Transcript Reactive Oxygen Species
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
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The Earth was originally anoxic
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Metabolism was anaerobic
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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 2 + 36ADP + 36P i 6CO 2 + 36ATP + 6H 2 O
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Ground-state oxygen has 2-unpaired electrons .
O:O .
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The unpaired electrons have parallel spins
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Oxygen molecule is minimally reactive due to spin restrictions
Basics of Redox Chemistry
Term
Oxidation Reduction Oxidant Reductant
Definition
Gain in oxygen Loss of hydrogen Loss of electrons Loss of oxygen Gain of hydrogen Gain of electrons Oxidizes another chemical by taking electrons, hydrogen, or by adding oxygen Reduces another chemical by supplying electrons, hydrogen, or by removing oxygen
Prooxidants
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 .
R 3 N .
R-O .
R-S .
Carbon-centered Nitrogen-centered Oxygen-centered Sulfur-centered
Non-Radicals:
Species that have strong oxidizing potential Species that favor the formation of strong oxidants (e.g., transition metals)
H 2 O 2 HOCl -
Hydrogen peroxide Hypochlorous acid
O 3 1 O 2 ONOO -
Ozone Singlet oxygen Peroxynitrite
Me n+
Transition metals
Radicals: O 2 . OH .
Superoxide Hydroxyl
RO 2 .
RO .
HO 2 .
Reactive Oxygen Species (ROS)
Peroxyl Alkoxyl Hydroperoxyl
Non-Radicals: H 2 O 2 HOCl -
Hydrogen peroxide Hypochlorous acid
O 3 1 O 2 ONOO -
Ozone Singlet oxygen Peroxynitrite
Reactive Nitrogen Species (RNS) Radicals: NO .
Nitric Oxide
NO 2 .
Nitrogen dioxide
Non-Radicals: ONOO -
Peroxynitrite
ROONO
Alkyl peroxynitrites
N 2 O 3 N 2 O 4 HNO 2 NO 2 + NO NO + NO 2 Cl
Dinitrogen trioxide Dinitrogen tetroxide Nitrous acid Nitronium anion Nitroxyl anion Nitrosyl cation Nitryl chloride
“Longevity” of reactive species
Half-life Reactive Species
Hydrogen peroxide Organic hydroperoxides Hypohalous acids Peroxyl radicals Nitric oxide Peroxynitrite Superoxide anion Singlet oxygen Alcoxyl radicals Hydroxyl radical ~ minutes ~ seconds ~ milliseconds ~ microsecond ~ nanosecond
Antioxidants
Oxidative Stress
Prooxidants
“An imbalance favoring prooxidants and/or disfavoring antioxidants, potentially leading to damage” -H. Sies
Radical-mediated reactions
Addition
R
.
+ H 2 C=CH 2 R-CH 2 -CH 2
.
Hydrogen abstraction
R
.
+ LH RH + L
.
Electron abstraction
R
.
+ ArNH 2
Termination
R
.
+ Y
.
R R-Y + ArNH 2
.
+
Disproportionation
CH 3 CH 2
.
+ CH 3 CH 2
.
CH 3 CH 3 + CH 2 =CH 2
Hydroxyl radical (
.
OH)
Fenton Haber-Weiss O 2 . + Fe 3+ H 2 O 2 + Fe 2+ O 2 . + H 2 O 2 O 2 + Fe 2+ (ferrous) OH + .
OH + Fe 3+ (ferric) OH + O 2 + .
OH
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Transition metal catalyzed
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Other reductants can make Fe 2+
(e.g., GSH, ascorbate, hydroquinones) •
Fe2+ is an extremely reactive oxidant
Important Enzyme-Catalyzed Reactions
Biological Pathways for Oxygen Reduction
II. Sources of ROS Endogenous sources of ROS and RNS
Microsomal Oxidation, Flavoproteins, CYP enzymes Xanthine Oxidase, NOS isoforms Endoplasmic Reticulum Myeloperoxidase (phagocytes) Transition metals Cytoplasm Lysosomes Fe Cu Oxidases, Flavoproteins Peroxisomes
Mitochondria
Plasma Membrane Lipoxygenases, Prostaglandin synthase NADPH oxidase Electron transport
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
Fatty acyl-CoA synthetase
H 2 O 2
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 PGH 2
PHS
Cytoplasmic sources of ROS and RNS
xanthine oxidase xanthine oxidase Nitric Oxide Synthases (NOS): neuronal nNOS (I) endothelial inducible eNOS (III) iNOS (II)
NO •
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
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).
Microsomes as a source of ROS (II)
Davydov, Trends Biochem Sci, 2001
Exogenous sources of free radicals
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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
H 2 O 2 UV B
UV radiation
OH .
+ OH .
UV A UV B UV C = 320-400 nm = 290-320 nm = 100-290 nm
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Primarily a concern in skin and eye
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Can also cause DNA damage
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Can form singlet oxygen in presence of a sensitizer 2H 2 O
Ionizing radiation
g
-rays H 2 O + e + H 2 O* H 2 O* H + .
OH
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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
O 3 Ozone + Chemicals that form peroxides O O O C C C C 1 O 2 + Singlet oxygen C C O C O C
Chemicals that promote O 2 . formation NAD(P)H NAD(P) + Flavoprotein H 3 C N+ Paraquat O 2 . N+ CH 3 H 3 C N+ O 2 N .
Paraquat radical cation CH 3
Chemicals that are metabolized to radicals Polyhalogenated alkanes Phenols, aminophenols
Chemicals that are metabolized to radicals
Chemicals that release iron
Ferretin + e Fe 2+ Fenton Chemistry
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Requires reductant
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Promotes .
OH formation
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Promotes lipid peroxidation in vitro
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
1.
2.
3.
Initiation LH + X • L • + XH Propagation L • + O 2 LOO • LOO • + LH L • + LOOH Termination 2 LOO • L • + LOO • L • + L • non-radical products non-radical products non-radical products Catalyzed by metals LOOH + Fe 2+ OH + LO .
+ Fe 3+
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
HS NH 2 CH 2 CHCOOH
Cysteine
Protein targets for ROS
H 3 C S H CH 2 CH 2 C COOH HO NH 2
Methionine Tyrosine
NH 2 CH 2 CHCOOH
Oxidized proteins and amino acids found in biological systems
HN N NH 2 CH 2 CHCOOH
Histidine
NH 2 CH 2 CHCOOH HN
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 2 o and 3 o structure
Increased susceptibility to proteolysis
HN O
DNA oxidation products
NH 2 NH 2 N H 2 N N N R 8-hydroxyguanine N N OH N N R 8-hydroxyadenine OH N N HO N H N R 2-hydroxyadenine OH NH 2 O N N H OH H OH 5,8-dihydroxycytosine O HN CH 3 OH O H OH H thymidine glycol N O CH 2 OH O N H 5-hydroxymethyluracil
R .
Oxidation of deoxyribose (DNA backbone)
O O O H O P O OR B O O .
O O P O OR B O 2 O O .
O O P O OR B Strand Breaks O O O O O P O OR + B Apurinic/apyriminic sites O .
OO O O O P O OR B O .
O + O O B Aldehyde products
Consequences of DNA oxidation
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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