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Reactive Oxygen Species I. Free radicals & ROS Defined II. Sources of ROS III. Oxidative damage in biological systems IV. Antioxidant Defense V. ROS signaling.

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Presentation on theme: "Reactive Oxygen Species I. Free radicals & ROS Defined II. Sources of ROS III. Oxidative damage in biological systems IV. Antioxidant Defense V. ROS signaling."— Presentation transcript:

1 Reactive Oxygen Species I. Free radicals & ROS Defined II. Sources of ROS III. Oxidative damage in biological systems IV. Antioxidant Defense V. ROS signaling and redox sensitive pathways VI. Oxidative stress and disease VII. Detection methods for ROS & oxidative stress

2 The Earth was originally anoxic Metabolism was anaerobic O 2 started appearing ~2.5 x 10 9 years ago Anaerobic metabolism-glycolysis Glucose + 2ADP + 2P i Lactate + 2ATP + 2H 2 O O 2 an electron acceptor in aerobic metabolism Glucose + 6O ADP + 36P i 6CO ATP + 6H 2 O I. Free Radicals & ROS Defined

3 Ground-state oxygen has 2-unpaired electrons O:O :: : :.. The unpaired electrons have parallel spins Oxygen molecule is minimally reactive due to spin restrictions

4 TermDefinition OxidationGain in oxygen Loss of hydrogen Loss of electrons ReductionLoss of oxygen Gain of hydrogen Gain of electrons OxidantOxidizes another chemical by taking electrons, hydrogen, or by adding oxygen ReductantReduces another chemical by supplying electrons, hydrogen, or by removing oxygen Basics of Redox Chemistry

5 Free Radicals Free Radicals:  Any species capable of independent existence that contains one or more unpaired electrons  A molecule with an unpaired electron in an outer valence shell R 3 C. Carbon-centered R 3 N. Nitrogen-centered R-O. Oxygen-centered R-S. Sulfur-centered Prooxidants Non-Radicals:  Species that have strong oxidizing potential  Species that favor the formation of strong oxidants (e.g., transition metals) H 2 O 2 Hydrogen peroxide HOCl - Hypochlorous acid O 3 Ozone 1 O 2 Singlet oxygen ONOO - Peroxynitrite Me n+ Transition metals

6 Reactive Oxygen Species (ROS) Radicals: O 2.- Superoxide OH. Hydroxyl RO 2. Peroxyl RO. Alkoxyl HO 2. Hydroperoxyl Non-Radicals: H 2 O 2 Hydrogen peroxide HOCl - Hypochlorous acid O 3 Ozone 1 O 2 Singlet oxygen ONOO - Peroxynitrite Reactive Nitrogen Species (RNS) Radicals: NO. Nitric Oxide NO 2. Nitrogen dioxide Non-Radicals: ONOO - Peroxynitrite ROONO Alkyl peroxynitrites N 2 O 3 Dinitrogen trioxide N 2 O 4 Dinitrogen tetroxide HNO 2 Nitrous acid NO 2 + Nitronium anion NO - Nitroxyl anion NO + Nitrosyl cation NO 2 ClNitryl chloride

7 “Longevity” of reactive species Reactive SpeciesHalf-life Hydrogen peroxide Organic hydroperoxides~ minutes Hypohalous acids Peroxyl radicals~ seconds Nitric oxide Peroxynitrite~ milliseconds Superoxide anion Singlet oxygen ~ microsecond Alcoxyl radicals Hydroxyl radical~ nanosecond

8 “An imbalance favoring prooxidants and/or disfavoring antioxidants, potentially leading to damage” -H. Sies Antioxidants Prooxidants Oxidative Stress

9 Radical-mediated reactions Addition R. +H 2 C=CH 2 R-CH 2 -CH 2. Hydrogen abstraction R. +LHRH +L. Electron abstraction R. +ArNH 2 R - +ArNH 2.+ Termination R. +Y. R-Y Disproportionation CH 3 CH 2. + CH 3 CH 2. CH 3 CH 3 + CH 2 =CH 2

10 Hydroxyl radical (. OH) O Fe 3+ O 2 + Fe 2+ (ferrous) H 2 O 2 + Fe 2+ OH - +. OH + Fe 3+ (ferric) O H 2 O 2 OH - + O 2 +. OH Haber-Weiss Fenton Transition metal catalyzed Other reductants can make Fe 2+ (e.g., GSH, ascorbate, hydroquinones) Fe2+ is an extremely reactive oxidant

11 Important Enzyme-Catalyzed Reactions

12 Biological Pathways for Oxygen Reduction From: McMurry and Castellion “Fundamentals of general, organic and biological chemistry”

13 Endogenous sources of ROS and RNS Mitochondria Lysosomes Peroxisomes Endoplasmic Reticulum Cytoplasm Microsomal Oxidation, Flavoproteins, CYP enzymes Myeloperoxidase (phagocytes) Electron transport Oxidases, Flavoproteins Plasma Membrane Lipoxygenases, Prostaglandin synthase NADPH oxidase Xanthine Oxidase, NOS isoforms Fe Cu Transition metals II. Sources of ROS

14 Mitochondria as a source of ROS Turrens, J Physiol, 2003 Localization of the main mitochondrial sources of superoxide anion Mitochondrial electron chain Quinone cycle Chandel & Budinger, Free Radical Biol Med, 2007

15 Peroxisomes as a source of ROS and RNS Enzymes in mammalian peroxisomes that generate ROS Schader & Fahimi, Histochem Cell Biol, 2004

16 NADPH oxidase as a source of ROS Present mainly in neutrophils (oxidative burst), but also in many other cell types Activation of the gp91phox (NOX2) containing NOX complex of phagocytes involves phosphorylation of the cytoplasmic regulator p47phox, with the translocation of the cytoplasmic p47phox, p67phox, and p40phox regulatory components to the plasma membrane to interact with flavocytochrome-b558, which is composed of gp91phox and p22phox. Activation of the complex also involves guanine nucleotide exchange on the GTP-binding protein RAC stimulated by guanine nucleotide exchange factors. Guanine nucleotide exchange on RAC is associated with release of RhoGDI and translocation of RAC from the cytosol to the NOX complex at the plasma membrane. ANTIOXIDANTS & REDOX SIGNALING Volume 8, Numbers 3 & 4, 2006

17 PHS Prostaglandin H Synthase (PHS) as a source of ROS Co-oxidation of xenobiotics (X) during arachidonic acid metabolism to PGH 2

18 xanthine oxidase Cytoplasmic sources of ROS and RNS NO Nitric Oxide Synthases (NOS): neuronal nNOS (I) endothelial eNOS (III) inducible iNOS (II)

19 Lysosome as a source of ROS and RNS Myeloperoxidase undergoes a complex array of redox transformations and produces HOCl, degrades H 2 O 2 to oxygen and water, converts tyrosine and other phenols and anilines to free radicals, and hydroxylates aromatic substrates via a cytochrome P450-like activity

20 Microsomes as a source of ROS (I) A scheme of the catalytic cycle of cytochrome P450-containing monooxygenases. The binding of the substrate (RH) to ferric P450 (a) results in the formation of the substrate complex (b). The ferric P450 then accepts the first electron from CPR (cytochrome P450 reductase), thereby being reduced to the ferrous intermediate (c). This intermediate then binds an oxygen molecule to form oxycomplex (d), which is further reduced to give peroxycomplex (e). The input of protons to this intermediate can result in the heterolytic cleavage of the O–O bond, producing H 2 O and the ‘oxenoid’ complex (f), the latter of which then inserts the heme-bound activated oxygen atom into the substrate molecule to produce ROH. In eukaryotic monooxygenases, reactive oxygen species (ROS) are produced by ‘leaky’ branches (red arrows). In one such branch, a superoxide anion radical is released owing to the decay of the one-electron-reduced ternary complex (d). The second ROS-producing branch is the protonation of the peroxycytochrome P450 (e), which forms of H2O2. In addition to these ROS-producing branches, another mechanism of electron leakage appears to be the four- electron reduction of the oxygen molecule with the production of water (Davydov, Trends Biochem Sci, 2001).

21 Davydov, Trends Biochem Sci, 2001 Microsomes as a source of ROS (II)

22 Exogenous sources of free radicals Radiation UV light, x-rays, gamma rays Chemicals that react to form peroxides Ozone and singlet oxygen Chemicals that promote superoxide formation Quinones, nitroaromatics, bipyrimidiulium herbicides Chemicals that are metabolized to radicals e.g., polyhalogenated alkanes, phenols, aminophenols Chemicals that release iron ferritin

23 UV radiation H 2 O 2 OH. + OH. UV B UV A = nm UV B = nm UV C = nm Primarily a concern in skin and eye Can also cause DNA damage Can form singlet oxygen in presence of a sensitizer Ionizing radiation  -rays 2H 2 OH 2 O + e - + H 2 O* H 2 O*H +. OH High energy radiation will result in. OH

24 Quinone redox cycling as a mechanism to generate ROS “Premarin (Wyeth–Ayerst) is the most common drug used for hormone replacement therapy (HRT) and is composed of approximately 50% estrogens and 40% equine estrogens [equilenin (EN) and equilin (EQ)] (9). In vitro experiments have shown that equine estrogens are successively metabolized and are capable of forming various types of DNA damage (9–11) (Figure 1). Like estrogen, EN and EQ are metabolized by cytochrome P450 enzymes (CYP) to their 4-hydroxy and 2-hydroxy forms (9,10). 4-Hydroxyequilenin (4- OHEN) is rapidly auto-oxidized to an o-quinone (4- OHEN-o-quinone) which in turn readily reacts with DNA, resulting in the formation of unique dC, dA and dG adducts (4-OHEN–DNA adducts) with four possible stereoisomers for each base adduct (9,11,12). 4- Hydroxyequilin (4-OHEQ) is also autoxidized to an o- quinone which isomerizes to 4-OHEN-o-quinone. As a result, 4-OHEQ and 4-OHEN produce the same 4- OHEN–DNA adduct (13). Simultaneously, oxidative DNA damage, such as 7,8-dihydro-8-oxodeoxyguanine (8-oxodG), is also generated by reactive oxygen species through redox cycling between the o-quinone of 4- OHEN and its semiquinone radicals (14).”99–11Figure Nucl. Acids Res. (210) 38 (12):e133

25 O O 2 + Ozone Singlet oxygen Chemicals that form peroxides

26 Chemicals that promote O 2.- formation Flavoprotein NAD(P)HNAD(P) + O 2.- O2O2 Paraquat radical cation

27 Chemicals that are metabolized to radicals Phenols, aminophenols Polyhalogenated alkanes

28 Chemicals that are metabolized to radicals

29 Chemicals that release iron Ferretin Fe 2+ Fenton Chemistry + e - Requires reductant Promotes. OH formation Promotes lipid peroxidation in vitro

30 III. Oxidative Damage in Biological Systems Oxidative stress and cell damage High doses: directly damage/kill cells Low doses/chronic overproduction of oxidants: activation of cellular pathways stimulation of cell proliferation damage to cellular proteins, DNA and lipids

31 Classic lipid peroxidation 1.Initiation LH + X L + XH 2.Propagation L + O 2 LOO LOO + LH L + LOOH 3.Termination 2 LOO non-radical products L + LOO non-radical products L + L non-radical products Catalyzed by metals LOOH + Fe 2+ OH - + LO. + Fe 3+

32 Consequences of lipid peroxidation Structural changes in membranes alter fluidity and channels alter membrane-bound signaling proteins increases ion permeability Lipid peroxidation products form adducts/crosslinks with non lipids e.g., proteins and DNA Cause direct toxicity of lipid peroxidation products e.g., 4-hydroxynonenal toxicity Disruptions in membrane-dependent signaling DNA damage and mutagenesis

33 Protein targets for ROS Cysteine Methionine Tyrosine Histidine Tryptophan Oxidized proteins and amino acids found in biological systems

34 Consequences of protein thiol oxidation Oxidation of catalytic sites on proteins loss of function/abnormal function BUT(!): sometimes it is gain in function! Formation of mixed sulfide bonds Protein-protein linkages (RS-SR) Protein-GSH linkages (RS-SG) Alteration in 2 o and 3 o structure Increased susceptibility to proteolysis

35 8-hydroxyguanine 8-hydroxyadenine 2-hydroxyadenine 5,8-dihydroxycytosine thymidine glycol 5-hydroxymethyluracil DNA oxidation products

36 Oxidation of deoxyribose (DNA backbone) + B + Strand Breaks Aldehyde products Apurinic/apyriminic sites O2O2

37 Consequences of DNA oxidation DNA adducts/AP sites/Strand breaks mutations initiation of cancer Stimulation of DNA repair can deplete energy reserves (PARP) imbalanced induction of DNA repair enzymes induction of error prone polymerases activation of other signaling pathways

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