THESIS SUBMITTED FOR THE DEGREE OF DOCTORATE 3

University of Constantine 1 Faculty of Life and Natural Sciences

Department of Animal Biology

THESIS SUBMITTED FOR THE DEGREE OF DOCTORATE 3

CYCLE Option: Immuno-oncology

Presented by: ASSIA BENMEBAREK

Examination board:

President: D. SATTA

University of Constantine 1 Supervisor: S. ZERIZER

University of Constantine 1 Examiner: S. TEBIBEL

University of Constantine 1 Examiner: M. SAKA SAAD

University of Annaba

Examiner: L. ARRAR

University of Sétif

Guest : Z. KABOUCHE

University of Constantine 1

2013-2014

Effect of bioactive molecules extracted from medicinal plants on inflammation induced by

hyperhomocysteinemia and tumoral process

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This thesis is dedicated to my family.

My dad, mum, brother and sister for their continued love, support and encouragement to pursue my Doctorate,

I could not have made it without you all.

My husband for helping me and supporting me during all the hard periods of research and for always being patient and supportive, regardless of the many

time consuming hours I spent in front of my computer.

Before all else, I bow my head with reverence and express my gratitude to God for his kind benediction on me during all the stages of this project.

I wish to express my sincere thanks and deep gratitude to my supervisor Mrs. S.

Zerizer, professor at the Faculty of Life and Natural Sciences at the University of Constantine 1, for believing in me and for her continued support, guidance, expertise and counsel during the duration of my research degree.

I would like to thank all the jury members, Prof. D. SATTA (president of the jury), Prof. S. TEBIBEL (examiner of the doctorate thesis), Prof. M. SAKA SAAD (examiner of the doctorate thesis), and Prof. L. ARRAR (examiner of the doctorate thesis), for their participation and interest in my doctorate work.

My cordial thanks are due to Mrs Z. Kabouche, professor at the Faculty of pure Sciences, University of Constantine 1, for allowing me to access Laboratoire d'Obtention de Substances Thérapeutiques (LOST) in order to perform parts of my project and for providing me with the required plant materials for my experiments. I also extend my sincere thanks to the whole laboratory team.

I am grateful to all those who helped me, in one way or other, with the achievement of this thesis.

LIST OF PUBLICATIONS...ix

LIST OF ABBREVIATIONS...xi

LIST OF FIGURES...xiv

LIST OF TABLES...xix

INTRODUCTION...1

CHAPTER I: LITERATURE REVIEW I.1. Atherogenesis...5

I.1.1. Endothelium activation and mononuclear phagocytes recruitment...5

I.1.2. Macrophages activation and expression of scavenger receptors...6

I.1.3. Proatherogenic and antiatherogenic cytokines...9

I.1.4. Atheroma progression and complication...10

I.2. Homocysteine origin, structure and metabolism...13

I.2.1. Origin and forms of homocysteine...13

I.2.2. Metabolism of homocysteine...14

a. Remethylation pathway...15

b. Transsulfuration pathway...16

I.2.3. Classification of hyperhomocysteinemia...17

I.2.4. Pathogenicity of hyperhomocysteinemia...17

a. Chemokine production...17

b. Oxidative stress production...18

c. MPO production...19

d. NO production and metabolism...20

e. Coagulation...21

I.3. Homocysteine and Carcinogenesis...21

I.3.1. Homocysteine as a cancer marker...21

I.3.2. Homocysteine as a cancer risk factor...23

I.3.3.Carcinogenesis through folate deficiency...25

I.3.4. Carcinogenesis through oxidative stress...26

I.3.5. Carcinogenesis through aberrant DNA methylation...27

I.3.6. Carcinogenesis through homocysteine thiolactone production...28

I.4. Immunomodulation...30

I.4.1. Definition...30

I.4.2. Immunomodulators...30

I.4.3. Immunomodulators classification...30

a. Immunoadjuvants...30

b. Immunostimmulants...31

c. Immunosuppressants...31

I.4.4. Immunomodulation of atherosclerosis... ....31

I.5. Stachys species medicinal plants...32

I.5.1. Stachys mialhesi de Noé...32

a. Description...32

b. Botanical classification...33

c. Pharmacological properties...34

I.5.2. Stachys ocymastrum...34

a. Description...34

b. Botanical classification...35

c. Pharmacological properties...36

CHAPTER II: MATERIALS AND METHODS II.1. In vitro experiment II.1.1. Plant material...37

II.1.2. Evaluation of antioxidant activity...37

II.1.2.1. DPPH scavenging assay... 37

a. Principle...37

b. Procedure...39

 Reaction...39

 Measurement...39

II.2. In vivo experiments

II.2.1. Effect of Stachys mialhesi on homocysteine induced inflammation...39

II.2.1.1. Used materials...39

a. Chemical products...40

b. Animals...40

c. Blood and organs collection...40

II.2.1.2. Procedure...41

a. Experimental diet...41

b. Blood Biochemistry...42

c. Dissection protocol...42

d. Histological sections preparation...42

 Tissue fixation...42

 Infiltration and embedding in paraffin...43

 Staining...43

II.2.2. Evaluation of the immunomodulatory activity of the plant extracts...43

II.2.2.1. Used material...43

a. Stachys mialhesi extract...43

 Plant material...43

 Plant extraction...44

b. Stachys ocymastrum extract...44

 Plant material...44

 Plant extraction...44

c. Animals...45

d. Chemical products...45

II.2.2. 2. Experimental procedure...45

 Macrophage Phagocytosis by Carbon Clearance Assay...45

 Blood samples...46

 Results calculations...47

II.2.3. Statistical analysis...47

CHAPTER III: RESULTS AND DISCUSSION

III.1. Evaluation of the antioxidant activity of medicinal plants...48

III.1.1. Evaluation of Stachys mialhesi butanolic extract (Aerial parts)...48

III.1.2. Evaluation of Stachys Ocymastrum butanolic extract...49

III.1.3. Evaluation of Stachys mialhesi roots...50

Discussion of the results...53

III.2. Weight and diet experiments...53

III.2.1. Effect of L-methionine intake on weight and diet...53

a. weight variation...53

b. Diet variation...54

III.2.2. Effect of Stachys mialhesi intake on weight and diet...55

a. weight variation...55

b. Diet variation...55

Discussion of the results...56

III.3. hs-CRP measurement results...57

III.4. Histological studies...58

III.4.1. Microscope observations...58

III.4.1.1. Aorta...58

III.4.1.2. Cardiac muscle...72

III.4.1.3. Liver...72

III.4.1.4. Thymus...77

III.4.1.5. Spleen...77

Discussion of the results...83

III.5. Evaluation of the immunomodulatory activity of medicinal plants...87

III.5.1. Stachys mialhesi butanolic extract (Aerial parts)...87

Discussion of the results...88

III.5.2. Stachys ocymastrum butanolic extract (Aerial parts)...89

Discussion of the results...94

III. 6. Comparison of Stachys mialhesi and Stachys ocymastrum extracts...97

CONCLUSION AND PERSPECTIVES...99

REFERENCES...102

PAPERS...129

APPENDICES...158

FRENCH THESIS...168

ix List of publications

International publications

Benmebarek A., Zerizer S., Laggoune S., and Kabouche Z. (2013). Immunostimulatory activity of Stachys mialhesi de Noé. Allergy Asthma & Clinical Immunology 9:2 doi:10.1186/1710-1492-9-2.

Benmebarek A., Zerizer S., Laggoune S., and Kabouche Z. (2013). Effect of Stachys mialhesi de Noé on the inflammation induced by hyperhomocysteinemia in cardivascular diseases. Der Pharmacia Lettre 5 (2):212-223.

Benmebarek A., Zerizer S., Lakhel H., and Kabouche Z. (2014). Biphasic Dose Response Effect of Stachys Ocymastrum on the Reticuloendothelial System Phagocytic Activity. International Journal of Pharmacy and Pharmaceutical Sciences 6 (2): 534-537.

Benmebarek A., and Zerizer S. Homocysteine as a potential cancer marker and risk factor: review of possible carcinogenic mechanisms. Submitted.

National conferences

Zerizer S., Benmebarek A., Nacer M., Aribi B. Homocysteine as a potential cancer marker and risk factor: review of possible carcinogenic mechanisms. 2^èmes Journnées scientifiques LOST. Valorisation de plantes médicinales Algériennes. Université Constantine 1- Constantine, 24-25 Février 2014.

International conferences

Benmebarek A., Zerizer S., Laggoune S., and Kabouche Z.Immunostimulatory activity of Stachys mialhesi de Noé. 3^ème Journnée scientifique de l'ATT "Toxicologie, Environnement et Santé". Tabarka, Tunisia 03-05 February 2012.

x List of abbreviations

BHMT: Betaine-homocysteine methyl transferase CAT: Catalase

CCR2: Chemokine receptor CGL: Cystathione γ-lyase Cl^-: Chlorine

DMG: Dimethylglycine DNA: Deoxyribonucleic acid

dTMP:Deoxythymidine monophosphate acid DPPH: 1,1-Diphényl-2-Picryl-Hydrazyl free radical dTTP:Deoxythymidine triphosphate

dUMP:Deoxyuridine monophosphate EDRF: Endothelium derived relaxing factor EDTA:Ethylenediaminetetraacetic

FAD: Flavin adenine dinucleotide

GM-CSF: Granulocyte-macrophage colony-stimulating factor H2O2: Hydrogen peroxide

HAECs: Human aortic endothelial cells Hcy: Homocysteine

HDL:High-density lipoprotein HHcy: Hyperhomocysteinemia HLA: Human leukocyte antigen HOCL: Hypochlorous acid

hs-CRP: Hyper sensitive C-reactive proteine HTL: Homocysteine thiolactone

ICAM-1:Intercellular Adhesion Molecule 1 INF: Interferon

i.p: Intraperitoneal

LDL: Low density lipoproteins

xi MAT: Methionine adenosyl transferase MCP-1: Monocyte chemoattractant protein-1 M-CSF: Macrophage colony-stimulating factor MDA:Malondialdehyde

MPO: Myeloperoxidase MS: Methionine synthase

MTHF: Methylenetetrahydrofolate reductase

NADPH:Nicotinamide adenine dinucleotide phosphate NF-κB: Nuclear factor κ

NK: Natural killer NO: Nitric oxide

Nrf-2: Nuclear factor-erythroid 2-related factor 2 O2-: Superoxide anion

OH^-: Hydroxyl radicals

PAMP: Pathogen-associated molecular patterns PBS: Phosphate Buffer Saline

PDGF: Platelet derived growth factor PEG: Polyethylene glycol

PRR: Patern recognition receptor RES: Reticulo-endothelial system RNA:Ribonucleic acid

ROS: Reactive oxygen species S: Stachys

SAH: S-adenosylhomocysteine SAM: S-adenosylmethionine

SAHH: S-adenosylhomocysteine hydrolase SMCs: Smooth muscle cells

SOD: Superoxide dismutase TGF: Transforming growth factor tHcy: Total homocysteine

xii THF: Tetrahydrofolate reductase

THP-1: Human monocytic leukemia cell line TF: Tissue Factor

TNF: Tumor necrosis factor

VCAM-1: Vascular cell adhesion molecule 1 VEGF: Vascular endothelium growth factor VSMCs: Vascular smooth muscle cells

xiii List of figures

Figure 01. Mononuclear phagocytes in atherogenesis...6

Figure 02. The roles of T lymphocytes in atherogenesis...8

Figure 03. Life history of an atheroma...12

Figure 04. Molecular formula of homocysteine...13

Figure 05. Molecular formula of homocysteine forms...14

Figure 06. Homocysteine metabolism...15

Figure 07. Potential carcinogenic molecular effect of high homocysteine...24

Figure 08. Photography of Stachys mialhesi de Noé...33

Figure 09. Photography of Stachys ocymastrum...35

Figure 10. Chemical structure of DPPH free radical...37

Figure 11. DPPH radical reaction with a phenol...38

Figure 12. DPPH radical scavenging activity of S.mialhesi (Aerial parts) and quercetin...48

Figure 13. Graph of the IC50 value of S.mialhesi (Aerial parts)...49

Figure 14. DPPH radical scavenging activity of S.ocymastrum (Aerial parts) and quercetin...49

Figure 15. Graph of the IC50 value of S.ocymastrum (Aerial parts)...50

Figure 16. DPPH radical scavenging activity of S.mialhesi roots extract and quercetin...50

Figure 17. Graph of the IC50 value of S.mialhesi roots extract...51

Figure 18. Comparison of DPPH radical scavenging activity of of S.mialhesi, S.ocymastrum, S.mialhesi roots, and quercetin...52

Figure 19. Effect of L-methionine intake on mice weight during 21 days application...54

Figure 20. Effect of L-methionine intake on mice diet during 21 days application...54

Figure 21. Effect of L-methionine and S.mialhesi intake on mice weight during 21 days application...55

Figure 22. Effect of L-methionine and S.mialhesi intake on mice diet during 21 days application...56

Figure 23. The interaction of L-methionine and S.mialhesi on the plasm ultra sensitive CRP in mice during 21 days application...57

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Figure 24. Histological section of arch aorta, 21 days of oral methionine application (200 mg/kg/day), hematoxylin eosin staining (x100)...59 Figure 25. Histological section of arch aorta, control’s hematoxylin eosin staining

(x400)………59 Figure 26. Histological section of arch aorta, 21 days of oral methionine application (200 mg/kg/day), hematoxylin eosin staining (x1000)...60 Figure 27. Histological section of arch aorta, control’s hematoxylin eosin staining

(x400)...60 Figure 28. Histological section of arch aorta, oral methionine (200 mg/kg/day) + S.mialhesi extract (50mg/kg/day) application, hematoxylin eosin staining (x400)...61 Figure 29. Histological section of arch aorta, positive control's hematoxylin eosin staining (x400)...61 Figure 30. Histological section of thoracic aorta, 21 days of oral methionine application (200 mg/kg/day), hematoxylin eosin staining (x400)...62 Figure 31. Histological section of thoracic aorta, control's hematoxylin eosin staining (x100)...62 Figure 32. Histological section of thoracic aorta, 21 days of oral methionine application (200 mg/kg/day), hematoxylin eosin staining (x100)...63 Figure 33. Histological section of thoracic aorta, control's hematoxylin eosin staining (x100)...63 Figure 34. Histological section of thoracic aorta, oral methionine (200 mg/kg/day) + S.mialhesi extract (50mg/kg/day) application, hematoxylin eosin staining (x100)...64 Figure 35. Histological section of thoracic aorta, positive control’s hematoxylin eosin staining (x400)...64 Figure 36. Histological section of the abdominal aorta, 21 days of oral methionine

application (200 mg/kg/day), hematoxylin eosin staining (x100)...65 Figure 37. Histological section of the abdominal aorta, control's hematoxylin eosin

staining (x400)...65 Figure 38. Histological section of abdominal aorta, 21 days of oral methionine application (200 mg/kg/day), hematoxylin eosin staining (x400)...66

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Figure 39. Histological section of the abdominal aorta, control’s hematoxylin eosin

staining (x400)...66 Figure 40. Histological section of the abdominal aorta, oral methionine (200 mg/kg/day) + S.mialhesi extract (50mg/kg/day) application, hematoxylin eosin staining (x400)...67 Figure 41. Histological section of the abdominal aorta, positive control’s hematoxylin eosin staining (x400)...67 Figure 42. Histological section of the iliac aorta, 21 days of oral methionine application (200 mg/kg/day), hematoxylin eosin staining (x100)...68 Figure 43. Histological section of the iliac aorta, control's hematoxylin eosin staining (x400)...68 Figure 44. Histological section of the iliac aorta, 21 days of oral methionine application (200 mg/kg/day), hematoxylin eosin staining (x400)...69 Figure 45. Histological section of the iliac aorta, control’s hematoxylin eosin staining (x400)...69 Figure 46. Histological section of the iliac aorta, oral methionine (200 mg/kg/day) + S.mialhesi extract (50mg/kg/day) application, hematoxylin eosin staining (x400)...70 Figure 47. Histological section of the iliac aorta, positive control’s hematoxylin eosin staining (x400)...70 Figure 48. Histological section of the iliac aorta, oral methionine (200 mg/kg/day) + S.mialhesi extract (50mg/kg/day) application, hematoxylin eosin staining (x400)...71 Figure 49. Histological section of the iliac aorta, positive control’s hematoxylin eosin staining (x400)...71 Figure 50. Histological section of the cardiac muscle. 21 days of oral methionine

application (200 mg/kg/day), hematoxylin eosin staining (x400)...73 Figure 51. Histological section of the cardiac muscle, control’s hematoxylin eosin

staining (x400)...73 Figure 52. Histological section of the cardiac muscle. 21 days of oral methionine (200 mg/kg/day) + S.mialhesi extract (50mg/kg/day) application, hematoxylin eosin staining (x400)...74

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Figure 53. Histological section of the cardiac muscle, positive ontrol’s hematoxylin eosin staining (x100)...74 Figure 54. Histological section of the Liver. 21 days of oral methionine application (200 mg/kg/day), hematoxylin eosin staining (x400)...75 Figure 55. Histological section of the liver, control’s hematoxylin eosin staining

(x400)...75 Figure 56. Histological section of the liver, 21 days of oral methionine (200 mg/kg/day) + S.mialhesi extract (50mg/kg/day) application, hematoxylin eosin staining (x100)...76 Figure 57. Histological section of the liver, positive control’s hematoxylin eosin staining (x400)...76 Figure 58. Histological section of the thymus. 21 days of oral methionine application (200 mg/kg/day), hematoxylin eosin staining (x1000)...78 Figure 59. Histological section of the thymus, 21 days of oral methionine application (200 mg/kg/day), hematoxylin eosin staining (x400)...78 Figure 60. Histological section of the thymus, 21 days of oral methionine application (200 mg/kg/day), hematoxylin eosin staining (x400)...79 Figure 61. Histological section of the thymus, control’s hematoxylin eosin staining

(x100)...79 Figure 62. Histological section of the thymus, 21 days of oral methionine (200 mg/kg/day) + S.mialhesi extract (50mg/kg/day) application hematoxylin eosin staining (x400)...80 Figure 63. Histological section of the thymus, positive control’s hematoxylin eosin staining (x400)...80 Figure 64. Histological section of the spleen. 21 days of oral methionine application (200 mg/kg/day), hematoxylin eosin staining (x1000)...81 Figure 65. Histological section of the spleen, control’s hematoxylin eosin staining

(x100)………81 Figure 66. Histological section of the spleen, 21 days of oral methionine (200 mg/kg/day)

+ S.mialhesi extract (50mg/kg/day) application, hematoxylin eosin staining (x400)...82 Figure 67. Histological section of the spleen, positive control’s hematoxylin eosin staining

(x100)...82

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Figure 68. Effect of S. mialhesi extract on phagocytic activity...87

Figure 69. Effect of S. mialhesi extract on the carbon clearance rate...88

Figure 70. The phagocytic activity of mice treated with S. ocymastrum extract...90

Figure 71. The carbon clearance rate of mice treated with S.ocymastrum extract...92

Figure 72. The corrected phagocytic index α of mice treated with S.ocymastrum extract..92

Figure 73. Organ weight to body weight ratio (Liver, Spleen) of mice treated with S.ocymastrum extract...92

Figure 74. Biphasic dose-response effect of S.ocymastrum on the phagocytic activity of the reticuloendothelial system...93

Figure 75. Biphasic dose-response effect of S.ocymastrum on the carbon clearance rate...93

Figure 76. Biphasic dose-response effect of S.ocymastrum on the on the corrected phagocytic index α...94

Figure 77. Comparison of the effect of S.mialhesi and S.ocymastrum on the phagocytic activity of the reticuloendothelial system...98

Figure 78. Comparison of the effect of S.mialhesi and S.ocymastrum on the carbon clearance rate...98

xviii List of tables

Table 01. Classification of Hyperhomocysteinemia...17

Table 02. Mice food composition...41

Table 03. Treatment of mice...41

Table 04. Treatment of mice...46

Table 05. Treatment of mice...46

Table 06. Plants extracts IC50 Values...51

Table 07. Extracts inhibition pourcentage...52

Table 08. 21 days control's group (F)...160

Table 09. 21 days of oral methionine application (M)...161

Table 10. 21 days of oral methionine and S.mialhesi extract application (MP)...162

Table 11. 21 days postitive control group (P)...163

Table 12. Carbon Clearance Essay by S.mialhesi...164

Table 13. Carbon Clearance Essay by S.ocymastrum...165

Table 14. Carbon Clearance Essay by S.ocymastrum...166

Table 15. Carbon Clearance Essay by S.ocymastrum...167

1 Introduction

Elevated levels of plasma homocysteine (Hcy) have been associated with cardiovascular risk in multiple epidemiological studies (Charalambos et al., 2009) and have been recognized as a strong, independent, and causal risk factor for atherosclerosis (McCully, 1996; Wald et al., 2002). Studies from in vitro and in vivo investigations have suggested that generation of potent reactive oxygen intermediates, such as superoxide anion radical, hydrogen peroxide and impaired production of endothelial nitric oxide, are central mechanisms by which vascular exposure to elevated levels of Hcy may mediate long-term oxidative damage at the vascular interface (Harker et al., 1974; Stamler, 1993). These reactive oxygen intermediates may be a target for therapy development and their inhibition might be a strategy in preventing cardiovascular diseases.

In addition, single C reactive protein (CRP) measurement is one of the predictors of cardiovascular events (Sheta et al., 2009) and CRP was established as a sensitive marker of inflammation a long time ago (Tillett and Francis, 2009). Accumulating evidence from various epidemiological prospective studies indicates that CRP is an important marker of future cardiovascular risk (Danesh et al., 1998; Koenig et al., 1999; Haidari et al., 2001;

Folsom et al., 2002; Speidl et al., 2002). Also, CRP deposition in human atherosclerotic lesions is now well established (Torzewski et al., 1998; Zhang et al., 1999; Yasojima et al., 2001). The molecule is detectable in the arterial intima in the earliest stages of atherogenesis and accumulates with lesion progression. Also, CRP has been identified as a powerful cardiovascular risk marker (Koenig et al., 1999; Ridker et al., 2000)and may also be causally involved in atherogenesis (Blake and Ridker, 2003; Manolov et al., 2003;

Ridker et al., 2003; Jialal et al., 2004; Guthikonda and Haynes, 2006).

Furthermore, treatment of hyperhomocysteinemia (HHcy) is primarily through vitamin supplementation; folic acid and vitamins B6 and B12 are the mainstay of therapy (Guthikonda and Haynes, 2006). It has been demonstrated in vivo that vitamin therapy in rats has reduced Hcy plasma levels (Zerizer and Naimi, 2004), therefore, substituting

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vitamin therapy by phytotherapy or combining the two treatments could be a novel therapy in treating HHcy as Guthikonda and Haynes (2006) reported that atherogenesis is promoted through increased oxidant stress by elevated levels of plasma Hcy.

In addition to being an important risk factor in cardiovascular diseases HHcy may also be implicated in cancer biology. A review should be undertaken in order to provide a background for understanding the potential role of Hcy as a cancer risk factor and a cancer marker. A review should also present a summary of the mechanism aspects that may be implicated in HHcy induced cacinogenesis.

Moreover, the immune system protects against destructive forces either from outside the body (bacteria, viruses, and parasites) or from within (malignant and autoreactive cells). It comprises two functional divisions that work together in a coordinated manner (Goldsby et al., 2000). The innate immune system consists of cellular components, soluble factors, physical barriers and the reticuloendothelial system (RES) (Goldsby et al., 2000). It provides early host defense against infections before the development of an adaptive immune response (Borgdan et al., 2000). The adaptive immune system produces a specific reaction and immunologic memmory to each pathogen and comprises cellular components and soluble factors (Goldsby et al., 2000). The RES consists of the phagocytic cells such as monocytes and macrophages (Brannon-Peppas and Blanchette, 2004) that kill the invading organism by phagocytosis, and this latter is a multi- step process that begins by engulfing the organism and ends with modification and chemical breakdown of its structural components (Borgdan et al., 2000).

Further, immunomodulation is the regulation and modulation of immunity either by enhancing or by reducing the immune response (Shivaprasad et al., 2006). An immunomodulator can influence any constituent or function of the immune system in a specific or nonspecific manner including both innate or adaptive arms of the immune response (Agarwal and Singh, 1999). An immunomodulator can cause immunostimulation by stimulating effector cells or production of their metabolic inducers or by inhibiting the

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immunity limiting factors. Immunosuppression can be achieved by stimulating the inhibitor cells and humoral factors, or inhibition of effector cells (Katiyar et al., 1997).

Immunostimulators have been known to support T-cell function, activate macrophages, granulocytes, complement and natural killer cells apart from affecting the production of various effectors molecules generated by activated cells (Paraimmunity) (Dash et al., 2006). It is expected that non-specific effects offer protection against different pathogens, including bacteria, fungi, and viruses and constitute an alternative to conventional chemotherapy (Atal et al., 1986).

Furthermore, a large number of plants have been shown to have potential immunity.

Some medicinal plants have even been shown to exert immunomodulatory and anti-cancer activity (Verma et al., 2010a; Verma et al., 2010b; Verma et al., 2011). The genus Stachys (Lamiaceae) includes about 200 to 300 species in the world (Rechinger and Hedge, 1986) and is considered to be one of the largest genera of this family.

Pharmacological studies have confirmed that extract of plants belonging to the genus Stachys exert significant antibacterial, anti-inflammatory, antitoxic, anti-nephritic, antihepatitis, anti-anoxia and hypotensive activity, antispasmodic, anti-asthma and anti- rheumatic activities (Makhlouf et al., 2002; Vongtau et al., 2004). In Iran, the aerial parts of S. inflata Benth are used to treat infection, asthma, rheumatic and other inflammatory disorders (Maleki et al., 2001). S. lavandulifolia Vahl was used as an anxiolytic and sedative (Amin, 1991). The study of Amirghofran et al. (2007) showed inhibitory effects of S. obtusicrena on both cellular and humoral immune responses and suggests that this effect may in part be due to the induction of apoptosis in proliferative lymphocytes.

Accordingly, and because of the various biological interests in the secondary metabolites (flavonoids, diterpenes, phenylethanoid glycosides) of Stachys genus (Quezel and Santa, 1963), we can use their crude extracts to assesss their therapeutic effect and evaluate their potential immunomodulatory activity. Also, they can be used as therapeutic substances in order to prevent the occurence of different pathologies.

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To that end and in the present study, our objectives were to:

 Provide a background for understanding the potential role of Hcy as a cancer marker and a cancer risk factor, and review the potential mechanism aspects that may be implicated in HHcy induced carcinogenesis.

 Evaluate the antioxidant activity of the aerial parts of S. mialhesi and S. ocymastrum, and the roots of S. mialhesi in order to compare their activities with

reference molecules.

 Evaluate the effect of L-methionine and S. mialhesi on the weight and diet consumption of mice.

 Examine the atherogenic effect of L-methionine on the aortic, heart, liver, thymus and spleen histology of mice to confirm the angiotoxic and toxic action of Hcy.

 Examine the effect of Hcy on the vascular inflammation and atherogenesis through the measurement of the plasma hs-CRP marker.

 Evaluate the protective and preventive effect of S. mialhesi extract on the angiotoxic and toxic action of L-methionine and on hs-CRP as a biochemical inflammation marker.

 Assess the immunomodulatory effect of the butanolic extracts obtained from S. mialhesi, and S. ocymastrum using carbon clearance assay.

 Compare the immunomodulatory effect of S. mialhesi, and S. ocymastrum extracts in order to determine the most effective one as a therapeutic substance.

Chapter I:

Literature review

5 I.1. Atherogenesis

I.1.1. Endothelium activation and mononuclear phagocytes recruitment

The initial phase of the atherosclerotic plaque formation is characterized by the infiltration of the arterial wall by monocytes and lymphocytes. This recruitment requires the endothelium activation by expressing leukocyte adhesion molecules (Hansson, 2001).

These adhesion molecules, such as selectins (E-selectin and P-selectin) and adhesion molecules (ICAM-1 and VCAM-1 essentially), constitute receptors for integrins and glycoconjugates present on the surface of monocytes and T cells (Beaudeux et al., 2004).

Thus, the extravasation of low density lipoprotein (LDL) and their oxidation (Caligiuri, 2004), leads to the activation of endothelial cells and smooth muscle cells (SMC) through the expression of the transcription factor NF-kB. This mechanism amplifies the synthesis of different molecules including cytokines (IL-8), chemokines (MCP-1), and adhesion molecules. This cellular activation results in the attachment of leukocytes and monocytes/macrophages to the endothelilal cells and their access to the vascular wall (Lacolley et al., 2012) (Figure 01).

Another process that also results in the expression of factors such as VCAM-1 is the decrease in nitric oxide concentrations (NO), a potent vasodilator and an anti-inflammatory released by the endothelium and that inhibits the expression of VCAM-1. Other pro- inflammatory and pro-atherogenic changes that may occur; include the upregulation of intercellular adhesion molecule (ICAM-1) and an increase in the production of proteoglycan (Verma, 1996).

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Figure 01. Mononuclear phagocytes in atherogenesis (Libby, 2002)

I.1.2. Macrophages activation and expression of scavenger receptors

Monocytes/macrophages in the atherosclerotic plaque have the ability to multiply.

The M-CSF which is a hematopoietic factor that contributes to the differentiation and proliferation of monocytes is locally produced by the endothelial cells and smooth muscle cells (SMCs) of human atherosclerotic plaque and participates in the atherosclerotic process (Toussaint et al., 2003).

7

Thus, the macrophages are activated and secrete proliferation and growth factors (M-CSF), pro-inflammatory cytokines (IL-18), and proteases and free radicals (Lacolley et al., 2012). During this step there is an upregulation of the pattern recognition receptors (PRR) of the innate immune system, including scavenger receptors and toll-like receptors.

Scavenger receptors internalize a large repertoire of molecules and particles featuring Pathogen Associated Molecular Patterns motifs (PAMP). Bacterial endotoxins, apoptotic cell debris and oxidized LDL particles are internalized and destroyed following this path (Hansson, 2005). Accordingly, after fixation of oxidized LDL, macrophages internalize them in large quantities through scavenger receptors (Adams et al., 2001). In contrast to the conventional LDL receptors (Brown Goldstein receptors), the oxidized LDL receptors are not negatively regulated by the intracellular content in cholesterol (Adams et al., 2001).

Consequently, the quantity of oxidized LDL accumulated within macrophages exceeds their capacity of degradation. These macrophages are transformed into foam cells, that remain in the intima and are the first atherosclerotic lesions. Macrophages then trigger a slow apoptotic process that deposits cellular debris, microparticles containing tissue factor, and phosphatidylserine residues in the atherosclerotic plaque (Caligiuri, 2004).

Macrophages can contribute directly to lesions progression via the lipoproteins oxidation. The progression can also be through the production of growth factors that act on the SMCs proliferation, or via the extracellular matrix degradation by metalloprotease.

These effects are fatal because collagen degradation may lead to the rupture of the atherosclerotic plaque (Caligiuri, 2004). In the atheroma, macrophages produce and release IL-12, a cytokine that promotes Th1 differentiation pathway. Also, the endothelial cells express P- and E-selectin which recruit Th1 lymphocytes (Caligiuri, 2004). These cellular immune responses are involved in the atherosclerotic process, because CD4⁺ and CD8⁺ cells are present in the lesions at each step of the process (Beaudeux et al., 2004). T cells are activated during the antigen presentation by macrophages. This activation induces cytokines secretion, including IFN-γ (Beaudeux et al., 2004), a Th1 pathway cytokine (Caligiuri, 2004), and TNFα and β, which implifies the inflammatory response (Beaudeux et al., 2004).

8

The release of IFN-γ by Th1 cells may have complex effects on atherosclerosis progression . This is due to the fact that IFN-γ inhibits collagen synthesis and can thereby reduce the fibrous cap resistance and promote the atherosclerotic plaque rupture (Caligiuri, 2004). IFN-γ increases the synthesis of inflammatory cytokines such as TNF and IL-1.

Therefore, two types of responses are distinguished in the atherosclerotic lesion, while the first is a Th1 cell response that activates macrophages and initiate a pro-inflammatory response, the second one is a Th2 cell response that initiates an anti-inflammatory response (Hansson, 2005) (Figure 02).

Figure 02. The role of T lymphocytes in atherogenesis (Libby, 2002)

9

I.1.3. Proatherogenic and antiatherogenic cytokines

The presence of interleukins has been indicated in atherosclerotic lesions and circulating levels of certain interleukin have been correlated with atherosclerosis. In vivo studies showed the role of interleukins in processes that are associated with lesion formation. In addition, a causal contribution of several interleukins has been confirmed by changes in the extent of atherosclerosis in mice that are deficient or transgenic for specific interleukins (Hauer, 1977).

In a healthy artery the contribution of anti and pro-inflammatory interleukins are in balance, whereas in atherosclerosis affected arteries, the pro-inflammatory interleukins predominate over the anti-inflammatory cytokins, contributing to enhanced inflammation and atherosclerosis. Pro-inflammatory cytokines comprise TNFα, IL-1, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18 and IFNγ (Hauer, 1977; Tedgui and Mallat, 2004), whereas the anti-inflammatory cytoklines comprise IL-1 receptor antagonist, IL 9 and IL-10 (Hauer, 1977).

During the adaptive immune response the development of a dysbalance between pro- and anti-inflammatory cytokines, including interleukins is largely depending on the differentiation of CD4⁺ naive T helper cells (Th0) towards either T helper 1 (Th1) or T helper 2 (Th2) cells. The Th1 cells primarily produce pro-inflammatory cytokines, whereas Th2 cells primarily produce anti-inflammatory cytokines. One of the key factors that stimulate the differentiation of naive T helper cells toward Th1 cells is IL-12. Il-12 has been shown to play a causal role in the development of atherosclerosis in apoE^-/- mice. In addition to its contribution to Th1 cell development IL-12, especially in combination with the pro-inflammatory IL-18, is able to directly stimulate macrophages, resulting in enhanced production of cytokines (Hauer, 1977).

10

Another interleukin that may contribute to an enhanced inflammatory environment during atherosclerosis is IL-17. It induces the expression of many pro-inflammatory cytokines by a variety of cell types (Hauer, 1977). For instance, it enhances the production of IL-6 and IL-8 by stromal cells, and the expression of IL-1, IL-6 and TNF-α by macrophages. Furthermore, the binding of IL-17 to its receptor stimulates NF-κB activity, which is a nuclear factor that regulates the expression of many interleukins (IL-1, IL-2, IL- 3, IL-6, IL-8 and IL-12) (Hauer, 1977). Thus, activated immune cells in the plaque produce inflammatory cytokines that induce the production of substantial amounts of IL-6. IL-6, in turn, stimulates the production of large amounts of acute-phase reactants, including C- reactive protein (CRP), serum amyloid A, and fibrinogen, especially in the liver. Although cytokines at all steps have important biological effects, their amplification at each step of the cascade makes the measurement of downstream mediators such as CRP particularly useful for clinical diagnosis (Hansson, 2005).

The presence of C-reactive protein (CRP) is used clinically for several years as a nonspecific marker of inflammatory processes and supported by the arrival of the high- sensitive assays (hs-CRP). hs-CRP is emerging as one of the strongest independent predictors of cardiovascular disease. hs-CRP also predicts cardiovascular risk in various clinical settings, including men and women that have no cardiovascular disease manifestestations (Ridker et al., 2000a, 2000b). The human recombinant hs-CRP, has known concentrations that can predict vascular diseases. It has a multitude effects on the endothelial biology, promoting a pro-inflammatory and pro-atherosclerotic phenotype (Verma et al., 2004).

I.1.4. Atheroma progression and complication

The atherosclerotic plaque develops long before altering the vascular caliber, this is due to the fact that the vessel adapts through a compensatory enlargement called eccentric vascular remodeling. When the intimal mass exceeds 40% of the total wall surface, the vascular remodeling is no longer sufficient to hold the plaque. It develops then at the

11

expense of the arterial lumen which leads to progressive stenosis (Mann and Davies, 1996).

The plaque develops asymptomatically for years in the intima before reaching maturity. At this point, the lesion is a focal thickening, or plaque, that is formed of an atheroma, consisting of lipidic crystals and many cell debris, embedded in a thick layer of collagen (sclerosis) (Wyplosz, 1960). It essentially contains two cell types:

1) Smooth muscle cells that have lost most of their contractile phenotype and acquired a secretory phenotype;

2) Blood leukocytes, mostly monocytes/macrophages but also some rare T lymphocytes and granulocytes, plasma cells and monocytes (Wyplosz, 1960).

The migration, proliferation, and extracellular matrix synthesis by SMCs is involved in the plaque stenotic action (Mann and Davies, 1996). The SMCs experience an increased capacity of protein synthesis. They produce a large amount of connective tissue matrix, consisting of collagen, elastic fibers and proteoglycans. SMCs can also transform into foam cells, but the mechanism is still poorly understood (Léoni, 2001).

Unlike the normal arterial wall that is not vascularized, the plaque is irrigated by neovessels resulting from the adventitia; and the lumen. The mature plaque follows several types of intima alterations, and an evolution of four stages can be distinguished: the initial lesion (foam cell accumulation), the fatty streak (foam cells grouping in clusters), pre- atheroma (extracellular lipid deposition) and atheroma (foam cells and deposits grouping in the absence of multiple conjunctiva) (Wyplosz, 1960).

There are two major causes of coronary thrombosis: plaque rupture and endothelial erosion. Plaque rupture, which is detectable in 60 to 70% of cases, is dangerous because it exposes prothrombotic material from the core of the plaque phospholipids, tissue factor, and platelet-adhesive matrix molecules to the blood. Ruptures preferentially occur where the fibrous cap is thin and partly destroyed (Hansson, 2005).

12

At these sites, activated immune cells are abundant. They produce numerous inflammatory molecules and proteolytic enzymes that can weaken the cap and activate cells in the core, transforming the stable plaque into a vulnerable, unstable structure that can rupture, induce a thrombus, and elicit an acute coronary syndrome (Hansson, 2005). The reduction or interruption of the blood flow that results leads to ischemic events (myocardial infarction, stroke, angina pectoris, and intermittent claudication), which can be fatal to the myocardium or brain (Wyplosz, 1960) (Figure 03).

Figure 03. Atheroma progression (Libby, 2002)

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I.2. Homocysteine origin, structure and metabolism

I.2.1. Origin and forms of homocysteine

Hcy is a sulfur amino acid that is not found in the protein structure, but which is an important intermediate in the function of methyl donor in methionine metabolism. Hcy is also important in the metabolism of methionine to the other sulfur amino acids such as cysteine (EL Mabchour, 2010). Our body synthesizes about 20 mmol of Hcy per day but a very low concentration is found in the blood as free and protein-bound forms (Mouchabac, 2008).

Figure 04. Molecular formula of Hcy (Raisonnier, 2011)

A small amount exists as free reduced Hcy in plasma (Seshadri et al., 2002) (Figure 04), and constitutes only 1% -2% of total Hcy, whereas 98% -99% of total Hcy exists in the form of disulfide bonds: 75% -80% of total Hcy is linked to the reactive cysteine residues of proteins. While other Hcy are in the form of free disulfide bonds, homocysteine homocystine (Hcy-Hcy), dimeric oxidized form of Hcy and cysteine homocysteine (Hcy- Cys) (Chen, 2009). Homocysteine thiolactone (HTL), a Hcy cyclic thioester represents 0.29% to 28% of total Hcy in plasma and urine. Hcy can also bind to proteins by amide bonds (-N-Hcy protein). N-Hcy-hemoglobin and N-Hcy-albumin comprise 75% and 22%

of total N-Hcy-protein respectively (Chen, 2009). The Hcy is, therefore, the total plasma concentration of all forms of Hcy after its reduction (Seshadri et al., 2002). (Figure 05)

14

Figure 05. Molecular formula of Hcy forms (Chen, 2009)

I.2.2. Metabolism of homocysteine

Hcy is produced during the catabolism of methionine from plant and animal proteins (Caussé, 2008). It is synthesized by all cells of the body and can be catabolized via two pathways: remethylation pathway and transulfuration pathway (Grunitzky et al., 2008) (Figure 06).

15

Figure 06. Hcy Metabolism (Benmebarek and Zerizer, 2014)

a. Remethylation pathway

This pathway involves the transfer of a methyl group from 5-methyl- tetrahydrofolate (5-CH3THF) to Hcy, so that it is remethylated to methionine. This reaction is catalysed by methionine synthase (MS) and uses vitamin B12 as cofactor (Plazar and

16

Jurdana, 2010). The 5-CH3THF formation depends on the enzyme N5, N10 methylenetetrahydrofolate reductase (MTHFR), which catalyzes the reduction of

N5, N10 methylenetetrahydrofolate (5,10-CH2THF) formed from tetrahydrofolate (THF) (EL Mabchour, 2010). This enzyme uses the flavin adenine dinucleotide (FAD) as a cofactor (Chen, 2009).

The serine-glycine interconversion leads to the N5, N10 methylene THF synthesis by the hydroxymethylase serine in the presence of THF. The formed N5, N10 methylene THF is either a methyl donor for the synthesis of thymidylate or is reduced to N5 methyl THF. So, in the presence of N5, N10 methylene THF as a coenzyme, the methylation of desoxyuridilate monophosphate (dUMP) by thymidylate synthase leads to the formation of dTMP, which is incorporated into DNA (Carreras and Santi, 1995).

In the liver, a parallel remethylation pathway independent from folate and cobalamin uses the conversion of betaine to dimethylglycine (DMG) under the action of betaine-homocysteine methyl transferase (BHMT) (EL Mabchour, 2010). Methionine adenosyl transferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM) from ATP and methionine. All organisms have one or two of the three MAT isoforms: MATI, MATII and MATIII. The S-adenosylmethionine is the universal donor of the methyl group that is necessary for the maintenance of cellular methylation: DNA, RNA, proteins, and lipids. This methyl group transfer, which is catalyzed by methyltransferases, induces the synthesis of S-adenosylhomocysteine (SAH). S-adenosyl homocysteine hydrolase (SAHH) catalyzes the conversion of SAH to adenosine and Hcy. This is the only reversible reaction of all the chemical reactions involved in Hcy metabolism (Chen, 2009).

b. Transsulfuration pathway

In this step which takes place mainly in the liver and pancreas, and in the presence of excess methionine or increased need in cysteine, the thiol group of the Hcy side chain is transferred to a serine (Magné, 2009). This condensation will then lead to the formation of

17

cystathionine. This reaction catalyzed by cystathionine β-synthase, requires vitamin B6 as a cofactor (Buysschaert and Hermans, 2003). Cystathionine is cleaved and deaminated to cysteine and α-ketobutyrate by Cystathione γ-lyase (CGL). These irreversible reactions require the presence of pyridoxal 5 'phosphate or vitamin B6 cofactors (Chen, 2009).

Cysteine will form a major antioxidant sulfur amino acid, the glutathione, and other amino acids such as taurine, and / or it will be excreted in the urine (Mouchabac, 2008).

I.2.3. Classification of hyperhomocysteinemia

The plasma Hcy concentrations in a normal subject is between 5 and 15 micromol/l.

The elevation of that concentration determines HHcy which classification is illustrated in Table 01 (Mouchabac, 2008). Hcy plasma levels increase with age and are usually higher in males. In female subjects, Hcy rises significantly after menopause (Buysschaert and Hermans, 2003). In children (3-14 years) normal concentrations are lower and are around 6 mmol / l (Mouchabac, 2008).

Table 01. Classification of HHcy (Mouchabac, 2008) [Hcy] (μmol/l) Moderate HHcy

intermediate HHcy severe or major HHcy

15 à 30 30 à 100

> 100

I.2.4. Pathogenicity of hyperhomocysteinemia

a. Chemokine production

It has been shown in vitro that Hcy can stimulate the production of many proinflammatory factors such as MCP-1, a chemokine for monocytes, and IL-8, a chemokine for T lymphocytes and neutrophils (Peyrin-Biroulet, 2007). These chemokines induce the recruitment and attachment of leukocytes to the endothelium which explains

18

why high plasma concentrations of MCP-1 and IL-8 may contribute to the development of atherosclerotic lesions (Zeng et al., 2004; Wang and Karmin, 2001).

Hcy specifically stimulates the secretion of MCP-1 and IL-8 by monocytes in the culture of human whole blood (Zeng et al., 2004; Wang and Karmin, 2001), in human endothelial aortic cells (HAECs) (Poddar et al., 1997), and in cultured human vascular smooth muscle cells (Wang and Karmin, 2001). In cultures of human endothelial cells, MCP-1 are expressed via the activation of the signaling pathway p38 MAP kinase, which leads to monocyte chemotaxis. While in cultures of vascular smooth muscle cells (VSMCs), their expression is mediated by the activation of the nuclear factor NF-kB (Wang and Karmin, 2001). The action of MCP-1 is exerted following its interaction with CCR2 receptor. This receptor is located on the surface of monocytes and whose expression is stimulated in THP-1 cells and human peripheral blood monocytes by Hcy. Thus this stimulation may cause the recruitment and infiltration of monocytes to the artery wall during atherosclerosis (Wang and Karmin, 2001). However, The expression of TNF-α, GM-CSF, IL-1β and TGF-β is not affected by Hcy (Poddar et al., 1997).

b. Oxidative stress production

Oxidative stress is involved in Hcy mediated cardiovascular risk. Hcy induces the production of free radicals which are the causal mechanisms of cell destruction in the vascular wall (Wang and Karmin, 2001). Free radicals induce also an increase in chemokine secretion (Zeng et al., 2004). Hcy contains a thiol group (Del, 2000), it is quickly oxidized, leading to the formation of Hcy, mixed disulfides and HTL. The oxidation of the-SH group generates superoxide anion O2-, hydrogen peroxide H2O2 and hydroxyl radicals OH^- (Zittoun, 1998). These components are extremely important in the Hcy induced production of MCP-1 and IL-8 in the work of Zeng et al. (2004). Recent results suggested that Hcy increased the production of O2- in VSMCs. O2- and H2O2 also stimulate the expression of CCR2 receptor on monocytes (Wang and Karmin, 2001).

19

Superoxide dismutases enzymes are capable of removing superoxide anions by forming an oxygen molecule and a hydrogen peroxide molecule with two superoxides.

(Favier, 2003). Superoxide dismutase (SOD) can reduce the cytotoxic effect of Hcy caused by the formation of O2-. It can reverse the stimulatory effect of Hcy on the NF-κB activation, on the MCP-1 expression in VSMCs and MCP-1 and IL-8 expression in cultured human whole blood (Zeng et al., 2004).

Extracellular SOD elevation may represent an antioxidant that protects against oxidative stress generated by Hcy (Del, 2000). The addition of SOD could prevent from the elevation of Hcy induced O^2- cellular levels. It could also attenuate CCR2 expression on monocytes and reduces the Hcy stimulated high binding activity of CCR2 with MCP-1 (Wang and Karmin, 2001). Hcy induced MCP-1 and IL-8 secretion was completely reduced after treatment with De2SO4, an OH^- radical scavenger, but only partially reduced with PEGSOD or PEG-CAT, an O2- or H2O2 scavengers. This suggests that although the three types of free radicals are involved, the OH^- radicals have a more important role in Hcy induced MCP-1 and IL-8 secretion in the whole blood (Zeng et al., 2004).

c. MPO production

Elevated plasma Hcy levels induced increase myeloperoxidase (MPO) levels in whole blood cell culture (Zeng et al., 2004). MPO is considered a marker for cardiovascular diseases. It is particularly cytotoxic to endothelial cells and fibroblasts which are capable of binding and internalizing it. In contact with the endothelium, neutrophils can adhere and be activated in vivo. Neutrophils degranulation releases MPO in contact with neighboring cells such as endothelial cells or fibroblasts. Within endothelial cells, MPO remains active and can utilize the hydrogen peroxide to produce hypochlorous acid (HOCl), a strong oxidant capable of oxidizing and chlorinating many molecules (Serteyn et al., 2003).

20

In the inflammatory site, MPO activates enzymes such as progelatinase and procollagenase. This activation leads to connective tissues destruction , and is attributed to the oxidizing action of HOCl. HOCl and MPO also activate NF-kB that can increase the expression of inflammatory mediators (Serteyn et al., 2003). Thus, Hcy induced MPO elevation can be one of the mechanisms by which Hcy causes the initiation and progression of vascular disease. Because IL-8 was a strong promoter of MPO increase, the Hcy induced IL-8 secretion by monocytes in whole blood cell culture can play a vital role in the regulation of MPO levels. This regulation of MPO revealed a novel mechanism in Hcy induced cardiovascular diseases progression (Zeng et al., 2004).

d. NO production and metabolism

High levels of Hcy alter the vasodilatory properties of endothelial cells via decreasing EDRF (endothelium derived relaxing factor) (Zittoun, 1998). Also, their antithrombotic properties are altered through lowering nitric oxide bioavailability. Under normal conditions, endothelial cells detoxify Hcy via the release of nitrogen monoxide which interacts with its thiol group and generates the S-nitrosohomocystéine (Zittoun, 1998). The latter has a vasodilatory and anti-platelet action (Seshadri et al., 2002) and does not generate H2O2 (Laraqui, 2006). Exposure of cultured endothelial cells to Hcy has a biphasic effect on the production of nitric oxide (NO). Initially, there is a release of NO and S-nitrosohomocystéine. Then continued exposure induces a predominant oxidative stress that reduces the NO production (Khan et al., 2001). Also, Hcy stimulates the production of reactive oxygen species and decreases the H2O2 detoxification mediated by glutathione peroxidase in bovine aortic endothelial cells, which leads to a decrease in NO bioavailability (Upchurch, 1997a, 1997b). Furthermore, in the normal state, the endothelium-derived NO controls the proliferation and migration of the VSMCs. However, it has been shown in vitro that Hcy, even in moderate doses, increased collagen synthesis and proliferation of SMCs, which is one of the characteristics of the arteriosclerotic lesions (Zittoun, 1998).

21 e. Coagulation

HHcy can lead to an imbalance between pro-and anti clotting factors. Elevated Hcy concentrations induce the activation of V, X and XII factors and tissue factor which initiate clotting (El Mabchour, 2010). Hcy also causes a decrease in plasminogen activity and stimulates platelet thromboxane A2 secretion, an eicosanoid responsible for vasoconstriction and platelet aggregation (Lentz and Haynes, 2004). HHcy increases, therefore, platelet aggregation, which strongly suggests that it induces vascular inflammation and hypercoagulability. These factors are associated with atherosclerosis development and thrombotic event (Hofmann et al., 2001).

I.3. Homocysteine and Carcinogenesis

HHcy is an important risk factor in cardiovascular diseases (Miyaki, 2010), however, it may also participate in cancer biology. Recent studies have shown that besides being a marker for cardiovascular events, Hcy may also be a cancer marker. According to different data, there appear to be a mechanism by which HHcy, and thus elevated Hcy levels may be carcinogenic. This mechanism may happen namely through folate deficiency, oxidative stress, aberrant DNA methylation and the production of HTL (Wu and Wu, 2002). The up coming section describes the data supporting the involvement of Hcy in cancer as a marker and a risk factor. It also evaluates the evidence from cellular, animal and human studies about the mechanisms that may be involved in Hcy induced carcinogenesis

I.3.1. Homocysteine as a cancer marker

Recent studies have shown that Hcy is a marker for cardiovascular events, but several other studies suggest that it may also be a cancer marker. Higher Hcy levels were found in patients with ovarian (Corona et al., 1997), pancreatic (Sun et al., 2002), colorectal (Kato et al., 1999), head and neck squamous cell carcinomas (Almadori et al., 2002, 2005) and acute lymphoblastic leukemia (Ueland and Refsum, 1989).

22

Another study conducted on patients with different solid tumors (digestive, bronchial, breast, urologic, ovarien) and with hematologic malignancies (lymphoma and myeloma) showed that one patient in two had a significative increase in Hcy levels. These levels were not associated with folate deficiencies which suggests a high production of Hcy by tumor cells (Falvo et al., 2007).

In addition, it has been demonstrated in vitro that the rapid growth of tumor cells resulted in a much higher concentration of circulating total Hcy even though they were not treated with anti-folate drugs. The study demonstrated also that the concentrations of total Hcy and tumor markers increased during the growth of tumor cells, but only Hcy concentrations declined in response to tumor cell death. These results imply that Hcy could be used as a tumor marker in the diagnosis, staging and follow-up of cancers and in the evaluation of patients response to treatment (Sun et al., 2002).

Experimental and epidemiological evidence has shown an association between folate status and risk of cancer (Glynn and Albanes, 1994). Serum folate levels were significantly lower in patients with head and neck squamous cell carcinoma and in patients with laryngeal leukoplakia compared with the control group. In contrast, serum Hcy levels in patients with head and neck squamous cell cancer were significantly higher compared with Hcy levels both in the control groups and in patients with laryngeal leukoplakia (Almadori et al., 2005).

A recent study on lung cancer patients has shown an increased total Hcy, decreased total glutathione and folate levels when compared with healthy controls. Also, total Hcy levels were significantly higher and total glutathione and folate levels were significantly lower in the advanced-stage group compared with controls. The overall findings indicate that the inversely related folate and total glutathione status to plasma Hcy concentrations may be a marker and a risk factor for cancer (Özkan et al., 2007).

23 I.3.2. Homocysteine as a cancer risk factor

Prospective and retrospective clinical studies provide information on Hcy as a risk factor for cancer. In fact, serum Hcy levels were strongly and significantly predictive of invasive cervical cancer risk. This association reflected a potential folate, B12 and/or B6 inadequacy, or genetic polymorphisms affecting one-carbon metabolism (Weinstein et al., 2001). Also, high plasma Hcy was associated with increased risk of colorectal cancer, whereas high cysteine was associated with decreased risk (Selhub and Miller, 1992). In fact, individuals with a high serum folate level have an approximately 50% decrease in risk of colorectal cancer, whereas those with low folate levels and high Hcy levels are at increased risk (Mattson, 2002). In addition, low-folate status has been associated with increased risks of several cancer types, suggesting a chemopreventive role of folate (Luebeck et al., 2008). Another study demonstrated that increased Hcy was strongly associated with the risk of colorectal cancer independently of oxidative stress indicators and antioxidant capacities. However, cysteine and folate were not found to be related to oxidative stress, antioxidant capacities and the risk of colorectal cancer (Chiang et al., 2013).

Li et al. (2011) estimated that Hcy levels were not associated with overall risk for breast cancer but observed that there is a positive association between cysteine levels and breast cancer risk. Their study suggested that women with higher levels of Hcy and cysteine were at a greater risk for developing breast cancer when their folate levels were low. In another investigation, elevated plasma Hcy levels were significantly linked to increased risk of breast cancer. This pattern was observed in both pre-menopausal and post- menopausal women and did not differ substantially by level of dietary intake of B-group vitamins which suggested that plasma Hcy levels could be a metabolic risk factor for breast cancer (Chou et al., 2007). According to these different studies, high Hcy may be a risk factor for cancer, and there appear to be a mechanism by which HHcy, and thus elevated Hcy levels might be carcinogenic.

24

This mechanism may happen namely through folate deficiency, oxidative stress, aberrant DNA methylation and the production of HTL (Wu and Wu, 2002). (Figure 07)

Figure 07. Potential carcinogenic molecular effect of high Hcy (Benmebarek and Zerizer, 2014)

25 I.3.3. Carcinogenesis through folate deficiency

Folate is important in the synthesis and repair of deoxyribonucleic acid (DNA) through the generation of purine and pyrimidine nucleotides (Lucock, 2000). Folate acts as a methyl donor for the enzyme thymidilate synthetase which converts deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) (Eriksson and Arner, 1994). Deoxythymidine monophosphate (dTMP) is further phosphorylated to deoxythymidine triphosphate (dTTP) for DNA synthesis (Lamprecht and Lipkin, 2003).

The risk of HHcy in cancer is linked to folic acid deficiency which results in elevated Hcy levels (Wu, 2007). This deficiency can have destabilizing consequences during DNA replication. Inadequate folate availability during cell division can result in the misincorporation of uracil into DNA sequence by substituting thymidine (Lamprecht and Lipkin, 2003). This induces uracil DNA glycosylase to remove any misincorporated uracil from the DNA molecule (Lindah et al., 1977).

Successive DNA repair enzymes act upon the DNA to remove the misincorporated base, creating a small gap, and the break is then sealed by DNA ligase activity. However, under continual conditions of folate deficiency, uracil misincorporation and repair may occur repeatedly (Jacob et al., 1998; Goulian et al., 1980; Reidy, 1988). This may trigger attempts to repair the defect and increases the frequency of DNA double strand breaks which induces chromosome breakage (Lamprecht and Lipkin, 2003). A study on postmenopausal women with induced folate deficiency demonstrated a significantly elevated plasma Hcy. The folate depletion resulted in an increased ratio of dUTP/dTTP in mitogen-stimulated lymphocyte DNA which suggested a misincorporation of uracil into DNA and increased DNA repair activity (Jacob et al., 1998). Folate deficiency in Swiss mice increased the incidence of micronuclei in peripheral blood erythrocytes, indicating increased chromosomal damage in nucleated erythrocyte precursors (MacGregor et al., 1990). Also, low folic acid induced the formation of micronuclei in tissue culture which is indicadive of chromosome breakage (Kimura et al., 2004).

26

Vitamins B12 and B6 deficiency may also induce cancer development. A recent in vivo study has shown that a diet deficient in vitamin B12 disrupted the normal homeostasis of one-carbon metabolism in the colonic mucosa. It also resulted in diminished genomic DNA methylation and increased uracil misincorporation in DNA. These processes are two supposed mechanisms for one carbon metabolism-related colonic carcinogenesis (Friso and Sang Woon, 2005). In addition, it has been reported that folate and vitamin B6 may have the potential to be chemopreventive against breast cancer and ensuring adequate circulating levels of folate and vitamin B6 may contribute to a reduction in the risk of breast cancer (Zhang et al., 2003).

I.3.4. Carcinogenesis through oxidative stress

HHcy has been implicated as a risk factor for numerous diseases (Prasad, 1999; Lee and Prasad, 2002). Because Hcy and other thiols have pro-oxidant activity, the oxidant stress hypothesis is frequently invoked to explain the damaging effects of Hcy on vascular cells and tissues. It could also explain the existing possibility that elevated Hcy can lead to carcinogenesis through free oxygen radicals (McCully, 1996; Loscalzo, 1996). Hcy may invade the intracellular space of many tissues and locally generate in a time and concentration dependant manner (Yan et al., 2006) reactive oxygen species (ROS) after undergoing intracellular and extracellular autooxidation in the presence of molecular oxygen (Carmel and Jacobsen, 2001b). Hcy contains a reactive sulfhydryl group (-SH) and, like most thiols (RSH), can undergo oxidation to the disulfide (RSSR) at physiological pH in the presence of O2 which generates hydrogen peroxide (H2O2). The general reaction is catalized by transition metals and a variety of ROS can be produced including superoxide anion radical and hydrogen peroxide (Jacobsen, 2000).

Central cellular processes such as proliferation, apoptosis, senescence are implicated in the development of cancer and are influenced by ROS. Oxidative damage to cellular DNA can lead to mutations and may, therefore, play an important role in the initiation and progression of multistage carcinogenesis (Waris and Ahsan, 2006).