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Oxidative stress
Oxidative Stress
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Reactive oxygen species (ROS)
ROS and oxidative stress
Antioxidant system
Oxidative damage
Oxidative stress and apoptosis
Oxidative stress and aging
Oxidative stress and cancer
ROS as signaling molecules
Reactive oxygen species (ROS)
• ROS
– OH. (hyroxyl radical)
– O2-.
(superoxide radical)
– H2O2 (hydrogen peroxide)
– NO. (nitric oxide)
• Oxidative stress
• Oxidative damage
Toxic effects of ROS
• Protein oxidation
• Lipid peroxidation
• Nucleic acids damage
– Double-strand DNA breaks
– Single-strand DNA breaks
– Change DNA bases
• 8-oxoguanine
• Thymine glycol
Lipid peroxidation
• Measure the malondialdehyde formed
• Lipid peroxidation is a chain reaction.
• Each fatty acyl moiety that undergoes
peroxidaion generate a radical that can
initiate another peroxidation reaction.
Intracellular sources of free radicals
• Mitochondrial electron transport system
– Superoxide radical and semiquinone
radical
• Microsomal (ER) electron transport system
– Superoxide radical and H2O2
• Arachidonic acid metabolism
• Reactions within peroxisome
– Superoxide radical and H2O2
• H2O2 and O2-. may diffuse from their
subcellular sites of production and affect
the whole cell
• H2O2 can cross biological membranes
NO.
• Small
• Gas
• Synthesize by nitric oxide synthase (NOS)
– nNOS: constitutive expression
– eNOS: constitutive expression
– iNOS: inducible
Reactive nitrogen species (RNS)
• Inactivation of respiratory chain complexes;
inhibition of protein and DNA synthesis
• RNS are reduced or inactivated through the
generation of a disulfur bond between two
glutathione molecules to form oxidized
glutathione
Antioxidative system
• Antioxidant
– Glutathione, GSH
– Vitamin C, E
– Cysteine
– Protein-thiol
– Cerutoplasmin: important in reducing
Fe3+ release from ferritin
• Antioxidative enzyme
Glutathione (GSH)
Antioxidative enzyme
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Catalase
Superoxide dismutase
Glutathione peroxidase
Glutathione reductase
Gluththione S-transferase
Glucose-6-phosphate dehydrogenase
DT-diaphorase
Oxidants as stimulators of signal
transduction
• Oxidants
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Superoxide
Hydrogen peroxide
Hydroxyl radicals
Lipid hydroperoxides
ROS act as second messengers
• Ligand-receptor interactions produce ROS
and that antioxidants block receptormediated signal transduction led to a
proposal that ROS may be second
messengers
Reactive oxygen species (ROS) as
second messengers
• Generation of ROS by cytokines
Ligand
Tumor necrosis factor-
Interleukin 1
Transforming growth Factor-1
Platelet derived growth factor
Insulin
Angiotension II
Vitamin D3
Parathyroid hormone
ROS
H2O2/HO
H2O2/O2-
H2O2
H2O2
H2O2
H2O2/O2-
O2-
O2-
• TNF activates oxidative stress-responsive
transcription factors, NF-B and AP-1, and
also induces apoptosis.
ROS induce apoptosis
• Both H2O2 and menadione induce neuronal
cells apoptosis.
• Decreased superoxide dismutase activity was
found to cause apoptosis in neuronal cells
• Apoptosis induced by HIV infection was
inhibited by antioxidant such as Nacetylcysteine, catalase, vitamin E, and 2mercaptoethanol
ROS induce apoptosis
• Bcl-2 protects cells from TNF-induced
apoptosis in mouse L cells.
• Bcl-2 was ineffective in influencing TNF
signaling for NF-B activation in these cells.
• H2O2 activates the DNA binding activity of
p53. P53 is required for the induction of
apoptosis.
ROS measurement
2,7- Dichlorodihydrofluorescein
diacetate (DCFH/DA)
• DCFH/DA diffuses through the cell membrane
where it is enzymatically deacetylated by
intracellular esterases to the more hydrophilic
nonfluorescent reduced dye dichlorofluorescein.
• In the presence of reactive oxygen metabolites,
DCFH is rapidly oxidized to DCF.
• DCF, excitated with 503 nm and emission at
523 nm.
DCFH/DA
• Hydroxyl radical, hydrogen peroxide and
perhaps a ferryl species, but not superoxide,
may oxidize DCFH.
• The intracellular fluorescent measurements
using dichlorofluorescein diacetate may
reflect the ability of the test agent or
toxicant to generate hydroxyl radical.
DCFH/DA
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MW 487.3
Dissolved in 50% methanol
Did not dissolved in H2O or DMSO
Measurement of intracellular H2O2
• Cells incubated with 5 mM
dihydrorhodamine 123 for 45 min
• PBS wash
• Reduced form dihydrorhodamine 123 is
oxidized by intracellulr H2O2 to rhodamine
123
• Rhodamine 123
– 485 nm excitation
– 530 nm emission
Hydroethidium
• Measure superoxide anion concentration
• Superoxide anion can be measured by
hydroethidium oxidation into ethidium
Dihydroethidium
• Detect superoxide anion
Dihydroethidium
Oxidation
Ethidium
Blue fluorescent
Red fluorescent
Absorption/Emission
Absorption/Emission
355/420 nm
518/605 nm
Oxidative stress and aging
Oxidative damage to mitochondrial
DNA is inversely related to maximum
life span in the heart and brain of
mammals
• Oxidative damage marker 8-oxo-7,8-dihydro-2’deoxyguanosine (8-oxodG) in mitochondrial
DNA is inversely correlated with maximum life
span in the heart and brain of mammals. This
inverse relationship is restricted to mtDNA, not in
nuclear DNA.
Doxorubicin-induced Apoptosis is
associated with increased transcription
of endothelial nitric-oxide synthase
• Redox activation of DOX by eNOS
– The reductase domain of endothelial nitricoxide synthase (eNOS) activates doxorubicin
(DOX) by a reductive activation and forming
semiquinone and superoxide
• DOX-induced apoptosis is linked to the redox
activation of DOX by eNOS
• DOX-induced increase eNOS transcription and
protein expression in bovine aortic endothelial
cells (BAEC).
• DOX-induced H2O2 formation is responsible for
the increased transcription of eNOS. Treatment
with antioxidants restored the levels of
antiapoptotic proteins (Hsp70 and Bcl-2) in DOXtreated BAEC.
• DOX-induced intracellular oxidative stress was
inhibited by antisense eNOS oligonucleotide and
antioxidant treatment.
NFB and AP-1 mediate
transcriptional responses to oxidative
stress in skeletal muscle cells
• Oxidative challenges lead to an increase in
antioxidant enzymes, particularly
glutathione peroxidase (Gpx) and catalase
(CAT) in mouse skeletal muscle
• Mouse Gpx and CAT genes revealed
putative binding motifs for NFB and AP-1
NFB and AP-1 mediate transcriptional
responses to oxidative stress in skeletal
muscle cells
• Oxidative stress led to increases in the DNA
binding of NFB in differentiated muscle
cells. The NFB complexes included a
p50/p65 heterodimer, a p50 homodimer, and
a p50/RelB heterodimer
• Ap-1 is activated, but with slower kinetics
than that of NFB
Does oxidative damage to DNA
increase with age?
• The levels of 8-oxo-2-deoxyguanosine (oxo8dG)
in DNA isolated from tissues of rodents (male
F344 rats, male B6D2F1 mice, male C57BL/6
mice, and female C57BL/6 mice) of various ages
were measured.
• Oxo8dG was measured in nuclear DNA (nDNA)
isolated from liver, heart, brain, kidney, skeletal
muscle, and spleen and in mitochondrial DNA
(mtDNA) isolated from liver.
• A significant increase in oxo8dG levels in
nDNA with age in all tissues and strains of
rodents studied.
• Age-related increase in oxo8dG in mtDNA
isolated from the livers of the rats and mice.
Assay for protein oxidation
• 2,4-dinitrophenyl hydrazine (DNPH)
• carbonyl group in oxidized protein (10 g )
+ DNPH  Hydrazone derivatives
• SDS-PAGE
• Transfer to NC paper
• React with anti-dinitrophenylhydrazine
antibody
• Ref: J. Invest. Dermatol. 112: 1480-1487
(2004)
Assay for 8-OHdG
• Cells cytospun to slide
• Fixed in methacarn (methanol/chloroform/acetic acid,
6/3/1, v/v) for 1 h, RT
• Endogenous peroxidase block with H2O2 in methanol
30 min
• Nonspecific binding with 10% normal goat serum in
Tris-buffered saline 15 mins
(150 mM Tris/HCl and 150 mM NaCl, pH7.6)
• Cells treated with proteinase K (20 mg/ml in PBS) 15
min
• Cells reacted with anti-8-OHdG monoclonal
antibody
DNPH reacts with carbonyl group in
oxidized protein
Oxidative stress and diseases
Oxidative damage is the earliest
event in Alzheimer disease
• A significant increase of an oxidized
nucleoside derived from RNA, 8hydroxyguanosine (8OHG), and an oxidized
amino acid, nitrotyrosine in vulnerable
neurons of patients with Alzheimer disease
(AD).
• The increased oxidative damage is an early event
in AD that decreases with disease progression and
lesion formation.
Reactive oxygen species increase
risk of disease through
damage to key biological structures
Free radicals in disease
• The formation of ROS is a feature of many
degenerative diseases, such as atherosclerosis
and neurodegeneration
ROS involved in stroke
• Stroke is a severe and prevalent syndrome
for which there is a great need for treatment,
including agents to block the cascade of
brain injury that occurs in the hours after
the onset of ischemia. ROS have been
implicated in this destructive process
• EUK-134, a newly reported salenmanganese complex having greater catalase
and cytoprotective activities and equivalent
SOD activity compared with the prototype
EUK-8
Small molecules mimicing antioxidant
enzymes
• Mn(II) complex M40403 (a synzyme)
– possesses SOD activity approaching that
of the native Mn-SOD enzyme
– possessing outstanding chemical and
biological stability
• Removes superoxide without interfering
with other relevant biological oxidants, such
as nitric oxide, peroxynitrite, or hydrogen
peroxide