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

•

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

•

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

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

•

Transition metal catalyzed

•

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

• • • • •

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

•

Primarily a concern in skin and eye

•

Can also cause DNA damage

•

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

•

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

•

Requires reductant

•

Promotes .

OH formation

•

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

•

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