Reactive Oxygen Species

Slides:



Advertisements
Similar presentations
Pathways that Harvest and Store Chemical Energy
Advertisements

PAUL VI CATHOLIC HIGH SCHOOL
AGEING CAN BE DEFINED AS THE PROGRESSIVE LOSS OF FUNCTION ACCOMPANIED BY DECREASING FERTILITY AND INCREASING MORTALITY.
Overview of oxidative phosphorylation
Detection of reactive oxygen and nitrogen species using leuco dyes (DCFH 2 and DHR) Marta Wrona, Mark Burkitt and Peter Wardman Gray Cancer Institute,
BIOLOGICAL ROLE OF OXYGEN
Cell Injury and Cell Death
Oxidants and Aging Rolf J. Mehlhorn Lawrence Berkeley Laboratory
Oxidative Stress Concepts Donald Becker Redox Biology Center University of Nebraska Graduate Course 2214/938 (KI/UNL) June 14, 2010.
Reactive Oxygen and Nitrogen Species
Reactive Oxygen Species
Reactive Oxidative Species Generation and Neurodegenerative Disorders
Oxidative Stress.
Antioxidants & Free radicals. What are Reactive Oxygen Species?  ROS also known as Free oxygen radicals  Any molecule with an unpaired electron 
Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons.
Cell Injury and Cell Death
Introduction Microbes transfer energy by moving electrons.
General Principles of Cell Injury
Oxidative Stress and Atherosclerosis. Objectives: What is „free radical“? Reactive oxygen and nitrogen species (RONS) Are the RONS always dangerous? Well.
Oxidative Stress and Atherosclerosis
Chapter 18 Oxidative phosphorylation  the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH 2 to O 2 by a series.
Oxidative phosphorylation pathway Lecture 15 Modified from internet resources, journals and books.
AP Biology Ch. 9 – Cellular Respiration. Catabolic pathway Fermentation Aerobic respiration Anaerobic respiration Cellular respiration Redox reaction.
The most important structural feature of an atom for determining behaviour is the number of electrons in the outer shell. A substance that has a full.
Free radicals and Antioxidants. Objectives  Basics of Redox Chemistry.  Heterolytic and Homolytic fission.  Free Radical meaning.  Important characteristics.
“Other” detoxication mechanisms P-glycoprotein: ATP-dependent carrier that removes molecules from cells Multidrug resistance associated protein MDR Multispecific.
Ch 25 Metabolism and Energetics Introduction to Metabolism Cells break down organic molecules to obtain energy  Used to generate ATP Most energy production.
Pathways That Harvest Chemical Energy
Cellular Respiration: Harvesting Chemical Energy
صدق الله العظيم الروم ـ 54 Visible light and infra red RADIATION Non-ionizing radiation Ionizing radiation Particulate Alpha-, Beta-particles & Neutrons.
Luděk Bláha, PřF MU, RECETOX BIOMARKERS AND TOXICITY MECHANISMS 07 – Mechanisms Oxidative stress.
Pp 69 – 73 & Define cell respiration Cell respiration is the controlled release of energy from organic compounds in cells to form ATP Glucose.
Mitochondrial Electron Transport The cheetah, whose capacity for aerobic metabolism makes it one of the fastest animals.
1.Chemistry of reactive oxygen species (ROS) 2. Sources, defense mechanisms and pathological consequences 3. A survey of pathological conditions connected.
Coordination of Intermediary Metabolism. ATP Homeostasis Energy Consumption (adult woman/day) – kJ (>200 mol ATP) –Vigorous exercise: 100x rate.
Minerals as co-ezymes Dr. Shariq Syed Shariq AIKC/SYB/2014.
Twelfth lecture PROTECTIONREPAIRREGENERATION GENETICS Reduce concentration of reactive intermediates Restore molecular function Stimulate proliferation.
Chapter 07 Cellular Respiration Biology II – Dual Enrollment.
Fifth Lecture Oxidative damage by free radicals Oxidative damage by free radicals DNA Protein Lipids Cross- linkage Oxidized bases Strand breaks Hydroxylation.
Oxidant Mechanisms in Response to Ambient Air Particles Beatriz González-Flecha Department of Environmental Health Harvard School of Public Health Boston,
Biochemistry of free radicals, oxidative stress and aging
Introduction Peter Celec, MD, Dipl. Ing, MSc, PhD, MPH
بسم الله الرحمن الرحيم.
Vitamin C & its Antioxidant Chemistry Ascorbic Acid In the Name of The Most High.
METABOLISM OF OXYGEN AND FREE RADICALS. Oxygen acts as a substrate for approx. 200 enzymes. Based on the enzyme mechanism we discriminate: OXIDASES OXYGENASES.
CYTOCHROMES M.Prasad Naidu MSc Medical Biochemistry,
Ch. 6: Cellular Respiration Harvesting Chemical Energy.
Biochemical Basis of CVD:Part-1 Role of Free radicals & Antioxidants
Oxygen. Oxygen Terrestrial distribution: 3rd of the most frequently occurring elements: (H, He, O 2 ) 16 8 O (99 %) 18 8 O (izotóp) Bioinorganic importance.
Cytochrome P 450 Biochemistry Department. Cytochrome P 450 Unique family of heme proteins present in bacteria, fungi, insects, plants, fish, mammals and.
ELECTRON TRANSPORT CHAIN. An electron transport chain (ETC) couples electron transfer between an electron donor (such as NADH ) and an electron acceptor.
Lipid Peroxidation.
Reactive Oxygen Species in Foods
06 – Mechanisms Oxidative stress
Oxidative Stress and Atherosclerosis
Ubiquinol (QH2) is also the Entry Point for Electrons From FADH2 of Flavoproteins succinate dehydrogenase (integral membrane protein of inner mitochondrial.
Metabolism - Biotransformation
Oxidants and antioxidants in alcohol-induced liver disease
Copyright © 2001 American Medical Association. All rights reserved.
Mechanism of Cell Injury
Pathways that Harvest and Store Chemical Energy
Do reactive oxygen species play a role in myeloid leukemias?
Oxidants and antioxidants in alcohol-induced liver disease
METABOLISM OF OXYGEN AND FREE RADICALS.
Oxidative stress in Alzheimer's disease
by Sean X. Gu, Jeff W. Stevens, and Steven R. Lentz
Antioxidants & Free radicals
Metabolism of reactive species
Oxidative Stress in the Pathogenesis of Skin Disease
WBCs Metabolism By Dr. Samar Kassim.
Presentation transcript:

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

I. Free Radicals & ROS Defined The Earth was originally anoxic Metabolism was anaerobic O2 started appearing ~2.5 x 109 years ago Anaerobic metabolism-glycolysis Glucose + 2ADP + 2Pi Lactate + 2ATP + 2H2O O2 an electron acceptor in aerobic metabolism Glucose + 6O2 + 36ADP + 36Pi 6CO2 + 36ATP + 6H2O

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

Basics of Redox Chemistry Term Definition Oxidation Gain in oxygen Loss of hydrogen Loss of electrons Reduction Loss of oxygen Gain of hydrogen Gain of electrons Oxidant Oxidizes another chemical by taking electrons, hydrogen, or by adding oxygen Reductant Reduces another chemical by supplying electrons, hydrogen, or by removing oxygen

Prooxidants R3C. Carbon-centered R3N. Nitrogen-centered R-O. Oxygen-centered R-S. Sulfur-centered 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 H2O2 Hydrogen peroxide HOCl- Hypochlorous acid O3 Ozone 1O2 Singlet oxygen ONOO- Peroxynitrite Men+ Transition metals Non-Radicals: Species that have strong oxidizing potential Species that favor the formation of strong oxidants (e.g., transition metals)

Reactive Oxygen Species (ROS) Reactive Nitrogen Species (RNS) Radicals: O2.- Superoxide OH. Hydroxyl RO2. Peroxyl RO. Alkoxyl HO2. Hydroperoxyl Non-Radicals: H2O2 Hydrogen peroxide HOCl- Hypochlorous acid O3 Ozone 1O2 Singlet oxygen ONOO- Peroxynitrite Reactive Nitrogen Species (RNS) Non-Radicals: ONOO- Peroxynitrite ROONO Alkyl peroxynitrites N2O3 Dinitrogen trioxide N2O4 Dinitrogen tetroxide HNO2 Nitrous acid NO2+ Nitronium anion NO- Nitroxyl anion NO+ Nitrosyl cation NO2Cl Nitryl chloride Radicals: NO. Nitric Oxide NO2. Nitrogen dioxide

“Longevity” of reactive species Reactive Species Half-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

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

Radical-mediated reactions Addition R. + H2C=CH2 R-CH2-CH2. Hydrogen abstraction R. + LH RH + L. Electron abstraction R. + ArNH2 R- + ArNH2.+ Termination R. + Y. R-Y Disproportionation CH3CH2. + CH3CH2. CH3CH3 + CH2=CH2

Hydroxyl radical (.OH) O2.- + Fe3+ O2 + Fe2+ (ferrous) H2O2 + Fe2+ OH- + .OH + Fe3+ (ferric) O2.- + H2O2 OH- + O2 + .OH Fenton Haber-Weiss Transition metal catalyzed Other reductants can make Fe2+ (e.g., GSH, ascorbate, hydroquinones) Fe2+ is an extremely reactive oxidant

Important Enzyme-Catalyzed Reactions

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

Prostaglandin synthase II. Sources of ROS 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

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

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

NADPH oxidase as a source of ROS Present mainly in neutrophils (oxidative burst), but also in many other cell types ANTIOXIDANTS & REDOX SIGNALING Volume 8, Numbers 3 & 4, 2006 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.

Prostaglandin H Synthase (PHS) as a source of ROS Co-oxidation of xenobiotics (X) during arachidonic acid metabolism to PGH2 PHS

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

Lysosome as a source of ROS and RNS Myeloperoxidase undergoes a complex array of redox transformations and produces HOCl, degrades H2O2 to oxygen and water, converts tyrosine and other phenols and anilines to free radicals, and hydroxylates aromatic substrates via a cytochrome P450-like activity

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 H2O 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).

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

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

UV radiation Ionizing radiation H2O2 OH. + OH. g-rays UVB UVA = 320-400 nm UVB = 290-320 nm UVC = 100-290 nm Primarily a concern in skin and eye Can also cause DNA damage Can form singlet oxygen in presence of a sensitizer Ionizing radiation g-rays 2H2O H2O + e- + H2O* H2O* H + .OH High energy radiation will result in .OH

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).” Nucl. Acids Res. (210) 38 (12):e133

Chemicals that form peroxides Ozone 1O2 + Singlet oxygen

Chemicals that promote O2.- formation NAD(P)H NAD(P)+ Flavoprotein Paraquat radical cation Paraquat O2.- O2

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

Chemicals that are metabolized to radicals

Chemicals that release iron Ferretin Fe2+ Fenton Chemistry + e- Requires reductant Promotes .OH formation Promotes lipid peroxidation in vitro

Oxidative stress and cell damage 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

Classic lipid peroxidation Initiation LH + X• L• + XH 2. Propagation L• + O2 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 + Fe2+ OH- + LO. + Fe3+

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

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

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 2o and 3o structure Increased susceptibility to proteolysis

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

Oxidation of deoxyribose (DNA backbone) Strand Breaks O2 + B + Apurinic/apyriminic sites Aldehyde products

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