The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting

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Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor

Growth and Muscle Wasting

Mohamad Assi

To cite this version:

Mohamad Assi. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting.

Education. Université Rennes 2, 2016. English. �NNT : 2016REN20055�. �tel-01587650�

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THESE / UNIVERSITE RENNES 2

sous le sceau de l’Université Bretagne Loire pour obtenir le titre de DOCTEUR DE L’UNIVERSITE DE RENNES 2 Mention : STAPS Ecole doctorale Vie-Agro-Santé (VAS)

présentée par

Mohamad Assi

Préparée au laboratoire Mouvement, Sport, Santé M2S (EA1274, Rennes)

The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle

Crosstalk during Cancer

Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting

Thèse soutenue le 08 Décembre 2016 Devant le jury composé de :

Pr. Marc FRANCAUX

Directeur de recherches et professeur,

Université Catholique de Louvain, Belgique /rapporteur Pr. Stéphane SERVAIS

Professeur, Université François Rabelais-Tours, France /rapporteur

Pr. Odile SERGENT

Professeur, Université Rennes 1, France /examinatrice Pr. Françoise REDINI

Directrice de recherche et professeur, Université Nantes, France/examinatrice

Dr. Adrien ROSSARY

Maitre de conférences, Université Clermont-Ferrand, France /examinateur

Dr. Amélie REBILLARD

Maitre de conférences/HDR, Université Rennes 2, France/Directeur de thèse

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All Our Dreams Can Come True, If We Have The Courage To Pursue Them.

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I will start this section by gratefully thanking the jury members for accepting to examine my Ph.D. work. Namely, Professor Marc Francaux form the University Catholique de Louvain at Belgium and Professor Stéphane Servais from the University Francois Rabelais-Tours, for accepting to report this manuscript. Also, I would like to thank Professor Francoise Redini from the University of Nantes, Professor Odile Sergent from the University of Rennes 1 and Doctor Adrien Rossary from the University of Clermont-Ferrand, for accepting to be among the jury members.

Many thanks for the previous director of the laboratory “Movement, Sport and health Sciences” (M2S), Paul Delamarche and for the actual director Benoit Bideau, for receiving me as a member of the M2S family.

Today, if pursuing a career of a researcher is not a dream anymore, it is in a major part due to Amélie Rebillard who believed in my competences and capacities to conduct this work. I would like to sincerely thank Amélie for her in-science passion, patience, support, motivation and positive energy. From you I learned that we can find a solution for every complicated problem; to never give-up or despair when we fail to obtain positive results (recurrent phenomenon among Ph.D.

students); to never neglect literature and reading at the expense on performing experiments; and to explore new fields and ideas, where others don’t dare, which is the real meaning of research and being a researcher. I can say that in addition to the phenotypic changes that I have undergone during these three years, where my beard started to grow spontaneously, I have also gained in maturity at both scientific and personal plan.

I also appreciated my discussions with Frédéric Derbré, who transmitted to me the passion to train mice on treadmill and to explore muscle physiology. How can I forget the animated talks with Nicolas Pierre on RNA extraction and PCR?! But also the great moments of jokes and laughs that we have shared with the future doctor Kévin Nay. It was a pleasure to share with you my laboratory experiments, meals and after-noon jogging, even if I was unlikely to follow the rhythm of Frédéric and Nicolas when they were decided to do fractioned exercise!

Although I did not understand exactly what the “Biomechanics guys” Camille, Pierre (also known as, Plantouille), Sébastien, Rémy and Sean, were trying to do during these three years, I often saw them seated in front of their computers, while I was shuttling between my office, the biochemistry Lab, the histopathology platform and the animal care facility. However, I enjoyed

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Acknowledgments

IV | P a g e our coffee breaks to discuss golf with Camille, politics with Pierre and English with Sean! I would say that the concept of “foot-chaise” (playing football while seating on the chair in your office) was innovating and funny, apart the pain that I felt the next day of the match. A special thank for the road partner Jordan with whom I passed more than two weeks of daily travel Rennes-Nantes, to perform radiotherapy on mice. I think that I am a fan of your coaching spirit, especially when you try to gently communicate with your mice and motivate them to pursue the exercise till the end.

I would like to thank Dany and Luz for their help and technical advices in animal handling, gavage, injections and sacrifice. I appreciated chemistry discussions with Brice, which were useful in performing my cell culture experiments. I am also thankful for the Master’s students Florian, Moussa and Suzanne for their technical help but also for the energy and the positive ambiance that they deployed in the laboratory, it was a pure pleasure for me to work with such motivated students. I also hope a good luck for the new Ph.D. students Charles and Suzanne in their projects and certainly a lot of scientific publications.

My best friends, Ali Farhat, Mohamad Chamas, Stanlislas Zafihita and Yvan Dietrich, I met you at different moments in my life; Ali and Mohamad we shared the same bench for several years at the school and the university in Lebanon; Since my arrival to France, I first met Stanlislas during my Master’s studies at the Réunion Island and, then, Yvan at the Veterinary School of Nantes. Because of you I knew that true friendship exits and I simply want to thank each one of you.

I address my gratitude to my mother Abla and father Nabil for their unconditional love and support. I have the chance to be born in a family that encourages their children to learn science and provides them with all the means to realize their own ambitions. This work is the culmination of several years of study, research, perseverance and patience; thus, I dedicate it for them as a sign of recognition. Finally, I want to thank my brothers Ali, Hassan and Hussein for being a part of my life and making it more joyful and meaningful.

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This work is dedicated to:

All those who fought cancer: either lost their battle or survived…

My parents Abla and Nabil

My brothers Ali, Hassan and Hussein

My small country Lebanon

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A-Introduction and Literature Overview

-Introduction

……….03

Chapter I. Reactive Oxygen Species

1. Reactive oxygen and nitrogen species……….. 09

2. Antioxidant defense……….. 11

2.1. Enzymatic antioxidant system……….. 11

2.2. Non-enzymatic: low molecular weight antioxidants and micronutrients……… 15

3. Oxidative stress and related macromolecules damage……….. 19

3.1. Lipid peroxidation………... 21

3.2. Protein oxidation and peroxidation……….. 22

3.3. Oxidative DNA damage……….... 22

4. Biological role of ROS………... 23

Chapter II. The Role of ROS in Cancer

1. Oxidative stress and cancer: an introduction………. 29

2. Sources of ROS in cancer………. 30

2.1. ROS-producing enzymes……….. 31

2.2. Mitochondrial ROS generation……….... 34

3. Antioxidant defense in cancer……….. 36

4. The double face of ROS in cancer………... 38

4.1. Mechanisms promoting tumor growth and progression………. 38

4.2. Mechanisms suppressing cancer growth and progression……….. 42

Chapter III. The Role of ROS in Cancer-Related Muscle Wasting

1. Defining cancer cachexia………... 51

2. Muscle wasting: key feature of cachexia……….. 52

3. Tumor-muscle crosstalk: a principle axis for ROS production and muscle wasting….. 53

4. Sources of ROS in cancer-related muscle wasting……….. 55

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Table of Content

VIII | P a g e

4.1. ROS-producing enzymes……….... 55

4.2. Mitochondrial dysfunction……….... 58

5. Defective antioxidant response………. 59

6. ROS-dependent mechanisms in cancer-related muscle wasting………... 61

6.1. Anabolic pathways……….... 61

6.2. Catabolic pathways……….. 63

7. Tumor-muscle crosstalk: what about adjuvant therapies?... 67

Chapter IV. Physical Activity in Cancer and Related Muscle Wasting

1. Cancer progression, muscle wasting and related physical inactivity……… 73

2. Molecular mechanisms modulated by physical activity in healthy conditions………... 74

2.1. Antioxidant response, mitochondrial biogenesis and angiogenesis……… 74

2.2. Insulin sensitivity, hormonal changes and muscle-related secretory phenotype…………. 76

3. Exercise-oncology: the impact of physical activity on cancer progression………. 79

4. Paucity of data: the impact of physical activity on intramuscular cancers, taking liposarcoma as an example……… 85

5. Limited application of physical activity in cancer cachectic patients: need for an alternative solution………... 89

Chapter V. Antioxidants in Cancer and Related Muscle Wasting

1. The use of antioxidants in cancer and related cachexia……… 93

2. Antioxidant vitamins……….. 95

3. Polyphenols……….. 98

4. Oligoelements: zinc and selenium……… 101

5. Antioxidant mixture and pharmacological agents………... 104

B- Aims of the Thesis………...

111

C- Experimental Studies and Personal Contribution

- Preview on the First Experimental Study………... 115 - First Experimental Study: Maintaining a regular physical activity promotes tumor growth

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in an orthotopic model of mice bearing intramuscular liposarcoma………...120

- Preview on the Second Experimental Study………...149

- Second Experimental Study: Antioxidant supplementation accelerates cachexia development by promoting tumor growth in C26 tumor-bearing mice………154

- Review Article………... 182

D- General Discussion and Future Directions……….

223

E- Bibliography……….………

243

-Annexes: Curriculum vitae………...

277

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Table of Content

X | P a g e

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4E-BP: 4-E-binding protein 4-HNE : 4-hydroxynonenal

8-oxoG : 8-oxo-7,8-dihydroguanine ALS: Amyotrophic lateral sclerosis AO: Antioxidants

ARE: Antioxidant responsive element ATP: Adenosine triphosphate

C26: Murine colon cancer cell line C2C12: Murine skeletal muscle cells CAF: Cancer-associated fibroblasts Cap: Caplain

CAT: Catalase CK: Creatine kinase

COPD: Chronic obstructive pulmonary diseases

ECM: Extracellular matrix

EGCG: Epigallocatechin-3-gallate EGF: Epidermal growth factor

eIF4E: Eukaryotic translational initiation factor 4E

FOXO: Forkhead box

GLUT-4: Glucose transporter-4 GPx: Glutathione peroxidase

GSH: Glutathione

GSSG: Oxidized glutathione H2O2: Hydrogen peroxide HIF: Hypoxia inducible factor HSP: Heat shock protein IGF: Insulin-like growth factor IGFBP: IGF binding protein IgG: Immunoglobulin

IL: Interleukin

INCa: French national cancer institute iNOS : Inducible NOS

LC3: Light chain-3

LMWA: Low molecular weight antioxidants LS: Liposarcoma

MAFbx: Muscle atrophy F-box

MAPK: Mitogen-activated protein kinase MCK: Myosine creatinine kinase

MDA: Malondialdehyde mDNA: Mitochondrial DNA MET: Metabolic equivalent task mGSH: Mitochondrial glutathione MMP: Metalloproteinase

MRC: Mitochondrial respiratory complex

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Abbreviation List

XII | P a g e MSC: Mesenchyme stem cells

Mstn: Myostatin

MuRF-1: Muscle ringer finger protein-1 NAC: N-acetylcysteine

NF-κB: Nuclear factor-κB NK: Natural killer

NO^.: Nitric oxide

NOS: Nitric oxide synthase NOX: Nicotinamide phosphate dinucleotide oxidase

NSCLC: Non-small-cell lung carcinoma ONOO^.: Peroxynitrite

OS: Oxidative stress PA: Physical activity

PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha

PI3K: Phosphoinositide-3 kinase

PIF: Proteolysis-inducing growth factor PKR: Double-stranded RNA dependent protein kinase

PPP: Pentose phosphate pathway PRDX: Peroxiredoxin

PSA: Prostate-specific antigen

PTEN: TENsin homolog QOL: Quality of life

R^.: Carbon-centered radicals RNS: Reactive nitrogen species ROO^.: Peroxyl radical

ROS: Reactive oxygen species RS^.: Thiyl radicals

SASP: Senescence-associated secretory phenotype SBP-1: Selenium-binding protein-1

SCLC: Small-cell lung carcinoma SecH: Seleno-cysteine

SeOH: Selenic acid

Se-SG: Glutathiolated selenol

SFNEP: French speaking society of clinical and metabolism

SH: Thiol

SOD: Superoxide dismutase SOR: Superoxide reductase SR: Sarcoplasmic reticulum

STAT3: Signal transducer and activator of transcription -3

SW872: Human liposarcoma cell line TFAM: Mitochondrial transcription factor

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TGF-β : Transforming growth factor-beta TNF-α: Tumor necrosis factor-alpha TRX: Thioredoxin

UPS: Ubiquitin proteasome system

VDAC: Voltage-dependent anion channel VEGF: Vascular endothelial growth factor WCRF: world cancer research fund

WHO: World health organization XDH: Xanthine dehydrogenase XO: Xanthine oxidase

XOR: Xanthine oxidoreductase

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FIGURE I: Introductive scheme showing the different interactions between tumor and skeletal

muscle, which could be modulated by factors like, PA and antioxidants ... 05

FIGURE 1: Disproportionation of O2.- by the “Ping-Pong” reaction; O2.- reduces the oxidized metal ion and then oxidizes the reduced metal ion. ... 12

FIGURE 2: Main enzymatic reactions involved in ROS neutralization ... 13

FIGURE 3: Reduction of free radicals by the GPx-GSH system ... 14

FIGURE 4: Reduction of disulfide bonds by TRX ... 15

FIGURE 5: Interrelationship between GSH, vitamin C and vitamin E. ... 19

FIGURE 6: OS profiles in biological system ... 20

FIGURE 7: Three main steps of lipid peroxidation. ... 21

FIGURE 8: Example of oxidative modifications that proteins can undergo in response to high ROS production. ... 23

FIGURE 9: The biological cellular response in relation to intracellular ROS levels and redox potential ... 24

FIGURE 10: Oxidative stress as a hallmark of cancer ... 29

FIGURE 11: Cancer cells engage different biological responses according to the doses of nitric oxide produced by iNOS. Low-to-moderate doses enhance tumor growth but higher doses produce the opposite effects ... 33

FIGURE 12: Alteration of mitochondrial bioenergetics profiles maintains redox homeostasis in tumor cell and promotes its growth. ... 36

FIGURE 13: Main ROS-dependent mechanisms driving tumor growth and progression ... 41

FIGURE 14: Main ROS-dependent mechanisms driving tumor regression and death ... 44

FIGURE 15: The impact of tumor derived-factors on multiple organs during cancer cachexia...52

FIGURE 16: The main axis by which tumor-derived chemicals induce ROS production, oxidative damage and muscle degradation ... 54

FIGURE 17: The identified pathways for iNOS/NO. in muscle atrophy and dedifferentiation ... 57

FIGURE 18: Mitochondrial dysfunction in wasted muscles ... 20

FIGURE 19: Regeneration of GSH molecules through the pentose phosphate pathway ... 60

FIGURE 20: Main protein synthesis pathways inhibited in skeletal muscle during cancer cachexia ... 62

FIGURE 21: ROS as second messenger in the activation of catabolic pathways within skeletal muscle in response to inflammatory and atrophic stimuli. ... 64

FIGURE 22: Main ROS-dependent molecular events leading to calcium accumulation and calpain activation in skeletal muscle cells, in response to inflammatory or catabolic stimuli ... 67

FIGURE 23: Scheme recapitulating the central role of ROS in both carcinogenesis and related skeletal muscle wasting. ... 69

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Figures and Tables Index

XVI | P a g e FIGURE 24: The vicious circle between physical inactivity, tumor development and muscle wasting ... 73 FIGURE 25: Hypothetical scheme representing the potential beneficial and harmful effects of ROS in a dose-dependent manner within skeletal muscle ... 75 FIGURE 26: Exercise-induced antioxidant, metabolic, hormonal and angiogenic adaptations as well as myokines production ... 78 FIGURE 27: Main factors modulating by physical activity, leading to reduced cancer

progression and muscle wasting ... 85 FIGURE 28: PubMed search with “Cancer” & “Antioxidants” showing the production of

scientific papers in the field over more than two decades ... 94 FIGURE 29: The ambivalence and dual role of ROS between tumor and skeletal muscle as well as the poor selectivity of antioxidant vitamins for tumor tissues, complicates their use in cancer cachexia ... 97 FIGURE 30: Polyphenols as promising compounds in the treatment of tumor growth and muscle wasting ... 100 FIGURE 31: Redistribution of selenium and zinc between tumor and skeletal muscle, promotes the progression of cancer cachexia ... 102 FIGURE 32: Simplified model showing (●) the plasmatic AO values corresponding to each patient and (■) the suitable measure that could be applied to avoid undesirable side-effects .... 105 TABLE 1: List of main oxygen, nitrogen and chlorine derivatives in the biological system ... 10 TABLE 2: Main characteristics of chosen antioxidant enzymes ... 11 TABLE 3: Studies showing the double-faced role of ROS in sarcoma-bearing mice ... 47 TABLE 4: Clinical studies emphasizing the impact of sarcoma on physical function and the potential effects of PA in sarcoma patients ... 88 TABLE 5: List of main clinical studies with antioxidants alone or in combination with

pharmacological agents on cancer patients with muscle atrophy (cachectic patients). ... 107 Box 1: Role of creatine kinase in skeletal muscle ... 56

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A-Introduction and Literature

Overview

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The burden of non-communicable diseases including: cardiovascular dysfunction, type 2 diabetes, and certain types of cancer is increasing in developed countries and account for more than 60% of the 56 million deaths that occur annually (WHO, 2015). Cancer is the first cause of mortality in France and the second after, heart diseases, worldwide (INCa, 2015). Cancer care is a major issue at both health and socioeconomic plan. Each year the French government disburses more than 5 milliard euros on cancer treatment and clinical care. In 2014, 155 million euros were allocated for cancer research by institutional organizations and charitable national foundations.

According to the recent report released, in 2015, by the French national cancer institute (INCa, 2015), 385 000 new cases were diagnosed with cancer at a median age of 67-68 year-old. The most frequent types are lung, prostate and colorectal cancer in men. Breast cancer remains the most recurrent in women, followed by colorectal and lung cancer. In 2015, 149 500 persons (56% men) have succumbed to cancer in France. Among those, lung and breast cancer were the principal cause leading to death in men and women respectively (INCa, 2015).

A shift in mortality toward an increase could be due to a number of health complications associated to cancer that reduce the tolerance to anticancer therapies (Fearon et al., 2011). The two key limiting factors are the alteration of both nutritional and activity status of patients (WHO, 2004). In fact, advanced stages of cancer are often associated with malnutrition and excessive loss of skeletal muscle mass leading to functional impairment and high risk of inactivity (Fearon et al., 2011). Indeed, the “Plan of cancer 2014-2019” launched by the French government, underscored the importance of improving patients’ quality of life (QOL) and physical performance in the completion of anticancer treatments. From this perspective, adjuvant therapies like physical activity (PA) and nutritional support could be helpful when used in combination with conventional treatments.

In this light, the crosstalk between tumor and skeletal muscle is a major event occurring during cancer (Jones et al., 2012). We suppose that two main routes could drive the crosstalk between tumor and skeletal muscle; in this manuscript, we have chosen to term them the direct or indirect crosstalk. An indirect tumor-muscle interaction occurs through the mutual release of tumor or muscle-specific factors into systemic circulation. In such condition, tumor could secrete mediators that induce muscle atrophy (Arthur et al., 2008) or inversely muscle may produce factors that act on tumor and modulate its behavior (Aoi et al., 2013; Fontes-Oliveira et al., 2014; Siems

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Introduction

4 | P a g e Tumor-Skeletal Muscle Crosstalk in Cancer- Ph.D. thesis-Mohamad Assi, 2016

and Grune, 2003; Tan et al., 2015). A direct tumor-muscle crosstalk could take place in particular cases when tumor develops in skeletal muscle itself. The evidence that PA can reduce cancer growth, progression, recurrence and related mortality is accumulating in patients with breast, prostate, colon and esophageal cancer (illustrated in green arrows; Figure IB) (Friedenreich et al., 2016; Meyerhardt et al., 2006b; Wang et al., 2016a; Wu et al., 2016). Physically active patients exhibited also an improvement in overall QOL and physical performance. These findings affirmed the conviction that PA must constitute a part of the primary anticancer treatment. Little is known about the exact molecular mechanisms through which exercise could mediate its protective effects.

However, changes in systemic inflammation and the ability of PA to mimic an antioxidant response may play a key role (de Sousa et al., 2016; McTiernan, 2008). Intriguingly, although skeletal muscle is the first organ solicited during physical exercise, the impact of PA on tumors that develop inside muscle itself and, therefore, directly affect its function has not received much attention (Figure IA). As a result, patients with lower-extremities cancers like liposarcoma, don’t benefit from advises regarding the level of PA that could be practiced during the treatment period.

In some cases, the capacity of patients with advanced stages of cancer (cachectic patients) to pursue a hospital-based PA program could be limited owing to anemia, cardiovascular dysfunction and advanced pro-catabolic state (Argiles et al., 2012). Therefore, an alternative strategy to PA could be useful to slow the deterioration of patients’ overall health.

From a molecular point of view, such condition is usually associated with chronic inflammation due to the sustained release of tumor-derived chemicals, which deregulates the redox balance in the body (illustrated in red arrows; Figure IB) (Mantovani et al., 2002b). This promotes excessive reactive oxygen species (ROS) formation within skeletal muscle and its subsequent degradation (Li et al., 1998). We might underscore that ROS are also produced within tumor and control its biological response (Schieber and Chandel, 2014; Sabharwal and Schumacker, 2014).

Antioxidants have been proposed to counteract these ROS-dependent mechanism and reduce tumor growth as well as the magnitude of muscle wasting. Unfortunately, the obtained results from preclinical and clinical studies of cancer were controversial (Figure IIB) (Lucas et al., 2016; Sayin et al., 2014; Schmidt et al., 2016; Shadfar et al., 2011). The heterogeneity in these outcomes were largely depending on (1) the type/localization of tumor, (2) the type/dose of antioxidants, (3) basal antioxidant status of the patient and (4) other factors that may distort evidence-based conclusion

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such as, self-prescribe antioxidant supplements, a practice highly recurrent among lung and colon cancer patients (Sandler et al., 2001).

In this Ph.D. thesis we tested the impact of PA and antioxidants in two distinct model of cancer, designed to be as close as possible to the clinical context nowadays. In the first study we addressed the impact of regular PA on the development of intramuscular liposarcoma and in the second model we evaluated the effectiveness of antioxidant supplementation in cachectic mice bearing subcutaneous colon cancer. These models allowed us to understand the impact of two adjuvant strategies on: tumor growth, skeletal muscle mass/function and the direct/indirect crosstalk between tumor and skeletal muscle.

F^{IGURE I}: Introductive scheme showing the different interactions between tumor and skeletal muscle, which could be modulated by factors like, PA and antioxidants

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Chapter I

The Reactive Oxygen Species

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1. Reactive oxygen and nitrogen species

The life of humans on earth is intimately linked and conditioned by the levels of oxygen (O2) in the atmosphere. Molecular O2 is required for the transformation of carbohydrates, lipids and proteins provided by our diet into energy within cells through the Krebs cycle that takes place within mitochondria, a process known as oxidative phosphorylation (Ernster and Schatz, 1981).

Quantum chemistry rules indicate that molecular O2 becomes reactive in the organism and may exert several biological functions through the formation of intermediary oxygen derivatives.

Usually, chemical molecules are connected to each other by two electron pairs to insure stability.

The particularity of O2 is that both atoms are matched to each other by only one electron pair, meaning that each oxygen atom possesses one unmatched electron in its orbit (Halliwell, 1999a).

This characteristic is in fine responsible for the formation of reactive oxygen derivatives, also called pro-oxidant species.

Chemically, every compound that can accept electrons is an oxidant and every substance that can give electrons is a reductant (Cao and Prior, 1998; Prior and Cao, 1999). In biology, oxidizing and reducing agents are known as pro-oxidants and antioxidants, respectively. Generally, pro-oxidants are referred to reactive oxygen species (ROS). Another set of pro-oxidants, has been extensively studied the last twenty years, regroups the reactive nitrogen species (RNS) mainly produced by an enzyme called nitric oxide synthase (Patel et al., 1999). ROS and RNS can be classified to either radical or non-radical compounds (Table 1). Most radicals are highly reactive, unstable and short-lived molecules that possess one impaired electron in their outer valence shell (Kohen and Nyska, 2002). They include: anion superoxide (O2.-), hydroxyl radical (OH^.) and nitric oxide (NO^.). Non-radicals are also highly reactive, among these species we cite: hydrogen peroxide (H2O2), hypochlorous acid (HClO) and ozone (O3) (Halliwell, 1999a). The cell could be exposed to various amounts of ROS from exogenous and endogenous sources. Ionizing and non-ionizing radiations (i.e. γ-irradiation and UV), air pollutants, drugs, xenobiotic and food are major exogenous factors that lead to the production of a variety of ROS/RNS in living organisms (Elsayed et al., 1992; Kanner and Lapidot, 2001; Pentland, 1994). Interestingly, the exposure to endogenous sources is more important, as it is a part of a continuous process in every single cell (Kohen, 1999).

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Chapter I. Reactive Oxygen Species

Mitochondria is considered as the major source for O2.- which could be then rapidly converted into H2O2 molecules. During oxidative phosphorylation, 98% of O2 are transformed to H2O and 2% are transformed initially to O2.- (Chance et al., 1970). The rates of O2.- production depend on the (1) level of O2 in the tissue, (2) concentration of proteins carrying electron donors, (3) proportion of electron carriers able to react with O2 to give O2.- and (4) the rate constant reaction of electron carriers (Murphy, 2009). The two major sites of O2.- production within mitochondria are complex I and III. The evidence that complex I generates O2.- and H2O2 molecules was first demonstrated in isolated sub-mitochondrial particles, for which a decrease in O2 concentration or NADH pool blunted oxidants generation (Kussmaul and Hirst, 2006). In addition to mitochondria, enzymes such as xanthine oxidase (XO), nicotinamide phosphate dinucleotide oxidase (NOX) and nitric oxide synthase (NOS) are also an important source for endogenous ROS and RNS generation (Canas, 1999; Shaul, 2002).

T^ABLE1: List of main oxygen, nitrogen and chlorine derivatives in the biological system

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2. Antioxidant defense

Apparently, the antioxidant system was developed throughout the evolutionary process to maintain the redox balance in living organisms and protect them against the toxic effects related to O2 augmentation in the atmosphere, also termed as the “great oxidation event” (Kohen and Nyska, 2002). To maintain redox homeostasis, ROS and RNS are continuously buffered by several enzymatic and non-enzymatic antioxidant compounds; hereafter we describe the most important.

2.1. Enzymatic antioxidant system

A number of enzymes are involved in the direct detoxification of ROS and RNS, these include: superoxide dismutase (SOD), superoxide reductase (SOR), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRX).

SOR and SOD:

Chemically, SOR catalyzes the one-electron reduction of O2.- to form H2O2. This reaction needs two protons (H⁺) and one external electron-donor (e^-). SOD catalyzes the disproportionation of O2.- into O2 and H2O2, a reaction that needs one H⁺ per O2.- (Sheng et al., 2014). Interestingly, SOD enzymes are very effective to adapt to an urgent environmental

TABLE 2: Main characteristics of a number of chosen antioxidant enzymes

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change and maintain low physiological concentrations of O2.- at ~ 2 × 10^-10 M. This was demonstrated by the early observation of Imlay et al. showing that SODs are able to dismutate 10^-

9 or 10^-6 M of O2.-with the same half-time reaction of 175ms (Imlay and Fridovich, 1991). Contrary to SOR enzymes that contain only iron, the SODs differ in their structure and cofactors. They include Nickel-containing SOD (NiSOD), iron or manganese SOD (FeSOD/MnSOD) and copper and zinc SOD (CuZnSOD). In eukaryote, NiSOD and CuZnSOD are localized in the cytoplasm, while MnSOD and FeSOD are found in the mitochondria (Halliwell, 1999a). The obvious similarities between these enzymes, is that they all contain a redox-active metal at their active site and can catalyze O2.- disproportionation by a so-called “Ping-Pong” mechanism (Figure 1). Three conditions must be regrouped to optimize O2.- dismutation: (1) adjustment of metal level, (2) control of O2.- access to the active site and (3) availability of a source of H⁺ for the formation of H2O2 (Sheng et al., 2014). From a physiological point of view, the deletion of SOD was associated with the development of several disorders. The best known to be linked to CuZnSOD-deficiency, are axonal injury and amyotrophic lateral sclerosis (ALS) in motor neurons (Reaume et al., 1996).

The expression of MnSOD but not CuZnSOD is indispensable for survival, since MnSOD-deficient mice exhibited neurodegeneration, myocardial dysfunction and perinatal death (Lebovitz et al., 1996).

CAT:

Two H2O2 molecules obtained from the SOD system, are then transformed to 2H2O and oxygen thanks to the activity of CAT (Chance et al., 1979). CAT possesses a tetramer structure (Lardinois, 1995). In each subunit, the ferric ions of the haem group undergo oxidation after interaction with H2O2, yielding a radical called “porphyrin”. Next, another molecule of H2O2 acts as an electron donor and reacts with “porphyrin”, resulting in the destruction of two H2O2

molecules per reaction (Lardinois, 1995) (Figure 2). Even though that CAT is not essential under FIGURE 1: Disproportionation of O2.- by the “Ping-Pong” reaction; O2.- reduces the oxidized metal ion

and then oxidizes the reduced metal ion. From (Sheng et al., 2014)

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physiological condition, it plays an important role to counteract ROS-mediated harmful effects.

For example, overexpression of CAT prevented H2O2-mediated skeletal muscle atrophy in rats (Dodd et al., 2010). CAT is also highly used in different production chains going from food, to textile and cosmetic industry to insure that the products are H2O2-free (Wadhwani, 2012).

GPx:

GPx-1 is a protein of tetrameric structure more abundantly expressed compared to other GPx members, it can be found in the cytosol and mitochondrial compartment (Flohe et al., 1971). Compared to CAT, GPx-1 has a greater affinity for H2O2 elimination even at low concentrations (Chance et al., 1979), but the reaction costs too expensive for cells, as the neutralization of one H2O2 molecule requires two molecules of reduced glutathione (GSH) (Kohen and Nyska, 2002) (Figure 2). Indeed, GPx-1 levels in the body are close to that of GSH. The direct link between GPx enzymatic action and reduced/oxidized GSH ratio was illustrated in cultured cells; thus, treatment with H2O2 led to an increase in oxidized GSH in wild type but not in GPx-1- deficient cells (Liddell et al., 2006). GPx-1 contains one seleno-cysteine (SecH) residue (active site) in each of the four subunits (Speranza et al., 1993). The detoxification of oxidative radicals by GPx-1 is a reaction involving a total of three steps: (1) H2O2 reacts with the active site SecH, giving rise to selenic acid (SeOH), (2) then one molecule of GSH reduces SeOH leading to the formation of glutathiolated selenol (Se-SG) and (3) finally, a second molecule of GSH allows the reduction of Se-SG and the restoration of the active site (Figure 3) (Lubos et al., 2011).

FIGURE 2: Main enzymatic reactions involved in ROS neutralization

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In addition to H2O2, GPx-1 can effectively remove lipid peroxidation adducts, like hydroperoxides from the intracellular compartment (Marinho et al., 1997). GPx-4 preferentially localized at the membrane is also able to reduce membrane phospholipids hydroperoxide and oxidized lipoproteins (Lubos et al., 2011). As for SOD and CAT, disturbance in the GPx-1 function has been studied in several diseases including atherosclerosis and human cancer (Lubos et al., 2011).

TRX:

Despite the presence of GSH at high concentrations inside the cells to maintain a redox buffering, the prevention of disulfide bonds formation needs a more effective system like TRX. The antioxidant role of TRX was demonstrated by redox proteomic technics in human intestinal epithelial cells; thus, the inhibition of TRX with auranofin increased the oxidation of cysteine residues over control (Go et al., 2013). TRX catalyzes disulfide reduction via two N- and C-terminal cysteine residues conserved in the active catalytic site. Under physiological conditions, the N-terminal sulfur group of cysteine is present in the form of thiolate (S^-), allowing TRX to attack disulfide on target proteins (Foloppe et al., 2001). Subsequently, a disulfide bond is formed between TRX and the target protein. The sulfur group of the C-terminal cysteine is present in the form of thiol (SH). After formation of double bond between TRX and the target protein, C-terminal SH is activated into S^- to react with the N-terminal side, allowing TRX to dissociate from target

FIGURE 3: Reduction of free radicals by the GPx-GSH system. From (Lubos et al., 2011)

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protein (Collet and Messens, 2010). Reduced target protein and oxidized TRX with disulfide bond are the end-products of the reaction (Figure 4). TRX affects several functional vital pathways like glycolysis, cytoskeleton remodeling, protein synthesis and folding process (Floen et al., 2014;

Yang and Lee, 2015). Other roles in cell death signaling, and protection of proteins from oxidative aggregation are also attributed to TRX (Ravi et al., 2005).

2.2. Non-enzymatic: low molecular weight antioxidants and micronutrients

Low molecular weight antioxidants (LMWA) family contains numerous compounds, which are principally obtained from dietary origin. LMWA act directly or indirectly as electron donors to scavenge oxidative radicals or as metal chelating agents that prevent them to participate in the metal-dependent Haber-Weiss reaction (Weiss, 1934). Micronutrients like zinc and selenium are also indispensable cofactors for the functioning and stabilization of some antioxidant enzymes.

Zinc and selenium:

Zinc and selenium are two important cofactors for CuZnSOD and GPx respectively. Zinc could be required for the function of over 200 enzymes and, therefore, its deficiency may affect multiple systems. Since zinc is considered as a redox inert ion, it is more likely seen as a stabilizing component for the CuZnSOD structure, while copper ions are more

FIGURE 4: Reduction of disulfide bonds by TRX. Adapted from (Collet and Messens, 2010)

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involved in direct radicals detoxification (Parge et al., 1992). Zinc may also bind to and stabilize the thiol groups in proteins like metallothioneins and zinc-finger transcription factors (Klotz et al., 2003). The role of zinc in stabilizing thiols was clearly demonstrated in δ-aminolevulinate dehydratase, which became less prone to oxidative damage and maintained its activity (Powell, 2000). The antioxidant-like effects of selenium are driven by its ability to reduce hydroperoxides and peroxynitrite by three main routes. The first, is through forming the selenocysteine residue at the active site of GPx, which enables it to transform the above-mentioned radicals into nitrite and alcohol, indicating that selenium is indispensable for GPx activity (Lubos et al., 2011). The second, is mimicking the reaction of GPx via low-molecular-weight selenomethionine compounds that are very effective in neutralizing peroxynitrite and protect DNA from oxidative single-strand breaks (Roussyn et al., 1996). It is interesting to mention that oxidized selenomethionine could be recycled to its reduced (active) form by zinc-stabilized metallothioneins, highlighting the interrelationship between zinc and selenium to maintain an effective antioxidant defense. The third, involves the TRX reductase that is a selenoenzyme able to reduce oxidized selenocompounds, formed after the interaction with reactive radicals (Klotz et al., 2003).

Glutathione:

Among LMWA, GSH is considered as the master of antioxidants and it is present at high concentrations reaching the millimolar level in the intracellular compartment (Kohen and Nyska, 2002). GSH exerts indirect antioxidant functions by acting as a cofactor for GPx to reduce H2O2 molecules and produce oxidized GSH (GSSG). Additionally, GSH intervenes directly in the (1) regeneration metabolism of ascorbic acid, (2) as a scavenger of OH^. and ROO^. radicals and (3) a chelating agent for copper ions (Halliwell, 1999a). Approximately, 15% of GSH are found in the mitochondria (mGSH) (Mari et al., 2009). This pool of mGSH is essential for the stabilization of mitochondria structure and protecting it from oxidative insults (Mari et al., 2009).

It is also interesting to know that GSH can across the plasma membrane to the extracellular space.

Liver is the main source producing and releasing GSH in the bloodstream (Lushchak, 2012).

Circulating GSH can primarily reach lungs, which are continuously exposed to O2/inhaled toxins, and detoxify external pro-oxidant derivatives to prevent damage (Lushchak, 2012). Similar protective antioxidant actions are exerted by GSH in liver, intestine and kidney, which could be exposed to ROS from food origin (post-prandial oxidative stress), xenobiotic and other high intensity O2-based energy mechanisms (Song et al., 2000). In addition to its antioxidant function GSH plays distinct roles at different cellular compartments. At the sarcoplasmic reticulum (SR)

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the ratio GSSG/GSH is much higher compared to the cytosol, due to the efficient formation of disulfide bonds in nascent polypeptides, which is an unavoidable step for proteins to acquire functional conformation (Cooper et al., 1980). In the nucleus, GSH could be also useful for the reduction of ribonucleotides into deoxyribonucleotides and DNA replication (Holmgren and Sengupta, 2010).

Cysteine-based compounds:

Maintaining an optimal pool of thiol within the cell is an efficient way of protection against accumulation of oxidative damages. In continuity with the role of GSH, N-acetylcysteine (NAC), α-lipoic acid and carbocysteine are also important in maintaining cellular redox state. Particularly, NAC has been mainly studied as an antioxidant agent that improves respiratory and skeletal muscle performance (Reid et al., 1994; Supinski et al., 1997).

Redox molecular analysis revealed that NAC induced its protective effects via increasing the pool of GSH and preventing apoptosis commitment within muscles (Supinski et al., 1997). Thanks to their antioxidant virtues, NAC and carbocysteine are today used clinically in patients with chronic obstructive pulmonary disease (COPD) as a mucolytic agent, able to degrade the three-dimensional structure of mucus by reducing the disulfide bonds and, thus, mediating airways clearance (King and Rubin, 2002). Additionally, carbocysteine and α-lipoic acid are used to improve overall performance and QOL in conditions evoking high ROS levels including, advanced stages of cancer (Maccio et al., 2012).

Vitamin C

: Hydrosoluble vitamin C in the form of ascorbic acid is also considered as a powerful scavenger of HClO, OH^., ROO^.and SH^., given its capacity to donate two electrons. After losing the first electron, ascorbic acid is transformed to ascorbyl radical that could be either further oxidized to produce dehydroascorbate or regenerated by a reductant such as, GSH (Kohen and Nyska, 2002). A link between vitamin C deficiency and oxidative damage was established in Gulo

-/- mice for which lipid peroxidation adducts were much elevated in the liver, compared to control (Harrison et al., 2010). The direct antioxidant capacity of ascorbic acid was validated in vitro but there is no convincing evidence about the efficacy of its scavenging effects in animals and humans (Paolini et al., 1999). The results of clinical trials were disappointing, rising doubts about the safety of “vitamin C overdosing” in the treatment of patients with cancer and cardiovascular diseases (Loria et al., 2000). Supplementation with vitamin C seems beneficial in particular conditions evoking high levels of lipid peroxidation, inflammation and oxidized low-density lipoprotein

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(LDL), such as endothelial dysfunction, or when serum levels of vitamin C are opposed to vitamin C intake (Loria et al., 2000). Vitamin C may improve endothelial function by increasing NO^. bioactivity (Williams et al., 1994). NO^. can be eliminated by reacting with O2.- radicals to produce peroxynitrite. At this level, ascorbic acid may stop O2.- production either (1) directly through scavenging O2.- molecules produced mainly by NOX or (2) indirectly by filling in the “uncoupled”

active site of eNOS in the absence of arginine substrates (in the absence of arginine, eNOS produces O2.- instead of NO^.) (Traber and Stevens, 2011).

Vitamin E:

Another antioxidant that has been extensively used in animal and human studies is the liposoluble vitamin E in the form of α-tocopherol (Halliwell, 1999a). In the human body, various β, γ and δ-tocopherols are absorbed and transferred, but only α-tocopherol is maintained in plasma and tissues (Traber, 2007). This long half-life is due the hepatic α-tocopherol transfer protein (α-TTP) and the rapid recirculation between liver and blood, allowing a daily replacement of the entire α-tocopherol pool (Traber et al., 1994). The α-tocopherol interacts with radicals and give rise to α-tocopherolquinone and subsequently to α-tocopherylquinone. Then, the α-tocopherylquinone is recycled to its reduced form by ascorbic acid (Herrera and Barbas, 2001).

Given its bioavailability and pharmacokinetics proprieties, α-tocopherol has the most effective role in stopping the lipid peroxidation process. Its antioxidant effects are largely depend on the concentrations of vitamin C. For example, lipid peroxidation and the great disappearance rate of vitamin E, observed in the presence of high ROS amounts, were normalized by vitamin C supplementation (Bruno et al., 2006). This emphasizes the importance of vitamin C as a

“quarterback” for vitamin E regeneration-metabolism. Deficiency in vitamin E may cause severe anemia, neurological disorder and negatively affect the immune system function (Traber and Stevens, 2011). Figure 5 illustrates the overlapping cycles of regeneration between different antioxidants, which is crucial to maintain an effective antioxidant defense.

Polyphenols:

The family of polyphenols may hold thousands of compounds, among these we cite: catechins (e.g. green tea extracts), flavonols (e.g. quercetin), stilbens (e.g.

resveratrol), and the list is too long. They could be obtained from various alimentary sources like, green/black tea, coffee, fruits, vegetables, olive, red wine and chocolate (Perron and Brumaghim, 2009). Unlike vitamin E, polyphenols have negligible scavenging effects in vivo, and mediate their antioxidant response via the Keap-Nrf2 pathway (Erlank et al., 2011). In the absence of stressor

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stimuli, the Cul3-Keap ubiquitin ligase, maintains Nrf-2 sequestrated in the cytoplasm, promotes its ubiquitination and subsequent degradation by the proteasome (Sekhar et al., 2002). In the presence of ROS, the thiol groups on Keap become oxidized, thus altering the interaction within complex and liberating Nrf-2 (Satoh et al., 2013). Then, Nrf-2 will translocate into the nucleus and bind the antioxidant responsive element (ARE) to promote the expression of antioxidant genes including, SOD and TRX (Itoh et al., 2004). Paradoxically, given their cyclic-benzyl chemical structure, polyphenols could undergo spontaneous oxidation to produce moderate levels of ROS and activate the Keap-Nrf2 system (Erlank et al., 2011). In some diseases, like cancer, an increase in the nuclear accumulation of Nrf2 could be observed due to activating mutations on Nrf2 or silencing of Keap (Kansanen et al., 2013). However, the health-related benefits from polyphenols in cancer, are not restricted to their antioxidant virtues but expand to modulate pathways involved in senescence, proliferation and epigenetic modifications (Mileo and Miccadei, 2016a).

3. Oxidative stress and related macromolecules damage

Even though that ROS are essential for body functioning under normal conditions, excessive and sustained production of ROS has been involved in the development or progression of more than 200 clinical disorders (Kohen and Nyska, 2002). In pathological conditions, a shift in the redox balance toward an increase in the pro-oxidant power leads to damage and the development of oxidative stress (OS). OS is defined as a “disruption of the redox balance towards an increase in pro-oxidant over the capacity of antioxidants, leading to a perturbation of redox signaling and control and/or molecular damage” (Jones, 2006). A redox deregulation in the FIGURE 5: Interrelationship between GSH, vitamin C and vitamin E. From (Kohen and Nyska, 2002)

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opposite side is called reductive stress (antioxidants over pro-oxidants), but this condition has not been studied in depth.

Despite the relative short half-life of ROS, some species can diffuse and reach sensitive locations, where they can interact with essential biological targets (i.e. lipids, proteins and DNA) and cause damage. Indeed, we can distinguish between three different profiles of OS. Acute OS in response to an external physiological stimuli, which could be reduced by an effective antioxidant defense. In the case of ineffective antioxidant response, the cell is exposed to a more prolonged production of ROS, giving way to the development of chronic OS. Contrary to acute OS, chronic OS could have dangerous consequences on health and it is associated with the development of several diseases including, cancer (Sabharwal and Schumacker, 2014). The third OS profile is called “quasistationary”, it occurs when ROS levels do not regain the initial steady state; thus, it is somehow a new redefined level of ROS in the cell, usually much higher than the normal range (Lushchak, 2012) (Figure 6).

F^IGURE 6: OS profiles in biological system. From (Lushchak, 2012)

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3.1. Lipid peroxidation (Cillard, 2006; Halliwell, 1999a; Meral et al., 2000)

The lipids of cellular membrane are particularly sensitive to oxidation. The interaction of ROS with lipids induces damage through a process composed of three main stages, known as lipid peroxidation. The first stage called initiation, in which the derivatives of oxygen remove an atom of hydrogen from a methylene (-CH2) group adjacent to a double bound (because of the double bound, the interaction between carbon and hydrogen is weakened and it can be removed easily by oxygen radicals). The formed fatty acid radical is stabilized by rearrangement of the structure to form a conjugated diene. Then, it reacts with oxygen giving place to a lipid peroxyl radical (ROO^.).

During the propagation stage, ROO^. can abstract the hydrogen of other neighboring lipids and, therefore, produce further fatty acid radicals.

The interaction of ROO^.with a new fatty acid produces a lipid hydroperoxide radical (ROOH) that can be further decomposed into aldehydes, hydrocarbons and isoprostanes. The last stage, termination, takes place when ROO^. enters in contact with an antioxidant, like vitamin E.

The major end-products of the reaction include malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) (Figure 7). It has been shown that ω-6 polyunsaturated fatty acids and mitochondrial

F^IGURE7: Three main steps of lipid peroxidation. From (Neda Mimica-Dukić, 2012)

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phospholipid cardiolipin are important sources of 4-HNE formation (Zhong et al, 2015). When produced at high levels in the cell, 4-HNE preferentially reacts with histidine, cysteine and lysine residues of intracellular proteins, resulting in structural modifications of “Schiff-base” type or Michael addition (Negre-Salvayre et al, 2008). The formed HNE-protein adducts are more resistant to removal by the proteasome and favor the aggregation of oxidized protein in the cell; such condition is associated with ageing and cancer (Friguet, 2006). Moreover, 4-HNE may directly oxidize proteasome subunits and reduce its activity (Keller et al, 2000). Nucleic acids are also an important target of 4-HNE. For example, 4-HNE-DNA adduct is a known mutagen in cells mainly found at the p53 gene in human cancers (Hussain et al, 2000). Even more important, 4-HNE may compromise DNA repair mechanisms such as nucleotide excision repair (NER) within human cells, in response to mutagenic agents or exposure to UV lights (Feng et al, 2004). This could be attributed to the ability of 4-HNE to react with essential proteins involved in NER (Feng et al, 2004).

3.2. Protein oxidation and peroxidation

Proteins are also important targets for biological oxidants due to their important concentration and high rate of constant reaction with numerous oxidant (Davies, 2016). For example, amino acids like cysteine can undergo an abstraction of hydrogen from the thiol (SH) group by OH^., resulting in the formation of thiyl radicals (RS^.). These radicals are able to abstract a hydrogen from α (backbone) and 𝛽-carbon (side-chain) at the same or neighboring amino acid, to give carbon-centered radicals (R^.). Carbon-centered radicals undergo peroxidation, fragmentation, dimerization, disproportionation and isomerization, which results in dramatic effects on protein structure and function (Fradkin et al., 2014; Jiskoot et al., 2012). The predominant reaction is the interaction of R^. with O2 that results in the formation of ROO^.radicals.

One of the possible pathways is the cross-reaction of two ROO^. radicals (ROO-OOR) that decay to yield aldehydes/ketones, through a succession of reactions. These reactions lead to the formation of protein carbonyls, which is a widely used biomarker for protein oxidation (Dalle-Donne et al., 2006; Dalle-Donne et al., 2003). However, more than 70% of initial ROO^. are transformed into ROOH, given the abundance of C-H and S-H bonds in proteins. ROOH radicals can react with more susceptible amino acids like, methionine and cysteine, resulting in the inactivation of several enzymes and alteration of energy metabolism as well as many cellular functions (Nagy and Erhardt,

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2010) (Figure 8). Interestingly, oxidized cysteine could undergo reparation by TRX and glutaredoxin (GRDX) (Holmgren et al, 2005). Both TRX and GRDX contain two cysteine residues in their redox active site, which allow them to reduce oxidized cysteine on the damaged protein.

Upon reparation of target proteins, TRX and GRDX become oxidized and are then recycled to their reduced form by the TRX reductase and GSH reductase, respectively (Holmgren et al, 2011).

Protein function could be also restored after the reversion of oxidized methionine to its reduced form under the action of methionine sulfoxide reductase enzymes (Moskovitz, 2005; Sreekumar et al, 2011). Additionally, other small chaperone proteins such as heat shock protein (HSP) could serve as cytoplasmic antioxidants, by binding to and hiding the sensitive amino acids on the target protein (Dahl et al, 2015). HSPs may also hold oxidized/denatured proteins until their refolding or degradation in order to prevent accumulation of protein aggregates (Dahl et al, 2015). Nevertheless, some products like carbonylated amino acid derivatives are irreversible and typically undergo degradation by the proteasome 20 and 26 S in the cytoplasm and by the ATP-dependent Lon protease within mitochondrial matrix (Shang et al, 2001; Bota & Davies, 2002).

F^IGURE8: Example of oxidative modifications that proteins can undergo in response to high ROS production. Adapted from (Davies, 2016)

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3.3. Oxidative DNA damage

Although DNA is a well-protected molecule, some ROS can reach this location to interact with nucleotides and induce damage. Indeed, OH^. possesses a high rate constant reaction with DNA bases and ribose sugar (k~ 5-8×10⁹ M^-1 s^-1) (Cadet et al., 2012). Hydroxyl radical reacts indistinctly with DNA either by addition or hydrogen abstraction and, then, the formed products can undergo a secondary reaction with O2 or O2.- (Jaruga et al., 2012). The DNA-OH^. interaction gives rise to a large plethora of end-products depending on the: nature of modified bases, type of the initial reaction and the amounts of pro-oxidants (Cobley et al., 2015). For example, the oxidation of guanine yields more than 20 DNA-adducts, with the 8-oxo-7,8-dihydroguanine (8-oxoG) being the most assayed product to study DNA oxidative damage (Sayin et al., 2014). Interestingly, H2O2, NO^. and O2.- are unlikely to induce direct DNA damage (Halliwell, 1999b), but could serve as a source to produce reactive intermediates that can attack DNA. For example, NO^.and O2.- can react to produce peroxynitrite molecules extremely damaging for DNA. Base excision repair and NER are two important pathways involved in DNA damages repair. NER is constituted of two sub- pathways: (1) transcription-coupled NER (TC-NER) that recognizes stalled polymerase II during transcription events because of DNA helix distortions and (2) global genome-NER (GG-NER) which is a less efficient mechanism that screens the whole genome to detect secondary DNA products and the chemical structure of helix-distorting lesions (Melis et al, 2013). Both NER and BER could be considered as a part of the intracellular antioxidant defense, given their ability to prevent or repair oxidative DNA damage. In fact, the inactivation of key components of the TC- NER or GG-NER system, like cockayne syndrome A and B factors (CSA/B) and XPC, in animals and cells increases their sensitivity to oxidative stressors and causes the accumulation of DNA damages (de Waard, 2003; Pascucci et al, 2012; Hollander et al, 2005). At the mitochondria, it is the BER system that will recognize and remove 8-oxoG adducts via a specific glycosylase called OGG1 (Melis et al, 2013). The damaged base is then released, keeping a free unbound site known as an apurinic/apyrimidinic (AP) site. This is cleaved by an endonuclease and the resulting single- strand break could be ligated with a single-to-ten nucleotides (Melis et al, 2013). Additionally, NER provides support to BER function. For example, CSB regulates the expression of OGG1.

Deficiency in CSB causes weak OGG1 transcription and inefficient 8-oxoG repair (Javeri et al, 2011).All together these findings emphasize the importance of BER and NER to contest oxidative DNA damage and, thus, to reduce cancer incidence.

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4. Biological role of ROS

The mechanisms of cellular signaling are intimately controlled by the redox state of the cell. ROS act as a second messenger to regulate the activity of several transcriptional factors and, therefore, the expression of various genes involved in cell cycle regulation, inflammation, cell death, autophagy, DNA damage repair and the list is too long (Alfadda and Sallam, 2012). This indicates that ROS control the fate of the cell, whether the cell will proliferate or not; will undergo repair and live or dead; will maintain its function or not, are largely dependent on ROS levels inside the cell. Accordingly, the half-cell reduction potential (Ehc) seems to correlate with the biological response of the cell (Schafer and Buettner, 2001). From a mathematical point of view, it could be estimated with the Nernst equation {redox environment = Σ Ei × (reduced species)i} where, Ei is the half-cell reduction potential for a given redox pair (i.e. GSH) and (reduced species)i represents the concentration of the reduced species in the redox pair. Indeed, the redox potential of a normal cell is approximately, Ehc ≈ -240 mV, and associated with cell proliferation. In response to progressive increase in intracellular ROS levels, the cell will switch into (1) differentiation (Ehc ≈ -200 mV), (2) then to apoptosis when the redox potential reaches Ehc ≈ -170 mV and (3) finally, to necrosis (Ehc ≈ -150 mV) (Schafer and Buettner, 2001) (Figure 9). In addition to controlling quiescence, proliferation, differentiation and apoptosis, ROS could be needed for proper cell migration, chemotaxis and structure stabilization (Munnamalai and Suter, 2009). ROS are crucial for the polymerization of F-actin monomers and α/β-tubulin as well as the regulation of microtubules dynamics and vesicles trafficking (Wilson and Gonzalez-Billault, 2015).

Physiological amounts of ROS are essential for a healthy body functioning. For example, a role for mitochondrial O2.- and H2O2 in the regulation of vascular endothelial diameter in response to stressor stimuli has been previously unveiled (Liu et al., 2003). ROS may also act as O2 sensor in response to hypoxia. When hypoxic non-transformed normal cells suffer from a lack of O2, the intracellular levels of ROS increase and induce the activation of hypoxia-inducible factor -1 (HIF- 1), resulting in angiogenesis and tissue oxygenation (Guzy and Schumacker, 2006). The so-called oxidative burst reaction is one of the most effective defense system against pathogens (Droge, 2002). Phagocytes will mainly produce O2.-, H2O2 and HClO via the membrane-anchored NOX, leading to direct pathogens destruction and alarming acquired immune cells (Droge, 2002).

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Interestingly, ROS may also intervene in other vital mechanisms, like: neurotransmission, skeletal muscle contraction and redox cell signaling (Halliwell, 2006a, b; Schieber and Chandel, 2014). All these data provide convincing evidence about a fine tuning of main cellular vital processes by ROS, in which a simple deregulation may lead to catastrophic disorder.

►Key Messages from Chapter I — O

xygen derivatives are produced inside the cells as a part of a continuous vital process. ROS are essential for healthy body functioning as they intervene in various physiological mechanisms including, but not limited to, innate immune defense, endothelial function, oxygenation, proliferation, differentiation, structure stabilization, adhesion, migration, DNA repair and apoptosis. Antioxidants act as a “cup” that survey ROS to avoid any undesirable biological effects. The antioxidant enzymes share some similarities in their mechanisms of action; they all possess metals or sulfur groups conserved in their active catalytic sites, which enable them to attack free oxidative radicals. Non-enzymatic LMWA including, vitamins, polyphenols and cysteine-based compounds participate to such mechanism either by direct radical-scavenging or indirectly as a second messenger in cellular signaling pathways.

However, the deregulation of this redox balance and the subsequent development of OS lead to cellular dysfunction and disease development. For example, inside skeletal muscle cell, basal levels of ROS promote protein synthesis, whereas excessive amounts of ROS induce muscle F^IGURE9: The biological cellular response in relation to intracellular ROS levels and redox potential