Oxygen Radicals and Reactive Oxygen Species

1. Oxygen Radicals and Reactive Oxygen Species ENRIQUE CADENAS PSC 616 Buettner GR SFRBM 2009 Sunrise Free Radical School Peter Day

2. Free Radicals • A free radical is an atom (or group of atoms) capable of independent existence (hence the term “free”) that contains one or more unpaired electrons. • This definition includes the hydrogen atom and most of the transition metal ions. It also includes the oxygen molecule, which is biradical since its outer two electrons are in different orbitals and have parallel spins (they are not paired). • Free radicals are • small • diffusible • unstable • very reactive • short-lived

3. Free Radicals • Free radicals may be electrically neutral or either positively or negatively charged. • They attack sites of increased electron density such as: • the nitrogen atom present in proteins and DNA predominantly • and carbon-carbon double bonds present in polyunsaturated fatty acids and phospholipids to produce additional free radical, often reactive, intermediates. • Free radicals can participate in chain reactions in which a single free radical initiation event can be propagated to damage multiple molecules. Knight J, in Free Radicals, Antioxidants, Aging and Disease 1999 Halliwell B and Gutteridge J in Free Radicals in Biology and Medicine 2007

4. Free Radical Notation • Denoted by a superscript dot to the right. • Examples (Note: dot, then charge) • H,Cl,HO, or (HO) • O2 or O22 dioxygen • O2- , CO2-, Asc-, PQ+ Buettner GR SFRBM 2007 Sunrise Free Radical School

5. Common Notations and Abbreviations Species Systematic IUPAC Name Alternative/Comments O  oxide(1-) hydroxyl radical without proton O2   dioxide(1-) superoxide O3 trioxygen ozone O3  trioxide(1-) ozonide HO hydroxyl not hydroxy, hydroxide is OH HO2 hydrogen dioxide hydrodioxyl, or hydroperoxyl, but perhydroxyl does not make sense HO2  hydrogen dioxide(1-) hydrogenperoxide(1-) H2O2 hydrogen peroxide RO alkoxyl not alkoxy ROO alkyldioxyl alkylperoxyl not peroxy ROOH alkyl hydroperoxide ONOO oxoperoxonitrate (1-) peroxynitrite ONOOH hydrogen oxoperoxonitrate peroxynitrous acid NO nitrogen monoxide nitric oxide Buettner GR SFRBM 2007 Sunrise Free Radical School

6. Types of radicals Sigma,  pi-delocalized,  Mixture of sigma and pi Carbon-centered, H3C O2–centered, H3COO Sulfur-centered, GS Nitrogen-centered, R2NO Reducing radicals, CO2-, PQ+ Oxidizing radicals, HO, LOO, CO3- Buettner GR SFRBM 2007 Sunrise Free Radical School

7. Free Radicals: Positive Effects • The presence of low concentrations of free radicals is important for normal cellular redox status, immune function, and intracellular signalling. • Immune system: neutrophils and macrophages use ROS to destroy engulfed microorganisms. O2.- and HOCL are powerful oxidizing agents that degrade microbes. • Can serve as second messengers or modify oxidation-reduction (redox) states. • Involved in some enzyme activation. • Play an essential role in muscle contraction. Temple M etal; Trends in Cell Biology 2005, 15 (6) 319-326 Gomez-Cabera M; Free Radical Biology and Medicine 2008, 44: 126-131 Finaud J et al; Sports Med, 2006: 36(4) 327-358

8. Free Radicals: Negative Effects • However, excessive production can provoke inflammation or altered cellular functions through: • lipid peroxidation • protein modification • DNA modification Which compromises cell function leading to cell death by necrosis or apoptosis.

9. Measurement of Free Radicals • Direct measurement • Biomarkers of free radical damage • Cytotoxicity Assays • GSH/GSSG • Total Antioxidant Activity • Antioxidant Enzyme Activity Assays • Lipid peroxidation • 4-hydroxy-2-nonenal (HNE) • Malondialdehyde (MDA) • 2-propenal (acrolein) • Isoprostanes • Protein modification • Protein Carbonyls • Tyrosine oxidation, nitration and halogenation • DNA Modification • 8-hydroxy-2’-deoxyguanine (8-OHdG) Dalle-Donne I etal; Clin Chem (2006), 52:4, 601-623 Finaud Jetal; Sports Medicine (2006) 36:4, 327-358 Halliwell B & Whiteman M; British Journal of Pharmacology (2004), 142, 231-255

10. Oxygen • Toxicity • Higher oxygen levels are toxic • Varies with species, age, physiological state, and diet • Reason • Enhances damaging effects of ionizing radiations • Damaging effects • Acute effects • Due to inactivation of enzymes • Slower effects • Due to formation of free radicals/ROS

11. Reactive Oxygen Species • Oxygen is a relatively unreactive compound • Can be metabolized in vivo to form highly reactive oxidants known as Reactive Oxygen Species (ROS). • Increasing evidence suggests that the generation of these ROS plays an important role in the pathophysiology of a number of diseased states.

12. Reactive Oxygen Species : Clinical conditions • Skin - porphyria - solar radiation • Eye - cataract - retrolentalfibroplasia • Cardiovascular system - Keshan disease (selenium deficiency) - atherosclerosis - adriamycincardiotoxicity) • Brain - Parkinson’s disease - Alzheimer’s disease - Multiple sclerosis - neurotoxins • Ischemia-reperfusion states - myocardial infarction / stroke - organ transplantation - frostbite • Red blood cells - hemolytic anemia - protoporphyrinphotooxidation - lead poisoning - phenylhydrazine toxicity - primaquine and related drugs • Lung - emphysema - bleomycin toxicity - paraquat toxicity - asbestos carcinogenicity

13. Reactive Oxygen Species What are Reactive Oxygen Species ? How reactive are Reactive Oxygen Species ? How are they generated in the cell? How do they mediate cellular damage? How do cells protect themselves against oxygen radicals?

14. Oxygen • A free radical is defined as • Any species that contains one or more unpaired electron occupying an atomic or molecular orbital by itself. • The box diagram configuration for O2 shows that in itself oxygen is a diradical, because it possesses two unpaired electrons; the Lewis dot diagram on the right also shows the diradical character of molecular oxygen

15. Oxygen • Stable in its ground-state • Very unlikely to participate in reactions with organic molecules unless it is "activated". • The requirement for activation occurs because the two unpaired electrons in oxygen have parallel spins. • According to Pauli's exclusion principle, this precludes reactions unless the reactant also has two unpaired electrons with parallel spin opposite to that of the oxygen, which is a very rare occurrence.

16. Oxygen • Spin restriction makes O2 prefer to accept electrons one at a time • Reacts sluggishly with most non-radicals • Reacts fast with other radicals by single electron transfers • Fortunately, most molecules in human body are non-radicals.

17. Oxygen

20. Reactive Oxygen Species • Singlet states: • Can be generated by an input of energy. • Spin restriction is removed • increases oxidizing ability

21. Reactive Oxygen Species If triplet oxygen absorbs sufficient energy to reverse the spin of one of its unpaired electrons, it will form the singlet state, in which the two electrons have opposite spins This activation overcomes the spin restriction and singlet oxygen can consequently participate in reactions involving the simultaneous transfer of two electrons (divalent reduction). Since paired electrons are common in organic molecules, singlet oxygen is much more reactive towards organic molecules than its triplet counterpart.

22. Generation of ROS Temple Metal; Trends in Cell Biology 2005, 15 (6): 319-326

23. Reactive Oxygen Species : Mechanisms of Formation • Oxygen radicals or reactive oxygen species may be generated by • electron-transfer reactions • energy-transfer reactions • Both types of reactions are important in a biological milieu and account partly for different types of cellular injury and toxicity. • The reactive oxygen species (not all of them are free radicals) generated by either process outlined above are: • electron–transfer reactions • - superoxide anion radical • - hydrogen peroxide • - hydroxyl radicals • lipid alkoxyl and peroxyl radicals • energy–transfer reactions • - singlet oxygen

24. Reactive Oxygen Species : Mechanisms of Formation Formation of Oxidants by Electron Transfer Reactions The following scheme illustrates the sequential univalent reduction of oxygen to water with formation of different intermediates: superoxide anion radical (O2.–), hydrogen peroxide (H2O2), and hydroxyl radical (HO.):

25. Bonding in diatomic oxygen molecule and its derivatives

26. Reactive Oxygen Species : Mechanisms of Formation • Superoxide anion radical (O2.–) • Formed by the addition of one electron to molecular oxygen • Not a very reactive species and its chemical reactivity depends on • its site of generation in the cell, • the possibility of being protonated to a stronger oxidant (perhydroxyl radical), • collision with suitable substrates.

27. Reactive Oxygen Species : Mechanisms of Formation • Hydrogen peroxide (H2O2) • Formed upon • two-electron reduction of molecular oxygen • or • one-electron reduction of superoxide anion (O2.–):

28. Reactive Oxygen Species : Mechanisms of Formation Univalent reduction of molecular oxygen to hydrogen peroxide (H2O2) encompasses superoxide anion (O2.–)as an intermediate:

29. Reactive oxygen species Remember the definition of a free radical as a species that contains one or more unpaired electron occupying an atomic or molecular orbital by itself. Therefore,

30. Reactive Oxygen Species : Mechanisms of Formation • Hydroxyl radical (HO.) • The most reactive oxygen species • originating from a reaction between superoxide anion radical (O2.–) and hydrogen peroxide (H2O2). • Chemically, one-electron reduction of hydrogen peroxide yields hydroxyl radical and water (hydroxyl anion).

31. Reactive Oxygen Species : Mechanisms of Formation Formation of Oxidants by Energy Transfer Reactions Transfer of energy from an excited sensitizer to ground state molecular oxygen is known as photosensitization. The sensitizer (S) absorbs energy upon irradiation and transfers it to molecular oxygen (O2) with formation of singlet oxygen (1O2): Examples of sensitizers are methylene blue, rose bengal, acridine orange, and several biological molecules, such as riboflavin, bilirubin, retinal, porphyrins, chlorophylls, etc.

32. Reactive Oxygen Species : Mechanisms of Formation Absorption of energy by a sensitizer in the ground state (S0) is associated with promotion of an electron to the next energy level (box diagram), thereby yielding the excited state of the sensitizer (S1). Depending on the amount of energy absorbed, the singlet states may be S1, S2, etc.

33. Reactive Oxygen Species : Mechanisms of Formation The electron promoted to the next energy level in a singlet state has a different spin. That promoted to the next energy level in a triplet state has the same spin. Singlet states can release a modest amount of energy and be transformed into triplet states (which involves change of the electron spin). The process is known as intersystem crossing:

34. Reactive Oxygen Species : Mechanisms of Formation Molecular oxygen in the ground state is a triplet (in the p2 boxes, two electrons have the same spin). Energy transferred to ground state molecular oxygen from an excited sensitizer is used to promote the electron to the next energy level as well as a change of spin, thereby the name singlet oxygen.

35. Reactivity of ROS The chemical reactivity of a variety of reactive species, whether of free radical character or not, varies substantially; regardless of their source. Reactivity of Superoxide Anion The reactivity of superoxide radical is dependent on the cellular environment. Two reactions are important in a cellular setting, which change the chemical reactivity of superoxide anion(O2.–): a. Reactivity of superoxide anion with itself Superoxide anion (O2.–) is short lived and tends to react with itself, a process known as dismutation or disproportionation and which yields molecular oxygen and hydrogen peroxide (H2O2). b. Protonation of superoxide anion This process may take place in the vicinity of membranes (with an increased proton gradient). The protonation of superoxide anion (O2.–) yields perhydroxyl radical (HO2.), which is an extremely reactive species.

36. Reactivity of ROS Hence, these two process are important to understand the chemical reactivity of superoxide anion radical in cells: • its dismutation yields non-radical products: oxygen and hydrogen peroxide (H2O2), thereby decreasing the reactivity of superoxide radical • its protonation increases the reactivity by generating perhydroxyl radical.

37. Reactivity of ROS Reactivity of Hydrogen Peroxide Hydrogen peroxide (H2O2) is not a free radical but it may be considered as an oxidant. Per se, hydrogen peroxide (H2O2) is little reactive. Its reactivity in biological systems depends on two properties: • first, it can diffuse long distances crossing membranes • second, it reacts with transition metals by a homolytic cleavage yielding the highly reactive hydroxyl radical (HO.).

38. Reactivity of ROS • Reactivity of Hydroxyl radical • Hydroxyl radical (HO.) is the most powerful oxidant and unlike superoxide anion (O2.–) and hydrogen peroxide (H2O2), it indiscriminately reacts with almost all biological compounds. • The extremely reactive nature of hydroxyl radical (HO.) suggests • that it will only mediate direct effects close to its site of generation • it cannot diffuse long distances

39. Reactivity of ROS Reactivity of Hydroxyl radical • The chemical reactivity of hydroxyl radical (HO.) may be assumed to encompass two main reactions: • Hydrogen Abstraction • Addition Reaction • • Hydrogen abstraction • Hydroxyl radical (HO.) may react with almost any compound abstracting a hydrogen and yielding a free radical species of the compound. Abstraction of a hydrogen by hydroxyl radical (HO.) results in its reduction to water: Again, because of its reactivity, RH could be any type of molecule within the cell, for example polyunsaturated fatty acids, DNA, glutathione, and certain amino acids.

40. Reactivity of ROS Reactivity of Hydroxyl radical • Addition reactions One of the most important addition reactions of hydroxyl radical (HO.) pertains the generation of 8-hydroxy-desoxyguanosine (a DNA base). Oxidation of this base, which can be detected in vivo, is a fingerprint of free radical attack to informational molecules.

41. Reactivity of ROS Reactivity of Hydroxyl radical Remember the two type of products observed upon hydroxyl radical (HO.) attack on DNA: Hydrogen abstraction reactions lead to DNA strand breaks • DNA strand break leads to decreased • levels of NAD+ • Repair enzyme: poly (ADP-ribose) • synthetase uses NAD+ • Eventually cell death (no TCA, no Glycolysis)

42. Reactivity of ROS Reactivity of Singlet Oxygen Singlet oxygen reacts efficiently with several molecules of biological importance: • vitamin E or α-tocopherol • vitamin C or ascorbic acid • bilirubin • DNA • cholesterol • β-carotene • tryptophan • methionine • cysteine • NADPH • polyunsaturated fatty acids

43. Reactivity of ROS Reactivity of Singlet Oxygen The chemical reactivity of singlet oxygen is rather specific comprising five types of reactions, of which ene addition to fatty acids and dioxetane formation are of biological interest: In the ene addition a lipid hydroperoxide is formed (RH, unsaturated fatty acid; ROOH, unsaturated fatty acid hydroperoxide). Dioxetanes are formed when singlet oxygen adds across a double bond (also important in fatty acid oxidation).

44. Sources of ROS In cells, there are two main sources of superoxide anion (O2.–) and hydrogen peroxide (H2O2). Hydroxyl radical (HO.) is generated from superoxide anion (O2.–) and hydrogen peroxide (H2O2).

46. Cellular sources of ROS Leopold J and Loscalzo J; Arterioscler Thromb Vasc Biol. 2005;25:1102–1111

47. Sources of ROS • Mitochondria are major cellular sources of reactive oxygen species. • Mitochondria consume oxygen associated with the process of oxidative phosphorylation. • Under normal conditions, approximately 95-97% of the oxygen is reduced to water; a small fraction of the oxygen consumed (3-5%) is reduced univalently to superoxide anion (O2.–).

48. Sources of ROS • Coenzyme Q or ubiquinone is a mobile electron carrier in the respiratory chain and it collects electrons from complex I and complex II. • The coenzyme Q pool faces both the intermembrane space and the mitochondrial matrix (outer coenzyme Q pool (QO) and inner coenzyme Q pool (QI), respectively).

49. Sources of ROS • Coenzyme Q or ubiquinone is reduced by Complex I and Complex II and donates electrons to complex III (the bc1 segment). • Because of these redox transitions, ubiquinone exists as • a quinone (fully oxidized), • semiquinone, and • hydroquinone (fully reduced)

50. Sources of ROS Electron leakage, accounting for about 3-5% of the total oxygen consumed by mitochondria, is associated with the generation of oxygen radicals: Ubisemiquinone donates one electron to molecular oxygen yielding superoxide anion and ubiquinone; this is known as autoxidation of ubisemiquinone.