1. OXYGEN TOXICITY Dr. Saidunnisa M.D., Professor and chairperson Biochemistry

2. Objectives  At the end of session student shall be able to: 1. Define reactive oxygen species 2. Explain the mechanism of generation of free radicals 3. Discuss the damage produced by free radicals 4. Describe the free radical scavenger enzyme system 5. Explain the clinical significance 6. Define and discuss lipid peroxidation and its mechanism 7. Define antioxidants and there mechanism of action

3. Introduction  Electrons prefer to be in pairs and when an electron is alone in its orbital in will try to take an electron from another atom to become more stable.  When the other atom loses its electron it tries in turn to steal an electron from another atom, often resulting in a dangerous chain reaction.

4. Free radical  A free radical is a molecule that contains one or more unpaired electrons in its outer orbital.  Free radical is conventionally represented by a superscript dot (R. )

5. Oxygen is essential but toxic also  Oxygen has two unpaired electrons in two different orbitals spinning parallely.

6. Recap  Oxidation of molecular oxygen is completely reduced to water.  Products of partial reduction of oxygen are highly reactive and damage the living tissues.  Hence they are called Reactive oxygen species (ROS).

7. Reduction of oxygen by four one electron steps Progressively when oxygen accepts one electron generates superoxide, when it accepts an electron it is reduced to hydrogen peroxide, it accepts another electron forming hyroxyl radical finally accepts the last electron forming water.

8. Reactive oxygen species Name of ROSsymbolHalf life Superoxide anion radical O 2. 10-6 Hydroperoxyl radicalHOO. sec Hydrogen peroxideH2O2H2O2 Min (is lipid soluble it can diffuse through membranes and generate (OH.) Hydroxyl radicalOH. 10-9secis (probably the most potent ROS). Lipid peroxide radical ROO. sec Singlet oxygenO210-6 sec Nitric oxideNO. Sec Peroxy nitriteONOO --. 10-3

9. Characteristics of Reactive Oxygen Species (ROS) 1. Highly reactive 2. Short life span 3. Generation of new ROS by chain reaction 4. Damage to various tissues.

10. Generation of free radicals  Non enzymatically: 1. Exposing to ionizing radiation. 2. Cigarette smoking. 3. CoQ (ETC) 4. Highly toxic hydroxyl radical formed by:  Fenton and  Haber weiss reaction. 6. O 2. can be formed during methaemoglobin formation.

11. Generation of free radicals

12. Fenton Reaction  Hydroxyl radical (OH. ) can be formed from Hydrogen Peroxide (H 2 O 2 )n the presence of Fe 3+ or Cu + (Fenton reaction)

13. Haber-weiss reaction  The Superoxide forms the highly toxic Hydroxyl radical (OH. ) non enzymatically by reacting with hydrogen peroxide (Haber - Weiss Reaction )

14. Generation of free radicals Enzymatically: Cytochrome p 450 –leakage of electrons during detoxification Mono amine oxidase, fatty acid oxidase & Xanthine oxidase present in the peroxisomes generates H 2 O 2.

15. Enzymatic Generation of free radicals

16. Infection & Inflammation Phagocytic Killing Microorganisms can enter our body – skin, respiratory, GI tracts etc. Next line of defense? Phagocytes Phagocytes – WBCs that ingest microbes, other cells and foreign particles Neutrophils, Eosinophil's and Macrophages (Inflammatory cells)

17. Phagocytic Killing Oxygen dependent killing Activated Macrophages produce ROI and RNI species Q. How are these ROI/RNIs made?? A. Respiratory Burst

18. Respiratory Burst Occurs in macrophages during phagocytosis Abrupt rise in oxygen consumption Increased glucose consumption HMP Shunt (Pentose phosphate pathway) Large amounts of reactive oxygen intermediates to destroy the infecting agents. Enzyme – NADPH Oxidase

19. Respiratory Burst  During phagocytosis inflammatory cells (macrophages, neutrophils) produce superoxide anion by the action of NADPH oxidase.  The superoxide is converted to H 2 O 2 and hypochlorous acid (HCLO) by the action of SOD and myeloperoxidase (MPO) which have bactericidal action.  This is deliberate production of free radical by the body for defense.

20. Respiratory Burst

21. Formation of phagocytic vesicle

22. Reactive Nitrogen Species Activated macrophages express high levels of Nitric oxide synthase (NOS) NOS catalyzes: L-arginine + O 2 + NADPH NO + L-citrulline +NADP + NO has potent antimicrobial activity. Can combine with O 2 ¯ to yield more potent antimicrobial substances (Peroxynitrites) NO + 2O 2 ¯ ONOO¯

23. ROI vs RNI Microbial killing mainly ROI dependent in normal neutrophils. RNI may play role in cells with deficiencies of NADPH oxidase/MPO pathways.

24. Chronic Granulomatous Disease (CGD) Inherited immunodeficiency Individuals have impaired or completely absent oxidative burst Suffer from recurrent and life threatening infections Incidence – 1 in 200,000 to 1 in 250,000 live births Persistent infection of soft tissue, lungs, granulomas in multiple organs Pneumonia and septicemia, sometimes leading to death Organisms – S.aureus, salmonella, Pseudomonas, Candida albicans, aspergillus

25. Conclusions ROI/RNIs play an important role in our immune system. Consequences of lack of ROS production in neutrophils can be seen in CGD.

26. Antioxidants  Damage produced by ROS may be prevented by anti-oxidants.  Classified into two types: 1. Enzymatic anti oxidants 2. Non enzymatic antioxidants

27. Antioxidant scavenging enzymes  Enzymatic anti oxidants:  Superoxide dismutase, (removes superoxides)  Catalase and  Glutathione peroxidase (removes hydrogen peroxides as well as lipid peroxides)

28. SOD  2O 2 - SOD H 2 O 2 (superoxide anion) 2H+------O2  SOD converts superoxide to H 2 O 2 which is non toxic unless converted to other ROS

29. Catalase  2H 2 O 2 Catalase 2H 2 O + O2 (peroxisomes)

30. Glutathione peroxidase and reductase  Gluta. Peroxidase  H 2 O 2 2H 2 O  2GSH GSSG

31. Non enzymatic antioxidants  Vitamin E, Vitamin C, carotenoids and plant flavanoids (green tea, chocolate, red wine) and some endogenous substances like uric acid, allopurinol, melatonin, act as antioxidants.  Used in food industry:  VitE, BHA (butylated hydroxy anisole), BHT (butylated hydroxy toulene) to increase the shelf – life of products.

32. Lipid peroxidation  Free radicals damage on cellular components  A chain of reactions involving free radicals and the lipids in the membranes forms the major ROS induced injury.  Damage of cell membrane will cause increase permeability to sodium ions, rapid influx of calcium, osmotic entrance of water into the cell leading to cell damage.  It involves four stages –initiation, propagation, degradation and termination.

33. Initiation  Lipid peroxidation is initiated by a hydroxyl or other radical that extracts a hydrogen atom from PUFA (LH) there by forming a lipid radical (L. ) LH +. OH L. + OH (lipid radical)

34. Propagation The free radical chain reaction is propagated by reaction with O2 forming the lipid peroxy radical (LOO. ) and lipid peroxide (LOOH). L. + O 2 LOO. (peroxy radical) LOO. + LH LOOH + L. (lipid peroxide ) Degradation: Rearrangements of the single electron result in degradation of the lipid. Malondialdehyde one of the compounds formed is soluble and appears in blood

35. Termination The chain reaction can be terminated by vit.E and other lipid-soluble antioxidants that donate single electrons subsequently forming oxidized antioxidant (stable). This will bring the reaction to an end. Loo. + L. LOOH + LH OR L. + VitE LH + Vit E. Vit E. + L. LH + VitE ox

36. Oxidative stress & disease  When this defense process is inefficient, the cells are under oxidative stress.  The oxidative stress is the underlying cause for major diseases like :

37. Lipid peroxidation  Also, the lipid peroxides form cross linkages with proteins forming such compounds called as lipofuscins and the accumulation of these compounds inside the cells are responsible for aging.

38. Respiratory diseases  Breathing 100% oxygen for more than 24 hrs releases free radicals and produces destruction of endothelium of lung and oedema.  In premature newborn infants causes bronchopulmonary dysplasia in adults (ARDS) respiratory distress syndrome.

39. Disease of eye  Retrolental fibroplasia or retinopathy of prematurity: premature infants on prolong exposure to oxygen release free radicals which inturn release thromboxane causing vascular constriction and cellular injury.

40. Atherosclerosis and MI  LDL is deposited under the endothelial cells which undergo oxidation by free radicals released from these cells attract macrophages and initiate atherosclerotic plaque and promote atherosclerosis.

41. Peptic ulcer  Produced due to erosion of gastric mucosa by HCL. Superoxide anions are involved.  Helicobacter pylori infection increases the disease leading to tissue destruction by macrophage oxidative burst.

42. Cancer treatment  Free radicals damage DNA and accumulated damage lead to mutations and malignancy.  Cancer is treated by radiation which produces ROS in the cells and trigger cell death.

43. Ischemia- reperfusion injury  Ischemia decreased blood flow the ability of heart to generate ATP from O.P is decreased.  During ischemia COQ and other single electron components of ETC become saturated with electrons.  When oxygen is introduced (reperfusion) electron donation to O2 to form superoxide is increased this results in enhanced formation of H 2 O 2 and hydroxyl radical.

44. Ischemia- reperfusion injury  Macrophages in that area to clear cell debris from ischemia produces nitric oxide which further releases RNOS thus increasing the infarct size.  Currently an intense study on ischemic insults by preventing reperfusion injury include exo and endogenous antioxidants administered before reperfusion would prevent its injurious effects.

45. RNOS (Reactive nitrogen –oxygen species)  Nitric oxide(NO) is an oxygen containing free radical which is both essential and toxic to life.  NO has a single electron and therefore binds to other compounds containing single electrons.  As gas it diffuses through the cytosol and lipid membranes and into cells.  At low concentrations functions physiologically as a neurotransmitter and hormone causing vasodilatation.

46. RNOS  At high concentrations it combines with O2 or superoxide to form RNOS.  These are involved in parkinson’s and chronic inflamamtory disease like rheumatoid arthritis.

47. RNOS  Arginine No synthase citrulline No. (nitric oxide)  Clinical application: Nitroglycerin in tablet form often given to patients with CAD who experience chest pain (angina).  This nitroglycerine converts in the blood forming NO a potent vasodilator which increases blood flow to the heart and relieves the angina.