Free Radicals

1
Free Radicals Antioxidants

• 614 351 Toxicology
• Supatra Porasuphatana, Ph.D.

2
Contents

• Introduction of oxygen toxicity
• Free radicals/Reactive oxygen species
• Sources of free radical formation
• Types of free radicals
• Free radical toxicity
• Free radical and diseases
• Antioxidants

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Oxygen Toxicity

• Evidences
• High pressure oxygen inhibits bacterial growth
• High pressure oxygen causes acute CNS toxicity
• Oxygen exposure in premature babies
• Experiments Tissue damages by oxygen

4
Free Radicals

• any species capable of independent existent that
  contains one or more unpaired electrons
• Example
• Radicals can be formed by
• The LOSS of a single electron from a non-radical,
  or by the GAIN of a single electron by a
  non-radical
• The breakage of covalent bond homolytic fission

?
?
?
?
?
A B A B
H2O H OH
Example
?
5
Free Radical Nomenclature

• A free radical is denoted by a superscript dot to
  the oxygen (or carbon)
• e.g., HO?, NO?, ?CH3
• If a free radical is a charged species, the dot
  is put and then the charge
• e.g., O2?-
• (See Free Radical Nomenclature, Suggestions by
  
• Buettner, G.R., Schafer, F.Q.)

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Free Radicals
Singlet O2 (1?gO2)
Singlet O2 (1?gO2)
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Reactive Oxygen Species

• Radicals Hydroxyl radical
• Molecules Hydrogen peroxide
• Ions Hypochlorite ion
• Superoxide anion which is both ion and radical

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Types of Free Radicals

• Oxygen-centered radicals
• Singlet oxygen, superoxide, hydroxyl radicals
• Sulfur-centered radicals
• Thiyl radical (RS)
• Carbon-centered radicals
• CCl3, CH2CHOH
• Nitrogen-centered radicals
• NO, R2NO

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Oxygen-Centered Radicals

• Reactive Oxygen Species (ROS)

Radicals
Non-Radicals
Superoxide, O2?- Hydroxyl, HO? Peroxyl,
ROO? Alkoxyl, RO? Hydroperoxyl, HOO?
Hydrogen peroxide, H2O2 Hypochlorous acid,
HOCl Ozone, O3 Singlet oxygen, 1?g Peroxynitrite,
ONOO-
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Superoxide Radicals

• Generation of superoxide (O2?-)
• The addition a single electron to the
  ground-state molecule (O2 e- O2?-)
• Biological generation of O2?-
• Mitochondrial electron transport chain
• Enzymatic reduction of oxygen (O2)
• Xenobiotic metabolisms (redox cycling)
• Respiratory burst (phagocytes)

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Mitochondrial Electron Transport Chain

• The most important source of O2?- in vivo in most
  aerobic cells
• Mitochondrial functions
• Oxidation of NADH, FADH2, ?-oxidation of fatty
  acids, other metabolic pathway
• Electron transport chain
  in the inner mitochondrial
  membrane
• Energy released is used for
• ATP synthesis

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Superoxide Production from Mitochondrial Electron
Transport Chain
Leaking of electron (to oxygen) during electron
transport leads to the formation of O2?- (O2 e-
O2?-)
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Biological Generation of Superoxide

• Enzymatic reduction of oxygen
• Xanthine/hypoxanthine
  Uric acid
• Redox cycling Paraquat

XOD
O2?-
O2
XOD xanthine oxidase
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Respiratory Burst

• Myeloperoxidase
• Oxidizes Cl- to hypochlorous acid
• Chronic granulomatous disease
• NADPH oxidase enzyme

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Respiratory Burst

• NADPH oxidase complex
• Cytoplasmic proteins
• (p47, p67, gp91, p22)
• NADPH NADP H
• Electron is transferred
• from NADPH to O2, resulting
• in the formation of O2?-

NADPH Reduced Nicotinamide Adenine
Dinucleotide Phosphate
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Hydroxyl Radical (HO?)

• Highly reactive oxygen radicals
• Formation of hydroxyl radicals in biological
  systems
• Ionizing radiation
• Reaction of metal ions with hydrogen peroxide
  (Fenton reaction)
• Formation of hydroxyl radical from ozone (O3)
• Reactions of hydroxyl radicals
• Hydrogen atom abstraction
• Addition
• Electron transfer

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Fenton Reaction

• Discovered by Fenton (1894)
• A mixture of hydrogen peroxide and an iron(II)
  salts causes the formation of hydroxyl radical
• Fe2 H2O2 intermediate complex
  Fe3 OH- HO?
• Fe3 H2O2 intermediate complex
  Fe2 O2? - 2H
• Haber-Weiss reaction
• Fe2 H2O2 Fe3 OH- HO?
• Fe3 O2? - Fe2 O2
• Net O2?- H2O2 O2 HO? OH-

metal catalyst
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Nitrogen-Centered Radicals

• Nitric oxide (NO?)
• Endothelial derived-relaxing factor (EDRF)
• Generated from the catalysis of L-arginine by
  nitric oxide synthase (NOS) enzymes
• Functions
• Vascular function, platelet aggregation, immune
  response, neurotransmitter, signal transduction
• cytotoxicity
• NO? O2?- ONOO- (highly toxic)

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Sources of Free Radicals

• Endogenous sources of free radicals
• Oxidative metabolic transformation
• Mitochondrial respiratory chain
• Oxygen burst (respiratory burst) during
  phagocytosis
• Eicosanoid synthesis
• Enzymatic reactions (oxygenases, oxidases)
• Xenobiotic metabolism (redox cycling)

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Sources of Free Radicals

• Exogenous sources of free radicals
• Ionizing radiation
• Ultraviolet radiation
• Ultrasound
• Chemicals, tobacco smoke, etc

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Roles of Free Radicals in Biological Systems

• Enzyme-catalyzed reactions
• Electron transport in mitochondria
• Signal transduction gene expression
• Activation of nuclear transcription factors
• Oxidative damages of molecules, cells, tissues
• Antimicrobial actions
• Aging diseases

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Oxidative Stress

• Damages caused by free radicals/reactive oxygen
  species
• Cellular damages at different levels (membrane,
  proteins, DNA, etc) lead to cell death, tissue
  injury, cellular toxicity, etc
• Reduction of antioxidants
  (cause consequence ?)
• Prevented by the reduction of free radicals,
  inhibition of free radical formation

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Oxidative Stress
O2
Non-enzymatic sources Mitochondrial respiratory
chain Glucose autoxidation Enzymatic
sources NADPH oxidase Xanthine oxidase Cyclooxygen
ase
?OH
Fenton reaction (Fe or Cu)
SOD
H2O2
O2?-
GSH
NO?
GPx
Catalase
GSSG
ONOO-
H2O O2
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Free Radical Toxicity

• Causes of free radical toxicity
• Increase production of free radicals
• Decrease level of defense system (e.g.,
  antioxidants)
• Lipid peroxidation
• DNA damage
• Protein oxidation

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Lipid Peroxidation

• Initiation of first-chain reaction
• Abstraction of H by ROS (OH)
• Formation of lipid radical (LH)
• Formation of peroxyl radical (LOO, ROO)
• Propagation
• H abstraction by lipid peroxyl radical (LOO)
• Termination
• Radical interaction non-radical product

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I
Hydrogen abstraction

-H

Molecular rearrangement
Conjugated diene
O2 Oxygen uptake
Peroxy radical abstract H rom another fatty
acid causing an autocatalytic chain reactions
Lipid hydroperoxide Cyclic peroxide Cyclic
endoperoxide
Initiation Propagation
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Products of Lipid Peroxidation
Reactive Oxygen Species
Lipid peroxides
Aldehyde products
Alkanes
?,?-unsaturated aldehydes
Conjugated dienes
Malondialdehyde (MDA)
n-aldehydes
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PGF2?-Isoprostane

• A group of prostaglandin (PGF2)-like compounds
  produced by non-enzymatic free radical-induced
  peroxidation of arachidonic acid
• Reliable biomarker of oxidative stress in vivo
• Specific product of lipid peroxidation
• Stable compound
• Detectable level in biological samples
• Level increases during oxidative injury
• Formation is modulated by antioxidant status
• Not affected by lipid contents from diet

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Oxidative DNA Damage

• Correlation with cancers and diseases
• Oxidative DNA lesions by
• Direct attack
• Indirect activation of endonuclease enzymes
• Oxidative modification of bases mutation
• Oxidative modification of sugar moieties DNA
  strand break

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A computer image depicts a hydroxyl radical
attacking the sugar on the back bone of a DNA
molecule
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Oxidative DNA Damage
Base Moieties
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Oxidative DNA Damage
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Oxidative DNA Products
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Oxidative DNA Damage

• 8-Hydroxyguanine (8-OH-Gua)
• GC TA transversions (frequently detected
  in p53 gene and ras protooncogene)
• 2-Hydroxyadenine (2-OH-Ade)
• 8-Hydroxyadenine (8-OH-Ade)
• 5-Hydroxycytosine (5-OH-Cyt)
• 5-Hydroxyuracil (5-OHUra)

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Oxidative DNA Damage

• Abstraction of H atom from carbon atoms of sugar
  molecules
• Disproportionations and rearrangement lead to
  C-C bond fragmentation and DNA strand
  break

Sugar Moieties
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Protein Oxidation

• Protein targets
• Receptors, transport proteins, enzymes, etc
• Secondary damage autoimmunity
• Protein oxidation products
• Protein carbonyl group, 3-nitrotyrosine, other
  oxidized amino acids
• Most susceptible amino acids
• Tyrosine, histidine, cysteine, methionine

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Protein Oxidation
Oxidative protein degradations
Modifications of prosthetic group of enzymes
Modifications of amino acid chain
Protein aggregation Protein fragmentation
Activations of protease enzymes
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Protein Oxidation
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Free Radical Toxicity
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Free Radicals and Diseases

• Cancer
• Inflammation/Infection
• Ischemia-reperfusion injury
• Cardiac ischemia-reperfusion injury
• Cerebral ischemia-reperfusion injury
• Neurodegenerative diseases
• Cardiovascular diseases
• Aging
• Others (e.g., drug/chemical-induced toxicity, etc)

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Oxidative DNA Damage Cancer
ROS can attack deoxyribose, purine, and
pyrimidine bases in DNA resulting in DNA strand
breaks DNA strand breaks induced by ?OH cause
deletions and point mutations
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Oxidative DNA Damage Cancer
Oxidative DNA damage
Point mutation Chromosomal aberrations DNA
strand breaks Oxidative modification of DNA Base
modifications Sequence change Activation of
kinases Activation of protooncogenes Inactivation
of tumor suppressor genes
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Oxidative DNA Damage Cancer

• Hydroxyl radical-induced
• DNA oxidative damage
• Hydroxylation of guanine residue (dG) to produce
  8-OH-dG is the most common biomarker of
  OH-induced DNA damage
• Detectable in cell, tissues, urine
• Levels can be modulated by antioxidants

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Oxidative DNA Damage Cancer
Figure The individual value of the urinary
modified base (8-OH-Gua) (Ref. Rozalski, R.
et al. Cancer Epidemiol. Biomarkers
Prev.2002111072-5.)
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Oxidative Stress Cancer
(Ref. Olinski, R., et al. Mutation Res.
2003531177-90)
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Acquired Immunodeficiency Syndromes Evidences

• Cells infected with HIV can enhance production of
  superoxide anion
• HIV-infected patients Studies
• Deficiency in SOD and catalase enzymes
• Decreased concentrations of antioxidant vitamins
• A significant increase in the level of 8-OHGua
  and 5-OHUra in lymphocytes -- apoptosis
• Vitamin supplementations lead to the reduction of
  the level of oxidative DNA damage

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Oxidative Stress in HIV-Infected Patients
Study Results Supplementation with antioxidant
vitamins (vitamin A, E and C) prevents oxidative
modification of DNA in lymphocytes of
HIV-infected patients
(Ref. Jaruga, P., et al. Free Radic. Biol. Med.
200232414-20)
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Ischemia-Reperfusion Injury

• Ischemic reoxygenation
• Cardiac ischemic-reperfusion
• Cerebral ischemic-reperfusion
• Tissue damages caused by excessive production of
  free radicals
• High concentration of oxygen
• Low levels of antioxidants
• Prevented by antioxidant supplementation

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Ischemia-Reperfusion Injury
ATP
Xanthine dehydrogenase
i s c h e m i a
Ca2-dependent protease
AMP
TISSUE INJURY
Xanthine oxidase
Adenosine
Hypoxanthine/Purine
REPERFUSION
Fe2
O2 H2O2 OH-
O2
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Neurodegenerative Diseases/Neurotoxicity
Diseases Evidences
Acute Cerebral ischemia/ O2.- and ONOO-
increased, impaired reperfusion
mitochondrial function Traumatic brain
injury ROS increased, lipid peroxidation,
protein oxidation increased, antioxidant
decreased Chronic Alzheimers
disease Oxidation of lipids, DNA, proteins
increased, induction of ROS by amyloid-?
Parkinsons disease Oxidation of lipids,
DNA, proteins increased in substantia
nigra Huntingtons disease Oxidative damage
increased in the basal ganglia, ROS
levels increased Amyotrophic lateral
sclerosis ROS increased, oxidation of lipids, DNA
and proteins increased, mutant of SOD
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Cardiovascular Diseases

• Elevated level of 8-OHGua in the lesion of the
  aorta wall in atherosclerotic patients
• Level of 8-OHGua in lymphocytes of
  atherosclerotic patients was significantly higher
  than in the DNA of control group
• Formation of oxidized LDL
• Direct action Foam cell formation
• Indirect actions Down regulates the base
  excision repair (BER) pathway, leading to higher
  level of 8-OHGua prevented by antioxidant
  vitamins

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Atherosclerosis

• (A) Oxidized LDL stimulates monocyte chemotaxis
• (B) Oxidized LDL inhibits monocyte egress from
  the vascular wall

(C) Monocytes differentiate into macrophages that
internalize oxidized LDL, leading to foam
cell formation (D) Oxidized LDL also causes
endothelial dysfunction and injury (E) Oxidized
LDL causes foam cell necrosis, resulting in the
release of lysosomal enzymes and necrotic
debris
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Base Excision Repair (BER)

1. Removal of the incorrect base by an appropriate
   DNA N-glycosylate to create an AP site (abasic
   site the position of the modified (damaged)
   base)
2. Nicking of the damaged DNA strand by AP
   endonuclease upstream of the AP site, thus
   creating a 3-OH terminus of adjacent to the AP
   site
3. Extension of the 3-OH terminus by a DNA
   polymerase, accompanied by excision of the AP site

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Aging

• AGING a progressive accumulation of changes
  overtime that increases the probability of
  disease and death.
• Two main theories of aging Aging theories
• Programmed theory a genetic timetable
• Damage theory injuries that build up overtime

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Free Radical Theory of Aging

• Aging the cumulative consequences of free
  radical reactions
• Life-span experiments
• Relationship between antioxidants,
  redox-sensitive transcription factors and free
  radical levels
• Age-related decline in activation tresholds of
  transcription factors and its normalization by
  antioxidants

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Drug-Induced Toxicity
58
Chemical-Induced Toxicity

• Environmental pollutants tobacco smoke, dust,
  etc
• Organic solvents benzene, carbon tetrachloride,
  etc
• Other chemicals
• Detection of oxidative biomarkers as an index of
  chemical exposure

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Measurement of Oxidative Stress

• Oxygen consumption
• Oxidative markers footprints
• Lipid peroxidation products (TBARs, lipid
  hydroperoxides, etc)
• DNA hydroxylation products (8-OHGua,
• Protein hydroxylation products (nitrosation
  products)
• Free radical detection
• Single photon counting
• Chemiluminescence
• Fluorescent probe
• Electron paramagnetic resonance spectroscopy (EPR)

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ANTIOXIDANTS

• 614 351 Toxicology
• Supatra Porasuphatana, Ph.D.

61
Contents

• Oxidant-Antioxidant balance
• Biological actions of antioxidant defense system
• Antioxidant defense system
• Superoxide dismutase (SOD)
• Catalase
• Glutathione cycle/Glutathione peroxidase
• Diet-derived antioxidants Low molecular weight
  antioxidants
• Roles in the cellular protection against
  oxidative stress oxidative stress-related
  diseases

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Oxidant-Antioxidant Balance
Damage (Pro-oxidants)
Defense (Antioxidants)
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Oxidant-Antioxidant Balance
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Cellular Defense Mechanisms

• Isolation of generation sites of reactive oxygen
  species
• Inhibition of propagation phase of reactive
  oxygen species
• Scavenging of reactive oxygen species
• Repair of the damage caused by reactive oxygen
  species

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Protection Against ROS Damage

• Direct protection against ROS
• Superoxide dismutase, Glutathione peroxidase,
  Catalase
• Non-specific reduction system
• Glutathione, Vitamin C
• Protection against lipid peroxidation
• Glutathione peroxidase, Vitamin E, ?-Carotene
• Sequestration of metals
• Transferrin, Lactoferrin, Ferritin,
  Metalothionein
• Repair systems
• DNA repair enzymes, Macroxyproteinases,
  Glutathione transferase

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Antioxidant Defense System

• Antioxidant Enzymes
• Superoxide dismutase (SOD)
• Catalase (CAT)
• Glutathione peroxidase (GPx)
• Endogenous non-enzymatic antioxidants
• GSH, bilirubin

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Antioxidant Defense System

• Exogenous antioxidant molecules
• a-Tocopherol -- prevents oxidation of fatty
  acids
• Carotenoids (b-carotene, leutin, lycopene, etc)
  -- destroy a particularly damaging form of
  singlet oxygen
• Ascorbic acid -- radical scavenging, recycling
  of vitamin E
• Bioflavonoids -- potent antioxidant activity

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Superoxide Dismutase (SOD)
Function
k 2-4 x 109 M-1s-1

• Only enzyme known to react with radical
• The presence of SOD implies O2.- produced in
• cell during normal metabolism
• SOD is a primary antioxidant enzyme

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Forms of SOD
Procaryotic SOD MW/Da Subunits Fe-SOD 40,000
2 Mn-SOD 40,000 2 80,000
4 Eucaryotic SOD MW/Da Subunits Mn-SOD 88,000
4 CuZn-SOD 32,000 2 EC
(CuZn) 135,000 4 EC Mn-SOD 150,000
2,4
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Intracellular Location of SOD

• CuZn-SOD
• Cytoplasm, nucleus, lysosomes
• Mn-SOD
• Mitochondrial matrix
• EC (CuZn)
• Plasma membrane, extracellular
• EC Mn-SOD
• Plasma membrane

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Structure and Properties of SOD

• CuZn-SOD
• One of the most stable protein
• Inactivated by guanidine HCl, CN-,
  diethyldithiocarbamate (DETC)
• EC-SOD
• Inhibited by CN-, azide, H2O2, SDS
• Located in extracellular fluids
• Suppresses inflammation
• Fe/Mn-SOD
• Not stable

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Catalase (CAT)

• Function Removes H2O2
• 2 H2O2 2 H2O O2
• Prevents lipid peroxidation
• and protein oxidation

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Glutathione Cycle
Glutathione Glu-Cys-Gly
Reduced glutathione (GSH) Oxidized glutathione
(GSSG)
Function gets rid of H2O2 or ROOH
(hydroperoxide)
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Glutathione Biosynthesis
Two Step-Mechanism

• By enzyme g-glutamylcysteine synthetase
• L-glutamate L-cysteine ATP
  L-g-glutamylcysteine ADP Pi
• 2. By enzyme glutathione synthetase
• L-g-glutamylcysteine glycine ATP
  GSH ADP Pi

Buthionine sulphoximine (BSO) inhibits
g-glutamylcysteine synthetase
Cellular GSH increase sensitivity to
toxicants
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Glutathione Peroxidase (GPx)
Function Removes H2O2 ROOH ROOH 2
GSH ROH H2O
GSSG Deficiency in GPX leads to oxidative
hemolysis Protects against lipid peroxidation
Selenium
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Low Molecular Mass Agents

• Compounds synthesized in vivo
• bilirubin, melatonin, lipoic acid, uric acid,
  etc.
• Compounds derived from the diet
• Ascorbic acid
• Vitamin E

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Ascorbic Acid

• Antioxidant Function
• Donate 1 e- semidehydroascorbate
  (ascorbyl radical)

Relatively unreactive
78
Ascorbate-Glutathione Cycle
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Tocopherol
Chain-breaking antioxidant
Scavenges peroxy radical Inhibits chain
reaction of lipid peroxidation
Eight naturally-occurring substances d-a-, d-b-,
d-g-tocopherols d-a-, d-b-, d-g-tocotrienols
80
Antioxidant Network

• Antioxidant network

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Biological Properties of Natural Antioxidants

• Natural antioxidants
• Polyphenols (phenolic, flavonoids), carotenoids,
  lycopene, etc
• Electron donor property
• Ability of antioxidant to donate
  an electron to a species (free
  radical) reducing property
• Antioxidant remains stable

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Basic Ring System of Flavonoids
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Basic Structure of Flavonoids
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Two-Stage Oxidation of Quercetin
(J. Agric. Food Chem. 2003511684-90)
85
Roles of Antioxidants in Protection Against
Oxidative Damage

• Animal models
• Transgenic mice overexpressing SOD, CAT, GPx show
  an increase tolerance in oxidative damage
  (ischemia-reperfusion, heart brain injury,
  hyperoxia, adriamycin and paraquat toxicity)
• Antioxidant gene knockout mice increased
  susceptibility to oxidative damage
  (ischemia-reperfusion, free radical generation)

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Roles of Antioxidants in Protection Against
Oxidative Damage

• Human studies
• Aging (mitochondrial dysfunctions leads to
  excessive production of free radicals tissue
  damage), age-related diseases (cataract, cancer,
  etc)
• Chronic diseases (cancer, cardiovascular disease,
  diabetes, neurodegenerative diseases,
  inflammation, etc)
• Oxidative injury caused by chemicals, drugs, etc

87
SUMMARY

• Characteristics of free radicals/reactive oxygen
  species
• Endogenous/Exogenous formation of free radicals
• Oxidative cell damage (lipids, DNA, proteins)
• Oxidative damage-related carcinogenesis
• Antioxidants (types, functions)
• Antioxidant network
• Roles in the preventions against oxidative damage

88
References/Suggesting Readings

• Halliwell, B., Gutteridge, J.C.M. (eds.) Free
  Radical in Biology and Medicine. 3rd ed.
• Packer, J., Hiramatsu, M., Yoshikawa, T. (eds.)
  Antioxidant food supplements in human health.
  Academic Press.
• De Zwart, L.L., et al. Free Radic. Biol. Med.
  199926202-26.
• Roberts, L.J. II, Morrow, J.D. Free Radic. Biol.
  Med. 200028505-13.
• Olinski, R., et al. Free Radic. Biol. Med.
  200233192-200.
• Mayne, S.T. J Nutr. 2003 133 (Suppl 3)
  933S-940S.