pro-oxidants or antioxidants

8/13/2019 Pro-oxidants or Antioxidants

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PRO-OXIDANTS OR ANTIOXIDANTS

Christine Winterbourn, Ph.D.

University of OtagoChristchurch School of Medicine

Dept. of PathologyPO Box 4345

Christchurch, New Zealand

Phone: 643/364-0564

Fax: 643/364-1083Email: [email protected]


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Pro-oxidants and AntioxidantsWhat Characterizes an Antioxidant

Christine WinterbournUniversity of Otago, Christchurch, New Zealandg , ,

Free Radical School, SFRBM, San Francisco 2009


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What is an Antioxidant?hat is an Antioxidant?Meaning can depend on context

Definition (Halliwell Gutteridge 2007)A substance that, when present at a low concentrationcompared with that of an oxidizable substrate, inhibitsoxidation of the substrate.


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Pro-oxidantA species that causes or promotes oxidation.

Depending on the circumstances, a compound mayexhibit pro- or antioxidant activityxhibit pro- or antioxidant activity.Examples include:polyphenols, thiols, -tocopherol


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Antioxidant Classificationntioxidant ClassificationFunctionunction Free radical scavengers Scavengers of non-radical oxidantscavengers of non radical oxidants

C d th t i hibit ti f id t Compounds that inhibit generation of oxidants

Compounds that induce the production of antioxidants


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Antioxidant Classificationntioxidant ClassificationFunctionunction Free radical scavengers

Ascorbicacid

Scavengers of non-radical oxidantscavengers of non radical oxidantsCatalase(H2O2);thiols(HOCl)

C d th t i hibit ti f id t Compounds that inhibit generation of oxidantsMetalchelators

Compounds that induce the production of antioxidantsIsothiocyanates(sulforaphane)


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Antioxidant Classificationntioxidant ClassificationStructure

Enzymaticnzymatic Small molecule

Locality Water soluble Lipophilicipophilic


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Antioxidants act in different ways What is effective against one oxidant may be ineffectiveagainst another Localization is important Compounds may enhance the antioxidant capacity ofcells but be ineffective in test tube assays


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You need to know your oxidant

ROS is not an entityReactive oxygen species are individuals


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One electron Two electron (Non-radical)Free radicals

( )Hydrogen peroxideP i ieroxynitriteHypochlorous Acid

InterconversionH2O2 + Fe2+(chelate) Fe3+(chelate) + OH- + OH

ONOO- + CO2 NO2 + CO2 -H2O2 + tyrosine peroxidase H2O + tyrosyl radical


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Requirements for a Reactionequirements for a Reaction

Thermodynamics Free energy change must be negative

Kinetics Activation energy must be overcomectivation energy must be overcome

Q


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Qinghai-Tibet

Railway, highest

po n , m

above sea level

But in between, the Himalayas

G is

-ve

Lucknow,India 128 mThanks to Henry Forman


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Thermodynamics v KineticsHydrogen Peroxide (H2O2) v Hypochlorous Acid (HOCl)g ( 2 2) yp ( )

H2O2 is the stronger oxidant (higher reduction potential) Activation energy for most 2e oxidation reactions of H2O2 is high

Activation energy is much lower for reactions of HOClctivation energy is much lower for reactions of HOCl HOCl is a much more reactive oxidant


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But for most radical reactions -

Activationener low

Pecking order based on reduction potentialecking order based on reduction potentialRadical Reduction potential (V) *

Hydroxyl OH,H+/H2O 2.31

Carbonate CO3-

,H+

/CO32-

1.78 .

Superoxide O2-/2H+/H2O2 0.94

Phenoxyl PhO,H+/PhOH 0.90 -

Ascorbyl Asc -,H+/Asc2- 0.28

*pH 7 (Relative to NHE) Buettner 1993


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Identifying Oxidant Targets Reactions occur in competition.

y g gp

Determined by rate constants and concentrations.

For two substrates, the ratio of the amounts ofoxidant reacting with each is given byda t eact g t eac s g e byk1 [substrate 1]1k2 [substrate 2]


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Radical Scavengers as Antioxidants

One radical begets another radical

Radicals propagate chain reactions

Whether a scavenger is an antioxidant depends onhether a scavenger is an antioxidant depends onthe fate of the secondary radical


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AscorbateR + ascorbate RH + ascorbyl radical

2 ascorbyl radical ascorbate + dehydroascorbateAntioxidant

Reduced Glutathione (GSH)R + GSH RH + GS oxidizing radical

lipid peroxidation

GSNO

Pro-oxidant?


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GSH as an AntioxidantR + GSH RH + GS

GS-strong reductantSSG-

GS

Oxygen drives reaction O2Superoxide is radical sink GSSG + O2 -

H O + OSOD

H2O2 + O2

Winterbourn 1993


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Radical SinksSuperoxide R GS GSSG- O2 -R GS GSSG O2

Ascorbylradical Asc -Asc

Interplay with 2e Chemistry2 A b d h d bAsc - ascorbate + dehydroascorbate

dehydroascorbate + GSSG ascorbate + GSH


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Vitamin E - Pro- or Antioxidant?Antioxidant: Lipid peroxidationp

LOO + Toc LOOH + TocToc + LOO chain termination

Pro-oxidant: In LDL particles

Toc TocToc Toc + Lipid-H Lipid

TocToc

Toc

Toc

UNLESS T Li id H Li idNLESS Toc + Lipid-H Lipid Thomas & Stocker 2000


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Hydrogen PeroxideCommon assumptions

Oxidative damageActs via 1e (radical) mechanism

Metal centres

Redox signallingUndergoes 2e oxidations

Thiol targets


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Antioxidant Protection against H2O2Ie mechanisms

ChelatorsRadical scavengersd ca sca e ge sEnzymatic breakdown

2e mechanismsLow molecular weight antioxidants react slowly with H2O2R di l i ff tiadical scavengers are ineffectiveEnzymatic breakdownnzymatic breakdown


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Defences against hydrogen peroxideefences against hydrogen peroxide

Catalase Glutathione peroxidase / GSH cycle Peroxiredoxin / thioredoxin cycle


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Peroxiredoxins

Ubiquitous class of antioxidant or signaling proteinsbiquitous class of antioxidant or signaling proteinsPresent in cells at high copy numbersresent in cells at high copy numbersHighly reactive with H O (k>107 M 1s 1) Highly reactive with H2O2 (k>107 M-1s-1)2 d 1 f 2-cys and 1-cys forms


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2-Cys Peroxiredoxins

Peroxiredoxin / thioredoxin cycle

H2O2SpH SrH

SrH SpH

SOH SrH

SrH SOH

SO2H SrH

SrH SO2H

H2O2

Thioredoxin,

Thioredoxin

verox at on

re uc ase,NADPH S S


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2 2

ase on ra e cons an s an es ma e ce u ar concen ra ons, anassuming homogeneous system (Winterbourn 2008)


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Di A i id ietary Antioxidants


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pprox ma e p asma concen ra ons o an ox an s

ascorbic acid 20 - 80 M-tocopherol 50 Mcarotenoids 0.1 - 0.4 Mpolyphenols 0.01 - 0.1 Misothiocyanates 0 1 1 Msothiocyanates 0.1 - 1 M


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To be effective physiologicallyMust be able to compete with endogenous scavengersust be able to compete with endogenous scavengers

Must interact with endogenous pathways May have alternative effect


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Many dietary antioxidants act as chemoprotectantsActivate NRF2/KEAP pathway to induce Phase II responseIncludes induction of antioxidant and repair enzymes

Activity may reflect radical scavenging properties but is notctivity may reflect radical scavenging properties but is notdue to antioxidant action


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-

. . .

Are the best antioxidants poisons?

Is a little of something bad good for you?

Final Comments


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Final Comments

Antioxidant efficacy depends on the oxidant

Different strategies are needed for 1 electron and 2

-

radical sink

Effective scavenging of H2O2 is generally enzymatic

Selectivity of H2O2 for cell signaling proteins is likely to

require site co-localization

References


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Halliwell, B. and Gutteridge, J. M. C. (2007) Free Radicals in Biology and Medicine. 3rd Ed, Oxford University Press, Oxford

Buettner, G. R. (1993) The pecking order of free radicals and antioxidants: Lipid peroxidation, -tocopherol, and ascorbate. ArchBiochem Biophys. 300, 535-543

Winterbourn C. C. 2008 Reconcilin the chemistr and biolo of reactive ox en s ecies. Nat Chem Biol 4 278-286

Winterbourn, C. C. (1993) Superoxide as an intracellular radical sink. Free Radic Biol Med 14, 85-90

Wardman, P. and von Sonntag, C. (1995) Kinetic factors that control the fate of thiyl radicals in cells. Methods Enzymol 251, 31-45

Sturgeon, B. E., Sipe, H. J., Jr, Barr, D. P., Corbett, J. T., Martinez, J. G. and Mason, R. P. (1998) The fate of the oxidizing tyrosylradical in the presence of glutathione and ascorbate. Implications for the radical sink hypothesis. J Biol.Chem. 273, 30116-30121

Thomas, S. R. and Stocker, R. (2000) Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis. Free

Radic Biol Med 28, 1795-1805

Winterbourn, C. C. and Metodiewa, D. (1999) Reactivity of biologically relevant thiol compounds with superoxide and hydrogen

peroxide. Free Radic Biol.Med. 27, 322-328

Forman, H. J., Fukuto, J. M. and Torres, M. (2004) Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen

species can act as second messengers. Am J Physiol Cell Physiol 287, C246-256

Winterbourn, C. C. and Ham ton, M. B. 2008 Thiol chemistr and s ecificit in redox si nalin . Free Radic Biol Med 45, 549-561

Rhee, S. G., Chae, H. Z. and Kim, K. (2005) Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and

emerging concepts in cell signaling. Free Radic Biol Med 38, 1543-1552

Hall, A., Karplus, P. A. and Poole, L. B. (2009) Typical 2-Cys peroxiredoxins--structures, mechanisms and functions. FEBS J 276,-

Sies, H. (2007) Total antioxidant capacity: appraisal of a concept. J Nutr 137, 1493-1495

Dinkova-Kostova, A. T. and Talalay, P. (2008) Direct and indirect antioxidant properties of inducers of cytoprotective proteins. Mol Nutr

Food Res 52 Suppl 1, S128-138

Galati, G. and O'Brien, P. J. (2004) Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive

and anticancer properties. Free Radic Biol Med 37, 287-303

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