• ROS (Reactive Oxygen Species)
• RNS (Reactive Nitrogen Species)
90% of oxygen is used in the oxidative phosphorilation
9% is consumed by enzymes which catalizes reactions of
hydroxilation and oxidation
1% ROS
ROS
•O - (superoxide anion),
2
•
O2H (hydroperoxide radical);
•
OH (hydroxyl radical);
These species do not diffuse within the cell
H2O2
HClO
They are not radicalic species but they can produce
hydroxyl radical
H2O2 can diffuse within the cell and between different
cells. It can act as second messanger
In biological systems, ROS are generated and eliminated
.
Fenton e Haber-Weiss
Fenton:
H2O2 + Fe2+ (Cu+ ) à Fe3+ (Cu2+) + HO• + OH¯
Haber-Weiss:
H2O2 + •O2- à HO• + OH¯ + O2
Haber-Weiss is catalized by metals
The release of metals due to necrosis or inflammation causes
oxidative stress
Mithocondrion is the principal source of ROS
An important contribution in the production of
superoxide • O2- derived from the plasma membrane
multienzyme complex of NAD (P) H oxidase
In the smooth endoplasmic reticulum, the complex of the
cytochrome P-450, that catalyzes hydroxylation reactions
of organic xenobiotics or substrates can generate the
superoxide anion by means of direct oxygen reduction
O2 + CitP450 (Fe2 +) à P450 (Fe3+) + •O2-
In Peroxisomes, oxidation of fatty acids and oxidative
deamination of certain amino acids produces H2O2
R-CH(NH2)-COOH + O2 + H2O à R-CO-COOH + NH3 + H2O2
In addition the H2O2 can also be produced by some
cytosolic oxidases such as cyclooxygenase, monoamine
oxidase, xanthine oxidase, lipoxygenase
1
Inner
mitochondrial
complex I -complex Oxidative
membrane-
III phoshorilation
electron transport
chain
2
Plasma Mechanisms of cell-
NAD (P) H oxidase
membrane mediated defense
smooth
3 Detoxification
endoplasmic citocrome P450
processes
reticulum
4
Peroxisomes oxidases Fatti acid oxidation
5 Cytosol xantina ossidasi Tissue riperfusion
At moderate concentrations, ROS actively participate to a variety of
complex biological processes involved in normal cell growth such as
signal transduction, the control of gene expression, cell senescence,
apoptosis
They contribute to the killing of bacteria in neutrophils
A ROS high concentrations are harmful to the body because they attack
the major constituents of the cell (proteins, nucleic acids, lipids,
carbohydrates).
Some aspects of the vascular damage in atherosclerosis are linked to
ROS
Many aspects of carcinogenesis involving reactions between free radicals
and DNA lead to mutations.
The ROS induced by alcohol metabolism contribute to
liver damage
ROS are also implicated in disease with progressive
loss of the cellular population: Parkinson's, senile
dementia, Alzheimer's.
Hemochromatosis, a iron metabolism disease is
associated with formation of free radicals in the skin,
nervous system, pancreas, as well as in the liver
Endogenous Exogenous
respiratory chain (mitochondria) xenobiotics
NADPH oxidase Ionizing radiations
cytochrome P450 ultraviolet light
peroxisomes Ros sources electric fields
xanthine oxidase
cyclooxygenase chemotherapeutic agents
lipoxygenase
monoamine oxidase
•
O 2- HClO
•
O 2H
•
OH H2O2
10 -8 - 10 -5 M 10 -3 - 10 -4 M
Oxidative damage to
ROS macromolecules
Signal transduction
functions Inflammation
gene expression control
cellular senescence uncontrolled proliferation
apoptosis Aging
chemotherapy resistance
Necrosis
polyunsaturated fatty acids
The hydroxyl radical •OH reacts with biomolecules
One of the most sensitive sites to the damage caused by ROS is
the plasma membrane, in particular the target is at the level of
polyunsaturated fatty acids.
The hydroxyl radical •OH subtracts an hydrogen atom to a
polyunsaturated fatty acid, thus starting a chain reaction of lipid
peroxidation.
The lipid peroxides formed in this reaction are degraded to form
the characteristic products such as malondialdehyde (MDA) or the
4-hydroxynonenal (HNE)
Malondialdehyde (MDA) or 4-hydroxynonenal (HNE) react with
the proteins forming chemical adducts which are named advanced
lipooxidatin end products -ALE that are biological markers of
oxidative stress
The hydroxyl radical •OH reacts with various amino acids to form
hydroxylated derivatives (p. Example Phe)
The H2O2 and HClO react with methionine and tyrosine in particular
to form methionine sulphoxide, ditirosine, and tirosine + clorine
Cys can be oxidised
•OH reacts with nitrogen bases. The major oxidation products are the
8-ossiguanosine (the main indicator of DNA damage), timinaglicole,
5-idrossimetiluracile
In this case you can have repair for excision of bases or nucleotide
excision
ROS can cause breakage in double stranded or single-stranded,
and crosslinking.
These types of damage are difficult to repair.
The cells exposed to DNA damage activates DNA Damage
Response DDR. The DDR causes a transient growth arrest to
facilitate an efficient DNA repair, or if the damage is not
repairable cause permanent arrest, cellular senescence or
alternatively apoptosis.
ROS also react with carbohydrates to form dicarbonyl
compounds which react with proteins, nucleic acids
and lipids, forming crosslinks or adducts, known as
advanced glycosilation end products, AGE
Antioxidant defenses
defense system against ROS can be enzyme or not
("scavenger").
The main enzymes responsible for redox homeostasis are:
superoxide dismutase (SOD)
catalase
glutathione peroxidase
thioredoxin
peroxiredoxin
These enzymes act as "scavengers" of ROS, but they cannot
act directly on biological macromolecules to remove a
damage already occurred.
SOD
2 •O2- + 2H+ à H2O2 + O2
• various isoenzymes.
• mitochondrial isoenzymes uses as a cofactor manganese (MnSOD).
• Other isoenzymes, have cytosolic or nuclear localization and use as
cofactors copper or zinc (Cu / ZnSOD).
• There is also a secreted extracellular isozyme Cu / ZnSOD (EC-SOD)
Catalase e glutathione peroxidase
• Catalase is present in peroxisomes :
2H2O2 à O2 + 2H2O
• Glutathione peroxidase uses reduced glutathione (GSH):
H2O2 + 2GSH à 2H2O + GSSG
thioredoxin and peroxiredoxin
Thioredoxin is a Small ubiquitous protein with redox activity
which contains two cysteines.
It carries hydrogen atoms, provided by NADPH.
This protein is part of a multiprotein system involving two other
enzymes such as
the thioredoxin reductase and thioredoxin peroxidase.
Peroxiredoxin, uses only one cysteine residue which is
oxidized to sulfenic acid RSOH.
Non enzymatic system
Vitamin C (or ascorbic acid), vitamin E (or a and g-
tocopherol), vitamin A (or b-carotene), and the GSH
Vitamin C and vitamin E act in a coordinated and synergistic
way
Vitamin E, a fat-soluble, protects the membranes from
oxidative damage by blocking the chain reactions
characteristics of the lipid peroxidation process of biological
membranes.
vitamin C, water-soluble, regenerates vitamin E becoming
ascorbyl radical, and is converted back to ascorbic acid by
an NADPH-dependent reductase.
Vitamina E
Vitamina C
NO
NO is formed in many tissues:
1) Brain (neuronal and glial cells)
3) Retina
5) Adrenal Medullary
6) Vascular Endothelial Cells
NO, stimulates the activity of guanilate ciclase (GC)
producing cGMP
Monossigenasi Ossidazione a 5 elettroni
idrossiarginina(C=N-OH);
1/2 NADPH, O2--->1/2 NADP+ + H2O
NOS
constitutive form present in the nervous cells and endothelium
induces a low and transient production of NO.
Calcium / CaM-dependent
The constitutive form also requires tetrahydrobiopterin (BH4)
Low production of NO modulates vascular tone, contributes to the
regulation of blood pressure and platelet aggregation and acts as a
neuromodulator
inducible form: present in various cell types including
macrophages, muscles, liver. Calcium / CaM-independent; it is
induced by endotoxins, interferon g, by tumor necrosis factor
(TNF a), by IL-1 and produce sensitive amounts of NO for
considerably long periods
