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OXYGEN AND LIFE
biochemistry of reactive oxygen(ROS),
mechanisms of formation & detoxification of ROS,
their role in human health and disease
동아의대 생리학교실
서덕준 [email protected]
OXYGEN
SOURCE
OF ROS:
A
N
T
I
O
X
I
D
A
N
T
OXIDATIVE
DAMAGE to:
CAUSAL
CONTRIBUTION to:
PATHOLOGY
Mechanism of
cell injury
O2 HOMEOSTASIS:
Physiological systems have
evolved to ensure the
optimal oxygenation of all
cells in each organism.
In aerobic organism, O2 is converted to H2O
at the end of the respiratory chain in mitochondria.
Mitochondria are the “power plants” in our cells that provide
the energy needed to maintain normal body function and metabolism.
However, in the same mitochondria respiratory chain,
O2 is “partially reduces” to form superoxide.
Superoxide is radical. Radicals usually are very reactive species.
Because of its radical character, superoxide is also called a
“Reactive Oxygen Species”(ROS).
LEARNING OBJECTIVES
• Identify the major reactive oxygen species
(ROS) and their sources in the cell.
• Identify targets of reactive oxygen damage
and describe the effects of ROS on
biomolecules.
• Identify the major antioxidant enzymes,
vitamins, and biomolecules that provide
protection against ROS.
• Describe the role of reactive oxygen in
regulatory biology and immunologic defenses.
• Explain the role of ROS in development of
chronic and inflammatory diseases.
• Describe the functions of the antioxidant
response element in protection against
nucleophiles and ROS.
Introduction
At body temperature, oxygen is a relatively sluggish
oxidant
The element oxygen (O2) is essential for the life of aerobic
organisms.
Although it is highly reactive in combustion reactions at high
temperature, oxygen is relatively inert at body temperature; it
has a high activation energy for oxidation reactions.
This is fortunate, otherwise we might spontaneously combust.
About 90% of our O2 usage is committed to oxidative
phosphorylation.
Enzymes that use O2 for hydroxylation and oxygenation
reactions consume another 10%,
and a residual fraction, <1%,
is converted to reactive oxygen species (ROS),
such as superoxide and hydrogen peroxide, which are reactive
forms of oxygen.
ROS are important in metabolism
– some enzymes use H2O2 as a substrate.
ROS also play a role in regulation of metabolism and in
immunologic defenses against infection.
However, ROS are also a source of chronic damage to tissue
biomolecules.
One of the risks of harnessing O2 as a substrate for energy
metabolism is that we may, and do, get burned.
For this reason, we have a range of antioxidant defenses that
protect us against ROS.
This chapter will deal with:
the biochemistry of reactive oxygen,
the mechanisms of formation and detoxification of ROS,
and their role in human health and disease.
The inertness of oxygen
At body temperature, O2 is a biradical, a molecule with two unpaired
electrons.
These electrons have parallel spins and are unpaired.
Since most organic oxidation reactions, are two-electron oxidation
reactions, O2 is generally not very reactive in these reactions.
In fact, it is completely stable, even in the presence of a strong reducing
agent such as H2.
When enough heat (activation energy) is applied, one of the unpaired
electrons flips to form an electron pair, which then participates in the
combustion reaction.
Once started, the combustion provides the heat needed to propagate
the reaction, sometimes explosively.
Oxygen is activated by transition metal ions, such as
iron or copper, in the active of metalloenzymes
Metabolic reactions are conducted at body temperature, far below
the temperature required to activate free oxygen.
In biological redox reactions involving O2, the oxygen is always
activated by redox active metal ions, such as iron and copper; these
metals also have unpaired electrons and form reactive metal–oxo
complexes.
All enzymes that use O2 in vivo are metalloenzymes and, in fact,
even the oxygen transport proteins, hemoglobin and myoglobin,
contain iron in the form of heme.
These metal ions provide one electron at a time to oxygen,
activating O2 for metabolism.
Because iron and copper, and sometimes manganese and other ions,
activate oxygen, these redox-active metal ions are kept at very low
(sub-micromolar) free concentrations in vivo.
Normally, they are tightly sequestered (compartmentalized) in
storage or transport proteins, and they are locally activated at the
active sites of enzymes where oxidation chemistry can be contained
and focused on a specific substrate.
Free redox-active metal ions are dangerous in biological systems
because, in free form, they activate O2, and ROS formed in these
reactions cause oxidative damage to biomolecules.
Damage to proteins is often site specific, occurring at sites of metal
binding to proteins, indicating that metal–oxo complexes participate
in ROS-mediated damage in vivo.
CLINICAL BOX
Iron overload increases risk for diabetes and cardiomyopathy
Patients with hematologic disorders such as hereditary
hemochromatosis, thalassemias and sickle cell disease, or who
receive frequent blood transfusions, gradually develop iron
overload, a condition that increases the risk for development
of cardiomyopathy and diabetes.
The heart and β-cells are rich in mitochondria.
The development of secondary disease in iron overload is
considered the result of iron-mediated enhancement of
mitochondrial ROS production in these tissues.
Mutations in the mitochondrial genome may lead to
progressive mitochondrial dysfunction, compromising cardiac
and β-cell function.
Reactive oxygen species and
oxidative stress
ROS are reactive, strongly oxidizing forms of oxygen
Oxidative stress:
condition in which the rate of
generation of ROS exceeds our
ability to protect ourselves
against them, resulting in an
increase in oxidative damage to
biomolecules.
Oxidative stress is a characteristic
feature of inflammatory diseases
in which cells of the immune
system produce ROS in response
to challenge.
Oxidative stress may be localized,
for instance in the joints in
arthritis or in the vascular wall in
atherosclerosis, or can be
systemic, e.g. in systemic lupus
erythematosus (SLE) or diabetes.
Among the ROS, hydrogen peroxide(H2O2) is
present at highest concentration in blood and
tissues, albeit at micromolar or lower conc.
H2O2 is relatively stable; it can be stored in the
laboratory or medicine cabinet for years but decomposes in the
presence of redox-active metal ions.
The hydroxyl radical (OH•) is the most reactive and damaging species;
its half-life, measured in nanoseconds, is diffusion limited, i.e.
determined by the time to collision with a target biomolecule.
Superoxide (O2•) is intermediate in stability and may actually serve as
either an oxidizing or reducing agent, forming H2O2 or O2, respectively.
At physiologic pH, the hydroperoxyl radical (HOO•, pKa < 4.5), the
protonated form of superoxide, represents only a small fraction of
total (about 0.1%), but this radical is intermediate in reactivity,
between O2• and OH•. HOO• and H2O2 are small, uncharged molecules
and readily diffuse through cell membranes.
ROS are formed by three major mechanisms in vivo:
1.
2.
3.
by reaction of oxygen with
decompartmentalized metal
ions;
as a side reaction of
mitochondrial electron
transport;
by normal enzymatic reactions,
e.g. formation of H2O2 by fatty
acid oxidases in the
peroxisome.
Secondary ROS are also formed by
enzymatic reactions, e.g.
myeloperoxidase in the
macrophage catalyzes the reaction
of H2O2 with Cl– to produce
another ROS, hypochlorous acid
(HOCl).
HOCl, which is the major oxidizing
species in chlorine-based bleaches,
is also part of the bactericidal
machinery of the macrophage.
ADVANCED CONCEPT BOX
Radiotherapy: Medical application of reactive oxygen
Radiation therapy uses a focused beam of high-energy electrons
or γ-rays from an X-ray or cobalt-60 source to destroy tumor tissue.
The radiation produces a flux of hydroxyl radicals (from water)
and organic radicals at the site of the tumor.
The localized oxidative stress causes damage to all biomolecules
in the tumor cell, but the damage to DNA is critical – it prevents
tumor cell replication, inhibiting tumor growth.
Irradiation is also used as a method of sterilization of food,
destroying viral or bacterial contaminants or insect infestations and
preserving food products during long-term storage.
Exposure to ionizing radiation from nuclear explosions or
accidents, or breathing or ingestion of radioactive elements, such as
radon gas or strontium-90, also causes oxidative damage to DNA.
Cells that survive the damage may have mutations in DNA that
eventually lead to development of cancers.
Leukemias are particularly prominent because of the rapid division
of bone marrow cells.
CLINICAL BOX
Toxicity of hyperoxia(1/2)
Supplemental oxygen therapy may be used for treatment of
patients with hypoxemia, respiratory distress, or following
exposure to carbon monoxide.
Under normobaric conditions, the fraction of oxygen in air can be
increased to nearly 100% using a facial mask or nasal cannula.
However, patients develop chest pain, cough and alveolar damage
within a few hours of exposure to 100% oxygen.
Edema gradually develops and compromises pulmonary function.
The damage results from overproduction of ROS in the lung.
Rats can be protected from oxygen toxicity by gradually
increasing the oxygen tension over a period of several days.
During this time, antioxidant enzymes, such as superoxide
dismutase, are induced in the lung and provide increased
protection against oxygen toxicity.
CLINICAL BOX
Toxicity of hyperoxia(2/2)
The lung is not the only tissue affected by hyperoxia.
Premature infants, especially those with acute respiratory distress
syndrome (IRDS), often require supplemental oxygen for survival.
During the 1950s, it was recognized that the high oxygen tension
used in incubators for premature infants increased the risk for
blindness, resulting from retinopathy of prematurity (retrolental
fibroplasia).
CLINICAL BOX
Ischemia/reperfusion injury: A patient with myocardial infarction(1/3)
A patient suffered a severe myocardial infarction, which was treated with
tissue plasminogen activator, a clot-dissolving (thrombolytic) enzyme.
During the days following hospitalization, the patient experienced
palpitations, irregular rapid heartbeat, associated with weakness and
faintness.
The patient was treated with antiarrhythmic agents.
CLINICAL BOX
Ischemia/reperfusion injury: A patient with myocardial infarction(2/3)
Comment
Ischemia, meaning limited blood flow, is a condition in which a tissue is
deprived of oxygen and nutrients.
Damage to heart tissue during a myocardial infarction occurs not during
the hypoxic or ischemic phase, but during reoxygenation of the tissue.
This type of damage also occurs following transplantation, and
cardiovascular surgery.
ROS are thought to play a major role in reperfusion injury.
CLINICAL BOX
Ischemia/reperfusion injury: A patient with myocardial infarction(3/3)
Comment(cont.)
When cells are deprived of oxygen, they must rely on anaerobic glycolysis and
glycogen stores for ATP synthesis.
NADH and lactate accumulate, and all the components of the mitochondrial
electron transport system are saturated with electrons (reduced), because the
electrons cannot be transferred to oxygen.
The mitochondrial membrane potential is increased (hyperpolarized), and
when oxygen is reintroduced, great quantities of ROS are rapidly produced,
overwhelming antioxidant defenses.
ROS flood throughout the cell, damaging membrane lipids, DNA and other
vital cellular constituents, leading to necrosis.
Antioxidant supplements and drugs are being evaluated for use during
recovery from myocardial infarction and stroke, during surgery, and for
protection of tissues prior to transplantation.
Reactive nitrogen species
and nitrosative stress
Peroxynitrite is a strongly oxidizing reactive N2 species
Nitric oxide synthases (NOS) catalyze the production of the
free radical nitric oxide (NO•) from the amino acid L-arginine.
There are three isoforms of NOS:
1. nNOS in neuronal tissue,
where NO• serves a neurotransmitter function;
2. iNOS in the immune system,
where it is involved in regulation of the immune response;
3. eNOS in endothelial cells,
where NO•, known as endothelium-derived relaxation factor (EDRF),
has a role in the regulation of vascular tone.
In a side reaction at sites of inflammation, NO• reacts with to form
the strong oxidant, peroxynitrite (ONOO–), a reactive nitrogen
species (RNS).
Like ROS, which produce oxidative stress, RNS produce nitrosative
stress by reaction with biomolecules. ONOOH has many of the
strong oxidizing properties of OH• but has a longer biological halflife.
It is also a potent nitrating agent, producing nitrotyrosine in proteins,
nitrated phospholipids in membranes and nucleotides in DNA.
Simultaneous production of NO• and , with the concomitant increase
in ONOO– and a decrease in NO•, is thought to limit vasodilatation
and exacerbate hypoxia and oxidative stress in the vascular wall
during ischemia-reperfusion injury, setting the stage for vascular
disease.
ONOOH degrades, in part, by homolytic cleavage to produce two
very reactive species, OH• and NO2•. NO2• is also formed by
eosinophil peroxidase or myeloperoxidase-catalyzed oxidation of
NO• by H2O2.
The nature of oxygen radical
damage
hydroxyl radical is the most reactive & damaging ROS
The reaction of ROS with biomolecules produces characteristic products,
described as biomarkers of oxidative stress.
These compounds may be formed directly in the oxidation reaction with
the ROS, or by secondary reactions between oxidation products and other
biomolecules.
The hydroxyl radical reacts with biomolecules primarily by hydrogen
abstraction and addition reactions.
One of the most sensitive sites of free radical damage are cell membranes,
which are rich in readily oxidized polyunsaturated fatty acids (PUFA).
Peroxidative damage to the plasma membrane affects the integrity and
function of the membrane, compromising the cell’s ability to maintain ion
gradients and membrane phospholipid asymmetry.
When OH• abstracts a hydrogen atom from a
PUFA, it initiates a chain of lipid peroxidation
reaction, producing secondary oxidation
products, lipid peroxides and lipid peroxyl
radicals.
The lipid oxidation products formed in this
reaction degrade to form reactive compounds,
such as malondialdehyde (MDA)
and hydroxynonenal (HNE).
These compounds react with proteins to form
adducts and crosslinks, known as advanced
lipoxidation end products (ALE).
Increased levels of MDA and HNE adducts to
lysine residues have been measured in
lipoproteins in plasma and the vascular wall in
atherosclerosis and in amyloid plaque in
Alzheimer’s disease, implicating oxidative stress
and damage in the pathogenesis of these
diseases.
Hydroxyl radicals also react by
addition to phenylalanine, tyrosine
and nucleic acid bases to form
hydroxylated derivatives and
crosslinks.
Other ROS and RNS leave tell-tale
tracks, such as nitro- and
chlorotyrosine, formed from
ONOOH and HOCl, respectively,
and methionine sulfoxide, formed
by reaction of H2O2 or HOCl with
methionine residues in proteins.
Nitrotyrosine, like ALEs, is increased
in atherosclerotic and Alzheimer’s
plaques.
Antioxidant defenses
Antioxidant:
A substance when present in trace (small) amounts
inhibits oxidation of the bulk.
There are several levels of protection
against oxidative damage
ROS damage to lipids and proteins is repaired largely by degradation
and resynthesis.
Oxidized proteins, for example, are preferred targets for proteasomal
degradation, and damaged DNA is repaired by a number of excision-repair
mechanisms.
The process is not perfect. Some proteins, such as collagens and crystallins,
turn over slowly, so that damage accumulates and function may be
impaired, e.g. age-dependent browning of lens proteins, crosslinking of
collagen and elastin, and loss of elasticity or changes in permeability of the
vascular wall and renal basement membrane.
The association between chronic inflammation and cancer indicates that
chronic exposure to ROS causes cumulative damage to the genome in the
form of nonlethal mutations in DNA.
ADVANCED CONCEPT BOX
Sentinel function of methionine
Methionine (Met) residues in protein may be oxidized to methionine sulfoxide
(MetSO) by H2O2, HOCl or lipid peroxides.
Met is generally on the surface of proteins and rarely has a role in the active
site or mechanism of action of enzymes.
However, there is evidence that it serves as an ‘antioxidant pawn’, protecting
the active site of enzymes.
Half of the Met residues of glutamine synthetase can be oxidized without
affecting the enzyme’s specific activity.
These residues are physically arranged in an array that ‘guards’ the entrance to
the active site, protecting the enzyme from inactivation by ROS.
MetSO can be reduced back to methionine by methionine sulfoxide reductases,
providing a catalytic amplification of the antioxidant potential of each
methionine residue.
CLINICAL BOX
Methionine oxidation and emphysema(1/2)
α1-Antitrypsin (A1AT) is a plasma protein, synthesized and
secreted by liver.
It is a potent inhibitor of elastase and protects tissues from
damage by the neutrophil enzyme secreted during
inflammation.
Deficiency of this protein (about 1 in 4000 worldwide) is
commonly associated with emphysema, progressive lung
disease, and also hepatic damage resulting from accumulation
of protein aggregates.
The lung damage is attributed to failure of A1AT to inhibit
elastase released by alveolar macrophages during
phagocytosis of airborne particulate matter.
CLINICAL BOX
Methionine oxidation and emphysema(2/2)
Therapy includes replacement therapy by weekly intravenous
infusion of a purified plasma concentrate or recombinant protein.
Cigarette smoking and exposure to mineral dust (coal, silica)
exacerbate pathology in patients with A1AT deficiency, but are
also independent risk factors for emphysema and pulmonary
fibrosis.
Cigarette smoke and microparticulate materials activate lung
macrophages, leading to release of proteolytic enzymes and
increased production of ROS as a result of inflammation.
The ROS cause oxidation of a specific Met residue in A1AT,
irreversibly inhibiting the anti-elastase activity of this protein.
Increased levels of inactive, Met(O)-containing A1AT are present
in plasma of chronic smokers.
ADVANCED CONCEPT BOX
Selenium, an antioxidant micronutrient
Selenocysteine is an unusual amino acid in proteins, found in only 25
proteins in the human proteome.
It is encoded by UGA, which is normally a STOP codon, under direction
of a SElenoCysteine Insertion Sequence (SECIS), a 50-nucleotide stemloop structure in the mRNA.
The 25-member selenoproteome includes five glutathione peroxidase
isozymes, three thioredoxin reductases, methionine sulfoxide reductase,
one of three enzymes that reduces methionine sulfoxide back to
methionine, and three iodothyronine deiodinases.
About a third of the selenoproteins have no known function, but
appear to be involved in antioxidant defense mechanisms.
Selenium is essential for life, in part because of severe
hypothyroidism and oxidative stress in its absence.
Selenium deficiency in adults is associated with cardiomyopathy in
Keshan disease, osteoarthropathy (cartilage degeneration) in Kashin–
Beck disease, and with symptoms of hypothyroidism, including chronic
fatigue and goiter.
ADVANCED CONCEPT BOX
The antioxidant response element(1/2)
Cells adapt to oxidative stress by induction of antioxidant enzymes.
Many of these are controlled by the antioxidant response element (ARE),
also known as the electrophile response element.
The central regulator of the ARE is the transcription factor Nrf2, which is
retained in inactive form in the cytoplasmic compartment by binding to a
cysteine-rich protein, Keap1. Under normal conditions, Keap1 directs
ubiquitination and proteasomal degradation of Nrf2.
During oxidative stress, modification of the sulfhydryl groups of Keap1 by
nucleophiles, such as the lipid peroxidation products, hydroxynonenal or
acrolein, causes dissociation of Nrf2 from Keap1. Nrf2 then translocates to
the nucleus and activates ARE-dependent genes.
Keap1 also reacts with exogenous nucleophiles, including carcinogens that
would otherwise react with DNA.
ADVANCED CONCEPT BOX
The antioxidant response element(2/2)
ARE-dependent enzymes include
catalase (CAT) and superoxide
dismutase (SOD), and enzymes that
catalyze the oxidation and
conjugation of carcinogens and
oxidants for excretion.
One of these enzymes, hepatic
glutathione S-transferase, catalyzes
conjugation with GSH.
The conjugates are then excreted in
urine as mercapturic acid, which
are S-substituted N-acetyl-cysteine
derivatives.
Our first line of defense against oxidative damage is
sequestration or chelation of redox-active metal ions
Endogenous chelators include a number of metal-binding proteins
that sequester iron and copper in inactive form, such as transferrin
and ferritin, the transport and storage forms of iron.
The plasma protein haptoglobin binds to hemoglobin from ruptured
red cells, and delivers the hemoglobin molecule to the liver for
catabolism.
Plasma hemopexin binds heme, the lipid-soluble form of iron,
which catalyzes ROS formation in lipid environments; it delivers the
heme to the liver for catabolism.
Albumin, the major plasma protein, has a strong binding site for
copper and effectively inhibits copper-catalyzed oxidation reactions
in plasma.
Carnosine (β-alanyl-L-histidine) and related peptides are present in
muscle and brain at millimolar concentrations; they are potent
copper chelators and may have a role in intracellular antioxidant
protection.
Despite these manifold and potent metal chelation
systems, ROS are formed continuously in the body,
both by enzymes and spontaneous metal-catalyzed
reactions.
In these cases, there are a group of enzymes that
act to detoxify ROS and their precursors.
These include superoxide dismutase (SOD), catalase
(CAT), and glutathione peroxidase (GPx).
SOD converts to the less toxic H2O2. There are two
classes of SOD: an MnSOD isozyme which is found
in mitochondria, and CuZnSOD isozyme which is
widely distributed throughout the cell. An
extracellular, secreted glycoprotein isoform of
CuZnSOD (EC-SOD) binds to proteoglycans in the
vascular wall and is thought to protect against O2•
and ONOO– injury.
CAT, which inactivates H2O2, is found largely in
peroxisomes, the major site of H2O2 generation in
the cell.
GPx (glutathione peroxidase) is widely
distributed in the cytosol, in mitochondria
and the nucleus.
It reduces H2O2 and lipid hydroperoxides to
water and a lipid alcohol, respectively, using
reduced glutathione (GSH) as a co-substrate.
GSH is a tripeptide that is present at 1–
5 mM concentration in all cells. The GSH is
recycled by an NADPH-dependent enzyme,
GSH reductase(GR). The NADPH, provided
by the pentose phosphate pathway,
maintains about a 100 : 1 ratio of GSH : GSSG
in the cell.
GPx is actually a family of seleniumcontaining isozymes; a phospholipid
hydroperoxide glutathione peroxidase will
reduce lipid hydroperoxides in phospholipids
and membranes, while other isozymes are
specific for free fatty acid or cholesterol
ester hydroperoxides.
There is also an isoform of GPx in intestinal
epithelial cells, which is thought to have a
role in detoxification of dietary
hydroperoxides, e.g. in fried foods.
Vitamin C is the outstanding antioxidant
in biological systems
Three antioxidant vitamins, A, C and
E, provide the third line of defense
against oxidative damage.
These vitamins, primarily vitamin C
(ascorbate) in the aqueous phase
and vitamin E (α- and γ-tocopherol)
in the lipid phase, act as chainbreaking antioxidants.
They act as reducing agents,
donating a hydrogen atom (H•) and
quenching organic radicals formed
by reaction of ROS with biomolecules.
The vitamin C and E radicals
produced in this reaction are
unreactive, resonance-stabilized
species; they do not propagate
radical damage and are enzymatically
recycled, e.g. by dehydroascorbate
reductase
Vitamin C reduces superoxide and lipid peroxyl radicals, but also has a
special role in reduction and recycling of vitamin E.
In response to severe oxidative stress. vitamin C recycles vitamin E, so that
vitamin E is maintained at constant concentration in the lipid phase until all
the vitamin C is consumed.
These antioxidants work together to inhibit lipid peroxidation reactions in
plasma lipoproteins and membranes.
Vitamin A is also a lipophilic antioxidant. Although best understood for its
role in vision, it is a potent singlet oxygen scavenger and protects against
damage from sunlight in the retina and skin.
Glutathionylation of proteins
– protection against ROS under stress
Despite the multiplicity of defensive mechanisms, there is always some
evidence of ongoing oxidative damage in tissues.
Under physiologic conditions, when proteins are exposed to O2, their
sulfhydryl groups gradually oxidize to form disulfides, either
intramolecularly or intermolecularly with other proteins.
This is multi-step process. First, a protein sulfhydryl group is oxidized to
a sulfenic acid (PrSOH) by an ROS, such as H2O2 or HOCl; then the sulfenic
acid reacts with another PrSH to form a crosslinked protein PrS-SPr. These
crosslinked proteins are reduced by glutathione to form oxidized glutathione
and regenerate the native protein with free sulfhydryl groups.
The reaction sequence is
PrSH + ROS → PrSOH
PrSOH + PrSH → PrS-SPr + H2O
PrS-SPr + 2 GSH → 2 PrSH + 2 GSSG
During oxidative stress, there is a significant increase in S-glutathionylated
proteins (PrS-SG) in the cell. In this case, the reaction sequence is
PrSH + ROS → PrSOH
PrSOH + GSH → PrS-SG+ H2O
PrS-SG+ GSH → PrSH + GSSG
S-Glutathionylation is reversed by nonenzymatic reduction by GSH
or by enzymes using thiol protein cofactors (thioredoxin,
glutaredoxin).
This pathway inhibits the formation of crosslinked protein aggregates,
such as Heinz bodies, which are hemoglobin precipitates that
develop in red cells in glucose-6-phosphate dehydrogenase
deficiency, characterized by decreased levels of GSH.
S-glutathionylation is thought to have a dual role, not only in
protecting cysteine against irreversible oxidation to the sulfinic or
sulfonic acid during oxidative and/or nitrosative stress but also in
modulating cellular metabolism (redox regulation).
Target proteins include a wide range of enzymes with active site or
regulatory –SH groups, such as glyceraldehyde-3-phosphate
dehydrogenase in glycolysis and protein kinases in signaling
cascades, as well as chaperones and transport proteins.
S-glutathionylation also appears to protect proteins from ubiquitinmediated proteasomal degradation during oxidative stress.
CLINICAL BOX
Peroxidase activity for detection of occult blood
Peroxidases, such as the glutathione peroxidase (GPx), are
enzymes that catalyze the oxidation of a substrate using H2O2.
Hemoglobin and heme have a pseudoperoxidase activity in
vitro.
In the guaiac-based test for occult fecal blood, a stool
sample is applied to a small card containing guaiac acid.
Hemoglobin in a stool specimen oxidizes phenolic compounds
in guaiac acid to quinones.
A positive test is indicated by a blue stain along the edge of
the fecal smear. Incompletely digested hemoglobin and
myoglobin from animal meat and some plant peroxidases may
cause false positives.
Similar peroxidase-dependent assays are used to identify
bloodstains at crime scenes.
The beneficial effects of
reactive oxygen species
ROS are essential
for many metabolic and signaling pathways
While this chapter has focused thus far on the dangerous aspects of
reactive oxygen, it is worth closing with some recognition of the
beneficial effects of ROS.
Among these are the regulatory functions of NO, the role of ROS in
activation of the ARE, the requirement for ROS in the bactericidal activity
of macrophages, and the use of ROS as substrates for enzymes, e.g.
H2O2 for the hemeperoxidases involved in iodination of thyroid hormone.
There is also increasing evidence that ROS, particularly H2O2, are
important signaling molecules involved in regulation of metabolism.
The tissue concentration of H2O2 is estimated to be in the submicromolar range; estimates vary widely, from 1 to 700 nmol/L.
However, significant changes in H2O2 concentration occur in response to
cytokines, growth factors and biomechanical stimulation.
The fact that these signaling events are inhibited by peroxide scavengers
or by overexpression of catalase implicates H2O2 in the signaling
cascade.
Insulin signaling, for example, appears to involve H2O2 as part of
the mechanism for reversible inactivation of some protein tyrosine
phosphatases, at the same time that protein tyrosine kinases are
activated through the insulin receptor.
As the evidence for the signaling role of H2O2 has become
convincing, there is increasing interest in research on the
regulatory role of superoxide.
활성 수용체티로신인산화효소는
세포내 신호전달 단백질의 복합체를 형성한다
활성 수용체티로신인산화효소는
세포내 신호전달 단백질의 복합체를 형성한다
활성화된 티로신인산화효소 수용체에 결합하는
세포내 신호전달단백질들은 물리적 어댑터 역할을 하는
어댑터 단백질에 의해 서로 복합체가 형성됨.
이들 단백질은 수용체와 다른 단백질을 연결시켜
더 큰 복합체를 이루게 하고, 이와 같은 커다란 복합체는
또 다른 단백질을 활성화신호 신호전달과정이 진행됨.
ADVANCED CONCEPT BOX
The glyoxalase pathway: A special role for glutathione(1/2)
A small fraction of triose phosphates produced
during metabolism spontaneously degrades
to methylglyoxal (MGO), a reactive dicarbonyl sugar.
MGO is also formed during metabolism of glycine
and threonine, and as a product of nonenzymatic
oxidation of carbohydrates and lipids – it is a
significant precursor of advanced glycation and
lipoxidation end products (AGE/ALEs).
MGO reacts primarily with arginine residues in
proteins, but also with lysine, histidine and cysteine,
leading to enzyme inactivation and protein
crosslinking.
MGO is inactivated by enzymes of the glyoxalase
pathway, a GSH-dependent system found in all cells
in the body.
The glyoxalase pathway consists of two enzymes
that catalyze an internal redox reaction in which
carbon-1 of MGO is oxidized from an aldehyde to a
carboxylic acid group and carbon-2 is reduced from
a ketone to a secondary alcohol.
The end product, D-lactate, does not react with
proteins; D-lactate is distinct from L-lactate, the
product of glycolysis, but may be converted into Llactate for further metabolism.
ADVANCED CONCEPT BOX
The glyoxalase pathway: A special role for glutathione(2/2)
Levels of MGO and D-lactate are increased in blood of diabetic
patients, because levels of glucose and glycolytic intermediates,
including triose phosphates, are increased intracellularly in
diabetes.
The glyoxalase system also inactivates glyoxal, and other
dicarbonyl sugars produced during nonenzymatic oxidation of
carbohydrates and lipids.
Glyoxalase inhibitors are being evaluated for chemotherapy
because cancer cells appear to be more sensitive to glyoxalase
inhibitors, perhaps because of their increased reliance on
glycolysis.
ADVANCED CONCEPT BOX
The respiratory burst in macrophages(1/2)
Macrophages launch a sequence of ROSproducing reactions during the burst of
oxygen consumption accompanying
phagocytosis.
NADPH oxidase in the macrophage
plasma membrane is activated to
produce , which is then converted to
H2O2 by superoxide dismutase.
The H2O2 is used by another macrophage
enzyme, myeloperoxidase (MPO), to
oxidize chloride ion, ubiquitous in body
fluids, to hypochlorous acid (HOCl).
H2O2 and HOCl mediate bactericidal
activity by oxidative degradation of
microbial lipids, proteins, and DNA.
The macrophage has a high intracellular
concentration of antioxidants, especially
ascorbate, to protect itself during ROS
production, but its relatively short life
span, 2-4 months, suggests that it is not
immune to oxidative damage.
ADVANCED CONCEPT BOX
The respiratory burst in macrophages(2/2)
The consumption of O2 by NADPH oxidase is responsible for the
‘respiratory burst’, the sharp increase in O2 consumption for
production of ROS, which accompanies phagocytosis.
One of the end products of this reaction sequence, HOCl, is also
the active oxidizer in chlorine-containing laundry bleaches.
Intravenous infusion of dilute HOCl solutions was actually used
for treatment of bacterial sepsis in battlefield hospitals during
World War I, before the advent of penicillin and other antibiotics.
Chronic granulomatous disease (CGD) is an inherited disease
resulting from a genetic defect in NADPH oxidase. The inability
to produce superoxide leads to chronic life-threatening bacterial
and fungal infections.
ADVANCED CONCEPT BOX
Antioxidant defenses in the red blood cell (RBC)(1/2)
The RBC does not use oxygen for metabolism, nor is it involved
in phagocytosis.
However, because of the high O2 tension in arterial blood and
the heme iron content of RBCs, ROS are formed continuously in
the RBC.
Hb spontaneously produces superoxide (O2∙) in a minor side
reaction associated with binding of O2.
The occasional reduction of O2 to O2∙ is accompanied by
oxidation of normal (ferro) Hb to methemoglobin
(ferrihemoglobin), a rust-brown protein that does not bind or
transport O2.
Methemoglobin may release heme, which reacts with and
H2O2 in Fenton-type reactions to produce hydroxyl radical (OH•)
and reactive iron-oxo species.
These ROS initiate lipid peroxidation reactions that can lead to
loss of membrane integrity and cell death.
ADVANCED CONCEPT BOX
Antioxidant defenses in the red blood cell (RBC)(2/2)
The RBC is well fortified with antioxidant defenses to protect
itself against oxidative stress.
These include catalase (CAT), superoxide dismutase (SOD) and
glutathione peroxidase (GPx), as well as a methemoglobin
reductase activity that reduces methemoglobin back to normal
ferrohemoglobin.
Normally, less than 1% of Hb is present as methemoglobin.
However, persons with congenital methemoglobinemia,
resulting from methemoglobin reductase deficiency, typically
have a dark and cyanotic appearance.
Treatment with large doses of ascorbate (vitamin C) is used to
reduce their methemoglobin to functional hemoglobin.
GSH, present at ~2 mmol/L in the RBC, not only supports
antioxidant defenses but also is an important sulfhydryl buffer,
maintaining –SH groups in hemoglobin and enzymes in the
reduced state.
Summary
Reactive oxygen species (ROS) are the sparks produced by oxidative metabolism,
and oxidative stress may be viewed as the price we pay for using oxygen for
metabolism.
ROS and RNS, such as superoxide, peroxide, hydroxyl radical, and peroxynitrite,
are reactive and toxic, sometimes difficult to contain, but their production is
important for regulation of metabolism, turnover of biomolecules and protection
against microbial infection.
ROS and RNS cause oxidative damage to all classes of biomolecules: proteins,
lipids and DNA.
There are a number of protective antioxidant mechanisms, including
sequestration of redox-active metal ions, enzymatic inactivation of major ROS,
inactivation of organic radicals by small molecules, such as GSH and vitamins,
and, when all else fails, repair and/or turnover, and, in extremis, apoptosis.
Biomarkers of oxidative stress are readily detected in tissues in inflammation, and
oxidative stress is increasingly implicated in the pathogenesis of age-related,
chronic disease.
Despite their damaging actions, ROS are also essential for the normal functions
of the immune system, and for many enzymes and cell signaling pathways.
Active learning
Review the evidence that atherosclerosis is an
inflammatory disease resulting from overproduction of
ROS in the vascular wall.
Discuss the evidence that hyperglycemia in diabetes
induces a state of oxidative stress that leads to renal and
vascular complications.
Review the data on use of antioxidants in therapy for
atherosclerosis and diabetes. Based on these studies,
how strong is the evidence that these diseases are the
result of increased oxidative stress?
Discuss recent advances in the use of antioxidants for
organ and tissue protection during surgery and
transplantation.
Further reading
•
•
•
•
•
•
•
Burgoyne JR, Oka SI, Ale-Agha N, et al.: Hydrogen peroxide sensing
and signaling by protein kinases in the cardiovascular system. Antioxid
Redox Signal. 18:1042-10522013 22867279
Ferrari CK, Souto PC, França EL, et al.: Oxidative and nitrosative stress
on phagocytes’ function: from effective defense to immunity evasion
mechanisms. Arch Immunol Ther Exp. 59:441-448 2011
Greenough MA, Camakaris J, Bush AI: Metal dyshomeostasis and
oxidative stress in Alzheimer’s disease. Neurochem Int. 62:540555 2013 22982299
Riccioni G, D’Orazio N, Salvatore C, et al.: Carotenoids and vitamins C
and E in the prevention of cardiovascular disease. Int J Vitam Nutr
Res. 82:15-26 2012 22811373
Tkachev VO, Menshchikova EB, Zenkov NK: Mechanism of the
Nrf2/Keap1/ARE signaling system. Biochemistry (Mosc). 76:407422 2011 21585316
Zhang H, Forman HJ: Glutathione synthesis and its role in redox
signaling. Semin Cell Dev Biol. 23:722-728 2012 22504020
Zhang Y, Tocchetti CG, Krieg T, et al.: Oxidative and nitrosative stress in
the maintenance of myocardial function. Free Radic Biol Med. 53:15311540 2012 22819981
Websites
• Antioxidants and cancer.
www.cancer.gov/cancertopics/factsheet/antioxidantsprevention
• Free radical lectures.
http://web.mst.edu/~nercal/documents/chem464/lectures/Lec0
1_FreeRadicals.pdf
• Oxidative stress and disease.
www.oxidativestressresource.org/
• Reactive oxygen species and antioxidant vitamins.
http://lpi.oregonstate.edu/f-w97/reactive.html
• Virtual Free Radical School
www.sfrbm.org/sections/education/frs-presentations
The generation, removal, and role of reactive oxygen species(ROS) in cell injury
SOURCE
OF ROS:
mitochondrial
Resp. chain
activated
leukocytes
A
N
T
I
OXIDATIVE
DAMAGE to:
DNA
O
lipids
X
proteins
indoor &
outdoor air
(cigarette,
radon, ozone)
D
UV
A
etc.
N
I
T
CAUSAL
CONTRIBUTION to:
aging
heart dis.
cancer
Alzheimer’s dis.
inflammatory-immune dis.
autoimmune dis.
(rheumatoid arthritis,
lupus, DM)
AIDS
ARDS
etc.
“I would rather know
the person who has the disease
than know the disease the person has.”
Hippocarates
“병이나 장기를 고치는 기술자가 아니라,
병을 앓고 있는 인간을 전인적으로 돌보는
인격적인 존재이다.”
냉철한 이성과 따뜻한 인성을 갖춘
다양한 분야와 소통할 수 있는 학도
(유연한 사고와 따뜻한 마음을 가진 학생)
인문학적 소양
도덕적이며, 협동적이고, 실천적인 인간이어야 한다.
우수한 진료 능력, 바른 직업관, 신뢰를 받는 사람.
교육의 삼층 구조
세 차원의 교육
1. 생명/종교 교육
(원리: 합명(合命), 의무)
2. 생활/교양 교육
(원리: 합의(合義), 자유)
3. 전문/기술 교육
(원리: 합리(合理), 재능)
운명(destiny)
professionalism
(전문가 정신)
인격(character)
습관(habits)
행동(deeds)
말(words)
생각(thoughts)
배움과 가르침(敎學相長)
• 최근에는 강의식 수업의 부적절성을 지적
하며 토론식 수업만이 최상의 수업방식이
라는 분위기가 형성되고 있는 것 같다.
• 그러나, 토론을 하기 위해서는 그 주제에
대한 기본적 지식이 필요한데, 이것은 토
론으로 얻는 것보다는 강의를 통해 얻는
것이 훨씬 효과적이다.
啐啄同時
http://blog.hankyung.com/?mid=blog&category=
815699&vid=jsyoon&document_srl=182239
새도 가끔 날개짓을 쉽니다.
철새가 수만리를 비행할 수 있는 것은
 쉬어가야 할 때와
 쉬는 방법을 알기 때문입니다.