What Is A Free Radical - Ústav lékařské chemie a biochemie

Reactive Oxygen Species
in the Body
Antioxidant Defence
Jan Pláteník MD, PhD
Ústav lékařské biochemie 1.LF UK
What is a free radical ?
- any chemical species (molecule, atom, ion)
capable of independent existence that contains
at least one unpaired electron
radical
O 2·–
ion
molecule
1

For what do we need oxygen?
Transfer of electrons (oxidation) from organic substances to
oxygen releases huge amount of energy
E.g. Glucose:
C 6 H 12 O 6 + 6 O 2 → 6 CO 2 + 6 H 2 O
∆G o ’ = – 2820 kJ/ mol (180 g of glucos
ose)
In addition, reactivity of oxygen can be controlled by catalysis
with transition metals (iron, copper)
Does sugar burn ?
2

Spin restriction of dioxygen
• Normal (triplet) O 2 is a
biradical, with high
affinity to electrons
• But gain of an electron
requires one of the
unpaired electrons
changes its spin, which is
a slow process.
• That’s why we do not
burn in the air
• Singlet O 2 is an excited,
highly reactive species
Respiratory chain in the
mitochondrial inner membrane
4

The respiratory chain:
..ele
electrons flow ‘downhill’ and
terminate at oxygen
Overall reaction:
NADH + H + + 1/2 O 2 → NAD + + H 2 O
∆G o ’ = –219.25 kJ mol -1
..and
resulting proton gradient powersp
ATP synthesis
5

Reactive oxygen species (ROS, ‘oxygen radicals’) can be
derived from intermediates of oxygen reduction to water:
O 2
+4 e-
(+4 H + )
+1 e-
O 2·–
superoxide
+2 e-
(+2 H + )
H 2 O 2
hydrogen peroxide
+3 e-
(+3 H + )
2H 2 O
OH·
hydroxyl
radical
( +H 2 O )
Superoxide
O 2·–
• Sources in the body:
– Escape of electrons to oxygen
- Respiratory chain in mitochondria
- Other similar redox carrier systems, e.g. microsomal
cytochrome P450 monooxygenase
– NAD(P)H Oxidase:
- Phagocytes (”respiratory burst”)
- Non-phagocytic cells
– Some enzymes:
- Xanthine oxidase
- Cyclooxygenase
- Lipoxygenase
– Reaction of FeII-hemoglobin with oxygen
– Autoxidation (reaction with oxygen) of various compounds
(ascorbate, glutathione and other thiols, catecholamines)
6

Superoxide originates in the
mitochondrial respiratory chain
• 1-2 % from total
oxygen consumption
• Escape of electrons
from redox centers of
complexes I and III,
mostly from
semiquinone
Respiratory burst of phagocyte
7

Origin of superoxide from oxyhemoglobin
Štípek S et al.: Antioxidanty a volné radikály ve zdraví a nemoci. Grada
Publishing, Praha, 2000.
Superoxide
• Properties:
- Moderate reactivity, both oxidising and reducing
agent
- Limited ability to cross biological membranes (only
through anion channels or in its protonated form)
- Releases iron from Fe-S clusters
• Fate:
– Dismutation:
O 2·–
O 2·–
+ O 2·–
+ 2 H + → O 2 + H 2 O 2
– Reaction with nitric oxide:
NO· + O 2·–
→ OONO – (peroxynitrite)
8

Hydrogen peroxide
H 2 O 2
• Formation in the body:
– Dismutation of superoxide (spontaneous or
catalyzed by superoxide dismutase)
O 2·–
+ O 2·–
+ 2 H + → O 2 + H 2 O 2
– Directly by action of some enzymes:
• Xanthine oxidase
• Monoamine oxidase (MAO)
Hydrogen peroxide
• Properties:
- No radical
- Freely crosses biological membranes
- Fairly unreactive as such
- But rapidly reacts with reduced transition metals such
as iron, copper (the Fenton reaction)
• Fate:
– The Fenton reaction:
H 2 O 2
hydroxyl radical
H 2 O 2 + Fe 2+ → OH – + OH· + Fe 3+
– Safe decomposition by glutathione peroxidase or
catalase
9

Hydroxyl radical
• Formation in the body:
– Ionising radiation:
H 2 O → H· + OH·
OH ·
– The Fenton reaction:
H 2 O 2 + Fe 2+ → OH – + OH· + Fe 3+
• Properties:
- Extremely high reactivity. Damages biomolecules
near its site of formation.
Reactive Oxygen Species (ROS)
• Radicals:
– Superoxide, O 2·–
– Hydroperoxyl radical, HO 2·
– Hydroxyl radical, OH·
– Peroxyl radicals, ROO·
– Alkoxyl radicals, RO·
• Non-radicals:
– Hydrogen peroxide, H 2 O 2
– Hypochlorous acid, HClO
– Ozone O 3
– Singlet oxygen, 1 O 2
10

Nitric oxide
• Formation in the body:
NO ·
– NO synthase-catalysed reaction:
L-Arginine + O 2
+ NADPH → L-Citrulline + NADP + +NO·
– Three different NO synthases:
- NOS I (neuronal, constitutive)
- NOS II (phagocytes, inducible)
- NOS III (endothelial, constitutive)
Nitric oxide
• Properties:
- Gaseous radical
- Reaction with heme iron of guanylate cyclase
(…physiological effects)
- Reaction with heme iron of hemoglobin (…physiological
inactivation)
- Reaction with sulfhydryl groups on glutathione etc. to
nitrosothiol (…transport NO)
- Reaction with superoxide to peroxynitrite and finally
hydroxyl radical (…toxicity):
NO· + O 2·–
→ OONO – peroxynitrite
NO ·
OONO – + H + → HOONO → OH· + NO 2 ·
peroxynitrous acid
hydroxyl radical
11

NO causes relaxation of smooth muscles in blood
vessel wall:
Reactive Nitrogen Species (RNS)
• Radicals:
– Nitric oxide, NO·
– Nitric dioxide, NO 2 ·
• Non-radicals:
– Nitrosonium, NO +
– Nitroxyl, NO –
– Nitrous acid, HNO 2
– Dinitrogen trioxide, N 2 O 3
– Dinitrogen tetraoxide, N 2 O 4
– Nitronium, NO 2
+
– Peroxynitrite, ONOO –
– Alkylperoxynitrite, ROONO
12

Oxidative damage to biomolecules
• Lipids: peroxidation of polyunsaturated
fatty acids in membranes
• Proteins: oxidation of -SH, carbonylation -
NH 2 , hydroxylation/nitrosylation of
aromatic AA, cross-linking, degradation
• Nucleic acids: strand breaks, hydroxylation
of bases ... mutation, cancerogenesis
Lipid peroxidation
(rancidity)
13

Further fate of lipid peroxides
Ionising radiation:
Hydroxyl radical originates from ionisation of water:
H 2 O + hν → H· + OH·
Reactive oxygen species in the body:
One-electron reduction of oxygen (mitochondria,
NADPH oxidase) forms superoxide O 2·–
Dismutation of superoxide produces hydrogen peroxide:
O 2·–
+ O 2·–
+ 2 H + → O 2 + H 2 O 2
Fenton reaction with Fe or Cu converts peroxide to
hydroxyl radical:
H 2 O 2 + Fe 2+ → OH – + OH· + Fe 3+
14

Where RONS are beneficial:
Monooxygenases (cytochrome P450): hydroxylation
of steroids, xenobiotics etc.
Mechanism: basically a controlled Fenton reaction
Fe 3+ + SH + 2 e- + O 2 + H + → FeOOH SH
FeOOH SH → FeO 3+ SH + H 2 O
FeO 3+ SH → FeOH 3+ S• → Fe 3+ SOH → Fe 3+ + SOH
Where RONS are beneficial:
• Monooxygenases: hydroxylation of steroids,
xenobiotics etc.
• Phagocyte weapons: killing microbes or cancer cells
• Mediators of inflammatory reaction
15

Beneficial ROS:
Respiratory
burst of
neutrophilic granulocyte
© 1998 Garland Publishing
Where RONS are beneficial:
• Monooxygenases: hydroxylation of steroids,
xenobiotics etc.
• Phagocyte weapons: killing microbes or cancer cells
• Mediators of inflammatory reaction
• Local hormones/mediators:
- NO:
- smooth muscle relaxation in blood vessels, gastrointestinal tract,
corpus cavernosum penis
- neurotransmitter/ neuromodulator in the CNS, function in
synaptic plasticity, learning & memory
- inhibition of thrombocyte adhesion & aggregation, leucocyte
adhesion
Superoxide as well with effects opposing NO ?
16

Concept of redox signaling
• Redox environment inside the cell –
determined mostly by ratios GSH/GSSG
and NADH/NAD
• Redox sensors on proteins:
– critical -SH groups
– Fe-S centers
• Targets of redox signaling:
– transcription factors
– protein kinases and phosphatases
Redox
signaling
• Oxidative stress activates
certain protein kinases and
transcription factors
• Outcome: e.g. stimulation
of proliferation, or cellular
senescence (ageing)
• Actions of
ROS/antioxidants in the
cell can be very specific
(T. Finkel & N.J. Holbrook, Nature 408 (2000), 239-247)
17

…and very complex
Oxidative stress
• Levels of reactive oxygen species are kept
within certain limits by mechanisms of
antioxidant defence
• Oxidative stress occurs if the
oxidant/antioxidant balance shifts to
oxidation
18

Antioxidant defence
• prevention of ROS/RNS formation
(regulation of producing enzymes,
sequestration of metals)
• scavenging, trapping and quenching of
radicals
• reparation systems (phospholipases,
proteasome, DNA repair)
Antioxidant in food chemistry
• Reducing agent capable
of terminating chain
reaction of lipid
peroxidation
…“chain-breaking” ...
• E.g.:
– Butylated
hydroxytoluene (BHT)
– Butylated hydroxyanisol
(BHA)
– Tocopherol (Vitamin E)
19

Antioxidant defence of human body
• Anatomy of the body limiting tissue oxygen
• Antioxidant enzymes
• Sequestration of redox active metals
• Antioxidant substrates (scavengers)
• Stress response
• (Repair systems)
O 2
First organism
(anaerobic)
?
Develop
antioxidant
defence ?
Die out ?
Resort to
anaerobic
condition ?
20

Clump together !
O 2
O 2
Antioxidant defence I
Regulation of tissue O 2
Inhaled air: 160 mmHg O 2
Lung capillaries: 100 mmHg O 2
Arterial blood: 85 mmHg O 2
Arterioles: 70 mmHg O 2
Capillaries: 50 mmHg O 2
Cells: 1-10 mmHg O 2
Mitochondria: < 0,5 mmHg O 2
Fig: Wikipedia
21

Mitochondria originated from
phagocyted/parasitic bacteria ...
Antioxidant defence II
Antioxidant enzymes
• Superoxide dismutase:
O 2·–
+ O 2·–
+ 2 H + → O 2 + H 2 O 2
• Catalase:
2 H 2 O 2 → 2 H 2 O + O 2
• Glutathione peroxidase, peroxiredoxin:
H 2 O 2 + 2 R-SH → 2 H 2 O + RS-SR
22

Superoxide dismutase (SOD)
• Catalyses dismutation of superoxide:
O 2·–
+ O 2·–
+ 2 H + → O 2 + H 2 O 2
• Absolutely required for life in oxygen
• SOD1: Cu+Zn (cytosol of eukaryotic cells)
• SOD2:
– Mn (mitochondrial matrix)
– Fe (bacteria)
• EC-SOD (SOD3): extracelullar, Cu+Zn,
– MW 135,000; binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)
• Reduction of peroxides coupled to oxidation of
glutathione:
2 GSH + H 2 O 2 → GS-SG + 2 H 2 O
(glutathione is subsequently regenerated by
glutathione reductases)
• Active site contains selenocysteine
• Cytosolic glutathione peroxidase:
– reduces H 2 O 2 and LOOH after release from
phospholipids
• Phospholipide hydroperoxide-GSH-peroxidase:
– reduces LOOH even in membranes
23

Glutathione (GSH/GSSG)
• tripeptide, in every cell
1-10 mM
• keeps ICT reduced
• substrate for GPX, etc.
• also non-enzymatic
reactions with ROS and
mixed disulfides with
proteins ... products of
GSH oxidation are toxic
for cell
• in oxidative stress the
cell exports GSSG out
Catalase
• Tetramer, every subunit contains heme with
Fe
• Dismutation of hydrogen peroxide:
2 H 2 O 2 → 2 H 2 O + O 2
• Red blood cells, peroxisomes
• Also peroxidase activity:
H 2 O 2 + ROOH → H 2 O + ROH + O 2
(in comparison to GPX less significant)
24

Oxidation
of very long chain fatty acids in peroxisome
oxisomes:
O 2·–
Superoxide dismutase
H 2 O 2
H 2 O
Glutathione
peroxidase
GSH
Reduced glutathione
(GSH)
GS-SG Oxidised glutathione NADP +
(GS-SG)
Glutathione
reductase
NADPH+H +
Transhydrogenase
NAD +
NADH+H +
Pentose cycle
ATP
25

Peroxiredoxin/Thioredoxin
• Recently discovered antioxidant system,
more important for removal of hydrogen
peroxide than GPX
O 2·–
Superoxide dismutase
NADPH+H +
H 2 O 2
Peroxiredoxin
RED
Thioredoxin
RED
Thioredoxin
reductase RED
SH HS
SH HS
FADH 2 (Se)
GSH
Peroxiredoxin
OX
Thioredoxin
OX
Thioredoxin
reductase OX
H 2 O
S
S
S
S
FAD (Se)
NADP +
26

Antioxidant defence III
Sequestration of metals
• Redox-active transition metals (Fe, Cu)
accept/donate one electron easily
– ... alleviation of spin restriction of dioxygen
– ... metals are in active centers of all oxygen handlingenzymes
• But, the same properties of Fe, Cu are deleterious
if uncontrolled
– the Fenton oxidant:
H 2 O 2 + Fe 2+ → OH – + OH· + Fe 3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metals
• Iron/copper handling proteins:
– transferrin: binds 2 atoms Fe 3+ (transport)
– lactoferrin: analogous to transferrin, but no Fe
release (... only sequestration), leucocytes
– ferritin: H and L subunits, H is ferroxidase, Fe
storage (up to 4500 atoms Fe 3+ )
– haptoglobin: binds hemoglobin in circulation
– hemopexin: binds heme in circulation
– ceruloplasmin: contains Cu, function:
ferroxidase (export Fe from the cells)
– albumin: transport of Cu
27

ICT
ECT
Superoxide
Peroxide
Fe/Cu
Superoxide
Peroxide
Fe/Cu
Superoxide dismutase
Glutathione peroxidase
Catalase
Glutathione
Tocopherol
Ascorbate
Labile iron pool (LIP) present
Antioxidant enzymes &
glutathione levels very low
Tocopherol
Ascorbate
Carotenoids, uric acid, albumin,
glucose, bilirubin...
Sequestration of iron and copper:
- transferrin, lactoferrin
- hemopexin
- haptoglobin
- ceruloplasmin (ferroxidase)
- Cu bound to albumin
Antioxidant defence IV
Low-molecular-weight antioxidant substrates
• THIOLS:
– Glutathione
– Thioredoxin
• OTHER ENDOGENOUS METABOLITES:
– Bilirubin
– Uric acid
– Lipoic acid
• DIETARY:
– Ascorbate (Vitamin C)
– α-Tocopherol (Vitamin E)
– Carotenoids
– Plant phenols
28

Tocopherols (Vitamin E)
• group of 8 isomers,
α-tocopherol most
effective
• antioxidant of
membranes
(lipophilic)
• “chain-breaking” ...
terminates the
chain reaction of
lipid peroxidation
Ascorbate (Vitamin C)
• Redox-active saccharide
• In most animals synthesized from glucuronic acid
• Vitamin for humans, other primates, bats and guinea pigs
• Deficit causes scurvy (scorbut)
29

Ascorbate in the body:
• Main function is pro-oxidant: cofactor of hydroxylases
– Hydroxylation of Pro and Lys in collagen synthesis
– Synthesis of noradrenaline from dopamine
– Synthesis of carnitine (… role in oxidation of fat)
– Activation of hypothalamic peptidic hormones by
amidation (CRH, GRH, oxytocin, vasopressin,
substance P)
• Reductant for iron: promotes its intestinal absorption
• Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) (?)
• Daily need 70-100 mg, high doses p.o. excreted by
urine (renal threshold cca 200 mg/24 hours)
membrane compartment:
hydrophilic compartment:
LH
Tocopherol
Semidehydroascorbate
LOO·
+e -
chain reaction of
lipid peroxidation
Dehydroascorbate
LOOH
L·
Tocopheryl
radical
Ascorbate
-e - dehydroascorbate
GSSG
reductase
2GSH
30

Activated neutrophiles
accumulate
dehydroascorbate (DHA)
DHA
GLUT1
GSH
Glutaredoxin
Ascorbate
GSSG
Protects membrane of the neutrophile
from its own ROS ...
• Selenium:
– Trace element (daily need 55 µg), possibility of
deficiency as well as intoxication
– Component of several antioxidant enzymes
(glutathione peroxidase, thioredoxin reductase)
and also e.g. 5‘-dejodase
• β-carotene (provitamin A) is precursor for
synthesis of:
– Retinal … vision
– Retinoic acid …regulator of gene expression,
cellular growth and differentiation
31

Antioxidant defence V
Stress response
Oxidation or nitrosylation of sensor -SH
Transcription factors (NFκB, Nrf-2…):
activation, nuclear translocation
Induction of gene expression:
• chaperones (heat shock proteins)
• antioxidant enzymes
• metallothionein
• hemoxygenase 1
…→ more resistant to further
oxidative stress
Apoptosis as the
ultimate antioxidant defence ?
32

Why red blood cells need
pentose phosphate cycle?
2 O 2·–
+2H +
H 2 O 2 + O 2
Superoxide
dismutase
NADP +
H 2 O
Glutathione
peroxidase
Glutathione
reductase
Methemoglobin
reductase
Hb-FeII
NADPH+H +
Hb-FeIII
Pentose phosphate pathway
33

Free radicals in pathogenesis of human diseases
• Cause of disease, e.g.:
• cancerogenesis due to exposition to ionising radiation
• retinopathy of the newborn (fibroplasia retrolentalis)
• Contribute to pathogenesis, e.g:
• atherosclerosis
• diabetes mellitus
• hypertension
• some kinds of cancer
• brain trauma/hemorrhage
• ischemia/reperfusion injury of heart and other organs
• Parkinson disease
• Alzheimer disease
• ageing
• Merely an epiphenomenon (general consequence of
tissue damage)
34