Oxidative Stress (Lecture 5, Exam 2 Content)

Reactive Oxygen Species (ROS) Radicals

O2 HO HO2

Reactive Nitrogen Species (RNS) Radicals

NO HNO

Reactive Nitrogen Species (RNS) Non-Radicals

NO2 ONOO- generally more stable

Reactive Oxygen Species (ROS) Non-Radicals

HClO H2O2 HBrO O3 delta g

Supraoxide radical

O2*-
The addition a single electron to the ground-state
molecule (O2 + e- = O2.-)

Generation of superoxide (O2
.-)

Mitochondrial electron transport chain
• Enzymatic reduction of oxygen (O2 )
• Xenobiotic metabolism (redox cycling)
• Respiratory burst (phagocytes)
Superoxide radical

Hydroxyl radical

HO.
Highly reactive oxygen radicals
• Short-lived species with high affinity
toward other molecules.
• Formation of hydroxyl radicals in
biological systems:
• Ionizing radiation
• Formation of hydroxyl radical from ozone (O3)
• Reaction of metal ions with hydrogen peroxide
(Fenton Haber-Weiss reaction)
Hydroxyl radical (HO.)

Hydrogen peroxide

H2O2
not radical but reactive with metals (fenton rxn)
Supraoxide Dismutase H30 - H202 - H20

Nitric oxide

NO.

Endothelial derived-relaxing factor (EDRF) -
vasodilator
• Functions
• Vascular function, platelet aggregation,
immune response, neurotransmitter, signal
transduction
• Cytotoxicity

NO. + O2 =
.- ONOO- (highly toxic)

Role of free radicals

enzyme reactions

e- transport chain in mitochondria

signal transduction - gene expression

activation of nuclear transcription factors

oxydative damage of molecules, cells, tissues

antimicrobia,

aging and disease

Molecular effects of oxidative stress

Lipid peroxidation
• DNA damage
• Protein oxidation
• Inactivation of enzymes
• Release of Ca2+ ions from intracellular stores
• Cytoskeletal damage
• Chemotaxis

PUFAs

polyunsaturated fatty acids

potential toxic effects

antioxidants

cellular defense mechanisms against
oxidative stress: Antioxidants
• Enzymes and repair systems
• Superoxide dismutase
• Catalase
• Glutathione system
• DNA repair enzymes
• Macroxyproteinases – degrades oxidatively
denatured proteins in red blood cells.
• Sequestration of metals
• Transferrin, lactoferrin, ferritin, metallothionein
• Vitamins
• Vitamin A
• Vitamin B
• Vitamin C 26

Nrf2-ARE pathway

antioxidant response element

nrf2 is a multiorgan protector

Supraoxide dismutase

in the cytosol - SOD1 (Cu +Zn)

in mitochondria - SOD2 (MnSOD)

h30 - h2o2

Gluthathion Redox Cycle

Major reducing power - processes hydrogen peroxide

reduced form: GSH

Oxidized form : GSSG

mediated by gluthathion peroxidase (GPx)

fascilitated by NADPH - NADP

Metal sequestration agents

• Ferritin, transferrin, and ceruloplasmin:
- Ensures extremely low levels of free cytosolic metals with a potential for valence changes.

• Metallothioneins:
- Sequester several metals with a high affinity for –SH groups (e.g., mercury and cadmium).
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Measurement of oxidative stress

Oxygen consumption
• Activity and levels of antioxidants
• Oxidative markers
– Lipid peroxidation products (TBARs, lipid hydroperoxides,etc.)
– DNA hydroxylation products
– Protein hydroxylation products (nitration products)

Free Radical detection

Single photon counting
• Chemiluminescence
• Fluorescent probe
• Electron paramagnetic resonance spectroscopy (EPR)

Dopamine oxidation

mediated by monoamine oxidase MAO) to 3,4-dihydroxyphenylacetic acid
(DOPAC or DPA) and H2O2

Dopamine auto-oxidation

Auto-oxidation in the presence of O2 or trace
multivalence metal ions