Biochemistry of aging – Free radicals and antioxidants Petr Tůma and Eva Samcová

Oxygen Origin of O2 – photosynthesis 6CO2 + 12H2O → C6H12O6 + 6H2O + 6O2 Cyanobacteria produce oxygen first 2 billion years ago aerobic metabolism Two basic equilibrium Acid-base – proton transfer base + H+ ↔ acid Oxidation-reduction – electron transfer oxidation form + e- ↔ reducing form

Reactive oxygen species (ROS) Reactive oxygen species are involved in releasing and conversion of energy necessary for life processes, are part of enzyme mechanisms, and some are also signaling molecules. Damages of organisms only when there is a loss of control.

Reactive oxygen species - ROS Gradual 4 electrons reduction of O2 to water superoxide (radical) O2 + e- → O2·- Hydrogen peroxide O2·- + e- + 2H+ → H2O2 Hydroxyl radical H2O2 + e- → OH- + HO· water HO· + e- → OH-

Source of superoxide 1. Respiration chain in mitochondrion 1-4% O2 is reduced incompletely to ROS complex I (NADH – dehydrogenase) complex III (ubiquinol: cytochrome c- reductase) 2. Cytochrome P-450 in endoplasmic reticulum ROS bound to enzyme – biotransformation, ethanol oxidation 3. Specialized cells – leukocytes, macrophages NADPH – oxidase in cytoplasmic membrane – baktericidal prophylactic system myeloperoxidase – production of HClO 4. Oxidation of hemoglobin to methemoglobin

Source of H2O2 Dismutation of superoxide: 2 O2·- + 2H+ = O2 + H2O2 spontaneous Enzyme Superoxide Dismutase Direct reduction of O2 action of oxidases Monoamine oxidase, Glutathione oxidase, Xanthine oxidase Peroxisomes Equipped with several enzymes, which are used for oxidation of diferent organic substrates (ethanol, phenols, formaldehyde)→ H2O2 Oxidation of long and side-chain fatty acids

Nonenzymic sources of ROS Besides enzymes, the oxygen is reduced in cells by small endogenous and exogenous molecules (they transfer electron to O2 from different reductases (e.g. from NADPH-cytochrom-P450 reductase and others) Quinone antibiotics adriamycine, daunomycine, streptonigrin, - cardiotoxic... 2. Pyridine herbicides paraquat, diquat – lung injury 3. Low-molecular complexes of Fe with phosphates, nucleotides (other toxic carrier of electrons) Complexes with ATP, ADP

Reactive nitrogen species – nitric oxide important second messenger antimicrobial effects – macrophages vasodilator Nitric oxide synthases – NOS NOS I – neuronal(brain) NOS II – macrophage NOS III – endothelial Peroxynitrite NO· + O2·- = OONO- Important powerful oxidant – oxidation of amino acids in proteins Antimicrobial effects – macrophages O2 + NADPH NO·

Physiological functions of free radicals Free radicals are a tool of oxidases and oxygenases Respiratory chain Inner mitochondrial membrane Aerobic phosphorylation Biotransformation of xenobiotics Mitochondrial cytochrome oxidase – P450 Superoxide and peroxide bound to enzyme Synthesis in cells Monooxygenases in endoplasmic reticulum of hepatocyte or in mitochondria of adrenal gland Hydroxylation of xenobiotics, synthesis of cholesterol and bile acids

ROS and RNS as an effective weapon of phagocytes against germs Neutrophils and macrophages Bactericidal prophylactic system (removes dead cells and kills bacteria) NADPH-oxidase (enzyme membrane complex) Activated after absorption of foreign particle →reduction of oxygen to superoxide→H2O2 Fenton reaction Myeloperoxidase Synthesis of HClO from H2O2 and Cl- Synthetase of NO (NOS II) NO concentration increases by several orders of magnitude Synthesis of peroxynitrite - NO + superoxide – OONO-

ROS and RNS as signal molecules Information net Primary messenger, secondary messenger Protein kinases – influencing activities of enzyme, transcription factors → gene expression Sensitivity of information net depends on redox state of cell (influencing of protein kinases) Redox state Capacity of antioxidant system (accessibility of reducing equivalents) Intensity of oxidation load (RONS) Nitric oxide NO (nitrogen oxide) Secondary messenger Neurotransmitter in CNS and autonomic nervous system (vegetative) Vasodilatation of vessels

Antioxidant protective system Restriction of excessive formation of ROS and RNS Regulation of enzyme activity Trap of transition elements from reactive sites Trap and elimination of radicals scavengers, trappers, quenching enzymes, substances which form with radicals more stable products General reparative mechanisms of injured macromolecules phospholipases reparative enzymes of DNA proteolysis of proteins injured by oxidative stress

Enzyme antioxidant systems H2O + ½ O2 catalase SOD + Fe2+ O2·- H2O2 ·OH + Fe3+ + OH- 2 GSH NADPH+H+ GSHPx 2 H2O GSSG NADP+

Superoxide dismutase – SOD accelerates the dismutation of superoxide by 4 orders present in most of aerobic cells and in extracellular fluid several isoenzymes with different cofactors: Cu, Zn, Mn, Fe Types of superoxide dismutases : mitochondrial (SOD2 = Mn-SOD, Fe-SOD) tetramer in prokaryotes and in mitochondria matrix cytoplasmic (SOD1 =CuZn-SOD) dimer, atom Cu and Zn in each subunit(also intermembrane space) elimination of SOD1 decreases life time, and causes the development of degenerative disease associated with old age – carcinogenesis extracellular (SOD3 = EC-SOD) elimination has only minimal effect

Glutathione peroxidase Removal of intracellular hydroperoxides Proteins with selen – selenocystein in active center 2 GSH + ROOH = GSSH + H2O + ROH cytosol GSH – glutathione peroxidase (cGPx) decomposes hydroperoxides of fatty acids after releasing from lipids by phospholipase A2, H2O2 Phospholipid hydro peroxide-GSH-peroxidase (PHGPx) reduces phospholipid hydroperoxides directly in plasmatic membrane without releasing of fatty acids from phospholipids

Catalase Two-electron dismutation of hydrogen peroxide 2H2O2 = 2 H2O + O2 Inactivation of H2O2 – peroxisomes and mitochondria of hepatocyte, cytoplasm of erythrocyte

High-molecular endogenic antioxidants Proteins which bind transition elements Fe and Cu = inactivation of these elements for catalysis transferrin – binds Fe3+ in blood lactoferrin – binds Fe3+ in leukocytes ferritin – intracellular, storage of Fe in the cell haptoglobin – uptake of extracellular hemoglobin ceruloplazmin – binds Cu in blood plasma albumins – bind on its –SH groups Cu2+oxidation to Cu3+ and damage the surrounding structures of albumin metalothioneins– proteins with many cysteins and via sulfur atoms form chelates with metal ions in the nucleus

Low-molecular endogenic antioxidants Soluble in water ascorbic acid – vitamin C glutathione uric acid lipoic acid Soluble in fat carotenoids and vitamin A α-tocopherol – vitamin E ubiquinol – coenzym Q

Ascorbic acid – vitamin C Derivative of monosaccharides occuring in animals and plants Essential for synthesis of collagen, hydroxylation of proline and lysine and in conversion of dopamine to norepinephrine Reduces radicals – O2·-, HOO·, HO·, ROO·, NO2 Transfer to hydroascorbate (ascorbyl radical) Regeneration by NADH In combination with Fe – prooxidative effect reduces Fe3+ to Fe2+ (absorption of Fe in intestine) HO· + H2O

a – tocopherol and vitamin E Group of 8 isomers –most significant a-tocopherol Most important lipophilic antioxidant Antioxidant of biological membranes Reduces alkylperoxyl radicals LOO· of lipids to hydrogen peroxides, which after are reduced by glutathione peroxidase From tocopherol arises slightly reactive tocopheryl radical Regeneration by ascorbate R-O-O· + R-O-O-H

Ubiquinone/ubiquinol – coenzyme Q10 Transfer of reducing equivalents in respiratory chain in the mitochondria It serves as an antioxidant in mitochondria and membranes (together with tocopherol) Partly is synthesized, partly accepted by diet Its level decreases in the mitochondria with increased age (old age). Then Heart failure Myocardial infarction Atherosclerosis

Carotenoids, b-carotene and vitamin A chemically isoprenoids Remove radicals centered on carbon and alkylperoxyl radicals ROO· in lipids

Glutathione GSH Glutathione (tripeptide- g-glutamylcysteinylglycine) is synthesized in the body The most significant intracellular redox buffer 2 GSH = GSSG + 2H+ + 2e- Nonenzymatic removal of ROS – HO·, RO·, ROO· Keeps in reduced form –SH groups of proteins, cysteine, CoA, regenerates ascorbate, Its regeneration is catalyzed by glutathione reductase Necessary substrate of glutathione peroxidase

Uric acid (urate) Lipoic acid (lipoate) End product of purine catabolism in human and primates Most plentiful antioxidant in blood plasma Traps RO·, HClO, binds Fe a Cu Hyperuricemia – gout Lipoic acid (lipoate) cofactor pyruvate dehydrogenase and a-oxoglutarate dehydrogenase multienzyme complex (cytric acid cycle) antioxidant ROO·, ascorbyl radical, HO·, NO·, O2·- Uric acid

Breaking antioxidant protection Oxidative stress – unbalancing between formation and removal of ROS a RNS excessive production of radicals inadequate antioxidant protection Causes of excessive production of ROS and RNS reoxygenation of tissue after ischemia after receiving redox active xenobiotics release of chemical bonds of Fe a Cu from bonds to storage proteins excessive production of NO and congestion load of SOD NO + O2·- = peroxynitrite - strong oxidant

Key role of Fe in oxidative damage to the body Fenton reaction Fe2+ + H2O2 = Fe3+ + HO· + OH- Catalytic ability of Fe in active enzyme centers (minute amount)– reactivity of Fe is rectified in favor of life events Fe reacts as in the case of nonspecific protein, lipid, and NA binding, and also after release from transferrin and ferritin – Damage to biomolecules Human body – 4 grams of Fe in oxidoreductases only tiny part 70% in hemoglobin, 10% in myoglobinu

Lipid peroxidation (LPO) in vitro – rancidity of oils – auto-oxidation radical reaction in vivo – lipid peroxidation – polyunsaturated fatty acids (PUFA) nonenzymatic Caused by non-specific pathological factors FFA cleavage on hydrocarbons - ethane, pentane and aldehydes→decrease of membrane fluidity 2. Enzymatic takes place at the active centers of hydroperoxidases and endoperoxidases → prostaglandins and leukotrienes

lipid HO· - H2O alkyl radical O2 peroxyl radical Fe alkoxyl radical C2H6 alkane alkenal

DNA damage Reaction with HO· radical removal of deoxyribose H atom - interrupts chain addition of HO· to bases - hydroxy and oxo derivatives

Protein damage Oxidation of amino acid residues methionine – methionine sulfoxide cysteine – cysteic acid arginine – aldehyde of glutamic acid proline – glutamic acid Hydroxylation of amino acids Aromatic amino acids Products of lipid peroxidation - ROS and RNS react with membrane proteins and proteins lipoprotein particles - other products LPO (reactive aldehydes MDA…) is covalently linked to the ɛ- lysine group → aggregation, networking

Non-enzymatic glycation of proteins and Diabetes mellitus Hyperglycemia is a major symptom of diabetes- high concentration of glucose – reactive molecule Covalent bond of aldehyde group glucose to amine group of proteins = glycation (Shiff base) Non-enzymatic glycation -early stage-hours-Shiff´s bases – ketomines -transitional stage-days-Amadori products – fructosamines - advanced stage –weeks, months – linking chains(transversal covalent bonds) – Advanced glycation end products (AGE = Maillard´s products) Glycation is accompanied glykooxidation (AGE and glucose are oxidized → ROS

Diabetes mellitus Glycated hemoglobin long-term blood glucose control in diabetics the percentage of glycated hemoglobin formed is directly proportional to the glucose concentration and the time that the red cells have been exposed to glucose.Measurement of it gives an integrated picture of the mean blood glucose during preceding 60 days. Physiological level – less than 4% Controlled diabetes ( DM) – 5% Impulse to change therapy – more than 6%