather non-specific for MDA [33]. High-pressure liquid chromatogra-phywould be expected to be highly specific, and perhaps, more accuratethan the spectrophotometric procedures [34]. More recently, an ELISA-based method for the detection of isoprostane, which is considered tobe themost reliablemarker of lipid oxidation [35], has become commer-cially available and might be able to shed more light on the role of lipidperoxidation during the peripartum period in ruminants.

3. Potential antioxidant biomarkers

Epidemiologic literature that focuses on antioxidant status andchronic disease risk has in the past relied primarily upon biomarkersof exposure to antioxidant nutrients. However, this approach is inessence using exposure estimates to a select group of nutrients as a sur-rogate for estimating actual oxidative defense or oxidative stress status.Emphasis is now being placed on developing functional biomarkers ofoxidative stress status, that is, biomarkers that integrate the effect ofexposure to oxidants coupled with the full range of antioxidantprotectivemechanisms in vivo. Many of such biomarkers are beingstudied including various measures of oxidation products of lipid,DNA, and protein (Table 1). Some of these biomarkers are now beingapplied in research of pathologies related to oxidative stress.

3.1. Superoxide dismutase (SOD)

The term superoxide dismutase characterizes a family of enzymaticproteins differing in their structure and cofactors, among them beingMn-SOD and Cu–Zn-SOD. SOD activity enhances the spontaneousdismutation of superoxide radicals to H2O2 [52]. SOD can be measuredby utilizing the technique of Misra and Fridovich [43], which is basedon inhibition of the formation of nicotine amide adenine dinucleotide,phenazine methosulfate, and amino blue tetrazolium formazan.

3.2. Catalase (CAT)

The end product of the dismutation reaction, i.e., H2O2, can be re-moved by the activity of the enzyme catalase. CAT is an enzyme witha very high KM for its substrate and can remove H2O2 present in highconcentrations [52]. The activity of CAT can be measured through the

Table 1Biomarkers of oxidative stress.

Biomarkers Location Reference

Carbonyls Extracellular Mohanty et al. [36]Lipid peroxidation•Malondialdehyde Extracellular Ohkawa et al. [37]•F2-isoprostane Extracellular Collins et al. [38]•4-Hydroxynonenal Extracellular Halliwell and Gutteridge [39]

Ferric reducing ability of plasma Extracellular Benzie and Strain [40]Plasma vitamins•Vitamin C Extracellular Roe and Kuether [41]•Vitamin E Extracellular Teissier [42]

Antioxidant enzymes•Superoxide dismutase Intracellular Misra and Fridovich [43]•Catalase Intracellular Aebi and Bergmeyer [44]•Glutathione peroxidase Intracellular Floh'e andG¨unzler [45]•GSH/GSSG ratio in erythrocyte Intracellular Hissin and Hilf [46]

Prooxidant enzymes•Xanthine oxidase Intracellular Haining and Legan [47]•NADPH oxidase Intracellular Nauseef [48]

Others•Endothelial microparticles Extracellular Burger and Touyz [49]•Endothelial progenitor cells Extracellular Touyz and Schiffrin [50]•Ischemia modified albumin Extracellular Sinha et al. [51]

non spontaneous decomposition of hydrogen peroxide, by the methoddescribed by Aebi [44].

3.3. Glutathione peroxidase (GPx)

The biochemical function of glutathione peroxidase is to reduce lipidhydroperoxides to their corresponding alcohols and to reduce free H2O2

to water. In contrast to catalase, peroxidase possesses a high affinity forand can remove H2O2 even when it is present in a low concentration[52]. Its activity can be estimated by the method described by Floheand Gunzler [45].

3.4. Thiol index

The GSH/GSSG index is a parameter of the intracellular redox status.GSH is the major endogenous antioxidant produced by cells; and itparticipates directly in the neutralization of free radicals and reactive-oxygen compounds, as well as in maintaining exogenous antioxidantssuch as vitamins C and E in their reduced forms [53]. GSH and GSSGlevels can be measured in erythrocytes by the method described byHissin and Hilf [46].

3.5. Xanthine oxidase (XO)

XO is a key enzyme involved in the formation of reactive oxygenspecies, and it plays a major role in cell oxidative stress. This enzymecatalyzes the oxidation of hypoxanthine to xanthine and can furthercatalyze the oxidation of xanthine to uric acid [54]. Xanthine oxidasecan be estimated by the method of Haining and Legan [47].

3.6. NADPH Oxidase

TheNADPH-oxidase complex utilizes electrons to produce superoxideradicals from the oxygen molecule. NADPH oxidase may be measured bya chemiluminescence [55] or electrochemical method among others.Approaches to quantitate oxygen consumption, extracellular release ofO2∙ or H2O2, and intracellular O2

∙ production provide reliable assessmentof NADPH oxidase activity in a given population of cells [56].

3.7. A biomarker of cardiovascular damage

Endothelial microparticles (EMPs) are nuclear fragments of cellularmembrane shed from oxidatively stressed or damaged cells. Typicallydefined as having a diameter of 0.1 to 1.0mm, thesemicroparticles con-tain surface proteins and cytoplasmic material of the parental cells [57].

Many biomonitoring studies have investigated the role of antioxi-dants in reducing oxidatively generated DNA damage in urine andwhite blood cells. A collective interpretation is difficult because manystudies lack sufficient control and have unreasonably high baselinelevels of oxidatively damaged DNA. to investigating antioxidant effectshas been the use of biomarkers of oxidatively damaged DNA. This isbased on the mechanistic rationale that dietary antioxidants inhibitthe oxidation of DNA. With the possibility of detecting 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), the first of many antioxidantsupplementation studies with focus on this lesion in WBC appeared inthe beginning of the 1990s. At the same time, reliable detection of uri-nary 8-oxodG excretion was achieved, and this was soon followed byantioxidant supplementation studies using urinary 8-oxodG excretionas key biomarker. However, by far the most popular method in antiox-idant intervention trials has been the comet assay that detects DNAstrand breaks (SB). An enzyme-modified version of the comet assayhas been developed to detect oxidatively altered nucleobases byincluding a DNA digestion step with DNA glycosylase or endonucleaseenzymes [58]. The characteristics of the intervention studies areoutlined in Table 2.

Table 2Administrations of dietary antioxidant with assessment of oxidatively DNA damage in white blood cells and urine.

Supplement per day Subjects Age(years)

Effect References

β-carotene (20 mg) for 14 weeks 122 M (S) 39 (NR) No effect on urinary excretion of 8-oxodG/24-h (HPLC) [59]β-carotene (30 mg) for 1 months 14 M (NS) 19–22 No effect on urinary excretion of 8-oxodG/24-h (HPLC) [60]Carotenoidsa for 3 weeks 32 MF(NS) 32 ± 11 No effect versus baseline, but decreased urinary excretion of 8-oxodG ELISA)

in active group post-supplementation[61]

VitaminC (500 mgas plain or slow release formulations)and vitamin E (182 mg) for 4 weeks

48 M (S) 39 ± 12 Decreased ENDOIII and FPG sites (Comet) in the group that ingested tabletswith the slowrelease vitamin C formulation

[62]

Multi-vitamin tablet (100 mg vitamin C, 280 mg vitaminE, and 25 mg b-carotene) for 20 weeks

100 M (50S) 50–59 Decreased ENDOIII sites (Comet) after 20 weeks in WBC [63]

Vegetable consumption (3.6 vs 12 servings) for2 weeks

64 F (NR) 23–81 Lower 8-oxodG in WBC (HPLC) among those subjects eating 12 servings per day [64]

Kiwi fruit (1–3 pieces) for 3 weeks 14 MF (NS) 26–54 Decreased ENDOIII and FPG (Comet) sites [65]Cranberry juice (750 ml) for 2 weeks 20 F (2S) 18–40 No effect on ENDOIII (Comet) in WBC [66]Blackcurrant juice or anthocyanine drink(475–1000 ml) for 3 weeks

57 MF (6S) 19–52 No effect on ENDOIII and FPG sites (Comet) in WBC [67]

Green tea or black tea (four cups) for 1–4 months 120 MF(S) 18–79 Decreased excretion of 8-oxodG (ELISA) in spot urine samples after 4 moin green tea group, but not before

[68,69]

Two interventions of green tea with 300 ml for 7 daysor 32 oz for 7 days

68MF (13S) 18–45 Decreased 12-h urinary excretion of 8-oxodG (HPLC) [70]

Green tea extract b for 3 weeks 16 M (8S) 20–31 No effect related to supplementation on 24-h urinary excretion of 8-oxodG(HPLC) but a decreased excretion during the study in all groups

[71]

Green tea (500 or 1000 mg) among high-risk subjectsof liver cancer for 3 months

124MF(NR)

NR Lower 8-oxodG/24-h (HPLC) after 3 months supplementation but not after1 month

[72]

Soya-hypocotyl tea (N1000 ml) for 1 months 38 F (NR) NR Decreased excretion of 8-oxodG (ELISA) in active group (statisticaltest not reported)

[73]

Soy milk, rice milk, or cow milk (1 l) for 4 weeks 10 M (NS) 20–50 Decreased ENDOIII sites (Comet) for soy milk [74]Polyphenol-rich olive oils (25 ml) for 40 days 12 M (NS) 20–22 Decreased excretion of 8-oxodG (HPLC) in spot urinary samples following

supplementation in a dose-dependent manner[75]

Olive oil (25 ml) with three different content of phenoliccompounds for 3 weeks

182 M (NS) 20–60 Decreased 8-oxodG/24-h urinary excretion, but no difference in urinary excretionof 8-oxoGua/24-h. No difference in the effect of the different olive oils

[76]

Antioxidant rich diet or tabletsc for 5 weeks 55 MF (NR) 71 ± 6 No effect versus baseline, but decreased 24-h urinary excretion of 8-oxodG(ELISA) in active group post-supplementation

[77]

Number of subjects indicated as males (M) and females (F). Smokers (S) and non-smokers (NS) are indicated in brackets. Lacking information is indicated as NR (not reported) Age isshown as range or mean ± standard deviation. aSupplement constitute b-carotene (6 mg), a-carotene (1.4 mg), lycopene (4.5 mg), bixin (11.7 mg), lutein (4.4 mg), and paprika carot-enoids (2.2 mg), b Consisted of 1000 mg extract/kg bodyweight inmeat patties (total phenolics were 23.5 mg/10 MJ), cConsisted of tablets with vitamin antioxidants (400 mg vitamin C,150 mg vitamin E, 4 mg b-carotene), capsules (90 mg vitamin C, 18 IU vitamin E, 2.4 mg b-carotene, and powder or extract of fruits and berries), or a carotenoid-rich diet.

335P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347

4. Mechanism of antioxidant action and nutrients

It is reported that antioxidants can execute protective role againstfree radicals by a variety of different mechanism including the catalyticsystems to neutralize or divert ROS, binding or inactivation ofmetal ionsprevents generation of ROS by Haber–Weiss reaction, suicidal and chainbreaking antioxidants scavenge and destroy ROS or absorb energy,electron and quenching of ROS. In 21st century, demand for intake ofantioxidant food or dietary antioxidant increasing with the hope tokeep body healthy and free from diseases [78,79] and the potentialbeneficial effects of antioxidants in protecting against disease havebeen well established. It is increasingly thought that nutrition mayplay a vital role in helping to defend against oxidative stress and damage

Table 3Effects of the health associated to the intake of Antioxidants.

Antioxidants Effect of health

Vitamin C Protects against cancers, Protects from heart disease, Improvement of tcartilage, joints and skin, Maintaining a healthy immune system, Improantibody production, Increase in the absorption of nutrients, Increasesagainst H2O2-induced DNA strand breaks

Vitamin E Prevents coronary heart disease, Prevents the formation of blood clotsincidence of breast and prostate cancers, Brain protection, Reduces lonof dementia Decreases risk of Parkinson's disease

Polyphenol Inhibit oxidation of LDL, Inhibit platelet aggregation, Improve endothelLower risk of myocardial infarction, Effect anticarcinogenic Prevent neudiseases, Protect against neurotoxic drugs, treatment of diabetes, treatmosteoporosis, Inhibit non-heme iron absorption

Cu, Zn, Mn, Se Cofactors of antioxidant enzymes SOD-Cu/Zn, Mn-SOD and GSH-PoxOther carotenoids(lycopene)

Protection against oxidation of lipids, LDL, proteins and DNA. Abductioradical scavenging

induced by free radicals. Therefore, certain nutrients and dietary com-ponents with antioxidant properties are important for the protectionagainst oxidative stress injury of the body. Food consumption is amajor source of exogenous antioxidants and has been estimated that atypical diet provides more than 25,000 bioactive food constituents asnutrients and many of this may modify a multitude of processes thatare related to different diseases. Generally, antioxidants are abundantin vegetables and fruits and are also found in grain cereals, peas,legumes, nuts and other food products. At this juncture, a systematicsurvey has also identified more than 3100 antioxidants in foods, likebeverages, spices, herbs and supplements which are regularlyconsumed by different cultures [80]. It has been speculated that the de-crease in the intake of nutritional and antioxidants rich food may

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increase the chances of oxidative stress whichmay lead to cell damage,therefore intake of such natural antioxidants may give protective effectagainst free radical induced diseases [80,81]. In view of the importanceof antioxidants, Suntres [82] reported that antioxidant liposomes willhold an important role in future research on antioxidants, and alsoreports that it facilitates the delivery of antioxidants to specific sites aswell as achieving prophylactic and therapeutic action. In addition,Bouayed and Bohn [83] have suggested that the balance between oxida-tion and anti-oxidation is critical in maintaining a healthy biologic sys-tem and low doses of antioxidants may be favorable to this system,but high quantities may disrupt the balance. It is true that antioxidantsare beneficial and display a useful role in the maintenance of the ho-meostasis in human ROS system, but so are pro-oxidants, thereforethe scientific community should search deeper into the kinetics andin vivo mechanisms of antioxidants to uncover the optimal concentra-tions or desired functions in order to push forward against cancer,neurodegenerative and cardiovascular diseases [84] (Table 3). Variousstudies related to free radicals, oxidative stress and antioxidant activityof food reveals the prominent beneficial role of antioxidant and its spe-cific role against different diseases individually. However the collectiveand concise data on the role of antioxidants in human diseases will bebetter and most appropriate one in order to know the exact role of an-tioxidants in all kinds of human diseases (Fig. 1). Hence, an attempthas been made in this review, to summarize the detailed role andimpact of antioxidants in certain selected dreadful human diseases.

Fig. 1. Antioxidants/oxidative s

5. Antioxidants vs human diseases

5.1. Antioxidant vs arthritis

Chronic autoimmune inflammation,which is commonly observed inrheumatoid arthritis (RA), disrupts the delicate balance between boneresorption and calcification causing the destruction of the bone andjoints. Multiple aetiologies are suspected to contribute to the formationof RA, including defective articular cartilage structure and biosynthesis,joint trauma, joint instability, congenital and developmental abnormal-ities, and inflammatory conditions [109,110]. Oxidative stress induceddamage to essential cell components caused by oxygen free radicals isa mechanism in the patho-biology of degenerative rheumatoid arthritis[109]. Hydroxyl radicals cause degradation of isolated proteoglycans,and hypochlorous acid (HOCl) fragments of collagen. Hydrogen perox-ide, which is highly diffusible, readily inhibits cartilage proteoglycansynthesis, by interfering with ATP synthesis, in part by inhibiting theglycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase inchondrocytes, aggravating the effects of proteolytic and free-radical-mediated cartilage degradation. Peroxynitrite and HOCl may facilitatecartilage damages by inactivating TIMPs. TIMP-1 inhibits stromelysins,collagenases and gelatinasesand this ability is lost after nitrusoxide (NO3

−) or HOCl treatment. HOCl can also activate latent forms ofneutrophil collagenases and gelatinase with obvious consequences.Hypochlorous acid, NO3

− andO2− reactwith ascorbate, which is essential

tress and health diseases.

Table 4Clinical trials (variables observed: vitamins C, E, selenium, and β-carotene, ORAC).

Study Sample size anddemographics

Cancer agents Dosage and mode Study design Results Strengths/weaknesses

Hematologic malignanciesClemens et al. [155] Subjects: n = 19 CY, MEL, VP16, TBI α-Toc 9 mg (TPN) Cohort study Subjects (TBI+): serum α-Toc an -Car 2

(baseline v day +12); TBI-α-Toc er time(P b .001), no significant changes β-Car

Significant differences between two groups atbaseline; nonrandomized, small cohort, nocontrols; intervention assessed the effects ofRDA only

Clemens et al. [156] Subjects: (TBI+)N = 16, mean age:30.5 y, Controls: (TBI+)n = 10, mean age:37.5 y

CY, VP16, BU, TBI TBI+: α-Toc 825 mg, β-Car45 mg, AA 450 mg, (PO); TBI+:AA 530 mg, α-Toc 9 mg (TPN)

Nonrandomized,nonblinded, controlledtrial

↓ Hepatotoxicity; no significant di rences inrates of relapse

Study groups received different TPN regimens;significant differences were not observed ateach time point; P values NR; small cohort, dif-ferent anticancer regimens; TBI+ also received130 mg AA and 13.1 mg α-Toc by TPN

Hunnisett et al.,[157] Controls: n = 19, meanage: 33 y; Subjects:n = 10, mean age: 37 y

High-dose chemotx,±TBI

VC 25 mg; VE 1000 IU (TPN) 20 sequential patients Acute 2 in VC and VE (P b. .001; P .001); nosignificant differences after day 0

Nonrandomized, small cohort; cancerdiagnosis and agents NR

Lloyd et al. [158] Controls: n = 26, agerange: 18–28 y; subjects:n = 16, intervention:n = 6

NR AA 1 g X 4 days (mode NR) Cohort study Prior to intervention: mean platele and plasma↓AA In CML controls (P b .001; P 05), meanplatelet AA↓ in CML v controls (P .05); afterintervention: ↑ in platelet and pla a AA in AL& CML (P b .025) ↑ plasma AA in L (P b .05)

Inquired about dietary intake/Cancer therapyNR; time of blood withdrawal, length ofobservation, and timing of intervention NR;methodology NR

Gynecologic cancerSundstrom et al. [159] Subjects: n = 56;

controls: n = 44, meanage: 58.7 y

DXR, CY, CDDP, MELPH Four arms: 1) VE 300 mg, Se96 μg; 2) Se 96 μg; 3) VE300 mg; 4) placebo (PO)

Controlled trial ↑ in serum Se in Se group 1 and g up 2 vcontrols (P b .001; P b .001)

Placebo controlled trial/multiple gynecologiccancers, stages, and regimens

Lung cancerJaakola et al. [160] Subjects: n = 18, age

range: 51.8–69.6 yCY, ADR, VP16, VCR,DXR, EPI CBPT, RT

MV β-Car 10,000–20,000 U,α-Toc 300–800 U, AA 2000–5000 U, Se 856 μg (PO)

Nonrandomizedcontrolled trial

Study group had increased surviv l ratescompared to historical controls ( alue NR);plasma Se, α-Toc NS, plasma AA, -Car NR

Variable doses administered depending on“individual”micronutrient analysis(methodology NR); multiple stages combined;one subject received no cancer therapy

Breast cancerLockwood et al. [161] Subjects: n = 32, age

range: 32–81 yChemotx, RT, Surgery VC 2850 mg, VE 2500 IU, β-Car

32,500 U, Se 387 μg (PO)Nonrandomized,noncontrolled trial

Whole blood β-Car, VE, and Se ↑ ter 3 and12 months (P b .01), P b .05), P b 01); nodeaths, expected four; no subject ad weightloss, ↓ use of pain killers, ↑ QOL

Whole blood VC NR, no controls, chemother-apy agents NR; subjects also administered MVcontaining Co-Q10 and essential fatty acids; Pvalues not consistently reported

Abbreviations: ORAC, Oxygen radical absorbance capacity; CY, cyclophosphamide;MELPH,melphalan; VP16, etoposide; 2, decrease; 1, increase; TBI, total body irradiation;α-Toc, alpha copherol; TPN, total parenteral nutrition;β-Car, beta carotene;RDA, recommended dietary allowances; y, years; BU, busulfan: PO, orally; AA, ascorbic acid; NR, not reported; VC, vitamin C; VE, vitamin E; DZ, diaziquone; TP, thiotepa; Ara-C, cytosin rabinoside; k-Cal, total caloric intake; CML, chronic myelocyticleukemia; AL, acute leukemia; CLL, chronic lymphocytic leukemia; VCR, vincristine; DXR, doxorubicin; EPI, epirubicin;CBPT, carboplatin; RT, radiation therapy;MV,multivitamin; NS, nonsignificant; CDDP, cisplatinumdiammine dichloride; Se, selenium; chemotx, chemotherapy; QOL, quality of life; Co 10, coenzyme Q 10; y-TOC, gamma tocopherol (Modified fromElena J. Ladas et al., 2004).

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338 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347

for cartilage function, leading to low levels of ascorbate in synovial fluid.Low concentrations of hydrogen peroxide (H2O2), O2

.− or both, acceler-ate bone resorption by osteoclasts, whereas nitric oxide (NO.) inhibits itand promotes chondrocyte apoptosis, thereby inhibits proteoglycansynthesis and activates latent metalloproteinases and cyclooxygenase.ROS, produced by activated phagocytes, could alter the antigenic behav-ior of immunoglobulin G, producing fluorescent protein aggregates thatcan further activate phagocytic cells. Radical-exposed IgG is able to bindrheumatoid factor and results in the generation of C3a. This reactionmay be self-perpetuating within the rheumatoid joint, suggesting thatfree radicals play a role in the chronicity of the inflammatory reactionwhich is a key question regarding to which extent free radicals contrib-ute to the consequences of inflammation, such as the cartilage andbone destruction. Reactive oxygen intermediates can also function assignaling messengers to activate transcription factors, like NF-κBChange all to NF-κBand activator protein 1(AP-1), and induce geneexpression [111]. All this knowledge might serve to apply a rationalselection of antioxidants for possible therapeutic purposes, of theinflammatory joint disease.

Many raw foods contains natural antioxidants, including enzymessuch as superoxide dismutase, glutathione peroxidase and catalase,which are usually inactivated during food processing, and non-enzymic antioxidants such as carotenoids (e.g. canthaxanthin andastaxanthin in some farmed fish), β-carotene, lutein, lycopene, tocoph-erols (in oils) and other compounds in plants. The latter include othercarotenoids and ascorbate (vitamin C). The plasma concentrations ofthese are largely determined by dietary intake and it is possible thattwo or more antioxidants can act together synergistically [112]. Inview of the high antioxidant content of the French diet which is richin fruit, vegetables and red wine [113,114], it is intriguing to note therelatively low incidence of arthritis [115]. More work is required on tis-sue distributions and bioavailability of antioxidant molecules withinjoints since lipophilic antioxidant molecules, such as vitamin E orβ-carotene, may not have the same access to tissues as hydrophilic an-tioxidants, such as vitamin C. Perhaps, the differences in their effects indisease processes may depend on the hydrophilicity of the antioxidantmolecules concerned and the resulting pattern of tissue distribution indifferent tissue areas. One major problem is that there is no assaycurrently available to measure oxidant activity within joints and possi-bly the activity could occur from an alternative mechanism.

5.2. Anti oxidant vs cancer

Reactive Oxygen Species is a hallmark of human cancer. ROS andtheir functions with respect to the cancer initiation and signalling incancer cells is a prime concern of cancer research. Tobacco smoke alsoplays a very important role in increasing the risk for inflammation andcancer due to its high carcinogenic potential and the synergistic effectswith other respirable particulate to generate ROS and catalyse redox re-actions in the cells of humans, leading to oxidative stress and increasedproduction ofmediators of inflammation [116–118]. Inflammatory cellsare particularly effective in generating most of the reactive oxygenspecies. The activation of the redox metabolism of the inflammatorycells generates a highly oxidative environment within an organ of aero-bic organisms. Much of the oxygen biochemistry, through the activationof plasma membrane NADPH oxidase, of macrophages and neutrophilsis directed towards the release of superoxide anion, hydrogen peroxideand hydroxyl radicals [119–121]. Inflammation acts through the forma-tion of ROS and reactive nitro species (RNS) which cause oxidativedamage to cellular components. Many pro-inflammatory mediators,especially cytokines, chemokines and prostagladins, turn on the angio-genesis switches mainly controlled by vascular endothelial growthfactors [122]. Cancer-associated inflammation is also linked withimmune-suppression that allows cancer cells to evade detection bythe immune system. Inflammation is a critical component of tumor pro-gression. Many cancers arise from sites of infection, chronic irritation

and inflammation. It is now becoming clear that the tumor microenvi-ronment, which is largely orchestrated by inflammatory cells, is anindispensable participant in the neoplastic process, fostering prolifera-tion, survival and migration. In addition, tumor cells have co-optedsome of the signalling molecules of the innate immune system such asselectins, chemokines and their receptors for invasion, migration andmetastasis. These insights are fostering new anti-inflammatory thera-peutic approaches to cancer development [123]. Pathological angiogen-esis is a hallmark of cancer and various ischaemic and inflammatorydiseases. Chronic inflammation is associated with angiogenesis, aprocess that helps cancer cells to grow. Angiogenesis is necessary forexpansion of tumor mass and macrophage, platelets, fibroblasts andtumor cells are a major source of angiogenic factors (vascular endothe-lial growth factors, chemokines, NO, etc.) [124,125]. The inflammationin the tumor microenvironment is characterized by leukocyte infiltra-tion, ranging in size, distribution and composition, as: tumor-associated macrophages (TAM), mast cells, dendritic cells, natural killer(NK) cells, neutrophils, eosinophils and lymphocytes. These cells producea variety of cytotoxic mediators such as ROS and RNS, serine and cysteineproteases, membrane perforating agents, matrix metalloproteinase(MMP), tumor necrosis factor α (TNFα), interleukins (IL-1, IL-6, IL-8),interferons (IFNs) and enzymes, likecyclooxygenase-2 (COX-2),lipooxygenase-5 (LOX-5) and phospholipase A2 (PLA2). Other re-searchers discovered that tumor-associated macrophages are key regula-tors of the link between inflammation and carcinogenesis [126] andexperimental studies showed that, tobacco smoke produce ROS thathave been associated with activation of mitogen-activated protein kinase(MAPK) family members and activation of transcription factors such asNF-κβ and AP-1. These signaling pathways have been implicated in pro-cesses of inflammation, apoptosis, proliferation, transformation and dif-ferentiation [127]. The activation protein-1 (AP-1) contributes to basalgene expression in biological systems. ROS can activate AP-1 throughseveral mechanisms and the effect of AP-1 activation is an increased cellproliferation due to increased expression of growth stimulatory genessuch as cyclin D1 and suppression of the protein p21waf. Studies showedthat AP-1 and NF-kB, inducible by tumor promoters of oxidative stimuli,show differential protein levels or activation in response to tumor pro-moters in JB6 cells. Researchers suggest that as long as oxidative eventsregulate AP-1 and NF-kβ transactivation, these oxidative events can beimportant molecular targets for cancer prevention [128,129]. NF-kB isexpressed and participates in a wide range of biological processes, suchas cell survival, differentiation, inflammation and growth [130]. NF-kB ac-tivation has been linked to a wide spectrum of extracellular stimuli andoxidants and subsequent involvement in the carcinogenic processthrough promotion of angiogenesis and tumor cell invasion andmetasta-sis [131,132].

Dietary factors like chlorogenic acid have also been found to protectagainst environmental carcinogen induced carcinogenesis by their up-regulation of phase II conjugating enzymes and suppression of ROS-mediated correct, AP-1, and MAPK activation [133]. Cellular defencesystem is comprised of several Phase II detoxification enzymes such asglutathione-S-transferases (GSTs), NADP (H) quinoneoxidoreductase(NQO1), Glutathione peroxidases (GPx), Catalase, superoxidedismutases (SODs), epoxide hydrolase, hemeoxygenase (HO-1), UDP-glucuronosyltransferases (UGTs), gammaglutamylcysteinesynthetaseandmanyothers. Expression of these proteins protects cells fromoxida-tive damage and can prevent mutagenesis and cancer [134–137]. Tocounterbalance the oxidative damage from ROS, aerobic organismshave created a variety of antioxidant mechanisms to maintain theirgenomic stability. These mechanisms include phase II detoxificationand other anti-oxidation enzymes that act in cellular defence such ascatalase and SOD, GPx, GST, other constitutive and inducible antioxi-dants, DNA repair enzymes, and other cellular mechanisms of genomicsurveillance, such as cell cycle checkpoint control systems [133]. More-over, several growth factors such as serum, insulin like growth factor I,or fibroblast growth factor 2 generates ROS in MIA PaCa-2 and PANC-1

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cells, which are human pancreatic adenocarcinoma cells that promotescell survival. Inhibiting ROS generation with the antioxidants or NADPHoxidase (Nox4) antisense, or MnSODover expression would stimulateapoptosis in PaCa-2 and PANC-1 cells. This mechanism might play animportant role in pancreatic cancer resistance for treatment and thusrepresent a novel therapeutic target. In gastrointestinal and coloncancer, oxidative pentose pathway (OPP) and the glutathione (GSH) an-tioxidant defense system play an important role in the regulation of cellgrowth and apoptosis. The OPPmodulate intracellular redox status andprovides NADPH for the synthesis of GSH, which is responsible for theinactivation of intracellular ROS that induce apoptosis and cell injury.Depletion of GSH increases the sensitivity of cells to ROS. Therapeuticinhibition of the OPP and/or the GSH defense system might increasethe sensitivity of gastric and colon cancer cells to anti-cancer therapy[138]. Recent studies have shown that the enzymatic product of thymi-dine phosphorylase (TP) generated ROS within cancer cells that help inmaintaining the growth of colon cancer cells thus may provide im-proved therapeutic results as well as a preventative effect on carcino-genesis of the colorectum [124]. Emerging evidence also suggests thatsupranutritional doses of selenite could induce typical apoptosis incolorectal cancer cells in vitro and in xenografttumors by inducingROS and it indicates that antioxidants can activate the apoptoticmachinery through redox-dependent activation and could be useful incancer therapy [139].

The MAPK signalling cascade is activated by a wide variety of recep-tors involved in growth and differentiation including receptor tyrosinekinases (RTKs), integrins and ion channels. This pathway has been re-ported to be activated in over 50% of acute myelogenous leukemia,acute lymphocytic leukemia and are frequently activated in breast,prostate cancers and other cancer types [140,141]. Under optimalgrowth conditions, transiently elevated ROS levels confer a growth ad-vantage to tumor cells. However, exposure of these cells to anticanceragents induces a prolonged increase in ROS levels resulting in potentiat-ing of apoptosis. Thus ROS modulates the ability of stress kinases tostimulate cell growth or cell death and it depends on signal intensityand signal duration [142]. This idea has been nurtured to explain thecomplex nature of ERK signaling in cell cycle regulation and in stress ac-tivated protein kinase signalling (SAPK). Transient, reduced activity ofSAPK promotes cell proliferation, whereas persistent, increased activityresults in cell killing [143]. Conversely, in drug-resistant tumors, phase IIenzymes or other antioxidant defences are often up regulated, shieldingcells from apoptosis. ASK1, an upstream regulator of SAPKs, is inhibitedin normal cells through its association with thioredoxin, which is anantioxidant capable of metabolizing ROS. Increased levels of ROS leadto the dissociation of this complex and thereby facilitate the activationof ASK1 and subsequently SAPKs [144]. A similar redox control hasbeen identified for JNKwhere increased level of ROS triggers thedetach-ment of JNK associated glutathione-S-transferase-π (GSTp) and therebyfacilitating JNK activation. Moreover, ROS-dependent activation of JNKmay also lead to knock down of a JNK phosphatase via an unknownmechanism linking ROS and stress induced kinases an important playerin cancer cell growth [145]. Growth inhibition of pancreatic cells is de-pendent on the efficiency of scavenging ROS as well as effective inhibi-tion of ERK1/2 signaling pathway activation as demonstrated by usingthe free radical scavenger N-Acetyl Cysteine (NAC) or the MEK/ERK1/2 inhibitor (PD98059). Moreover, ERK1/2 induction is dependent onROS production as demonstrated by a complete removal of ERK1/2phosphorylation by NAC. Thus ROS act as pro-survival, anti-apoptoticfactor in pancreatic cancer cells [52]. Moreover, treatment of SW620(colon cancer cells) with berberine, which is a major constituents ofCoptidisthizoma, resulted in activation of apoptosis by phosphorylationof JNK and p38 MAPK, as well as generation of the ROS [146]. Further-more, proline oxidase (POX), that often considered as a ‘housekeepingenzyme’, induce apoptosis through both intrinsic and extrinsicpathways and is involved in nuclear factor of activated T cells (NFAT)signaling and regulation of the MEK/ERK pathway in colon cancer

cells. It has been suggested that, as a nutrition factor, POXmaymodulateapoptosis signals induced by p53 or other anti-cancer agents andenhance apoptosis in stress situations [147].

Components of the cell signaling network, especially those whichconverge on the ubiquitous eukaryotic redox-sensitive transcriptionfactor nuclear factor-kappa B (NF-κB), have been implicated in patho-genesis of many inflammation-associated disorders. Under normalphysiologic conditions, NF-κB is sequestered in the cytoplasm by bind-ing to the inhibitory protein called IκBa. Phosphorylation and subse-quent ubiquitination results in degradation of IκBa by proteasomes.Phosphorylation of IκBa is mainly mediated by the IκB kinase (IKK)complex. Phosphorylation of specific serine residues of p65 subunit ofNF-κB has been considered to facilitate the translocation of NF-κB tonucleus and interaction with the coactivator CBP/p300. Induction ofκphase-2 detoxifying or antioxidant genes represents an important cel-lular defense in response to oxidative and electrophilic insults. Nucleartranscription factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2)plays a crucial role in regulating phase-2 detoxifying/antioxidant geneinduction. Like NF-κB, Nrf2 is present in the cytoplasm as an inactivecomplexwith the inhibitory protein subunit, in this case Keap1. Dissoci-ation of Nrf2 from Keap1 is prerequisite for nuclear translocation andsubsequent transactivational activity. Once translocated into nucleus,Nrf2 interacts with a small Maf protein, forming a heterodimer thatbinds to antioxidant responsive elements (ARE) or electrophile respon-sive elements (EpRE) present in the promoter/enhancer regions ofgenes encodingmany antioxidant and detoxifying enzymes. Keap1 con-tains several cysteine residues that function as sensors of redox changes.Oxidation or covalent modification of these critical cysteine thiolsdiminishes the affinity of Nrf2 for Keap1, releasing Nrf2 for nucleartranslocation. Dissociation of the Nrf2-Keap1 complex is also assumedto be stimulated through the phosphorylation of Nrf2 by distinct up-stream kinases such as MAPKs, PKC, PI3K, etc. As in the case of NF-κB,phosphorylation of Nrf2 is also considered to facilitate the interactionof this redox-sensitive transcription factor with CBP/p300.NRF2 isa known regulator of the antioxidant response [148,149]. NRF2-regulated phase II enzymes protect against the development of cancerby catalyzing reactions that convert highly reactive, carcinogenicchemicals to less reactive products [150,151]. Singh et al. (2012) [152]recently demonstrated that vitamin C and BHA provide protectionagainst E2-mediated oxidative DNA damage but the mechanism is notwell understood.Many antioxidants derived fromdietary andmedicinalplants have been found to modulate Nrf2-Keap1 signaling, therebypotentiating cellular antioxidant capacity or facilitating detoxificationof carcinogens and other toxicants [153,154]. Table 4 describes 7 obser-vational studies reporting changes in serum, plasma, or whole bloodlevels of vitamins Cand E, selenium,β-carotene, and total radical antiox-idant parameter (TRAP), which represents total body antioxidant status,in patients undergoing anticancer therapy. Information is grouped bymalignancy and includes subject demographics, cancer data, cancertreatment, end points evaluated, results, and comments. No specifictreatment was consistently associated with changes in the individualantioxidants (Table 4) [162].

5.3. Antioxidant vs diabetes

Glucose overload may damage the cells through oxidative stress.This is currently the basis of the “unifying hypothesis” thathyperglycemia-induced oxidative stress may account for the pathogen-esis of all diabetic complications [163,164]. Increasing evidence in bothexperimental and clinical studies suggests that oxidative stress plays amajor role in the pathogenesis of both types of diabetes mellitus. Freeradicals are formed disproportionately in diabetes by glucose oxidation,non-enzymaticglycation of proteins, and the subsequent oxidativedegradation of glycatedproteins [165]. This increased superoxideproduction causes the activation of 5 major pathways involved inthe pathogenesis of complications: polyol pathway flux, increased

Fig. 2. Shows the involvement of cellular antioxidant enzymes in cardiovascular diseases [216–227].

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formation of AGEs (advanced glycation end products), increased ex-pression of the receptor for AGEs and its activating ligands, activationof protein kinase C isoforms, and overactivity of the hexosamine path-way. It also directly inactivates 2 critical anti-atherosclerotic enzymes,endothelial nitric oxide synthase and prostacyclin synthase. Throughthese pathways, increased intracellular reactive oxygen species causedefective angiogenesis in response to ischemia, activate a number ofpro-inflammatory pathways, and cause long-lasting epigenetic changesthat drive persistent expression of proinflammatory genes after glyce-mia is normalized (“hyperglycemic memory”). Atherosclerosis and car-diomyopathy in diabetes are caused in part by pathway-selectiveinsulin resistance, which increases mitochondrial ROS productionfrom free fatty acids and by inactivation of anti-atherosclerosis enzymesby ROS. Over expression of antioxidant enzymes in diabetic patientsprevents diabetic retinopathy, nephropathy, and cardiomyopathy[166]. Antioxidants have proven to play a minimal if any role in thetreatment of diabetic complications in humans [167]. Decreases in ex-pression, and in some instances the activity of antioxidant enzymes,has been previously reported in diabetic microvascular disease [168].Indeed, the over expression of Cu+Zn2+ superoxide dismutase (SOD)protects against end organ damage in models of type II diabetic ne-phropathy [169]. Other studies inmicewith genetic deletions of variousantioxidant enzymeshave also provided insight into the specific relativecontributions of Mn2+SOD [170] to the development of diabetes com-plications. Mn2+SODmimetics such asMnTBAP have also shown effica-cy in preventing ROS-induced injury in vitro, although the utility in vivoof such drugs may be limited [171,172]. Further strengthening a poten-tial role for the antioxidant Mn2+SOD, specific polymorphisms of theMn2+SOD gene are associated with the development of diabetic com-plications [173]. Interestingly, GPx-1-deficient mice have no increased

risk for microvascular disease, in particular diabetic complications[174], most likely because of redundancy with respect to GPx isoforms.Over-expression of catalase in experimental models of type 2 diabeticappears to be protective the diabetic complications [175]. In contrastto Mn2+SOD, however, studies in humans have indicated no relation-ship between catalase gene polymorphisms that interfere with itscellular expression and the incidence of type 2 diabetic patients [176].

Kunisaki et al. [177] investigated the effects of treatments with clas-sical antioxidants such as vitamin E,vitamin C and lipoic acid. Specifical-ly, vitamin E normalizes retinal blood flow and protein kinase C (PKC)activity in the vascular tissue of diabetic rats. One short-term experi-mental study showed that high doses of vitamin C can improve some as-pects of endothelial dysfunction in diabetes. Another experimentalstudy has demonstrated that intra-peritoneal administration of α-lipoic acid to streptozotocin (STZ) diabetic Wistar rats normalisesTBARS levels in plasma, and the retina, liver, and pancreas [178]. Fur-thermore, it has been reported that α-lipoic acid leads to a decrease inthe severity of diabetic neuropathy by maintaining GSH levels and/orby its direct antioxidant properties [179]. However, lipoic acid adminis-tration improved endothelial function in subjects with metabolic syn-drome. In another study to determine the effects of vitamin E onoxidative stress and cell membrane fluidity in the brains of diabetes-induced experimental rats, Hong et al. [180] reported that vitamin Ewas found to be effective for strengthening the antioxidant defense sys-tem. They noted a reduction of the accumulation of ROS such as super-oxide radicals, a decrease in the generation of oxidative damagingsubstances such as the carbonyl value, a significantly improved lipidcomposition, and maintenance of membrane fluidity in the brains ofthe rats. Coleman suggested that triple antioxidant therapy (vitamin E,lipoic acid and vitamin C) in diabetic volunteers attenuates the

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experimental oxidative stress ofmet-hemoglobin formation in vitro andreduces haemoglobin glycation in vivo [181]. Panjwani et al. [182]suggested that vitamin C administered alone or in combination withvitamin E reduced the fall in ulnar nerve conduction velocity. Sivanand Reece [183] investigated whether dietary supplementation withvitamin E would reduce the incidence of diabetic embryopathy in anin vivo rat model. Fifty pregnant rats were designated as the controlgroup and received a normal diet while the diabetic group received vita-min E supplements (400 mg/day). This experimental study found thatsupplementation with vitamin E reduces the incidence of neural tubedefects bymore than 75% [184]. These findings suggest that vitamin E re-duces this oxidative load, confers a protective effect against diabeticembryopathy, and thus may potentially serve as a dietary prophylaxis inthe future. These results are in line with the reports of Chang et al. [184]which showed that diabetic embryopathy of rat or mouse embryos isprevented by vitamin C, vitamin E, SOD, N-acetyl-cysteine, or glutathioneethyl ester. Another study by Otero et al. [185] showed the effects ofsupplementation with vitamin E to diabetic mice. The beneficial effectof vitamin E observed in this model was shown to retard coronaryatherosclerosis accelerated by DM, and was demonstrated to be due to areduction in oxidative stress, and not secondary to a decrease in plasmaglucose or cholesterol since their respective plasma concentrationsremained unchanged in the diabetic mice supplemented with vitamin E[186]. Furthermore, it has been recently reported that macrophagesfrom diabetic mice are under excess oxidative stress, and that the antiox-idant vitamin E can attenuatemacrophage oxidative stresswhich exists inDM and leads to accelerated atherosclerosis development. In a placebo-controlled, randomized trial of diabetic patients, Reaven investigatedthe effect of 1600 IU of RRR-α-tocopherol supplementation daily for10 weeks on hyperglycemia-induced LDL modifications. The result ofthe study pointed to a reduction of approximately 60% of plasma LDLoxidation in diabetic patients, which was statistically significant whencompared to healthy controls [187]. These results are supported and con-firmed by other similar clinical data. Salonen et al. [188] found that dosesequal to or higher than 450 IU are sufficient to significantly amelioratethe susceptibility of LDL to oxidation, indicating that relativelyhigh doses of RRR-α-tocopherol for supplementation are needed.Furthermore, Bellomo and others have shown that the effects of RRR-α-tocopherolsupplementation on LDL oxidation are accompanied by aconcomitant reduction in autoantibody levels againsthyperglycemia-induced LDL modifications. Moreover, clinical studies have shownan inhibitory effect of RRR-α-tocopherol supplementation onthehyperglycemia-induced LDL modifications inT1DM and T2DM dia-betic patients. Based on nutrition recommendations and interventionsfor diabetes, there is no clear evidence of benefits that can be derivedfrom vitamin or mineral supplementation in people with diabetes.Routine supplementation with antioxidants, such as vitamins E and Cand carotene, is not advised because of lack of evidence of efficacy, andconcern related to long term safety, and therefore cannot be recom-mended [189]. The majority of studies included in this review supporta possible role of antioxidant supplementation in reducing the risk ofdiabetic complications

5.4. Antioxidant vs arthrosclerosis

The mechanisms of oxidative stress and antioxidants are in patientswith atherosclerosis have been evaluated in extensive animal experi-ments and clinical research. There is a relationship between atheroscle-rotic risk factors (ARF) and increased vascular production of ROS; themost important ARFs are heredity, age, hypertension, dyslipidemia,diabetes and smoking [190]. It has been reported that in in-vitro studiesan increased vascular production of ROS, particularly superoxide causesdeleterious effects in cell death [191,192]. ROS and reactive nitrogenspecies (RNS) are closely linked to the disease process. Incomplete scav-enging of ROS and RNS influences the mitochondrial lipid cardiolipin,stimulates the release of mitochondrial cytochrome c and finally

activates the intrinsic death pathway [193]. Local generation of RNSmay contribute to vascular tissue injury. Therefore, ROS and RNS partic-ipate as signalling molecules that regulate diverse pathophysiologicalsignallingpathways. High levels of ROS are potent inducers of the intrin-sic apoptotic pathway and tissue injury in pathophysiological condi-tions as an integral part of atherosclerotic plaque stabilization. Themost important sources of ROS production associated with CVD pathol-ogy are the mitochondrial respiratory chain, nicotinamide adeninedinucleotide phosphate oxidases (NADP oxidase). The pathogenesis ofatherosclerosis is further related to inflammation, immune responseand the proliferative process. Endothelial denuding injury leads toplatelet aggregation and releases platelet-derived growth factor,which triggers the proliferation of smooth muscle cells forming thenidus of the atherosclerotic plaque in the arterial intima, implicating in-flammatory changes in the development of the disease [194,195].Supplementing treatment with antioxidants might be helpful to pa-tients. Vitamin E reduces the consequences of lipid peroxide contentin mouse peritoneal macrophages (MPMs) [196]. E-selectin has beenproposed as an important factor in the development of the inflammato-ry process underlying atherothrombosis. The expression of E-selectin isdecreased by vitamin E in the diabetic rats [197]. Vitamin C has alsobeen investigated in studies on the prevention of atherosclerosis. Thefollowing proposals have been put forward regarding the mechanismsof vitamin C in preventing atherosclerosis: First, vitamin C has beenshown to prevent apoptosis caused by cytokines in cultured endothelialcells [198]. It also decreases the release of micro-particles derived fromendothelial cells and suppresses pro-apoptotic activity in congestiveheart failure patients in vivo [199]. Second, vitamin C stimulates alltypes of collagen synthesis by specific hydroxylase enzymes [200].Endothelial cell proliferation is, in part, associated with the synthesisof type IV collagen [201]. Thus, the lack of vitamin C prevents thegeneration of type IV collagen in cultured endothelial cells [202].Third, vitamin C protects the vascular endothelium by enhancing endo-thelial NO synthase. Endothelial NO synthase activity is inhibited byROSthat oxidize and deplete the co-factor tetrahydrobiopterin [203]. There-fore, vitamin C prevents the loss of NO synthase activity bymaintainingtetrahydrobiopterin [204]. Glutathione monoethyl ester, but not ascor-bic acid, exerted protective effects against ischemia–reperfusion injury.Interestingly, the protective effects of glutathione monoethyl ester areenhanced by co-administration with vitamin C in rat hearts subjectedto ischemia and reperfusion [205]. The oxidized LDL leads to increasedplatelet-endothelial cell adhesion, which can be prevented by superox-ide dismutase (SOD) and catalase [206]. In fact, the vascular extracellu-lar expression of SOD is stimulated by NO [207]. Narang et al. [208]reported that dietary palmolein oil, which is rich in monounsaturatedfatty acid and antioxidant vitamins, “protected rat heart from oxidativestress associated with ischemiareperfusion injury”. Das et al. [209]found “a role for c-Src [a family of proto-oncogenic tyrosine kinases]in post ischemic cardiac injury and dysfunction and demonstrate directcardio protective effects of [the tocotrienol-rich fraction of palm oil(TRF)]. The cardio protectiveproperties of TRF appear to be due toinhibition of c-Src activation and proteasome stabilization”. Carlsonet al. [210] using a rat model, reported: “Antioxidant vitamin therapy[vitamin C, vitamin E, vitamin A and zinc] abrogatedmyocardial inflam-matory cytokine signaling and attenuated sepsis-related contractiledysfunction, suggesting that antioxidant vitamin therapy may be apotential approach to treat injury and disease states characterized bymyocardial dysfunction”. Hypertension is directly regulated by thekidneys and cardiovascular system and adversely affects these organs.Tian et al. [211] found that in salt sensitive rat model of hypertension,vitamin C and vitamin E treatments “decreased renal inflammatorycytokines and chemokines, renal immune cells, NF-κB, and arterial pres-sure and improved renal function and damage”. As noted above, chroniczidovudine administration promotes cardiovascular damage, and vita-min C has been found to combat this effect in rats [212]. Diabetesmellitus may initiate increased myocardial vulnerability to ischemia–

342 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347

reperfusion injury and pro/antioxidant imbalance. Resistance toischemia-induced ventricular arrhythmias and levels of endogenous an-tioxidants (α-tocopherol) has been found to be increased in diabetic ratmyocardium [213]. The currently available evidence needs to be con-firmed in clinical trials. Consumption of fruit and vegetable diets richin antioxidant nutrients can be recommended for all individuals, andthere is some indication that such a diet can be beneficial in the effectsof cardiovascular events. Fig. 2 shows the involvement of cellular anti-oxidant enzymes in cardiovascular diseases.

5.5. Anti oxidant vs neurodegerative diseases

Neurodegenerative disorders are a heterogeneous group of diseasesof the nervous system, including the brain, spinal cord, and peripheralnerves that have much different aetiology. ROS are particularly activein the brain and neuronal tissue as the excitatory amino acids andneurotransmitters, whose metabolism is factory of ROS, which areunique to the brain and serve as sources of oxidative stress. ROS attackglial cells and neurons, which are post-mitotic cells and therefore,they are particularly sensitive to free radicals, leading to neuronal dam-age [226]. It has been reported that deleterious effects of ROS on humancells may end in oxidative injury leading to programmed cell death i.e.apoptosis [227]. A multitude of genes involved in redox status, anti-inflammation and detoxification are transcribed by Nrf2–ARE pathwayactivation. These genes are known to be involved in cytoprotectionfrom various oxidative insult and cellular injuries in numerous differenttissues and organs including brain. Intracellular peroxidases are clearedby a group of enzymes that are transcribed by Nrf2–ARE pathway. Theperoxisomal CAT catalyses the conversion of H2O2 to water and molec-ular oxygen. However, the specific activity of CAT ismuch lower in brainthan peripheral tissue [228]. Glutathione-s-peroxidase (Gpx) is anotherenzyme that metabolizes H2O2 and depends on reduced glutathione(GSH). The oxidized GSH (GS-SG) is recycled to GSH by glutathionereductase (GR). Glutathione (GSH), a tripeptide γ-glutamyl-cysteinyl-glycine, is the most abundant low molecular weight thiol expressedubiquitously. It is widely recognized as an endogenous non-enzymaticantioxidant and an oxyradical scavenger, and is thereby critical tomain-taining a reducing environment in the cell and protect against oxidativedamage by ROS [229–233]. GSH has been implicated in a wide range ofmetabolic processes, including cell division, DNA repair, regulationof enzyme activity, and activation of transcription factors, modulationof anion and cation homeostasis, and protection against oxidativedamage [234].

GSTs are key detoxification enzymes that catalyze the conjugation ofvarious electrophiles, reactive alkenals, and numerous other xenobioticto GSH. These GSH-S-conjugates are removed from cells by the multi-drug resistant protein-1 (MRP-1), an ATP binding cassette (ABC) familyprotein [235,236]. MRP-1 is an integral plasma membrane protein thatexports glutathione-S conjugates out of the cell in an ATP-dependentmanner [237,238]. Studies have shown reduced GST activity in brainand ventricular fluids in AD [239]. Increased expression of GST leadsto increased resistance towards oxidative stress in neuroblastoma cellsand provides protection against HNE-mediated toxicity in neuronalcell culture [239]. Growing evidence demonstrates that the AD brain isunder tremendous oxidative stress. A significantly increased HO-1 ex-pression was reported in post-mortem AD temporal cortex and hippo-campus compared to aged-matched control [240]. Additionally, anincreased Nqo1 activity and expression was found in astrocytes andneurons of AD brain [229] and Nrf2 was predominantly localized incytoplasm in AD hippocampal neurons [241]. Furthermore, there is in-creased protein oxidation [242,243] and lipid peroxidation [244–246]in AD brain when compared to aged matched controls. Recent studiesin aged APP/PS1 AD mouse models showed reduced Nrf2, Nqo1, GCLcatalytic subunit (GCLC) and GCL modifier subunit (GCLM) mRNA andNrf2 protein levels [247]. Additionally, in a triple transgenic ADmouse, the GSH/GSSG ratio was reported to be reduced [248].

The hallmark of PD is a severe reduction of dopamine in all compo-nents of the basal ganglia. Dopamine and its metabolites are depletedin the caudate nucleus, putamen, globuspallidus, nucleus accumbens,the ventral tegmental area, and the substantianigra pars compacta andreticulata. Moderate losses of dopamine are found in the lateral hypo-thalamus, medial olfactory region and amygdaloid nucleus [249]. Inearly parkinsonism, there appears to be a compensatory increase indopamine receptors to accommodate the initial loss of dopamineneurons [250]. As the disease progresses, the number of dopamine re-ceptors are decreases, apparently due to the concomitant degenerationof dopamine target sites on striatal neurons. In the remainingneurons inpatients with PD, dopamine turnover seems greatly increased, judgingfrom the concentrations of homovannilic acid [HVA] in the nerve termi-nals in the striatum and the cell bodies and dendrites in the substantianigra [251] and the ROS production may very well increase in conse-quence. This hypothesis is strengthened by a study showing that theconcentrations of GSH decrease when dopamine turnover increaseafter reserpine treatment in rats, indicating increased activity of theperoxide scavenging enzyme GSH-Px [252]. If the increase in ROSproduction due to increased dopamine turnover is not buffered by thescavenging enzymes (SOD, catalase, and GSH-Px), the compensatoryhyperactivity of the dopaminergic neurons may become self-destructive. Chronic administration of L-DOPA would then only exacer-bate the production of destructive ROS [253]. The administration ofL-DOPA itself has been postulated to enhance the accumulation of ROS[254]. Another index of oxidative stress in PD might be the evidenceof a robust increase of NF-κB in the nuclei of dopaminergic neurons inthe substantia nigra of PD patients [255]. This clinical finding is consis-tent with in vitro data showing that oxidative stress induced by C2-ceramide treatment causes nuclear translocation of NF-κB in culturedmesencephalic neurons [256]. More recently, it has been shown thatthe neurotoxin 6-OHDA activates NF-κB in PC12 cells by enhancing in-tracellular ROS levels [257]. Interestingly, in this experimental model,NF-κB seems to sustain cell survival by stimulating the expression ofthe anti-apoptotic proteins Bcl-2 and Bfl-1144. Moreover, as alreadymentioned, the potent green tea polyphenol antioxidant EGCG exertsa neuroprotective effect in a MPTP mouse model of PD [258]. However,there are still few and controversial epidemiological data on this impor-tant point, which might be partly due to the intrinsic difficulties inperforming epidemiological surveys regarding the dietary habits oflarge populations. Nevertheless, it is desirable that future studiesaimed at investigating the relationship between dietary antioxidantintake and the relative risk for neurodegenerative disorders such asAD, PD, and ALS will throw more light on this very important aspect ofpublic health.

6. Conclusion

In summary the reactive oxygen species and oxidative stress play animportant role in the aetiology and progression of major human degen-erative diseases has triggered enormous and worldwide interest inendogenous and exogenous antioxidants. There is now abundant evi-dence that substances in fruit and vegetables are potent preventives ofvarious diseases, especially arthritis, cancer, heart disease, diabetesand neurodegenerative diseases. With these recent developments inscientific knowledge which firmly establish the links between foodfactors and the prevention of disease. The present review reveals avast number of relevant studies related to oxidative stress and selectedhuman diseases. The research field actually presents a variety ofapproaches that have been produced suggestive evidence that antioxi-dants might have an impact against many diseases. However, moresystematic research in this line is imperative in order to reveal the rela-tionship and involvement of antioxidants with other diseases. The largediversity in the molecular mechanisms and the methodology of antiox-idants discussed here are factors probably responsible for the consistentfindings of their prevention of diseases. This shows that it will be very

343P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347

useful in controlling many oxidative stress mediated diseases asdiscussed above. In comparison with the effects of the antioxidantsanalysed in the previous study we can expect that many plant derivedcompounds which have excellent antioxidant property and were suc-cessfully investigated here and validates or corroborates the antioxidanthypothesis of many diseases [165,259–263]. Current review highlightsthe perspectives and possibilities of unrevealing the uncapped poten-tials of antioxidants in herbal plants for disease cure and warrants theneed for systematic research in the field to clear the insights in thisvery complex matter. However, recent research work with antioxidanttherapy suggests another promising area i.e., combining potential anti-oxidants and using them during the early onset of the disease as aprophylactic measure to prevent the disease that may eventuallyprove to be more effective in treating several devastating diseases.

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