Oxidative Stress

1
Oxidative Stress
ANTIOXIDANTS
OXIDANTS
Oxygen Free Radicals Nitrogen Free Radicals
Specific Enzymes Vitamin E, Vitamin
C, Carotenoids, Selenium
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Some Basic Tenets of Oxidative Stress

• Free radicals are produced in normal processes
  through the bodys use of oxygen
• Environmental pollutants and many drugs cause
  free radical production
• Free radicals can damage cell structure and
  function in a chain reaction
• Antioxidants protect cells against free radicals
  by scavenging these species

Antioxidants
Oxidants
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Free Radical Involvement in Pathophysiological
Conditions

• Adriamycin cardiotoxicity
• AIDS
• Adult Respiratory Distress Syndrome
• Aging
• Alcoholism
• Alzheimers Disease
• Amyotrophic Lateral Sclerosis
• Atherosclerosis
• Diabetes
• Cancer

• Exercise
• Favism
• Iron Overload
• Myocardial Infarction
• Oxygen Toxicity
• Parkinsons Disease
• Radiation Therapy
• Smoking
• Stroke
• Trauma

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Oxidative Stress
Oxidants
Antioxidants
1. What are Oxygen Radicals or Reactive
Oxygen Species? 2. How reactive are reactive
oxygen species? 3. How are they generated in
the cell?
4. How do cells protect themselves against
reactive oxygen species?
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1. What are Oxygen Radicals or Reactive Oxygen
Species
Oxidants
Definition and Properties of Free Radicals and
Oxidants
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1.1. Free Radicals Definition
A free radical is defined as any species that
contains one or more unpaired electron occupaying
an atomic or molecular orbital by itself.
Box diagram
Lewis dot diagram
Based on the above definition, molecular oxygen
is a diradical, for it contains two unpaired
electrons
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1.1. Free Radicals Definition Nitrogen-centered
radicals
A free radical is defined as any species that
contains one or more unpaired electron occupaying
an atomic or molecular orbital by itself.
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1.3. Formation of Oxidants Electron-Transfer
Reactions
O2 ? O2. ??? H2O2 ? HO. ? H2O
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1.3. Formation of Oxidants Electron-Transfer
Reactions Superoxide Anion
O2 ? O2. ??? H2O2 ? HO. ? H2O
The addition of one electron to molecular oxygen
results in the formation of superoxide anion
radical
.
.
.


.
OO
.


1 e
O2 ????????? O2.
1 e
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1.3. Formation of Oxidants Electron-Transfer
Reactions Hydrogen Peroxide
Hydrogen peroxide can be formed by addition of
two electrons to molecular oxygen addition
of one electron to superoxide anion
2 e
1 e
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1.3. Formation of Oxidants Electron-Transfer
Reactions Hydroxyl Radical
Hydroxyl radical is formed upon one-electron
reduction of hydrogen peroxide
.
.
.
.



.
.
.
.
OO
H
H
O
H
.




1e
H2O2 HO. HO
1e
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1.3. Formation of Oxidants by Electron-Transfer
Reactions
O2 ? O2. ??? H2O2 ? HO. ? H2O
molecular oxygen Has two unpaired electrons It
is a diradical
superoxide anion Has one unpaired electron It is
a radical
hydrogen peroxide Has no unpaired electrons It
is not a radical
hydroxyl radical Has one unpaired electron It is
a radical
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2. How Reactive are Oxygen Radicals or Oxidants
Oxidants
Biochemical Reactivity of Oxidants
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2.1. Superoxide Anion Radical

• Two reactions are important in a cellular
  setting
• Disproportionation or dismutation reaction of
  superoxide anion with itself
• O2. O2. 2H ? H2O2 O2
• Protonation of superoxide anion formation of
  perhydroxyl radical
• O2. H ? HO2.

.
.
.
.
.
.
.
.








.
.
.-
.-
OO
OO
OO
H
H
OO










2H
.
.
.
.




.
.-
OO
OO
H
H




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2.2. Hydrogen Peroxide

• Hydrogen peroxide is not a free radical and per
  se is very little reactive.
• Its reactivity in biological systems depends on
  two properties
• It can diffuse long distances crossing membranes
• It reacts with transition metals by homolytic
  cleavage yielding the
• highly reactive hydroxyl radical

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2.3. Hydroxyl Radical

• Hydroxyl radical is the most powerful oxidant and
  it reacts undiscrimina-
• tely with all biomolecules.
• The biochemical reactivity of hydroxyl radical
  encompasses two reactions
• Hydrogen abstraction
• Addition

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2.3. Hydroxyl Radical

• Hydrogen abstraction
• HO. may react with any compound abstracting a
  hydrogen and yielding
• a free radical species of the compound and
  water
• RH HO. ?
  R. HO
• Example of H abstraction on DNA yields strand
  breaks

Cu
Cu
Cu
H2O2
HO.
strand breaks
hydrogen abstraction
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2.3. Hydroxyl Radical

• Addition
• HO. may add to desoxyguanosine (a DNA base)
  with formation of 8-
• hydroxy-desoxyguanosine (8dG). This oxidized
  base, which can be
• isolated in vivo, is a fingerprint of free
  radical attack on DNA.

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2.4. Nitric Oxide Summary of Nitric Oxide
Chemistry Prooxidant and Antioxidant Reactions
H. Rubo R. Radi In Handbook of
Antioxidants Cadenas Packer, Eds., pp. 689-707
(2001)
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2.4. Nitric Oxide

• Binding of NO to ferrous heme iron of guanilate
  cyclase
• Reaction with oxyhemoglobin
• Reaction with superoxide anion
• Reaction with oxygen

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2.4. Nitric Oxide

• The second reaction and certainly the major
  route
• for the destruction of nitric oxide in vivo is
  the fast
• and irreversible reaction with oxyhemoglobin
  to yield
• nitrate

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2.4. Nitric Oxide

• The fourth reaction, that of nitric oxide with
  oxygen,
• is slow, but it gains significance when the
  steady-
• state levels of oxygen and nitric oxide are
  considered

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Prooxidant Properties of Nitric Oxide

• Nitric oxide reaction with oxygen

Nitrogen dioxide E 0.99 V

• Nitric oxide reaction with superoxide anion

O2. .NO ONOO
k 1.9 x 1010 M1s1
Peroxynitrite E 1.4 V
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Prooxidant Properties of Nitric Oxide

• Peroxynitrite is in rapid protonation
  equilibrium
• with peroxynitrous acid

• Peroxynitrous acid may decompose to HO. and NO2.

• Chemical reactivity includes oxidants and
  nitrations

R. NO2. RNO2
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Prooxidant Properties of Nitric Oxide

• 3-Nitrotyrosine, a fingerprint of peroxynitrite
  reactivity

Tyr.
TyrNO2
Tyr
HO. . NO2.
NO2.
H2O
O2.
.NO
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3. How are oxidants generated in the cell
Oxidants
Cellular Sources of Oxidants
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3.1. Sources of Superoxide Anion

• Enzymic reactions
• NADH oxidase
• NADPH-P450 reductase
• xanthine oxidase
• Cellular sources
• leukocytes and macrophages
• mitochondrial electron transfer
• microsomal monooxygenase

• Environmental factors
• ultraviolet light
• X-rays
• toxic chemicals
• aromatic hydroxylamines
• aromatic nitro compounds
• insecticides
• chemotherapeutic agents

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3.3. Sources of Hydroxyl Radical
Most of the HO. generated in vivooriginates from
the breakdown of H2O2 via a Fenton reaction
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3.3. Sources of Nitric Oxide
Nitric oxide is generated by a
Ca-dependent mixed function oxidase nitric oxide
synthase (NOS) in mammalian tissues
NOS
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3.3. Sources of Nitric Oxide
Nitric Oxide Synthases (NOS) in mammalian tissues
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3.3. Sources of Nitric Oxide
Nitric Oxide Synthases (NOS) in mammalian
tissues Mayor Domains
calmodulin
oxygenase domain
reductase domain
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3.5. Summary of Sources of Oxidants

• Enzymic reactions
• NADH oxidase
• NADPH-P450 reductase
• xanthine oxidase
• Cellular sources
• leukocytes and macrophages
• mitochondrial electron transfer
• microsomal monooxygenase
• Environmental sources

• No cellular sources
• Fenton reaction from
• O2. and H2O2

O2 ? O2. ??? H2O2 ? HO. ? H2O
Dismutation
.NO
NOS

• Enzymic generation
• (non radical)
• glycolate oxidase
• acetyl-CoA oxidase
• D-amino acid oxidase
• NADH oxidase
• urate oxidase
• monoamine oxidase

ONOO
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4. How do cells protect themselves against
oxidants
Antioxidants
Specific and non-specific antioxidant defenses
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4. Primary Antioxidant Defenses

• Specific Antioxidant Enzymes
• Superoxide dismutase
• Catalase
• Glutathione Peroxidases
• Glutathione Transferases

• Small Antioxidant Molecules
• Vitamin E
• Vitamin C
• Coenzyme Q
• Uric Acid
• Carotenoids

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4.1. Specific Antioxidant Enzymes Removal of
Superoxide Anion

• Spontaneous, nonenzymic dismutation
• O2. O2. 2H ????????????H2O2
  O2 105 M1 s1
• Enzyme-catalyzed dismutation
• O2. O2. 2H ????????????H2O2
  O2 109 M1 s1

Superoxide dismutase (SOD)
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4.1. Specific Antioxidant Enzymes Removal of
Hydrogen Peroxide

• Catalase peroxisomes
• H2O2 H2O2???????2 H2O O2
• Glutathione peroxidase cytosol, mitochondria
• H2O2 2GSH??????? H2O GSSG

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4.1. Summary of Specific Antioxidant Defenses
catalase
O2 ? O2. ??? H2O2 ? HO. ? H2O
superoxide dismutase
glutathione peroxidase
2 GSH
GSSG
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