REACTIVE OXGEN AND NITROGEN SPECIES AND THEIR BIOLOGICAL EFFECTS Peter Kaplán

Reactive oxygen and nitrogen species ROS/RNS Free radical – each molecule or its fragment, which can exists independently And contains one or two unpaired electrons Reactive oxygen species - species, which contain one or more oxygen atoms and are much more reactive than molecular oxygen ROS/RNS Free radicals superoxide radical hydroperoxyl radical hydroxyl radical nitric oxide hydrogen peroxide

Some characteristics of ROS powerful oxidizing agent 10 -6 s 1 O 2 Singlet oxygen weak oxidant, high diffusability 10 -2 s LOO • Peroxyl radical less reactive than OH • , but more than ROO • 10 -6 s LO • Alkoxyl radical extremely reactive, diffuses only to very low distance 10 -9 s OH • Hydroxyl radical oxidant, diffuses across membranes minúty H 2 O 2 Hydrogen peroxide stronger oxidant than O 2 •- HO 2 • Hydroperoxyl radical poor oxidant 10 -6 s O 2 •- Superoxide radical Properties Half-life Symbol ROS

Sources of ROS, RNS and other oxidants Ionizing radiation: homolytic break of covalent bonds in water, DNA and other biomolecules

Cellular sources of ROS xanthine oxidase hemoglobin riboflavin catecholamines Cytochrome P450 electron transport chain lipid peroxidation NADPH oxidase (oxidative burst: phagocytes) oxidases flavoproteins myeloperoxidase (oxidative burst: phagocytes) transient metals

Cellular sources of ROS - examples Electron transport system: Autooxidation of hemoglobin: Fenton reaction:

Cellular sources of RNS Formation of nitric oxide From arginine: NO and superoxide radical combine to form strong oxidizing agent peroxynitrite : Peroxynitrous acid breaks down to form additional ROS/RNS (hydroxyl radical and nitrogen dioxide): Three tissue-specific NO synthases: neuronal (nNOS) inducible (iNOS) –in cells of the immune system, macrophages – responsible for the overproduction of NO endothelial (eNOS)

Three tissue-specific NO synthases:

neuronal (nNOS)

inducible (iNOS) –in cells of the immune system, macrophages – responsible for the overproduction of NO

endothelial (eNOS)

Formation of ROS and peroxynitrous acid in phagocytic vacuole of phagocytes SOD – superoxid dismutase MPO - myeloperoxidase

Formation of lipid (alkyl) radical initiated by ROS: Alkyl radical react with O 2 to produce peroxyl radical: Peroxyl radical attacks another poly- unsturated FA to produce new alkyl radical and lipid peroxide: Oxidative damage to lipids - Lipid peroxidation (LPO) ROS

malondialdehyde 4-hydroxynonenal HNE – highly toxic aldehyd, which reacts with free –SH and -NH 2 groups of proteins and -NH 2 groups of DNA MDA - toxic aldehyde, which reacts with free -NH 2 groups of proteins and DNA Loss of biological activity DNA mutation Oxidative damage to proteins and DNA - Modification by LPO products. Formation of intra- and intermolecular cross-links.

Oxidative modification of proteins dityrosine and 3-nitrotyrosine – markers of protein damage by ROS and RNS

Oxidative damage to DNA Formation of thymine dimers Modification of purines – 8-hydroxy-deoxyguanosine Marker of the oxidative damage to DNA

Oxidative damage to DNA – DNA mutation

Glucose and cell oxidative injury Hyperglycemia Glucose autooxidation Oxidative stress Formation of AGE products Polyol pathway Angiopathy Nefropathy Neuropathy Retinopathy

Glucose and oxidative damage Formation of AGE products AGE – advanced glycation end-products (pentosidine and others)

Mechanisms of cell injury mediated by ROS and RNS

Antioxidants and secondary defense systems Prevent ROS formation Eliminate radicals by formation of nonradicals or less reactive radicals Repair dameged molecules and cell structures Expression of genes coding for antioxidant enzymes Antioxidants and secondary defense systems Enzyme antioxidants Nonenzymatic antioxidants Chelating agents Enzymes of repair and de novo synthesis of damaged molecules Water-soluble Lipid-soluble Endogenous Present in diet

Prevent ROS formation

Eliminate radicals by formation of nonradicals or less reactive radicals

Repair dameged molecules and cell structures

Expression of genes coding for antioxidant enzymes

Antioxidant enzymes

Antioxidant enzymes SOD scavenges superoxide radical: Catalase decomposes hydrogen peroxid in peroxisomes : Glutathione peroxidase (GPx) decomposes H 2 O 2 and lipid peroxides in cytosol and mitochondria by help of GSH, NADPH and glutathionereductase (GRed):

Nonenzymatic antioxidants Endogenous antioxidants - Synthesized in the body bilirubín glutathione and other thiocompounds (thioredoxin) uric acid coenzyme Q (Ubichinon-10/Ubichinol-10) lipooic acid melatonin sex hormones 2-oxoacids (pyruvate, 2-oxoglutarate) dipeptides containig His (carnosine, anserine) albumin (-SH groups) Dietary antioxidants ascorbic acid vitamine E carotenoids flavonoids – plant phenols (catechin, quercetin etc) Synthetic antioxidants N-acetylcystein (scavenger of ROS), deferoxamine (chelator), alopurinol (inhibitor of XO), acetyl salicylic acid (feritine synthesis)

Nonenzymatic antioxidants – regeneration of α -tocopherol by ascorbic acid dehydroascorbic acid ascorbic acid

Chelating agents and secondary defenses

ROS, RNS and signal transmission – redox signaling ROS/RNS ionic channels and pumps thioredoxin GSH transcription factors protein kinases protein phosphatases Enzymes of CTC respiratory chain cell growth/apoptosis/survival Molecular mechanisms: modification of the redox state of protein (protein thiol groups) nitrosylation of protein

Molecular mechanisms:

modification of the redox state of protein

(protein thiol groups)

nitrosylation of protein

NO – signaling molecule, which difuses through water and lipid membranes At low concentrations serves as a second messenger and neurotrasmitter and participates in: smooth muscle relaxation lowering blood pressure inhibition of pletelet aggregation inflammatory and immune responses

At low concentrations serves as a second messenger and neurotrasmitter and participates in:

smooth muscle relaxation

lowering blood pressure

inhibition of pletelet aggregation

inflammatory and immune responses

Pro-oxidants, antioxidants and oxidative stress Oxidative stress – imbalance of pro-oxidants and antioxidants in favor of the former Oxidative stress – disruption of redox signaling and control

Major diseases/disorders linked to ROS/RNS Respiratory system Inhalation of oxidants (SO 2 , NO 2 , O 3 ) Smoking Brain Alzheimer `s disease Parkinson ` s disease Amyotrophic lateral sclerosis Down syndrome Traumatic injury Cardiovascular system Atherosclerosis Ischemia-reperfusion injury Myocardial infarction and heart failure Selenium deficiency (Keshan disease) Skin Ionizing radiation Thermal injury Porphyrias Photosenzitizers and other reagents Muscle Exercise Muscular dystrophy Others Aging Cancer Cataracts Diabetes mellitus Inflamatory and autoimmune diseases Liver damage by endotoxins or halogen derivatives of hydrocarbons Kidney diseases/disorders AIDS

Formation of ROS during ischemia and reperfusion ATP ADP AMP adenosine inosine hypoxanthine Xanthine dehydrogenase Xanthine oxidase Xanthine + •O 2 - + H 2 O 2 I S C H E M I A R E P E R F USION Ca 2+ proteolysis