1. Introduction Peter Celec, MD, Dipl. Ing, MSc, PhD, MPH petercelec@gmail.com www.imbm.sk

2. Literature  Journals Indexed CC  Current contents  Impact factor

3. Literature  Systematic searching  Logical operators  Specific operators [ti], [au] Review Date range limit Type of publication

4. Literature  PubMed/Medline  ISI Web of Science  Publishers Elsevier/Sciencedirect Springer Willey Interscience

5. Literature  Access to journals  Libraries  Google

6. Literature  Open Access  Biomednet Central  PubMed Central  Public Library of Science

7. Clinical studies  Empirical medicine  Expert opinion  Evidence-based medicine Guidelines  Individualized medicine

8. Meta-analyses  Cohorts from several clinical studies  Statistical analysis  High informative value  The weight of large numbers

10. Oxidative and carbonyl stress Peter Celec, MD, Dipl. Ing, MSc, PhD, MPH petercelec@gmail.com www.imbm.sk

11. Introduction  Oxidative and carbonyl stress Definitions Sources and causes Consequences  Markers  The role in the pathogenesis of diseases  Therapeutic influences

12. Definitions  Free radical Atom or molecule with an unpaired – free electron  Reactive oxygen/nitrogen species – ROS/RNS Highly reactive molecule

13. ROS  Free radicals Superoxide. O 2 - Hydroxyl. OH Peroxyl ROO. Alkoxyl RO. Hydroperoxyl HO 2. Molecular oxygen O 2  Others Hydrogen peroxide H 2 O 2 Hypochlorous acid HClO Ozone O 3 Singlet oxygen 1 O 2

14. ROS

16. Fenton reaction  Fe 2+ + H 2 O 2 → Fe 3+ +. OH + OH - . O 2 - + Fe 3+ → O 2 + Fe 2+ . O 2 - + H 2 O 2 → O 2 +. OH + OH -  Catalytic effect of metal ions

17. RNS  Free radicals Nitric oxide. NO Nitric dioxide. NO 2  Others Nitrozyl NO + Nitrous acid HNO 2 Nitric trioxide N 2 O 3 Peroxynitrite ONOO - Alkylperoxynitrite ROONO

18. Endogen sources of ROS – reactions  Autooxidation (Fenton reaction) Hemoglobin, myoglobin, reduced thiols, cytochrome C, catecholamines  Respiratory burst NADPH oxidase  Other enzymatic production Prostaglandin synthase, lipoxygenase, myeloperoxidase, xantin oxidase

19. Endogen sources of ROS – organels  Mitochondria Terminal oxidation & Oxidative phosphorylation Electron transport system – cytochrome oxidase Superoxide anion radical Hyperoxia / hypoxia

20. Endogen sources of ROS – organels  Endoplasmatic reticulum Oxidation-reduction reactions Metabolism of xenobiotics Cytochrome P450  Microsomes – Peroxisomes Metabolism of fatty acids Hydrogen peroxide

21. Exogen sources of ROS  Drugs Nitrofurantoin, antracyclins, metotrexate, sulfasalasine  Radiation X-rays, radiotherapy, UV  Smoking  Ozone

22. Sources of RNS  NO synthases Endothelial Inducible Neuronal  Reaction of. NO and. O 2 -

23. Physiological functions of ROS  Detoxification of xenobiotics Monoxygenases, cytochrome oxidases  Immune response & Phagocytosis Myeloperoxidases NADPH oxidase  Signalling pathways

24. Physiological functions of RNS  Currently known only for. NO  Vasodilatation  Paracrine communication  Neuromediator  Nitrosylation

25. Damage caused by ROS  Lipids Lipoperoxidation  XNA Damage of bases – mutations  Proteins Carbonylation of proteins Cross-linking

26. Antioxidants  Antioxidative status Inhibition of production Scavenging Correction mechanisms

27. Antioxidants  Endogen  Exogen  Low molecular weight (LMW; non- enzymatic)  High molecular weight (HMW; enzymatic)

28. HMW antioxidants Enzymatic antioxidants Superoxide dismutase SOD Catalase CAT Glutathione peroxidase/reductase

29. HMW antioxidants Superoxide dismutase Mitochondrial MnSOD Cytoplasmatic CuZn Extracellular CuZn

30. SOD  In all aerobic organisms  Even in procaryotic organisms . O 2 - +. O 2 - + 2 H + → H 2 O 2 + O 2

31. CAT  In all eucaryotic aerobic organisms  Dismutation of hydrogen peroxide  2 H 2 O 2 → 2 H 2 O + O 2

32. Glutathione system  Glutathione (GSH) Glu-Cys-Gly  -SH group on cysteine residue  LMW antioxidant  Scavenger of free radicals  2 GSH +. O 2 - → GSSG + H 2 O  Most important cytoplasmatic antioxidant

33. Glutathione system  Only in animals  Glutathione peroxidase Contains selenocysteine Reduces hydrogen peroxide using glutathione 2 GSH + H 2 O 2 → GSSG + 2 H 2 O  Glutathione reductase GSSG + NADPH + H + → 2 GSH + NADP +

34. Antioxidant metabolic pathways

35. LMW antioxidants  Glutathione  Vitamins C, E  Karotenoids  Coenzyme Q 10  Tanins, Flavonoids  Proline ...

36. Balance OxidantsAntioxidants

37. Oxidative stress  Disbalance beteen the production of free radicals and antioxidative mechanisms  In the pathogenesis......of everything

38. Markers of oxidative stress  EPR  Damage of lipids Malondialdehyde, 4-OH-2-nonenal, 8-isoprostane  Damage of DNA 8-OH-guanine  Damage of proteins Dityrosine, carbonylated proteins

39. Diabetes mellitus

40. Carbonyl stress  Advanced glycation end products  AGEs

41. Carbonyl stress  Diabetic hyperglycemia (glycated hemoglobin)  Alcohol  Renal failure  Melanoidins  Oxidative stress

42. Synergism

44. Down syndrome  Trisomy of 21  21q22.11 – SOD gene 150% gene dose  Increased oxidative stress  Why?

45. Atherosclerosis

46. Aging  Decreased antioxidative status  Lipoperoxidation  Lipofuscin – Age pigment  Cross-linking of proteins  Induction of chronic inflammation  Cumulative DNA damage  Decreased effectivity of mitochondria

47. How to slow down the time?

49. Future outlook  New antioxidants  Combinatory approaches  Antioxidative gene therapy  Aminoguanidine and AGE-breakers  Clinical studies...