Oxidative stress and the role of antioxidative treatment ...

Oxidants and Antioxidants in Medical Science ISSN 2146-8389

Year : 3 Volume : 3 Issue : 1 AIMS & SCOPE Owner & Publisher: GESDAV (Foundation for the Education, Health, Social Cooperation and Solidarity of the People of Gülhane) Managing editor: Bilal Bakır; M.D., Professor Editorial office: Mithatpaşa Cad. No: 71/4 Yenişehir- Ankara - Türkiye Phone: (+90) 312 4305883 Fax: (+90) 312 4354466 Type of publication: Scientific periodical Printed in: SAGE Yayıncılık, Matbaacılık San. ve Tic. Ltd. Şti. Kazım Karabekir Caddesi Kültür Han No: 7/101-102 (2.Kat) Ulus, Ankara, Türkiye Phone: (+90) 312 3410002 Print date: March 31, 2014

Oxidants and Antioxidants in Medical Science is an international peer-reviewed scientific periodical dedicated to research around redox interactions in all fields of medicine. The Journal is published quarterly and aims to publish high-quality full-length research papers or brief communications, comprehensive or ‘mini’review articles as well as original hypotheses reflecting new sights and ideas in following areas: • Oxidation/reduction cycles in biological systems • Role of reactive molecules in signaling • Mechanistic approach to endogenous antioxidant systems • Efficacy of dietary or synthetic antioxidants and their relevance to health and disease • Actual aspects around classic terms like (nitro)oxidative stress, oxidative damage, lipid/protein (per)oxidation, reactive oxygen/nitrogen species, radical scavenging, etc in redox science Both experimental and clinical research studies are welcome. By this way, one of the main aims of the Journal is to supply original information from bench to bedside. Thus, submitted manuscripts should provide a significant advance to the field supported by evident data.

Oxidants and Antioxidants in Medical Science Volume 3 – Issue 1 – March 2014

EDITORIAL BOARD JOURNAL MANAGER Bilal Bakir; Ankara, Turkey

EDITOR-IN-CHIEF Sukru Oter; Ankara, Turkey

ASSOCIATE EDITORS Abdullah Kilic; Ankara, Turkey Akilesh K. Pandey; Lubbock, Texas, United States Apollina Goel; Iowa City, Iowa, United States Carlos G. Perez-Plasencia; Mexico City, Mexico Esma R. Isenovic; Belgrade, Serbia Francesco Marotta; Milan, Italy Gjumrakch Aliev; San Antonio, Texas, United States Hugh Jude Damien Dorman; Helsinki, Finland Hakan Yaren; Ankara, Turkey Haleagrahara S. Nagaraja; Townsville, Queensland, Australia Igor Yakymenko; Kiev, Ukraine Ionel Alexandru Checherita; Bucharest, Romania Joanna Elzbieta Rybka; Bydgoszcz, Poland Kazem M. Azadzoi; Boston, Massachusetts, United States Korivi Mallikarjuna; Taoyuan, Taiwan Liang-Jun Yan; Fort Worth, Texas, United States Lorenzo Loffredo; Rome, Italy Luca Cucullo; Amarillo, Texas, United States Madalina Minciu Macrea; Salem, Virgina, United States Madhusudhanan Narasimhan; Lubbock, Texas, United States Milan Terzic; Belgrade, Serbia Mirjana Mihailovic; Belgrade, Serbia Murat Gulsun; Ankara, Turkey Pasquale Pagliaro; Bologna, Italy Rajeev Bhat; Penang, Malaysia Si Jin; Wuhan, Hubei, People's Republic of China Swaran J. S. Flora; Gwalior, Madhya Pradesh, India Ying-Yong Zhao; Xi’an, Shaanxi, People's Republic of China Youssef Al-Tonbary; Mansoura, Egypt EDITOR FOR STATISTICS & EPIDEMIOLOGY Turker Turker; Ankara, Turkey LANGUAGE EDITOR Zehra Coskun; Istanbul, Turkey http://www.oamsjournal.com


GUEST EDITORS & ADVISORY BOARD Abhishek Yadav; Gwalior, Madhya Pradesh, India Ahmed E. Abdel Moneim; Cairo, Egypt Anthony C. Cemaluk Egbuonu; Umudike, Umuahia, Abia, Nigeria Apostolos Zarros; Glasgow, Scotland, United Kingdom Ashish Mehta; Singapore, Singapore Ata Abbas; Huntington, West Virgina, United States Chung Yu Chen; Taipei City, Taiwan Dailiah P. Roopha; Tirunelveli, Tamil Nadu, India Diane Henshel; Bloomington, Indiana, United States Emilio Rojas; Mexico City, Mexico Etsuo Niki; Ikedo, Osaka, Japan Evgeniy Sidorik; Kiev, Ukraine Gargi Ghosal; Houston, Texas, United States Guanghua Zhao; Haidian, Beijing, People's Republic of China Gulcin Sagdicoglu Celep; Ankara, Turkey Hisakazu Ogita; Otsu, Shiga, Japan Hosam El Din Hussein Osman; Taif, Saudi Arabia Ibrahim Aydin; Ankara, Turkey Kartick C. Pramanik; State College, Pennsylvania, United States Keyvan Dastmalchi; New York, New York, United States Kuldip Singh; Amritsar, Punjab, India L. Jack Windsor; Indianapolis, Indiana, United States Lokanatha Valluru; Kuppam, Andhra Pradesh, India Manoj A. Suva; Jamnagar, India Megha Mittal; Gwalior, Madhya Pradesh, India Monica Rosa Loizzo; Arcavacata Rende, Italy Nitesh Mohan; Bareilly, India Ola Ali Gharib; Cairo, Egypt Paul Gideon Thomes; Omaha, Nebraska, United States Sergiy Kyrylenko; Campinas, Sao Paulo, Brazil Shanshan Wan; Ann Arbor, Michigan, United States Swadesh Malhotra; Lucknow, Uttar Pradesh, India Tatsuji Haneji; Kuramoto, Tokushima, Japan Upendra N. Dwivedi; Lucknow, Uttar Pradesh, India Xixi Cao; Houston, Texas, United States Ymera Pignochino; Candiolo, Torino, Italy Yolanda Chirino; Tlalnepantla, Morelos, Mexico http://www.oamsjournal.com


TABLE OF CONTENTS EDITORIAL Low intensity radiofrequency radiation: a new oxidant for living cells Igor Yakymenko, Evgeniy Sidorik, Diane Henshel, Sergiy Kyrylenko

EDITORIAL / MINI REVIEW Oxidants and antioxidants in health and disease Gulcin Sagdicoglu Celep, Francesco Marotta

1-3

5-8

INVITED REVIEWS Oxidative stress and the role of antioxidative treatment in diabetes mellitus Dragana Nikolic, Julijana Stanimirovic, Predrag Bjelogrlic, Esma R. Isenovic

9-14

Role of free radicals and antioxidants in gynecological cancers: current status and future prospects Lokanatha Valluru, Subramanyam Dasari, Rajendra Wudayagiri

15-26

REVIEW ARTICLE Mild mitochondrial uncoupling as potentially effective intervention to slow aging Vladimir Illich Padalko

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ORIGINAL ARTICLES Hepatoprotective effect of green propolis is related with antioxidant action in vivo and in vitro Niraldo Paulino, Aguinaldo P. Barbosa, Amarilis S. Paulino, Maria C. Marcucci

43-50

The protective effect of Podophyllum hexandrum on hepato-pulmonary toxicity in irradiated 51-64 mice Savita Verma, Bhargab Kalita, Rashmi Saini, Manju Lata Gupta Antioxidant and anti-inflammatory potential of some dietary cucurbits Indu Rawat, Dhara Sharma, Harish Chandra Goel

65-72

Biological investigations of antioxidant, antimicrobial properties and chemical composition of essential oil from Warionia saharae Khalid Sellam, Mhamed Ramchoun, Farid Khalouki, Chakib Alem, Lhoussaine El-Rhaffari

73-78

BRIEF REPORT Dietary protection by garlic extract against lead induced oxidative stress and genetic birth defects Oladimeji S. Tugbobo, Omotade I. Oloyede, Olusola B. Adewale

http://www.oamsjournal.com

79-82

Oxid Antioxid Med Sci 2014; 3(1):1-3

ISSN: 2146-8389

EDITORIAL

Low intensity radiofrequency radiation: a new oxidant for living cells Igor Yakymenko1, Evgeniy Sidorik1, Diane Henshel2, Sergiy Kyrylenko3 1

Institute of Experimental Pathology, Oncology and Radiobiology, Kiev, Ukraine School of Public and Environmental Affairs, Indiana University Bloomington, IN, United States 3 Department of Structural and Functional Biology, University of Campinas, Campinas, Brazil

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Received March 12, 2014 Accepted March 24, 2014

intensity RFR. Consequently, the recent epidemiological studies unexpectedly indicated a significant increase in the occurrence of various tumors among long-term and “heavy” users of cellular phones. These include brain tumors [2, 3], acoustic neuromas [4, 5], tumors of parotid glands [6], seminomas [7], melanomas [8] and lymphomas [9]. Similarly, an increase in tumor incidence among people living nearby cellular base transmitting stations was also reported [10, 11]. As a result, in 2011 the World Health Organization/ International Agency for Research on Cancer classified radiofrequency radiation as a possible carcinogen to humans [12].

Published Online March 29, 2014 DOI 10.5455/oams.240314.ed.002 Corresponding Author Igor Yakymenko Vasylkivska 45, Kyiv, 03022 Ukraine. [email protected] Key Words Cancer; Electrohypersensitivity; Oxidative stress; Radiofrequency radiation © 2014 GESDAV

Radiofrequency radiation (RFR), e.g. electromagnetic waves emitted by our cell phones and Wi-Fi, are referred to as non-ionizing. This means that in contrast to the ionizing radiation, which does induce ionization of water and biologically important macromolecules, RFR does not have a capacity for such effects. Unlike, for example X-rays, the energy of RFR is not enough to break electrons off the molecules. However, is RFR completely safe for public health? Traditionally, the industry and the public bodies said yes. Nevertheless, new research data change this perception. Oxidative stress is an induced imbalance between prooxidant and antioxidant systems resulting in oxidative damage to proteins, lipids and DNA; and is closely connected to overproduction of reactive oxygen species (ROS) in living cells [1]. The notion that the low intensity RFR can bring about significant oxidative stress in living cells has been doubted for years. The logic is simple: as low intensity radiofrequency electromagnetic waves are not able to ionize molecules, they can do nothing wrong for the living tissues. However, during the last decades a worldwide increase in penetration of wireless communication systems, including cellular telephony and Wi-Fi, attracted massive attention to possible biological effects of low


To that, a new medical condition, so-called electrohypersensitivity, in which subjects suffer due to RFR exposure has been described. Typically these people suffer from skin and mucosa related symptoms (itching, smarting, pain, heat sensation), or heart and nervous system disorders after exposure to computer monitors, cell phones and other electromagnetic devices [13]. This malady is growing continuously: starting from 0.06% of the total population in 1985 this category now includes as much as 9-11% of the European population [14]. A number of experimental studies demonstrate metabolic effects induced by low intensity RFR [15-17]. Notwithstanding the non-ionizing nature of RFR, profound mutagenic effects and features of significant oxidative stress in living cells under low intensity RFR exposure were detected using various biological models [18, 19]. Some of the papers however still show an absence of biological effects [20]. To clarify the picture, we analyzed peer-reviewed publications on oxidative effects of RFR and found altogether 80 currently available papers, of which a remarkable part, 76 papers (92.5%), reported the detection of significant oxidative stress. These effects most often included overproduction of ROS, lipid peroxidation/increased concentrations of malondialdehyde, protein peroxidation, increased concentrations of nitric oxide (NO) and changes in the activity of

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Yakymenko et al: Radiofrequency radiation: a new oxidant for living cells antioxidant enzymes [21-26]. Some papers point to the role of particular ROS and the ROS related pathways. For example, the mitochondrial pathways of superoxide/ROS generation have been shown to be activated in living cells during exposure to low intensity RFR [17, 27]. Importantly, a non-phagocyte NADH oxidase, a known enzymatic source of ROS, was shown to be significantly activated just after a few minutes of exposure to low intensity RFR [16]. More to that, a possibility of mechanochemical disruption of water molecule clusters with dissociation of water molecules due to low intensity microwave exposure was demonstrated already many years ago [28]. Unexpectedly, a strong non-thermal character of biological effects of RFR has been documented. As low as 0.1 µW/cm2 intensity of RFR and absorbed energy (specific absorption rate, SAR) of 0.3 µW/kg were demonstrated to be effective in inducing significant oxidative stress in living cells [27, 29]. This observation is particularly important as the modern international safety limits on RFR exposure are based solely on the thermal effects of the radiation and only restrict RFR intensity to 450-1000 µW/cm2 and SAR to 2 W/kg [30]. Moreover, studies where thermal intensities of RFR have been used could not reveal oxidative effects [31-33], which might point to the variety of molecular mechanisms of action of radiation induced by different radiation intensities. It is indicative that many studies demonstrated the effectiveness of different antioxidants to reverse the oxidative stress caused by RFR exposure. Such effects have been reported for melatonin [34-37], vitamins E and C [24, 38], caffeic acid phenethyl ester [36], selenium and L-carnitine [39], and garlic extract [40]. It is still a question how low intensity RFR could activate superoxide-generating enzyme NADH oxidase or significantly increase the level of NO in a cell (e.g., possibly due to activation of NO synthase). But what is understood at the moment is that significantly increased levels of ROS in living cells caused by low intensity RFR exposure could lead to mutagenic effects through expressive oxidative damage of DNA [17, 27, 41]. It is also well documented nowadays that in biological systems, oxidants are not necessarily always the triggers for oxidative damage, and that oxidants such as H2O2 could actually serve as signaling messengers and drive several aspects of cellular signaling [42]. This leads to a hypothesis that overproduction of ROS/free radical species in living cells under low intensity RFR exposure can lead to disturbances in cell signaling cascades, which in turn may result in various pathologic consequences. Whatever the particular first-step molecular mechanisms, it is clear that the substantial overproduction of ROS in living cells under low

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intensity RFR exposure could cause a broad spectrum of health disorders and diseases, including cancer in humans. Undoubtedly, this calls for the further intensive research in the area, as well as to a precautionary approach in routine usage of wireless devices.

COMPETING INTERESTS The authors report no conflicts of interest.

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ISSN: 2146-8389

EDITORIAL / MINI REVIEW

Oxidants and antioxidants in health and disease Gulcin Sagdicoglu Celep1, Francesco Marotta2 1

Gazi University, Family and Consumer Sciences Department, Industrial Arts Education Faculty, Food and Nutrition Technology, Ankara, Turkey 2 ReGenera Research Group for Aging Intervention, Italy and WHO-Center for Traditional Medicine and Biotechnology, University of Milano, Italy

Received February 12, 2014 Accepted February 23, 2014 Published Online March 6, 2014 DOI 10.5455/oams.230214.rv.013 Corresponding Author Gulcin Sagdicoglu Celep Gazi Universitesi, Endustriyel Sanatlar Egitim Fakultesi, Aile ve Tuketici Bilimleri Egitimi Bolumu, Besin ve Beslenme Teknolojisi Egitimi Anabilim Dalı, 06500 Teknikokullar, Ankara, Turkey. [email protected] Key Words Disease; Free radicals; Health; Polyphenols; Reactive oxygen species

Abstract Free radicals are highly reactive chemical compounds that can be formed in biological systems as a part of regular metabolic reactions or with environmental reasons such as ultraviolet radiation and smoke. Even though they are necessary for several reactions as well as cellular defense mechanisms, they can generate oxidative stress if they exceed the antioxidant capacity of the cells. Therefore, their concentrations are strictly regulated with a variety of enzymes and antioxidant molecules as well as with our genes. Free radicals can react with many biological molecules such as proteins, lipids or nucleic acids and consequently damage the cells in a number of ways. The impact of free radical damage has been well established in many diseases including cardiovascular diseases, Alzheimer’s disease, cancer and aging. Dietary polyphenols make an important contribution to human health not only with their antioxidant properties but also other structural implications. Antioxidants are expected to be useful in the treatment of related degenerative diseases and also for healthy aging.

FREE RADICALS AND OXIDATIVE STRESS Oxygen is a critical element for the living organisms since it is necessary for several metabolic functions including cellular respiration and it can also be deleterious for the cells [1]. Species capable of existing independently with one or more unpaired electrons are called free radicals [2]. These highly reactive compounds are dangerous for the cells when their cellular production exceeds the antioxidant capacity. In biological systems, generally called reactive oxygen species (ROS) can exist in radical forms such as superoxide (O2•–), hydroxyl (OH•), alkoxyl (LO•, R–O•), peroxyl (LOO•, ROO•), nitric oxide (NO•) or in non-radical forms such as peroxynitrite (ONOO–), hypochlorite (HOCl), hydrogen peroxide (H 2O2), ozone (O3), singlet oxygen (1O2) and hydroperoxide (ROOH). Atmospheric O2 is also classified as free radical due to its chemical structure; therefore it also functions as a strong oxidizing agent in cells [3]. Sources of free radicals ROS can be produced in the cells by various chemical processes including enzymatic reactions, toxic compounds, tobacco smoke, ultraviolet and ionizing radiation and other environmental factors [4]. The most important sources of O2•– in aerobic cells are the electron transport chains of mitochondria where approximately 1-5% of all oxygen used in metabolic processes escapes as free radicals [2, 5]. Oxidative


© 2014 GESDAV

reactions catalyzed by Cytochrome P450, cyclooxygenases, lipoxygenases, dehydrogenases and peroxidases have the potential for generating free radicals. Fe-S proteins and NADH dehydrogenases are possible sites of O2•– and H2O2 formation [6]. Xanthine oxidase produces superoxide anions during oxidation of xanthine to uric acid [7]. NAD(P)H oxidase in the plasma membrane of neutrophiles produces O2•– within the plasma membrane or on its outer surface. Hydroxyl radical can be generated when H2O2 comes into contact with certain transition metal ion chelates, especially with ferrous iron (Fe2+) and cuprous copper (Cu+) [8]. A summary of sources of ROS is given in Fig.1. Mechanisms of free radical damage and diseases Rebecca Gerschman and coworkers were the first to propose the relation of oxygen toxicity with free radicals in 1954 [9]. Within the progressive years, modification of biological molecules by ROS and its impact on cells has been better understood. Free radicals can react with biological molecules such as sugars, amino acids, phospholipids and nucleotides and can induce cellular damage [10]. OH• is one of the most reactive chemical species known to be able to react with purine and pyrimidine bases present in DNA and RNA. It plays role in the pathogenesis of many diseases including atherosclerosis, neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases, cancer as well as in the aging process [11]. ONOO- can

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Celep & Marotta: Oxidants and antioxidants in health and disease initiate the peroxidation reactions of lipids in the membranes or initiate DNA breakage. DNA damage can cause also cause strand breaks, DNA-protein crosslinks and base-free sites [12]. ONOO- can further react with thiols while exerting protein modification especially by oxidation on methionine, cysteine, tryptophane or tyrosine residues and nitration of tyrosine or tryptophane residues [13]. In addition, several forms of oxidatively modified proteins are reported to be accumulating during aging process. The role of oxidative stress in autoimmune, infectious, metabolic, cardiovascular, neurodegenerative diseases and cancer is summarized in Fig.2. Free radicals can react with non radical molecules to give radicals and it can further cause chain reactions. Lipid peroxidation is the most studied free radical chain reaction and it has many consequences in pathobiology of several diseases [14]. They can abstract a hydrogen atom from an unsaturated fatty acid and initiate the chain reaction, resulting in formation of a carbon centered radical which reacts with oxygen to form a peroxy radical. Sequentially, it can abstract a hydrogen atom from an unsaturated fatty acid, leaving a carbon centered radical and a lipid hydroperoxide [15, 16]. Malondialdehyde is formed during the peroxidation of membrane fractions which is an important contributor to DNA damage and mutation [17, 18]. ANTIOXIDANT PROTECTION MECHANISMS Although free radicals are necessary for certain biological reactions in many processes including cellular defense and signaling, they can be very dangerous when their levels are not regulated by cellular antioxidants. Enzymes such as superoxide dismutase (SOD) in mitochondria and cytosol, catalase (CAT) in peroxisomes and glutathione peroxidase (GPX) in cytosol are important antioxidants that balance the redox status of the cells. Other antioxidant molecules are the hydrophilic antioxidants such as vitamin C, uric acid, bilirubin, albumin and thiols, and lipophilic α-tocopherol (vitamine E), ubiquinol, retinoic acid and carotene that can scavenge free radicals and prevent lipid peroxidation to protect membrane integrity [19, 20]. Glutathione (GSH; L-g-glutamyl-Lcysteinylglycine) is the principal non-protein thiol involved in the antioxidant cellular defense against ROS. In association with the ‘French paradox’ theory coined by Serge Renaud in the early 1990s [21], the health benefits of consumption of fruit- and vegetable-rich diets have been better established. Consequently, the research to reveal the protective effects of dietary polyphenols and related plant derived phenolic compounds, particularly their antioxidant properties, have notably accelerated [22]. Antioxidant therapy is

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expected to be useful in the treatment of related diseases since critical steps in the signal transduction cascades are sensitive to oxidants and antioxidants [23, 24]. The capacity of polyphenols to scavenge free radicals and/or chelate metals has been often claimed to be responsible for their antioxidant actions. Nevertheless, after consumption of polyphenol rich foods, the physiological concentrations of these polyphenols in animals and human tissues are reported to be incompatible with the kinetic requirements necessary to reach physiologically relevant reaction rates by means of thermodynamic principles; therefore other biochemical mechanisms related to polyphenollipid and polyphenol-protein interactions due to the presence of OH groups of dietary polyphenols are expected to clarify their antioxidant mechanisms [22]. Health benefits of dietary antioxidants Beyond their free radical scavenging effects, several different bioactivities are pointed out for polyphenols and other antioxidants related with health and disease states. For instance, regarding cardiovascular diseases, angiotensin converting enzyme (ACE) is a possible target for prevention and treatment of hypertension to assess the therapeutic effects of antioxidant molecules such as (-)-epicatechin, the antioxidant compund found in grapes, tea, etc. It can efficiently inhibit the activity of ACE while modulating the overall angiotensin II signaling pathways and redox balance of the cells [25]. Beneficiary effects of ‘sage tea’ (Salvia fruticosa) commonly growing in Turkey were attributed to its phenolic constituents and their antioxidant activities. Besides, sage was recently evaluated for its anticholinesterase inhibitory activity, which is a preventive and therapeutic methodology in neurological diseases such as Alzheimer’s disease [26]. Glutathione S-transferase (GST) enzymes are important phase II drug metabolizing enzymes that catalyze the conjugation of the reduced form of GSH to xenobiotic substrates for the purpose of detoxification.

Figure 1. Sources of reactive oxygen species

DOI 10.5455/oams.230214.rv.013

Oxidants and Antioxidants in Medical Science 2014; 3(1):5-8 Particularly, GST activity increases in certain cancers causing multidrug resistancy which is an obstacle for chemotherapy. Dietary flavonoids are considered as potential antioxidants to overcome the multidrug resistancy by inhibiting specific GST isozymes [27]. In the last few years, with the progression of technological developments in molecular biology research systems such as bioinformatics and microarrays, the view of oxidative stress has changed more through genes, and the ways in which gene expression is regulated by oxidants, antioxidants, and the redox state that has promising therapeutic implications. Well-defined transcription factors, nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 have been identified to be regulated by the intracellular redox state and are directly involved in the pathogenesis of diseases such as acquired immunodeficiency syndrome (AIDS), cancer, atherosclerosis and diabetic complications [28]. The transcription factor nuclear factor (erythroidderived 2)-like 2 (Nrf2) is referred to as the “master regulator” of the antioxidant response since it modulates the expression of hundreds of genes, including the antioxidant enzymes, and others involved in immune and inflammatory responses, tissue remodeling and fibrosis, carcinogenesis and metastasis, and even cognitive dysfunction and addictive behavior. Furthermore, dysregulation of Nrf2-regulated genes are reported to be linking oxidative stress and around 200 human diseases including colon cancer, cardiovascular disease, and Alzheimer’s disease [29].

Figure 2. Oxidative stress and its general contribution to diseases

CONCLUSION

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There has been an accelerative discovery of the roles of free radicals in the development of several diseases like cancer, diabetes, cardiovascular, neurodegenerative and inflammatory diseases, as well as the roles of the protective effects of antioxidants from these disases [12].

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Nevertheless, there are still a number of disease mechanisms and treatment strategies waiting to be enlighted regarding the oxidants and antioxidants with related pathways including all the biomolecules including genes.

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In this issue of Oxidants and Antioxidants in Medical Science, as in the previous ones, you will find articles having significant contribution to this interdisciplinary field. We are very proud to announce the new issue which incorporates many research professionals on the platform of redox research.


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Celep & Marotta: Oxidants and antioxidants in health and disease 8.

Jomova K, Valko M. Advances in metal-induced oxidative stress and human disease. Toxicology 2011; 283:65-87.

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10. Slater T. Free radical mechanism in tissue injury. Biochem J 1984; 222:1-15. 11. Gruber J, Fong S, Chen CB, Yoong S, Pastorin G, Schaffer S, Cheah I, Halliwell B. Mitochondria-targeted antioxidants and metabolic modulators as pharmacological interventions to slow ageing. Biotechnol Adv 2013; 31:563-92. 12. Pham-Huy LA, He H, Pham-Huy C. Free radicals, antioxidants in disease and health. Int J Biomed Sci 2008; 4: 89-96.

21. Renaud S, de Lorgeril M. Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet 1992; 33:1523-6. 22. Fraga CG, Celep GS, Galleano M. Biochemical actions of plant phenolics compounds: thermodynamic and kinetic aspects. In: Fraga CG (ed) Plant Phenolics And Human Health – Biochemistry, Nutritional and Pharmacology, John Wiley & Sons, Hoboken, NJ, 2009. 23. Marotta F, Yoshida C, Barreto R, Naito Y, Packer L. Oxidativeinflammatory damage in cirrhosis: effect of vitamin E and a fermented papaya preparation. J Gastroenterol Hepatol 2007; 22:697-703.

13. Virag L, Szabp E, Gergely P, Szabo C. Peroxynitrite-induced cytotoxicity: mechanism and opportunities for intervention. Toxicol Lett 2003; 140-141:113-24.

24. Litterio MC, Jaggers G, Sagdicoglu Celep G, Adamo AM, Costa MA, Oteiza PI, Fraga CG, Galleano M. Blood pressure-lowering effect of dietary (-)-epicatechin administration in L-NAMEtreated rats is associated with restored nitric oxide levels. Free Radic Biol Med 2012; 53:1894-902.

14. Njie-Mbye YF, Kulkarni-Chitnis M, Opere CA, Barrett A, Ohia SE. Lipid peroxidation: pathophysiological and pharmacological implications in the eye. Front Physiol 2013; 4:366.

25. Actis-Goretta L, Ottaviani JI, Fraga CG. Inhibition of angiotensin converting enzyme activity by flavanol-rich foods. J Agric Food Chem 2006;Jan 11;54(1):229-34.

15. Gutteridge JM. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem 1995; 41:1819-28.

26. Senol FS, Celep F, Erdem SA, Kahraman A, Kan Y, Kartal M, Orhan I, Sener B, Dogan M. Evaluation of cholinesterase inhibitory and antioxidant activities of wild and cultivated samples of sage (Salvia fruticosa) by activity-guided fractionation. J Med Food 2011; 14:1476-83.

16. Pizzimenti S, Ciamporcero E, Daga M, Pettazzoni P, Arcaro A, Cetrangolo G, Minelli R, Dianzani C, Lepore A, Gentile F, Barrera G. Interaction of aldehydes derived from lipid peroxidation and membrane proteins. Front Physiol 2013; 4:242. 17. Jomova K, Valko M. Advances in metal-induced oxidative stress and human disease. Toxicology 2011; 283:65-87. 18. Han Y, Chen JZ. Oxidative stress induces mitochondrial DNA damage and cytotoxicity through independent mechanisms in human cancer cells. Biomed Res Int 2013; 2013:825065. 19. Singal AK, Jampana SC, Weinman SA. Antioxidants as therapeutic agents for liver disease. Liver Int 2011; 31:1432-48.

27. Wu JY, Cheng CC, Wang JY, Wu DC,Hsieh JS, Lee SC, Wang WM. Discovery of tumor markers for gastric cancer by proteomics. PloS One 2014; 9:e84158. 28. Sen CK, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J 1996; 10:709-20. 29. Hybertson BM, Gao B, Bose SK, McCord JM. Oxidative stress in health and disease: the therapeutic potential of Nrf2 activation. Mol Aspects Med 2011; 32:234-46.

20. Victor VM, Rocha M, Fuente MD. Immune cells: free radicals and antioxidants in sepsis. Int Immunopharmacol 2004; 4:32747. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided that the work is properly cited.

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DOI 10.5455/oams.230214.rv.013


ISSN: 2146-8389

INVITED REVIEW

Oxidative stress and the role of antioxidative treatment in diabetes mellitus Dragana Nikolic1, Julijana Stanimirovic2, Predrag Bjelogrlic3, Esma R. Isenovic2 1

Biomedical Department of Internal Medicine and Medical Specialties, University of Palermo, Italy Institute Vinca, Laboratory of Radiobiology and Molecular Genetics, University of Belgrade, Belgrade, Serbia 3 School of Medicine, Clinical Skills, University of St. Andrews, Scotland, United Kingdom

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Received March 3, 2014 Accepted March 7, 2014 Published Online March 28, 2014 DOI 10.5455/oams.070314.rv.014 Corresponding Author Esma R. Isenovic Laboratory of Radiobiology and Molecular Genetics, University of Belgrade, P.O. Box 522, Belgrade, Serbia. [email protected] Key Words Antioxidative agents; Diabetes Mellitus; Free radicals; Oxidative stress

Abstract It is well known that increased free radical (FR) production or decreased activity of antioxidative system (AOS) lead to an imbalance between pro-oxidants and antioxidants called oxidative stress (OxS). Oxidative stress is involved in numerous diseases including diabetes mellitus (DM). Elevated blood glucose level and other biochemical disorders accompanied with an inappropriate insulin secretion or improper insulin action are known features of DM. The antioxidative enzyme catalase (CAT) diminishes the production of hydrogen peroxide which is highly toxic for pancreatic cells. The increased activity of this enzyme found in DM type 1 (DMT1) patients signifies the importance of OxS in the pathogenesis of this autoimmune disease with excessive OxS. Additionally, hyperglycemia induces the generation of highly reactive FR and leads to the development of OxS which accelerates the development of DM and its complications associated to the decreased activity of AOS. It is important to point out that high doses of antioxidant agents could paradoxically have pro-oxidant effect. In this article, we present literature data related to relationship between OxS and DM with focus on non-enzymatic antioxidants as a potential novel therapeutical approach in treatment of DM. Dietary supplementation with antioxidant nutritional factors such as micronutrients and vitamins could be used as a novel strategy in both prevention and control of DM type 2 (DMT2). © 2014 GESDAV

INTRODUCTION In physiological conditions, oxidative stress (OxS) occurs in all cells which breathe and on this way release free radicals (FR), which can be harmful for the organism, while the FR occurred in pathological conditions are responsible for the damage of biomolecules [1]. During evolution, in response to the creation of FR, protective antioxidant defense mechanisms have been created. In a healthy body, there is a balance between oxidative processes and its antioxidant capacity; but when the equilibrium is disturbed, biologically ‘hyper’-active molecules of generated OxS act as signaling agents through various pathways called “redox signaling” [2]. It is believed that the OxS is one of the important factors responsible for cell disorders in diabetes mellitus (DM), firstly initiated by hyperglycemia [3-5]; however, some authors indicate no direct link between hyperglycemia and OxS [6]. The pathogenesis of diabetic complications is related to the increased production in FR [7] and decreased antioxidants [8]. In this context, the mechanisms that may contribute to the formation of reactive oxygen species (ROS) in DM type 2 (DMT2) include also increased non-enzymatic glycosylation [9], glucose auto-oxidation, metabolic


stress, levels of mediators of inflammation and antioxidant defense status, that overall lead to dysfunction and cell damage [10]. The main part of the contents of ROS and reactive nitrogen species (RNS), are superoxide (O2•-), hydroxyl (•OH), peroxy (ROO•-), and nitric oxide free radical (•NO) and products of the reactions of these FR, such as hydrogen peroxide (H2O2) and and peroxynitrite (ONOO-) [11]. As known, O2•- may cause an increased production of hydroperoxyl radicals and H2O2 in the presence of superoxide dismutase (SOD), which easily cross cell membranes and initiate oxidative reactions [10]. Very similar happened during lipid peroxidation where lipids are oxidized by FR produced in diabetes. Further, catalase (CAT) and glutathione peroxidase (GSH-Px) are able to convert (reduce) H2O2 to water (H2O) and oxygen (O2). In the presence of high ferric iron (Fe3+), H2O2 can be reduced to the generation of •OH, a mechanism known as the “Fenton reaction”, but also by Haber-Weiss reaction [12], leading to the conversion of O2•- to •OH. Some of the drugs currently used in treating DM have been reported to have antioxidant properties [13-15]. However, it is not clear whether such effects are mediated by control of glucose or by the drug itself

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Nikolic et al: Oxidative stress, diabetes mellitus and antioxidants [16, 17]. On the other hand, therapeutic methods that directly target the reduction in toxicity of OxS in vascular cells could be a therapeutic approach in patients with DM, in addition to treatments that regulate glucose levels [18]. In this context, treatment with substances which act as antioxidants, in the highglucose conditions, showed that they inhibit a production of FR and reduce OxS as well as cell damages. Therefore, antioxidants are themselves potential therapeutics in DM treatment [18, 19].

glycation end products (AGEs pathway) that influence the transcription of pro-inflammatory genes to promote OxS [10, 27, 29]. Also, excessive production of FR in hyperglycemia state is ascribed to auto-oxidation of glucose, non-enzymatic glycation of proteins, activation of NAD(P)H oxidases and nitric oxide synthase [30, 31]. Different signaling mechanisms have been described in different complications of DM such as cardiac pathophysiology, renal injury, liver dysfunction [21].

This review was undertaken in order to summarize current knowledge about OxS in DM including possible signaling pathways, with focus on nonenzymatic antioxidants as potential novel approach in the treatment of this disease.

It has been reported that plasma levels of extracellular (EC)-SOD is associated with insulin resistance in DM and its concentrations are significantly higher in diabetic subjects [32]. Further, serum EC-SOD levels positively correlate with the severity of diabetic vascular complications, such as nephropathy and retinopathy [33]. Recent in vivo study reported that diabetic skin tissues express a relatively small amount of EC-SOD protein that may be related to elevated ROS production [32].

OXIDATIVE STRESS AND DIABETES There is increasing evidence that OxS plays a major role in the onset and progression of diabetes, and even its complications [20, 21]. Due to the lack of regulation, a high amount of glucose in the blood increases oxygen and releases O2•- which easily reacts with the present nitric oxide (•NO) disabling its action as endothelial vasodilator [19, 22]. Consequently, there is a reduction in endothelium-dependent relaxation and cell synthesis in the wall of blood vessels, resulting in micro- and macro-pathological changes [23, 24]. Hyperglycemia, increases the levels of free fatty acids (FFA), and together with hyperinsulinemia lead to increased production of ROS and RNS [25]. ROS and RNS activate nuclear factor-kappaB (NF-κB), a proinflammatory transcription factor, that further cause a signaling cascade leading to a continued synthesis of oxidative species and to inflammation [26]. Increased FFA causes dysfunction by two mechanisms: (1) the activation of peroxisome proliferator-activated receptor (PPAR)-α which hampers mitochondrial oxidative phosphorylation; and (2) the production of lipotoxic substances during FFA metabolism leading to opening of K+ channels which impairs Ca2+ homeostasis. Diminished expression of the pyruvate dehydrogenase results in the accumulation of glycolytic intermediate and ceramide inducing apoptosis and consequently leads to cardiomyopathy in DM [21]. Further, hyperglycemia modifies the redox balance through the polyol pathway (reducing glucose to sorbitol, with subsequent decreases in NADPH and reduced glutathione), activates oxidases, and interferes with the mitochondrial electron transport chain [27]. These processes may trigger various signaling cascades, such as activation of protein kinase C, hexosamine pathway that further increase the synthesis of ROS [10, 28]. Non-enzymatically, glucose auto-oxidation generates hydroxyl radicals and leads to the formation of

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DIABETES MELLITUS AND ANTIOXIDANTS It is considered that a normalization of activity of any OxS markers, such as enzymes, thiobarbituric acid reactive substances (TBARS) and FR, and finally the balance of FR/removal, represent an effective way to reduce the harmful effects of ROS [34, 35]. Based on the latest results, it is clear that the goal is to block the formation of ROS by antioxidants and those results suggest the need for possible use of antioxidants in the treatment of DM. Even more it has been suggested that antioxidant therapy may inhibit the onset of DM and also prevent the development of DM complications [36, 37]. The HOPE (Heart Outcomes Prevention Evaluation trial) study is the largest study dealt with the use of antioxidants in DM. This study has lasted for 4.5 years, and demonstrated that ramipril (a drug used for the treatment of hypertension and heart failure) decreases the possibility of occurrence of diabetic nephropathy in DM patients, as opposed to vitamin E, which did not lead to a significant reduction of cardiovascular risk [19, 38]. Regarding vitamin E several epidemiological studies demonstrated its inverse association with markers of oxidation, inflammation, and DMT2 incidence [39], although other studies did not support such findings [40, 41], including the Women’s Health Study [42]. The European Prospective Investigation of Cancer-Norfolk Prospective Study investigated association between fruit and vegetable intake and plasma levels of vitamin C with risk of DMT2, during a 12year follow-up of 735 participants. A significant inverse association was found between plasma levels of vitamin C and risk of diabetes, as well as between fruit and vegetable intake and DMT2 risk [43]. However,

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Oxidants and Antioxidants in Medical Science 2014; 3(1):9-14 some randomized, crossover, double-blind intervention trials reported no benefit effects after supplementation of vitamin C (3000 mg/day) for 2 weeks in DMT2 subjects or 800 mg/day for 4 weeks, respectively [44 45]. In contrast, it has been found benefit of 1000 mg/day of vitamin C for 4 months [46], as well as the reduced red blood cell sorbitol/plasma glucose ratio after 2 weeks of the same doses [47]. Several investigations showed a positive effect of vitamin C on a cardiovascular system in DMT1 patients [19]. Also, a combination of vitamins C and E in these subjects improves renal function [4]. Some other studies showed that simultaneous treatment with both vitamins C and E has a positive effect on the cardiovascular system in DMT1 patients, but a negative effect in subjects with DMT2 [48, 49]. The studies focused on the use of vitamin E, unfortunately, do not provide sufficient evidence that the vitamin E reaches the target cells [38]. Furthermore, it has been shown that the α-lipoic acid enhances the function of the nerve and gives better results in the treatment of DM compared to vitamin E. Also, the vitamin C was not able to provide greater protection from the occurrence of cardiovascular complications, in comparison with the α-lipoic acid which use in the prevention of cardiovascular complications is considering [38]. However, the results of the vitamins should not be generalized for all antioxidants. Treatment with vitamins, as a class of compounds with the expected effects, ignores a wide range of their chemical and pharmacological properties [50]. Vitamin D and calcium homeostasis have also been reported to be associated to DM [51, 52]. The presence of vitamin D receptors (VDR) in the pancreatic β-islet cells support the role of vitamin D in subjects with DMT2 [53]. Furthermore, reduced overall risk of the disease in subjects who ingest 800 IU/day of vitamin D has been reported [51, 54]. In this context, data from the Women’s Health Study showed that among women taking more than 511 IU/day of vitamin D reduced the risk of DMT2 when compared to ingesting 159 IU/day [55]. Therefore, clinical trials with antioxidants in DM are limited and focused majority on the use of vitamins E and C, and in recent years, α-lipoic acid [19, 56]. In any case, choice and dosage of the used antioxidant is very important [38] and it is recommended that a high dose of antioxidants should not be given as monotherapy, but in combination with other antidiabetic drugs, due to the possibilities of disruption of antioxidant/prooxidant balance [57, 58]. Hopefully, further studies related to the pathophysiology of OxS and to the role of antioxidants in the treatment of DM, will lead for sure to a number of clinical trials which will confirm therapeutic effects of antioxidants.


Several cross-sectional and interventional studies reported that dietary intake of micronutrients can lead to reduced levels of OxS, proinflammatory cytokines, and could be a risk for DMT2 [43, 49, 59]. Such an approach may represent a novel strategy for the prevention of DMT2. In this context, some researchers have investigated the dietary antioxidants from plant food materials [60], and they found a strong antioxidative activity in curcuminoids, the main yellow pigments in Curcuma longa (turmeric), known to possess antioxidant activity many years ago [61]. Dietary antioxidants possess the direct scavenging activity of ROS and also induction of antioxidative enzymes including detoxification enzymes and may prevent or delay diabetes complications including renal and neural dysfunction [60]. Also, supplementation with Allium sativum (garlic), Panax quinquefolius (American ginseng), and Panax ginseng (Asian ginseng), with antioxidant, antiinflammatory, and adaptogenic properties, were reported to down-regulate the OxS and the synthesis of pro-inflammatory cytokines [62]. In this context, Rashid et al [21] recently reviewed the therapeutic effects of naturally occurring antioxidants. An antioxidative action of microelements such as zinc (Zn) has been reported too [63, 64]. Moghaddam et al [65], although on small number of subjects, show that exercise training might contribute to an improvement of the antioxidative defense against ROS in men suffering from non-insulin-dependent DMT2. Even more, Venkatasamy et al [66] reported that multiple mechanisms through which physical exercise acts, including up-regulating mechanisms governing physiological anti-oxidant generation. In animals, many drugs that are already used in the treatment of DM have antioxidant properties, in addition to their primary pharmacological activity (e.g. N-acetylcysteine) [67] and those antioxidant properties may be a key factor in the effectiveness of these drugs [67-69]. In addition, the treatment of diabetic rats with vitamins C, E and beta-carotene results in a significant reduction in the level of TBARS and GSH-Px activity, an increase in SOD activity, while CAT activity does not change. Vitamins C and E, lower the level of TBARS and GSH-Px, while the activity of CAT and SOD were increased [70]. On the other hand, numerous studies have demonstrated a reduced SOD activity [34, 70], other studies show increased [23] or there was no change in the activity of this enzyme [71]. Of interest, the reduced SOD activity in the heart of diabetic rats is normalized by α-lipoic acid; in addition, an intraperitoneal application of α-lipoic acid in diabetic rats, normalized the level of TBARS in plasma, liver and pancreas [56]. Earlier, it was observed that the reduction of the activity of GST does not change with the α-lipoic acid [72]. However, other

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Nikolic et al: Oxidative stress, diabetes mellitus and antioxidants studies showed an increase of GSH-Px activity in the aorta in diabetic rats, which was normalized by treatment with α-lipoic acid [56, 73]. A recent in vitro study showed that antioxidant treatment attenuated high glucose induced increased OxS in primary rat pancreatic stellate cells (PSC) [74], a potential underlying mechanism of islet fibrosis, which may contribute to progressive β-cell failure in DMT2. Earlier, it was reported that antioxidants reduced fibrosis and α-smooth muscle actin, the most commonly used index of PSC activation, expression in the islets in Otsuka Long-Evans Tokushima Fatty rats [75].

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CONCLUDING REMARKS Based on previous studies of OxS in DM, today it became clear that OxS is a cause but also at the same time a consequence in both genesis and pathogenesis of DM. It could be concluded that OxS in DM occurs at the initial stage of disease, and progressively increases with the development of disorders in DM, wherein the antioxidative system (AOS) more exhausts. Therefore, it is necessary to maintain functional AOS, in terms of both prevention and therapy of DM. Further studies are needed to elucidate either direct or indirect antioxidative effects of drugs currently used in the treatment of DM and also to differentiate such effects from those on glycemia. Depletion of individual components of AOS during DM, suggesting an increased intake of antioxidants necessary for certain compensation, as well as to prevent and/or mitigate the occurrence of pathological changes caused by insufficient antioxidant protection [35, 76]. When application of antioxidants is planned in the treatment of DM, special attention should be paid to the applying dose of chosen antioxidants, in both cases as monotherapy and in combination with the main drugs used in the treatment of DM. Applying nutritional intervention to decrease OxS, as well as inflammation, might represent the first-line strategy for prevention and treatment of DMT2, including lifestyle change and exercise [77].

ACKNOWLEDGEMENTS This work was supported by the project number 173033 funded by the Ministry of Science and Environmental Protection. COMPETING INTERESTS The authors declare that they have no conflict of interest.

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41. Wu JH, Ward NC, Indrawan AP, Almeida CA, Hodgson JM, Proudfoot JM, Puddey IB, Croft KD. Effects of alpha-tocopherol and mixed tocopherol supplementation on markers of oxidative stress and inflammation in type 2 diabetes. Clin Chem 2007; 53:511-9. 42. Liu S, Lee IM, Song Y, Van Denburgh M, Cook NR, Manson JE, Buring JE. Vitamin E and risk of type 2 diabetes in the women's health study randomized controlled trial. Diabetes 2006; 55:2856-62. 43. Harding AH, Wareham NJ, Bingham SA, Khaw K, Luben R, Welch A, Forouhi NG. Plasma vitamin C level, fruit and vegetable consumption, and the risk of new-onset type 2 diabetes mellitus: the European prospective investigation of cancer-Norfolk prospective study. Arch Intern Med 2008; 168:1493-9. 44. Lu Q, Bjorkhem I, Wretlind B, Diczfalusy U, Henriksson P, Freyschuss A. Effect of ascorbic acid on microcirculation in patients with Type II diabetes: a randomized placebo-controlled cross-over study. Clin Sci (Lond) 2005; 108:507-13. 45. Chen H, Karne RJ, Hall G, Campia U, Panza JA, Cannon RO, 3rd, Wang Y, Katz A, Levine M, Quon MJ. High-dose oral vitamin C partially replenishes vitamin C levels in patients with Type 2 diabetes and low vitamin C levels but does not improve endothelial dysfunction or insulin resistance. Am J Physiol Heart Circ Physiol 2006; 290:H137-45. 46. Paolisso G, Balbi V, Volpe C, Varricchio G, Gambardella A, Saccomanno F, Ammendola S, Varricchio M, D'Onofrio F. Metabolic benefits deriving from chronic vitamin C supplementation in aged non-insulin dependent diabetics. J Am Coll Nutr 1995; 14:387-92. 47. Wang H, Zhang ZB, Wen RR, Chen JW. Experimental and clinical studies on the reduction of erythrocyte sorbitol-glucose ratios by ascorbic acid in diabetes mellitus. Diabetes Res Clin Pract 1995; 28:1-8. 48. Beckman JA, Goldfine AB, Gordon MB, Garrett LA, Keaney JF, Jr., Creager MA. Oral antioxidant therapy improves endothelial function in Type 1 but not Type 2 diabetes mellitus. Am J Physiol Heart Circ Physiol 2003; 285:H2392-8. 49. Rizzo MR, Abbatecola AM, Barbieri M, Vietri MT, Cioffi M, Grella R, Molinari A, Forsey R, Powell J, Paolisso G. Evidence for anti-inflammatory effects of combined administration of vitamin E and C in older persons with impaired fasting glucose: impact on insulin action. J Am Coll Nutr 2008; 27:505-11. 50. Gutteridge JM, Halliwell B. Antioxidants: Molecules, medicines, and myths. Biochem Biophys Res Commun 2010; 393:561-4. 51. Pittas AG, Lau J, Hu FB, Dawson-Hughes B. The role of vitamin D and calcium in type 2 diabetes. A systematic review and metaanalysis. J Clin Endocrinol Metab 2007; 92:2017-29. 52. Teegarden D, Donkin SS. Vitamin D: emerging new roles in insulin sensitivity. Nutr Res Rev 2009; 22:82-92. 53. Holick MF. Diabetes and the vitamin d connection. Curr Diab Rep 2008; 8:393-8.

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Nikolic et al: Oxidative stress, diabetes mellitus and antioxidants 54. Pittas AG, Dawson-Hughes B, Li T, Van Dam RM, Willett WC, Manson JE, Hu FB. Vitamin D and calcium intake in relation to type 2 diabetes in women. Diabetes Care 2006; 29:650-6. 55. Liu S, Song Y, Ford ES, Manson JE, Buring JE, Ridker PM. Dietary calcium, vitamin D, and the prevalence of metabolic syndrome in middle-aged and older U.S. women. Diabetes Care 2005; 28:2926-32. 56. Bitar MS, Ayed AK, Abdel-Halim SM, Isenovic ER, Al-Mulla F. Inflammation and apoptosis in aortic tissues of aged type II diabetes: amelioration with alpha-lipoic acid through phosphatidylinositol 3-kinase/Akt- dependent mechanism. Life Sci 2010; 86:844-53. 57. Haidara M, Mikhailidis DP, Yassin HZ, Dobutovic B, Smiljanic KT, Soskic S, Mousa SA, Rizzo M, Isenovic ER. Evaluation of the possible contribution of antioxidants administration in metabolic syndrome. Curr Pharm Des 2011; 17:3699-712. 58. Opara EC. Oxidative stress, micronutrients, diabetes mellitus and its complications. J R Soc Promot Health 2002; 122:28-34. 59. Bartlett HE, Eperjesi F. Nutritional supplementation for type 2 diabetes: a systematic review. Ophthalmic Physiol Opt 2008; 28:503-23. 60. Osawa T, Kato Y. Protective role of antioxidative food factors in oxidative stress caused by hyperglycemia. Ann N Y Acad Sci 2005; 1043:440-51. 61. Sugiyama Y, Kawakishi S, Osawa T. Involvement of the betadiketone moiety in the antioxidative mechanism of tetrahydrocurcumin. Biochem Pharmacol 1996; 52:519-25. 62. Weisberg SP, Leibel R, Tortoriello DV. Dietary curcumin significantly improves obesity-associated inflammation and diabetes in mouse models of diabesity. Endocrinology 2008; 149:3549-58.

67. Kamboj SS, Sandhir R. Protective effect of N-acetylcysteine supplementation on mitochondrial oxidative stress and mitochondrial enzymes in cerebral cortex of streptozotocintreated diabetic rats. Mitochondrion 2011; 11:214-22. 68. Prior SL, Gable DR, Cooper JA, Bain SC, Hurel SJ, Humphries SE, Stephens JW. Association between the adiponectin promoter rs266729 gene variant and oxidative stress in patients with diabetes mellitus. Eur Heart J 2009; 30:1263-9. 69. Robertson RP. Antioxidant drugs for treating beta-cell oxidative stress in type 2 diabetes: glucose-centric versus insulin-centric therapy. Discov Med 2010; 9:132-7. 70. Kedziora-Kornatowska K, Szram S, Kornatowski T, SzadujkisSzadurski L, Kedziora J, Bartosz G. Effect of vitamin E and vitamin C supplementation on antioxidative state and renal glomerular basement membrane thickness in diabetic kidney. Nephron Exp Nephrol 2003; 95:e134-43. 71. Maritim AC, Sanders RA, Watkins JB 3rd. Effects of alpha-lipoic acid on biomarkers of oxidative stress in streptozotocin-induced diabetic rats. J Nutr Biochem 2003; 14:288-94. 72. Obrosova IG, Fathallah L, Greene DA. Early changes in lipid peroxidation and antioxidative defense in diabetic rat retina: effect of DL-alpha-lipoic acid. Eur J Pharmacol 2000; 398:13946. 73. Kocak G, Aktan F, Canbolat O, Ozogul C, Elbeg S, YildizogluAri N, Karasu C. Alpha-lipoic acid treatment ameliorates metabolic parameters, blood pressure, vascular reactivity and morphology of vessels already damaged by streptozotocindiabetes. Diabetes Nutr Metab 2000; 13:308-18. 74. Ryu GR, Lee E, Chun HJ, Yoon KH, Ko SH, Ahn YB, Song KH. Oxidative stress plays a role in high glucose-induced activation of pancreatic stellate cells. Biochem Biophys Res Commun 2013; 439:258-63.

63. Hashemipour M, Kelishadi R, Shapouri J, Sarrafzadegan N, Amini M, Tavakoli N, Movahedian-Attar A, Mirmoghtadaee P, Poursafa P. Effect of zinc supplementation on insulin resistance and components of the metabolic syndrome in prepubertal obese children. Hormones (Athens) 2009; 8:279-85.

75. Lee E, Ryu GR, Ko SH, Ahn YB, Yoon KH, Ha H, Song KH. Antioxidant treatment may protect pancreatic beta cells through the attenuation of islet fibrosis in an animal model of type 2 diabetes. Biochem Biophys Res Commun 2011; 414:397-402.

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77. Garcia-Bailo B, El-Sohemy A, Haddad PS, Arora P, Benzaied F, Karmali M, Badawi A. Vitamins D, C, and E in the prevention of type 2 diabetes mellitus: modulation of inflammation and oxidative stress. Biologics 2011; 5:7-19.

66. Venkatasamy VV, Pericherla S, Manthuruthil S, Mishra S, Hanno R. Effect of physical activity on insulin resistance, inflammation and oxidative stress in diabetes mellitus. J Clin Diagn Res 2013; 7:1764-6. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided tha the work is properly cited.

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DOI 10.5455/oams.070314.rv.014


ISSN: 2146-8389

INVITED REVIEW

Role of free radicals and antioxidants in gynecological cancers: current status and future prospects Lokanatha Valluru1, Subramanyam Dasari1, Rajendra Wudayagiri2 1

Department of Biotechnology, Dravidian University, Kuppam; Andhra Pradesh, India Department of Zoology, Sri Venkateswara University, Tirupati; Andhra Pradesh, India

2

Received July 15, 2013 Accepted November 10, 2013 Published Online December 18, 2013 DOI 10.5455/oams.201113.rv.011 Corresponding Author Lokanatha Valluru Department of Biotechnology, Dravidian University, Kuppam-517 426, Andhra Pradesh, India. [email protected] Key Words Cervical cancer; Endometrial cancer; Free radicals; Reactive species

Abstract The potential role of free radicals and associated oxidative stress has been well documented in the development of many diseases. Free radicals are mainly derived from oxygen (reactive oxygen species, ROS) and nitrogen (reactive nitrogen species, RNS), under various physicochemical or pathological conditions. Excessive amount of free radicals eventually attack biomolecules including proteins, lipids and DNA; thus result in increased oxidative damage, lead to alter the physiological functions of the cell, play role in the activation of transcription factors, and trigger a number of human diseases including carcinogenesis. Apart from many dietary components, mammalian cells have endowed with protective antioxidant defense system, which includes enzymatic (superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase) and non-enzymatic (glutathione, vitamin E (tocotrienols and tocopherols), vitamin C) antioxidants. The present review describes the role of the free radicals in the development of gynecological cancers and their key factors of the non-specific immune defense mechanism (antioxidants). The review also emphasizes the different potential applications of antioxidant/free radical manipulations in prevention and/or control of cancer. The novel and future approaches for better control of diseases are including gene therapy to produce more antioxidants, genetically engineered plant products with higher level of antioxidants, artificial antioxidant enzymes, novel biomolecules and the use of foods enriched with antioxidants. © 2014 GESDAV

INTRODUCTION In the last decade there has been a growing interest in understanding the role of free radicals in biomedicine. Free radicals are atoms with unpaired electrons such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). 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 halflives in milli-, micro- or nanoseconds. Some of the biologically important reactive species are described in Table 1. ROS are molecules that contain oxygen and have higher reactivity than ground state molecular oxygen [1]. The reactive oxygen species includes: the hydroxyl radical (•OH), the most damaging of this chemical species; which also include superoxide anions (O2•-); singlet oxygen (1O2); and hydrogen peroxide (H2O2). ROS can be formed under the aerobic condition not only during oxidative phosphorylation, through the action of mixed function oxidases, and as by-products of normal metabolism by enzymes such as superoxide dismutase (SOD), NADPH oxidase, and xanthine oxidase (XO) in neutrophils, but can also be generated from redox cycling of certain drugs and by radiation [2]. A well known example for RNS is nitric oxide


(NO), a short-lived endogenous gas that acts as a signaling molecule in the body. NO was synthesized by nitric oxide synthase (NOS) and produced by almost all mammalian cells. Excessive or unregulated NO synthesis has been implicated as causal or contributor to some pathophysiological conditions including cancer. Expression of NOS has been distinguished in various cancers including cervical, breast, central nervous system, laryngeal, and head and neck cancers [3-7]. Providentially, the mammalian cells have endowed by an antioxidant defense mechanism that allows equilibrium between the generation of oxidants and antioxidants. The interrupted condition in between these two factors develops an oxidative stress, and despite the antioxidant defense mechanism to counteract the reactive species-related deleterious effects, damage to macromolecules occurs as a result of these reactions. Oxidative damage accumulates during the life cycle and lead to different pathological progressions like myocardial infection, atherosclerosis, neurodegenerative disorders, rheumatoid arthritis, and cancer [8]. Globally, among several diseases, cancer has become a big threat to human beings. As per Indian census data, the rate of mortality due to cancer was high and

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Valluru et al: Oxidant and antioxidant status in gynecological cancers alarming with about 806,000 existing cases by the end of the last century. Cancer is the second most public disease in India responsible for high mortality with about 0.3 million deaths per year. This is owing to the poor availability of prevention, diagnosis and therapy of the disease. All cancers have been reported in Indian population including the cancers of skin, breast, lungs, rectum, prostate, stomach, cervix, liver, esophagus, bladder, mouth and blood, etc. [9]. Gynecological (ovarian, endometrial and cervical) cancers are the most common malignancies among females in many developing countries. It has been noticed that 90,000 new cases of gynecological cancer are reported every year in India [10]. Gynecological cancers represent a great clinical challenge in oncology. Since most cases are asymptomatic until the disease has metastasized, two-thirds are diagnosed with advanced stage. Hence, most of the gynecological cancers have the highest fatality-to-case ratio of all women malignancies [11]. Ovarian carcinoma often is lethal gynecologic malignancy with epithelial neoplasms in adult women. In India, approximately 15% of all gynecological cancers are ovarian malignancy [12] and it represents the greatest clinical challenge. Riley and Behrman [13] stated that the role of ROS and antioxidant enzymes, i.e. copper/zinc SOD (Cu/ZnSOD), manganese SOD (MnSOD) and glutathione peroxidase (GSH-Px), in oocyte maturation. It is documented that, oxidative stress play a major role in ovarian function through the intensified lipid peroxidation in the pre-ovulatory Graafian follicle [14]. Paszkowski et al [15] also demonstrated that GSH-Px may help in maintaining low levels of hydroperoxides inside follicle, suggesting a significant role of oxidative stress in ovarian function. Oxidative stress and inflammatory process have roles in the pathophysiology of polycystic ovarian disease and drugs such as Rosiglitazone may be effective by decreasing the levels of oxidative stress [16]. Endometrial carcinoma is the next most common subtype of gynecological malignancies representing 15% of cases [17]. Augmented generation of ROS by peritoneal fluid macrophages, with improved lipid peroxidation in patients with endometriosis, has been demonstrated, whereas other researchers have reported contrary findings [18]. It is well established that markers of lipid peroxidation such as diminished peritoneal fluid antioxidants, elevated oxidized lipoproteins and lysophosphatidyl choline provide further evidence of oxidative stress in the peritoneal microenvironment of patients with endometriosis [19]. Increased production of autoantibodies to oxidatively modified lipoproteins that are antigenic has been reported in patients with endometriosis. The investigations of various biomarkers have then revealed

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presence of oxidative stress locally and systemically in patients with endometriosis [20]. Cervical cancer refers to the epithelial malignancy that arises from the cervix. Cervical cancer is the most common cancer among women worldwide after breast cancer. According to the World Health Organization (WHO) report, globally, cervical cancer comprises 12% of all cancers in women and it is the leading gynecological malignancy in the world. Approximately 20,000 new cases were detected in India [21]. Particularly, in Southern India, carcinoma of the uterine cervix is the most common form of cancer in females [22]. Recently a report says that there are an estimated 132,000 new cases and 74,000 deaths annually in India [23]. Carcinogenesis is a multi-step process leading a cell from normal to pre-cancerous stage and finally to an early stage of cancer [2]. Cancer development is characterized by cumulative action of multiple events occurring in a single cell and can be described as initiation, promotion and progression. Free radicals can acts in all the stages of carcinogenesis [24] (Fig.1). In view of these facts, the present review describes the role of various types of free radicals or reactive species and their defense mechanisms in gynecological cancers. In addition to this, efforts have also been made to predict new-fangled approaches include gene therapy to produce more antioxidants, genetically engineered plant products with increased levels of antioxidants, artificial antioxidant enzymes, biomolecules with antioxidant and the use of functional foods enriched with antioxidants. FREE RADICAL GENESIS

MEDIATED

CARCINO-

Development of cancer is a multistage process requiring the cumulative action of multiple events that occur in one cell clone. The role of free radicals in carcinogenesis and their contribution to the initiation and progression of the cancer process is well established [25]. The overall process includes a three stage model: -first (initiation), a permanent change occurs in the genetic material (one somatic cell); -secondly (promotion), the mutated cell clone expants; -and finally (progression), malignant conversion into cancer develops (Fig.1). ROS can stimulate carcinogenesis by acting at all three stages [26]. There is a complex interplay of cytokines, hormones and other stressors that affects cellular generation of free radicals; these molecules act further through the modulation of many transcription factors and gene expression [19].

DOI 10.5455/oams.201113.rv.011

Oxidants and Antioxidants in Medical Science 2014; 3(1):15-26

Figure 1. Three-stage model of carcinogenesis: (a) initiation (attack by ROS/RNS as carcinogens), accumulation of carcinogenic mutations; (b) progresses through pre-neoplastic (reversible) stages by the acquisition of more mutations and up regulated cell signaling (promotion); (c) progression by tumor promoters and development of angiogenic potential leading to the expression of neoplastic stage which is an irreversible condition.

Initiation step In the initial step of the cancer development, a permanent change in genetic material of one cell is achieved by DNA mutation. Oxidative DNA damage can occur through hydroxyl radical generated from H2O2. Increased oxidative stress, which may in turn deplete the endogenous antioxidant reserves, is an important signal leading to Ca2+ mobilization. ROSmediated Ca2+ changes lead to the activation of endonucleases which can cause DNA fragmentation during apoptosis [2]. There is a steady formation of DNA lesions in living cells. Hydroxyl radical and other free radicals attack upon DNA and generate a series of DNA damage by a variety of mechanisms. These include sugars and base modifications, strand breaks and DNA protein crosslinks. Modified DNA base (pyrimidine and purine) constitute one of the most lesions which has mutagenic properties being potentially able to damage the genome integrity [27]. One of the most studied lesions generated by the modified DNA bases is the guaninecytosine → thymine-adenine (G-C → T-A) transversion mutagenesis [28]. Several modified bases which have also been shown to possess miscoding potentials and thus perhaps premutagenic properties. Most of these DNA damages are thought to be prepared mainly by base excision repair [29]. A large body of evidences indicated that a direct correlation between 8-OH-Gua (8-hydroxyguanine) generation and carcinogenesis in vivo [30]. Furthermore, the G-C → T-A transversion have been frequently deleted in the tumor suppressor p53 gene and proto-oncogene ras either through the inactivation of tumor suppressor gene or the activation of oncogene; thus, ROS-related mutations may lead to the initial step in the development of cancer. Tumor promotion The effect of oxidative stress is strongly involved in the promotion of carcinogenesis. A number of tumor promoters are thought to stimulate endogenous oxygen radical production by altering cellular metabolic processes [2]. ROS/RNS can stimulate the expression of mutated cell clones by temporarily modulating the


gene related to proliferation or cell death. While an overload from high levels of oxidative stress halts proliferation by cytotoxic effects, low levels can stimulate cell division and promote the growth of tumor cells [31]. Hence, the stimulation of intracellular production of reactive species is considered the main way to promote the free radical mediated tumors [32]. In in vitro experiments of Toyokuni et al [33] reported that, the DNA bindings of p53, activator protein (AP)-1 and nuclear factor (NF)-κB are activated in a reductive condition and repressed in an oxidative condition. However certain transcription factors are activated by oxidation while others are repressed by oxidation [34]. Tumor progression The progression of carcinogenesis comprises the acquisition of malignant properties to the tumor cell. Progression is characterized by accelerated cell proliferation, escape from immune surveillance, tissue invasion and metastasis [34]. The generation of large amounts of free radicals, together with the increase in the level of oxidatively modified DNA bases, may attribute to the ability of some tumors to transform (mutate), inhibit anti-proteases and damage local tissues [35]. Conversely, the increased levels of modified DNA bases may contribute to the genetic instability and metastatic potential of the tumor cells in fully developed cancers cells [36]. However, another study reported that, an intense oxidative stress may kill cells; on the other hand, cancer development does not ensue in response to increased levels of oxidative DNA damage [37]. Hence, it has been suggested that oxidative DNA base damage alone may be insufficient to cause cancer development, or damage over only a certain range is active and excessive damage may have an anti-cancer effect by promoting apoptosis [38]. It is a fact that, the healthy individuals, under the normal conditions produce ROS through their aerobic metabolism. Hence, cells have developed a wide range of antioxidant mechanisms to prevent and inactivate the activity of ROS and then repair cell damage [39]. Under normal health conditions, the unbalance between ROS and antioxidants leads to the development of oxidative stress. This oxidative stress plays a key role in female reproductive tract. Agarwal and Said [39] reported that, ROS have important roles in the normal functioning of female reproductive system and in the pathogenesis of female infertility. ROS can regulate cellular functions and can impair the intracellular environment resulting in diseased cells. The excessive levels of ROS can result in the pathogenesis of female reproduction leading to development carcinogenesis through modifying gene expression. ROS such as O2•- are able to diffuse through cell membranes and alter cellular molecules such as lipids, proteins and nucleic acids. As a result, multiple consequences like embryo cell block, mitochondrial modifications, ATP

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Valluru et al: Oxidant and antioxidant status in gynecological cancers Table 1. Reactive oxygen and nitrogen species involved in Carcinogenesis and their interactions Half-life Reactive Species Production Interaction (seconds) ROS

Associated cancer

Hydroxyl radical (•OH)

10-9

Produced by increased iron concentration in body

Superoxide (O2•-)

10-6

Generated in mitochondria and cardiovascular system Produced during large number of metabolic reactions

Cellular compounds like carbohydrates, nucleic acids, lipids and proteins Inactivates enzymes containing iron-sulfur clusters Intercats with lipids, protiens and nucleic acids

Reacts with transient metal ions to yield reactive species

Intercats with lipid peroxidation of poly-unsaturated fatty acids

Melanoma and other skin cancers

Generated during photosensitization and chemical reaction

Cellular proteins and lipids

*Ovarian cancer

Breast cancer, *Brain cancer Breast cancer, Cervical cancer *Gastric cancer

Hydrogen peroxide (H2O2) Organic hydroperoxide (ROOH)

Stable Stable

Singlet oxygen (1O2)

10-6

Bronchogenic and colorectal carcinoma Colorectal carcinoma *Hepatocellular carcinoma

RNS Nitric oxide (NO•)

5

Neurotransmitter and blood pressure regulator

Peroxynitrite (ONOO-) Peroxynitrous acid (ONOOH)

10-3

Formed from NO• and O2•-

Breakdown and deamination of nucleic acids Activation of cyclooxygenase gene

Fairly stable

Protonated form of ONOO-

Intercats with neurotransmitters

*Indicates the indirect role of reactive species in the development of cancer.

diminution and apoptosis take place [40]. ROS also induces lipid peroxidation with related effects in cell division, metabolic transport and mitochondrial dysfunction. Table 1 shows the involvement of ROS and RNS in carcinogenesis and their interactions with different target cells. FREE RADICALS CANCERS

IN

GYNECOLOGICAL

Free radicals in ovarian and endometrial cancers Recent epidemiological studies described that oxidative stress has a causal role in the carcinogenesis of two histological subtypes of ovarian cancer, namely clear cell carcinoma (CCC) and endometrioid adenocarcinoma (EAC). Because of recurrent hemorrhage in endometrial cysts, excess of ROS are produced due to increased iron ions, which results in direct genome mutation of epithelial cells and exaggeration of oxidative stress by stromal cells. In endometrial associated ovarian cancer, genomic changes in specific genes such as p53, ARID1A, K-ras, PTEN and PI3CA have been reported [41]. Endometrial cysts are well known lesions in endometriosis that contain fluid with excess of ferric iron (Fe+3) because of recurrent hemorrhage in cyst. In 1925, Sampson [42] mentioned the first time on endometriosis associated cancer. The deposition of hemosiderin, heme or iron in endometriotic lesions has been assumed to trigger oxidative damage and chronic inflammation [43]. In particular, intracellular iron activates the NF-kB pathway and exaggerates chronic

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inflammation [44]. As a result, prominent oxidative stress or an excess of ROS, is consistently produced. This process leads to have a causative role in endometriosis development and progression leading to carcinogenesis [45]. The high concentration of free iron in endometrial cysts may directly provide oxidative stress that induces genomic mutation in epithelial cells [41]. The excess of iron in experimental animals enhances the epithelial cell proliferation [46] and causes malignant tumors with genomic abnormalities [47], which suggest a similar mechanism leading to carcinogenesis in human endometriosis. Hence, there is scientific evidence in the literature that minimal levels of ROS may be necessary for the development of gynecological cancers (Fig.2). However, further studies are awaited to elucidate the precise role of irondeposition induced oxidative stress in carcinogenesis of endometriosis-associated cancer. Free radicals and oxidative stress have been inconsistently associated with ovarian cancer risk. The women’s health initiative (WHI) study performed on post-menopausal ovarian cancer patients demonstrated that the intake of dietary antioxidants, carotenoids, and vitamin A are not associated with a reduction in ovarian cancer risk [48]. Several risk factors including usage of oral contraceptives, tobacco products may influence disease risk [49]. However, these factors are not modifiable. Experimental evidence suggested that ovarian cancer patients exhibit significantly elevated levels of oxidative stress [50]. Patients diagnosed with ovarian cancers have been shown to have significantly reduced plasma antioxidants [51] and vitamin A concentrations. On the other hand these patients show

DOI 10.5455/oams.201113.rv.011


Figure 2. Oxidative stress mediated gynecological disorders in female reproduction. Oxidative stress causes most of the gynecological disorders namely tubal fertility, polycystic ovarian, endometrial cyst and embryopathies which finally lead to the neoplastic condition through the increased risk factors.

increased levels of oxidative stress and induce the free radical mediated carcinogenesis. A great effort has been made to understand the effects of ROS on DNA damage, induction of mutations and the role of ROS on epigenetics [52]. Most of the signal transduction factors are highly prone to free radical damage resulting in altered function and have been implicated in the activation of transcription factors. Through their ability to stimulate cell proliferation and either positive or negative control of apoptosis, transcription factors can arbitrate many of the physiological and pathological exposures. Hypoxia-inducible factor (HIF)-1 is a heterodimeric transcription factor that plays an important role in signaling and cellular oxygen levels. HIF-1 has been implicated in ROS-induced carcinogenesis of breast, bladder, colon, ovarian, hepatocellular, pancreatic, prostate and renal cancers [53]. Rankin and Giaccia [54] reported that, elevated HIF-1 expression correlates with poor outcome in patients with nasopharyngeal, colorectal, pancreatic, breast, cervical, endometrial, ovarian, bladder, gastric carcinoma and glioblastoma. Some of the findings highlighted that HIF-1 activation is a common event in cancer. Emerging evidence indicates that ROS produced by mitochondrial complex-III are required for hypoxic activation of HIF-1 [55]. Hence, ROS are considered to be activators of HIF-I in hypoxic tumors. Free radicals in cervical cancer A large number of evidence indicates that infection with the sexually acquired human papillomavirus (HPV) is the primary risk factor for cervical cancer and plays a key role in cervical carcinogenesis [56]. Few


prospective studies suggests that HPV infection alone may not be sufficient to promote cervical carcinogenesis and a number of co-factors such as smoking, oral contraceptives, deficiency in antioxidants and inflammation are involved in the development of cancer [57]. Risk factors like cigarette smoking [58] co-infection with bacteria (Chlamydia trachomatis) [57] and cervical inflammation [59] are associated with elevated load of oxidative stress and have been shown to be independently associated with either HPV persistence or grade 2/3 cervical intraepithelial neoplasia (CIN II/III) and cancer. Oxidative stress is implicated in the pathogenesis of cancer; generally two mechanisms contribute to an increase in oxidant load, either excessive generation of ROS or inadequate antioxidant defense [60]. ROS appear to have a key role in cell signaling by activating AP-1 and NF-kB (transcription factors), cell proliferation and apoptosis [61] (Fig.3). These findings are particularly relevant to cervical carcinogenesis when viral replication, expression of HPV-16 E6 and E7 proteins, cell proliferation and apoptosis are important events in cervical carcinogenesis. Using in vitro and in vivo models, scientists have demonstrated that ROS increases the viral titer [62] and infectivity of the influenza virus. In in vitro studies, increases in the cellular oxidative load have been shown to increase the human immunodeficiency virus (HIV) replication [63].

Figure 3: Oxidative stress mediated cancer development in gynecological tissues. Biological, chemical and physical factors mediated free radicals, damages the biomolecules of gynecological tissues/cells (epithelial and connective tissues). The damaged biomolecules initiates the neoplastic cells through the up-regulation of transcriptional factors and inactivation of tumor suppressor genes. The damaged biomolecules also alters the functions of DNA repair proteins, apoptic modulators, metabolic enzymes and signaling pathways induces the neoplastic condition.

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Valluru et al: Oxidant and antioxidant status in gynecological cancers AP-1 is a central transcription factor for the expression of oncoproteins E6 and E7 of the oncogenic HPV which can be activated by ROS. It is believed that this effect is to be due to the ROS-activated NF-kB, as a nuclear transcriptional factor that is required for the replication of HIV [63]. It is well documented that the NF-kB has a functional binding site in the HPV upstream regulatory region (URR) and its effect on HPV gene expression is currently being investigated. Previous findings suggest that inhibition of NF-kB will up-regulate HPV gene expression and, vice versa, activation of NF-kB will down-regulate expression [64]. One of the recent study also reported that the levels of ROS and expression of NF-κB and p53 were higher in nasopharyngeal carcinoma tissue than those in normal nasopharyngeal tissue [65]. Wang et al [66] reported that 25.4% of cervical cancers, 48.4% of endometrial cancers, 21.9% of ovarian cancers and 29.4% of breast cancers shows that one or more mitochondrial microsatellite instability (mtMSI), which was frequently detected in the D-loop region but rarely occurred in the coding region [67] reported that the mutation in the D-loop takes part in carcinogenesis and progression of cervical cancer through the effect of increased ROS. Wei et al [68] reported that, NO acts as a molecular co-factor with HPV infection in cervical carcinogenesis. In another study, Wei et al [69] also found that the presence of HR-HPV is associated with an increased release of NO in the human uterine cervix and that physiological dose of NO could endorse malignant progression of HPV-infected cells in vivo. They also stated that, various epidemiologically defined co-factors for cervical cancer increase NO levels in the cervical microenvironment of cervix [68]. This increase in NO induces earlier mRNA expression, declined retinoblastoma protein (pRb) and p53 levels, low p53 activity, and apoptotic indices in HPV-infected cells in the cervix, consequently resulting in increased survival of mutant cells and leading to carcinogenesis. Epidemiological studies have revealed a number of risk factors like smoking, multiparity, long-time usage of oral contraceptives pills, chronic inflammation and other sexually transmitted infections (e.g., Chlamydia trachomatis and herpes simplex virus (HSV) type 2) [70]. Interestingly, all these co-factors increase the NO levels in the microenvironment of uterine cervix [71]. Significantly increased levels of NO were observed in serum of patients with cervical cancer as compared to healthy controls [72]. Increased NO levels and markers of NO-mediated mutagenesis have been observed in the cervixes of women with CIN [73]. All these findings suggested that NO has potential mutagenic and carcinogenic activity in cervical cancer.

radicals. In the 19th and early 20th century the term antioxidant has been referred specifically to a chemical that prevents the consumption of molecular oxygen. Literally the term antioxidant defines the ‘against to the oxygen’ or so called antioxygen. A well-established research has been developed on the antioxidants which protect cells against the damaging effects of ROS, such as singlet oxygen, peroxyl radicals, superoxide and hydroxyl radicals, and RNS, such as peroxynitrite and nitric oxide [74]. There are two types of antioxidants: enzymatic and non-enzymatic: enzymatic antioxidants are also known as natural antioxidants, act by neutralizing excessive reactive oxygen species, and prevent it from damaging the cellular structure. They are composed of catalase (CAT), glutathione redutase (GR), GSH-Px and SOD, which causes reduction of hydrogen peroxide to water and alcohol [39]. Superoxide dismutase and glutathione peroxidase are natural antioxidants present in organisms which eliminate some ROS and glutathione peroxidase catalyzes the reduction of peroxide by oxidizing glutathione (GSH) to oxidized glutathione [74]. Nonenzymatic antioxidants are synthetic antioxidants or dietary supplements [39]. The body’s antioxidant system is influenced by dietary intake of antioxidant, vitamins and minerals such as vitamin C, vitamin E, selenium, zinc, taurin, hypotaurin, glutathione (GSH), beta carotene and carotene [75] Vitamins C and E are not produced in the body but must be obtained through diet. GSH is produced by the body, but levels of this antioxidant decline with age [76]. Vitamin E has been considered as a natural antioxidant that reacts with soluble free radicals in lipids membranes which prevents the process of lipid peroxidation, as antioxidants reduce agents that disrupt the oxidative chain reactions, often by scavenging ROS before they can cause damage to the cells [77]. This vitamin protects lipids from peroxidation, while being oxidized to tocopheryl quinone or into tocopheroxyl free radical. In both cases, it is reduced by ascorbate (vitamin C), which is afterwards oxidized into dehydroascorbate or αascorbate free radical. Either enzymatic or non-enzymatic antioxidant mechanisms are rapidly attacking the radical and thereby terminate its damaging pathways. This mechanism presumes that the resulting antioxidant derived radical is a ‘harmless’ one, i.e. the reactivity of the antioxidant radical toward typical biomolecules must be low [78].

Antioxidants against to free radicals Nature has endowed each biological cell with adequate protective mechanism against any harmful free

There is large epidemiological evidence that have shown inverse correlation between the levels of established antioxidants/phytonutrients present in

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ANTIOXIDANT ENZYME LEVELS IN CANCER

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Oxidants and Antioxidants in Medical Science 2014; 3(1):15-26 tissue/blood samples and occurrence of cancer. However, some recent meta-analysis shows that supplementation with single antioxidants may not be that effective [79]. Based on the majority of epidemiological and case control studies recommendations were made for the daily dietary intake of some established antioxidants like vitamin E and C. Antioxidants may act in vivo to decrease oxidative damage to DNA, protein and lipids; thus, drop the cancer risk [80]. These include SOD that catalyses the dismutation of superoxide to H 2O2, and CAT that breaks H2O2 down to water [81]. How antioxidant mediated treatment could protect normal cells against damage from treatment, while often increasing their cytotoxic effect against cancer cells? There are two concepts which explain this question; one is the recent evidence that radiation and chemotherapy often harm DNA to a relatively small extent, which causes the cells to undergo the process of apoptosis, rather than necrosis [82]. Hence, many antioxidant treatments induce apoptotic pathways [83]; the potential exists for a synergistic effect with radiation or chemotherapy with antioxidants. A second concept is that the defensive mechanisms of many cancer cells are known to be decreased. This apparently makes tumor cells unable to use the extra antioxidants in a repair capacity; this has been illustrated in vitro [83]. The cellular changes would ideally, enhance tumor cell killing, largely by apoptosis, and reduce the probability of normal cell death. Antioxidant enzymes and detoxifiers have the ability to inhibit tumor initiation and promotion in vivo and in vitro [1]. Other studies in association with antioxidant status in human cervical carcinoma showed a significant reduction in the content of GSH, vitamin E and C, GSH-Px and SOD when compared to normal controls. The reduction was more marked in late stages (II, IV) than in early stages (I, II) [26]. However, at early stages, the activities of antioxidant defense enzymes SOD and CAT in Jamaican women with cervical cancer, showed no substantial changes in the GSH and SOD levels in patients compared to that of controls (normal healthy women). On the contrary, CAT activity was significantly higher in patients than that of the controls [2]. Dasari et al [84] also reported similar results indicating elevated lipid peroxidation and impaired antioxidant status in cervical cancer patients. The increased activity of SOD in some tumor cells is not a characteristic of all tumors [85]. Thus, MnSOD is reduced in a variety of tumor cells and the lowest activity of total SOD (Cu/ZnSOD and MnSOD) has been associated with fastest growing tumor [86]. MnSOD constitutes an enzyme with variable activity in


tumors. A significant overexpression of MnSOD has been found in colorectal and gastric adenocarcinoma. Similarly, other studies have revealed a significant increase of MnSOD mRNA in both esophageal and gastric cancers, compared to normal tissue [87]. It is well established that the individual variability of SODs due to polymorphisms may predispose to carcinogenesis. MnSOD polymorphisms have been investigated in several types of malignancies namely lung cancer, mesothelioma, breast cancer and colon carcinoma. The primary MnSOD polymorphism studied is the presence of valine or alanine (Val/Ala) at position 16 in the MnSOD-targeting sequence for the mitochondria [88]. Therefore MnSOD polymorphism seems to be the main role in the abnormal expression of SOD in different cancers. Several investigations have reported the preventive role of selenium against cancer in a variety of organs and species. In fact, regarding the association between low selenium level and advanced tumor disease, it needs yet to be decided whether this phenomenon is more likely to be a consequence or a causative factor for development and course of the disease [89]. Subramanyam et al [90] also reported that, the antioxidant activity was directly proportional to the levels of serum selenium, which indicates that selenium is one of the key components of the antioxidative mechanism. Selenium exerts its chemopreventive defense mechanism against oxidative damage by scavenging the ROS and improves the synthesis of enzymatic antioxidant GSH-Px [91]. Decreased levels of reduced/oxidized glutathione (GSH/GSSG) ratio in blood were observed in patients with breast and colon cancers. These decreased levels of GSSG were especially recorded in advanced stages of cancer progression. This is may be due to the increased peroxide generation, which leads to an affectation in the GSH-related enzymes, and an increased GSSG release from different tissues within the red blood cells [92]. In fact, these high GSH and peroxide levels in the cells have been reported when a substantial proliferative activity exists. On the other hand, this antioxidant content decreases when cell proliferation and the rate of protein synthesis in the tumor decreases [93]. Antioxidants in cancer therapy Antioxidants enzymes prevent cellular damage by reacting with and eliminating oxidizing free radicals. Usage of antioxidant in cancer treatment is a rapidly developing area, because they have been extensively studied for their ability to prevent cancer in humans [94]. However, in cancer treatment, a mode of action of certain chemotherapeutic agents involves the generation of free radicals to cause cellular damage and necrosis of tumor cells. Hence, a concern has logically

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Valluru et al: Oxidant and antioxidant status in gynecological cancers developed as to whether exogenous antioxidant compounds taken concurrently during chemotherapy could reduce the beneficial effect of chemotherapy on tumor cells. The importance of this concern is underlined by a recent study which estimates 23% of cancer patients take antioxidants [95]. The primary focus of either chemo- or radiotherapy is to produce irreversible DNA damage in tumor cells that will prevent their replication and lead to their demise [96]. Cancer patients with antioxidant supplementation can alleviate the toxicity and improve long-term outcome. The modulating effects of antioxidants in treatment depend on a wide range of factors, including the metabolic state of the patient, the stage and site of the disease, and the modality being used. Another course of action is to alter cellular homeostasis and modify signal transduction pathways and disposition to apoptosis. Antioxidants in chemotherapy Anticancer or chemotherapeutic drugs work by affecting DNA synthesis; they do not kill resting cells unless those cells divide soon after exposure to the drug. Therefore, the efficacy of anticancer drugs used in chemotherapy is limited by the fraction of actively dividing cells. Most anticancer drugs do not rely on ROS, although a few produce free radicals that play a role in treatment; these include bleomycin, doxorubicin (adriamycin) and cisplatin. Although bleomycin is more toxic to oxygenated cells, similar to x- and γ-rays, doxorubicin is preferentially toxic to hypoxic cells [96]. Antioxidant protection of normal cells in all treatments, even when the mechanism of the chemotherapeutic drug is independent of free radical action, help to maintain the health of normal tissues and protect them from the toxic effects of free radical-producing cytokines that circulate in cancer patients and increase with the severity of the disease [97]. Gautam et al [98] reported that, decreased antioxidants were increased in ovarian cancer patients after 6 weeks of chemotherapy treatment. Antioxidants in radiotherapy Radiotherapy uses ionizing radiation (x- and γ-rays) to induce cancer cell death through free radical formation. There are two mechanisms; the first mechanism is apoptosis which results in cell death within a few hours of radiation, and the second one is radiation-induced failure of mitosis and the inhibition of cellular proliferation which kills cancer cells. The principal target of radiation is considered to be cellular DNA. However, experimental studies indicate the signals for apoptosis can be generated by the effect of radiation on cell membranes through lipid peroxidation. This indicates an alternate mechanism to the hypothesis that DNA damage is required for cell death [99]. About two thirds of x- and γ-ray damage is caused by free radicals that kill tumor cells but threaten the integrity and

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survival of surrounding normal cells. Radiation induces mitotic cell death in dividing cells and activates pathways that lead to death by apoptosis in interphase cells and differentiated cells. Response to radiation depends on the type, dosage and time intervals of radiation, inherent tissue sensitivity, and intracellular factors that include position in the cell cycle, concentration of oxygen, thiols and other antioxidants [100]. Combinations of antioxidants Combinations of antioxidants have been shown synergistic anti-tumor effects in vivo. Combinations of antioxidants with chemotherapy and radiation have been shown to increase survival time and reduce toxicity in humans. Whelan et al [101] reported that coadministration of beta carotene and alpha-tocopherol led to much greater tumor regression than either agent alone. During the treatment of radiation, the cancer infected epithelial cells were shrinkage and tumor size was reduced. Hence, the radiotherapy along with chemotherapy kills and decreases the size of cancer cells which facilitate the significant alterations (increased) in the development of antioxidant system, which is not possible in case of chemotherapy alone. Dasari et al [84] reported that significant upsurge was observed in antioxidant levels between the patients treated with radiotherapy and chemotherapy than the patients treated with chemotherapy alone. Hence, the combinational treatment of radiation with chemotherapy in cervical cancer causes sensitization to antioxidants. An open trial of combination antioxidant treatment along with chemotherapy and radiation in patients with small-cell lung cancer had encouraging results with greater two-year survival rate than that of patients received normal treatment [99].

NOVEL APPROACHES TO REDUCE FREE RADICAL DAMAGE AND FUTURE PROSPECTS Several new approaches were developed for the study of free radicals/antioxidants for the improvement of human health. A number of physiological and genetical changes occur in cancer patients, even in the absence of degenerative conditions. Recent studies have found associations between the decline of free radicals and lower status of antioxidants. The experimental, clinical, and epidemiological studies support the notion that consumption of foods obtaining high levels of dietary antioxidants (Table 2), in addition to exerting several health benefits, may reduce the risk of free radical mediated cancer or other diseases. Different naturally derived SOD mimetics are well suited for a drug because of having much lower molecular weight, being more stable, and seeming not to elicit an immune response in the body. SOD

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Oxidants and Antioxidants in Medical Science 2014; 3(1):15-26 Table 2. Dietary antioxidants and their interactions Antioxidants Mode of action on cancers Chemical drugs as antioxidant supplements Regenerates active α-tocopherol (vitamin E) by reducing its radical form 1 Vitamin C Transport and storage depend on selenium; absorption is reduced when vitamin A 2 Vitamin E and β-carotene levels are high – gynecological cancers Conversion to vitamin A requires vitamin E 3 β-carotene Synergistic with vitamin E – gynecological cancers 4 Selenium Dietary antioxidant supplements (vegetables and fruits) Carotenoids - breast cancer 5 Carrots, green vegetables Cruciferous, vegetables, yellow Rich in lycopene – prostate cancer 6 vegetables and tomatoes High levels of antioxidant organosulfur compounds – gastrointestinal cancer 7 Allium vegetables (garlic, onions) Rich in polyphenols – breast, ovarian and cervical cancers 8 Green tea Quercetin, an antioxidant abundant in apples (equals to vitamin C) 9 Apples The high β-carotene content 10 Apricots Vitamins B and C 11 Banana Antioxidants – reduce the side effects of chemotherapy 12 Mustard apple Extracted from Borek [96].

mimetics also have ability to increase antitumor effects of interleukins, besides being efficient radioprotectors [8]. Development of genetically engineered plants, to yield vegetables with higher level of compounds is another approach to increase antioxidant availability. Tomatoes with up to three times lycopene concentration as well as with longer shelf life were developed. ‘Orange cauliflower’ is found to be rich in carotene. Intake of fruits and vegetables with ORAC (oxygen radical absorbance capacity) values between 3000 and 5000 per day is recommended to have significant impact of the beneficial effect of antioxidants [102]. One of the major applications of nanotechnology in biomedicine was the production of bioactive nanoparticles. Nanoparticles can be engineered as nanoplatforms for effective and targeted delivery of drugs and imaging labels by overcoming the many biological, biophysical, and biomedical barriers [103]. To avoid problems of cancer chemotherapy and increase the absorption of drugs in the exact target sites, nanotechnological targeted cancer chemotherapy has been proposed. Nanotechnological based system includes nanoparticles, nanofibers, nanocapsules, nanorods, nanocrystals, nanotubes, stealth nanoparticles, liposomes, stealth liposomes, pH and temperature sensitive liposomes, etc. All such delivery materials implies selective and effective localization of pharmacological active moiety at pre-identified (e.g. over expressed receptors in cancer) targets in therapeutic concentration while restricting its access to non-target sites, thus, reduces toxicity, maximizes therapeutic index as well as improves the biodistribution of the drug, which is a major factor in success of cancer chemotherapy [104].


CONCLUSION Free radicals have been implicated in the etiology of large number of cancers, especially gynecological cancers. They can adversely alter many crucial biological molecules leading to loss of structure and function. Such undesirable changes in the women reproductive system can lead to conditions through ROS or RNS, known as gynecological cancers. Antioxidants can protect against the damage induced by free radicals at various levels. Either enzymatic or non-enzymatic antioxidants act as scavengers against free radical induced damage. Excessive intake of foods with functional attributes including high level of antioxidants is one strategy that is gaining importance in advanced countries and is making its appearance in our country (India). It is important to note that the traditional Indian diet and medicinal plants are rich bases of natural antioxidants. Harmonized research involving scientists, nutritionists and physicians can make significant variance to human health in the coming decades. Investigation on free radicals and antioxidants is one such effort in the right direction.

ACKNOWLEDGEMENTS: The second author (SD) is thankful to the University Grants Commission (UGC), New Delhi for providing the Basic Scientific Research (BRS)-Non-SAP (Special Assistance Programme) fellowship.

COMPETING INTERESTS The authors declare that they have no conflict of interest.

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81. Sies H. Antioxidants in Disease, Mechanisms and Therapy, Academic Press, New York, NY, pp 165-170, 1996.

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82. Schmitt CA, Lowe SW. Apoptosis and therapy. J Pathol 1999; 187:127-37. 83. Mediavilla MD, Cos S, Sanchez-Barcelo EJ. Melatonin increases p53 and p21WAF1 expression in MCF-7 human breast cancer cells in vitro. Life Sci 1999; 65:415-20. 84. Dasari S, Wudayagiri R, Valluru L. Efficacy of treatment on antioxidant status in cervical cancer patients: a case control study. Free Radic Antioxid 2013; 3:87-92. 85. Kong Q, Lillehei KO. Antioxidant inhibitors for cancer therapy. Med Hypotheses; 1998; 51:405-9. 86. Bertrand JS. The molecular biology of cancer. Mol Aspects Med 2000; 21:167-223 87. Izutani R, Asano S, Imano M. Expression of manganese superoxide dismutase in esophageal and gastric cancers. J Gastroenterol 1998; 33:816-22. 88. Vuokko LK, James DC Superoxide dismutases in malignant cells and human tumors. Free Radic Biol Med 2004; 36:718-44. 89. El-Bayoumi K, Narayanan BA, Desi DH, Narayanan NK, Pittman B, Amin SG, Schwartz J, Nixon DW. Elucidation of molecular targets of mammary cancer chemoprevention in the rats by organoselenium compounds using cDNA microarray. Carcinogenesis 2003; 24:1505-14. 90. Subramanyam D, Subbaiah KV, Rajendra W, Lokanatha V. Serum selenium concentration and antioxidant activity in cervical cancer patients before and after treatment. Exp Oncol 2013; 35:97-100. 91. Patrick L. Selenium biochemistry and cancer: a review of the literature. Altern Med Rev 2004; 9:239-58. 92. Carretero J, Obrador E, Anasagasti MJ, Martin JJ, VidalVanaclocha F, Estrela JM. Growth-associated changes in glutathione content correlate with liver metastatic activity of B 16 melanoma cells. Clin Exp Metastasis 1999; 17:567-74.

95. VandeCreek L, Rogers E, Lester J. Use of alternative therapies among breast cancer outpatients compared with the general population. Altern Ther Health Med 1999; 5:71-6. 96. Borek C. Dietary Antioxidants and human cancer. Integr Cancer Ther 2004; 3:333-41. 97. Sozen S, Coskun U, Sancak B, Bukan N, Gunel N, Tunc L, Bozkirli I. Serum levels of interleukin-18 and nitrite+nitrate in renal cell carcinoma patients with different tumor stage and grade. Neoplasma 2004; 51:25-9. 98. Gautam S, Bhatt MLB, Singh R, Mehrotra S, Singh U, Saxena JK, Singh RK. Impact of therapeutic intervention on oxidants and antioxidants status in patients with ovarian malignancy. Biomed Res 2011; 22:259-62. 99. Lamson DW, Brignall MS. Antioxidants in cancer therapy; their actions and interactions with oncologic therapies. Altern Med Rev 1999; 4:304-29. 100.Coia LR, Moyland DJ. Introduction to Clinical Radiation Oncology. Medical Physics Publishing, Madison, WI, pp 15-19, 1998. 101.Whelan RL, Horvath KD, Gleason NR, Forde KA, Treat MD, Teitelbaum SL, Bertram A, Neugut AI. Vitamin and calcium supplement use is associated with decrease adenoma recurrence in patients with a previous history of neoplasia. Dis Colon Rectum 1999; 42:212-7. 102.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. 103.Grodzinski P, Silver M, Molnar LK. Nanotechnology for cancer diagnostics: promises and challenges. Expert Rev Mol Diagn 2006; 6:307-18. 104.Kakde D, Jain D, Shrivastava V, Kakde R, Patil AT. Cancer therapeutics - opportunities, challenges and advances in drug delivery. J Appl Pharm Sci 2011; 1:1-10.


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DOI 10.5455/oams.201113.rv.011


ISSN: 2146-8389

REVIEW ARTICLE

“Mild” mitochondrial uncoupling as potentially effective intervention to slow aging Vladimir Illich Padalko Department of Membrane Biophysics, Research Institute of Biology, V. N. Karazin Kharkiv National University, Kharkiv, Ukraine Received November 17, 2013 Accepted December 16, 2013 Published Online January 27, 2014 DOI 10.5455/oams.161213.rv.012 Corresponding Author Vladimir Illich Padalko Department of Membrane Biophysics, Research Institute of Biology, V. N. Karazin Kharkiv National University, Svobody sq. 4, Kharkiv 61022, Ukraine [email protected] Key Words Aging; Mitochondrial uncoupling; Oxidative stress

Abstract The main objective of this review is to elucidate the role of endogenous reactive oxygen species (primarily mitochondrial origin) in the aging process. We have attempted to highlight the findings from several investigations about the relationship between reduction in mitochondrial production of free radicals (using specific antioxidants or “mild” mitochondrial uncoupling) and life span. Several studies on animal models have shown that aging rates and life expectancy could be modified using mitochondria-targeted antioxidants and uncouplers. In particular, different uncoupling strategies were able to extend life span in models ranging from yeast to mammals. These findings are in line with the “uncoupling to survive” hypothesis, suggesting that uncoupling could be an approach to promote life span extension due to its ability to prevent the formation of reactive oxygen species. Obviously, because of the high toxicity, 2,4-dinitrophenol and other uncouplers themselves can not be applied in practical geriatrics, but their low toxic analogues having controlled and “soft” action either agonists affecting the natural way of uncoupling (uncoupling proteins, UCPs) are promising for the development of means for control of tissue redox state and animal life span. © 2014 GESDAV

INTRODUCTION Great current interest in the aging process, both in scientific and public settings, has been stimulated by a number of factors. First of all, advances in medicine and public health have essentially increased average life expectancy over the past 200 years. According to the data from the National Institute of Aging (NIA), United Nations and Statistical Office of the European Communities [1-3] there are several trends in global aging: -a considerable aging of the population is now occurring in economically developed countries, i.e., the fraction of elderly persons has increased especially during the last quarter of the XX century; -the number of oldest old is rising. People aged 85 and over are now the fastest growing portion of many national populations. Average human life expectancy has progressively increased largely due to the improvements in nutrition, vaccination, antimicrobial agents and effective treatment/prevention of cardiovascular disease, cancer, etc; -all these trends have serious economic consequences, since population aging will have dramatic effects on social entitlement programs, labor supply, trade and savings around the globe. Altogether, this global demographic change represents one of the main social, scientific and economic challenges of our time. On the one hand this indicates


essential achievements of mankind in health care and solution of social problems, but on the other hand it is associated with many problems which require fundamental understanding of aging mechanisms and novel therapeutic developments. An enormous effort has been expended to understand how the aging process is regulated at the molecular and cellular levels, but unfortunately the final aging mechanisms are not established. MECHANISMS OF AGING As known, modern understanding of the mechanisms of aging are based on two most important aspects of the aging process: (1) aging is characterized as a progressive decline in biological functions with time, and (2) aging results in a decreased resistance to multiple forms of stress, as well as an increased susceptibility to numerous diseases [2]. But despite well-described definition, aging remains one of the most poorly understood phenomena, due in large part to its inherent complex and integrative nature, as well as the difficulty in dissociating the effects of normal aging from those manifested as a consequence of age-associated disease conditions [2]. As a result, it is proposed a large number of theories that attempt to explain why we age, and no single theory has been completely successful in explaining the aging process [4, 5].

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Padalko: Mild mitochondrial uncoupling slows aging For example, more than a century ago, Max Rubner coupled the observation that maximal life span of mammalia increases with body size and the massspecific rate of metabolism of mammalia decreases with body size. Later, this perspective was elaborated and extended by Raymond Pearl, who proposed that life span differences of animals maintained at different temperatures could be explained by differences in metabolic rate and gave the “rate-ofliving theory of aging” its name [6]. But one of the prevalent theories in the current literature revolves around free radicals, as causal agents in the process of aging. “Free radical theory”, formulated by Harman [7] in 1950s is one of the leading hypotheses because it rather satisfactorily explains many observations of modern gerontology. It is established that generation of reactive oxygen species (ROS), which are molecules containing unpaired, highly reactive electrons, contributes to the accumulation of oxidative damage to cellular constituents (nucleic acids, proteins and lipids, among others), resulting in the process of “aging”. Thus, a more modern version of this tenet is the “oxidative stress theory” of aging. The term “oxidative stress”, popularized by Helmut Sies [8], was defined as “a disturbance in the pro-oxidant/ antioxidant balance in favor of the former, leading to potential damage”. In the context of the aging process, oxidative stress was interpreted to connote a cellular state in which the antioxidant defenses are insufficient for the complete eradication of various ROS, thereby resulting in the age-dependent accrual of macromolecular structural damage [9]. Nevertheless, there are relatively few substantive differences between the basic propositions of the original “free radical” and the later “oxidative stress” hypotheses. Both postulate that the rate of aging, i.e., the progression of age related deleterious alterations, is a function of the imbalance between ROS fluxes and antioxidant defenses and both predict that the narrowing of this gap should reduce the amount of structural damage and prolong the life span. Therefore, from this historical perspective, the postulated mechanism implicating ROS in the aging process according to Sohal and Orr [9] can be characterized as the “structural damage-based oxidative stress hypothesis”. So, today the variants of “oxidative stress theory” are among the most widely accepted mechanistic explanations of aging and life span, and there is much evidence to support it.

OXIDATIVE STRESS IN NORMAL AGING AND EFFECTS OF ANTIOXIDANT MANIPULATION OR CALORIE RESTRICTION ON LONGEVITY One of the central themes of the oxidative stress hypothesis is that ROS are the primary causal underlying aging associated declines in physiological functions. Several sets of direct and indirect evidences generated over the past two decades have demonstrated a strong correlation between aging and an increase in oxidative damage to tissues in species ranging from Caenorhabditis elegans to humans [10-12]. But although numerous studies have demonstrated a correlation between in vivo oxidative damage and aging, a more insightful test of the oxidative stress theory would be to assess the direct effects of antioxidants on aging processes. Antioxidants Over the past few decades, research in natural dietary compounds with use of various animal models provides a new strategy for anti-aging. Natural dietary compounds act through a variety of mechanisms to extend life span and prevent age-related diseases [13]. For example, a polyphenolic antioxidant resveratrol can reduce oxidative damage and cognitive impairment in senescence-accelerated mouse [14]. Chondrogianni et al [15] have identified quercetin and its derivative, namely quercetin caprylate, as a proteasome activator with antioxidant properties that consequently influence cellular life span, survival and viability of primary human fibroblasts (HFL-1, human fetal lung fibroblasts). Grape intakes, especially grape pomace with the highest content of flavonoids, betacarotene, tocopherols and dietary fiber, showed the effective capacity of inhibiting age-related increase of lipid peroxidation and DNA damage [16]. These and many other results suggest that the pharmacological action of natural antioxidants may offer a perspective therapeutic strategy for the treatment of age-related conditions. And now many people consume antioxidants (synthetic or natural) in order to decrease oxidative stress, to modulate the aging process, and to extend the health-span. For example, the data from the Food and Drug Administration (FDA) of the United States suggest that more than 29,000 different nutritional supplements (many of which are antioxidants) are available to consumers. It also appears that in the USA more than 12 billion US$ per year may be spent on supplements, and supplements represent a market of over 30 billion $ worldwide [17, 18]. Calorie restricted diets Probably one of the most important arguments in favor of the free radical concept of aging are the

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DOI 10.5455/oams.161213.rv.012

Oxidants and Antioxidants in Medical Science 2014; 3(1):27-42 results obtained from keeping animals on a calorie restricted diets (CR). CR is the only non-genetic treatment that has been shown clearly to increase maximal life span in most, if not all, species where it has been applied [19]. A growing body of evidence supports the hypothesis that CR, with no malnutrition and an adequate mineral intake, also works by decreasing oxidative stress [10, 20]. In particular, Zheng et al’s investigation [21] and our own results [22], obtained in the study with Drosophila, support the universal character of the CR geroprotective action on organisms of different levels of organization and show that CR in flies, as in mammalia, slows the accumulation of oxidatively damaged macromolecules. Thus, a large body of evidences supports the notion that ROS are produced in cells and can manifest damage, but a causal link between ROS and aging has still not been clearly established [6]. Furthermore, in addition to being potentially toxic, ROS were considered to be physiologically important regulatory molecules. Accordingly, oxygen utilization by aerobes was regarded as both vital for their survival, by virtue of its capacity to accept electrons during respiration, and potentially deleterious: a phenomenon that was referred to as the “oxygen paradox” [9]. Cellular sources of reactive oxygen species: mitochondrial theory of aging As generally known, ROS include a number of molecular species derived from oxygen that are relatively reactive, but biologically most of them are derived from either superoxide (O 2•-) and/or hydrogen peroxide (H2O2). In mammalian cells, a number of endogenous sources of ROS are known including mitochondria (mainly complex I & III, but also monoamino oxidase and α-ketoglutarate dehydrogenase), endoplasmic reticulum (mainly cytochrome P-450 and b5 enzymes), peroxisomes (mainly fatty acid oxidation and D-amino acid oxidase), cytosol (NO synthases and lipoxygenases), plasma membrane (NADPH oxidases, lipoxygenases), and extracellular space (xanthine oxidase) [23]. Mitochondrial energy metabolism is recognized today as one of the most quantitatively important source of ROS in the eukaryotes. Hydrogen peroxide production from mitochondria has been first recorded more than 40 years ago [24]. In these pioneering works it was recognized that H2O2 production in mitochondria accounts for up to 2% of the total oxygen consumption and the rate of mitochondrial H2O2 production is strongly dependent on the metabolic state: it is high in state 4, when the NADH/NAD + and ubiquinol/ubiquinone pools are largely reduced; and it


is low in state 3, when the steady-state concentrations of the potential oxygen reductants are decreased [24, 25]. However, the other point of view, according to which the frequent assertion that 1-4% of mitochondrial O 2 consumption is diverted to ROS in vivo is wrong. It is based on the maximum rate of superoxide production from complex III in mitochondria in the presence of antimycin, at saturating substrate and O 2. Less unrealistic conditions predict lower values, only 0.15%, and which of the specific sites of mitochondrial ROS production is the most active in cells in the absence of inhibitors of the electron transport chain is probably unclear [26]. Generally, little is known about the regulation of mitochondrial function in vivo; thus it is unknown how much superoxide is produced by mitochondria in vivo. But it would still be considered, that under certain physiologically relevant conditions, mitochondria are thought to contribute significantly to the steady-state level of H2O2 in cells [27]. As it is well known, the mitochondrial electron transport chain (ETC) links the transfer of electrons from reduced cofactors to the ultimate electron acceptor of the ETC (i.e., oxygen) with the simultaneous transport of protons from the mitochondrial matrix across the inner mitochondrial membrane and into the intermembrane space. Movement of protons out of the matrix and into the intermembrane space results in an electrochemical potential (ΔΨ) which can then be used to drive protons back into the mitochondrial matrix through the ATP synthase (complex V), facilitating the synthesis of adenosine-5′-triphosphate [12, 28]. However, at various sites within the ETC, electrons may occasionally leak to oxygen, forming O 2•- via single electron reduction [29]. About half of the total ROS (O2•- and H2O2) produced by heart mitochondria at high NADH/NAD + ratio can be accounted for as being generated by the respiratory chain components (complexes I and III) [27]. Inhibitory analysis suggests that the major part of superoxide produced by the respiratory chain (up to 70%) is generated by complex I [30]. The reductant of oxygen to produce O2•- in complex I is not known and published reports are highly conflicting [31]. However, recent studies of Russian scientists [32] have shown that complex I bears at least two NADH (NAD +) specific sites. One site (F) has high affinity to NADH and low affinity to NAD +, serves as the entry point for NADH oxidation by ubiquinone and coupled translocation of 4 H + per molecule of NADH oxidized, and the other site (R) is specifically involved in univalent reduction of oxygen. The presence of the specific site R

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Padalko: Mild mitochondrial uncoupling slows aging participating in the ΔµH+-dependent ubiquinol:NAD + reductase reaction (reverse electron transfer) and in O2•- generation by complex I raises the question of the physiological significance of these reactions. According to Vinogradov and Grivennikova [32] of the above mentioned study, succinate, the most widely used substrate, is not the only and, most likely, not the major electron donor at ubiquinone level. Each first dehydrogenation in the fatty acid β-oxidation cycle, oxidation of α-glycerophosphate, choline, sarcosine, and dihydroorotic acid (in eukaryotes) is coupled with direct ubiquinone reduction, and site R may participate in “universalization” of the reducing equivalents in the form of NADH via reverse electron transfer. But in general, despite the large amount of scientific results, there is still much uncertainty as to the mechanisms and physiological role of generation of ROS in the mitochondrial membranes. As previously stated in this review the proportion of oxygen consumed that emerges as ROS is highest under nonphosphorylating conditions rather than phosphorylating ones, most determinations of mitochondrial ROS production are made under state 4 conditions. This is combined with the fact that in intact cells, mitochondria normally respire in metabolic states that are intermediate between state 3 and state 4. Consequently, more studies of ROS generation are needed to elucidate the sites and rates of ROS production in vivo [31]. In addition, according to the report of Vinogradov and Grivennikova [32] physiological significance of O2•- generation by the respiratory chain (mainly by complex I) seems to be of minor importance, at least under normal mitochondrial functioning. The rate of O2•- formation is quite low and combined operation of antioxidant enzymes in matrix is expected to diminish or neglect completely the potential ability of the respiratory chain to create a significant amount of O2•-. This, by no mean, makes O2•- production by mitochondria unimportant [32]. But this process may become of great importance under some extreme conditions (such as reoxygenation after anoxia), and the relative “leakage” of the respiratory chain for univalent oxygen reduction may be tissue specific; therefore comparative quantitative analysis of mitochondria from different tissues and species would be the most interesting [32]. At present, the analysis of relative contribution of different enzymes into overall superoxide and hydrogen peroxide production by mitochondria remains strongly dependent on reliability of the methods for quantitative determination of these analytically “difficult” compounds [32]. According to Brand [26], there are two major problems hindering

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identification of the specific mitochondrial sites of ROS production in cells. The first one is the methods to quantify ROS production; fluorescent probes such as dichlorodihydrofluorescein or dihydrorhodamine have interpretational problems: their specificity is often unclear and they may themselves cause ROS production. The second problem is the use of electron transport inhibitors to identify sites of ROS production, which poses the same problems as with isolated mitochondria. Overall, despite the lack of consensus in the interpretation of results obtained in different laboratories, probably it can be considered that mitochondria are still attracting the attention of researchers as an important intracellular source of ROS, which are known to be important determinants in cell function, participating in many signaling networks and also in a variety of degenerative processes [33]. For example, mitochondrial ROS are considered to be totally or partially responsible for several diseases including Alzheimer's disease, Parkinson's disease and several human cancers [34]. Furthermore the “mitochondrial free radical theory of aging” (MFRTA) hypothesizes that mitochondria are the critical component in control of aging. It is proposed that electrons leaking from the ETC produce ROS and that these molecules can then damage ETC components and mitochondrial DNA, leading to further increases in intracellular ROS levels and a decline in mitochondrial function [2]. In support of MFRTA, there are comparative data showing that the production of ROS is low in mitochondria isolated from large long-lived mammals compared to small short-lived ones, with mitochondria from mammals of intermediate life span and body mass having intermediate rates of ROS production, and ROS production rates of mitochondria from long-lived birds are lower than those from similar-sized short-lived mammals (rats and mice) [35]. Lambert et al [35] critically tested the hypothesis that longevity correlates with mitochondria radical production. Authors investigated this correlation by comparing rates of H2O2 production by heart mitochondria isolated from groups or pairs of species selected to have very different maximum life span but similar body masses (small mammals, medium-sized mammals, birds). It was shown that during succinate oxidation, H2O2 production rates were generally lower in the longer-lived species. Additional data were obtained from large species and the final dataset comprised mouse, rat, white-footed mouse, naked mole-rat, Damara mole-rat, guinea pig, baboon, little brown bat, Brazilian free-tailed bat, ox, pigeon and

DOI 10.5455/oams.161213.rv.012

Oxidants and Antioxidants in Medical Science 2014; 3(1):27-42 quail. In this dataset, maximum life span was negatively correlated with H 2O2 production at complex I during reverse electron transport [35]. These findings indicate that enhanced longevity may be causally associated with low free radical production by mitochondria across species over two classes of vertebrate homeotherms [35]. However, further studies with greater numbers of species are still required to test the generality of the hypothesis, particularly since one exception was found: heart mitochondria from long-lived naked mole-rats produced more ROS than expected (or the naked mole-rats lived longer than expected) compared to the other species that were examined [35]. Thus, the present data from different laboratories suggest that complex I can probably play a central role in the regulation of longevity. Stefanatos and Sanz [36] propose that complex I regulates aging through at least two mechanisms: (1) ROS-dependent mechanism that leads to mitochondrial DNA damage, and (2) ROS-independent mechanism through the control of the NAD + to NADH ratio. In summary, “mitochondrial free radical theory of aging” is currently mainly supported by indirect data which show a negative correlation between free radical production in isolated mitochondria and life span in several different model organisms. However, correlations can suggest but not demonstrate causality. In fact, the only definitive way to test MFRTA is to specifically decrease (or increase) mitochondrial ROS production and to study the effect of such modification on life span. CONDITIONS MODIFYING MITOCHONDRIAL FUNCTION AND ANIMALS’ SURVIVAL It does not cause a big surprise that the selected life conditions that are able to improve survival in animals are similar to the recommendations that are commonly followed by many aging humans, i.e., moderate physical exercise, antioxidant supplementation, CR and so on [12]. It is known that CR rats showed structural and functional liver mitochondrial properties (fatty acid pattern, respiratory chain activities, antioxidant level, and hydroperoxide content) similar to those of younger rats [37]. It was reported that long-term CR led to an essential decreasing in the rate of mitochondrial H 2O2 generation and in oxidative damage to mitochondrial DNA in the rat skeletal muscle [38]. These and other results support the possibility that CR increases maximum life span at least in part through decreases in mitochondrial oxidative stress. At the same time although mitochondria can produce ROS at complexes I and III, it was shown that CR decreases ROS production exclusively at complex I,


because the decrease in oxygen radical generation occurs with pyruvate plus malate, but not with succinate plus rotenone, as substrate [20]. Furthermore, according to Barja [39], the mechanism allowing the decrease in ROS production during CR probably is not a simple decline in mitochondrial oxygen consumption because it stays unchanged. Instead, the percentage of total electron flow directed to ROS generation (the free radical leakage) is decreased with CR. And recent data indicate that the decrease in mitochondrial ROS generation may be also due to protein restriction rather than to calorie restriction, and more specifically to dietary methionine restriction. According to Pamplona and Barja [40], lowering of methionine levels is involved in the control of mitochondrial oxidative stress and vertebrate longevity by at least two different mechanisms: (1) decreasing the sensitivity of proteins to oxidative damage, and (2) lowering of the rate of ROS generation at mitochondria level. So, at present the MFRTA probably should be considered as one of the most widely believed and supported theories of aging. But in spite of its attractiveness, MFRTA has received some recent criticism [41]. For example, there is a standing criticism by Nohl and colleagues that mitochondria in vivo are not an effective source of O2•- and H2O2 and that the determined rates are artifactual [42]. According to Brown and Borutaite [23], mitochondria are a significant source of ROS, but not the main source at least in mammalian liver. Long-lived animals were shown to produce fewer free radicals and to have lower oxidative damage levels in their tissues. However, it does not prove that free radical generation determines life span. In fact, the longest-living rodent, naked mole rat Heterocephalus glaber, produces high levels of free radicals and has significant oxidative damage levels in proteins, lipids and DNA [35, 43]. So in summary, available data concerning the role of free radicals in longevity control are sometimes contradictory and do not always prove MFRTA. In fact, the only way to test this theory is by specifically decreasing mitochondrial free radical production without altering other physiological parameters. If MFRTA is true animals producing fewer mitochondrial ROS must have the ability to live much longer than their experimental controls. Some alternatives will be discussed which might be effective in decreasing mitochondrial oxidative stress. It seems more promising to reduce the free radical formation in mitochondrion than trying to neutralize free radicals after they have been produced. Recent evidences discussed below show that such possibilities exist.

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Padalko: Mild mitochondrial uncoupling slows aging PROBABLE WAYS TO TEST THE MITOCHONDRIAL FREE RADICAL THEORY OF AGING BY SPECIFICALLY DECREASING FREE RADICAL PRODUCTION Over-expression enzymes

of

mitochondrial

antioxidant

One approach to protect mitochondria from oxidative damage is targeting antioxidant compounds, including low molecular weight antioxidants and antioxidant enzymes, specifically to mitochondria. In support of this strategy, mitochondrially-targeted catalase mitigates multiple age-related pathologies, including cardiac tissue pathology, hearing loss, and comorbidity factors including tumor burden [44]. In contrast to these mitochondria-targeted catalase mice, over-expression of the same enzyme in either the nucleus or peroxisomes did not result in significant life span effects, and in other experiments, over-expression of the mitochondrial matrix MnSOD decreased ROS without a corresponding life span increase [28, 45]. In accordance with the study of Jang et al [45], Perez et al [46] collected life span data from multiple studies using mice either under- or over-expressing different antioxidant system components. Of these, only SOD1-/- mice had a significantly shorter life span. However, these mice also displayed levels of oxidative damage 4-5 fold higher than in aged wild type mice, and had a high incidence of hepatocellular carcinoma, suggesting that their life span deficit may not represent normal mechanisms of aging [44]. According to Mookerjee et al [44] two major considerations emerge from these studies: in addition to the amount of ROS, both the species of ROS and the subcellular location of antioxidant activity, and not simply ROS levels, can strongly affect whether increased ROS production correlates with decreased life span. At the same time the mitochondria-targeted catalase mice probably may be considered proof-ofprinciple for the potentially significant, multi-factorial efficacy of targeted antioxidant interventions in the context of age-dependent diseases and aging [28]. Mitochondria-targeted antioxidants Penetrating cations It is known that a well-established approach to targeting small molecules to mitochondria utilizes the negative charge of the mitochondrial matrix against to the cytoplasm to facilitate accumulation of positively charged molecules (cations) in the mitochondrial matrix [47]. However, small charged molecules are typically hydrophilic in nature and moving them from the aqueous extracellular or cytosolic compartment into the hydrophobic interior of cell or mitochondrial membranes is energetically unfavorable. Only if a

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cation is combined with suitably hydrophobic groups the overall molecule become sufficiently hydrophobic to allow entry into the hydrophobic interior of biological membranes. Such lipophilic or “penetrating” cations are then able to cross through the hydrophobic interior of the phospholipid bilayer of biological membranes. As a result, lipophilic cations will accumulate inside mitochondria [47, 48]. While a wide range of lipophilic cations could in principle be utilized for drug targeting purposes (for example commonly used fluorescent dyes such as rhodamine 123), to date only one such compound has been extensively characterized in this context. Triphenylphosphonium (TPP) consists of a positively charged phosphorus atom surrounded by three hydrophobic phenyl groups, giving it an extended hydrophobic surface despite the positive charge of the phosphorus atom. TPP and TPP-conjugated compounds have been reported to firstly accumulate 5-10 fold inside cells relative to the extracellular space, and then to further accumulate up to 1,000 fold inside energized mitochondria both in vitro and in vivo [48, 49]. A range of antioxidant moieties has been conjugated to TPP for targeting to mitochondria. Using aliphatic linkers of various lengths between TPP and the antioxidant moiety, the degree of hydrophobicity of such compounds can be modified, and their efficacies have been explored both in vitro and in vivo [48, 49]. Mito-compounds As it is known, one of the first proof-of-principle compounds for the feasibility of utilizing the TPP moiety to target antioxidants to mitochondria was mitochondria-targeted α-tocopherol, called MitoVit E: [2-(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1benzopyran-2-yl)ethyl]triphenylphosphoniumbromide [43]. While in vivo data show that MitoVit E can be chronically administered to animals in drinking water, has good bioavailability and limited toxicity [50], it has not been investigated for its therapeutic potential in humans. To date, most intervention studies have focused on TPP conjugated to ubiquinone via a 10-carbon aliphatic carbon chain, namely ubiquinonyl-decyltriphenylphosphonium or MitoQ. Ubiquinol was chosen as the antioxidant moiety because of its ability to inhibit lipid peroxidation and because it can be regenerated by the mitochondrial ETC following oxidation [50]. Multiple lines of evidences indicate that most MitoQ and related TPP compounds found in tissue indeed localize to mitochondria [48, 50]. But were reported in vitro evidence that MitoQ, at high enough concentration, can undergo redox cycling and act as pro-oxidant [48, 51]. At the same time, some authors

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Oxidants and Antioxidants in Medical Science 2014; 3(1):27-42 have also reported no evidence that such pro-oxidant effects were observed in vivo, possibly because MitoQ under physiological conditions was not able to interact sufficiently with the flavin site of complex I to cause such pro-oxidant effects [28]. Nevertheless, MitoQ's potential as a modulator of aging has been examined in Drosophila melanogaster. In life span experiments using wild type Drosophila maintained under normal culture conditions, MitoQ was reported to have no [52] or at best a marginal positive effect [48] on life span. When administered to a short-lived mutant strain, deficient in the mitochondrial form of the antioxidant enzyme superoxide dismutase, MitoQ significantly extended life span in adult female but not male flies [52]. While MitoQ does not seem to modulate aging rate, at least in those model organisms that have been investigated (flies, nematodes) [52, 53], it might be suitable as an intervention into age-dependent conditions that are associated with increased oxidative stress and elevated mitochondrial damage, but an obstacle to the widespread use of this drug may be a “slight difference” between anti- and pro-oxidant doses of the MitoQ. Mitochondria-targeted plastoquinone (SkQ) Probably, the most active research groups in mitochondria-targeted antioxidants investigation are from Russia and some other countries. They are headed by Vladimir Skulachev, who developed an alternative series of mitochondria-targeted antioxidant compounds based on the hydrophobic cation targeting approach. Skulachev et al [48] used in their research plastoquinone, a quinone in the electron transfer chain of chloroplasts, in place of the ubiquinone antioxidant moiety of MitoQ, reasoning that plastoquinone might provide better antioxidant activity. Several related “Sk” compounds were synthesized, exploring a range of plastoquinone derivatives, as well as ubiquinol as antioxidant moieties, and also including some alternative lipophilic targeting cations [48]. Using planar bilayer phospholipid membrane, autors [54, 55] selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyltriphenylphosphonium (SkQ1), plastoquinonyl-decylrhodamine 19 (SkQR1), and methylplastoquinonyldecyl-triphenylphosphonium (SkQ3). Anti- and pro-oxidant properties of these substances and also of MitoQ were tested in aqueous solution, detergent micelles, liposomes, isolated mitochondria, and cell cultures [54]. In mitochondria, cationic quinone derivatives in micromolar concentrations were found to be pro-oxidant, but at lower (submicromolar) concentrations they displayed antioxidant activity decreasing in the series SkQ1 = SkQR1 > SkQ3 >MitoQ. SkQ1 was reduced


by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Essentially than in cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria [54]. So, the most important feature of the cationic derivatives of antioxidant plastoquinones is the extremely low concentrations required for their effects to be seen: in experiments on cells this is the range of 10–12 to 10–9 M; in the case of treatment of senile ophthalmic diseases, one drop of 2.5•10–7 M solution daily; in therapy of heart arrhythmia, 1•10–10 mol/kg per day; in experiments on life span elongation, 5•10–10 to 5•10–9 mol/kg per day [56]. Another factor providing the high efficiency of SkQ1 is its ability to recover from its oxidized form to the initial reduced (working) form. All the above-said allows authors [56] to conclude that SkQ1 is an artificial rechargeable antioxidant addressed to the inner mitochondrial membrane. The greatest interest for the purposes of this review is the evidence that in the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, fly Drosophila and mice, SkQ1 prolonged life span, being especially effective at early and middle stages of aging [54]. In particular, addition of SkQ1 to Drosophila melanogaster growing medium has been reported to extend life span in virgin [53] but not in mated flies [57]. SkQ1 exerted its protective effect in virgin flies predominantly by protecting flies from early death and the observed life span benefit was small (~15%) yet significant [53]. Using long-term monitoring of SkQ1 effects on the Drosophila life span, Krementsova et al [58] analyzed different integral parameters of Drosophila survival and mortality under SkQ1 treatment. Adding SkQ1 to fly food was associated with a reduction in early mortality and a decrease in random variation in life span. Analysis of the Gompertz function parametric plane demonstrated significant differences between points corresponding to experimental and control cohorts. These findings indicated that the SkQ1 effect on life span was associated with both elevation of life quality and slowing of aging. Furthermore, according to Krementsova et al [58], the results presented in their work show that the nature of the SkQ1 effect on Drosophila longevity was constant for six years, regardless of fluctuations in the control life span, differences in preparation and administration of SkQ1 solution, or the year and season when the experiments were conducted. What is more, SkQ1 affected the life span of not only inbred and outbred laboratory mice but also wild rodents housed in outdoor cages or kept under conditions that simulated natural seasonal changes in

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Padalko: Mild mitochondrial uncoupling slows aging temperature and illumination, the dwarf hamster Phodopus campbelli and the mole vole Ellobius talpinus [59]. Taking the data together, the authors concluded that SkQ1 increased the survival of young flies and primarily improved the quality of life. The same tendency of predominant early-in-life SkQ1 effect was observed for other animals, for example, mice. SkQ1 prolonged the median life span of dwarf hamsters and mole-voles, and the effect being especially great in the case of mole-voles treated with SkQ1 from an early age [59]. There are some reports suggesting that SkQ1 may reduce baseline levels of oxidative damage in young, healthy animals. For instance, protein carbonyls in muscle tissue have been reported to be reduced by SkQ1 treatment in healthy wild type rats [60]. It was shown that SkQ1 not only prevented ageassociated hormonal alterations but partially reversed them [61]. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc [54]. Thus, the involvement of mitochondrial ROS in senescence was confirmed in Skulachev group’s experiments showing that cationic derivatives of antioxidant SkQs selectively accumulated in mitochondria, lengthen the life span of a wide circle of eukaryotes (from fungi to mammals), and retard the development of typical symptoms of senescence and senile diseases [48, 56]. So, SkQs look promising as potential tools for treatment of senescence and age-related diseases [48]. In this context, it is worth remembering the fact that TPP compounds can facilitate mitochondrial uncoupling [55], an intervention that has itself been explored as a possible life span modulator. “Mild” mitochondrial uncoupling modulators In practice, the two approaches are possible to reduce the generation of ROS in the mitochondria. One of these is the use of highly specific antioxidants which would work directly in the mitochondria (e.g., penetrating SkQ1 cation or antioxidants such MitoQ [48]) as mentioned above. However, an alternative approach would be to modulate mitochondrial function with the aim of producing less ROS while maintaining adequate ATP production. As noted earlier in this review, mitochondria are believed to be one of the major sources of ROS, although 98% of oxygen consumed by mitochondria is converted into water and only 2% produce ROS during side chemical reactions in chain of respiratory enzymes [24, 25].

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According to Skulachev [62] this “parasitic” chemical reaction appears to be inevitable since the initial and middle steps of the respiratory chain contain very reactive electron carriers of negative redox potential that are chemically competent in the one electron reduction of oxygen. The carriers in question are short-lived under conditions of active respiration but seem to become long-lived in the resting state when the respiration rate is limited by ADP availability. As a result of these relationships, the rate of the O2•- production by mitochondria increases when ADP is exhausted. And in 1994 Skulachev suggested that mitochondria possess a special mechanism called “mild” uncoupling, which prevents a strong increase in ΔµH+, and hence, in O2•- formation under resting conditions [62, 63]. Later Skulachev’s group showed that 15% decrease in membrane potential ΔΨ under resting conditions caused by an uncoupler, a respiratory inhibitor or the oxidative phosphorylation substrates (ADP + Pi) results in 10 fold decrease in the H 2O2 production by heart mitochondria [64]. In addition, for undefined reasons linked to the proton-pumping mechanism of complex I, ROS generation is more dependent on changes in the transmembrane pH gradient (ΔpH) than on the membrane potential (ΔΨ) [65]. As mild uncoupling decreases proton motive force (Δp) by lowering both ΔpH and ΔΨ, it is an effective means to lower mitochondrial superoxide production at the cost of efficient ATP synthesis [29]. Thus, “mild” mitochondrial uncoupling probably can be regarded as the reduction of the efficiency of energy conversion without compromising intracellular high energy phosphate levels [66]. Uncoupling also increases respiration, which decreases the local concentration of O 2 and therefore decreases the rate of ROS production [67]. Based on these observations, it has been suggested that a “soft” uncoupling should be a mechanism preventing excessive production of ROS [68] and as a result (in the frames of the free radical aging theory) should prevent shortening of the life span. This theoretical postulate argues in favor of the fact that mild uncoupling will decrease ROS production and thereby extend life span even if it increases the “rate of living”. To test this, Speakman et al [69] separated mice into quartiles of metabolic intensity and then investigated their longevity. They found that mice in the highest quartile lived longer than those in the lowest. In another study, a tightly-coupled human first dorsal interosseus muscle showed greater deterioration with age than a relatively uncoupled tibialis anterior one [70]. Also, in a comparison of strains of Caenorhabditis elegans carrying life span extending mutations, a

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Oxidants and Antioxidants in Medical Science 2014; 3(1):27-42 lower mitochondrial membrane potential was associated with increased worm life span and the effect could be replicated using a chemical uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP) [71]. These results are in line with a series of findings on an increase of longevity by mild uncoupling as well they are contradicting to others [72]. For example, although some experiments nicely illustrate the ΔΨ dependence of mitochondrial superoxide production [31], there are authors who argue that the conditions used to demonstrate this effect may not reflect conditions occurring in vivo. A recent study [73] has challenged the hypothesis that mild uncoupling could be neuroprotective by decreasing oxidative stress and found no significant change in the level of matrix superoxide in cultured rat cerebellar granuler neurons on addition of low protonophore (FCCP) concentrations. According to Shabalina and Nedergaard [74] it is only ROS production from succinate under reverse electron-flow conditions that is sensitive to membrane potential fluctuations, and so, only this type of ROS production could be affected in uncoupling investigation; however, the conditions under which succinate-supported ROS production is observed include succinate concentrations that are supraphysiological. Any decrease in membrane potential, even 'mild uncoupling', must necessarily lead to large increases in respiration, i.e. it must be markedly thermogenic. Mitochondria within cells are normally ATP-producing and thus already have a diminished membrane potential, and treatment of cells, organs or animals with small amounts of artificial uncoupler does not seem to have beneficial effects that are explainable via reduced ROS production. Although it has been suggested that members of the uncoupling protein family (UCPs) may mediate a mild uncoupling, authors evidence does not unequivocally support such an effect, e.g. the absence of the truly uncoupling protein UCP1 is not associated with increased oxidative damage. Generally, the authors conclude that present evidence does not support mild uncoupling as a physiologically relevant alleviator of oxidative damage [74]. So, further experimental data are required to verify the hypothesis that a physiological uncoupling acts as a protective mechanism against oxidative stress. In general, it should be noted that although there is no single point of view on the problem, the possibility that “mild” uncoupling could be protective against oxidative damage by diminishing ROS production has attracted much interest during the last decade. And according to Cunha et al [66] mild mitochondrial uncoupling by activation of mitochondrial uncoupling pathways may therefore represent a plausible


mitochondrial-targeted strategy to modulate ROS production. Perhaps these ideas are the most clearly articulated in the “uncoupling to survive” hypothesis: the attenuation of ROS production by partial or mild mitochondrial uncoupling while maintaining sufficient ATP production is a potential mechanism for delaying cellular senescence (since reduce oxidative damage to DNA, proteins and lipids) and may extend life span [75]. So given results of experiments from several laboratories demonstrated that reduction of ROS generation in the mitochondria could be achieved by dissipation of the mitochondrial membrane potential using mild mitochondrial uncoupling. Uncoupling of mitochondria occurs naturally but may be induced by exogenous chemical protonophores such as "classical" protonophore 2,4-dinitrophenol (DNP) and by activating endogenous innate mitochondrial uncoupling pathways involving, for instance, UCPs [28]. Uncoupling proteins (UCPs) As noted, ROS, natural by-products of aerobic respiration, are important cell signaling molecules, which can also severely impair cellular functions and induce cell death. Hence, cells have developed a series of systems to keep ROS in the nontoxic range. It is advisable in this connection to draw attention to the existence of endogenous regulators of respiration and oxidative phosphorylation, a family of uncoupling proteins, which are the likely natural modulators of mitochondrial functions. The “mild” uncoupling that they catalyze has been proposed to function as an evolutionarily conserved mechanism to attenuate ROS production [44]. The UCPs represent a family of transporters belonging to the mitochondrial carrier protein superfamily, which is found in all eukaryotic organisms. They transport substrates across the mitochondrial inner membrane and can dissipate the proton gradient [76]. This transporters family also includes the adenine nucleotide translocase (ANT), an ATP/ADP antiporter, and multiple metabolite and ion transporters [44]. UCP1 was the first identified uncoupling protein able to mediate non-shivering thermogenesis by brown adipose tissue [77]. But according to Ricquier [78], the ancient function of the UCPs may rather be associated with adaptation to oxygen and control of free radicals than to thermogenesis. UCP2 and UCP3 are the closest in amino acid identity to UCP1 (57% and 59%, respectively). But due to their relatively low abundance, the degree of uncoupling by UCP2 and UCP3 in cells is much lower than UCP1 [44]. A negative feedback mechanism has been suggested in which increased level of ROS induce UCPs

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Padalko: Mild mitochondrial uncoupling slows aging uncoupling to decrease the formation of oxygen toxic species inside mitochondria [79]. For example the lipid peroxidation product 4-hydroxy-2-nonenal can stimulate inhibitor-sensitive proton conductance through the UCPs and ANT [80]. Mailloux and Harper [79] not only confirmed that ROS activate UCP2 and UCP3, but also demonstrated that UCP2 and UCP3 are controlled by covalent modification by glutathione. In more recent studies, Mailloux et al [81] have identified glutaredoxin-2 (Grx2) as the enzyme responsible for regulating proton leak through UCP3 by glutathionylation. Specifically, by conjugating glutathione to UCP3, Grx2 inhibits proton leak in skeletal muscle mitochondria. Moreover, despite the identification of UCP2 and UCP3 in 1997, the physiological functions of the mitochondrial uncoupling proteins are still under debate. A wide variety of roles for these proteins including fatty acid transport and metabolism, efflux of mitochondrial ROS by-products, glucose metabolism, and calcium homeostasis has been identified [31]. However, for the purposes of this paper the most important is the ability of UCP‘s to cause mild uncoupling and so diminish mitochondrial superoxide production, hence protecting against oxidative damage. The findings of Mailloux and Harper [79] are consistent with the conclusion that UCP2 and UCP3 function as the “first line of defense” against mitochondrial ROS production. Thus, at the moment, multiple recent reviews discuss the putative biochemical and physiological functions of the UCPs [44]. For the purposes of this review, the most interesting is the application of these proteins to life span modulation. One approach to assess the importance of mitochondrial UCP function in aging and life span is to examine the correlation between the level of mitochondrial uncoupling and life span and if more uncoupled mitochondria may be favorable for long life span. There are few studies that directly manipulate UCP levels and measure life span. For instance, fruit fly life span could be lengthened approximately 10-30% by over-expression of human UCP2 in the nervous system [82]. Closer inspection indicated that the production of ROS was reduced in human UCP2expressing flies, and the transgenic flies were more resistant to exogenously applied oxidative stress than non-transgenic controls. While human UCP2 overexpression was associated with increased life span in Drosophila, the deletion of one of the endogenous Drosophila UCP homologs, DmUCP5, could also extend life span [82]. This evidence may support the hypothesis that high UCP activity may augment organism antioxidant defenses and increase

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life span. But other studies found no change in life span of UCP1-/-, UCP2-/-, UCP3-/-, or transgenic UCP3over-expressing mice relative to the wild type [83, 84]. However, Gates et al [85] observed reduced incidence of lymphoma and atherosclerosis in UCP1overexpressing mice, suggesting that despite a lack of life span extension, an increase in “survival potential” could be attributed to UCP1-dependent uncoupling. Chemical uncouplers It is important to keep in mind that the mammalian uncoupling proteins are tissue specific and insufficiently active, and effective pharmacological agonists of these proteins in mammals are not known [86]. That's why unlike modulation of the UCPs’ abundance or function within naturally occurring mitochondrial uncoupling pathways, direct uncoupling of mitochondria using chemical uncouplers is may be more specific since they act directly to facilitate transport of protons across the inner mitochondrial membrane, from the intermembrane space into the mitochondrial matrix, thus dissipating the proton-motive force. Mild mitochondrial uncoupling using chemical uncouplers may therefore provide a stronger test for the “uncoupling to survive” hypothesis. One of the chemical uncouplers is the “classic” DNP, which was extensively used as an obesity treatment in the 1930s, and recently has been increasingly utilized as a putative anti-aging substance. Known, that Drosophila melanogaster is an excellent model organism to study aging due to its short generation time and life span, the availability of the genome sequence and an enormous catalogue of genetic tools. In insects, as in mammals, there is a negative correlation between free radical production in isolated mitochondria and life span, and the extreme longevity of queen ants and bees is correlated with a resistance to oxidative stress [34]. That’s why in our laboratory the effect of a moderate (“soft”) DNP uncoupling of mitochondria on the life span and some parameters of biological age of Drosophila melanogaster Oregon-R strain was studied [87]. It was found that the insects treated with approximately 40 mM DNP during the larval stage had longer life span. For example, on the 30 th day about 50% of the flies were dead in the control, whereas in the experiment not more than 35% of the flies had died. However, the maximal life spans of flies in both groups were not different [87]. In other available investigation [88], 0.1% DNP prolonged the average life span of Drosophila by 12.3%. In various series of our experiments, the increase in the average life span was not less than 20%. This and other obtained results allowed us to assume

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Oxidants and Antioxidants in Medical Science 2014; 3(1):27-42 that DNP can determine subsequent events: the soft uncoupling of mitochondria is accompanied by preventing the excess production of ROS by the mitochondrial respiratory chain, and this can be associated with a significant decrease in the amount of cell reserves of NADH- and FADH2-providing substrates and ATP. This should decrease the total intensity of metabolic processes; moreover, just the increased consumption of O 2 without production of ATP lowers the level of free molecular oxygen capable for producing O2•- [68, 87]. This is likely to reduce oxidative damage of the cell and increase the life span. In other series of experiments, we studied the effect of DNP and sodium nitroprusside (SNP; known donor of nitric oxide radicals) on protein oxidative damage and life span of Drosophila melanogaster, Oregon-R strain. It was shown that SNP had negative effect on flies viability connected, probably, with activation of processes of proteins oxidative damage. At the same time, DNP essentially corrected the SNP negative action on insects’ survival rates and the “normalizing” action was revealed both at the level of sensitivity of flies to exogenic stresses and protein carbonyl levels, and at a level of insect life span as a whole. DNP was supposed to protect from SNP negative action on flies viability by reduction of intensity of free radicals production [89]. So, the results of our Drosophila experiments [87, 89] probably favor the point of view that “soft” DNP uncoupling of mitochondria has a positive effects on oxidative proteins damage degree and may increase the life span of flies. Not so long ago, it was shown that in yeast Saccharomyces cerevisiae, treatment with 10 nM DNP was sufficient to increase both the chronological and replicative life span of yeast mother cells, the latter by approximately 15% [90]. At the same time mild uncoupling using low concentrations of DNP or FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) was revealed to strongly promote premature senescence in yeast according to the data of other authors [91]. Extension of life span by mild uncoupling was also described for mice. Animals treated with 5 μM DNP in drinking water had larger median (771 vs 722 days in controls) and mean (769.7 vs 718.8 days in controls) life spans than untreated mice [86], although the mice used in the study were of a short-lived strain. Nevertheless, this life span extension was accompanied by decreases in ROS production and biomarkers of oxidative damage for DNA (measured as 8-hydroxydeoxyguanosine levels) and protein carbonyl levels, in mouse brain, liver and heart tissues [86]. In other investigation, Cerqueira et al [92]


demonstrated that murine life span can be extended by low doses of the DNP in a manner accompanied by weight loss, activation of eNOS and Akt-dependent pathways leading to mitochondrial biogenesis, lower serological levels of glucose, insulin and triglycerides as well as a strong decrease in biomarkers of oxidative damage and tissue ROS release. The effect of uncoupler DNP on the oxidative processes intensity in liver biomembranes of rats of different age during longitudinal experiment was also studied in our laboratory [93]. On the young, 3-month old males it was shown that long-term xenobiotic administration had been accompanied by intensification of the rate of oxygen consumption, decrease of the rate of ROS formation in microsomal redox-chain, decline in lipid hydroperoxides and protein carbonyl levels in blood serum and liver microsomes, and (at the end of the long-term experiment) also by increase of their mean life span. Indeed, the average life expectancy at 50% mortality of animals was increased by 19.6% (677 ± 23 days vs 566 ± 55 days in control) [93]. An interesting fact is that the increase in the median life expectancy in the group treated with DNP doesn’t depend on the level of model organization, as the fruit flies and rats exhibit the same increase of median life expectancy (20-25%) [87, 93]. It is important that other chemical uncouplers such as carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and FCCP were found to extend the life span of wild type Caenorhabditis elegans also [71]. Attempts to study the effect of mild uncoupling on cellular aging in culture have produced mixed results, with one study supporting the “uncoupling to survive” hypothesis [94], whereas others do not [91]. Overall, despite some contradictory research results it was established that uncouplers (in particular DNP) at certain doses could increase the life span of a wide range of model organisms including yeast [90], flies [87], mice [86] and rats [93]. Thus, the literature and our own results demonstrate that mild mitochondrial uncoupling is a highly effective in vivo antioxidant strategy, and show significant efficacy of DNP and other uncouplers in animal experiments as potential geroprotectors, and confirm the reasonability of further studies on the role of uncoupling in the regulation of development and aging of organisms. Is it possible to use DNP in clinical practice? As well known, dinitrophenols (DNPs) are a class of synthetic chemicals which do not occur in nature. All DNPs are highly toxic and the mechanism for toxicity common to all DNPs is uncoupling of mitochondrial oxidative phosphorylation. By mitochondrial uncoupling, the drug causes a marked increase in fat

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Padalko: Mild mitochondrial uncoupling slows aging metabolism. This shift in metabolism led to the use of DNPs as an ingredient in weight-loss pills in the early 1930s for the treatment of obesity [95, 96]. It should be mentioned that uncoupling agents could have severe health risks. As such, DNP is a metabolic poison that acts by uncoupling oxidative phosphorylation, leading to cataracts, renal failure, hyperthermia, tachycardia, diaphoresis and tachypnea, eventually leading to death [95, 97, 98] and as a result, it was banned for weight-loss purposes in 1938 [96]. In spite of this, fatalities related to exposure to DNP have been reported since the turn of the twentieth century. To date, there have been 62 published deaths in the medical literature attributed to DNP [95]. For example, the published data describe toxicological findings from two deaths, one in Tacoma, WA and a second in San Diego, CA, following the ingestion of DNP. In the Tacoma case, DNP was identified in capsules the decedent was taking for weight loss, while in the San Diego case, DNP was identified in a yellow powder found at the scene [97]. Another study reports the cases of two patients whose deaths were attributed to occupational and non-oral exposure of DNP. They were all poisoned through skin absorption and respiratory tract inhalation; common features were excessive sweating, hyperthermia, tachycardia, clouded consciousness and asystole [99]. Unfortunately, review of information available on the Internet [98] suggests that DNP is still illicitly promoted for weight loss, for example, among body building enthusiasts. Although, the drug is illegal in the United States, it can be purchased on the Internet under such names as “Sulfo Black”, “Nitro Klenup”, or “Caswell No.392” from commercial web sites which sell and promote the use of anabolic steroids [98]. Some of the websites promoting use of the DNP give no information about its dangers, though others include a disclaimer that it is not safe for human consumption. So, DNP and other nitrophenols have long been known to be toxic at high concentrations (the ‘bad’ face of DNP), an effect that appears essentially related to interference with cellular energy metabolism due to uncoupling of mitochondrial oxidative phosphorylation. But at the same time other studies have provided evidence of beneficial actions of DNP (at low concentrations), including neuroprotection against different types of insult, blockade of amyloid aggregation, stimulation of neurite outgrowth and neuronal differentiation [100]. These results indicate that treatment with mitochondrial uncoupling agent DNP may provide a novel approach for the treatment of secondary injury following spinal cord contusion [101].

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Moreover DNP uncoupling of the mitochondrial electron-transport chain may be the strategy to prevent intracellular oxidative stress during liver cold preservation/warm reperfusion injury. The results of Petrenko et al [102] suggest that reversible uncoupling may be one way to influence oxidative stress associated with hepatic cold preservation. In other investigation on rhesus monkeys with low cryoresistant ejaculates, reducing ROS through mild mitochondrial uncoupling had statistically significant beneficial effects on sperm cryopreservation [103]. Individuals or species that have higher sensitivity to cryodamage may derive the most benefit from these treatments. Thus, despite a number of positive effects of mild uncoupling on the living organism, the use of DNP and other chemical uncouplers in clinical practice should be considered impossible due to the high toxicity. But the possibility of correction of oxidative processes intensity in tissues of mammals and their life span by means of modulation of ROS production in membrane electron transport chains does exist. In this connection, the search of non-toxic uncouplers (preferably of natural origin) is of great interest for the further studies on the role of uncoupling in the regulation of development and aging of organisms. CONCLUDING REMARKS Aging is a universal process commonly defined as the accumulation of diverse deleterious changes occurring in cells and tissues with advancing age which are responsible for the increased risk of disease and death. Understanding of the aging mechanisms is of major interest to scientists, physicians and general population as well. But attempts at understanding the causes of aging are limited by the complexity of the problem. More than 300 theories have been postulated and free radical theory of aging is one of the most prominent and well studied. The free radical theory of aging hypothesizes that oxygen-derived free radicals are responsible for the age-related damage at the cellular and tissue levels. In a normal situation, equilibrium exists among oxidants, antioxidants and biomolecules. Excessive generation of free radicals may overwhelm natural cellular antioxidant defences leading to undesirable oxidation of biomolecules and further contributing to cellular functional impairment. The identification of free radical reactions as promoters of the aging process implies that interventions aimed at limiting or inhibiting them should be able to reduce the rate of formation of aging changes. The main objective of this review is to elucidate the role of endogenous (primarily mitochondrial origin)

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Oxidants and Antioxidants in Medical Science 2014; 3(1):27-42 ROS in the aging process. We have attempted to highlight the findings from several investigations about the connections between reduction in mitochondrial production of free radicals (using specific antioxidants or mild mitochondrial uncoupling) and life span.

REFERENCES

It should be noted that the conclusions from this review do not provide entirely clear picture of the effect of mitochondria-targeted antioxidants and uncouplers on life span and it is presently unclear if mild mitochondrial uncoupling is useful as a therapeutic strategy for achieving extended human longevity. Further investigation of these interesting agents is necessary. Nevertheless, several studies on animal models have shown that aging rates and life expectancy could be modified using mitochondriatargeted antioxidants and uncouplers.

3. Iliadi KG, Knight D, Boulianne GL. Healthy aging - insights from Drosophila. Front Physiol 2012; 3:106.

In particular, different uncoupling strategies were able to extend life span in models ranging from yeast to mammals [86, 87, 90, 93]. These findings are in line with Brand’s “uncoupling to survive” hypothesis [29], suggesting that uncoupling could be an approach to promote life span extension due to its ability to prevent the formation of ROS. Indeed, mild mitochondrial uncoupling can be a highly effective intervention to prevent the formation of ROS. Obviously, because of the high toxicity DNP and other uncouplers, themselves cannot be applied in practical geriatrics, but their low toxic analogues having controlled and “soft” action either agonists affecting the natural way of uncoupling (UCPs) are promising for the development of means for control of tissue redox state and animal life span. In this regard, we support Harman’s point of view [7] regarding the fact that the extensive studies based on the free radical theory of aging hold promise that average life expectancy at birth and the maximum life span in principle can be extended, although we cannot yet answer the question of “When?”.

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ACKNOWLEDGMENT The author is deeply grateful to E. Kozlova and I. Leonova (V. N. Karazin Kharkiv National University) for their valuable help in performing the present work.

COMPETING INTERESTS This work presents no conflict of interest.


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DOI 10.5455/oams.161213.rv.012


ISSN: 2146-8389

ORIGINAL ARTICLE

Hepatoprotective effect of green propolis is related with antioxidant action in vivo and in vitro Niraldo Paulino1,2, Aguinaldo P. Barbosa1, Amarilis S. Paulino2, Maria C. Marcucci1 1

Biomedication Research and Development Group, Professional Masters Program in Pharmacy, Bandeirante University of Sao Paulo, Sao Paulo, SP; 2 MEDLEX Information Management & Courses Ltd, Florianopolis, SC, Brasil

Received October 10, 2013 Accepted February 15, 2014 Published Online February 27, 2014 DOI 10.5455/oams.150214.or.058 Corresponding Author Niraldo Paulino Grupo de Pesquisa e Desenvolvimento de Biomedicamentos (BIOMED), Programa de Mestrado Profissional em Farmacia e Programa de Mestrado e Doutorado em Biotecnologia e Inovacao em Saude, Universidade Anhanguera de Sao Paulo (UNIAN), Rua Maria Candida, 1813, Vila Guilherme, Sao Paulo, SP, Brasil. [email protected] Key Words Artepillin C; Green propolis; Hepatoprotective; Propolis

Abstract Objective: Propolis is a natural product produced by bees. In this study, the free radical scavenger and hepatoprotective activity of green propolis extract (G1) was investigated. Methods: In vitro experiments on guinea pig isolated trachea tissues and in vivo study on rat liver tissues were performed. Hepatic damage was induced by oral administration of carbon tetrachloride (CCl4) to rats. Hepatoprotective effect was monitored by histological analysis of neutrophil margination (NM) on liver, aspartate and alanine transaminases (AST, ALT), and gamma-glutamil transferase (γ-GT) activity. Results: Chemical constitution of G1 by high performance liquid chromatography analysis resulted in the presence of phenolic compounds. G1 produced a reduction of the relative activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. G1 also exhibited high superoxide radical and potent hydroxyl radical scavenging activity. On guinea pig isolated trachea tissues, G1 inhibited the superoxide radical-induced contraction in vitro. After CCl4 administration, AST, ALT and -GT activities were found to be increased; these levels were reduced with G1 treatment. Conclusion: Taken together, our results suggest a potent antioxidant effect of G1, related with hepatoprotective action on liver damage induced by CCl4.

INTRODUCTION Propolis or bee glue, is a natural resine produced by bees, which is produced by mixing the exudates collected from various plants by honeybees [1, 2]. Propolis is a traditional remedy, in folk medicine, and is widely used around the world for the treatment of numerous diseases, such as inflammatory airway affections, cutaneo-mucosal infections, viral infections, etc [3]. It has been reported that water extract of propolis showed hepato-protective activity in both chemical and immunological liver injury models [4], antiviral activity, inhibition of platelet aggregation [5], and antiinflammatory activity [6]. However, there are few studies of propolis extracts from South of Brazil on reactive oxygen species (ROS) in and against autoxidation and free radicals, such as superoxide anion, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals and their relation with the hepatoprotective effect of propolis. The Brazilian propolis G1, classified as BRG [11] from Shouteast of Brazil, induces a potent anti-nociceptive effect in the chemical models of nociception in mice [8]. In addition to the analgesic effect, we have demonstrated anti-edematogenic properties induced by Brazilian propolis G1 [9]. Recently, we have


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demonstrated, that Brazilian propolis G1 induce relaxation in the guinea pig isolated trachea in vitro by mean modulation of the several cellular signaling pathways, such as potassium channels, vasoactive intestinal polypeptide and nitric oxide (NO) [10]. Chemical analysis of propolis show more than 150 polyphenolic compounds including flavonoids, cinnamic acid derivatives and prenylated compounds [10, 11]. It has been reported that propolis and/or its active constituents exert potent biological actions such as free radical scavenging and antioxidant properties [12-14], anti-carcinogenic action [15-16], antiviral [3] and antibacterial [17] effects, anti-protozoan action against Tripanossoma cruzi [18], immunomodulatory and anti-inflammatory properties [19], which are related or not to propolis antioxidant properties. Recently, there has been growing interest in the involvement of ROS in several pathological situations, such as cell membrane disintegration, membrane protein damage and DNA mutation, which can further initiate or propagate the development of many diseases involved in the genesis of cancer, cardiovascular disease and liver injury [20, 21]. In the current study we therefore investigated the ability of standardized Brazilian propolis extract (G1), to act as scavenger of ROS, superoxide and hydroxyl

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Paulino et al: Antioxidant and hepatoprotective effect of green propolis radicals in vitro and on guinea pig isolated trachea organ bath system in vitro. We investigated also, the hepatoprotective effect of G1 on hepatic damage induced by toxic concentration of carbon tetrachloride (CCl4) in rats. MATERIALS AND METHODS Standards and reagents Nitroblue tetrazolium (NBT) chloride, 2-thiobarbituric acid (TBA), TBA deoxyribose, 2-deoxy-d-ribose, xanthine, xanthine oxidase from butter milk (XOD; 0.34 U/mg powder), 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, ferric chloride anhydrous (FeCl3), βnicotinamide adenine dinucleotide (NADH), ethylenediaminetetraacetic acid (EDTA) disodium salt, ascorbic acid, CCl4, and acetaminophen were obtained from Sigma (St. Louis, MO, USA). All other reagents were of analytical grade. Ultra pure milli Q water was used through-out. Propolis extract were supplied by Pharmanectar Ltda (Belo Horizonte, MG, Brazil). Propolis extract preparations Standardized ethanolic extract of propolis, G1, obtained from commercial preparations available in Brazil, supplied by Pharmanectar Ltda, was prepared as follows: the alcohol of this preparation was evaporated and the dry resin was diluted in stock solution at a concentration of 10% (w/v). The propolis was collected from the beehive on March near Caeta city, in the Minas Gerais state, Brazil (following a sample frozen stocked in our labaratory). Propolis was triturated and mixed with an extractive solution containing 96GL alcohol. The mixture was left for 10 days, with a single mixing of 10 min once a day. After this period, the mixture was concentrated in Soxhlet extractor and the alcohol was removed from the solution to make a dry residue. The product of this extraction was diluted in a concentration of 10% (w/v) in 96GL alcohol. High performance liquid chromatography The ethanolic extracts of propolis were analyzed by means of an HPLC (Merck-Hitachi; Darmstadt, Germany), equipped with a pump (Merck-Hitachi: model L-6200) and a diode array detector (MerckHitachi: model L-3000). Separation was achieved on a Lichrochart 125-4 column (Merck, Darmstadt, Germany; RP-18, 12.5 x 0.4 cm, 5 mm particle size) using water, formic acid (95:5, v/v) (solvent A) and methanol (solvent B). The elution was carried out with a linear gradient and a flow rate of 1 ml/min The detection was monitored at 280 nm and the compounds were identified and quantified by a method described previously [11]. For data analysis, the Merck-Hitachi D-6000 Chromatography Data Station-DAD Manager was used. The classification of propolis type was measured using commercial software. The exact

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concentration of major compounds of propolis was calculated by means of comparison with authentic standards previously isolated from Brazilian green propolis. Effect of propolis G1 on DPPH free radicals The effect of propolis G1 (1-100 g/ml) on DPPH radical was evaluated by the following assay: the mixture contained 0.3 ml of 1 mM DPPH radical solution, 2.4 ml of 99% ethanol, and 0.3 ml of sample propolis G1 solution. The solution was rapidly mixed and scavenging capacity was measured spectrophotometrically by monitoring the decrease in absorbance at 517 nm. Ascorbic acid (1 M) was used as positive control. Effect of propolis G1 on superoxide anion radical The effect of propolis G1 (0.1-10 g/ml) on superoxide anion radical production was evaluated by the method described by Nagai et al [22]. This system contained 0.48 ml of 0.05 M sodium carbonate buffer (pH 10.5), 0.02 ml of 3 mM xanthine, 0.02 ml of 3 mM ethylenediaminetetraacetic acid disodium salt (EDTA), 0.02 ml of 0.15% bovine serum albumin, 0.02 ml of 0.75 mM NBT and 0.02 ml of sample solution. The reaction was started by adding 6 mU XOD and carried out at 25ºC for 20 min. After this time, the reaction was stopped by adding 0.02 ml of 6 mM CuCl. The absorbance of the reaction mixture was measured at 560 nm and the inhibition rate was calculated by measuring the amount of the formazan that was reduced from NBT by superoxide. Ascorbic acid (1 M) was used as positive control. Effect of propolis G1 on hydroxyl radical The effect of propolis G1 on hydroxyl radical production was assayed by using the deoxyribose method. The reaction mixture contained 0.45 ml of 0.2 M sodium phosphate buffer (pH 7), 0.15 ml of 10 mM 2-deoxyribose, 0.15 ml of 10 mM FeSO4EDTA, 0.15 ml of 10 mM H2O2, 0.525 ml of H2O, and 0.075 ml of sample propolis G1 solution in an eppendorf tube. The reaction was started by the addition of H2O2. After incubation at 37ºC for 4 h, the reaction was stopped by adding 0.75 ml of 2.8% trichloroacetic acid and 0.75 ml of 1% of TBA in 50 mM NaOH; the solution was boiled for 10 min, and then cooled in water. The absorbance of the solution was measured at 520 nm using a Hitachi u2010 spetrophotometer. Hydroxyl radical scavenging ability was evaluated as the inhibition rate of 2-deoxyribose oxidation by hydroxyl radical [23]. Ascorbic acid (1 M) was used as positive control. Inhibition of superoxide-induced contraction of isolated guinea pig trachea Tissue preparations: guinea pigs (250-400 g) of both sexes were anesthetized and killed by cervical

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Oxidants and Antioxidants in Medical Science 2014; 3(1):43-50 dislocation [24]. This protocol was approved by the ethical committee at Universidade do Sul de Santa Catarina (UNISUL). The trachea was rapidly removed, and after being freed from connective tissue, each trachea was cut into six transverse rings (3-4 mm wide), each containing 3 cartilages as described previously [25]. The rings were opened (usually 6 strips of 8-10 mm in length were obtained from the same animal) and were suspended in individual 10 ml jacketed organ baths containing Krebs-Henseleit solution maintained at 37ºC, pH 7.8, and gassed with a mixture of 95% O2 and 5% CO2. The Krebs solution had the following composition (mM): NaCl 118, KCl 4.4, MgSO4 1.1, CaCl2 2.5, NaHCO3 25, KH2PO4 1.2, glucose 11. Tissues were allowed to equilibrate for at least 60 min before drug addition, during which time the fresh buffer solution was renewed every 15 min, under a resting tension of 1 g. Isometric responses were measured by means of TRI201 force displacement transducers (Panlab apparatus; Barcelona, Spain) and were recorded on a polygraph (Letica Scientific Instruments; Barcelona, Spain). In most experiments, the epithelial layer of the trachea was gently removed with a cotton-tipped applicator. The integrity of the epithelium was assessed by the ability of bradykinin to induce relaxation [24]. The animals were used in accordance with current ethical guidelines for the care of laboratory animals. Experimental procedure; after the equilibration period of at least 60 min, the preparations without epithelium were contracted with histamine (1 mM, approx. the EC50) to evaluate the tonic responsivity of the smooth muscle. After 60 min and when tonic baseline became stable (usually after 5 min) they were exposed to the superoxide radicals producing system, in absence or in presence of G1 (0.1, 1 or 10 g/ml) or superoxide dismutase. Superoxide was generated in the organ bath by the xanthine/xanthine oxidase system following the reaction mixtures: xanthine (44 mM), xanthine oxidase (0.29 U/ml) in a final volume of 2.5 ml. Xanthine was dissolved in NaOH (1 mM) and after in phosphate buffered saline plus EDTA 0.1 mM, in pH 7.8; xanthine oxidase in EDTA 0.1 mM. Usually, two to three complete cumulative concentration-response curves were obtained in each preparation at 60 min intervals between curves.

investigations of experimental conscious animals [26] and the current ethical guideline to use of animals approved by Committee at UNISUL 021/2006. The animals were treated orally (p.o.) with propolis G1 (1, 3 or 10 mg/kg) during 7 consecutive days, once a day. After treatment the animals received carbon tetrachloride (1.5 ml/Kg, p.o.) twice during 48 h. During the experiments, toxical effect and compartmental reactions were monitored. After the treatment, the rats were anesthetized and killed by cervical dislocation, and the liver and kidney were isolated by histological analysis. Slices of liver and kidney (near microvascular place) were cored by means of hematoxylin-eosin method and analyzed by mean optical microscopy. We evaluate the neutrophil migration and Kupffer cells on micro-vascular liverand kidney-cored slices, respectively. In this time the blood (3 ml) was collected by cardiac pucture using sterile disposable syringes. Serum was separated by centrifugation (3000 rpm) and aspartate transaminase (AST), alanine trasnaminase (ALT) and gamma glutamyl transferase (-GT) were estimated on the same day using Merck Diagnostic kits on UNISUL Biochemical Laboratory.

Hepatoprotective effect of propolis G1 on hepatic damage induced by carbon tetrachloride and acetaminophen in rats Male rats (180-250 g), from UNISUL facilities, were housed at 22 ± 2ºC under a 12 h light-dark cycle. Food and water were offered ad libitum. The animals were acclimatized to the laboratory for at least 1 day before testing and the experiments were carried out in accordance with current guidelines for the care of laboratory animals and ethical guidelines for

Antioxidant effect of Brazilian propolis G1 DPPH radical scavenging assay: DPPH is a free radical compound and has been widely used to test the free radical scavenging ability of various chemicals and natural products around the world. Our results showed Brazilian propolis G1 produced a significant and dosedependent reduction of the relative activity of DPPH with mean IC50 of 0.96  0.4 g/ml. In these experiments, ascorbic acid (0.1 or 1 mM) was used as positive control (Fig.2).


Statistical analysis Responses were expressed as means ± SD. Statistical analysis of the results was carried out by means of the unpaired Student’s t-test (Graph Pad InStat software), by comparison of individual points of the treated groups with the control groups, during the experiments. P < 0.05 was considered as indicative of significance. The IC50 values were determined from individual experiments for the complete dose-response by graphical interpretation test. The IC50 values are reported as geometric means accompanied by their respective 95% confidence limits.

RESULTS Phenolic composition of Brazilian propolis G1 The chemical composition of green propolis was evaluated by HPLC analysis (Fig.1), showing high levels of phenolic compounds. The total content of phenolic compounds is 151.69 mg/g of dried extract.

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Paulino et al: Antioxidant and hepatoprotective effect of green propolis of 10 g/ml completely inhibited the production of superoxide in this system. The activities of propolis G1 were higher than that of 1 mM ascorbic acid (Fig.3).

Figure 1. The chemical composition of G1 has been determined by high performance liquid chromatography: The amount of phenolic compounds was estimated as follows (mg/g): (1) coumaric acid 3.81, (2) rutin 9.87, (3) pinobanksin 3.48, (4) quercetin 2.15, (5) kaempferol 0.78, (6) apigenin 1.86, (7) pinocembrin 22.55, (8) pinobanksin-3-acetate 4.1, (9) chrysin 2.49, (10) galangin 4.14, (11) kaempferide 5.59, (12) tectochrysin 2.90, (13) artepillin C 87.97.

Hydroxyl radical scavenging activity of propolis We used the Fenton reaction to determine the scavenging effect of propolis G1 (1-100 g/ml) against hydroxyl radical. We have shown here that propolis G1 present a potent hydroxyl radical scavenging activity and its activity was increased with concentration of the sample and completely abolished the hydroxyl radical when 100 g/ml was add on solution. The mean IC50 of this effect was 15.7  2.5g/ml (Fig.4). Effect of propolis on superoxide radical-induced contraction in the guinea pig isolated trachea Cumulative addition of the standardized propolis extract (G1) (0.1, 1 or 10 g/ml) inhibited the superoxide radical-induced contraction in the guinea pig isolated trachea, with significant inhibition rate of 56.6 ± 4.2% or 97.3 ± 2.2% to 1 or 10 mg/ml, respectively, and with IC50 mean of 0.79 ± 0.2 g/ml (Fig.5).

Figure 2. Effect of propolis (1, 10 or 100 g/ml) or ascorbic acid (AA, 1 M) on DPPH relative activity in vitro. The results represent the mean of three experiments. *P < 0.05 for control vs treated group.

Figure 4. Effect of propolis (1, 10 or 100 g/ml) or ascorbic acid (AA, 1 M) on hydroxil production in vitro. The results represent the mean of three experiments. *P < 0.05 for control vs treated group.

Figure 3. Effect of propolis (1, 10 or 100 g/ml) or ascorbic acid (AA, 1 M) on superoxide production in vitro. The results represent the mean of three experiments. *P < 0.05 for control vs treated group.

Superoxide-scavenging activity of propolis Superoxide-scavenging activity of Brazilian propolis extract G1 was measured using the xanthine–xanthine oxidase system and these results are indicated as the superoxide productivity. Our results showed that propolis G1 (0.1-10 g/ml) exhibited high superoxidescavenging activity by dose dependent manner with mean IC50 of 0.28  0.09 g/ml and the pre-incubation

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Figure 5. Effect of propolis (1, 10 or 100 g/ml) or superoxide dismutase (SOD) on guinea pig trachea contraction induced by superoxide system in vitro. The results represent the mean of 3 experiments. *P < 0.05 for control vs treated group.

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Figure 6. Effect of propolis (1, 3 or 10 mg/Kg) on neutrophil migration on rat’s liver after oral treatment with CCL4. The results represent the mean of five experiments and * P < 0.05 are significant difference between control and treated group.

Effect of propolis on CCl4-induced neutrophil margination on liver CCl4-induced hepatic injuries are commonly used models for hepatoprotective drug screening. CCl4 can be converted into halogenated free radicals that spread propagation of the alkylation as well as peroxidation, causing damage to macromolecules in membrane, focal neutrophil margination and inflamation. In in vivo experiments, propolis extract G1 (1, 3 or 10 mg/kg, p.o., during 7 consecutive days), inhibited the hepatic neutrophil margination induced by CCl4 with IC50 mean of 5.78  0.9 mg/kg. Our results showed that propolis G1 can be a potent lipoperoxide free radical scavenger and this effect can be related with hepatoprotective action on liver damage induced by CCl4. Effect of propolis G1 on liver biochemical funtion during CCl4-induced hepatotoxicity The transaminases and -GT levels were determined in rat’s serum before induction of. The initial values of serum AST, ALT and -GT in control (saline + vehicle) group (169  25, 70  3 and 0.25  0.02 U/l, respectively) were found to be increased (755  72, 475  32 and 3.55  0.25 U/l, respectively) after administration of the toxic dose of CCl4 (1.5 mg/kg, p.o.). Treatment with propolis G1 (1, 10 and 100 mg/kg, p.o., during 7 consecutive days) reduced the AST (578  43, 354  30 and 184  12 U/l, respectively), ALT (325  29, 170  18 and 77  5 U/l, respectively) and -GT (2.8  0.2, 1.8  0.2, and 1.2  0.1 U/l, respectively) levels significantly (Fig.7).

DISCUSSION Propolis is a natural product produced by bees and used in the folk medicine to treat many pathologies, including pain, inflammatory diseases, cancer, etc. We have recently shown that Brazilian propolis can induce a potent relaxant effect on guinea pig isolated trachea by means of potassium channel, NO and VIP receptor modulations [9]. In a recent review Marcucci and Bankova [7] described that Brazilian propolis have a complex chemical composition with a majority of compounds linked to phenolic and prenylated compounds family.

Figure 7. Effect of propolis (1, 10 or 100 mg/kg) on AST, ALT or -GT activity in blood from rats treated with CCl4. The results represent the mean of five experiments. *P < 0.05 for control vs treated group.


Phenolic compounds are substances of low molecular weight and are present in several plants and other natural products. It was previously shown that phenolic compounds isolated from Brazilian propolis, such as 3-prenyl-4-hydroxycinnamic acid, 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran, 3,5-diprenyl-4-hydroxycinnamic acid and 2,2-dimethyl-6-carboxyethenyl8-prenyl-2H-1-benzopyran, have potent anti-protozoan, antibacterial and relaxant effect on guinea pig isolated

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Paulino et al: Antioxidant and hepatoprotective effect of green propolis trachea [27]. Kimoto et al [28, 29] showed that 3,5-diprenyl-4-hydroxycinnamic acid has anti-tumoral, anti-leukemic and anti-carcinogenic effects in isolated cell line. In addition, flavonoids and phenolic compounds possess anti-inflammatory, antioxidant, anti-allergic, hepatoprotective, anti-thrombotic, antiviral and anti-carcinogenic activities [30]. In this work we showed that Brazilian propolis, named G1, present a chemical constitution based on phenolic and prenylated compounds. This propolis sample show antioxidant effect, scavenging ability on free radicals and inhibitory effect on the superoxide radical mediated contractile activity of guinea pig isolated trachea. It has been shown that free radicals can induce several pathologies including aging, atherosclerosis, neurodegenerative diseases [31], hepatic damage and inflammatory response [32]. Some flavonoids and phenolic compounds act by antioxidant mechanisms including the inhibition of enzymes involved in the formation of ROS (xanthine oxidase, protein kinase C, lipoxygenase, cyclooxygenase, NADH oxidase, etc) or the chelation of trace elements (free iron or copper) which are potential enhancers of free radical generation or stabilizing free radicals involved in oxidative processes by complexing with them [33, 34]. The pharmacological effect of propolis G1 on free radical system, i.e. inhibiting DPPH, superoxide and hydroxyl radicals can establish its relationship with anti-inflammatory and hepatoprotective properties, shown here on hepatic toxicity experiments. We have demonstrated that the treatment with propolis G1, orally, can induce a potent reduction of the CCl 4induced inflammatory response and hepatotoxicity, clearly mediated by lipoperoxide free radical reaction. On the other hand, when the hepatoxicity were paracetamol-mediated, the treatment with propolis G1 was poorly effective to prevent the neutrophil migration on liver or on the enzymatic hepatic function. The increase in serum levels of ASP, ALT and -GT has been attributed to the damaged structural integrity of the liver induced by CCl4, because these are cytoplasmic in location and are released into circulation after cellular damage. The treatment of the animals with propolis G1 seems to preserve the structural integrity of the hepatocellular membrane producing a significant reduction in the paracetamol and CCl4induced increase in serum enzymes of rats. The results of this study indicate that propolis G1, a common folk remedy in several countries, exhibits hepatoprotective activity, related with antioxidant effect, and the presence of phenolic and prenylated compounds in this propolis extract confirmed, at least in part, the folkloric use of propolis in hepatic damage or to treat other pathologies with free radical-mediated inflammation.

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In fact, our research group has studied several samples of propolis and recently published a report on the antiinflammatory and analgesic effects of green propolis from Brazil. We have shown that green propolis produces potent anti-edematogenic, anti-inflammatory and analgesic effects using several animal models and molecular biology methods. Propolis G1 inhibits prostaglandin E2 production during the acute inflammation induced by carrageenin, the NO production in the murine macrophage cell line RAW 264.7 and the nuclear factor kappa B (NF-κB) overexpression in human embryonic kidney (HEK) cells. This inhibitory effect reduced the transcription and expression of the inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX2). These results are also reproducible for artepillin C [35]. Therefore, the incubation of green propolis on vascular endothelial cells did not affect the activity or expression of endothelial nitric oxide synthase (eNOS), but it did have a dual effect on the protein kinase B (PKB)/Akt activity in smooth muscle cells from rat aorta in the presence of angiotensin II: in low concentration it induced phosphorylation and the activation of this system, and in high concentration it decrease the phosphorylated form and the activity. This pharmacological effect may indicate that green propolis from Baccharis dracunculifolia induces the analgesic and anti-inflammatory effect, at least in part, by means of NF-B modulation. We also have shown that artepillin C, the main compound identified in G1, reduced NF-κB expression suggesting anti-inflammatory activity, particularly during acute inflammation. Lastly, artepillin C was absorbed after an oral dose (10 mg/kg) with maximal peaks found at 1 h [36]. In addition, Fonseca et al [37] suggest the potential applicability of propolis extracts for preventing UVinduced skin damages. Green propolis extracts exhibited considerable antioxidant activity and inhibited UV irradiation-induced GSH depletion. In aggreement of this effect demonstrated that B.dracunculifolia exhibit potent antioxidant activity protecting liver mitochondria against oxidative damage and such action probably contribute to the antioxidant and hepatoprotective effects of green propolis [38]. The antioxidant effect is directly linked to the antiinflammatory action, as demonstrated by Szliszka et al [39] showed that propolis exerted strong antioxidant activity and significantly inhibited the production of ROS, reactive nitrogen species (RNS), NO, cytokines IL-1α, IL-1β, IL-4, IL-6, IL-12p40, IL-13, TNF-α, G-CSF, GM-CSF, monocyte chemotactic protein(MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES in stimulated J774A.1 macrophages.

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Oxidants and Antioxidants in Medical Science 2014; 3(1):43-50 Collectively, our results showed for the first time that propolis G1 can protect the liver from oxidative stress and that its effect can be modulated by antioxidant and anti-inflammatory action mediated, at least in part, by prostaglandin E2 and NO inhibition through NF-κB modulation. This effect was produced by phenolic compounds that exhibited bioavailability after oral administration, such as artepillin C. Taken togheter, our results suggest a strong evidence to use propolis G1 like an antioxidant and anti-inflammatory natural remedy.

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ACKNOWLEDGEMENTS The authors are grateful to MEDLEX Gestao de Informacoes & Cursos Ltda and to the Pharmanectar Ltda for providing propolis sample.

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ISSN: 2146-8389

ORIGINAL ARTICLE

The protective effect of Podophyllum hexandrum on hepato-pulmonary toxicity in irradiated mice Savita Verma, Bhargab Kalita, Rashmi Saini, Manju Lata Gupta Institute of Nuclear Medicine and Allied Sciences, Defence Research and Development Organisation, Delhi, India

Received September 4, 2013 Accepted December 26, 2013 Published Online February 25, 2014 DOI 10.5455/oams.261213.or.057 Corresponding Author Manju Lata Gupta Deparment of Radioprotective Drug Development Research, Institute of Nuclear Medicine and Allied Sciences (INMAS), Defence Research and Development Organisation (DRDO), SK Mazumdar Marg, Delhi -110054, India. [email protected] Key Words Extracellular superoxide dismutase; Immunohistochemistry; Podophyllum hexandrum; Radiation; Radioprotection; Reactive oxygen species

Abstract Objective: Present study reports the modulatory effect of a novel formulation (G-002M), prepared from three isolated molecules of Podophyllum hexandrum rhizomes on radiation induced oxidative stress in lung and liver tissues of strain A mice. Methods: Mice, administered with G-002M formulation 1 h prior to lethal (9Gy) radiation exposure dose, were sacrificed at different time intervals. Lung and liver tissues were processed to assess the expression of extracellular superoxide dismutase (EC-SOD) by immunohistochemistry and Western blotting techniques. Histological alterations along with the biochemical determinations, using as reported markers of hepatic and lung oxidative stress were recorded. Preadministration of G-002M resulted in significant restoration of radiation induced decline in the expression of EC-SOD in both lung and liver tissues. Results: In lungs, radiation mediated histological alterations like alveolar edema, interalveolar septa thickening, inflammation and infiltration of inflammatory cells into alveolar spaces were significantly prevented by G-002M pre-treatment. Histopathological features of liver in response to radiation such as steatosis, deformed cell structure, vacuolization of the cytoplasm and injury of hepatocyte membranes were also minimized by formulation pre-treatment. The formulation could successfully inhibit radiation induced malondialdehyde (MDA) content and counter the radiation induced depletion in SOD, catalase, glutathione reductase, glutathione S-transferase and glutathione, in serum, lung and liver tissues. Radiation induced alterations in serum aspartate aminotransferase, alanine tranaminase, cholesterol, high-density lipoprotein, low-density lipoprotein, triglycerides, alkaline phosphatase, total protein, albumin and lactate dehydrogenase were maintained by G-002M pre-treatment. Conclusion: These results demonstrated that the bioactive phyto-constituents of Podophyllum hexandrum could significantly protect lung and liver against radiation, predominantly by reduction in lipid peroxidation and elevation of EC-SOD along with a set of endogenous defense enzymes. © 2013 GESDAV

INTRODUCTION Natural radiation mediated oxidative stress is carefully regulated by endogenous defence enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione S-transferase (GST), etc. The non enzymatic entities, i.e. glutathione (GSH), vitamin C, α-tocopherol etc, also play important role in minimizing this stress. Under adverse conditions, when the production of free radicals is in excess, there is a redox imbalance in the cell leading to oxidative stress which may lead to irreversible recovery. Liver is the organ where antioxidant enzymes involved in the redox reactions in the cell leading to oxidative stress are synthesized and broken down. Being the main organ of metabolic reaction as well as detoxification, the reported radiosensitivity [1] and the slow regeneration capacity of liver cells makes the damages more threatened to life [2]; therefore, protection of liver from radiation induced damage is inevitable.


Presence of large concentrations of dissolved oxygen in lung enhances the probability of reactive oxygen species (ROS) generation [3]. This organ, being minimally regenerative, does not undergo fast recovery, hence can not tolerate high doses of radiation. Radiation mediated onset of complex reactions in lung is known to lead to pneumonitis, fibrosis and carcinogenesis. Extracellular (EC)-SOD, an antioxidant enzyme highly expressed in the lung, is mainly located in the extracellular matrix of tissue by heparin/matrix binding domain. The role of EC-SOD has been well established against radiation induced oxidative stress causing lung pathogenesis [4-6]. Oxidant/antioxidant imbalance mediated by extracellularly produced ROS which contributes to pathogenesis of acute lung injury can be significantly inhibited by EC-SOD. EC-SOD is also reported to inhibit inflammation [7, 8] and development of fibrosis [6]; however, knowledge related to lung radiobiology is still limited [9]. The search for development of radioprotectors started many decades ago, however, a success remained elusive till today. Only a thiol compound, namely

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Verma et al: Radioprotection by Podophyllum hexandrum in lung and liver WR-2721 (amifostine; S-2-(3-aminopropylamino) ethyl phosphorothioic acid) has been approved by the US Food and Drug Administration (FDA) as a radioprotector and chemoprotector. Basically, undesired toxicity of chemical radioprotectors and their analogues have barred them from clinical use and imposed the need to search the safe agents which could regulate the signaling cascades leading to major organs protection without causing any stress [10].

sterilized rice husk under the controlled environmental conditions (24 ± 2°C; 12 h alternating dark and light cycle). The experiments were conducted strictly adhering to the guidelines of the institutional animal ethics committee. Mice were lethally exposed to 9 Gy dose in 60Co gamma chamber (Cobalt Teletherapy Bhabhatron-II) at the dose rate of 0.925-0.828 Gy/min. Radiation dose calibration was done by Fricke’s dosimetry method.

Use of natural resources in Ayurveda and Chinese medication system has prompted radiation biologists to study herbs against radiation. Various plants from different geographical locations have been evaluated for their radioprotective efficacy [11]. Among the high altitude plants screened for their protective potential against radiation, Podophyllum hexandrum was found to be a potent counter agent in crude [12-14] and semipurified [15, 16] form. The extracts/formulations prepared from P.hexandrum rhizome have been found to deliver protection to the hematopoietic system [14, 16, 17], cellular macromolecules [18, 19] and the gastrointestinal system [20].

Plant material and preparation of G-002M The formulation (G-002M) was prepared by combining of three active molecules isolated from dried rhizomes of Podophyllum hexandrum. The plant material was collected from high altitude regions of Leh and Ladakh (Jammu and Kashmir, India) and was authenticated by plant taxonomist from the center of Plant Taxonomy, University of Kashmir, Srinagar, India. The shade dried rhizomes were crushed to obtain fine powder which was processed further and extracted with petroleum ether. The three active principles isolated after elaborate processing, were analyzed on HPLC for their chemical identification and purity. The active principles were identified as podophyllotoxin, podophyllotoxin-β-D-glucoside and rutin. All the molecules were in their > 97% purity. G-002M (3.5 mg/kg by weight of animal) was prepared freshly at the time of administration by dissolving in DMSO (10% of final concentration) which was diluted further in distilled water.

The present study is focused on evaluating the radioprotective potential of G-002M, a formulation prepared by combination of three active principles isolated from the rhizome of P.hexandrum. Expression of EC-SOD, histological alterations, changes in the oxidative stress marker enzymes and serum biochemical profile are the integral parts of the current analysis.

MATERIALS AND METHODS Reagents and antibodies Goat anti-rabbit horseradish peroxidase (HRP) was procured from Santa Cruz (Cat. No. Sc-2030; CA, USA). Polyclonal antibody of EC-SOD (Cat. No. S4946), goat anti rabbit fluorescein-5-isothiocyanate (FITC) (Cat. No. F4018), SOD standard (Cat. No. S9636), dimethyl sulfoxide (DMSO), trichloroacetic acid (TCA), thiobarbituric acid (TBA), phenazine methosulphate (PMS), nitroblue tetrazolium (NBT), nicotinamide adenine dinucleotide phosphate (NADPH), bovine serum albumin (BSA) and all other required chemicals were obtained from Sigma Aldrich (St Louis, MO, USA). Mayer’s Hematoxylin and Eosin stain was purchased from Fisher Scientific (Pittsburgh, PA, USA). Animals and gamma-ray irradiation Strain A female mice (26 ± 2 g), 8-10 weeks old, maintained at standard laboratory conditions, fed with standard food pellet (Amrut Laboratory Animal Feed; Pune, India) and water ad libitum, were used for study. They were housed in polypropylene cages bedded with

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Experimental design The animals were divided into four groups with three animals in each group: control group (C), G-002M group (D), irradiated group (R) and G-002M plus irradiated group (DR). The experiments were performed in triplicate having 3 animals in each group (3 x 3 = 9). Control group mice were administered 200 µl normal saline while G-002M group was administered the formulation only, intramuscularly. Irradiated group animals were irradiated with a dose of 9 Gy. In G-002M plus irradiated group, mice were administered with G-002M intramuscularly one hour prior to 9 Gy radiation exposure. The animals were dissected at different time intervals (24 h, 72 h and 5th day) by cervical dislocation and blood was collected by heart puncture. Lung and liver were excised out immediately after dissection and processed for biochemical and other studies. Immunohistochemical staining Mice lung and liver tissue, fixed with 10% buffered formalin, dehydrated through graded series of alcohol, paraffinized and were cut in 3-5 µm sections. Sections were deparaffinized, rehydrated, rinsed in distilled water and washed in Tris buffer saline (TBS). Immunohistochemistry was then performed as

DOI 10.5455/oams.261213.or.057

Oxidants and Antioxidants in Medical Science 2014; 3(1):51-64 described by Midgley et al [21]. To inactivate endogenous peroxidases, sections were incubated with 6% H2O2. Antigen retrieval done by heating the sections immersed in citrate buffer (pH 6) in a domestic microwave oven at 600 Watt. Slides were then washed in TBS for 3 min and immunostained using EC-SOD in a dilution of 1:200. After incubation with fluorescence labelled (FITC tagged) secondary antibody for 2 h, the sections were counterstained with 4’,6-diamidino-2phenylindole dihydrochloride (DAPI) at room temperature and visualized under fluorescence microscope (Olympus; Model: BX 63). Western blot analysis Proteins from frozen liver and lung tissue were isolated by homogenizing in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate , 1% Nonidet-P40, and a mixture of protease inhibitors). Proteins, quantified by Bradford method [22], were denatured, subjected to SDS-PAGE on 12% polyacrylamide gels and electrophoretically transferred to Whatman PROTRAN Nitrocellulose Transfer Membrane (Sigma) [4]. After blocking nonspecific sites with 5% (w/v) skimmed milk for 2 h, the membranes were incubated with anti-mouse ECSOD antibody overnight at 4ºC. These membranes, after washing three times, were incubated with HRPconjugated goat anti-rabbit IgG for 2 h at room temperature. EC-SOD expression signal was detected using chemiluminescence detection system (Sigma). Densitometry was performed on the resulting autoradiograph using Image Lab software of BioRad Gel Documentation System (Gel Doc XR; Cat. No. 1708195). Histological studies For histological evaluation, mice were sacrificed on 5 th day of experimentation. Tissues were fixed in 10% buffered formalin, dehydrated through graded series of ethanol, cleared in xylene and infiltered with melted paraffin wax. Paraffin blocks were cut into 3-5 µm serial sections and stained with hematoxylin and eosin for microscopic examination. Biochemical studies The lung and liver tissues from differentially treated mice were isolated and homogenized in ice cold phosphate buffer saline to make 10% homogenate. The samples were centrifuged to obtain clear supernatants and were subjected to following biochemical analysis. Lipid peroxidation (LPx) was estimated by thiobarbituric acid reaction; malondialdehyde (MDA), the end product of lipidperoxidation, reacts with thiobarbituric acid to form pink coloured substances which were measured at 535 nm [23]. Superoxide dismutase activity was measured spectrophotometrically at 560 nm using NBT [24]. Catalase activity was assayed by the method of Sinha [25] using dichromate


acetic acid reagent which reacts with the hydrogen peroxide to form blue precipitate of perchromic acid and decomposed to give green solution. Reduced GSH concentration was determined following the method described by Beutler [26]; the yellow colour obtained by reacting 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) with the GSH present in the sample was measured at 412 nm against the reagent blank. Glutathione reductase activity was determined by procedure of Carlberg and Mannervik [27]; addition of NADPH and oxidized glutathione (GSSG) resulted in changes in the enzyme activities which was measured at 340 nm. Glutathione S-transferase activity was estimated spectrophotometrically at 340 nm using 1-chloro 2,4-dinitrobenzene and GSH [28]. Protein determination Protein contents were measured by Bradford method [22]. Standard curve was plotted by using different known concentrations of BSA as a standard. Assessment of serum biochemistry profile The mice were sacrificed on 5th day after treatment. Blood samples were collected in plain vials and serum was separated by centrifugation at 5000 rpm for 10 min at 4ºC. The sera were stored at -20ºC until analysis. The serum level of aspartate aminotransferase (AST), alanine tranaminase (ALT), cholesterol (CHO), highdensity lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG), alkaline phosphatase (ALP), total protein (TP), lactate dehydrogenase (LDH) and albumin were measured in all the experimental mice by using a full-automatic Biochemistry Analyzer (Erba; Model No: EM-360). Statistical analysis: The results were expressed as mean ± SEM of three replicates. Statistical analysis was performed using analysis of variance (ANOVA) to test the significant difference between the groups. A value of P < 0.05 was considered as statistically significant. RESULTS Immunohistochemical studies Figs.1A&B depict lung tissue sections from control mice stained with DAPI showing nucleus and FITC indicating prominent localisation of EC-SOD in alveolar parenchyma and matrix, respectively. In radiation exposed group FITC staining, indicating the presence of EC-SOD, was found diminished in comparison to controls, indicating loss in radiation mediated EC-SOD expression (Fig.1D). G-002M pretreatment could significantly retain EC-SOD expression both in extracellular spaces and cell lining of bronchioles, expressed with more prominent staining (Fig.1F). Lungs of G-002M group mice showed localization of EC-SOD comparable to

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Verma et al: Radioprotection by Podophyllum hexandrum in lung and liver controls (Fig.1H). DAPI in this study was used to confirm the existence of cells in all the experimental groups (Figs.1ACEG). Fig.2 indicates immunolabeling of EC-SOD in liver tissues. In control mice (Fig.2B), FITC staining in sinusoidal lining and vascular wall was quite visible indicating rich existence of EC-SOD in these areas. Radiation exposure in these animals has significantly descended the presence of EC-SOD which is indicated

in the figure in the form of faintly stained sections (Fig.2D). In G-002M plus irridiated group, we could see enhanced intensity of the stain expressing increase in EC-SOD expression in comparison to radiation exposed group (Fig.2F). G-002M group animals showed intense staining of EC-SOD in liver sections which was comparable to controls (Fig.2H). Sections stained with DAPI (Figs.2ACEG) have confirmed the existence of nucleus in hepatocytes.

Figure 1. EC-SOD immunostaining in the lung tissue sections. (B) Controls showing bronchiole (*), alveoli with large air spaces and EC-SOD deep staining in extracellular portions. (D) Irradiated group showing diffuse staining in bronchiolar (*) and alveolar cells. (F) G-002M pre-treated irradiated mice showing bronchioles (*) and overexpression of EC-SOD in extracellular spaces. (H) Enhanced expression of EC-SOD in G-002M alone group. Nucleus (DAPI) staining in different groups: (A) controls; (C) irradiated; (E) G-002M pre-treated irradiated; (G) G-002M alone. The lung sections were studied under fluorescence microscope (x200).

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Figure 2. Images showing EC-SOD immunostaining in liver tissue sections. (B) Controls showing intense immunostaining of EC-SOD. (D) Irradiated groups showing diffused staining in the sinusoidal lining and vascular wall. (F) G-002M pretreated irradiated group showing restored EC-SOD expression. (H) G-002M group showing prominent ECSOD staining. Nucleus (DAPI) staining in different groups: (A) controls; (C) irradiated; (E) G-002M pre-treated irradiated; (G) G-002M alone. The liver sections were observed under fluorescence microscope (x400).

EC-SOD expression by Western blotting Figs.3A&B demonstrate EC-SOD expression by Western blotting in lung and liver tissues, respectively, of mice sacrificed on 5th study day. In radiation exposed group we observed significant decrease in EC-SOD band intensity. The same was confirmed by densitometry and represented in the same figure in histogram form. G-002M pre-treatment could significantly enhance EC-SOD expression in lungs (Fig.3A) when compared with irradiated group. However, band intensity in this group was still less than control. In G-002M group, we found also insignificantly increased intensity of EC-SOD band compared to controls.


Also in liver study we found more or less similar results. Formulation pre-treatment could significantly enhance the expression of EC-SOD when compared to irradiated group (Fig.3B). In liver we could also see that G-002M treatment alone had increased EC-SOD existence in comparison to controls. Histological studies Figs.4A-D depict lung histology of controls, irradiated, G-002M pre-treated irradiated, and G-002M alone groups of mice, respectively. In control tissue sections we observed visible thin walled alveoli and numerous bronchioles lined with ciliated epithelium. In radiation exposed mice, the lung architecture was found severely affected.

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Verma et al: Radioprotection by Podophyllum hexandrum in lung and liver

Figure 3. Expression of EC-SOD in lung and liver tissues detected by Western blotting. Exposure of gamma radiation (9 Gy) resulted in significant loss of EC-SOD in both the tissues. [A] EC-SOD expression in lung of controls (C), G-002M administered (D), irradiated (R), and G-002M pretreated irriadiated (DR) mice; [B] EC-SOD expression in liver of control, G-002M administered, irradiated, and G-002M pretreated irriadiated mice.

Alveolar edema, hemorrhage and interalveolar septa thickening were commonly seen. Lung inflammation and infiltration of inflammatory cells into interstitium and residual alveolar spaces due to damaged endothelium was also seen (Fig.4B). In G-002M pre-treated mice, thickening of alveolar wall was mild (Fig.4C). Interstitial edema was also significantly reduced in this group. Infiltration of inflammatory cells was much less in comparison to radiation exposed mice. Damage to endothelial cells was also found significantly reduced. G-002M group mice showed normal alveoli, bronchioles and no pathological alterations in lung architecture (Fig.4D). Histological examination of the control liver section had shown hexagonal hepatic lobule having hepatocytes, central vein, vascular channels, i.e. sinusoids along with its empty spaces and Kupffer cells, lining the sinusoids (Fig.5A). Portal tract consisted of portal vein, hepatic artery and bile ducts were also apparent in the control liver sections. Exposure of lethal dose of gamma radiation resulted in severe steatosis, deformed cell structure, vacuolization of the cytoplasm, injury of hepatocyte membranes, and cloudy swelling of sinusoidal and endothelial cells (Figs.5C&D). Portal vessels were also found to be expanded and bile ducts were highly damaged in radiation exposed mice. In G-002M pretreated group the effect of radiation was significantly less than irradiated animals. Steatosis which was severe in radiation exposed group was still visible in formulation treated group but significantly less. Cell morphology, vacuoles formation in cytoplasm, swelling of sinusoidal, endothelial cells and hepatocyte membrane injury

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was more or less corresponding to controls (Figs.5E&F). Bile ducts and portal vessels were also minimally damaged than irradiated group. Liver architecture was comparable to controls in G-002M group mice on 5 th day of experimentat (Figs.5G&H). Biochemical studies Lipid peroxidation: Fig.6 indicates MDA formation in liver and lung of controls, G-002M, radiation exposed and G-002M pre-treated groups. In radiation exposed group MDA formation in liver was found approximately 2 folds increased at 24 h (P < 0.001; radiation vs controls) while in lungs increase in MDA at the same time interval was not significant. In G-002M group, MDA at the same time interval, was corresponding to controls. In G-002M pre-treated group, MDA concentration in liver at 24 h was observed to be significantly declined (P < 0.001; radiation vs G-002M pre-treated). At 72 h study, MDA was found still increased in comparison to controls in both liver and lung but the values got decreased in liver when compared to 24 h irriadiated group. In G-002M pre-treatment samples, MDA concentration at 72 h was less in both the organs when compared with irradiated group. On 5th day study (Fig.6), there was a sharp decline in liver MDA concentration of radiation exposed group. However, in lungs the values further increased. In G-002M pre-treatment group, MDA in both liver and lung was corresponding to controls at this time point also. Superoxide dismutase: Fig.7 depicts SOD level in mice serum, liver and lung of different experimental groups processed at different time intervals. At 24 h of study, SOD level was found significantly declined in both serum (P < 0.001) and liver (P < 0.01) of irradiated mice when compared with controls, however,

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Figure 4. Histological examination of lungs in differentially treated mice on 5th day of experiment. (A) Control group showing normal architecture with alveoli (arrow) and no pathological changes; (B) irradiated mice showing edema (*), wall thickenings (arrow) and infiltration of inflammatory cells (arrow head); (C) G-002M pretreated irradiated mice showing normal alveoli (arrow head), minimum interstitial edema, no inflammatory infiltration and bronchioles with large air spaces (arrows) as compared to irradiated group; (D) G-002M group mice showing normal architecture. The lung sections were studied under light microscope (x100).


Figure 5. Histological examination of liver in differentially treated mice on 5th day of experimentation. (A) Control showing normal hepatocytes, central vein and sinusoids; (B) inset image showing magnified view indicating the normal hepatocytes (arrows); (C) irradiated mice showing steatosis and structural anomalies; (D) magnified view showing steatosis (*), deformed cell structure, vacuolization of the cytoplasm, injury of hepatocyte nucleus and membranes (arrow head); (E) G-002M pre-treated irradiated mice showing mild steatosis nearly normal structure; (F) inset shows magnified view indicating mild steatosis (*) and normal hepatocyte nucleus (arrows); (G) G-002M group mice showing normal hepatocytes and no pathological changes; (H) inset image showing magnified view indicating normal hepatocytes by arrows. The sections were studied under light microscope (A, C, E and G x100; B, D, F and H x400).

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Verma et al: Radioprotection by Podophyllum hexandrum in lung and liver the fall was not significant in lungs. The values of SOD at 72 h in the same group had fallen faster in comparison to 24 h in liver and blood. At this time point, decline in SOD concentration was noticed in lung tissue also. Formulation treatment had enhanced SOD level in all the three tissues under study, when compared with irradiated group (Fig.7). On 5 th day fall in blood, liver and lung SOD in radiation exposed group was severe. G-002M pre-treatment though could enhance SOD concentration when compared with corresponding irradiated group, but fall was apparent in comparison to controls (Fig.7). G-002M administration alone did not alter SOD expression either in liver or lung and blood at any time point of study. Catalase activity: Concentration of catalase in serum, liver and lung of differentially treated experimental mice is expressed in Fig.8. We observed continuous regression in catalase activity in serum, liver and lung of irradiated mice, measured at 24h, 72h and 5 th day of study. G-002M pre-treatment, though significantly countered the fall in catalase activity at all the time points in all studied tissues (serum, liver and lung), still the values are not comparable to controls. G-002M treatment alone did not induce any variation in catalase activity studied at all the time points and in all studied tissues. Reduced glutathione; Fig.9 expresses the level of GSH in differentially treated mice sacrificed at 24 h, 72 h and 5th day. In irradiated group, we found constant fall in GSH concentration in blood, liver and lung, studied at different time intervals. G-002M pre-treatment could significantly enhance GSH level when compared in serum, liver and lung of irradiated animals. However, the values of GSH were still less on 5th day of study when compared with controls. G-002M alone did not induce any effect on GSH level of treated mice at any

time interval. Glutathione reductase; Fig.10 depicts GR activity of serum, liver and lung tissues of differentially treated experimental mice. Radiation exposure significantly declined GR activity at all the time points of study. Fall was continuous up to 5th day. G-002M pre-treatment could enhance the activity of this enzyme in comparison to irradiated mice; however, the concentration still could not reach the control level even on 5th day of study. G-002M treatment alone did not alter GR activity at any time point of study in any of the studied tissues. Glutathione S-transferase; GST activity in serum, liver and lung of experimental mice is shown in Fig.11. Radiation has significantly declined the activity of this enzyme at all the time intervals of study in all the three tissues. The regression of GST in this group was constantly increased upto 5th day of the study. G-002M treatment alone did not induce any alteration in GST activity in any of the tissue studied currently. G-002M pre-treatment could significantly counter the activity of this enzyme at all the time points in all the tissues; however, the values were not comparable to the controls at any time interval even on 5th day of study. Serum biochemistry profile Table 1 reflects the levels of CHO, HDL, LDL, TG, ALP, AST, ALT, LDH, TP and albumin in sera of differentially treated experimental mice. We observed that radiation exposure could significantly decrease the level of CHO, HDL, LDL, TG and ALP on 5 th day of experimentation. The values of AST (148.80 ± 8.66 U/l and 112.07 ± 7.8 in irradiated and control groups, respectively; P < 0.01), ALT (86.65 ± 3.15 IU/l and 62.5 ± 5.01 in irradiated and control groups,

Figure 6. Effect of G-002M on radiation (9 Gy) induced lipid peroxidation in liver and lung tissues of mice. Mice were dissected at different time intervals. Experiments were performed in triplicate with 3 animals in each group. Tissue homogenates were prepared from excised lungs and liver. MDA values measured at 535 nm, are expressed as nanomoles of MDA formed/mg of protein. Error bars are SEM for n = 9. aR vs C of the respective tissue; b24 h DR vs R of the respective tissue; c72 h DR vs R of the respective tissue; d5th day DR vs R of the respective tissue. *P < 0.01, **P < 0.001, NS = not significant.

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Figure 7. Level of SOD in differentially treated mice. Mice were dissected at different time intervals. Experiments were performed in triplicate with 3 animals in each group. Homogenates were prepared from excised lungs and liver tissues. SOD activity measured at 560 nm, is expressed as U/mg protein in tissues and U/µl in serum. Error bars are SEM for n = 9. aR vs C of the respective tissue; b24 h DR vs R of the respective tissue; c72 h DR vs R of the respective tissue; d5th day DR vs R of the respective tissue. *P < 0.01, **P < 0.001.

Figure 8. Catalase activity of differentially treated mice. Mice were dissected at different time intervals. Experiments were performed in triplicate with 3 animals in each group. Tissues homogenates were prepared from excised lungs and liver. The enzyme activity measured at 570 nm, is expressed as U/mg protein in tissues and U/ml x 10 in serum. Error bars are SEM for n = 9. aR vs C of the respective tissue; b24 h DR vs R of the respective tissue; c72 h DR vs R of the respective tissue; d5th day DR vs R of the respective tissue. *P < 0.01, **P < 0.001.

Figure 9. GSH level in differentially treated mice. Mice were dissected at different time intervals. Experiments were performed in triplicate with 3 animals in each group. Tissues homogenates were prepared from excised lungs and liver. Glutathione concentration measured at 412 nm, is expressed as µM GSH/mg protein in tissues and µM GSH/ml x 10 2 in blood. The experiment was performed in triplicate with 3 animals in each group. Error bars are SEM for n = 9. aR vs C of the respective tissue; b24 h DR vs R of the respective tissue; c72 h DR vs R of the respective tissue; d5th day DR vs R of the respective tissue. *P < 0.01, **P < 0.001, NS = not significant.


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Verma et al: Radioprotection by Podophyllum hexandrum in lung and liver

Figure 10. GR activity in differentially treated mice. Mice were dissected at different time intervals. Experiments were performed in triplicate with 3 animals in each group. Tissues homogenates were prepared from excised lungs and liver. GR activity measured at 340 nm is expressed as mU/min/mg protein and mU/min/ml x 102 in serum. Error bars are SEM for n = 9. aR vs C of the respective tissue; b24 h DR vs R of the respective tissue; c72 h DR vs R of the respective tissue; d5th day DR vs R of the respective tissue. *P < 0.01, **P < 0.001, NS = not significant.

Figure 11. GST activity in differentially treated mice. Mice were dissected at different time intervals. Experiments were performed in triplicate with 3 animals in each group. Tissues homogenates were prepared from excised lungs and liver. GST activity measured at 340 nm is expressed as mM GSH-CDNB conjugate formed/min/mg protein in tissues and mM GSH-CDNB conjugate formed/min/ml x 10-2 in serum. Error bars are SEM for n = 9. aR vs C of the respective tissue; b24 h DR vs R of the respective tissue; c72 h DR vs R of the respective tissue; d5th day DR vs R of the respective tissue. *P < 0.01, **P < 0.001, NS = not significant.

DISCUSSION respectively; P < 0.01) and LDH (462.67 ± 16.75 U/l and 301 ± 18.18 in irradiated and control groups, respectively; P < 0.001) got increased after radiation exposure. TP and albumin were not found altered in any of the experimental group. With G-002M pretreatment, the radiation induced increased level of LDH, ALT and AST reduced to normal level, while CHO, HDL, LDL, TG and ALP got normalized. However in our study we found that TG was still higher than normal on 5th day of study. G-002M treatment alone did not induce any alteration in any of the studied parameter.

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Free radicals generated by ionizing radiation induce damage to the critical macromolecules of the cell. The ROS such as superoxide, peroxynitrite, hydroxyl radical, hydrogen peroxide, etc lead to pathogenesis of many organs including lung and liver [29-32]. Chances of lung and liver cancer are also known to be augmented by over exposure of ROS [33]. Various reports have conveyed that treating liver and lung with radiation exposure induced fibrosis, malignancies and pneumonitis [30, 34]. Though in routine process, radiation mediated damages to the tissues are taken care by natural defense mechanisms; however, in severe conditions exogenous support to minimize

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Oxidants and Antioxidants in Medical Science 2014; 3(1):51-64 Table 1. Levels of serum cholesterol (CHO), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG), total protein (TP), albumin, aspartate aminotransferase (AST), alanine tranaminase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) of various experimental groups of strain A mice Control group CHO (mg/dl)

75.64 ± 9.84

G-002M group

Irradiated group a

G-002M+irradiated group

70.35 ± 6.85

54 ± 5.14 *

65.66 ± 4.11b*

a

HDL (mg/dl)

111.73 ± 10.96

89.63 ± 8.25

58.85 ± 4.11 **

64.16 ± 3.97b*

LDL (mg/dl)

37.26 ± 5.61

41.23 ± 3.65

20.3 ± 3.24a*

31.09 ± 4.04b*

TG (mg/dl)

76.33 ± 6.9

80.35 ± 6.36

51 ± 5.05a*

108.33 ± 9.74b**

TP (g/dl)

5.65 ± 0.35

5.24 ± 0.47

4.54 ± 0.14

5.17 ± 0.48

Albumin

3.59 ± 0.19

3.25 ± 0.12

3.09 ± 0.11

3.18 ± 0.09

AST (U/l)

112.07 ± 7.8

125.36 ± 9.64

148.8 ± 8.66a*

111.66 ± 2.17b*

ALT (IU/l)

62.5 ± 5.01

60.25 ± 4.56

86.65 ± 3.15a*

68.66 ± 4.61b*

ALP (U/l)

112 ± 14.48

102.36 ± 12.25

41.66 ± 6.94a**

69.33 ± 5.43b*

LDH (U/l)

301 ± 18.18

298.45 ± 12.3

a

462.67 ± 16.75 **

325 ± 7.07b**

Values are expressed as mean ± SEM of serum collected individually from the groups of experimental animals. Experiments were performed in triplicates having 3 animals in each group. aIrradiated group vs controls, bG-002M + irradiated group vs irradiated group. *P < 0.01, **P < 0.001.

radiation induced damage is inevitable [2]. P.hexandrum has been extensively reported by our group to render protection against radiation mediated damages to various vital organs of lethally irradiated mice [14, 16-18]. In the present study, mode of extending radioprotection by P.hexandrum (G-002M) to lung and liver tissues of lethally irradiated mice could be via increased expression of antioxidant defensive enzymes resulting into minimal tissue injuries, has been experimentally evidenced. In lungs, EC-SOD, the key antioxidant enzyme, is well reported to prevent free radicals mediated tissue damage [4, 7, 8]. Since lungs have large quantity of connective tissues and vessels, the enzyme EC-SOD is highly expressed as compared to the other tissues. This enzyme contains a heparin or matrix binding domain, which enables it to bind to the matrix and to cell surfaces in tissue. This matrix-binding domain is sensitive to proteolysis [35]. In the current study, whole body exposure to lethal dose of gamma radiation to mice has resulted in significant loss of EC-SOD from extracellular matrix of the lung (Figs.1D&3A). This indicates that radiation has either triggered the release of some proteases in extracellular matrix which catalyze the proteolysis of heparin binding domain or over-utilized this enzyme during superoxide radical scavenging process. Protection against functional and tissue damage in the lungs has been demonstrated earlier also by administration of manganese (Mn)SOD and SOD mimetic [9]. In consonance, present study confirms protection delivered to the lung tissues of lethally irradiated mice by pre-administration of G-002M. Observations demonstrate that this protection could have been due to G-002M mediated enhanced expression of EC-SOD (Figs.1F&3A) along with the up-regulation of other endogenous antioxidant enzymes


in blood and lung tissues (Figs.7,8,10,11). Current study also is in corroboration to the previous findings where EC-SOD has been shown to extend protection to the lung inflammation and fibrosis induced by oxidative stress [6, 7, 8, 35]. In irradiated animals the values of MDA at 24 h were comparable to controls which may be due to abundant availability of EC-SOD in the lungs. EC-SOD is amply reported for inhibition in generation of superoxide radicals which prevents the formation of secondary radicals required to initiate lipid peroxidation. However on 3rd and 5th day marked increase in MDA formation was noticed which may be due to ROS mediated damage to vascular endothelial cells resulting into release of free iron from red blood cells. Free iron is known to aggravate Fenton reaction leading to enhanced lipid peroxidation. The glucoside and rutin present in G-002M, known for their free radical scavenging potential [36], might have certainly lowered ROS production leading to minimize lipid peroxidation. G-002M pre-administration also protected radiation induced lung injuries like inflammation and edema which are known to be caused predominantly due to free radicals mediated vasculature damage leading to excessive release of cellular fluid into alveolar spaces. Radiation has also been amply reported for causing significant loss of antioxidant enzymes in liver leading in acceleration of lipid peroxidation which may result into oxidative damage to the tissues [2, 13, 29]. Enhanced oxidative stress under severe conditions may lead to hepatic fibrosis by degeneration of hepatic cells and their replacement by fibrotic tissues. This has been confirmed in our current investigation through biochemical and histological analysis. Whole body exposure to lethal radiation in our experimental mice

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Verma et al: Radioprotection by Podophyllum hexandrum in lung and liver has also shown loss of EC-SOD along with other antioxidant enzymes in the liver (Figs.2D,3B,7,8,10,11) which has aggravated lipid peroxidation leading to damage of the hepatocyte membranes and changes into nuclear morphology (Figs.5C&D). G-002M pretreatment could successfully up-regulate/maintain the release of these antioxidants and also declined MDA formation resulting into minimal damage to the liver tissues as revealed by our histological analysis (Figs.5E&F). Marginal decline in GSH level in G-002M pre-treated irradiated mice (Fig.9) had confirmed less consumption of this endogenous antioxidant which might be due to reduction in radiation induced ROS generation. Reduction in ROS formation could be attributed to proton donating and free radical stabilizing property of the flavonoid and glucoside present in our formulation. The similar kind of facts has been elaborately mentioned in our earlier study [36]. Significant availability of GSH and SOD, the key determinant of tissue integrity, in G-002M pretreated mice was able to inhibit extracellular and intracellular ROS generation resulting into decreased tissue injuries. In our study, antioxidant enzymes’ decline rate in G-002M pretreated group, was significantly less than irradiated group; however, the values were lower than controls even on 5th day of the study. Delay in recovery may be predominantly because of lethal radiation induced severe damage to the major organ systems and slow cell turnover rate in the liver resulting into its compromised functionality. Reformation in tissue pathology and EC-SOD level, analyzed in the liver during current investigation, are also in consonance with antioxidant enzymes’ status. Studies on antioxidant status with lethal dose of radiation are very limited, therefore we found it difficult to corroborate. Variations in the level of hepatic enzymes like AST, ALT and LDH are also used as diagnostic indicators of hepatic injury [37]. In the current investigation, levels of all the three enzymes in sera of radiation exposed animals were found elevated which could be due to radiation induced protein and lipid modification of the hepatocytes resulted in increased permeability and cellular leakage of these enzymes. As reported earlier and in our current study also, significant decrease in the level of serum CHO, HDL, LDL and TG after radiation exposure, could be due to altered liver lipid metabolism and oxidative damage of lipids in membrane of hepatocytes. These changes are confirmed in our histological findings where steatosis has been visualized in the liver of irradiated mice (Fig.5C). The protective action of G-002M on liver structural damage might be due to scavenging of oxidation-initiating radicals and minimizing modification in the protein and lipid backbone of cellular membrane. Significant protection to radiation induced hepatotoxicity and

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alterations in liver architecture like cellular morphology, vacuolization of cytoplasm and hepatocytes’ membrane damage (Figs.5E&F) by G-002M pretreatment is due to reduced oxidative stress. Hepatoprotective role of P.hexandrum by elevating a set of endogenous antioxidant enzymes has been reported earlier also [13, 16, 38]. In the current study we have demonstrated that the P.hexandrum formulation (G-002M) has radioprotective potential and is able to provide significant protection against radiation mediated cytotoxicity in lung and liver tissues by significantly maintaining the level of EC-SOD and other antioxidant enzymes and minimizing lipid peroxidation. Podophyllotoxin, present in the formulation, temporarily induces cell cycle arrest (G2/M) by inhibiting tubulin polymerization, therefore, leading to minimal DNA damage and providing sufficient time for repair. The two other constituents such as podophyllotoxin-β-D-glucoside and rutin have also been shown in our other studies for significantly stabilizing free radicals by donating hydrogen atoms from their attached hydroxyl groups [36]. In addition, the immunomodulatory action of glucoside supports in fast recovery of the immune system by enhancing the level of anti-inflammatory cytokines and granulocytes growth stimulating factors. The three active principles of G-002M, having different properties, present in the formulation might have acted in synergism to extend radioprotection against lethal doses of radiation in mice. Further studies, besides antioxidant, anti lipid peroxidation, DNA damage repair property and antioxidant proteins status in G-002M treated samples should be done to explore the other possible role of these phyto-constituents of Podophyllum hexandrum by which it renders radioprotection, are underway.

ACKNOWLEDGEMENTS The research work was supported by grants from Defense Research Development and Organization (DRDO). The authors duly acknowledge Dr. R. P. Tripathi, Director of the Institute of Nuclear Medicine and Allied Sciences (INMAS), for his administrative support. The authors also appreciate the assistance of Ms. Renu Bansh for experimental work. COMPETING INTERESTS The authors declare that they have no conflict of interests.

DOI 10.5455/oams.261213.or.057

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DOI 10.5455/oams.261213.or.057


ISSN: 2146-8389

ORIGINAL ARTICLE

Antioxidant and anti-inflammatory potential of some dietary cucurbits Indu Rawat, Dhara Sharma, Harish Chandra Goel Amity Center for Radiation Biology, Amity University, Noida, Uttar Pradesh, India

Received November 26, 2013 Accepted March 5, 2014 Published Online March 26, 2014 DOI 10.5455/oams.050314.or.059 Corresponding Author Harish Chandra Goel Amity Center for Radiation Biology, Amity University, Sector-125, Noida, Uttar Pradesh, 201303, India. [email protected] Key Words Cucurbita pepo; Cyclooxygenases; Free radicals; Lagenaria siceraria; Luffa cylindrica

Abstract Objective: The inflammatory problems and associated diseases in the gastrointestinal tract are known to exist in a sizable fraction of the population. It results due to complex interaction between food, enteric microbes and the host. Therefore, the cucurbits as an easily digestible diet were evaluated for their antioxidant and anti-inflammatory activities. Methods: Antioxidant properties of cucurbits (Lagenaria siceraria, Ls; Cucurbita pepo, Cp; Luffa cylindrical, Lc) were evaluated in vitro following xylene orange or Amplex® Red dye method. Anti-inflammatory properties of cucurbits with respect to cyclooxygenase (COX)-1 and 2 were studied and interleukin (IL)-1β and tumor necrosis factor (TNF)-α in serum samples of lipopolysaccharide (LPS)-inflamed mice. Results: The inhibition rate of superoxide radicals by Ls, Cp and Lc was 40, 36 and 31%, respectively, at a concentration of 0.08 mg/ml. The perhydroxyl radical scavenging activity was in a declining order and maximum effect was exhibited by Ls. With respect to COX-1 inhibition, IC50 for indomethacin, Cp, Lc and Ls were estimated to be 0.0154, 0.026, 0.0337 and 0.0335 mg/µl; and IC50 against COX-2 were 0.0427, 0.023, 0.0183 and 0.0150 mg/µl, respectively. In vivo study revealed that Ls, Lc, indomethacin and Cp correspondingly downregulated the expression of IL-1β by 52, 68, 75 and 126 pg/100 µl, significantly less than control (260 pg/100 µl). TNF-α was also reduced significantly more by Ls, Lc, indomethacin and Cp by 12, 16, 20 and 26 pg/100 µl, respectively, than control (46 pg/100 µl). Conclusion: The dietary cucurbits seem to have potential for developing a non-toxic therapeutic product. © 2014 GESDAV

INTRODUCTION Reactive oxygen species (ROS) are regularly generated in the body during metabolic activities. Pollutants, radiations, pathogens and gut microbiome also generate free radicals in our bodies. The free radicals interact with biomolecules like DNA, proteins and membranes. Interaction of radicals with biological membranes generates various peroxides and malonaldehyde [1], and alters membrane permeability, signal transduction, and even leads to the cell rupture. The free radicals activate various pro-inflammatory mediators and transcription factors like tumor necrüosis factor (TNF)-α, interleukin (IL)-1β, IL-6, nuclear factor (NF)-κB etc, which in turn activate various cytokines and subsequent inflammatory events at initial levels [2]. In the gastrointestinal (GI) tract, free radicals interact with gut lining and alter the intracellular redox status. Free radicals activate receptors and non-receptor kinase cascades like tyrosine kinase, protein kinase C and mitogen-activated protein kinase (MAPK), which in turn activates various inflammatory cytokines, tumor growth factors, lymphocytes, neutrophils and prostaglandins. These inflammatory molecules generate pain and fever [3]. During inflammation, membrane bound enzymes cyclooxygenase (COX)-1 and COX-2


are activated. COX-1 is a constitutive and cytoprotective enzyme [4], while COX-2 activates phospholipase A2, releases arachidonic acid from cell membrane and biosynthesizes prostaglandins (PG) which activates inflammation. There are in fact a large number of natural and synthetic compounds namely SAID (steroidal antiinflammatory drugs) and NSAID (non-steroidal antiinflammatory drugs) used for relieving inflammation. Unfortunately, both classes of these agents have adverse reactions which may sometimes prove even fatal. NSAID is indeed a weak inhibitor of COX-2 but strong inhibitor of COX-1 and therefore leads to GI toxicity. For controlling inflammation in the GI tract inhibition of COX-1 should be as low as possible in comparison to COX-2. Some dietary cucurbits, i.e. Lagenaria siceraria (Molina) Standl, Cucurbita pepo Linn and Luffa cylindrica (Linn) M. Roem, were investigated for antiinflammatory activities. Extracts of these cucurbits contain biomolecules like flavonoids, terpenoids, alkaloids etc, which have been well documented free radical scavenging properties. Such agents have been reported for enhancing the potential of endogenous antioxidants such as glutathione peroxidase (GPx),

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Rawat et al: Antioxidant and anti-inflammatory potential of cucurbits superoxide-dismutase (SOD) and catalase (CAT) [5]. Lagenaria siceraria (Ls), has been reported for antioxidant and anti-inflammatory attributes [6, 7]. Similarly, Cucurbita pepo (Cp) has been shown to have antioxidant [8], anti-inflammatory [9] and anti-ulcer properties [10]. Luffa cylindrica (Lc) has been documented for antioxidant [11], anti-inflammatory [12], anti-bacterial and anti-fungal properties [13].

in 100 ml solvent (TDW and absolute alcohol; 50:50, v/v for 24 h) at ambient temperature. Twenty four hours later the homogenate was centrifuged for 15-20 min at 5000 rpm and supernatant was collected and filtered using 0.22 µ membrane filters and further concentrated by using Rotavapor. Finally the concentrated extract of each cucurbit fruit was stored at 4ºC [15].

The present study aims to investigate the effect of some dietary cucurbits (fruit extracts) both in vitro and in vivo against lipopolysaccharide (LPS)-induced inflammation as a challenge in the GI tract.

Isolation of human neutrophils Three milliliters of Histopaque®-1119 was added to 15 ml capped centrifuge tubes. 3 ml of Histopaque®1077 was poured gently on the Histopaque ®-1119 and thereafter 6 ml of anticoagulated blood collected from healthy volunteers was added. The blood on the Histopaque® solutions was centrifuged at 700 rpm for 30 min at room temperature (18-26ºC). Neutrophils precipitated and formed a layer (2 ml from the bottom) in the centrifuge tube. These neutrophils were carefully separated using a micropipette and were washed with 10 ml of phosphate buffer saline. This mixture was further centrifuged for 10 min at 200 rpm and the concentrated cell mass was converted into pellet. The pellets were re-suspended in Hank’s balanced salt solution (HBSS) and the viability of cells was determined by trypan blue [16].

MATERIAL AND METHODS Reagents Nutrient broth, eosin methylene blue, trypan blue, gallic acid, and xylene orange were procured from HiMedia (Mumbai, India); Tri-sodium citrate, citric acid, sodium chloride, disodium phosphate hydrogen phosphate, calcium chloride, magnesium chloride, sodium-potassium tartrate, dimethyl sulphoxide (DMSO), methanol and ethanol were procured from Merck (Mumbai); Sodium dihydrogen phosphate, aluminum chloride, dextrose, potassium chloride, potassium dihydrogen phosphate, hydrogen peroxide (H2O2), sodium carbonate and sodium bicarbonate were procured from Qualigens (Mumbai); WST-1 (2-(4Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)2H-tetrazolium, monosodium salt) was procured from Interchim (Montlucon, France); Indomethacin was procured from MP Biomedicals (Strasbourg, France); RPMI-1640, Histopaque®-1077 and Histopaque®-1119, were procured from Sigma Aldrich (St. Louis, MO, USA); Quercetin was procured from Sisco Research Laboratories (Mumbai); Folin-Ciocalteu was procured from Loba Chemie (Mumbai); Amplex® Red assay kit was procured from Molecular Probes® (Germany); TMPD (N’,N’,N’,N’-tetramethyl-p-phenylenediamine), COX inhibitor screening kit, TNF-α and IL-1β kits were procured from Cayman (Ann Arbor, MI, USA). Bacterial culture Escherichia coli procured from Jamia Milia Islamia University (New Delhi, India) were grown on selective growth media eosin methylene blue at 37ºC in a sterilized environment and were harvested after 24 h of inoculation. E.coli were identified by gram reaction, cell morphology and catalase reaction [14]. Preparation of cucurbits extracts Fresh fruits of cucurbits Ls (bottle gourd), Cp (pumpkin) and Lc (sponge gourd) procured from local market were washed thoroughly with triple distilled water (TDW) several times and 1 kg of each plant material was crushed and spun in homogenizer. The homogenate of each plant material was kept separately

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LPS induced inflammation in mice LPS from E.coli (1 mg/ml) was stored as stock solution at -20ºC. For experiments the solution was diluted (20 µg/100 µl saline) immediately before intraperitoneal (i.p.) injection. Control mice received 20 µg of LPS in 100 µl. In double control, 100 µl of saline was injected to mice. In experimental group 2 h after LPS injection, prebiotic was administered orally (100 µl) wherever needed. In double control, control and test samples, after 4 h of LPS injection, mice were anesthetized and blood samples were taken by cardiac puncture and kept overnight at 4ºC for clot formation [17]. Serum was collected after centrifugation (5000 rpm) for 5 min and stored at -20ºC until used for the measurement of IL-1β and TNF-α using ELISA. Determination of phenols and flavonoids The phenolic contents were determined as equivalent to gallic acid and expressed as mg/ml. Following the method as adopted by Hodzic et al [18], 1 ml aliquot of a cucurbit extract (Ls, Lc or Cp) taken in a test tube was made up to a volume of 3 ml by adding TDW. To this solution, Folin-Ciocalteau solution (0.5 ml of 1 part Folin-Ciocalteau and 1 part TDW) and 2 ml of 20% sodium carbonate solution in TDW were added in each test tube and was kept 40ºC for 1 min. Each test tube was cooled and absorbance was measured at 650 nm. Gallic acid was taken as a reference for the determination of total phenolic contents. Flavonoid content was determined as equivalent to

DOI 10.5455/oams.050314.or.059

Oxidants and Antioxidants in Medical Science 2014; 3(1):65-72 quercetin and expressed as mg/ml. Following the method as adopted by Khatiwora et al [19], 1 ml aliquot of each cucurbit extract (Ls, Lc and Cp) was taken in different test tubes and made up to the volume of 3 ml with methanol (analytical grade). 0.1 ml of 10% aqueous solution of aluminum chloride (10%) and 0.1 ml of sodium-potassium tartrate (1 M aqueous solution) and 2.8 ml TDW were added. Contents in each test tube were shaken and were incubated for 30 min at ambient temperature and absorbance was measured at 415 nm. Quercetin was taken as reference for determination of flavonoids. Superoxide radical scavenging assay The superoxide radicals in neutrophils were determined following the method adopted by Jalil et al [20]. The neutrophils were stressed by addition of the cells of E.coli, three times of the number of neutrophils. The stressed neutrophils were transferred to the microplate well. WST-1 dye was added at this stage and maintained at ambient temperature. Ten minutes later different concentration of a cucurbit extract (Ls, Cp or Lc) or indomethacin was transferred separately to a microplate well having activated neutrophils. The stressed neutrophils were incubated for 30 min at 37ºC and the concentration of decomposed superoxide radicals were measured at 450 nm using microplate reader. Catalase estimation Xylene orange method: The decomposition of perhydroxyl radicals by endogenous catalase present in neutrophils was evaluated by using xylene orange as an indicator following the method adopted by Zmijewski et al [21]. Neutrophils (620 cells/250 µl of HBSS) were stressed by addition of hydrogen peroxide (50 µl of 150 mM), E.coli (34 x 103/50 µl broth) or both the agents together. The effect of a cucurbit extract (Ls/Cp/Lc; 0.05 mg/ml) or NSAID (indomethacin) on scavenging the concentration of perhydroxyl radicals was investigated by adding these agents individually to neutrophils after 10 minutes of stress induction. This aliquot thereafter was incubated at 37ºC for 60 min and the perhydroxyl radicals were measured spectrophotometrically at 585 nm. Amplex® Red method: The production of hydrogen peroxide in neutrophils (750 cells /250µl of Hank’s solution) was induced by the stressors hydrogen peroxide (40 mM) or E.coli (11 x 103/50 µl in medium) individually or in combination in equal volumes. The decomposition of hydrogen peroxide by cucurbit extracts or indomethacin was determined by using Amplex® Red dye and horse radish peroxidase following the method adopted by Zhao et al [22]. Cucurbit extracts (Ls/Cp/Lc) or indomethacin was added in microplate well after 10 min of induction of stress in neutrophils. This was then incubated at 37ºC


for 30 min and the decomposition of hydrogen peroxide was measured spectrophotometrically at 585 nm. COX-1 and COX-2 inhibition assay 10 µl of COX-1 or COX-2 was transferred to each well of the microplate followed by addition of 10 µl of each agents (Ls, Cp , Lc or indomethacin) separately and was incubated for 5 min at 25ºC. Thereafter, 20 µl of TMPD dye was added and read at 585 nm using microplate reader as the method followed by Alberto et al [23]. The data was represented as percent inhibition of COX-1 or COX-2. The IC50 for COX-1and COX-2 as a result of the addition of various agents was calculated as follows: IC50 of cucurbit / IC50 of indomethacin x 100

Measurement of IL-1β and TNF-α in LPS induced inflammation in mice serum (in vivo) In 96 well plates, each well was pre-coated with monoclonal antibody specific for IL-1β. 50 µl of serum samples was added in a well followed by addition of 50 µl of IL-1β-acetylcholinesterase Fab’ conjugate and incubated overnight at 4ºC. Non-adherent cells from well plates were rinsed using wash buffer and 100 µl of Ellman’s reagent was added and incubated for 30 min in dark. Absorbance was measured at 415 nm. For TNF-α measurement, a monoclonal antibody specific for TNF-α was pre-coated in 96 well plates. Fifty microliter of serum samples were added in different wells followed by addition of 50 µl of acetylcholinesterase Fab’ conjugate and incubated overnight at 4ºC. Non adherent cells were removed by rinsing with wash buffer and 100 µl of Ellman’s reagent was added and incubated for 30 min in dark and absorbance was measured at 415 nm as the method followed by Jaegle et al [17]. Statistical analysis Each experiment was an average of three sets of experiments and the data has been calculated as the mean ± standard error of the mean (SEM). One way ANOVA was applied to test the significance which was defined as P < 0.05. RESULTS Total phenols and flavonoids Spectrophotometric measurement revealed that Lc has maximum phenolic contents followed by Ls and Cp in a decreasing order (Table 1). On the other hand, Ls has maximum flavonoid concentration as compared to Lc and Cp. Superoxide radical scavenging activity In the unstressed and stressed neutrophils the values for superoxide radicals were 0.123 ± 0.009 and 1.623 ± 0.1, respectively, and the data obtained for

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Rawat et al: Antioxidant and anti-inflammatory potential of cucurbits stressed neutrophils was used as the ‘control’ for this study. With the increasing concentration of the cucurbits extracts (0.01, 0.02, 0.06 and 0.08 mg/ml) added to a well containing 250 µl of the neutrophils in HBSS, the SOD activity was enhanced and the superoxide radicals were appreciably scavenged and reduced. Addition of 0.08 mg/ml of each of the cucurbit extracts reduced the superoxide radicals as follows: Ls 40%, Cp 36% and Lc 31% (Fig.1). However, an increase in the concentration of cucurbit extract to the extent of 0.12 mg/ml did not increase the SOD further and rather decreased to some extent. The superoxide radicals therefore remained in more abundance at this concentration of cucurbit. Administration of indomethacin also followed the same pattern. An increase in the concentration of indomethacin from 0.01 to 0.08 mg/ml rendered progressive scavenging of superoxide radicals up to 44%. However, the presence of 0.12 mg/ml of indomethacin decreased SOD to some extent and therefore the decrease in scavenging of superoxide radicals was observed as 40% only. Catalase activity (in presence of xylene orange) The absorbance of neutrophils without stress measured as 0.738 ± 0.009 was taken as control. The stressed neutrophils exhibited the absorbances 0.662 ± 0.01 for H2O2, 0.578 ± 0.04 for E.coli and in case of H2O2 and E.coli given together the value was 0.63 ± 0.003 (Fig.2). Presence of Ls or Cp had scavenged the perhydroxyl radical formed in stressed neutrophils within 30 min and almost completely neutralized within 60 min (Ls 0.736 ± 0.01; Cp 0.735 ± 0.007). Stressed neutrophils were partially neutralized by Lc (0.7 ± 0.009) or indomethacin (0.649 ± 0.01) in 60 min (Fig.2). Statistical analysis revealed that presence of Ls, Cp and Lc in stressed neutrophils enhanced the CAT activity significantly compared to control (P < 0.05). Catalase activity (in presence of Amplex® Red) The excess of H2O2 formed in unstressed neutrophils was measured as absorbance and found to be 0.108 ± 0.005 which acted as its control. However, interaction with stressors like E.coli, H2O2 or both together, the presence of H2O2 in neutrophils increased to 0.166 ± 0.01, 0.161 ± 0.02 and 0.15 ± 0.01, respectively. On addition of cucurbit extract or indomethacin the presence of excess of H2O2 was found to decrease; maximum effect was observed by addition of Ls (0.124 ± 0.008) followed by Cp (0.134 ± 0.01), Lc (0.136 ± 0.005) and indomethacin (0.146 ± 0.003) as depicted in Fig.3. The statistical analysis by ANOVA revealed that the presence of Ls Lc and Cp in stressed neutrophils enhanced the CAT activity significantly compared to control (P < 0.05).

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Figure 1. Effect of the change of concentration of various agents on inhibition of superoxide radicals generated in stressed neutrophils The data were averaged for three independent experiments. The Ls and Cp were highly significant in scavenging superoxide radicals than control (P < 0.05). [The agents in columns are listed in following order; indomethacin, Ls, Cp, Lc.]

Figure 2. Diminution of peroxide radicals by CAT (using xylene orange method) generated in neutrophils stressed by addition of a combination of H2O2 or E.coli or a combination of the two in equal quantities of the culture medium in equal volume. The modulatory effect of various cucurbit extracts and indomethacin was observed at different post-treatment time. Data from three independent experiments were averaged. The Ls, Cp and Lc were significant in scavenging peroxide radicals compared to the control (P < 0.05). [The black column represents the control group; for the other columns, the order is the same as in Fig.1.]

Figure 3. Estimation of CAT activity generated in neutrophils due to the addition of stressors H2O2 or E.coli individually or their combination in equal quantity. Data from three independent experiments were averaged. The Ls, Cp and Lc were significantly effective in scavenging peroxide radicals than control (P < 0.05). [I, double control (un-stressed neutrophils); II, E.coli; III, H2O2; IV, H2 H2O2 + E.coli (control); V: H2O2 + E.coli + indomethacin; VI, H2O2 + E.coli + Cp; VII, H2O2 + E.coli + Lc; and VIII, H2O2 + E.coli + Ls]

DOI 10.5455/oams.050314.or.059

Oxidants and Antioxidants in Medical Science 2014; 3(1):65-72 COX-1 and COX-2 inhibition Fig.4 depicts the IC50 values for various agents studied here. Indomethacin rendered IC50 for COX-1 as 0.0154 mg/µl, followed by Cp (0.026 mg/µl), Lc (0.0337 mg/µl) and Ls (0.0335 mg/µl). Similarly, as to see in Fig.5, indomethacin revealed an IC50 for COX-2 as 0.0427 mg/µl, followed by Cp (0.023 mg/µl), Lc (0.0183 mg/µl) and Ls (0.015 mg/µl). Statistical analysis revealed that the IC50 of COX-1 by Ls, Lc and Cp were 2.17, 2.18 and 1.68 times less effective than indomethacin. However the IC50 of COX-2 by Ls, Lc and Cp were 0.35, 0.428 and 0.53 times more effective than indomethacin (P < 0.05). In vivo studies in LPS induced mice serum The specific cytokines involved in inflammation were studied in response to the administration of cucurbit extracts orally. Various assays were done to evaluate the concentration of IL-1β and TNF-α in LPS-inflamed mice serum. IL-1β: the amount of IL-1β in control (mice serum) was 260 pg. However, IL-1β measured in serum of mice which were subjected to inflammation by LPS administration (i.p.) revealed that treatment with Cp, Lc, Ls or indomethacin individually was 126, 68, 52 or 75 pg, respectively, as displayed in Fig.6. Each experiment was performed three times independently and statistical analysis revealed that Ls was most effective in reducing the expression of IL-1β followed by Lc, indomethacin and Cp than control (P < 0.05). TNF-α: the amount of TNF-α quantified in control (mice serum) was 46 pg. However, the mice which were administered LPS, revealed that treatment with Cp, Lc, Ls or indomethacin rendered 26, 16, 12 or 20 pg of TNF-α, respectively, as displayed in Fig.7. Each experiment was performed three times independently and statistical analysis revealed that Ls was most effective in reducing the expression of TNF-α followed by Lc, indomethacin and Cp than control (P < 0.05).

Figure 4. IC50 of COX-1 activity demonstrated by various cucurbits or indomethacin. The data of three independent experiments were averaged. The indomethacin was highly significant (P < 0.05) than Lc and Ls for inhibiting COX-1.


Figure 5. IC50 of COX-2 activity demonstrated by various cucurbits or indomethacin. The data of three independent experiments were averaged. The Ls, Lc and Cp were highly significant (P < 0.05) than indomethacin for inhibiting COX-2.

Figure 6. Effect of cucurbits and indomethacin on the expression of IL-1β. These agents were administered to LPS-inflamed serum of the data represented to average of three independent experiments. Ls was highly significant in reducing the expression of IL-1β followed by Lc, indomethacin and Cp than control (P < 0.05).

Figure 7. Effect of cucurbits and indomethacin on the expression of TNF-α. These agents were administered to LPS-inflamed serum of the data represented to average of three independent experiments. Ls was highly significant in reducing the expression of TNF-α followed by Lc, indomethacin and Cp than control (P < 0.05).

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Rawat et al: Antioxidant and anti-inflammatory potential of cucurbits Table 1. Total Phenols and flavonoids of various cucurbit extracts Total Total Flavonoids Cucurbit phenols flavonoids (% of total species (mg/ml) (mg/ml) phenols) Lagenaria siceraria

0.256 ± 0.005

0.052 ± 0.003

20%

Luffa cylindrica

0.295 ± 0.007

0.042 ± 0.001

14%

Cucurbita pepo

0.234 ± 0.003

0.018 ± 0.001

7%

DISCUSSION The oxidative stress to an organism is responded by scavenging or neutralization of oxidative radicals with the help of endogenous antioxidants such as SOD, GPx and CAT [24]. In the gut the prebiotic microbiota remains in a dynamic state and keeps generating oxidative species. The prebiotics interact with the oxidative and antioxidant molecules in the metabolome and in the body. The cucurbit extracts (prebiotics) display high antioxidant activities through variations in hydroxylic groups and spatial arrangement of phenolic compounds could be looked in for explaining variations in the inhibition potential of oxidative radicals (Table 1). The total phenolic contents of Lc were higher than that of Ls. However in Ls the higher antioxidant activity may be attributed to the increased proportion of flavonoids as compared to Lc and Cp (Table 1). The free radical scavenging efficacy of various cucurbits as seen above may be attributed to the differences in the genetic composition also. The addition of cucurbits extract to the stressed neutrophils led to enhancement of free radical scavenging activity (Fig.1). This activity of cucurbits may be attributed to the phenolic compounds like flavonoids [25-28] which are known to act as antioxidants because of the donation of an electron or hydrogen atom [29]. The increase in concentrations of different cucurbit extracts (from 0.01 to 0.08 mg/ml) quenched the superoxide radicals in stressed neutrophils in a proportionate manner. Further increase in the concentration of cucurbit extracts (0.12 mg/ml) however did not inhibit the superoxide radicals in an increasing order. It appears that active biomolecules up to in 0.08 mg/ml of the cucurbit extracts scavenged superoxide radicals in stressed neutrophils almost completely (Fig.1). At 0.12 mg/ml the presence of some other molecules in the whole extract did not enhance the effect and rather decreased in this context bringing down the SOD activity in comparison to the 0.08 mg/ml concentration. The CAT activity in neutrophils induced by E.coli was more pronounced in terms of peroxides in comparison to the one induced by hydrogen peroxide or E.coli

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alone or in combination (Fig.2). In fact, hydrogen peroxide is a known per-oxidizing agent [30]; yet, in the present study it evoked much less effect than was done by E.coli. The stress generated by the presence of E.coli (LPS) augmented the production of endogenous CAT (Fig.2) for neutralizing the perhydroxyl radicals. In stressed neutrophils, neutralization of perhydroxyl radicals by endogenous catalase could not be achieved completely within 15 or 30 min (post-treatment). The addition of cucurbits to neutrophils at this concentration of hydrogen peroxide increased the CAT activity for scavenging of perhydroxyl radicals. This study also indicates that some biomolecules present in cucurbit extracts (Ls, Cp, Lc) could have enhanced the scavenging activity of perhydroxyl radicals independently or in synergy with catalases [31]. Our results indicate that for production of endogenous CAT there has to be some time delay; therefore, up to 30 min the scavenging of perhydroxyl radicals displayed an increasing trend. However this activity becomes stable at 45 min and beyond. Bioactive molecules may require some time to activate a phenomenon and this time dependence varies from product to product. Many bioactive molecules have been shown to act in the time dependent manner [32]. The Amplex® Red assay also corroborates the results of CAT activity as shown above. The activity of CAT was exhausted in the process of perhydroxyl radicals scavenging. However, addition of cucurbits (Ls, Cp and Lc) enhanced the peroxide scavenging activity further (Fig.3) in a more effective manner than indomethacin and thus decreased the peroxide stress. The study implies that Ls has maximum flavonoid contents (Table 1) out of the three cucurbits studied here. Therefore Ls had shown maximum anti-peroxide activity. Cp though had less flavonoid contents, yet enhanced the catalase activity comparable to Ls and it implies that molecules other than flavonoids present in Cp could have also enhanced the CAT activity. The cyclooxygenases form another set of important bioactive molecules: COX-1, a constitutive and cytoprotective enzyme; and COX-2, an inducible enzyme regulated by various cytokines are two important cyclooxygenases. COX-2 leads to the formation of prostaglandins which further induce the inflammatory response [33]. The results revealed that indomethacin inhibits COX-1 [34] substantially more than the one manifested by administration of Ls, Lc and Cp. The cucurbits selectively inhibited COX-2 activity more efficiently than indomethacin. Therefore, cucurbits can provide very useful clues towards the production of anti-inflammatory agents for the cure of chronic inflammatory disorders. LPS induces free radical generation which activates the cellular kinase cascades [3]. Activated kinases

DOI 10.5455/oams.050314.or.059

Oxidants and Antioxidants in Medical Science 2014; 3(1):65-72 phosphorylate IκB, a nuclear factor (NF)- κB inhibitor, and detach IκB from the IκB/NF-κB complex. The released NF-kB acts as a transcription factor and induces the expression of many cytokines such as IL-1β and TNF-α, and prostaglandins [35]. For the present investigations, gut was used as a model system for inflammation because we intended to study the antiinflammatory effects of some dietary cucurbits in the GI tract. Administration of LPS (20 µg/100 µl) to mice (Figs.6&7) induced the production of IL-1β (260 pg) and TNF-α (46 pg). These indicated that immunocompetent cells (macrophages, lymphocytes, etc) followed the caspase-1 pathway which induced the production of IL-1β. The secretion of IL-1β and TNF-α behave independently and their regulatory control with respect to each other could not be evidently observed. Post-treatment with Ls significantly reduced the expression of both IL-1β (52 pg) and TNF-α (12 pg). This effect may be attributed to the presence of various phenolic compounds like flavonoids, triterpenoids (cucurbitacin), etc. The Ls acts as a primary free radical scavenger and thus inhibits various inflammatory molecules like cytokines (IL-1β, IL-6, TNF-α) and prostaglandins [6, 36, 37]. Therefore, the cucurbits need to be looked for reduction of gut inflammation and related disorders. The above study concluded that commonly consumed dietary cucurbits (fruits) are rich in various pharmacologically active molecules like phenols, flavonoids, terpenes and saponins having a wide spectrum of biological functions and may be exploited for developing non-toxic anti-inflammatory and antioxidant products.

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ACKNOWLEDGEMENTS Authors are grateful to Life Science Research Board (Defense Research and Development Organization, Ministry of Defense, Government of India) for financial support through the project LSRB-195 and to Amity University, for providing laboratory facilities to conduct research work.


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DOI 10.5455/oams.050314.or.059


ISSN: 2146-8389

ORIGINAL ARTICLE

Biological investigations of antioxidant, antimicrobial properties and chemical composition of essential oil from Warionia saharae Khalid Sellam1, Mhamed Ramchoun2, Farid Khalouki2, Chakib Alem2, Lhoussaine El-Rhaffari1 1

Laboratory of Environment and Health, and 2Laboratory of Biochemistry, Department of Biology, Faculty of Sciences & Techniques, Errachidia, Morocco

Received September 18, 2013 Accepted November 12, 2013 Published Online December 26, 2013 DOI 10.5455/oams.121113.or.056 Corresponding Author Khalid Sellam Laboratory of Environment and Health, Department of Biology, Faculty of Sciences & Techniques, 52000 Errachidia, Morocco. [email protected] Key Words Antimicrobial activity; Antioxidant activity; Chemical composition; Essential Oil; Warionia saharae

Abstract Objective: Several aromatic plants and their essential oils are known to possess antimicrobial and antioxidant properties. Warionia saharae Benth & Coss, an endemic species of North Africa, is traditionally used in the treatment of inflammatory diseases such as rheumatoid arthritis and for gastrointestinal disorders. The aims of this study were to examine the chemical composition of the essential oil isolated from W.saharae, and to test the efficacy of the essential oil as a potential antimicrobial and antioxidant. Methods: The essential oil was investigated by gas chromatography-mass spectrometry (GC-MS). Thirty-six compounds, accounting 96.8% of total oil with 1.1% oil yield were identified. The major compents of W.saharae essential oils were β-eudesmol (24.6%), trans-nerolidol (18.2%), linalool (16.8%), 1,8 cineole (6.2%), camphor (4.6%), p-cymene (3.7%) and terpinen-4-ol (3.6%). In this study, we analyzed biological activities of Warionia essential oil from Errachidia region, Morocco. Indeed, we investigated mainly, the antimicrobial activity against four referenced and representative human diseases health bacteria. Also this essential oil was tested against phytopathogenic fungi. Results: The results showed that W.saharae oil exhibited significant antibacterial and antifungal activities; with minimum inhibitory concentrations (MIC) ranging between 0.039 and 0.156 mg/ml for all bacteria and remarkable antifungal effect that exceeds 50% inhibition of mycelial growth for all fungal strains. We also checked whether this oil exhibited an antioxidant property via radical scavenging ability and antioxidant activity, determined by 2,2-diphenyl-1picrylhydrazyl (DPPH) assay and β-carotene bleaching test. Conclusion: Our results show an important antioxidant property for W.saharae essential oil. © 2014 GESDAV

INTRODUCTION The Warionia saharae, which belongs to the important Asteraceae’s family, is an endemic species of North Africa, characterized by a discerning odor [1]. W.saharae was reported for the first time in the Oranais Sahara (Beni Ounif in Algeria) by Dr. Warrion as a shrub of 1 to 3 m of height. The thick trunk, is covered of a gray peel, structural of very wavy terminal leaf bouquets, and of capitulate of yellow flowers. The picking of stems leafed of this bush, clear a very heady and spicy odor, the latex that flows out of injuries of the peel, glue to hands in a very tenacious way [2]. In Morocco, W.saharae is growing wild in various regions [3]. The habitat is between schistose rocks [4]. W.saharae is known in Morocco by the Berber vernacular names of ‘afessas’ and ‘tazart n-ifiss’. In the Moroccan traditional medicine, the leaves of the plant are used to treat inflammatory diseases such as rheumatoid arthritis, and for gastrointestinal disorders [5].


The chemical composition of W.saharae leaves has been investigated and thus twelve new guaianolide type sesquiterpene lactones were identified. Cytotoxic and anti-inflammatory sesquiterpene lactones effects were showed [6, 7]. The chemical composition of the hexanoic extract from W.saharae leaves prepared using soxhlet apparatus was reported by Essaqui et al [8]. In addition, several studies reported the chemical composition of W.saharae essential oil from the leaves [9, 10]. This study deals with the valorization of medicinal and aromatic plants of the oasis Moroccan flora, in order to find new bioactive natural products. Information concerning in vitro antioxidant, antimicrobial activities of the essential oil from the W.saharae has not been reported earlier. The aims of this work were to examine the chemical composition of the the essential oil obtained from aerial part of W.saharae originated from Southern Moroccan Sahara and investigate their antimicrobial and antioxidant activities.

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Sellam et al: Biological investigation of Warionia saharae MATERIAL AND METHODS Plant material Flowering parts of Warionia saharae were collected in May from natural populations in Errachidia (East of Morocco). The botanical identification was achieved by the National Scientific Institute (Rabat) where voucher specimens were deposited in the Herbarium. The dried plant material is stored in the laboratory at room temperature (25°C) and in the shade before the extraction. Isolation of the essential oil The extraction of essential oil of the aerial part of W.saharae was conducted by hydrodistillation. The essential oil obtained was dried under anhydrous sodium sulfate and stored at 4°C in the dark before analysis. Gas chromatography-mass spectrometry The GC-MS analysis was done on a Trio 1000 mass spectrometer coupled with a model 8000 gas chromatograph (Thermo Scientific, Fisons Instruments) equipped with a HP-5ms capillary spectrometer (30 m long x 0.25 mm diameter, 0.25 µm film tickness). The column temperature program was 60°C for 6 min, with 5°C increases per min to 150°C; which was maintained for 10 min. The carrier gas was helium at a flow rate of 2 ml/min (splitless mode). The detector and injector temperature were maintained at 250 and 225°C respectively. The quadrupole mass spectrometer was scanned over the range 28-400 atomic mass unit at 1 scan per second, with an ionizing voltage of 70 eV, an ionization current of 150 µA. Kovats retention indices were calculated using co-chromatographed standards hydrocarbons. The individual compounds were identified by MS and their identity was confirmed by comparing their retention indices relatives to C8-C32 n-alkanes and by comparing their mass spectra and retention times with those of authentic samples or with data already available in the National Institute of Standards and Technology (NIST) library and literature [11]. Antibacterial activity Microorganisms; the microorganisms used in this study consisted of two Gram(+) bacteria, i.e. Staphylococcus aureus (ATCC 29213) and Bacillus cereus (ATCC 29213); and two Gram(-) bacteria, i.e. Escherichia coli (ATCC 35218) and Pseudomonas aeruginosa (ATCC 27853). Diffusion method; the qualitative antimicrobial assay of the volatile fraction of W.saharae was carried out by the disc diffusion method [12]. It was performed using culture growth at 37°C for 18 h and adjusted to approximately 108 colony forming unit per milliliter (CFU/ml). The culture medium used for the bacteria was Mueller Hinton Agar (MHA). Five hundred

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microliters of the inoculums were spread over plates containing MHA and a Whatman paper disc (6 mm) impregnated with 5, 10, 15 µl of the volatile fraction was placed on the surface of the media. The plates were left 30min at room temperature to allow the diffusion of the oil. They were incubated 24 h at 37°C for the bacteria. After incubation period, the inhibition zone obtained around the disc was measured. Two controls were also included in the test, the first was involving the presence of microorganisms without test material and the second was standard antibiotic: ampicillin used to control the sensitivity of the tested bacteria. The experiments were run in triplicate, and the developing inhibition zones were compared with those of reference discs. Dilution method; the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of tested volatile fractions were determined using the Mueller Hinton broth (MHB) dilution method [12]. All tests were performed in MHB supplemented with Tween 80 (1%) [10]. Bacterial strains were cultured overnight in MHB at 37°C. Tubes of MHB containing various concentrations of volatile fractions were inoculated with 10 µl of 108 CFU/ml of standardized microorganism’s suspensions. Control tubes without tested samples were assayed simultaneously. All samples were tested in triplicate. The MIC was defined as the lowest concentration preventing visible growth [13, 14]. Antifungal activity Fungal strains; four agricultural pathogenic fungi were obtained from the culture collection at Faculty of Sciences and Techniques, Errachidia. The fungal strains used in the experiments are Alternaria sp, Pencillium expansum, Rhizopus stolonifer and Botrytis cinerea. Antifungal activity tests; the antifungal activity is evaluated as described by Chang et al [15]. The quantities of essential oil (100 and 200 µl) are added to 20 ml of sterile potato dextrose agar (PDA). The mixtures were cast on Petri dish. Afterwards, the discs of mycelium each mold 5 mm in diameter cut the device from a culture of 7 days are inoculated in the center of the boxes and then incubated at 25 ± 2°C for 3 days for Rhizopus and 7 days for others. Measuring diameters of hyphal growth relative to the control results in applying the following formula: Antifungal index (I) = [1 - (Da / Db)] x 100 -Da; diameter growth dish treated (mm) -Db; diameter growth control (mm).

Antioxidant activity The antioxidant activity was assessed by 1,1-diphenyl2-picrylhydrazyl (DPPH) assay and β-carotene bleaching method systems. Data collected for each assay was an average of three experiments.

DOI 10.5455/oams.121113.or.056

Oxidants and Antioxidants in Medical Science 2014; 3(1):73-78 Free radical-scavenging assay; the method is based on the reduction of alcoholic DPPH solutions in the presence of a hydrogen donating antioxidant. DPPH solutions show a strong absorption band at 517 nm with a deep violet color. The absorption vanishes and the resulting discoloration is stochiometric with respect to degree of reduction. The remaining DPPH, measured after a certain time, corresponds inversely to the radical scavenging activity of the antioxidant [16]. 50 µl of the extracted oil dilutions in ethanol was added to 1 ml of 100 µM solution of DPPH. After 30 min of incubation at room temperature, the absorbance was read against a blank at 517 nm (Jenway, UV/Vis 6000). Inhibition (I) of DPPH free radical in percent was calculated as follows: I (%) = (1 – Asample/Ablank) × 100

-Ablank; absorbance of the control reaction (containing all reagents except the test compound) -Asample; absorbance of the test compound. Extract concentration providing 50% inhibition IC50 was calculated from the graph plotting inhibition percentage against extract concentration. All tests were carried out in triplicate. The synthetic antioxidant butylated hydroxytoluene (BHT) was used as positive control. Beta-carotene bleaching assay; the β-carotene method was carried out according Shahidi et al [17]. Two milliliters of β-carotene solution (0.2 mg/ml in chloroform) were pipetted into a round-bottomed flask containing 20 µl linoleic acid and 200 µl Tween 20. The mixture was then evaporated at 40°C for 10 min to remove the solvent. Then, the addition of distilled water (100 ml) followed immediately. After agitating the mixture, 1.5 ml aliquot of the resulting emulsion was transferred into test tubes containing 150 µl of extract and the absorbance was measured at 470 nm against a blank consisting of an emulsion without β-carotene. The tubes were placed in a water bath at 50°C and the oxidation of the emulsion was monitored by measuring absorbance at 470 nm after 2 h using spectrophotometry. The same procedure was repeated with BHT as positive control. The antioxidant capacity (AA%) of the solutions tested was calculated via the following formula: β-carotene content after 2 h assay AA% = ---------------------------------------------- x100 Initial β-carotene content

Statistical analysis The data were analyzed using analysis of variance (ANOVA) and the significance of the differences between means was determined at P < 0.05 using Duncan's multiple range tests. Results were expressed as means ± standard deviation of three independent tests. All tests were carried out in an identical condition.


RESULTS AND DISCUSSION Chemical composition of the essential oil The essential oil of W.saharae was extracted by hydrodistillation appearing as blue-green color viscous liquid with a percentage yield of 1.1% (w/w). The volatile components identified by GC-MS, their relative area percentages and their retention times are summarized in Table 1. Table 1. Constituents of Warionia saharae essential oil Compounds KIa ??? % α-thujene 934 0.4 α-pinene 938 0.5 Camphene 953 0.8 Sabinene 973 2.4 β-pinene 980 0.1 β-myrcene 993 0.1 para-cymene 1025 3.7 Limonene 1030 0.2 1,8-cineole 1034 6.2 β-ocimene 1042 0.1 Linalool 1098 16.8 α-thujone 1105 0.3 β-thujone 1115 0.2 Camphor 1145 4.6 Borneol 1167 0.2 Terpinen-4-ol 1176 3.6 Terpineol 1189 0.5 trans carveol 1217 0.2 Gernaiol 1235 0.6 Pulegone 1237 0.8 Neral 1240 0.7 Linalyl acetate 1259 1.4 Bornyl acetate 1284 0.9 Carvacrol 1299 1.6 α-terpinyl acetate 1333 0.8 Eugenol 1353 0.6 β-elemene 1387 0.3 β-caryophyllene 1415 0.1 β-farnesene 1452 2.2 δ-cadinene 1526 1.2 Trans-nerolidol 1566 18.2 Caryophyllene oxide 1580 0.7 α-cadinol 1640 0.8 β-eudesmol 1650 24.6 α-eudesmol 1652 0.2 14-hydroxy-α-humulene 1780 0.2 Monoterpene hydrocarbons 8.3 Oxygenated monoterpenes 40 Sesquiterpenes hydrocarbons 3.8 Oxygenated sesquiterpenes 44.3 Total identified 96.4 a

Compounds are listed in order of their elution from an HP-5ms capillary column using the homologous series of n-alkanes.

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Sellam et al: Biological investigation of Warionia saharae In this study, 36 components representing 96.8% of the W.saharae leaves oil were identified. The chemical composition of the essential oil was dominated highly by the oxygenated sesquiterpenes (44.3%) followed by oxygenated monoterpenes (40.0%). The most abundant compounds were β-eudesmol (24.6%), trans-nerolidol (18.2%) and linalool (16.8%). These three compounds represent 59.6% of the total oils (Fig.1). These results were in accordance with those previously reported in literature [8, 9, 10]. Indeed, Znini et al identified only 3 compounds such as eudesmol, linalool and nerolidol [9]. Thirty compounds amounting 91% of the oil, were identified by Essaqui et al [8]; the major components were β-eudesmol, trans-nerolidol and linalool [10]. Among the other chemical components were linalool, 1,8-cineole, camphor, p-cymene, terpinen-4-ol and sabinene. The differences recorded for the chemical composition of the essential oil of W.saharae can be according to the genetic characteristics and climatic, seasonal, geographical and geological differences where the plant is collected. Among identified compound, nerolidol showed antileishmanial activity [18] and exhibits antineoplastic activity [19]. This compound is a sesquiterpene present in essential oils of several plants, approved by the US Food and Drug Administration (FDA) as a food flavoring agent. Beta-eudesmol has multiple pharmacological effects; the anti-inflammatory effect of β-eudesmol was shown recently [20].

Figure 1. Chemical molecular structure of three major constituents of W.saharae essential oil.

Antibacterial activity The antibacterial activity of W.saharae essential oil were evaluated by a paper disc diffusion method against bacterial strains including Gram positive and Gram negative bacteria, as to see in Table 2. W.saharae essential oil showed an important antibacterial activity against S.aureus, B.cereus and P.aeruginosa, while the growth of E.coli was less inhibited. The MICs confirmed this observation with values ranging between 0.039 and 0.156 mg/ml for all strains used; MBC values varied between 0.312 and 1.25 mg/ml. S.aureus and B.cereus were strongly inhibited by essential oil of W.saharae, since MBCs values were lower (0.312 and 0.625 mg/ml, respectively). The antibacterial effect of the oil could be explained by instead of through the disruption of bacteria membrane integrity. Indeed, previous findings revealed that tea tree oil damages the cell membrane structure of E.coli, S.aureus and Candida albicans [21]. It is also possible that the minor components might be involved in some type of synergism with the other active compounds [22]. Antifungal activity The results of antifungal activity assays showed that the essential oils of W.saharae had inhibitory effects on the growth of fungi (Table 3). Botrytis cinerea was most suppressed as its growth was mostly reduced, followed by Penicillium expansum, Rhizopus stolonifer and Alternaria sp at 50 ppm; the essential oils of W.saharae appeared to be effective against growth of these phytopathogens above 60%. The volatile oils consist of complex mixtures of numerous components. The major or trace compound(s) might give rise to the antifungal activity. Possible synergistic and antagonistic effects of compounds also play an important role in fungi inhibition. Previous papers on the antifungal activities of essential oils of some species of various genera have shown that they have varying degrees of growth inhibition effects against some agricultural pathogenic fungal species [23].

Table 2. Antibacterial activity of Warionia saharae essential oil Inhibition zone diametera EO (µg/disc) Microorganism Amp (µg/disc) 5 10 15 Gram(-) bacteria E.coli ATCC 25922 16.4 ± 0.6 18.7 ± 0.4 20.3 ± 1.2 27.7 ± 1.5 P.aeruginosa ATCC 27853 12.5 ± 0.6 13.1 ± 1.5 17.3 ± 0.4 21 ± 1.2 Gram(+) bacteria S.aureus ATCC 25923 B.cerus ATCC 29213

20 ± 1.5 18.9 ± 0.4

24.7 ± 0.4 20.5 ± 0

28.4 ± 0 22.7 ± 1.5

32.7 ± 0.6 24 ± 1

EO

Amp

MIC

MBC

MIC

MBC

0.156 0.078

1.25 > 0.625

1.95 3.9

> 3.9 7.81

0.039 0.078

0.312 0.625

6.25 3.9

15.62 7.81

a Diameter of the zone of inhibition (mm) including disk diameter of 6 mm; Amp, ampicillin; EO, essential oil; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; values given as mg/ml for the essential oils and as µg/ml for antibiotics.

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DOI 10.5455/oams.121113.or.056

Oxidants and Antioxidants in Medical Science 2014; 3(1):73-78 Table 3. Antifungal activity of Warionia saharae essential oil Fungal species Alternaria sp Penicillium expansum Rhizopus stolonifer Botrytis cinerea

Oil growth (mm) 21 ± 1 23.5 ± 2.3 25 ± 0.2 18.6 ± 1.5

Index antifungal (%) d

44.44 ± 0.7 63.22 ± 0.7b 60.75 ± 1c 71.02 ± 0.4a

Control growth (mm) 37.8 ± 3.7 63.9 ± 7.3 63.7 ± 7.2 64.2 ± 2.5

Values are means ± standard deviation of three separate experiments; means with different letters are significantly different at P < 0.05.

The higher observed activity of essential oil could be attributed to the presence of sesquiterpenes, monoterpene hydrocarbons and oxygenated monoterpenes in the leaves essential oil. The essential oils containing terpenes are reported to possess antimicrobial activity [24], which is in part consistent with our present study. Antioxidant activity The principle of antioxidant activity is based on the availability of electrons to neutralize free radicals. In this study, the antioxidant activity of W.saharae oil was evaluated by two complementary tests: scavenging of DPPH+ free radicals and the β-carotene bleaching test. The results are shown in Table 4. DPPH is a free radical compound which has been widely used to test the free-radical scavenging ability of various samples. The model of scavenging the stable DPPH radical is a widely used method to evaluate the free radical scavenging ability of various samples [16]. The antioxidant effect of essential oil on DPPH radical scavenging may be due to their hydrogen donating ability and it reduce the stable violet DPPH radical to the yellow DPPH-H. Substances which are able to perform this reaction can be considered as antioxidants and therefore radical scavengers [25]. DPPH scavenging activity is usually presented by IC50 value, defined as the concentration of the antioxidant needed to scavenge 50% of DPPH present in the test solution. Comparison of the DPPH scavenging activity of the W.saharae essential oil (26.23 ± 1.25 μg/ml) and those expressed by BHT (7.73 ± 0.11) showed that the essential oil exhibited weaker antioxidant effects than BHT; the antioxidant effect of the oil was about 3 times lower than that of the synthetic antioxidant BHT. The DPPH scavenging ability of this oil can be attributed to the presence of phenolic constituents and to the free radical scavenging activity of some volatile oils [26]. The β-carotene bleaching method is based on the loss of the yellow color of β-carotene due to its reaction with radicals that are formed by linoleic acid oxidation in an emulsion. In this assay, antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxide arising from linoleic acid oxidation. Beta-carotene undergoes rapid discoloration in the absence of an antioxidant; however, the presence of antioxidant will be minimizing its oxidation. This test


Table 4. Antioxidant capacity of Warionia saharae W.saharae Butylated essential oil hydroxytoluene DPPH (IC50 µg/ml 26.23 ± 1.25 6.2 ± 0.21 DPPH solution) Inhibition in linoleic 60.4 ± 1.02 86.5 ± 0.5 acid system (%) Values are means ± standard deviation of three separate experiments.

measures the potential of the plant to inhibit conjugated diene hydroperoxide formation from linoleic acid oxidation. As can be seen from Table 4, the potential of W.saharae to inhibit lipid peroxidation was evaluated using the β-carotene/linoleic acid bleaching test by measuring the antioxidant capacity (AA%), with a value of 60.4 ± 1.02% and 86.5 ± 0.5%, obtained for the oil and the positive control BHT, respectively at the same concentration of 100 µg/ml. This activity was moderately lower than that of BHT and was attributed to the presence of appreciable amount of antioxidant compounds such as 1,8-cineole, α-pinene and carvacrol and to the presence of phenolic compounds [25]. These research findings lead us to conclude that W.saharae essential oil could be considered as potential alternatives for synthetic bactericides and natural antioxidants for use in the food industry along with their possible applications in the pharmaceutical industry for the prevention or treatment of pathogenesis caused by microorganisms and free radicals.


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15. Chang C, Yang M, Wen H, Chern J. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal 2002; 10:178-82. 16. Mensor LL, Menezes FS, Leitao GG, Reis AS, Dos Santos TC, Coube CS, Leitao SG. Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytother Res 2001; 15:127-30.

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Benabid A, Fennane M. Connaissance sur la vegetation du Maroc: phytogeographie, phytosociologie et series de vegetations. Lazaroa 1994; 14:21-97.

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Watillon C, Gaspar T, Hofinger M, Ramaut JL. La micropropagation de Warionia saharae Benth & Coss. Al Biruniya 1987; 4:35-8.

17. Shahidi F, Chavan UD, Naczk M, Amarowicz R. Nutrient distribution and phenolic antioxidants in air-classified fractions of beach pea (Lathyrus maritimus L.). J Agric Food Chem 2001; 49:926-33.

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Bellakhdar J. La Pharmacopee Marocaine Traditionnelle, Medecine Arabe Ancienne et Savoirs Populaires, Ibis Press, pp 208-209, 1997.

18. Arruda DC, D’Aledandri FL, Katzin AM, Uliana SRB. Antileishmanial activity of the terpene nerolidol. Antimicrob Agents Chemother 2005; 49:1679-87.

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Hilmi F, Sticher O, Heilmann J. New cytotoxic 6,7-cis and 6,7trans configured guaianolides from Warionia saharae. J Nat Prod 2002; 65:523-6.

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Hilmi F, Sticher O, Heilmann J. New cytotoxic sesquiterpene lactones from Warionia saharae. Planta Medica 2003; 69:462-4.

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Essaqui A, Elamrani A, Benaissa M, Rodrigues AI, Yoongho L. Chemical composition of the leaves extract of Warionia saharae of Morocco. JEOBP 2004; 7:250-4.

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Znini M, Cristofari G, Majidi L, El Harrek A, Paolini J, Costa J. In vitro antifungal activity and chemical composition of Warionia saharae essential oil against 3 apple phytopathogenic fungi. Food Sci Biotechnol 2013; 22:113-9.

10. Amezouar F, Badri W, Hsaine M, Bourhim N, Fougrach H. Chemical composition, antioxidant and antibacterial activities of leaves essential oil and ethanolic extraction of Moroccan Warionia saharae Benth & Coss. J Appl Pharm Sci 2012; 2:2127. 11. Adams RP. Identification of Essential Oil components by Gas Chromatography/Quadruple Mass Spectroscopy. Allured Publishing, Illinois, 2001. 12. National Committee for Clinical Laboratory Standards, Performance Standards for Antimicrobial Susceptibility Testing, 6th edition, Approved Standards. M2-A6, Wayne, PA, pp 220221, 1999. 13. May J, Chan CH, King A, Williams L. French GL. Time-kill studies of tea tree oils on clinical solates. J Antimicrobial Chemoter 2000; 45:639-43.

19. Wattenberg LW. Inhibition of azoxymethane-induced neoplasia of the large bowel by 3-hydroxy-3,7,11-trimethyl-1,6,10dodecatriene (nerolidol). Carcinogenesis 1991; 12:151-2. 20. Seo MJ, Kim SJ, Kang TH, Rim HK, Jeong HJ, Um JY, Hong SH, Kim HM. The regulatory mechanism of β-eudesmol is, through the suppression of caspase-1 activation in mast cellmediated inflammatory response. Immunopharmacol Immunotoxicol 2011; 33:178-85. 21. Cox SD, Mann CM, Markham JL, Bell HC, Gustafson JE, Warmington JR, Wyllie SG. The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil). J Appl Microbiol 2000; 88:170-5. 22. Marino M, Bersani C, Comi G. Impedance measurements to study the antimicrobial activity of essential oils from Lamiaceace and Compositae. Int J Food Microbiol 2001; 67:187-95. 23. Alvarez-Castellanos PP, Bishop CD, Pascual-Villalobos MJ. Antifungal activity of the essential oil of flowerheads of garland chrysanthemum (Chrysanthemum coronarium) against agricultural pathogens. Phytochemistry 2001; 57:99-102. 24. Dorman HJ, Deans SG. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J Appl Microbiol 2000; 88:308-16. 25. Ebrahimzadeh MA, Nabavi SF, Nabavi SM. Antioxidant Activities of Methanol Extract of Sambucus ebulus L. Flower. Pak J Biol Sci 2009; 12:447-50. 26. Edris AE. Pharmaceutical and therapeutic potentials of essential oils and their individual volatile constituents: a review. Phytother Res 2007; 21:308-23.

14. Burt S. Essential oils: their antibacterial properties and potential applications in foods. Int J Food Microbiol 2004; 94:223-53. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided that the work is properly cited.

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DOI 10.5455/oams.121113.or.056


ISSN: 2146-8389

BRIEF REPORT

Dietary protection by garlic extract against lead induced oxidative stress and genetic birth defects Oladimeji S. Tugbobo1, Omotade I. Oloyede2, Olusola B. Adewale3 1

Biochemistry Unit, Department of Science Technology, Federal Polytechnic; 2 Department of Biochemistry, Ekiti State University; 3 Biochemistry Unit, Department of Chemical Sciences, Afe Babalola University; Ado Ekiti, Nigeria Received August 30, 2013 Accepted December 6, 2013 Published Online January 25, 2014 DOI 10.5455/oams.061213.br.008 Corresponding Author Omotade Ibidun Oloyede Department of Biochemistry, Ekiti State University, Ado Ekiti, Nigeria. [email protected] Key Words Chromosomal aberration; Garlic; Lead; Oxidative stress

Abstract The antioxidant potentials of Allium sativum extract was studied in bone marrow cells of albino rats using micronucleus assay with the use of 100 mg/ml crude garlic extract as dietary supplement via oral gavage. The rats were divided into three groups: A, distilled water; B, lead acetate; C, garlic extract + lead acetate. After the short-term exposure, rats were sacrificed by cervical dislocation and chromosomal preparations were made from bone marrow according to colchicines-hypotonic-fixation-air-drying-Giemsa schedule. The cytogenic end points observed were chromosomal aberrations and tissue damage. The chromosomal aberration induced by lead was reduced significantly in animals fed with the extract in group C while lead acetate administered to animals in group B was highly mutagenic. Besides, the antioxidant properties of garlic was further demonstrated in the in vitro experiment where it provided significant protection to thiobarbituric reactive substances inhibition, and reduced and oxidized glutathione contents in liver tissues. This suggests their ability to act as free-radical scavengers and in protecting the cell against oxidative stress for normal cellular functions. The results further harp on the involvement of reactive oxygen species in lead toxicity and also revealed the beneficial role of garlic therapeutic efficacy which indicates the antimutagenic and antioxidant potentials of garlic against oxidative stress and mutation. © 2014 GESDAV

INTRODUCTION Chronic exposure to lead leads to its accumulation in vital organs with maximum concentrations reported in kidneys [1]. Developing brain is particularly vulnerable to its toxic effects ranging from behavioral abnormalities, learning impairment, decreased hearing and impaired cognitive functions in human and experimental animals [2]. Oxidative stress has been reported as one of the important mechanisms of lead toxicity [3]. This suggests that changes in glutathione levels as well as antioxidant enzyme activities implicate oxidative stress in lead toxicity. Some earlier studies also indicate that the disruption of reducing status of tissue leads to formation of reactive oxygen species (ROS) which may damage essential biomolecules such as protein, lipids and DNA [4]. This further emphasizes that at high levels; these ROS could be toxic to cells and may possibly contribute to cellular dysfunction and poisoning. On the other hand, the roles of garlic as dietary supplements cannot be overemphasized as regards restoration and maintenance of the body physiological well-being. Its roles in the diets have been reported to reduce cholesterol levels. Furthermore, the antimicrobial, antithrombotic, and antibiotic properties of garlic have been proven over different diseases such as stroke, atherosclerosis, infertility and prostate cancer [5].


The present investigation was designed to study the degree of protection offered by garlic extract against lead-induced oxidative stress and mutagenic effects as it had been reported that lead is a mutagen capable of inducing chromosomal aberrations in man and animals [6]. Besides, it is also aimed to justify the hypothesis that most, if not all, cancer cells are characterized by chromosomal changes or alterations that are frequently specific to a particular tumor. MATERIALS AND METHODS Plant extract Crude aqueous extract of garlic (Allium sativum L) of single clove variety was prepared from bulbs purchased from the market in Ado Ekiti, Nigeria. The clove was sliced, ground into paste and then dissolved in distilled water; 10 mg/ml corresponding to 100 mg/kg of animals was used for the in vivo experiment. The garlic concentration was however, varied for the in vitro experiment relative to the liver tissue used. Experimental animals The in vivo experiment was performed using twelve male Wistar rats weighing 120 ± 10 g housed in stainless cages with temperature maintained at 25 ± 2ºC and 12 h alternating day/night cycle, and the rats were

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Tugbobo et al: Dietary protection with garlic extract fed standard pellets and water ad libitum. The handling and use of the animals were in strict compliance with NIH guide for the care and use of laboratory animals. Experimental protocols The animals were divided into three groups with four animals in each group: rats in group A serve as control and were treated with distilled water only; those in group B received 2.5 mg/kg lead acetate; group C rats were fed simultaneously with 2.5 mg/kg lead acetate and 100 mg/kg garlic extract (1:1). The concentration of the lead salt was made equivalent to 1/10 of the LD50 [7]. The dose of garlic extract was equivalent to the exact concentration used for beneficial effects against specific disease conditions [8]. Each dose was administered via oral gavage to the animals on daily basis consecutively for four weeks. Chromosomal aberrations Chromosomes were studied from bone marrow cells following the usual colchicines-hypotonic-fixation-airdrying technique [9]. Animals were sacrificed by cervical dislocation 24 h after the last treatment; 90 min prior to sacrifice, each animal was injected with 1% colchicine. Femurs were removed and the bone marrow was flushed out into centrifuge tubes with freshly prepared and pre warmed (36ºC) solution of 0.56% KCl into 8 ml centrifuge tubes. The fat lumps were removed with fine tipped pipette and was allowed to stand for 1 h to allow cells swell in the hypotonic solution. The cells were later pelleted by centrifugation (1000 rpm) for 5 min and supernatant discarded by the use of a Pasteur pipette [10]. Freshly prepared Clerk’s fixative (glacial acetic acid:methanol, 1:3) was added to the cells and was suspended by vigorously agitating the centrifuge tubes [11]. The slides were coded and scored blind and were stained with Giemsa for 15 min, rinsed, dried at room temperature. Each slide was placed on the microscope and scanned carefully under objective (10x) for metaphase spreads while better views were observed under 40x for identifying clearly mitotic spreads of the chromosomes. The identified spreads were viewed using 100x oil immersion objective. The end points scored were chromosomal aberrations and damage cells. Thiobarbituric acid reactive substances (TBARS) Production of TBARS was determined by the method of Ohkawa et al [12]. The rats were anesthetized with ether, sacrificed by decapitation, and the liver tisssue were quickly removed and placed on ice-blocks. 1 g of the tissue was homogenized in cold Tris-HCl buffer at pH 7.4 (1:10 w/v). The homogenate was centrifuged for 10 min at 1400g and the supernatant was used for the assay. The supernatant was incubated with or without 50 mµ of freshly prepared lead acetate (2.5 mg) and at different concentrations (10-160 mg/ml) of the plant extract, together with an appropriate volume of

80

deionized water amounted to total volume of 300 mµ at 37ºC for 1 h. The color reaction was carried out by adding 200, 250 and 500 mµ each of 8.1% sodium dodecyl sulphate (SDS), acetic acid (pH 3.4), and 0.6% TBA, respectively, and were further incubated at 97ºC for 1 h. The absorbance was read after cooling at a wavelength of 532 nm in a spectrophotometer. The experimental design involves the basal containing no extract and pro-oxidant (normal), control containing pro-oxidant without the extract, while the other testtubes contain both the extract and pro-oxidant. Reduced glutathione (GSH) 0.2 ml of sample homogenate (liver tissue) was added to 1.8 ml of distilled water and 3 ml of the precipitating agent sulphosalicylic acid was mixed with the 2.5 ml garlic extract. This was centrifuged at 3000g for 4 min and 0.5 ml of the supernatant was added to 4.5 ml of Ellman’s reagent. A blank was prepared with 0.5 ml of the diluted precipitating agent and 4 ml of phosphate buffer and 0.5 ml of Ellman’s reagent. The absorbance of the reaction mixture was taken within 30 min of color development at 412 nm against a reagent blank. The concentration of GSH was extrapolated from the GSH standard curve [13]. Oxidized glutathione (GSSG) The reaction mixture containing 500 mµ phosphate buffer, 100 mµ sodium azide, 200 mµ GSH and 100 mµ H2O2 were added to 500 mµ of the sample, after which 600 mµ of distilled water was added and mixed thoroughly. The mixture was incubated at 37ºC for 3 min after which 0.5 ml of TCA was added and centrifuged at 3000 rpm for 5 min. Two milliliters of K2HPO4 and 1 ml of dinitrothiocyanobenzene (DNTB) was added each to 1 ml of the supernatants and the absorbance was read at 412 nm against blank. Statistical analysis The data from the groups were pooled and analyzed statistically using ANOVA [14] and Duncan’s multiple range tests in order to compare significance of differences where P values less than 0.05 were considered significant. RESULTS Lead acetate-induced chromosomal aberrations such as chromosomes and chromatic breaks, gap and chromosomal rearrangement were analyzed. Fig.1 shows the frequencies of total chromosomal aberrations and the mean frequencies per cell as well as percentage of damaged cells in rats exposed to lead acetate in vivo. The frequency of damaged cells significantly reduced (2%) in group C animals fed simultaneously with both the toxicant and garlic extract. Group B animals showed highly mutagenicity with a total chromosomal aberration rate of 25% while no significant difference was observed in animals of group A.

DOI 10.5455/oams.061213.br.008

Oxidants and Antioxidants in Medical Science 2014; 3(1):79-82 Table 1. The inhibitory effect of garlic extract on lead acetate induced lipid peroxidation in rat liver Extract (mg/ml)

Absorbance

Inhibition (%)

Basal (normal)

0.082 ± 0.00041

-

Control

0.498 ± 0.00041

-

10

0.324 ± 0.00048

34.9

20

0.31 ± 0.00054

37.8

40

0.208 ± 0.00149

58.2

80

0.201 ± 0.00325

59.6

160

0.128 ± 0.012

74.3

Results are Mean ± SD of two determinations

Figure 1. Chromosomal aberrations following treatment with lead acetate and garlic extract [G, chromosomal gap; B, chromosome break; B”, chromatid break; RR, chromosomal rearrangement; P < 0.05 for the total chromosomal aberrations].

Table 1 shows the interaction (inhibition) of garlic extract with lead acetate induced lipid peroxidation in the liver. There was a statistically significant (P < 0.05) increase in the formation of TBARS in lead acetate in control group (B) that contained no extract when compared to normal group (A). Garlic extract significantly inhibited lipid peroxidation in a dose dependent manner from 10 to 160 mg/ml; it was observed that with increased concentration of the extract, there was marked progressive increase in the inhibitory effect ranging from 34% to 74%. Table 2 and 3 shows the effect of garlic extract on the levels of GSH and GSSG, respectively. It was observed that the aqueous extract caused marked increase in their levels. DISCUSSION The results in Fig.1 show that allicin, which is the principal constituent of the extract, formed from its precursor allin via enzymatic degradation when crushed and macerated, destroyed the cellular structure releasing the antimutagens. The toxicity of divalent lead ions to the animals is caused by its binding to thiol or suflhydryl group; thus, inhibiting some enzymatic reactions in the body tissue [15]. Hence, the significant reduction of the mutagenic effects of lead by crude garlic extract could be attributed to the activity of allicin. The anti-mutagenic effect observed in this experiment, indicates that garlic is a viable protective dietary supplement against DNA damage and associated cancer diseases which could later manifest as birth defects, and as it has since been known that every mutant cell is highly susceptible to developing cancer. Besides, generation of highly ROS like hydroxyl radical,


Table 2. Effect of garlic extract on reduced glutathione (GSH) in the liver tissue Extract GSH Absorbance concentration concentration 1 mg/ml 0.824 175.32 µg/ml 2 mg/ml

0.824

175.32 µg/ml

3 mg/ml

0.823

175.11 µg/ml

Table 3. Effect of garlic extract on oxidized glutathione (GSSG) in liver tissue Extract GSSG Absorbance concentration concentration 1 mg/ml 0.58 123.4 µg/ml 2 mg/ml

0.584

124.26 µg/ml

3 mg/ml

0.583

124.04 µg/ml

hydrogen peroxide, superoxide anions and lipid peroxide aftermath of lead exposure may result in systematic mobilization and depletion of cell intrinsic antioxidant defenses. The results of the in vitro experiment also show a significant increase in the levels of TBARS on lead administration serving as a pro-oxidant. This increase in TBARS levels, especially in the control group, may be a key factor in oxidative deterioration of membrane polyunsaturated fatty acid as well as assault on membrane integrity of the tissue [16]. However, there was a marked recovery in the altered TBARS and GSH levels due to the effects of the garlic extract on the liver tissue. The results obtained show that the effect of the extract is in synergy with the increase in the antioxidant defense system characterized by GSH and GSSG levels, respectively, and as well supporting the hypothesis that administrations of thiol-containing dietary supplements counteract both in vivo and in vitro oxidative stress posed by lead exposures [17]. Besides, heavy metals such as lead have been reported to cause oxidative stress due to production of ROS and to resist

81

Tugbobo et al: Dietary protection with garlic extract the oxidative damage, the antioxidant enzymes and certain metabolites present in animal tissues playing an important role leading to adaptation and ultimate survival of the animals during period of stress. This action of the antioxidant enzymes has been complemented by garlic extract fed to the experimental animals in this study where it caused marked increase in the enzymes activities.

6.

Poddar S, Mukherjee P, Talukder G, Sharma A. Dietary protection by iron against clastogenic effects of short-term exposure to arsenic in mice in vivo. Food Chem Toxicol 2000; 38:735-7.

7.

Choudhury AR, Das T, Sharma A, Talukder G. Inhibition of clastogenic effects of arsenic through continued oral administration of garlic extract in mice in vivo. Mutat Res 1997; 392:237-42.

8.

Mansell P, Reckless JP. Garlic. Br Med J 1991; 303:379-80.

In conclusion, this study re-emphasizes the needful consumption of garlic as dietary supplement in protecting the body against oxidative stress which could mutate and render the cancerous cells [18].

9.

Sharma AK, Sharma A. Chromosome Techniques – A Manual. 6th edition, Harwood Academic Publishers, Chur, Switzerland, p 368, 1994.


10. Alasoadura OH. Cytogenic study in a species of anurans (amphibians) Bufo regularis. Dissertation, Department of Zoology, Obafemi Awolowo University, Ile-Ife, Nigeria, 1996. 11. Al-Shehri AH, Al-Saleh AA. Karyotype of amphibian in Saudi Arabia. 1.The karyotype of Rana ridibunda. J Biol Sci 2005; 5:335-8. 12. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95:351-8. 13. Beutler E, Gelbart T. Improved assay of the enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase and glutathione synthetase. Clin Chim Acta 1986; 158:115-23.

REFERENCES 1.

Humphreys DJ. Effects of exposure to excessive quantities of lead on animals. Br Vet J 1991; 147:18-30.

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Brautbar MD. Industrial solvents and kidney disease. Int J Occup Environ Health 2004; 10:79-83.

15. Sharma A, Talukder G. Effects of metals on chromosomes of higher organisms. Environ Mutagen 1987; 9:191-226.

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Ercal N, Treeratphan P, Hammond TC, Matthews RH, Grannemann NH, Spitz DR. In vivo indices of oxidative stress in lead-exposed C57BL/6 mice are reduced by treatment with meso-2,3-dimercaptosuccinic acid or N-acetylcysteine. Free Radic Biol Med 1996; 21:157-61.

16. Monteiro HP, Bechara EJ, Abdalla DS. Free radicals involvement in neurological porphyrias and lead poisoning. Mol Cell Biochem 1991; 103:73-83.

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Halliwell B. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 1994; 344:721-4.

17. Flora SJ, Pande M, Mehta A. Beneficial effect of combined administration of some naturally occurring antioxidant and thiol chelators in lead treatment. Chem Biol Interact 2003; 145:26780.

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Hilderbrand DC, Der R, Griffin WT, Fahim MS. Effect of lead acetate on reproduction. Am J Obstet Gynecol 1973; 115:105865.

18. Tugbobo OS, Oloyede OI, Daramola AO. Protection by garlic extract against lead induced tissue atrophy in albino rats in vivo. Arch Appl Sci Res 2012; 4:65-71.


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DOI 10.5455/oams.061213.br.008

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