• The Earth was originally anoxic
Reactive Oxygen and
Nitrogen Species
• Metabolism was anaerobic
• O2 started appearing ~2.5 x 109 years ago
Anaerobic metabolism-glycolysis
Glucose + 2ADP + 2Pi
Lactate + 2ATP + 2H2O
O2 an electron acceptor in aerobic metabolism
Glucose + 6O2 + 36ADP + 36Pi
6CO2 + 36ATP + 6H2O
Before 1950s: “golden age” of chemistry
• Ground-state oxygen has 2-unpaired electrons
: :
: :
. O:O .
• The unpaired electrons have parallel spins
Many free radicals have been discovered and described
Haber & Weiss (1934): Superoxide + Hydrogen peroxide
Baeyer & Villiger (1901): discovered peroxynitrite
Hydroxyl radical
Early 1950s: presence of radicals in biological materials
Commoner et al. (1954) Nature 174: 689-691
1956: Radicals may be formed as by-products of
enzymatic reactions in vivo?
Harman (1956): radicals are “pandora’s box” of evils (aging, mutations, cancer,…)
1969: Discovery of superoxide dismutase (SOD)
• Oxygen molecule is minimally reactive
due to spin restrictions
McCord & Fridovich (1969) JBC 244: 6049-6055
Free radicals must be important for biology if the body has a defense enzyme(s)!
Intensive investigations on the role of oxidants in disease begun
Since late 70s: Radicals are important for normal biology
Mittal & Murad (1977): superoxide anion stimulates formation of cGMP
Ignarro & Kadowitz (1985) and Moncada (1987): NO• is important in circulation
1
Prooxidants
Basics of Redox Chemistry
Free Radicals:
Radicals
Term
Definition
Oxidation
Gain in oxygen
Loss of hydrogen
Loss of electrons
Reduction
Oxidant
Reductant
ƒ Any species capable of independent
existence that contains one or more
unpaired electrons
ƒ A molecule with an unpaired electron
in an outer valence shell
Loss of oxygen
Gain of hydrogen
Gain of electrons
Non-Radicals:
Oxidizes another chemical by taking
electrons, hydrogen, or by adding oxygen
Reduces another chemical by supplying
electrons, hydrogen, or by removing oxygen
Reactive Oxygen Species (ROS)
ƒ Species that have strong oxidizing
potential
ƒ Species that favor the formation of
strong oxidants (e.g., transition
metals)
Non-Radicals:
O2.- Superoxide
.OH Hydroxyl
H2O2
HOCl-
Hydrogen peroxide
RO2 Peroxyl
O3
Ozone
Radicals:
NO. Nitric Oxide
RO. Alkoxyl
1O
2
Singlet oxygen
NO2. Nitrogen dioxide
HO2. Hydroperoxyl
ONOO-
Peroxynitrite
Hypochlorous acid
R3N. Nitrogen-centered
R-O. Oxygen-centered
R-S. Sulfur-centered
H2O2 Hydrogen peroxide
HOCl- Hypochlorous acid
O3
Ozone
1O
2
Singlet oxygen
ONOO- Peroxynitrite
Men+ Transition metals
Reactive Nitrogen Species (RNS)
Radicals:
.
R3C. Carbon-centered
Non-Radicals:
ONOOPeroxynitrite
ROONO Alkyl peroxynitrites
N2O3
Dinitrogen trioxide
N2O4
Dinitrogen tetroxide
HNO2
Nitrous acid
NO2+
Nitronium anion
NONitroxyl anion
+
NO
Nitrosyl cation
NO2Cl
Nitryl chloride
2
Oxidative Stress
“Longevity” of reactive species
Reactive Species
Half-life
Antioxidants
Hydrogen peroxide
Organic hydroperoxides
Hypohalous acids
~ minutes
Peroxyl radicals
Nitric oxide
~ seconds
Peroxynitrite
~ milliseconds
Superoxide anion
Singlet oxygen
Alcoxyl radicals
~ microsecond
Hydroxyl radical
~ nanosecond
Radical-mediated reactions
Addition
R.
+
H2C=CH2
RH
Electron abstraction
R.
+
ArNH2
R-
Termination
+
Y.
R.
“An imbalance favoring prooxidants and/or disfavoring
antioxidants, potentially leading to damage” -H. Sies
Endogenous sources of ROS and RNS
Microsomal Oxidation,
Flavoproteins, CYP enzymes
R-CH2-CH2.
Hydrogen abstraction
+
LH
R.
Prooxidants
+
L.
+
ArNH2.+
Xanthine Oxidase,
NOS isoforms
Transition
metals
Endoplasmic Reticulum
Cytoplasm
R-Y
Myeloperoxidase
(phagocytes)
Lysosomes
Fe
Cu
Oxidases,
Flavoproteins
Peroxisomes
Mitochondria
Plasma Membrane
Disproportionation
CH3CH2. + CH3CH2.
CH3CH3 + CH2=CH2
Electron transport
Lipoxygenases,
Prostaglandin synthase
NADPH oxidase
3
Mitochondria as a source of ROS
PEROXISOME
•
•
•
•
•
β-oxidation of fatty acids
bile acid synthesis
purine and polyamine catabolism
amino acid catabolism
oxygen metabolism
Fatty acyl-CoA
synthetase
Fatty Acid
Acyl-CoA
H2O2
Acyl-CoA oxidase
Enoyl-CoA
Enoyl-CoA hydrolase
Hydroxyacyl-CoA
Hydroxyacyl-CoA
dehydrogenase
Ketoacyl-CoA
Thiolase
The source of mitochondrial ROS appears to involve a non-heme iron protein that transfers
electrons to oxygen. This occurs primarily at Complex I (NADH-coenzyme Q) and, to a lesser
extent, following the auto-oxidation of coenzyme Q from the Complex II (succinate-coenzyme Q)
and/or Complex III (coenzyme QH2-cytochrome c reductases) sites. The precise contribution of
each site to total mitochondrial ROS production is probably determined by local conditions
including chemical or physical damage to the mitochondria, oxygen availability and the presence
of xenobiotics.
From Kehrer JP (2000) Toxicology 149: 43-50
Acetyl-CoA
NADPH oxidase as a source of ROS
Acyl-CoA shortened
by two carbons
Cytoplasmic sources of ROS
Mainly in neutrophils (oxidative burst), but also in many other cell types
xanthine oxidase
xanthine oxidase
Nitric Oxide Synthases (NOS):
neuronal
nNOS (I)
endothelial
eNOS (III)
inducible
iNOS (II)
NO•
4
Lysosome as a source of ROS (Chlorox
!)
(Chlorox!)
Microsomes as a source of ROS
oxidase
NADPH + H+ + O2
NADP+ + H2O2
monooxygenase
-
O2•
-
O2•
RH + NADPH + H+ + O2
ROH + NADP+ + H2O
-
O2•
RH + XOOH
peroxidase
ROH + XOH
Myeloperoxidase undergoes a complex array of redox transformations and produces
HOCl, degrades H2O2 to oxygen and water, converts tyrosine and other phenols and
anilines to free radicals, and hydroxylates aromatic substrates via a cytochrome
P450 type activity.
Exogenous sources of free radicals
•
Radiation
UV light, x-rays, gamma rays
•
Chemicals that react to form peroxides
Ozone and singlet oxygen
•
Chemicals that promote superoxide formation
Quinones, nitroaromatics, bipyrimidiulium herbicides
•
Chemicals that are metabolized to radicals
e.g., polyhalogenated alkanes, phenols, aminophenols
•
Chemicals that release iron
ferritin
Oxidative stress and cell damage
• High doses:
directly damage/kill cells
• Low doses/chronic overproduction of oxidants:
activation of cellular pathways
stimulation of cell proliferation
damage to cellular proteins, DNA and lipids
5
Free Radicals/Reactive Intermediates
Lipids
Proteins
DNA
Oxidation of
vitamin E
Thiol oxidation
Carbonyl formation
DNA damage
Lipid peroxidation
Damage to Ca2+ and
other ion transport
systems
Membrane damage
Disruption of normal
ion gradients
Activation/deactivation
of various enzyme systems
Adapted from: Kehrer JP, 1993
Altered gene
expression
Depletion of ATP
and NAD(P)H
Consequences of lipid peroxidation
• Structural changes in membranes
alter fluidity and channels
alter membrane-bound signaling proteins
increases ion permeability
• Lipid peroxidation products form adducts/crosslinks
with non lipids
e.g., proteins and DNA
• Cause direct toxicity of lipid peroxidation products
e.g., 4-hydroxynonenal toxicity
• Disruptions in membrane-dependent signaling
• DNA damage and mutagenesis
Cell Injury
Consequences of protein thiol oxidation
Oxidation of catalytic sites on proteins
loss of function/abnormal function
BUT(!): sometimes it is gain in function!
Formation of mixed sulfide bonds
Protein-protein linkages (RS-SR)
Protein-GSH linkages (RS-SG)
Alteration in 2o and 3o structure
Increased susceptibility to proteolysis
Consequences of DNA oxidation
• DNA adducts/AP sites/Strand breaks
mutations
initiation of cancer
• Stimulation of DNA repair
can deplete energy reserves (PARP)
imbalanced induction of DNA repair enzymes
induction of error prone polymerases
activation of other signaling pathways
6
Pathological conditions that involve
oxidative stress
Defense against Prooxidants
1. Prevention of prooxidant formation
•
•
•
•
•
Inflammation
Atherosclerosis
Ischemia/reperfusion injury
Cancer
Aging
Prevention of prooxidant formation
2. Interception of prooxidants
3. Breaking the chain of radical reactions
4. Repair of damage caused by prooxidants
ANTIOXIDANT: a substance that is able, at relatively low concentrations,
to compete with other oxidizable substrates and, thus, to significantly deley
or inhibit the oxidation of other substrates
Examples of preventative ‘antioxidants’
Physical prevention:
Behavioral:
Barriers:
- avoidance
- organismal level
- organ level
- cellular level
Biochemical prevention:
Control of prooxidant molecules:
- transition metal chelators
- catalytic control of O2 reduction
Control of prooxidant enzymes:
- blockade of stimuli
- inhibition of enzymes
Anti-inflammatory agents
Nitric oxide synthase inhibitors
Metal chelators:
- Metallothionein
- Transferrin
- Lactoferrin
NADPH oxidase inhibitors
Xanthine oxidase inhibitors
7
Interception of prooxidants
Chain breaking antioxidants
Example of radical chain-reaction: lipid peroxidation
‘Classical’ antioxidant:
ROO• (peroxyl radicals) are often the chain-carrying radicals
Intercepts species, once formed
Chain-breaking oxidants act by reacting with peroxyl radicals:
Excludes from further damaging activity
“Donor” antioxidants (tocopherol, ascorbate, uric acid,…)
Transfers species from critical parts of cell
LOO• + TOH
“Sacrificial” antioxidants (Nitric oxide):
Important considerations for interception reactions:
LOO• + NO•
Speed of reaction (rate constant)
Concentration of intercepting species in vivo
Is reaction truly a detoxication pathway?
Is reaction catalytically recyclable?
‘Antioxidant Network’
Catalytic maintenance of antioxidant defense
Non-scavenging enzymes (re-reduce antioxidants)
Dependence on energy status of cell
Glucose is the most important ‘antioxidant’
Small Molecules
glutathione, uric acid, ascorbate (Vit. C)
α-tocopherol (Vit. E), β-carotene, coenzyme Q
Catalytic reduction of peroxides
Proteins
-Intracellular:
-Cell membrane:
-Extracellular:
LOONO
Good chain-breaking antioxidant:
both ANT and ANT• should be relatively UNreactive
ANT• decays to harmless products
does not add O2 to make a new peroxyl radical
is renewed (recycled)
Cellular antioxidants
-Water soluble:
-Lipid soluble:
LOOH + TO•
SOD (I and II), glutathione peroxidase, catalase
SOD (III), ecGPx, plasma proteins (e.g. albumin)
phospholipid hydroperoxide GPx (PHGPx)
ROOH
ROH
G-SeH
G-SeOH
GSSG
2 GSH
Catalytic reduction of lipid radicals
LOO.
Tocopherol
LOO
Tocopheroxyl radical
GPx
GSSG
reductase
Ascorbate
Ubiquinone
Ubiquinol
NADPH
Dehydroascorbate
NADP+
G6PDH
6-phosphogluconate glucose-6-phosphate
GSH
NAD(P)+
GSSG
NAD(P)H
8
Repair of damage caused by prooxidants
Protection not perfect
Repair of damaged products
proteins and lipids
-rereduction and degradation
DNA
-repair enzymes
Cell death (apoptosis/necrosis)
How free radicals can be involved in signaling?
• Heme oxidation
• Oxidation of iron-sulfur centers in proteins
• Changes in thiol/disulfide redox state of the cell
• Change in conformation Æ change in activity
• Oxidative modification of proteins: degradation,
loss of function, or gain of function
Can free radicals be second messengers?
Second messengers should be:
• Short lived (concentrations can change rapidly)
• Enzymatically generated in response to stimulant
• Enzymatically degraded
• Specific in action (?)
Some free radicals fit these criteria!
O2.-, H2O2, NO., ONOO-
NO• signaling in physiology
Nitric Oxide Synthase
O2-•
NO•
ONOO-
Binds to heme moiety of
guanylate cyclase
Conformational change of
the enzyme
Increased activity
(production of cGMP)
• Oxidative modification of DNA: activation of
repair, and/or apoptosis
Modulation of activity of
other proteins (protein
kinases, phosphodiesterases, ion channels)
• Oxidative modification of lipids: disruption of
membrane-associated signaling, DNA damage,
and formation of protein adducts
Physiological response
(relaxation of smooth
muscles, inhibition of
platelet aggregation, etc.)
9