Pathophysiology and Pharmacology of Reactive Oxygen Species ROS

1. Pathophysiology and Pharmacology of Reactive Oxygen Species (ROS) V. Bauer, Š. Mátyás, S. Štolc, R. Sotníková, V. Nosálová

2. The beginnings

3. Evidence on the existence of ROS

4. Free radicals have one or more unpaired electrons in their outer orbital, indicated in formulas as [?]. As a consequence they have an increased reactivity with other molecules. This reactivity is determined by the ease with which a species can accept or donate electrons. The prevalence of oxygen in biological systems means that oxygen centered radicals are the most common type found. O2 acts in a process that is central to metabolism in aerobic life, as a terminal electron acceptor, being reduced to water. Transfer of electron to oxygen yields the reactive intermediates.

5. The term reactive oxygen species (ROS) rather than oxygen radicals is now generally preferred because singlet oxygen (its one form), hydrogen peroxide, hypochlorous acid, peroxide, hydroperoxide and epoxide metabolites of endogenous lipids and xenobiotics have chemically reactive oxygen containing functional groups, but are not radicals and do not necessarily interact with biological tissues via radical reactions. Molecular oxygen is a biradical, having two unpaired electrons of parallel spin. As it is a terminal electron acceptor being reduced to water, oxygen acts in processes that are central to metabolism in aerobic life.

7. Half-life of some reactive species

8. ROS present in mammalian tissues have both endogenous and exogenous origin. Their production is essential to normal function or metabolism of most mammalian cells. Approximately, 90% of all oxygen consumed by mammalian cells is catalytically reduced by four electrons to yield two molecules of water. It is now clear that oxygen may also be reduced by less than four electrons in enzymatic and nonenzymatic reactions. ROS are, however, also destructive unless tightly controlled. Mammalian cells have developed a battery of defenses to prevent and repair the injuries caused by oxidative stress.

9. Origin of ROS H+ .NO OONO- HOONO NO2. e- e- e- e- O2 O2.- H2O2 .OH H2O O2.- O2 Fe2+ Fe3+ H+ Cl- Myelo- peroxidase H2O HOCl 1O2 + Cl- H2O2 H2O Sources endogenous exogenous prostaglandin synth. radiation, ultrasound respiratory chain cigarette smoke autooxidation drugs FREE RADICAL S phagocytes heat oxyhemoglobin pesticides oxidative enzymes infections accumul reduced.metab. hyperoxia, exercise air pollution (NOx, O3)

10. Enzymatic sources of ROS Xanthine oxidase Hypoxanthine + 2O2 ® Xanthine + O2.- + H2O2 NADPH oxidase NADPH + O2 ® NADP+ + O2.- Amine oxidases R-CH2-NH2 + H2O + O2 ® R-CHO + NH3 + H2O2 Myeloperoxidase Hypohalous acid formation H2O2 + X- + H+ ® HOX + H2O NADH oxidase reaction Hb(Mb)-Fe3+ + ROOH ® Compound I + ROH Compound I + NADPH ® NAD· + Compound II Compound II + NADH ® NAD· + E-Fe3+ NAD· + O2 ® NAD+ + O2.- Aldehyde oxidase 2R-CHO + 2O2 ® 2R-COOH + O2.- Dihydroorotate dehydrogenase Dihydroorotate + NAD· + O2 ® NADH + O2.- + Orotic acid

12. Nonenzymatic sources of ROS and autooxidation reactions Fe2+ + O2 ® Fe3++ O2.- Hb(Mb)-Fe2+ + O2 ® Hb(Mb)-Fe3++ O2.- Catecholamines + O2 ® Melanin + O2.- Reduced flavin Leukoflavin + O2 ® Flavin semiquinone + O2.- Coenzyme Q-hydroquinone + O2 ® Coenzyme Q (ubiquinone) + O2 .- Tetrahydropterin + 2 O2 ® Dihydropterin + 2 O2.-

13. Until the 1960s, free radicals were not considered particularly relevant for mammalian physiology and pathology. The discoveries of the existence of superoxide dismutase (SOD) activity in mammalian cells in 1969 by McCord and Fridovich and association of bactericidal activity of neutrophils with production of the superoxide radical (O2.-) by Babior and coworkers in 1973, linked free radicals to numerous physiological and pathophysiological processes. One decade later, in 1981, Granger and coworkers established a hypothesis on the role of these reactive species in the reperfusion injury after intestinal ischemia.

14. ROS are tightly controlled resulting in a physiological balance between their production and elimination

15. Biological antioxidant defense mechanisms

16. Under pathological condition the physiological balance is lost

17. Disbalance between production and elimination of ROS develops during inflammation, ischemia/reperfusion, altered metabolism, action of drugs, pollutants, etc. Such disbalance causes pathology of brain, heart, vessels, gut, airways, muscle, parenchy- matous organs (liver, kidney, pancreas), eye, skin, joints, etc. Exposure of the tissues to ROS in a variety of biological systems has documented their ability to damage lipids, proteins and DNA. The resulting damage potentiated by increased free intracellular Ca2+ causes activation/deacti-vation of various enzyme systems and cell injury or death.

18. Mechanisms of ROS induced cell injury Lipid Oxidation of thiols DNA damage Schiff bases peroxidation Carbonyl formation Damage to Ca2+ and Poly ADP Altered gene other ion transport ribosylation expression systems Amadori products Membrane Instability to maintain Depletion of ATP damage normal ion gradients and NAD(P)(H) Activation/Deactivation of AGEs various enzyme systems (Advanced glycation end products) Cell injury

19. Involvement of ROS in APOPTOSIS NOXA (trauma, hypoxia under homeostatic metabolic insufficiency control to a certain activation of excitatory receptors) limit Ion disbalance caspase/calpaine ROS generation Mitochondrial failure activation Bcl-2 / Bax disbalance CELL DEATH (necrosis / apoptosis)

20. Currently it is believed that free radicals are definitely paticipating in several health disorders. There are different pathologic conditions where extracellular, intracellular or both ROS act at least in part. However, in spite of the extensive studies our knowledge concerning the role and action of free radicals and ROS is still incomplete and changing.

21. Pathological conditions that may have a free radical component and sites of ROS actions

22. ROS generation during ischemia and reperfusion

23. ROS in the sequence of events in STROKE HYPOXIA ATP depletion Cell depolarization (? Mg block of NMDA rec.) Excitatory aminoacid release Ca2+ influx into the cells Slow accumul. Ca2+ in mitochondria Activation of phosholipases, MPT pore opening in mitochondria proteinkinases, proteases, H+ gradient collapse in mitochondria endonucl., phosphatases etc. ROS generation ONOO- generation Devastatory effect in cells NEURONAL DEATH Therapeutic interventions: cyklosporine (specific MPT pore inhibitor), antioxidants (lazaroids, deferoxamine, SOD in liposomes, allopurinol)

24. Frequent targets of ROS

25. ROS affect different tissues and tissue components. They affect e.g. not only the smooth muscle cells, but also their epithelium, endothelium, innervation, membrane lipids, receptors, transmitter systems, prostanoid production, Ca2+ homeo- stasis, etc.)

26. Effects of H2O2 on guinea pig ileum

29. ?NO reacting with O2?- gives rise to unstable peroxynitrite, which decom-poses also to the most toxic ?OH. Because of the large energy gain of the reduction of ?OH to H2O, this radical reacts instantaneously with any biological molecule in its immediate environment by abstracting hydrogen atom.

30. Production of ROS in endothelium and neutrophils

31. Elimination by SOD with CAT of the effects of FMLP activated neutrophils (NEUT) generating O2 ?- on noradrenaline (NA) precontracted rat aorta

32. Effects of ROS on the endothelium and development of atherosclerosis atherosclerotic lesion cell proliferation release of growth factor active oxygen, recruitment of collagenase, elastase, adherence macrophages lipases, proteases of platelets Plasma endothelial cells LDL Intima activated oxygen Fatty Streak iron/copper Oxidatively modified LDL apoB-bound 4-hydoxynonenal, oxidized lipids, fatty acid hydroperoxides

33. Diseases that may have ROS related pathogenesisI Airways Normobaric hyperoxic injury Bronchopulmonary dysplasia Idiopathic pulmonary fibrosis Respiratory distress syndromes (ARDS, IRDS) Emphysema Chronic bronchitis & asthma bronchiale Asbestosis Inhaled pollutants, smoke, chemicals (e.g. paraquat, bleomycin) & oxidants (e.g. SO2, NOx, O3) Gut Ischemia/reperfusion Crohn’s disease Ulcerative colitis & necrotizing enterocolitis Gastric & intestinal ulcers Chemicals (e.g. NSAID)

34. II Heart and vessels Ischemia/reperfusion (after infarction, transplantation) Chemicals (e.g. ethanol, doxorubicin) Atherosclerosis/hypertension Selenium deficiency Vasculitis Brain and nerves Hyperbaric hyperoxic injury Parkinson's disease Alzheimer’s disease (details see in the next panel) Amyotrophic lateral scleroses Neuropathies (e.g. diabetic) Neurotoxins (e.g. 6-hydroxydopamine, MPTP) Vitamin E deficiency Neuronal ceroid lipofuscinoses Traumatic injury/hemorrhage/inflammation Ischemia/reperfusion HIV-dementia Multiple sclerosis

35. ALZHEIMER DISEASE and oxidative stress ? Protein oxidation (carbonyls) - „crosslinking“ ? Fe in neurons with fibrilary aggregates (?-hyperphosphoryl.protein) ? Content of aluminium in neurons with fibrilllary aggregates ?-amyloid generation (direct cytotoxic action,? Cai, generation of ROS even in the absence of Me2+) ? Activity of microglia (brain macrophages = ROS source) ? Activity of CAT without ? SOD activity resulting in ? H2O2 and ??OH Generation of lipid hydroperoxides and reactive cytotoxic aldehydes (e.g. HNE) Therapeutic interventions: antioxidants and ROS scavengers (e.g. U-74500A, U-78517F, U-83836E, vitamines E,C), chelators, CAT, deprenyl

36. III Blood Chemicals (e.g. phenylhydrazine, primaquine, sulphonamides, lead) Protoporphyrine photooxidation Malaria Anemias (sickle cell, favism) Liver Ischemia/reperfusion Chemicals (e.g. halogenated hydrocarbons, quinones, ethanol, acetaminophen) Accumulation of iron or copper Endotoxin Kidney Autoimmune nephrosis (inflammation, e.g. glomerulonephritis) Chemicals (e.g. aminoglycosides, heavy metals)

37. IV Pancreas Acute & chronic pancreatitis Diabetes mellitus Eye Retinopathy of prematurity Photic retinopathy Cataracts Laser photoablation Skin Radiation (solar, ionising) Thermal injury Chemicals (photosensitizers, e.g. tetracyclines) Contact dermatitis Porphyria

38. V Muscle Muscular dystrophy Multiple sclerosis Exercise Others Aging Pregnancy and newborn complications Radiation injury Cancer Chemicals (e.g. alloxan, iron overload, radiosensitizers) Autoimmune diseases (e.g. rheumatoid arthritis, lupus erythematodes) Inflammation (in general)

39. Potential antioxidant therapy I Inhibitors of ROS synthesis NADPH-oxidase Inhibitors Flavoprotein inhibitors (FAD analogs, antibodies of cytP450 reductase) Agents forming complexes with Fe2+ in cyt b (butylisocyanide, imidazole, pyridine) Mg2+(enabling FAD binding),Fe2+ ,Cu2+ chelators (bathophenantroline, EDTA, EGTA, deferoxamine, bilirubin) Thiol reagents (N-ethylmaleimide, 1-naphtol, 1,4- naphtoquinone) NADPH analogs (NADPH 2,3-dialdehyde) Inhibitors of metabolism of AA and PLA2 IMAO (Deprenyl) Others (corticosteroids, diphenyliodonium) Inhibitors of xanthine oxidase (tungsten, oxypurinol, allopurinol, pterinaldehyde, folic acid) Antibodies against leukocytes

40. II Agents supporting and complementing enzymatic protective systems Superoxide dismutase (SOD) SOD (Lip-SOD,PEG-SOD) Copper diisopropylsalicylate SOD mimetics Catalase (Cat) Cat (Lip-CatTP, Peg-CatTP) Glutathionperoxidase (GTPx) GSH, GSH methylester, GSH diethylmaleate Low m.w. thiols (e.g. cystein) High m.w. thiols (e.g. albumin) L-2-oxothiazidolidine-4-carboxylate N-acetylcysteine Ebselen Selenium Lactoperoxidase & DT-diaphorase

41. III Drugs interfering with iron and copper metabolism (deferoxamine, hemopexine, ferritin, transferrin, lactoferrin, ceruloplasmin, serum albumin) Antioxidants Vitamins and their analogues (vitamin E, vitamin C, carotenoids, oxycarotenoids) Phenol derivatives (eugenol, guajacol, probucol, N,N-diphenyl- phenylendiamine) Flavone derivatives (flavonoids, isoflavonoids, allirazine, green tea) Indol derivatives (stobadine, carvedilol, melatonin, ?-carbolines) Xanthine derivatives (allopurinol, oxypurinol, uric acid) 21-amino steroids (lazaroids) Antiinflammatory drugs (piroxicam, flufenamic acid, mefenamic acid, hydroquinone, sulindac, fenylbutazone, indomethacin, ibuprofen, naproxen, levamisole, sulfasalazine, acetylsalicylic acid) Hypolipidemics (lovastatin) Proteins (albumin)

42. IV Agents containing sulfur (cysteine, cysteamine, GSH, dithiothreitol, N-acetylcysteine, ACE inhibitors, dimethylthiourea, thiourea, thiomalate, hypotaurine, taurine, penicillamine, 2-amino-2-thiazole, dihydrolipoate, a-mercaptopropionyl glycine, N-2-mercaptopropionyl glycine, b-mercaptoethanole, D,L-methionine, other low and high m.w. thiols) Nitroso compounds ( .NO, nitrosopine) Other drugs (b-adrenolytics, H2-antihistaminics, calcium channel blockers, pentoxyphylline, carbanilates, urea, bilirubin, glucans, manitol, glucose, 2-methylaminochromans, DMSO, BHT, BHA, 2-MEA, etoxiquin, a-lipoic acid, Zn2+)

43. V Inhibition of O2.- formation Nonsteroid antiflogistics Antiasthmatics (b-adrenomimetics, corticoids, methylxanthines) Prostaglandins Flavonoids Antibiotics (e.g. minocycline) Antimalarics Inhibitors of ACE Dipyridamol

44. VI/a Scavenging or removal of ROS Scavenging of generated O2.- Flavonoids & other natural products Vitamins E, C, A(?-carotene) Synthetic analogs of PGB2 Dipyridamol Pentoxiphylline Antibiotics .NO donors 5-acetylsalicylic acid Uric acid Scavenging HOCl Uric acid Taurine, hypotaurine Scavenging or quenching of 1O2 Silymarine ?-carotene Vitamin E Stobadine

45. VI/b Scavenging or removal of ROS Removal of H2O2 Catalase (not working in the presence of .NO) N-acetylcysteine Elimination of OH. Manitol Thiourea Stobadine Melatonin Probucol 5-acetylsalicylic acid Lazaroids DMSO, DMTU, BHT Uric acid Glucose

46. VI/c Scavenging or removal of ROS Lipid oxidation chain breaking antioxidants (anti LO. and LOO.) Bilirubin Vitamins E Vitamin C ?-carotenoids and oxycarotenoids Stobadine Melatonin ?-lipoic acid Uric acid Lazaroids BHT, BHA Ehoxyquin 2-methylaminochroman

47. Protection by STOBADINE (STB) of the acetylcholine induced relaxation in rat aortic rings caused by reversible occlusion of aorta in vivo (I/R)

48. STOBADINE (STB) effect on experimetal myocardial infarction (MI) in dogs 3hr occlusion of the anterior descendent branch of the left coronary artery Stobadine (1 mg/kg iv) given 30 min after the occlusion Reduction of the infarction area by 28% (? P < 0.05)

49. STOBADINE effect on transmission in rat hippocampal slices during hypoxia/reoxygenation

52. Therapeutic relevance of the use of antioxidantsI

53. II

54. ROS in the sequence of events in NEUROTRAUMA TRAUMA Excitatory aminoacid release (GLU) Ca2+ influx into the cells Activ. of inflam. cascade Protease/lipase activation (PAF, eikosanoids, Cell depolarization cytokines, PMN activ.) (? Mg block of NMDA rec.) ROS generation Na+influx cell devastation Edema NEURONAL DEATH TRIAD : EXCITOTOXICITY, Ca-OVERLOAD, OXIDATIVE STRESS Therapeutic interventions: -SH donors (N-acetylcysteine), lazaroids, steroids, deferoxamine, SOD, vitamines A,E,C, pyridoindoles, stobadine, PBN, flavonoids (quercetine), PAF antag. (BN 520210)

55. III

56. IV

57. Antianginal effect of STOBADINE (STB) Phase II clinical study Patients with angina pectoris (stable and effort) (n = 13) Effect of 4 week treatment with STOBADINE (up to 100 mg/day p.o.) Significant decrease in the No. of anginal attacks Significant (* P<0.05) decrease in the No. of selfadministered nitroglycerine tablets

58. Conclusions IROS act by:

59. Conclusions IIThe effects of ROS could be prevented or stopped by:

60. Conclusions IIITherapeutic success with the use of antioxidants, quenchers and scavengers