ANTIOXIDANTS in health and diseases

1. ANTIOXIDANTSin health and diseases MUHAMMAD IQBAL CHOUDHARY Dr. Panjwani Center for Molecular Medicine and Drug Research International Center for Chemical and Biological Sciences University of Karachi, Karachi-75270

2. Oxidation and Human Health One of the paradoxes of life on this planet is that the molecule that sustain aerobic life, oxygen, is not only fundamentally essential for energy metabolism and respiration, but implicated in many diseases and degenerative conditions. Marx, Science, 235, 529-531 (1985).

3. Learning Objectives Understanding the relationship between oxidative stress, and health and diseases… What are oxidants or ROS, types, sources, and activities? Their role in normal physiological process Their detrimental role in the onset and progression of diseases, chemical basis of oxidative damage! Biomarkers of oxidative damage to the vital biomolecules and their analysis What are antioxidants, types, sources and activities? Perceived role of antioxidants in the preservation of health and prevention of diseases Anti-oxidant drug development- challenges and opportunities

4. CONTENT What is oxidation? Oxidation in biological system What are free radicals? Sources of free radicals Harmful effects of free radicals Damage to proteins and associated diseases Damage to DNA and associated diseases Damage to lipids and associated diseases Damage to carbohydrates and associated diseases What are antioxidants? Nature’s antioxidants system Dietary sources of antioxidants Oxidative Stress- Imbalance between oxidation and anti-oxidation Types of antioxidants Mechanism of anti-oxidation Bioassays used to discover new antioxidants

5. Why This Topic? If you search for Antioxidants… Sci-Finder 305,081 Articles (April 2013) Pubmed 384,341 Hits (April 2013) Google Search Over 7,000,000Web pages (Jan. 2014) Chemical Abstracts 131,961 Publications (2003-2013)

6. What is Oxidation? Combination of substrate with oxygen. Reaction in which the atoms in a compound lose electrons. Any compound, including oxygen, that can accept electrons is an oxidant or oxidizing agent (pro-oxidant), while a substance that donates electrons is a reductant or reducing agent (antioxidant).

7. Oxidation in Biological System We live in an aerobic environment Oxygen is the life sustaining element We consume approximately 3.5 kilograms of oxygen every day 2.8 percent of the oxygen is not properly used and forms free radicals Several kilograms of peroxides (harmful oxidized lipids) are produced in our body every year

8. What are Free Radicals? Free radicals (pro-oxidants) are any chemical species, capable of independent (although extremely short) existence with one or more unpaired electrons Highly unstable and reactive Looking for electrons from other sources to stabilize themselves. In this process they initiate a chain reaction of oxidation Most commons are Reactive Oxygen Species (ROS) Reactive Nitrogen Species (RNS) (NO., ONOO-, etc) or Reactive Sulfur Species (RSS)

9. What are Reactive Oxygen Species (ROS)? ROS are: oxygen derived radicals (O2.-, .OH, ROO., 1O2, RO.) oxygen-derived non radical species (O2, H2O2, O3, ROOH, HOCl) They are now considered as major players in biochemical reactions, cellular response, and clinical outcome

12. Common Free Radicals

13. Common Free Radicals

14. What Free Radical does? Free radicals are cellular renegades; they wreak havoc by damaging DNA, altering biochemical compounds, corroding cell membranes and killing cells outright. Such molecular mayhem, scientists increasingly believe, plays a major role in the development of ailments like cancer, heart or lung diseases and cataracts. Many researchers are convinced that the cumulative effects of free radicals also underlie the gradual deterioration that is the hallmark of aging in all individuals, healthy as well as sick. TIME, April 6, 1992 publications

15. Internal Sources of Free Radicals Mitochondria Phagocytes (macrophages) Xanthine oxidase Reaction involving iron and other transition metals Arachidonate pathways Peroxisomes Exercise Inflammation Ischaemia/Reperfusion

16. External Sources of Free Radicals Cigarette smoke Environmental pollutants Radiations Ultraviolet radiations Ozone Certain drugs, pesticides, anesthetics and industrial solvents

17. Useful Functions of Free Radicals Necessary in the maturation processes of cellular structures Necessary in the antibacterial activity- White blood cells (phagocytes) releases free radicals to destroy invading pathogenic microbes as part of the body’s defense mechanism Necessary in the immune system Necessary in the prostaglandin biosynthesis Some of them play an important role in cell signaling

18. History: Harmful Effect of Free Radicals 1775 Priestly- Toxicity of oxygen to the organism similar to burning of candle 1954 Gilbert and Gersham- Free radicals are important player in biological environment and responsible for deleterious process in the cell 1969 Mc Cord and Fridovich- Superoxide theory of toxicity

19. Harmful Effect of Free Radicals- Perception Free radicals can damage all cellular macromolecules including proteins, carbohydrates, lipids and nucleic acids Destructive effect play a role in the onset and progression of different diseases and in normal aging.

20. Oxidative Damage to Organs

21. Ageing is one of the major consequences of oxidative damage 1956 Denham- Free Radical Theory of Ageing

22. Human Ailments Associated with Oxidative Damage Neurological Alzheimer’s Disease Parkinson‘s Disease Endocrine Diabetes Gastrointestinal Acute Pancreatitis

23. Human Ailments Associated with Oxidative Damage Others Conditions Obesity Loss of catalytic functions of proteins Toxicity Chronic Inflammation and arthritis

24. Diseases Related to Oxidative Damage

25. Exogenous v/s Endogenous Sources of Free Radicals Exogenous ROS are extremely high Exposure to endogenous oxidants is much more important and extensive, because it is a continued process during the entire life span Mitochondria play an extremely important role in endogenous ROS production Presence of metals (iron, copper, chromium, cobalt, vanadium) in un-complexed form significantly increase the level of oxidative stress.

26. Damage to Lipids Lipids are highly prone to get oxidized. Polyunsaturated fatty acid (PUFA)- major part of the low-density lipoprotein (LDL) in blood.

27. Damage to Lipids Lipid peroxidation, if not terminated rapidly, can cause damage to cell membranes. Removal of lipid peroxides is essential for mammalian life (Glutathione peroxidase IV knock-out mouse doesn’t survive beyond embryonic state). Malondialdehyde (MDA) is an important biomarker of oxidative stress. It reacts with DNA bases to for DNA-adduct

28. End Products of Lipid Peroxidation These end products are the markers for lipid peroxidation determination. For example malondialdehyde (MDA) is detected in TBARS (Thiobarb- ituric Acid Reactive Substance) assay, specific for lipid peroxidation determination. 13-HPODE= 13- Hydroperxy- 9Z,11E-octdecdienoic acid 4-HNE= Hydroxynonenal

29. Damage to Lipids-Associated Diseases Alterations in the structures of lipid molecules lead to change in their physical properties, such as permeability, surface adhesion, etc. It cause damage to the cell membrane, made up of mainly lipids. Risk of cardiovascular diseases (CVD), including atherosclerosis.

30. Damage to Lipids-Associated Diseases End products of lipid peroxidation can also cause mutagenesis and carcinogenesis. For example malondialdehyde reacts with deoxyadenosine and deoxyguanosine in DNA to form a variety of DNA adducts. Body has evolved a range of molecules, such as Vitamin E, and enzymes such as SOD, catalase, and peroxidase to control lipid peroxidation. Knockout animals (which can not produce anti-oxidant enzymes), generally do not survive.

31. Atherosclerosis and Oxidative Stress Compelling evidence points oxidative stress as an important trigger in the complex chain of events leading to atherosclerosis. It involves accumulation of macrophages in the arterial wall. Which then promptly incorporate oxidized LDL to form foam cells. ROS can lead to platelet activation and thrombosis formation. Probucol has shown reduced progression of carotid atherosclerosis in clinical trials.

32. Oxidative Damage to Proteins .OH and RO. and cause damage to proteins Direct damage include peroxidation, damage to specific amino acid residues, change in tertiary structures, degradation and fragmentation No efficient mechanism of repair of protein damage exists Proteolytic enzymes play an important role in the removal of damaged proteins

33. Protein Oxidation Products Aldehydes, keto and other carbonyl compounds 3-Nitrotyrosine, produced by interaction of tyrosine and ONOO-, is a useful biomarker of oxidative protein damage Ortho- and meta-tyrosines from phenylalanine. Other damaged products include hydroxyproline, glutamyl semialdehyde, etc Crossed linked proteins

34. Damage to Proteins- Associated Problems Modified oxidized proteins are susceptible to many changes in their functions This include chemical fragmentation, inactivation and increased proteolytic degradation Oxidative changes in the structures of catalytic proteins lead to loss of enzyme activity Altered cellular functions such as energy production, interference with the creation of membrane potential and change in the type and level of cellular proteins Non- enzymatic glycation of proteins lead to multiple poteopathic disorders Serum protein carbonyl concentration is directly related to muscle dysfunctions.

35. Mechanism of Glycation of Protein- Role of ROS Catalyzed by transition metals (M) and the superoxide radical generated are converted to the hydroxyl radical via the Fenton reaction.

36. Oxidative Damage to DNA DNA is stable, well protected molecules ROS, specially .OH, can interact with it and cause several types of damage Including modification of DNA bases, single- and double helical breaks, loss of purines, damage to deoxyribose sugar, DNA protein cross linkage and DNA repair system Out of four bases, guanine is the most easily oxidizable nucleic acid base.

37. Oxidative Damage to DNA

38. Oxidative Damage to DNA Oxidative products of guanosine serve as biomarkers of damage to DNA molecule.

39. Oxidative Damage to DNA ROS in the cells lead to DNA damage, cause stable DNA lesions which are mutagenic, if un-repaired Damaged DNA provide the wrong genetic code leading to unregulated protein synthesis and/or cell growth which results in cancer. Presence of 8-oxo-2-deoxyguanosine (oxo8dG) in DNA is an important indicator of oxidative damage to DNA Oxidative damage to DNA accumulate with ageing, increasing the possibilities of cancers and other disorders

40. Damage to DNA- Associated Problems Number of oxidative hits to DNA per cell per day is about 100,000 in the rat and about 10,000 in the human (Reason????) There is an inherent mechanism (specific repair glycosylases, etc.) to repair most of the DNA damage caused by ROS Oxidative lesions in DNA accumulate with age and eventually lead to serious health challenges (well established relationship between onset of cancers and age)

41. Oxidative Stress MarkersOxidative stress end products detection Lipoperoxidation markers: malondialdehyde (MDA), conjugated dienes,isoprostanes Oxidative damage to protein markers : protein hydroperoxides Oxidative damage to DNA : modified nucleosides

43. Potentialities of oxidative/nitrosative stress-related biomarkers Acta Medica Okayama, 61 (4), 181-189, 2007

44. What are Antioxidants? Antioxidants (reductants or reducing agents) are compounds capable of preventing the pro-oxidation process or biological oxidative damage by scavenging or stabilizing reactive oxidative species.

45. An antioxidant is a molecule stable enough to donate an electron to a rampaging free radical and neutralize it, thus reducing its capacity to damage.

46. What are Antioxidants? Antioxdiants produced during normal metabolism include glutathione, ubiquinol and uric acid Antioxidant enzymes include glutathione peroxidases, superoxide dismutases and catalase Antioxidants from dietary sources such as Vitamins E and C and carotenoids Antioxidants from non-dietary sources include phenolic or polyphenolic compounds as well as selenium

47. What Antioxidant Do???

48. WHY ARE ANTIOXIDANTS IMPORTANT ? • They inhibit the conversion of nitrites to nitrosamines (which are tumor promoters) and enhance the immune response. • Vitamins E, and C, ubiquinones, etc. remove free radicals from the epidermis of the skin and counteract their potentially damaging effect. • They terminate free radical- induced cellular damage and functional degeneration (aging). • They trap and neutralize free radicals and protect our body tissues from environmental pollutants.

49. Sources of Antioxidants More than 4,000 antioxidants are known Endogenous- Antioxidant enzymes include glutatione peroxidases, superoxide dismutases and catalase Antioxidants from dietary sources, such as Vitamin E, Vitamin C and carotenoids Antioxidants from non-dietary sources include phenolic or polyphenolic compounds

50. Antioxidant Enzymes Glutathione peroxidases (Seleno proteins) catalyze the reduction of lipid hydroperoxides to their corresponding alcohols Superoxide dismutases, a family of metal-containing enzymes (Mn, Fe, Zn, Cu), catalyze the dismutation of superoxide into oxygen and hydrogen peroxide Catalases catalyze the decomposition of hydrogen peroxide to water and oxygen