Introduction Peter Celec, MD, Dipl. Ing, MSc, PhD, MPH

Literature  Journals Indexed CC  Current contents  Impact factor

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

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

Literature  Access to journals  Libraries  Google

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

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

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

Oxidative and carbonyl stress Peter Celec, MD, Dipl. Ing, MSc, PhD, MPH

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

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

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

ROS

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

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

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

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

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

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

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

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

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

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

Antioxidants  Antioxidative status Inhibition of production Scavenging Correction mechanisms

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

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

HMW antioxidants Superoxide dismutase Mitochondrial MnSOD Cytoplasmatic CuZn Extracellular CuZn

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

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

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

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 +

Antioxidant metabolic pathways

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

Balance OxidantsAntioxidants

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

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

Diabetes mellitus

Carbonyl stress  Advanced glycation end products  AGEs

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

Synergism

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

Atherosclerosis

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

How to slow down the time?

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